Guiding plant virus particles to integrin-displaying cells

NASA Astrophysics Data System (ADS)

Hovlid, Marisa L.; Steinmetz, Nicole F.; Laufer, Burkhardt; Lau, Jolene L.; Kuzelka, Jane; Wang, Qian; Hyypiä, Timo; Nemerow, Glen R.; Kessler, Horst; Manchester, Marianne; Finn, M. G.

2012-05-01

Viral nanoparticles (VNPs) are structurally regular, highly stable, tunable nanomaterials that can be conveniently produced in high yields. Unmodified VNPs from plants and bacteria generally do not show tissue specificity or high selectivity in binding to or entry into mammalian cells. They are, however, malleable by both genetic and chemical means, making them useful scaffolds for the display of large numbers of cell- and tissue-targeting ligands, imaging moieties, and/or therapeutic agents in a well-defined manner. Capitalizing on this attribute, we modified the genetic sequence of the Cowpea mosaic virus (CPMV) coat protein to display an RGD oligopeptide sequence derived from human adenovirus type 2 (HAdV-2). Concurrently, wild-type CPMV was modified via NHS acylation and Cu(i)-catalyzed azide-alkyne cycloaddition (CuAAC) chemistry to attach an integrin-binding cyclic RGD peptide. Both types of particles showed strong and selective affinity for several different cancer cell lines that express RGD-binding integrin receptors.Viral nanoparticles (VNPs) are structurally regular, highly stable, tunable nanomaterials that can be conveniently produced in high yields. Unmodified VNPs from plants and bacteria generally do not show tissue specificity or high selectivity in binding to or entry into mammalian cells. They are, however, malleable by both genetic and chemical means, making them useful scaffolds for the display of large numbers of cell- and tissue-targeting ligands, imaging moieties, and/or therapeutic agents in a well-defined manner. Capitalizing on this attribute, we modified the genetic sequence of the Cowpea mosaic virus (CPMV) coat protein to display an RGD oligopeptide sequence derived from human adenovirus type 2 (HAdV-2). Concurrently, wild-type CPMV was modified via NHS acylation and Cu(i)-catalyzed azide-alkyne cycloaddition (CuAAC) chemistry to attach an integrin-binding cyclic RGD peptide. Both types of particles showed strong and selective affinity for several different cancer cell lines that express RGD-binding integrin receptors. Electronic supplementary information (ESI) available: Synthetic procedures and compound characterization data; assay procedures; additional confocal micrographs at different time points. See DOI: 10.1039/c2nr30571b

Synthesis and evaluation of two NIR fluorescent cyclic RGD penta-peptides for targeting integrins

NASA Astrophysics Data System (ADS)

Ye, Yunpeng; Bloch, Sharon; Xu, Baogang; Achilefu, Samuel

2006-02-01

Interest in novel RGD peptides has been increasingly growing as the interactions between RGD peptides and integrins are the basis for a variety of cellular functions and medical applications such as modulation of cell adhesion, invasion, tumor angiogenesis, and metastasis. In particular, we have been interested in novel NIR fluorescent RGD peptides as potential optical contrast agents for in vivo tumor optical imaging. Therefore, two cyclic RGD penta-peptides conjugated with a NIR fluorescent carbocyanine (Cypate), i.e. lactam-based cyclo[RGDfK(Cypate)] (1) and disulfide-containing Cypate-cyclo(CRGDC)-NH II (2), were designed and synthesized. The competitive binding assay between the purified α vβ 3 integrin and the peptide ligands using 125I-echistatin as a tracer showed that 1 had a higher receptor binding affinity (IC 50~10 -7 M) than 2 (IC 50~10 -6 M). Furthermore, the internalization of 1 in A549 cells in vitro was less than 2, as revealed by fluorescence microscopy. These results suggest that both the lactam- and disulfide-based cyclic RGD penta-peptides should be further studied structurally and functionally to elucidate the advantages of each class of compounds.

The use of one-bead one-compound combinatorial library technology to discover high-affinity αvβ3 integrin and cancer targeting arginine-glycine-aspartic acid ligands with a built-in handle.

PubMed

Xiao, Wenwu; Wang, Yan; Lau, Edmond Y; Luo, Juntao; Yao, Nianhuan; Shi, Changying; Meza, Leah; Tseng, Harry; Maeda, Yoshiko; Kumaresan, Pappanaicken; Liu, Ruiwu; Lightstone, Felice C; Takada, Yoshikazu; Lam, Kit S

2010-10-01

The αvβ3 integrin, expressed on the surface of various normal and cancer cells, is involved in numerous physiologic processes such as angiogenesis, apoptosis, and bone resorption. Because this integrin plays a key role in angiogenesis and metastasis of human tumors, αvβ3 integrin ligands are of great interest to advances in targeted therapy and cancer imaging. In this report, one-bead one-compound (OBOC) combinatorial libraries containing the arginine-glycine-aspartic acid (RGD) motif were designed and screened against K562 myeloid leukemia cells that had been transfected with the human αvβ3 integrin gene. Cyclic peptide LXW7 was identified as a leading ligand with a built-in handle that binds specifically to αvβ3 and showed comparable binding affinity (IC(50) = 0.68 ± 0.08 μmol/L) to some of the well-known RGD "head-to-tail" cyclic pentapeptide ligands reported in the literature. The biotinylated form of LXW7 ligand showed similar binding strength as LXW7 against αvβ3 integrin, whereas biotinylated RGD cyclopentapeptide ligands revealed a 2- to 8-fold weaker binding affinity than their free forms. LXW7 was able to bind to both U-87MG glioblastoma and A375M melanoma cell lines, both of which express high levels of αvβ3 integrin. In vivo and ex vivo optical imaging studies with the biotinylated ligand/streptavidin-Cy5.5 complex in nude mice bearing U-87MG or A375M xenografts revealed preferential uptake of biotinylated LXW7 in tumor. When compared with biotinylated RGD cyclopentapeptide ligands, biotinylated LXW7 showed higher tumor uptake but lower liver uptake.

Integrin Engagement by the Helical RGD Motif of the Helicobacter pylori CagL Protein Is Regulated by pH-induced Displacement of a Neighboring Helix*

PubMed Central

Bonsor, Daniel A.; Pham, Kieu T.; Beadenkopf, Robert; Diederichs, Kay; Haas, Rainer; Beckett, Dorothy; Fischer, Wolfgang; Sundberg, Eric J.

2015-01-01

Arginine-aspartate-glycine (RGD) motifs are recognized by integrins to bridge cells to one another and the extracellular matrix. RGD motifs typically reside in exposed loop conformations. X-ray crystal structures of the Helicobacter pylori protein CagL revealed that RGD motifs can also exist in helical regions of proteins. Interactions between CagL and host gastric epithelial cell via integrins are required for the translocation of the bacterial oncoprotein CagA. Here, we have investigated the molecular basis of the CagL-host cell interactions using structural, biophysical, and functional analyses. We solved an x-ray crystal structure of CagL that revealed conformational changes induced by low pH not present in previous structures. Using analytical ultracentrifugation, we found that pH-induced conformational changes in CagL occur in solution and not just in the crystalline environment. By designing numerous CagL mutants based on all available crystal structures, we probed the functional roles of CagL conformational changes on cell surface integrin engagement. Together, our data indicate that the helical RGD motif in CagL is buried by a neighboring helix at low pH to inhibit CagL binding to integrin, whereas at neutral pH the neighboring helix is displaced to allow integrin access to the CagL RGD motif. This novel molecular mechanism of regulating integrin-RGD motif interactions by changes in the chemical environment provides new insight to H. pylori-mediated oncogenesis. PMID:25837254

A natural variant of the cysteine protease virulence factor of group A Streptococcus with an arginine-glycine-aspartic acid (RGD) motif preferentially binds human integrins alphavbeta3 and alphaIIbbeta3.

PubMed

Stockbauer, K E; Magoun, L; Liu, M; Burns, E H; Gubba, S; Renish, S; Pan, X; Bodary, S C; Baker, E; Coburn, J; Leong, J M; Musser, J M

1999-01-05

The human pathogenic bacterium group A Streptococcus produces an extracellular cysteine protease [streptococcal pyrogenic exotoxin B (SpeB)] that is a critical virulence factor for invasive disease episodes. Sequence analysis of the speB gene from 200 group A Streptococcus isolates collected worldwide identified three main mature SpeB (mSpeB) variants. One of these variants (mSpeB2) contains an Arg-Gly-Asp (RGD) sequence, a tripeptide motif that is commonly recognized by integrin receptors. mSpeB2 is made by all isolates of the unusually virulent serotype M1 and several other geographically widespread clones that frequently cause invasive infections. Only the mSpeB2 variant bound to transfected cells expressing integrin alphavbeta3 (also known as the vitronectin receptor) or alphaIIbbeta3 (platelet glycoprotein IIb-IIIa), and binding was blocked by a mAb that recognizes the streptococcal protease RGD motif region. In addition, mSpeB2 bound purified platelet integrin alphaIIbbeta3. Defined beta3 mutants that are altered for fibrinogen binding were defective for SpeB binding. Synthetic peptides with the mSpeB2 RGD motif, but not the RSD sequence present in other mSpeB variants, blocked binding of mSpeB2 to transfected cells expressing alphavbeta3 and caused detachment of cultured human umbilical vein endothelial cells. The results (i) identify a Gram-positive virulence factor that directly binds integrins, (ii) identify naturally occurring variants of a documented Gram-positive virulence factor with biomedically relevant differences in their interactions with host cells, and (iii) add to the theme that subtle natural variation in microbial virulence factor structure alters the character of host-pathogen interactions.

An Rgd Sequence in the P2y2 Receptor Interacts with αVβ3 Integrins and Is Required for Go-Mediated Signal Transduction

PubMed Central

Erb, Laurie; Liu, Jun; Ockerhausen, Jonathan; Kong, Qiongman; Garrad, Richard C.; Griffin, Korey; Neal, Chris; Krugh, Brent; Santiago-Pérez, Laura I.; González, Fernando A.; Gresham, Hattie D.; Turner, John T.; Weisman, Gary A.

2001-01-01

The P2Y2 nucleotide receptor (P2Y2R) contains the integrin-binding domain arginine-glycine-aspartic acid (RGD) in its first extracellular loop, raising the possibility that this G protein–coupled receptor interacts directly with an integrin. Binding of a peptide corresponding to the first extracellular loop of the P2Y2R to K562 erythroleukemia cells was inhibited by antibodies against αVβ3/β5 integrins and the integrin-associated thrombospondin receptor, CD47. Immunofluorescence of cells transfected with epitope-tagged P2Y2Rs indicated that αV integrins colocalized 10-fold better with the wild-type P2Y2R than with a mutant P2Y2R in which the RGD sequence was replaced with RGE. Compared with the wild-type P2Y2R, the RGE mutant required 1,000-fold higher agonist concentrations to phosphorylate focal adhesion kinase, activate extracellular signal–regulated kinases, and initiate the PLC-dependent mobilization of intracellular Ca2+. Furthermore, an anti-αV integrin antibody partially inhibited these signaling events mediated by the wild-type P2Y2R. Pertussis toxin, an inhibitor of Gi/o proteins, partially inhibited Ca2+ mobilization mediated by the wild-type P2Y2R, but not by the RGE mutant, suggesting that the RGD sequence is required for P2Y2R-mediated activation of Go, but not Gq. Since CD47 has been shown to associate directly with Gi/o family proteins, these results suggest that interactions between P2Y2Rs, integrins, and CD47 may be important for coupling the P2Y2R to Go. PMID:11331301

The fibronectin synergy site re-enforces cell adhesion and mediates a crosstalk between integrin classes

PubMed Central

Benito-Jardón, Maria; Klapproth, Sarah; Gimeno-LLuch, Irene; Petzold, Tobias; Bharadwaj, Mitasha; Müller, Daniel J; Zuchtriegel, Gabriele; Reichel, Christoph A; Costell, Mercedes

2017-01-01

Fibronectin (FN), a major extracellular matrix component, enables integrin-mediated cell adhesion via binding of α5β1, αIIbβ3 and αv-class integrins to an RGD-motif. An additional linkage for α5 and αIIb is the synergy site located in close proximity to the RGD motif. We report that mice with a dysfunctional FN-synergy motif (Fn1syn/syn) suffer from surprisingly mild platelet adhesion and bleeding defects due to delayed thrombus formation after vessel injury. Additional loss of β3 integrins dramatically aggravates the bleedings and severely compromises smooth muscle cell coverage of the vasculature leading to embryonic lethality. Cell-based studies revealed that the synergy site is dispensable for the initial contact of α5β1 with the RGD, but essential to re-enforce the binding of α5β1/αIIbβ3 to FN. Our findings demonstrate a critical role for the FN synergy site when external forces exceed a certain threshold or when αvβ3 integrin levels decrease below a critical level. DOI: http://dx.doi.org/10.7554/eLife.22264.001 PMID:28092265

Effect of P to A Mutation of the N-Terminal Residue Adjacent to the Rgd Motif on Rhodostomin: Importance of Dynamics in Integrin Recognition

PubMed Central

Chen, Yi-Chun; Chang, Yao-Tsung; Chang, Yung-Sheng; Huang, Chun-Hao; Chuang, Woei-Jer

2012-01-01

Rhodostomin (Rho) is an RGD protein that specifically inhibits integrins. We found that Rho mutants with the P48A mutation 4.4–11.5 times more actively inhibited integrin α5β1. Structural analysis showed that they have a similar 3D conformation for the RGD loop. Docking analysis also showed no difference between their interactions with integrin α5β1. However, the backbone dynamics of RGD residues were different. The values of the R2 relaxation parameter for Rho residues R49 and D51 were 39% and 54% higher than those of the P48A mutant, which caused differences in S2, Rex, and τe. The S2 values of the P48A mutant residues R49, G50, and D51 were 29%, 14%, and 28% lower than those of Rho. The Rex values of Rho residues R49 and D51 were 0.91 s−1 and 1.42 s−1; however, no Rex was found for those of the P48A mutant. The τe values of Rho residues R49 and D51 were 9.5 and 5.1 times lower than those of P48A mutant. Mutational study showed that integrin α5β1 prefers its ligands to contain (G/A)RGD but not PRGD sequences for binding. These results demonstrate that the N-terminal proline residue adjacent to the RGD motif affect its function and dynamics, which suggests that the dynamic properties of the RGD motif may be important in Rho's interaction with integrin α5β1. PMID:22238583

Dependence of cancer cell adhesion kinetics on integrin ligand surface density measured by a high-throughput label-free resonant waveguide grating biosensor.

PubMed

Orgovan, Norbert; Peter, Beatrix; Bősze, Szilvia; Ramsden, Jeremy J; Szabó, Bálint; Horvath, Robert

2014-02-07

A novel high-throughput label-free resonant waveguide grating (RWG) imager biosensor, the Epic® BenchTop (BT), was utilized to determine the dependence of cell spreading kinetics on the average surface density (v(RGD)) of integrin ligand RGD-motifs. v(RGD) was tuned over four orders of magnitude by co-adsorbing the biologically inactive PLL-g-PEG and the RGD-functionalized PLL-g-PEG-RGD synthetic copolymers from their mixed solutions onto the sensor surface. Using highly adherent human cervical tumor (HeLa) cells as a model system, cell adhesion kinetic data of unprecedented quality were obtained. Spreading kinetics were fitted with the logistic equation to obtain the spreading rate constant (r) and the maximum biosensor response (Δλmax), which is assumed to be directly proportional to the maximum spread contact area (Amax). r was found to be independent of the surface density of integrin ligands. In contrast, Δλmax increased with increasing RGD surface density until saturation at high densities. Interpreting the latter behavior with a simple kinetic mass action model, a 2D dissociation constant of 1753 ± 243 μm(-2) (corresponding to a 3D dissociation constant of ~30 μM) was obtained for the binding between RGD-specific integrins embedded in the cell membrane and PLL-g-PEG-RGD. All of these results were obtained completely noninvasively without using any labels.

Variation in One Residue Associated with the Metal Ion-Dependent Adhesion Site Regulates αIIbβ3 Integrin Ligand Binding Affinity

PubMed Central

Wu, Xue; Xiu, Zhilong; Li, Guohui; Luo, Bing-Hao

2013-01-01

The Asp of the RGD motif of the ligand coordinates with the β I domain metal ion dependent adhesion site (MIDAS) divalent cation, emphasizing the importance of the MIDAS in ligand binding. There appears to be two distinct groups of integrins that differ in their ligand binding affinity and adhesion ability. These differences may be due to a specific residue associated with the MIDAS, particularly the β3 residue Ala252 and corresponding Ala in the β1 integrin compared to the analogous Asp residue in the β2 and β7 integrins. Interestingly, mutations in the adjacent to MIDAS (ADMIDAS) of integrins α4β7 and αLβ2 increased the binding and adhesion abilities compared to the wild-type, while the same mutations in the α2β1, α5β1, αVβ3, and αIIbβ3 integrins demonstrated decreased ligand binding and adhesion. We introduced a mutation in the αIIbβ3 to convert this MIDAS associated Ala252 to Asp. By combination of this mutant with mutations of one or two ADMIDAS residues, we studied the effects of this residue on ligand binding and adhesion. Then, we performed molecular dynamics simulations on the wild-type and mutant αIIbβ3 integrin β I domains, and investigated the dynamics of metal ion binding sites in different integrin-RGD complexes. We found that the tendency of calculated binding free energies was in excellent agreement with the experimental results, suggesting that the variation in this MIDAS associated residue accounts for the differences in ligand binding and adhesion among different integrins, and it accounts for the conflicting results of ADMIDAS mutations within different integrins. This study sheds more light on the role of the MIDAS associated residue pertaining to ligand binding and adhesion and suggests that this residue may play a pivotal role in integrin-mediated cell rolling and firm adhesion. PMID:24116162

[68Ga]FSC-(RGD)3 a trimeric RGD peptide for imaging αvβ3 integrin expression based on a novel siderophore derived chelating scaffold—synthesis and evaluation

PubMed Central

Knetsch, Peter A.; Zhai, Chuangyan; Rangger, Christine; Blatzer, Michael; Haas, Hubertus; Kaeopookum, Piriya; Haubner, Roland; Decristoforo, Clemens

2015-01-01

Over the last years Gallium-68 (68Ga) has received tremendous attention for labeling of radiopharmaceuticals for positron emission tomography (PET). 68Ga labeling of biomolecules is currently based on bifunctional chelators containing aminocarboxylates (mainly DOTA and NOTA). We have recently shown that cyclic peptide siderophores have very good complexing properties for 68Ga resulting in high specific activities and excellent metabolic stabilities, in particular triacetylfusarinine-C (TAFC). We postulated, that, starting from its deacetylated form (Fusarinine-C (FSC)) trimeric bioconjugates are directly accessible to develop novel targeting peptide based 68Ga labeled radiopharmaceuticals. As proof of principle we report on the synthesis and 68Ga-radiolabeling of a trimeric FSC-RGD conjugate, [68Ga]FSC-(RGD)3, targeting αvβ3 integrin, which is highly expressed during tumor-induced angiogenesis. Synthesis of the RGD peptide was carried out applying solid phase peptide synthesis (SPPS), followed by the coupling to the siderophore [Fe]FSC via in situ activation using HATU/HOAt and DIPEA. Subsequent demetalation allowed radiolabeling of FSC-(RGD)3 with 68Ga. The radiolabeling procedure was optimized regarding peptide amount, reaction time, temperature as well buffer systems. For in vitro evaluation partition coefficient, protein binding, serum stability, αvβ3 integrin binding affinity, and tumor cell uptake were determined. For in vitro tests as well as for the biodistribution studies αvβ3 positive human melanoma M21 and αvβ3 negative M21-L cells were used. [68Ga]FSC-(RGD)3 was prepared with high radiochemical yield (> 98%). Distribution coefficient was − 3.6 revealing a hydrophilic character, and an IC50 value of 1.8 ± 0.6 nM was determined indicating a high binding affinity for αvβ3 integrin. [68Ga]FSC-(RGD)3 was stable in PBS (pH 7.4), FeCl3- and DTPA-solution as well as in fresh human serum at 37 °C for 2 hours. Biodistribution assay confirmed the receptor specific uptake found in vitro. Uptake in the αvβ3 positive tumor was 4.3% ID/g 60 min p.i. which was 3-fold higher than the monomeric [68Ga]NODAGA-RGD. Tumor to blood ratio of approx. 8 and tumor to muscle ratio of approx. 7 were observed. [68Ga]FSC-(RGD)3 serves as an example for the feasibility of a novel class of bifunctional chelators based on cyclic peptide siderophores and shows excellent targeting properties for αvβ3 integrin in vivo for imaging tumor-induced neovascularization. PMID:25459110

Genetic Retargeting of Adenovirus: Novel Strategy Employing “Deknobbing” of the Fiber

PubMed Central

Magnusson, Maria K.; Hong, Saw See; Boulanger, Pierre; Lindholm, Leif

2001-01-01

For efficient and versatile use of adenovirus (Ad) as an in vivo gene therapy vector, modulation of the viral tropism is highly desirable. In this study, a novel method to genetically alter the Ad fiber tropism is described. The knob and the last 15 shaft repeats of the fiber gene were deleted and replaced with an external trimerization motif and a new cell-binding ligand, in this case the integrin-binding motif RGD. The corresponding recombinant fiber retained the basic biological functions of the natural fiber, i.e., trimerization, nuclear import, penton formation, and ligand binding. The recombinant fiber bound to integrins but failed to react with antiknob antibody. For virus production, the recombinant fiber gene was rescued into the Ad genome at the exact position of the wild-type (WT) fiber to make use of the native regulation of fiber expression. The recombinant virus Ad5/FibR7-RGD yielded plaques on 293 cells, but the spread through the monolayer was two to three times delayed compared to WT, and the ratio of infectious to physical particles was 20 times lower. Studies on virus tropism showed that Ad5/FibR7-RGD was able to infect cells which did not express the coxsackie-adenovirus receptor (CAR), but did express integrins. Ad5/FibR7-RGD virus infectivity was unchanged in the presence of antiknob antibody, which neutralized the WT virus. Ad5/FibR7-RGD virus showed an expanded tropism, which is useful when gene transfer to cells not expressing CAR is needed. The described method should also make possible the construction of Ad genetically retargeted via ligands other than RGD. PMID:11462000

Molecular imaging of alpha v beta3 integrin expression in atherosclerotic plaques with a mimetic of RGD peptide grafted to Gd-DTPA.

PubMed

Burtea, Carmen; Laurent, Sophie; Murariu, Oltea; Rattat, Dirk; Toubeau, Gérard; Verbruggen, Alfons; Vansthertem, David; Vander Elst, Luce; Muller, Robert N

2008-04-01

The integrin alpha v beta3 is highly expressed in atherosclerotic plaques by medial and intimal smooth muscle cells and by endothelial cells of angiogenic microvessels. In this study, we have assessed non-invasive molecular magnetic resonance imaging (MRI) of plaque-associated alpha v beta3 integrin expression on transgenic ApoE-/- mice with a low molecular weight peptidomimetic of Arg-Gly-Asp (mimRGD) grafted to gadolinium diethylenetriaminepentaacetate (Gd-DTPA-g-mimRGD). The analogous compound Eu-DTPA-g-mimRGD was employed for an in vivo competition experiment and to confirm the molecular targeting. The specific interaction of mimRGD conjugated to Gd-DTPA or to 99mTc-DTPA with alpha v beta3 integrin was furthermore confirmed on Jurkat T lymphocytes. The mimRGD was synthesized and conjugated to DTPA. DTPA-g-mimRGD was complexed with GdCl3.6H2O, EuCl3.6H2O, or with [99mTc(CO)3(H2O)3]+. MRI evaluation was performed on a 4.7 T Bruker imaging system. Blood pharmacokinetics of Gd-DTPA-g-mimRGD were assessed in Wistar rats and in c57bl/6j mice. The presence of angiogenic blood vessels and the expression of alpha v beta3 integrin were confirmed in aorta specimens by immunohistochemistry. Gd-DTPA-g-mimRGD produced a strong enhancement of the external structures of the aortic wall and of the more profound layers (possibly tunica media and intima). The aortic lumen seemed to be restrained and distorted. Pre-injection of Eu-DTPA-g-mimRGD diminished the Gd-DTPA-g-mimRGD binding to atherosclerotic plaque and confirmed the specific molecular targeting. A slower blood clearance was observed for Gd-DTPA-g-mimRGD, as indicated by a prolonged elimination half-life and a diminished total clearance. The new compound is potentially useful for the diagnosis of vulnerable atherosclerotic plaques and of other pathologies characterized by alpha v beta3 integrin expression, such as cancer and inflammation. The delayed blood clearance, the significant enhancement of the signal-to-noise ratio, and the low immunogenicity of the mimetic molecule highlight its potential for an industrial and clinical implementation.

Novel Approach to Prepare {sup 99m}Tc-Based Multivalent RGD Peptides

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shuang Liu

2012-10-24

This project presents a novel approach to prepare the {sup 99m}Tc-bridged multivalent RGD (arginine-glycine-aspartate) peptides. This project will focus on fundamentals of {sup 99m}Tc radiochemistry. The main objective of this project is to demonstrate the proof-of-principle for the proposed radiotracers. Once a kit formulation is developed for preparation of the {sup 99m}Tc-bridged multivalent RGD peptides, various tumor-bearing animal models will be used to evaluate their potential for SPECT (single photon-emission computed tomography) imaging of cancer. We have demonstrated that (1) multimerization of cyclic RGD peptides enhances the integrin {alpha}{sub v}{beta}{sub 3} bonding affinity and radiotracer tumor uptake; (2) addition ofmore » G{sub 3} or PEG{sub 4} linkers makes it possible for two RGD motifs in 3P-RGD{sub 2} and 3G-RGD{sub 2} to achieve simultaneous integrin {alpha}{sub v}{beta}{sub 3} binding; and (3) multimers are actually bivalent (not multivalent), the presence of extra RGD motifs can enhance the tumor retention time of the radiotracer.« less

Development of pre-implantation porcine blastocysts cultured within alginate hydrogel systems either supplemented with secreted phosphoprotein 1 or conjugated with Arg-Gly-Asp Peptide.

PubMed

Laughlin, Taylor D; Miles, Jeremy R; Wright-Johnson, Elane C; Rempel, Lea A; Lents, Clay A; Pannier, Angela K

2017-11-01

Although deficiencies in porcine blastocyst elongation play a significant role in early embryonic mortality and establishment of within-litter developmental variation, the exact mechanisms of elongation are poorly understood. Secreted phosphoprotein 1 (SPP1) is increased within the uterine milieu during early porcine pregnancy and contains an Arg-Gly-Asp (RGD) peptide sequence that binds to cell surface integrins on the uterine endometrium and trophectoderm, promoting cell adhesion and migration. The aim of the present study was to evaluate the development of preimplantation porcine blastocysts encapsulated and cultured within alginate hydrogels either supplemented with SPP1 or conjugated with RGD. Blastocysts encapsulated within alginate hydrogels supplemented with SPP1 or conjugated with RGD had increased survival compared with non-encapsulated control blastocysts. In addition, the percentage of blastocysts encapsulated within RGD hydrogels that underwent morphological changes was greater than that of blastocysts encapsulated within standard alginate hydrogels or SPP1-supplemented hydrogels. Finally, only blastocysts encapsulated within RGD hydrogels had both increased expression of steroidogenic and immune responsiveness transcripts and increased 17β-oestradiol production, consistent with blastocysts undergoing elongation in vivo. These results illustrate the importance of the integrin-binding RGD peptide sequence for stimulating the initiation of blastocyst elongation.

In vivo imaging of tumour angiogenesis in mice with the alpha(v)beta (3) integrin-targeted tracer 99mTc-RAFT-RGD.

PubMed

Sancey, Lucie; Ardisson, Valérie; Riou, Laurent M; Ahmadi, Mitra; Marti-Batlle, Danièle; Boturyn, Didier; Dumy, Pascal; Fagret, Daniel; Ghezzi, Catherine; Vuillez, Jean-Philippe

2007-12-01

The molecular imaging of tumour neoangiogenesis currently represents a major field of research for the diagnostic and treatment strategy of solid tumours. Endothelial cells from tumour neovessels overexpress the alpha(v)beta(3) integrin, which selectively binds to Arg-Gly-Asp (RGD)-containing peptides. We evaluated the potential of the novel radiotracer (99m)Tc-RAFT-RGD for the non-invasive molecular imaging of alpha(v)beta(3) integrin expression in mice models of tumour development. (99m)Tc-RAFT-RGD, (99m)Tc-cRGD (specific control) and (99m)Tc-RAFT-RAD (non-specific control) were injected intravenously to mice bearing B16F0 or TS/A-pc tumours. In vivo whole-body tomographic imaging and post-mortem biodistribution studies were performed 60 min following tracer injection. Adjacent tumour slices were used to compare the localisation of neovessels from immunostaining and the pattern of (99m)Tc-RAFT-RGD uptake from autoradiographic ex vivo imaging. Biodistribution studies indicated that (99m)Tc-RAFT-RGD tumour uptake was significantly higher than that of (99m)Tc-RAFT-RAD in B16F0 (2.4+/-0.5 vs 1.0+/-0.1%ID/g, respectively) and in TS/A-pc tumours (2.7+/-0.8 vs 0.7+/-0.1%ID/g, respectively). Immunohistochemical and autoradiographic studies indicated that (99m)Tc-RAFT-RGD intratumoural uptake preferentially occurred in angiogenic areas. Tomographic imaging allowed tumour visualisation following injection of (99m)Tc-RAFT-RGD and (99m)Tc-cRGD with similar tumour-to-contralateral muscle (T/CM) ratios in B16F0 and in TS/A-pc tumours whereas (99m)Tc-RAFT-RAD T/CM ratios did not allow tumour imaging. In accordance with the higher level of alpha(v)beta(3) integrin expression on TS/A-pc tumours than on B16F0 tumours as determined from western blot and immunoprecipitation analyses, the (99m)Tc-RAFT-RGD T/CM ratio was significantly higher in TS/A-pc than in B16F0 tumours. (99m)Tc-RAFT-RGD allowed the in vivo imaging of alpha(v)beta(3) integrin tumour expression.

Identification of Equine Lactadherin-derived Peptides That Inhibit Rotavirus Infection via Integrin Receptor Competition.

PubMed

Civra, Andrea; Giuffrida, Maria Gabriella; Donalisio, Manuela; Napolitano, Lorenzo; Takada, Yoshikazu; Coulson, Barbara S; Conti, Amedeo; Lembo, David

2015-05-08

Human rotavirus is the leading cause of severe gastroenteritis in infants and children under the age of 5 years in both developed and developing countries. Human lactadherin, a milk fat globule membrane glycoprotein, inhibits human rotavirus infection in vitro, whereas bovine lactadherin is not active. Moreover, it protects breastfed infants against symptomatic rotavirus infections. To explore the potential antiviral activity of lactadherin sourced by equines, we undertook a proteomic analysis of milk fat globule membrane proteins from donkey milk and elucidated its amino acid sequence. Alignment of the human, bovine, and donkey lactadherin sequences revealed the presence of an Asp-Gly-Glu (DGE) α2β1 integrin-binding motif in the N-terminal domain of donkey sequence only. Because integrin α2β1 plays a critical role during early steps of rotavirus host cell adhesion, we tested a minilibrary of donkey lactadherin-derived peptides containing DGE sequence for anti-rotavirus activity. A 20-amino acid peptide containing both DGE and RGD motifs (named pDGE-RGD) showed the greatest activity, and its mechanism of antiviral action was characterized; pDGE-RGD binds to integrin α2β1 by means of the DGE motif and inhibits rotavirus attachment to the cell surface. These findings suggest the potential anti-rotavirus activity of equine lactadherin and support the feasibility of developing an anti-rotavirus peptide that acts by hindering virus-receptor binding. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Dynamic PET and Optical Imaging and Compartment Modeling using a Dual-labeled Cyclic RGD Peptide Probe

PubMed Central

Zhu, Lei; Guo, Ning; Li, Quanzheng; Ma, Ying; Jacboson, Orit; Lee, Seulki; Choi, Hak Soo; Mansfield, James R.; Niu, Gang; Chen, Xiaoyuan

2012-01-01

Purpose: The aim of this study is to determine if dynamic optical imaging could provide comparable kinetic parameters to that of dynamic PET imaging by a near-infrared dye/64Cu dual-labeled cyclic RGD peptide. Methods: The integrin αvβ3 binding RGD peptide was conjugated with a macrocyclic chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) for copper labeling and PET imaging and a near-infrared dye ZW-1 for optical imaging. The in vitro biological activity of RGD-C(DOTA)-ZW-1 was characterized by cell staining and receptor binding assay. Sixty-min dynamic PET and optical imaging were acquired on a MDA-MB-435 tumor model. Singular value decomposition (SVD) method was applied to compute the dynamic optical signal from the two-dimensional optical projection images. Compartment models were used to quantitatively analyze and compare the dynamic optical and PET data. Results: The dual-labeled probe 64Cu-RGD-C(DOTA)-ZW-1 showed integrin specific binding in vitro and in vivo. The binding potential (Bp) derived from dynamic optical imaging (1.762 ± 0.020) is comparable to that from dynamic PET (1.752 ± 0.026). Conclusion: The signal un-mixing process using SVD improved the accuracy of kinetic modeling of 2D dynamic optical data. Our results demonstrate that 2D dynamic optical imaging with SVD analysis could achieve comparable quantitative results as dynamic PET imaging in preclinical xenograft models. PMID:22916074

Dynamic PET and Optical Imaging and Compartment Modeling using a Dual-labeled Cyclic RGD Peptide Probe.

PubMed

Zhu, Lei; Guo, Ning; Li, Quanzheng; Ma, Ying; Jacboson, Orit; Lee, Seulki; Choi, Hak Soo; Mansfield, James R; Niu, Gang; Chen, Xiaoyuan

2012-01-01

The aim of this study is to determine if dynamic optical imaging could provide comparable kinetic parameters to that of dynamic PET imaging by a near-infrared dye/(64)Cu dual-labeled cyclic RGD peptide. The integrin α(v)β(3) binding RGD peptide was conjugated with a macrocyclic chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) for copper labeling and PET imaging and a near-infrared dye ZW-1 for optical imaging. The in vitro biological activity of RGD-C(DOTA)-ZW-1 was characterized by cell staining and receptor binding assay. Sixty-min dynamic PET and optical imaging were acquired on a MDA-MB-435 tumor model. Singular value decomposition (SVD) method was applied to compute the dynamic optical signal from the two-dimensional optical projection images. Compartment models were used to quantitatively analyze and compare the dynamic optical and PET data. The dual-labeled probe (64)Cu-RGD-C(DOTA)-ZW-1 showed integrin specific binding in vitro and in vivo. The binding potential (Bp) derived from dynamic optical imaging (1.762 ± 0.020) is comparable to that from dynamic PET (1.752 ± 0.026). The signal un-mixing process using SVD improved the accuracy of kinetic modeling of 2D dynamic optical data. Our results demonstrate that 2D dynamic optical imaging with SVD analysis could achieve comparable quantitative results as dynamic PET imaging in preclinical xenograft models.

RGD based peptide amphiphiles as drug carriers for cancer targeting

NASA Astrophysics Data System (ADS)

Saraf, Poonam S.

Specific interactions of ligands with receptors is one of the approaches for active targeting of anticancer drugs to cancer cells. Over expression of integrin receptors is a physiological manifestation in several cancers and is associated with cancer progression and metastasis, which makes it an attractive target for cancer chemotherapy. The peptide sequence for this integrin recognition is the Arg-Gly-Asp (RGD). Self-assembly offers a unique way of presenting ligands to target receptors for recognition and binding. This study focuses on development of integrin specific peptide amphiphile self-assemblies as carriers for targeted delivery of paclitaxel to αvbeta 3 integrin overexpressing cancers. Amphiphiles composed of conjugates of different analogs of RGD (linear, cyclic or glycosylated) and aliphatic fatty acid with or without 8-amino-3,6-dioxaoctanoic acid (ADA) as linker were synthesized and characterized. The amphiphiles exhibited Critical Micellar Concentration in the range of 7-30 μM. Transmission electron microscopy images revealed the formation of spherical micelles in the size range of 10-40 nm. Forster Resonance Energy Transfer studies revealed entrapment of hydrophobic dyes within a tight micellar core and provided information regarding the cargo exchange within micelles. The RGD micelles exhibited competitive binding with 55% displacement of a bound fluorescent probe by the cyclic RGD micelles. The internalization of fluorescein isothiocynate (FITC) loaded RGD micelles was significantly higher in A2058 melanoma cells compared to free FITC within 20 minutes of incubation at 37°C. The same micelles showed significantly lower internalization at 4°C and on pretreatment with 0.45M sucrose confirming endocytotic uptake of the RGD micellar carriers. The IC50 of paclitaxel in A2058 melanoma cells was lower when treated within RGD micelles as compared to treatment of free drug. On the other hand, IC50 values increased by 2 to 9 fold for micellar treatment in comparison to free drug in Detroit 551 cells. In A2058 melanoma xenograft mice model, the Paclitaxel-RGD micelles exhibited a significant inhibition of tumor growth in comparison to control treatment for both alternate day and twice weekly treatments. The studies showed the feasibility of using the non covalent peptide based self-assemblies as vehicles for targeted delivery in cancer.

Collagen Scaffolds in Bone Sialoprotein-Mediated Bone Regeneration

PubMed Central

Kruger, Thomas E.; Miller, Andrew H.; Wang, Jinxi

2013-01-01

Decades of research in bioengineering have resulted in the development of many types of 3-dimentional (3D) scaffolds for use as drug delivery systems (DDS) and for tissue regeneration. Scaffolds may be comprised of different natural fibers and synthetic polymers as well as ceramics in order to exert the most beneficial attributes including biocompatibility, biodegradability, structural integrity, cell infiltration and attachment, and neovascularization. Type I collagen scaffolds meet most of these criteria. In addition, type I collagen binds integrins through RGD and non-RGD sites which facilitates cell migration, attachment, and proliferation. Type I collagen scaffolds can be used for bone tissue repair when they are coated with osteogenic proteins such as bone morphogenic protein (BMP) and bone sialoprotein (BSP). BSP, a small integrin-binding ligand N-linked glycoprotein (SIBLING), has osteogenic properties and plays an essential role in bone formation. BSP also mediates mineral deposition, binds type I collagen with high affinity, and binds αvβ 3 and αvβ 5 integrins which mediate cell signaling. This paper reviews the emerging evidence demonstrating the efficacy of BSP-collagen scaffolds in bone regeneration. PMID:23653530

Collagen scaffolds in bone sialoprotein-mediated bone regeneration.

PubMed

Kruger, Thomas E; Miller, Andrew H; Wang, Jinxi

2013-01-01

Decades of research in bioengineering have resulted in the development of many types of 3-dimentional (3D) scaffolds for use as drug delivery systems (DDS) and for tissue regeneration. Scaffolds may be comprised of different natural fibers and synthetic polymers as well as ceramics in order to exert the most beneficial attributes including biocompatibility, biodegradability, structural integrity, cell infiltration and attachment, and neovascularization. Type I collagen scaffolds meet most of these criteria. In addition, type I collagen binds integrins through RGD and non-RGD sites which facilitates cell migration, attachment, and proliferation. Type I collagen scaffolds can be used for bone tissue repair when they are coated with osteogenic proteins such as bone morphogenic protein (BMP) and bone sialoprotein (BSP). BSP, a small integrin-binding ligand N-linked glycoprotein (SIBLING), has osteogenic properties and plays an essential role in bone formation. BSP also mediates mineral deposition, binds type I collagen with high affinity, and binds α v β 3 and α v β 5 integrins which mediate cell signaling. This paper reviews the emerging evidence demonstrating the efficacy of BSP-collagen scaffolds in bone regeneration.

Aerosol Stable Peptide-Coated Liposome Nanoparticles: A Proof-of-Concept Study with Opioid Fentanyl in Enhancing Analgesic Effects and Reducing Plasma Drug Exposure

PubMed Central

HOEKMAN, JOHN D; SRIVASTAVA, PRAMOD; HO, RODNEY J Y

2014-01-01

Previous we reported a novel pressurized olfactory drug (POD) delivery device that deposit aerosolized drug preferentially to upper nasal cavity. This POD device provided sustained CNS levels of soluble morphine analgesic effects. However, analgesic onset of less soluble fentanyl was more rapid but brief, likely due to hydrophobic fentanyl redistribution readily back to blood. To determine whether fentanyl incorporated into an aerosol stable liposome that binds to nasal epithelial cells will enhance CNS drug exposure and analgesic effects and reduce plasma exposure, we constructed RGD liposomes anchored with acylated integrin binding peptides (palmitoyl-GRGDS). The RGD liposomes, which assume gel-phase membrane structure at 25°C were stable under the stress of aerosolization as only 2.2 ± 0.5 % calcein leakage detected. The RGD mediated integrin binding of liposome is also verified to be unaffected by aerosolization. Rats treated with fentanyl in RGD-liposome and POD device exhibited greater analgesic effect, compared to the free drug counterpart (AUCeffect = 1387.l vs. 760.1 %MPE*min); while ~20% reduced plasma drug exposure was noted (AUC0-120 = 208.2 vs 284.8 ng*min/ml). Collectively, fentanyl incorporated in RGD-liposomes are physically and biologically stable under aerosolization, enhanced the overall analgesic effects and reduced plasma drug exposure for the first 2 hours. PMID:24909764

Gd-EDDA/HYNIC-RGD as an MR molecular probe imaging integrin alphanubeta3 receptor-expressed tumor-MR molecular imaging of angiogenesis.

PubMed

Huo, Tianlong; Du, Xiangke; Zhang, Sen; Liu, Xia; Li, Xubing

2010-02-01

The aim of this study is to develop a novel MR probe containing arginine-glycine-aspartic acid (RGD) motif for imaging integrin alphanubeta3 receptor-expressed tumor. Commercially available HYNIC-RGD conjugated with co-ligand EDDA was labeled with Gd(3+), and the mixture was isolated and purified by solid phase extract (SPE) to get the entire probe Gd-EDDA/HYNIC-RGD. Human hepatocellular carcinoma (HHCC) cell line BEL-7402 was cultured and the cells harvested and suspended in serum-free Dulbecco's modified Eagle medium (DMEM) were subcutaneously inoculated into athymic nude mice for tumor growth. In vitro cell binding assay to integrin alphanubeta3 receptor and cell viability experiments were conducted. The in vivo imaging of the three arms of xenografts were performed by MR scan with a dedicated animal coil at time points of 0, 30, 60, 90min and 24-h post-intravenous injection (p.i.). Three arms of nude mice then were sacrificed for histological examination to confirm the imaging results. Gd-EDDA/HYNIC-RGD was successfully isolated by SPE and validity was verified on signal enhancement through in vitro and in vivo experiments. The nude mice model bearing HHCC was well established. There was approx. 30% signal enhancement on T1WI FSE images at 90min post-intravenous injection of the Gd-EDDA/HYNIC-RGD compared with baseline, and the signal to time curve is straightforward over time in the span of 0-90min p.i., while the control arms do not show this tendency. Gd-EDDA/HYNIC-RGD has the potential to serve as an MR probe detecting integrin alphanubeta3 receptor-expressed tumor. Copyright (c) 2008 Elsevier Ireland Ltd. All rights reserved.

Integrin activation and focal complex formation in cardiac hypertrophy.

PubMed

Laser, M; Willey, C D; Jiang, W; Cooper, G; Menick, D R; Zile, M R; Kuppuswamy, D

2000-11-10

Cardiac hypertrophy is characterized by both remodeling of the extracellular matrix (ECM) and hypertrophic growth of the cardiocytes. Here we show increased expression and cytoskeletal association of the ECM proteins fibronectin and vitronectin in pressure-overloaded feline myocardium. These changes are accompanied by cytoskeletal binding and phosphorylation of focal adhesion kinase (FAK) at Tyr-397 and Tyr-925, c-Src at Tyr-416, recruitment of the adapter proteins p130(Cas), Shc, and Nck, and activation of the extracellular-regulated kinases ERK1/2. A synthetic peptide containing the Arg-Gly-Asp (RGD) motif of fibronectin and vitronectin was used to stimulate adult feline cardiomyocytes cultured on laminin or within a type-I collagen matrix. Whereas cardiocytes under both conditions showed RGD-stimulated ERK1/2 activation, only collagen-embedded cells exhibited cytoskeletal assembly of FAK, c-Src, Nck, and Shc. In RGD-stimulated collagen-embedded cells, FAK was phosphorylated only at Tyr-397 and c-Src association occurred without Tyr-416 phosphorylation and p130(Cas) association. Therefore, c-Src activation is not required for its cytoskeletal binding but may be important for additional phosphorylation of FAK. Overall, our study suggests that multiple signaling pathways originate in pressure-overloaded heart following integrin engagement with ECM proteins, including focal complex formation and ERK1/2 activation, and many of these pathways can be activated in cardiomyocytes via RGD-stimulated integrin activation.

Integrin activation and focal complex formation in cardiac hypertrophy

NASA Technical Reports Server (NTRS)

Laser, M.; Willey, C. D.; Jiang, W.; Cooper, G. 4th; Menick, D. R.; Zile, M. R.; Kuppuswamy, D.

2000-01-01

Cardiac hypertrophy is characterized by both remodeling of the extracellular matrix (ECM) and hypertrophic growth of the cardiocytes. Here we show increased expression and cytoskeletal association of the ECM proteins fibronectin and vitronectin in pressure-overloaded feline myocardium. These changes are accompanied by cytoskeletal binding and phosphorylation of focal adhesion kinase (FAK) at Tyr-397 and Tyr-925, c-Src at Tyr-416, recruitment of the adapter proteins p130(Cas), Shc, and Nck, and activation of the extracellular-regulated kinases ERK1/2. A synthetic peptide containing the Arg-Gly-Asp (RGD) motif of fibronectin and vitronectin was used to stimulate adult feline cardiomyocytes cultured on laminin or within a type-I collagen matrix. Whereas cardiocytes under both conditions showed RGD-stimulated ERK1/2 activation, only collagen-embedded cells exhibited cytoskeletal assembly of FAK, c-Src, Nck, and Shc. In RGD-stimulated collagen-embedded cells, FAK was phosphorylated only at Tyr-397 and c-Src association occurred without Tyr-416 phosphorylation and p130(Cas) association. Therefore, c-Src activation is not required for its cytoskeletal binding but may be important for additional phosphorylation of FAK. Overall, our study suggests that multiple signaling pathways originate in pressure-overloaded heart following integrin engagement with ECM proteins, including focal complex formation and ERK1/2 activation, and many of these pathways can be activated in cardiomyocytes via RGD-stimulated integrin activation.

Extracellular-Matrix-Based and Arg-Gly-Asp–Modified Photopolymerizing Hydrogels for Cartilage Tissue Engineering

PubMed Central

Kim, Hwan D.; Heo, Jiseung; Hwang, Yongsung; Kwak, Seon-Yeong; Park, Ok Kyu; Kim, Hyunbum; Varghese, Shyni

2015-01-01

Articular cartilage damage is a persistent and increasing problem with the aging population. Strategies to achieve complete repair or functional restoration remain a challenge. Photopolymerizing-based hydrogels have long received an attention in the cartilage tissue engineering, due to their unique bioactivities, flexible method of synthesis, range of constituents, and desirable physical characteristics. In the present study, we have introduced unique bioactivity within the photopolymerizing-based hydrogels by copolymerizing polyethylene glycol (PEG) macromers with methacrylated extracellular matrix (ECM) molecules (hyaluronic acid and chondroitin sulfate [CS]) and integrin binding peptides (RGD peptide). Results indicate that cellular morphology, as observed by the actin cytoskeleton structures, was strongly dependent on the type of ECM component as well as the presence of integrin binding moieties. Further, CS-based hydrogel with integrin binding RGD moieties increased the lubricin (or known as superficial zone protein [SZP]) gene expression of the encapsulated chondrocytes. Additionally, CS-based hydrogel displayed cell-responsive degradation and resulted in increased DNA, GAG, and collagen accumulation compared with other hydrogels. This study demonstrates that integrin-mediated interactions within CS microenvironment provide an optimal hydrogel scaffold for cartilage tissue engineering application. PMID:25266634

Evaluation of RGD-targeted albumin carriers for specific delivery of auristatin E to tumor blood vessels.

PubMed

Temming, Kai; Meyer, Damon L; Zabinski, Roger; Dijkers, Eli C F; Poelstra, Klaas; Molema, Grietje; Kok, Robbert J

2006-01-01

Induction of apoptosis in endothelial cells is considered an attractive strategy to therapeutically interfere with a solid tumor's blood supply. In the present paper, we constructed cytotoxic conjugates that specifically target angiogenic endothelial cells, thus preventing typical side effects of apoptosis-inducing drugs. For this purpose, we conjugated the potent antimitotic agent monomethyl-auristatin-E (MMAE) via a lysosomal cleavable linker to human serum albumin (HSA) and further equipped this drug-albumin conjugate with cyclic c(RGDfK) peptides for multivalent interaction with alphavbeta3-integrin. The RGD-peptides were conjugated via either an extended poly(ethylene glycol) linker or a short alkyl linker. The resulting drug-targeting conjugates RGDPEG-MMAE-HSA and RGD-MMAE-HSA demonstrated high binding affinity and specificity for alphavbeta3-integrin expressing human umbilical vein endothelial cells (HUVEC). Both types of conjugates were internalized by endothelial cells and killed the target cells at low nM concentrations. Furthermore, we observed RGD-dependent binding of the conjugates to C26 carcinoma. Upon i.v. administration to C26-tumor bearing mice, both drug-targeting conjugates displayed excellent tumor homing properties. Our results demonstrate that RGD-modified albumins are suitable carriers for cell selective intracellular delivery of cytotoxic compounds, and further studies will be conducted to assess the antivascular and tumor inhibitory potential of RGDPEG-MMAE-HSA and RGD-MMAE-HSA.

αvß3-Integrin-Targeted Magnetic Resonance Imaging for the Assessment of Early Antiangiogenic Therapy Effects in Orthotopic Breast Cancer Xenografts.

PubMed

Kazmierczak, Philipp Maximilian; Schneider, Moritz; Habereder, Thomas; Hirner-Eppeneder, Heidrun; Eschbach, Ralf S; Moser, Matthias; Reiser, Maximilian F; Lauber, Kirsten; Nikolaou, Konstantin; Cyran, Clemens C

2016-11-01

The aim of this study was to investigate magnetic resonance imaging (MRI) with αvß3-integrin-targeted ultrasmall superparamagnetic iron oxide nanoparticles (RGD-USPIO) for the in vivo monitoring of early antiangiogenic therapy effects in experimental breast cancer. Orthotopic human breast cancer (MDA-MB-231) xenograft-bearing severe combined immunodeficiency mice were imaged before and after a 1-week therapy with the vascular endothelial growth factor receptor-antibody bevacizumab or placebo (n = 10 per group, daily intraperitoneal injections of bevacizumab or a volume-equivalent placebo solution, respectively) on a clinical 3 T scanner (Magnetom Skyra; Siemens Healthcare, Erlangen, Germany) before and 60 minutes after the intravenous injection of RGD-USPIO (P04000; Guerbet, Villepinte, France). R2 relaxometry employing a T2-weighted spin-echo sequence with 4 echo times (echo time, 20/40/60/80 milliseconds; repetition time, 3800 milliseconds; matrix, 128 × 128; field of view, 50 × 50; slice thickness, 1.2 mm; time to acquisition, 25 minutes) was used as semiquantitative measure to determine RGD-USPIO endothelial binding. In addition, the T2-weighted images were used to perform volumetric tumor response assessments. Imaging results were validated by ex vivo multiparametric immunohistochemistry with regard to αvß3-integrin expression, microvascular density (CD31), proliferation (Ki-67), and apoptosis (TUNEL). RGD-USPIO endothelial binding was significantly reduced after vascular endothelial growth factor inhibition, compared with the control group in which an increased endothelial binding was detected ([INCREMENT]R2Therapy = -0.80 ± 0.78 s; [INCREMENT]R2Control = +0.27 ± 0.59 s; P = 0.002). Correspondingly, immunohistochemistry revealed a significantly lower αvß3-integrin expression (91 ± 30 vs 357 ± 72; P < 0.001), microvascular density (CD31, 109 ± 46 vs 440 ± 208; P < 0.001), tumor cell proliferation (Ki-67, 4040 ± 1373 vs 6530 ± 1217; P < 0.001), as well as significantly higher apoptosis (TUNEL, 11186 ± 4387 vs 4017 ± 1191; P = 0.004) in the therapy compared with the control group. Contrary to the changes in αvß3-integrin expression detected by RGD-USPIO MRI, morphology-based tumor response assessments did not show a significant intergroup difference in tumor volume development over the course of the experiment (ΔVolTherapy +71 ± 40 μL vs ΔVolControl +125 ± 81 μL; P > 0.05). RGD-USPIO MRI allows for the noninvasive assessment of αvß3-integrin expression in the investigated breast cancer model. RGD-USPIO MRI may be applicable for the in vivo monitoring of early antiangiogenic therapy effects in experimental breast cancer, generating possible complementary molecular imaging biomarkers to morphology-based tumor response assessments.

A heterodimeric [RGD-Glu-[(64)Cu-NO2A]-6-Ahx-RM2] αvβ3/GRPr-targeting antagonist radiotracer for PET imaging of prostate tumors.

PubMed

Durkan, Kubra; Jiang, Zongrun; Rold, Tammy L; Sieckman, Gary L; Hoffman, Timothy J; Bandari, Rajendra Prasad; Szczodroski, Ashley F; Liu, Liqin; Miao, Yubin; Reynolds, Tamila Stott; Smith, Charles J

2014-02-01

In the present study, we describe a (64)Cu-radiolabeled heterodimeric peptide conjugate for dual αvβ3/GRPr (αvβ3 integrin/gastrin releasing peptide receptor) targeting of the form [RGD-Glu-[(64)Cu-NO2A]-6-Ahx-RM2] (RGD: the amino acid sequence [Arg-Gly-Asp], a nonregulatory peptide used for αvβ3 integrin receptor targeting; Glu: glutamic acid; NO2A: 1,4,7-triazacyclononane-1,4-diacetic acid; 6-Ahx: 6-amino hexanoic acid; and RM2: (D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2), an antagonist analogue of bombesin (BBN) peptide used for GRPr targeting). RGD-Glu-6Ahx-RM2] was conjugated to a NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid) complexing agent to produce [RGD-Glu-[NO2A]-6-Ahx-RM2], which was purified by reversed-phase high-performance liquid chromatography (RP-HPLC) and characterized by electrospray ionization-mass spectrometry (ESI-MS). Radiolabeling of the conjugate with (64)Cu produced [RGD-Glu-[(64)Cu-NO2A]-6-Ahx-RM2 in high radiochemical yield (≥95%). In vivo behavior of the radiolabeled peptide conjugate was investigated in normal CF-1 mice and in the PC-3 human prostate cancer experimental model. A competitive displacement receptor binding assay in human prostate PC-3 cells using (125)I-[Tyr(4)]BBN as the radioligand showed high binding affinity of [RGD-Glu-[(nat)Cu-NO2A]-6-Ahx-RM2] conjugate for the GRPr (3.09±0.34 nM). A similar assay in human, glioblastoma U87-MG cells using (125)I-Echistatin as the radioligand indicated a moderate receptor-binding affinity for the αvβ3 integrin (518±37.5 nM). In vivo studies of [RGD-Glu-[(64)Cu-NO2A]-6-Ahx-RM2] showed high accumulation (4.86±1.01 %ID/g, 1h post-intravenous injection (p.i.)) and prolonged retention (4.26±1.23 %ID/g, 24h p.i.) of tracer in PC-3 tumor-bearing mice. Micro-positron emission tomography (microPET) molecular imaging studies produced high-quality, high contrast images in PC-3 tumor-bearing mice at 4h p.i. The favorable pharmacokinetics and enhanced tumor uptake of (64)Cu-NOTA-RGD-Glu-6Ahx-RM2 warrant further investigations for dual integrin and GRPr-positive tumor imaging and possible radiotherapy. Copyright © 2014 Elsevier Inc. All rights reserved.

Micro-positron emission tomography/contrast-enhanced computed tomography imaging of orthotopic pancreatic tumor-bearing mice using the αvβ₃ integrin tracer ⁶⁴Cu-labeled cyclam-RAFT-c(-RGDfK-)₄.

PubMed

Aung, Winn; Jin, Zhao-Hui; Furukawa, Takako; Claron, Michael; Boturyn, Didier; Sogawa, Chizuru; Tsuji, Atsushi B; Wakizaka, Hidekatsu; Fukumura, Toshimitsu; Fujibayashi, Yasuhisa; Dumy, Pascal; Saga, Tsuneo

2013-09-01

The purpose of this study was to develop a clinically relevant orthotopic xenotransplantation model of pancreatic cancer and to perform a preclinical evaluation of a new positron emission tomography (PET) imaging probe, ⁶⁴Cu-labeled cyclam-RAFT-c(-RGDfK-)₄ peptide (⁶⁴Cu-RAFT-RGD), using this model. Varying degrees of αvβ₃ integrin expression in several human pancreatic cancer cell lines were examined by flow cytometry and Western blotting. The cell line BxPC-3, which is stably transfected with a red fluorescence protein (RFP), was used for surgical orthotopic implantation. Orthotopic xenograft was established in the pancreas of recipient nude mice. An in vivo probe biodistribution and receptor blocking study, preclinical PET imaging coregistered with contrast-enhanced computed tomography (CECT) comparing ⁶⁴Cu-RAFT-RGD and ¹⁸F-fluoro-2-deoxy-d-glucose (¹⁸F-FDG) accumulation in tumor, postimaging autoradiography, and histologic and immunohistochemical examinations were done. Biodistribution evaluation with a blocking study confirmed that efficient binding of probe to tumor is highly αvβ₃ integrin specific. ⁶⁴Cu-RAFT-RGD PET combined with CECT provided for precise and easy detection of cancer lesions. Autoradiography, histologic, and immunohistochemical examinations confirmed the accumulation of ⁶⁴Cu-RAFT-RGD in tumor versus nontumor tissues. In comparative PET studies, ⁶⁴Cu-RAFT-RGD accumulation provided better tumor contrast to background than ¹⁸F-FDG. Our results suggest that ⁶⁴Cu-RAFT-RGD PET imaging is potentially applicable for the diagnosis of αvβ₃ integrin-expressing pancreatic tumors.

Effect of linkers on the αvβ3 integrin targeting efficiency of cyclic RGD-conjugates

NASA Astrophysics Data System (ADS)

Karmakar, Partha; Grabowska, Dorota; Sudlow, Gail; Ziabrev, Kostiantyn; Sanyal, Nibedita; Achilefu, Samuel

2018-02-01

Cyclic arginine-glycine-aspartic acid (cRGD) peptides are well known to target ανβ3 integrin expressed on cancer cells and neovasculature. Conjugation of these peptides with dyes, drugs, antibodies and other biomolecules through covalent linkers provides a facile way to deliver these products to tumor cells for targeted cancer therapy and diagnosis. Click chemistry and acid-amine couplings are widely used conjugation strategies. However, the effects of different linkers and the distance between the cRGD and the conjugates on the binding of cRGD ligand with ανβ3 has been underexplored. In this present study, we prepared cRGD-conjugates using different linkers and determined how they altered the tumor targeting efficiency in vitro and in vivo. The results demonstrate that different linkers significantly altered the pharmacokinetics of the cRGD conjugates and the tumor uptake kinetics. Unlike large antibodies, this preliminary finding shows that linkers used to attach drugs and fluorescent molecular probes to small peptides play a major role in the accuracy of tumor targeting and treatment outcomes. As a result, considerable attention should be paid to the nature of linkers used in the design of molecular probes and targeted therapeutics.

Ligand-induced Epitope Masking

PubMed Central

Mould, A. Paul; Askari, Janet A.; Byron, Adam; Takada, Yoshikazu; Jowitt, Thomas A.; Humphries, Martin J.

2016-01-01

We previously demonstrated that Arg-Gly-Asp (RGD)-containing ligand-mimetic inhibitors of integrins are unable to dissociate pre-formed integrin-fibronectin complexes (IFCs). These observations suggested that amino acid residues involved in integrin-fibronectin binding become obscured in the ligand-occupied state. Because the epitopes of some function-blocking anti-integrin monoclonal antibodies (mAbs) lie near the ligand-binding pocket, it follows that the epitopes of these mAbs may become shielded in the ligand-occupied state. Here, we tested whether function-blocking mAbs directed against α5β1 can interact with the integrin after it forms a complex with an RGD-containing fragment of fibronectin. We showed that the anti-α5 subunit mAbs JBS5, SNAKA52, 16, and P1D6 failed to disrupt IFCs and hence appeared unable to bind to the ligand-occupied state. In contrast, the allosteric anti-β1 subunit mAbs 13, 4B4, and AIIB2 could dissociate IFCs and therefore were able to interact with the ligand-bound state. However, another class of function-blocking anti-β1 mAbs, exemplified by Lia1/2, could not disrupt IFCs. This second class of mAbs was also distinguished from 13, 4B4, and AIIB2 by their ability to induce homotypic cell aggregation. Although the epitope of Lia1/2 was closely overlapping with those of 13, 4B4, and AIIB2, it appeared to lie closer to the ligand-binding pocket. A new model of the α5β1-fibronectin complex supports our hypothesis that the epitopes of mAbs that fail to bind to the ligand-occupied state lie within, or very close to, the integrin-fibronectin interface. Importantly, our findings imply that the efficacy of some therapeutic anti-integrin mAbs could be limited by epitope masking. PMID:27484800

Human immunodeficiency virus type 1 Tat binds to Candida albicans, inducing hyphae but augmenting phagocytosis in vitro

PubMed Central

Gruber, Andreas; Lell, Claudia P; Speth, Cornelia; Stoiber, Heribert; Lass-Flörl, Cornelia; Sonneborn, Anja; Ernst, Joachim F; Dierich, Manfred P; Würzner, Reinhard

2001-01-01

Tat, the human immunodeficiency virus type 1 (HIV-1) transactivating protein, binds through its RGD-motif to human integrin receptors. Candida albicans, the commonest cause of mucosal candidiasis in subjects infected with HIV-1, also possesses RGD-binding capacity. The present study reveals that Tat binds to C. albicans but not to C. tropicalis. Tat binding was markedly reduced by laminin and to a lesser extent by a complement C3 peptide containing the RGD motif, but not by a control peptide. The outgrowth of C. albicans was accelerated following binding of Tat, but phagocytosis of opsonized C. albicans was also increased after Tat binding. Thus, Tat binding promotes fungal virulence by inducing hyphae but may also reduce it by augmenting phagocytosis. The net effect of Tat in vivo is difficult to judge but in view of the many disease-promoting effects of Tat we propose that accelerating the formation of hyphae dominates over the augmentation of phagocytosis. PMID:11899432

Fibronectins containing extradomain A or B enhance osteoblast differentiation via distinct integrins

PubMed Central

Sens, Carla; Huck, Katrin; Pettera, Stefan; Uebel, Stephan; Wabnitz, Guido; Moser, Markus; Nakchbandi, Inaam A.

2017-01-01

Fibronectin is a multidomain protein secreted by various cell types. It forms a network of fibers within the extracellular matrix and impacts intracellular processes by binding to various molecules, primarily integrin receptors on the cells. Both the presence of several isoforms and the ability of the various domains and isoforms to bind to a variety of integrins result in a wide range of effects. In vivo findings suggest that fibronectin isoforms produced by the osteoblasts enhance their differentiation. Here we report that the isoform characterized by the presence of extradomain A activates α4β1 integrin and augments osteoblast differentiation. In addition, the isoform containing extradomain B enhances the binding of fibronectin through the RGD sequence to β3-containing integrin, resulting in increased mineralization by and differentiation of osteoblasts. Our study thus reveals novel functions for two fibronectin isoforms and the mediating receptors in osteoblast differentiation. PMID:28325836

Interaction between Fibronectin and β1 Integrin Is Essential for Tooth Development

PubMed Central

Yamada, Aya; Yuasa, Kenji; Yoshizaki, Keigo; Iwamoto, Tsutomu; Saito, Masahiro; Nakamura, Takashi; Fukumoto, Satoshi

2015-01-01

The dental epithelium and extracellular matrix interact to ensure that cell growth and differentiation lead to the formation of teeth of appropriate size and quality. To determine the role of fibronectin in differentiation of the dental epithelium and tooth formation, we analyzed its expression in developing incisors. Fibronectin mRNA was expressed during the presecretory stage in developing dental epithelium, decreased in the secretory and early maturation stages, and then reappeared during the late maturation stage. The binding of dental epithelial cells derived from postnatal day-1 molars to a fibronectin-coated dish was inhibited by the RGD but not RAD peptide, and by a β1 integrin-neutralizing antibody, suggesting that fibronectin-β1 integrin interactions contribute to dental epithelial-cell binding. Because fibronectin and β1 integrin are highly expressed in the dental mesenchyme, it is difficult to determine precisely how their interactions influence dental epithelial differentiation in vivo. Therefore, we analyzed β1 integrin conditional knockout mice (Intβ1lox-/lox-/K14-Cre) and found that they exhibited partial enamel hypoplasia, and delayed eruption of molars and differentiation of ameloblasts, but not of odontoblasts. Furthermore, a cyst-like structure was observed during late ameloblast maturation. Dental epithelial cells from knockout mice did not bind to fibronectin, and induction of ameloblastin expression in these cells by neurotrophic factor-4 was inhibited by treatment with RGD peptide or a fibronectin siRNA, suggesting that the epithelial interaction between fibronectin and β1 integrin is important for ameloblast differentiation and enamel formation. PMID:25830530

Positively Charged Residues at the Five-Fold Symmetry Axis of Cell Culture-Adapted Foot-and-Mouth Disease Virus Permit Novel Receptor Interactions

PubMed Central

Berryman, Stephen; Clark, Stuart; Kakker, Naresh K.; Silk, Rhiannon; Seago, Julian; Wadsworth, Jemma; Chamberlain, Kyle; Knowles, Nick J.

2013-01-01

Field isolates of foot-and-mouth disease virus (FMDV) have a restricted cell tropism which is limited by the need for certain RGD-dependent integrin receptors. In contrast, cell culture-adapted viruses use heparan sulfate (HS) or other unidentified molecules as receptors to initiate infection. Here, we report several novel findings resulting from cell culture adaptation of FMDV. In cell culture, a virus with the capsid of the A/Turkey/2/2006 field isolate gained the ability to infect CHO and HS-deficient CHO cells as a result of a single glutamine (Q)-to-lysine (K) substitution at VP1-110 (VP1-Q110K). Using site-directed mutagenesis, the introduction of lysine at this same site also resulted in an acquired ability to infect CHO cells by type O and Asia-1 FMDV. However, this ability appeared to require a second positively charged residue at VP1-109. CHO cells express two RGD-binding integrins (α5β1 and αvβ5) that, although not used by FMDV, have the potential to be used as receptors; however, viruses with the VP1-Q110K substitution did not use these integrins. In contrast, the VP1-Q110K substitution appeared to result in enhanced interactions with αvβ6, which allowed a virus with KGE in place of the normal RGD integrin-binding motif to use αvβ6 as a receptor. Thus, our results confirmed the existence of nonintegrin, non-HS receptors for FMDV on CHO cells and revealed a novel, non-RGD-dependent use of αvβ6 as a receptor. The introduction of lysine at VP1-110 may allow for cell culture adaptation of FMDV by design, which may prove useful for vaccine manufacture when cell culture adaptation proves intractable. PMID:23740982

Ligand-induced Epitope Masking: DISSOCIATION OF INTEGRIN α5β1-FIBRONECTIN COMPLEXES ONLY BY MONOCLONAL ANTIBODIES WITH AN ALLOSTERIC MODE OF ACTION.

PubMed

Mould, A Paul; Askari, Janet A; Byron, Adam; Takada, Yoshikazu; Jowitt, Thomas A; Humphries, Martin J

2016-09-30

We previously demonstrated that Arg-Gly-Asp (RGD)-containing ligand-mimetic inhibitors of integrins are unable to dissociate pre-formed integrin-fibronectin complexes (IFCs). These observations suggested that amino acid residues involved in integrin-fibronectin binding become obscured in the ligand-occupied state. Because the epitopes of some function-blocking anti-integrin monoclonal antibodies (mAbs) lie near the ligand-binding pocket, it follows that the epitopes of these mAbs may become shielded in the ligand-occupied state. Here, we tested whether function-blocking mAbs directed against α5β1 can interact with the integrin after it forms a complex with an RGD-containing fragment of fibronectin. We showed that the anti-α5 subunit mAbs JBS5, SNAKA52, 16, and P1D6 failed to disrupt IFCs and hence appeared unable to bind to the ligand-occupied state. In contrast, the allosteric anti-β1 subunit mAbs 13, 4B4, and AIIB2 could dissociate IFCs and therefore were able to interact with the ligand-bound state. However, another class of function-blocking anti-β1 mAbs, exemplified by Lia1/2, could not disrupt IFCs. This second class of mAbs was also distinguished from 13, 4B4, and AIIB2 by their ability to induce homotypic cell aggregation. Although the epitope of Lia1/2 was closely overlapping with those of 13, 4B4, and AIIB2, it appeared to lie closer to the ligand-binding pocket. A new model of the α5β1-fibronectin complex supports our hypothesis that the epitopes of mAbs that fail to bind to the ligand-occupied state lie within, or very close to, the integrin-fibronectin interface. Importantly, our findings imply that the efficacy of some therapeutic anti-integrin mAbs could be limited by epitope masking. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Novel 64Cu Labeled RGD2-BBN Heterotrimers for PET Imaging of Prostate Cancer.

PubMed

Lucente, Ermelinda; Liu, Hongguang; Liu, Yang; Hu, Xiang; Lacivita, Enza; Leopoldo, Marcello; Cheng, Zhen

2018-05-16

Bombesin receptor 2 (BB 2 ) and integrin α v β 3 receptor are privileged targets for molecular imaging of cancer because of their overexpression in a number of tumor tissues. The most recent developments in heterodimer-based radiopharmaceuticals concern BB 2 - and integrin α v β 3 -targeting compounds, consisting of bombesin (BBN) and cyclic arginine-glycine-aspartic acid peptides (RGD), connected through short length linkers. Molecular imaging probes based on RGD-BBN heterodimer design exhibit improved tumor targeting efficacy compared to the single-receptor targeting peptide monomers. However, their application in clinical study is restricted because of inefficient synthesis or unfavorable in vivo properties, which could depend on the short linker nature. Thus, the aim of the present study was to develop a RGD 2 -BBN heterotrimer, composed of (7-14)BBN-NH 2 peptide (BBN) linked to the E[ c(RGDyK)] 2 dimer peptide (RGD 2 ), bearing the new linker type [Pro-Gly] 12 . The heterodimer E[c(RGDyK)] 2 -PEG 3 -Glu-(Pro-Gly) 12 -BBN(7-14)-NH 2 (RGD 2 -PG 12 -BBN) was prepared through conventional solid phase synthesis, then conjugated with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) or 1,4,7-triazacyclononane-1-glutaric acid-4,7-diacetic acid (NODA-GA). In 64 Cu labeling, the NODA-GA chelator showed superior radiochemical characteristics compared to DOTA (70% vs 40% yield, respectively). Both conjugates displayed dual targeting ability, showing good α v β 3 affinities and high BB 2 receptor affinities which, in the case of the NODA-GA conjugate, were in the same range as the best RGD-BBN heterodimer ligands reported to date ( K i = 24 nM). 64 Cu-DOTA and 64 Cu-NODA-GA probes were also found to be stable after 1 h incubation in mouse serum (>90%). In a microPET study in prostate cancer PC-3 xenograft mice, both probes showed low tumor uptake, probably due to poor pharmacokinetic properties in vivo. Overall, our study demonstrates that novel RGD-BBN heterodimer with long linker can be prepared and they preserve high binding affinities to BB 2 and integrin α v β 3 receptor binding ability. The present study represents a step forward in the design of effective heterodimer or heterotrimer probes for dual targeting.

Targeting of tumor endothelium by RGD-grafted PLGA-nanoparticles.

PubMed

Danhier, Fabienne; Pourcelle, Vincent; Marchand-Brynaert, Jacqueline; Jérôme, Christine; Feron, Olivier; Préat, Véronique

2012-01-01

The destruction of the neovessels in solid tumors can cause the death of tumor cells resulting from the lack of oxygen and nutrients. Peculiarities of the tumor vasculature, however, also position angiogenic endothelial cells as obvious targets to address cytotoxic drugs into the tumor. In particular, the identification of a three-amino acids sequence, arginine-glycine-aspartate (RGD), as a fundamental recognition site for proliferating endothelial attachment to the extracellular matrix leads to the development of tumor-targeting ligands for nanoparticles. The RGD peptide can target the α(v)β(3) integrin overexpressed by the tumor endothelium, and thereby increases the accumulation of drug-loaded RGD-grafted nanoparticles. RGD-nanoparticles may thus extravasate more efficiently and enter the tumor via the enhanced permeability and retention (EPR) effect. This combination of active and passive processes leads to the penetration of nanoparticles into the tumor tissue, followed by cellular uptake and intracellular delivery of the cytotoxic payload. Since cancer cells may also express α(v)β(3) integrin, the entrapping of RGD-nanoparticles into the tumor interstitial fluid may yet be facilitated through direct binding to cancer cells. Here, we describe methods used for the preparation of RGD-nanoparticles and for the validation of their potential of tumor endothelium targeting both in vitro and in vivo. We also illustrate how RGD-nanoparticles may be more suited than nontargeted modalities for the tumor delivery of poorly soluble and/or highly cytotoxic drugs, using different mouse tumor xenograft models. Copyright © 2012 Elsevier Inc. All rights reserved.

Evaluating the Usefulness of a Novel 10B-Carrier Conjugated With Cyclic RGD Peptide in Boron Neutron Capture Therapy

PubMed Central

Masunaga, Shin-ichiro; Kimura, Sadaaki; Harada, Tomohiro; Okuda, Kensuke; Sakurai, Yoshinori; Tanaka, Hiroki; Suzuki, Minoru; Kondo, Natsuko; Maruhashi, Akira; Nagasawa, Hideko; Ono, Koji

2012-01-01

Background To evaluate the usefulness of a novel 10B-carrier conjugated with an integrin-binding cyclic RGD peptide (GPU-201) in boron neutron capture therapy (BNCT). Methods GPU-201 was synthesized from integrin-binding Arg-Gly-Asp (RGD) consensus sequence of matrix proteins and a 10B cluster 1, 2-dicarba-closo-dodecaborane-10B. Mercaptododecaborate-10B (BSH) dissolved in physiological saline and BSH and GPU-201 dissolved with cyclodextrin (CD) as a solubilizing and dispersing agent were intraperitoneally administered to SCC VII tumor-bearing mice. Then, the 10B concentrations in the tumors and normal tissues were measured by γ-ray spectrometry. Meanwhile, tumor-bearing mice were continuously given 5-bromo-2’-deoxyuridine (BrdU) to label all proliferating (P) cells in the tumors, then treated with GPU-201, BSH-CD, or BSH. Immediately after reactor neutron beam or γ-ray irradiation, during which intratumor 10B concentrations were kept at levels similar to each other, cells from some tumors were isolated and incubated with a cytokinesis blocker. The responses of the Q and total (= P + Q) cell populations were assessed based on the frequency of micronuclei using immunofluorescence staining for BrdU. Results The 10B from BSH was washed away rapidly in all these tissues and the retention of 10B from BSH-CD and GPU-201 was similar except in blood where the 10B concentration from GPU-201 was higher for longer. GPU-201 showed a significantly stronger radio-sensitizing effect under neutron beam irradiation on both total and Q cell populations than any other 10B-carrier. Conclusion A novel 10B-carrier conjugated with an integrin-binding RGD peptide (GPU-201) that sensitized tumor cells more markedly than conventional 10B-carriers may be a promising candidate for use in BNCT. However, its toxicity needs to be tested further. PMID:29147290

Optimized multimodal nanoplatforms for targeting αvβ3 integrins

NASA Astrophysics Data System (ADS)

Bolley, Julie; Lalatonne, Yoann; Haddad, Oualid; Letourneur, Didier; Soussan, Michael; Pérard-Viret, Joelle; Motte, Laurence

2013-11-01

Magnetic Resonance Imaging (MRI) using contrast agents is a very powerful technique for diagnosis in clinical medicine and biomedical research. The synthesis of ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles targeting αvβ3 integrins and acting as new MRI contrast agents seems to be a promising way for cancer diagnosis. Indeed, it is well established that αvβ3 integrin plays a key role in tumor angiogenesis acting like a receptor for the extracellular matrix proteins like vitronectin, fibronectin through the arginine-glycine-aspartic acid (RGD) sequence. Up-regulation of αvβ3 has been found to be associated with a wide range of cancers, making it a broad-spectrum tumor-marker. In this study, USPIO nanocrystals were synthesized and surface passivated with caffeic acid. The large number of the carboxylic acid functions at the outer surface of the nanoplatforms was used for the covalent coupling of Rhodamine123, polyethylene glycol (PEG) and cyclic RGD. Soluble carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were used to crosslink carboxylic acid with the amino group of the ligands. We examined the design of the nanoplatforms with each individual entity and then the combination of two and three of them. Several methods were used to characterize the nanoparticle surface functionalization and the magnetic properties of these contrast agents were studied using a 1.5 T clinical MRI scanner. The affinity towards integrins was evidenced by surface plasmon resonance and solid-phase receptor-binding assay.Magnetic Resonance Imaging (MRI) using contrast agents is a very powerful technique for diagnosis in clinical medicine and biomedical research. The synthesis of ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles targeting αvβ3 integrins and acting as new MRI contrast agents seems to be a promising way for cancer diagnosis. Indeed, it is well established that αvβ3 integrin plays a key role in tumor angiogenesis acting like a receptor for the extracellular matrix proteins like vitronectin, fibronectin through the arginine-glycine-aspartic acid (RGD) sequence. Up-regulation of αvβ3 has been found to be associated with a wide range of cancers, making it a broad-spectrum tumor-marker. In this study, USPIO nanocrystals were synthesized and surface passivated with caffeic acid. The large number of the carboxylic acid functions at the outer surface of the nanoplatforms was used for the covalent coupling of Rhodamine123, polyethylene glycol (PEG) and cyclic RGD. Soluble carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were used to crosslink carboxylic acid with the amino group of the ligands. We examined the design of the nanoplatforms with each individual entity and then the combination of two and three of them. Several methods were used to characterize the nanoparticle surface functionalization and the magnetic properties of these contrast agents were studied using a 1.5 T clinical MRI scanner. The affinity towards integrins was evidenced by surface plasmon resonance and solid-phase receptor-binding assay. Electronic supplementary information (ESI) available: TEM image and size distribution of γFe2O3@CA nanoplatforms, determination of CA number per nanoparticle, dye R123, NH2-PEG-COOH and cRGD derivative coupling, biological stability, SPR analysis - theoretical analyte binding capacity, solid phase binding assay - determination of echistatin Kd. See DOI: 10.1039/c3nr03763k

Targeted radionuclide therapy with RAFT-RGD radiolabelled with (90)Y or (177)Lu in a mouse model of αvβ3-expressing tumours.

PubMed

Bozon-Petitprin, A; Bacot, S; Gauchez, A S; Ahmadi, M; Bourre, J C; Marti-Batlle, D; Perret, P; Broisat, A; Riou, L M; Claron, M; Boturyn, D; Fagret, D; Ghezzi, Catherine; Vuillez, J P

2015-02-01

The αvβ3 integrin plays an important role in tumour-induced angiogenesis, tumour proliferation, survival and metastasis. The tetrameric RGD-based peptide, regioselectively addressable functionalized template-(cyclo-[RGDfK])4 (RAFT-RGD), specifically targets the αvβ3 integrin in vitro and in vivo. The aim of this study was to evaluate the therapeutic potential of RAFT-RGD radiolabelled with β(-) emitters in a nude mouse model of αvβ3 integrin-expressing tumours. Biodistribution and SPECT/CT imaging studies were performed after injection of (90)Y-RAFT-RGD or (177)Lu-RAFT-RGD in nude mice subcutaneously xenografted with αvβ3 integrin-expressing U-87 MG cells. Experimental targeted radionuclide therapy with (90)Y-RAFT-RGD or (177)Lu-RAFT-RGD and (90)Y-RAFT-RAD or (177)Lu-RAFT-RAD (nonspecific controls) was evaluated by intravenous injection of the radionuclides into mice bearing αvβ3 integrin-expressing U-87 MG tumours of different sizes (small or large) or bearing TS/A-pc tumours that do not express αvβ3. Tumour volume doubling time was used to evaluate the efficacy of each treatment. Injection of 37 MBq of (90)Y-RAFT-RGD into mice with large αvβ3-positive tumours or 37 MBq of (177)Lu-RAFT-RGD into mice with small αvβ3-positive tumours caused significant growth delays compared to mice treated with 37 MBq of (90)Y-RAFT-RAD or 37 MBq of (177)Lu-RAFT-RAD or untreated mice. In contrast, injection of 30 MBq of (90)Y-RAFT-RGD had no effect on the growth of αvβ3-negative tumours. (90)Y-RAFT-RGD and (177)Lu-RAFT-RGD are potent agents targeting αvβ3-expressing tumours for internal targeted radiotherapy.

Osteopontin Signals through Calcium and Nuclear Factor of Activated T Cells (NFAT) in Osteoclasts

PubMed Central

Tanabe, Natsuko; Wheal, Benjamin D.; Kwon, Jiyun; Chen, Hong H.; Shugg, Ryan P. P.; Sims, Stephen M.; Goldberg, Harvey A.; Dixon, S. Jeffrey

2011-01-01

Osteopontin (OPN), an integrin-binding extracellular matrix glycoprotein, enhances osteoclast activity; however, its mechanisms of action are elusive. The Ca2+-dependent transcription factor NFATc1 is essential for osteoclast differentiation. We assessed the effects of OPN on NFATc1, which translocates to nuclei upon activation. Osteoclasts from neonatal rabbits and rats were plated on coverslips, uncoated or coated with OPN or bovine albumin. OPN enhanced the proportion of osteoclasts exhibiting nuclear NFATc1. An RGD-containing, integrin-blocking peptide prevented the translocation of NFATc1 induced by OPN. Moreover, mutant OPN lacking RGD failed to induce translocation of NFATc1. Thus, activation of NFATc1 is dependent on integrin binding through RGD. Using fluorescence imaging, OPN was found to increase the proportion of osteoclasts exhibiting transient elevations in cytosolic Ca2+ (oscillations). OPN also enhanced osteoclast survival. The intracellular Ca2+ chelator 1,2-bis(O-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) suppressed Ca2+ oscillations and inhibited increases in NFATc1 translocation and survival induced by OPN. Furthermore, a specific, cell-permeable peptide inhibitor of NFAT activation blocked the effects of OPN on NFATc1 translocation and osteoclast survival. This is the first demonstration that OPN activates NFATc1 and enhances osteoclast survival through a Ca2+-NFAT-dependent pathway. Increased NFATc1 activity and enhanced osteoclast survival may account for the stimulatory effects of OPN on osteoclast function in vivo. PMID:21940634

Surface potential-governed cellular osteogenic differentiation on ferroelectric polyvinylidene fluoride trifluoroethylene films.

PubMed

Tang, Bolin; Zhang, Bo; Zhuang, Junjun; Wang, Qi; Dong, Lingqing; Cheng, Kui; Weng, Wenjian

2018-07-01

Surface potential of biomaterials can dramatically influence cellular osteogenic differentiation. In this work, a wide range of surface potential on ferroelectric polyvinylidene fluoride trifluoroethylene (P(VDF-TrFE)) films was designed to get insight into the interfacial interaction of cell-charged surface. The P(VDF-TrFE) films poled by contact electric poling at various electric fields obtained well stabilized surface potential, with wide range from -3 to 915 mV. The osteogenic differentiation level of cells cultured on the films was strongly dependent on surface potential and reached the optimum at 391 mV in this system. Binding specificity assay indicated that surface potential could effectively govern the binding state of the adsorbed fibronectin (FN) with integrin. Molecular dynamic (MD) simulation further revealed that surface potential brought a significant difference in the relative distance between RGD and synergy PHSRN sites of adsorbed FN, resulting in a distinct integrin-FN binding state. These results suggest that the full binding of integrin α5β1 with both RGD and PHSRN sites of FN possesses a strong ability to activate osteogenic signaling pathway. This work sheds light on the underlying mechanism of osteogenic differentiation behavior on charged material surfaces, and also provides a guidance for designing a reasonable charged surface to enhance osteogenic differentiation. The ferroelectric P(VDF-TrFE) films with steady and a wide range of surface potential were designed to understand underlying mechanism of cell-charged surface interaction. The results showed that the charged surface well favored upregulation of osteogenic differentiation of MC3T3-E1 cells, and more importantly, a highest level occurred on the film with a moderate surface potential. Experiments and molecular dynamics simulation demonstrated that the surface potential could govern fibronectin conformation and then the integrin-fibronectin binding. We propose that a full binding state of integrin α5β1 with fibronectin induces effective activation of integrin-mediated FAK/ERK signaling pathway to upregulate cellular osteogenic differentiation. This work provides a guidance for designing a reasonable charged surface to enhance osteogenic differentiation. Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Optical imaging of head and neck squamous cell carcinoma in vivo using arginine-glycine-aspartic acid peptide conjugated near-infrared quantum dots.

PubMed

Huang, Hao; Bai, Yun-Long; Yang, Kai; Tang, Hong; Wang, You-Wei

2013-01-01

Molecular imaging plays a key role in personalized medicine and tumor diagnosis. Quantum dots with near-infrared emission spectra demonstrate excellent tissue penetration and photostability, and have recently emerged as important tools for in vivo tumor imaging. Integrin αvβ3 has been shown to be highly and specifically expressed in endothelial cells of tumor angiogenic vessels in almost all types of tumors, and specifically binds to the peptide containing arginine-glycine-aspartic acid (RGD). In this study, we conjugated RGD with quantum dots with emission wavelength of 800 nm (QD800) to generate QD800-RGD, and used it via intravenous injection as a probe to image tumors in nude mice bearing head and neck squamous cell carcinoma (HNSCC). Twelve hours after the injection, the mice were still alive and were sacrificed to isolate tumors and ten major organs for ex vivo analysis to localize the probe in these tissues. The results showed that QD800-RGD was specifically targeted to integrin αvβ3 in vitro and in vivo, producing clear tumor fluorescence images after the intravenous injection. The tumor-to-background ratio and size of tumor image were highest within 6 hours of the injection and declined significantly at 9 hours after the injection, but there was still a clearly visible tumor image at 12 hours. The greatest amount of QD800-RGD was found in liver and spleen, followed by tumor and lung, and a weak fluorescence signal was seen in tibia. No detectable signal of QD800-RGD was found in brain, heart, kidney, testis, stomach, or intestine. Our study demonstrated that using integrin αvβ3 as target, it is possible to use intravenously injected QD800-RGD to generate high quality images of HNSCC, and the technique offers great potential in the diagnosis and personalized therapy for HNSCC.

The group B streptococcal alpha C protein binds alpha1beta1-integrin through a novel KTD motif that promotes internalization of GBS within human epithelial cells.

PubMed

Bolduc, Gilles R; Madoff, Lawrence C

2007-12-01

Group B Streptococcus (GBS) is the leading cause of bacterial pneumonia, sepsis and meningitis among neonates and a cause of morbidity among pregnant women and immunocompromised adults. GBS epithelial cell invasion is associated with expression of alpha C protein (ACP). Loss of ACP expression results in a decrease in GBS internalization and translocation across human cervical epithelial cells (ME180). Soluble ACP and its 170 amino acid N-terminal region (NtACP), but not the repeat protein RR', bind to ME180 cells and reduce internalization of wild-type GBS to levels obtained with an ACP-deficient isogenic mutant. In the current study, ACP colocalized with alpha(1)beta(1)-integrin, resulting in integrin clustering as determined by laser scanning confocal microscopy. NtACP contains two structural domains, D1 and D2. D1 is structurally similar to fibronectin's integrin-binding region (FnIII10). D1's (KT)D146 motif is structurally similar to the FnIII10 (RG)D1495 integrin-binding motif, suggesting that ACP binds alpha(1)beta(1)-integrin via the D1 domain. The (KT)D146A mutation within soluble NtACP reduced its ability to bind alpha(1)beta(1)-integrin and inhibit GBS internalization within ME180 cells. Thus ACP binding to human epithelial cell integrins appears to contribute to GBS internalization within epithelial cells.

Hypertonicity-induced transmitter release at Drosophila neuromuscular junctions is partly mediated by integrins and cAMP/protein kinase A

NASA Technical Reports Server (NTRS)

Suzuki, Kazuhiro; Grinnell, Alan D.; Kidokoro, Yoshiaki

2002-01-01

The frequency of quantal transmitter release increases upon application of hypertonic solutions. This effect bypasses the Ca(2+) triggering step, but requires the presence of key molecules involved in vesicle fusion, and hence could be a useful tool for dissecting the molecular process of vesicle fusion. We have examined the hypertonicity response at neuromuscular junctions of Drosophila embryos in Ca(2+)-free saline. Relative to wild-type, the response induced by puff application of hypertonic solution was enhanced in a mutant, dunce, in which the cAMP level is elevated, or in wild-type embryos treated with forskolin, an activator of adenylyl cyclase, while protein kinase A (PKA) inhibitors decreased it. The response was also smaller in a mutant, DC0, which lacks the major subunit of PKA. Thus the cAMP/PKA cascade is involved in the hypertonicity response. Peptides containing the sequence Arg-Gly-Asp (RGD), which inhibit binding of integrins to natural ligands, reduced the response, whereas a peptide containing the non-binding sequence Arg-Gly-Glu (RGE) did not. A reduced response persisted in a mutant, myospheroid, which expresses no integrins, and the response in DC0 was unaffected by RGD peptides. These data indicate that there are at lease two components in the hypertonicity response: one that is integrin mediated and involves the cAMP/PKA cascade, and another that is not integrin mediated and does not involve the cAMP/PKA cascade.

Alpha-V Integrin Targeted PET Imagining of Breast Cancer Angiogenesis and Lose-Dose Metronomic Anti-Angiogenic Chemotherapy Efficacy

DTIC Science & Technology

2005-08-01

Chen X, Plasencia C, Hou Y, Neamati N. Synthesis and Biological Evaluation of Dimeric RGD Peptide-Paclitaxel Conjugate as Model for Integrin Targeted...Plasencia C, Hou Y, Neamati N. Synthesis and biological evaluation of dimeric RGD peptide-paclitaxel conjugate as a model for integrin-targeted drug...Targeted Imaging of Lung Cancer. Neoplasia 2005;7:271-279. 6. Chen X, Plasencia C, Hou Y, Neamati N. Synthesis and Biological Evaluation of Dimeric RGD

Triazole RGD antagonist reverts TGFβ1-induced endothelial-to-mesenchymal transition in endothelial precursor cells.

PubMed

Bianchini, Francesca; Peppicelli, Silvia; Fabbrizzi, Pierangelo; Biagioni, Alessio; Mazzanti, Benedetta; Menchi, Gloria; Calorini, Lido; Pupi, Alberto; Trabocchi, Andrea

2017-01-01

Fibrosis is the dramatic consequence of a dysregulated reparative process in which activated fibroblasts (myofibroblasts) and Transforming Growth Factor β1 (TGFβ1) play a central role. When exposed to TGFβ1, fibroblast and epithelial cells differentiate in myofibroblasts; in addition, endothelial cells may undergo endothelial-to-mesenchymal transition (EndoMT) and actively participate to the progression of fibrosis. Recently, the role of αv integrins, which recognize the Arg-Gly-Asp (RGD) tripeptide, in the release and signal transduction activation of TGFβ1 became evident. In this study, we present a class of triazole-derived RGD antagonists that interact with αvβ3 integrin. Above different compounds, the RGD-2 specifically interferes with integrin-dependent TGFβ1 EndoMT in Endothelial Colony-Forming Cells (ECPCs) derived from circulating Endothelial Precursor Cells (ECPCs). The RGD-2 decreases the amount of membrane-associated TGFβ1, and reduces both ALK5/TGFβ1 type I receptor expression and Smad2 phosphorylation in ECPCs. We found that RGD-2 antagonist reverts EndoMT, reducing α-smooth muscle actin (α-SMA) and vimentin expression in differentiated ECPCs. Our results outline the critical role of integrin in fibrosis progression and account for the opportunity of using integrins as target for anti-fibrotic therapeutic treatment.

Optimizing cardiovascular gene therapy: increased vascular gene transfer with modified adenoviral vectors.

PubMed

Kibbe, M R; Murdock, A; Wickham, T; Lizonova, A; Kovesdi, I; Nie, S; Shears, L; Billiar, T R; Tzeng, E

2000-02-01

Adenovirus is widely used as a vector for gene transfer to the vasculature. However, the efficiency of these vectors can be limited by ineffective viral-target cell interactions. Viral attachment, which largely determines adenoviral tropism, is mediated through binding of the adenoviral fiber coat protein to the Coxsackievirus and adenovirus receptor, while internalization follows binding of the adenoviral RGD motif to alpha(v)-integrin receptors. Modifications of the fiber coat protein sequence have been successful for targeting the adenovirus to more prevalent receptors in the vasculature, including heparan sulfate-containing receptors and alpha(v)-integrin receptors. Modified adenoviral vectors targeted to receptors more prevalent in the vasculature result in an increased transfer efficiency of the virus in vitro and in vivo even in the presence of clinically relevant doses of heparin. We tested 2 modified E1- and E3-deleted Ad5 type adenoviral vectors containing the beta-galactosidase gene. AdZ.F(pK7) contains multiple positively charged lysines in the fiber coat protein that target the adenovirus to heparan sulfate receptors, while AdZ.F(RGD) contains an RGD integrin-binding sequence in the fiber coat protein that allows binding to alpha(v)-integrin receptors. The gene transfer efficiency of these modified viruses was compared in rat aortic smooth muscle cells in vitro and in an in vivo porcine model of balloon-induced arterial injury. Because of the use of heparin during most vascular surgical procedures and the concern that heparin might interfere with the binding of AdZ.F(pK7) to heparan sulfate receptors, the effect of heparin on the in vitro and in vivo transfer efficiency of these 2 modified adenoviruses was evaluated. In vitro infection of rat aortic smooth muscle cells with AdZ.F(pK7) and AdZ.F(RGD) resulted in significantly higher levels of beta-galactosidase expression compared with the unmodified adenovirus (mean +/- SEM, 1766.3 +/- 89.1 and 44.8 +/- 3.4 vs 10.1 +/- 0.7 mU per milligram of protein; P<.001). Following heparin administration, the gene transfer efficiency achieved with AdZ.F(pK7) diminished slightly in a concentration-dependent manner. However, the transfer efficiency was still greater than with the unmodified virus (mean +/- SEM, 1342.3 +/- 101.8 vs 4.8 +/- 0.4 mU per milligram of protein; P<.001). In vivo, following injury to the pig iliac artery with a 4F Fogarty balloon catheter, we found that AdZ.F(pK7) transduced the artery approximately 35-fold more efficiently than AdZ.F and 3-fold more efficiently than AdZ.F(RGD) following the administration of intravenous heparin, 100 U/kg body weight, and heparinized saline irrigation. Modifications of the adenovirus that lead to receptor targeting resulted in significantly improved gene transfer efficiencies. These improvements in transfer efficiencies observed with the modified vectors decreased slightly in the presence of heparin. However, AdZ.F(pK7) was still superior to AdZ.F(RGD) and AdZ.F despite heparin administration. These data demonstrate that modifications of adenoviral vectors that enhance binding to heparan sulfate receptors significantly improve gene transfer efficiency even in the presence of heparin and suggest an approach to optimize gene transfer into blood vessels.

microPET Imaging of Glioma Integrin (alpha-v, beta-3) Expression Using Cu-64-Labeled Tetrameric RGD Peptide

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Yun; Zhang, , Xianzhong; Xiong, , Zhengming

2005-10-01

Integrins ?v?3 and ?v?5 play a critical role in tumor-induced angiogenesis and metastasis, and have become promising diagnostic indicators and therapeutic targets of tumors. Radiolabeled RGD peptides that are integrin-specific may be used for non-invasive imaging of integrin expression level as well as for integrin-targeted radionuclide therapy. We previously conjugated a series of mono- and dimeric RGD peptides with 1,4,7,10-tetraazacyclododecane-N, N?,N??,N???-tetraacetic acid (DOTA) and labeled these with copper-64 for microPET imaging in various mouse xenograft models. The copper-64 tracers showed ?v?3-selective tumor uptake, but the magnitude of tumor uptake was relatively low, the tumor washout was rapid, and non-target organ/tissuemore » retention was high. In this study we developed a tetrameric RGD peptide tracer 64Cu-DOTA-E{l_brace}E[c(RGDfK)]2{r_brace}2 for positron emission tomography (PET) imaging of integrin ?v?3 expression in a subcutaneous U87MG glioma xenograft model in female athymic nude mice. The RGD tetramer showed significantly higher integrin binding affinity than the corresponding mono- and dimeric RGD analogs, most likely due to polyvalency effect. The radiolabeled peptide showed rapid blood clearance (0.61 ? 0.01%ID/g at 30 min and 0.21 ? 0.01 %ID/g at 4 h postinjection (p.i.), respectively) and predominantly renal excretion. Tumor uptake was rapid and high and the tumor washout was slow (9.93 ? 1.05 %ID/g at 30 min p.i. and 4.56 ? 0.51 %ID/g at 24 h post-injection). The metabolic stability of 64Cu-DOTA-E{l_brace}E[c(RGDfK)]2{r_brace}2 was determined in mouse blood, urine, and liver and kidney homogenates at different times after tracer injection. The average fractions of intact tracer in these organs at 1 h were approximately 70, 58, 51 and 26 percent, respectively. Non-invasive microPET imaging studies showed significant tumor uptake and good contrast in the subcutaneous tumor-bearing mice, which agreed well with the biodistribution results. Integrin ?v?3 specificity was demonstrated by successful blocking of tumor uptake of 64Cu-DOTA-E{l_brace}E[c(RGDfK)]2{r_brace}2 in the presence of excess amount of c(RGDyK) at 1 h postinjection. The highest absorbed radiation doses determined for the human reference adult were received by the urinary bladder wall (0.263 mGy/MBq), kidneys (0.0298 mGy/MBq), and liver (0.0244 mGy/MBq). Assuming 0.5-g U87MG glioma tumors in man, we calculated an absorbed dose of 65.3 mGy/MBq (242 rad/mCi) following a single injection of 64Cu-DOTA-E{l_brace}E[c(RGDfK)]2{r_brace}2. In conclusion, the high integrin avidity and favorable biokinetics make 64Cu-DOTA-E{l_brace}E[c(RGDfK)]2{r_brace}2 a promising agent for peptide receptor radionuclide imaging therapy of integrin-positive tumors.« less

Vitronectin-Based, Biomimetic Encapsulating Hydrogel Scaffolds Support Adipogenesis of Adipose Stem Cells

PubMed Central

Clevenger, Tracy N.; Hinman, Cassidy R.; Ashley Rubin, Rebekah K.; Smither, Kate; Burke, Daniel J.; Hawker, Craig J.; Messina, Darin; Van Epps, Dennis

2016-01-01

Soft tissue defects are relatively common, yet currently used reconstructive treatments have varying success rates, and serious potential complications such as unpredictable volume loss and reabsorption. Human adipose-derived stem cells (ASCs), isolated from liposuction aspirate have great potential for use in soft tissue regeneration, especially when combined with a supportive scaffold. To design scaffolds that promote differentiation of these cells down an adipogenic lineage, we characterized changes in the surrounding extracellular environment during adipogenic differentiation. We found expression changes in both extracellular matrix proteins, including increases in expression of collagen-IV and vitronectin, as well as changes in the integrin expression profile, with an increase in expression of integrins such as αVβ5 and α1β1. These integrins are known to specifically interact with vitronectin and collagen-IV, respectively, through binding to an Arg-Gly-Asp (RGD) sequence. When three different short RGD-containing peptides were incorporated into three-dimensional (3D) hydrogel cultures, it was found that an RGD-containing peptide derived from vitronectin provided strong initial attachment, maintained the desired morphology, and created optimal conditions for in vitro 3D adipogenic differentiation of ASCs. These results describe a simple, nontoxic encapsulating scaffold, capable of supporting the survival and desired differentiation of ASCs for the treatment of soft tissue defects. PMID:26956095

Regulation of collagenase-3 and osteocalcin gene expression by collagen and osteopontin in differentiating MC3T3-E1 cells

NASA Technical Reports Server (NTRS)

D'Alonzo, Richard C.; Kowalski, Aaron J.; Denhardt, David T.; Nickols, G. Allen; Partridge, Nicola C.

2002-01-01

Both collagenase-3 and osteocalcin mRNAs are expressed maximally during the later stages of osteoblast differentiation. Here, we demonstrate that collagenase-3 mRNA expression in differentiating MC3T3-E1 cells is dependent upon the presence of ascorbic acid, is inhibited in the presence of the collagen synthesis inhibitor, 3,4-dehydroproline, and is stimulated by growth on collagen in the absence of ascorbic acid. Transient transfection studies show that collagenase-3 promoter activity increases during cell differentiation and requires the presence of ascorbic acid. Additionally, we show that, in differentiating MC3T3-E1 cells, collagenase-3 gene expression increases in the presence of an anti-osteopontin monoclonal antibody that binds near the RGD motif of this protein, whereas osteocalcin expression is inhibited. Furthermore, an RGD peptidomimetic compound, designed to block interaction of ligands to the alpha(v) integrin subunit, increases osteocalcin expression and inhibits collagenase-3 expression, suggesting that the RGD peptidomimetic initiates certain alpha(v) integrin signaling in osteoblastic cells. Overall, these studies demonstrate that stimulation of collagenase-3 expression during osteoblast differentiation requires synthesis of a collagenous matrix and that osteopontin and alpha(v) integrins exert divergent regulation of collagenase-3 and osteocalcin expression during osteoblast differentiation.

RGD peptide-mediated chitosan-based polymeric micelles targeting delivery for integrin-overexpressing tumor cells.

PubMed

Cai, Li-Li; Liu, Ping; Li, Xi; Huang, Xuan; Ye, Yi-Qing; Chen, Feng-Ying; Yuan, Hong; Hu, Fu-Qiang; Du, Yong-Zhong

2011-01-01

Solid tumors need new blood vessels to feed and nourish them as well as to allow tumor cells to escape into the circulation and lodge in other organs, which is termed "angiogenesis." Some tumor cells within solid tumors can overexpress integrins α(v)β(3) and α(v)β(5), which can specifically recognize the peptide motif Arg-Gly-Asp (RGD). Thus, the targeting of RGD-modified micelles to tumor vasculature is a promising strategy for tumor-targeting treatment. RGD peptide (GSSSGRGDSPA) was coupled to poly(ethylene glycol)-modified stearic acid-grafted chitosan (PEG-CS-SA) micelles via chemical reaction in the presence of N,N'-Disuccinimidyl carbonate. The critical micelle concentration of the polymeric micelles was determined by measuring the fluorescence intensity of pyrene as a fluorescent probe. The micelle size, size distribution, and zeta potential were measured by light scattering and electrophoretic mobility. Doxorubicin (DOX) was chosen as a model anticancer drug to investigate the drug entrapment efficiency, in vitro drug-release profile, and in vitro antitumor activities of drug-loaded RGD-PEG-CS-SA micelles in cells that overexpress integrins (α(ν)β(3) and α(ν)β(5)) and integrin-deficient cells. Using DOX as a model drug, the drug encapsulation efficiency could reach 90%, and the in vitro drug-release profiles suggested that the micelles could be used as a controlled-release carrier for the hydrophobic drug. Qualitative and quantitative analysis of cellular uptake indicated that RGD-modified micelles could significantly increase the DOX concentration in integrin-overexpressing human hepatocellular carcinoma cell line (BEL-7402), but not in human epithelial carcinoma cell line (Hela). The competitive cellular-uptake test showed that the cellular uptake of RGD-modified micelles in BEL-7402 cells was significantly inhibited in the presence of excess free RGD peptides. In vitro cytotoxicity tests demonstrated DOX-loaded RGD-modified micelles could specifically enhance the cytotoxicity against BEL-7402 compared with DOX-loaded PEG-CS-SA and doxorubicin hydrochlorate. This study suggests that RGD-modified PEG-CS-SA micelles are promising drug carriers for integrin-overexpressing tumor active targeting therapy.

Growth promoting in vitro effect of synthetic cyclic RGD-peptides on human osteoblast-like cells attached to cancellous bone.

PubMed

Magdolen, Ursula; Auernheimer, Jörg; Dahmen, Claudia; Schauwecker, Johannes; Gollwitzer, Hans; Tübel, Jutta; Gradinger, Reiner; Kessler, Horst; Schmitt, Manfred; Diehl, Peter

2006-06-01

In tissue engineering, the application of biofunctional compounds on biomaterials such as integrin binding RGD-peptides has gained growing interest. Anchorage-dependent cells like osteoblasts bind to these peptides thus ameliorating the integration of a synthetic implant. In case sterilized bone grafts are used as substitutes for reconstruction of bone defects, the ingrowth of the implanted bone is often disturbed because of severe pretreatment such as irradiation or autoclaving, impairing the biological and mechanical properties of the bone. We report for the first time on the in vitro coating of the surface of freshly resected, cleaned bone discs with synthetic, cyclic RGD-peptides. For this approach, two different RGD-peptides were used, one containing two phosphonate anchors, the other peptide four of these binding moieties to allow efficient association of these reactive RGD-peptides to the inorganic bone matrix. Human osteoblast-like cells were cultured on RGD-coated bone discs and the adherence and growth of the cells were analyzed. Coating of bone discs with RGD-peptides did not improve the adhesion rate of osteoblast-like cells to the discs but significantly (up to 40%) accelerated growth of these cells within 8 days after attachment. This effect points to pretreatment of bone implants, especially at the critical interface area between the implanted bone and the non-resected residual bone structure, before re-implantation in order to stimulate and enhance osteointegration of a bone implant.

C-TERMINAL FRAGMENT OF TRANSFORMING GROWTH FACTOR BETA-INDUCED PROTEIN (TGFBIp) IS REQUIRED FOR APOPTOSIS IN HUMAN OSTEOSARCOMA CELLS

PubMed Central

Zamilpa, Rogelio; Rupaimoole, Rajesha; Phelix, Clyde F.; Somaraki-Cormier, Maria; Haskins, William; Asmis, Reto; LeBaron, Richard G.

2009-01-01

Transforming growth factor beta induced protein (TGFBIp), is secreted into the extracellular space. When fragmentation of C-terminal portions is blocked, apoptosis is low, even when the protein is overexpressed. If fragmentation occurs, apoptosis is observed. Whether full-length TGFBIp or integrin-binding fragments released from its C-terminus is necessary for apoptosis remains equivocal. More importantly, the exact portion of the C-terminus that conveys the pro-apoptotic property of TGFBIp is uncertain. It is reportedly within the final 166 amino acids. We sought to determine if this property is dependent upon the final 69 amino acids containing the integrin-binding, EPDIM and RGD, sequences. With MG-63 osteosarcoma cells, transforming growth factor (TGF)-β1 treatment increased expression of TGFBIp over 72 hours (p<0.001). At this time point, apoptosis was significantly increased (p<0.001) and was prevented by an anti-TGFBIp, polyclonal antibody (p<0.05). Overexpression of TGFBIp by transient transfection produced a 2-fold increase in apoptosis (p<0.01). Exogenous purified TGFBIp at concentrations of 37 to 150 nM produced a dose dependent increase in apoptosis (p<0.001). Mass spectrometry analysis of TGFBIp isolated from conditioned medium of cells treated with TGF-β1 revealed truncated forms of TGFBIp that lacked integrin-binding sequences in the C-terminus. Recombinant TGFBIp truncated, similarly, at amino acid 614 failed to induce apoptosis. A recombinant fragment encoding the final 69 amino acids of the TGFBIp C-terminus produced significant apoptosis. This apoptosis level was comparable to that induced by TGF-β1 upregulation of endogenous TGFBIp. Mutation of the integrin-binding sequence EPDIM, but not RGD, blocked apoptosis (p<0.001). These pro-apoptotic actions are dependent on the C-terminus most likely to interact with integrins. PMID:19505574

Improved tumor-targeting MRI contrast agents: Gd(DOTA) conjugates of a cycloalkane-based RGD peptide

DOE Office of Scientific and Technical Information (OSTI.GOV)

Park, Ji-Ae, E-mail: jpark@kirams.re.kr; Lee, Yong Jin; Ko, In Ok

2014-12-12

Highlights: • Development of improved tumor-targeting MRI contrast agents. • To increase the targeting ability of RGD, we developed cycloalkane-based RGD peptides. • Gd(DOTA) conjugates of cycloalkane-based RGD peptide show improved tumor signal enhancement in vivo MR images. - Abstract: Two new MRI contrast agents, Gd-DOTA-c(RGD-ACP-K) (1) and Gd-DOTA-c(RGD-ACH-K) (2), which were designed by incorporating aminocyclopentane (ACP)- or aminocyclohexane (ACH)-carboxylic acid into Gd-DOTA (gadolinium-tetraazacyclo dodecanetetraacetic acid) and cyclic RGDK peptides, were synthesized and evaluated for tumor-targeting ability in vitro and in vivo. Binding affinity studies showed that both 1 and 2 exhibited higher affinity for integrin receptors than cyclic RGDyKmore » peptides, which were used as a reference. These complexes showed high relaxivity and good stability in human serum and have the potential to improve target-specific signal enhancement in vivo MR images.« less

β1 Integrins Mediate Attachment of Mesenchymal Stem Cells to Cartilage Lesions

PubMed Central

Zwolanek, Daniela; Flicker, Magdalena; Kirstätter, Elisabeth; Zaucke, Frank; van Osch, Gerjo J.V.M.; Erben, Reinhold G.

2015-01-01

Abstract Mesenchymal stem cells (MSC) may have great potential for cell-based therapies of osteoarthritis. However, after injection in the joint, only few cells adhere to defective articular cartilage and contribute to cartilage regeneration. Little is known about the molecular mechanisms of MSC attachment to defective articular cartilage. Here, we developed an ex vivo attachment system, using rat osteochondral explants with artificially created full-thickness cartilage defects in combination with genetically labeled MSC isolated from bone marrow of human placental alkaline phosphatase transgenic rats. Binding of MSC to full-thickness cartilage lesions was improved by serum, but not hyaluronic acid, and was dependent on the presence of divalent cations. Additional in vitro tests showed that rat MSC attach, in a divalent cation-dependent manner, to collagen I, collagen II, and fibronectin, but not to collagen XXII or cartilage oligomeric matrix protein (COMP). RGD peptides partially blocked the adhesion of MSC to fibronectin in vitro and to cartilage lesions ex vivo. Furthermore, the attachment of MSC to collagen I and II in vitro and to cartilage lesions ex vivo was almost completely abolished in the presence of a β1 integrin blocking antibody. In conclusion, our data suggest that attachment of MSC to ex vivo full-thickness cartilage lesions is almost entirely β1 integrin-mediated, whereby both RGD- and collagen-binding integrins are involved. These findings suggest a key role of integrins during MSC attachment to defective cartilage and may pave the way for improved MSC-based therapies in the future. PMID:26309781

Nanoparticle Imaging of Integrins on Tumor Cells1

PubMed Central

Montet, Xavier; Montet-Abou, Karin; Reynolds, Fred; Weissleder, Ralph; Josephson, Lee

2006-01-01

Abstract Nanoparticles 10 to 100 nm in size can deliver large payloads to molecular targets, but undergo slow diffusion and/or slow transport through delivery barriers. To examine the feasibility of nanoparticles targeting a marker expressed in tumor cells, we used the binding of cyclic arginine-glycine-aspartic acid (RGD) nanoparticle targeting integrins on BT-20 tumor as a model system. The goals of this study were: 1) to use nanoparticles to image αvβ3 integrins expressed in BT-20 tumor cells by fluorescence-based imaging and magnetic resonance imaging, and, 2) to identify factors associated with the ability of nanoparticles to target tumor cell integrins. Three factors were identified: 1) tumor cell integrin expression (the αvβ3 integrin was expressed in BT-20 cells, but not in 9L cells); 2) nanoparticle pharmacokinetics (the cyclic RGD peptide cross-linked iron oxide had a blood half-life of 180 minutes and was able to escape from the vasculature over its long circulation time); and 3) tumor vascularization (the tumor had a dense capillary bed, with distances of <100 µm between capillaries). These results suggest that nanoparticles could be targeted to the cell surface markers expressed in tumor cells, at least in the case wherein the nanoparticles and the tumor model have characteristics similar to those of the BT-20 tumor employed here. PMID:16611415

Direct contact with perivascular tumor cells enhances integrin αvβ3 signaling and migration of endothelial cells

PubMed Central

Burgett, Monica E.; Lathia, Justin D.; Roth, Patrick; Nowacki, Amy S.; Galileo, Deni S.; Pugacheva, Elena; Huang, Ping; Vasanji, Amit; Li, Meizhang; Byzova, Tatiana; Mikkelsen, Tom; Bao, Shideng; Rich, Jeremy N.; Weller, Michael; Gladson, Candece L.

2016-01-01

The secretion of soluble pro-angiogenic factors by tumor cells and stromal cells in the perivascular niche promotes the aggressive angiogenesis that is typical of glioblastoma (GBM). Here, we show that angiogenesis also can be promoted by a direct interaction between brain tumor cells, including tumor cells with cancer stem-like properties (CSCs), and endothelial cells (ECs). As shown in vitro, this direct interaction is mediated by binding of integrin αvβ3 expressed on ECs to the RGD-peptide in L1CAM expressed on CSCs. It promotes both EC network formation and enhances directed migration toward basic fibroblast growth factor. Activation of αvβ3 and bone marrow tyrosine kinase on chromosome X (BMX) is required for migration stimulated by direct binding but not for migration stimulated by soluble factors. RGD-peptide treatment of mice with established intracerebral GBM xenografts significantly reduced the percentage of Sox2-positive tumor cells and CSCs in close proximity to ECs, decreased integrin αvβ3 and BMX activation and p130CAS phosphorylation in the ECs, and reduced the vessel surface area. These results reveal a previously unrecognized aspect of the regulation of angiogenesis in GBM that can impact therapeutic anti-angiogenic targeting. PMID:27270311

Selective Cell Adhesion and Biosensing Applications of Bio-Active Block Copolymers Prepared by CuAAC/Thiol-ene Double Click Reactions.

PubMed

Oyman Eyrilmez, Gizem; Doran, Sean; Murtezi, Eljesa; Demir, Bilal; Odaci Demirkol, Dilek; Coskunol, Hakan; Timur, Suna; Yagci, Yusuf

2015-09-01

N-Acetyl-l-cysteine (NAC)-capped poly(methyl methacrylate)-b-polycaprolactone block copolymer (PMMA-b-PCL-NAC) was prepared using the previously described one-pot photoinduced sequential CuAAC/thiol-ene double click procedure. PMMA-b-PCL-NAC had previously shown good applicability as a matrix for cell adhesion of cells from the Vero cell line (African green monkey kidney epithelial). Here, in this work, PMMA-b-PCL-NAC served as an excellent immobilization matrix for biomolecule conjugation. Covalent binding of RGD (R: arginine, G: glycine, and D: aspartic acid) peptide sequence onto the PMMA-b-PCL-NAC-coated surface was performed via EDC chemistry. RGD-modified PMMA-b-PCL-NAC (PMMA-b-PCL-NAC-RGD) as a non-toxic cell proliferation platform was used for selective "integrin αvβ3-mediated cell adhesion and biosensing studies. Both optical and electrochemical techniques were used to monitor the adhesion differences between "integrin αvβ3" receptor positive and negative cell lines on to the designed biofunctional surfaces. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Inhibition of melanoma cell motility by the snake venom disintegrin eristostatin

PubMed Central

Tian, Jing; Paquette-Straub, Carrie; Sage, E. Helene; Funk, Sarah E.; Patel, Vivek; Galileo, Deni; McLane, Mary Ann

2007-01-01

Eristostatin, an RGD-containing disintegrin isolated from the venom of Eristicophis macmahoni, inhibits lung or liver colonization of melanoma cells in a mouse model. In this study, transwell migration and in vitro wound closure assays were used to determine the effect of eristostatin on the migration of melanoma cells. Eristostatin significantly impaired the migration of 5 human melanoma cell lines. Furthermore, it specifically inhibited cell migration on fibronectin in a concentration-dependent manner, but not that on collagen IV or laminin. In contrast, eristostatin was found to have no effect on cell proliferation or angiogenesis. These results indicate that the interaction between eristostatin and melanoma cells may involve fibronectin-binding integrins that mediate cell migration. Mutations to alanine of seven residues within the RGD loop of eristostatin and four residues outside the RGD loop of eristostatin resulted in significantly less potency in both platelet aggregation and wound closure assays. For six of the mutations, however, decreased activity was found only in the latter assay. We conclude that a different mechanism and/or integrin is involved in these two cell activities. PMID:17316731

Suppression of Eosinophil Integrins Prevents Remodeling of Airway Smooth Muscle in Asthma

PubMed Central

Januskevicius, Andrius; Gosens, Reinoud; Sakalauskas, Raimundas; Vaitkiene, Simona; Janulaityte, Ieva; Halayko, Andrew J.; Hoppenot, Deimante; Malakauskas, Kestutis

2017-01-01

Background: Airway smooth muscle (ASM) remodeling is an important component of the structural changes to airways seen in asthma. Eosinophils are the prominent inflammatory cells in asthma, and there is some evidence that they contribute to ASM remodeling via released mediators and direct contact through integrin–ligand interactions. Eosinophils express several types of outer membrane integrin, which are responsible for cell–cell and cell–extracellular matrix interactions. In our previous study we demonstrated that asthmatic eosinophils show increased adhesion to ASM cells and it may be important factor contributing to ASM remodeling in asthma. According to these findings, in the present study we investigated the effects of suppression of eosinophil integrin on eosinophil-induced ASM remodeling in asthma. Materials and Methods: Individual combined cell cultures of immortalized human ASM cells and eosinophils from peripheral blood of 22 asthmatic patients and 17 healthy controls were prepared. Eosinophil adhesion was evaluated using eosinophil peroxidase activity assay. Genes expression levels in ASM cells and eosinophils were measured using quantitative real-time PCR. ASM cell proliferation was measured using alamarBlue® solution. Eosinophil integrins were blocked by incubating with Arg-Gly-Asp-Ser peptide. Results: Eosinophils from the asthma group showed increased outer membrane α4β1 and αMβ2 integrin expression, increased adhesion to ASM cells, and overexpression of TGF-β1 compared with eosinophils from the healthy control group. Blockade of eosinophil RGD-binding integrins by Arg-Gly-Asp-Ser peptide significantly reduced adhesion of eosinophils to ASM cells in both groups. Integrin-blocking decreased the effects of eosinophils on TGF-β1, WNT-5a, and extracellular matrix protein gene expression in ASM cells and ASM cell proliferation in both groups. These effects were more pronounced in the asthma group compared with the control group. Conclusion: Suppression of eosinophil-ASM interaction via RGD-binding integrins attenuates eosinophil-induced ASM remodeling in asthma. Trial Registration: ClinicalTrials.gov Identifier: NCT02648074. PMID:28119625

Foot-and-Mouth Disease Virus Receptors: Comparison of Bovine αV Integrin Utilization by Type A and O Viruses

PubMed Central

Duque, Hernando; Baxt, Barry

2003-01-01

Three members of the αV integrin family of cellular receptors, αVβ1, αVβ3, and αVβ6, have been identified as receptors for foot-and-mouth disease virus (FMDV) in vitro. The virus interacts with these receptors via a highly conserved arginine-glycine-aspartic acid (RGD) amino acid sequence motif located within the βG-βH (G-H) loop of VP1. Other αV integrins, as well as several other integrins, recognize and bind to RGD motifs on their natural ligands and also may be candidate receptors for FMDV. To analyze the roles of the αV integrins from a susceptible species as viral receptors, we molecularly cloned the bovine β1, β5, and β6 integrin subunits. Using these subunits, along with previously cloned bovine αV and β3 subunits, in a transient expression assay system, we compared the efficiencies of infection mediated by αVβ1, αVβ3, αVβ5, and αVβ6 among three strains of FMDV serotype A and two strains of serotype O. While all the viruses could infect cells expressing these integrins, they exhibited different efficiencies of integrin utilization. All the type A viruses used αVβ3 and αVβ6 with relatively high efficiency, while only one virus utilized αVβ1 with moderate efficiency. In contrast, both type O viruses utilized αVβ6 and αVβ1 with higher efficiency than αVβ3. Only low levels of viral replication were detected in αVβ5-expressing cells infected with either serotype. Experiments in which the ligand-binding domains among the β subunits were exchanged indicated that this region of the integrin subunit appears to contribute to the differences in integrin utilizations among strains. In contrast, the G-H loops of the different viruses do not appear to be involved in this phenomenon. Thus, the ability of the virus to utilize multiple integrins in vitro may be a reflection of the use of multiple receptors during the course of infection within the susceptible host. PMID:12551988

Suppression of murine collagen-induced arthritis by targeted apoptosis of synovial neovasculature

PubMed Central

Gerlag, Danielle M; Borges, Eric; Tak, Paul P; Ellerby, H Michael; Bredesen, Dale E; Pasqualini, Renata; Ruoslahti, Erkki; Firestein, Gary S

2001-01-01

Because angiogenesis plays a major role in the perpetuation of inflammatory arthritis, we explored a method for selectively targeting and destroying new synovial blood vessels. Mice with collagen-induced arthritis were injected intravenously with phage expressing an RGD motif. In addition, the RGD peptide (RGD-4C) was covalently linked to a proapoptotic heptapeptide dimer, D(KLAKLAK)2, and was systemically administered to mice with collagen-induced arthritis. A phage displaying an RGD-containing cyclic peptide (RGD-4C) that binds selectively to the αvβ3 and αvβ5 integrins accumulated in inflamed synovium but not in normal synovium. Homing of RGD-4C phage to inflamed synovium was inhibited by co-administration of soluble RGD-4C. Intravenous injections of the RGD-4C–D(KLAKLAK)2 chimeric peptide significantly decreased clinical arthritis and increased apoptosis of synovial blood vessels, whereas treatment with vehicle or uncoupled mixture of the RGD-4C and the untargeted proapoptotic peptide had no effect. Targeted apoptosis of synovial neovasculature can induce apoptosis and suppress clinical arthritis. This form of therapy has potential utility in the treatment of inflammatory arthritis. PMID:11714389

Reduction of renal uptake of 111In-DOTA-labeled and A700-labeled RAFT-RGD during integrin αvβ3 targeting using single photon emission computed tomography and optical imaging.

PubMed

Briat, Arnaud; Wenk, Christiane H F; Ahmadi, Mitra; Claron, Michael; Boturyn, Didier; Josserand, Véronique; Dumy, Pascal; Fagret, Daniel; Coll, Jean-Luc; Ghezzi, Catherine; Sancey, Lucie; Vuillez, Jean-Philippe

2012-06-01

Integrin α(v)β(3) expression is upregulated during tumor growth and invasion in newly formed endothelial cells in tumor neovasculature and in some tumor cells. A tetrameric RGD-based peptide, regioselectively addressable functionalized template-(cyclo-[RGDfK])4 (RAFT-RGD), specifically targets integrin α(v)β(3) in vitro and in vivo. When labeled with indium-111, the RAFT-RGD is partially reabsorbed and trapped in the kidneys, limiting its use for further internal targeted radiotherapy and imaging investigations. We studied the effect of Gelofusine on RAFT-RGD renal retention in tumor-bearing mice. Mice were imaged using single photon emission computed tomography and optical imaging 1 and 24 h following tracer injection. Distribution of RAFT-RGD was further investigated by tissue removal and direct counting of the tracer. Kidney sections were analyzed by confocal microscopy. Gelofusine significantly induced a >50% reduction of the renal reabsorption of (111)In-DOTA-RAFT-RGD and A700-RAFT-RGD, without affecting tumor uptake. Injection of Gelofusine significantly reduced the renal retention of labeled RAFT-RGD, while increasing the tumor over healthy tissue ratio. These results will lead to the development of future therapeutic approaches. © 2012 Japanese Cancer Association.

Cyclic RGD peptidomimetics containing 4- and 5-amino-cyclopropane pipecolic acid (CPA) templates as dual αVβ3 and α5β1 integrin ligands.

PubMed

Sernissi, Lorenzo; Trabocchi, Andrea; Scarpi, Dina; Bianchini, Francesca; Occhiato, Ernesto G

2016-02-15

4-Amino- and 5-amino-cyclopropane pipecolic acids (CPAs) with cis relative stereochemistry between the carboxylic and amino groups were used as templates to prepare cyclic peptidomimetics containing the RGD sequence as possible integrin binders. The peptidomimetic c(RGD8) built on the 5-amino-CPA displayed an inhibition activity (IC50=2.4nM) toward the αvβ3 integrin receptor (expressed in M21 human melanoma cell line) comparable to that of the most potent antagonists reported so far and it was ten times more active than the corresponding antagonist c(RGD7) derived from the isomeric 4-amino-CPA. Both compounds were also nanomolar ligands of the α5β1 integrin (expressed in human erythroleukemia cell line K562). These results suggest that the CPA-derived templates are suitable for the preparation of dual αvβ3 and α5β1 ligands to suppress integrin-mediated events as well as for targeted drug delivery in cancer therapy. Copyright © 2015 Elsevier Ltd. All rights reserved.

Oxidation-induced Structural Changes of Ceruloplasmin Foster NGR Motif Deamidation That Promotes Integrin Binding and Signaling

PubMed Central

Barbariga, Marco; Curnis, Flavio; Spitaleri, Andrea; Andolfo, Annapaola; Zucchelli, Chiara; Lazzaro, Massimo; Magnani, Giuseppe; Musco, Giovanna; Corti, Angelo; Alessio, Massimo

2014-01-01

Asparagine deamidation occurs spontaneously in proteins during aging; deamidation of Asn-Gly-Arg (NGR) sites can lead to the formation of isoAsp-Gly-Arg (isoDGR), a motif that can recognize the RGD-binding site of integrins. Ceruloplasmin (Cp), a ferroxidase present in the cerebrospinal fluid (CSF), contains two NGR sites in its sequence: one exposed on the protein surface (568NGR) and the other buried in the tertiary structure (962NGR). Considering that Cp can undergo oxidative modifications in the CSF of neurodegenerative diseases, we investigated the effect of oxidation on the deamidation of both NGR motifs and, consequently, on the acquisition of integrin binding properties. We observed that the exposed 568NGR site can deamidate under conditions mimicking accelerated Asn aging. In contrast, the hidden 962NGR site can deamidate exclusively when aging occurs under oxidative conditions, suggesting that oxidation-induced structural changes foster deamidation at this site. NGR deamidation in Cp was associated with gain of integrin-binding function, intracellular signaling, and cell pro-adhesive activity. Finally, Cp aging in the CSF from Alzheimer disease patients, but not in control CSF, causes Cp deamidation with gain of integrin-binding function, suggesting that this transition might also occur in pathological conditions. In conclusion, both Cp NGR sites can deamidate during aging under oxidative conditions, likely as a consequence of oxidative-induced structural changes, thereby promoting a gain of function in integrin binding, signaling, and cell adhesion. PMID:24366863

RGD-Targeted Ultrasound Contrast Agent for Longitudinal Assessment of Hep-2 Tumor Angiogenesis In Vivo.

PubMed

Hu, Qiao; Wang, Xiao-Yan; Kang, Li-Ke; Wei, Hai-Ming; Xu, Chun-Mei; Wang, Tao; Wen, Zong-Hua

2016-01-01

To prepare arginine-glycine-aspartate (RGD)-targeted ultrasound contrast microbubbles (MBs) and explore the feasibility of their use in assessing dynamic changes in αvβ3 integrin expression in a murine model of tumor angiogenesis. RGD peptides were conjugated to the surfaces of microbubbles via biotin-avidin linkage. Microbubbles bearing RADfK peptides were prepared as controls. The RGD-MBs were characterized using an Accusizer 780 and optical microscopy. The binding specificity of the RGD-MBs for ανβ3-expressing endothelial cells (bEnd.3) was demonstrated in vitro by a competitive inhibition experiment. In an in vivo study, mice bearing tumors of three different stages were intravenously injected with RGD-MBs and subjected to targeted, contrast-enhanced, high-frequency ultrasound. Subsequently, tumors were harvested and sectioned for immunofluorescence analysis of ανβ3 expression. The mean size of the RGD-MBs was 2.36 ± 1.7 μm. The RGD-MBs showed significantly higher adhesion levels to bEnd.3 cells compared to control MBs (P < 0.01). There was rarely binding of RGD-MBs to αvβ3-negative MCF-7 cells. Adhesion of the RGD-MBs to the bEnd.3 cells was significantly inhibited following treatment with anti-alpha(v) antibodies. The quantitative acoustic video intensity for high-frequency, contrast-enhanced ultrasound imaging of subcutaneous human laryngeal carcinoma (Hep-2) tumor xenografts was significantly higher in small tumors (19.89 ± 2.49) than in medium tumors (11.25 ± 2.23) and large tumors (3.38 ± 0.67) (P < 0.01). RGD-MBs enable noninvasive in vivo visualization of changes in tumor angiogenesis during tumor growth in subcutaneous cancer xenografts.

RGD-Targeted Ultrasound Contrast Agent for Longitudinal Assessment of Hep-2 Tumor Angiogenesis In Vivo

PubMed Central

Hu, Qiao; Wang, Xiao-Yan; Kang, Li-Ke; Wei, Hai-Ming; Xu, Chun-Mei; Wang, Tao; Wen, Zong-Hua

2016-01-01

Objective To prepare arginine-glycine-aspartate (RGD)-targeted ultrasound contrast microbubbles (MBs) and explore the feasibility of their use in assessing dynamic changes in αvβ3 integrin expression in a murine model of tumor angiogenesis. Methods RGD peptides were conjugated to the surfaces of microbubbles via biotin-avidin linkage. Microbubbles bearing RADfK peptides were prepared as controls. The RGD-MBs were characterized using an Accusizer 780 and optical microscopy. The binding specificity of the RGD-MBs for ανβ3-expressing endothelial cells (bEnd.3) was demonstrated in vitro by a competitive inhibition experiment. In an in vivo study, mice bearing tumors of three different stages were intravenously injected with RGD-MBs and subjected to targeted, contrast-enhanced, high-frequency ultrasound. Subsequently, tumors were harvested and sectioned for immunofluorescence analysis of ανβ3 expression. Results The mean size of the RGD-MBs was 2.36 ± 1.7 μm. The RGD-MBs showed significantly higher adhesion levels to bEnd.3 cells compared to control MBs (P < 0.01). There was rarely binding of RGD-MBs to αvβ3-negative MCF-7 cells. Adhesion of the RGD-MBs to the bEnd.3 cells was significantly inhibited following treatment with anti-alpha(v) antibodies. The quantitative acoustic video intensity for high-frequency, contrast-enhanced ultrasound imaging of subcutaneous human laryngeal carcinoma (Hep-2) tumor xenografts was significantly higher in small tumors (19.89 ± 2.49) than in medium tumors (11.25 ± 2.23) and large tumors (3.38 ± 0.67) (P < 0.01). Conclusions RGD-MBs enable noninvasive in vivo visualization of changes in tumor angiogenesis during tumor growth in subcutaneous cancer xenografts. PMID:26862757

Characterization and evaluation of DOTA-conjugated Bombesin/RGD-antagonists for prostate cancer tumor imaging and therapy.

PubMed

Stott Reynolds, Tamila J; Schehr, Rebecca; Liu, Dijie; Xu, Jingli; Miao, Yubin; Hoffman, Timothy J; Rold, Tammy L; Lewis, Michael R; Smith, Charles J

2015-02-01

Here we present the metallation, characterization, in vivo and in vitro evaluations of dual-targeting, peptide-based radiopharmaceuticals with utility for imaging and potentially treating prostate tumors by virtue of their ability to target the αVβ3 integrin or the gastrin releasing peptide receptor (GRPr). [RGD-Glu-6Ahx-RM2] (RGD: Arg-Gly-Asp; Glu: glutamic acid; 6-Ahx: 6-amino hexanoic acid; RM2: (D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2)) was conjugated to a DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) bifunctional chelator (BFCA) purified via reversed-phase high-performance liquid chromatography (RP-HPLC), characterized by electrospray ionization-mass spectrometry (ESI-MS), and radiolabeled with (111)In or (177)Lu. Natural-metallated compounds were assessed for binding affinity for the αVβ3 integrin or GRPr in human glioblastoma U87-MG and prostate PC-3 cell lines and stability prior to in vivo evaluation in normal CF-1 mice and SCID mice xenografted with PC-3 cells. Competitive displacement binding assays with PC-3 and U87-MG cells revealed high to moderate binding affinity for the GRPr or the αVβ3 integrin (IC50 range of 5.39±1.37 nM to 9.26±0.00 nM in PC-3 cells, and a range of 255±47 nM to 321±85 nM in U87-MG cells). Biodistribution studies indicated high tumor uptake in PC-3 tumor-bearing mice (average of 7.40±0.53% ID/g at 1h post-intravenous injection) and prolonged retention of tracer (mean of 4.41±0.91% ID/g at 24h post-intravenous injection). Blocking assays corroborated the specificity of radioconjugates for each target. Micro-single photon emission computed tomography (microSPECT) confirmed favorable radiouptake profiles in xenografted mice at 20h post-injection. [RGD-Glu-[(111)In-DO3A]-6-Ahx-RM2] and [RGD-Glu-[(177)Lu- DO3A]-6-Ahx-RM2] show favorable pharmacokinetic and radiouptake profiles, meriting continued evaluation for molecular imaging in murine U87-MG/PC-3 xenograft models and radiotherapy studies with (177)Lu and (90)Y conjugates. These heterovalent, peptide-targeting ligands perform comparably with many mono- and multivalent conjugates with the potential benefit of increased sensitivity for detecting cancer cells exhibiting differential expression of target receptors. Published by Elsevier Inc.

Modulation of Cell Proliferation and Differentiation through Substrate-dependent Changes in Fibronectin Conformation

PubMed Central

García, Andrés J.; Vega, María D.; Boettiger, David

1999-01-01

Integrin-mediated cell adhesion to extracellular matrices provides signals essential for cell cycle progression and differentiation. We demonstrate that substrate-dependent changes in the conformation of adsorbed fibronectin (Fn) modulated integrin binding and controlled switching between proliferation and differentiation. Adsorption of Fn onto bacterial polystyrene (B), tissue culture polystyrene (T), and collagen (C) resulted in differences in Fn conformation as indicated by antibody binding. Using a biochemical method to quantify bound integrins in cultured cells, we found that differences in Fn conformation altered the quantity of bound α5 and β1 integrin subunits but not αv or β3. C2C12 myoblasts grown on these Fn-coated substrates proliferated to different levels (B > T > C). Immunostaining for muscle-specific myosin revealed minimal differentiation on B, significant levels on T, and extensive differentiation on C. Differentiation required binding to the RGD cell binding site in Fn and was blocked by antibodies specific for this site. Switching between proliferation and differentiation was controlled by the levels of α5β1 integrin bound to Fn, and differentiation was inhibited by anti-α5, but not anti-αv, antibodies, suggesting distinct integrin-mediated signaling pathways. Control of cell proliferation and differentiation through conformational changes in extracellular matrix proteins represents a versatile mechanism to elicit specific cellular responses for biological and biotechnological applications. PMID:10069818

RGD-containing peptides activate S6K1 through beta3 integrin in adult cardiac muscle cells.

PubMed

Balasubramanian, Sundaravadivel; Kuppuswamy, Dhandapani

2003-10-24

The enzyme p70S6 kinase (S6K1) is critical for cell growth, and we have reported its activation during cardiac hypertrophy. Because cardiac hypertrophy also involves integrin activation, we analyzed whether integrins could contribute to S6K1 activation. Using adult feline cardiomyocytes, here we report that integrin-interacting Arg-Gly-Asp (RGD) peptides activate S6K1 as observed by band shifting, kinase activity and phosphorylation at Thr-389 and Thr-421/Ser-424 of S6K1, and S6 protein phosphorylation. Perturbation of specific integrin function with blocking antibodies and by overexpressing the beta1A cytoplasmic tail revealed that beta3 but not beta1 integrin mediates the RGD-induced S6K1 activation. This activation is focal adhesion complex-independent and is accompanied by the activation of extracellular signal-regulated kinases 1/2 (ERK) and mammalian target of rapamycin (mTOR). Studies using specific inhibitors and dominant negative c-Raf expression in cardiomyocytes indicate that the S6K1 activation involves mTOR, MEK/ERK, and phosphatidylinositol 3-kinase pathways and is independent of protein kinase C and c-Raf. Finally, addition of fluorescent-labeled RGD peptide to cardiomyocytes exhibits its internalization and localization to the endocytic vesicles, and pretreatment of cardiomyocytes with endocytic inhibitors reduced the S6K1 activation. These data suggest that RGD interaction with beta3 integrin and its subsequent endocytosis trigger specific signaling pathway(s) for S6K1 activation in cardiomyocytes and that this process may contribute to hypertrophic growth and remodeling of myocardium.

Clustered Integrin Ligands as a Novel Approach for the Targeting of Non-Viral Vectors

NASA Astrophysics Data System (ADS)

Ng, Quinn Kwan Tai

Gene transfer or gene delivery is described as the process in which foreign DNA is introduced into cells. Over the years, gene delivery has gained the attention of many researchers and has been developed as powerful tools for use in biotechnology and medicine. With the completion of the Human Genome Project, such advances in technology allowed for the identification of diseases ranging from hereditary disorders to acquired ones (cancer) which were thought to be incurable. Gene therapy provides the means necessary to treat or eliminate genetic diseases from its origin, unlike traditional medicine which only treat symptoms. With ongoing clinical trials for gene therapy increasing, the greatest difficulty still lies in developing safe systems which can target cells of interest to provide efficient delivery. Nature, over millions of years of evolution, has provided an example of one of the most efficient delivery systems: viruses. Although the use of viruses for gene delivery has been well studied, the safety issues involving immunogenicity, insertional mutagenesis, high cost, and poor reproducibility has provided problems for their clinical application. From understanding viruses, we gain insight to designing new systems for non-viral gene delivery. One of these techniques utilized by adenoviruses is the clustering of ligands on its surface through the use of a protein called a penton base. Through the use of nanotechnology we can mimic this basic concept in non-viral gene delivery systems. This dissertation research is focused on developing and applying a novel system for displaying the integrin binding ligand (RGD) in a constrained manner to form a clustered integrin ligand binding platform to be used to enhance the targeting and efficiency of non-viral gene delivery vectors. Peptide mixed monolayer protected gold nanoparticles provides a suitable surface for ligand clustering. A relationship between the peptide ratios in the reaction solution used to form these ligand clusters compared to the reacted amounts on the surface of the particle was studied. This provided us the ability to control the size of the clusters formed and the spacing between the integrins for gold nanoparticles of various sizes. We then applied the clustered ligand binding system for targeting of DNA/PEI polyplexes and demonstrated that the use of RGD nanoclusters enhances gene transfer up to 35-fold which was dependent on the density of alphavbeta3 integrins on the cell surface. Cell integrin sensitivity was shown in which cells with higher alpha vbeta3 densities resulting in higher luciferase transgene expression. The targeting of RGD nanoclusters for DNA/PEI polyplexes was further shown in vivo using PET/CT technology which displayed improved targeting towards high level alphavbeta3 integrin expression (U87MG) tumors over medium level alphavbeta 3 integrin expression (HeLa). In addition to studying the clustered integrin binding system, the current non-viral vectors used suffer from stability and toxicity issues in vitro and in vivo. We have applied a new chemistry for synthesizing nanogels utilizing a Traut's reagent initiated Michael addition reaction for modification of diamine containing crosslikers which will allow for the development of stable and cell demanded release of oligonucleotides. We have shown bulk gels made were capable of encapsulating and holding DNA within the gel and were able to synthesize them into nanogels. The combined research shown here using clustered integrin ligands and a new type of nanogel synthesis provides an ideal system for gene delivery in the future.

Pathogenetic role of Arg-Gly-Asp-recognizing integrins in acute renal failure. off.

PubMed Central

Goligorsky, M S; DiBona, G F

1993-01-01

Reorientation of the alpha 3 subunit of integrins from predominantly basal to the apical cell surface of cultured renal tubular epithelial cells subjected to oxidant stress has previously been demonstrated. The present study was designed to assess functional competence of ectopically expressed apical integrins. Cell-cell adhesion assay revealed enhanced cytoatractant properties of stressed cells. Stressed epithelial cells exhibited specific recognition and binding of laminin-coated latex beads. These processes were inhibited with the peptide Gly-Arg-Gly-Asp-Asn-Pro (GRGDNP) suggesting a role of RGD-recognizing integrins in augmented adhesion to stressed cells. Given that such enhanced adhesion in in vivo acute renal failure may govern tubular obstruction by desquamated epithelium, a physiological marker of patency of tubular lumen, proximal tubular pressure, was monitored in rats subjected to 60 min of renal ischemia followed by reperfusion. Proximal tubular pressure increased 2-fold after 2 hr of reperfusion in animals that had undergone 60 min of ischemia. Infusion of GRGDNP into the renal artery during reperfusion period virtually abolished an increase in proximal tubular pressure observed in ischemic acute renal failure. These in vitro and in vivo findings are consistent with the hypothesis that RGD-recognizing integrins play an important role in the pathogenesis of tubular obstruction in ischemic acute renal failure. Images Fig. 2 Fig. 3 PMID:8516318

Mechanisms underlying the attachment and spreading of human osteoblasts: from transient interactions to focal adhesions on vitronectin-grafted bioactive surfaces.

PubMed

Brun, Paola; Scorzeto, Michele; Vassanelli, Stefano; Castagliuolo, Ignazio; Palù, Giorgio; Ghezzo, Francesca; Messina, Grazia M L; Iucci, Giovanna; Battaglia, Valentina; Sivolella, Stefano; Bagno, Andrea; Polzonetti, Giovanni; Marletta, Giovanni; Dettin, Monica

2013-04-01

The features of implant devices and the reactions of bone-derived cells to foreign surfaces determine implant success during osseointegration. In an attempt to better understand the mechanisms underlying osteoblasts attachment and spreading, in this study adhesive peptides containing the fibronectin sequence motif for integrin binding (Arg-Gly-Asp, RGD) or mapping the human vitronectin protein (HVP) were grafted on glass and titanium surfaces with or without chemically induced controlled immobilization. As shown by total internal reflection fluorescence microscopy, human osteoblasts develop adhesion patches only on specifically immobilized peptides. Indeed, cells quickly develop focal adhesions on RGD-grafted surfaces, while HVP peptide promotes filopodia, structures involved in cellular spreading. As indicated by immunocytochemistry and quantitative polymerase chain reaction, focal adhesions kinase activation is delayed on HVP peptides with respect to RGD while an osteogenic phenotypic response appears within 24h on osteoblasts cultured on both peptides. Cellular pathways underlying osteoblasts attachment are, however, different. As demonstrated by adhesion blocking assays, integrins are mainly involved in osteoblast adhesion to RGD peptide, while HVP selects osteoblasts for attachment through proteoglycan-mediated interactions. Thus an interfacial layer of an endosseous device grafted with specifically immobilized HVP peptide not only selects the attachment and supports differentiation of osteoblasts but also promotes cellular migration. Copyright © 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Comparison of three dimeric 18F-AlF-NOTA-RGD tracers.

PubMed

Guo, Jinxia; Lang, Lixin; Hu, Shuo; Guo, Ning; Zhu, Lei; Sun, Zhongchan; Ma, Ying; Kiesewetter, Dale O; Niu, Gang; Xie, Qingguo; Chen, Xiaoyuan

2014-04-01

RGD peptide-based radiotracers are well established as integrin αvβ3 imaging probes to evaluate tumor angiogenesis or tissue remodeling after ischemia or infarction. In order to optimize the labeling process and pharmacokinetics of the imaging probes, we synthesized three dimeric RGD peptides with or without PEGylation and performed in vivo screening. Radiolabeling was achieved through the reaction of F-18 aluminum-fluoride complex with the cyclic chelator, 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA). Three imaging probes were synthesized as (18)F-AlF-NOTA-E[c(RGDfK)]2, (18)F-AlF-NOTA-PEG4-E[c(RGDfK)]2, and (18)F-AlF-NOTA-E[PEG4-c(RGDfk)]2. The receptor binding affinity was determined by competitive cell binding assay, and the stability was evaluated by mouse serum incubation. Tumor uptake and whole body distribution of the three tracers were compared through direct tissue sampling and PET quantification of U87MG tumor-bearing mice. All three compounds remained intact after 120 min incubation with mouse serum. They all had a rapid and relatively high tracer uptake in U87MG tumors with good target-to-background ratios. Compared with the other two tracers, (18)F-AlF-NOTA-E[PEG4-c(RGDfk)]2 had the highest tumor uptake and the lowest accumulation in the liver. The integrin receptor specificity was confirmed by co-injection of unlabeled dimeric RGD peptide. The rapid one-step radiolabeling strategy by the complexation of (18)F-aluminum fluoride with NOTA-peptide conjugates was successfully applied to synthesize three dimeric RGD peptides. Among the three probes developed, (18)F-AlF-NOTA-E[PEG4-c(RGDfk)]2 with relatively low liver uptake and high tumor accumulation appears to be a promising candidate for further translational research.

Radiolabeled Cyclic RGD Peptides as Radiotracers for Imaging Tumors and Thrombosis by SPECT

PubMed Central

Zhou, Yang; Chakraborty, Sudipta; Liu, Shuang

2011-01-01

The integrin family is a group of transmembrane glycoprotein comprised of 19 α- and 8 β-subunits that are expressed in 25 different α/β heterodimeric combinations on the cell surface. Integrins play critical roles in many physiological processes, including cell attachment, proliferation, bone remodeling, and wound healing. Integrins also contribute to pathological events such as thrombosis, atherosclerosis, tumor invasion, angiogenesis and metastasis, infection by pathogenic microorganisms, and immune dysfunction. Among 25 members of the integrin family, the αvβ3 is studied most extensively for its role of tumor growth, progression and angiogenesis. In contrast, the αIIbβ3 is expressed exclusively on platelets, facilitates the intercellular bidirectional signaling (“inside-out” and “outside-in”) and allows the aggregation of platelets during vascular injury. The αIIbβ3 plays an important role in thrombosis by its activation and binding to fibrinogen especially in arterial thrombosis due to the high blood flow rate. In the resting state, the αIIbβ3 on platelets does not bind to fibrinogen; on activation, the conformation of platelet is altered and the binding sites of αIIbβ3 are exposed for fibrinogen to crosslink platelets. Over the last two decades, integrins have been proposed as the molecular targets for diagnosis and therapy of cancer, thrombosis and other diseases. Several excellent review articles have appeared recently to cover a broad range of topics related to the integrin-targeted radiotracers and their nuclear medicine applications in tumor imaging by single photon emission computed tomography (SPECT) or a positron-emitting radionuclide for positron emission tomography (PET). This review will focus on recent developments of αvβ3-targeted radiotracers for imaging tumors and the use of αIIbβ3-targeted radiotracers for thrombosis imaging, and discuss different approaches to maximize the targeting capability of cyclic RGD peptides and improve the radiotracer excretion kinetics from non-cancerous organs. Improvement of target uptake and target-to-background ratios is critically important for target-specific radiotracers. PMID:21547153

Integrin αvβ3-targeted dynamic contrast-enhanced magnetic resonance imaging using a gadolinium-loaded polyethylene gycol-dendrimer-cyclic RGD conjugate to evaluate tumor angiogenesis and to assess early antiangiogenic treatment response in a mouse xenograft tumor model.

PubMed

Chen, Wei-Tsung; Shih, Tiffany Ting Fang; Chen, Ran-Chou; Tu, Shin-Yang; Hsieh, Wen-Yuen; Yang, Pang-Chyr

2012-01-01

The purpose of this study was to validate an integrin αvβ3-targeted magnetic resonance contrast agent, PEG-G3-(Gd-DTPA)6-(cRGD-DTPA)2, for its ability to detect tumor angiogenesis and assess early response to antiangiogenic therapy using dynamic contrast-enhanced (DCE) magnetic resonance imaging (MRI). Integrin αvβ3-positive U87 cells and control groups were incubated with fluorescein-labeled cRGD-conjugated dendrimer, and the cellular attachment of the dendrimer was observed. DCE MRI was performed on mice bearing KB xenograft tumors using either PEG-G3-(Gd-DTPA)6-(cRGD-DTPA)2 or PEG-G3-(Gd-DTPA)6-(cRAD-DTPA)2. DCE MRI was also performed 2 hours after anti-integrin αvβ3 monoclonal antibody treatment and after bevacizumab treatment on days 3 and 6t. Using DCE MRI, the 30-minute contrast washout percentage was significantly lower in the cRGD-conjugate injection groups. The enhancement patterns were different between the two contrast injection groups. In the antiangiogenic therapy groups, a rapid increase in 30-minute contrast washout percentage was observed in both the LM609 and bevacizumab treatment groups, and this occurred before there was an observable decrease in tumor size. The integrin αvβ3 targeting ability of PEG-G3-(Gd-DTPA)6-(cRGD-DTPA)2 in vitro and in vivo was demonstrated. The 30-minute contrast washout percentage is a useful parameter for examining tumor angiogenesis and for the early assessment of antiangiogenic treatment response.

Cell Adhesion on RGD-Displaying Knottins with Varying Numbers of Tryptophan Amino Acids to Tune the Affinity for Assembly on Cucurbit[8]uril Surfaces.

PubMed

Sankaran, Shrikrishnan; Cavatorta, Emanuela; Huskens, Jurriaan; Jonkheijm, Pascal

2017-09-05

Cell adhesion is studied on multivalent knottins, displaying RGD ligands with a high affinity for integrin receptors, that are assembled on CB[8]-methylviologen-modified surfaces. The multivalency in the knottins stems from the number of tryptophan amino acid moieties, between 0 and 4, that can form a heteroternary complex with cucurbit[8]uril (CB[8]) and surface-tethered methylviologen (MV 2+ ). The binding affinity of the knottins with CB[8] and MV 2+ surfaces was evaluated using surface plasmon resonance spectroscopy. Specific binding occurred, and the affinity increased with the valency of tryptophans on the knottin. Additionally, increased multilayer formation was observed, attributed to homoternary complex formation between tryptophan residues of different knottins and CB[8]. Thus, we were able to control the surface coverage of the knottins by valency and concentration. Cell experiments with mouse myoblast (C2C12) cells on the self-assembled knottin surfaces showed specific integrin recognition by the RGD-displaying knottins. Moreover, cells were observed to elongate more on the supramolecular knottin surfaces with a higher valency, and in addition, more pronounced focal adhesion formation was observed on the higher-valency knottin surfaces. We attribute this effect to the enhanced coverage and the enhanced affinity of the knottins in their interaction with the CB[8] surface. Collectively, these results are promising for the development of biomaterials including knottins via CB[8] ternary complexes for tunable interactions with cells.

Fibrinogen Motif Discriminates Platelet and Cell Capture in Peptide-Modified Gold Micropore Arrays.

PubMed

Adamson, Kellie; Spain, Elaine; Prendergast, Una; Moran, Niamh; Forster, Robert J; Keyes, Tia E

2018-01-16

Human blood platelets and SK-N-AS neuroblastoma cancer-cell capture at spontaneously adsorbed monolayers of fibrinogen-binding motifs, GRGDS (generic integrin adhesion), HHLGGAKQAGDV (exclusive to platelet integrin α IIb β 3 ), or octanethiol (adhesion inhibitor) at planar gold and ordered 1.6 μm diameter spherical cap gold cavity arrays were compared. In all cases, arginine/glycine/aspartic acid (RGD) promoted capture, whereas alkanethiol monolayers inhibited adhesion. Conversely only platelets adhered to alanine/glycine/aspartic acid (AGD)-modified surfaces, indicating that the AGD motif is recognized preferentially by the platelet-specific integrin, α IIb β 3 . Microstructuring of the surface effectively eliminated nonspecific platelet/cell adsorption and dramatically enhanced capture compared to RGD/AGD-modified planar surfaces. In all cases, adhesion was reversible. Platelets and cells underwent morphological change on capture, the extent of which depended on the topography of the underlying substrate. This work demonstrates that both the nature of the modified interface and its underlying topography influence the capture of cancer cells and platelets. These insights may be useful in developing cell-based cancer diagnostics as well as in identifying strategies for the disruption of platelet cloaks around circulating tumor cells.

Molecular Magnetic Resonance Imaging of Angiogenesis In Vivo using Polyvalent Cyclic RGD-Iron Oxide Microparticle Conjugates

PubMed Central

Melemenidis, Stavros; Jefferson, Andrew; Ruparelia, Neil; Akhtar, Asim M; Xie, Jin; Allen, Danny; Hamilton, Alastair; Larkin, James R; Perez-Balderas, Francisco; Smart, Sean C; Muschel, Ruth J; Chen, Xiaoyuan; Sibson, Nicola R; Choudhury, Robin P

2015-01-01

Angiogenesis is an essential component of tumour growth and, consequently, an important target both therapeutically and diagnostically. The cell adhesion molecule αvβ3 integrin is a specific marker of angiogenic vessels and the most prevalent vascular integrin that binds the amino acid sequence arginine-glycine-aspartic acid (RGD). Previous studies using RGD-targeted nanoparticles (20-50 nm diameter) of iron oxide (NPIO) for magnetic resonance imaging (MRI) of tumour angiogenesis, have identified a number of limitations, including non-specific extravasation, long blood half-life (reducing specific contrast) and low targeting valency. The aim of this study, therefore, was to determine whether conjugation of a cyclic RGD variant [c(RGDyK)], with enhanced affinity for αvβ3, to microparticles of iron oxide (MPIO) would provide a more sensitive contrast agent for imaging of angiogenic tumour vessels. Cyclic RGD [c(RGDyK)] and RAD [c(RADyK)] based peptides were coupled to 2.8 μm MPIO, and binding efficacy tested both in vitro and in vivo. Significantly greater specific binding of c(RGDyK)-MPIO to S-nitroso-n-acetylpenicillamine (SNAP)-stimulated human umbilical vein endothelial cells in vitro than PBS-treated cells was demonstrated under both static (14-fold increase; P < 0.001) and flow (44-fold increase; P < 0.001) conditions. Subsequently, mice bearing subcutaneous colorectal (MC38) or melanoma (B16F10) derived tumours underwent in vivo MRI pre- and post-intravenous administration of c(RGDyK)-MPIO or c(RADyK)-MPIO. A significantly greater volume of MPIO-induced hypointensities were found in c(RGDyK)-MPIO injected compared to c(RADyK)-MPIO injected mice, in both tumour models (P < 0.05). Similarly, administration of c(RGDyK)-MPIO induced a greater reduction in mean tumour T2* relaxation times than the control agent in both tumour models (melanoma P < 0.001; colorectal P < 0.0001). Correspondingly, MPIO density per tumour volume assessed immunohistochemically was significantly greater for c(RGDyK)-MPIO than c(RADyK)-MPIO injected animals, in both melanoma (P < 0.05) and colorectal (P < 0.0005) tumours. In both cases, binding of c(RGDyK)-MPIO co-localised with αvβ3 expression. Comparison of RGD-targeted and dynamic contrast enhanced (DCE) MRI assessment of tumour perfusion indicated sensitivity to different vascular features. This study demonstrates specific binding of c(RGDyK)-MPIO to αvβ3 expressing neo-vessels, with marked and quantifiable contrast and rapid clearance of unbound particles from the blood circulation compared to NPIO. Combination of this molecular MRI approach with conventional DCE MRI will enable integrated molecular, anatomical and perfusion tumour imaging. PMID:25767618

Distinct Effects of RGD-glycoproteins on Integrin-Mediated Adhesion and Osteogenic Differentiation of Human Mesenchymal Stem Cells

PubMed Central

Schwab, Elisabeth H.; Halbig, Maria; Glenske, Kristina; Wagner, Alena-Svenja; Wenisch, Sabine; Cavalcanti-Adam, Elisabetta A.

2013-01-01

The detailed interactions of mesenchymal stem cells (MSCs) with their extracellular matrix (ECM) and the resulting effects on MSC differentiation are still largely unknown. Integrins are the main mediators of cell-ECM interaction. In this study, we investigated the adhesion of human MSCs to fibronectin, vitronectin and osteopontin, three ECM glycoproteins which contain an integrin-binding sequence, the RGD motif. We then assayed MSCs for their osteogenic commitment in the presence of the different ECM proteins. As early as 2 hours after seeding, human MSCs displayed increased adhesion when plated on fibronectin, whereas no significant difference was observed when adhering either to vitronectin or osteopontin. Over a 10-day observation period, cell proliferation was increased when cells were cultured on fibronectin and osteopontin, albeit after 5 days in culture. The adhesive role of fibronectin was further confirmed by measurements of cell area, which was significantly increased on this type of substrate. However, integrin-mediated clusters, namely focal adhesions, were larger and more mature in MSCs adhering to vitronectin and osteopontin. Adhesion to fibronectin induced elevated expression of α5-integrin, which was further upregulated under osteogenic conditions also for vitronectin and osteopontin. In contrast, during osteogenic differentiation the expression level of β3-integrin was decreased in MSCs adhering to the different ECM proteins. When MSCs were cultured under osteogenic conditions, their commitment to the osteoblast lineage and their ability to form a mineralized matrix in vitro was increased in presence of fibronectin and osteopontin. Taken together these results indicate a distinct role of ECM proteins in regulating cell adhesion, lineage commitment and phenotype of MSCs, which is due to the modulation of the expression of specific integrin subunits during growth or osteogenic differentiation. PMID:24324361

Distinct effects of RGD-glycoproteins on Integrin-mediated adhesion and osteogenic differentiation of human mesenchymal stem cells.

PubMed

Schwab, Elisabeth H; Halbig, Maria; Glenske, Kristina; Wagner, Alena-Svenja; Wenisch, Sabine; Cavalcanti-Adam, Elisabetta A

2013-01-01

The detailed interactions of mesenchymal stem cells (MSCs) with their extracellular matrix (ECM) and the resulting effects on MSC differentiation are still largely unknown. Integrins are the main mediators of cell-ECM interaction. In this study, we investigated the adhesion of human MSCs to fibronectin, vitronectin and osteopontin, three ECM glycoproteins which contain an integrin-binding sequence, the RGD motif. We then assayed MSCs for their osteogenic commitment in the presence of the different ECM proteins. As early as 2 hours after seeding, human MSCs displayed increased adhesion when plated on fibronectin, whereas no significant difference was observed when adhering either to vitronectin or osteopontin. Over a 10-day observation period, cell proliferation was increased when cells were cultured on fibronectin and osteopontin, albeit after 5 days in culture. The adhesive role of fibronectin was further confirmed by measurements of cell area, which was significantly increased on this type of substrate. However, integrin-mediated clusters, namely focal adhesions, were larger and more mature in MSCs adhering to vitronectin and osteopontin. Adhesion to fibronectin induced elevated expression of α₅-integrin, which was further upregulated under osteogenic conditions also for vitronectin and osteopontin. In contrast, during osteogenic differentiation the expression level of β₃-integrin was decreased in MSCs adhering to the different ECM proteins. When MSCs were cultured under osteogenic conditions, their commitment to the osteoblast lineage and their ability to form a mineralized matrix in vitro was increased in presence of fibronectin and osteopontin. Taken together these results indicate a distinct role of ECM proteins in regulating cell adhesion, lineage commitment and phenotype of MSCs, which is due to the modulation of the expression of specific integrin subunits during growth or osteogenic differentiation.

A novel solid lipid nanoparticle formulation for active targeting to tumor α(v) β(3) integrin receptors reveals cyclic RGD as a double-edged sword.

PubMed

Shuhendler, Adam J; Prasad, Preethy; Leung, Michael; Rauth, Andrew M; Dacosta, Ralph S; Wu, Xiao Yu

2012-09-01

The overexpression of α(v) β(3) integrin receptors on tumor cells and tumor vascular endothelium makes it a useful target for imaging, chemotherapy and anti-angiogenic therapy. However integrin-targeted delivery of therapeutics by nanoparticles have provided only marginal, if any, enhancement of therapeutic effect. This work was thus focused on the development of novel α(v) β(3) -targeted near infrared light-emitting solid lipid nanoparticles (SLN) through conjugation to the α(v) β(3) integrin-specific ligand cyclic Arg-Gly-Asp (cRGD), and the assessment of the effects of α(v) β(3) targeting on nanoparticle biodistribution. Since our previously developed non-targeted "stealth" SLN showed little hepatic accumulation, unlike most reported liposomes and micelles, they served as a reference for quantifying the effects of cRGD-conjugation on tumor uptake and whole animal biodistribution of SLN. Non-targeted SLN, actively targeted (RGD-SLN) and blocked RGD-SLN were prepared to contain near infrared quantum dots for live animal imaging. They were injected intravenously to nude mice bearing xenograft orthotopic human breast tumors or dorsal window chamber breast tumors. Tumor micropharmacokinetics of various SLN formulations were determined using intravital microscopy, and whole animal biodistribution was followed over time by optical imaging. The active tumor targeting with cRGD was found to be a "double-edged sword": while the specificity of RGD-SLN accumulation in tumor blood vessels and their tumor residence time increased, their distribution in the liver, spleen, and kidneys was significantly greater than the non-targeted SLN, leaving a smaller amount of nanoparticles in the tumor tissue. Nevertheless the enhanced specificity and retention of RGD-SLN in tumor neovasculature could make this novel formulation useful for tumor neovascular-specific therapies and imaging applications. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Human Metapneumovirus (HMPV) Binding and Infection Are Mediated by Interactions between the HMPV Fusion Protein and Heparan Sulfate

PubMed Central

Chang, Andres; Masante, Cyril; Buchholz, Ursula J.

2012-01-01

Human metapneumovirus (HMPV) is a major worldwide respiratory pathogen that causes acute upper and lower respiratory tract disease. The mechanism by which this virus recognizes and gains access to its target cell is still largely unknown. In this study, we addressed the initial steps in virus binding and infection and found that the first binding partner for HMPV is heparan sulfate (HS). While wild-type CHO-K1 cells are permissive to HMPV infection, mutant cell lines lacking the ability to synthesize glycosaminoglycans (GAGs), specifically, heparan sulfate proteoglycans (HSPGs), were resistant to binding and infection by HMPV. The permissiveness to HMPV infection was also abolished when CHO-K1 cells were treated with heparinases. Importantly, using recombinant HMPV lacking both the G and small hydrophobic (SH) proteins, we report that this first virus-cell binding interaction is driven primarily by the fusion protein (HMPV F) and that this interaction is needed to establish a productive infection. Finally, HMPV binding to cells did not require β1 integrin expression, and RGD-mediated interactions were not essential in promoting HMPV F-mediated cell-to-cell membrane fusion. Cells lacking β1 integrin, however, were less permissive to HMPV infection, indicating that while β1 integrins play an important role in promoting HMPV infection, the interaction between integrins and HMPV occurs after the initial binding of HMPV F to heparan sulfate proteoglycans. PMID:22238303

Carbodiimide versus click chemistry for nanoparticle surface functionalization: a comparative study for the elaboration of multimodal superparamagnetic nanoparticles targeting αvβ3 integrins.

PubMed

Bolley, Julie; Guenin, Erwann; Lievre, Nicole; Lecouvey, Marc; Soussan, Michael; Lalatonne, Yoann; Motte, Laurence

2013-11-26

Superparamagnetic fluorescent nanoparticles targeting αvβ3 integrins were elaborated using two methodologies: carbodiimide coupling and click chemistries (CuACC and thiol-yne). The nanoparticles are first functionalized with hydroxymethylenebisphonates (HMBP) bearing carboxylic acid or alkyne functions. Then, a large number of these reactives functions were used for the covalent coupling of dyes, poly(ethylene glycol) (PEG), and cyclic RGD. Several methods were used to characterize the nanoparticle surface functionalization, and the magnetic properties of these contrast agents were studied using a 1.5 T clinical MRI. The affinity toward integrins was evidenced by solid-phase receptor-binding assay. In addition to their chemoselective natures, click reactions were shown to be far more efficient than the carbodiimide coupling. The grafting increase was shown to enhance targeting affinity to integrin without imparing MRI and fluorescent properties.

PET imaging of αvβ3 integrin expression in tumours with 68Ga-labelled mono-, di- and tetrameric RGD peptides

PubMed Central

Yim, Cheng-Bin; Franssen, Gerben M.; Schuit, Robert C.; Luurtsema, Gert; Liu, Shuang; Oyen, Wim J. G.; Boerman, Otto C.

2010-01-01

Purpose Due to the restricted expression of αvβ3 in tumours, αvβ3 is considered a suitable receptor for tumour targeting. In this study the αvβ3-binding characteristics of 68Ga-labelled monomeric, dimeric and tetrameric RGD peptides were determined and compared with their 111In-labelled counterparts. Methods A monomeric (E-c(RGDfK)), a dimeric (E-[c(RGDfK)]2) and a tetrameric (E{E[c(RGDfK)]2}2) RGD peptide were synthesised, conjugated with DOTA and radiolabelled with 68Ga. In vitro αvβ3-binding characteristics were determined in a competitive binding assay. In vivo αvβ3-targeting characteristics of the compounds were assessed in mice with subcutaneously growing SK-RC-52 xenografts. In addition, microPET images were acquired using a microPET/CT scanner. Results The IC50 values for the Ga(III)-labelled DOTA-E-c(RGDfK), DOTA-E-[c(RGDfK)]2 and DOTA-E{E[c(RGDfK)]2}2 were 23.9 ± 1.22, 8.99 ± 1.20 and 1.74 ± 1.18 nM, respectively, and were similar to those of the In(III)-labelled mono-, di- and tetrameric RGD peptides (26.6 ± 1.15, 3.34 ± 1.16 and 1.80 ± 1.37 nM, respectively). At 2 h post-injection, tumour uptake of the 68Ga-labelled mono-, di- and tetrameric RGD peptides (3.30 ± 0.30, 5.24 ± 0.27 and 7.11 ± 0.67%ID/g, respectively) was comparable to that of their 111In-labelled counterparts (2.70 ± 0.29, 5.61 ± 0.85 and 7.32 ± 2.45%ID/g, respectively). PET scans were in line with the biodistribution data. On all PET scans, the tumour could be clearly visualised. Conclusion The integrin affinity and the tumour uptake followed the order of DOTA-tetramer > DOTA-dimer > DOTA-monomer. The 68Ga-labelled tetrameric RGD peptide has excellent characteristics for imaging of αvβ3 expression with PET. Electronic supplementary material The online version of this article (doi:10.1007/s00259-010-1615-x) contains supplementary material, which is available to authorized users. PMID:20857099

RGD peptide-targeted polyethylenimine-entrapped gold nanoparticles for targeted CT imaging of an orthotopic model of human hepatocellular carcinoma

NASA Astrophysics Data System (ADS)

Zhou, Benqing; Wang, Meng; Zhou, Feifan; Song, Jun; Qu, Junle; Chen, Wei R.

2018-02-01

We report the synthesis and characterization of arginine-glycine-aspartic acid (RGD) peptide-targeted polyethylenimine (PEI)-entrapped gold nanoparticles (RGD-Au PENPs) for targeted CT imaging of hepatic carcinomas in situ. In this work, PEI sequentially modified with polyethylene glycol (PEG), and RGD linked-PEG was used as a nanoplatform to prepare AuNPs, followed by complete acetylation of PEI surface amines. We showed that the designed RGD-Au PENPs were colloidally stable and biocompatible in the given concentration range, and could be specifically taken up by αvβ3 integrin-overexpressing liver cancer cells in vitro. Furthermore, in vivo CT imaging results revealed that the particles displayed a great contrast enhancement of hepatic carcinomas region, and could target to hepatic carcinomas region in situ. With the proven biodistribution and histological examinations in vivo, the synthesized RGD-Au PENPs show a great formulation to be used as a contrast agent for targeted CT imaging of different αvβ3 integrin receptoroverexpressing tumors.

Chemical functionalization of bioceramics to enhance endothelial cells adhesion for tissue engineering.

PubMed

Borcard, Françoise; Staedler, Davide; Comas, Horacio; Juillerat, Franziska Krauss; Sturzenegger, Philip N; Heuberger, Roman; Gonzenbach, Urs T; Juillerat-Jeanneret, Lucienne; Gerber-Lemaire, Sandrine

2012-09-27

To control the selective adhesion of human endothelial cells and human serum proteins to bioceramics of different compositions, a multifunctional ligand containing a cyclic arginine-glycine-aspartate (RGD) peptide, a tetraethylene glycol spacer, and a gallate moiety was designed, synthesized, and characterized. The binding of this ligand to alumina-based, hydroxyapatite-based, and calcium phosphate-based bioceramics was demonstrated. The conjugation of this ligand to the bioceramics induced a decrease in the nonselective and integrin-selective binding of human serum proteins, whereas the binding and adhesion of human endothelial cells was enhanced, dependent on the particular bioceramics.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Steiner, B.; Cousot, D.; Trzeciak, A.

The platelet glycoprotein IIb-IIIa complex (GP IIb-IIIa) is a member of the integrin receptor family that recognizes adhesive proteins containing the Arg-Gly-Asp (RGD) sequence. In the present study the binding characteristics of the synthetic hexapeptide Tyr-Asn-Arg-Gly-Asp-Ser (YNRGDS, a sequence present in the fibrinogen alpha-chain at position 570-575) to purified GP IIb-IIIa were determined by equilibrium dialysis. The binding of 125I-YNRGDS to GP IIb-IIIa was specific, saturable, and reversible. The apparent dissociation constant was 1.0 +/- 0.2 microM, and the maximal binding capacity was 0.92 +/- 0.02 mol of 125I-YNRGDS/mol of GP IIb-IIIa, indicating that GP IIb-IIIa contains a single bindingmore » site for RGD peptides. The binding of 125I-YNRGDS to purified GP IIb-IIIa showed many of the characteristics of fibrinogen binding to activated platelets: the binding was inhibited by fibrinogen, by the monoclonal antibody A2A9, and by the dodecapeptide from the C terminus of the fibrinogen gamma-chain. In addition, the binding of 125I-YNRGDS to GP IIb-IIIa was divalent cation-dependent. Our data suggest that two divalent cation binding sites must be occupied for YNRGDS to bind: one site is specific for calcium and is saturated at 1 microM free Ca2+, whereas the other site is less specific and reaches saturation at millimolar concentrations of either Ca2+ or Mg2+. The results of the present study support the hypothesis that the RGD domains within the adhesive proteins are responsible for their binding to GP IIb-IIIa.« less

RGD-functionalized ultrasmall iron oxide nanoparticles for targeted T1-weighted MR imaging of gliomas

NASA Astrophysics Data System (ADS)

Luo, Yu; Yang, Jia; Yan, Yu; Li, Jingchao; Shen, Mingwu; Zhang, Guixiang; Mignani, Serge; Shi, Xiangyang

2015-08-01

We report a convenient approach to prepare ultrasmall Fe3O4 nanoparticles (NPs) functionalized with an arginylglycylaspartic acid (RGD) peptide for in vitro and in vivo magnetic resonance (MR) imaging of gliomas. In our work, stable sodium citrate-stabilized Fe3O4 NPs were prepared by a solvothermal route. Then, the carboxylated Fe3O4 NPs stabilized with sodium citrate were conjugated with polyethylene glycol (PEG)-linked RGD. The formed ultrasmall RGD-functionalized nanoprobe (Fe3O4-PEG-RGD) was fully characterized using different techniques. We show that these Fe3O4-PEG-RGD particles with a size of 2.7 nm are water-dispersible, stable, cytocompatible and hemocompatible in a given concentration range, and display targeting specificity to glioma cells overexpressing αvβ3 integrin in vitro. With the relatively high r1 relaxivity (r1 = 1.4 mM-1 s-1), the Fe3O4-PEG-RGD particles can be used as an efficient nanoprobe for targeted T1-weighted positive MR imaging of glioma cells in vitro and the xenografted tumor model in vivo via an active RGD-mediated targeting pathway. The developed RGD-functionalized Fe3O4 NPs may hold great promise to be used as a nanoprobe for targeted T1-weighted MR imaging of different αvβ3 integrin-overexpressing cancer cells or biological systems.We report a convenient approach to prepare ultrasmall Fe3O4 nanoparticles (NPs) functionalized with an arginylglycylaspartic acid (RGD) peptide for in vitro and in vivo magnetic resonance (MR) imaging of gliomas. In our work, stable sodium citrate-stabilized Fe3O4 NPs were prepared by a solvothermal route. Then, the carboxylated Fe3O4 NPs stabilized with sodium citrate were conjugated with polyethylene glycol (PEG)-linked RGD. The formed ultrasmall RGD-functionalized nanoprobe (Fe3O4-PEG-RGD) was fully characterized using different techniques. We show that these Fe3O4-PEG-RGD particles with a size of 2.7 nm are water-dispersible, stable, cytocompatible and hemocompatible in a given concentration range, and display targeting specificity to glioma cells overexpressing αvβ3 integrin in vitro. With the relatively high r1 relaxivity (r1 = 1.4 mM-1 s-1), the Fe3O4-PEG-RGD particles can be used as an efficient nanoprobe for targeted T1-weighted positive MR imaging of glioma cells in vitro and the xenografted tumor model in vivo via an active RGD-mediated targeting pathway. The developed RGD-functionalized Fe3O4 NPs may hold great promise to be used as a nanoprobe for targeted T1-weighted MR imaging of different αvβ3 integrin-overexpressing cancer cells or biological systems. Electronic supplementary information (ESI) available: Additional experimental results. See DOI: 10.1039/c5nr04003e

Preparation and evaluation of a 68Ga-labeled RGD-containing octapeptide for noninvasive imaging of angiogenesis: biodistribution in non-human primate

PubMed Central

Velikyan, Irina; Lindhe, Örjan

2018-01-01

Monitoring general disease marker such as angiogenesis may contribute to the development of personalized medicine and improve therapy outcome. Readily availability of positron emitter based imaging agents providing quantification would expand clinical positron emission tomography (PET) applications. Generator produced 68Ga provides PET images of high resolution and the half-life time frame is compatible with the pharmacokinetics of small peptides comprising arginine-glycine-aspartic acid (RGD) sequence specific to αvβ3 integrin receptors. The main objective of this study was to develop a method for 68Ga-labeling of RGD containing bicyclic octapeptide ([68Ga]Ga-DOTA-RGD) with high specific radioactivity and preclinically assess its imaging potential. DOTA-RGD was labeled using generator eluate preconcentration technique and microwave heating. The binding and organ distribution properties of [68Ga]Ga-DOTA-RGD were tested in vitro by autoradiography of frozen tumor sections, and in vivo in mice carrying a Lewis Lung carcinoma graft (LL2), and in non-human primate (NHP). Another peptide with aspartic acid-glycine-phenylalanine sequence was used as a negative control. The full 68Ga radioactivity eluted from two generators was quantitatively incorporated into 3-8 nanomoles of the peptide conjugates. The target binding specificity was confirmed by blocking experiments. The specific uptake in the LL2 mice model was observed in vivo and confirmed in the corresponding ex vivo biodistribution experiments. Increased accumulation of the radioactivity was detected in the wall of the uterus of the female NHP probably indicating neovascularization. [68Ga]Ga-DOTA-RGD demonstrated potential for the imaging of angiogenesis. PMID:29531858

VE-cadherin RGD motifs promote metastasis and constitute a potential therapeutic target in melanoma and breast cancers.

PubMed

Bartolomé, Rubén A; Torres, Sofía; Isern de Val, Soledad; Escudero-Paniagua, Beatriz; Calviño, Eva; Teixidó, Joaquín; Casal, J Ignacio

2017-01-03

We have investigated the role of vascular-endothelial (VE)-cadherin in melanoma and breast cancer metastasis. We found that VE-cadherin is expressed in highly aggressive melanoma and breast cancer cell lines. Remarkably, inactivation of VE-cadherin triggered a significant loss of malignant traits (proliferation, adhesion, invasion and transendothelial migration) in melanoma and breast cancer cells. These effects, except transendothelial migration, were induced by the VE-cadherin RGD motifs. Co-immunoprecipitation experiments demonstrated an interaction between VE-cadherin and α2β1 integrin, with the RGD motifs found to directly affect β1 integrin activation. VE-cadherin-mediated integrin signaling occurred through specific activation of SRC, ERK and JNK, including AKT in melanoma. Knocking down VE-cadherin suppressed lung colonization capacity of melanoma or breast cancer cells inoculated in mice, while pre-incubation with VE-cadherin RGD peptides promoted lung metastasis for both cancer types. Finally, an in silico study revealed the association of high VE-cadherin expression with poor survival in a subset of melanoma patients and breast cancer patients showing low CD34 expression. These findings support a general role for VE-cadherin and other RGD cadherins as critical regulators of lung and liver metastasis in multiple solid tumours. These results pave the way for cadherin-specific RGD targeted therapies to control disseminated metastasis in multiple cancers.

Is vitronectin the velcro that binds the gametes together?

PubMed

Fusi, F M; Bernocchi, N; Ferrari, A; Bronson, R A

1996-11-01

Evidence has been presented that the adhesion of human spermatozoa to the oolemma is mediated by integrins recognizing the Arg-Gly-Asp sequence (RGD). Fibronectin and vitronectin, glycoproteins that contain functional RGD sequences, are both present on human spermatozoa, and integrins that recognize these ligands have been detected on spermatozoa and eggs. In this work, we studied the effects of oligopeptides specifically designed to block fibronectin or vitronectin receptors on the interaction of human spermatozoa with zona-free hamster oocytes. GRGDdSP, a peptide blocking cell attachment to fibronectin, was without effect, while GdRGDSP, which blocks both fibronectin and vitronectin receptors, significantly inhibited the binding of human spermatozoa to the oolemma of zona-free hamster eggs, in a concentration-dependent manner, over a range 1-100 microM. As these experiments suggested that a vitronectin receptor plays a role in sperm-oolemmal adhesion, we performed a series of experiments studying the effects of exogenous vitronectin, when added to spermatozoa and oocytes, on gamete interactions. Sperm-oolemmal adherence, as well as sperm aggregation, was promoted by vitronectin, over range of 2.2 nM to 1 microM, but only in the presence of calcium ions. We propose that vitronectin released during the sperm acrosome reaction is recognized by both gametes and plays a role in their adhesion.

Arginylglycylaspartic Acid-Surface-Functionalized Doxorubicin-Loaded Lipid-Core Nanocapsules as a Strategy to Target Alpha(V) Beta(3) Integrin Expressed on Tumor Cells

PubMed Central

Antonow, Michelli B.; Franco, Camila; Prado, Willian; Beckenkamp, Aline; Silveira, Gustavo P.; Buffon, Andréia; Guterres, Sílvia S.

2017-01-01

Doxorubicin (Dox) clinical use is limited by dose-related cardiomyopathy, becoming more prevalent with increasing cumulative doses. Previously, we developed Dox-loaded lipid-core nanocapsules (Dox-LNC) and, in this study, we hypothesized that self-assembling and interfacial reactions could be used to obtain arginylglycylaspartic acid (RGD)-surface-functionalized-Dox-LNC, which could target tumoral cells overexpressing αvβ3 integrin. Human breast adenocarcinoma cell line (MCF-7) and human glioblastoma astrocytoma (U87MG) expressing different levels of αvβ3 integrin were studied. RGD-functionalized Dox-LNC were prepared with Dox at 100 and 500 mg·mL−1 (RGD-MCMN (Dox100) and RGD-MCMN (Dox500)). Blank formulation (RGD-MCMN) had z-average diameter of 162 ± 6 nm, polydispersity index of 0.11 ± 0.04, zeta potential of +13.2 ± 1.9 mV and (6.2 ± 1.1) × 1011 particles mL−1, while RGD-MCMN (Dox100) and RGD-MCMN (Dox500) showed respectively 146 ± 20 and 215 ± 25 nm, 0.10 ± 0.01 and 0.09 ± 0.03, +13.8 ± 2.3 and +16.4 ± 1.5 mV and (6.9 ± 0.6) × 1011 and (6.1 ± 1.0) × 1011 particles mL−1. RGD complexation was 7.73 × 104 molecules per nanocapsule and Dox loading were 1.51 × 104 and 7.64 × 104 molecules per nanocapsule, respectively. RGD-functionalized nanocapsules had an improved uptake capacity by U87MG cells. Pareto chart showed that the cell viability was mainly affected by the Dox concentration and the period of treatment in both MCF-7 and U87MG. The influence of RGD-functionalization on cell viability was a determinant factor exclusively to U87MG. PMID:29271920

Effects on in vitro and in vivo angiogenesis induced by small peptides carrying adhesion sequences.

PubMed

Conconi, Maria Teresa; Ghezzo, Francesca; Dettin, Monica; Urbani, Luca; Grandi, Claudio; Guidolin, Diego; Nico, Beatrice; Di Bello, Carlo; Ribatti, Domenico; Parnigotto, Pier Paolo

2010-07-01

It is well known that tumor growth is strictly dependent on neo-vessel formation inside the tumor mass and that cell adhesion is required to allow EC proliferation and migration inside the tumor. In this work, we have evaluated the in vitro and in vivo effects on angiogenesis of some peptides, originally designed to promote cell adhesion on biomaterials, containing RGD motif mediating cell adhesion via integrin receptors [RGD, GRGDSPK, and (GRGDSP)(4)K] or the heparin-binding sequence of human vitronectin that interacts with HSPGs [HVP(351-359)]. Cell adhesion, proliferation, migration, and capillary-like tube formation in Matrigel were determined on HUVECs, whereas the effects on in vivo angiogenesis were evaluated using the CAM assay. (GRGDSP)(4)K linear sequence inhibited cell adhesion, decreased cell proliferation, migration and morphogenesis in Matrigel, and induced anti-angiogenic responses on CAM at higher degree than that determined after incubation with RGD or GRGDSPK. Moreover, it counteracted both in vitro and in vivo the pro-angiogenic effects induced by the Fibroblast growth factor (FGF-2). On the other hand, HVP was not able to affect cell adhesion and appeared less effective than (GRGDSP)(4)K. Our data indicate that the activity of RGD-containing peptides is related to their adhesive properties, and their effects are modulated by the number of cell adhesion motifs and the aminoacidic residues next to these sequences. The anti-angiogenic properties of (GRGDSP)(4)K seem to depend on its interaction with integrins, whereas the effects of HVP may be partially due to an impairment of HSPGs/FGF-2.

(18)F-glyco-RGD peptides for PET imaging of integrin expression: efficient radiosynthesis by click chemistry and modulation of biodistribution by glycosylation.

PubMed

Maschauer, Simone; Haubner, Roland; Kuwert, Torsten; Prante, Olaf

2014-02-03

Glycosylation frequently improves the biokinetics and clearance properties of macromolecules in vivo and could therefore be used for the design of radiopharmaceuticals for positron emission tomography (PET). Recently, we have developed a click chemistry method for (18)F-fluoroglycosylation of alkyne-bearing RGD-peptides targeting the integrin receptor. To investigate whether this strategy could yield an (18)F-labeled RGD glycopeptide with favorable biokinetics, we generated a series of new RGD glycopeptides, varying the 6-fluoroglycosyl residue from monosaccharide to disaccharide units, which provided the glucosyl ([(19)F]6Glc-RGD, 4b), galactosyl ([(19)F]Gal-RGD, 4c), maltosyl ([(19)F]Mlt-RGD, 4e), and cellobiosyl ([(19)F]Cel-RGD, 4f) conjugated peptides in high yields and purities of >97%. All of these RGD glycopeptides showed high affinity to αvβ3 (11-55 nM), αvβ5 (6-14 nM), and to αvβ3-positive U87MG cells (90-395 nM). (18)F-labeling of the various carbohydrate precursors (1a-f) using cryptate-assisted reaction conditions (CH3CN, 85 °C, 10 min) gave (18)F-labeled glycosyl azides in radiochemical yields (RCYs) of up to 84% ([(18)F]2b). The deacetylation and subsequent click reaction with the alkyne-bearing cyclic RGD peptide proceeded in one-pot reactions with RCYs as high as 81% in 15-20 min at 60 °C, using a minimal amount of peptide precursor (100 nmol). Optimization of the radiosynthesis strategy gave a decay-uncorrected RCY of 16-24% after 70-75 min (based on [(18)F]fluoride). Due to their high-yield radiosyntheses, the glycopeptides [(18)F]6Glc-RGD and [(18)F]Mlt-RGD were chosen for comparative biodistribution studies and dynamic small-animal PET imaging using U87MG tumor-bearing nude mice. [(18)F]6Glc-RGD and [(18)F]Mlt-RGD showed significantly decreased liver and kidney uptake by PET relative to the 2-[(18)F]fluoroglucosyl analog [(18)F]2Glc-RGD, and showed specific tumor uptake in vivo. Notably, [(18)F]Mlt-RGD revealed uptake and retention in the U87MG tumor comparable to that of [(18)F]Galacto-RGD. Both [(18)F]6Glc-RGD and [(18)F]Mlt-RGD were obtained by a reliable and easy click chemistry-based procedure, much more rapidly than was [(18)F]Galacto-RGD. Due to its favorable biodistribution and tissue clearance in vivo, [(18)F]Mlt-RGD represents a viable alternative radiotracer for imaging integrin expression in solid tumors by PET.

Connection between integrins and cell activation in rat adrenal glomerulosa cells: a role for Arg-Gly-Asp peptide in the activation of the p42/p44(mapk) pathway and intracellular calcium.

PubMed

Campbell, Shirley; Otis, Melissa; Côté, Mylène; Gallo-Payet, Nicole; Payet, Marcel Daniel

2003-04-01

Integrins are responsible for adhesion and activation of several intracellular cascades. The present study was aimed at determining whether the interaction between fibronectin and integrins could generate pathways involved in physiological functions of rat adrenal glomerulosa cells. Immunofluorescence studies and adhesion assays showed that fibronectin was the best matrix in promoting the formation of focal adhesion. Binding of glomerulosa cells to fibronectin, but not to collagen I or poly-L-lysine, involved the integrin-binding sequence Arg-Gly-Asp (RGD). Activation of glomerulosa cells with Arg-Gly-Asp-Ser (RGDS) induced an increase in [Ca(2+)](i), whereas fibronectin triggered a release of Ca(2+) from InsP(3)-sensitive Ca(2+) stores. Aldosterone secretion induced by ACTH, angiotensin II, and RGDS and proliferation were improved on fibronectin, compared with poly-L-lysine. The RGDS peptide induced a transient increase in the activity of the p42/p44(mapk), independent of phosphatidylinositol-3 kinase and protein kinase C. Integrins alpha(5) and alpha(V) as well as their fibronectin receptor partners beta(1) and beta(3), were identified. These results suggest that in rat adrenal glomerulosa cells, binding of the alpha(5)beta(1), alpha(v)beta(1), or alpha(v)beta(3) integrins to fibronectin is involved in the generation of two important signaling events, increase in intracellular calcium, and activation of the p42/p44(mapk) cascade, leading to cell proliferation and aldosterone secretion.

Polymerisation of fibrin αC-domains promotes endothelial cell migration and proliferation.

PubMed

Yakovlev, S; Mikhailenko, I; Tsurupa, G; Belkin, A M; Medved, L

2014-12-01

Upon conversion of fibrinogen into fibrin, fibrinogen αC-domains containing the RGD recognition motif form ordered αC polymers. Our previous study revealed that polymerisation of these domains promotes integrin-dependent adhesion and spreading of endothelial cells, as well as integrin-mediated activation of the FAK and ERK1/2 signalling pathways. The major goal of this study was to test the impact of αC-domain polymerisation on endothelial cell migration and proliferation during wound healing, and to clarify the mechanism underlying superior activity of αC polymers toward endothelial cells. In an in vitro wound healing assay, confluent endothelial cell monolayers on tissue culture plates coated with the αC monomer or αC polymers were wounded by scratching and wound closure was monitored by time-lapse videomicroscopy. Although the plates were coated with equal amounts of αC species, as confirmed by ELISA, wound closure by the cells occurred much faster on αC polymers, indicating that αC-domain polymerisation promotes cell migration and proliferation. In agreement, endothelial cell proliferation was also more efficient on αC polymers, as revealed by cell proliferation assay. Wound closure on both types of substrates was equally inhibited by the integrin-blocking GRGDSP peptide and a specific antagonist of the ERK1/2 signalling pathway. In contrast, blocking the FAK signaling pathway by a specific antagonist decreased wound closure only on αC polymers. These results indicate that polymerisation of the αC-domains enhances integrin-dependent endothelial cell migration and proliferation mainly through the FAK signalling pathway. Furthermore, clustering of integrin-binding RGD motifs in αC polymers is the major mechanism triggering these events.

Density-tunable conjugation of cyclic RGD ligands with polyion complex vesicles for the neovascular imaging of orthotopic glioblastomas

NASA Astrophysics Data System (ADS)

Kawamura, Wataru; Miura, Yutaka; Kokuryo, Daisuke; Toh, Kazuko; Yamada, Naoki; Nomoto, Takahiro; Matsumoto, Yu; Sueyoshi, Daiki; Liu, Xueying; Aoki, Ichio; Kano, Mitsunobu R.; Nishiyama, Nobuhiro; Saga, Tsuneo; Kishimura, Akihiro; Kataoka, Kazunori

2015-06-01

Introduction of ligands into 100 nm scaled hollow capsules has great potential for diagnostic and therapeutic applications in drug delivery systems. Polyethylene glycol-conjugated (PEGylated) polyion complex vesicles (PICsomes) are promising hollow nano-capsules that can survive for long periods in the blood circulation and can be used to deliver water-soluble macromolecules to target tissues. In this study, cyclic RGD (cRGD) peptide, which is specifically recognized by αVβ3 and αvβ5 integrins that are expressed at high levels in the neovascular system, was conjugated onto the distal end of PEG strands on PICsomes for active neovascular targeting. Density-tunable cRGD-conjugation was achieved using PICsomes with definite fraction of end-functionalized PEG, to substitute 20, 40, and 100% of PEG distal end of the PICsomes to cRGD moieties. Compared with control-PICsomes without cRGD, cRGD-PICsomes exhibited increased uptake into human umbilical vein endothelial cells. Intravital confocal laser scanning microscopy revealed that the 40%-cRGD-PICsomes accumulated mainly in the tumor neovasculature and remained in the perivascular region even after 24 h. Furthermore, we prepared superparamagnetic iron oxide (SPIO)-loaded cRGD-PICsomes for magnetic resonance imaging (MRI) and successfully visualized the neovasculature in an orthotopic glioblastoma model, which suggests that SPIO-loaded cRGD-PICsomes might be useful as a MRI contrast reagent for imaging of the tumor microenvironment, including neovascular regions that overexpress αVβ3 integrins.

Tumor targeting RGD conjugated bio-reducible polymer for VEGF siRNA expressing plasmid delivery

PubMed Central

Kim, Hyun Ah; Nam, Kihoon; Kim, Sung Wan

2014-01-01

Targeted delivery of therapeutic genes to the tumor site is critical for successful and safe cancer gene therapy. The arginine grafted bio-reducible poly (cystamine bisacrylamide-diaminohexane, CBA-DAH) polymer (ABP) conjugated poly (amido amine) (PAMAM), PAM-ABP (PA) was designed previously as an efficient gene delivery carrier. To achieve high efficacy in cancer selective delivery, we developed the tumor targeting bio-reducible polymer, PA-PEG1k-RGD, by conjugating cyclic RGDfC (RGD) peptides, which bind αvβ3/5 integrins, to the PAM-ABP using polyethylene glycol (PEG,1kDa) as a spacer. Physical characterization showed nanocomplex formation with bio-reducible properties between PA-PEG1k-RGD and plasmid DNA (pDNA). In transfection assays, PA-PEG1k-RGD showed significantly higher transfection efficiency in comparison with PAM-ABP or PA-PEG1k-RGD in αvβ3/5 positive MCF7 breast cancer and PANC-1 pancreatic cancer cells. The targeting ability of PA-PEG1k-RGD was further established using a competition assay. To confirm the therapeutic effect, the VEGF siRNA expressing plasmid was constructed and then delivered into cancer cells using PA-PEG1k-RGD. PA-PEG1k-RGD showed 20-59% higher cellular uptake rate into MCF7 and PANC-1 than that of non-targeted polymers. In addition, MCF7 and PANC-1 cancer cells transfected with PA-PEG1k-RGD/pshVEGF complexes had significantly decreased VEGF gene expression (51-71%) and cancer cell viability (35-43%) compared with control. These results demonstrate that a tumor targeting bio-reducible polymer with an anti-angiogenic therapeutic gene could be used for efficient and safe cancer gene therapy. PMID:24894645

A cyclic-RGD-BioShuttle functionalized with TMZ by DARinv “Click Chemistry” targeted to αvβ3 integrin for therapy

PubMed Central

Braun, Klaus; Wiessler, Manfred; Pipkorn, Rüdiger; Ehemann, Volker; Bäuerle, Tobias; Fleischhacker, Heinz; Müller, Gabriele; Lorenz, Peter; Waldeck, Waldemar

2010-01-01

Clinical experiences often document, that a successful tumor control requires high doses of drug applications. It is widely believed that unavoidable adverse reactions could be minimized by using gene-therapeutic strategies protecting the tumor-surrounding healthy tissue as well as the bone-marrow. One new approach in this direction is the use of “Targeted Therapies” realizing a selective drug targeting to gain effectual amounts at the target site, even with drastically reduced application doses. MCF-7 breast cancer cells expressing the αvβ3 [alpha(v)beta(3)] integrin receptor are considered as appropriate candidates for such a targeted therapy. The modularly composed BioShuttle carrier consisting of different units designed to facilitate the passage across the cell membranes and for subcellular addressing of diagnostic and/or therapeutic molecules could be considered as an eligible delivery platform. Here we used the cyclic RGD-BioShuttle as a carrier for temozolomide (TMZ) at the αvβ3 integrin receptor realizing local TMZ concentrations sufficient for cell killing. The IC50 values are 12 µMol/L in the case of cRGD-BioShuttle-TMZ and 100 µMol/L for underivatized TMZ, which confirms the advantage of TMZ reformulation to realize local concentrations sufficient for cell killing. Our paper focuses on the design, synthesis and application of the cRGD-BioShuttle conjugate composed of the cyclic RGD, a αvβ3 integrin-ligand, ligated to the cytotoxic drug TMZ. The ligation was carried out by the Diels Alder Reaction with inverse electron demand (DARinv). PMID:20922134

The Dentin Sialoprotein (DSP) Domain Regulates Dental Mesenchymal Cell Differentiation through a Novel Surface Receptor

PubMed Central

Wan, Chunyan; Yuan, Guohua; Luo, Daoshu; Zhang, Lu; Lin, Heng; Liu, Huan; Chen, Lei; Yang, Guobin; Chen, Shuo; Chen, Zhi

2016-01-01

Dentin sialophosphoprotein (DSPP) is a dentin extracellular matrix protein that is processed into dentin sialoprotein (DSP), dentin glycoprotein (DGP) and dentin phosphoprotein (DPP). DSP is mainly expressed in odontoblasts. We hypothesized that DSP interacts with cell surface receptors and subsequently activates intracellular signaling. Using DSP as bait for screening a protein library, we demonstrate that DSP acts as a ligand and binds to integrin β6. The 36 amino acid residues of DSP are sufficient to bind to integrin β6. This peptide promoted cell attachment, migration, differentiation and mineralization of dental mesenchymal cells. In addition, DSP aa183-219 stimulated phosphorylation of ERK1/2 and P38 kinases. This activation was inhibited by an anti-integrin β6 antibody and siRNA. Furthermore, we demonstrate that this DSP fragment induces SMAD1/5/8 phosphorylation and nuclear translocation via ERK1/2 and P38 signaling. SMAD1/5/8 binds to SMAD binding elements (SBEs) in the DSPP gene promoter. SBE mutations result in a decrease in DSPP transcriptional activity. Endogenous DSPP expression was up-regulated by DSP aa183-219 in dental mesenchymal cells. The data in the current study demonstrate for the first time that this DSP domain acts as a ligand in a RGD-independent manner and is involved in intracellular signaling via interacting with integrin β6. The DSP domain regulates DSPP expression and odontoblast homeostasis via a positive feedback loop. PMID:27430624

The Dentin Sialoprotein (DSP) Domain Regulates Dental Mesenchymal Cell Differentiation through a Novel Surface Receptor.

PubMed

Wan, Chunyan; Yuan, Guohua; Luo, Daoshu; Zhang, Lu; Lin, Heng; Liu, Huan; Chen, Lei; Yang, Guobin; Chen, Shuo; Chen, Zhi

2016-07-19

Dentin sialophosphoprotein (DSPP) is a dentin extracellular matrix protein that is processed into dentin sialoprotein (DSP), dentin glycoprotein (DGP) and dentin phosphoprotein (DPP). DSP is mainly expressed in odontoblasts. We hypothesized that DSP interacts with cell surface receptors and subsequently activates intracellular signaling. Using DSP as bait for screening a protein library, we demonstrate that DSP acts as a ligand and binds to integrin β6. The 36 amino acid residues of DSP are sufficient to bind to integrin β6. This peptide promoted cell attachment, migration, differentiation and mineralization of dental mesenchymal cells. In addition, DSP (aa183-219) stimulated phosphorylation of ERK1/2 and P38 kinases. This activation was inhibited by an anti-integrin β6 antibody and siRNA. Furthermore, we demonstrate that this DSP fragment induces SMAD1/5/8 phosphorylation and nuclear translocation via ERK1/2 and P38 signaling. SMAD1/5/8 binds to SMAD binding elements (SBEs) in the DSPP gene promoter. SBE mutations result in a decrease in DSPP transcriptional activity. Endogenous DSPP expression was up-regulated by DSP (aa183-219) in dental mesenchymal cells. The data in the current study demonstrate for the first time that this DSP domain acts as a ligand in a RGD-independent manner and is involved in intracellular signaling via interacting with integrin β6. The DSP domain regulates DSPP expression and odontoblast homeostasis via a positive feedback loop.

Dual targeting of integrin αvβ3 and matrix metalloproteinase-2 for optical imaging of tumors and chemotherapeutic delivery

PubMed Central

Crisp, Jessica L.; Savariar, Elamprakash N.; Glasgow, Heather L.; Ellies, Lesley G.; Whitney, Michael A.; Tsien, Roger. Y.

2014-01-01

Activatable cell penetrating peptides (ACPPs) provide a general strategy for molecular targeting by exploiting the extracellular protease activities associated with disease. Previous work used a matrix metalloproteinase (MMP-2 and 9) cleavable sequence in the ACPP to target contrast agents for tumor imaging and fluorescence guided surgery. To improve specificity and sensitivity for MMP-2, an integrin αvβ3 binding domain, cyclic-RGD, was covalently linked to the ACPP. This co-targeting strategy relies on the interaction of MMP-2 with integrin αvβ3, which are known to associate via MMP-2’s hemopexin domain. In U87MG glioblastoma cells in culture, dual targeting greatly improved ACPP uptake compared to either MMP or integrin αvβ3 targeting alone. In vivo, dual-targeted ACPP treatment resulted in tumor contrast of 7.8±1.6, a 10 fold higher tumor fluorescence compared to the negative control peptide, and increased probe penetration into the core of MDA-MB-231 tumors. This platform also significantly improved efficacy of the chemotherapeutic monomethylauristatin E (MMAE) in both MDA-MB-231 orthotopic human and syngeneic Py230 murine breast tumors. Treatment with cyclic-RGD-PLGC(Me)AG-MMAE-ACPP resulted in complete tumor regression in one quarter of MDA-MB-231 tumor bearing mice, compared to no survival in the control groups. This rational mechanism for amplified delivery of imaging and potent chemotherapeutic agents avoids the use of antibodies and may be of considerable generality. PMID:24737028

Dual targeting of integrin αvβ3 and matrix metalloproteinase-2 for optical imaging of tumors and chemotherapeutic delivery.

PubMed

Crisp, Jessica L; Savariar, Elamprakash N; Glasgow, Heather L; Ellies, Lesley G; Whitney, Michael A; Tsien, Roger Y

2014-06-01

Activatable cell-penetrating peptides (ACPP) provide a general strategy for molecular targeting by exploiting the extracellular protease activities associated with disease. Previous work used a matrix metalloproteinase (MMP-2 and 9)-cleavable sequence in the ACPP to target contrast agents for tumor imaging and fluorescence-guided surgery. To improve specificity and sensitivity for MMP-2, an integrin α(v)β(3)-binding domain, cyclic-RGD, was covalently linked to the ACPP. This co-targeting strategy relies on the interaction of MMP-2 with integrin α(v)β(3), which are known to associate via the hemopexin domain of MMP-2. In U87MG glioblastoma cells in culture, dual targeting greatly improved ACPP uptake compared with either MMP or integrin α(v)β(3) targeting alone. In vivo, dual-targeted ACPP treatment resulted in tumor contrast of 7.8 ± 1.6, a 10-fold higher tumor fluorescence compared with the negative control peptide, and increased probe penetration into the core of MDA-MB-231 tumors. This platform also significantly improved efficacy of the chemotherapeutic monomethylauristatin E (MMAE) in both MDA-MB-231 orthotopic human and syngeneic Py230 murine breast tumors. Treatment with cyclic-RGD-PLGC(Me)AG-MMAE-ACPP resulted in complete tumor regression in one quarter of MDA-MB-231 tumor-bearing mice, compared with no survival in the control groups. This rational mechanism for amplified delivery of imaging and potent chemotherapeutic agents avoids the use of antibodies and may be of considerable generality. ©2014 American Association for Cancer Research.

Multiple alpha subunits of integrin are involved in cell-mediated responses of the Manduca immune system.

PubMed

Zhuang, Shufei; Kelo, Lisha; Nardi, James B; Kanost, Michael R

2008-01-01

The cell-mediated responses of the insect innate immune system-phagocytosis, nodulation, encapsulation-involve multiple cell adhesion molecules of hemocyte surfaces. A hemocyte-specific (HS) integrin and a member of the immunoglobulin (Ig) superfamily (neuroglian) are involved in the encapsulation response of hemocytes in Manduca sexta. In addition, two new integrin alpha (alpha) subunits have been found on these hemocytes. The alpha2 subunit is mainly expressed in epidermis and Malphigian tubules, whereas the alpha3 subunit is primarily expressed on hemocytes and fat body cells. Of the three known alpha subunits, the alpha1 subunit found in HS integrin is the predominant subunit of hemocytes. Cell adhesion assays indicate that alpha2 belongs to the integrin family with RGD-binding motifs, confirming the phylogenetic analysis of alpha subunits based on the amino-acid sequence alignment of different alpha subunits. Double-stranded RNAs (dsRNAs) targeting each of these three integrin alpha subunits not only specifically decreased transcript expression of each alpha subunit in hemocytes, but also abolished the cell-mediated encapsulation response of hemocytes to foreign surfaces. The individual alpha subunits of M. sexta integrins, like their integrin counterparts in mammalian immune systems, have critical, individual roles in cell-substrate and cell-cell interactions during immune responses.

Activatable iRGD-based peptide monolith: Targeting, internalization, and fluorescence activation for precise tumor imaging.

PubMed

Cho, Hong-Jun; Lee, Sung-Jin; Park, Sung-Jun; Paik, Chang H; Lee, Sang-Myung; Kim, Sehoon; Lee, Yoon-Sik

2016-09-10

A disulfide-bridged cyclic RGD peptide, named iRGD (internalizing RGD, c(CRGDK/RGPD/EC)), is known to facilitate tumor targeting as well as tissue penetration. After the RGD motif-induced targeting on αv integrins expressed near tumor tissue, iRGD encounters proteolytic cleavage to expose the CendR motif that promotes penetration into cancer cells via the interaction with neuropilin-1. Based on these proteolytic cleavage and internalization mechanism, we designed an iRGD-based monolithic imaging probe that integrates multiple functions (cancer-specific targeting, internalization and fluorescence activation) within a small peptide framework. To provide the capability of activatable fluorescence signaling, we conjugated a fluorescent dye to the N-terminal of iRGD, which was linked to the internalizing sequence (CendR motif), and a quencher to the opposite C-terminal. It turned out that fluorescence activation of the dye/quencher-conjugated monolithic peptide probe requires dual (reductive and proteolytic) cleavages on both disulfide and amide bond of iRGD peptide. Furthermore, the cleavage of the iRGD peptide leading to fluorescence recovery was indeed operative depending on the tumor-related angiogenic receptors (αvβ3 integrin and neuropilin-1) in vitro as well as in vivo. Compared to an 'always fluorescent' iRGD control probe without quencher conjugation, the dye/quencher-conjugated activatable monolithic peptide probe visualized tumor regions more precisely with lower background noise after intravenous injection, owing to the multifunctional responses specific to tumor microenvironment. All these results, along with minimal in vitro and in vivo toxicity profiles, suggest potential of the iRGD-based activatable monolithic peptide probe as a promising imaging agent for precise tumor diagnosis. Copyright © 2016 Elsevier B.V. All rights reserved.

Self assembling bioactive materials for cell adhesion in tissue repair

NASA Astrophysics Data System (ADS)

Hwang, Julia J.

This work involved the study of biodegradable and biocompatible materials that have the potential to modify tissue engineering scaffolds through self assembly, generating multiple layers that deliver bioactivity. Diblock biomaterials containing cholesteryl moieties and oligomers of lactic acid units were found to form single crystals when precipitated from hot ethanol and smectic liquid crystalline phases when cast as a film. Cell culture experiments on these films with 3T3 and 3T6 fibroblasts indicated that these ordered materials form surfaces with specific chemistries that favored cell adhesion, spreading, and proliferation suggesting the potential of mediating human tissue repair. The author believes the cholesteryl moieties found on the surface play a key role in determining cell behavior. Cholesteryl-(L-lactic acid) diblock molecules were then functionalized with moieties including vitamin Bx, cholesterol, and the anti-inflammatory drug indomethacin. An unstable activated ester between indomethacin and the diblock molecule resulted in the release of indomethacin into the culture medium which inhibited the proliferation of 3T3 fibroblasts. Finally, a series of molecules were designed to incorporate dendrons based on amino acids at the termini of the diblock structures. It was determined that lysine, a basic amino acid, covalently coupled to cholesteryl-(L-lactic acid) can promote cell adhesion and spreading while negatively charged and zwitterionic 2nd generation dendrons based on aspartic acid do not. Incorporation of the well known arginine-glycine-aspartic acid (RGD) sequence, which is found in many adhesive proteins, to the dendrons imparted integrin-mediated cell adhesion as evidenced by the formation of stress fibers. We also explored the capacity of integrin receptors to bind to ligands that are not the linear form of RGD, but have R, G, and D spatially positioned to mimic the linear RGD environments. For this purpose, the arms of the 2 nd generation lysine dendrons were functionalized with R, G, and D to yield an 'R,G,D library' of molecules. These materials were found to promote adhesion of 3T3 fibroblasts through integrin receptors. A dendron is multifunctional and allows a large degree of functionality in chemical design.

Long residence time of ultrasound microbubbles targeted to integrin in murine tumor model.

PubMed

Jun, Hong Young; Park, Seong Hoon; Kim, Hun Soo; Yoon, Kwon-Ha

2010-01-01

The aim of this study was to evaluate the intratumoral residence time of microbubbles (MBs) targeted to alpha(v)beta(3) integrin expressed in the endothelial cells of mice during the process of tumor angiogenesis. For the preparation of MBs, decafluorobutane gas was sonically dispersed in phosphate buffer saline containing L-A-phosphatidylcholine-distearoyl, polyethylene glycol 40 stearate, and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)2000] in a 77:15:8 molar ratio. Avidin-fluorescein isothiocyanate and biotin-cyclic arginine-glycine-aspartate-D-tyrosine-lysine (cRGD) or biotin-alanine-glycine-aspartate (AGD) conjugates were added to the reaction mixture. Adhesion testing of the targeting MBs was performed for the MS-1 cell line expressing alpha(v)beta(3) integrin in vitro. The in vivo acoustic properties of the MBs were assessed by clinical ultrasound on the HT1080 fibrosarcoma model (n = 8) for 1 hour. Cryosections were stained with hematoxylin and eosin and by immunohistochemical staining to identify expression of alpha(v)beta(3) integrin in the HT1080 tumor. The adherence of the MBs conjugated to cRGD was significantly greater than the adherence of the MBs conjugated to biotin-AGD (P < .01) for the MS-1 endothelial cell line. The acoustic enhancement on ultrasound was observed as a stable imaging window until 1 hour after injection of the MB conjugates in the mice. The MBs targeted via cRGD preferentially adhered to the vascular endothelium of the HT-1080 tumors. The findings of ultrasound imaging were correlated with immunohistochemical findings for the expression of alpha(v)beta(3) integrin on the vascular endothelium of the tumors. The prepared MBs conjugated with cRGD demonstrated a sufficient residence time to attach to the target integrin of tumor tissues. This finding suggests that the MBs are a potential molecular contrast agent that enables characterization of tumor angiogenesis and the monitoring of antitumor and antiangiogenic therapy.

Suppression of tumour growth by orally administered osteopontin is accompanied by alterations in tumour blood vessels.

PubMed

Rittling, S R; Wejse, P L; Yagiz, K; Warot, G A; Hui, T

2014-03-04

The integrin-binding protein osteopontin is strongly associated with tumour development, yet is an abundant dietary component as a constituent of human and bovine milk. Therefore, we tested the effect of orally administered osteopontin (o-OPN) on the development of subcutaneous tumours in mice. Bovine milk osteopontin was administered in drinking water to tumour-bearing immune-competent mice. Tumour growth, proliferation, necrosis, apoptosis and blood vessel size and number were measured. Expression of the α₉ integrin was determined. o-OPN suppressed tumour growth, increased the extent of necrosis, and induced formation of abnormally large blood vessels. Anti-OPN reactivity detected in the plasma of OPN-null mice fed OPN suggested that tumour-blocking peptides were absorbed during digestion, but the o-OPN effect was likely distinct from that of an RGD peptide. Expression of the α₉ integrin was detected on both tumour cells and blood vessels. Potential active peptides from the α₉ binding site of OPN were identified by mass spectrometry following in vitro digestion, and injection of these peptides suppressed tumour growth. These results suggest that peptides derived from o-OPN are absorbed and interfere with tumour growth and normal vessel development. o-OPN-derived peptides that target the α₉ integrin are likely involved.

Integrin αvβ3 as a Promising Target to Image Neoangiogenesis Using In-House Generator-Produced Positron Emitter (68)Ga-Labeled DOTA-Arginine-Glycine-Aspartic Acid (RGD) Ligand.

PubMed

Vatsa, Rakhee; Bhusari, Priya; Kumar, Sunil; Chakraborty, Sudipta; Dash, Ashutosh; Singh, Gurpreet; Dhawan, Devinder Kumar; Shukla, Jaya; Mittal, Bhagwant Rai

2015-06-01

For the growth and spread of a tumor beyond 2 mm, angiogenesis plays a crucial role, and association of various integrins with angiogenesis is evidential. The aim of the study was radiolabeling of DOTA-chelated RGD (arginine-glycine-aspartic acid) peptide with (68)Ga for PET imaging in locally advanced breast carcinoma. DOTA-RGD was incubated with (68)GaCl3, eluted in 0.05 m HCl. Elution volume, peptide amount, and reaction pH were studied. Radio-ITLC, gas chromatography, endotoxin, and sterility testing were performed. Serial (n=3) and whole-body (n=2) PET/CT imaging was done on patients post i.v. injection of 111-185 MBq of (68)Ga-DOTA-RGD. Maximum radiolabeling yield was achieved with 3 mL elution volume of 15-20 μg peptide at pH 3.5-4.0 with 10 minutes of incubation at 95°C. Product samples were sterile having 99.5% radiochemical purity with residual ethanol content and endotoxins in injectable limits. Intense radiotracer uptake was noticed in the tumor with SUVmax 15.3 at 45 minutes in serial images. Physiological radiotracer uptake was seen in the liver, spleen, ventricles, and thyroid with excretion through the kidneys. The authors concluded that (68)Ga-DOTA-RGD has the potential for imaging α,vβ3 integrin-expressing tumors.

Artificial Chemical Reporter Targeting Strategy Using Bioorthogonal Click Reaction for Improving Active-Targeting Efficiency of Tumor.

PubMed

Yoon, Hong Yeol; Shin, Min Lee; Shim, Man Kyu; Lee, Sangmin; Na, Jin Hee; Koo, Heebeom; Lee, Hyukjin; Kim, Jong-Ho; Lee, Kuen Yong; Kim, Kwangmeyung; Kwon, Ick Chan

2017-05-01

Biological ligands such as aptamer, antibody, glucose, and peptide have been widely used to bind specific surface molecules or receptors in tumor cells or subcellular structures to improve tumor-targeting efficiency of nanoparticles. However, this active-targeting strategy has limitations for tumor targeting due to inter- and intraheterogeneity of tumors. In this study, we demonstrated an alternative active-targeting strategy using metabolic engineering and bioorthogonal click reaction to improve tumor-targeting efficiency of nanoparticles. We observed that azide-containing chemical reporters were successfully generated onto surface glycans of various tumor cells such as lung cancer (A549), brain cancer (U87), and breast cancer (BT-474, MDA-MB231, MCF-7) via metabolic engineering in vitro. In addition, we compared tumor targeting of artificial azide reporter with bicyclononyne (BCN)-conjugated glycol chitosan nanoparticles (BCN-CNPs) and integrin α v β 3 with cyclic RGD-conjugated CNPs (cRGD-CNPs) in vitro and in vivo. Fluorescence intensity of azide-reporter-targeted BCN-CNPs in tumor tissues was 1.6-fold higher and with a more uniform distribution compared to that of cRGD-CNPs. Moreover, even in the isolated heterogeneous U87 cells, BCN-CNPs could bind artificial azide reporters on tumor cells more uniformly (∼92.9%) compared to cRGD-CNPs. Therefore, the artificial azide-reporter-targeting strategy can be utilized for targeting heterogeneous tumor cells via bioorthogonal click reaction and may provide an alternative method of tumor targeting for further investigation in cancer therapy.

Timely Visualization of the Collaterals Formed during Acute Ischemic Stroke with Fe3 O4 Nanoparticle-based MR Imaging Probe.

PubMed

Wang, Ting; Hou, Yi; Bu, Bo; Wang, Wenxin; Ma, Tiancong; Liu, Chunyan; Lin, Lan; Ma, Lin; Lou, Xin; Gao, Mingyuan

2018-04-17

Ischemic stroke is one of the major leading causes for long-term disability and mortality. Collateral vessels provide an alternative pathway to protect the brain against ischemic injury after arterial occlusion. Aiming at visualizing the collaterals occurring during acute ischemic stroke, an integrin α v β 3 -specific Fe 3 O 4 -Arg-Gly-Asp (RGD) nanoprobe is prepared for magnetic resonance imaging (MRI) of the collaterals. Rat models are constructed by occluding the middle cerebral artery for imaging studies of cerebral ischemia and ischemia-reperfusion on 7.0 Tesla MRI using susceptibility-weighted imaging sequence. To show the binding specificity to the collaterals, the imaging results acquired with the Fe 3 O 4 -RGD nanoprobe and the Fe 3 O 4 mother nanoparticles, respectively, are carefully compared. In addition, an RGD blocking experiment is also carried out to support the excellent binding specificity of the Fe 3 O 4 -RGD nanoprobe. Following the above experiments, cerebral ischemia-reperfusion studies show the collateral dynamics upon reperfusion, which is very important for the prognosis of various revascularization therapies in the clinic. The current study has, for the first time, enabled the direct observation of collaterals in a quasi-real time fashion and further disclosed that the antegrade flow upon reperfusion dominates the blood supply of primary ischemic tissue during the early stage of infarction, which is significantly meaningful for clinical treatment of stroke. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Force measurements on the molecular interactions between ligand (RGD) and human platelet α IIbβ 3 receptor system

NASA Astrophysics Data System (ADS)

Lee, ImShik; Marchant, Roger E.

2001-10-01

The peptide sequence arginine-glycine-aspartate (RGD) found in fibrinogen, von Willebrand factor, fibronectin, and vitronectin, plays a critical role in platelet adhesion and thrombus formation, when bound to the platelet α IIbβ 3 integrin receptor. Using atomic force microscopy (AFM), we have measured the debonding interaction between an RGD peptide-modified AFM probe tip and a human platelet surface from pN to nN levels of force. The peptide sequence, GSSSGRGDSPA, which contains the biologically active RGDSP sequence with a hydrophilic spacer sequence (GSSSG), was covalently coupled to AFM probe tips. Direct measurements on the debonding force for the RGD ligand - α IIbβ 3 platelet receptor system were carried out in Tyrode buffer at room temperature. Our results show three distinct distributions of debonding forces at a loading rate of 12 nN/s, from which we estimate the debonding force for the single ligand-receptor to be ˜93 pN. The results also show evidence for considerable extension in the flexible sample surface during the debonding process, and a linear correlation between the debonding force and the logarithm of the rate of loading. From our analysis, the zero kinetic off-rate Koff(0), the single molecular binding energy Eb, and the transition state xB, assuming rigid binding, were extracted from the data, and estimated to be 22.6 s -1, -2.64×10 -20 J and 0.1 nm, respectively.

Tumor-targeted inhibition by a novel strategy - mimoretrovirus expressing siRNA targeting the Pokemon gene.

PubMed

Tian, Zhiqiang; Wang, Huaizhi; Jia, Zhengcai; Shi, Jinglei; Tang, Jun; Mao, Liwei; Liu, Hongli; Deng, Yijing; He, Yangdong; Ruan, Zhihua; Li, Jintao; Wu, Yuzhang; Ni, Bing

2010-12-01

Pokemon gene has crucial but versatile functions in cell differentiation, proliferation and tumorigenesis. It is a master regulator of the ARF-HDM2-p53 and Rb-E2F pathways. The facts that the expression of Pokemon is essential for tumor formation and many kinds of tumors over-express the Pokemon gene make it an attractive target for therapeutic intervention for cancer treatment. In this study, we used an RNAi strategy to silence the Pokemon gene in a cervical cancer model. To address the issues involving tumor specific delivery and durable expression of siRNA, we applied the Arg-Gly-Asp (RGD) peptide ligand and polylysine (K(18)) fusion peptide to encapsulate a recombinant retrovirus plasmid expressing a siRNA targeting the Pokemon gene and produced the 'mimoretrovirus'. At charge ratio 2.0 of fusion peptide/plasmid, the mimoretrovirus formed stable and homogenous nanoparticles, and provided complete DNase I protection and complete gel retardation. This nanoparticle inhibited SiHa cell proliferation and invasion, while it promoted SiHa cell apoptosis. The binding of the nanoparticle to SiHa cells was mediated via the RGD-integrin α(v)β(3) interaction, as evidenced by the finding that unconjugated RGD peptide inhibited this binding significantly. This tumor-targeting mimoretrovirus exhibited excellent anti-tumor capacity in vivo in a nude mouse model. Moreover, the mimoretrovirus inhibited tumor growth with a much higher efficiency than recombinant retrovirus expressing siRNA or the K(18)/P4 nanoparticle lacking the RGD peptide. Results suggest that the RNAi/RGD-based mimoretrovirus developed in this study represents a novel anti-tumor strategy that may be applicable to most research involving cancer therapy and, thus, has promising potential as a cervical cancer treatment.

A novel dendritic nanocarrier of polyamidoamine-polyethylene glycol-cyclic RGD for “smart” small interfering RNA delivery and in vitro antitumor effects by human ether-à-go-go-related gene silencing in anaplastic thyroid carcinoma cells

PubMed Central

Li, Guanhua; Hu, Zuojun; Yin, Henghui; Zhang, Yunjian; Huang, Xueling; Wang, Shenming; Li, Wen

2013-01-01

The application of RNA interference techniques is promising in gene therapeutic approaches, especially for cancers. To improve safety and efficiency of small interfering RNA (siRNA) delivery, a triblock dendritic nanocarrier, polyamidoamine-polyethylene glycol-cyclic RGD (PAMAM-PEG-cRGD), was developed and studied as an siRNA vector targeting the human ether-à-go-go-related gene (hERG) in human anaplastic thyroid carcinoma cells. Structure characterization, particle size, zeta potential, and gel retardation assay confirmed that complete triblock components were successfully synthesized with effective binding capacity of siRNA in this triblock nanocarrier. Cytotoxicity data indicated that conjugation of PEG significantly alleviated cytotoxicity when compared with unmodified PAMAM. PAMAM-PEG-cRGD exerted potent siRNA cellular internalization in which transfection efficiency measured by flow cytometry was up to 68% when the charge ratio (N/P ratio) was 3.5. Ligand-receptor affinity together with electrostatic interaction should be involved in the nano-siRNA endocytosis mechanism and we then proved that attachment of cRGD enhanced cellular uptake via RGD-integrin recognition. Gene silencing was evaluated by reverse transcription polymerase chain reaction and PAMAM-PEG-cRGD-siRNA complex downregulated the expression of hERG to 26.3% of the control value. Furthermore, gene knockdown of hERG elicited growth suppression as well as activated apoptosis by means of abolishing vascular endothelial growth factor secretion and triggering caspase-3 cascade in anaplastic thyroid carcinoma cells. Our study demonstrates that this novel triblock polymer, PAMAM-PEG-cRGD, exhibits negligible cytotoxicity, effective transfection, “smart” cancer targeting, and therefore is a promising siRNA nanocarrier. PMID:23569377

RGD peptide-modified multifunctional dendrimer platform for drug encapsulation and targeted inhibition of cancer cells.

PubMed

He, Xuedan; Alves, Carla S; Oliveira, Nilsa; Rodrigues, João; Zhu, Jingyi; Bányai, István; Tomás, Helena; Shi, Xiangyang

2015-01-01

Development of multifunctional nanoscale drug-delivery systems for targeted cancer therapy still remains a great challenge. Here, we report the synthesis of cyclic arginine-glycine-aspartic acid (RGD) peptide-conjugated generation 5 (G5) poly(amidoamine) dendrimers for anticancer drug encapsulation and targeted therapy of cancer cells overexpressing αvβ3 integrins. In this study, amine-terminated G5 dendrimers were used as a platform to be sequentially modified with fluorescein isothiocyanate (FI) via a thiourea linkage and RGD peptide via a polyethylene glycol (PEG) spacer, followed by acetylation of the remaining dendrimer terminal amines. The developed multifunctional dendrimer platform (G5.NHAc-FI-PEG-RGD) was then used to encapsulate an anticancer drug doxorubicin (DOX). We show that approximately six DOX molecules are able to be encapsulated within each dendrimer platform. The formed complexes are water-soluble, stable, and able to release DOX in a sustained manner. One- and two-dimensional NMR techniques were applied to investigate the interaction between dendrimers and DOX, and the impact of the environmental pH on the release rate of DOX from the dendrimer/DOX complexes was also explored. Furthermore, cell biological studies demonstrate that the encapsulation of DOX within the G5.NHAc-FI-PEG-RGD dendrimers does not compromise the anticancer activity of DOX and that the therapeutic efficacy of the dendrimer/DOX complexes is solely related to the encapsulated DOX drug. Importantly, thanks to the role played by RGD-mediated targeting, the developed dendrimer/drug complexes are able to specifically target αvβ3 integrin-overexpressing cancer cells and display specific therapeutic efficacy to the target cells. The developed RGD peptide-targeted multifunctional dendrimers may thus be used as a versatile platform for targeted therapy of different types of αvβ3 integrin-overexpressing cancer cells. Copyright © 2014 Elsevier B.V. All rights reserved.

In-vitro and in-vivo phenotype of type Asia 1 foot-and-mouth disease viruses utilizing two non-RGD receptor recognition sites

PubMed Central

2011-01-01

Background Foot-and-mouth disease virus (FMDV) uses a highly conserved Arg-Gly-Asp (RGD) triplet for attachment to host cells and this motif is believed to be essential for virus viability. Previous sequence analyses of the 1D-encoding region of an FMDV field isolate (Asia1/JS/CHA/05) and its two derivatives indicated that two viruses, which contained an Arg-Asp-Asp (RDD) or an Arg-Ser-Asp (RSD) triplet instead of the RGD integrin recognition motif, were generated serendipitously upon short-term evolution of field isolate in different biological environments. To examine the influence of single amino acid substitutions in the receptor binding site of the RDD-containing FMD viral genome on virus viability and the ability of non-RGD FMDVs to cause disease in susceptible animals, we constructed an RDD-containing FMDV full-length cDNA clone and derived mutant molecules with RGD or RSD receptor recognition motifs. Following transfection of BSR cells with the full-length genome plasmids, the genetically engineered viruses were examined for their infectious potential in cell culture and susceptible animals. Results Amino acid sequence analysis of the 1D-coding region of different derivatives derived from the Asia1/JS/CHA/05 field isolate revealed that the RDD mutants became dominant or achieved population equilibrium with coexistence of the RGD and RSD subpopulations at an early phase of type Asia1 FMDV quasispecies evolution. Furthermore, the RDD and RSD sequences remained genetically stable for at least 20 passages. Using reverse genetics, the RDD-, RSD-, and RGD-containing FMD viruses were rescued from full-length cDNA clones, and single amino acid substitution in RDD-containing FMD viral genome did not affect virus viability. The genetically engineered viruses replicated stably in BHK-21 cells and had similar growth properties to the parental virus. The RDD parental virus and two non-RGD recombinant viruses were virulent to pigs and bovines that developed typical clinical disease and viremia. Conclusions FMDV quasispecies evolving in a different biological environment gained the capability of selecting different receptor recognition site. The RDD-containing FMD viral genome can accommodate substitutions in the receptor binding site without additional changes in the capsid. The viruses expressing non-RGD receptor binding sites can replicate stably in vitro and produce typical FMD clinical disease in susceptible animals. PMID:21711567

Fibronectin on extracellular vesicles from microvascular endothelial cells is involved in the vesicle uptake into oligodendrocyte precursor cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Osawa, Sho; Department of Molecular and Cellular Neurobiology, Gunma University Graduate School of Medicine, 3-39-22 Showa-machi, Maebashi, Gunma 371-8511; Kurachi, Masashi

We previously reported transplantation of brain microvascular endothelial cells (MVECs) into cerebral white matter infarction model improved the animal's behavioral outcome by increasing the number of oligodendrocyte precursor cells (OPCs). We also revealed extracellular vesicles (EVs) derived from MVECs promoted survival and proliferation of OPCs in vitro. In this study, we investigated the mechanism how EVs derived from MVECs contribute to OPC survival and proliferation. Protein mass spectrometry and enzyme-linked immunosorbent assay revealed fibronectin was abundant on the surface of EVs from MVECs. As fibronectin has been reported to promote OPC survival and proliferation via integrin signaling pathway, we blocked themore » binding between fibronectin and integrins using RGD sequence mimics. Blocking the binding, however, did not attenuate the survival and proliferation promoting effect of EVs on OPCs. Flow cytometric and imaging analyses revealed fibronectin on EVs mediates their internalization into OPCs by its binding to heparan sulfate proteoglycan on OPCs. OPC survival and proliferation promoted by EVs were attenuated by blocking the internalization of EVs into OPCs. These lines of evidence suggest that fibronectin on EVs mediates their internalization into OPCs, and the cargo of EVs promotes survival and proliferation of OPCs, independent of integrin signaling pathway. - Highlights: • Fibronectin exists on the surface of extracellular vesicles from endothelial cells. • Integrin signaling is not involved in effects of extracellular vesicles on OPCs. • Fibronectin on the surface of extracellular vesicles mediates their uptake into OPCs.« less

Imaging of alpha(v)beta(3) expression by a bifunctional chimeric RGD peptide not cross-reacting with alpha(v)beta(5).

PubMed

Zannetti, Antonella; Del Vecchio, Silvana; Iommelli, Francesca; Del Gatto, Annarita; De Luca, Stefania; Zaccaro, Laura; Papaccioli, Angela; Sommella, Jvana; Panico, Mariarosaria; Speranza, Antonio; Grieco, Paolo; Novellino, Ettore; Saviano, Michele; Pedone, Carlo; Salvatore, Marco

2009-08-15

To test whether a novel bifunctional chimeric peptide comprising a cyclic Arg-Gly-Asp pentapeptide covalently bound to an echistatin domain can discriminate alpha(v)beta(3) from alpha(v)beta(5) integrin, thus allowing the in vivo selective visualization of alpha(v)beta(3) expression by single-photon and positron emission tomography (PET) imaging. The chimeric peptide was preliminarily tested for inhibition of alpha(v)beta(3)-dependent cell adhesion and competition of 125I-echistatin binding to membrane of stably transfected K562 cells expressing alpha(v)beta(3) (Kalpha(v)beta(3)) or alpha(v)beta(5) (Kalpha(v)beta(5)) integrin. The chimeric peptide was then conjugated with diethylenetriaminepentaacetic acid and labeled with 111In for single-photon imaging, whereas a one-step procedure was used for labeling the full-length peptide and a truncated derivative, lacking the last five C-terminal amino acids, with 18F for PET imaging. Nude mice bearing tumors from Kalpha(v)beta(3), Kalpha(v)beta(5), U87MG human glioblastoma, and A431 human epidermoid cells were subjected to single-photon and PET imaging. Adhesion and competitive binding assays showed that the novel chimeric peptide selectively binds to alpha(v)beta(3) integrin and does not cross-react with alpha(v)beta(5). In agreement with in vitro findings, single-photon and PET imaging studies showed that the radiolabeled chimeric peptide selectively localizes in tumor xenografts expressing alphavbeta3 and fails to accumulate in those expressing alpha(v)beta(5) integrin. When 18F-labeled truncated derivative was used for PET imaging, alphavbeta3- and alpha(v)beta(5)-expressing tumors were visualized, indicating that the five C-terminal amino acids are required to differentially bind the two integrins. Our findings indicate that the novel chimeric Arg-Gly-Asp peptide, having no cross-reaction with alphavbeta5 integrin, allows highly selective alphavbeta3 expression imaging and monitoring.

Mesoporous silica nanoparticles functionalized with fluorescent and MRI reporters for the visualization of murine tumors overexpressing αvβ3 receptors

NASA Astrophysics Data System (ADS)

Hu, He; Arena, Francesca; Gianolio, Eliana; Boffa, Cinzia; di Gregorio, Enza; Stefania, Rachele; Orio, Laura; Baroni, Simona; Aime, Silvio

2016-03-01

A novel fluorescein/Gd-DOTAGA containing nanoprobe for the visualization of tumors by optical and Magnetic Resonance Imaging (MRI) is reported herein. It is based on the functionalization of the surface of small mesoporous silica nanoparticles (MSNs) (~30 nm) with the arginine-glycine-aspartic (RGD) moieties, which are known to target αvβ3 integrin receptors overexpressed in several tumor cells. The obtained nanoprobe (Gd-MSNs-RGD) displays good stability, tolerability and high relaxivity (37.6 mM-1 s-1 at 21.5 MHz). After a preliminary evaluation of their cytotoxicity and targeting capability toward U87MG cells by in vitro fluorescence and MR imaging, the nanoprobes were tested in vivo by T1-weighted MR imaging of xenografted murine tumor models. The obtained results demonstrated that the Gd-MSNs-RGD nanoprobes are good reporters both in vitro and in vivo for the MR-visualization of tumor cells overexpressing αvβ3 integrin receptors.A novel fluorescein/Gd-DOTAGA containing nanoprobe for the visualization of tumors by optical and Magnetic Resonance Imaging (MRI) is reported herein. It is based on the functionalization of the surface of small mesoporous silica nanoparticles (MSNs) (~30 nm) with the arginine-glycine-aspartic (RGD) moieties, which are known to target αvβ3 integrin receptors overexpressed in several tumor cells. The obtained nanoprobe (Gd-MSNs-RGD) displays good stability, tolerability and high relaxivity (37.6 mM-1 s-1 at 21.5 MHz). After a preliminary evaluation of their cytotoxicity and targeting capability toward U87MG cells by in vitro fluorescence and MR imaging, the nanoprobes were tested in vivo by T1-weighted MR imaging of xenografted murine tumor models. The obtained results demonstrated that the Gd-MSNs-RGD nanoprobes are good reporters both in vitro and in vivo for the MR-visualization of tumor cells overexpressing αvβ3 integrin receptors. Electronic supplementary information (ESI) available: Absorption and emission spectra, energy dispersive X-ray analysis (EDXA) and XPS spectra, TGA, zeta-potential and the molecular structures of the Gd-complexes. See DOI: 10.1039/c5nr08878j

Arginine-glycine-aspartic acid motif is critical for human parechovirus 1 entry.

PubMed

Boonyakiat, Y; Hughes, P J; Ghazi, F; Stanway, G

2001-10-01

The human parechovirus 1 RGD motif in VP1 was studied by mutagenesis. An RGD-to-RGE change gave only revertant viruses with a restored RGD, while deletion of GD was lethal and nonrevertable. Mutations at the +1 and +2 positions had some effect on growth properties and a +1 M-to-P change was lethal. These studies indicate that the RGD motif plays a critical role in infectivity, presumably by interacting with integrins, and that downstream amino acids can have an influence on function.

Association of high microvessel αvβ3 and low PTEN with poor outcome in stage 3 neuroblastoma: rationale for using first in class dual PI3K/BRD4 inhibitor, SF1126

PubMed Central

Singh, Alok R.; Joshi, Shweta; Vega, Francisco M.; Guo, Pinzheng; Xu, Jingying; Groshen, Susan; Ye, Wei; Millard, Melissa; Campan, Mihaela; Morales, Guillermo; Garlich, Joseph R.; Laird, Peter W.; Seeger, Robert C.; Shimada, Hiroyuki

2017-01-01

Neuroblastoma (NB) is the most common extracranial solid tumor in children. Our previous studies showed that the angiogenic integrin αvβ3 was increased in high-risk metastatic (stage 4) NB compared with localized neuroblastomas. Herein, we show that integrin αvβ3 was expressed on 68% of microvessels in MYCN-amplified stage 3 neuroblastomas, but only on 34% (means) in MYCN-non-amplified tumors (p < 0.001; n = 54). PTEN, a tumor suppressor involved in αvβ3 signaling, was expressed in neuroblastomas either diffusely, focally or not at all (immunohistochemistry). Integrin αvβ3 was expressed on 60% of tumor microvessels when PTEN was negative or focal, as compared to 32% of microvessels in tumors with diffuse PTEN expression (p < 0.001). In a MYCN transgenic mouse model, loss of one allele of PTEN promoted tumor growth, illustrating the potential role of PTEN in neuroblastoma pathogenesis. Interestingly, we report the novel dual PI-3K/BRD4 activity of SF1126 (originally developed as an RGD-conjugated pan PI3K inhibitor). SF1126 inhibits BRD4 bromodomain binding to acetylated lysine residues with histone H3 as well as PI3K activity in the MYCN amplified neuroblastoma cell line IMR-32. Moreover, SF1126 suppressed MYCN expression and MYCN associated transcriptional activity in IMR-32 and CHLA136, resulting in overall decrease in neuroblastoma cell viability. Finally, treatment of neuroblastoma tumors with SF1126 inhibited neuroblastoma growth in vivo. These data suggest integrin αvβ3, MYCN/BRD4 and PTEN/PI3K/AKT signaling as biomarkers and hence therapeutic targets in neuroblastoma and support testing of the RGD integrin αvβ3-targeted PI-3K/BRD4 inhibitor, SF1126 as a therapeutic strategy in this specific subgroup of high risk neuroblastoma. PMID:28881723

Integrin-extracellular matrix interactions in connective tissue remodeling and osteoblast differentiation

NASA Technical Reports Server (NTRS)

Globus, R. K.; Moursi, A.; Zimmerman, D.; Lull, J.; Damsky, C.

1995-01-01

The differentiaton of bone cells is a complex multistep process. Bone is somewhat unusual in that it is very actively and continually remodeled in the adult and that maintenance of its mass in the mature organism is exquisitely sensitive to mechanical as well as chemical signals. Bone is also unique because it consists of a very large amount of extracellular matrix (ECM) that is mineralized. The integrin family of ECM receptors has been shown to play an important role in tissue morphogenesis in several systems. Our studies on the regulation of matrix remodeling enzymes by integrins in rabbit synovial fibroblasts show that two b1 integrin fibronectin (FN) receptor complexes (alpha 5 beta 1 and alpha 4 beta 1) cooperate in detecting subtle changes in the composition of the ECM. As a result of signal transduction by these integrins, the levels of mRNA and protein for several members of the metalloproteinase family are regulated in these cells. We have also used antibody and RGD peptide perturbation studies to determine the significance of cell/ECM interactions to normal osteogenesis. We found that interactions between the cell binding domain of FN and integrins are required for both normal morphogenesis and gene expression in cultured osteoblasts that differentiate to form bone-like tissue in culture. These data lead us to propose that beta 1 integrins play an important role in osteoblast differentiation as well as in bone remodeling.

Radiolabeled arginine-glycine-aspartic acid peptides to image angiogenesis in swine model of hibernating myocardium.

PubMed

Johnson, Lynne L; Schofield, Lorraine; Donahay, Tammy; Bouchard, Mark; Poppas, Athena; Haubner, Roland

2008-07-01

Our aim was to image angiogenesis produced by endomyocardial injection of phVEGF165 in a swine model of hibernating myocardium using [123I]Gluco-arginine-glycine-aspartic acid (RGD) targeting the alphavbeta3 integrins. A noninvasive test to monitor the efficacy of therapy inducing angiogenesis is needed. The interaction between extracellular matrix and endothelial cells in sprouting capillaries is effected primarily by alphavbeta3 integrins that bind through RGD motifs. At 21 +/- 4 days, after left circumflex coronary artery ameroid constrictor placement, 8 swine received endomyocardial injection of 1.2 mg phVEGF165 divided into 6 sites and 6 swine received saline (S) using nonfluoroscopic 3-dimensional endocardial mapping system (Noga)-guided delivery. After 20 +/- 6 days, 13 animals were injected with 6.4 +/- 1.7 mCi [123I]Gluco-RGD, 1 VEGF (vascular endothelial growth factor)-injected animal with I-123-labeled peptide control, and all animals with 2.5 +/- 0.4 mCi of Tl-201 and underwent single-photon emission computed tomography imaging. Blood flow and echocardiographic measurements were made at both time points and tissue analyzed for fibrosis and capillary density by lectin staining. Hibernating myocardium in the ameroid constrictor territory at time of injections was documented by reduced wall thickening compared with remote. Ratio of myocardial blood flow in left circumflex coronary artery/left anterior descending coronary artery territories increased by 15 +/- 11% in the VEGF animals and fell 13 +/- 12% in S-injected (p < 0.01). There was a small increase in wall thickening in constrictor territory after VEGF (8 +/- 17%) while in S-injected animals wall thickening fell by 23 +/- 31% (p = 0.01 vs. VEGF). Lectin staining as percent positive tissue staining for ameroid territory was higher in VEGF-injected compared with S-injected animals (2.5 +/- 1.5% vs. 0.87 +/- 0.52%, p = 0.01). Focal uptake of [123I]Gluco-RGD corresponding to Tl-201 defects was seen in VEGF-injected but not in S-injected animals. [123I]Gluco-RGD uptake in the ameroid territory as percent injected dose correlated with lectin staining (R2 = 0.80, p = 0.002). These data suggest that single-photon emission computed tomography imaging of radiolabeled RGD peptides may be a useful noninvasive method to monitor therapy that induces angiogenesis in the heart.

Integrin – Dependent Mechanotransduction in Mechanically Stimulated Human Annulus Fibrosus Cells: Evidence for an Alternative Mechanotransduction Pathway Operating with Degeneration

PubMed Central

Gilbert, Hamish T. J.; Nagra, Navraj S.; Freemont, Anthony J.; Millward-Sadler, Sarah J.; Hoyland, Judith A.

2013-01-01

Intervertebral disc (IVD) cells derived from degenerate tissue respond aberrantly to mechanical stimuli, potentially due to altered mechanotransduction pathways. Elucidation of the altered, or alternative, mechanotransduction pathways operating with degeneration could yield novel targets for the treatment of IVD disease. Our aim here was to investigate the involvement of RGD-recognising integrins and associated signalling molecules in the response to cyclic tensile strain (CTS) of human annulus fibrosus (AF) cells derived from non-degenerate and degenerate IVDs. AF cells from non-degenerate and degenerate human IVDs were cyclically strained with and without function blocking RGD – peptides with 10% strain, 1.0 Hz for 20 minutes using a Flexercell® strain device. QRT-PCR and Western blotting were performed to analyse gene expression of type I collagen and ADAMTS -4, and phosphorylation of focal adhesion kinase (FAK), respectively. The response to 1.0 Hz CTS differed between the two groups of AF cells, with decreased ADAMTS -4 gene expression and decreased type I collagen gene expression post load in AF cells derived from non-degenerate and degenerate IVDs, respectively. Pre-treatment of non-degenerate AF cells with RGD peptides prevented the CTS-induced decrease in ADAMTS -4 gene expression, but caused an increase in expression at 24 hours, a response not observed in degenerate AF cells where RGD pre-treatment failed to inhibit the mechano-response. In addition, FAK phosphorylation increased in CTS stimulated AF cells derived from non-degenerate, but not degenerate IVDs, with RGD pre-treatment inhibiting the CTS – dependent increase in phosphorylated FAK. Our findings suggest that RGD -integrins are involved in the 1.0 Hz CTS – induced mechano-response observed in AF cells derived from non-degenerate, but not degenerate IVDs. This data supports our previous work, suggesting an alternative mechanotransduction pathway may be operating in degenerate AF cells. PMID:24039840

Evidence for the evolution of tenascin and fibronectin early in the chordate lineage.

PubMed

Tucker, Richard P; Chiquet-Ehrismann, Ruth

2009-02-01

Fibronectin and tenascin are extracellular matrix glycoproteins that play important roles in cell adhesion and motility. In a previous study we provided evidence that tenascin first appeared early in the chordate lineage. As tenascin has been proposed to act, in part, through modulation of cell-fibronectin interactions, we sought here to identify fibronectin genes in non-vertebrate chordates and other invertebrates to determine if tenascin and fibronectin evolved separately or together, and to identify phylogenetically conserved features of both proteins. We found that the genome of the urochordate Ciona savignyi contains both a tenascin gene and a gene encoding a fibronectin-like protein with fibronectin type 1, 2 and 3 repeats. The genome of the cephalochordate Branchiostoma floridae (amphioxus) also has a tenascin gene. However, we could not identify a fibronectin-like gene in B. floridae, nor could we identify fibronectin or tenascin genes in echinoderms, protostomes or cnidarians. If urochordates are more closely related to vertebrates, tenascin may have evolved before fibronectin in an ancestor common to tunicates and amphioxus. Alternatively, tenascin and fibronectin may have evolved in an ancestor common to B. floridae and C. savignyi and the fibronectin gene was subsequently lost in the cephalochordate lineage. The fibronectin-like gene from C. savignyi does not encode the RGD motif for integrin binding found in all vertebrate fibronectins, and it lacks most of the fibronectin type 1 domains believed to be critical for fibrillogenesis. In contrast, the tenascin gene in B. floridae encodes multiple RGD motifs, suggesting that integrin binding is fundamental to tenascin function.

Enhanced In Vivo Tumor Detection by Active Tumor Cell Targeting Using Multiple Tumor Receptor-Binding Peptides Presented on Genetically Engineered Human Ferritin Nanoparticles.

PubMed

Kwon, Koo Chul; Ko, Ho Kyung; Lee, Jiyun; Lee, Eun Jung; Kim, Kwangmeyung; Lee, Jeewon

2016-08-01

Human ferritin heavy-chain nanoparticle (hFTH) is genetically engineered to present tumor receptor-binding peptides (affibody and/or RGD-derived cyclic peptides, named 4CRGD here) on its surface. The affibody and 4CRGD specifically and strongly binds to human epidermal growth factor receptor I (EGFR) and human integrin αvβ3, respectively, which are overexpressed on various tumor cells. Through in vitro culture of EGFR-overexpressing adenocarcinoma (MDA-MB-468) and integrin-overexpressing glioblastoma cells (U87MG), it is clarified that specific interactions between receptors on tumor cells and receptor-binding peptides on engineered hFTH is critical in active tumor cell targeting. After labeling with the near-infrared fluorescence dye (Cy5.5) and intravenouse injection into MDA-MB-468 or U87MG tumor-bearing mice, the recombinant hFTHs presenting either peptide or both of affibody and 4CRGD are successfully delivered to and retained in the tumor for a prolonged period of time. In particular, the recombinant hFTH presenting both affibody and 4CRGD notably enhances in vivo detection of U87MG tumors that express heterogeneous receptors, integrin and EGFR, compared to the other recombinant hFTHs presenting either affibody or 4CRGD only. Like affibody and 4CRGD used in this study, other multiple tumor receptor-binding peptides can be also genetically introduced to the hFTH surface for actively targeting of in vivo tumors with heterogenous receptors. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Biodistribution and Radiation Dosimetry of the Integrin Marker 64Cu-BaBaSar-RGD2 Determined from Whole-Body PET/CT in a Non-Human Primate

NASA Astrophysics Data System (ADS)

Liu, Shuanglong; Vorobyova, Ivetta; Park, Ryan; Conti, Peter S.

2017-10-01

Introduction: 64Cu-BaBaSar-RGD2 is a positron emission radiotracer taken up by integrin αvβ3, which is overexpressed in many malignancies. The aim of this study was to evaluate the biodistribution of 64Cu-BaBaSar-RGD2 in a non-human primate with positron emission tomography and to estimate the absorbed doses in major organs for human. Materials and methods: Whole-body PET imaging was done in a Siemens Biograph scanner in a male macaque monkey. After an i.v. injection of 13.1–19.7 MBq/kg of 64Cu-BaBaSar-RGD2, whole body scan was collected for a total duration of 180 min. Attenuation and scatter corrections were applied to reconstruction of the whole-body emission scan. After image reconstruction, three-dimensional volumes of interest (VOI) were hand-drawn on the PET transaxial or coronal slices of the frame where the organ was most conspicuous. Time-activity curves for each VOI were obtained, and residence time of each organ was calculated by integration of the time-activity curves. Human absorbed doses were estimated using the standard human model in OLINDA/EXM software. Results: Injection of 64Cu-BaBaSar-RGD2 was well tolerated in the macaque monkey, with no serious tracer-related adverse events observed. 64Cu-BaBaSar-RGD2 was cleared rapidly from the blood pool, with a 12.1-min biological half-time. Increased 64Cu-BaBaSar-RGD2 uptake was observed in the kidneys, and bladder, with mean percentage injected dose (ID%) values at 1 h after injection approximately 35.50 ± 6.47 and 36.89 ± 5.48, respectively. The calculated effective dose was 15.30 ± 2.21 µSv/MBq, and the kidneys had the highest absorbed dose at 108.43 ± 16.41 µGy/MBq using the non-voiding model. For an injected activity of 925 MBq 64Cu for human, the effective dose would be 14.2 ± 2.1 mSv. Discussion: Due to the limitation of the monkey number, we evaluated 64Cu-BaBaSar-RGD2 in the same monkey of three imaging sessions. Measured absorbed doses and effective doses of 64Cu-BaBaSar-RGD2 are comparable to other reported RGD-derived radiopharmaceuticals labeled with 64Cu and 18F. Therefore, 64Cu-BaBaSar-RGD2 can be safely injected into humans for studying integrin αvβ3 expression non-invasively.

Simultaneous Targeting of RGD-Integrins and Dual Murine Double Minute Proteins in Glioblastoma Multiforme.

PubMed

Merlino, Francesco; Daniele, Simona; La Pietra, Valeria; Di Maro, Salvatore; Di Leva, Francesco Saverio; Brancaccio, Diego; Tomassi, Stefano; Giuntini, Stefano; Cerofolini, Linda; Fragai, Marco; Luchinat, Claudio; Reichart, Florian; Cavallini, Chiara; Costa, Barbara; Piccarducci, Rebecca; Taliani, Sabrina; Da Settimo, Federico; Martini, Claudia; Kessler, Horst; Novellino, Ettore; Marinelli, Luciana

2018-05-18

In the fight against Glioblastoma Multiforme, recent literature data have highlighted that integrin α5β1 and p53 are part of convergent pathways in the control of glioma apoptosis. This observation prompted us to seek a molecule able to simultaneously modulate both target families. Analyzing the results of a previous virtual screening against murine double minute 2 protein (MDM2), we envisaged that Arg-Gly-Asp (RGD)-mimetic molecules could be inhibitors of MDM2/4. Herein we present the discovery of compound 7, which inhibits both MDM2/4 and α5β1/αvβ3 integrins. A lead optimization campaign was carried out on 7 with aim to preserve the activities on integrins while improving those on MDM proteins. Compound 9 turned out to be a potent MDM2/4, and α5β1/αvβ3 blocker. In p53-wild type glioma cells, 9 arrested cell cycle and proliferation and strongly reduced cell invasiveness, emerging as the first molecule of a novel class of integrin/MDM inhibitors, which might be especially useful in subpopulations of patients with glioblastoma expressing a functional p53 concomitantly with a high level of α5β1 integrin.

PAMAM-RGD Conjugates Enhance siRNA Delivery Through a Multicellular Spheroid Model of Malignant Glioma

PubMed Central

Waite, Carolyn L.; Roth, Charles M.

2011-01-01

Generation 5 poly(amidoamine) (PAMAM) dendrimers were modified by the addition of cyclic RGD targeting peptides and were evaluated for their ability to associate with siRNA and mediate siRNA delivery to U87 malignant glioma cells. PAMAM-RGD conjugates were able to complex with siRNA to form complexes of approximately 200 nm in size. Modest siRNA delivery was observed in U87 cells using either PAMAM or PAMAM-RGD conjugates. PAMAM-RGD conjugates prevented the adhesion of U87 cells to fibrinogen coated plates, in a manner that depends on the number of RGD ligands per dendrimer. The delivery of siRNA through three-dimensional multicellular spheroids of U87 cells was enhanced using PAMAM-RGD conjugates compared to the native PAMAM dendrimers, presumably by interfering with integrin-ECM contacts present in a three-dimensional tumor model. PMID:19775120

STAT3 activation in pressure-overloaded feline myocardium: role for integrins and the tyrosine kinase BMX.

PubMed

Willey, Christopher D; Palanisamy, Arun P; Johnston, Rebecca K; Mani, Santhosh K; Shiraishi, Hirokazu; Tuxworth, William J; Zile, Michael R; Balasubramanian, Sundaravadivel; Kuppuswamy, Dhandapani

2008-06-27

Growth, survival and cytoskeletal rearrangement of cardiomyocytes are critical for cardiac hypertrophy. Signal transducer and activator of transcription-3 (STAT3) activation is an important cardioprotective factor associated with cardiac hypertrophy. Although STAT3 activation has been reported via signaling through Janus Kinase 2 (JAK2) in several cardiac models of hypertrophy, the importance of other nonreceptor tyrosine kinases (NTKs) has not been explored. Utilizing an in vivo feline right ventricular pressure-overload (RVPO) model of hypertrophy, we demonstrate that in 48 h pressure-overload (PO) myocardium, STAT3 becomes phosphorylated and redistributed to detergent-insoluble fractions with no accompanying JAK2 activation. PO also caused increased levels of phosphorylated STAT3 in both cytoplasmic and nuclear fractions. To investigate the role of other NTKs, we used our established in vitro cell culture model of hypertrophy where adult feline cardiomyocytes are embedded three-dimensionally (3D) in type-I collagen and stimulated with an integrin binding peptide containing an Arg-Gly-Asp (RGD) motif that we have previously shown to recapitulate the focal adhesion complex (FAC) formation of 48 h RVPO. RGD stimulation of adult cardiomyocytes in vitro caused both STAT3 redistribution and activation that were accompanied by the activation and redistribution of c-Src and the TEC family kinase, BMX, but not JAK2. However, infection with dominant negative c-Src adenovirus was unable to block RGD-stimulated changes on either STAT3 or BMX. Further analysis in vivo in 48 h PO myocardium showed the presence of both STAT3 and BMX in the detergent-insoluble fraction with their complex formation and phosphorylation. Therefore, these studies indicate a novel mechanism of BMX-mediated STAT3 activation within a PO model of cardiac hypertrophy that might contribute to cardiomyocyte growth and survival.

RGD capsid modification enhances mucosal protective immunity of a non-human primate adenovirus vector expressing Pseudomonas aeruginosa OprF

PubMed Central

Krause, A; Whu, W Z; Qiu, J; Wafadari, D; Hackett, N R; Sharma, A; Crystal, R G; Worgall, S

2013-01-01

Replication-deficient adenoviral (Ad) vectors of non-human serotypes can serve as Ad vaccine platforms to circumvent pre-existing anti-human Ad immunity. We found previously that, in addition to that feature, a non-human primate-based AdC7 vector expressing outer membrane protein F of P. aeruginosa (AdC7OprF) was more potent in inducing lung mucosal and protective immunity compared to a human Ad5-based vector. In this study we analysed if genetic modification of the AdC7 fibre to display an integrin-binding arginine–glycine–aspartic acid (RGD) sequence can further enhance lung mucosal immunogenicity of AdC7OprF. Intratracheal immunization of mice with either AdC7OprF.RGD or AdC7OprF induced robust serum levels of anti-OprF immunoglobulin (Ig)G up to 12 weeks that were higher compared to immunization with the human vectors Ad5OprF or Ad5OprF.RGD. OprF-specific cellular responses in lung T cells isolated from mice immunized with AdC7OprF.RGD and AdC7OprF were similar for T helper type 1 (Th1) [interferon (IFN)-γ in CD8+ and interleukin (IL)-12 in CD4+], Th2 (IL-4, IL-5 and IL-13 in CD4+) and Th17 (IL-17 in CD4+). Interestingly, AdC7OprF.RGD induced more robust protective immunity against pulmonary infection with P. aeruginosa compared to AdC7OprF or the control Ad5 vectors. The enhanced protective immunity induced by AdC7OprF.RGD was maintained in the absence of alveolar macrophages (AM) or CD1d natural killer T cells. Together, the data suggest that addition of RGD to the fibre of an AdC7-based vaccine is useful to enhance its mucosal protective immunogenicity. PMID:23607394

ß3 integrin modulates transforming growth factor beta induced (TGFBI) function and paclitaxel response in ovarian cancer cells.

PubMed

Tumbarello, David A; Temple, Jillian; Brenton, James D

2012-05-28

The extracellular matrix (ECM) has a key role in facilitating the progression of ovarian cancer and we have shown recently that the secreted ECM protein TGFBI modulates the response of ovarian cancer to paclitaxel-induced cell death. We have determined TGFBI signaling from the extracellular environment is preferential for the cell surface αvß3 integrin heterodimer, in contrast to periostin, a TGFBI paralogue, which signals primarily via a ß1 integrin-mediated pathway. We demonstrate that suppression of ß1 integrin expression, in ß3 integrin-expressing ovarian cancer cells, increases adhesion to rTGFBI. In addition, Syndecan-1 and -4 expression is dispensable for adhesion to rTGFBI and loss of Syndecan-1 cooperates with the loss of ß1 integrin to further enhance adhesion to rTGFBI. The RGD motif present in the carboxy-terminus of TGFBI is necessary, but not sufficient, for SKOV3 cell adhesion and is dispensable for adhesion of ovarian cancer cells lacking ß3 integrin expression. In contrast to TGFBI, the carboxy-terminus of periostin, lacking a RGD motif, is unable to support adhesion of ovarian cancer cells. Suppression of ß3 integrin in SKOV3 cells increases resistance to paclitaxel-induced cell death while suppression of ß1 integrin has no effect. Furthermore, suppression of TGFBI expression stimulates a paclitaxel resistant phenotype while suppression of fibronectin expression, which primarily signals through a ß1 integrin-mediated pathway, increases paclitaxel sensitivity. Therefore, different ECM components use distinct signaling mechanisms in ovarian cancer cells and in particular, TGFBI preferentially interacts through a ß3 integrin receptor mediated mechanism to regulate the response of cells to paclitaxel-induced cell death.

Fibrous Hydrogels for Cell Encapsulation: A Modular and Supramolecular Approach.

PubMed

Włodarczyk-Biegun, Małgorzata K; Farbod, Kambiz; Werten, Marc W T; Slingerland, Cornelis J; de Wolf, Frits A; van den Beucken, Jeroen J J P; Leeuwenburgh, Sander C G; Cohen Stuart, Martien A; Kamperman, Marleen

2016-01-01

Artificial 3-dimensional (3D) cell culture systems, which mimic the extracellular matrix (ECM), hold great potential as models to study cellular processes under controlled conditions. The natural ECM is a 3D structure composed of a fibrous hydrogel that provides both mechanical and biochemical cues to instruct cell behavior. Here we present an ECM-mimicking genetically engineered protein-based hydrogel as a 3D cell culture system that combines several key features: (1) Mild and straightforward encapsulation meters (1) ease of ut I am not so sure.encapsulation of the cells, without the need of an external crosslinker. (2) Supramolecular assembly resulting in a fibrous architecture that recapitulates some of the unique mechanical characteristics of the ECM, i.e. strain-stiffening and self-healing behavior. (3) A modular approach allowing controlled incorporation of the biochemical cue density (integrin binding RGD domains). We tested the gels by encapsulating MG-63 osteoblastic cells and found that encapsulated cells not only respond to higher RGD density, but also to overall gel concentration. Cells in 1% and 2% (weight fraction) protein gels showed spreading and proliferation, provided a relative RGD density of at least 50%. In contrast, in 4% gels very little spreading and proliferation occurred, even for a relative RGD density of 100%. The independent control over both mechanical and biochemical cues obtained in this modular approach renders our hydrogels suitable to study cellular responses under highly defined conditions.

Novel Bifunctional Cyclic Chelator for 89Zr Labeling–Radiolabeling and Targeting Properties of RGD Conjugates

PubMed Central

2015-01-01

Within the last years 89Zr has attracted considerable attention as long-lived radionuclide for positron emission tomography (PET) applications. So far desferrioxamine B (DFO) has been mainly used as bifunctional chelating system. Fusarinine C (FSC), having complexing properties comparable to DFO, was expected to be an alternative with potentially higher stability due to its cyclic structure. In this study, as proof of principle, various FSC-RGD conjugates targeting αvß3 integrins were synthesized using different conjugation strategies and labeled with 89Zr. In vitro stability, biodistribution, and microPET/CT imaging were evaluated using [89Zr]FSC-RGD conjugates or [89Zr]triacetylfusarinine C (TAFC). Quantitative 89Zr labeling was achieved within 90 min at room temperature. The distribution coefficients of the different radioligands indicate hydrophilic character. Compared to [89Zr]DFO, [89Zr]FSC derivatives showed excellent in vitro stability and resistance against transchelation in phosphate buffered saline (PBS), ethylenediaminetetraacetic acid solution (EDTA), and human serum for up to 7 days. Cell binding studies and biodistribution as well as microPET/CT imaging experiments showed efficient receptor-specific targeting of [89Zr]FSC-RGD conjugates. No bone uptake was observed analyzing PET images indicating high in vivo stability. These findings indicate that FSC is a highly promising chelator for the development of 89Zr-based PET imaging agents. PMID:25941834

Blocking immunosuppression by human Tregs in vivo with antibodies targeting integrin αVβ8.

PubMed

Stockis, Julie; Liénart, Stéphanie; Colau, Didier; Collignon, Amandine; Nishimura, Stephen L; Sheppard, Dean; Coulie, Pierre G; Lucas, Sophie

2017-11-21

Human regulatory T cells (Tregs) suppress other T cells by converting the latent, inactive form of TGF-β1 into active TGF-β1. In Tregs, TGF-β1 activation requires GARP, a transmembrane protein that binds and presents latent TGF-β1 on the surface of Tregs stimulated through their T cell receptor. However, GARP is not sufficient because transduction of GARP in non-Treg T cells does not induce active TGF-β1 production. RGD-binding integrins were shown to activate TGF-β1 in several non-T cell types. Here we show that αVβ8 dimers are present on stimulated human Tregs but not in other T cells, and that antibodies against αV or β8 subunits block TGF-β1 activation in vitro. We also show that αV and β8 interact with GARP/latent TGF-β1 complexes in human Tregs. Finally, a blocking antibody against β8 inhibited immunosuppression by human Tregs in a model of xenogeneic graft-vs.-host disease induced by the transfer of human T cells in immunodeficient mice. These results show that TGF-β1 activation on the surface of human Tregs implies an interaction between the integrin αVβ8 and GARP/latent TGF-β1 complexes. Immunosuppression by human Tregs can be inhibited by antibodies against GARP or against the integrin β8 subunit. Such antibodies may prove beneficial against cancer or chronic infections.

A novel role for the integrin-binding III-10 module in fibronectin matrix assembly.

PubMed

Hocking, D C; Smith, R K; McKeown-Longo, P J

1996-04-01

Fibronectin matrix assembly is a cell-dependent process which is upregulated in tissues at various times during development and wound repair to support the functions of cell adhesion, migration, and differentiation. Previous studies have demonstrated that the alpha 5 beta 1 integrin and fibronectin's amino terminus and III-1 module are important in fibronectin polymerization. We have recently shown that fibronectin's III-1 module contains a conformationally sensitive binding site for fibronectin's amino terminus (Hocking, D.C., J. Sottile, and P.J. McKeown-Longo. 1994. J. Biol. Chem. 269: 19183-19191). The present study was undertaken to define the relationship between the alpha 5 beta 1 integrin and fibronectin polymerization. Solid phase binding assays using recombinant III-10 and III-1 modules of human plasma fibronectin indicated that the III-10 module contains a conformation-dependent binding site for the III-1 module of fibronectin. Unfolded III-10 could support the formation of a ternary complex containing both III-1 and the amino-terminal 70-kD fragment, suggesting that the III-1 module can support the simultaneous binding of III-10 and 70 kD. Both unfolded III-10 and unfolded III-1 could support fibronectin binding, but only III-10 could promote the formation of disulfide-bonded multimers of fibronectin in the absence of cells. III-10-dependent multimer formation was inhibited by both the anti-III-1 monoclonal antibody, 9D2, and amino-terminal fragments of fibronectin. A fragment of III-10, termed III-10/A, was able to block matrix assembly in fibroblast monolayers. Similar results were obtained using the III-10A/RGE fragment, in which the RGD site had been mutated to RGE, indicating that III-I0/A was blocking matrix assembly by a mechanism distinct from disruption of integrin binding. Texas red-conjugated recombinant III-1,2 localized to beta 1-containing sites of focal adhesions on cells plated on fibronectin or the III-9,10 modules of fibronectin. Monoclonal antibodies against the III-1 or the III-9,10 modules of fibronectin blocked binding of III-1,2 to cells without disrupting focal adhesions. These data suggest that a role of the alpha 5 beta 1 integrin in matrix assembly is to regulate a series of sequential self-interactions which result in the polymerization of fibronectin.

Alpha-v Integrin Targeted PET Imaging of Breast Cancer Angiogenesis and Low-Dose Metronomic Anti-Angiogenic Chemotherapy Efficacy

DTIC Science & Technology

2008-08-01

AD_________________ Award Number: W81XWH-04-1-0697 TITLE: Alpha -v Integrin Targeted PET Imaging of...SUBTITLE 5a. CONTRACT NUMBER Alpha -v Integrin Targeted PET Imaging of Breast Cancer Angiogenesis and Low- Dose Metronomic Anti-Angiogenic...Evaluation of biodistribution and anti-tumor effect of a dimeric RGD peptide-paclitaxel conjugate in mice with breast cancer” was published in Eur J Nucl

Preparation of arginine-glycine-aspartic acid-modified biopolymeric nanoparticles containing epigalloccatechin-3-gallate for targeting vascular endothelial cells to inhibit corneal neovascularization.

PubMed

Chang, Che-Yi; Wang, Ming-Chen; Miyagawa, Takuya; Chen, Zhi-Yu; Lin, Feng-Huei; Chen, Ko-Hua; Liu, Guei-Sheung; Tseng, Ching-Li

2017-01-01

Neovascularization (NV) of the cornea can disrupt visual function, causing ocular diseases, including blindness. Therefore, treatment of corneal NV has a high public health impact. Epigalloccatechin-3-gallate (EGCG), presenting antiangiogenesis effects, was chosen as an inhibitor to treat human vascular endothelial cells for corneal NV treatment. An arginine-glycine-aspartic acid (RGD) peptide-hyaluronic acid (HA)-conjugated complex coating on the gelatin/EGCG self-assembly nanoparticles (GEH-RGD NPs) was synthesized for targeting the α v β 3 integrin on human umbilical vein endothelial cells (HUVECs) in this study, and a corneal NV mouse model was used to evaluate the therapeutic effect of this nanomedicine used as eyedrops. HA-RGD conjugation via COOH and amine groups was confirmed by 1 H-nuclear magnetic resonance and Fourier-transform infrared spectroscopy. The average diameter of GEH-RGD NPs was 168.87±22.5 nm with positive charge (19.7±2 mV), with an EGCG-loading efficiency up to 95%. Images of GEH-RGD NPs acquired from transmission electron microscopy showed a spherical shape and shell structure of about 200 nm. A slow-release pattern was observed in the nanoformulation at about 30% after 30 hours. Surface plasmon resonance confirmed that GEH-RGD NPs specifically bound to the integrin α v β 3 . In vitro cell-viability assay showed that GEH-RGD efficiently inhibited HUVEC proliferation at low EGCG concentrations (20 μg/mL) when compared with EGCG or non-RGD-modified NPs. Furthermore, GEH-RGD NPs significantly inhibited HUVEC migration down to 58%, lasting for 24 hours. In the corneal NV mouse model, fewer and thinner vessels were observed in the alkali-burned cornea after treatment with GEH-RGD NP eyedrops. Overall, this study indicates that GEH-RGD NPs were successfully developed and synthesized as an inhibitor of vascular endothelial cells with specific targeting capacity. Moreover, they can be used in eyedrops to inhibit angiogenesis in corneal NV mice.

Preparation of arginine–glycine–aspartic acid-modified biopolymeric nanoparticles containing epigalloccatechin-3-gallate for targeting vascular endothelial cells to inhibit corneal neovascularization

PubMed Central

Chang, Che-Yi; Wang, Ming-Chen; Miyagawa, Takuya; Chen, Zhi-Yu; Lin, Feng-Huei; Chen, Ko-Hua; Liu, Guei-Sheung; Tseng, Ching-Li

2017-01-01

Neovascularization (NV) of the cornea can disrupt visual function, causing ocular diseases, including blindness. Therefore, treatment of corneal NV has a high public health impact. Epigalloccatechin-3-gallate (EGCG), presenting antiangiogenesis effects, was chosen as an inhibitor to treat human vascular endothelial cells for corneal NV treatment. An arginine–glycine–aspartic acid (RGD) peptide–hyaluronic acid (HA)-conjugated complex coating on the gelatin/EGCG self-assembly nanoparticles (GEH-RGD NPs) was synthesized for targeting the αvβ3 integrin on human umbilical vein endothelial cells (HUVECs) in this study, and a corneal NV mouse model was used to evaluate the therapeutic effect of this nanomedicine used as eyedrops. HA-RGD conjugation via COOH and amine groups was confirmed by 1H-nuclear magnetic resonance and Fourier-transform infrared spectroscopy. The average diameter of GEH-RGD NPs was 168.87±22.5 nm with positive charge (19.7±2 mV), with an EGCG-loading efficiency up to 95%. Images of GEH-RGD NPs acquired from transmission electron microscopy showed a spherical shape and shell structure of about 200 nm. A slow-release pattern was observed in the nanoformulation at about 30% after 30 hours. Surface plasmon resonance confirmed that GEH-RGD NPs specifically bound to the integrin αvβ3. In vitro cell-viability assay showed that GEH-RGD efficiently inhibited HUVEC proliferation at low EGCG concentrations (20 μg/mL) when compared with EGCG or non-RGD-modified NPs. Furthermore, GEH-RGD NPs significantly inhibited HUVEC migration down to 58%, lasting for 24 hours. In the corneal NV mouse model, fewer and thinner vessels were observed in the alkali-burned cornea after treatment with GEH-RGD NP eyedrops. Overall, this study indicates that GEH-RGD NPs were successfully developed and synthesized as an inhibitor of vascular endothelial cells with specific targeting capacity. Moreover, they can be used in eyedrops to inhibit angiogenesis in corneal NV mice. PMID:28115846

Non-Invasive Detection of Lung Inflammation by Near-Infrared Fluorescence Imaging Using Bimodal Liposomes.

PubMed

Desu, Hari R; Wood, George C; Thoma, Laura A

2016-01-01

Acute lung injury (ALI) and its more severe form, acute respiratory distress syndrome results in respiratory obstruction and severe lung inflammation. Critical characteristics of ALI are alveolar edema, infiltration of leukocytes (neutrophils and monocytes), release of pro-inflammatory cytokines and chemokines into broncho-alveolar lavage fluid, and activation of integrin receptors. The purpose of the study was to demonstrate non-invasive detection of lung inflammation using integrin receptor targeted fluorescence liposomes. An inflammation similar to that observed in ALI was elicited in rodents by intra-tracheal instillation of interleukin-1beta (IL-1beta). Cyclic arginine glycine-(D)-aspartic acid-peptide (cRGD-peptide) grafted fluorescence liposomes were administered to ALI induced male Sprague-Dawley rats for targeting lung integrin receptors. Near-infrared fluorescence imaging (NIRFI) was applied for visualization and quantitation of lung inflammation. NIRFI signals were correlated with inflammatory cellular and biochemical markers of lungs. A positive correlation was observed between NIRF signals and lung inflammation markers. Compared to control group, an intense NIRF signal was observed in ALI induced rats in the window 6-24 h post-IL-1beta instillation. Interaction of integrin receptors with targeted liposomes was assumed to contribute to intense NIRF signal. RT-PCR studies showed an elevated lung expression of alphavbeta5 integrin receptors, 12 h post-IL-1beta instillation. In vitro studies demonstrated integrin receptor specificity of targeted liposomes. These targeted liposomes showed binding to alphavbeta5 integrin receptors expressed on alveolar cells. Non-invasive detection of lung inflammation was demonstrated using a combination of integrin receptor targeting and NIRFI.

Dynamic Seeding of Perfusing Human Umbilical Vein Endothelial Cells (HUVECs) onto Dual-Function Cell Adhesion Ligands: Arg-Gly-Asp (RGD)-Streptavidin and Biotinylated Fibronectin

PubMed Central

Anamelechi, Charles C.; Clermont, Edward C.; Novak, Matthew T.; Reichert, William M.

2014-01-01

Surfaces decorated with high affinity ligands can be used to facilitate rapid attachment of endothelial cells; however, standard equilibrium cell detachment studies are poorly suited for assessing these initial adhesion events. Here, a dynamic seeding and cell retention method was used to examine the initial attachment of perfusing human umbilical vein endothelial cells (HUVECs) to bare Teflon-AF substrates, substrates pre-adsorbed with fibronectin alone, or substrates co-pre-adsorbed with two dual-function cell-adhesion ligands: biotinylated fibronectin (bFN) and RGD-streptavidin mutant (RGD-SA). Cell attachment was evaluated as a function of cell trypsinization (integrin digestion), surface protein formulation, and cell perfusion rate. Surfaces co-pre-adsorbed with bFN and RGD-SA showed the highest density of attached cells after 8 min of perfusion and the highest percent retention when subjected to shear flow at 60 dynes/cm2 for 2 min. Surfaces with no ligand treatment showed the lowest cell attachment and retention under flow in all cases. HUVECs trypsinized with mild 0.025% trypsin/ethylenediaminetetraacetic acid (EDTA) showed greater cell adhesion after perfusion and higher percent retention after shear flow than those trypsinized using harsher 0.05% trypsin/EDTA. The preferential affinities of the two dual-function ligands for α5β1 and αvβ3 integrins were also examined by surface plasmon resonance (SPR) spectroscopy. The dynamic cell seeding studies confirmed that the dual-function ligand system promotes HUVEC adhesion and retention at short time points when tested using a perfusion assay. SPR studies showed that the two ligands exhibited equal affinity for both α5β1 and αvβ3 integrins but that the combined ligands bound more total integrins than the two ligands tested separately. PMID:19348476

Development of a novel cyclic RGD peptide for multiple targeting approaches of liposomes to tumor region.

PubMed

Amin, Mohamadreza; Mansourian, Mercedeh; Koning, Gerben A; Badiee, Ali; Jaafari, Mahmoud Reza; Ten Hagen, Timo L M

2015-12-28

Liposomes containing cytotoxic agents and targeted with Arg-Gly-Asp based peptides have frequently been used against αvβ3 integrin on tumor neovasculature. However, like many other ligand modified liposomes these preparations suffered from enhanced uptake by the reticulo endothelial system (RES) and off-targeted interaction with integrin receptors vastly expressed in normal organs causing poor biodistribution and toxic effects. Here we mainly focus on development of a RGD-modified liposomal delivery system to enhance both targeting selectivity and tumor uptake. First, sterically stabilized liposomal doxorubicin (SSLD) prepared and decorated with cRGDfK and RGDyC peptides differ in their physical properties. Stability assessments as well as in vitro and in vivo studies revealed that increasing the peptide hydrophobicity promotes the therapeutic efficacy of RGD-SSLD in a C-26 tumor model due to decreased recognition by RES and opsonization and limited off-targeted interactions. Then a novel N-methylated RGD peptide was designed and its capability in targeting integrin presenting cells was comprehensively assessed both in vitro and in vivo. RGDf[N-methyl]C promotes the liposome internalization by HUVEC via integrin mediated endocytosis. Intravital microscopy in window chamber bearing mice illustrated the capability of RGDf[N-methyl]C-liposomes in targeting both tumor vasculature and tumor cells in murine B16F0 and human BLM tumor models. Quantitative biodistribution in mice bearing B16F0 tumor revealed its high affinity to tumor with no considerable affinity to normal organs. Treatment by high dose of RGDf[N-methyl]C-SSLD was found more effective than non-targeted SSLD and no toxic side effect was observed. In conclusion, the RGDf[N-methyl]C-liposome was found promising in targeting tumor vasculature as well as other cells inside the tumor. Copyright © 2015 Elsevier B.V. All rights reserved.

Activation of the mitogen-activated protein kinase pathway by bone sialoprotein regulates osteoblast differentiation.

PubMed

Gordon, Jonathan A R; Hunter, Graeme K; Goldberg, Harvey A

2009-01-01

Bone sialoprotein (BSP) is an abundant protein in the extracellular matrix of bone that has been suggested to have several different physiological functions, including the nucleation of hydroxyapatite (HA), promotion of cell attachment and binding of collagen. Studies in our lab have demonstrated that increased expression of BSP in osteoblast cells can increase expression of the osteoblast-related genes Runx2 and Osx as well as alkaline phosphatase and osteocalcin and increase matrix mineralization. To determine the molecular mechanisms responsible for the BSP-mediated increase in osteoblastic differentiation, several functional domain mutants of BSP were expressed in primary rat bone osteoblastic cells, including the contiguous glutamic acid sequences (polyGlu) and the arginine-glycine-aspartic acid (RGD) motif. Markers of osteoblast differentiation, including matrix mineralization and alkaline phosphatase staining, were increased in cells expressing BSP mutants of the polyGlu sequences but not in cells expressing RGD-mutated BSP. We also determined the dependence on integrin-associated pathways in promoting BSP-mediated differentiation responses in osteoblasts by demonstrating the activation of focal adhesion kinase, MAP kinase-associated proteins ERK1/2, ribosomal s6 kinase 2 and the AP-1 protein cFos. Thus, the mechanism regulating osteoblast differentiation by BSP was determined to be dependent on integrin-mediated intracellular signaling pathways. Copyright 2008 S. Karger AG, Basel.

Fluorescence lifetime imaging to differentiate bound from unbound ICG-cRGD both in vitro and in vivo

NASA Astrophysics Data System (ADS)

Stegehuis, Paulien L.; Boonstra, Martin C.; de Rooij, Karien E.; Powolny, François E.; Sinisi, Riccardo; Homulle, Harald; Bruschini, Claudio; Charbon, Edoardo; van de Velde, Cornelis J. H.; Lelieveldt, Boudewijn P. F.; Vahrmeijer, Alexander L.; Dijkstra, Jouke; van de Giessen, Martijn

2015-03-01

Excision of the whole tumor is crucial, but remains difficult for many tumor types. Fluorescence lifetime imaging could be helpful intraoperative to differentiate normal from tumor tissue. In this study we investigated the difference in fluorescence lifetime imaging of indocyanine green coupled to cyclic RGD free in solution/serum or bound to integrins e.g. in tumors. The U87-MG glioblastoma cell line, expressing high integrin levels, was cultured to use in vitro and to induce 4 subcutaneous tumors in a-thymic mice (n=4). Lifetimes of bound and unbound probe were measured with an experimental time-domain single-photon avalanche diode array (time resolution <100ps). In vivo measurements were taken 30-60 minutes after intravenous injection, and after 24 hours. The in vitro lifetime of the fluorophores was similar at different concentrations (20, 50 and 100μM) and showed a statistically significant higher lifetime (p<0.001) of bound probe compared to unbound probe. In vivo, lifetimes of the fluorophores in tumors were significantly higher (p<0.001) than at the control site (tail) at 30-60 minutes after probe injection. Lifetimes after 24 hours confirmed tumor-specific binding (also validated by fluorescence intensity images). Based on the difference in lifetime imaging, it can be concluded that it is feasible to separate between bound and unbound probes in vivo.

Bone sialoprotein does not interact with pro-gelatinase A (MMP-2) or mediate MMP-2 activation.

PubMed

Hwang, Queena; Cheifetz, Sela; Overall, Christopher M; McCulloch, Christopher A; Sodek, Jaro

2009-04-22

A recent model for activation of the zymogen form of matrix metalloproteinase 2 (MMP-2, also known as gelatinase A) has suggested that interactions between the SIBLING protein bone sialoprotein (BSP) and MMP-2 leads to conformational change in MMP-2 that initiates the conversion of the pro-enzyme into a catalytically active form. This model is particularly relevant to cancer cell metastasis to bone since BSP, bound to the alphavbeta3 integrin through its arginine-glycine-aspartic acid motif, could recruit MMP-2 to the cell surface. We critically assessed the relationship between BSP and proMMP-2 and its activation using various forms of recombinant and purified BSP and MMP-2. Gelatinase and collagenase assays, fluorescence binding assays, real-time PCR, cell culture and pull-down assays were employed to test the model. Studies with a fluorogenic substrate for MMP-2 showed no activation of proMMP-2 by BSP. Binding and pull-down assays demonstrated no interaction between MMP-2 and BSP. While BSP-mediated invasiveness has been shown to depend on its integrin-binding RGD sequence, analysis of proMMP-2 activation and the level of membrane type 1 (MT1)-MMP in cells grown on a BSP substratum showed that the BSP-alphavbeta3 integrin interaction does not induce the expression of MT1-MMP. These studies do not support a role for BSP in promoting metastasis through interactions with pro-MMP-2.

Matched rabbit monoclonal antibodies against αv-series integrins reveal a novel αvβ3-LIBS epitope, and permit routine staining of archival paraffin samples of human tumors

PubMed Central

Goodman, Simon L.; Grote, Hans Juergen; Wilm, Claudia

2012-01-01

Summary The relationship between integrin expression and function in pathologies is often contentious as comparisons between human pathological expression and expression in cell lines is difficult. In addition, the expression of even integrins αvβ6 and αvβ8 in tumor cell lines is not comprehensively documented. Here, we describe rabbit monoclonal antibodies (RabMabs) against the extracellular domains of αv integrins that react with both native integrins and formalin fixed, paraffin embedded (FFPE) human tissues. These RabMabs, against αvβ3 (EM22703), αvβ5 (EM09902), αvβ6 (EM05201), αvβ8 (EM13309), and pan-αv (EM01309), recognize individual integrin chains in Western blots and in flow cytometry. EM22703 detected a ligand-induced binding site (LIBS), reporting an epitope enhanced by the binding of an RGD-peptide to αvβ3. αvβ8 was rarely expressed in human tumor specimens, and weakly expressed in non-small-cell lung carcinoma (NSCLC). However, ovarian carcinoma cell lines expressed αvβ8, as did some melanoma cells, whereas U87MG glioma lacked αvβ8 expression. We observed an unexpected strong expression of αvβ6 in tumor samples of invasive ductal breast adenoma, colorectal carcinoma (CRC), and NSCLC. αvβ3 was strongly expressed in some invasive NSCLC cohorts. Interestingly, PC3 prostate cell and human prostate tumors did not express αvβ3. The RabMabs stained plasma membranes in FFPE-immunohistochemistry (IHC) samples of tumor cell lines from lung, ovary, colon, prostate, squamous cell carcinoma of head and neck (SCCHN), breast, and pancreas carcinomas. The RabMabs are unique tools for probing αv integrin biology, and suggest that especially αvβ6 and αvβ8 biologies still have much to reveal. PMID:23213423

Modulating macrophage response to biomaterials

NASA Astrophysics Data System (ADS)

Zaveri, Toral

Macrophages recruited to the site of biomaterial implantation are the primary mediators of the chronic foreign body response to implanted materials. Since foreign body response limits performance and functional life of numerous implanted biomaterials/medical devices, various approaches have been investigated to modulate macrophage interactions with biomaterial surfaces to mitigate this response. In this work we have explored two independent approaches to modulate the macrophage inflammatory response to biomaterials. The first approach targets surface integrins, cell surface receptors that mediate cell adhesion to biomaterials through adhesive proteins spontaneously adsorbed on biomaterial surfaces. The second approach involves surface modification of biomaterials using nanotopographic features since nanotopography has been reported to modulate cell adhesion and viability in a cell type-dependent manner. More specifically, Zinc Oxide (ZnO) nanorod surface was investigated for its role in modulating macrophage adhesion and survival in vitro and foreign body response in vivo. For the first approach, we have investigated the role of integrin Mac-1 and RGD-binding integrins in the in-vivo osteolysis response and macrophage inflammatory processes of phagocytosis as well as inflammatory cytokine secretion in response to particulate biomaterials. We have also investigated the in vivo foreign body response (FBR) to subcutaneously implanted biomaterials by evaluating the thickness of fibrous capsule formed around the implants after 2 weeks of implantation. The role of Mac-1 integrin was isolated using a Mac-1 KO mouse and comparing it to a WT control. The role of RGD binding integrins in FBR was investigated by coating the implanted biomaterial with ELVAX(TM) polymer loaded with Echistatin which contains the RGD sequence. For the in-vivo osteolysis study and to study the in-vitro macrophage response to particulate biomaterials, we used the RGD peptide encapsulated in ELVAX(TM) and dissolved in macrophage media respectively. By studying the phagocytosis, inflammatory and FBR of macrophages from integrin knockout mice, as well as using various integrin blocking techniques we aim to identify the role of various integrins in macrophage inflammatory response. These integrins can serve as therapeutic targets for mitigating this inflammatory response and improve functional life of implanted biomaterials. Zinc oxide (ZnO) has been investigated in a number of biomedical applications and surfaces presenting well-controlled nanorod structures of ZnO have recently been developed. In order to investigate the influence of nanotopography on macrophage adhesive response, we evaluated macrophage adhesion and viability on ZnO nanorods, compared to a relatively flat sputtered ZnO controls and using glass substrates for reference. We found that although macrophages are capable of initially adhering to and spreading on ZnO nanorod substrates, the number of adherent macrophages on ZnO nanorods was reduced compared to ZnO flat substrate and glass. While these data suggest nanotopography may modulate macrophage adhesion, reduced cell viability on both sputtered and nanorod ZnO substrate indicates appreciable toxicity associated with ZnO. In order to determine long-term physiological responses, ZnO nanorodcoated and sputtered ZnO-coated polyethylene terephthalate (PET) discs were implanted subcutaneously in mice for 14 days. Upon implantation, both ZnO-coated discs resulted in a discontinuous cellular fibrous capsule indicative of unresolved inflammation, in contrast to uncoated PET discs, which resulted in typical foreign body capsule formation. Hence although ZnO substrates presenting nanorod topography have previously been shown to modulate cellular adhesion in a topography-dependent fashion for specific cell types, this work demonstrates that for primary murine macrophages, cell adhesion and viability correlate to both nanotopography and toxicity of dissolved Zn, parameters which are likely interdependent. Considering the toxicity of ZnO nanorod surface towards macrophages, their role as an antibacterial surface was explored. Antibacterial coating approaches are being investigated to modify implants to reduce bacterial adhesion and viability in order to reduce implant-associated infection. To assess the efficacy of ZnO nanorod surfaces as an anti-bacterial coating, we evaluated bacterial adhesion and viability, compared to sputtered ZnO and glass substrates. Common implant-associated pathogens, Pseudomonas aeruginosa and Staphylococcus epidermidis were investigated. ZnO nanorod surface and sputtered ZnO demonstrated a significant bactericidal effect, killing respectively 2.5x and 1.7x times the number of bacteria dead on glass. A similar bactericidal effect of ZnO substrates on S. epidermidis was also evident, with sputtered ZnO and ZnO nanorod substrates killing respectively 22x and 32x times bacteria dead on glass. These data support the further investigation of ZnO nanorod coatings for bacterial adhesion resistance and bactericidal properties.

A Low Molecular Weight Protein from the Sea Anemone Anemonia viridis with an Anti-Angiogenic Activity

PubMed Central

Loret, Erwann P.; Luis, José; Nuccio, Christopher; Villard, Claude; Mansuelle, Pascal; Lebrun, Régine; Villard, Pierre Henri

2018-01-01

Sea anemones are a remarkable source of active principles due to a decentralized venom system. New blood vessel growth or angiogenesis is a very promising target against cancer, but the few available antiangiogenic compounds have limited efficacy. In this study, a protein fraction, purified from tentacles of Anemonia viridis, was able to limit endothelial cells proliferation and angiogenesis at low concentration (14 nM). Protein sequences were determined with Edman degradation and mass spectrometry in source decay and revealed homologies with Blood Depressing Substance (BDS) sea anemones. The presence of a two-turn alpha helix observed with circular dichroism and a trypsin activity inhibition suggested that the active principle could be a Kunitz-type inhibitor, which may interact with an integrin due to an Arginine Glycin Aspartate (RGD) motif. Molecular modeling showed that this RGD motif was well exposed to solvent. This active principle could improve antiangiogenic therapy from existing antiangiogenic compounds binding on the Vascular Endothelial Growth Factor (VEGF). PMID:29671760

The anti-tumour activity of rLj-RGD4, an RGD toxin protein from Lampetra japonica, on human laryngeal squamous carcinoma Hep-2 cells in nude mice.

PubMed

Shao, Fangyu; Lv, Mei; Zheng, Yuanyuan; Jiang, Junshu; Wang, Yue; Lv, Li; Wang, Jihong

2015-12-01

The objective of this study is to investigate the antiproliferative activity and mechanism of integrin-binding rLj-RGD4 in a Hep-2 human laryngeal carcinoma-bearing nude mouse model. Human laryngeal squamous carcinoma cells (Hep-2) were inoculated subcutaneously into the axilla of nude mice to generate a Hep-2 human laryngeal carcinoma-bearing nude mouse model. When the Hep-2 xenograft model was successfully established, the animals were randomly separated into five groups. Three groups were treated with different dosages of rLj-RGD4. Cisplatin was administered to the positive control group, and normal saline (NaCl) was administered to the negative control group for 3 weeks. The body weights and the survival of the nude mice were evaluated, and the volumes and weights of the solid tumours were measured. The mechanism underlying rLj-RGD4 inhibition of tumour growth in transplanted Hep-2 human laryngeal carcinoma-bearing nude mice was evaluated by haematoxylin-eosin (HE) staining, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling (TUNEL), measurement of intratumoural microvessel density (MVD), Western blotting, and quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The tumour volumes and weights of the treatment groups were reduced compared with the model group, and survival times were improved by rLj-RGD4 treatment in Hep-2 human laryngeal carcinoma-bearing nude mice. The number of apoptotic Hep-2 human cells and intratumoural MVD significantly decreased after the administration of rLj-RGD4. In the xenograft tissue of animals treated with rLj-RGD4, FAK, PI3K, and Akt expression was unaltered, whereas P-FAK, P-PI3K, Bcl-2, P-Akt, and VEGF levels were down-regulated. In addition, activated caspase-3, activated caspase-9, and Bax levels were up-regulated. rLj-RGD4 exhibits potent in vivo activity and inhibits the growth of transplanted Hep-2 human laryngeal carcinoma cells in a nude mouse model. Thus, these results indicate that the recombinant RGD toxin protein rLj-RGD4 may serve as a potent clinical therapy for human laryngeal squamous carcinoma. Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Hyaluronic acid and Arg-Gly-Asp peptide modified Graphene oxide with dual receptor-targeting function for cancer therapy.

PubMed

Guo, Yufeng; Xu, Haixing; Li, Yiping; Wu, Fengzheng; Li, Yixuan; Bao, Yun; Yan, Xiumei; Huang, Zhijun; Xu, Peihu

2017-07-01

Graphene oxide (GO) modified with hyaluronic acid (HA) and Arg-gly-asp peptide (RGD) was designed as a dual-receptor targeting drug delivery system to enhance the specificity and efficiency of anticancer drug delivery. Firstly, GO-HA-RGD conjugate was characterized to reveal its structure and morphology. Whereafter, doxorubicin (Dox) as a model drug was loaded on GO-HA-RGD carrier, which displayed a high loading rate (72.9%, GO:Dox (w/w) = 1:1), pH-response and sustained drug release behavior. Cytotoxicity experiments showed that GO-HA-RGD possessed excellent biocompatibility towards SKOV-3 and HOSEpiC cells. Additionally, the GO-HA-RGD/Dox had a stronger cytotoxicity for SKOV-3 cells than either GO-HA/Dox (single receptor) or GO/Dox (no receptor). Moreover, celluar uptake studies illustrated that GO-HA-RGD conjugate could be effectively taken up by SKOV-3 cells via a synergic effect of CD44-HA and integrin-RGD mediated endocytosis. Hence, GO-HA-RGD nanocarrier is able to be a promising platform for targeted cancer therapeutic.

Investigation of early cell–surface interactions of human mesenchymal stem cells on nanopatterned β-type titanium–niobium alloy surfaces

PubMed Central

Medda, Rebecca; Helth, Arne; Herre, Patrick; Pohl, Darius; Rellinghaus, Bernd; Perschmann, Nadine; Neubauer, Stefanie; Kessler, Horst; Oswald, Steffen; Eckert, Jürgen; Spatz, Joachim P.; Gebert, Annett; Cavalcanti-Adam, Elisabetta A.

2014-01-01

Multi-potent adult mesenchymal stem cells (MSCs) derived from bone marrow have therapeutic potential for bone diseases and regenerative medicine. However, an intrinsic heterogeneity in their phenotype, which in turn results in various differentiation potentials, makes it difficult to predict the response of these cells. The aim of this study is to investigate initial cell–surface interactions of human MSCs on modified titanium alloys. Gold nanoparticles deposited on β-type Ti–40Nb alloys by block copolymer micelle nanolithography served as nanotopographical cues as well as specific binding sites for the immobilization of thiolated peptides present in several extracellular matrix proteins. MSC heterogeneity persists on polished and nanopatterned Ti–40Nb samples. However, cell heterogeneity and donor variability decreased upon functionalization of the gold nanoparticles with cyclic RGD peptides. In particular, the number of large cells significantly decreased after 24 h owing to the arrangement of cell anchorage sites, rather than peptide specificity. However, the size and number of integrin-mediated adhesion clusters increased in the presence of the integrin-binding peptide (cRGDfK) compared with the control peptide (cRADfK). These results suggest that the use of integrin ligands in defined patterns could improve MSC-material interactions, not only by regulating cell adhesion locally, but also by reducing population heterogeneity. PMID:24501674

The rescue and evaluation of FLAG and HIS epitope-tagged Asia 1 type foot-and-mouth disease viruses.

PubMed

Yang, Bo; Yang, Fan; Zhang, Yan; Liu, Huanan; Jin, Ye; Cao, Weijun; Zhu, Zixiang; Zheng, Haixue; Yin, Hong

2016-02-02

The VP1 G-H loop of the foot-and-mouth disease virus (FMDV) contains the primary antigenic site, as well as an Arg-Gly-Asp (RGD) binding motif for the αv-integrin family of cell surface receptors. We anticipated that introducing a foreign epitope tag sequence downstream of the RGD motif would be tolerated by the viral capsid and would not destroy the antigenic site of FMDV. In this study, we have designed, generated, and characterized two recombinant FMDVs with a FLAG tag or histidine (HIS) inserted in the VP1 G-H loop downstream of the RGD motif +9 position. The tagged viruses were genetically stable and exhibited similar growth properties with their parental virus. What is more, the recombinant viruses rFMDV-FLAG and rFMDV-HIS showed neutralization sensitivity to FMDV type Asia1-specific mAbs, as well as to polyclonal antibodies. Additionally, the r1 values of the recombinant viruses were similar to that of the parental virus, indicating that the insertion of FLAG or HIS tag sequences downstream of the RGD motif +9 position do not eradicate the antigenic site of FMDV and do not affect its antigenicity. These results indicated that the G-H loop of Asia1 FMDV is able to effectively display the foreign epitopes, making this a potential approach for novel FMDV vaccines development. Copyright © 2015 Elsevier B.V. All rights reserved.

Adhesion of MC3T3-E1 cells to bone sialoprotein and bone osteopontin specifically bound to collagen I.

PubMed

Bernards, Matthew T; Qin, Chunlin; Ratner, Buddy D; Jiang, Shaoyi

2008-09-01

Bone sialoprotein (BSP) and bone osteopontin (OPN) are members of the SIBLING (small integrin-binding ligand, N-linked glycoproteins) family of proteins commonly found in mineralized tissues. Previously, OPN was shown to exhibit a preferential orientation for MC3T3-E1 cell adhesion when it was specifically bound to collagen. In this work, the orientation of BSP under similar circumstances is examined and compared with OPN. Radiolabeled adsorption isotherms were obtained for BSP bound to both tissue culture polystyrene (TCPS) and collagen-coated TCPS. The results show that collagen has the capacity to bind almost twice as much OPN under identical conditions. An in vitro MC3T3-E1 cell adhesion assay was then performed to compare the cell binding ability of BSP on either TCPS or collagen-coated TCPS with identical amounts of adsorbed protein. It was found that there is no significant difference in the cell binding ability of BSP on either of the substrates. For cell binding studies on collagen-coated TCPS, it was shown that there are a greater number of cells bound to substrates with adsorbed OPN as compared with BSP. The preferable orientation of OPN for cell binding coupled with the higher binding capability of collagen for OPN indicates that OPN is more important than BSP for osteoblast adhesion to the collagen matrix. In addition, a cell inhibition assay was performed to show that all of the cell binding that occurred throughout these studies was dependent upon integrin interactions with the RGD cell binding moiety.

Adhesion of MC3T3-E1 cells to bone sialoprotein and bone osteopontin specifically bound to collagen I

PubMed Central

Bernards, Matthew T.; Qin, Chunlin; Ratner, Buddy D.; Jiang, Shaoyi

2009-01-01

Bone sialoprotein (BSP) and bone osteopontin (OPN) are members of the SIBLING (small integrin-binding ligand, N-linked glycoproteins) family of proteins commonly found in mineralized tissues. Previously, OPN was shown to exhibit a preferential orientation for MC3T3-E1 cell adhesion when it was specifically bound to collagen. In this work, the orientation of BSP under similar circumstances is examined and compared with OPN. Radiolabeled adsorption isotherms were obtained for BSP bound to both tissue culture polystyrene (TCPS) and collagen-coated TCPS. The results show that collagen has the capacity to bind almost twice as much OPN under identical conditions. An in vitro MC3T3-E1 cell adhesion assay was then performed to compare the cell binding ability of BSP on either TCPS or collagen-coated TCPS with identical amounts of adsorbed protein. It was found that there is no significant difference in the cell binding ability of BSP on either of the substrates. For cell binding studies on collagen-coated TCPS, it was shown that there are a greater number of cells bound to substrates with adsorbed OPN as compared with BSP. The preferable orientation of OPN for cell binding coupled with the higher binding capability of collagen for OPN indicates that OPN is more important than BSP for osteoblast adhesion to the collagen matrix. In addition, a cell inhibition assay was performed to show that all of the cell binding that occurred throughout these studies was dependent upon integrin interactions with the RGD cell binding moiety. PMID:18041732

Toxicity assessment of repeated intravenous injections of arginine-glycine-aspartic acid peptide conjugated CdSeTe/ZnS quantum dots in mice.

PubMed

Wang, You-Wei; Yang, Kai; Tang, Hong; Chen, Dan; Bai, Yun-Long

2014-01-01

Nanotechnology-based near-infrared quantum dots (NIR QDs) have many excellent optical properties, such as high fluorescence intensity, good fluorescence stability, and strong tissue-penetrating ability. Integrin αvβ3 is highly and specifically expressed in tumor angiogenic vessel endothelial cells of almost all carcinomas. Recent studies have shown that NIR QDs linked to peptides containing the arginine-glycine-aspartic acid (RGD) sequence (NIR QDs-RGD) can specifically target integrin αvβ3 expressed in endothelial cells of tumor angiogenic vessels in vivo, and they offer great potential for early cancer diagnosis, in vivo tumor imaging, and tumor individualized therapy. However, the toxicity profile of NIR QDs-RGD has not been reported. This study was conducted to investigate the toxicity of NIR QDs-RGD when intravenously administered to mice singly and repeatedly at the dose required for successful tumor imaging in vivo. A NIR QDs-RGD probe was prepared by linking NIR QDs with the maximum emission wavelength of 800 nm (QD800) to the RGD peptide (QD800-RGD). QD800-RGD was intravenously injected to BALB/C mice once or twice (200 pmol equivalent of QD800 for each injection). Phosphate-buffered saline solution was used as control. Fourteen days postinjection, toxicity tests were performed, including complete blood count (white blood cell, red blood cell, hemoglobin, platelets, lymphocytes, and neutrophils) and serum biochemical analysis (total protein, albumin, albumin/globulin, aspartate aminotransferase, alanine aminotransferase, and blood urea nitrogen). The coefficients of liver, spleen, kidney, and lung weight to body weight were measured, as well as their oxidation and antioxidation indicators, including superoxide dismutase, glutathione, and malondialdehyde. The organs were also examined histopathologically. After one or two intravenous injections of QD800-RGD, as compared with control, no significant differences were observed in the complete blood count; biochemical indicators of blood serum, organ coefficient, and oxidation and antioxidation indicators; and no cell necrosis or inflammation were seen in the liver, spleen, kidney, or lung through histopathological examination. Our data demonstrate that the single and repeated intravenous injection of QD800-RGD at a dose needed for successful tumor imaging in vivo is not toxic to mice. Our work lays a solid foundation for further biomedical applications of NIR QDs-RGD.

[In situ visual imaging of oral squamous cell carcinoma in mice by using near-infrared quantum dots conjugated with arginine-glycine-aspartic acid peptide fluorescent probes].

PubMed

Yunlong, Bai; Hao, Huang; Kai, Yang; Hong, Tang

2014-10-01

To investigate in situ visualization using near-infrared quantum dots (QDs) conjugated with arginine- glycine-aspartic acid (ROD) peptide fluorescent probes in oral squamous cell carcinoma (08CC). QDs with emission wavelength of 800 nm (QD800) were conjugated with RGD peptides to produce QD800-RGD fluorescent probes. Human OSCC cell line BcaCD885 was inoculated in nude mice cheeks to establish OSCC mouse models. Frozen BcaCD885 tumor slices were immunofluorescence double stained by using QD800-RGD and CD105 monoclonal antibody and were observed using a laser scanning confocal microscope. QD800-RGD was injected into the OSCC models through the tail veins, and the in situ visualization was analyzed at different time points. The mice were sacrificed 12 h after injection to isolate tumors for the ex vivo analysis of probe localization in the tumors. QD800-RGD specifically targeted the integrin avβ3 expressed in the endothelial cells of tumor angiogenic vessels in vitro and in vivo, producing clear tumor fluorescence images after intravenous injection. The most complete tumor images with maximal signal-to-noise ratios were observed 0.5 h to 6 h after injection of the probe and significantly reduced 9 h after the injection. However, the tumor image was still clearly visible at 12 h. Using intravenously injected QD800-RGD generates high quality OSCC images when integrin avβ3, which is expressed in the endothelial cells of tumor angiogenic vessels, is used as the target. The technique offers great potential in the diagnosis and individual treatment of OSCC.

The synthesis and biological evaluation of integrin receptor targeting molecules as potential radiopharmaceuticals

NASA Astrophysics Data System (ADS)

Pellegrini, Paul

This thesis reports on the synthesis, characterisation and biological evaluation of a number of metal complexes designed to interact with the alphavbeta3 integrin receptor, an important biological target that is heavily involved in angiogenesis, and thus cancer related processes. Two approaches were used to synthesise the integrin-avid targets. The first was to attach a variety of bifunctional chelators (BFC's) for the incorporation of different metal centres to a known integrin antagonist, L-748,415, developed by Merck. The BFC's used were the hydrazinonicotinamide (HYNIC) and monoamine monoamide dithiol (MAMA) systems for coordination to Tc-99m and rhenium of which was used as a characterization surrogate for the unstable Tc core. The 1,4,7,10-tetraazacyclotridecanetetraacetic acid (TRITA) BFC was attached for the inclusion of copper and lutetium. This 'conjugate' approach was designed to yield information on how the BFC and the linker length would affect the affinity for the integrin receptor. The second approach was an 'integrated' method where the chelation moiety was integral to the biologically relevant part of the molecule, which in the case of the alphavbeta3 integrin receptor, is the arginine-glycine-aspartic acid (RGD) mimicking sequence. Two complexes were created with a modified MAMA derivative placed between a benzimidazole moiety (arginine mimick) and the aspartic acid mimicking terminal carboxylic acid to see how it would affect binding while keeping the molecular weight relatively low. The molecules were tested in vitro against purified human alphavbeta3 integrin receptor protein in a solid phase receptor binding assay to evaluate their inhibition constants against a molecule of known high affinity and selectivity in [I125]L-775,219, the I125 labelled alphavbeta3 integrin antagonist. The radiolabelled analogues were also tested in vivo against the A375 human melanoma cell line transplanted into balb/c nude mice as well as Fischer rats implanted with MAT BIII rat mammary adenocarcinoma cells. Animals were imaged on a SPECT camera at various time points and compared to normal tissue to yield tumour/non-tumour uptake ratios.

CLONING, EXPRESSION, AND HEMOSTATIC ACTIVITIES OF A DISINTEGRIN, r-MOJASTIN 1, FROM THE MOHAVE RATTLESNAKE (Crotalus scutulatus scutulatus)

PubMed Central

Sánchez, Elda E.; Lucena, Sara E.; Reyes, Steven; Soto, Julio G.; Cantu, Esteban; Lopez-Johnston, Juan Carlos; Guerrero, Belsy; Salazar, Ana Maria; Rodríguez-Acosta, Alexis; Galán, Jacob A.; Tao, W. Andy; Pérez, John C.

2012-01-01

Interactions with exposed subendothelial extracellular proteins and cellular integrins (endothelial cells, platelets and lymphocytes) can cause alterations in the hemostatic system associated with atherothrombotic processes. Many molecules found in snake venoms induce pathophysiological changes in humans, cause edema, hemorrhage, and necrosis. Disintegrins are low molecular weight, non-enzymatic proteins found in snake venom that mediate changes by binding to integrins of platelets or other cells and prevent binding of the natural ligands such as fibrinogen, fibronectin or vitronectin. Disintegrins are of great biomedical importance due to their binding affinities resulting in the inhibition of platelet aggregation, adhesion of cancer cells, and induction of signal transduction pathways. RT-PCR was used to obtain a 216 bp disintegrin cDNA from a C. s. scutulatus snake venom gland. The cloned recombinant disintegrin called r-mojastin 1 codes for 71 amino acids, including 12 cysteines, and an RGD binding motif. r-Mojastin 1 inhibited platelet adhesion to fibronectin with an IC50 of 58.3 nM and ADP-induced platelet aggregation in whole blood with an IC50 of 46 nM. r-Mojastin 1 was also tested for its ability to inhibit platelet ATP release using PRP resulting with an IC50 of 95.6 nM. MALDI-TOF mass spectrum analysis showed that r-mojastin has a mass of 7.9509 kDa. PMID:20598348

Fibronectin regulates calvarial osteoblast differentiation

NASA Technical Reports Server (NTRS)

Moursi, A. M.; Damsky, C. H.; Lull, J.; Zimmerman, D.; Doty, S. B.; Aota, S.; Globus, R. K.

1996-01-01

The secretion of fibronectin by differentiating osteoblasts and its accumulation at sites of osteogenesis suggest that fibronectin participates in bone formation. To test this directly, we determined whether fibronectin-cell interactions regulate progressive differentiation of cultured fetal rat calvarial osteoblasts. Spatial distributions of alpha 5 integrin subunit, fibronectin, osteopontin (bone sialoprotein I) and osteocalcin (bone Gla-protein) were similar in fetal rat calvaria and mineralized, bone-like nodules formed by cultured osteoblasts. Addition of anti-fibronectin antibodies to cultures at confluence reduced subsequent formation of nodules to less than 10% of control values, showing that fibronectin is required for normal nodule morphogenesis. Anti-fibronectin antibodies selectively inhibited steady-state expression of mRNA for genes associated with osteoblast differentiation; mRNA levels for alkaline phosphatase and osteocalcin were suppressed, whereas fibronectin, type I collagen and osteopontin were unaffected. To identify functionally relevant domains of fibronectin, we treated cells with soluble fibronectin fragments and peptides. Cell-binding fibronectin fragments (type III repeats 6-10) containing the Arg-Gly-Asp (RGD) sequence blocked both nodule initiation and maturation, whether or not they contained a functional synergy site. In contrast, addition of the RGD-containing peptide GRGDSPK alone did not inhibit nodule initiation, although it did block nodule maturation. Thus, in addition to the RGD sequence, other features of the large cell-binding fragments contribute to the full osteogenic effects of fibronectin. Nodule formation and osteoblast differentiation resumed after anti-fibronectin antibodies or GRGDSPK peptides were omitted from the media, showing that the inhibition was reversible and the treatments were not cytotoxic. Outside the central cell-binding domain, peptides from the IIICS region and antibodies to the N terminus did not inhibit nodule formation. We conclude that osteoblasts interact with the central cell-binding domain of endogenously produced fibronectin during early stages of differentiation, and that these interactions regulate both normal morphogenesis and gene expression.

Bone sialoprotein does not interact with pro-gelatinase A (MMP-2) or mediate MMP-2 activation

PubMed Central

2009-01-01

Background A recent model for activation of the zymogen form of matrix metalloproteinase 2 (MMP-2, also known as gelatinase A) has suggested that interactions between the SIBLING protein bone sialoprotein (BSP) and MMP-2 leads to conformational change in MMP-2 that initiates the conversion of the pro-enzyme into a catalytically active form. This model is particularly relevant to cancer cell metastasis to bone since BSP, bound to the αvβ3 integrin through its arginine-glycine-aspartic acid motif, could recruit MMP-2 to the cell surface. Methods We critically assessed the relationship between BSP and proMMP-2 and its activation using various forms of recombinant and purified BSP and MMP-2. Gelatinase and collagenase assays, fluorescence binding assays, real-time PCR, cell culture and pull-down assays were employed to test the model. Results Studies with a fluorogenic substrate for MMP-2 showed no activation of proMMP-2 by BSP. Binding and pull-down assays demonstrated no interaction between MMP-2 and BSP. While BSP-mediated invasiveness has been shown to depend on its integrin-binding RGD sequence, analysis of proMMP-2 activation and the level of membrane type 1 (MT1)-MMP in cells grown on a BSP substratum showed that the BSP-αvβ3 integrin interaction does not induce the expression of MT1-MMP. Conclusion These studies do not support a role for BSP in promoting metastasis through interactions with pro-MMP-2. PMID:19386107

STAT3 Activation in Pressure-Overloaded Feline Myocardium: Role for Integrins and the Tyrosine Kinase BMX

PubMed Central

Willey, Christopher D.; Palanisamy, Arun P.; Johnston, Rebecca K.; Mani, Santhosh K.; Shiraishi, Hirokazu; Tuxworth, William J.; Zile, Michael R.; Balasubramanian, Sundaravadivel; Kuppuswamy, Dhandapani

2008-01-01

Growth, survival and cytoskeletal rearrangement of cardiomyocytes are critical for cardiac hypertrophy. Signal transducer and activator of transcription-3 (STAT3) activation is an important cardioprotective factor associated with cardiac hypertrophy. Although STAT3 activation has been reported via signaling through Janus Kinase 2 (JAK2) in several cardiac models of hypertrophy, the importance of other nonreceptor tyrosine kinases (NTKs) has not been explored. Utilizing an in vivo feline right ventricular pressure-overload (RVPO) model of hypertrophy, we demonstrate that in 48 h pressure-overload (PO) myocardium, STAT3 becomes phosphorylated and redistributed to detergent-insoluble fractions with no accompanying JAK2 activation. PO also caused increased levels of phosphorylated STAT3 in both cytoplasmic and nuclear fractions. To investigate the role of other NTKs, we used our established in vitro cell culture model of hypertrophy where adult feline cardiomyocytes are embedded three-dimensionally (3D) in type-I collagen and stimulated with an integrin binding peptide containing an Arg-Gly-Asp (RGD) motif that we have previously shown to recapitulate the focal adhesion complex (FAC) formation of 48 h RVPO. RGD stimulation of adult cardiomyocytes in vitro caused both STAT3 redistribution and activation that were accompanied by the activation and redistribution of c-Src and the TEC family kinase, BMX, but not JAK2. However, infection with dominant negative c-Src adenovirus was unable to block RGD-stimulated changes on either STAT3 or BMX. Further analysis in vivo in 48 h PO myocardium showed the presence of both STAT3 and BMX in the detergent-insoluble fraction with their complex formation and phosphorylation. Therefore, these studies indicate a novel mechanism of BMX-mediated STAT3 activation within a PO model of cardiac hypertrophy that might contribute to cardiomyocyte growth and survival. PMID:18612371

Inhibition of untransformed prostaglandin H(2) production and stretch-induced contraction of rabbit pulmonary arteries by indoxam, a selective secretory phospholipase A(2) inhibitor.

PubMed

Tanabe, Yoshiyuki; Saito, Maki; Morikawa, Yuki; Kamataki, Akihisa; Sawai, Takashi; Hirose, Masamichi; Nakayama, Koichi

2011-01-01

Involvement of secretory phospholipase A(2) (sPLA(2)) in the stretch-induced production of untransformed prostaglandin H(2) (PGH(2)) in the endothelium of rabbit pulmonary arteries was investigated. The stretch-induced contraction was significantly inhibited by indoxam, a selective inhibitor for sPLA(2), and NS-398, a selective inhibitor for cyclooxygenase-2 (COX-2). Indoxam inhibited the RGD-sensitive-integrin-independent production of untransformed PGH(2), but did not affect the RGD-sensitive-integrin-dependent production of thromboxane A(2) (TXA(2)). These results suggest that the stretch-induced contraction and untransformed PGH(2) production was mediated by sPLA(2)-COX-2 pathway, making it a new possible target for pharmacological intervention of pulmonary artery contractility.

Tumor radiosensitization by monomethyl auristatin E: mechanism of action and targeted delivery.

PubMed

Buckel, Lisa; Savariar, Elamprakash N; Crisp, Jessica L; Jones, Karra A; Hicks, Angel M; Scanderbeg, Daniel J; Nguyen, Quyen T; Sicklick, Jason K; Lowy, Andrew M; Tsien, Roger Y; Advani, Sunil J

2015-04-01

Intrinsic tumor resistance to radiotherapy limits the efficacy of ionizing radiation (IR). Sensitizing cancer cells specifically to IR would improve tumor control and decrease normal tissue toxicity. The development of tumor-targeting technologies allows for developing potent radiosensitizing drugs. We hypothesized that the anti-tubulin agent monomethyl auristatin E (MMAE), a component of a clinically approved antibody-directed conjugate, could function as a potent radiosensitizer and be selectively delivered to tumors using an activatable cell-penetrating peptide targeting matrix metalloproteinases and RGD-binding integrins (ACPP-cRGD-MMAE). We evaluated the ability of MMAE to radiosensitize both established cancer cells and a low-passage cultured human pancreatic tumor cell line using clonogenic and DNA damage assays. MMAE sensitized colorectal and pancreatic cancer cells to IR in a schedule- and dose-dependent manner, correlating with mitotic arrest. Radiosensitization was evidenced by decreased clonogenic survival and increased DNA double-strand breaks in irradiated cells treated with MMAE. MMAE in combination with IR resulted in increased DNA damage signaling and activation of CHK1. To test a therapeutic strategy of MMAE and IR, PANC-1 or HCT-116 murine tumor xenografts were treated with nontargeted free MMAE or tumor-targeted MMAE (ACPP-cRGD-MMAE). While free MMAE in combination with IR resulted in tumor growth delay, tumor-targeted ACPP-cRGD-MMAE with IR produced a more robust and significantly prolonged tumor regression in xenograft models. Our studies identify MMAE as a potent radiosensitizer. Importantly, MMAE radiosensitization can be localized to tumors by targeted activatable cell-penetrating peptides. ©2015 American Association for Cancer Research.

Tumor radiosensitization by monomethyl auristatin E: mechanism of action and targeted delivery

PubMed Central

Crisp, Jessica L.; Jones, Karra A.; Hicks, Angel M.; Scanderbeg, Daniel J.; Nguyen, Quyen T.; Sicklick, Jason K.; Lowy, Andrew M.; Tsien, Roger Y.; Advani, Sunil J.

2015-01-01

Intrinsic tumor resistance to radiotherapy limits the efficacy of ionizing radiation (IR). Sensitizing cancer cells specifically to IR would improve tumor control and decrease normal tissue toxicity. The development of tumor targeting technologies allows for developing potent radiosensitizing drugs. We hypothesized that the anti-tubulin agent monomethyl auristatin E (MMAE), a component of a clinically approved antibody-directed conjugate, could function as a potent radiosensitizer and be selectively delivered to tumors using an activatable cell penetrating peptide targeting matrix metalloproteinases and RGD binding integrins (ACPP-cRGD-MMAE). We evaluated the ability of MMAE to radiosensitize both established cancer cells and a low passage cultured human pancreatic tumor cell line using clonogenic and DNA damage assays. MMAE sensitized colorectal and pancreatic cancer cells to IR in a schedule and dose dependent manner correlating with mitotic arrest. Radiosensitization was evidenced by decreased clonogenic survival and increased DNA double strand breaks in irradiated cells treated with MMAE. MMAE in combination with IR resulted in increased DNA damage signaling and activation of CHK1. To test a therapeutic strategy of MMAE and IR, PANC-1 or HCT-116 murine tumor xenografts were treated with non-targeted free MMAE or tumor targeted MMAE (ACPP-cRGD-MMAE). While free MMAE in combination with IR resulted in tumor growth delay, tumor targeted ACPP-cRGD-MMAE with IR produced a more robust and significantly prolonged tumor regression in xenograft models. Our studies identify MMAE as a potent radiosensitizer. Importantly, MMAE radiosensitization can be localized to tumors by targeted activatable cell penetrating peptides. PMID:25681274

The angiogenic factor CCN1 promotes adhesion and migration of circulating CD34+ progenitor cells: potential role in angiogenesis and endothelial regeneration.

PubMed

Grote, Karsten; Salguero, Gustavo; Ballmaier, Matthias; Dangers, Marc; Drexler, Helmut; Schieffer, Bernhard

2007-08-01

Tissue regeneration involves the formation of new blood vessels regulated by angiogenic factors. We reported recently that the expression of the angiogenic factor CCN1 is up-regulated under various pathophysiologic conditions within the cardiovascular system. Because CD34+ progenitor cells participate in cardiovascular tissue regeneration, we investigated whether CCN1-detected for the first time in human plasma-promotes the recruitment of CD34+ progenitor cells to endothelial cells, thereby enhancing endothelial proliferation and neovascularization. In this study, we demonstrated that CCN1 and supernatants from CCN1-stimulated human CD34+ progenitor cells promoted proliferation of endothelial cells and angiogenesis in vitro and in vivo. In addition, CCN1 induced migration and transendothelial migration of CD34+ cells and the release of multiple growth factors, chemokines, and matrix metalloproteinase-9 (MMP-9) from these cells. Moreover, the CCN1-specific integrins alpha(M)beta(2) and alpha(V)beta(3) are expressed on CD34+ cells and CCN1 stimulated integrin-dependent signaling. Furthermore, integrin antagonists (RGD-peptides) suppressed both binding of CCN1 to CD34+ cells and CCN1-induced adhesion of CD34+ cells to endothelial cells. These data suggest that CCN1 promotes integrin-dependent recruitment of CD34+ progenitor cells to endothelial cells, which may contribute to paracrine effects on angiogenesis and tissue regeneration.

The promotion of osseointegration of titanium surfaces by coating with silk protein sericin.

PubMed

Nayak, Sunita; Dey, Tuli; Naskar, Deboki; Kundu, Subhas C

2013-04-01

A promising strategy to influence the osseointegration process around orthopaedic titanium implants is the immobilization of bioactive molecules. This recruits appropriate interaction between the surface and the tissue by directing cells adhesion, proliferation, differentiation and active matrix remodelling. In this study, we aimed to investigate the functionalization of metallic implant titanium with silk protein sericin. Titanium surface was immobilized with non-mulberry Antheraea mylitta sericin using glutaraldehyde as crosslinker. To analyse combinatorial effects the sericin immobilized titanium was further conjugated with integrin binding peptide sequence Arg-Gly-Asp (RGD) using ethyl (dimethylaminopropyl) carbodiimide and N-hydroxysulfosuccinimide as coupling agents. The surface of sericin immobilized titanium was characterized biophysically. Osteoblast-like cells were cultured on sericin and sericin/RGD functionalized titanium and found to be more viable than those on pristine titanium. The enhanced adhesion, proliferation, and differentiation of osteoblast cells were observed. RT-PCR analysis showed that mRNA expressions of bone sialoprotein, osteocalcin and alkaline phosphatase were upregulated in osteoblast cells cultured on sericin and sericin/RGD immobilized titanium substrates. Additionally, no significant amount of pro-inflammatory cytokines TNF-α, IL-1β and nitric oxide production were recorded when macrophages cells and osteoblast-macrophages co culture cells were grown on sericin immobilized titanium. The findings demonstrate that the sericin immobilized titanium surfaces are potentially useful bioactive coated materials for titanium-based medical implants. Copyright © 2013 Elsevier Ltd. All rights reserved.

Identification of second arginine-glycine-aspartic acid motif of ovine vitronectin as the complement C9 binding site and its implication in bacterial infection.

PubMed

Prasada, Rao T; Lakshmi, Prasanth T; Parvathy, R; Murugavel, S; Karuna, Devi; Paritosh, Joshi

2017-02-01

Vitronectin (Vn), a multifunctional protein of blood and extracellular matrix, interacts with complement C9. This interaction may modulate innate immunity. Details of Vn-C9 interactions are limited. Vn-C9 interactions were assessed by employing a goat homologous system and observing Vn binding to C9 in three different assays. Using recombinant fragments, C9 binding was mapped to the N-terminus of Vn. Site directed mutagenesis was performed to alter the second arginine glycine aspartic acid (RGD) sequence (RGD-2) of Vn. Changing R to G or D to A in RGD-2 caused significant decrease in Vn binding to C9 whereas changing of R to G in the first RGD motif (RGD-1) had no effect on Vn binding to C9. These results imply that the RGD-2 of goat Vn is involved in C9 binding. In a competitive binding assay, the presence of soluble RGD peptide inhibited Vn binding to C9 whereas heparin had no effect. Vn binding to C9 was also evaluated in terms of bacterial pathogenesis. Serum dependent inhibition of Escherichia coli growth was significantly reverted when Vn or its N-fragment were included in the assay. The C-fragment, which did not support C9 binding, also partly nullified serum-dependent inhibition of bacterial growth, probably through other serum component(s). © 2017 The Societies and John Wiley & Sons Australia, Ltd.

Evidence that a functional fertilin-like ADAM plays a role in human sperm-oolemmal interactions.

PubMed

Bronson, R A; Fusi, F M; Calzi, F; Doldi, N; Ferrari, A

1999-05-01

Fertilin is a protein initially identified in guinea pig spermatozoa; it is the prototype of a larger family of conserved, proteins designated as a disintegrin and a metalloproteinase (ADAM). These heterodimers which consist of alpha and beta subunits, containing metalloproteinase-like and disintegrin-like domains, appear to play a role in mammalian fertilization. Peptides derived from the disintegrin domains of two ADAMs, fertilin and cyritestin, interfere with gamete adhesion and sperm-egg membrane fusion in non-human species. It has been suggested that fertilin-beta binds to an oolemmal integrin, and it is proposed that the tripeptide FEE (Phe-Glu-Glu) is the integrin recognition sequence in human fertilin-beta. We evaluated whether fertilin beta plays a role in human fertilization by studying the effects of a linear octapeptide containing the FEE sequence, SFEECDLP, and a scrambled octapeptide with the same amino acids, SFPCEDEL, on the incorporation of human spermatozoa by human zona-free eggs. The effects of G4120, a potent RGD-containing (Arg-Gly-Asp) thioether-bridged cyclic peptide which blocks both fibronectin and vitronectin receptors, and the relationship between FEE- and RGD-receptor interactions on sperm-egg interactions were also studied. The FEE-containing peptide, but not the scrampled peptide, inhibited sperm adhesion to oocytes and their penetration, over the range 1-5 microM. The inhibition induced by SFEECDLP was reversible and occurred only in the presence of peptide itself. The G4120 peptide exhibited 10-fold less inhibitory effects on sperm adhesion and penetration than did SFEECDLP. When combined, SFEECDLP and G4120 exhibited strong inhibition of both adhesion and penetration at concentrations that individually had been ineffective, suggesting co-operation between the two receptor-ligand interactions during fertilization. We propose that a fertilin-like molecule is functionally active on human spermatozoa and that its interaction with an oolemmal integrin receptor plays a role in fertilization in humans.

Nectinepsin: a new extracellular matrix protein of the pexin family. Characterization of a novel cDNA encoding a protein with an RGD cell binding motif.

PubMed

Blancher, C; Omri, B; Bidou, L; Pessac, B; Crisanti, P

1996-10-18

We report the isolation and characterization of a novel cDNA from quail neuroretina encoding a putative protein named nectinepsin. The nectinepsin cDNA identifies a major 2.2-kilobase mRNA that is detected from ED 5 in neuroretina and is increasingly abundant during embryonic development. A nectinepsin mRNA is also found in quail liver, brain, and intestine and in mouse retina. The deduced nectinepsin amino acid sequence contains the RGD cell binding motif of integrin ligands. Furthermore, nectinepsin shares substantial homologies with vitronectin and structural protein similarities with most of the matricial metalloproteases. However, the presence of a specific sequence and the lack of heparin and collagen binding domains of the vitronectin indicate that nectinepsin is a new extracellular matrix protein. Furthermore, genomic Southern blot studies suggest that nectinepsin and vitronectin are encoded by different genes. Western blot analysis with an anti-human vitronectin antiserum revealed, in addition to the 65- and 70-kDa vitronectin bands, an immunoreactive protein of about 54 kDa in all tissues containing nectinepsin mRNA. It seems likely that the form of vitronectin found in chick egg yolk plasma by Nagano et al. ((1992) J. Biol. Chem. 267, 24863-24870) is the protein that corresponds to the nectinepsin cDNA. This new protein could be an important molecule involved in the early steps of the development.

Novel Aggregation Properties of Candida albicans Secreted Aspartyl Proteinase Sap6 Mediate Virulence in Oral Candidiasis

PubMed Central

Kumar, Rohitashw; Saraswat, Darpan; Tati, Swetha

2015-01-01

Candida albicans, a commensal fungus of the oral microbiome, causes oral candidiasis in humans with localized or systemic immune deficiencies. Secreted aspartic proteinases (Saps) are a family of 10 related proteases and are virulence factors due to their proteolytic activity, as well as their roles in adherence and colonization of host tissues. We found that mice infected sublingually with C. albicans cells overexpressing Sap6 (SAP6 OE and a Δsap8 strain) had thicker fungal plaques and more severe oral infection, while infection with the Δsap6 strain was attenuated. These hypervirulent strains had highly aggregative colony structure in vitro and higher secreted proteinase activity; however, the levels of proteinase activity of C. albicans Saps did not uniformly match their abilities to damage cultured oral epithelial cells (SCC-15 cells). Hyphal induction in cells overexpressing Sap6 (SAP6 OE and Δsap8 cells) resulted in formation of large cell-cell aggregates. These aggregates could be produced in germinated wild-type cells by addition of native or heat-inactivated Sap6. Sap6 bound only to germinated cells and increased C. albicans adhesion to oral epithelial cells. The adhesion properties of Sap6 were lost upon deletion of its integrin-binding motif (RGD) and could be inhibited by addition of RGD peptide or anti-integrin antibodies. Thus, Sap6 (but not Sap5) has an alternative novel function in cell-cell aggregation, independent of its proteinase activity, to promote infection and virulence in oral candidiasis. PMID:25870228

Novel Aggregation Properties of Candida albicans Secreted Aspartyl Proteinase Sap6 Mediate Virulence in Oral Candidiasis.

PubMed

Kumar, Rohitashw; Saraswat, Darpan; Tati, Swetha; Edgerton, Mira

2015-07-01

Candida albicans, a commensal fungus of the oral microbiome, causes oral candidiasis in humans with localized or systemic immune deficiencies. Secreted aspartic proteinases (Saps) are a family of 10 related proteases and are virulence factors due to their proteolytic activity, as well as their roles in adherence and colonization of host tissues. We found that mice infected sublingually with C. albicans cells overexpressing Sap6 (SAP6 OE and a Δsap8 strain) had thicker fungal plaques and more severe oral infection, while infection with the Δsap6 strain was attenuated. These hypervirulent strains had highly aggregative colony structure in vitro and higher secreted proteinase activity; however, the levels of proteinase activity of C. albicans Saps did not uniformly match their abilities to damage cultured oral epithelial cells (SCC-15 cells). Hyphal induction in cells overexpressing Sap6 (SAP6 OE and Δsap8 cells) resulted in formation of large cell-cell aggregates. These aggregates could be produced in germinated wild-type cells by addition of native or heat-inactivated Sap6. Sap6 bound only to germinated cells and increased C. albicans adhesion to oral epithelial cells. The adhesion properties of Sap6 were lost upon deletion of its integrin-binding motif (RGD) and could be inhibited by addition of RGD peptide or anti-integrin antibodies. Thus, Sap6 (but not Sap5) has an alternative novel function in cell-cell aggregation, independent of its proteinase activity, to promote infection and virulence in oral candidiasis.

Access to site-specific Fc-cRGD peptide conjugates through streamlined expressed protein ligation.

PubMed

Frutos, S; Jordan, J B; Bio, M M; Muir, T W; Thiel, O R; Vila-Perelló, M

2016-10-12

An ideal drug should be highly effective, non-toxic and be delivered by a convenient and painless single dose. We are still far from such optimal treatment but peptides, with their high target selectivity and low toxicity profiles, provide a very attractive platform from which to strive towards it. One of the major limitations of peptide drugs is their high clearance rates, which limit dosage regimen options. Conjugation to antibody Fc domains is a viable strategy to improve peptide stability by increasing their hydrodynamic radius and hijacking the Fc recycling pathway. We report the use of a split-intein based semi-synthetic approach to site-specifically conjugate a synthetic integrin binding peptide to an Fc domain. The strategy described here allows conjugating synthetic peptides to Fc domains, which is not possible via genetic methods, fully maintaining the ability of both the Fc domain and the bioactive peptide to interact with their binding partners.

Tuning the autophagy-inducing activity of lanthanide-based nanocrystals through specific surface-coating peptides

NASA Astrophysics Data System (ADS)

Zhang, Yunjiao; Zheng, Fang; Yang, Tianlong; Zhou, Wei; Liu, Yun; Man, Na; Zhang, Li; Jin, Nan; Dou, Qingqing; Zhang, Yong; Li, Zhengquan; Wen, Long-Ping

2012-09-01

The induction of autophagy on exposure of cells to a variety of nanoparticles represents both a safety concern and an application niche for engineered nanomaterials. Here, we show that a short synthetic peptide, RE-1, identified by means of phage display, binds to lanthanide (LN) oxide and upconversion nanocrystals (UCN), forms a stable coating layer on the nanoparticles’ surface, and effectively abrogates their autophagy-inducing activity. Furthermore, RE-1 peptide variants exhibit a differentially reduced binding capability, and correspondingly, a varied ability to reduce the autophagic response. We also show that the addition of an arginine-glycine-aspartic acid (RGD) motif to RE-1 enhances autophagy for LN UCN through the interaction with integrins. RE-1 and its variants provide a versatile tool for tuning material-cell interactions to achieve the desired level of autophagy, and may prove useful for the various diagnostic and therapeutic applications of LN-based nanomaterials and nanodevices.

Arginine-glycine-aspartic acid-polyethylene glycol-polyamidoamine dendrimer conjugate improves liver-cell aggregation and function in 3-D spheroid culture.

PubMed

Chen, Zhanfei; Lian, Fen; Wang, Xiaoqian; Chen, Yanling; Tang, Nanhong

The polyamidoamine (PAMAM) dendrimer, a type of macromolecule material, has been used in spheroidal cell culture and drug delivery in recent years. However, PAMAM is not involved in the study of hepatic cell-spheroid culture or its biological activity, particularly in detoxification function. Here, we constructed a PAMAM-dendrimer conjugate decorated by an integrin ligand: arginine-glycine-aspartic acid (RGD) peptide. Our studies demonstrate that RGD-polyethylene glycol (PEG)-PAMAM conjugates can promote singly floating hepatic cells to aggregate together in a sphere-like growth with a weak reactive oxygen species. Moreover, RGD-PEG-PAMAM conjugates can activate the AKT-MAPK pathway in hepatic cells to promote cell proliferation and improve basic function and ammonia metabolism. Together, our data support the hepatocyte sphere treated by RGD-PEG-PAMAM conjugates as a potential source of hepatic cells for a biological artificial liver system.

Crystal structure of the zymogen form of the group A Streptococcus virulence factor SpeB: an integrin-binding cysteine protease.

PubMed

Kagawa, T F; Cooney, J C; Baker, H M; McSweeney, S; Liu, M; Gubba, S; Musser, J M; Baker, E N

2000-02-29

Pathogenic bacteria secrete protein toxins that weaken or disable their host, and thereby act as virulence factors. We have determined the crystal structure of streptococcal pyrogenic exotoxin B (SpeB), a cysteine protease that is a major virulence factor of the human pathogen Streptococcus pyogenes and participates in invasive disease episodes, including necrotizing fasciitis. The structure, determined for the 40-kDa precursor form of SpeB at 1.6-A resolution, reveals that the protein is a distant homologue of the papain superfamily that includes the mammalian cathepsins B, K, L, and S. Despite negligible sequence identity, the protease portion has the canonical papain fold, albeit with major loop insertions and deletions. The catalytic site differs from most other cysteine proteases in that it lacks the Asn residue of the Cys-His-Asn triad. The prosegment has a unique fold and inactivation mechanism that involves displacement of the catalytically essential His residue by a loop inserted into the active site. The structure also reveals the surface location of an integrin-binding Arg-Gly-Asp (RGD) motif that is a feature unique to SpeB among cysteine proteases and is linked to the pathogenesis of the most invasive strains of S. pyogenes.

Immunological characterization of eristostatin and echistatin binding sites on alpha IIb beta 3 and alpha V beta 3 integrins.

PubMed Central

Marcinkiewicz, C; Rosenthal, L A; Mosser, D M; Kunicki, T J; Niewiarowski, S

1996-01-01

Two disintegrins with a high degree of amino acid sequence similarity, echistatin and eristostatin, showed a low level of interaction with Chinese hamster ovary (CHO) cells, but they bound to CHO cells transfected with alpha IIb beta 3 genes (A5 cells) and to CHO cells transfected with alpha v beta 3 genes (VNRC3 cells) in a reversible and saturable manner. Scatchard analysis revealed that eristostatin bound to 816000 sites per A5 cell (Kd 28 nM) and to 200000 sites (Kd 14 nM) per VNRC3 cell respectively. However, VNRC3 cells did not bind to immobilized eristostatin. Echistatin bound to 495000 sites (Kd 53 nM) per A5 cell and to 443000 sites (Kd 20 nM) per VNRC3 cell. As determined by flow cytometry, radiobinding assay and adhesion studies, binding of both disintegrins to A5 cells and resting platelets and binding of echistatin to VNRC3 cells resulted in the expression of ligand-induced binding sites (LIBS) on the beta 3 subunit. Eristostatin inhibited, more strongly than echistatin, the binding of three monoclonal antibodies: OPG2 (RGD motif dependent), A2A9 (alpha IIb beta 3 complex dependent) and 7E3 (alpha IIb beta 3 and alpha v beta 3 complex dependent) to A5 cells, to resting and to activated platelets and to purified alpha IIb beta 3. Experiments in which echistatin and eristostatin were used alone or in combination to inhibit the binding of 7E3 and OPG2 antibodies to resting platelets suggested that these two disintegrins bind to different but overlapping sites on alpha IIb beta 3 integrin. Monoclonal antibody LM 609 and echistatin seemed to bind to different sites on alpha v beta 3 integrin. However, echistatin inhibited binding of 7E3 antibody to VNRC3 cells and to purified alpha v beta 3 suggesting that alpha v beta 3 and alpha IIb beta 3 might share the same epitope to which both echistatin and 7E3 bind. Eristostatin had no effect in these systems, providing further evidence that it binds to a different epitope on alpha v beta 3. PMID:8760368

Cyclic RGD peptide-modified liposomal drug delivery system for targeted oral apatinib administration: enhanced cellular uptake and improved therapeutic effects.

PubMed

Song, Zhiwang; Lin, Yun; Zhang, Xia; Feng, Chan; Lu, Yonglin; Gao, Yong; Dong, Chunyan

2017-01-01

Apatinib is an oral tyrosine kinase inhibitor, which selectively targets vascular endothelial growth factor receptor 2 and has the potential to treat many tumors therapeutically. Cyclic arginylglycylaspartic acid (cRGD)- and polyethylene glycol (PEG)-modified liposomes (cRGD-Lipo-PEG) were constructed to act as a targeted delivery system for the delivery of apatinib to the human colonic cancer cell line, HCT116. These cRGD-modified liposomes specifically recognized integrin α v β 3 and exhibited greater uptake efficiency with respect to delivering liposomes into HCT116 cells when compared to nontargeted liposomes (Lipo-PEG), as well as greater death of tumor cells and apoptosis. The mechanism by which cRGD-Lipo-PEG targets cells was elucidated further with competition assays. To determine the anticancer efficacy in vivo, nude mice were implanted with HCT116 xenografts and treated with apatinib-loaded liposomes or free apatinib intravenously or via intragastric administration. The active and passive targeting of cRGD-Lipo-PEG led to significant tumor treatment targeting ability, better inhibition of tumor growth, and less toxicity when compared with treatments using uncombined apatinib. The results presented strongly support the case for cRGD-Lipo-PEG representing a targeted delivery system for apatinib in the treatment of colonic cancer.

Cyclic RGD peptide-modified liposomal drug delivery system for targeted oral apatinib administration: enhanced cellular uptake and improved therapeutic effects

PubMed Central

Song, Zhiwang; Lin, Yun; Zhang, Xia; Feng, Chan; Lu, Yonglin; Gao, Yong; Dong, Chunyan

2017-01-01

Apatinib is an oral tyrosine kinase inhibitor, which selectively targets vascular endothelial growth factor receptor 2 and has the potential to treat many tumors therapeutically. Cyclic arginylglycylaspartic acid (cRGD)- and polyethylene glycol (PEG)-modified liposomes (cRGD-Lipo-PEG) were constructed to act as a targeted delivery system for the delivery of apatinib to the human colonic cancer cell line, HCT116. These cRGD-modified liposomes specifically recognized integrin αvβ3 and exhibited greater uptake efficiency with respect to delivering liposomes into HCT116 cells when compared to nontargeted liposomes (Lipo-PEG), as well as greater death of tumor cells and apoptosis. The mechanism by which cRGD-Lipo-PEG targets cells was elucidated further with competition assays. To determine the anticancer efficacy in vivo, nude mice were implanted with HCT116 xenografts and treated with apatinib-loaded liposomes or free apatinib intravenously or via intragastric administration. The active and passive targeting of cRGD-Lipo-PEG led to significant tumor treatment targeting ability, better inhibition of tumor growth, and less toxicity when compared with treatments using uncombined apatinib. The results presented strongly support the case for cRGD-Lipo-PEG representing a targeted delivery system for apatinib in the treatment of colonic cancer. PMID:28331317

Use of arginine-glycine-aspartic acid adhesion peptides coupled with a new collagen scaffold to engineer a myocardium-like tissue graft.

PubMed

Schussler, O; Coirault, C; Louis-Tisserand, M; Al-Chare, W; Oliviero, P; Menard, C; Michelot, R; Bochet, P; Salomon, D R; Chachques, J C; Carpentier, A; Lecarpentier, Y

2009-03-01

Cardiac tissue engineering might be useful in treatment of diseased myocardium or cardiac malformations. The creation of functional, biocompatible contractile tissues, however, remains challenging. We hypothesized that coupling of arginine-glycine-aspartic acid-serine (RGD+) adhesion peptides would improve cardiomyocyte viability and differentiation and contractile performance of collagen-cell scaffolds. Clinically approved collagen scaffolds were functionalized with RGD+ cells and seeded with cardiomyocytes. Contractile performance, cardiomyocyte viability and differentiation were analyzed at days 1 and 8 and/or after culture for 1 month. The method used for the RGD+ cell-collagen scaffold coupling enabled the following features: high coupling yields and complete washout of excess reagent and by-products with no need for chromatography; spectroscopic quantification of RGD+ coupling; a spacer arm of 36 A, a length reported as optimal for RGD+-peptide presentation and favorable for integrin-receptor clustering and subsequent activation. Isotonic and isometric mechanical parameters, either spontaneous or electrostimulated, exhibited good performance in RGD+ constructs. Cell number and viability was increased in RGD+ scaffolds, and we saw good organization of cell contractile apparatus with occurrence of cross-striation. We report a novel method of engineering a highly effective collagen-cell scaffold based on RGD+ peptides cross-linked to a clinically approved collagen matrix. The main advantages were cell contractile performance, cardiomyocyte viability and differentiation.

Culturing INS-1 cells on CDPGYIGSR-, RGD- and fibronectin surfaces improves insulin secretion and cell proliferation.

PubMed

Kuehn, Carina; Dubiel, Evan A; Sabra, Georges; Vermette, Patrick

2012-02-01

Rat insulinoma cells (INS-1), an immortalized pancreatic beta cell line, were cultured on low-fouling carboxymethyl-dextran (CMD) layers bearing fibronectin, the tripeptide Arg-Gly-Asp (RGD) or CDPGYIGSR, a laminin nonapeptide. INS-1 cells were non-adherent on CMD and RGE but adhered to fibronectin- and peptide-coated CMD surfaces and to tissue culture polystyrene (TCPS). On CMD bearing fibronectin and the peptides, INS-1 cells showed higher glucose-stimulated insulin secretion compared to those on TCPS, bare CMD and RGE. INS-1 cells experienced a net cell growth, with the lowest found after 7 days on CMD and the highest on fibronectin. Similarly, cells on RGD and CDPGYIGSR showed lower net growth rates than those on fibronectin. Expression of E-cadherin and integrins αvβ3 and α5 were similar between the conditions, except for α5 expression on fibronectin, RGD and CDPGYIGSR. Larger numbers of Ki-67-positive cells were found on CDPGYIGSR, TCPS, fibronectin and RGD. Cells in all conditions expressed Pdx1. Copyright © 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

cRGD Peptide-Conjugated Pyropheophorbide-a Photosensitizers for Tumor Targeting in Photodynamic Therapy.

PubMed

Li, Wenjing; Tan, Sihai; Xing, Yutong; Liu, Qian; Li, Shuang; Chen, Qingle; Yu, Min; Wang, Fengwei; Hong, Zhangyong

2018-04-02

Pyropheophorbide-a (Pyro) is a highly promising photosensitizer for tumor photodynamic therapy (PDT), although its very limited tumor-accumulation ability seriously restricts its clinical applications. A higher accumulation of photosensitizers is very important for the treatment of deeply seated and larger tumors. The conjugation of Pyro with tumor-homing peptide ligands could be a very useful strategy to optimize the physical properties of Pyro. Herein, we reported our studies on the conjugation of Pyro with a cyclic cRGDfK (cRGD) peptide, an integrin binding sequence, to develop highly tumor-specific photosensitizers for PDT application. To further reduce the nonspecific uptake and, thus, reduce the background distribution of the conjugates in normal tissues, we opted to add a highly hydrophilic polyethylene glycol (PEG) chain and an extra strongly hydrophilic carboxylic acid group as the linker to avoid the direct connection of the strongly hydrophobic Pyro macrocycle and cRGD ligand. We reported here the synthesis and characterization of these conjugates, and the influence of the hydrophilic modification on the biological function of the conjugates was carefully studied. The tumor-accumulation ability and photodynamic-induced cell-killing ability of these conjugates were evaluated through both in vitro cell-based experiment and in vivo distribution and tumor therapy experiments with tumor-bearing mice. Thus, the synthesized conjugate significantly improved the tumor enrichment and tumor selectivity of Pyro, as well as abolished the xenograft tumors in the murine model through a one-time PDT treatment.

Exosome release of ADAM15 and the functional implications of human macrophage-derived ADAM15 exosomes.

PubMed

Lee, Hee Doo; Koo, Bon-Hun; Kim, Yeon Hyang; Jeon, Ok-Hee; Kim, Doo-Sik

2012-07-01

A disintegrin and metalloproteinase 15 (ADAM15), the only ADAM protein containing an Arg-Gly-Asp (RGD) motif in its disintegrin-like domain, is a widely expressed membrane protein that is involved in tumor progression and suppression. However, the underlying mechanism of ADAM15-mediated tumor suppression is not clearly understood. This study demonstrates that ADAM15 is released as an exosomal component, and ADAM15 exosomes exert tumor suppressive activities. We found that exosomal ADAM15 release is stimulated by phorbol 12-myristate 13-acetate, a typical protein kinase C activator, in various tumor cell types, and this results in a corresponding decrease in plasma membrane-associated ADAM15. Exosomes rich in ADAM15 display enhanced binding affinity for integrin αvβ3 in an RGD-dependent manner and suppress vitronectin- and fibronectin-induced cell adhesion, growth, and migration, as well as in vivo tumor growth. Exosomal ADAM15 is released from human macrophages, and macrophage-derived ADAM15 exosomes have tumor inhibitory effects. This work suggests a primary role of ADAM15 for exosome-mediated tumor suppression, as well as functional significance of exosomal ADAM protein in antitumor immunity.

A Kinase-Independent Function of c-Src Mediates p130Cas Phosphorylation at the Serine-639 Site in Pressure Overloaded Myocardium

PubMed Central

Palanisamy, Arun P.; Suryakumar, Geetha; Panneerselvam, Kavin; Willey, Christopher D.; Kuppuswamy, Dhandapani

2017-01-01

Early work in pressure overloaded (PO) myocardium shows that integrins mediate focal adhesion complex formation by recruiting the adaptor protein p130Cas (Cas) and nonreceptor tyrosine kinase c-Src. To explore c-Src role in Cas-associated changes during PO, we used a feline right ventricular in vivo PO model and a three-dimensional (3D) collagen-embedded adult cardiomyocyte in vitro model that utilizes a Gly-Arg-Gly-Asp-Ser (RGD) peptide for integrin stimulation. Cas showed slow electrophoretic mobility (band-shifting), recruitment to the cytoskeleton, and tyrosine phosphorylation at 165, 249, and 410 sites in both 48 h PO myocardium and 1 h RGD-stimulated cardiomyocytes. Adenoviral mediated expression of kinase inactive (negative) c-Src mutant with intact scaffold domains (KN-Src) in cardiomyocytes did not block the RGD stimulated changes in Cas. Furthermore, expression of KN-Src or kinase active c-Src mutant with intact scaffold function (A-Src) in two-dimensionally (2D) cultured cardiomyocytes was sufficient to cause Cas band-shifting, although tyrosine phosphorylation required A-Src. These data indicate that c-Src’s adaptor function, but not its kinase function, is required for a serine/threonine specific phosphorylation(s) responsible for Cas band-shifting. To explore this possibility, Chinese hamster ovary cells that stably express Cas were infected with either β-gal or KN-Src adenoviruses and used for Cas immunoprecipitation combined with mass spectrometry analysis. In the KN-Src expressing cells, Cas showed phosphorylation at the serine-639 (human numbering) site. A polyclonal antibody raised against phospho-serine-639 detected Cas phosphorylation in 24–48 h PO myocardium. Our studies indicate that c-Src’s adaptor function mediates serine-639 phosphorylation of Cas during integrin activation in PO myocardium. PMID:25976166

Identification of an Endogenously Generated Cryptic Collagen Epitope (XL313) That May Selectively Regulate Angiogenesis by an Integrin Yes-associated Protein (YAP) Mechano-transduction Pathway*

PubMed Central

Ames, Jacquelyn J.; Contois, Liangru; Caron, Jennifer M.; Tweedie, Eric; Yang, Xuehui; Friesel, Robert; Vary, Calvin; Brooks, Peter C.

2016-01-01

Extracellular matrix (ECM) remodeling regulates angiogenesis. However, the precise mechanisms by which structural changes in ECM proteins contribute to angiogenesis are not fully understood. Integrins are molecules with the ability to detect compositional and structural changes within the ECM and integrate this information into a network of signaling circuits that coordinate context-dependent cell behavior. The role of integrin αvβ3 in angiogenesis is complex, as evidence exists for both positive and negative functions. The precise downstream signaling events initiated by αvβ3 may depend on the molecular characteristics of its ligands. Here, we identified an RGD-containing cryptic collagen epitope that is generated in vivo. Surprisingly, rather than inhibiting αvβ3 signaling, this collagen epitope promoted αvβ3 activation and stimulated angiogenesis and inflammation. An antibody directed to this RGDKGE epitope but not other RGD collagen epitopes inhibited angiogenesis and inflammation in vivo. The selective ability of this RGD epitope to promote angiogenesis and inflammation depends in part on its flanking KGE motif. Interestingly, a subset of macrophages may represent a physiologically relevant source of this collagen epitope. Here, we define an endothelial cell mechano-signaling pathway in which a cryptic collagen epitope activates αvβ3 leading to an Src and p38 MAPK-dependent cascade that leads to nuclear accumulation of Yes-associated protein (YAP) and stimulation of endothelial cell growth. Collectively, our findings not only provide evidence for a novel mechano-signaling pathway, but also define a possible therapeutic strategy to control αvβ3 signaling by targeting a pro-angiogenic and inflammatory ligand of αvβ3 rather than the receptor itself. PMID:26668310

Guinea Pig-Adapted Foot-and-Mouth Disease Virus with Altered Receptor Recognition Can Productively Infect a Natural Host▿

PubMed Central

Núñez, José I.; Molina, Nicolas; Baranowski, Eric; Domingo, Esteban; Clark, Stuart; Burman, Alison; Berryman, Stephen; Jackson, Terry; Sobrino, Francisco

2007-01-01

We report that adaptation to infect the guinea pig did not modify the capacity of foot-and-mouth disease virus (FMDV) to kill suckling mice and to cause an acute and transmissible disease in the pig, an important natural host for this pathogen. Adaptive amino acid replacements (I248→T in 2C, Q44→R in 3A, and L147→P in VP1), selected upon serial passages of a type C FMDV isolated from swine (biological clone C-S8c1) in the guinea pig, were maintained after virus multiplication in swine and suckling mice. However, the adaptive replacement L147→P, next to the integrin-binding RGD motif at the GH loop in VP1, abolished growth of the virus in different established cell lines and modified its antigenicity. In contrast, primary bovine thyroid cell cultures could be productively infected by viruses with replacement L147→P, and this infection was inhibited by antibodies to αvβ6 and by an FMDV-derived RGD-containing peptide, suggesting that integrin αvβ6 may be used as a receptor for these mutants in the animal (porcine, guinea pig, and suckling mice) host. Substitution T248→N in 2C was not detectable in C-S8c1 but was present in a low proportion of the guinea pig-adapted virus. This substitution became rapidly dominant in the viral population after the reintroduction of the guinea pig-adapted virus into pigs. These observations illustrate how the appearance of minority variant viruses in an unnatural host can result in the dominance of these viruses on reinfection of the original host species. PMID:17522230

Guinea pig-adapted foot-and-mouth disease virus with altered receptor recognition can productively infect a natural host.

PubMed

Núñez, José I; Molina, Nicolas; Baranowski, Eric; Domingo, Esteban; Clark, Stuart; Burman, Alison; Berryman, Stephen; Jackson, Terry; Sobrino, Francisco

2007-08-01

We report that adaptation to infect the guinea pig did not modify the capacity of foot-and-mouth disease virus (FMDV) to kill suckling mice and to cause an acute and transmissible disease in the pig, an important natural host for this pathogen. Adaptive amino acid replacements (I(248)-->T in 2C, Q(44)-->R in 3A, and L(147)-->P in VP1), selected upon serial passages of a type C FMDV isolated from swine (biological clone C-S8c1) in the guinea pig, were maintained after virus multiplication in swine and suckling mice. However, the adaptive replacement L(147)-->P, next to the integrin-binding RGD motif at the GH loop in VP1, abolished growth of the virus in different established cell lines and modified its antigenicity. In contrast, primary bovine thyroid cell cultures could be productively infected by viruses with replacement L(147)-->P, and this infection was inhibited by antibodies to alphavbeta6 and by an FMDV-derived RGD-containing peptide, suggesting that integrin alphavbeta6 may be used as a receptor for these mutants in the animal (porcine, guinea pig, and suckling mice) host. Substitution T(248)-->N in 2C was not detectable in C-S8c1 but was present in a low proportion of the guinea pig-adapted virus. This substitution became rapidly dominant in the viral population after the reintroduction of the guinea pig-adapted virus into pigs. These observations illustrate how the appearance of minority variant viruses in an unnatural host can result in the dominance of these viruses on reinfection of the original host species.

Twisting integrin receptors increases endothelin-1 gene expression in endothelial cells

NASA Technical Reports Server (NTRS)

Chen, J.; Fabry, B.; Schiffrin, E. L.; Wang, N.; Ingber, D. E. (Principal Investigator)

2001-01-01

A magnetic twisting stimulator was developed based on the previously published technique of magnetic twisting cytometry. Using ligand-coated ferromagnetic microbeads, this device can apply mechanical stresses with varying amplitudes, duration, frequencies, and waveforms to specific cell surface receptors. Biochemical and biological responses of the cells to the mechanical stimulation can be assayed. Twisting integrin receptors with RGD (Arg-Gly-Asp)-containing peptide-coated beads increased endothelin-1 (ET-1) gene expression by >100%. In contrast, twisting scavenger receptors with acetylated low-density lipoprotein-coated beads or twisting HLA antigen with anti-HLA antibody-coated beads did not lead to alterations in ET-1 gene expression. In situ hybridization showed that the increase in ET-1 mRNA was localized in the cells that were stressed with the RGD-coated beads. Blocking stretch-activated ion channels with gadolinium, chelating Ca2+ with EGTA, or inhibiting tyrosine phosphorylation with genistein abolished twist-induced ET-1 mRNA elevation. Abolishing cytoskeletal tension with an inhibitor of the myosin ATPase, with an inhibitor of myosin light chain kinase, or with an actin microfilament disrupter blocked twisted-induced increases in ET-1 expression. Our results are consistent with the hypothesis that the molecular structural linkage of integrin-cytoskeleton is an important pathway for stress-induced ET-1 gene expression.

Biomimetic Hydrogels Incorporating Polymeric Cell-Adhesive Peptide to Promote the 3D Assembly of Tumoroids

PubMed Central

Hao, Ying; Zerdoum, Aidan B.; Stuffer, Alexander J.; Rajasekaran, Ayyappan K.; Jia, Xinqiao

2016-01-01

Towards the goal of establishing physiologically relevant in vitro tumor models, we synthesized and characterized a biomimetic hydrogel using thiolated hyaluronic acid (HA-SH) and an acrylated copolymer carrying multiple copies of cell adhesive peptide (PolyRGD-AC). PolyRGD-AC was derived from a random copolymer of tert-butyl methacrylate (tBMA) and oligomeric (ethylene glycol) methacrylate (OEGMA), synthesized via atom transfer radical polymerization (ATRP). Acid hydrolysis of tert-butyl moieties revealed the carboxylates, through which acrylate groups were installed. Partial modification of the acrylate groups with a cysteine-containing RGD peptide generated PolyRGD-AC. When PolyRGD-AC was mixed with HA-SH under physiological conditions, a macroscopic hydrogel with an average elastic modulus of 630 Pa was produced. LNCaP prostate cancer cells encapsulated in HA-PolyRGD gels as dispersed single cells formed multicellular tumoroids by day 4 and reached an average diameter of ~95 μm by day 28. Cells in these structures were viable, formed cell-cell contacts through E-cadherin (E-CAD and displayed cortical organization of F-actin. Compared to the control gels prepared using PolyRDG, multivalent presentation of the RGD signal in the HA matrix increased cellular metabolism, promoted the development of larger tumoroids and enhanced the expression of E-CAD and integrins. Overall, hydrogels with multivalently immobilized RGD is a promising 3D culture platform for dissecting principles of tumorigenesis and for screening anticancer drugs. PMID:27723964

Targeting nanoparticles to M cells with non-peptidic ligands for oral vaccination.

PubMed

Fievez, Virginie; Plapied, Laurence; des Rieux, Anne; Pourcelle, Vincent; Freichels, Hélène; Wascotte, Valentine; Vanderhaeghen, Marie-Lyse; Jerôme, Christine; Vanderplasschen, Alain; Marchand-Brynaert, Jacqueline; Schneider, Yves-Jacques; Préat, Véronique

2009-09-01

The presence of RGD on nanoparticles allows the targeting of beta1 integrins at the apical surface of human M cells and the enhancement of an immune response after oral immunization. To check the hypothesis that non-peptidic ligands targeting intestinal M cells or APCs would be more efficient for oral immunization than RGD, novel non-peptidic and peptidic analogs (RGD peptidomimitic (RGDp), LDV derivative (LDVd) and LDV peptidomimetic (LDVp)) as well as mannose were grafted on the PEG chain of PCL-PEG and incorporated in PLGA-based nanoparticles. RGD and RGDp significantly increased the transport of nanoparticles across an in vitro model of human M cells as compared to enterocytes. RGD, LDVp, LDVd and mannose enhanced nanoparticle uptake by macrophages in vitro. The intraduodenal immunization with RGDp-, LDVd- or mannose-labeled nanoparticles elicited a higher production of IgG antibodies than the intramuscular injection of free ovalbumin or intraduodenal administration of either non-targeted or RGD-nanoparticles. Targeted formulations were also able to induce a cellular immune response. In conclusion, the in vitro transport of nanoparticles, uptake by macrophages and the immune response were positively influenced by the presence of ligands at the surface of nanoparticles. These targeted-nanoparticles could thus represent a promising delivery system for oral immunization.

Alpha-v Integrin Targeted PET Imaging of Breast Cancer Angiogenesis and Low-Dose Metronomic Anti-Angiogenic Chemotherapy Efficacy

DTIC Science & Technology

2007-08-01

He L, Neamati N, Chen X. Evaluation of Biodistribution and Anti-tumor Effect of a Dimeric RGD Peptide-paclitaxel Conjugate in Mice with Breast...Chemistry-Anti-Cancer Agents (CMC-ACA). 2006;6:407-428. 11. Dayam R, Aiello F, Wu Y, Garofalo A, Chen X, Neamati N. Discovery of Small Molecule...Tumor Integrin αvβ3 Expression with [18F]FRGD2. J Nucl Med. 2006;47:113-121. 18 16. Chen X, Plasencia C, Hou Y, Neamati N. Synthesis and

Recombinant Nonstructural 3 Protein, rNS3, of Hepatitis C Virus Along With Recombinant GP96 Induce IL-12, TNFα and α5integrin Expression in Antigen Presenting Cells

PubMed Central

Hajizadeh, Mohammad Reza; Mokarram, Pooneh; Kamali sarvestani, Eskandar; Bolhassani, Azam; Mostafavi Pour, Zohreh

2013-01-01

Background Hepatitis C virus (HCV) infection is the main cause of chronic liver disease and to date there has been no vaccine development to prevent this infection. Among non-structural HCV proteins, NS3 protein is an excellent goal for a therapeutic vaccine, due to its large size and less variation in conserved regions. The immunogenic properties of heat shock proteins (HSPs) for instance GP96 have prompted investigations into their function as strong adjuvant to improve innate and adaptive immunity. Objectives The aim of this study was to examine additive effects of recombinant GP96 (rGP96) fragments accompanied by rNS3 on expression levels of α5integrin and pro-inflammatory cytokines, IL-12 and TNFα, in Antigen Presenting Cells (APCs). Materials and Methods Recombinant viral proteins (rNS3 and rRGD-NS3), N-terminal and C-terminal fragments of GP96 were produced and purified from E. coli in order to treat the cells; mouse spleen Dendritic Cells (DCs) and THP-1 macrophages. Results Our results showed that rNT-GP96 alone significantly increases the expression level of IL-12, TNFα and α5integrin in THP-1 macrophages and DCs, while IL-12 and TNFα expression levels were unaffected by either rNS3 or rRGD-NS3. Interestingly, the co-addition of these recombinant proteins with rNT-GP96 increased IL-12, TNFα and α5integrin expression. Pearson Correlation showed a direct association between α5integrin with IL-12 and TNF-α expression. Conclusions we have highlighted the role of rNS3 plus rNT-GP96 mediated by α5integrin in producing IL-12 and TNFα. It can be suggested that rNT-GP96 could enhance immunity characteristic of rNS3 protein via production of pro-inflammatory cytokines. PMID:24032046

GARP regulates the bioavailability and activation of TGFβ.

PubMed

Wang, Rui; Zhu, Jianghai; Dong, Xianchi; Shi, Minlong; Lu, Chafen; Springer, Timothy A

2012-03-01

Glycoprotein-A repetitions predominant protein (GARP) associates with latent transforming growth factor-β (proTGFβ) on the surface of T regulatory cells and platelets; however, whether GARP functions in latent TGFβ activation and the structural basis of coassociation remain unknown. We find that Cys-192 and Cys-331 of GARP disulfide link to the TGFβ1 prodomain and that GARP with C192A and C331A mutations can also noncovalently associate with proTGFβ1. Noncovalent association is sufficiently strong for GARP to outcompete latent TGFβ-binding protein for binding to proTGFβ1. Association between GARP and proTGFβ1 prevents the secretion of TGFβ1. Integrin α(V)β(6) and to a lesser extent α(V)β(8) are able to activate TGFβ from the GARP-proTGFβ1 complex. Activation requires the RGD motif of latent TGFβ, disulfide linkage between GARP and latent TGFβ, and membrane association of GARP. Our results show that GARP is a latent TGFβ-binding protein that functions in regulating the bioavailability and activation of TGFβ.

Access to site-specific Fc–cRGD peptide conjugates through streamlined expressed protein ligation†

PubMed Central

Frutos, S.; Jordan, J. B.; Bio, M. M.; Muir, T. W.; Thiel, O. R.; Vila-Perelló, M.

2018-01-01

An ideal drug should be highly effective, non-toxic and be delivered by a convenient and painless single dose. We are still far from such optimal treatment but peptides, with their high target selectivity and low toxicity profiles, provide a very attractive platform from which to strive towards it. One of the major limitations of peptide drugs is their high clearance rates, which limit dosage regimen options. Conjugation to antibody Fc domains is a viable strategy to improve peptide stability by increasing their hydrodynamic radius and hijacking the Fc recycling pathway. We report the use of a split-intein based semi-synthetic approach to site-specifically conjugate a synthetic integrin binding peptide to an Fc domain. The strategy described here allows conjugating synthetic peptides to Fc domains, which is not possible via genetic methods, fully maintaining the ability of both the Fc domain and the bioactive peptide to interact with their binding partners. PMID:27722696

Platelet-camouflaged nanococktail: Simultaneous inhibition of drug-resistant tumor growth and metastasis via a cancer cells and tumor vasculature dual-targeting strategy.

PubMed

Jing, Lijia; Qu, Haijing; Wu, Dongqi; Zhu, Chaojian; Yang, Yongbo; Jin, Xing; Zheng, Jian; Shi, Xiangsheng; Yan, Xiufeng; Wang, Yang

2018-01-01

Multidrug resistance (MDR) poses a great challenge to cancer therapy. It is difficult to inhibit the growth of MDR cancer due to its chemoresistance. Furthermore, MDR cancers are more likely to metastasize, causing a high mortality among cancer patients. In this study, a nanomedicine RGD-NPVs@MNPs/DOX was developed by encapsulating melanin nanoparticles (MNPs) and doxorubicin (DOX) inside RGD peptide (c(RGDyC))-modified nanoscale platelet vesicles (RGD-NPVs) to efficiently inhibit the growth and metastasis of drug-resistant tumors via a cancer cells and tumor vasculature dual-targeting strategy. Methods: The in vitro immune evasion potential and the targeting performance of RGD-NPVs@MNPs/DOX were examined using RAW264.7, HUVECs, MDA-MB-231 and MDA-MB-231/ADR cells lines. We also evaluated the pharmacokinetic behavior and the in vivo therapeutic performance of RGD-NPVs@MNPs/DOX using a MDA-MB-231/ADR tumor-bearing nude mouse model. Results: By taking advantage of the self-recognizing property of the platelet membrane and the conjugated RGD peptides, RGD-NPVs@MNPs/DOX was found to evade immune clearance and target the αvβ3 integrin on tumor vasculature and resistant breast tumor cells. Under irradiation with a NIR laser, RGD-NPVs@MNPs/DOX produced a multipronged effect, including reversal of cancer MDR, efficient killing of resistant cells by chemo-photothermal therapy, elimination of tumor vasculature for blocking metastasis, and long-lasting inhibition of the expressions of VEGF, MMP2 and MMP9 within the tumor. Conclusion: This versatile nanomedicine of RGD-NPVs@MNPs/DOX integrating unique biomimetic properties, excellent targeting performance, and comprehensive therapeutic strategies in one formulation might bring opportunities to MDR cancer therapy.

Molecular imaging-guided photothermal/photodynamic therapy against tumor by iRGD-modified indocyanine green nanoparticles.

PubMed

Yan, Fei; Wu, Hao; Liu, Hongmei; Deng, Zhiting; Liu, Hong; Duan, Wanlu; Liu, Xin; Zheng, Hairong

2016-02-28

Multifunctional near-infrared (NIR) nanoparticles demonstrate great potential in tumor theranostic applications. To achieve the sensitive detection and effective phototherapy in the early stage of tumor genesis, it is highly desirable to improve the targeting of NIR theranostic agents to biomarkers and to enhance their accumulation in tumor. Here we report a novel targeted multifunctional theranostic nanoparticle, internalized RGD (iRGD)-modified indocyanine green (ICG) liposomes (iRGD-ICG-LPs), for molecular imaging-guided photothermal therapy (PTT) and photodynamic therapy (PDT) therapy against breast tumor. The iRGD peptides with high affinity to αvβ3 integrin and effective tumor-internalized property were firstly used to synthesize iRGD-PEG2000-DSPE lipopeptides, which were further utilized to fabricate the targeted ICG liposomes. The results indicated that iRGD-ICG-LPs exhibited excellent stability and could provide an accurate and sensitive detection of breast tumor through NIR fluorescence molecular imaging. We further employed this nanoparticle for tumor theranostic application, demonstrating significantly higher tumor accumulation and tumor inhibition efficacy through PTT/PDT effects. Histological analysis further revealed much more apoptotic cells, confirming the advantageous anti-tumor effect of iRGD-ICG-LPs over non-targeted ICG-LPs. Notably, the targeting therapy mediated by iRGD provides almost equivalent anti-tumor efficacy at a 12.5-fold lower drug dose than that by monoclonal antibody, and no tumor recurrence and obvious treatment-induced toxicity were observed in our study. Our study provides a promising strategy to realize the sensitive detection and effective treatment of tumors by integrating molecular imaging into PTT/PDT therapy. Copyright © 2015. Published by Elsevier B.V.

Coadministration of iRGD with Multistage Responsive Nanoparticles Enhanced Tumor Targeting and Penetration Abilities for Breast Cancer Therapy.

PubMed

Hu, Chuan; Yang, Xiaotong; Liu, Rui; Ruan, Shaobo; Zhou, Yang; Xiao, Wei; Yu, Wenqi; Yang, Chuanyao; Gao, Huile

2018-06-21

Limited tumor targeting and poor penetration of nanoparticles are two major obstacles to improving the outcome of tumor therapy. Herein, coadministration of tumor-homing peptide iRGD and multistage-responsive penetrating nanoparticles for the treatment of breast cancer are reported. This multistage-responsive nanoparticle, IDDHN, was comprised of an NO donor-modified hyaluronic acid (HN) shell and a small-sized dendrimer, namely, dendri-graft-l-lysine conjugated with doxorubicin and indocyanine (IDD). The results showed that IDDHN could be degraded rapidly from about 330 nm to a smaller size that was in a size range of 35 to 150 nm (most at 35-60 nm) after hyaluronidase (HAase) incubation for 4 h; in vitro cellular uptake demonstrated that iRGD could mediate more endocytosis of IDDHN into 4T1 cells, which was attributed to the overexpression of α v β 3 integrin receptor. Multicellular spheroids penetration results showed synergistically enhanced deeper distribution of IDDHN into tumors, with the presence of iRGD, HAase incubation, and NO release upon laser irradiation. In vivo imaging indicated that coadministration with iRGD markedly enhanced the tumor targeting and penetration abilities of IDDHN. Surprisingly, coadministration of IDDHN with iRGD plus 808 nm laser irradiation nearly suppressed all tumor growth. These results systematically revealed the excellent potential of coadministration of iRGD with multistage-responsive nanoparticles for enhancing drug delivery efficiency and overcoming the 4T1 breast cancer.

Modulation of adhesion-dependent cAMP signaling by echistatin and alendronate

NASA Technical Reports Server (NTRS)

Fong, J. H.; Ingber, D. E.

1996-01-01

We measured intracellular cAMP levels in cells during attachment and spreading on different extracellular matrix (ECM) proteins. Increases in cAMP were observed within minutes when cells attached to fibronectin, vitronectin, and a synthetic RGD-containing fibronectin peptide (Petite 2000), but not when they adhered to another integrin alpha nu beta 3 ligand, echistatin. Because echistatin also inhibits bone resorption, we measured the effects of adding another osteoporosis inhibitor, alendronate, in this system. Alendronate inhibited the cAMP increase induced by ligands that primarily utilize integrin alpha nu beta 3 (vitronectin, Peptite 2000), but not by fibronectin which can also use integrin alpha 5 beta 1. These results show that cell adhesion to ECM can increase intracellular cAPM levels and raise the possibility that inhibitors of osteoporosis may act, in part, by preventing activation of this pathway by integrins.

Potency of Fish Collagen as a Scaffold for Regenerative Medicine

PubMed Central

Yamamoto, Kohei; Yanagiguchi, Kajiro

2014-01-01

Cells, growth factors, and scaffold are the crucial factors for tissue engineering. Recently, scaffolds consisting of natural polymers, such as collagen and gelatin, bioabsorbable synthetic polymers, such as polylactic acid and polyglycolic acid, and inorganic materials, such as hydroxyapatite, as well as composite materials have been rapidly developed. In particular, collagen is the most promising material for tissue engineering due to its biocompatibility and biodegradability. Collagen contains specific cell adhesion domains, including the arginine-glycine-aspartic acid (RGD) motif. After the integrin receptor on the cell surface binds to the RGD motif on the collagen molecule, cell adhesion is actively induced. This interaction contributes to the promotion of cell growth and differentiation and the regulation of various cell functions. However, it is difficult to use a pure collagen scaffold as a tissue engineering material due to its low mechanical strength. In order to make up for this disadvantage, collagen scaffolds are often modified using a cross-linker, such as gamma irradiation and carbodiimide. Taking into account the possibility of zoonosis, a variety of recent reports have been documented using fish collagen scaffolds. We herein review the potency of fish collagen scaffolds as well as associated problems to be addressed for use in regenerative medicine. PMID:24982861

Peptide-Mediated Platelet Capture at Gold Micropore Arrays.

PubMed

Adamson, Kellie; Spain, Elaine; Prendergast, Una; Moran, Niamh; Forster, Robert J; Keyes, Tia E

2016-11-30

Ordered spherical cap gold cavity arrays with 5.4, 1.6, and 0.98 μm diameter apertures were explored as capture surfaces for human blood platelets to investigate the impact of surface geometry and chemical modification on platelet capture efficiency and their potential as platforms for surface enhanced Raman spectroscopy of single platelets. The substrates were chemically modified with single-constituent self-assembled monolayers (SAM) or mixed SAMs comprised of thiol-functionalized arginine-glycine-aspartic acid (RGD, a platelet integrin target) with or without 1-octanethiol (adhesion inhibitor). As expected, platelet adhesion was promoted and inhibited at RGD and alkanethiol modified surfaces, respectively. Platelet adhesion was reversible, and binding efficiency at the peptide modified substrates correlated inversely with pore diameter. Captured platelets underwent morphological change on capture, the extent of which depended on the topology of the underlying substrate. Regioselective capture of the platelets enabled study for the first time of the surface enhanced Raman spectroscopy of single blood platelets, yielding high quality Raman spectroscopy of individual platelets at 1.6 μm diameter pore arrays. Given the medical importance of blood platelets across a range of diseases from cancer to psychiatric illness, such approaches to platelet capture may provide a useful route to Raman spectroscopy for platelet related diagnostics.

Engineered phage-based therapeutic materials inhibit Chlamydia trachomatis intracellular infection

PubMed Central

Bhattarai, Shanta Raj; Yoo, So Young; Lee, Seung-Wuk; Dean, Deborah

2012-01-01

Developing materials that are effective against sexually transmitted pathogens such as Chlamydia trachomatis (Ct) and HIV-1 is challenging both in terms of material selection and improving bio-membrane and cellular permeability at desired mucosal sites. Here, we engineered the prokaryotic bacterial virus (M13 phage) carrying two functional peptides, integrin binding peptide (RGD) and a segment of the polymorphic membrane protein D (PmpD) from Ct, as a phage-based material that can ameliorate Ct infection. Ct is a globally prevalent human pathogen for which there are no effective vaccines or microbicides. We show that engineered phage stably express both RGD motifs and Ct peptides and traffic intracellularly and into the lumen of the inclusion in which the organism resides within the host cell. Engineered phage were able to significantly reduce Ct infection in both HeLa and primary endocervical cells compared with Ct infection alone. Polyclonal antibodies raised against PmpD and co-incubated with constructs prior to infection did not alter the course of infection, indicating that PmpD is responsible for the observed decrease in Ct infection. Our results suggest that phage-based design approaches to vector delivery that overcome mucosal cellular barriers may be effective in preventing Ct and other sexually transmitted pathogens. PMID:22494890

Poly(amido-amine)-based hydrogels with tailored mechanical properties and degradation rates for tissue engineering.

PubMed

Martello, Federico; Tocchio, Alessandro; Tamplenizza, Margherita; Gerges, Irini; Pistis, Valentina; Recenti, Rossella; Bortolin, Monica; Del Fabbro, Massimo; Argentiere, Simona; Milani, Paolo; Lenardi, Cristina

2014-03-01

Poly(amido-amine) (PAA) hydrogels containing the 2,2-bisacrylamidoacetic acid-4-amminobutyl guanidine monomeric unit have a known ability to enhance cellular adhesion by interacting with the arginin-glycin-aspartic acid (RGD)-binding αVβ3 integrin, expressed by a wide number of cell types. Scientific interest in this class of materials has traditionally been hampered by their poor mechanical properties and restricted range of degradation rate. Here we present the design of novel biocompatible, RGD-mimic PAA-based hydrogels with wide and tunable degradation rates as well as improved mechanical and biological properties for biomedical applications. This is achieved by radical polymerization of acrylamide-terminated PAA oligomers in both the presence and absence of 2-hydroxyethylmethacrylate. The degradation rate is found to be precisely tunable by adjusting the PAA oligomer molecular weight and acrylic co-monomer concentration in the starting reaction mixture. Cell adhesion and proliferation tests on Madin-Darby canine kidney epithelial cells show that PAA-based hydrogels have the capacity to promote cell adhesion up to 200% compared to the control. Mechanical tests show higher compressive strength of acrylic chain containing hydrogels compared to traditional PAA hydrogels. Copyright © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

A targeted nanoglobular contrast agent from host-guest self-assembly for MR cancer molecular imaging

PubMed Central

Zhou, Zhuxian; Han, Zhen; Lu, Zheng-Rong

2016-01-01

The clinical application of nanoparticular Gd(III) based contrast agents for tumor molecular MRI has been hindered by safety concerns associated with prolonged tissue retention, although they can produce strong tumor enhancement. In this study, a targeted well-defined cyclodextrin-based nanoglobular contrast agent was developed through self-assembly driven by host-guest interactions for safe and effective cancer molecular MRI. Multiple β-cyclodextrins attached POSS (polyhedral oligomeric silsesquioxane) nanoglobule was used as host molecule. Adamantane–modified macrocyclic Gd(III) contrast agent, cRGD (cyclic RGDfK peptide) targeting ligand and fluorescent probe was used as guest molecules. The targeted host-guest nanoglobular contrast agent cRGD-POSS-βCD-(DOTA-Gd) specifically bond to αvβ3 integrin in malignant 4T1 breast tumor and provided greater contrast enhancement than the corresponding non-targeted agent. The agent also provided significant fluorescence signal in tumor tissue. The histological analysis of the tumor tissue confirmed its specific and effective targeting to αvβ3 integrin. The targeted imaging agent has a potential for specific cancer molecular MR and fluorescent imaging. PMID:26874280

A Kinase-Independent Function of c-Src Mediates p130Cas Phosphorylation at the Serine-639 Site in Pressure Overloaded Myocardium.

PubMed

Palanisamy, Arun P; Suryakumar, Geetha; Panneerselvam, Kavin; Willey, Christopher D; Kuppuswamy, Dhandapani

2015-12-01

Early work in pressure overloaded (PO) myocardium shows that integrins mediate focal adhesion complex formation by recruiting the adaptor protein p130Cas (Cas) and nonreceptor tyrosine kinase c-Src. To explore c-Src role in Cas-associated changes during PO, we used a feline right ventricular in vivo PO model and a three-dimensional (3D) collagen-embedded adult cardiomyocyte in vitro model that utilizes a Gly-Arg-Gly-Asp-Ser (RGD) peptide for integrin stimulation. Cas showed slow electrophoretic mobility (band-shifting), recruitment to the cytoskeleton, and tyrosine phosphorylation at 165, 249, and 410 sites in both 48 h PO myocardium and 1 h RGD-stimulated cardiomyocytes. Adenoviral mediated expression of kinase inactive (negative) c-Src mutant with intact scaffold domains (KN-Src) in cardiomyocytes did not block the RGD stimulated changes in Cas. Furthermore, expression of KN-Src or kinase active c-Src mutant with intact scaffold function (A-Src) in two-dimensionally (2D) cultured cardiomyocytes was sufficient to cause Cas band-shifting, although tyrosine phosphorylation required A-Src. These data indicate that c-Src's adaptor function, but not its kinase function, is required for a serine/threonine specific phosphorylation(s) responsible for Cas band-shifting. To explore this possibility, Chinese hamster ovary cells that stably express Cas were infected with either β-gal or KN-Src adenoviruses and used for Cas immunoprecipitation combined with mass spectrometry analysis. In the KN-Src expressing cells, Cas showed phosphorylation at the serine-639 (human numbering) site. A polyclonal antibody raised against phospho-serine-639 detected Cas phosphorylation in 24-48 h PO myocardium. Our studies indicate that c-Src's adaptor function mediates serine-639 phosphorylation of Cas during integrin activation in PO myocardium. © 2015 Wiley Periodicals, Inc.

Pyropheophorbide A and c(RGDyK) comodified chitosan-wrapped upconversion nanoparticle for targeted near-infrared photodynamic therapy.

PubMed

Zhou, Aiguo; Wei, Yanchun; Wu, Baoyan; Chen, Qun; Xing, Da

2012-06-04

Near-infrared (NIR)-to-visible upconversion nanoparticle (UCNP) has shown promising prospects in photodynamic therapy (PDT) as a drug carrier or energy donor. In this work, a photosensitizer pyropheophorbide a (Ppa) and RGD peptide c(RGDyK) comodified chitosan-wrapped NaYF(4):Yb/Er upconversion nanoparticle UCNP-Ppa-RGD was developed for targeted near-infrared photodynamic therapy. The properties of UCNP-Ppa-RGD, such as morphology, stability, optical spectroscopy and singlet oxygen generation efficiency, were investigated. The results show that covalently linked pyropheophorbide a molecule not only is stable but also retains its spectroscopic and functional properties. In vitro studies confirm a stronger targeting specificity of UCNP-Ppa-RGD to integrin α(v)β(3)-positive U87-MG cells compared with that in the corresponding negative group. The photosensitizer-attached nanostructure exhibited low dark toxicity and high phototoxicity against cancer cells upon 980 nm laser irradiation at an appropriate dosage. These results represent the first demonstration of a highly stable and efficient photosensitizer modified upconversion nanostructure for targeted near-infrared photodynamic therapy of cancer cells. The novel UCNP-Ppa-RGD nanoparticle may provide a powerful alternative for near-infrared photodynamic therapy with an improved tumor targeting specificity.

Arginine-Glycine-Aspartic Acid-Modified Lipid-Polymer Hybrid Nanoparticles for Docetaxel Delivery in Glioblastoma Multiforme.

PubMed

Shi, Kairong; Zhou, Jin; Zhang, Qianyu; Gao, Huile; Liu, Yayuan; Zong, Taili; He, Qin

2015-03-01

Hybrid nanoparticles consisting of lipids and the biodegradable polymer, poly (D,L-lactide-co-glycolide) (PLGA), were developed for the targeted delivery of the anticancer drug, docetaxel. Transmission electron microscopic observations confirmed the presence of a lipid coating over the polymeric core. Using coumarin-6 as a fluorescent probe, the uptake efficacy of RGD conjugated lipid coated nanoparticles (RGD-L-P) by C6 cells was increased significantly, compared with that of lipid-polymer hybrid nanoparticles (L-P; 2.5-fold higher) or PLGA-nanoparticles (PLGA-P; 1.76-fold higher). The superior tumor spheroid penetration of RGD-L-P indicated that RGD-L-P could target effectively and specifically to C6 cells overexpressing integrin α(v)β3. The anti-proliferative activity of docetaxel-loaded RGD-L-P against C6 cells was increased 2.69- and 4.13-fold compared with L-P and PLGA-P, respectively. Regarding biodistribution, the strongest brain-localized fluorescence signals were detected in glioblastoma multiforme (GBM)-bearing rats treated with 1,10-Dioctadecyl-3,3,30,30-tetramethylindotricarb-ocyanine iodide (DiR)-loaded RGD-L-P, compared to rats treated with DiR-loaded L-P or PLGA-P. The median survival time of GBM-bearing rats treated with docetaxel-loaded RGD-L-P was 57 days, a fold increase of 1.43, 1.78, 3.35, and 3.56 compared with animals given L-P (P < 0.05), PLGA-P (P < 0.05), Taxotere (P < 0.01) and saline (P < 0.01), respectively. Collectively, these results support RGD-L-P as a promising drug delivery system for the specific targeting and the treatment of GBM.

Dual targeting luminescent gold nanoclusters for tumor imaging and deep tissue therapy.

PubMed

Chen, Dan; Li, Bowen; Cai, Songhua; Wang, Peng; Peng, Shuwen; Sheng, Yuanzhi; He, Yuanyuan; Gu, Yueqing; Chen, Haiyan

2016-09-01

Dual targeting towards both extracellular and intracellular receptors specific to tumor is a significant approach for cancer diagnosis and therapy. In the present study, a novel nano-platform (AuNC-cRGD-Apt) with dual targeting function was initially established by conjugating gold nanocluster (AuNC) with cyclic RGD (cRGD) that is specific to αvβ3integrins over-expressed on the surface of tumor tissues and aptamer AS1411 (Apt) that is of high affinity to nucleolin over-expressed in the cytoplasm and nucleus of tumor cells. Then, AuNC-cRGD-Apt was further functionalized with near infrared (NIR) fluorescence dye (MPA), giving a NIR fluorescent dual-targeting probe AuNC-MPA-cRGD-Apt. AuNC-MPA-cRGD-Apt displays low cytotoxicity and favorable tumor-targeting capability at both in vitro and in vivo level, suggesting its clinical potential for tumor imaging. Additionally, Doxorubicin (DOX), a widely used clinical chemotherapeutic drug that kill cancer cells by intercalating DNA in cellular nucleus, was immobilized onto AuNC-cRGD-Apt forming a pro-drug, AuNC-DOX-cRGD-Apt. The enhanced tumor affinity, deep tumor penetration and improved anti-tumor activity of this pro-drug were demonstrated in different tumor cell lines, tumor spheroid and tumor-bearing mouse models. Results in this study suggest not only the prospect of non-toxic AuNC modified with two targeting ligands for tumor targeted imaging, but also confirm the promising future of dual targeting AuNC as a core for the design of prodrug in the field of cancer therapy. Copyright © 2016 Elsevier Ltd. All rights reserved.

Non-invasive assessment of inter-and intrapatient variability of integrin expression in metastasized prostate cancer by PET.

PubMed

Beer, Ambros J; Schwarzenböck, Sarah M; Zantl, Niko; Souvatzoglou, Michael; Maurer, Tobias; Watzlowik, Petra; Kessler, Horst; Wester, Hans-Jürgen; Schwaiger, Markus; Krause, Bernd Joachim

2016-05-10

Due to the high expression of the integrin αvβ3 not only on endothelial cells, but also on mature osteoclasts and prostate cancer cells, imaging of osseous metastases with αvβ3-targeted tracers seems promising. However, little is known about the patterns of αvβ3-expression in metastasized prostate cancer lesions in-vivo. Thus we evaluated the uptake of the αvβ3-specific PET tracer [18F]Galacto-RGD for assessment of bone metastases in prostate cancer patients. [18F]Galacto-RGD PET identified 58/74 bone-lesions (detection rate of 78.4%) and lymph node metastases in 2/5 patients. The SUVmean was 2.12+/-0.94 (range 0.70-4.38; tumor/blood 1.36+/-0.53; tumor/muscle 2.82+/-1.31) in bone-lesions and 2.21+/-1.18 (range 0.75-3.56) in lymph node metastases. Good visualization and detection of bone metastases was feasible due to a low background activity of the surrounding normal bone tissue. 12 patients with known metastasized prostate cancer according to conventional staging (including bone-scintigraphy and contrast-enhanced CT; median PSA 68.63 ng/ml, range 3.72-1935) were examined with PET after i.v.-injection of [18F]Galacto-RGD. Two blinded nuclear-medicine physicians evaluated the PET-scans in consensus concerning lesion detectability. Volumes-of-interest were drawn in the PET-scans over all metastases defined by conventional staging (maximum of 11 lesions/patient), over the left ventricle, liver and muscle and standardized-uptake-values (SUVs) were calculated. Our data show generally elevated uptake of [18F]Galacto-RGD in bone metastases from prostate cancer with a marked inter- and intrapatient variability. While [18F]Galacto-RGD PET is inferior to bone scintigraphy for detection of osseous metastases, it might be valuable in patient screening and monitoring of αvβ3-targeted therapies due to the high variability of αvβ3-expression.

Effect of grafting RGD and BMP-2 protein-derived peptides to a hydrogel substrate on osteogenic differentiation of marrow stromal cells.

PubMed

He, Xuezhong; Ma, Junyu; Jabbari, Esmaiel

2008-11-04

Osteogenic differentiation and mineralization of bone marrow stromal (BMS) cells depends on the cells' interactions with bioactive peptides associated with the matrix proteins. The RGD peptides of ECM proteins interact with BMS cells through integrin surface receptors to facilitate cell spreading and adhesion. The BMP peptide corresponding to residues 73-92 of bone morphogenetic protein-2 promotes differentiation and mineralization of BMS cells. The objective of this work was to investigate the effects of RGD and BMP peptides, grafted to a hydrogel substrate, on osteogenic differentiation and mineralization of BMS cells. RGD peptide was acrylamide-terminated by reacting acrylic acid with the N-terminal amine group of the peptide to produce the functionalized Ac-GRGD peptide. The PEGylated BMP peptide was reacted with 4-carboxybenzenesulfonazide to produce an azide functionalized Az-mPEG-BMP peptide. Poly (lactide-co-ethylene oxide- co-fumarate) (PLEOF) macromer was cross-linked with Ac-GRGD peptide and propargyl acrylate to produce an RGD conjugated hydrogel. Az-mPEG-BMP peptide was grafted to the hydrogel by "click chemistry". The RGD and BMP peptide density on the hydrogel surface was 1.62+/-0.37 and 5.2+/-0.6 pmol/cm2, respectively. BMS cells were seeded on the hydrogels and the effect of RGD and BMP peptides on osteogenesis was evaluated by measuring ALPase activity and calcium content with incubation time. BMS cells cultured on RGD conjugated, BMP peptide grafted, and RGD+BMP peptide modified hydrogels showed 3, 2.5, and 5-fold increase in ALPase activity after 14 days incubation. BMS cells seeded on RGD+BMP peptides modified hydrogel showed 4.9- and 11.8-fold increase in calcium content after 14 and 21 days, respectively, which was significantly higher than RGD conjugated or BMP grafted hydrogels. These results demonstrate that RGD and BMP peptides, grafted to a hydrogel substrate, act synergistically to enhance osteogenic differentiation and mineralization of BMS cells. These findings are potentially useful in developing engineered scaffolds for bone regeneration.

Coupling Gd-DTPA with a bispecific, recombinant protein anti-EGFR-iRGD complex improves tumor targeting in MRI

PubMed Central

XIN, XIAOYAN; SHA, HUIZI; SHEN, JINGTAO; ZHANG, BING; ZHU, BIN; LIU, BAORUI

2016-01-01

Recombinant anti-epidermal growth factor receptor-internalizing arginine-glycine-aspartic acid (anti-EGFR single-domain antibody fused with iRGD peptide) protein efficiently targets the EGFR extracellular domain and integrin αvβ/β5, and shows a high penetration into cells. Thus, this protein may improve penetration of conjugated drugs into the deep zone of gastric cancer multicellular 3D spheroids. In the present study, a novel tumor-targeting contrast agent for magnetic resonance imaging (MRI) was developed, by coupling gadolinium-diethylene triamine pentaacetate (Gd-DTPA) with the bispecific recombinant anti-EGFR-iRGD protein. The anti-EGFR-iRGD protein was extracted from Escherichia coli and Gd was loaded onto the recombinant protein by chelation using DTPA anhydride. Single-targeting agent anti-EGFR-DTPA-Gd, which served as the control, was also prepared. The results of the present study showed that anti-EGFR-iRGD-DTPA-Gd exhibited no significant cyto toxicity to human gastric carcinoma cells (BGC-823) under the experimental conditions used. Compared with a conventional contrast agent (Magnevist), anti-EGFR-iRGD-DTPA-Gd showed higher T1 relaxivity (10.157/mM/sec at 3T) and better tumor-targeting ability. In addition, the signal intensity and the area under curve for the enhanced signal time in tumor, in vivo, were stronger than Gd-DTPA alone or the anti-EGFR-Gd control. Thus, Gd-labelled anti-EGFR-iRGD has potential as a tumor-targeting contrast agent for improved MRI. PMID:27035336

RGD conjugates of the H2dedpa scaffold: synthesis, labeling and imaging with 68Ga.

PubMed

Boros, Eszter; Ferreira, Cara L; Yapp, Donald T T; Gill, Rajanvir K; Price, Eric W; Adam, Michael J; Orvig, Chris

2012-08-01

The rekindled interest in the (68)Ga generator as an attractive positron emission tomography generator system has led us and others to investigate novel chelate systems for (68)Ga. We have previously reported our findings with the acyclic, rapidly coordinating chelate H(2)dedpa and its model derivatives. In this report, we describe the synthesis of the corresponding bifunctional chelate scaffolds (H(2)dp-bb-NCS and H(2)dp-N-NCS) as well as the radiolabeling properties, transferrin stability, binding to the target using in vitro cell models and in vivo behavior the corresponding conjugates with the α(v)β(3) targeting cyclic pentapeptide cRGDyK (monomeric H(2)RGD-1 and dimeric H(2)RGD-2). The ability of the conjugated ligands to coordinate Ga isotopes within 10 min at room temperature at concentrations of 1 nmol was confirmed. Complex [(67)Ga(RGD-1)](+) was more stable (92% after 2 h) than [(67)Ga(RGD-2)](+) (73% after 2 h) in a transferrin challenge experiment. IC(50) values for both conjugates (H(2)RGD-1 and H(2)RGD-2) and nonconjugated RGD were determined in a cell-based competitive binding assay with (125)I-echistatin using U87MG cells, where enhanced specific binding was observed for the multivalent H(2)RGD-2 conjugate compared to the monovalent H(2)RGD-1 and nonconjugated cRGDyK. The U87MG cell line was also used to generate subcutaneous xenograft tumors on RAG2M mice, which were used to evaluate the in vivo properties of [(68)Ga(RGD-1)](+) and [(68)Ga(RGD-2)](+). After 2 h of dynamic imaging, both block and nonblock mice were sacrificed to collect select organs at the 2-h time point. Although the uptake is specific, as judged from the ratios of nonblock to block (2.36 with [(67)Ga(RGD-1)](+), 1.46 with [(67)Ga(RGD-2)](+)), both conjugates display high uptake in blood. We have successfully synthesized and applied the first bifunctional versions of H(2)dedpa for conjugation to a targeting vector and subsequent imaging of the corresponding conjugates. Copyright © 2012 Elsevier Inc. All rights reserved.

Integrin-targeted pH-responsive micelles for enhanced efficiency of anticancer treatment in vitro and in vivo

NASA Astrophysics Data System (ADS)

Liu, Jinjian; Deng, Hongzhang; Liu, Qiang; Chu, Liping; Zhang, Yumin; Yang, Cuihong; Zhao, Xuefei; Huang, Pingsheng; Deng, Liandong; Dong, Anjie; Liu, Jianfeng

2015-02-01

The key to developing more nanocarriers for the delivery of drugs in clinical applications is to consider the route of the carrier from the administration site to the target tissue and to look for a simple design to complete this whole journey. We synthesized the amphiphilic copolymer cRGDfK-poly(ethylene glycol)-b-poly(2,4,6-trimethoxybenzylidene-1,1,1-tris(hydroxymethyl) ethane methacrylate) (cRGD-PETM) to construct multifunctional micelles. These micelles combined enhanced drug-loading efficiency with tumor-targeting properties, visual detection and controllable intracellular drug release, resulting in an improved chemotherapeutic effect in vivo. Doxorubicin (DOX) was encapsulated within the cRGD-PETM micelles as a model drug (termed as cRGD-PETM/DOX Ms). The size and morphology of the micelles were characterized systematically. As a result of the hydrophobic interaction and the π-π conjugation between the DOX molecules and the PTTMA copolymers, the cRGD-PETM/DOX Ms showed an excellent drug-loading capacity. The results of in vitro drug-release studies indicated that the cumulative release of DOX from cRGD-PETM/DOX Ms at pH 5.0 was twice that at pH 7.4. The results of fluorescent microscopic analysis showed that the cRGD-PETM/DOX Ms could be internalized by 4T1 and HepG2 cells via receptor-mediated endocytosis with rapid intracellular drug release, which resulted in increased cytotoxicity compared with free DOX. Ex vivo imaging studies showed that the cRGD-PETM/DOX Ms improved the accumulation and retention of the drug in tumor tissues. Studies of the in vivo anticancer effects showed that the cRGD-PETM/DOX Ms had a significantly higher therapeutic efficacy with lower side-effects than free DOX and PETM/DOX Ms. These results show that the multifunctional cRGD-PETM/DOX Ms have great potential as vehicles for the delivery of hydrophobic anticancer drugs.

Endothelium-derived fibronectin regulates neonatal vascular morphogenesis in an autocrine fashion.

PubMed

Turner, Christopher J; Badu-Nkansah, Kwabena; Hynes, Richard O

2017-11-01

Fibronectin containing alternatively spliced EIIIA and EIIIB domains is largely absent from mature quiescent vessels in adults, but is highly expressed around blood vessels during developmental and pathological angiogenesis. The precise functions of fibronectin and its splice variants during developmental angiogenesis however remain unclear due to the presence of cardiac, somitic, mesodermal and neural defects in existing global fibronectin KO mouse models. Using a rare family of surviving EIIIA EIIIB double KO mice, as well as inducible endothelial-specific fibronectin-deficient mutant mice, we show that vascular development in the neonatal retina is regulated in an autocrine manner by endothelium-derived fibronectin, and requires both EIIIA and EIIIB domains and the RGD-binding α5 and αv integrins for its function. Exogenous sources of fibronectin do not fully substitute for the autocrine function of endothelial fibronectin, demonstrating that fibronectins from different sources contribute differentially to specific aspects of angiogenesis.

Design of RGD-ATWLPPR peptide conjugates for the dual targeting of αVβ3 integrin and neuropilin-1.

PubMed

Thoreau, Fabien; Vanwonterghem, Laetitia; Henry, Maxime; Coll, Jean-Luc; Boturyn, Didier

2018-05-18

Targeting the tumour microenvironment is a promising strategy to detect and/or treat cancer. The design of selective compounds that co-target several receptors frequently overexpressed in solid tumours may allow a reliable and selective detection of tumours. Here we report the modular synthesis of compounds encompassing ligands of αVβ3 integrin and neuropilin-1 that are overexpressed in the tumour microenvironment. These compounds were then evaluated through cellular experiments and imaging of tumours in mice. We observed that the peptide that displays both ligands is more specifically accumulating in the tumours than in controls. Simultaneous interaction with αVβ3 integrin and NRP1 induces NRP1 stabilization at the cell membrane surface which is not observed with the co-injection of the controls.

Molecular dynamics simulations of certain RGD-based peptides from Kistrin provide insight into the higher activity of REI-RGD34 protein at higher temperature.

PubMed

Upadhyay, Sanjay K

2014-05-01

To determine the bioactive conformation required to bind with receptor aIIbb3, the peptide sequence RIPRGDMP from Kistrin was inserted into CDR 1 loop region of REI protein, resulting in REI-RGD34. The activity of REI-RGD34 was observed to increase at higher temperature towards the receptor aIIbb3. It could be justified in either way: the modified complex forces the restricted peptide to adapt bioactive conformation or it unfolds the peptide in a way that opens its binding surface with high affinity for receptor. Here, we model the conformational preference of RGD sequence in RIPRGDMP at 25 and 42 °C using multiple MD simulations. Further, we model the peptide sequence RGD, PRGD and PRGDMP from kistrin to observe the effect of flanking residues on conformational sampling of RGD. The presence of flanking residues around RGD peptide greatly influenced the conformational sampling. A transition from bend to turn conformation was observed for RGD sequence at 42 °C. The turn conformation shows pharmacophoric parameters required to recognize the receptor aIIbb3. Thus, the temperaturedependent activity of RIPRGDMP when inserted into the loop region of REI can be explained by the presence of the turn conformation. This study will help in designing potential antagonist for the receptor aIIbb3.

Electronic structure, dielectric response, and surface charge distribution of RGD (1FUV) peptide.

PubMed

Adhikari, Puja; Wen, Amy M; French, Roger H; Parsegian, V Adrian; Steinmetz, Nicole F; Podgornik, Rudolf; Ching, Wai-Yim

2014-07-08

Long and short range molecular interactions govern molecular recognition and self-assembly of biological macromolecules. Microscopic parameters in the theories of these molecular interactions are either phenomenological or need to be calculated within a microscopic theory. We report a unified methodology for the ab initio quantum mechanical (QM) calculation that yields all the microscopic parameters, namely the partial charges as well as the frequency-dependent dielectric response function, that can then be taken as input for macroscopic theories of electrostatic, polar, and van der Waals-London dispersion intermolecular forces. We apply this methodology to obtain the electronic structure of the cyclic tripeptide RGD-4C (1FUV). This ab initio unified methodology yields the relevant parameters entering the long range interactions of biological macromolecules, providing accurate data for the partial charge distribution and the frequency-dependent dielectric response function of this peptide. These microscopic parameters determine the range and strength of the intricate intermolecular interactions between potential docking sites of the RGD-4C ligand and its integrin receptor.

A targeted nanoglobular contrast agent from host-guest self-assembly for MR cancer molecular imaging.

PubMed

Zhou, Zhuxian; Han, Zhen; Lu, Zheng-Rong

2016-04-01

The clinical application of nanoparticular Gd(III) based contrast agents for tumor molecular MRI has been hindered by safety concerns associated with prolonged tissue retention, although they can produce strong tumor enhancement. In this study, a targeted well-defined cyclodextrin-based nanoglobular contrast agent was developed through self-assembly driven by host-guest interactions for safe and effective cancer molecular MRI. Multiple β-cyclodextrins attached POSS (polyhedral oligomeric silsesquioxane) nanoglobule was used as host molecule. Adamantane-modified macrocyclic Gd(III) contrast agent, cRGD (cyclic RGDfK peptide) targeting ligand and fluorescent probe was used as guest molecules. The targeted host-guest nanoglobular contrast agent cRGD-POSS-βCD-(DOTA-Gd) specifically bond to αvβ3 integrin in malignant 4T1 breast tumor and provided greater contrast enhancement than the corresponding non-targeted agent. The agent also provided significant fluorescence signal in tumor tissue. The histological analysis of the tumor tissue confirmed its specific and effective targeting to αvβ3 integrin. The targeted imaging agent has a potential for specific cancer molecular MR and fluorescent imaging. Copyright © 2016 Elsevier Ltd. All rights reserved.

The influence of surface integrin binding patterns on specific biomaterial-cell interactions

NASA Astrophysics Data System (ADS)

Beranek, Maggi Marie

As the future of biomaterials progresses toward bioactivity, the biomaterial surface must control non-specific protein adsorption and encourage selective protein and cell adsorption. Integrins alphavbeta3, alpha 1beta1, alpha5beta1 and alpha Mbeta2 are expressed on cells involved in endothelialization, inflammation, and intimal hyperplasia. These cellular events play a vital role in biomaterial biocompatibility, especially in the vascular environment. The overall hypothesis of these studies is that biomaterial surfaces exhibit selective integrin binding, which then specifies differential cell binding. To test this hypothesis, four specific aims were developed. The first aim was designed to determine whether metal and polymeric biomaterials exhibit selective integrin binding. The tested materials included 316L stainless steel, nitinol, gold, Elgiloy RTM, poly(D, L-lactide-co-glycolide), polycarbonate urethane and expanded polytetrafluoroethylene. Discrete integrin binding patterns were detected microscopically using integrin specific fluorescent antibodies. Stainless steel exhibited high level integrin alpha1beta 1 and low level integrin alphaMbeta2 binding pattern. This suggests that this metal surface should selectively encourage endothelial cell to inflammatory cell binding. In contrast, gold bound ten times the amount of integrin alphaMbeta2 compared to integrin alpha1beta1, which should encourage inflammatory cell adhesion. The 65/35 poly(D, L-lactide-co-glycolide) was the only polymeric biomaterial tested that had integrin binding levels comparable to metal biomaterials. Based on these observations, a combinational biomaterial with a surface pattern of 65/35 poly(D, L-lactide-co-glycolide) dots on a 316L stainless steel background was created. A pattern of high level integrin alpha1beta1 binding and low level integrin alpha Mbeta2 binding on this combinational surface indicates that this surface should selectively favor endothelial cell binding. In the second aim, the response of surface-bound integrins to flow-related shear stress was examined. Based on fluorescent analysis, total alphavbeta 3, alpha1beta1, and alpha5beta 1 appeared to increase on stainless steel after 90-minute low shear stress exposure, whereas only alpha5beta1 appeared to increase when exposed to high shear. 65/35 poly(D, L-lactide-co-glycolide) exhibited increased total binding of alpha5beta1 and alphaMbeta2, when exposed to either shear stress level. Exposure to either shear stress regimen appeared to increase binding of all integrins on the combinational surface. These responses to shear stress suggest differential integrin binding affinity compared to stainless steel. Using antibodies specific to the integrin subunits, the apparent increase in surface-bound integrins was found to be related to a surface disassociation of alpha and beta subunits. The third aim evaluated human aortic endothelial cells and acute monocytic leukemia cells (THP-1) cell binding to the tested biomaterial surfaces under both static and flow conditions. Both stainless steel and the combinational surface had increased endothelial cell binding compared to monocyte attachment. Pre-incubation of the surface with the specific integrins significantly inhibited human aortic endothelial cell binding. Aim four was designed to investigate the influence of surface bound integrins on human aortic endothelial cell migration under shear stress. If biomaterial surface integrin binding patterns are specific, then pre-bound surface integrins should competitively inhibit binding of cellular integrins to the surface. Cell migration distance on to alphavbeta3, alpha 1beta1, and alpha5beta1 pre-incubated stainless steel was decreased ten-fold, and decreased by three-fold on both 65/35 poly(D, L-lactide-coglycolide) and combinational surfaces compared to the respective bare surfaces. In contrast, migration distance on to alphaMbeta2 pre-coated stainless steel and combinational surface was decreased by only sixty percent and only fifty percent on alphaMbeta2 precoated 65/35 poly(D, L -lactide-co-glycolide). These results suggested that surface binding sites are selective and critical in governing endothelial cell migration. In conclusion, these results support the hypothesis that a surface that encourages specific integrin binding would promote differential cell binding. The novel integrin binding model used in this investigation may be a methodology that can be employed to evaluate potential vascular biomaterials.

The regulation of integrin function by divalent cations

PubMed Central

Zhang, Kun; Chen, JianFeng

2012-01-01

Integrins are a family of α/β heterodimeric adhesion metalloprotein receptors and their functions are highly dependent on and regulated by different divalent cations. Recently advanced studies have revolutionized our perception of integrin metal ion-binding sites and their specific functions. Ligand binding to integrins is bridged by a divalent cation bound at the MIDAS motif on top of either α I domain in I domain-containing integrins or β I domain in α I domain-less integrins. The MIDAS motif in β I domain is flanked by ADMIDAS and SyMBS, the other two crucial metal ion binding sites playing pivotal roles in the regulation of integrin affinity and bidirectional signaling across the plasma membrane. The β-propeller domain of α subunit contains three or four β-hairpin loop-like Ca2+-binding motifs that have essential roles in integrin biogenesis. The function of another Ca2+-binding motif located at the genu of α subunit remains elusive. Here, we provide an overview of the integrin metal ion-binding sites and discuss their roles in the regulation of integrin functions. PMID:22647937

Targeted Cancer Therapy: Correlative Light-Electron Microscopy Shows RGD-Targeted ZnO Nanoparticles Dissolve in the Intracellular Environment of Triple Negative Breast Cancer Cells and Cause Apoptosis with Intratumor Heterogeneity (Adv. Healthcare Mater. 11/2016).

PubMed

Othman, Basmah A; Greenwood, Christina; Abuelela, Ayman F; Bharath, Anil A; Chen, Shu; Theodorou, Ioannis; Douglas, Trevor; Uchida, Maskai; Ryan, Mary; Merzaban, Jasmeen S; Porter, Alexandra E

2016-06-01

On page 1310 J. S. Merzaban, A. E. Porter, and co-workers present fluorescently labeled RGD-targeted ZnO nanoparticles (NPs; green) for the targeted delivery of cytotoxic ZnO to integrin αvβ3 receptors expressed on triple negative breast cancer cells. Correlative light-electron microscopy shows that NPs dissolve into ionic Zn(2+) (blue) upon uptake and cause apoptosis (red) with intra-tumor heterogeneity, thereby providing a possible strategy for targeted breast cancer therapy. Cover design by Ivan Gromicho. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Engineered peptide-based nanobiomaterials for electrochemical cell chip.

PubMed

Kafi, Md Abdul; Cho, Hyeon-Yeol; Choi, Jeong-Woo

2016-01-01

Biomaterials having cell adhesion ability are considered to be integral part of a cell chip. A number of researches have been carried out to search for a suitable material for effective immobilization of cell on substrate. Engineered ECM materials or their components like collagen, Poly-l-Lysine (PLL), Arg-Gly-Asp (RGD) peptide have been extensively used for mammalian cell adhesion and proliferation with the aim of tissue regeneration or cell based sensing application. This review focuses on the various approaches for two- and three-dimensionally patterned nanostructures of a short peptide i.e. RGD peptide on chip surfaces together with their effects on cell behaviors and electrochemical measurements. Most of the study concluded with positive remarks on the well-oriented engineered RGD peptide over their homogenous thin film. The engineered RGD peptide not only influences cell adhesion, spreading and proliferation but also their periodic nano-arrays directly influence electrochemical measurements of the chips. The electrochemical signals found to be enhanced when RGD peptides were used in well-defined two-dimensional nano-arrays. The topographic alteration of three-dimensional structure of engineered RGD peptide was reported to be suitably contacted with the integrin receptors of cellular membrane which results indicated the enhanced cell-electrode adhesion and efficient electron exchange phenomenon. This enhanced electrochemical signal increases the sensitivity of the chip against the target analytes. Therefore, development of engineered cellular recognizable peptides and its 3D topological design for fabrication of cell chip will provide the synergetic effect on bio-affinity, sensitivity and accuracy for the in situ real-time monitoring of analytes.

Protein Disulfide Isomerase Directly Interacts with β-Actin Cys374 and Regulates Cytoskeleton Reorganization*

PubMed Central

Sobierajska, Katarzyna; Skurzynski, Szymon; Stasiak, Marta; Kryczka, Jakub; Cierniewski, Czeslaw S.; Swiatkowska, Maria

2014-01-01

Recent studies support the role of cysteine oxidation in actin cytoskeleton reorganization during cell adhesion. The aim of this study was to explain whether protein disulfide isomerase (PDI) is responsible for the thiol-disulfide rearrangement in the β-actin molecule of adhering cells. First, we showed that PDI forms a disulfide-bonded complex with β-actin with a molecular mass of 110 kDa. Specific interaction of both proteins was demonstrated by a solid phase binding assay, surface plasmon resonance analysis, and immunoprecipitation experiments. Second, using confocal microscopy, we found that both proteins colocalized when spreading MEG-01 cells on fibronectin. Colocalization of PDI and β-actin could be abolished by the membrane-permeable sulfhydryl blocker, N-ethylmaleimide, by the RGD peptide, and by anti-αIIbβ3 antibodies. Consequently, down-regulation of PDI expression by antisense oligonucleotides impaired the spreading of cells and initiated reorganization of the cytoskeleton. Third, because of transfection experiments followed by immunoprecipitation and confocal analysis, we provided evidence that PDI binds to the β-actin Cys374 thiol. Formation of the β-actin-PDI complex was mediated by integrin-dependent signaling in response to the adhesion of cells to the extracellular matrix. Our data suggest that PDI is released from subcellular compartments to the cytosol and translocated toward the periphery of the cell, where it forms a disulfide bond with β-actin when MEG-01 cells adhere via the αIIbβ3 integrin to fibronectin. Thus, PDI appears to regulate cytoskeletal reorganization by the thiol-disulfide exchange in β-actin via a redox-dependent mechanism. PMID:24415753

Effects of arginine-glycine-aspartic acid peptide-conjugated quantum dots-induced photodynamic therapy on pancreatic carcinoma in vivo.

PubMed

Li, Ming-Ming; Cao, Jia; Yang, Jia-Chun; Shen, Yu-Jie; Cai, Xiao-Lei; Chen, Yuan-Wen; Qu, Chun-Ying; Zhang, Yi; Shen, Feng; Xu, Lei-Ming

2017-01-01

Quantum dots (QDs) conjugated with integrin antagonist arginine-glycine-aspartic acid (RGD) peptides (QDs-RGD) are novel nanomaterials with a unique optical property: a high molar extinction coefficient. Previously, we have shown that QDs-RGD demonstrate a photodynamic therapy (PDT) effect as new photosensitizers for the pancreatic cancer cell line SW1990 in vitro. Here, we investigate the application of QDs-RGD in mice bearing pancreatic tumors using PDT. To ensure that more photosensitizers accumulated in tumors, QDs-RGD were injected intratumorally. After selection of an adequate dosage for injection from analyses of biodistribution images captured by an IVIS system, PDT was initiated. Three groups were created according to different PDT procedures. In group 1, mice were injected with QDs-RGD intratumorally, and an optical fiber connected to a laser light was inserted directly into the tumor. Irradiation was sustained for 20 min with a laser light (630 nm) at 100 mW/cm 2 . In group 2, the laser optical fiber was placed around, and not inserted into, tumors. In group 3, PDT was conducted as in group 1 but without injection of QDs-RGD. After 28 days of observation, tumors on the back of mice in group 1 grew slowly (V/V 0 =3.24±0.70) compared with the control groups, whose tumors grew quickly, and the mean V/V 0 reached 6.08±0.50 (group 2) and 7.25±0.82 (group 3). Histology of tumor tissues showed more necrotic tissues, more inflammatory cells, and less vascular tissue in the PDT group than those in the control groups. These results suggest that QDs-RGD-mediated PDT, with illumination using an optical fiber inserted directly into the tumor, can inhibit the growth of SW1990 tumors with high efficiency in nude mice.

PET-Based Human Dosimetry of the Dimeric αvβ3 Integrin Ligand 68Ga-DOTA-E-[c(RGDfK)]2, a Potential Tracer for Imaging Tumor Angiogenesis.

PubMed

López-Rodríguez, Victoria; Galindo-Sarco, Carlos; García-Pérez, Francisco O; Ferro-Flores, Guillermina; Arrieta, Oscar; Ávila-Rodríguez, Miguel A

2016-03-01

Peptides containing the Arg-Gly-Asp (RGD) sequence have high affinity for αvβ3 integrin receptors overexpressed in tumor cells. The objective of this research was to determine the biodistribution and estimate the radiation dose from (68)Ga-DOTA-E-[c(RGDfK)]2 using whole-body PET scans in humans. Five healthy volunteers (2 women, 3 men; mean age ± SD, 37.2 ± 15.6 y; range, 28-65 y; mean weight, 79.2 ± 21.0 kg; range, 64-115 kg) were included. After intravenous injection of the tracer (198.3 ± 3.3 MBq), 3 successive whole-body (vertex to mid thigh) PET/CT scans at 3 time points (30, 60, and 120 min) were obtained on a 16-slice PET/CT scanner. The subjects did not void the bladder until the entire series of images was completed. Low-dose CT without contrast agent was used for anatomic localization and attenuation correction. OLINDA/EXM software was applied to calculate human radiation doses using the reference adult model. The highest uptake was in the urinary bladder, followed by the liver, kidneys, and spleen, in descending order. The critical organ was the urinary bladder wall. The mean effective doses (all subjects, men and women) were 34.1 ± 4.9, 31.0 ± 2.4, and 20.9 ± 5.2 μSv/MBq for the no-voiding, 2.5-h-voiding, and 1-h-voiding models, respectively. Of particular interest in this research was the visualization of the choroid plexus and ventricular system, which seems to be a characteristic of RGD-dimeric peptides. Measured absorbed doses and effective doses are comparable to other previously reported RGD-based radiopharmaceuticals labeled with (68)Ga and (18)F. Therefore, (68)Ga-DOTA-E-[c(RGDfK)]2 can safely be used for imaging integrin αVβ3 expression. © 2016 by the Society of Nuclear Medicine and Molecular Imaging, Inc.

Near-infrared fluorescent peptide probes for imaging of tumor in vivo and their biotoxicity evaluation.

PubMed

Liu, Liwei; Lin, Guimiao; Yin, Feng; Law, Wing-Cheung; Yong, Ken-Tye

2016-04-01

Optical imaging techniques are becoming increasingly urgent for the early detection and monitoring the progression of tumor development. However, tumor vasculature imaging has so far been largely unexplored because of the lack of suitable optical probes. In this study, we demonstrated the preparation of near-infrared (NIR) fluorescent RGD peptide probes for noninvasive imaging of tumor vasculature during tumor angiogenesis. The peptide optical probes combined the advantages of NIR emission and RGD peptide, which possesses minimal biological absorption and specially targets the integrin, which highly expressed on activated tumor endothelial cells. In vivo optical imaging of nude mice bearing pancreatic tumor showed that systemically delivered NIR probes enabled us to visualize the tumors at 24 hours post-injection. In addition, we have performed in vivo toxicity study on the prepared fluorescent RGD peptide probes formulation. The blood test results and histological analysis demonstrated that no obvious toxicity was found for the mice treated with RGD peptide probes for two weeks. These studies suggest that the NIR fluorescent peptide probes can be further designed and employed for ultrasensitive fluorescence imaging of angiogenic tumor vasculature, as well as imaging of other pathophysiological processes accompanied by activation of endothelial cells. © 2016 Wiley Periodicals, Inc.

The improvement of fibroblast growth on hydrophobic biopolyesters by coating with polyhydroxyalkanoate granule binding protein PhaP fused with cell adhesion motif RGD.

PubMed

Dong, Ying; Li, Ping; Chen, Chong-bo; Wang, Zhi-hui; Ma, Ping; Chen, Guo-Qiang

2010-12-01

Polyhydroxyalkanoates (PHA), a family of biopolyesters, have been studied as tissue engineering biomaterials due to their adjustable mechanical properties, biodegradability and tissue compatibility. Amphiphilic PHA granule binding protein PhaP has been shown to be able to bind to hydrophobic surfaces of polymers, especially PHA, via strong hydrophobic interaction. Genes of PhaP and RGD peptides, which are a cell adhesion motif recognized by many cell surface receptors, were successfully expressed and obtained as a pure fusion protein PhaP-RGD in Escherichia coli DH5α. When films of poly(3-hydroxybutyrate-co-3-hydroxy- hexanoate) (PHBHHx), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and polylactic acid (PLA) were coated with PhaP-RGD, their surface hydrophilicities were all increased compared with their corresponding naked (non-coated) films, respectively. Among the three biopolyesters, PHBHHx demonstrated the strongest affinity to PhaP. In vitro study showed that mouse fibroblasts L929 and mouse embryonic fibroblasts NIH/3T3 attached better and grew faster on all three PhaP-RGD coated films compared with their related behaviors on PhaP coated and non-coated films, respectively. Both fibroblasts attached and grew very well on PhaP-RGD coated PHBHHx, PHBV and PLA, even in their serum-free medium, while the non-coated and PhaP coated biopolyesters poorly supported the cell growth if the two fibroblasts were incubated in their serum free medium. These results indicated that PhaP-RGD could be used as a coating material to improve cell growth on hydrophobic biopolyesters for implant tissue engineering purposes. Copyright © 2010 Elsevier Ltd. All rights reserved.

Electronic Structure, Dielectric Response, and Surface Charge Distribution of RGD (1FUV) Peptide

PubMed Central

Adhikari, Puja; Wen, Amy M.; French, Roger H.; Parsegian, V. Adrian; Steinmetz, Nicole F.; Podgornik, Rudolf; Ching, Wai-Yim

2014-01-01

Long and short range molecular interactions govern molecular recognition and self-assembly of biological macromolecules. Microscopic parameters in the theories of these molecular interactions are either phenomenological or need to be calculated within a microscopic theory. We report a unified methodology for the ab initio quantum mechanical (QM) calculation that yields all the microscopic parameters, namely the partial charges as well as the frequency-dependent dielectric response function, that can then be taken as input for macroscopic theories of electrostatic, polar, and van der Waals-London dispersion intermolecular forces. We apply this methodology to obtain the electronic structure of the cyclic tripeptide RGD-4C (1FUV). This ab initio unified methodology yields the relevant parameters entering the long range interactions of biological macromolecules, providing accurate data for the partial charge distribution and the frequency-dependent dielectric response function of this peptide. These microscopic parameters determine the range and strength of the intricate intermolecular interactions between potential docking sites of the RGD-4C ligand and its integrin receptor. PMID:25001596

Role of Altered Sialylation of the I-Like Domain of β1 Integrin in the Binding of Fibronectin to β1 Integrin: Thermodynamics and Conformational Analyses

PubMed Central

Pan, Di; Song, Yuhua

2010-01-01

Abstract N-glycosylation of the I-like domain of β1 integrin plays an essential role in integrin structure and function, and the altered sialylation of β1 integrin regulates β1 integrin binding to fibronectin. However, the structural basis underlying the effect of altered sialylation of the β1 I-like domain on β1 integrin binding to fibronectin remains largely unknown. In this study, we used a combination of molecular dynamics simulations and binding free energy analyses to investigate changes in binding thermodynamics and in conformation of the glycosylated β1 I-like domain-FN-III9-10 complex caused by altered sialylation of the β1 I-like domain. Binding free energy analyses showed that desialylation of β1 I-like domain increased β1 integrin binding to fibronectin, consistent with experimental results. Interaction analyses showed that altered sialylation of the β1 I-like domain resulted in significant changes in the interaction of the N-glycans of the I-like domain with both the I-like domain and fibronectin, and these changes could directly affect the allosteric regulation of the interaction between the I-like domain and fibronectin. Altered sialylation of the β1 I-like domain caused significant conformational changes in key functional sites of both the β1 I-like domain and fibronectin. In addition, altered sialylation of the β1 I-like domain resulted in changes in the degree of correlated motions between residues in the I-like domain and residues in fibronectin, and in the degree of motion changes in fibronectin, which could affect β1 integrin binding to fibronectin. We believe results from this study provide thermodynamic and structural evidence for a role of altered sialylation of β1 integrin in regulating β1 integrin binding to fibronectin and it's induced cellular activities. PMID:20655849

Engineering Factor Xa Inhibitor with Multiple Platelet-Binding Sites Facilitates its Platelet Targeting

NASA Astrophysics Data System (ADS)

Zhu, Yuanjun; Li, Ruyi; Lin, Yuan; Shui, Mengyang; Liu, Xiaoyan; Chen, Huan; Wang, Yinye

2016-07-01

Targeted delivery of antithrombotic drugs centralizes the effects in the thrombosis site and reduces the hemorrhage side effects in uninjured vessels. We have recently reported that the platelet-targeting factor Xa (FXa) inhibitors, constructed by engineering one Arg-Gly-Asp (RGD) motif into Ancylostoma caninum anticoagulant peptide 5 (AcAP5), can reduce the risk of systemic bleeding than non-targeted AcAP5 in mouse arterial injury model. Increasing the number of platelet-binding sites of FXa inhibitors may facilitate their adhesion to activated platelets, and further lower the bleeding risks. For this purpose, we introduced three RGD motifs into AcAP5 to generate a variant NR4 containing three platelet-binding sites. NR4 reserved its inherent anti-FXa activity. Protein-protein docking showed that all three RGD motifs were capable of binding to platelet receptor αIIbβ3. Molecular dynamics simulation demonstrated that NR4 has more opportunities to interact with αIIbβ3 than single-RGD-containing NR3. Flow cytometry analysis and rat arterial thrombosis model further confirmed that NR4 possesses enhanced platelet targeting activity. Moreover, NR4-treated mice showed a trend toward less tail bleeding time than NR3-treated mice in carotid artery endothelium injury model. Therefore, our data suggest that engineering multiple binding sites in one recombinant protein is a useful tool to improve its platelet-targeting efficiency.

Approaches to utilize mesenchymal progenitor cells as cellular vehicles.

PubMed

Pereboeva, L; Komarova, S; Mikheeva, G; Krasnykh, V; Curiel, D T

2003-01-01

Mammalian cells represent a novel vector approach for gene delivery that overcomes major drawbacks of viral and nonviral vectors and couples cell therapy with gene delivery. A variety of cell types have been tested in this regard, confirming that the ideal cellular vector system for ex vivo gene therapy has to comply with stringent criteria and is yet to be found. Several properties of mesenchymal progenitor cells (MPCs), such as easy access and simple isolation and propagation procedures, make these cells attractive candidates as cellular vehicles. In the current work, we evaluated the potential utility of MPCs as cellular vectors with the intent to use them in the cancer therapy context. When conventional adenoviral (Ad) vectors were used for MPC transduction, the highest transduction efficiency of MPCs was 40%. We demonstrated that Ad primary-binding receptors were poorly expressed on MPCs, while the secondary Ad receptors and integrins presented in sufficient amounts. By employing Ad vectors with incorporated integrin-binding motifs (Ad5lucRGD), MPC transduction was augmented tenfold, achieving efficient genetic loading of MPCs with reporter and anticancer genes. MPCs expressing thymidine kinase were able to exert a bystander killing effect on the cancer cell line SKOV3ip1 in vitro. In addition, we found that MPCs were able to support Ad replication, and thus can be used as cell vectors to deliver oncolytic viruses. Our results show that MPCs can foster expression of suicide genes or support replication of adenoviruses as potential anticancer therapeutic payloads. These findings are consistent with the concept that MPCs possess key properties that ensure their employment as cellular vehicles and can be used to deliver either therapeutic genes or viruses to tumor sites.

Akt/protein kinase B activation by adenovirus vectors contributes to NFkappaB-dependent CXCL10 expression.

PubMed

Liu, Qiang; White, Lindsay R; Clark, Sharon A; Heffner, Daniel J; Winston, Brent W; Tibbles, Lee Anne; Muruve, Daniel A

2005-12-01

In gene therapy, the innate immune system is a significant barrier to the effective application of adenovirus (Ad) vectors. In kidney epithelium-derived (REC) cells, serotype 5 Ad vectors induce the expression of the chemokine CXCL10 (IP-10), a response that is dependent on NFkappaB. Compared to the parental vector AdLuc, transduction with the RGD-deleted vector AdL.PB resulted in reduced CXCL10 activation despite increasing titers, implying that RGD-alpha(V) integrin interactions contribute to adenovirus induction of inflammatory genes. Akt, a downstream effector of integrin signaling, was activated within 10 min of transduction with Ad vectors in a dose-dependent manner. Akt activation was not present following transduction with AdL.PB, confirming the importance of capsid-alpha(V) integrin interactions in Ad vector Akt activation. Inhibition of the phosphoinositide-3-OH kinase/Akt pathway by Wortmannin or Ly294002 compounds decreased Ad vector induction of CXCL10 mRNA. Similarly, adenovirus-mediated overexpression of the dominant negative AktAAA decreased CXCL10 mRNA expression compared to the reporter vector AdLacZ alone. The effect of Akt on CXCL10 mRNA expression occurred via NFkappaB-dependent transcriptional activation, since AktAAA overexpression and Ly294002 both inhibited CXCL10 and NFkappaB promoter activation in luciferase reporter experiments. These results show that Akt plays a role in the Ad vector activation of NFkappaB and CXCL10 expression. Understanding the mechanism underlying the regulation of host immunomodulatory genes by adenovirus vectors will lead to strategies that will improve the efficacy and safety of these agents for clinical use.

Integrin-assisted drug delivery of nano-scaled polymer therapeutics bearing paclitaxel.

PubMed

Eldar-Boock, Anat; Miller, Keren; Sanchis, Joaquin; Lupu, Ruth; Vicent, María J; Satchi-Fainaro, Ronit

2011-05-01

Angiogenesis plays a prominent role in cancer progression. Anti-angiogenic therapy therefore, either alone or in combination with conventional cytotoxic therapy, offers a promising therapeutic approach. Paclitaxel (PTX) is a widely-used potent cytotoxic drug that also exhibits anti-angiogenic effects at low doses. However, its use, at its full potential, is limited by severe side effects. Here we designed and synthesized a targeted conjugate of PTX, a polymer and an integrin-targeted moiety resulting in a polyglutamic acid (PGA)-PTX-E-[c(RGDfK)(2)] nano-scaled conjugate. Polymer conjugation converted PTX to a macromolecule, which passively targets the tumor tissue exploiting the enhanced permeability and retention effect, while extravasating via the leaky tumor neovasculature. The cyclic RGD peptidomimetic enhanced the effects previously seen for PGA-PTX alone, utilizing the additional active targeting to the α(v)β(3) integrin overexpressed on tumor endothelial and epithelial cells. This strategy is particularly valuable when tumors are well-vascularized, but they present poor vascular permeability. We show that PGA is enzymatically-degradable leading to PTX release under lysosomal acidic pH. PGA-PTX-E-[c(RGDfK)(2)] inhibited the growth of proliferating α(v)β(3)-expressing endothelial cells and several cancer cells. We also showed that PGA-PTX-E-[c(RGDfK)(2)] blocked endothelial cells migration towards vascular endothelial growth factor; blocked capillary-like tube formation; and inhibited endothelial cells attachment to fibrinogen. Orthotopic studies in mice demonstrated preferential tumor accumulation of the RGD-bearing conjugate, leading to enhanced anti-tumor efficacy and a marked decrease in toxicity as compared with free PTX-treated mice. Copyright © 2011 Elsevier Ltd. All rights reserved.

The bioactive acidic serine- and aspartate-rich motif peptide.

PubMed

Minamizaki, Tomoko; Yoshiko, Yuji

2015-01-01

The organic component of the bone matrix comprises 40% dry weight of bone. The organic component is mostly composed of type I collagen and small amounts of non-collagenous proteins (NCPs) (10-15% of the total bone protein content). The small integrin-binding ligand N-linked glycoprotein (SIBLING) family, a NCP, is considered to play a key role in bone mineralization. SIBLING family of proteins share common structural features and includes the arginine-glycine-aspartic acid (RGD) motif and acidic serine- and aspartic acid-rich motif (ASARM). Clinical manifestations of gene mutations and/or genetically modified mice indicate that SIBLINGs play diverse roles in bone and extraskeletal tissues. ASARM peptides might not be primary responsible for the functional diversity of SIBLINGs, but this motif is suggested to be a key domain of SIBLINGs. However, the exact function of ASARM peptides is poorly understood. In this article, we discuss the considerable progress made in understanding the role of ASARM as a bioactive peptide.

Structure-Guided Design of a High-Affinity Platelet Integrin αIIbβ3 Receptor Antagonist That Disrupts Mg2+ Binding to the MIDAS | Center for Cancer Research

Cancer.gov

A Better Fit. An improved anticoagulant drug called RUC-2 (ball and stick structure) fits snugly into its binding pocket on integrin (blue), a protein found on the surface of platelets. RUC-2 binds both subunits of integrin, inhibiting the excessive blood coagulation that can lead to strokes and heart attacks. Unlike similar drugs that alter integrin's structure when they bind

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kawamoto, Eiji; Emergency and Critical Care Center, Mie University Hospital, 2-174 Edobashi, Tsu 514-8507; Okamoto, Takayuki, E-mail: okamotot@doc.medic.mie-u.ac.jp

LFA-1 (αLβ2) and Mac-1 (αMβ2) integrins regulate leukocyte trafficking in health and disease by binding primarily to IgSF ligand ICAM-1 and ICAM-2 on endothelial cells. Here we have shown that the anti-coagulant molecule thrombomodulin (TM), found on the surface of endothelial cells, functions as a potentially new ligand for leukocyte integrins. We generated a recombinant extracellular domain of human TM and Fc fusion protein (TM-domains 123-Fc), and showed that pheripheral blood mononuclear cells (PBMCs) bind to TM-domains 123-Fc dependent upon integrin activation. We then demonstrated that αL integrin-blocking mAb, αM integrin-blocking mAb, and β2 integrin-blocking mAb inhibited the binding ofmore » PBMCs to TM-domains 123-Fc. Furthermore, we show that the serine/threonine-rich domain (domain 3) of TM is required for the interaction with the LFA-1 (αLβ2) and Mac-1 (αMβ2) integrins to occur on PBMCs. These results demonstrate that the LFA-1 and Mac-1 integrins on leukocytes bind to TM, thereby establishing the molecular and structural basis underlying LFA-1 and Mac-1 integrin interaction with TM on endothelial cells. In fact, integrin-TM interactions might be involved in the dynamic regulation of leukocyte adhesion with endothelial cells. - Highlights: • LFA-1 and Mac-1 integrins bind to the anti-coagulant molecule thrombomodulin. • The serine/threonine-rich domain of thrombomodulin is essential to interact with the LFA-1 and Mac-1 integrins on PBMCs. • Integrin-TM interactions might be involved in the dynamic regulation of leukocyte adhesion with endothelial cells.« less

Integrin-specific mechanoresponses to compression and extension probed by cylindrical flat-ended AFM tips in lung cells.

PubMed

Acerbi, Irene; Luque, Tomás; Giménez, Alícia; Puig, Marta; Reguart, Noemi; Farré, Ramon; Navajas, Daniel; Alcaraz, Jordi

2012-01-01

Cells from lung and other tissues are subjected to forces of opposing directions that are largely transmitted through integrin-mediated adhesions. How cells respond to force bidirectionality remains ill defined. To address this question, we nanofabricated flat-ended cylindrical Atomic Force Microscopy (AFM) tips with ~1 µm(2) cross-section area. Tips were uncoated or coated with either integrin-specific (RGD) or non-specific (RGE/BSA) molecules, brought into contact with lung epithelial cells or fibroblasts for 30 s to form focal adhesion precursors, and used to probe cell resistance to deformation in compression and extension. We found that cell resistance to compression was globally higher than to extension regardless of the tip coating. In contrast, both tip-cell adhesion strength and resistance to compression and extension were the highest when probed at integrin-specific adhesions. These integrin-specific mechanoresponses required an intact actin cytoskeleton, and were dependent on tyrosine phosphatases and Ca(2+) signaling. Cell asymmetric mechanoresponse to compression and extension remained after 5 minutes of tip-cell adhesion, revealing that asymmetric resistance to force directionality is an intrinsic property of lung cells, as in most soft tissues. Our findings provide new insights on how lung cells probe the mechanochemical properties of the microenvironment, an important process for migration, repair and tissue homeostasis.

Integrin-Using Rotaviruses Bind α2β1 Integrin α2 I Domain via VP4 DGE Sequence and Recognize αXβ2 and αVβ3 by Using VP7 during Cell Entry

PubMed Central

Graham, Kate L.; Halasz, Peter; Tan, Yan; Hewish, Marilyn J.; Takada, Yoshikazu; Mackow, Erich R.; Robinson, Martyn K.; Coulson, Barbara S.

2003-01-01

Integrins α2β1, αXβ2, and αVβ3 have been implicated in rotavirus cell attachment and entry. The virus spike protein VP4 contains the α2β1 ligand sequence DGE at amino acid positions 308 to 310, and the outer capsid protein VP7 contains the αXβ2 ligand sequence GPR. To determine the viral proteins and sequences involved and to define the roles of α2β1, αXβ2, and αVβ3, we analyzed the ability of rotaviruses and their reassortants to use these integrins for cell binding and infection and the effect of peptides DGEA and GPRP on these events. Many laboratory-adapted human, monkey, and bovine viruses used integrins, whereas all porcine viruses were integrin independent. The integrin-using rotavirus strains each interacted with all three integrins. Integrin usage related to VP4 serotype independently of sialic acid usage. Analysis of rotavirus reassortants and assays of virus binding and infectivity in integrin-transfected cells showed that VP4 bound α2β1, and VP7 interacted with αXβ2 and αVβ3 at a postbinding stage. DGEA inhibited rotavirus binding to α2β1 and infectivity, whereas GPRP binding to αXβ2 inhibited infectivity but not binding. The truncated VP5* subunit of VP4, expressed as a glutathione S-transferase fusion protein, bound the expressed α2 I domain. Alanine mutagenesis of D308 and G309 in VP5* eliminated VP5* binding to the α2 I domain. In a novel process, integrin-using viruses bind the α2 I domain of α2β1 via DGE in VP4 and interact with αXβ2 (via GPR) and αVβ3 by using VP7 to facilitate cell entry and infection. PMID:12941907

Neural Cell Chip Based Electrochemical Detection of Nanotoxicity

PubMed Central

Kafi, Md. Abdul; Cho, Hyeon-Yeol; Choi, Jeong Woo

2015-01-01

Development of a rapid, sensitive and cost-effective method for toxicity assessment of commonly used nanoparticles is urgently needed for the sustainable development of nanotechnology. A neural cell with high sensitivity and conductivity has become a potential candidate for a cell chip to investigate toxicity of environmental influences. A neural cell immobilized on a conductive surface has become a potential tool for the assessment of nanotoxicity based on electrochemical methods. The effective electrochemical monitoring largely depends on the adequate attachment of a neural cell on the chip surfaces. Recently, establishment of integrin receptor specific ligand molecules arginine-glycine-aspartic acid (RGD) or its several modifications RGD-Multi Armed Peptide terminated with cysteine (RGD-MAP-C), C(RGD)4 ensure farm attachment of neural cell on the electrode surfaces either in their two dimensional (dot) or three dimensional (rod or pillar) like nano-scale arrangement. A three dimensional RGD modified electrode surface has been proven to be more suitable for cell adhesion, proliferation, differentiation as well as electrochemical measurement. This review discusses fabrication as well as electrochemical measurements of neural cell chip with particular emphasis on their use for nanotoxicity assessments sequentially since inception to date. Successful monitoring of quantum dot (QD), graphene oxide (GO) and cosmetic compound toxicity using the newly developed neural cell chip were discussed here as a case study. This review recommended that a neural cell chip established on a nanostructured ligand modified conductive surface can be a potential tool for the toxicity assessments of newly developed nanomaterials prior to their use on biology or biomedical technologies. PMID:28347059

Green tea polyphenol tailors cell adhesivity of RGD displaying surfaces: multicomponent models monitored optically

PubMed Central

Peter, Beatrix; Farkas, Eniko; Forgacs, Eniko; Saftics, Andras; Kovacs, Boglarka; Kurunczi, Sandor; Szekacs, Inna; Csampai, Antal; Bosze, Szilvia; Horvath, Robert

2017-01-01

The interaction of the anti-adhesive coating, poly(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) and its Arg-Gly-Asp (RGD) functionalized form, PLL-g-PEG-RGD, with the green tea polyphenol, epigallocatechin-gallate (EGCg) was in situ monitored. After, the kinetics of cellular adhesion on the EGCg exposed coatings were recorded in real-time. The employed plate-based waveguide biosensor is applicable to monitor small molecule binding and sensitive to sub-nanometer scale changes in cell membrane position and cell mass distribution; while detecting the signals of thousands of adhering cells. The combination of this remarkable sensitivity and throughput opens up new avenues in testing complicated models of cell-surface interactions. The systematic studies revealed that, despite the reported excellent antifouling properties of the coatings, EGCg strongly interacted with them, and affected their cell adhesivity in a concentration dependent manner. Moreover, the differences between the effects of the fresh and oxidized EGCg solutions were first demonstrated. Using a semiempirical quantumchemical method we showed that EGCg binds to the PEG chains of PLL-g-PEG-RGD and effectively blocks the RGD sites by hydrogen bonds. The calculations supported the experimental finding that the binding is stronger for the oxidative products. Our work lead to a new model of polyphenol action on cell adhesion ligand accessibility and matrix rigidity. PMID:28186133

Green tea polyphenol tailors cell adhesivity of RGD displaying surfaces: multicomponent models monitored optically.

PubMed

Peter, Beatrix; Farkas, Eniko; Forgacs, Eniko; Saftics, Andras; Kovacs, Boglarka; Kurunczi, Sandor; Szekacs, Inna; Csampai, Antal; Bosze, Szilvia; Horvath, Robert

2017-02-10

The interaction of the anti-adhesive coating, poly(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) and its Arg-Gly-Asp (RGD) functionalized form, PLL-g-PEG-RGD, with the green tea polyphenol, epigallocatechin-gallate (EGCg) was in situ monitored. After, the kinetics of cellular adhesion on the EGCg exposed coatings were recorded in real-time. The employed plate-based waveguide biosensor is applicable to monitor small molecule binding and sensitive to sub-nanometer scale changes in cell membrane position and cell mass distribution; while detecting the signals of thousands of adhering cells. The combination of this remarkable sensitivity and throughput opens up new avenues in testing complicated models of cell-surface interactions. The systematic studies revealed that, despite the reported excellent antifouling properties of the coatings, EGCg strongly interacted with them, and affected their cell adhesivity in a concentration dependent manner. Moreover, the differences between the effects of the fresh and oxidized EGCg solutions were first demonstrated. Using a semiempirical quantumchemical method we showed that EGCg binds to the PEG chains of PLL-g-PEG-RGD and effectively blocks the RGD sites by hydrogen bonds. The calculations supported the experimental finding that the binding is stronger for the oxidative products. Our work lead to a new model of polyphenol action on cell adhesion ligand accessibility and matrix rigidity.

Green tea polyphenol tailors cell adhesivity of RGD displaying surfaces: multicomponent models monitored optically

NASA Astrophysics Data System (ADS)

Peter, Beatrix; Farkas, Eniko; Forgacs, Eniko; Saftics, Andras; Kovacs, Boglarka; Kurunczi, Sandor; Szekacs, Inna; Csampai, Antal; Bosze, Szilvia; Horvath, Robert

2017-02-01

The interaction of the anti-adhesive coating, poly(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) and its Arg-Gly-Asp (RGD) functionalized form, PLL-g-PEG-RGD, with the green tea polyphenol, epigallocatechin-gallate (EGCg) was in situ monitored. After, the kinetics of cellular adhesion on the EGCg exposed coatings were recorded in real-time. The employed plate-based waveguide biosensor is applicable to monitor small molecule binding and sensitive to sub-nanometer scale changes in cell membrane position and cell mass distribution; while detecting the signals of thousands of adhering cells. The combination of this remarkable sensitivity and throughput opens up new avenues in testing complicated models of cell-surface interactions. The systematic studies revealed that, despite the reported excellent antifouling properties of the coatings, EGCg strongly interacted with them, and affected their cell adhesivity in a concentration dependent manner. Moreover, the differences between the effects of the fresh and oxidized EGCg solutions were first demonstrated. Using a semiempirical quantumchemical method we showed that EGCg binds to the PEG chains of PLL-g-PEG-RGD and effectively blocks the RGD sites by hydrogen bonds. The calculations supported the experimental finding that the binding is stronger for the oxidative products. Our work lead to a new model of polyphenol action on cell adhesion ligand accessibility and matrix rigidity.

A peptide affinity column for the identification of integrin alpha IIb-binding proteins.

PubMed

Daxecker, Heide; Raab, Markus; Bernard, Elise; Devocelle, Marc; Treumann, Achim; Moran, Niamh

2008-03-01

To understand the regulation of integrin alpha(IIb)beta(3), a critical platelet adhesion molecule, we have developed a peptide affinity chromatography method using the known integrin regulatory motif, LAMWKVGFFKR. Using standard Fmoc chemistry, this peptide was synthesized onto a Toyopearl AF-Amino-650 M resin on a 6-aminohexanoic acid (Ahx) linker. Peptide density was controlled by acetylation of 83% of the Ahx amino groups. Four recombinant human proteins (CIB1, PP1, ICln and RN181), previously identified as binding to this integrin regulatory motif, were specifically retained by the column containing the integrin peptide but not by a column presenting an irrelevant peptide. Hemoglobin, creatine kinase, bovine serum albumin, fibrinogen and alpha-tubulin failed to bind under the chosen conditions. Immunodetection methods confirmed the binding of endogenous platelet proteins, including CIB1, PP1, ICln RN181, AUP-1 and beta3-integrin, from a detergent-free platelet lysate. Thus, we describe a reproducible method that facilitates the reliable extraction of specific integrin-binding proteins from complex biological matrices. This methodology may enable the sensitive and specific identification of proteins that interact with linear, membrane-proximal peptide motifs such as the integrin regulatory motif LAMWKVGFFKR.

Hydrogels with precisely controlled integrin activation dictate vascular patterning and permeability

NASA Astrophysics Data System (ADS)

Li, Shuoran; Nih, Lina R.; Bachman, Haylee; Fei, Peng; Li, Yilei; Nam, Eunwoo; Dimatteo, Robert; Carmichael, S. Thomas; Barker, Thomas H.; Segura, Tatiana

2017-09-01

Integrin binding to bioengineered hydrogel scaffolds is essential for tissue regrowth and regeneration, yet not all integrin binding can lead to tissue repair. Here, we show that through engineering hydrogel materials to promote α3/α5β1 integrin binding, we can promote the formation of a space-filling and mature vasculature compared with hydrogel materials that promote αvβ3 integrin binding. In vitro, α3/α5β1 scaffolds promoted endothelial cells to sprout and branch, forming organized extensive networks that eventually reached and anastomosed with neighbouring branches. In vivo, α3/α5β1 scaffolds delivering vascular endothelial growth factor (VEGF) promoted non-tortuous blood vessel formation and non-leaky blood vessels by 10 days post-stroke. In contrast, materials that promote αvβ3 integrin binding promoted endothelial sprout clumping in vitro and leaky vessels in vivo. This work shows that precisely controlled integrin activation from a biomaterial can be harnessed to direct therapeutic vessel regeneration and reduce VEGF-induced vascular permeability in vivo.

Hydrogels with precisely controlled integrin activation dictate vascular patterning and permeability

PubMed Central

Li, Shuoran; Nih, Lina R.; Bachman, Haylee; Fei, Peng; Li, Yilei; Nam, Eunwoo; Dimatteo, Robert; Carmichael, S. Thomas; Barker, Thomas H.; Segura, Tatiana

2017-01-01

Integrin binding to bioengineered hydrogel scaffolds is essential for tissue regrowth and regeneration, yet not all integrin binding can lead to tissue repair. Here, we show that through engineering hydrogel materials to promote α3/α5β1 integrin binding, we can promote the formation of a space filling and mature vasculature compared to hydrogel materials that promote a αvβ3 integrin binding. In vitro, α3/α5β1 scaffolds promoted endothelial cells to sprout and branch, forming organized extensive networks that eventually reached and anastomosed with neighboring branches. In vivo, α3/α5β1 scaffolds delivering vascular endothelial growth factor (VEGF) promoted non-tortuous blood vessel formation and non-leaky blood vessels by 10-days post stroke. In contrast, materials that promote αvβ3 integrin binding promoted endothelial sprout clumping in vitro and leaky vessels in vivo. This work shows that precisely controlled integrin activation from a biomaterial can be harnessed to direct therapeutic vessel regeneration and reduce VEGF induced vascular permeability in vivo. PMID:28783156

Evaluation of a novel Arg-Gly-Asp-conjugated α-melanocyte stimulating hormone hybrid peptide for potential melanoma therapy.

PubMed

Yang, Jianquan; Guo, Haixun; Gallazzi, Fabio; Berwick, Marianne; Padilla, R Steven; Miao, Yubin

2009-08-19

The purpose of this study was to determine whether Arg-Gly-Asp (RGD)-conjugated α-melanocyte stimulating hormone (α-MSH) hybrid peptide could be employed to target melanocortin-1 (MC1) receptor for potential melanoma therapy. The RGD motif {cyclic(Arg-Gly-Asp-DTyr-Asp)} was coupled to [Cys(3,4,10), DPhe(7), Arg(11)]α-MSH(3-13) {(Arg(11))CCMSH} to generate RGD-Lys-(Arg(11))CCMSH hybrid peptide. The MC1 receptor binding affinity of RGD-Lys-(Arg(11))CCMSH was determined in B16/F1 melanoma cells. The internalization and efflux, melanoma targeting and pharmacokinetic properties and single photon emission computed tomography/CT (SPECT/CT) imaging of (99m)Tc-RGD-Lys-(Arg(11))CCMSH were determined in B16/F1 melanoma cells and melanoma-bearing C57 mice. Clonogenic cytotoxic effect of RGD-Lys-(Arg(11))CCMSH was examined in B16/F1 melanoma cells. RGD-Lys-(Arg(11))CCMSH displayed 2.1 nM MC1 receptor binding affinity. (99m)Tc-RGD-Lys-(Arg(11))CCMSH showed rapid internalization and extended retention in B16/F1 cells. The cellular uptake of (99m)Tc-RGD-Lys-(Arg(11))CCMSH was MC1 receptor-mediated. (99m)Tc-RGD-Lys-(Arg(11))CCMSH exhibited high tumor uptake (14.83 ± 2.94% ID/g 2 h postinjection) and prolonged tumor retention (7.59 ± 2.04% ID/g 24 h postinjection) in B16/F1 melanoma-bearing mice. Nontarget organ uptakes were generally low except for the kidneys. Whole-body clearance of (99m)Tc-RGD-Lys-(Arg(11))CCMSH was rapid, with approximately 62% of the injected radioactivity cleared through the urinary system by 2 h postinjection. Flank melanoma tumors were clearly imaged by small animal SPECT/CT using (99m)Tc-RGD-Lys-(Arg(11))CCMSH as an imaging probe 2 h postinjection. Single treatment (3 h incubation) with 100 nM of RGD-Lys-(Arg(11))CCMSH significantly (p < 0.05) decreased the clonogenic survival of B16/F1 cells by 65% compared to the untreated control cells. Favorable melanoma targeting property of (99m)Tc-RGD-Lys-(Arg(11))CCMSH and remarkable cytotoxic effect of RGD-Lys-(Arg(11))CCMSH in B16/F1 cells warranted the further evaluation of (188)Re-labeled α-MSH hybrid peptides as novel therapeutic peptides for melanoma treatment once the strategies of amino acid coinjection or structural modification of peptide sequence substantially reduce the renal uptake.

Kit preparation and biokinetics in women of 99mTc-EDDA/HYNIC-E-[c(RGDfK)]2 for breast cancer imaging.

PubMed

Ortiz-Arzate, Zareth; Santos-Cuevas, Clara L; Ocampo-García, Blanca E; Ferro-Flores, Guillermina; García-Becerra, Rocío; Estrada, Gisela; Gómez-Argumosa, Edgar; Izquierdo-Sánchez, Vanessa

2014-04-01

In breast cancer, α(ν)β(3) and/or α(ν)β(5) integrins are overexpressed in both endothelial and tumour cells. Radiolabelled peptides based on the Arg-Gly-Asp (RGD) sequence are radiopharmaceuticals with high affinity and selectivity for these integrins. The RGD-dimer peptide (E-[c(RGDfK)]2) radiolabelled with (99m)Tc has been reported as a radiopharmaceutical with a 10-fold higher affinity for the α(ν)β(3) integrin compared with the RGD-monomer. Ethylenediamine-N,N'-diacetic acid (EDDA) is a hydrophilic molecule that may favour renal excretion when used as coligand in the (99m)Tc labelling of hydrazinonicotinamide (HYNIC) peptides and can easily be formulated in a lyophilized kit. The aim of this study was to establish a biokinetic model for (99m)Tc-EDDA/HYNIC-E-[c(RGDfK)]2 prepared from lyophilized kits and evaluate its dosimetry as a tumour-imaging agent in seven healthy women and three breast cancer patients. (99m)Tc labelling was performed by adding sodium pertechnetate solution and 0.2 mol/l phosphate buffer (pH 7.0) to a lyophilized formulation containing E-[c(RGDfK)]2, EDDA, tricine, mannitol and stannous chloride. The radiochemical purity was evaluated using reverse-phase high-performance liquid chromatography and instant thin-layer chromatography on silica gel analyses. Stability studies in human serum were carried out using size-exclusion high-performance liquid chromatography. In-vitro cell uptake was tested using breast cancer cells (MCF7, T47D and MDA-MB-231) with blocked and nonblocked receptors. Biodistribution and tumour uptake were determined in MCF7 tumour-bearing nude mice with blocked and nonblocked receptors, and images were obtained using a micro-SPECT/PET/CT. Whole-body images from seven healthy women were acquired at 0.5, 1, 3, 6 and 24 h after (99m)Tc-EDDA/HYNIC-E-[c(RGDfK)]2 administration with radiochemical purities greater than 94%. Regions of interest were drawn around the source organs at each time frame. Each region of interest was converted to activity using the conjugate view counting method. The image sequence was used to extrapolate the (99m)Tc-EDDA/HYNIC-E-[c(RGDfK)]2 time-activity curves of each organ to adjust the biokinetic model and calculate the total number of disintegrations (N) that occurred in the source regions. N data were the input for the OLINDA/EXM code to calculate internal radiation dose estimates. In three breast cancer patients with histologically confirmed cancer, static images were obtained at 1 h in the supine position with hands placed behind the head. (99m)Tc-EDDA/HYNIC-E-[c(RGDfK)]2 obtained from lyophilized kits demonstrated high stability in human serum and specific cell receptor binding. The biodistribution data from mice showed rapid blood clearance, with both renal and hepatobiliary excretion, and specific binding towards α(ν)β(3) integrins in the MCF7 tumours. In women, the blood activity showed a half-life value of 1.60 min for the fast component (T1/2α = ln2/26.01) and half-life values of 1.0 h for the first slow component (T1/2β = ln2/0.69) and 4.03 h for the second slow component (T1/2γ = ln2/0.16). Images from patients showed an average tumour/heart (blood) ratio of 3.61 ± 0.62 at 1 h. The average equivalent doses calculated for a study using 740 MBq were 4.9, 6.2, 20.7, 34.5 and 57.0 mSv for the liver, intestines, spleen, kidneys and thyroid, respectively, and the effective dose was 6.1 mSv. All absorbed doses were comparable to those known from most of the (99m)Tc studies. (99m)Tc-EDDA/HYNIC-E-[c(RGDfK)]2 obtained from kit formulations showed high tumour uptake in patients with malignant lesions, making it a promising imaging radiopharmaceutical for targeting site-specific breast cancer. The results obtained in this study warrant further clinical studies to determine the specificity and sensitivity of (99m)Tc-EDDA/HYNIC-E-[c(RGDfK)]2.

Rab-coupling protein coordinates recycling of alpha5beta1 integrin and EGFR1 to promote cell migration in 3D microenvironments.

PubMed

Caswell, Patrick T; Chan, May; Lindsay, Andrew J; McCaffrey, Mary W; Boettiger, David; Norman, Jim C

2008-10-06

Here we show that blocking the adhesive function of alphavbeta3 integrin with soluble RGD ligands, such as osteopontin or cilengitide, promoted association of Rab-coupling protein (RCP) with alpha5beta1 integrin and drove RCP-dependent recycling of alpha5beta1 to the plasma membrane and its mobilization to dynamic ruffling protrusions at the cell front. These RCP-driven changes in alpha5beta1 trafficking led to acquisition of rapid/random movement on two-dimensional substrates and to a marked increase in fibronectin-dependent migration of tumor cells into three-dimensional matrices. Recycling of alpha5beta1 integrin did not affect its regulation or ability to form adhesive bonds with substrate fibronectin. Instead, alpha5beta1 controlled the association of EGFR1 with RCP to promote the coordinate recycling of these two receptors. This modified signaling downstream of EGFR1 to increase its autophosphorylation and activation of the proinvasive kinase PKB/Akt. We conclude that RCP provides a scaffold that promotes the physical association and coordinate trafficking of alpha5beta1 and EGFR1 and that this drives migration of tumor cells into three-dimensional matrices.

From a Helix to a Small Cycle: Metadynamics-Inspired αvβ6 Integrin Selective Ligands.

PubMed

Di Leva, Francesco Saverio; Tomassi, Stefano; Di Maro, Salvatore; Reichart, Florian; Notni, Johannes; Dangi, Abha; Marelli, Udaya Kiran; Brancaccio, Diego; Merlino, Francesco; Wester, Hans-Jürgen; Novellino, Ettore; Kessler, Horst; Marinelli, Luciana

2018-04-16

The RGD-recognizing αvβ6 integrin has only recently emerged as a major target for cancer diagnosis and therapy. Thus, the development of selective, low-molecular-weight ligands of this receptor is still in great demand. Here, a metadynamics-driven design strategy allowed us to successfully convert a helical nonapeptide into a cyclic pentapeptide (6) showing remarkable potency and αvβ6 specificity. NMR and docking studies elucidated the reasons for the high affinity and selectivity of this compound, setting the ground for the rational design of new αvβ6-specific small peptides or even peptidomimetics. In vivo PET imaging studies demonstrated the potential use of 6 for medical applications. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Probing the acidic residue within the integrin binding site of laminin-511 that interacts with the metal ion-dependent adhesion site of α6β1 integrin.

PubMed

Taniguchi, Yukimasa; Li, Shaoliang; Takizawa, Mamoru; Oonishi, Eriko; Toga, Junko; Yagi, Emiko; Sekiguchi, Kiyotoshi

2017-06-03

Laminins are major cell-adhesive proteins of basement membranes that interact with integrins in a divalent cation-dependent manner. Laminin-511 consists of α5, β1, and γ1 chains, of which three laminin globular domains of the α5 chain (α5/LG1-3) and a Glu residue in the C-terminal tail of chain γ1 (γ1-Glu1607) are required for binding to integrins. However, it remains unsettled whether the Glu residue in the γ1 tail is involved in integrin binding by coordinating the metal ion in the metal ion-dependent adhesion site of β1 integrin (β1-MIDAS), or by stabilizing the conformation of α5/LG1-3. To address this issue, we examined whether α5/LG1-3 contain an acidic residue required for integrin binding that is as critical as the Glu residue in the γ1 tail; to achieve this, we undertook exhaustive alanine substitutions of the 54 acidic residues present in α5/LG1-3 of the E8 fragment of laminin-511 (LM511E8). Most of the alanine mutants possessed α6β1 integrin binding activities comparable with wild-type LM511E8. Alanine substitution for α5-Asp3198 and Asp3219 caused mild reduction in integrin binding activity, and that for α5-Asp3218 caused severe reduction, possibly resulting from conformational perturbation of α5/LG1-3. When α5-Asp3218 was substituted with asparagine, the resulting mutant possessed significant binding activity to α6β1 integrin, indicating that α5-Asp3218 is not directly involved in integrin binding through coordination with the metal ion in β1-MIDAS. Given that substitution of γ1-Glu1607 with glutamine nullified the binding activity to α6β1 integrin, these results, taken together, support the possibility that the critical acidic residue coordinating the metal ion in β1-MIDAS is Glu1607 in the γ1 tail, but no such residue is present in α5/LG1-3. Copyright © 2017 Elsevier Inc. All rights reserved.

Direct binding of F actin to the cytoplasmic domain of the alpha 2 integrin chain in vitro

NASA Technical Reports Server (NTRS)

Kieffer, J. D.; Plopper, G.; Ingber, D. E.; Hartwig, J. H.; Kupper, T. S.

1995-01-01

The transmembrane integrins have been shown to interact with the cytoskeleton via noncovalent binding between cytoplasmic domains (CDs) of integrin beta chains and various actin binding proteins within the focal adhesion complex. Direct or indirect integrin alpha chain CD binding to the actin cytoskeleton has not been reported. We show here that actin, as an abundant constituent of focal adhesion complex proteins isolated from fibroblasts, binds strongly and specifically to alpha 2 CD, but not to alpha 1 CD peptide. Similar specific binding to alpha 2 CD peptide was seen for highly purified F actin, free of putative actin-binding proteins. The bound complex of actin and peptide was visualized directly by coprecipitation, and actin binding was abrogated by removal of a five amino acid sequence from the alpha 2 CD peptide. Our findings may explain the earlier observation that, while integrins alpha 2 beta 1 and alpha 1 beta 1 both bind to collagen, only alpha 2 beta 1 can mediate contraction of extracellular collagen matrices.

Novel tumor-targeted RGD peptide-camptothecin conjugates: synthesis and biological evaluation.

PubMed

Dal Pozzo, Alma; Ni, Ming-Hong; Esposito, Emiliano; Dallavalle, Sabrina; Musso, Loana; Bargiotti, Alberto; Pisano, Claudio; Vesci, Loredana; Bucci, Federica; Castorina, Massimo; Foderà, Rosanna; Giannini, Giuseppe; Aulicino, Concetta; Penco, Sergio

2010-01-01

Five RGD peptide-camptothecin (CPT) conjugates were designed and synthesized with the purpose to improve the therapeutic index of this antitumoral drug family. New RGD cyclopeptides were selected on the basis of their high affinity to alpha(v) integrin receptors overexpressed by tumor cells and their metabolic stability. The conjugates can be divided in two groups: in the first the peptide was attached to the drug through an amide bond, in the second through a hydrazone bond. The main difference between the two spacers lies in their acid stability. Affinity to the receptors was maintained for all conjugates and their internalization into tumor cells was demonstrated. The first group conjugates showed lower in vitro and in vivo activity than the parent drug, probably due to the excessive stability of the amide bond, even inside the tumor cells. Conversely, the hydrazone conjugates exhibited in vitro tumor cell inhibition similar to the parent drug, indicating high conversion in the culture medium and/or inside the cells, but their poor solubility hampered in vivo experiments. On the basis of these results, information was acquired for additional development of derivatives with different linkers and better solubility for in vivo evaluation. Copyright (c) 2009 Elsevier Ltd. All rights reserved.

Integrin binding and mechanical tension induce movement of mRNA and ribosomes to focal adhesions

NASA Technical Reports Server (NTRS)

Chicurel, M. E.; Singer, R. H.; Meyer, C. J.; Ingber, D. E.

1998-01-01

The extracellular matrix (ECM) activates signalling pathways that control cell behaviour by binding to cell-surface integrin receptors and inducing the formation of focal adhesion complexes (FACs). In addition to clustered integrins, FACs contain proteins that mechanically couple the integrins to the cytoskeleton and to immobilized signal-transducing molecules. Cell adhesion to the ECM also induces a rapid increase in the translation of preexisting messenger RNAs. Gene expression can be controlled locally by targeting mRNAs to specialized cytoskeletal domains. Here we investigate whether cell binding to the ECM promotes formation of a cytoskeletal microcompartment specialized for translational control at the site of integrin binding. High-resolution in situ hybridization revealed that mRNA and ribosomes rapidly and specifically localized to FACs that form when cells bind to ECM-coated microbeads. Relocation of these protein synthesis components to the FAC depended on the ability of integrins to mechanically couple the ECM to the contractile cytoskeleton and on associated tension-moulding of the actin lattice. Our results suggest a new type of gene regulation by integrins and by mechanical stress which may involve translation of mRNAs into proteins near the sites of signal reception.

Biologically engineered protein-graft-poly(ethylene glycol) hydrogels: A cell-adhesive and plasmin-degradable biosynthetic material for tissue repair

NASA Astrophysics Data System (ADS)

Halstenberg, Sven

2002-01-01

The goal of the research presented in this dissertation was to create a biomimetic artificial material that exhibits functions of extracellular matrix relevant for improved nerve regeneration. Neural adhesion peptides were photoimmobilized on highly crosslinked poly(ethylene glycol)-based substrates that were otherwise non-adhesive. Neurons adhered in two-dimensional patterns for eleven hours, but no neurites extended. To enable neurite extension and nerve regeneration in three dimensions, and to address the need for specifically cell adhesive and cell degradable materials for clinical applications in tissue repair in general, an artificial protein was recombinantly expressed and purified that consisted of a repeating amino acid sequence based on fibrinogen and anti-thrombin III. The recombinant protein contained integrin-binding RGD sites, plasmin degradation sites, heparin binding sites, and six thiol-containing cysteine residues as grafting sites for poly(ethylene glycol) diacrylate via Michael-type conjugate addition. The resulting protein-graft-poly(ethylene glycol)acrylates were crosslinked by photopolymerization to form hydrogels. Although three-dimensional, RGD mediated and serine protease-dependent ingrowth of human fibroblasts into protein-graft-poly(ethylene glycol) hydrogels occurred, only surface neurite outgrowth was observed from chick dorsal root ganglia. Axonal outgrowth depended on the concentration of matrix-bound heparin, suggesting that improved mechanical strength of the hydrogels and possible immobilization of neuroactive factors due to the presence of heparin promoted neurite outgrowth. Together, the above results show that specific biological functions can be harnessed by protein-graft-poly(ethylene glycol) hydrogels to serve as matrices for tissue repair and regeneration. In particular, the two design objectives, specific cell adhesion and degradability by cell-associated proteases, were fulfilled by the material. In the future, this and similar artificial protein-graft-poly(ethylene glycol) materials with varying protein elements for improved wound healing might serve as biosynthetic implant materials or wound dressings that degrade in synchrony with the formation of a variety of target tissues.

Disintegrins from Hematophagous Sources

PubMed Central

Assumpcao, Teresa C. F.; Ribeiro, José M. C.; Francischetti, Ivo M. B.

2012-01-01

Bloodsucking arthropods are a rich source of salivary molecules (sialogenins) which inhibit platelet aggregation, neutrophil function and angiogenesis. Here we review the literature on salivary disintegrins and their targets. Disintegrins were first discovered in snake venoms, and were instrumental in our understanding of integrin function and also for the development of anti-thrombotic drugs. In hematophagous animals, most disintegrins described so far have been discovered in the salivary gland of ticks and leeches. A limited number have also been found in hookworms and horseflies, and none identified in mosquitoes or sand flies. The vast majority of salivary disintegrins reported display a RGD motif and were described as platelet aggregation inhibitors, and few others as negative modulator of neutrophil or endothelial cell functions. This notably low number of reported disintegrins is certainly an underestimation of the actual complexity of this family of proteins in hematophagous secretions. Therefore an algorithm was created in order to identify the tripeptide motifs RGD, KGD, VGD, MLD, KTS, RTS, WGD, or RED (flanked by cysteines) in sialogenins deposited in GenBank database. The search included sequences from various blood-sucking animals such as ticks (e.g., Ixodes sp., Argas sp., Rhipicephalus sp., Amblyommasp.), tabanids (e.g., Tabanus sp.), bugs (e.g., Triatoma sp., Rhodnius prolixus), mosquitoes (e.g., Anopheles sp., Aedes sp., Culex sp.), sand flies (e.g., Lutzomyia sp., Phlebotomus sp.), leeches (e.g., Macrobdella sp., Placobdella sp.) and worms (e.g., Ancylostoma sp.). This approach allowed the identification of a remarkably high number of novel putative sialogenins with tripeptide motifs typical of disintegrins (>450 sequences) whose biological activity remains to be verified. This database is accessible online as a hyperlinked worksheet and displays biochemical, taxonomic, and gene ontology aspects for each putative disintegrin. It is also freely available for download (right click with the mouse) at links http://exon.niaid.nih.gov/transcriptome/RGD/RGD-Peps-WEB.xlsx (web version) and http://exon.niaid.nih.gov/transcriptome/RGD/RGD-sialogenins.zip (stand alone version). PMID:22778902

Disintegrins from hematophagous sources.

PubMed

Assumpcao, Teresa C F; Ribeiro, José M C; Francischetti, Ivo M B

2012-05-01

Bloodsucking arthropods are a rich source of salivary molecules (sialogenins) which inhibit platelet aggregation, neutrophil function and angiogenesis. Here we review the literature on salivary disintegrins and their targets. Disintegrins were first discovered in snake venoms, and were instrumental in our understanding of integrin function and also for the development of anti-thrombotic drugs. In hematophagous animals, most disintegrins described so far have been discovered in the salivary gland of ticks and leeches. A limited number have also been found in hookworms and horseflies, and none identified in mosquitoes or sand flies. The vast majority of salivary disintegrins reported display a RGD motif and were described as platelet aggregation inhibitors, and few others as negative modulator of neutrophil or endothelial cell functions. This notably low number of reported disintegrins is certainly an underestimation of the actual complexity of this family of proteins in hematophagous secretions. Therefore an algorithm was created in order to identify the tripeptide motifs RGD, KGD, VGD, MLD, KTS, RTS, WGD, or RED (flanked by cysteines) in sialogenins deposited in GenBank database. The search included sequences from various blood-sucking animals such as ticks (e.g., Ixodes sp., Argas sp., Rhipicephalus sp., Amblyommasp.), tabanids (e.g., Tabanus sp.), bugs (e.g., Triatoma sp., Rhodnius prolixus), mosquitoes (e.g., Anopheles sp., Aedes sp., Culex sp.), sand flies (e.g., Lutzomyia sp., Phlebotomus sp.), leeches (e.g., Macrobdella sp., Placobdella sp.) and worms (e.g., Ancylostoma sp.). This approach allowed the identification of a remarkably high number of novel putative sialogenins with tripeptide motifs typical of disintegrins (>450 sequences) whose biological activity remains to be verified. This database is accessible online as a hyperlinked worksheet and displays biochemical, taxonomic, and gene ontology aspects for each putative disintegrin. It is also freely available for download (right click with the mouse) at links http://exon.niaid.nih.gov/transcriptome/RGD/RGD-Peps-WEB.xlsx (web version) and http://exon.niaid.nih.gov/transcriptome/RGD/RGD-sialogenins.zip (stand alone version).

Potential of inherent RGD containing silk fibroin-poly (Є-caprolactone) nanofibrous matrix for bone tissue engineering.

PubMed

Bhattacharjee, Promita; Kundu, Banani; Naskar, Deboki; Kim, Hae-Won; Bhattacharya, Debasis; Maiti, T K; Kundu, S C

2016-02-01

The current study deals with the fabrication and characterization of blended nanofibrous scaffolds of tropical tasar silk fibroin of Antheraea mylitta and poly (Є-caprolactone) to act as an ideal scaffold for bone regeneration. The use of poly (Є-caprolactone) in osteogenesis is well-recognized. At the same time, the osteoconductive nature of the non-mulberry tasar fibroin is also established due to its internal integrin binding peptide RGD (Arg-Gly-Asp) sequences, which enhance cellular interaction and proliferation. Considering that the materials have the required and favorable properties, the blends are formed using an equal volume ratio of fibroin (2 and 4 wt%) and poly (Є-caprolactone) solution (10 wt%) to fabricate nanofibers. The nanofibers possess an average diameter of 152 ± 18 nm (2 % fibroin/PCL) and 175 ± 15 nm (4% fibroin/PCL). The results of Fourier transform infrared spectroscopy substantiates the preservation of the secondary structure of the fibroin in the blends indicating the structural stability of the neo-matrix. With an increase in the fibroin percentage, the hydrophobicity and thermal stability of the matrices as measured from melting temperature Tm (using DSC) decrease, while the mechanical strength is improved. The blended nanofibrous scaffolds are biodegradable, and support the viability and proliferation of human osteoblast-like cells as observed through scanning electron and confocal microscopes. Alkaline phosphatase assay indicates the cell proliferation and the generation of the neo-bone matrix. Taken together, these findings illustrate that the silk-poly (Є-caprolactone) blended nanofibrous scaffolds have an excellent prospect as scaffolding material in bone tissue engineering.

Biomimetic design of platelet adhesion inhibitors to block integrin α2β1-collagen interactions: I. Construction of an affinity binding model.

PubMed

Zhang, Lin; Sun, Yan

2014-04-29

Platelet adhesion on a collagen surface through integrin α2β1 has been proven to be significant for the formation of arterial thrombus. However, the molecular determinants mediating the integrin-collagen complex remain unclear. In the present study, the dynamics of integrin-collagen binding and molecular interactions were investigated using molecular dynamics (MD) simulations and molecular mechanics-Poisson-Boltzmann surface area (MM-PBSA) analysis. Hydrophobic interaction is identified as the major driving force for the formation of the integrin-collagen complex. On the basis of the MD simulation and MM-PBSA results, an affinity binding model (ABM) of integrin for collagen is constructed; it is composed of five residues, including Y157, N154, S155, R288, and L220. The ABM has been proven to capture the major binding motif contributing 84.8% of the total binding free energy. On the basis of the ABM, we expect to establish a biomimetic design strategy of platelet adhesion inhibitors, which would be beneficial for the development of potent peptide-based drugs for thrombotic diseases.

Crystal Structure of the Heterotrimeric Integrin-Binding Region of Laminin-111.

PubMed

Pulido, David; Hussain, Sadaf-Ahmahni; Hohenester, Erhard

2017-03-07

Laminins are cell-adhesive glycoproteins that are essential for basement membrane assembly and function. Integrins are important laminin receptors, but their binding site on the heterotrimeric laminins is poorly defined structurally. We report the crystal structure at 2.13 Å resolution of a minimal integrin-binding fragment of mouse laminin-111, consisting of ∼50 residues of α1β1γ1 coiled coil and the first three laminin G-like (LG) domains of the α1 chain. The LG domains adopt a triangular arrangement, with the C terminus of the coiled coil situated between LG1 and LG2. The critical integrin-binding glutamic acid residue in the γ1 chain tail is surface exposed and predicted to bind to the metal ion-dependent adhesion site in the integrin β1 subunit. Additional contacts to the integrin are likely to be made by the LG1 and LG2 surfaces adjacent to the γ1 chain tail, which are notably conserved and free of obstructing glycans. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Body distributioin of RGD-mediated liposome in brain-targeting drug delivery.

PubMed

Qin, Jing; Chen, DaWei; Hu, Haiyang; Qiao, MingXi; Zhao, XiuLi; Chen, Baoyu

2007-09-01

RGD conjugation liposomes (RGD-liposomes) were evaluated for brain-targeting drug delivery. The flow cytometric in vitro study demonstrated that RGD-liposomes could bind to monocytes and neutrophils effectively. Ferulic acid (4-hydroxy-3-methoxycinnamic, FA) was loaded into liposomes. Rats were subjected to intrastriatal microinjections of 100 units of human recombinant IL-1beta to produce brain inflammation and caudal vein injection of three formulations (FA solution, FA liposome and RGD-coated FA liposome). Animals were sacrificed 15, 30, 60 and 120 min after administration to study the body distribution of the FA in the three formulations. HPLC was used to determine the concentration of FA in vivo with salicylic acid as internal standard. The results of body distribution indicated that RGD-coated liposomes could be mediated into the brain with a 6-fold FA concentration compared to FA solution and 3-fold in comparison to uncoated liposome. Brain targeted delivery was achieved and a reduction in dosage might be allowed.

Neutrophil recruitment limited by high-affinity bent β2 integrin binding ligand in cis

PubMed Central

Fan, Zhichao; McArdle, Sara; Marki, Alex; Mikulski, Zbigniew; Gutierrez, Edgar; Engelhardt, Britta; Deutsch, Urban; Ginsberg, Mark; Groisman, Alex; Ley, Klaus

2016-01-01

Neutrophils are essential for innate immunity and inflammation and many neutrophil functions are β2 integrin-dependent. Integrins can extend (E+) and acquire a high-affinity conformation with an ‘open' headpiece (H+). The canonical switchblade model of integrin activation proposes that the E+ conformation precedes H+, and the two are believed to be structurally linked. Here we show, using high-resolution quantitative dynamic footprinting (qDF) microscopy combined with a homogenous conformation-reporter binding assay in a microfluidic device, that a substantial fraction of β2 integrins on human neutrophils acquire an unexpected E−H+ conformation. E−H+ β2 integrins bind intercellular adhesion molecules (ICAMs) in cis, which inhibits leukocyte adhesion in vitro and in vivo. This endogenous anti-inflammatory mechanism inhibits neutrophil aggregation, accumulation and inflammation. PMID:27578049

Hypertonic enhancement of transmitter release from frog motor nerve terminals: Ca2+ independence and role of integrins

NASA Technical Reports Server (NTRS)

Kashani, A. H.; Chen, B. M.; Grinnell, A. D.

2001-01-01

Hyperosmotic solutions cause markedly enhanced spontaneous quantal release of neurotransmitter from many nerve terminals. The mechanism of this enhancement is unknown. We have investigated this phenomenon at the frog neuromuscular junction with the aim of determining the degree to which it resembles the modulation of release by stretch, which has been shown to be mediated by mechanical tension on integrins.The hypertonicity enhancement, like the stretch effect, does not require Ca2+ influx or release from internal stores, although internal release may contribute to the effect. The hypertonicity effect is sharply reduced (but not eliminated) by peptides containing the RGD sequence, which compete with native ligands for integrin bonds.There is co-variance in the magnitude of the stretch and osmotic effects; that is, individual terminals exhibiting a large stretch effect also show strong enhancement by hypertonicity, and vice versa. The stretch and osmotic enhancements also can partially occlude each other.There remain some clear-cut differences between osmotic and stretch forms of modulation: the larger range of enhancement by hypertonic solutions, the relative lack of effect of osmolarity on evoked release, and the reported higher temperature sensitivity of osmotic enhancement. Nevertheless, our data strongly implicate integrins in a significant fraction of the osmotic enhancement, possibly acting via the same mechanism as stretch modulation.

Characterization of Laminin Binding Integrin Internalization in Prostate Cancer Cells.

PubMed

Das, Lipsa; Anderson, Todd A; Gard, Jaime M C; Sroka, Isis C; Strautman, Stephanie R; Nagle, Raymond B; Morrissey, Colm; Knudsen, Beatrice S; Cress, Anne E

2017-05-01

Laminin binding integrins α6 (CD49f) and α3 (CD49c) are persistently but differentially expressed in prostate cancer (PCa). Integrin internalization is an important determinant of their cell surface expression and function. Using flow cytometry, and first order kinetic modeling, we quantitated the intrinsic internalization rates of integrin subunits in a single cycle of internalization. In PCa cell line DU145, α6 integrin internalized with a rate constant (k actual ) of 3.25 min -1 , threefold faster than α3 integrin (1.0 min -1 ), 1.5-fold faster than the vitronectin binding αv integrin (CD51) (2.2 min -1 ), and significantly slower than the unrelated transferrin receptor (CD71) (15 min -1 ). Silencing of α3 integrin protein expression in DU145, PC3, and PC3B1 cells resulted in up to a 1.71-fold increase in k actual for α6 integrin. The internalized α6 integrin was targeted to early endosomes but not to lamp1 vesicles. Depletion of α3 integrin expression resulted in redistribution of α6β4 integrin to an observed cell-cell staining pattern that is consistent with a suprabasal distribution observed in epidermis and early PIN lesions in PCa. Depletion of α3 integrin increased cell migration by 1.8-fold, which was dependent on α6β1 integrin. Silencing of α6 integrin expression however, had no significant effect on the k actual of α3 integrin or its distribution in early endosomes. These results indicate that α3 and α6 integrins have significantly different internalization kinetics and that coordination exists between them for internalization. J. Cell. Biochem. 118: 1038-1049, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Characterization of Laminin Binding Integrin Internalization in Prostate Cancer Cells†

PubMed Central

Das, Lipsa; Anderson, Todd A.; Gard, Jaime M.C.; Sroka, Isis C.; Strautman, Stephanie R.; Nagle, Raymond B.; Morrissey, Colm; Knudsen, Beatrice S.; Cress, Anne E.

2017-01-01

Laminin binding integrins α6 (CD49f) and α3 (CD49c) are persistently but differentially expressed in prostate cancer (PCa). Integrin internalization is an important determinant of their cell surface expression and function. Using flow cytometry, and first order kinetic modelling, we quantitated the intrinsic internalization rates of integrin subunits in a single cycle of internalization. In PCa cell line DU145, α6 integrin internalized with a rate constant (kactual) of 3.25min−1, 3-fold faster than α3 integrin (1.0 min−1), 1.5-fold faster than the vitronectin binding αv integrin (CD51) (2.2 min−1), and significantly slower than the unrelated transferrin receptor (CD71) (15 min−1). Silencing of α3 integrin protein expression in DU145, PC3 and PC3B1 cells resulted in up to a 1.71-fold increase in kactual for α6 integrin. The internalized α6 integrin was targeted to early endosomes but not to lamp1 vesicles. Depletion of α3 integrin expression resulted in redistribution of α6β4 integrin to an observed cell-cell staining pattern that is consistent with a suprabasal distribution observed in epidermis and early PIN lesions in PCa. Depletion of α3 integrin increased cell migration by 1.8 fold, which was dependent on α6β1 integrin. Silencing of α6 integrin expression however, had no significant effect on the kactual of α3 integrin or its distribution in early endosomes. These results indicate that α3 and α6 integrins have significantly different internalization kinetics and that coordination exists between them for internalization. This article is protected by copyright. All rights reserved PMID:27509031

Extra-domain B in oncofetal fibronectin structurally promotes fibrillar head-to-tail dimerization of extracellular matrix protein.

PubMed

Schiefner, André; Gebauer, Michaela; Skerra, Arne

2012-05-18

The type III extra-domain B (ED-B) is specifically spliced into fibronectin (Fn) during embryogenesis and neoangiogenesis, including many cancers. The x-ray structure of the recombinant four-domain fragment Fn(III)7B89 reveals a tightly associated, extended head-to-tail dimer, which is stabilized via pair-wise shape and charge complementarity. A tendency toward ED-B-dependent dimer formation in solution was supported by size exclusion chromatography and analytical ultracentrifugation. When amending the model with the known three-dimensional structure of the Fn(III)10 domain, its RGD loop as well as the adhesion synergy region in Fn(III)9-10 become displayed on the same face of the dimer; this should allow simultaneous binding of at least two integrins and, thus, receptor clustering on the cell surface and intracellular signaling. Insertion of ED-B appears to stabilize overall head-to-tail dimerization of two separate Fn chains, which, together with alternating homodimer formation via disulfide bridges at the C-terminal Fn tail, should lead to the known macromolecular fibril formation.

Conservation of the glycoprotein B homologs of the Kaposi’s sarcoma-associated herpesvirus (KSHV/HHV8) and Old World primate rhadinoviruses of chimpanzees and macaques

PubMed Central

Bruce, A. Gregory; Horst, Jeremy A.; Rose, Timothy M.

2016-01-01

The envelope-associated glycoprotein B (gB) is highly conserved within the Herpesviridae and plays a critical role in viral entry. We analyzed the evolutionary conservation of sequence and structural motifs within the Kaposi’s sarcoma-associated herpesvirus (KSHV) gB and homologs of Old World primate rhadinoviruses belonging to the distinct RV1 and RV2 rhadinovirus lineages. In addition to gB homologs of rhadinoviruses infecting the pig-tailed and rhesus macaques, we cloned and sequenced gB homologs of RV1 and RV2 rhadinoviruses infecting chimpanzees. A structural model of the KSHV gB was determined, and functional motifs and sequence variants were mapped to the model structure. Conserved domains and motifs were identified, including an “RGD” motif that plays a critical role in KSHV binding and entry through the cellular integrin αVβ3. The RGD motif was only detected in RV1 rhadinoviruses suggesting an important difference in cell tropism between the two rhadinovirus lineages. PMID:27070755

Actin retrograde flow actively aligns and orients ligand-engaged integrins in focal adhesions

PubMed Central

Swaminathan, Vinay; Kalappurakkal, Joseph Mathew; Moore, Travis I.; Koga, Nobuyasu; Baker, David A.; Oldenbourg, Rudolf; Tani, Tomomi; Springer, Timothy A.; Waterman, Clare M.

2017-01-01

Integrins are transmembrane receptors that, upon activation, bind extracellular ligands and link them to the actin filament (F-actin) cytoskeleton to mediate cell adhesion and migration. Cytoskeletal forces in migrating cells generated by polymerization- or contractility-driven “retrograde flow” of F-actin from the cell leading edge have been hypothesized to mediate integrin activation for ligand binding. This predicts that these forces should align and orient activated, ligand-bound integrins at the leading edge. Here, polarization-sensitive fluorescence microscopy of GFP-αVβ3 integrins in fibroblasts shows that integrins are coaligned in a specific orientation within focal adhesions (FAs) in a manner dependent on binding immobilized ligand and a talin-mediated linkage to the F-actin cytoskeleton. These findings, together with Rosetta modeling, suggest that integrins in FA are coaligned and may be highly tilted by cytoskeletal forces. Thus, the F-actin cytoskeleton sculpts an anisotropic molecular scaffold in FAs, and this feature may underlie the ability of migrating cells to sense directional extracellular cues. PMID:29073038

Targeted gene delivery by polyplex micelles with crowded PEG palisade and cRGD moiety for systemic treatment of pancreatic tumors.

PubMed

Ge, Zhishen; Chen, Qixian; Osada, Kensuke; Liu, Xueying; Tockary, Theofilus A; Uchida, Satoshi; Dirisala, Anjaneyulu; Ishii, Takehiko; Nomoto, Takahiro; Toh, Kazuko; Matsumoto, Yu; Oba, Makoto; Kano, Mitsunobu R; Itaka, Keiji; Kataoka, Kazunori

2014-03-01

Adequate retention in systemic circulation is the preliminary requirement for systemic gene delivery to afford high bioavailability into the targeted site. Polyplex micelle formulated through self-assembly of oppositely-charged poly(ethylene glycol) (PEG)-polycation block copolymer and plasmid DNA has gained tempting perspective upon its advantageous core-shell architecture, where outer hydrophilic PEG shell offers superior stealth behaviors. Aiming to promote these potential characters toward systemic applications, we strategically introduced hydrophobic cholesteryl moiety at the ω-terminus of block copolymer, anticipating to promote not only the stability of polyplex structure but also the tethered PEG crowdedness. Moreover, Mw of PEG in the PEGylated polyplex micelle was elongated up to 20 kDa for expecting further enhancement in PEG crowdedness. Furthermore, cyclic RGD peptide as ligand molecule to integrin receptors was installed at the distal end of PEG in order for facilitating targeted delivery to the tumor site as well as promoting cellular uptake and intracellular trafficking behaviors. Thus constructed cRGD conjugated polyplex micelle with the elevated PEG shielding was challenged to a modeled intractable pancreatic cancer in mice, achieving potent tumor growth suppression by efficient gene expression of antiangiogenic protein (sFlt-1) at the tumor site. Copyright © 2013 Elsevier Ltd. All rights reserved.

Functionalized near-infrared quantum dots for in vivo tumor vasculature imaging

NASA Astrophysics Data System (ADS)

Hu, Rui; Yong, Ken-Tye; Roy, Indrajit; Ding, Hong; Law, Wing-Cheung; Cai, Hongxing; Zhang, Xihe; Vathy, Lisa A.; Bergey, Earl J.; Prasad, Paras N.

2010-04-01

In this paper, we report the use of near-infrared (NIR)-emitting alloyed quantum dots (QDs) as efficient optical probes for high contrast in vivo imaging of tumors. Alloyed CdTe1 - xSex/CdS QDs were prepared in the non-aqueous phase using the hot colloidal synthesis approach. Water dispersion of the QDs were accomplished by their encapsulation within polyethyleneglycol (PEG)-grafted phospholipid micelles. For tumor-specific delivery in vivo, the micelle-encapsulated QDs were conjugated with the cyclic arginine-glycine-aspartic acid (cRGD) peptide, which targets the αvβ3 integrins overexpressed in the angiogenic tumor vasculatures. Using in vivo NIR optical imaging of mice bearing pancreatic cancer xenografts, implanted both subcutaneously and orthotopically, we have demonstrated that systemically delivered cRGD-conjugated QDs, but not the unconjugated ones, can efficiently target and label the tumors with high signal-to-noise ratio. Histopathological analysis of major organs of the treated mice showed no evidence of systemic toxicity associated with these QDs. These experiments suggest that cRGD-conjugated NIR QDs can serve as safe and efficient probes for optical bioimaging of tumors in vivo. Furthermore, by co-encapsulating these QDs and anticancer drugs within these micelles, we have demonstrated a promising theranostic, nanosized platform for both cancer imaging and therapy.

Fibroblast surface-associated FGF-2 promotes contact-dependent colorectal cancer cell migration and invasion through FGFR-SRC signaling and integrin αvβ5-mediated adhesion

PubMed Central

Knuchel, Sarah; Anderle, Pascale; Werfelli, Patricia; Diamantis, Eva; Rüegg, Curzio

2015-01-01

Carcinoma-associated fibroblasts were reported to promote colorectal cancer (CRC) invasion by secreting motility factors and extracellular matrix processing enzymes. Less is known whether fibroblasts may induce CRC cancer cell motility by contact-dependent mechanisms. To address this question we characterized the interaction between fibroblasts and SW620 and HT29 colorectal cancer cells in 2D and 3D co-culture models in vitro. Here we show that fibroblasts induce contact-dependent cancer cell elongation, motility and invasiveness independently of deposited matrix or secreted factors. These effects depend on fibroblast cell surface-associated fibroblast growth factor (FGF) -2. Inhibition of FGF-2 or FGF receptors (FGFRs) signaling abolishes these effects. FGFRs activate SRC in cancer cells and inhibition or silencing of SRC in cancer cells, but not in fibroblasts, prevents fibroblasts-mediated effects. Using an RGD-based integrin antagonist and function-blocking antibodies we demonstrate that cancer cell adhesion to fibroblasts requires integrin αvβ5. Taken together, these results demonstrate that fibroblasts induce cell-contact-dependent colorectal cancer cell migration and invasion under 2D and 3D conditions in vitro through fibroblast cell surface-associated FGF-2, FGF receptor-mediated SRC activation and αvβ5 integrin-dependent cancer cell adhesion to fibroblasts. The FGF-2-FGFRs-SRC-αvβ5 integrin loop might be explored as candidate therapeutic target to block colorectal cancer invasion. PMID:25973543

Fibroblast surface-associated FGF-2 promotes contact-dependent colorectal cancer cell migration and invasion through FGFR-SRC signaling and integrin αvβ5-mediated adhesion.

PubMed

Knuchel, Sarah; Anderle, Pascale; Werfelli, Patricia; Diamantis, Eva; Rüegg, Curzio

2015-06-10

Carcinoma-associated fibroblasts were reported to promote colorectal cancer (CRC) invasion by secreting motility factors and extracellular matrix processing enzymes. Less is known whether fibroblasts may induce CRC cancer cell motility by contact-dependent mechanisms. To address this question we characterized the interaction between fibroblasts and SW620 and HT29 colorectal cancer cells in 2D and 3D co-culture models in vitro. Here we show that fibroblasts induce contact-dependent cancer cell elongation, motility and invasiveness independently of deposited matrix or secreted factors. These effects depend on fibroblast cell surface-associated fibroblast growth factor (FGF) -2. Inhibition of FGF-2 or FGF receptors (FGFRs) signaling abolishes these effects. FGFRs activate SRC in cancer cells and inhibition or silencing of SRC in cancer cells, but not in fibroblasts, prevents fibroblasts-mediated effects. Using an RGD-based integrin antagonist and function-blocking antibodies we demonstrate that cancer cell adhesion to fibroblasts requires integrin αvβ5. Taken together, these results demonstrate that fibroblasts induce cell-contact-dependent colorectal cancer cell migration and invasion under 2D and 3D conditions in vitro through fibroblast cell surface-associated FGF-2, FGF receptor-mediated SRC activation and αvβ5 integrin-dependent cancer cell adhesion to fibroblasts. The FGF-2-FGFRs-SRC-αvβ5 integrin loop might be explored as candidate therapeutic target to block colorectal cancer invasion.

Independent control of matrix adhesiveness and stiffness within a 3D self-assembling peptide hydrogel.

PubMed

Hogrebe, Nathaniel J; Reinhardt, James W; Tram, Nguyen K; Debski, Anna C; Agarwal, Gunjan; Reilly, Matthew A; Gooch, Keith J

2018-04-01

A cell's insoluble microenvironment has increasingly been shown to exert influence on its function. In particular, matrix stiffness and adhesiveness strongly impact behaviors such as cell spreading and differentiation, but materials that allow for independent control of these parameters within a fibrous, stromal-like microenvironment are very limited. In the current work, we devise a self-assembling peptide (SAP) system that facilitates user-friendly control of matrix stiffness and RGD (Arg-Gly-Asp) concentration within a hydrogel possessing a microarchitecture similar to stromal extracellular matrix. In this system, the RGD-modified SAP sequence KFE-RGD and the scrambled sequence KFE-RDG can be directly swapped for one another to change RGD concentration at a given matrix stiffness and total peptide concentration. Stiffness is controlled by altering total peptide concentration, and the unmodified base peptide KFE-8 can be included to further increase this stiffness range due to its higher modulus. With this tunable system, we demonstrate that human mesenchymal stem cell morphology and differentiation are influenced by both gel stiffness and the presence of functional cell binding sites in 3D culture. Specifically, cells 24 hours after encapsulation were only able to spread out in stiffer matrices containing KFE-RGD. Upon addition of soluble adipogenic factors, soft gels facilitated the greatest adipogenesis as determined by the presence of lipid vacuoles and PPARγ-2 expression, while increasing KFE-RGD concentration at a given stiffness had a negative effect on adipogenesis. This three-component hydrogel system thus allows for systematic investigation of matrix stiffness and RGD concentration on cell behavior within a fibrous, three-dimensional matrix. Physical cues from a cell's surrounding environment-such as the density of cell binding sites and the stiffness of the surrounding material-are increasingly being recognized as key regulators of cell function. Currently, most synthetic biomaterials used to independently tune these parameters lack the fibrous structure characteristic of stromal extracellular matrix, which can be important to cells naturally residing within stromal tissues. In this manuscript, we describe a 3D hydrogel encapsulation system that provides user-friendly control over matrix stiffness and binding site concentration within the context of a stromal-like microarchitecture. Binding site concentration and gel stiffness both influenced cell spreading and differentiation, highlighting the utility of this system to study the independent effects of these material properties on cell function. Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Nanolithographic control of the spatial organization of cellular adhesion receptors at the single-molecule level

PubMed Central

Schvartzman, Mark; Palma, Matteo; Sable, Julia; Abramson, Justin; Hu, Xian; Sheetz, Michael P.; Wind, Shalom J.

2011-01-01

The ability to control the placement of individual molecules promises to enable a wide range of applications and is a key challenge in nanoscience and nanotechnology. Many biological interactions, in particular, are sensitive to the precise geometric arrangement of proteins. We have developed a technique which combines molecular-scale nanolithography with site-selective biochemistry to create biomimetic arrays of individual protein binding sites. The binding sites can be arranged in heterogeneous patterns of virtually any possible geometry with a nearly unlimited number of degrees of freedom. We have used these arrays to explore how the geometric organization of the extracellular matrix (ECM) binding ligand RGD (Arg-Gly-Asp) affects cell adhesion and spreading. Systematic variation of spacing, density and cluster size of individual integrin binding sites was used to elicit different cell behavior. Cell spreading assays on arrays of different geometric arrangements revealed a dramatic increase in spreading efficiency when at least 4 liganded sites were spaced within 60 nm or less, with no dependence on global density. This points to the existence of a minimal matrix adhesion unit for fibronectin defined in space and stoichiometry. Developing an understanding of the ECM geometries that activate specific cellular functional complexes is a critical step toward controlling cell behavior. Potential practical applications range from new therapeutic treatments to the rational design of tissue scaffolds that can optimize healing without scarring. More broadly, spatial control at the single-molecule level can elucidate factors controlling individual molecular interactions and can enable synthesis of new systems based on molecular-scale architectures. PMID:21319842

A precision-guided MWNT mediated reawakening the sunk synergy in RAS for anti-angiogenesis lung cancer therapy.

PubMed

Su, Yujie; Hu, Yahui; Wang, Yu; Xu, Xiangting; Yuan, Yang; Li, Yunman; Wang, Zeyuan; Chen, Kerong; Zhang, Fangrong; Ding, Xuefang; Li, Min; Zhou, Jianping; Liu, Yuan; Wang, Wei

2017-09-01

Multi-walled carbon nanotube (MWNT) with its versatility has exhibited tremendous superiority in drug delivery. Despite plenty of researches on MWNT based delivery systems, precision-guided assistances to maximize their profitable properties are still lacking in substantive progress. We developed here a dual-targeting and co-delivery system based on MWNT for antiangiogenesis therapy in lung cancer which aimed at renin-angiotensin system (RAS) dysregulation by synergistically conducting angiotensin II type 1 receptor (AT 1 R) and type 2 receptor (AT 2 R) pathway. In this work, iRGD peptide connected to polyethyleneimine (PEI) was linked to MWNT skeleton, accompanying with candesartan (CD) conjugated to MWNT mediated by cystamine (SS). The functionalized MWNT is assembled with plasmid AT 2 (pAT 2 ) to form iRGD-PEI-MWNT-SS-CD/pAT 2 complexes. iRGD and CD act as pilots for complexes to dually target symbolic ανβ3-integrin and AT 1 R both overexpressed on tumor angiogenic endothelium and lung cancer cell. CD as chemotherapy showed synergistic downregulation of VEGF when combining of pAT 2 and efficiently inhibited angiogenesis. iRGD-PEI-MWNT-SS-CD/pAT 2 complexes greatly appreciated drug activities by changing drug distribution and exhibited remarkable tumor growth suppression in A549 xenograft nude mice. Our work presents that such dual-targeting strategy highly improves the delivery performance of MWNT and open a new avenue for RAS related lung cancer therapy. Copyright © 2017 Elsevier Ltd. All rights reserved.

The effects of an RGD-PAMAM dendrimer conjugate in 3D spheroid culture on cell proliferation, expression and aggregation.

PubMed

Jiang, Li-Yang; Lv, Bing; Luo, Ying

2013-04-01

By presenting biomolecular ligands on the surface in high density, ligand-decorated dendrimers are capable of binding to membrane receptors and cells with specificity and avidity. Despite the various uses, fundamental investigations on ligand-dendrimer conjugates have mainly focused on their binding behavior with cells, whereas their potential bioactivity and applications in multicellular systems, especially in three-dimensional (3D) culture systems, remains untapped. In this study, a typical adhesive peptide ligand - RGD - was modified to generation 4 polyamidoamine (PAMAM), and the bioactivity of suspended RGD-PAMAM conjugates was investigated on cells cultured as multicellular spheroids. Our results demonstrate that the RGD-PAMAM conjugates, after being incorporated into the 3D spheroids, were able to promote cellular proliferation and aggregation, and affect the mRNA expression of extracellular factors by NIH 3T3 cells. These bioactive functions were multivalency-dependent, as none of similar effects was observed for monovalent RGD ligand. Our study suggests that multivalent ligand-dendrimer conjugates may act as a unique type of artificial factors to mediate the cellular microenvironment in 3D culture, a property attributable to the spatial organization of the ligands and possible "cell-gluing" function of multivalent conjugates. This new finding opens the door for further exploring multivalent ligand-dendrimer conjugates for applications in 3D cell culture and tissue engineering. Copyright © 2013 Elsevier Ltd. All rights reserved.

VEGF-Iron Oxide Conjugate for Dual MR and PET Imaging of Breast Cancer Angiogenesis

DTIC Science & Technology

2007-09-01

with both VEGF121 and PET isotope 64Cu (t1/2 = 12.7 h) and test the dual probe in vitro. Aim 2: To test the PET and mMRI efficacy of the dual...iron oxide nanoparticles conjugated with macrocyclic chelating agent DOTA for 64Cu -labeling and cyclic RGD peptide for integrin alpha(v)beta(3...radionuclide 64Cu without loss of receptor affinity and functional activity of the protein. 64Cu -VEGF is also able to delineate small tumors that are

Retargeting of Rat Parvovirus H-1PV to Cancer Cells through Genetic Engineering of the Viral Capsid

PubMed Central

Allaume, Xavier; El-Andaloussi, Nazim; Leuchs, Barbara; Bonifati, Serena; Kulkarni, Amit; Marttila, Tiina; Kaufmann, Johanna K.; Nettelbeck, Dirk M.; Kleinschmidt, Jürgen; Rommelaere, Jean

2012-01-01

The rat parvovirus H-1PV is a promising anticancer agent given its oncosuppressive properties and the absence of known side effects in humans. H-1PV replicates preferentially in transformed cells, but the virus can enter both normal and cancer cells. Uptake by normal cells sequesters a significant portion of the administered viral dose away from the tumor target. Hence, targeting H-1PV entry specifically to tumor cells is important to increase the efficacy of parvovirus-based treatments. In this study, we first found that sialic acid plays a key role in H-1PV entry. We then genetically engineered the H-1PV capsid to improve its affinity for human tumor cells. By analogy with the resolved crystal structure of the closely related parvovirus minute virus of mice, we developed an in silico three-dimensional (3D) model of the H-1PV wild-type capsid. Based on this model, we identified putative amino acids involved in cell membrane recognition and virus entry at the level of the 2-fold axis of symmetry of the capsid, within the so-called dimple region. In situ mutagenesis of these residues significantly reduced the binding and entry of H-1PV into permissive cells. We then engineered an entry-deficient viral capsid and inserted a cyclic RGD-4C peptide at the level of its 3-fold axis spike. This peptide binds αvβ3 and αvβ5 integrins, which are overexpressed in cancer cells and growing blood vessels. The insertion of the peptide rescued viral infectivity toward cells overexpressing αvβ5 integrins, resulting in the efficient killing of these cells by the reengineered virus. This work demonstrates that H-1PV can be genetically retargeted through the modification of its capsid, showing great promise for a more efficient use of this virus in cancer therapy. PMID:22258256

Retargeting of rat parvovirus H-1PV to cancer cells through genetic engineering of the viral capsid.

PubMed

Allaume, Xavier; El-Andaloussi, Nazim; Leuchs, Barbara; Bonifati, Serena; Kulkarni, Amit; Marttila, Tiina; Kaufmann, Johanna K; Nettelbeck, Dirk M; Kleinschmidt, Jürgen; Rommelaere, Jean; Marchini, Antonio

2012-04-01

The rat parvovirus H-1PV is a promising anticancer agent given its oncosuppressive properties and the absence of known side effects in humans. H-1PV replicates preferentially in transformed cells, but the virus can enter both normal and cancer cells. Uptake by normal cells sequesters a significant portion of the administered viral dose away from the tumor target. Hence, targeting H-1PV entry specifically to tumor cells is important to increase the efficacy of parvovirus-based treatments. In this study, we first found that sialic acid plays a key role in H-1PV entry. We then genetically engineered the H-1PV capsid to improve its affinity for human tumor cells. By analogy with the resolved crystal structure of the closely related parvovirus minute virus of mice, we developed an in silico three-dimensional (3D) model of the H-1PV wild-type capsid. Based on this model, we identified putative amino acids involved in cell membrane recognition and virus entry at the level of the 2-fold axis of symmetry of the capsid, within the so-called dimple region. In situ mutagenesis of these residues significantly reduced the binding and entry of H-1PV into permissive cells. We then engineered an entry-deficient viral capsid and inserted a cyclic RGD-4C peptide at the level of its 3-fold axis spike. This peptide binds α(v)β(3) and α(v)β(5) integrins, which are overexpressed in cancer cells and growing blood vessels. The insertion of the peptide rescued viral infectivity toward cells overexpressing α(v)β(5) integrins, resulting in the efficient killing of these cells by the reengineered virus. This work demonstrates that H-1PV can be genetically retargeted through the modification of its capsid, showing great promise for a more efficient use of this virus in cancer therapy.

Tumor Necrosis Factor α‐Gene Therapy for an Established Murine Melanoma Using RGB (Arg‐Gly‐Asp) Fiber‐mutant Adenovirus Vectors

PubMed Central

Okada, Yuka; Nakagawa, Shinsaku; Mizuguchi, Hiroyuki; Takahashi, Koichi; Mizuno, Nobuyasu; Fujita, Takuya; Yamamoto, Akira; Hayakawa, Takao; Mayumi, Tadanori

2002-01-01

Although adenovirus vectors (Ad) provide high‐level transduction efficacy to many cell types, extremely high doses of Ad are required for sufficient gene transduction into several tumors, including melanoma. Here, we demonstrated that the expression of coxsackie‐adenovirus receptor, a primitive Ad‐receptor, was very low in murine and human melanoma cells. We also found that fiber‐mutant Ad containing the Arg‐Gly‐Asp (RGD) sequence in the fiber knob remarkably augmented gene transduction efficacy in melanoma cells by targeting αv‐integrins. In addition, intratumoral injection of RGD fiber‐mutant Ad containing the tumor necrosis factor α gene (AdRGD‐TNFα) revealed dramatic anti‐tumor efficacy through hemolytic necrosis in an established murine B16 BL6 melanoma model. Ad‐RGD‐TNFα required one‐tenth the dosage of Ad‐TNFα to induce an equal therapeutic effect. These results suggest that αv‐integrin‐targeted Ad will be a very powerful tool for the advancement of melanoma gene therapy. PMID:11985794

Inclusion of an Arg-Gly-Asp receptor-recognition motif into the capsid protein of rabbit hemorrhagic disease virus enables culture of the virus in vitro.

PubMed

Zhu, Jie; Miao, Qiuhong; Tan, Yonggui; Guo, Huimin; Liu, Teng; Wang, Binbin; Chen, Zongyan; Li, Chuanfeng; Liu, Guangqing

2017-05-26

The fact that rabbit hemorrhagic disease virus (RHDV), an important member of the Caliciviridae family, cannot be propagated in vitro has greatly impeded the progress of investigations into the mechanisms of pathogenesis, translation, and replication of this and related viruses. In this study, we have successfully bypassed this obstacle by constructing a mutant RHDV (mRHDV) by using a reverse genetics technique. By changing two amino acids (S305R,N307D), we produced a specific receptor-recognition motif (Arg-Gly-Asp; called RGD) on the surface of the RHDV capsid protein. mRHDV was recognized by the intrinsic membrane receptor (integrin) of the RK-13 cells, which then gained entry and proliferated as well as imparted apparent cytopathic effects. After 20 passages, the titers of RHDV reached 1 × 10 4.3 50% tissue culture infectious dose (TCID 50 )/ml at 72 h. Furthermore, mRHDV-infected rabbits showed typical rabbit plague symptoms and died within 48-72 h. After immunization with inactivated mRHDV, the rabbits survived wild-type RHDV infection, indicating that mRHDV could be a candidate virus strain for producing a vaccine against RHDV infection. In summary, this study offers a novel strategy for overcoming the challenges of proliferating RHDV in vitro Because virus uptake via specific membrane receptors, several of which specifically bind to the RGD peptide motif, is a common feature of host cells, we believe that this the strategy could also be applied to other RNA viruses that currently lack suitable cell lines for propagation such as hepatitis E virus and norovirus. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

At the Frontier; RXLR Effectors Crossing the Phytophthora-Host Interface.

PubMed

Bouwmeester, Klaas; Meijer, Harold J G; Govers, Francine

2011-01-01

Plants are constantly beset by pathogenic organisms. To successfully infect their hosts, plant pathogens secrete effector proteins, many of which are translocated to the inside of the host cell where they manipulate normal physiological processes and undermine host defense. The way by which effectors cross the frontier to reach the inside of the host cell varies among different classes of pathogens. For oomycete plant pathogens - like the potato late blight pathogen Phytophthora infestans - it has been shown that effector translocation to the host cell cytoplasm is dependent on conserved amino acid motifs that are present in the N-terminal part of effector proteins. One of these motifs, known as the RXLR motif, has a strong resemblance with a host translocation motif found in effectors secreted by Plasmodium species. These malaria parasites, that reside inside specialized vacuoles in red blood cells, make use of a specific protein translocation complex to export effectors from the vacuole into the red blood cell. Whether or not also oomycete RXLR effectors require a translocation complex to cross the frontier is still under investigation. For one P. infestans RXLR effector named IPI-O we have found a potential host target that could play a role in establishing the first contact between this effector and the host cell. This membrane spanning lectin receptor kinase, LecRK-I.9, interacts with IPI-O via the tripeptide RGD that overlaps with the RXLR motif. In animals, RGD is a well-known cell adhesion motif; it binds to integrins, which are membrane receptors that regulate many cellular processes and which can be hijacked by pathogens for either effector translocation or pathogen entry into host cells.

Integrin-mediated signal transduction linked to Ras pathway by GRB2 binding to focal adhesion kinase.

PubMed

Schlaepfer, D D; Hanks, S K; Hunter, T; van der Geer, P

The cytoplasmic focal adhesion protein-tyrosine kinase (FAK) localizes with surface integrin receptors at sites where cells attach to the extracellular matrix. Increased FAK tyrosine phosphorylation occurs upon integrin engagement with fibronectin. Here we show that adhesion of murine NIH3T3 fibroblasts to fibronectin promotes SH2-domain-mediated association of the GRB2 adaptor protein and the c-Src protein-tyrosine kinase (PTK) with FAK in vivo, and also results in activation of mitogen-activated protein kinase (MAPK). In v-Src-transformed NIH3T3, the association of v-Src, GRB2 and Sos with FAK is independent of cell adhesion to fibronectin. The GRB2 SH2 domain binds directly to tyrosine-phosphorylated FAK. Mutation of tyrosine residue 925 of FAK (YENV motif) to phenylalanine blocks GRB2 SH2-domain binding to FAK in vitro. Our results show that fibronectin binding to integrins on NIH3T3 fibroblasts promotes c-Src and FAK association and formation of an integrin-activated signalling complex. Phosphorylation of FAK at Tyr 925 upon fibronectin stimulation creates an SH2-binding site for GRB2 which may link integrin engagement to the activation of the Ras/MAPK signal transduction pathway.

Targeted theranostic platinum(IV) prodrug with a built-in aggregation-induced emission light-up apoptosis sensor for noninvasive early evaluation of its therapeutic responses in situ.

PubMed

Yuan, Youyong; Kwok, Ryan T K; Tang, Ben Zhong; Liu, Bin

2014-02-12

Targeted drug delivery to tumor cells with minimized side effects and real-time in situ monitoring of drug efficacy is highly desirable for personalized medicine. In this work, we report the synthesis and biological evaluation of a chemotherapeutic Pt(IV) prodrug whose two axial positions are functionalized with a cyclic arginine-glycine-aspartic acid (cRGD) tripeptide for targeting integrin αvβ3 overexpressed cancer cells and an apoptosis sensor which is composed of tetraphenylsilole (TPS) fluorophore with aggregation-induced emission (AIE) characteristics and a caspase-3 enzyme specific Asp-Glu-Val-Asp (DEVD) peptide. The targeted Pt(IV) prodrug can selectively bind to αvβ3 integrin overexpressed cancer cells to facilitate cellular uptake. In addition, the Pt(IV) prodrug can be reduced to active Pt(II) drug in cells and release the apoptosis sensor TPS-DEVD simultaneously. The reduced Pt(II) drug can induce the cell apoptosis and activate caspase-3 enzyme to cleave the DEVD peptide sequence. Due to free rotation of the phenylene rings, TPS-DEVD is nonemissive in aqueous media. The specific cleavage of DEVD by caspase-3 generates the hydrophobic TPS residue, which tends to aggregate, resulting in restriction of intramolecular rotations of the phenyl rings and ultimately leading to fluorescence enhancement. Such noninvasive and real-time imaging of drug-induced apoptosis in situ can be used as an indicator for early evaluation of the therapeutic responses of a specific anticancer drug.

Utilizing Fibronectin Integrin-Binding Specificity to Control Cellular Responses

PubMed Central

Bachman, Haylee; Nicosia, John; Dysart, Marilyn; Barker, Thomas H.

2015-01-01

Significance: Cells communicate with the extracellular matrix (ECM) protein fibronectin (Fn) through integrin receptors on the cell surface. Controlling integrin–Fn interactions offers a promising approach to directing cell behavior, such as adhesion, migration, and differentiation, as well as coordinated tissue behaviors such as morphogenesis and wound healing. Recent Advances: Several different groups have developed recombinant fragments of Fn that can control epithelial to mesenchymal transition, sequester growth factors, and promote bone and wound healing. It is thought that these physiological responses are, in part, due to specific integrin engagement. Furthermore, it has been postulated that the integrin-binding domain of Fn is a mechanically sensitive switch that drives binding of one integrin heterodimer over another. Critical Issues: Although computational simulations have predicted the mechano-switch hypothesis and recent evidence supports the existence of varying strain states of Fn in vivo, experimental evidence of the Fn integrin switch is still lacking. Future Directions: Evidence of the integrin mechano-switch will enable the development of new Fn-based peptides in tissue engineering and wound healing, as well as deepen our understanding of ECM pathologies, such as fibrosis. PMID:26244106

Stable coordination of the inhibitory Ca2+ ion at MIDAS in integrin CD11b/CD18 by an antibody-derived ligand aspartate: Implications for integrin regulation and structure-based drug design

PubMed Central

Mahalingam, Bhuvaneshwari; Ajroud, Kaouther; Alonso, Jose Luis; Anand, Saurabh; Adair, Brian; Horenstein, Alberto L; Malavasi, Fabio; Xiong, Jian-Ping; Arnaout, M. Amin

2011-01-01

A central feature of integrin interaction with physiologic ligands is the monodentate binding of a ligand carboxylate to a Mg2+ ion hexacoordinated at the metal-ion-dependent-adhesion site (MIDAS) in the integrin A-domain. This interaction stabilizes the A-domain in the high-affinity state, which is distinguished from the default low-affinity state by tertiary changes in the domain that culminate in cell adhesion. Small molecule ligand-mimetic integrin antagonists act as partial agonists, eliciting similar activating conformational changes in the A-domain, which has contributed to paradoxical adhesion and increased patient mortality in large clinical trials. As with other ligand-mimetic integrin antagonists, the function-blocking monoclonal antibody (mAb) 107 binds MIDAS of integrin CD11b/CD18 A-domain (CD11bA), but in contrast, it favors the inhibitory Ca2+ ion over Mg2+ at MIDAS. We determined the crystal structures of the Fab fragment of mAb 107 complexed to the low- and high-affinity states of CD11bA. Favored binding of Ca2+ at MIDAS is caused by the unusual symmetric bidentate ligation of a Fab-derived ligand Asp to a heptacoordinated MIDAS Ca2+. Binding of Fab 107 to CD11bA did not trigger the activating tertiary changes in the domain or in the full-length integrin. These data show that denticity of the ligand Asp/Glu can modify divalent cation selectivity at MIDAS and hence integrin function. Stabilizing the Ca2+ ion at MIDAS by bidentate ligation to a ligand Asp/Glu may provide one approach for designing pure integrin antagonists. PMID:22095715

A tumor-penetrating peptide enhances circulation-independent targeting of peritoneal carcinomatosis

PubMed Central

Sugahara, Kazuki N.; Scodeller, Pablo; Braun, Gary B.; de Mendoza, Tatiana Hurtado; Yamazaki, Chisato M.; Kluger, Michael D.; Kitayama, Joji; Alvarez, Edwin; Howell, Stephen B.; Teesalu, Tambet; Ruoslahti, Erkki; Lowy, Andrew M.

2015-01-01

Peritoneal carcinomatosis is a major source of morbidity and mortality in patients with advanced abdominal neoplasms. Intraperitoneal chemotherapy (IPC) is an area of intense interest given its efficacy in ovarian cancer. However, IPC suffers from poor drug penetration into peritoneal tumors. As such, extensive cytoreductive surgery is required prior to IPC. Here, we explore the utility of iRGD, a tumor-penetrating peptide, for improved tumor-specific penetration of intraperitoneal compounds and enhanced IPC in mice. Intraperitoneally administered iRGD significantly enhanced penetration of an attached fluorescein into disseminated peritoneal tumor nodules. The penetration was tumor-specific, circulation-independent, and mediated by the neuropilin-binding RXXK tissue-penetration peptide motif of iRGD. Q-iRGD, which fluoresces upon cleavage, including the one that leads to RXXK activation, specifically labeled peritoneal metastases displaying different growth patterns in mice. Importantly, iRGD enhanced intratumoral entry of intraperitoneally co-injected dextran to approximately 300% and doxorubicin to 250%. Intraperitoneal iRGD/doxorubicin combination therapy inhibited the growth of bulky peritoneal tumors and reduced systemic drug toxicity. iRGD delivered attached fluorescein and co-applied nanoparticles deep into fresh human peritoneal metastasis explants. These results indicate that intraperitoneal iRGD co-administration serves as a simple and effective strategy to facilitate tumor detection and improve the therapeutic index of IPC for peritoneal carcinomatosis. PMID:26071630

αV-class integrins exert dual roles on α5β1 integrins to strengthen adhesion to fibronectin

PubMed Central

Bharadwaj, Mitasha; Strohmeyer, Nico; Colo, Georgina P.; Helenius, Jonne; Beerenwinkel, Niko; Schiller, Herbert B.; Fässler, Reinhard; Müller, Daniel J.

2017-01-01

Upon binding to the extracellular matrix protein, fibronectin, αV-class and α5β1 integrins trigger the recruitment of large protein assemblies and strengthen cell adhesion. Both integrin classes have been functionally specified, however their specific roles in immediate phases of cell attachment remain uncharacterized. Here, we quantify the adhesion of αV-class and/or α5β1 integrins expressing fibroblasts initiating attachment to fibronectin (≤120 s) by single-cell force spectroscopy. Our data reveals that αV-class integrins outcompete α5β1 integrins. Once engaged, αV-class integrins signal to α5β1 integrins to establish additional adhesion sites to fibronectin, away from those formed by αV-class integrins. This crosstalk, which strengthens cell adhesion, induces α5β1 integrin clustering by RhoA/ROCK/myosin-II and Arp2/3-mediated signalling, whereas overall cell adhesion depends on formins. The dual role of both fibronectin-binding integrin classes commencing with an initial competition followed by a cooperative crosstalk appears to be a basic cellular mechanism in assembling focal adhesions to the extracellular matrix. PMID:28128308

Concepts in receptor optimization: targeting the RGD peptide.

PubMed

Chen, Wei; Chang, Chia-en; Gilson, Michael K

2006-04-12

Synthetic receptors have a wide range of potential applications, but it has been difficult to design low molecular weight receptors that bind ligands with high, "proteinlike" affinities. This study uses novel computational methods to understand why it is hard to design a high-affinity receptor and to explore the limits of affinity, with the bioactive peptide RGD as a model ligand. The M2 modeling method is found to yield excellent agreement with experiment for a known RGD receptor and then is used to analyze a series of receptors generated in silico with a de novo design algorithm. Forces driving binding are found to be systematically opposed by proportionate repulsions due to desolvation and entropy. In particular, strong correlations are found between Coulombic attractions and the electrostatic desolvation penalty and between the mean energy change on binding and the cost in configurational entropy. These correlations help explain why it is hard to achieve high affinity. The change in surface area upon binding is found to correlate poorly with affinity within this series. Measures of receptor efficiency are formulated that summarize how effectively a receptor uses surface area, total energy, and Coulombic energy to achieve affinity. Analysis of the computed efficiencies suggests that a low molecular weight receptor can achieve proteinlike affinity. It is also found that macrocyclization of a receptor can, unexpectedly, increase the entropy cost of binding because the macrocyclic structure further restricts ligand motion.

Collagen Gly missense mutations: Effect of residue identity on collagen structure and integrin binding.

PubMed

Qiu, Yimin; Mekkat, Arya; Yu, Hongtao; Yigit, Sezin; Hamaia, Samir; Farndale, Richard W; Kaplan, David L; Lin, Yu-Shan; Brodsky, Barbara

2018-05-11

Gly missense mutations in type I collagen, which replace a conserved Gly in the repeating (Gly-Xaa-Yaa) n sequence with a larger residue, are known to cause Osteogenesis Imperfecta (OI). The clinical consequences of such mutations range from mild to lethal, with more serious clinical severity associated with larger Gly replacement residues. Here, we investigate the influence of the identity of the residue replacing Gly within and adjacent to the integrin binding 502 GFPGER 507 sequence on triple-helix structure, stability and integrin binding using a recombinant bacterial collagen system. Recombinant collagens were constructed with Gly substituted by Ala, Ser or Val at four positions within the integrin binding region. All constructs formed a stable triple-helix structure with a small decrease in melting temperature. Trypsin was used to probe local disruption of the triple helix, and Gly to Val replacements made the triple helix trypsin sensitive at three of the four sites. Any mutation at Gly505, eliminated integrin binding, while decreased integrin binding affinity was observed in the replacement of Gly residues at Gly502 following the order Val > Ser > Ala. Molecular dynamics simulations indicated that all Gly replacements led to transient disruption of triple-helix interchain hydrogen bonds in the region of the Gly replacement. These computational and experimental results lend insight into the complex molecular basis of the varying clinical severity of OI. Copyright © 2018. Published by Elsevier Inc.

Identification of stepped changes of binding affinity during interactions between the disintegrin rhodostomin and integrin αIIbβ3 in living cells using optical tweezers

NASA Astrophysics Data System (ADS)

Hsieh, Chia-Fen; Chang, Bo-Jui; Pai, Chyi-Huey; Chen, Hsuan-Yi; Chi, Sien; Hsu, Long; Tsai, Jin-Wu; Lin, Chi-Hung

2004-10-01

Integrin receptors serve as both mechanical links and signal transduction mediators between the cell and its environment. Experimental evidence demonstrates that conformational changes and lateral clustering of the integrin proteins may affect their binding to ligands and regulate downstream cellular responses; however, experimental links between the structural and functional correlations of the ligand-receptor interactions are not yet elucidated. In the present report, we utilized optical tweezers to measure the dynamic binding between the snake venom rhodostomin, coated on a microparticle and functioned as a ligand, and the membrane receptor integrin alpha(IIb)beta(3) expressed on a Chinese Hamster Ovary (CHO) cell. A progressive increase of total binding affinity was found between the bead and CHO cell in the first 300 sec following optical tweezers-guided contact. Further analysis of the cumulative data revealed the presence of "unit binding force" presumably exerted by a single rhodostomin-integrin pair. Interestingly, two such units were found. Among the measurements of less total binding forces, presumably taken at the early stage of ligand-receptor interactions, a unit of 4.15 pN per molecule pair was derived. This unit force dropped to 2.54 pN per molecule pair toward the later stage of interactions when the total binding forces were relatively large. This stepped change of single molecule pair binding affinity was not found when mutant rhodostomin proteins were used as ligands (a single unit of 1.81 pN per pair was found). These results were interpreted along with the current knowledge about the conformational changes of integrins during the "molecule activation" process.

Plasma functionalization of polycarbonaturethane to improve endothelialization--Effect of shear stress as a critical factor for biocompatibility control.

PubMed

Lukas, Karin; Thomas, Ulrich; Gessner, André; Wehner, Daniel; Schmid, Thomas; Schmid, Christof; Lehle, Karla

2016-04-01

Medical devices made of polycarbonaturethane (PCU) combine excellent mechanical properties and little biological degradation, but restricted hemocompatibility. Modifications of PCU might reduce platelet adhesion and promote stable endothelialization. PCU was modified using gas plasma treatment, binding of hydrogels, and coupling of cell-active molecules (modified heparin, anti-thrombin III (ATIII), argatroban, fibronectin, laminin-nonapeptide, peptides with integrin-binding arginine-glycine-aspartic acid (RGD) motif). Biocompatibility was verified with static and dynamic cell culture techniques. Blinded analysis focused on improvement in endothelial cell (EC) adhesion/proliferation, anti-thrombogenicity, reproducible manufacturing process, and shear stress tolerance of ECs. EC adhesion and antithrombogenicity were achieved with 9/35 modifications. Additionally, 6/9 stimulated EC proliferation and 3/6 modification processes were highly reproducible for endothelialization. The latter modifications comprised immobilization of ATIII (A), polyethyleneglycole-diamine-hydrogel (E) and polyethylenimine-hydrogel connected with modified heparin (IH). Under sheer stress, only the IH modification improved EC adhesion within the graft. However, ECs did not arrange in flow direction and cell anchorage was restricted. Despite large variation in surface modification chemistry and improved EC adhesion under static culture conditions, additional introduction of shear stress foiled promising preliminary data. Therefore, biocompatibility testing required not only static tests but also usage of physiological conditions such as shear stress in the case of vascular grafts. © The Author(s) 2016.

Selective Modulation of Integrin-mediated Cell Migration by Distinct ADAM Family MembersV⃞

PubMed Central

Huang, Jing; Bridges, Lance C.; White, Judith M.

2005-01-01

A disintegrin and a metalloprotease (ADAM) family members have been implicated in many biological processes. Although it is recognized that recombinant ADAM disintegrin domains can interact with integrins, little is known about ADAM-integrin interactions in cellular context. Here, we tested whether ADAMs can selectively regulate integrin-mediated cell migration. ADAMs were expressed in Chinese hamster ovary cells that express defined integrins (α4β1, α5β1, or both), and cell migration on full-length fibronectin or on its α4β1 or α5β1 binding fragments was studied. We found that ADAMs inhibit integrin-mediated cell migration in patterns dictated by the integrin binding profiles of their isolated disintegrin domains. ADAM12 inhibited cell migration mediated by the α4β1 but not the α5β1 integrin. ADAM17 had the reciprocal effect; it inhibited α5β1- but not α4β1-mediated cell migration. ADAM19 and ADAM33 inhibited migration mediated by both α4β1 and α5β1 integrins. A point mutation in the ADAM12 disintegrin loop partially reduced the inhibitory effect of ADAM12 on cell migration on the α4β1 binding fragment of fibronectin, whereas mutations that block metalloprotease activity had no effect. Our results indicate that distinct ADAMs can modulate cell migration mediated by specific integrins in a pattern dictated, at least in part, by their disintegrin domains. PMID:16079176

Comparison of S. cerevisiae F-BAR domain structures reveals a conserved inositol phosphate binding site

PubMed Central

Moravcevic, Katarina; Alvarado, Diego; Schmitz, Karl R.; Kenniston, Jon A.; Mendrola, Jeannine M.; Ferguson, Kathryn M.; Lemmon, Mark A.

2015-01-01

SUMMARY F-BAR domains control membrane interactions in endocytosis, cytokinesis, and cell signaling. Although generally thought to bind curved membranes containing negatively charged phospholipids, numerous functional studies argue that differences in lipid-binding selectivities of F-BAR domains are functionally important. Here, we compare membrane-binding properties of the S. cerevisiae F-BAR domains in vitro and in vivo. Whereas some F-BAR domains (such as Bzz1p and Hof1p F-BARs) bind equally well to all phospholipids, the F-BAR domain from the RhoGAP Rgd1p preferentially binds phosphoinositides. We determined X-ray crystal structures of F-BAR domains from Hof1p and Rgd1p, the latter bound to an inositol phosphate. The structures explain phospholipid-binding selectivity differences, and reveal an F-BAR phosphoinositide binding site that is fully conserved in a mammalian RhoGAP called Gmip, and is partly retained in certain other F-BAR domains. Our findings reveal previously unappreciated determinants of F-BAR domain lipid-binding specificity, and provide a basis for its prediction from sequence. PMID:25620000

Targeting and destroying tumor vasculature with a near-infrared laser-activated "nanobomb" for efficient tumor ablation.

PubMed

Gao, Wen; Li, Shuangshuang; Liu, Zhenhua; Sun, Yuhui; Cao, Wenhua; Tong, Lili; Cui, Guanwei; Tang, Bo

2017-09-01

Attacking the supportive vasculature network of a tumor offers an important new avenue for cancer therapy. Herein, a near-infrared (NIR) laser-activated "nanobomb" was developed as a noninvasive and targeted physical therapeutic strategy to effectively disrupt tumor neovasculature in an accurate and expeditious manner. This "nanobomb" was rationally fabricated via the encapsulation of vinyl azide (VA) into c(RGDfE) peptide-functionalized, hollow copper sulfide (HCuS) nanoparticles. The resulting RGD@HCuS(VA) was selectively internalized into integrin α v β 3 -expressing tumor vasculature endothelial cells and dramatically increased the photoacoustic signals from the tumor neovasculature, achieving a maximum signal-to-noise ratio at 4 h post-injection. Upon NIR irradiation, the local temperature increase triggered VA to release N 2 bubbles rapidly. Subsequently, these N 2 bubbles could instantly explode to destroy the neovasculature and further induce necrosis of the surrounding tumor cells. A single-dose injection of RGD@HCuS(VA) led to complete tumor regression after laser irradiation, with no tumor regrowth for 30 days. More importantly, high-resolution photoacoustic angiography, combined with excellent biodegradability, facilitated the precise destruction of tumor neovasculature by RGD@HCuS(VA) without damaging normal tissues. These results demonstrate the great potential of this "nanobomb" for clinical translation to treat cancer patients with NIR laser-accessible orthotopic tumors. Copyright © 2017 Elsevier Ltd. All rights reserved.

Osteoclast size heterogeneity in rat long bones is associated with differences in adhesive ligand specificity.

PubMed

Hu, Yingwei; Ek-Rylander, Barbro; Karlström, Erik; Wendel, Mikael; Andersson, Göran

2008-02-01

Prothrombin (PT) is an RGD-containing bone-residing precursor to the serine protease thrombin (TH), which acts as an agonist for a variety of cellular responses in osteoblasts and osteoclasts. We show here that PT, TH, osteopontin (OPN) and fibronectin (FN) promoted adhesion of isolated neonatal rat long bone osteoclasts. However, the cells that adhered to PT and TH were smaller in size, rounded and contained 3-4 nuclei, in comparison to the cells adhering to OPN and FN, which were larger with extended cytoplasmic processes and 6-7 nuclei. Attachment of the larger osteoclasts to OPN and FN was inhibited by antibodies towards beta 3 and beta 1 integrin subunits, respectively. Whereas an RGD-containing peptide inhibited adhesion of the smaller osteoclasts to PT and TH, this was not seen with the beta 3 or beta 1 antibodies. In contrast, the beta 1 antibody augmented osteoclast adhesion to PT and TH in an RGD-dependent manner. Small osteoclasts were less efficient in resorbing mineralized bovine bone slices, as well as expressed lower mRNA levels of MMP-9 and the cathepsins K and L compared to large osteoclasts. The small osteoclast adhering to PT and TH may represent either an immature, less functional precursor to the large osteoclast or alternatively constitute a distinct osteoclast population with a specific role in bone.

Molecular characterization of amino acid deletion in VP1 (1D) protein and novel amino acid substitutions in 3D polymerase protein of foot and mouth disease virus subtype A/Iran87.

PubMed

Esmaelizad, Majid; Jelokhani-Niaraki, Saber; Hashemnejad, Khadije; Kamalzadeh, Morteza; Lotfi, Mohsen

2011-12-01

The nucleotide sequence of the VP1 (1D) and partial 3D polymerase (3D(pol)) coding regions of the foot and mouth disease virus (FMDV) vaccine strain A/Iran87, a highly passaged isolate (~150 passages), was determined and aligned with previously published FMDV serotype A sequences. Overall analysis of the amino acid substitutions revealed that the partial 3D(pol) coding region contained four amino acid alterations. Amino acid sequence comparison of the VP1 coding region of the field isolates revealed deletions in the highly passaged Iranian isolate (A/Iran87). The prominent G-H loop of the FMDV VP1 protein contains the conserved arginine-glycine-aspartic acid (RGD) tripeptide, which is a well-known ligand for a specific cell surface integrin. Despite losing the RGD sequence of the VP1 protein and an Asp(26)→Glu substitution in a beta sheet located within a small groove of the 3D(pol) protein, the virus grew in BHK 21 suspension cell cultures. Since this strain has been used as a vaccine strain, it may be inferred that the RGD deletion has no critical role in virus attachment to the cell during the initiation of infection. It is probable that this FMDV subtype can utilize other pathways for cell attachment.

Cocrystal structure of the ICAP1 PTB domain in complex with a KRIT1 peptide

PubMed Central

Liu, Weizhi; Boggon, Titus J.

2013-01-01

Integrin cytoplasmic domain-associated protein-1 (ICAP1) is a suppressor of integrin activation and directly binds to the cytoplasmic tail of β1 integrins; its binding suppresses integrin activation by competition with talin. Krev/Rap1 interaction trapped-1 (KRIT1) releases ICAP1 suppression of integrin activation by sequestering ICAP1 away from integrin cytoplasmic tails. Here, the cocrystal structure of the PTB domain of ICAP1 in complex with a 29-­amino-acid fragment (residues 170–198) of KRIT1 is presented to 1.7 Å resolution [the resolution at which 〈I/σ(I)〉 = 2.9 was 1.83 Å]. In previous studies, the structure of ICAP1 with integrin β1 was determined to 3.0 Å resolution and that of ICAP1 with the N-terminal portion of KRIT1 (residues 1–­198) was determined to 2.54 Å resolution; therefore, this study provides the highest resolution structure yet of ICAP1 and allows further detailed analysis of the interaction of ICAP1 with its minimal binding region in KRIT1. PMID:23695561

Fabrication of biomimetic nanomaterials and their effect on cell behavior

NASA Astrophysics Data System (ADS)

Porri, Teresa Jane

Cells in vivo respond to an intricate combination of chemical and mechanical signals. The corneal epithelium, a structure which prevents the admission of bacteria and undesirable molecules into the eye, grows on a basement membrane which presents both nanoscale topographic and adhesive chemical signals. An effective approach to biomaterials design takes advantage of the synergistic effects of the multiple cellular inputs which are available to engineer cell-substrate interactions. We have previously demonstrated the effects of nanoscale topography on a variety of corneal epithelial cell behaviors. To gain a better understanding of cell-level control in vivo, we employ a systems-level approach which looks at the effect of nanoscale topography in conjunction with a biomimetic surface chemistry. First, we discuss a novel method of fabricating nanoscale topography through templated electroless deposition of gold into PVP-coated polycarbonate membranes. This technique creates nanowires of gold with an uniform outer diameter that is dependent upon the size of the pores in the membrane used, and a nanowire length that is dependent upon the extent of etching into the polymer membrane. The gold nanowires can be modified with self-assembled monolayers (SAMs) of alkanethiols. Using these substrates, we study the effect of topographic length scale and surface chemistry on cells attached to a discontinuous nanoscale topography, and find a transition in cellular behavior at a length scale (between 600 and 2000 nm inter-wire spacing) that is commensurate with the transition length scale seen on surfaces presenting continuous grooves and ridges. Secondly, we study the effect of non-fouling peptide-modified SAMs on cellular behavior. We examine the effect of co-presented RGD and AG73 peptides and show that cell spreading is a function of the relative ratios of RGD and AG73 present on the surface. Finally, we explore the combinatorial effects of biologically relevant chemistry with anisotropic nanoscale topography with dimensions that vary from the micron to the nanoscale. We show that integrin binding, syndecan binding, and topographic length scale each independently influence epithelial cell response to nanoscale features, lending a high degree of control over cell morphologic responses.

Systemic Administration of siRNA via cRGD-containing Peptide.

PubMed

Huang, Yuanyu; Wang, Xiaoxia; Huang, Weiyan; Cheng, Qiang; Zheng, Shuquan; Guo, Shutao; Cao, Huiqing; Liang, Xing-Jie; Du, Quan; Liang, Zicai

2015-08-24

Although small interfering RNAs (siRNAs) have been demonstrated to specifically silence their target genes in disease models and clinical trials, in vivo siRNA delivery is still the technical bottleneck that limits their use in therapeutic applications. In this study, a bifunctional peptide named RGD10-10R was designed and tested for its ability to deliver siRNA in vitro and in vivo. Because of their electrostatic interactions with polyarginine (10R), negatively charged siRNAs were readily complexed with RGD10-10R peptides, forming spherical RGD10-10R/siRNA nanoparticles. In addition to enhancing their serum stability by preventing RNase from attacking siRNA through steric hindrance, peptide binding facilitated siRNA transfection into MDA-MB-231 cells, as demonstrated by FACS and confocal microscopy assays and by the repressed expression of target genes. When RGD10 peptide, a receptor competitor of RGD10-10R, was added to the transfection system, the cellular internalization of RGD10-10R/siRNA was significantly compromised, suggesting a mechanism of ligand/receptor interaction. Tissue distribution assays indicated that the peptide/siRNA complex preferentially accumulated in the liver and in several exocrine/endocrine glands. Furthermore, tumor-targeted delivery of siRNA was also demonstrated by in vivo imaging and cryosection assays. In summary, RGD10-10R might constitute a novel siRNA delivery tool that could potentially be applied in tumor treatment.

Systemic Administration of siRNA via cRGD-containing Peptide

PubMed Central

Huang, Yuanyu; Wang, Xiaoxia; Huang, Weiyan; Cheng, Qiang; Zheng, Shuquan; Guo, Shutao; Cao, Huiqing; Liang, Xing-Jie; Du, Quan; Liang, Zicai

2015-01-01

Although small interfering RNAs (siRNAs) have been demonstrated to specifically silence their target genes in disease models and clinical trials, in vivo siRNA delivery is still the technical bottleneck that limits their use in therapeutic applications. In this study, a bifunctional peptide named RGD10-10R was designed and tested for its ability to deliver siRNA in vitro and in vivo. Because of their electrostatic interactions with polyarginine (10R), negatively charged siRNAs were readily complexed with RGD10-10R peptides, forming spherical RGD10-10R/siRNA nanoparticles. In addition to enhancing their serum stability by preventing RNase from attacking siRNA through steric hindrance, peptide binding facilitated siRNA transfection into MDA-MB-231 cells, as demonstrated by FACS and confocal microscopy assays and by the repressed expression of target genes. When RGD10 peptide, a receptor competitor of RGD10-10R, was added to the transfection system, the cellular internalization of RGD10-10R/siRNA was significantly compromised, suggesting a mechanism of ligand/receptor interaction. Tissue distribution assays indicated that the peptide/siRNA complex preferentially accumulated in the liver and in several exocrine/endocrine glands. Furthermore, tumor-targeted delivery of siRNA was also demonstrated by in vivo imaging and cryosection assays. In summary, RGD10-10R might constitute a novel siRNA delivery tool that could potentially be applied in tumor treatment. PMID:26300278

The opposing roles of laminin-binding integrins in cancer.

PubMed

Ramovs, Veronika; Te Molder, Lisa; Sonnenberg, Arnoud

2017-01-01

Integrins play an important role in cell adhesion by linking the cytoskeleton of cells to components in the extracellular matrix. In this capacity, integrins cooperate with different cell surface receptors, including growth factor receptors and G-protein coupled receptors, to regulate intracellular signaling pathways that control cell polarization, spreading, migration, survival, and gene expression. A distinct subfamily of molecules in the integrin family of adhesion receptors is formed by receptors that mediate cell adhesion to laminins, major components of the basement membrane that lie under clusters of cells or surround them, separating them from other cells and/or adjacent connective tissue. During the past decades, many studies have provided evidence for a role of laminin-binding integrins in tumorigenesis, and both tumor-promoting and suppressive activities have been identified. In this review we discuss the dual role of the laminin-binding integrins α3β1 and α6β4 in tumor development and progression, and examine the factors and mechanisms involved in these opposing effects. Copyright © 2016 Elsevier B.V. All rights reserved.

The disintegrin, trimucrin, suppresses LPS-induced activation of phagocytes primarily through blockade of NF-κB and MAPK activation.

PubMed

Hung, Yu-Chun; Hsu, Chun-Chieh; Chung, Ching-Hu; Huang, Tur-Fu

2016-07-01

In addition to antiplatelet activity, disintegrin, a small-mass RGD-containing polypeptide, has been shown to exert anti-inflammatory effects but the mechanism involved remains unclear. In this study, we report that trimucrin, a disintegrin from the venom of Trimeresurus mucrosquamatus, inhibits lipopolysaccharide (LPS)-induced stimulation of THP-1 and RAW 264.7 cells. We also investigate the underlying mechanism. Trimucrin decreased the release of proinflammatory cytokines including tumor necrosis factor α (TNFα), interleukin-6 (IL-6), nitric oxide, and reactive oxygen species (ROS), and inhibited the adhesion and migration of LPS-activated phagocytes. Trimucrin significantly blocked the expression of nuclear factor kappaB (NF-κB)-related downstream inducible enzymes such as inducible nitric oxide synthase (iNOS) and COX-2. In addition, its anti-inflammatory effect was associated with the decreased mitogen-activated protein kinase (MAPK) phosphorylation. Furthermore, trimucrin concentration dependently inhibited LPS-induced phosphorylation of focal adhesion kinase (FAK), PI3K, and Akt. Trimucrin also reversed the DNA-binding activity of NF-κB by suppressing the LPS-induced nuclear translocation of p65 and the cytosolic IκB release. Flow cytometric analyses showed that trimucrin bound to cells in a concentration-dependent manner. The anti-αVβ3 mAb also specifically decreased the binding of fluorescein isothiocyanate (FITC)-conjugated trimucrin. Binding assays demonstrated that integrin αVβ3 was the binding site for trimucrin on THP-1 and RAW 264.7 cells. In conclusion, we showed that trimucrin decreases the inflammatory reaction through the attenuation of iNOS expression and nitric oxide (NO) production by blocking MAP kinase and the NF-κB activation in LPS-stimulated THP-1 and RAW 264.7 cells.

Selecting the correct cellular model for assessing of the biological response of collagen-based biomaterials.

PubMed

Davidenko, Natalia; Hamaia, Samir; Bax, Daniel V; Malcor, Jean-Daniel; Schuster, Carlos F; Gullberg, Donald; Farndale, Richard W; Best, Serena M; Cameron, Ruth E

2018-01-01

Accurate evaluation of the biological performance of biomaterials requires the correct assessment of their native-like cell ligation properties. However, cell attachment studies often overlook the details of the substrate-cell binding mechanisms, be they integrin-mediated or non-specific, and ignore the class- and species-specificities of the cell adhesion receptor involved. In this work we have used different collagen (Col) substrates (fibrillar collagens I, II and III and network-forming Col IV), containing different affinity cell-recognition motifs, to establish the influence of the receptor identity and species-specificity on collagen-cell interactive properties. Receptor expression was varied by using cells of different origin, or transfecting collagen-binding integrins into integrin-null cells. These include mouse C2C12 myoblasts transfected with human α1, α2, α10 or α11; human fibrosarcoma HT1080 cells which constitutively express only human α2β1, and rat glioma Rugli cells, with only rat α1β1. Using these lines, the nature of integrin binding sites was studied in order to delineate the bioactivity of different collagen substrates. Integrin ligation was studied on collagen coatings alongside synthetic (GFOGER/GLOGEN) and Toolkit (Col II-28/Col III-7) triple-helical peptides to evaluate (1) their affinity towards different integrins and (2) to confirm the activity of the inserted integrin in the transfected cells. Thin films of dermal and tendon Col I were used to evaluate the influence of the carbodiimide (EDC)-based treatment on the cellular response on Col of different origin. The results showed that the binding properties of transfected C2C12 cells to collagens depend on the identity of inserted integrin. Similar ligation characteristics were observed using α1+ and α10+ cells, but these were distinct from the similar binding features of α2+ and α11+ cells. Recombinant human and rat-α1 I domain binding to collagens and peptides correlated with the cell adhesion results, showing receptor class- and species-specificities. The understanding of the physiologically relevant cell anchorage characteristics of bio-constructs may assist in the selection of (1) the optimum collagen source for cellular supports and (2) the correct cellular model for their biological assessment. This, in turn, may allow reliable prediction of the biological performance of bio-scaffolds in vivo for specific TE applications. Integrins play a vital role in cellular responses to environmental cues during early-stage cell-substrate interaction. We describe physiologically relevant cell anchorage to collagen substrates that present different affinity cell-recognition motifs, to provide experimental tools to assist in understanding integrin binding. Using different cell types and recombinant integrin α1-I-domains, we found that cellular response was highly dependent on collagen type, origin and EDC-crosslinking status, as well as on the integrin class and species of origin. This comprehensive study establishes selectivity amongst the four collagen-binding integrins and species-specific properties that together may influence choice of cell type and receptor in different experimental settings. This work offers key guidance in selecting of the correct cellular model for the biological testing of collagen-based biomaterials. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Molecular Mechanotransduction: how forces trigger cytoskeletal dynamics

NASA Astrophysics Data System (ADS)

Ehrlicher, Allen

2012-02-01

Mechanical stresses elicit cellular reactions mediated by chemical signals. Defective responses to forces underlie human medical disorders, such as cardiac failure and pulmonary injury. Despite detailed knowledge of the cytoskeleton's structure, the specific molecular switches that convert mechanical stimuli into chemical signals have remained elusive. Here we identify the actin-binding protein, filamin A (FLNa) as a central mechanotransduction element of the cytoskeleton by using Fluorescence Loss After photoConversion (FLAC), a novel high-speed alternative to FRAP. We reconstituted a minimal system consisting of actin filaments, FLNa and two FLNa-binding partners: the cytoplasmic tail of ß-integrin, and FilGAP. Integrins form an essential mechanical linkage between extracellular and intracellular environments, with ß integrin tails connecting to the actin cytoskeleton by binding directly to filamin. FilGAP is a FLNa-binding GTPase-activating protein specific for Rac, which in vivo regulates cell spreading and bleb formation. We demonstrate that both externally-imposed bulk shear and myosin II driven forces differentially regulate the binding of integrin and FilGAP to FLNa. Consistent with structural predictions, strain increases ß-integrin binding to FLNa, whereas it causes FilGAP to dissociate from FLNa, providing a direct and specific molecular basis for cellular mechanotransduction. These results identify the first molecular mechanotransduction element within the actin cytoskeleton, revealing that mechanical strain of key proteins regulates the binding of signaling molecules. Moreover, GAP activity has been shown to switch cell movement from mesenchymal to amoeboid motility, suggesting that mechanical forces directly impact the invasiveness of cancer.

Probing the contractile vacuole as Achilles' heel of the biotrophic grapevine pathogen Plasmopara viticola.

PubMed

Tröster, Viktoria; Setzer, Tabea; Hirth, Thomas; Pecina, Anna; Kortekamp, Andreas; Nick, Peter

2017-09-01

The causative agent of Grapevine Downy Mildew, the oomycete Plasmopara viticola, poses a serious threat to viticulture. In the current work, the contractile vacuole of the zoospore is analysed as potential target for novel plant protection strategies. Using a combination of electron microscopy, spinning disc confocal microscopy, and video differential interference contrast microscopy, we have followed the genesis and dynamics of this vacuole required during the search for the stomata, when the non-walled zoospore is exposed to hypotonic conditions. This subcellular description was combined with a pharmacological study, where the functionality of the contractile vacuole was blocked by manipulation of actin, by Na, Cu, and Al ions or by inhibition of the NADPH oxidase. We further observe that RGD peptides (mimicking binding sites for integrins at the extracellular matrix) can inhibit the function of the contractile vacuole as well. Finally, we show that an extract from Chinese liquorice (Glycyrrhiza uralensis) proposed as biocontrol for Downy Mildews can efficiently induce zoospore burst and that this activity depends on the activity of NADPH oxidase. The effect of the extract can be phenocopied by its major compound, glycyrrhizin, suggesting a mode of action for this biologically safe alternative to copper products.

Entry and intracellular replication of Escherichia coli K1 in macrophages require expression of outer membrane protein A.

PubMed

Sukumaran, Sunil K; Shimada, Hiroyuki; Prasadarao, Nemani V

2003-10-01

Interactions between Escherichia coli K1, which causes meningitis in neonates, and macrophages have not been explored well. In this study we found that E. coli K1 was able to enter, survive, and replicate intracellularly in both murine and human macrophage cell lines, as well as in monocytes and macrophages of newborn rats. In addition, we demonstrated that OmpA (+) E. coli also enters and replicates in human peripheral blood monocytes in vitro. Outer membrane protein A (OmpA) expression on E. coli contributes to binding to macrophages, phagocytosis, and survival within macrophages. Opsonization with either complement proteins or antibody is not required for uptake and survival of the bacteria within the macrophages. Transmission electron microscopy and immunocytochemistry studies with the infected macrophages indicated that OmpA(+) E. coli multiplies enormously in a single phagosome and bursts the cell. Internalization of OmpA(+) E. coli by RAW 264.7 cells occurred by both actin- and microtubule-dependent processes, which are independent of RGD-mediated integrin receptors. Internalization and intracellular survival within phagocytic cells thus may play an important role in the development of bacteremia, which is crucial for E. coli crossing of the blood-brain barrier.

67Cu-Radiolabeling of a multimeric RGD peptide for αVβ3 integrin-targeted radionuclide therapy: stability, therapeutic efficacy, and safety studies in mice.

PubMed

Jin, Zhao-Hui; Furukawa, Takako; Ohya, Tomoyuki; Degardin, Mélissa; Sugyo, Aya; Tsuji, Atsushi B; Fujibayashi, Yasuhisa; Zhang, Ming-Rong; Higashi, Tatsuya; Boturyn, Didier; Dumy, Pascal; Saga, Tsuneo

2017-04-01

Copper-67 (Cu) is one of the most promising radionuclides for internal radiation therapy. Globally, several projects have recently been initiated for developing innovative approaches for the large-scale production of Cu. Encouraged by these, we performed Cu-radiolabeling of a tetrameric cyclic Arg-Gly-Asp (cRGD) peptide conjugate, cyclam-RAFT-c(-RGDfK-)4, which selectively targets αVβ3 integrin (αVβ3), the transmembrane receptor involved in tumor invasion, angiogenesis, and metastasis. We also evaluated the therapeutic potential and safety of this radiocompound. Cu, produced through the Ni(α, p)Cu reaction, was used for the radiolabeling of cyclam-RAFT-c(-RGDfK-)4 at 70°C for 10 min. The radiolabeling efficiency and product stability were assessed using reversed-phase high-performance liquid chromatography and/or thin-layer chromatography. Mice with subcutaneous αVβ3-positive U87MG-glioblastoma xenografts received a single intravenous injection of one of the following: Cu-cyclam-RAFT-c(-RGDfK-)4 (11.1 MBq), peptide control, or vehicle solution. The tumor volumes were measured, side effects were assessed in terms of body weight, routine hematology, and hepatic and renal functions, and the mouse radiation dosimetry was estimated. Cu-cyclam-RAFT-c(-RGDfK-)4 was produced with a radiochemical purity of 97.9±2.4% and a specific activity of 2.7±0.6 MBq/nmol and showed high in-vitro and in-vivo plasma stability. The administration of a single dose of Cu-cyclam-RAFT-c(-RGDfK-)4 resulted in significant tumor growth delay in comparison with that observed upon vehicle or peptide control administration, with an estimated tumor-absorbed dose of 0.712 Gy. No significant toxicity was observed in Cu-cyclam-RAFT-c(-RGDfK-)4-treated mice. Cu-cyclam-RAFT-c(-RGDfK-)4 would be a promising therapeutic agent for αVβ3 integrin-targeted internal radiotherapy.

Dissecting the role of matrix metalloproteinases (MMP) and integrin alpha(v)beta3 in angiogenesis in vitro: absence of hemopexin C domain bioactivity, but membrane-Type 1-MMP and alpha(v)beta3 are critical.

PubMed

Nisato, Riccardo E; Hosseini, Ghamartaj; Sirrenberg, Christian; Butler, Georgina S; Crabbe, Thomas; Docherty, Andrew J P; Wiesner, Matthias; Murphy, Gillian; Overall, Christopher M; Goodman, Simon L; Pepper, Michael S

2005-10-15

Matrix metalloproteinase (MMP)-2 and its hemopexin C domain autolytic fragment (also called PEX) have been proposed to be crucial for angiogenesis. Here, we have investigated the dependency of in vitro angiogenesis on MMP-mediated extracellular proteolysis and integrin alpha(v)beta3-mediated cell adhesion in a three-dimensional collagen I model. The hydroxamate-based synthetic inhibitors BB94, CT1399, and CT1847 inhibited endothelial cell invasion, as did neutralizing anti-membrane-type 1-MMP (MT1-MMP) antibodies and tissue inhibitor of MMP (TIMP)-2 and TIMP-3 but not TIMP-1. This confirmed the pivotal importance of MT1-MMP over other MMPs in this model. Invasion was also inhibited by a nonpeptidic antagonist of integrin alpha(v)beta3, EMD 361276. Although PEX strongly inhibited pro-MMP-2 activation, when contaminating lipopolysaccharide was neutralized, PEX neither affected angiogenesis nor bound integrin alpha(v)beta(3). Moreover, no specific binding of pro-MMP-2 to integrin alpha(v)beta3 was found, whereas only one out of four independently prepared enzymatically active MMP-2 preparations could bind integrin alpha(v)beta3 , and this in a PEX-independent manner. Likewise, integrin alpha(v)beta3 -expressing cells did not bind MMP-2-coated surfaces. Hence, these findings show that endothelial cell invasion of collagen I gels is MT1-MMP and alpha(v)beta3 - dependent but MMP-2 independent and does not support a role for PEX in alpha(v)beta3 integrin binding or in modulating angiogenesis in this system.

The effect of ligand affinity on integrins' lateral diffusion in cultured cells.

PubMed

Mainali, Dipak; Smith, Emily A

2013-04-01

The role of ligand affinity in altering αPS2CβPS integrins' lateral mobility was studied using single particle tracking (SPT) with ligand-functionalized quantum dots (QDs) and fluorescence recovery after photobleaching (FRAP) with fluorescent protein tagged integrins. Integrins are ubiquitous transmembrane proteins that are vital for numerous cellular functions, including bidirectional signaling and cell anchorage. Wild-type and high ligand affinity mutant (αPS2CβPS-V409D) integrins were studied in S2 cells. As measured by SPT, the integrin mobile fraction decreased by 22% and had a 4× slower diffusion coefficient for αPS2CβPS-V409D compared to wild-type integrins. These differences are partially the result of αPS2CβPS-V409D integrins' increased clustering. For the wild-type integrins, the average of all diffusion coefficients measured by SPT was statistically similar to the ensemble FRAP results. A 75% slower average diffusion coefficient was measured by SPT compared to FRAP for αPS2CβPS-V409D integrins, and this may be the result of SPT measuring only ligand-bound integrins, in contrast all ligand-bound and ligand-unbound integrins are averaged in FRAP measurements. Specific binding of the ligand-functionalized QDs was 99% for integrin expressing cells. The results prove that the ligand binding affinity affects the lateral dynamics of a subset of integrins based on the complementary SPT and FRAP data.

Phosphoinositide Signaling Regulates the Exocyst Complex and Polarized Integrin Trafficking in Directionally Migrating Cells

PubMed Central

Thapa, Narendra; Sun, Yue; Schramp, Mark; Choi, Suyoung; Ling, Kun; Anderson, Richard A

2011-01-01

Summary Polarized delivery of signaling and adhesion molecules to the leading edge is required for directional migration of cells. Here, we describe a role for the PIP2 synthesizing enzyme, PIPKIγi2, in regulation of exocyst complex control of cell polarity and polarized integrin trafficking during migration. Loss of PIPKIγi2 impaired directional migration, formation of cell polarity, and integrin trafficking to the leading edge. Upon initiation of directional migration PIPKIγi2 via PIP2 generation controls the integration of the exocyst complex into an integrin-containing trafficking compartment(s) that requires the talin-binding ability of PIPKIγi2, and talin for integrin recruitment to the leading edge. A PIP2 requirement is further emphasized by inhibition of PIPKIγi2-regulated directional migration by an Exo70 mutant deficient in PIP2 binding. These results reveal how phosphoinositide generation orchestrates polarized trafficking of integrin in coordination with talin that links integrins to the actin cytoskeleton, processes that are required for directional migration. PMID:22264730

Evidence that Distinct States of the Integrin α6β1 Interact with Laminin and an ADAM

PubMed Central

Chen, M.S.; Almeida, E.A.C.; Huovila, A.-P.J.; Takahashi, Y.; Shaw, L.M.; Mercurio, A.M.; White, J.M.

1999-01-01

Integrins can exist in different functional states with low or high binding capacity for particular ligands. We previously provided evidence that the integrin α6β1, on mouse eggs and on α6-transfected cells, interacted with the disintegrin domain of the sperm surface protein ADAM 2 (fertilin β). In the present study we tested the hypothesis that different states of α6β1 interact with fertilin and laminin, an extracellular matrix ligand for α6β1. Using α6-transfected cells we found that treatments (e.g., with phorbol myristate acetate or MnCl2) that increased adhesion to laminin inhibited sperm binding. Conversely, treatments that inhibited laminin adhesion increased sperm binding. Next, we compared the ability of fluorescent beads coated with either fertilin β or with the laminin E8 fragment to bind to eggs. In Ca2+-containing media, fertilin β beads bound to eggs via an interaction mediated by the disintegrin loop of fertilin β and by the α6 integrin subunit. In Ca2+-containing media, laminin E8 beads did not bind to eggs. Treatment of eggs with phorbol myristate acetate or with the actin disrupting agent, latrunculin A, inhibited fertilin bead binding, but did not induce laminin E8 bead binding. Treatment of eggs with Mn2+ dramatically increased laminin E8 bead binding, and inhibited fertilin bead binding. Our results provide the first evidence that different states of an integrin (α6β1) can interact with an extracellular matrix ligand (laminin) or a membrane-anchored cell surface ligand (ADAM 2). PMID:9971748

Coated platelets function in platelet-dependent fibrin formation via integrin αIIbβ3 and transglutaminase factor XIII

PubMed Central

Mattheij, Nadine J.A.; Swieringa, Frauke; Mastenbroek, Tom G.; Berny-Lang, Michelle A.; May, Frauke; Baaten, Constance C.F.M.J.; van der Meijden, Paola E.J.; Henskens, Yvonne M.C.; Beckers, Erik A.M.; Suylen, Dennis P.L.; Nolte, Marc W.; Hackeng, Tilman M.; McCarty, Owen J.T.; Heemskerk, Johan W.M.; Cosemans, Judith M.E.M.

2016-01-01

Coated platelets, formed by collagen and thrombin activation, have been characterized in different ways: i) by the formation of a protein coat of α-granular proteins; ii) by exposure of procoagulant phosphatidylserine; or iii) by high fibrinogen binding. Yet, their functional role has remained unclear. Here we used a novel transglutaminase probe, Rhod-A14, to identify a subpopulation of platelets with a cross-linked protein coat, and compared this with other platelet subpopulations using a panel of functional assays. Platelet stimulation with convulxin/thrombin resulted in initial integrin αIIbβ3 activation, the appearance of a platelet population with high fibrinogen binding, (independently of active integrins, but dependent on the presence of thrombin) followed by phosphatidylserine exposure and binding of coagulation factors Va and Xa. A subpopulation of phosphatidylserine-exposing platelets bound Rhod-A14 both in suspension and in thrombi generated on a collagen surface. In suspension, high fibrinogen and Rhod-A14 binding were antagonized by combined inhibition of transglutaminase activity and integrin αIIbβ3. Markedly, in thrombi from mice deficient in transglutaminase factor XIII, platelet-driven fibrin formation and Rhod-A14 binding were abolished by blockage of integrin αIIbβ3. Vice versa, star-like fibrin formation from platelets of a patient with deficiency in αIIbβ3 (Glanzmann thrombasthenia) was abolished upon blockage of transglutaminase activity. We conclude that coated platelets, with initial αIIbβ3 activation and high fibrinogen binding, form a subpopulation of phosphatidylserine-exposing platelets, and function in platelet-dependent star-like fibrin fiber formation via transglutaminase factor XIII and integrin αIIbβ3. PMID:26721892

Comparison of Saccharomyces cerevisiae F-BAR domain structures reveals a conserved inositol phosphate binding site.

PubMed

Moravcevic, Katarina; Alvarado, Diego; Schmitz, Karl R; Kenniston, Jon A; Mendrola, Jeannine M; Ferguson, Kathryn M; Lemmon, Mark A

2015-02-03

F-BAR domains control membrane interactions in endocytosis, cytokinesis, and cell signaling. Although they are generally thought to bind curved membranes containing negatively charged phospholipids, numerous functional studies argue that differences in lipid-binding selectivities of F-BAR domains are functionally important. Here, we compare membrane-binding properties of the Saccharomyces cerevisiae F-BAR domains in vitro and in vivo. Whereas some F-BAR domains (such as Bzz1p and Hof1p F-BARs) bind equally well to all phospholipids, the F-BAR domain from the RhoGAP Rgd1p preferentially binds phosphoinositides. We determined X-ray crystal structures of F-BAR domains from Hof1p and Rgd1p, the latter bound to an inositol phosphate. The structures explain phospholipid-binding selectivity differences and reveal an F-BAR phosphoinositide binding site that is fully conserved in a mammalian RhoGAP called Gmip and is partly retained in certain other F-BAR domains. Our findings reveal previously unappreciated determinants of F-BAR domain lipid-binding specificity and provide a basis for its prediction from sequence. Copyright © 2015 Elsevier Ltd. All rights reserved.

Comparison of Saccharomyces cerevisiae F-BAR Domain Structures Reveals a Conserved Inositol Phosphate Binding Site

DOE PAGES

Moravcevic, Katarina; Alvarado, Diego; Schmitz, Karl R.; ...

2015-01-22

F-BAR domains control membrane interactions in endocytosis, cytokinesis, and cell signaling. Although they are generally thought to bind curved membranes containing negatively charged phospholipids, numerous functional studies argue that differences in lipid-binding selectivities of F-BAR domains are functionally important. Here in this paper, we compare membrane-binding properties of the Saccharomyces cerevisiae F-BAR domains in vitro and in vivo. Whereas some F-BAR domains (such as Bzz1p and Hof1p F-BARs) bind equally well to all phospholipids, the F-BAR domain from the RhoGAP Rgd1p preferentially binds phosphoinositides. We determined X-ray crystal structures of F-BAR domains from Hof1p and Rgd1p, the latter bound tomore » an inositol phosphate. The structures explain phospholipid-binding selectivity differences and reveal an F-BAR phosphoinositide binding site that is fully conserved in a mammalian RhoGAP called Gmip and is partly retained in certain other F-BAR domains. In conclusion, our findings reveal previously unappreciated determinants of F-BAR domain lipid-binding specificity and provide a basis for its prediction from sequence.« less

Selective binding and lateral clustering of α5β1 and αvβ3 integrins: Unraveling the spatial requirements for cell spreading and focal adhesion assembly

PubMed Central

Schaufler, Viktoria; Czichos-Medda, Helmi; Hirschfeld-Warnecken, Vera; Neubauer, Stefanie; Rechenmacher, Florian; Medda, Rebecca; Kessler, Horst; Geiger, Benjamin; Spatz, Joachim P.; Cavalcanti-Adam, E. Ada

2016-01-01

ABSTRACT Coordination of the specific functions of α5β1 and αvβ3 integrins is crucial for the precise regulation of cell adhesion, spreading and migration, yet the contribution of differential integrin-specific crosstalk to these processes remains unclear. To determine the specific functions of αvβ3 and α5β1 integrins, we used nanoarrays of gold particles presenting immobilized, integrin-selective peptidomimetic ligands. Integrin binding to the peptidomimetics is highly selective, and cells can spread on both ligands. However, spreading is faster and the projected cell area is greater on α5β1 ligand; both depend on ligand spacing. Quantitative analysis of adhesion plaques shows that focal adhesion size is increased in cells adhering to αvβ3 ligand at 30 and 60 nm spacings. Analysis of αvβ3 and α5β1 integrin clusters indicates that fibrillar adhesions are more prominent in cells adhering to α5β1 ligand, while clusters are mostly localized at the cell margins in cells adhering to αvβ3 ligand. αvβ3 integrin clusters are more pronounced on αvβ3 ligand, though they can also be detected in cells adhering to α5β1 ligand. Furthermore, α5β1 integrin clusters are present in cells adhering to α5β1 ligand, and often colocalize with αvβ3 clusters. Taken together, these findings indicate that the activation of αvβ3 integrin by ligand binding is dispensable for initial adhesion and spreading, but essential to formation of stable focal adhesions. PMID:27003228

Transduction of beta3 integrin subunit cDNA confers on human keratinocytes the ability to adhere to gelatin.

PubMed

Kubo, Miyoko; Clark, Richard A F; Katz, Anne B; Taichman, Lorne B; Jin, Zaishun; Zhao, Ying; Moriguchi, Takahiko

2007-04-01

alphavbeta3 is a multiligand integrin receptor that interacts with fibrinogen (FG), fibrin (FB), fibronectin (FN), vitronectin (VN), and denatured collagen. We previously reported that cultured normal human keratinocytes, like in vivo keratinocytes, do not express alphavbeta3 on the cell surface, and do not adhere to and migrate on FG and FB. Furthermore, we reported that human keratinocytes transduced with beta3 integrin subunit cDNA by a retrovirus-mediated transduction method express alphavbeta3 on the cell surface and adhere to FG, FB, FN, and VN significantly compared with beta-galactosidase (beta-gal) cDNA-transduced keratinocytes (control). In this study, we determined whether these beta3 integrin subunit cDNA-transduced keratinocytes or normal human keratinocytes adhere to denatured collagen (gelatin) using a 1 h cell adhesion assay. beta3 cDNA-transduced keratinocytes adhered to gelatin, whereas no significant adhesion was observed with the control cells (beta-gal cDNA-transduced keratinocytes and normal human keratinocytes). The adhesion to gelatin was inhibited by LM609, a monoclonal antibody to alphavbeta3, and RGD peptides but not by normal mouse IgG1 nor RGE peptides. Thus, transduction of beta3 integrin subunit cDNA confers on human keratinocytes the ability to adhere to denatured collagen (gelatin) as well as to FG, FB, VN, and FN. Otherwise, normal human keratinocytes do not adhere to gelatin. These data support the idea that beta3 cDNA-transduced human keratinocytes can be a good material for cultured epithelium to achieve better take rate with acute or chronic wounds, in which FG, FB, and denatured collagen are abundantly present.

Sarcospan integration into laminin-binding adhesion complexes that ameliorate muscular dystrophy requires utrophin and α7 integrin

PubMed Central

Marshall, Jamie L.; Oh, Jennifer; Chou, Eric; Lee, Joy A.; Holmberg, Johan; Burkin, Dean J.; Crosbie-Watson, Rachelle H.

2015-01-01

Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene that result in loss of the dystrophin–glycoprotein complex, a laminin receptor that connects the myofiber to its surrounding extracellular matrix. Utrophin, a dystrophin ortholog that is normally localized to the neuromuscular junction, is naturally upregulated in DMD muscle, which partially compensates for the loss of dystrophin. Transgenic overexpression of utrophin causes broad sarcolemma localization of utrophin, restoration of laminin binding and amelioration of disease in the mdx mouse model of DMD. We previously demonstrated that overexpression of sarcospan, a dystrophin- and utrophin-binding protein, ameliorates mdx muscular dystrophy. Sarcospan boosts levels of utrophin to therapeutic levels at the sarcolemma, where attachment to laminin is restored. However, understanding the compensatory mechanism is complicated by concomitant upregulation of α7β1 integrin, which also binds laminin. Similar to the effects of utrophin, transgenic overexpression of α7 integrin prevents DMD disease in mice and is accompanied by increased abundance of utrophin around the extra-synaptic sarcolemma. In order to investigate the mechanisms underlying sarcospan ‘rescue’ of muscular dystrophy, we created double-knockout mice to test the contributions of utrophin or α7 integrin. We show that sarcospan-mediated amelioration of muscular dystrophy in DMD mice is dependent on the presence of both utrophin and α7β1 integrin, even when they are individually expressed at therapeutic levels. Furthermore, we found that association of sarcospan into laminin-binding complexes is dependent on utrophin and α7β1 integrin. PMID:25504048

Chondroitin Sulfate-E Binds to Both Osteoactivin and Integrin αVβ3 and Inhibits Osteoclast Differentiation.

PubMed

Miyazaki, Tatsuya; Miyauchi, Satoshi; Anada, Takahisa; Tawada, Akira; Suzuki, Osamu

2015-10-01

Integrins and their ligands have been suggested to be associated with osteoclast-mediated bone resorption. The present study was designed to investigate whether chondroitin sulfate E (CS-E), which is one of the sulfated glycosaminoglycans (GAGs), is involved in osteoactivin (OA) activity, and osteoclast differentiation. The binding affinity of sulfated GAGs to integrin and its ligand was measured using biotin-labeled CS-E, and the osteoclast differentiation was evaluated by tartrate-resistant acid phosphatase staining and a pit formation assay. CS-E as well as CS-B, synthetic chondroitin polysulfate, and heparin inhibited osteoclast differentiation of bone marrow-derived macrophages. Pre-coating of OA to synthetic calcium phosphate-coated plates enhanced the osteoclastic differentiation of RAW264 cells, and addition of a neutralizing antibody to OA inhibited its differentiation. CS-E bound not only to OA, fibronectin, and vitronectin, but also to its receptor integrin αVβ3, and inhibited the direct binding of OA to integrin αVβ3. Furthermore, CS-E blocked the binding of OA to cells and inhibited OA-induced osteoclastic differentiation. On the other hand, heparinase treatment of RAW264 cells inhibited osteoclastic differentiation. Since binding of OA to the cells was inhibited by the presence of heparan sulfate or heparinase treatment of cells, heparan sulfate proteoglycan (HSPG) was also considered to be an OA receptor. Taken together, the present results suggest that CS-E is capable of inhibiting OA-induced osteoclast differentiation by blocking the interaction of OA to integrin αVβ3 and HSPG. © 2015 Wiley Periodicals, Inc.

Equine herpesvirus 1 entry via endocytosis is facilitated by alphaV integrins and an RSD motif in glycoprotein D.

PubMed

Van de Walle, Gerlinde R; Peters, Sarah T; VanderVen, Brian C; O'Callaghan, Dennis J; Osterrieder, Nikolaus

2008-12-01

Equine herpesvirus 1 (EHV-1) is a member of the Alphaherpesvirinae, and its broad tissue tropism suggests that EHV-1 may use multiple receptors to initiate virus entry. EHV-1 entry was thought to occur exclusively through fusion at the plasma membrane, but recently entry via the endocytic/phagocytic pathway was reported for Chinese hamster ovary cells (CHO-K1 cells). Here we show that cellular integrins, and more specifically those recognizing RGD motifs such as alphaVbeta5, are important during the early steps of EHV-1 entry via endocytosis in CHO-K1 cells. Moreover, mutational analysis revealed that an RSD motif in the EHV-1 envelope glycoprotein D (gD) is critical for entry via endocytosis. In addition, we show that EHV-1 enters peripheral blood mononuclear cells predominantly via the endocytic pathway, whereas in equine endothelial cells entry occurs mainly via fusion at the plasma membrane. Taken together, the data in this study provide evidence that EHV-1 entry via endocytosis is triggered by the interaction between cellular integrins and the RSD motif present in gD and, moreover, that EHV-1 uses different cellular entry pathways to infect important target cell populations of its natural host.

Integrins are Mechanosensors That Modulate Human Eosinophil Activation

PubMed Central

Ahmadzai, Mustafa; Small, Mike; Sehmi, Roma; Gauvreau, Gail; Janssen, Luke J.

2015-01-01

Eosinophil migration to the lung is primarily regulated by the eosinophil-selective family of eotaxin chemokines, which mobilize intracellular calcium (Ca2+) and orchestrate myriad changes in cell structure and function. Eosinophil function is also known to be flow-dependent, although the molecular cognate of this mechanical response has yet to be adequately characterized. Using confocal fluorescence microscopy, we determined the effects of fluid shear stress on intracellular calcium concentration ([Ca2+]i) in human peripheral blood eosinophils by perfusing cells in a parallel-plate flow chamber. Our results indicate that fluid perfusion evokes a calcium response that leads to cell flattening, increase in cell area, shape change, and non-directional migration. None of these changes are seen in the absence of a flow stimulus, and all are blocked by chelation of intracellular Ca2+ using BAPTA. These changes are enhanced by stimulating the cells with eotaxin-1. The perfusion-induced calcium response (PICR) could be blocked by pre-treating cells with selective (CDP-323) and non-selective (RGD tripeptides) integrin receptor antagonists, suggesting that α4β7/α4β1 integrins mediate this response. Overall, our study provides the first pharmacological description of a molecular mechanosensor that may collaborate with the eotaxin-1 signaling program in order to control human eosinophil activation. PMID:26539194

Contribution of the 37-kDa laminin receptor precursor in the anti-metastatic PSP94-derived peptide PCK3145 cell surface binding

DOE Office of Scientific and Technical Information (OSTI.GOV)

Annabi, Borhane; Currie, Jean-Christophe; Bouzeghrane, Mounia

Purpose: PCK3145 is an anti-metastatic synthetic peptide with promising therapeutic efficacy against hormone-refractory prostate cancer. The characterization of the PCK3145 peptide cell surface binding/internalization mechanisms and of the receptors involved remained to be explored. Results: [{sup 14}C]PCK3145 cell surface binding assays showed rapid and transient kinetic profile, that was inhibited by RGD peptides, laminin, hyaluronan, and type-I collagen. RGD peptides were however unable to inhibit PCK3145 intracellular uptake. Far-Western ligand binding studies enabled the identification of the 37-kDa laminin receptor precursor (37LRP) as a potential ligand for PCK3145. Overexpression of the recombinant 37LRP indeed led to an increase in PCK3145more » binding but unexpectedly not to its uptake. Conclusions: Our data support the implication of laminin receptors in cell surface binding and in transducing PCK3145 anti-metastatic effects, and provide a rational for targeting cancers that express high levels of such laminin receptors.« less

Enhanced Cellular Adhesion on Titanium by Silk Functionalized with titanium binding and RGD peptides

PubMed Central

Vidal, Guillaume; Blanchi, Thomas; Mieszawska, Aneta J.; Calabrese, Rossella; Rossi, Claire; Vigneron, Pascale; Duval, Jean-Luc; Kaplan, David L.; Egles, Christophe

2012-01-01

Soft tissue adhesion on titanium represents a challenge for implantable materials. In order to improve adhesion at the cell/material interface we used a new approach based on the molecular recognition of titanium by specific peptides. Silk fibroin protein was chemically grafted with titanium binding peptide (TiBP) to increase adsorption of these chimeric proteins to the metal surface. Quartz Crystal Microbalance was used to quantify the specific adsorption of TiBP-functionalized silk and an increase in protein deposition by more than 35% was demonstrated due to the presence of the binding peptide. A silk protein grafted with TiBP and fibronectin-derived RGD peptide was then prepared. The adherence of fibroblasts on the titanium surface modified with the multifunctional silk coating demonstrated an increase in the number of adhering cells by 60%. The improved adhesion was demonstrated by Scanning Electron Microscopy and immunocytochemical staining of focal contact points. Chick embryo organotypic culture also revealed strong adhesion of endothelial cells expanding on the multifunctional silk-peptide coating. These results demonstrated that silk functionalized with TiBP and RGD represents a promising approach to modify cell-biomaterial interfaces, opening new perspectives for implantable medical devices, especially when reendothelialization is required. PMID:22975628

Correlative Light-Electron Microscopy Shows RGD-Targeted ZnO Nanoparticles Dissolve in the Intracellular Environment of Triple Negative Breast Cancer Cells and Cause Apoptosis with Intratumor Heterogeneity.

PubMed

Othman, Basmah A; Greenwood, Christina; Abuelela, Ayman F; Bharath, Anil A; Chen, Shu; Theodorou, Ioannis; Douglas, Trevor; Uchida, Maskai; Ryan, Mary; Merzaban, Jasmeen S; Porter, Alexandra E

2016-06-01

ZnO nanoparticles (NPs) are reported to show a high degree of cancer cell selectivity with potential use in cancer imaging and therapy. Questions remain about the mode by which the ZnO NPs cause cell death, whether they exert an intra- or extracellular effect, and the resistance among different cancer cell types to ZnO NP exposure. The present study quantifies the variability between the cellular toxicity, dynamics of cellular uptake, and dissolution of bare and RGD (Arg-Gly-Asp)-targeted ZnO NPs by MDA-MB-231 cells. Compared to bare ZnO NPs, RGD-targeting of the ZnO NPs to integrin αvβ3 receptors expressed on MDA-MB-231 cells appears to increase the toxicity of the ZnO NPs to breast cancer cells at lower doses. Confocal microscopy of live MDA-MB-231 cells confirms uptake of both classes of ZnO NPs with a commensurate rise in intracellular Zn(2+) concentration prior to cell death. The response of the cells within the population to intracellular Zn(2+) is highly heterogeneous. In addition, the results emphasize the utility of dynamic and quantitative imaging in understanding cell uptake and processing of targeted therapeutic ZnO NPs at the cellular level by heterogeneous cancer cell populations, which can be crucial for the development of optimized treatment strategies. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Bioconjugated PLGA-4-arm-PEG branched polymeric nanoparticles as novel tumor targeting carriers

NASA Astrophysics Data System (ADS)

Ding, Hong; Yong, Ken-Tye; Roy, Indrajit; Hu, Rui; Wu, Fang; Zhao, Lingling; Law, Wing-Cheung; Zhao, Weiwei; Ji, Wei; Liu, Liwei; Bergey, Earl J.; Prasad, Paras N.

2011-04-01

In this study, we have developed a novel carrier, micelle-type bioconjugated PLGA-4-arm-PEG branched polymeric nanoparticles (NPs), for the detection and treatment of pancreatic cancer. These NPs contained 4-arm-PEG as corona, and PLGA as core, the particle surface was conjugated with cyclo(arginine-glycine-aspartate) (cRGD) as ligand for in vivo tumor targeting. The hydrodynamic size of the NPs was determined to be 150-180 nm and the critical micellar concentration (CMC) was estimated to be 10.5 mg l - 1. Our in vitro study shows that these NPs by themselves had negligible cytotoxicity to human pancreatic cancer (Panc-1) and human glioblastoma (U87) cell lines. Near infrared (NIR) microscopy and flow cytometry demonstrated that the cRGD conjugated PLGA-4-arm-PEG polymeric NPs were taken up more efficiently by U87MG glioma cells, over-expressing the αvβ3 integrin, when compared with the non-targeted NPs. Whole body imaging showed that the cRGD conjugated PLGA-4-arm-PEG branched polymeric NPs had the highest accumulation in the pancreatic tumor site of mice at 48 h post-injection. Physical, hematological, and pathological assays indicated low in vivo toxicity of this NP formulation. These studies on the ability of these bioconjugated PLGA-4-arm-PEG polymeric NPs suggest that the prepared polymeric NPs may serve as a promising platform for detection and targeted drug delivery for pancreatic cancer.

Galectin-3 Induces Clustering of CD147 and Integrin-β1 Transmembrane Glycoprotein Receptors on the RPE Cell Surface

PubMed Central

Priglinger, Claudia S.; Szober, Christoph M.; Priglinger, Siegfried G.; Merl, Juliane; Euler, Kerstin N.; Kernt, Marcus; Gondi, Gabor; Behler, Jennifer; Geerlof, Arie; Kampik, Anselm; Ueffing, Marius; Hauck, Stefanie M.

2013-01-01

Proliferative vitreoretinopathy (PVR) is a blinding disease frequently occurring after retinal detachment surgery. Adhesion, migration and matrix remodeling of dedifferentiated retinal pigment epithelial (RPE) cells characterize the onset of the disease. Treatment options are still restrained and identification of factors responsible for the abnormal behavior of the RPE cells will facilitate the development of novel therapeutics. Galectin-3, a carbohydrate-binding protein, was previously found to inhibit attachment and spreading of retinal pigment epithelial cells, and thus bares the potential to counteract PVR-associated cellular events. However, the identities of the corresponding cell surface glycoprotein receptor proteins on RPE cells are not known. Here we characterize RPE-specific Gal-3 containing glycoprotein complexes using a proteomic approach. Integrin-β1, integrin-α3 and CD147/EMMPRIN, a transmembrane glycoprotein implicated in regulating matrix metalloproteinase induction, were identified as potential Gal-3 interactors on RPE cell surfaces. In reciprocal immunoprecipitation experiments we confirmed that Gal-3 associated with CD147 and integrin-β1, but not with integrin-α3. Additionally, association of Gal-3 with CD147 and integrin-β1 was observed in co-localization analyses, while integrin-α3 only partially co-localized with Gal-3. Blocking of CD147 and integrin-β1 on RPE cell surfaces inhibited binding of Gal-3, whereas blocking of integrin-α3 failed to do so, suggesting that integrin-α3 is rather an indirect interactor. Importantly, Gal-3 binding promoted pronounced clustering and co-localization of CD147 and integrin-β1, with only partial association of integrin-α3. Finally, we show that RPE derived CD147 and integrin-β1, but not integrin-α3, carry predominantly β-1,6-N-actyl-D-glucosamine-branched glycans, which are high-affinity ligands for Gal-3. We conclude from these data that extracellular Gal-3 triggers clustering of CD147 and integrin-β1 via interaction with β1,6-branched N-glycans on RPE cells and hypothesize that Gal-3 acts as a positive regulator for CD147/integrin-β1 clustering and therefore modifies RPE cell behavior contributing to the pathogenesis of PVR. Further investigations at this pathway may aid in the development of specific therapies for PVR. PMID:23922889

Effect of Physical Forces on the Metastatic Bone Microenvironment

DTIC Science & Technology

2014-12-01

phosphate dehydrogenase; HSP90, Heat shock protein 90; IBSP, Integrin binding sialoprotein ; bone sialoprotein ; IT, Intratibial; MEPE, Matrix...negative cell MLO-Y4 and DLM8, with lower expression in K12 and K7M2 cell lines. Bone sialoprotein (integrin binding sialoprotein ; Ibsp) is a

The lutheran/basal cell adhesion molecule promotes tumor cell migration by modulating integrin-mediated cell attachment to laminin-511 protein.

PubMed

Kikkawa, Yamato; Ogawa, Takaho; Sudo, Ryo; Yamada, Yuji; Katagiri, Fumihiko; Hozumi, Kentaro; Nomizu, Motoyoshi; Miner, Jeffrey H

2013-10-25

Cell-matrix interactions are critical for tumor cell migration. Lutheran (Lu), also known as basal cell adhesion molecule (B-CAM), competes with integrins for binding to laminin α5, a subunit of LM-511, a major component of basement membranes. Here we show that the preferential binding of Lu/B-CAM to laminin α5 promotes tumor cell migration. The attachment of Lu/B-CAM transfectants to LM-511 was slightly weaker than that of control cells, and this was because Lu/B-CAM disturbed integrin binding to laminin α5. Lu/B-CAM induced a spindle cell shape with pseudopods and promoted cell migration on LM-511. In addition, blocking with an anti-Lu/B-CAM antibody led to a flat cell shape and inhibited migration on LM-511, similar to the effects of an activating integrin β1 antibody. We conclude that tumor cell migration on LM-511 requires that Lu/B-CAM competitively modulates cell attachment through integrins. We suggest that this competitive interaction is involved in a balance between static and migratory cell behaviors.

Characterization of a conformationally sensitive murine monoclonal antibody directed to the metal ion-dependent adhesion site face of integrin CD11b.

PubMed

Li, Rui; Haruta, Ikuko; Rieu, Philippe; Sugimori, Takashi; Xiong, Jian-Ping; Arnaout, M Amin

2002-02-01

Integrin binding to physiologic ligands requires divalent cations and an inside-out-driven switch of the integrin to a high-affinity state. Divalent cations at the metal ion-dependent adhesion site (MIDAS) face of the alpha subunit-derived A domain provide a direct bridge between ligands and the integrin, and it has been proposed that activation dependency is caused by reorientation of the surrounding residues relative to the metal ion, forming an optimal binding interface. To gain more insight into the functional significance of the protein movements on the MIDAS face, we raised and characterized a murine mAb 107 directed against the MIDAS face of the A domain from integrin CD11b. We find that mAb 107 behaves as a ligand mimic. It binds in a divalent-cation-dependent manner to solvent-exposed residues on the MIDAS face of CD11b, blocks interaction of 11bA or the holoreceptor with ligands, and inhibits spreading and phagocytosis by human neutrophils. However, in contrast to physiologic ligands, mAb 107 preferentially binds to the inactive low-affinity form of the integrin, suggesting that its antagonistic effects are exerted in part by stabilizing the receptor in the low-affinity state. These data support a functional relevance of the protein movements on the MIDAS face and suggest that stabilizing the A domain in the low-affinity state may have therapeutic benefit.

Expression and putative function of fibronectin and its receptor (integrin alpha(5)beta(1)) in male and female gametes during bovine fertilization in vitro.

PubMed

Thys, Mirjan; Nauwynck, Hans; Maes, Dominiek; Hoogewijs, Maarten; Vercauteren, Dries; Rijsselaere, Tom; Favoreel, Herman; Van Soom, Ann

2009-09-01

Fibronectin (Fn) is a 440 kDa glycoprotein assumed to participate in sperm-egg interaction in human. Recently, it has been demonstrated that Fn--when present during bovine IVF--strongly inhibits sperm penetration. The present study was conducted firstly to evaluate the expression of Fn and its integrin receptor (alpha(5)beta(1)) on male and female bovine gametes using indirect immunofluorescence and secondly, to determine the function of Fn during bovine IVF. Endogenous Fn was detected underneath the zona pellucida (ZP) and integrin alpha(5) on the oolemma of cumulus-denuded oocytes. Bovine spermatozoa displayed integrin alpha(5) at their equatorial segment after acrosome reaction. We established that the main inhibitory effect of exogenously supplemented Fn was located at the sperm-oolemma binding, with a (concurrent) effect on fusion, and this can probably be attributed to the binding of Fn to spermatozoa at the equatorial segment, as shown by means of Alexa Fluor 488-conjugated Fn. Combining these results, the inhibitory effect of exogenously supplemented Fn seemed to be exerted on the male gamete by binding to the exposed integrin alpha(5)beta(1) receptor after acrosome reaction. The presence of endogenous Fn underneath the ZP together with integrin alpha(5) expression on oolemma and acrosome-reacted (AR) sperm cell surface suggests a 'velcro' interaction between the endogenous Fn ligand and corresponding receptors on both (AR) sperm cell and oolemma, initiating sperm-egg binding.

Dramatic and concerted conformational changes enable rhodocetin to block α2β1 integrin selectively

PubMed Central

Orriss, George L.; Niland, Stephan; Johanningmeier, Benjamin; Pohlentz, Gottfried; Meier, Markus; Karrasch, Simone; Estevão-Costa, Maria Inacia; Martins Lima, Augusto; Stetefeld, Jörg

2017-01-01

The collagen binding integrin α2β1 plays a crucial role in hemostasis, fibrosis, and cancer progression amongst others. It is specifically inhibited by rhodocetin (RC), a C-type lectin-related protein (CLRP) found in Malayan pit viper (Calloselasma rhodostoma) venom. The structure of RC alone reveals a heterotetramer arranged as an αβ and γδ subunit in a cruciform shape. RC specifically binds to the collagen binding A-domain of the integrin α2 subunit, thereby blocking collagen-induced platelet aggregation. However, until now, the molecular basis for this interaction has remained unclear. Here, we present the molecular structure of the RCγδ-α2A complex solved to 3.0 Å resolution. Our findings show that RC undergoes a dramatic structural reorganization upon binding to α2β1 integrin. Besides the release of the nonbinding RCαβ tandem, the RCγ subunit interacts with loop 2 of the α2A domain as result of a dramatic conformational change. The RCδ subunit contacts the integrin α2A domain in the “closed” conformation through its helix C. Combined with epitope-mapped antibodies, conformationally locked α2A domain mutants, point mutations within the α2A loop 2, and chemical modifications of the purified toxin protein, this molecular structure of RCγδ-α2A complex explains the inhibitory mechanism and specificity of RC for α2β1 integrin. PMID:28704364

A targeted IL-15 fusion protein with potent anti-tumor activity

PubMed Central

Chen, Siqi; Huang, Qiang; Liu, Jiayu; Xing, Jieyu; Zhang, Ning; Liu, Yawei; Wang, Zhong; Li, Qing

2015-01-01

IL-15 has been actively investigated for its potential in tumor immunotherapy. To enhance the anti-tumor activity of IL-15, the novel PFC-1 construct was designed, which comprises the following 3 parts: (1) IL-15Rα fused with IL-15 to enhance IL-15 activity, (2) an Fc fragment to increase protein half-life, and (3) an integrin-targeting RGD peptide to enhance tumor targeting. PFC-1 showed tumor cell targeting without compromising IL-15 activity. PFC-1 also had potent anti-tumor activities in xenograft models, suggesting the potential application of this multi-functional fusion protein in tumor therapy. PMID:26176990

Regulation of COX-2–mediated signaling by α3 type IV noncollagenous domain in tumor angiogenesis

PubMed Central

Boosani, Chandra Shekhar; Mannam, Arjuna P.; Cosgrove, Dominic; Silva, Rita; Hodivala-Dilke, Kairbaan M.; Keshamouni, Venkateshwar G.

2007-01-01

Human α3 chain, a noncollagenous domain of type IV collagen [α3(IV)NC1], inhibits angiogenesis and tumor growth. These biologic functions are partly attributed to the binding of α3(IV)NC1 to αVβ3 and α3β1 integrins. α3(IV)NC1 binds αVβ3 integrin, leading to translation inhibition by inhibiting focal adhesion kinase/phosphatidylinositol 3-kinase/Akt/mTOR/4E-BP1 pathways. In the present study, we evaluated the role of α3β1 and αVβ3 integrins in tube formation and regulation of cyclooxygenase-2 (COX-2) on α3(IV)NC1 stimulation. We found that although both integrins were required for the inhibition of tube formation by α3(IV)NC1 in endothelial cells, only α3β1 integrin was sufficient to regulate COX-2 in hypoxic endothelial cells. We show that binding of α3(IV)NC1 to α3β1 integrin leads to inhibition of COX-2–mediated pro-angiogenic factors, vascular endothelial growth factor, and basic fibroblast growth factor by regulating IκBα/NFκB axis, and is independent of αVβ3 integrin. Furthermore, β3 integrin–null endothelial cells, when treated with α3(IV)NC1, inhibited hypoxia-mediated COX-2 expression, whereas COX-2 inhibition was not observed in α3 integrin–null endothelial cells, indicating that regulation of COX-2 by α3(IV)NC1 is mediated by integrin α3β1. Our in vitro and in vivo findings demonstrate that α3β1 integrin is critical for α3(IV)NC1-mediated inhibition of COX-2–dependent angiogenic signaling and inhibition of tumor progression. PMID:17426256

Neisseria meningitidis Opc invasin binds to the sulphated tyrosines of activated vitronectin to attach to and invade human brain endothelial cells.

PubMed

Sa E Cunha, Claudia; Griffiths, Natalie J; Virji, Mumtaz

2010-05-20

The host vasculature is believed to constitute the principal route of dissemination of Neisseria meningitidis (Nm) throughout the body, resulting in septicaemia and meningitis in susceptible humans. In vitro, the Nm outer membrane protein Opc can enhance cellular entry and exit, utilising serum factors to anchor to endothelial integrins; but the mechanisms of binding to serum factors are poorly characterised. This study demonstrates that Nm Opc expressed in acapsulate as well as capsulate bacteria can increase human brain endothelial cell line (HBMEC) adhesion and entry by first binding to serum vitronectin and, to a lesser extent, fibronectin. This study also demonstrates that Opc binds preferentially to the activated form of human vitronectin, but not to native vitronectin unless the latter is treated to relax its closed conformation. The direct binding of vitronectin occurs at its Connecting Region (CR) requiring sulphated tyrosines Y(56) and Y(59). Accordingly, Opc/vitronectin interaction could be inhibited with a conformation-dependent monoclonal antibody 8E6 that targets the sulphotyrosines, and with synthetic sulphated (but not phosphorylated or unmodified) peptides spanning the vitronectin residues 43-68. Most importantly, the 26-mer sulphated peptide bearing the cell-binding domain (45)RGD(47) was sufficient for efficient meningococcal invasion of HBMECs. To our knowledge, this is the first study describing the binding of a bacterial adhesin to sulphated tyrosines of the host receptor. Our data also show that a single region of Opc is likely to interact with the sulphated regions of both vitronectin and of heparin. As such, in the absence of heparin, Opc-expressing Nm interact directly at the CR but when precoated with heparin, they bind via heparin to the heparin-binding domain of the activated vitronectin, although with a lower affinity than at the CR. Such redundancy suggests the importance of Opc/vitronectin interaction in meningococcal pathogenesis and may enable the bacterium to harness the benefits of the physiological processes in which the host effector molecule participates.

Cross-activating c-Met/β1 integrin complex drives metastasis and invasive resistance in cancer

PubMed Central

Jahangiri, Arman; Nguyen, Alan; Sidorov, Maxim K.; Yagnik, Garima; Rick, Jonathan; Han, Sung Won; Chen, William; Flanigan, Patrick M.; Schneidman-Duhovny, Dina; Mascharak, Smita; De Lay, Michael; Imber, Brandon; Park, Catherine C.; Matsumoto, Kunio; Lu, Kan; Bergers, Gabriele; Sali, Andrej; Weiss, William A.

2017-01-01

The molecular underpinnings of invasion, a hallmark of cancer, have been defined in terms of individual mediators but crucial interactions between these mediators remain undefined. In xenograft models and patient specimens, we identified a c-Met/β1 integrin complex that formed during significant invasive oncologic processes: breast cancer metastases and glioblastoma invasive resistance to antiangiogenic VEGF neutralizing antibody, bevacizumab. Inducing c-Met/β1 complex formation through an engineered inducible heterodimerization system promoted features crucial to overcoming stressors during metastases or antiangiogenic therapy: migration in the primary site, survival under hypoxia, and extravasation out of circulation. c-Met/β1 complex formation was up-regulated by hypoxia, while VEGF binding VEGFR2 sequestered c-Met and β1 integrin, preventing their binding. Complex formation promoted ligand-independent receptor activation, with integrin-linked kinase phosphorylating c-Met and crystallography revealing the c-Met/β1 complex to maintain the high-affinity β1 integrin conformation. Site-directed mutagenesis verified the necessity for c-Met/β1 binding of amino acids predicted by crystallography to mediate their extracellular interaction. Far-Western blotting and sequential immunoprecipitation revealed that c-Met displaced α5 integrin from β1 integrin, creating a complex with much greater affinity for fibronectin (FN) than α5β1. Thus, tumor cells adapt to microenvironmental stressors induced by metastases or bevacizumab by coopting receptors, which normally promote both cell migration modes: chemotaxis, movement toward concentrations of environmental chemoattractants, and haptotaxis, movement controlled by the relative strengths of peripheral adhesions. Tumor cells then redirect these receptors away from their conventional binding partners, forming a powerful structural c-Met/β1 complex whose ligand-independent cross-activation and robust affinity for FN drive invasive oncologic processes. PMID:28973887

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xiong, J.-P.; Stehle, T.; Zhang, R.

The structural basis for the divalent cation-dependent binding of heterodimeric alpha beta integrins to their ligands, which contain the prototypical Arg-Gly-Asp sequence, is unknown. Interaction with ligands triggers tertiary and quaternary structural rearrangements in integrins that are needed for cell signaling. Here we report the crystal structure of the extracellular segment of integrin alpha Vbeta 3 in complex with a cyclic peptide presenting the Arg-Gly-Asp sequence. The ligand binds at the major interface between the alpha V and beta 3 subunits and makes extensive contacts with both. Both tertiary and quaternary changes are observed in the presence of ligand. Themore » tertiary rearrangements take place in beta A, the ligand-binding domain of beta 3; in the complex, beta A acquires two cations, one of which contacts the ligand Asp directly and the other stabilizes the ligand-binding surface. Ligand binding induces small changes in the orientation of alpha V relative to beta 3.« less

β1 Integrin is an Adhesion Protein for Sperm Binding to Eggs

PubMed Central

Baessler, Keith A.; Lee, Younjoo; Sampson, Nicole S.

2009-01-01

We investigated the role of β1 integrin in mammalian fertilization and the mode of inhibition of fertilinβ-derived polymers. We determined that polymers displaying the Glu-Cys-Asp peptide from the fertilinβ disintegrin domain mediate inhibition of mammalian fertilization through a β1 integrin receptor on the egg surface. Inhibition of fertilization is a consequence of competition with sperm binding to the cell surface, not activation of an egg-signaling pathway. The presence of the β1 integrin on the egg surface increases the rate of sperm attachment, but does not alter the total number of sperm that can attach or fuse to the egg. We conclude that the presence of β1 integrin enhances the initial adhesion of sperm to the egg plasma membrane and that subsequent attachment and fusion are mediated by additional egg and sperm proteins present in the β1 integrin complex. Therefore, the mechanisms by which sperm fertilize wild-type and β1 knockout eggs are different. PMID:19338281

High-Throughput Screening based Identification of Small Molecule Antagonists of Integrin CD11b/CD18 Ligand Binding

PubMed Central

Faridi, Mohd Hafeez; Maiguel, Dony; Brown, Brock T.; Suyama, Eigo; Barth, Constantinos J.; Hedrick, Michael; Vasile, Stefan; Sergienko, Eduard; Schürer, Stephan; Gupta, Vineet

2010-01-01

Binding of leukocyte specific integrin CD11b/CD18 to its physiologic ligands is important for the development of normal immune response in vivo. Integrin CD11b/CD18 is also a key cellular effector of various inflammatory and autoimmune diseases. However, small molecules selectively inhibiting the function of integrin CD11b/CD18 are currently lacking. We used a newly described cell-based high throughput screening assay to identify a number of highly potent antagonists of integrin CD11b/CD18 from chemical libraries containing >100,000 unique compounds. Computational analyses suggest that the identified compounds cluster into several different chemical classes. A number of the newly identified compounds blocked adhesion of wild-type mouse neutrophils to CD11b/CD18 ligand fibrinogen. Mapping the most active compounds against chemical fingerprints of known antagonists of related integrin CD11a/CD18 shows little structural similarity, suggesting that the newly identified compounds are novel and unique. PMID:20188705

Integrin Clustering Matters: A Review of Biomaterials Functionalized with Multivalent Integrin-Binding Ligands to Improve Cell Adhesion, Migration, Differentiation, Angiogenesis, and Biomedical Device Integration.

PubMed

Karimi, Fatemeh; O'Connor, Andrea J; Qiao, Greg G; Heath, Daniel E

2018-03-25

Material systems that exhibit tailored interactions with cells are a cornerstone of biomaterial and tissue engineering technologies. One method of achieving these tailored interactions is to biofunctionalize materials with peptide ligands that bind integrin receptors present on the cell surface. However, cell biology research has illustrated that both integrin binding and integrin clustering are required to achieve a full adhesion response. This biophysical knowledge has motivated researchers to develop material systems biofunctionalized with nanoscale clusters of ligands that promote both integrin occupancy and clustering of the receptors. These materials have improved a wide variety of biological interactions in vitro including cell adhesion, proliferation, migration speed, gene expression, and stem cell differentiation; and improved in vivo outcomes including increased angiogenesis, tissue healing, and biomedical device integration. This review first introduces the techniques that enable the fabrication of these nanopatterned materials, describes the improved biological effects that have been achieved, and lastly discusses the current limitations of the technology and where future advances may occur. Although this technology is still in its nascency, it will undoubtedly play an important role in the future development of biomaterials and tissue engineering scaffolds for both in vitro and in vivo applications. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

WAVE2 Regulates High-Affinity Integrin Binding by Recruiting Vinculin and Talin to the Immunological Synapse▿

PubMed Central

Nolz, Jeffrey C.; Medeiros, Ricardo B.; Mitchell, Jason S.; Zhu, Peimin; Freedman, Bruce D.; Shimizu, Yoji; Billadeau, Daniel D.

2007-01-01

T-cell-receptor (TCR)-mediated integrin activation is required for T-cell-antigen-presenting cell conjugation and adhesion to extracellular matrix components. While it has been demonstrated that the actin cytoskeleton and its regulators play an essential role in this process, no mechanism has been established which directly links TCR-induced actin polymerization to the activation of integrins. Here, we demonstrate that TCR stimulation results in WAVE2-ARP2/3-dependent F-actin nucleation and the formation of a complex containing WAVE2, ARP2/3, vinculin, and talin. The verprolin-connecting-acidic (VCA) domain of WAVE2 mediates the formation of the ARP2/3-vinculin-talin signaling complex and talin recruitment to the immunological synapse (IS). Interestingly, although vinculin is not required for F-actin or integrin accumulation at the IS, it is required for the recruitment of talin. In addition, RNA interference of either WAVE2 or vinculin inhibits activation-dependent induction of high-affinity integrin binding to VCAM-1. Overall, these findings demonstrate a mechanism in which signals from the TCR produce WAVE2-ARP2/3-mediated de novo actin polymerization, leading to integrin clustering and high-affinity binding through the recruitment of vinculin and talin. PMID:17591693

WAVE2 regulates high-affinity integrin binding by recruiting vinculin and talin to the immunological synapse.

PubMed

Nolz, Jeffrey C; Medeiros, Ricardo B; Mitchell, Jason S; Zhu, Peimin; Freedman, Bruce D; Shimizu, Yoji; Billadeau, Daniel D

2007-09-01

T-cell-receptor (TCR)-mediated integrin activation is required for T-cell-antigen-presenting cell conjugation and adhesion to extracellular matrix components. While it has been demonstrated that the actin cytoskeleton and its regulators play an essential role in this process, no mechanism has been established which directly links TCR-induced actin polymerization to the activation of integrins. Here, we demonstrate that TCR stimulation results in WAVE2-ARP2/3-dependent F-actin nucleation and the formation of a complex containing WAVE2, ARP2/3, vinculin, and talin. The verprolin-connecting-acidic (VCA) domain of WAVE2 mediates the formation of the ARP2/3-vinculin-talin signaling complex and talin recruitment to the immunological synapse (IS). Interestingly, although vinculin is not required for F-actin or integrin accumulation at the IS, it is required for the recruitment of talin. In addition, RNA interference of either WAVE2 or vinculin inhibits activation-dependent induction of high-affinity integrin binding to VCAM-1. Overall, these findings demonstrate a mechanism in which signals from the TCR produce WAVE2-ARP2/3-mediated de novo actin polymerization, leading to integrin clustering and high-affinity binding through the recruitment of vinculin and talin.

Preparation and preclinical evaluation of (66)Ga-DOTA-E(c(RGDfK))2 as a potential theranostic radiopharmaceutical.

PubMed

Lopez-Rodriguez, V; Gaspar-Carcamo, R E; Pedraza-Lopez, M; Rojas-Calderon, E L; Arteaga de Murphy, C; Ferro-Flores, G; Avila-Rodriguez, M A

2015-02-01

Integrin αvβ3 plays an important role in angiogenesis and is over-expressed in tumoral endothelial cells and some other tumor cells. RGD (Arg-Gly-Asn) peptides labeled with (68)Ga (t1/2=68min) have showed good characteristics for imaging of αvβ3 expression using positron emission tomography (PET). Gallium-66 has been proposed as a PET imaging alternative to (68)Ga and given the unique high energy of its emitted positrons (Emax 4.15MeV) it may also be useful for therapy. The aim of this research is to prepare [(66)Ga]DOTA-E-[c(RGDfK)]2 and evaluate in mice its potential as a new theranostic radiopharmaceutical. High specific activity (66)Ga was produced via the (66)Zn(p,n) reaction, and the labelling method of DOTA-E-[c(RGDfK)]2 with (66)Ga was optimized. Radiochemical purity was determined by TLC, and in vitro stability and protein binding were determined. Serial microPET imaging and biodistribution studies were carried out in nude mice bearing C6 xenografts. Radiation absorbed dose estimates were based on the biodistribution studies, where tumor and organs of interest were collected at 0.5, 1, 3, 5 and 24h post-injection of [(66)Ga]DOTA-E-[c(RGDfK)]2. Our results have shown that [(66)Ga]DOTA-E-[c(RGDfK)]2 can be prepared with high radiochemical purity (>97%), specific activity (36-67GBq/μmol), in vitro stability, and moderate protein binding. MicroPET imaging up to 24 post-injection showed contrasting tumors reflecting αvβ3-targeted tracer accumulation. Biodistribution studies and dosimetry estimations showed a stable tumor uptake, rapid blood clearance, and favorable tumor-to-tissue ratios. The peptide conjugated DOTA-E-[c(RGDfK)]2 labeled with (66)Ga may be attractive as a theranostic agent for tumors over-expressing αvβ3 integrins. Copyright © 2014 Elsevier Inc. All rights reserved.

Binding of integrin α1 to bone morphogenetic protein receptor IA suggests a novel role of integrin α1β1 in bone morphogenetic protein 2 signalling.

PubMed

Zu, Yan; Liang, Xudong; Du, Jing; Zhou, Shuai; Yang, Chun

2015-11-05

Here, we observed that integrin α1β1 and bone morphogenetic protein receptor (BMPR) IA formed a complex and co-localised in several cell types. However, the molecular interaction between these two molecules was not studied in detail to date and the role of the interaction in BMPR signalling remains unknown; thus, these were investigated here. In a steered molecular dynamics (SMD) simulation, the observed development of the rupture force related to the displacement between the A-domain of integrin α1 and the extracellular domain of BMPR IA indicated a strong molecular interaction within the integrin-BMPR complex. Analysis of the intermolecular forces revealed that hydrogen bonds, rather than salt bridges, are the major contributors to these intermolecular interactions. By using Enzyme-linked immunosorbent assay (ELISA) and co-immunoprecipitation (co-IP) experiments with site-directed mutants, we found that residues 85-89 in BMPR IA play the most important role for BMPR IA binding to integrin α1β1. These residues are the same as those responsible for bone morphogenetic protein 2 (BMP-2)/BMPR IA binding. In our experiments, we also found that the interference of integrin α1β1 up regulated the level of phosphorylated Smad1, 5, 8, which is the downstream of BMP/BMPR signalling. Therefore, our results suggest that integrin α1β1/BMPR IA may block BMP-2/BMPR IA complex information and interfere with the BMP-2 signalling pathway in cells. Copyright © 2015 Elsevier Ltd. All rights reserved.

Relating conformation to function in integrin α5β1.

PubMed

Su, Yang; Xia, Wei; Li, Jing; Walz, Thomas; Humphries, Martin J; Vestweber, Dietmar; Cabañas, Carlos; Lu, Chafen; Springer, Timothy A

2016-07-05

Whether β1 integrin ectodomains visit conformational states similarly to β2 and β3 integrins has not been characterized. Furthermore, despite a wealth of activating and inhibitory antibodies to β1 integrins, the conformational states that these antibodies stabilize, and the relation of these conformations to function, remain incompletely characterized. Using negative-stain electron microscopy, we show that the integrin α5β1 ectodomain adopts extended-closed and extended-open conformations as well as a bent conformation. Antibodies SNAKA51, 8E3, N29, and 9EG7 bind to different domains in the α5 or β1 legs, activate, and stabilize extended ectodomain conformations. Antibodies 12G10 and HUTS-4 bind to the β1 βI domain and hybrid domains, respectively, activate, and stabilize the open headpiece conformation. Antibody TS2/16 binds a similar epitope as 12G10, activates, and appears to stabilize an open βI domain conformation without requiring extension or hybrid domain swing-out. mAb13 and SG/19 bind to the βI domain and βI-hybrid domain interface, respectively, inhibit, and stabilize the closed conformation of the headpiece. The effects of the antibodies on cell adhesion to fibronectin substrates suggest that the extended-open conformation of α5β1 is adhesive and that the extended-closed and bent-closed conformations are nonadhesive. The functional effects and binding sites of antibodies and fibronectin were consistent with their ability in binding to α5β1 on cell surfaces to cross-enhance or inhibit one another by competitive or noncompetitive (allosteric) mechanisms.

A fibronectin receptor on Candida albicans mediates adherence of the fungus to extracellular matrix

DOE Office of Scientific and Technical Information (OSTI.GOV)

Klotz, S.A.; Smith, R.L.

1991-03-01

Binding of fibronectin, an extracellular matrix (ECM) protein, to Candida albicans was measured, and adherence of the fungus to immobilized ECM proteins, fibronectin, laminin, types I and IV collagen, and subendothelial ECM was studied. 125I-labeled fibronectin was inhibited from binding to the fungus by unlabeled human plasma fibronectin and by Arg-Gly-Asp (RGD), Gly-Arg-Gly-Glu-Ser-Pro (GRGESP), and Gly-Arg-Gly-Asp-Thr-Pro (GRGDTP), but binding was not inhibited by Gly-Arg-Gly-Asp-Ser-Pro. Soluble fibronectin, RGD, GRGESP, and GRGDTP also inhibited fungal adherence to the individual immobilized ECM proteins in a complex pattern, but only soluble fibronectin (10(-7) M) inhibited fungal adherence to subendothelial ECM. Thus, C. albicans possessesmore » at least one type of cell surface receptor for binding soluble fibronectin that can be inhibited with peptides. This receptor apparently is used to bind the fungus to immobilized ECM proteins and to subendothelial ECM and may play a role in the initiation of disseminated disease by bloodborne fungi by providing for adherence of the microorganisms to ECM proteins.« less

Role of VLA-4 and VLA-5 in ex vivo maintenance of human and pig hematopoiesis in human stroma-supported long-term cultures.

PubMed

Giovino, Maria A; Wang, Hui; Sykes, Megan; Yang, Yong-Guang

2005-03-01

The advantage of recipient hematopoiesis over that of xenogeneic donors poses a fundamental obstacle to the induction of xenograft tolerance through mixed hematopoietic chimerism. Here we explore the role of beta1 integrins in maintenance of human vs porcine hematopoiesis within a human hematopoietic environment. Porcine and human c-kit+ bone marrow cells were purified and cultured on human bone marrow stroma for 6 weeks. The role of VLA-4 and VLA-5 in the maintenance of porcine vs human hematopoiesis in this human stroma-supported long-term bone marrow culture (LTBMC) system was evaluated by using blocking mAbs that bind to both species. Blocking VLA-4 with HP2/1 inhibited both human and porcine hematopoiesis, whereas anti-VLA-5 (SAM-1) suppressed the function of human, but not porcine, hematopoietic cells. In mixed LTBMC of porcine and human cells on a human stroma, porcine hematopoietic cells were at a competitive disadvantage, as seen by a rapid decline in cellularity, including clonogenic progenitors. This disadvantage was substantially overcome by the addition of SAM-1. Furthermore, human, but not porcine, cell adhesion to human fibronectin was inhibited by arginine-glycine-aspartic acid (RGD) peptides. Taken together, these results indicate that VLA-4 plays critical role for porcine hematopoiesis in a human hematopoietic environment, and raise the possibility that porcine VLA-5 might be unable to bind the respective human ligand and/or to initiate adequate post-ligand-binding signaling. Thus, VLA-5 may provide a potential target for developing approaches to improve porcine hematopoiesis in human recipients.

Identification of a novel cell culture adaptation site on the capsid of foot-and-mouth disease virus.

PubMed

Chamberlain, Kyle; Fowler, Veronica L; Barnett, Paul V; Gold, Sarah; Wadsworth, Jemma; Knowles, Nick J; Jackson, Terry

2015-09-01

Vaccination remains the most effective tool for control of foot-and-mouth disease both in endemic countries and as an emergency preparedness for new outbreaks. Foot-and-mouth disease vaccines are chemically inactivated virus preparations and the production of new vaccines is critically dependent upon cell culture adaptation of field viruses, which can prove problematic. A major driver of cell culture adaptation is receptor availability. Field isolates of foot-and-mouth disease virus (FMDV) use RGD-dependent integrins as receptors, whereas cell culture adaptation often selects for variants with altered receptor preferences. Previously, two independent sites on the capsid have been identified where mutations are associated with improved cell culture growth. One is a shallow depression formed by the three major structural proteins (VP1-VP3) where mutations create a heparan sulphate (HS)-binding site (the canonical HS-binding site). The other involves residues of VP1 and is located at the fivefold symmetry axis. For some viruses, changes at this site result in HS binding; for others, the receptors are unknown. Here, we report the identification of a novel site on VP2 where mutations resulted in an expanded cell tropism of a vaccine variant of A/IRN/87 (called A - ). Furthermore, we show that introducing the same mutations into a different type A field virus (A/TUR/2/2006) resulted in the same expanded cell culture tropism as the A/IRN/87 A -  vaccine variant. These observations add to the evidence for multiple cell attachment mechanisms for FMDV and may be useful for vaccine manufacture when cell culture adaptation proves difficult.

Identification of a novel cell culture adaptation site on the capsid of foot-and-mouth disease virus

PubMed Central

Chamberlain, Kyle; Fowler, Veronica L.; Barnett, Paul V.; Gold, Sarah; Wadsworth, Jemma; Knowles, Nick J.

2015-01-01

Vaccination remains the most effective tool for control of foot-and-mouth disease both in endemic countries and as an emergency preparedness for new outbreaks. Foot-and-mouth disease vaccines are chemically inactivated virus preparations and the production of new vaccines is critically dependent upon cell culture adaptation of field viruses, which can prove problematic. A major driver of cell culture adaptation is receptor availability. Field isolates of foot-and-mouth disease virus (FMDV) use RGD-dependent integrins as receptors, whereas cell culture adaptation often selects for variants with altered receptor preferences. Previously, two independent sites on the capsid have been identified where mutations are associated with improved cell culture growth. One is a shallow depression formed by the three major structural proteins (VP1–VP3) where mutations create a heparan sulphate (HS)-binding site (the canonical HS-binding site). The other involves residues of VP1 and is located at the fivefold symmetry axis. For some viruses, changes at this site result in HS binding; for others, the receptors are unknown. Here, we report the identification of a novel site on VP2 where mutations resulted in an expanded cell tropism of a vaccine variant of A/IRN/87 (called A − ). Furthermore, we show that introducing the same mutations into a different type A field virus (A/TUR/2/2006) resulted in the same expanded cell culture tropism as the A/IRN/87 A −  vaccine variant. These observations add to the evidence for multiple cell attachment mechanisms for FMDV and may be useful for vaccine manufacture when cell culture adaptation proves difficult. PMID:26296881

The presence of anti-Tat antibodies in HIV-infected individuals is associated with containment of CD4+ T-cell decay and viral load, and with delay of disease progression: results of a 3-year cohort study

PubMed Central

2014-01-01

Background Tat is a key HIV-1 virulence factor, which plays pivotal roles in virus gene expression, replication, transmission and disease progression. After release, extracellular Tat accumulates in tissues and exerts effects on both the virus and the immune system, promoting immune activation and virus spreading while disabling the host immune defense. In particular, Tat binds Env spikes on virus particles forming a virus entry complex, which favors infection of dendritic cells and efficient transmission to T cells via RGD-binding integrins. Tat also shields the CCR5-binding sites of Env rendering ineffective virus neutralization by anti-Env antibodies (Abs). This is reversed by the anti-Tat Abs present in natural infection or induced by vaccination. Findings Here we present the results of a cohort study, showing that the presence of anti-Tat Abs in asymptomatic and treatment-naïve HIV-infected subjects is associated with containment of CD4+ T-cell loss and viral load and with a delay of disease progression. In fact, no subjects with high anti-Tat Ab titers initiated antiretroviral therapy during the three years of follow-up. In contrast, no significant effects were seen for anti-Env and anti-Gag Abs. The increase of anti-Env Ab titers was associated with a reduced risk of starting therapy only in the presence of anti-Tat Abs, suggesting an effect of combined anti-Tat and anti-Env Abs on the Tat/Env virus entry complex and on virus neutralization. Conclusions Anti-Tat immunity may help delay HIV disease progression, thus, targeting Tat may offer a novel therapeutic intervention to postpone antiretroviral treatment or to increase its efficacy. PMID:24961156

Direct integrin alphavbeta6-ERK binding: implications for tumour growth.

PubMed

Ahmed, Nuzhat; Niu, Jun; Dorahy, Douglas J; Gu, Xinhua; Andrews, Sarah; Meldrum, Cliff J; Scott, Rodney J; Baker, Mark S; Macreadie, Ian G; Agrez, Michael V

2002-02-21

Blockade of the mitogen-activated protein (MAP) kinase pathway suppresses growth of colon cancer in vivo. Here we demonstrate a direct link between the extracellular signal-regulated kinase ERK2 and the growth-promoting cell adhesion molecule, integrin alphavbeta6, in colon cancer cells. Down-regulation of beta6 integrin subunit expression inhibits tumour growth in vivo and MAP kinase activity in response to serum stimulation. In alphavbeta6-expressing cells ERK2 is bound only to the beta6 subunit. The increase in cytosolic MAP kinase activity upon epidermal growth factor stimulation is all accounted for by beta6-bound ERK. Deletion of the ERK2 binding site on the beta6 cytoplasmic domain inhibits tumour growth and leads to an association between ERK and the beta5 subunit. The physical interaction between integrin alphavbeta6 and ERK2 defines a novel paradigm of integrin-mediated signalling and provides a therapeutic target for cancer treatment.

The role of β2 integrin associated heparin-binding protein release in ARDS.

PubMed

Liu, Yang; Ma, Shaolin; Wang, Xuebin; Feng, Yajing; Zhang, Shouqin; Wang, Sheng; Zhang, Xiangyu

2018-06-15

PMNs (polymorphonuclear neutrophil) play important roles in early stage of inflammation induced ARDS (Acute Respiratory Distress Syndrome). Both HBP (Heparin-Binding Protein) released from active PMNs and β2 integrins on the surface of PMNs are involved in vascular leakage. The role and relationship of HBP and β2 integrins on ARDS still requires study. We established ARDS model using C57BL/6 mice with cecal ligation and puncture and eliminating HBP and β2 integrin with respective antibodies. The mice were also challenged with HBP endotracheal instillation. Histopathology score, lung wet/dry ratio, bronchoalveolar lavage fluid protein, plasma HBP and β2 integrin on PMNs from all groups were measured. β2 integrin and HBP were analyzed after incubated PMNs with streptococcal and pretreat with anti-CD18, anti-HBP, 1-phosphatidylinositol 3-kinase (PI3K) inhibitor and p38 mitogen-activated protein kinase (MAPK) inhibitor. All lung injury indicatrix accompanied with HBP and β2 integrin elevated in CLP group, and HBP and β2 integrin were in correlation with each other and both were in correlation with the severity of lung injury. Endotracheal instillation HBP induced lung injury in CLP mice. Inhibiting both HBP and integrin ameliorated lung injury. HBP release was suppressed by inhibiting integrin and PI3K pathway, while integrin level did not decrease after eliminating HBP. Both HBP and β2 integrin play important roles in ARDS. HBP released from PMNs is β2 integrin-PI3K signaling pathway dependent process revealing potential novel therapeutic targets for ARDS treatment. Copyright © 2018 Elsevier Inc. All rights reserved.

The evolution of filamin – A protein domain repeat perspective

PubMed Central

Light, Sara; Sagit, Rauan; Ithychanda, Sujay S.; Qin, Jun; Elofsson, Arne

2013-01-01

Particularly in higher eukaryotes, some protein domains are found in tandem repeats, performing broad functions often related to cellular organization. For instance, the eukaryotic protein filamin interacts with many proteins and is crucial for the cytoskeleton. The functional properties of long repeat domains are governed by the specific properties of each individual domain as well as by the repeat copy number. To provide better understanding of the evolutionary and functional history of repeating domains, we investigated the mode of evolution of the filamin domain in some detail. Among the domains that are common in long repeat proteins, sushi and spectrin domains evolve primarily through cassette tandem duplications while scavenger and immunoglobulin repeats appear to evolve through clustered tandem duplications. Additionally, immunoglobulin and filamin repeats exhibit a unique pattern where every other domain shows high sequence similarity. This pattern may be the result of tandem duplications, serve to avert aggregation between adjacent domains or it is the result of functional constraints. In filamin, our studies confirm the presence of interspersed integrin binding domains in vertebrates, while invertebrates exhibit more varied patterns, including more clustered integrin binding domains. The most notable case is leech filamin, which contains a 20 repeat expansion and exhibits unique dimerization topology. Clearly, invertebrate filamins are varied and contain examples of similar adjacent integrin-binding domains. Given that invertebrate integrin shows more similarity to the weaker filamin binder, integrin β3, it is possible that the distance between integrin-binding domains is not as crucial for invertebrate filamins as for vertebrates. PMID:22414427

The evolution of filamin-a protein domain repeat perspective.

PubMed

Light, Sara; Sagit, Rauan; Ithychanda, Sujay S; Qin, Jun; Elofsson, Arne

2012-09-01

Particularly in higher eukaryotes, some protein domains are found in tandem repeats, performing broad functions often related to cellular organization. For instance, the eukaryotic protein filamin interacts with many proteins and is crucial for the cytoskeleton. The functional properties of long repeat domains are governed by the specific properties of each individual domain as well as by the repeat copy number. To provide better understanding of the evolutionary and functional history of repeating domains, we investigated the mode of evolution of the filamin domain in some detail. Among the domains that are common in long repeat proteins, sushi and spectrin domains evolve primarily through cassette tandem duplications while scavenger and immunoglobulin repeats appear to evolve through clustered tandem duplications. Additionally, immunoglobulin and filamin repeats exhibit a unique pattern where every other domain shows high sequence similarity. This pattern may be the result of tandem duplications, serve to avert aggregation between adjacent domains or it is the result of functional constraints. In filamin, our studies confirm the presence of interspersed integrin binding domains in vertebrates, while invertebrates exhibit more varied patterns, including more clustered integrin binding domains. The most notable case is leech filamin, which contains a 20 repeat expansion and exhibits unique dimerization topology. Clearly, invertebrate filamins are varied and contain examples of similar adjacent integrin-binding domains. Given that invertebrate integrin shows more similarity to the weaker filamin binder, integrin β3, it is possible that the distance between integrin-binding domains is not as crucial for invertebrate filamins as for vertebrates. Copyright © 2012 Elsevier Inc. All rights reserved.

Lipid raft regulates the initial spreading of melanoma A375 cells by modulating β1 integrin clustering.

PubMed

Wang, Ruifei; Bi, Jiajia; Ampah, Khamal Kwesi; Zhang, Chunmei; Li, Ziyi; Jiao, Yang; Wang, Xiaoru; Ba, Xueqing; Zeng, Xianlu

2013-08-01

Cell adhesion and spreading require integrins-mediated cell-extracellular matrix interaction. Integrins function through binding to extracellular matrix and subsequent clustering to initiate focal adhesion formation and actin cytoskeleton rearrangement. Lipid raft, a liquid ordered plasma membrane microdomain, has been reported to play major roles in membrane motility by regulating cell surface receptor function. Here, we identified that lipid raft integrity was required for β1 integrin-mediated initial spreading of melanoma A375 cells on fibronectin. We found that lipid raft disruption with methyl-β-cyclodextrin led to the inability of focal adhesion formation and actin cytoskeleton rearrangement by preventing β1 integrin clustering. Furthermore, we explored the possible mechanism by which lipid raft regulates β1 integrin clustering and demonstrated that intact lipid raft could recruit and modify some adaptor proteins, such as talin, α-actinin, vinculin, paxillin and FAK. Lipid raft could regulate the location of these proteins in lipid raft fractions and facilitate their binding to β1 integrin, which may be crucial for β1 integrin clustering. We also showed that lipid raft disruption impaired A375 cell migration in both transwell and wound healing models. Together, these findings provide a new insight for the relationship between lipid raft and the regulation of integrins. Copyright © 2013 Elsevier Ltd. All rights reserved.

Computer-aided design of peptide near infrared fluorescent probe for tumor diagnosis

NASA Astrophysics Data System (ADS)

Zhang, Congying; Gu, Yueqing

2014-09-01

Integrin αvβ3 receptors are expressed on activated endothelial cells during neovascularization to maintain tumor growth, so they become hot research tagets in cancer diagnosis. Peptides possess several attractive features when compared to protein and small molecule, such as small size and high structural compatibility with target proteins. Efficient design of high-affinity peptide ligands to Integrin αvβ3 receptors has been an important problem. Designed peptides in silico provide a valuable and high-selectivity peptide, meanwhile decrease the time of drug screening. In this study, we design peptide which can bind with integrin αvβ3 via computer, and then synthesis near infrared fluorescent probe. The characterization of this near infrared fluorescent probe was detected by UV. To investigate the tumor cell targeting of this probe, it was labeled with visible fluorescent dye Rhodamine B (RhB) for microscopy. To evaluate the targeting capability of this near infrared fluorescent probe, mice bearing integrin αvβ3 positive tumor xenografts were used. In vitro cellular experiments indicated that this probe have a clear binding affinity to αvβ3-positive tumor cells. In vivo experiments confirmed the receptor binding specificity of this probe. The peptide of computational design can bind with integrin αvβ3. Combined peptide near-infrared fluorescent probe with imaging technology use for clinical and tumor diagnosis have a greater development in future.

Compact solid-state CMOS single-photon detector array for in vivo NIR fluorescence lifetime oncology measurements.

PubMed

Homulle, H A R; Powolny, F; Stegehuis, P L; Dijkstra, J; Li, D-U; Homicsko, K; Rimoldi, D; Muehlethaler, K; Prior, J O; Sinisi, R; Dubikovskaya, E; Charbon, E; Bruschini, C

2016-05-01

In near infrared fluorescence-guided surgical oncology, it is challenging to distinguish healthy from cancerous tissue. One promising research avenue consists in the analysis of the exogenous fluorophores' lifetime, which are however in the (sub-)nanosecond range. We have integrated a single-photon pixel array, based on standard CMOS SPADs (single-photon avalanche diodes), in a compact, time-gated measurement system, named FluoCam. In vivo measurements were carried out with indocyanine green (ICG)-modified derivatives targeting the αvβ 3 integrin, initially on a genetically engineered mouse model of melanoma injected with ICG conjugated with tetrameric cyclic pentapeptide (ICG-E[c(RGD f K)4]), then on mice carrying tumour xenografts of U87-MG (a human primary glioblastoma cell line) injected with monomeric ICG-c(RGD f K). Measurements on tumor, muscle and tail locations allowed us to demonstrate the feasibility of in vivo lifetime measurements with the FluoCam, to determine the characteristic lifetimes (around 500 ps) and subtle lifetime differences between bound and unbound ICG-modified fluorophores (10% level), as well as to estimate the available photon fluxes under realistic conditions.

Differences in elasticity of vinculin-deficient F9 cells measured by magnetometry and atomic force microscopy

NASA Technical Reports Server (NTRS)

Goldmann, W. H.; Galneder, R.; Ludwig, M.; Xu, W.; Adamson, E. D.; Wang, N.; Ezzell, R. M.; Ingber, D. E. (Principal Investigator)

1998-01-01

We have investigated a mouse F9 embryonic carcinoma cell line, in which both vinculin genes were inactivated by homologous recombination, that exhibits defective adhesion and spreading [Coll et al. (1995) Proc. Natl. Acad. Sci. USA 92, 9161-9165]. Using a magnetometer and RGD-coated magnetic microbeads, we measured the local effect of loss and replacement of vinculin on mechanical force transfer across integrins. Vinculin-deficient F9Vin(-/-) cells showed a 21% difference in relative stiffness compared to wild-type cells. This was restored to near wild-type levels after transfection and constitutive expression of increasing amounts of vinculin into F9Vin(-/-) cells. In contrast, the transfection of vinculin constructs deficient in amino acids 1-288 (containing the talin- and alpha-actinin-binding site) or substituting tyrosine for phenylalanine (phosphorylation site, amino acid 822) in F9Vin(-/-) cells resulted in partial restoration of stiffness. Using atomic force microscopy to map the relative elasticity of entire F9 cells by 128 x 128 (n = 16,384) force scans, we observed a correlation with magnetometer measurements. These findings suggest that vinculin may promote cell adhesions and spreading by stabilizing focal adhesions and transferring mechanical stresses that drive cytoskeletal remodeling, thereby affecting the elastic properties of the cell.

Foot-and-mouth disease virus replicates only transiently in well-differentiated porcine nasal epithelial cells.

PubMed

Dash, Pradyot; Barnett, Paul V; Denyer, Michael S; Jackson, Terry; Stirling, Catrina M A; Hawes, Philippa C; Simpson, Jennifer L; Monaghan, Paul; Takamatsu, Haru-H

2010-09-01

Three-dimensional (3D) porcine nasal mucosal and tracheal mucosal epithelial cell cultures were developed to analyze foot-and-mouth disease virus (FMDV) interactions with mucosal epithelial cells. The cells in these cultures differentiated and polarized until they closely resemble the epithelial layers seen in vivo. FMDV infected these cultures predominantly from the apical side, primarily by binding to integrin alphav beta6, in an Arg-Gly-Asp (RGD)-dependent manner. However, FMDV replicated only transiently without any visible cytopathic effect (CPE), and infectious progeny virus could be recovered only from the apical side. The infection induced the production of beta interferon (IFN-beta) and the IFN-inducible gene Mx1 mRNA, which coincided with the disappearance of viral RNA and progeny virus. The induction of IFN-beta mRNA correlated with the antiviral activity of the supernatants from both the apical and basolateral compartments. IFN-alpha mRNA was constitutively expressed in nasal mucosal epithelial cells in vitro and in vivo. In addition, FMDV infection induced interleukin 8 (IL-8) protein, granulocyte-macrophage colony-stimulating factor (GM-CSF), and RANTES mRNA in the infected epithelial cells, suggesting that it plays an important role in modulating the immune response.

Modification to the Capsid of the Adenovirus Vector That Enhances Dendritic Cell Infection and Transgene-Specific Cellular Immune Responses

PubMed Central

Worgall, Stefan; Busch, Annette; Rivara, Michael; Bonnyay, David; Leopold, Philip L.; Merritt, Robert; Hackett, Neil R.; Rovelink, Peter W.; Bruder, Joseph T.; Wickham, Thomas J.; Kovesdi, Imi; Crystal, Ronald G.

2004-01-01

Adenovirus (Ad) gene transfer vectors can be used to transfer and express antigens and function as strong adjuvants and thus are useful platforms for the development of genetic vaccines. Based on the hypothesis that Ad vectors with enhanced infectibility of dendritic cells (DC) may be able to evoke enhanced immune responses against antigens encoded by the vector in vivo, the present study analyzes the vaccine potential of an Ad vector expressing β-galactosidase as a model antigen and genetically modified with RGD on the fiber knob [AdZ.F(RGD)] to more selectively infect DC and consequently enhance immunity against the β-galactosidase antigen. Infection of murine DC in vitro with AdZ.F(RGD) showed an eightfold-increased transgene expression following infection compared to AdZ (also expressing β-galactosidase, but with a wild-type capsid). Binding, cellular uptake, and trafficking in DC were also increased with AdZ.F(RGD) compared to AdZ. To determine whether AdZ.F(RGD) could evoke enhanced immune responses to β-galactosidase in vivo, C57BL/6 mice were immunized with AdZ.F(RGD) or AdZ subcutaneously via the footpad. Humoral responses with both vectors were comparable, with similar anti-β-galactosidase antibody levels following vector administration. However, cellular responses to β-galactosidase were significantly enhanced, with the frequency of CD4+ as well as the CD8+ β-galactosidase-specific gamma interferon response in cells isolated from the draining lymph nodes increased following immunization with AdZ.F(RGD) compared to Ad.Z (P < 0.01). Importantly, this enhanced cellular immune response of the AdZ.F(RGD) vector was sufficient to evoke enhanced inhibition of the growth of preexisting tumors expressing β-galactosidase: BALB/c mice implanted with the CT26 syngeneic β-galactosidase-expressing colon carcinoma cell line and subsequently immunized with AdZ.F(RGD) showed decreased tumor growth and improved survival compared to mice immunized with AdZ. These data demonstrate that addition of an RGD motif to the Ad fiber knob increases the infectibility of DC and leads to enhanced cellular immune responses to the Ad-transferred transgene, suggesting that the RGD capsid modification may be useful in developing Ad-based vaccines. PMID:14963160

Prostate Cancer Progression and Serum SIBLING (Small Integrin Binding N-Linked Glycoprotein)Levels

DTIC Science & Technology

2007-10-01

termed SIBLINGs (for small integrin binding ligand N-linked glycoproteins) whose members include bone sialoprotein (BSP), osteopontin (OPN), dentin...enzyme-linked immunosorbent assays (ELISAs) for quantitatively determining the levels of bone sialoprotein (BSP), osteopontin (OPN), dentin...synthesized as a chimeric protein, composed of three parts: dentin sialoprotein (DSP), dentin glycoprotein (DGP) and dentin phosphoprotein (DPP, also

Biomagnetics and Cell-Based Biochips

NASA Astrophysics Data System (ADS)

Ingber, Donald

2005-03-01

This presentation will review various micro- and nanotechnologies that we have developed over the past decade in our efforts to manipulate and probe living cells. In early studies, we used magnetic micro-particles to apply controlled mechanical forces to surface membrane receptors. We did this to probe cellular mechanical properties, and to investigate the molecular basis of mechanotransduction -- how mechanical forces are transduced into changes in intracellular biochemistry. The magnetic beads were coated with ligands for adhesion receptors, such as synthetic RGD (arginine-glycine-aspartate) peptides or antibodies that bind to membrane integrin receptors. Controlled twisting (torque) or pulling (tension) forces were exerted on the integrin-bound beads using magnetic twisting or pulling cytometry. To investigate the cellular response to dynamic forces, and to increase the level of stress applied, an electromagnetic needle was developed to apply a temporally varying magnetic field controlled by a user-defined solenoidal current; the end of the needle also was electropolished to produce a nanoscale pole tip. Magnetic forces applied to integrin receptors, but not other cell-surface receptors, induced force-dependent recruitment of cytoskeletal linker (focal adhesion) proteins to the site of bead binding, resulting in assembly and mechanical strengthening of the adhesions. Stress application to integrins also resulted in force-dependent increases in cAMP signaling and induction of gene transcription. These experiments revealed that integrins and the cytoskeleton play a central role in cellular mechanotransduction.studies in collaboration with George Whitesides (Harvard U.), we used microcontact printing techniques with self- assembled monolayers of alkanethiols to microfabricate extracellular matrix-coated adhesive islands of defined size, shape, and position on the micrometer scale. When cells were plated on these islands, the spread to take on the form of the island. These studies revealed that cells can be switched between growth, differentiation, and death (apoptosis) by varying the degree to which a cell physically can distend. When cells grown on islands with corners (e.g., squares, triangles) were stimulated with motility factors, the cells preferentially extended new motile processes from the corner regions, whereas cells on circular islands showed no bias. These findings demonstrated that much of cell behavior is controlled through physical interactions between cells and their adhesive substrate, and that microfabrication methods may be useful for tissue engineering, as well as creation of ``laboratories on a chip'' or biosensor devices that incorporate living mammalian cells. addition, in experiments with Bob Westervelt and Donhee Ham (Harvard U.), we have demonstrated the feasilibility of using microelectromagnetic circuits and CMOS technology to physically pull cells out from medium magnetically, and to move them in a directed manner. This approach may have great value for cell separation applications. Finally, with Whitesides group, we also demonstrated that microfluidics technologies may be used to deliver chemicals or probes to different regions of the same living cell under flow conditions. This provides a novel way to create chemical gradients at the subcellular scale and thereby probe the relation between cell structure and function. We also are currently exploring novel uses of microfluidics technologies, including their application for clinical cell separation applications. Taken together, this body of his work clearly demonstrates the great value of microsystem and microfluidic approaches for the analysis and manipulation of living cells. These approaches may have great value, both for fundamental scientific research and for clinical applications.

Mechanism of mast cell adhesion to human tenocytes in vitro.

PubMed

Behzad, Hayedeh; Tsai, Shu-Huei; Nassab, Paulina; Mousavizadeh, Rouhollah; McCormack, Robert G; Scott, Alex

2015-01-01

Mast cells and fibroblasts are two key players involved in many fibrotic and degenerative disorders. In the present study we examined the nature of binding interactions between human mast cells and tendon fibroblasts (tenocytes). In the mast cell-fibroblast co-culture model, mast cells were shown to spontaneously bind to tenocytes, in a process that was partially mediated by α5β1 integrin receptors. The same receptors on mast cells significantly mediated binding of these cells to tissue culture plates in the presence of tenocyte-conditioned media; the tenocyte-derived fibronectin in the media was shown to also play a major role in these binding activities. Upon binding to tenocytes or tissue culture plates, mast cells acquired an elongated phenotype, which was dependent on α5β1 integrin and tenocyte fibronectin. Additionally, tenocyte-derived fibronectin significantly enhanced mRNA expression of the adhesion molecule, THY1, by mast cells. Our data suggests that α5β1 integrin mediates binding of mast cells to human tenocyte and to tenocyte-derived ECM proteins, in particular fibronectin. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Shear-induced integrin signaling in platelet phosphatidylserine exposure, microvesicle release and coagulation.

PubMed

Pang, Aiming; Cui, Yujie; Chen, Yunfeng; Cheng, Ni; Delaney, M Keegan; Gu, Minyi; Stojanovic-Terpo, Aleksandra; Zhu, Cheng; Du, Xiaoping

2018-05-31

It is currently unclear why agonist-stimulated platelets require shear force to efficiently externalize the procoagulant phospholipid phosphatidylserine (PS) and release PS-exposed microvesicles (MVs). We reveal that integrin outside-in signaling is an important mechanism for this requirement. PS exposure and MV release were inhibited in β 3 -/- platelets or by integrin antagonists. The impaired MV release and PS exposure in β 3 -/- platelets were rescued by expressing wild type β 3 but not a Gα 13 binding-deficient β 3 mutant (E 733 EE to AAA), which blocks outside-in signaling but not ligand binding. Inhibition of Gα 13 or Src also diminished agonist/shear-dependent PS exposure and MV release, further indicating a role for integrin outside-in signaling. PS exposure in activated platelets was induced by application of pulling force via an integrin ligand, which was abolished by inhibiting Gα 13 -integrin interaction, suggesting that GGα 13 -dependent transmission of mechanical signals by integrins induces PS exposure. Inhibition of Gα 13 delayed coagulation in vitro. Furthermore, inhibition or platelet-specific knockout of Gα 13 diminished laser-induced intravascular fibrin formation in arterioles in vivo. Thus, β 3 integrins serve as a shear sensor activating the Gα 13 -dependent outside-in signaling pathway to facilitate platelet procoagulant function. Pharmacological targeting of Gα 13 -integrin interaction prevents occlusive thrombosis in vivo by inhibiting both coagulation and platelet thrombus formation. Copyright © 2018 American Society of Hematology.

Herpes simplex virus type 2 glycoprotein H interacts with integrin αvβ3 to facilitate viral entry and calcium signaling in human genital tract epithelial cells.

PubMed

Cheshenko, Natalia; Trepanier, Janie B; González, Pablo A; Eugenin, Eliseo A; Jacobs, William R; Herold, Betsy C

2014-09-01

Herpes simplex virus (HSV) entry requires multiple interactions at the cell surface and activation of a complex calcium signaling cascade. Previous studies demonstrated that integrins participate in this process, but their precise role has not been determined. These studies were designed to test the hypothesis that integrin αvβ3 signaling promotes the release of intracellular calcium (Ca2+) stores and contributes to viral entry and cell-to-cell spread. Transfection of cells with small interfering RNA (siRNA) targeting integrin αvβ3, but not other integrin subunits, or treatment with cilengitide, an Arg-Gly-Asp (RGD) mimetic, impaired HSV-induced Ca2+ release, viral entry, plaque formation, and cell-to-cell spread of HSV-1 and HSV-2 in human cervical and primary genital tract epithelial cells. Coimmunoprecipitation studies and proximity ligation assays indicated that integrin αvβ3 interacts with glycoprotein H (gH). An HSV-2 gH-null virus was engineered to further assess the role of gH in the virus-induced signaling cascade. The gH-2-null virus bound to cells and activated Akt to induce a small Ca2+ response at the plasma membrane, but it failed to trigger the release of cytoplasmic Ca2+ stores and was impaired for entry and cell-to-cell spread. Silencing of integrin αvβ3 and deletion of gH prevented phosphorylation of focal adhesion kinase (FAK) and the transport of viral capsids to the nuclear pore. Together, these findings demonstrate that integrin signaling is activated downstream of virus-induced Akt signaling and facilitates viral entry through interactions with gH by activating the release of intracellular Ca2+ and FAK phosphorylation. These findings suggest a new target for HSV treatment and suppression. Herpes simplex viruses are the leading cause of genital disease worldwide, the most common infection associated with neonatal encephalitis, and a major cofactor for HIV acquisition and transmission. There is no effective vaccine. These epidemiological findings underscore the urgency to develop novel HSV treatment or prevention strategies. This study addresses this gap by further defining the signaling pathways the virus usurps to enter human genital tract epithelial cells. Specifically, the study defines the role played by integrins and by the viral envelope glycoprotein H in entry and cell-to-cell spread. This knowledge will facilitate the identification of new targets for the development of treatment and prevention. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Triazine-Based Tool Box for Developing Peptidic PET Imaging Probes: Syntheses, Microfluidic Radiolabeling, and Structure–Activity Evaluation

PubMed Central

2015-01-01

This study was aimed at developing a triazine-based modular platform for targeted PET imaging. We synthesized mono- or bis-cyclo(RGDfK) linked triazine-based conjugates specifically targeting integrin αvβ3 receptors. The core molecules could be easily linked to targeting peptide and radiolabeled bifunctional chelator. The spacer core molecule was synthesized in 2 or 3 steps in 64–80% yield, and the following conjugation reactions with cyclo(RGDfK) peptide or bifunctional chelator were accomplished using “click” chemistry or amidation reactions. The DOTA-TZ-Bis-cyclo(RGDfK) 13 conjugate was radiolabeled successfully with 64Cu(OAc)2 using a microfluidic method, resulting in higher specific activity with above 95% labeling yields compared to conventional radiolabeling (SA ca. 850 vs 600 Ci/mmol). The dimeric cyclo(RGDfK) peptide was found to display significant bivalency effect using I125-Echistatin binding assay with IC50 value as 178.5 ± 57.1 nM, which displayed a 3.6-fold enhancement of binding affinity compared to DOTA-TZ-cyclo(RGDfK) 14 conjugate on U87MG human glioblastoma cell. Biodistribution of all four conjugates in female athymic nude mice were evaluated. DOTA-“Click”-cyclo(RGDfK) 15 had the highest tumor uptake among these four at 4 h p.i. with 1.90 ± 0.65%ID/g, while there was no clear bivalency effect for DOTA-TZ-BisRGD in vivo, which needs further experiments to address the unexpected questions. PMID:24661266

Positive evolutionary selection of an HD motif on Alzheimer precursor protein orthologues suggests a functional role.

PubMed

Miklós, István; Zádori, Zoltán

2012-02-01

HD amino acid duplex has been found in the active center of many different enzymes. The dyad plays remarkably different roles in their catalytic processes that usually involve metal coordination. An HD motif is positioned directly on the amyloid beta fragment (Aβ) and on the carboxy-terminal region of the extracellular domain (CAED) of the human amyloid precursor protein (APP) and a taxonomically well defined group of APP orthologues (APPOs). In human Aβ HD is part of a presumed, RGD-like integrin-binding motif RHD; however, neither RHD nor RXD demonstrates reasonable conservation in APPOs. The sequences of CAEDs and the position of the HD are not particularly conserved either, yet we show with a novel statistical method using evolutionary modeling that the presence of HD on CAEDs cannot be the result of neutral evolutionary forces (p<0.0001). The motif is positively selected along the evolutionary process in the majority of APPOs, despite the fact that HD motif is underrepresented in the proteomes of all species of the animal kingdom. Position migration can be explained by high probability occurrence of multiple copies of HD on intermediate sequences, from which only one is kept by selective evolutionary forces, in a similar way as in the case of the "transcription binding site turnover." CAED of all APP orthologues and homologues are predicted to bind metal ions including Amyloid-like protein 1 (APLP1) and Amyloid-like protein 2 (APLP2). Our results suggest that HDs on the CAEDs are most probably key components of metal-binding domains, which facilitate and/or regulate inter- or intra-molecular interactions in a metal ion-dependent or metal ion concentration-dependent manner. The involvement of naturally occurring mutations of HD (Tottori (D7N) and English (H6R) mutations) in early onset Alzheimer's disease gives additional support to our finding that HD has an evolutionary preserved function on APPOs.

Positive Evolutionary Selection of an HD Motif on Alzheimer Precursor Protein Orthologues Suggests a Functional Role

PubMed Central

Miklós, István; Zádori, Zoltán

2012-01-01

HD amino acid duplex has been found in the active center of many different enzymes. The dyad plays remarkably different roles in their catalytic processes that usually involve metal coordination. An HD motif is positioned directly on the amyloid beta fragment (Aβ) and on the carboxy-terminal region of the extracellular domain (CAED) of the human amyloid precursor protein (APP) and a taxonomically well defined group of APP orthologues (APPOs). In human Aβ HD is part of a presumed, RGD-like integrin-binding motif RHD; however, neither RHD nor RXD demonstrates reasonable conservation in APPOs. The sequences of CAEDs and the position of the HD are not particularly conserved either, yet we show with a novel statistical method using evolutionary modeling that the presence of HD on CAEDs cannot be the result of neutral evolutionary forces (p<0.0001). The motif is positively selected along the evolutionary process in the majority of APPOs, despite the fact that HD motif is underrepresented in the proteomes of all species of the animal kingdom. Position migration can be explained by high probability occurrence of multiple copies of HD on intermediate sequences, from which only one is kept by selective evolutionary forces, in a similar way as in the case of the “transcription binding site turnover.” CAED of all APP orthologues and homologues are predicted to bind metal ions including Amyloid-like protein 1 (APLP1) and Amyloid-like protein 2 (APLP2). Our results suggest that HDs on the CAEDs are most probably key components of metal-binding domains, which facilitate and/or regulate inter- or intra-molecular interactions in a metal ion-dependent or metal ion concentration-dependent manner. The involvement of naturally occurring mutations of HD (Tottori (D7N) and English (H6R) mutations) in early onset Alzheimer's disease gives additional support to our finding that HD has an evolutionary preserved function on APPOs. PMID:22319430

Structure of a double ubiquitin-like domain in the talin head: a role in integrin activation

PubMed Central

Goult, Benjamin T; Bouaouina, Mohamed; Elliott, Paul R; Bate, Neil; Patel, Bipin; Gingras, Alexandre R; Grossmann, J Günter; Roberts, Gordon C K; Calderwood, David A; Critchley, David R; Barsukov, Igor L

2010-01-01

Talin is a 270-kDa protein that activates integrins and couples them to cytoskeletal actin. Talin contains an N-terminal FERM domain comprised of F1, F2 and F3 domains, but it is atypical in that F1 contains a large insert and is preceded by an extra domain F0. Although F3 contains the binding site for β-integrin tails, F0 and F1 are also required for activation of β1-integrins. Here, we report the solution structures of F0, F1 and of the F0F1 double domain. Both F0 and F1 have ubiquitin-like folds joined in a novel fixed orientation by an extensive charged interface. The F1 insert forms a loop with helical propensity, and basic residues predicted to reside on one surface of the helix are required for binding to acidic phospholipids and for talin-mediated activation of β1-integrins. This and the fact that basic residues on F2 and F3 are also essential for integrin activation suggest that extensive interactions between the talin FERM domain and acidic membrane phospholipids are required to orientate the FERM domain such that it can activate integrins. PMID:20150896

The synthesis and coupling of photoreactive collagen-based peptides to restore integrin reactivity to an inert substrate, chemically-crosslinked collagen

PubMed Central

Malcor, Jean-Daniel; Bax, Daniel; Hamaia, Samir W.; Davidenko, Natalia; Best, Serena M.; Cameron, Ruth E.; Farndale, Richard W.; Bihan, Dominique

2016-01-01

Collagen is frequently advocated as a scaffold for use in regenerative medicine. Increasing the mechanical stability of a collagen scaffold is widely achieved by cross-linking using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS). However, this treatment consumes the carboxylate-containing amino acid sidechains that are crucial for recognition by the cell-surface integrins, abolishing cell adhesion. Here, we restore cell reactivity to a cross-linked type I collagen film by covalently linking synthetic triple-helical peptides (THPs), mimicking the structure of collagen. These THPs are ligands containing an active cell-recognition motif, GFOGER, a high-affinity binding site for the collagen-binding integrins. We end-stapled peptide strands containing GFOGER by coupling a short diglutamate-containing peptide to their N-terminus, improving the thermal stability of the resulting THP. A photoreactive Diazirine group was grafted onto the end-stapled THP to allow covalent linkage to the collagen film upon UV activation. Such GFOGER-derivatized collagen films showed restored affinity for the ligand-binding I domain of integrin α2β1, and increased integrin-dependent cell attachment and spreading of HT1080 and Rugli cell lines, expressing integrins α2β1 and α1β1, respectively. The method we describe has wide application, beyond collagen films or scaffolds, since the photoreactive diazirine will react with many organic carbon skeletons. PMID:26854392

Neurite outgrowth on a fibronectin isoform expressed during peripheral nerve regeneration is mediated by the interaction of paxillin with α4β1 integrins

PubMed Central

Vogelezang, Mariette; Forster, Ulrike B; Han, Jaewon; Ginsberg, Mark H; ffrench-Constant, Charles

2007-01-01

Background The regeneration of peripheral nerve is associated with a change in the alternative splicing of the fibronectin primary gene transcript to re-express embryonic isoforms containing a binding site for α4β1 integrins that promote neurite outgrowth. Here we use PC12 cells to examine the role of the interaction between paxillin and the α4 integrin cytoplasmic domain in neurite outgrowth. Results Expression of α4 with mutations in the paxillin-binding domain reduced neurite outgrowth on recombinant embryonic fibronectin fragments relative to wild type α4. Over-expression of paxillin promoted neurite outgrowth while a mutant isoform lacking the LD4 domain implicated in the regulation of ARF and Rac GTPases was less effective. Optimal α4-mediated migration in leucocytes requires spatial regulation of α4 phosphorylation at Ser988, a post-translational modification that blocks paxillin binding to the integrin cytoplasmic domain. In keeping with this α4(S988D), which mimics phosphorylated α4, did not promote neurite outgrowth. However, α4 was not phosphorylated in the PC12 cells, and a non-phosphorylatable α4(S988A) mutant promoted neurite outgrowth indistinguishably from the wild type integrin. Conclusion We establish the importance of the α4 integrin-paxillin interaction in a model of axonal regeneration and highlight differing dependence on phosphorylation of α4 for extension of neuronal growth cones and migration of non-neural cells. PMID:17603879

Crystal structure of isoflurane bound to integrin LFA-1 supports a unified mechanism of volatile anesthetic action in the immune and central nervous systems

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Hongmin; Astrof, Nathan S.; Liu, Jin-Huan

2009-09-15

Volatile anesthetics (VAs), such as isoflurane, induce a general anesthetic state by binding to specific targets (i.e., ion channels) in the central nervous system (CNS). Simultaneously, VAs modulate immune functions, possibly via direct interaction with alternative targets on leukocytes. One such target, the integrin lymphocyte function-associated antigen-1 (LFA-1), has been shown previously to be inhibited by isoflurane. A better understanding of the mechanism by which isoflurane alters protein function requires the detailed information about the drug-protein interaction at an atomic level. Here, we describe the crystal structure of the LFA-1 ligand-binding domain (I domain) in complex with isoflurane at 1.6more » {angstrom}. We discovered that isoflurane binds to an allosteric cavity previously implicated as critical for the transition of LFA-1 from the low- to the high-affinity state. The isoflurane binding site in the I domain involves an array of amphiphilic interactions, thereby resembling a 'common anesthetic binding motif' previously predicted for authentic VA binding sites. These results suggest that the allosteric modulation of protein function by isoflurane, as demonstrated for the integrin LFA-1, might represent a unified mechanism shared by the interactions of volatile anesthetics with targets in the CNS. Crystal structure of isoflurane bound to integrin LFA-1 supports a unified mechanism of volatile anesthetic action in the immune and central nervous systems.« less

The molecular mechanism of mediation of adsorbed serum proteins to endothelial cells adhesion and growth on biomaterials.

PubMed

Yang, Dayun; Lü, Xiaoying; Hong, Ying; Xi, Tingfei; Zhang, Deyuan

2013-07-01

To explore molecular mechanism of mediation of adsorbed proteins to cell adhesion and growth on biomaterials, this study examined endothelial cell adhesion, morphology and viability on bare and titanium nitride (TiN) coated nickel titanium (NiTi) alloys and chitosan film firstly, and then identified the type and amount of serum proteins adsorbed on the three surfaces by proteomic technology. Subsequently, the mediation role of the identified proteins to cell adhesion and growth was investigated with bioinformatics analyses, and further confirmed by a series of cellular and molecular biological experiments. Results showed that the type and amount of adsorbed serum proteins associated with cell adhesion and growth was obviously higher on the alloys than on the chitosan film, and these proteins mediated endothelial cell adhesion and growth on the alloys via four ways. First, proteins such as adiponectin in the adsorbed protein layer bound with cell surface receptors to generate signal transduction, which activated cell surface integrins through increasing intracellular calcium level. Another way, thrombospondin 1 in the adsorbed protein layer promoted TGF-β signaling pathway activation and enhanced integrins expression. The third, RGD sequence containing proteins such as fibronectin 1, vitronectin and thrombospondin 1 in the adsorbed protein layer bound with activated integrins to activate focal adhesion pathway, increased focal adhesion formation and actin cytoskeleton organization and mediated cell adhesion and spreading. In addition, the activated focal adhesion pathway promoted the expression of cell growth related genes and resulted in cell proliferation. The fourth route, coagulation factor II (F2) and fibronectin 1 in the adsorbed protein layer bound with cell surface F2 receptor and integrin, activated regulation of actin cytoskeleton pathway and regulated actin cytoskeleton organization. Copyright © 2013 Elsevier Ltd. All rights reserved.

Integrins in bone metastasis formation and potential therapeutic implications.

PubMed

Clëzardin, P

2009-11-01

Integrins constitute a family of cell surface receptors that are heterodimers composed of noncovalently associated alpha and beta subunits. Integrins bind to extracellular matrix proteins and immunogobulin superfamily molecules. They exert a stringent control on cell migration, survival and proliferation. However, their expression and functions are often deregulated in cancer, and many lines of evidence implicate them as key regulators during progression from primary tumor growth to metastasis. Here, we review the role of integrins in bone metastasis formation and present evidence that the use of integrin-targeted therapeutic agents may be an efficient strategy to block tumor metastasis.

Sequence-Specific Interaction between the Disintegrin Domain of Mouse ADAM 3 and Murine Eggs: Role of β1 Integrin-associated Proteins CD9, CD81, and CD98

PubMed Central

Takahashi, Yuji; Bigler, Dora; Ito, Yasuhiko; White, Judith M.

2001-01-01

ADAM 3 is a sperm surface glycoprotein that has been implicated in sperm-egg adhesion. Because little is known about the adhesive activity of ADAMs, we investigated the interaction of ADAM 3 disintegrin domains, made in bacteria and in insect cells, with murine eggs. Both recombinant proteins inhibited sperm-egg binding and fusion with potencies similar to that which we recently reported for the ADAM 2 disintegrin domain. Alanine scanning mutagenesis revealed a critical importance for the glutamine at position 7 of the disintegrin loop. Fluorescent beads coated with the ADAM 3 disintegrin domain bound to the egg surface. Bead binding was inhibited by an authentic, but not by a scrambled, peptide analog of the disintegrin loop. Bead binding was also inhibited by the function-blocking anti-α6 monoclonal antibody (mAb) GoH3, but not by a nonfunction blocking anti-α6 mAb, or by mAbs against either the αv or β3 integrin subunits. We also present evidence that in addition to the tetraspanin CD9, two other β1-integrin-associated proteins, the tetraspanin CD81 as well as the single pass transmembrane protein CD98 are expressed on murine eggs. Antibodies to CD9 and CD98 inhibited in vitro fertilization and binding of the ADAM 3 disintegrin domain. Our findings are discussed in terms of the involvement of multiple sperm ADAMs and multiple egg β1 integrin-associated proteins in sperm-egg binding and fusion. We propose that an egg surface “tetraspan web” facilitates fertilization and that it may do so by fostering ADAM–integrin interactions. PMID:11294888

Sequence-specific interaction between the disintegrin domain of mouse ADAM 3 and murine eggs: role of beta1 integrin-associated proteins CD9, CD81, and CD98.

PubMed

Takahashi, Y; Bigler, D; Ito, Y; White, J M

2001-04-01

ADAM 3 is a sperm surface glycoprotein that has been implicated in sperm-egg adhesion. Because little is known about the adhesive activity of ADAMs, we investigated the interaction of ADAM 3 disintegrin domains, made in bacteria and in insect cells, with murine eggs. Both recombinant proteins inhibited sperm-egg binding and fusion with potencies similar to that which we recently reported for the ADAM 2 disintegrin domain. Alanine scanning mutagenesis revealed a critical importance for the glutamine at position 7 of the disintegrin loop. Fluorescent beads coated with the ADAM 3 disintegrin domain bound to the egg surface. Bead binding was inhibited by an authentic, but not by a scrambled, peptide analog of the disintegrin loop. Bead binding was also inhibited by the function-blocking anti-alpha6 monoclonal antibody (mAb) GoH3, but not by a nonfunction blocking anti-alpha6 mAb, or by mAbs against either the alphav or beta3 integrin subunits. We also present evidence that in addition to the tetraspanin CD9, two other beta1-integrin-associated proteins, the tetraspanin CD81 as well as the single pass transmembrane protein CD98 are expressed on murine eggs. Antibodies to CD9 and CD98 inhibited in vitro fertilization and binding of the ADAM 3 disintegrin domain. Our findings are discussed in terms of the involvement of multiple sperm ADAMs and multiple egg beta1 integrin-associated proteins in sperm-egg binding and fusion. We propose that an egg surface "tetraspan web" facilitates fertilization and that it may do so by fostering ADAM-integrin interactions.

ADAP interactions with talin and kindlin promote platelet integrin αIIbβ3 activation and stable fibrinogen binding

PubMed Central

Kang, Jian; Kahner, Bryan; Ye, Feng; Ginsberg, Mark H.; Shattil, Sanford J.

2014-01-01

ADAP is a hematopoietic-restricted adapter protein that promotes integrin activation and is a carrier for other adapter proteins, Src kinase–associated phosphoprotein 1 (SKAP1) and SKAP2. In T lymphocytes, SKAP1 is the ADAP-associated molecule that activates integrins through direct linkages with Rap1 effectors (regulator of cell adhesion and polarization enriched in lymphoid tissues; Rap1-interacting adapter molecule). ADAP also promotes integrin αIIbβ3 activation in platelets, which lack SKAP1, suggesting an ADAP integrin–regulatory pathway different from those in lymphocytes. Here we characterized a novel association between ADAP and 2 essential integrin-β cytoplasmic tail-binding proteins involved in αIIbβ3 activation, talin and kindlin-3. Glutathione S-transferase pull-downs identified distinct regions in ADAP necessary for association with kindlin or talin. ADAP was physically proximal to talin and kindlin-3 in human platelets, as assessed biochemically, and by immunofluorescence microscopy and proximity ligation. Relative to wild-type mouse platelets, ADAP-deficient platelets exhibited reduced co-localization of talin with αIIbβ3, and reduced irreversible fibrinogen binding in response to a protease activated receptor 4 (PAR4) thrombin receptor agonist. When ADAP was heterologously expressed in Chinese hamster ovary cells co-expressing αIIbβ3, talin, PAR1, and kindlin-3, it associated with an αIIbβ3/talin complex and enabled kindlin-3 to promote agonist-dependent ligand binding to αIIbβ3. Thus, ADAP uniquely promotes activation of and irreversible fibrinogen binding to platelet αIIbβ3 through interactions with talin and kindlin-3. PMID:24523237

Astrocyte-to-neuron communication through integrin-engaged Thy-1/CBP/Csk/Src complex triggers neurite retraction via the RhoA/ROCK pathway.

PubMed

Maldonado, H; Calderon, C; Burgos-Bravo, F; Kobler, O; Zuschratter, W; Ramirez, O; Härtel, S; Schneider, P; Quest, A F G; Herrera-Molina, R; Leyton, L

2017-02-01

Two key proteins for cellular communication between astrocytes and neurons are αvβ3 integrin and the receptor Thy-1. Binding of these molecules in the same (cis) or on adjacent (trans) cellular membranes induces Thy-1 clustering, triggering actin cytoskeleton remodeling. Molecular events that could explain how the Thy-1-αvβ3 integrin interaction signals have only been studied separately in different cell types, and the detailed transcellular communication and signal transduction pathways involved in neuronal cytoskeleton remodeling remain unresolved. Using biochemical and genetic approaches, single-molecule tracking, and high-resolution nanoscopy, we provide evidence that upon binding to αvβ3 integrin, Thy-1 mobility decreased while Thy-1 nanocluster size increased. This occurred concomitantly with inactivation and exclusion of the non-receptor tyrosine kinase Src from the Thy-1/C-terminal Src kinase (Csk)-binding protein (CBP)/Csk complex. The Src inactivation decreased the p190Rho GTPase activating protein phosphorylation, promoting RhoA activation, cofilin, and myosin light chain II phosphorylation and, consequently, neurite shortening. Finally, silencing the adaptor CBP demonstrated that this protein was a key transducer in the Thy-1 signaling cascade. In conclusion, these data support the hypothesis that the Thy-1-CBP-Csk-Src-RhoA-ROCK axis transmitted signals from astrocytic integrin-engaged Thy-1 (trans) to the neuronal actin cytoskeleton. Importantly, the β3 integrin in neurons (cis) was not found to be crucial for neurite shortening. This is the first study to detail the signaling pathway triggered by αvβ3, the endogenous Thy-1 ligand, highlighting the role of membrane-bound integrins as trans acting ligands in astrocyte-neuron communication. Copyright © 2016 Elsevier B.V. All rights reserved.

Highly sensitive SERS analysis of the cyclic Arg-Gly-Asp peptide ligands of cells using nanogap antennas.

PubMed

Portela, Alejandro; Yano, Taka-Aki; Santschi, Christian; Martin, Olivier J F; Tabata, Hitoshi; Hara, Masahiko

2017-02-01

The cyclic RGD (cRGD) peptide ligands of cells have become widely used for treating several cancers. We report a highly sensitive analysis of c(RGDfC) using surface enhanced Raman spectroscopy (SERS) using single dimer nanogap antennas in aqueous environment. Good agreement between characteristic peaks of the SERS and the Raman spectra of bulk c(RGDfC) with its peptide's constituents were observed. The exhibited blinking of the SERS spectra and synchronization of intensity fluctuations, suggest that the SERS spectra acquired from single dimer nanogap antennas was dominated by the spectrum of single to a few molecules. SERS spectra of c(RGDfC) could be used to detect at the nanoscale, the cells' transmembrane proteins binding to its ligand. SERS of cyclic RGD on nanogap antenna. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

PI3Kδ promotes CD4(+) T-cell interactions with antigen-presenting cells by increasing LFA-1 binding to ICAM-1.

PubMed

Garçon, Fabien; Okkenhaug, Klaus

2016-05-01

Activation of T lymphocytes by peptide/major histocompatibility complex on antigen-presenting cells (APCs) involves dynamic contacts between the two cells, during which T cells undergo marked morphological changes. These interactions are facilitated by integrins. Activation of the T cells increases the binding of the integrin lymphocyte function-associated antigen 1 (LFA-1) expressed by T cells to intercellular adhesion molecule (ICAM)-1 and ICAM-2 expressed by APCs. The signalling pathways that control integrin affinities are incompletely defined. The phosphoinositide 3-kinases (PI3Ks) generate second-messenger signalling molecules that control cell growth, proliferation, differentiation and trafficking. Here we show that in T cells, PI3Kδ attenuates the activation of Rac1, but sustains the activation of Rap1. Consequently, PI3Kδ increases LFA-1-dependent adhesion to form stable conjugates with APCs. Increased Rap1 activity and LFA-1 adhesion were only in part mediated by the downstream kinase Akt, suggesting the involvement of additional phosphatidylinositol(3,4,5)P3-binding proteins. These results establish a link between PI3K activity, cytoskeletal changes and integrin binding and help explain the impaired T-cell-dependent immune responses in PI3Kδ-deficient mice.

The integrin-binding motif RGDS induces protein tyrosine phosphorylation without activation in Bufo arenarum (Amphibia) oocytes.

PubMed

Mouguelar, Valeria S; Cabada, Marcelo O; Coux, Gabriela

2011-05-01

Integrins are cell adhesion molecules that are thought to be involved in sperm-oocyte interaction. Nevertheless, their function in mammalian fertilization is still controversial, as different species behave differently. In amphibians, their role is mainly supported by Xenopus laevis studies, where RGDS peptide induces oocyte activation. We recently provided evidence suggesting the presence and involvement of integrins in the interaction of the oocyte plasma membrane (PM) with sperm in the amphibian Bufo arenarum. In order to understand the role of integrin homologs in oocytes and their possible contribution to egg activation mechanisms, we examined the presence of integrin subunits and the effect of RGDS peptide on oocytes and during fertilization. Western blot studies detected integrin subunits α5, αV and β1 in oocytes. In sperm, we could detect only the αV integrin subunit. We found that RGDS peptide was unable to elicit egg activation or MAPK dephosphorylation, but can induce reversible inhibition of fertilization. A similar partial inhibition was produced by an anti-β1 integrin antibody. Using an anti-phosphotyrosine antibody we found major changes in phosphotyrosine-containing proteins in egg extracts minutes after fertilization. Cytosol and PMs isolated from oocytes and fertilized eggs showed additional fertilization-induced phosphorylated proteins. Some of these were also present in cytosol and PMs from RGDS-treated oocytes (partially mimicking fertilization). These findings suggest that B. arenarum fertilization involves integrins (e.g. β1 subunit) as adhesion proteins. Our data support the view that RGDS-binding receptors may function as signaling receptors in B. arenarum oocytes, but integrin engagement by RGDS is not sufficient for oocyte activation.

Redox-Relevant Aspects of the Extracellular Matrix and Its Cellular Contacts via Integrins

PubMed Central

de Rezende, Flávia Figueiredo

2014-01-01

Abstract Significance: The extracellular matrix (ECM) fulfills essential functions in multicellular organisms. It provides the mechanical scaffold and environmental cues to cells. Upon cell attachment, the ECM signals into the cells. In this process, reactive oxygen species (ROS) are physiologically used as signalizing molecules. Recent Advances: ECM attachment influences the ROS-production of cells. In turn, ROS affect the production, assembly and turnover of the ECM during wound healing and matrix remodeling. Pathological changes of ROS levels lead to excess ECM production and increased tissue contraction in fibrotic disorders and desmoplastic tumors. Integrins are cell adhesion molecules which mediate cell adhesion and force transmission between cells and the ECM. They have been identified as a target of redox-regulation by ROS. Cysteine-based redox-modifications, together with structural data, highlighted particular regions within integrin heterodimers that may be subject to redox-dependent conformational changes along with an alteration of integrin binding activity. Critical Issues: In a molecular model, a long-range disulfide-bridge within the integrin β-subunit and disulfide bridges within the genu and calf-2 domains of the integrin α-subunit may control the transition between the bent/inactive and upright/active conformation of the integrin ectodomain. These thiol-based intramolecular cross-linkages occur in the stalk domain of both integrin subunits, whereas the ligand-binding integrin headpiece is apparently unaffected by redox-regulation. Future Directions: Redox-regulation of the integrin activation state may explain the effect of ROS in physiological processes. A deeper understanding of the underlying mechanism may open new prospects for the treatment of fibrotic disorders. Antioxid. Redox Signal. 20, 1977–1993. PMID:24040997

Two conformations of the integrin A-domain (I-domain): a pathway for activation?

PubMed

Lee, J O; Bankston, L A; Arnaout, M A; Liddington, R C

1995-12-15

Integrins are plasma membrane proteins that mediate adhesion to other cells and to components of the extracellular matrix. Most integrins are constitutively inactive in resting cells, but are rapidly and reversibly activated in response to agonists, leading to highly regulated cell adhesion. This activation is associated with conformational changes in their extracellular portions, but the nature of the structural changes that lead to a change in adhesiveness is not understood. The interactions of several integrins with their extracellular ligands are mediated by an A-type domain (generally called the I-domain in integrins). Binding of the I-domain to protein ligands is dependent on divalent cations. We have described previously the structure of the I-domain from complement receptor 3 with bound Mg2+, in which the glutamate side chain from a second I-domain completes the octahedral coordination sphere of the metal, acting as a ligand mimetic. We now describe a new crystal form of the I-domain with bound Mn2+, in which water completes the metal coordination sphere and there is no equivalent of the glutamate ligand. Comparison of the two crystal forms reveals a change in metal coordination which is linked to a large (10 A) shift of the C-terminal helix and the burial of two phenylalanine residues into the hydrophobic core of the Mn2+ form. These structural changes, analogous to those seen in the signal-transducing G-proteins, alter the electrophilicity of the metal, reducing its ability to bind ligand-associated acidic residues, and dramatically alter the surface of the protein implicated in binding ligand. Our observations provide the first atomic resolution view of conformational changes in an integrin domain, and suggest how these changes are linked to a change in integrin adhesiveness. We propose that the Mg2+ form represents the conformation of the domain in the active state and the Mn2+ form the conformation in the inactive state of the integrin.

Signal transduction by beta1 integrin receptors in human chondrocytes in vitro: collaboration with the insulin-like growth factor-I receptor.

PubMed

Shakibaei, M; John, T; De Souza, P; Rahmanzadeh, R; Merker, H J

1999-09-15

We have examined the mechanism by which collagen-binding integrins co-operate with insulin-like growth factor-I (IGF-I) receptors (IGF-IR) to regulate chondrocyte phenotype and differentiation. Adhesion of chondrocytes to anti-beta1 integrin antibodies or collagen type II leads to phosphorylation of cytoskeletal and signalling proteins localized at focal adhesions, including alpha-actinin, vinculin, paxillin and focal adhesion kinase (FAK). These stimulate docking proteins such as Shc (Src-homology collagen). Moreover, exposure of collagen type II-cultured chondrocytes to IGF-I leads to co-immunoprecipitation of Shc protein with the IGF-IR and with beta1, alpha1 and alpha5 integrins, but not with alpha3 integrin. Shc then associates with growth factor receptor-bound protein 2 (Grb2), an adaptor protein and extracellular signal-regulated kinase. The expression of the docking protein Shc occurs only when chondrocytes are bound to collagen type II or integrin antibodies and increases when IGF-I is added, suggesting a collaboration between integrins and growth factors in a common/shared biochemical signalling pathway. Furthermore, these results indicate that focal adhesion assembly may facilitate signalling via Shc, a potential common target for signal integration between integrin and growth-factor signalling regulatory pathways. Thus, the collagen-binding integrins and IGF-IR co-operate to regulate focal adhesion components and these signalling pathways have common targets (Shc-Grb2 complex) in subcellular compartments, thereby linking to the Ras-mitogen-activated protein kinase signalling pathway. These events may play a role during chondrocyte differentiation.

Peptides derived from central turn motifs within integrin αIIb and αV cytoplasmic tails inhibit integrin activation.

PubMed

Li, Xinlei; Liu, Yongqing; Haas, Thomas A

2014-12-01

We previously found that peptides derived from the full length of integrin αIIb and αV cytoplasmic tails inhibited their parent integrin activation, respectively. Here we showed that the cell-permeable peptides corresponding to the conserved central turn motif within αIIb and αV cytoplasmic tails, myr-KRNRPPLEED (αIIb peptide) and myr-KRVRPPQEEQ (αV peptide), similarly inhibited both αIIb and αV integrin activation. Pre-treatment with αIIb or αV peptides inhibited Mn(2+)-activated αIIbβ3 binding to soluble fibrinogen as well as the binding of αIIbβ3-expressing Chinese Hamster Ovary cells to immobilized fibrinogen. Our turn peptides also inhibited adhesion of two breast cancer cell lines (MDA-MB-435 and MCF7) to αV ligand vitronectin. These results suggest that αIIb and αV peptides share a same mechanism in regulating integrin function. Using αIIb peptide as a model, we found that replacement of RPP with AAA significantly attenuated the inhibitory activity of αIIb peptide. Furthermore, we found that αIIb peptide specifically bound to β-tubulin in cells. Our work suggests that the central motif of α tails is an anchoring point for cytoskeletons during integrin activation and integrin-mediated cell adhesion, and its function depends on the turn structure at RPP. However, post-treatment of peptides derived from the full-length tail or from the turn motif did not reverse αIIb and αV integrin activation. Copyright © 2014 Elsevier Inc. All rights reserved.

Conservation of the Human Integrin-Type Beta-Propeller Domain in Bacteria

PubMed Central

Chouhan, Bhanupratap; Denesyuk, Alexander; Heino, Jyrki; Johnson, Mark S.; Denessiouk, Konstantin

2011-01-01

Integrins are heterodimeric cell-surface receptors with key functions in cell-cell and cell-matrix adhesion. Integrin α and β subunits are present throughout the metazoans, but it is unclear whether the subunits predate the origin of multicellular organisms. Several component domains have been detected in bacteria, one of which, a specific 7-bladed β-propeller domain, is a unique feature of the integrin α subunits. Here, we describe a structure-derived motif, which incorporates key features of each blade from the X-ray structures of human αIIbβ3 and αVβ3, includes elements of the FG-GAP/Cage and Ca2+-binding motifs, and is specific only for the metazoan integrin domains. Separately, we searched for the metazoan integrin type β-propeller domains among all available sequences from bacteria and unicellular eukaryotic organisms, which must incorporate seven repeats, corresponding to the seven blades of the β-propeller domain, and so that the newly found structure-derived motif would exist in every repeat. As the result, among 47 available genomes of unicellular eukaryotes we could not find a single instance of seven repeats with the motif. Several sequences contained three repeats, a predicted transmembrane segment, and a short cytoplasmic motif associated with some integrins, but otherwise differ from the metazoan integrin α subunits. Among the available bacterial sequences, we found five examples containing seven sequential metazoan integrin-specific motifs within the seven repeats. The motifs differ in having one Ca2+-binding site per repeat, whereas metazoan integrins have three or four sites. The bacterial sequences are more conserved in terms of motif conservation and loop length, suggesting that the structure is more regular and compact than those example structures from human integrins. Although the bacterial examples are not full-length integrins, the full-length metazoan-type 7-bladed β-propeller domains are present, and sometimes two tandem copies are found. PMID:22022374

Two distinct roles of mitogen-activated protein kinases in platelets and a novel Rac1-MAPK–dependent integrin outside-in retractile signaling pathway

PubMed Central

Flevaris, Panagiotis; Li, Zhenyu; Zhang, Guoying; Zheng, Yi; Liu, Junling

2009-01-01

Mitogen-activated protein kinases (MAPK), p38, and extracellular stimuli-responsive kinase (ERK), are acutely but transiently activated in platelets by platelet agonists, and the agonist-induced platelet MAPK activation is inhibited by ligand binding to the integrin αIIbβ3. Here we show that, although the activation of MAPK, as indicated by MAPK phosphorylation, is initially inhibited after ligand binding to integrin αIIbβ3, integrin outside-insignaling results in a late but sustained activation of MAPKs in platelets. Furthermore, we show that the early agonist-induced MAPK activation and the late integrin-mediated MAPK activation play distinct roles in different stages of platelet activation. Agonist-induced MAPK activation primarily plays an important role in stimulating secretion of platelet granules, while integrin-mediated MAPK activation is important in facilitating clot retraction. The stimulatory role of MAPK in clot retraction is mediated by stimulating myosin light chain (MLC) phosphorylation. Importantly, integrin-dependent MAPK activation, MAPK-dependent MLC phosphorylation, and clot retraction are inhibited by a Rac1 inhibitor and in Rac1 knockout platelets, indicating that integrin-induced activation of MAPK and MLC and subsequent clot retraction is Rac1-dependent. Thus, our results reveal 2 different activation mechanisms of MAPKs that are involved in distinct aspects of platelet function and a novel Rac1-MAPK–dependent cell retractile signaling pathway. PMID:18957688

Effects of Arg-Gly-Asp sequence peptide and hyperosmolarity on the permeability of interstitial matrix and fenestrated endothelium in joints.

PubMed

Poli, A; Mason, R M; Levick, J R

2004-09-01

The aims were to assess the contribution of arg-gly-asp (RGD) mediated cell integrin-matrix bonds to interstitial hydraulic resistance and to fenestrated endothelial permeability in joints. Joint fluid is generated by filtration from fenestrated capillaries and drains through a fibronectin-rich synovial intercellular matrix. The role of parenchymal cell-matrix bonding in determining tissue hydraulic resistance is unknown. The knee cavity of anesthetized rabbits was infused with saline or the competitive hexapeptide blocker GRGDTP, with or without added osmotic stress (600 mosm saline). Intra-articular pressure Pj, net trans-synovial drainage rate s, and the permeation of Evans blue-labeled albumin (EVA) from plasma into the joint cavity were measured. GRGDTP increased the hydraulic conductance of the synovial drainage pathway, ds/dPj, by 71% (p =.02, paired t test, n = 6 animals). Synovial plasma EVA clearance (control 7.1 +/- 0.8 microL h-1, mean +/- SEM, n = 15) was unaffected by GRGDTP (7.0 +/- 2.3 microL h(-1), n = 6) or hyperosmolarity (4.9 +/- 1.5 microL h(-1), n = 8) but was increased by GRGDTP and hyperosmolarity together (15.9 +/- 4.8 microL h(-1), n = 5) (p =.01, ANOVA). Changes in dPj/dt evoked by GRGDTP plus hyperosmolarity, but neither alone, demonstrated increased microvascular filtration into the joint cavity (p <.001, ANOVA), as did changes in fluid absorption from the infusion system at fixed Pj. RGD-mediated bonds between the parenchymal cells and interstitial polymers reduce the interstitial hydraulic conductance by 42%. This helps to retain the lubricating fluid inside a joint cavity. RGD-mediated bonds also support the macromolecular barrier function of fenestrated endothelium, but in vivo this is evident only in stressed endothelium (cf. in vitro).

Launching a Novel Preclinical Infrastructure: Comparative Oncology Trials Consortium Directed Therapeutic Targeting of TNFα to Cancer Vasculature

PubMed Central

Mazcko, Christina; Hanna, Engy; Kachala, Stefan; LeBlanc, Amy; Newman, Shelley; Vail, David; Henry, Carolyn; Thamm, Douglas; Sorenmo, Karin; Hajitou, Amin; Pasqualini, Renata; Arap, Wadih

2009-01-01

Background Under the direction and sponsorship of the National Cancer Institute, we report on the first pre-clinical trial of the Comparative Oncology Trials Consortium (COTC). The COTC is a novel infrastructure to integrate cancers that naturally develop in pet dogs into the development path of new human drugs. Trials are designed to address questions challenging in conventional preclinical models and early phase human trials. Large animal spontaneous cancer models can be a valuable addition to successful studies of cancer biology and novel therapeutic drug, imaging and device development. Methodology/Principal Findings Through this established infrastructure, the first trial of the COTC (COTC001) evaluated a targeted AAV-phage vector delivering tumor necrosis factor (RGD-A-TNF) to αV integrins on tumor endothelium. Trial progress and data was reviewed contemporaneously using a web-enabled electronic reporting system developed for the consortium. Dose-escalation in cohorts of 3 dogs (n = 24) determined an optimal safe dose (5×1012 transducing units intravenous) of RGD-A-TNF. This demonstrated selective targeting of tumor-associated vasculature and sparing of normal tissues assessed via serial biopsy of both tumor and normal tissue. Repetitive dosing in a cohort of 14 dogs, at the defined optimal dose, was well tolerated and led to objective tumor regression in two dogs (14%), stable disease in six (43%), and disease progression in six (43%) via Response Evaluation Criteria in Solid Tumors (RECIST). Conclusions/Significance The first study of the COTC has demonstrated the utility and efficiency of the established infrastructure to inform the development of new cancer drugs within large animal naturally occurring cancer models. The preclinical evaluation of RGD-A-TNF within this network provided valuable and necessary data to complete the design of first-in-man studies. PMID:19330034

Conjugation of arginine-glycine-aspartic acid peptides to poly(ethylene oxide)-b-poly(epsilon-caprolactone) micelles for enhanced intracellular drug delivery to metastatic tumor cells.

PubMed

Xiong, Xiao-Bing; Mahmud, Abdullah; Uludağ, Hasan; Lavasanifar, Afsaneh

2007-03-01

An arginine-glycine-aspartic acid (RGD) containing model peptide was conjugated to the surface of poly(ethylene oxide)-block-poly(epsilon-caprolactone) (PEO-b-PCL) micelles as a ligand that can recognize adhesion molecules overexpressed on the surface of metastatic cancer cells, that is, integrins, and that can enhance the micellar delivery of encapsulated hydrophobic drug into a tumor cell. Toward this goal, PEO-b-PCL copolymers bearing acetal groups on the PEO end were synthesized, characterized, and assembled to polymeric micelles. The acetal group on the surface of the PEO-b-PCL micelles was converted to reactive aldehyde under acidic condition at room temperature. An RGD-containing linear peptide, GRGDS, was conjugated on the surface of the aldehyde-decorated PEO-b-PCL micelles by incubation at room temperature. A hydrophobic fluorescent probe, that is, DiI, was physically loaded in prepared polymeric micelles to imitate hydrophobic drugs loaded in micellar carrier. The cellular uptake of DiI loaded GRGDS-modified micelles by melanoma B16-F10 cells was investigated at 4 and 37 degrees C by fluorescent spectroscopy and confocal microscopy techniques and was compared to the uptake of DiI loaded valine-PEO-b-PCL micelles (as the irrelevant ligand decorated micelles) and free DiI. GRGDS conjugation to polymeric micelles significantly facilitated the cellular uptake of encapsulated hydrophobic DiI most probably by intergrin-mediated cell attachment and endocytosis. The results indicate that acetal-terminated PEO-b-PCL micelles are amenable for introducing targeting moieties on the surface of polymeric micelles and that RGD-peptide conjugated PEO-b-PCL micelles are promising ligand-targeted carriers for enhanced drug delivery to metastatic tumor cells.

Magnetic targeting of mechanosensors in bone cells for tissue engineering applications.

PubMed

Hughes, Steven; Dobson, Jon; El Haj, Alicia J

2007-01-01

Mechanical signalling plays a pivotal role in maintaining bone cell function and remodelling of the skeleton. Our previous work has highlighted the potential role of mechano-induction in tissue engineering applications. In particular, we have highlighted the potential for using magnetic particle techniques for tissue engineering applications. Previous studies have shown that manipulation of integrin attached magnetic particles leads to changes in intracellular calcium signalling within osteoblasts. However, due to the phenomenon of particle internalisation, previous studies have typically focused on short-term stimulation experiments performed within 1-2 h of particle attachment. For tissue engineering applications, bone tissue growth occurs over a period of 3-5 weeks. To date, no study has investigated the cellular responses elicited from osteoblasts over time following stimulation with internalised magnetic particles. Here, we demonstrate the long-term biocompatibility of 4.5 microm RGD-coated particles with osteoblasts up to 21 days in culture, and detail a time course of responses elicited from osteoblasts following mechanical stimulation with integrin attached magnetic particles (<2h post attachment) and internalised particles (>48h post attachment). Mechanical manipulation of both integrin attached and internalised particles were found to induce intracellular calcium signalling. It is concluded that magnetic particles offer a tool for applying controlled mechanical forces to osteoblasts, and can be used to stimulate intracellular calcium signalling over prolonged periods of time. Magnetic particle technology presents a potentially valuable tool for tissue engineering which permits the delivery of highly localised mechano-inductive forces directly to cells.

Trench-shaped binding sites promote multiple classes of interactions between collagen and the adherence receptors, alpha(1)beta(1) integrin and Staphylococcus aureus cna MSCRAMM.

PubMed

Rich, R L; Deivanayagam, C C; Owens, R T; Carson, M; Höök, A; Moore, D; Symersky, J; Yang, V W; Narayana, S V; Höök, M

1999-08-27

Most mammalian cells and some pathogenic bacteria are capable of adhering to collagenous substrates in processes mediated by specific cell surface adherence molecules. Crystal structures of collagen-binding regions of the human integrin alpha(2)beta(1) and a Staphylococcus aureus adhesin reveal a "trench" on the surface of both of these proteins. This trench can accommodate a collagen triple-helical structure and presumably represents the ligand-binding site (Emsley, J., King, S. L., Bergelson, J. M., and Liddington, R. C. (1997) J. Biol. Chem. 272, 28512-28517; Symersky, J., Patti, J. M., Carson, M., House-Pompeo, K., Teale, M., Moore, D., Jin, L., Schneider, A., DeLucas, L. J., Höök, M., and Narayana, S. V. L. (1997) Nat. Struct. Biol. 4, 833-838). We report here the crystal structure of the alpha subunit I domain from the alpha(1)beta(1) integrin. This collagen-binding protein also contains a trench on one face in which the collagen triple helix may be docked. Furthermore, we compare the collagen-binding mechanisms of the human alpha(1) integrin I domain and the A domain from the S. aureus collagen adhesin, Cna. Although the S. aureus and human proteins have unrelated amino acid sequences, secondary structure composition, and cation requirements for effective ligand binding, both proteins bind at multiple sites within one collagen molecule, with the sites in collagen varying in their affinity for the adherence molecule. We propose that (i) these evolutionarily dissimilar adherence proteins recognize collagen via similar mechanisms, (ii) the multisite, multiclass protein/ligand interactions observed in these two systems result from a binding-site trench, and (iii) this unusual binding mechanism may be thematic for proteins binding extended, rigid ligands that contain repeating structural motifs.

Nitric oxide/cGMP pathway signaling actively down-regulates α4β1-integrin affinity: an unexpected mechanism for inducing cell de-adhesion.

PubMed

Chigaev, Alexandre; Smagley, Yelena; Sklar, Larry A

2011-05-17

Integrin activation in response to inside-out signaling serves as the basis for rapid leukocyte arrest on endothelium, migration, and mobilization of immune cells. Integrin-dependent adhesion is controlled by the conformational state of the molecule, which is regulated by seven-transmembrane Guanine nucleotide binding Protein-Coupled Receptors (GPCRs). α4β1-integrin (CD49d/CD29, Very Late Antigen-4, VLA-4) is expressed on leukocytes, hematopoietic progenitors, stem cells, hematopoietic cancer cells, and others. VLA-4 conformation is rapidly up-regulated by inside-out signaling through Gαi-coupled GPCRs and down-regulated by Gαs-coupled GPCRs. However, other signaling pathways, which include nitric oxide-dependent signaling, have been implicated in the regulation of cell adhesion. The goal of the current report was to study the effect of nitric oxide/cGMP signaling pathway on VLA-4 conformational regulation. Using fluorescent ligand binding to evaluate the integrin activation state on live cells in real-time, we show that several small molecules, which specifically modulate nitric oxide/cGMP signaling pathway, as well as a cell permeable cGMP analog, can rapidly down-modulate binding of a VLA-4 specific ligand on cells pre-activated through three Gαi-coupled receptors: wild type CXCR4, CXCR2 (IL-8RB), and a non-desensitizing mutant of formyl peptide receptor (FPR ΔST). Upon signaling, we detected rapid changes in the ligand dissociation rate. The dissociation rate after inside-out integrin de-activation was similar to the rate for resting cells. In a VLA-4/VCAM-1-specific myeloid cell adhesion system, inhibition of the VLA-4 affinity change by nitric oxide had a statistically significant effect on real-time cell aggregation. We conclude that nitric oxide/cGMP signaling pathway can rapidly down-modulate the affinity state of the VLA-4 binding pocket, especially under the condition of sustained Gαi-coupled GPCR signaling, generated by a non-desensitizing receptor mutant. This suggests a fundamental role of this pathway in de-activation of integrin-dependent cell adhesion.

Talin-dependent integrin activation is required for fibrin clot retraction by platelets

PubMed Central

Haling, Jacob R.; Monkley, Susan J.; Critchley, David R.

2011-01-01

Talin functions both as a regulator of integrin affinity and as an important mechanical link between integrins and the cytoskeleton. Using genetic deletion of talin, we show for the first time that the capacity of talin to activate integrins is required for fibrin clot retraction by platelets. To further dissect which talin functions are required for this process, we tested clot retraction in platelets expressing a talin1(L325R) mutant that binds to integrins, but exhibits impaired integrin activation ascribable to disruption of the interaction between talin and the membrane-proximal region (MPR) in the β-integrin cytoplasmic domain. Talin-deficient and talin1(L325R) platelets were defective in retracting fibrin clots. However, the defect in clot retraction in talin1(L325R) platelets, but not talin-deficient platelets, was rescued by extrinsically activating integrins with manganese, thereby proving that integrin activation is required and showing that talin1(L325R) can form functional links to the actin cytoskeleton. PMID:20971947

The Novel α4B Murine α4 Integrin Protein Splicing Variant Inhibits α4 Protein-dependent Cell Adhesion*

PubMed Central

Kouro, Hitomi; Kon, Shigeyuki; Matsumoto, Naoki; Miyashita, Tomoe; Kakuchi, Ayaka; Ashitomi, Dai; Saitoh, Kodai; Nakatsuru, Takuya; Togi, Sumihito; Muromoto, Ryuta; Matsuda, Tadashi

2014-01-01

Integrins affect the motility of multiple cell types to control cell survival, growth, or differentiation, which are mediated by cell-cell and cell-extracellular matrix interactions. We reported previously that the α9 integrin splicing variant, SFα9, promotes WT α9 integrin-dependent adhesion. In this study, we introduced a new murine α4 integrin splicing variant, α4B, which has a novel short cytoplasmic tail. In inflamed tissues, the expression of α4B, as well as WT α4 integrin, was up-regulated. Cells expressing α4B specifically bound to VCAM-1 but not other α4 integrin ligands, such as fibronectin CS1 or osteopontin. The binding of cells expressing WT α4 integrin to α4 integrin ligands is inhibited by coexpression of α4B. Knockdown of α4B in metastatic melanoma cell lines results in a significant increase in lung metastasis. Expression levels of WT α4 integrin are unaltered by α4B, with α4B acting as a regulatory subunit for WT α4 integrin by a dominant-negative effect or inhibiting α4 integrin activation. PMID:24755217

Integrin α1β1 participates in Chondrocyte Transduction of Osmotic Stress.

PubMed Central

Jablonski, Christina L.; Ferguson, Samuel; Pozzi, Ambra; Clark, Andrea L.

2014-01-01

Background/purpose: The goal of this study was to determine the role of the collagen binding receptor integrin α1β1 in regulating osmotically induced [Ca2+]i transients in chondrocytes. Methods: The [Ca2+]i transient response of chondrocytes to osmotic stress was measured using real-time confocal microscopy. Chondrocytes from wildtype and integrin α1-null mice were imaged ex vivo (in the cartilage of intact murine femora) and in vitro (isolated from the matrix, attached to glass coverslips). Immunocytochemistry was performed to detect the presence of the osmosensor, transient receptor potential vanilloid-4 (TRPV4), and the agonist GSK1016790A (GSK101) was used to test for its functionality on chondrocytes from wildtype and integrin α1-null mice. Results/interpretation: Deletion of the integrin α1 subunit inhibited the ability of chondrocytes to respond to a hypo-osmotic stress with [Ca2+]i transients ex vivo and in vitro. The percentage of chondrocytes responding ex vivo was smaller than in vitro and of the cells that responded, more single [Ca2+]i transients were observed ex vivo compared to in vitro. Immunocytochemistry confirmed the presence of TRPV4 on wildtype and integrin α1-null chondrocytes, however application of GSK101 revealed that TRPV4 could be activated on wildtype but not integrin α1-null chondrocytes. Integrin α1β1 is a key participant in chondrocyte transduction of a hypo-osmotic stress. Furthermore, the mechanism by which integrin α1β1 influences osmotransduction is independent of matrix binding, but likely dependent on the chondrocyte osmosensor TRPV4. PMID:24495803

Integrin α1β1 participates in chondrocyte transduction of osmotic stress.

PubMed

Jablonski, Christina L; Ferguson, Samuel; Pozzi, Ambra; Clark, Andrea L

2014-02-28

The goal of this study was to determine the role of the collagen binding receptor integrin α1β1 in regulating osmotically induced [Ca(2+)]i transients in chondrocytes. The [Ca(2+)]i transient response of chondrocytes to osmotic stress was measured using real-time confocal microscopy. Chondrocytes from wildtype and integrin α1-null mice were imaged ex vivo (in the cartilage of intact murine femora) and in vitro (isolated from the matrix, attached to glass coverslips). Immunocytochemistry was performed to detect the presence of the osmosensor, transient receptor potential vanilloid-4 (TRPV4), and the agonist GSK1016790A (GSK101) was used to test for its functionality on chondrocytes from wildtype and integrin α1-null mice. Deletion of the integrin α1 subunit inhibited the ability of chondrocytes to respond to a hypo-osmotic stress with [Ca(2+)]i transients ex vivo and in vitro. The percentage of chondrocytes responding ex vivo was smaller than in vitro and of the cells that responded, more single [Ca(2+)]i transients were observed ex vivo compared to in vitro. Immunocytochemistry confirmed the presence of TRPV4 on wildtype and integrin α1-null chondrocytes, however application of GSK101 revealed that TRPV4 could be activated on wildtype but not integrin α1-null chondrocytes. Integrin α1β1 is a key participant in chondrocyte transduction of a hypo-osmotic stress. Furthermore, the mechanism by which integrin α1β1 influences osmotransduction is independent of matrix binding, but likely dependent on the chondrocyte osmosensor TRPV4. Copyright © 2014 Elsevier Inc. All rights reserved.

Structure-guided design of a high-affinity platelet integrin αIIbβ3 receptor antagonist that disrupts Mg²⁺ binding to the MIDAS.

PubMed

Zhu, Jieqing; Choi, Won-Seok; McCoy, Joshua G; Negri, Ana; Zhu, Jianghai; Naini, Sarasija; Li, Jihong; Shen, Min; Huang, Wenwei; Bougie, Daniel; Rasmussen, Mark; Aster, Richard; Thomas, Craig J; Filizola, Marta; Springer, Timothy A; Coller, Barry S

2012-03-14

An integrin found on platelets, α(IIb)β(3) mediates platelet aggregation, and α(IIb)β(3) antagonists are effective antithrombotic agents in the clinic. Ligands bind to integrins in part by coordinating a magnesium ion (Mg(2+)) located in the β subunit metal ion-dependent adhesion site (MIDAS). Drugs patterned on the integrin ligand sequence Arg-Gly-Asp have a basic moiety that binds the α(IIb) subunit and a carboxyl group that coordinates the MIDAS Mg(2+) in the β(3) subunits. They induce conformational changes in the β(3) subunit that may have negative consequences such as exposing previously hidden epitopes and inducing the active conformation of the receptor. We recently reported an inhibitor of α(IIb)β(3) (RUC-1) that binds exclusively to the α(IIb) subunit; here, we report the structure-based design and synthesis of RUC-2, a RUC-1 derivative with a ~100-fold higher affinity. RUC-2 does not induce major conformational changes in β(3) as judged by monoclonal antibody binding, light scattering, gel chromatography, electron microscopy, and a receptor priming assay. X-ray crystallography of the RUC-2-α(IIb)β(3) headpiece complex in 1 mM calcium ion (Ca(2+))/5 mM Mg(2+) at 2.6 Å revealed that RUC-2 binds to α(IIb) the way RUC-1 does, but in addition, it binds to the β(3) MIDAS residue glutamic acid 220, thus displacing Mg(2+) from the MIDAS. When the Mg(2+) concentration was increased to 20 mM, however, Mg(2+) was identified in the MIDAS and RUC-2 was absent. RUC-2's ability to inhibit ligand binding and platelet aggregation was diminished by increasing the Mg(2+) concentration. Thus, RUC-2 inhibits ligand binding by a mechanism different from that of all other α(IIb)β(3) antagonists and may offer advantages as a therapeutic agent.

Peptide functionalized gold nanoparticles: the influence of pH on binding efficiency

NASA Astrophysics Data System (ADS)

Harrison, Emma; Hamilton, Jeremy W. J.; Macias-Montero, Manuel; Dixon, Dorian

2017-07-01

We report herein on the synthesis of mixed monolayer gold nanoparticles (AuNPs) capped with both polyethylene glycol (PEG) and one of three peptides. Either a receptor-mediated endocytosis peptide, an endosomal escape pathway (H5WYG) peptide or the Nrp-1 targeting RGD peptide (CRGDK) labeled with FITC. All three peptides have a thiol containing cysteine residue which can be used to bind the peptides to the AuNPs. In order to investigate the influence of pH on peptide attachment, PEGylated AuNPs were centrifuged, the supernatant removed, and the nanoparticles were then re-suspended in a range of pH buffer solutions above, below and at the respective isoelectric points of the peptides before co-functionalization. Peptide attachment was investigated using dynamic light scattering, Ultra-violet visible spectroscopy (UV/Vis), FTIR and photo luminescence spectroscopy. UV/Vis analysis coupled with protein assay results and photoluminescence of the FITC tagged RGD peptide concluded that a pH of ∼8 optimized the cysteine binding and stability, irrespective of the peptide used.

HPV binding assay to Laminin-332/integrin α6β4 on human keratinocytes.

PubMed

Brendle, Sarah A; Christensen, Neil D

2015-01-01

Human papillomaviruses (HPVs) have been shown to bind to Laminin-332 (Ln-332) on the extracellular matrix (ECM) secreted by human keratinocytes. The assay described here is an important tool to study HPV receptor binding to the ECM. The assay can also be modified to study the receptors required for HPV infection and for binding to tissues. We previously showed that Ln-332 is essential for the binding of HPV11 to human keratinocytes and that infectious entry of HPV11 requires α6β4 integrin for the transfer of HPV11 from ECM to host cells (Culp et al., J Virol 80:8940-8950, 2006). We also demonstrated that several of the high-risk HPV types (16, 18, 31 and 45) bind to Ln-332 and/or other components of the ECM in vitro (Broutian et al., J Gen Virol 91:531-540, 2010). The exact binding and internalization mechanism(s) for HPV are still under investigation. A better understanding of these mechanisms will aid in the design of therapeutics against HPVs and ultimately help prevent many cancers. In this chapter, we describe the HPV binding assay to Ln-332/integrin α6β4 on human keratinocytes (ECM). We also present data and suggestions for modifying the assay for testing the specificity of HPV for receptors (by blocking receptors) and binding to human tissues (basement membrane, BM) in order to study binding mechanisms.

Novel peptide-based platform for the dual presentation of biologically active peptide motifs on biomaterials.

PubMed

Mas-Moruno, Carlos; Fraioli, Roberta; Albericio, Fernando; Manero, José María; Gil, F Javier

2014-05-14

Biofunctionalization of metallic materials with cell adhesive molecules derived from the extracellular matrix is a feasible approach to improve cell-material interactions and enhance the biointegration of implant materials (e.g., osseointegration of bone implants). However, classical biomimetic strategies may prove insufficient to elicit complex and multiple biological signals required in the processes of tissue regeneration. Thus, newer strategies are focusing on installing multifunctionality on biomaterials. In this work, we introduce a novel peptide-based divalent platform with the capacity to simultaneously present distinct bioactive peptide motifs in a chemically controlled fashion. As a proof of concept, the integrin-binding sequences RGD and PHSRN were selected and introduced in the platform. The biofunctionalization of titanium with this platform showed a positive trend towards increased numbers of cell attachment, and statistically higher values of spreading and proliferation of osteoblast-like cells compared to control noncoated samples. Moreover, it displayed statistically comparable or improved cell responses compared to samples coated with the single peptides or with an equimolar mixture of the two motifs. Osteoblast-like cells produced higher levels of alkaline phosphatase on surfaces functionalized with the platform than on control titanium; however, these values were not statistically significant. This study demonstrates that these peptidic structures are versatile tools to convey multiple biofunctionality to biomaterials in a chemically defined manner.

The Talin Head Domain Reinforces Integrin-Mediated Adhesion by Promoting Adhesion Complex Stability and Clustering

PubMed Central

Ellis, Stephanie J.; Lostchuck, Emily; Goult, Benjamin T.; Bouaouina, Mohamed; Fairchild, Michael J.; López-Ceballos, Pablo; Calderwood, David A.; Tanentzapf, Guy

2014-01-01

Talin serves an essential function during integrin-mediated adhesion in linking integrins to actin via the intracellular adhesion complex. In addition, the N-terminal head domain of talin regulates the affinity of integrins for their ECM-ligands, a process known as inside-out activation. We previously showed that in Drosophila, mutating the integrin binding site in the talin head domain resulted in weakened adhesion to the ECM. Intriguingly, subsequent studies showed that canonical inside-out activation of integrin might not take place in flies. Consistent with this, a mutation in talin that specifically blocks its ability to activate mammalian integrins does not significantly impinge on talin function during fly development. Here, we describe results suggesting that the talin head domain reinforces and stabilizes the integrin adhesion complex by promoting integrin clustering distinct from its ability to support inside-out activation. Specifically, we show that an allele of talin containing a mutation that disrupts intramolecular interactions within the talin head attenuates the assembly and reinforcement of the integrin adhesion complex. Importantly, we provide evidence that this mutation blocks integrin clustering in vivo. We propose that the talin head domain is essential for regulating integrin avidity in Drosophila and that this is crucial for integrin-mediated adhesion during animal development. PMID:25393120

Identification and characterization of integrin-binding sialoprotein (IBSP) genes in reptile and amphibian.

PubMed

Shintani, Seikou; Kamakura, Naofumi; Kobata, Mitsuhiko; Toyosawa, Satoru; Onishi, Tomoyuki; Sato, Akie; Kawasaki, Kazuhiko; Weiss, Kenneth M; Ooshima, Takashi

2008-11-15

Integrin-binding sialoprotein (IBSP) is a member of the small integrin-binding ligand N-linked glycoprotein (SIBLING) family; and the whole SIBLING family is further included in a larger secretory calcium-binding phosphoprotein (SCPP) family. SIBLING proteins are known to construct a part of the non-collagenous extracellular matrices of calcified tissues, and considered to have arisen by duplication and subsequent divergent evolution of a single ancient gene. To understand the alterations of SIBLING molecules associated with the evolution of calcified tissues in vertebrates, we initiated a search for lower vertebrate orthologs of SIBLING genes. In the present study, an IBSP ortholog from a reptile (caiman) and two distinct orthologs from an amphibian (African clawed toad) were identified and characterized. As expected, the toad IBSP genes were transcribed only in calcified tissue (jaw and tibia), as also seen in mammals. The caiman, toad, avian, and mammalian IBSPs share several unique features specific for IBSP and apparently have similar properties. Furthermore, analysis of the sequences suggested that the IBSP molecule might have gradually intensified its functions related to calcification during its evolutionary process through tetrapods.

Connective tissue growth factor inhibits gastric cancer peritoneal metastasis by blocking integrin α3β1-dependent adhesion.

PubMed

Chen, Chiung-Nien; Chang, Cheng-Chi; Lai, Hong-Shiee; Jeng, Yung-Ming; Chen, Chia-I; Chang, King-Jeng; Lee, Po-Huang; Lee, Hsinyu

2015-07-01

Connective tissue growth factor (CTGF) plays important roles in normal and pathological conditions. The aim of this study was to investigate the role of CTGF in peritoneal metastasis as well as the underlying mechanism in gastric cancer progression. CTGF expression levels for wild-type and stable overexpression clones were determined by Western blotting and quantitative polymerase chain reaction (Q-PCR). Univariate and multivariate analyses, immunohistochemistry, and survival probability analyses were performed on gastric cancer patients. The extracellular matrix components involved in CTGF-regulated adhesion were determined. Recombinant CTGF was added to cells or coinoculated with gastric cancer cells into mice to evaluate its therapeutic potential. CTGF overexpression and treatment with the recombinant protein significantly inhibited cell adhesion. In vivo peritoneal metastasis demonstrated that CTGF-stable transfectants markedly decreased the number and size of tumor nodules in the mesentery. Statistical analysis of gastric cancer patient data showed that patients expressing higher CTGF levels had earlier TNM staging and a higher survival probability after the surgery. Integrin α3β1 was the cell adhesion molecule mediating gastric cancer cell adhesion to laminin, and blocking of integrin α3β1 prevented gastric cancer cell adhesion to recombinant CTGF. Coimmunoprecipitation results indicated that CTGF binds to integrin α3. Coinoculation of recombinant CTGF and gastric cancer cell lines in mice showed effective inhibition of peritoneal dissemination. Our results suggested that gastric cancer peritoneal metastasis is mediated through integrin α3β1 binding to laminin, and CTGF effectively blocks the interaction by binding to integrin α3β1, thus demonstrating the therapeutic potential of recombinant CTGF in gastric cancer patients.

Very late antigen integrins are involved in the adhesive interaction of lymphoid cells to human gingival fibroblasts.

PubMed Central

Murakami, S; Saho, T; Shimabukuro, Y; Isoda, R; Miki, Y; Okada, H

1993-01-01

To date, it is still unclear how the trafficking and retention of activated lymphocytes in periodontal lesions are regulated. In this study, we investigated the molecular basis for the adhesive interactions between lymphocytes and human gingival fibroblasts (HGF). Peripheral blood T lymphocytes (PBT) exhibited binding ability, but only when the calls were activated with phorbol 12-myristate 13-acetate (PMA). Among several human cell lines tested, PMA-stimulated Molt-4, a human T-cell leukaemia line, also displayed significant binding ability to HGF. In order to clarify the molecule(s) involved in this cell-cell interaction, a panel of monoclonal antibodies (mAb) was prepared to PMA-activated Molt-4 and one clone, 4-145, was selected on the basis of its ability to block the binding of PMA-activated Molt-4 to HGF. Moreover, 4-145 inhibited the binding of not only activated Molt-4 but also activated PBT and other cell types to HGF. Biochemical and flow cytometric analyses revealed that 4-145 probably recognizes the beta 1 chain of very late antigen (VLA) integrins. Blocking experiments using mAb specific for the alpha-chain of VLA integrins demonstrated the involvement of alpha 4 (VLA-4) and, to a lesser extent, alpha 5 (VLA-5) chains in the adhesive interactions between T cells and HGF. Despite the significant involvement of VLA integrins in the adhesive interaction between PBT and HGF, the binding of PBT to human dermal fibroblasts (HDF) was not abrogated by 4-145, suggesting that HGF and HDF differ in their requirement of VLA integrins for adhesion to activated PBT. Furthermore, the fact that vascular cell adhesion molecule-1 (VCAM-1), one of the ligands of VLA-4, was not detected on HGF by flow cytometry and anti-fibronectin (FN) Ab did not block the adhesive interaction to HGF suggests that not-yet-identified ligand(s) for VLA-4 might be present on HGF. Images Figure 4 PMID:8406571

Platelets lacking PIP5KIγ have normal integrin activation but impaired cytoskeletal-membrane integrity and adhesion

PubMed Central

Wang, Yanfeng; Zhao, Liang; Suzuki, Aae; Lian, Lurong; Min, Sang H.; Wang, Ziqian; Litvinov, Rustem I.; Stalker, Timothy J.; Yago, Tadayuki; Klopocki, Arkadiusz G.; Schmidtke, David W.; Yin, Helen; Choi, John K.; McEver, Rodger P.; Weisel, John W.; Hartwig, John H.; Abrams, Charles S.

2013-01-01

Three isoforms of phosphatidylinositol-4-phosphate 5-kinase (PIP5KIα, PIP5KIβ, and PIP5KIγ) can each catalyze the final step in the synthesis of phosphatidylinositol-4,5-bisphosphate (PIP2), which in turn can be either converted to second messengers or bind directly to and thereby regulate proteins such as talin. A widely quoted model speculates that only p90, a longer splice form of platelet-specific PIP5KIγ, but not the shorter p87 PIP5KIγ, regulates the ligand-binding activity of integrins via talin. However, when we used mice genetically engineered to lack only p90 PIP5KIγ, we found that p90 PIP5KIγ is not critical for integrin activation or platelet adhesion on collagen. However, p90 PIP5KIγ-null platelets do have impaired anchoring of their integrins to the underlying cytoskeleton. Platelets lacking both the p90 and p87 PIP5KIγ isoforms had normal integrin activation and actin dynamics, but impaired anchoring of their integrins to the cytoskeleton. Most importantly, they formed weak shear-resistant adhesions ex vivo and unstable vascular occlusions in vivo. Together, our studies demonstrate that, although PIP5KIγ is essential for normal platelet function, individual isoforms of PIP5KIγ fulfill unique roles for the integrin-dependent integrity of the membrane cytoskeleton and for the stabilization of platelet adhesion. PMID:23372168

Developing Targeted Hybrid Imaging Probes by Chelator Scaffolding

PubMed Central

2017-01-01

Positron emission tomography (PET) as well as optical imaging (OI) with peptide receptor targeting probes have proven their value for oncological applications but also show restrictions depending on the clinical field of interest. Therefore, the combination of both methods, particularly in a single molecule, could improve versatility in clinical routine. This proof of principle study aims to show that a chelator, Fusarinine C (FSC), can be utilized as scaffold for novel dimeric dual-modality imaging agents. Two targeting vectors (a minigastrin analogue (MG11) targeting cholecystokinin-2 receptor overexpression (CCK2R) or integrin αVβ3 targeting cyclic pentapeptides (RGD)) and a near-infrared fluorophore (Sulfo-Cyanine7) were conjugated to FSC. The probes were efficiently labeled with gallium-68 and in vitro experiments including determination of logD, stability, protein binding, cell binding, internalization, and biodistribution studies as well as in vivo micro-PET/CT and optical imaging in U-87MG αVβ3- and A431-CCK2R expressing tumor xenografted mice were carried out. Novel bioconjugates showed high receptor affinity and highly specific targeting properties at both receptors. Ex vivo biodistribution and micro-PET/CT imaging studies revealed specific tumor uptake accompanied by slow blood clearance and retention in nontargeted tissues (spleen, liver, and kidneys) leading to visualization of tumors at early (30 to 120 min p.i.). Excellent contrast in corresponding optical imaging studies was achieved especially at delayed time points (24 to 72 h p.i.). Our findings show the proof of principle of chelator scaffolding for hybrid imaging agents and demonstrate FSC being a suitable bifunctional chelator for this approach. Improvements to fine-tune pharmacokinetics are needed to translate this into a clinical setting. PMID:28462989

Novel Regulation of Integrin Trafficking by Rab11-FIP5 in Aggressive Prostate Cancer.

PubMed

Das, Lipsa; Gard, Jaime M C; Prekeris, Rytis; Nagle, Raymond B; Morrissey, Colm; Knudsen, Beatrice S; Miranti, Cindy K; Cress, Anne E

2018-05-14

The laminin-binding integrins, α3β1 and α6β1, are needed for tumor metastasis and their surface expression is regulated by endocytic recycling. β1 integrins share the Rab11 recycling machinery but the trafficking of α3β1 and α6β1 are distinct by an unknown mechanism. Using a mouse PDX tumor model containing human metastatic prostate cancer, Rab11 family interacting protein 5 (Rab11-FIP5) was identified as a lead candidate for α6β1 trafficking. Rab11-FIP5 and its membrane binding domain were required for α6β1 recycling, without affecting the other laminin-binding integrin (i.e., α3β1) or unrelated membrane receptors like CD44, transferrin receptor, or E-cadherin. Depletion of Rab11-FIP5 resulted in the intracellular accumulation of α6β1 in the Rab11 recycling compartment, loss of cell migration on laminin, and an unexpected loss of α6β1 recycling in cell-cell locations. Taken together, these data demonstrate that α6β1 is distinct from α3β1 via Rab11-FIP5 recycling and recycles in an unexpected cell-cell location. Rab11-FIP5 dependent a6b1 integrin recycling may be selectively targeted to limit migration of prostate cancer cells into laminin-rich tissues. Copyright ©2018, American Association for Cancer Research.

Adhesion signaling promotes protease‑driven polyploidization of glioblastoma cells.

PubMed

Mercapide, Javier; Lorico, Aurelio

2014-11-01

An increase in ploidy (polyploidization) causes genomic instability in cancer. However, the determinants for the increased DNA content of cancer cells have not yet been fully elucidated. In the present study, we investigated whether adhesion induces polyploidization in human U87MG glioblastoma cells. For this purpose, we employed expression vectors that reported transcriptional activation by signaling networks implicated in cancer. Signaling activation induced by intercellular integrin binding elicited both extracellular signal‑regulated kinase (ERK) and Notch target transcription. Upon the prolonged activation of both ERK and Notch target transcription induced by integrin binding to adhesion protein, cell cultures accumulated polyploid cells, as determined by cell DNA content distribution analysis and the quantification of polynucleated cells. This linked the transcriptional activation induced by integrin adhesion to the increased frequency of polyploidization. Accordingly, the inhibition of signaling decreased the extent of polyploidization mediated by protease‑driven intracellular invasion. Therefore, the findings of this study indicate that integrin adhesion induces polyploidization through the stimulation of glioblastoma cell invasiveness.

Osteoblast mineralization requires β1 integrin/ICAP-1–dependent fibronectin deposition

PubMed Central

Brunner, Molly; Millon-Frémillon, Angélique; Chevalier, Genevieve; Nakchbandi, Inaam A.; Mosher, Deane; Block, Marc R.

2011-01-01

The morphogenetic and differentiation events required for bone formation are orchestrated by diffusible and insoluble factors that are localized within the extracellular matrix. In mice, the deletion of ICAP-1, a modulator of β1 integrin activation, leads to severe defects in osteoblast proliferation, differentiation, and mineralization and to a delay in bone formation. Deposition of fibronectin and maturation of fibrillar adhesions, adhesive structures that accompany fibronectin deposition, are impaired upon ICAP-1 loss, as are type I collagen deposition and mineralization. Expression of β1 integrin with a mutated binding site for ICAP-1 recapitulates the ICAP-1–null phenotype. Follow-up experiments demonstrated that ICAP-1 negatively regulates kindlin-2 recruitment onto the β1 integrin cytoplasmic domain, whereas an excess of kindlin-2 binding has a deleterious effect on fibrillar adhesion formation. These results suggest that ICAP-1 works in concert with kindlin-2 to control the dynamics of β1 integrin–containing fibrillar adhesions and, thereby, regulates fibronectin deposition and osteoblast mineralization. PMID:21768292

Cbl Associates with Pyk2 and Src to Regulate Src Kinase Activity, αvβ3 Integrin-Mediated Signaling, Cell Adhesion, and Osteoclast Motility

PubMed Central

Sanjay, Archana; Houghton, Adam; Neff, Lynn; DiDomenico, Emilia; Bardelay, Chantal; Antoine, Evelyne; Levy, Joan; Gailit, James; Bowtell, David; Horne, William C.; Baron, Roland

2001-01-01

The signaling events downstream of integrins that regulate cell attachment and motility are only partially understood. Using osteoclasts and transfected 293 cells, we find that a molecular complex comprising Src, Pyk2, and Cbl functions to regulate cell adhesion and motility. The activation of integrin αvβ3 induces the [Ca2+]i-dependent phosphorylation of Pyk2 Y402, its association with Src SH2, Src activation, and the Src SH3-dependent recruitment and phosphorylation of c-Cbl. Furthermore, the PTB domain of Cbl is shown to bind to phosphorylated Tyr-416 in the activation loop of Src, the autophosphorylation site of Src, inhibiting Src kinase activity and integrin-mediated adhesion. Finally, we show that deletion of c Src or c-Cbl leads to a decrease in osteoclast migration. Thus, binding of αvβ3 integrin induces the formation of a Pyk2/Src/Cbl complex in which Cbl is a key regulator of Src kinase activity and of cell adhesion and migration. These findings may explain the osteopetrotic phenotype in the Src−/− mice. PMID:11149930

The adapter protein SLP-76 mediates "outside-in" integrin signaling and function in T cells.

PubMed

Baker, R G; Hsu, C J; Lee, D; Jordan, M S; Maltzman, J S; Hammer, D A; Baumgart, T; Koretzky, G A

2009-10-01

The adapter protein SH2 domain-containing leukocyte protein of 76 kDa (SLP-76) is an essential mediator of signaling from the T-cell antigen receptor (TCR). We report here that SLP-76 also mediates signaling downstream of integrins in T cells and that SLP-76-deficient T cells fail to support adhesion to integrin ligands. In response to both TCR and integrin stimulation, SLP-76 relocalizes to surface microclusters that colocalize with phosphorylated signaling proteins. Disruption of SLP-76 recruitment to the protein named LAT (linker for activation of T cells) inhibits SLP-76 clustering downstream of the TCR but not downstream of integrins. Conversely, an SLP-76 mutant unable to bind ADAP (adhesion and degranulation-promoting adapter protein) forms clusters following TCR but not integrin engagement and fails to support T-cell adhesion to integrin ligands. These findings demonstrate that SLP-76 relocalizes to integrin-initiated signaling complexes by a mechanism different from that employed during TCR signaling and that SLP-76 relocalization corresponds to SLP-76-dependent integrin function in T cells.

Method of increasing radiation sensitivity by inhibition of beta one integrin

DOEpatents

Park, Catherine [San Francisco, CA; Bissell, Mina J [Berkeley, CA

2009-11-17

A method for increasing or monitoring apoptosis in tumor cells by the co-administration of ionizing radiation and an anti-integrin antibody. Increasing apoptosis reduces tumor growth in vivo and in a cell culture model. The antibody is directed against the beta-1 integrin subunit and is inhibitory of beta-1 integrin signaling. Other molecules having an inhibitory effect on beta-1 integrin, either in signaling or in binding to its cognate extracellular receptors may also be used. The present method is particularly of interest in treatment of tumor cells associated with breast cancer, wherein radiation is currently used alone. The present method further contemplates a monoclonal antibody suitable for human administration that may further comprise a radioisotope attached thereto.

ADAM disintegrin-like domain recognition by the lymphocyte integrins α4β1 and α4β7

PubMed Central

Bridges, Lance C.; Sheppard, Dean; Bowditch, Ron D.

2004-01-01

The ADAM (a disintegrin and metalloprotease) family of proteins possess both proteolytic and adhesive domains. We have established previously that the disintegrin domain of ADAM28, an ADAM expressed by human lymphocytes, is recognized by the integrin α4β1. The present study characterizes the integrin binding properties of the disintegrin-like domains of human ADAM7, ADAM28 and ADAM33 with the integrins α4β1, α4β7 and α9β1. Cell-adhesion assays demonstrated that, similar to ADAM28, the ADAM7 disintegrin domain supported α4β1-dependent Jurkat cell adhesion, whereas the ADAM33 disintegrin domain did not. The lymphocyte integrin α4β7 was also found to recognize both disintegrin domains of ADAM7 and ADAM28, but not of ADAM33. This is the first demonstration that mammalian disintegrins are capable of interacting with α4β7. All three disintegrin domains supported α9β1-dependent cell adhesion. Recognition by both α4β1 and α4β7 of ADAM7 and ADAM28 was activation-dependent, requiring either the presence of Mn2+ or an activating monoclonal antibody for cell attachment. Charge-to-alanine mutagenesis experiments revealed that the same residues within an individual ADAM disintegrin domain function in recognizing multiple integrins. However, the residues within a specific region of each ADAM disintegrin-like domain required for integrin binding were distinct. These results establish that ADAM7 and ADAM28 are recognized by the leucocyte integrins α4β1, α4β7 and α9β1. ADAM33 exclusively supported only α9β1-dependent adhesion. PMID:15504110

Expression of an Activated Integrin Promotes Long-Distance Sensory Axon Regeneration in the Spinal Cord

PubMed Central

Cheah, Menghon; Chew, Daniel J.; Moloney, Elizabeth B.; Verhaagen, Joost; Fässler, Reinhard

2016-01-01

After CNS injury, axon regeneration is blocked by an inhibitory environment consisting of the highly upregulated tenascin-C and chondroitin sulfate proteoglycans (CSPGs). Tenascin-C promotes growth of axons if they express a tenascin-binding integrin, particularly α9β1. Additionally, integrins can be inactivated by CSPGs, and this inhibition can be overcome by the presence of a β1-binding integrin activator, kindlin-1. We examined the synergistic effect of α9 integrin and kindlin-1 on sensory axon regeneration in adult rat spinal cord after dorsal root crush and adeno-associated virus transgene expression in dorsal root ganglia. After 12 weeks, axons from C6–C7 dorsal root ganglia regenerated through the tenascin-C-rich dorsal root entry zone into the dorsal column up to C1 level and above (>25 mm axon length) through a normal pathway. Animals also showed anatomical and electrophysiological evidence of reconnection to the dorsal horn and behavioral recovery in mechanical pressure, thermal pain, and ladder-walking tasks. Expression of α9 integrin or kindlin-1 alone promoted much less regeneration and recovery. SIGNIFICANCE STATEMENT The study demonstrates that long-distance sensory axon regeneration over a normal pathway and with sensory and sensory–motor recovery can be achieved. This was achieved by expressing an integrin that recognizes tenascin-C, one of the components of glial scar tissue, and an integrin activator. This enabled extensive long-distance (>25 mm) regeneration of both myelinated and unmyelinated sensory axons with topographically correct connections in the spinal cord. The extent of growth and recovery we have seen would probably be clinically significant. Restoration of sensation to hands, perineum, and genitalia would be a significant improvement for a spinal cord-injured patient. PMID:27383601

The role of a conserved membrane proximal cysteine in altering αPS2CβPS integrin diffusion.

PubMed

Syed, Aleem; Arora, Neha; Bunch, Thomas A; Smith, Emily A

2016-11-15

Cysteine residues (Cys) in the membrane proximal region are common post-translational modification (PTM) sites in transmembrane proteins. Herein, the effects of a highly conserved membrane proximal α-subunit Cys 1368 on the diffusion properties of αPS2CβPS integrins are reported. Sequence alignment shows that this cysteine is palmitoylated in human α3 and α6 integrin subunits. Replacing Cys 1368 in wild-type integrins with valine (Val 1368 ) putatively blocks a PTM site and alters integrins' ligand binding and diffusion characteristics. Both fluorescence recovery after photobleaching (FRAP) and single particle tracking (SPT) diffusion measurements show Val 1368 integrins are more mobile compared to wild-type integrins. Approximately 33% and 8% more Val 1368 integrins are mobile as measured by FRAP and SPT, respectively. The mobile Val 1368 integrins also exhibit less time-dependent diffusion, as measured by FRAP. Tandem mass spectrometry data suggest that Cys 1368 contains a redox or palmitoylation PTM in αPS2CβPS integrins. This membrane proximal Cys may play an important role in the diffusion of other alpha subunits that contain this conserved residue.

Near-infrared optical guided surgery of highly infiltrative fibrosarcomas in cats using an anti-αvß3 integrin molecular probe.

PubMed

Wenk, Christiane H F; Ponce, Frédérique; Guillermet, Stéphanie; Tenaud, Corinne; Boturyn, Didier; Dumy, Pascal; Watrelot-Virieux, Dorothée; Carozzo, Claude; Josserand, Véronique; Coll, Jean-Luc

2013-07-01

We investigated how near-infrared imaging could improve highly infiltrative spontaneous fibrosarcoma surgery in 12 cats in a clinical veterinary phase. We used an RGD-based nanoprobe at different doses and times before surgery and a portable clinical grade imaging system. All tumours were labelled by the tracer and had an overall tumour-to-healthy tissue ratio of 14±1 during surgery. No false negatives were found, and the percentage of tumour cells was linearly correlated with the fluorescence intensity. All cats recovered well and were submitted to long-term follow-up that is currently on-going 1year after the beginning of the study. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Usage of heparan sulfate, integrins, and FAK in HPV16 infection

PubMed Central

Abban, Cynthia Y.; Meneses, Patricio I.

2010-01-01

Human Papillomavirus Type 16 (HPV16) is the major causative agent of cervical cancer. Studies regarding the early binding and signaling molecules that play a significant role in infection are still lacking. The current study analyses the role of heparan sulfate, integrins, and the signaling molecule FAK in HPV16 infection of human adult keratinocytes cell line (HaCaTs). Our data demonstrate that infection requires the binding of viral particles to heparan sulfate followed by activation of focal adhesion kinase through an integrin. Infections were reduced in the presence of the FAK inhibitor, TAE226. TAE226 was observed to inhibit viral entry to the early endosome a known infectious route. These findings suggest that FAK can serve as a novel target for antiviral therapy. PMID:20441998

Functional Interaction of CD154 Protein with α5β1 Integrin Is Totally Independent from Its Binding to αIIbβ3 Integrin and CD40 Molecules*

PubMed Central

El Fakhry, Youssef; Alturaihi, Haydar; Yacoub, Daniel; Liu, Lihui; Guo, Wenyan; Leveillé, Claire; Jung, Daniel; Khzam, Lara Bou; Merhi, Yahye; Wilkins, John A.; Li, Hongmin; Mourad, Walid

2012-01-01

In addition to its classical CD40 receptor, CD154 also binds to αIIbβ3, α5β1, and αMβ2 integrins. Binding of CD154 to these receptors seems to play a key role in the pathogenic processes of chronic inflammation. This investigation was aimed at analyzing the functional interaction of CD154 with CD40, αIIbβ3, and α5β1 receptors. We found that the binding affinity of CD154 for αIIbβ3 is ∼4-fold higher than for α5β1. We also describe the generation of sCD154 mutants that lost their ability to bind CD40 or αIIbβ3 and show that CD154 residues involved in its binding to CD40 or αIIbβ3 are distinct from those implicated in its interaction to α5β1, suggesting that sCD154 may bind simultaneously to different receptors. Indeed, sCD154 can bind simultaneously to CD40 and α5β1 and biologically activate human monocytic U937 cells expressing both receptors. The simultaneous engagement of CD40 and α5β1 activates the mitogen-activated protein kinases, p38, and extracellular signal-related kinases 1/2 and synergizes in the release of inflammatory mediators MMP-2 and -9, suggesting a cross-talk between these receptors. PMID:22461623

SEL1L Regulates Adhesion, Proliferation and Secretion of Insulin by Affecting Integrin Signaling

PubMed Central

Diaferia, Giuseppe R.; Cirulli, Vincenzo; Biunno, Ida

2013-01-01

SEL1L, a component of the endoplasmic reticulum associated degradation (ERAD) pathway, has been reported to regulate the (i) differentiation of the pancreatic endocrine and exocrine tissue during the second transition of mouse embryonic development, (ii) neural stem cell self-renewal and lineage commitment and (iii) cell cycle progression through regulation of genes related to cell-matrix interaction. Here we show that in the pancreas the expression of SEL1L is developmentally regulated, such that it is readily detected in developing islet cells and in nascent acinar clusters adjacent to basement membranes, and becomes progressively restricted to the islets of Langherans in post-natal life. This peculiar expression pattern and the presence of two inverse RGD motifs in the fibronectin type II domain of SEL1L protein indicate a possible interaction with cell adhesion molecules to regulate islets architecture. Co-immunoprecipitation studies revealed SEL1L and ß1-integrin interaction and, down-modulation of SEL1L in pancreatic ß-cells, negatively influences both cell adhesion on selected matrix components and cell proliferation likely due to altered ERK signaling. Furthermore, the absence of SEL1L protein strongly inhibits glucose-stimulated insulin secretion in isolated mouse pancreatic islets unveiling an important role of SEL1L in insulin trafficking. This phenotype can be rescued by the ectopic expression of the ß1-integrin subunit confirming the close interaction of these two proteins in regulating the cross-talk between extracellular matrix and insulin signalling to create a favourable micro-environment for ß-cell development and function. PMID:24324549

Synthesis and evaluation of a radioiodinated peptide probe targeting αvβ6 integrin for the detection of pancreatic ductal adenocarcinoma

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ueda, Masashi; Department of Patho-Functional Bioanalysis, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501; Fukushima, Takahiro

Highlights: • We developed a radioiodinated peptide probe targeting αvβ6 integrin ({sup 123}I-IFMDV2). • {sup 123}I-IFMDV2 had a high affinity and selectivity for αvβ6 integrin. • {sup 123}I-IFMDV2 showed a specific binding to αvβ6 integrin in vivo. • {sup 123}I-IFMDV2 enabled clear visualization of the αvβ6-integrin-positive tumor. - Abstract: Introduction: Pancreatic ductal adenocarcinoma (PDAC) remains a major cause of cancer-related death. Since significant upregulation of αvβ6 integrin has been reported in PDAC, this integrin is a promising target for PDAC detection. In this study, we aimed to develop a radioiodinated probe for the imaging of αvβ6 integrin-positive PDAC with single-photonmore » emission computed tomography (SPECT). Methods: Four peptide probes were synthesized and screened by competitive and saturation binding assays using 2 PDAC cell lines (AsPC-1, αvβ6 integrin-positive; MIA PaCa-2, αvβ6 integrin-negative). The probe showing the best affinity was used to study the biodistribution assay, an in vivo blocking study, and SPECT imaging using tumor bearing mice. Autoradiography and immunohistochemical analysis were also performed. Results: Among the 4 probes examined in this study, {sup 125}I-IFMDV2 showed the highest affinity for αvβ6 integrin expressed in AsPC-1 cells and no affinity for MIA PaCa-2 cells. The accumulation of {sup 125}I-IFMDV2 in the AsPC-1 xenograft was 3–5 times greater than that in the MIA PaCa-2 xenograft, consistent with the expression of αvβ6 integrin in each xenograft, and confirmed by immunohistochemistry. Pretreatment with excess amounts of A20FMDV2 significantly blocked the accumulation of {sup 125}I-IFMDV2 in the AsPC-1 xenograft, but not in the MIA PaCa-2 xenograft. Furthermore, {sup 123}I-IFMDV2 enabled clear visualization of the AsPC-1 xenograft. Conclusion: {sup 123}I-IFMDV2 is a potential SPECT probe for the imaging of αvβ6 integrin in PDAC.« less

Peripheral myelin protein-22 (PMP22) modulates alpha 6 integrin expression in the human endometrium.

PubMed

Rao, Rajiv G; Sudhakar, Deepthi; Hogue, Claire P; Amici, Stephanie; Gordon, Lynn K; Braun, Jonathan; Notterpek, Lucia; Goodglick, Lee; Wadehra, Madhuri

2011-04-25

PMP22, a member of the GAS3 family of tetraspan proteins, is associated with a variety of neurological diseases. Previous studies have shown that PMP22 is expressed in proliferative endometrium, but its function within this tissue is poorly understood. In this study, we first characterized the expression of PMP22 in the human menstrual cycle and began to characterize its function in the endometrium. Using a combination of immunohistochemistry and quantitative PCR, we characterized the expression of PMP22 in both proliferative and secretory endometrium. Differences in PMP22 expression between proliferative and secretory endometrium were determined using a Mann-Whitney U test. In order to investigate the influence of PMP22 on α6 integrin expression, cells were created that ectopically overexpressed PMP22 or expressed a siRNA to inhibit its expression. These cells were analyzed for changes in integrins and binding to extracellular matrices. In this study, we show that PMP22 expression is higher in proliferative phase than secretory phase. Functionally, we have begun to characterize the functional significance of this expression. Previous studies have suggested a link between PMP22 and α6 integrin, and therefore we asked whether PMP22 could associate or potentially modulate the expression of α6 integrin. Expression of both PMP22 and α6 integrin were detectable in endometrial epithelial and stromal cells, and we show that both proteins can associate and colocalize with each other. To understand if PMP22 directly altered the expression of a6 integrin, we examined cell lines with modulated levels of the protein. Overexpression of PMP22 was sufficient to increase α6 integrin surface expression with a concominant increase in binding to the extracellular matrix laminin, while a reduction in PMP22 suppressed α6 integrin surface expression. These findings suggest a physiologic role for PMP22 on the expression of α6 integrin. We predict that this may be important for the maintainence of endometrial integrity and to the disease biology associated with altered levels of α6 integrin expression in the endometrium.

Distinct roles of beta1 metal ion-dependent adhesion site (MIDAS), adjacent to MIDAS (ADMIDAS), and ligand-associated metal-binding site (LIMBS) cation-binding sites in ligand recognition by integrin alpha2beta1.

PubMed

Valdramidou, Dimitra; Humphries, Martin J; Mould, A Paul

2008-11-21

Integrin-ligand interactions are regulated in a complex manner by divalent cations, and previous studies have identified ligand-competent, stimulatory, and inhibitory cation-binding sites. In collagen-binding integrins, such as alpha2beta1, ligand recognition takes place exclusively at the alpha subunit I domain. However, activation of the alphaI domain depends on its interaction with a structurally similar domain in the beta subunit known as the I-like or betaI domain. The top face of the betaI domain contains three cation-binding sites: the metal-ion dependent adhesion site (MIDAS), the ADMIDAS (adjacent to MIDAS), and LIMBS (ligand-associated metal-binding site). The role of these sites in controlling ligand binding to the alphaI domain has yet to be elucidated. Mutation of the MIDAS or LIMBS completely blocked collagen binding to alpha2beta1; in contrast mutation of the ADMIDAS reduced ligand recognition but this effect could be overcome by the activating monoclonal antibody TS2/16. Hence, the MIDAS and LIMBS appear to be essential for the interaction between alphaI and betaI, whereas occupancy of the ADMIDAS has an allosteric effect on the conformation of betaI. An activating mutation in the alpha2 I domain partially restored ligand binding to the MIDAS and LIMBS mutants. Analysis of the effects of Ca(2+), Mg(2+), and Mn(2+) on ligand binding to these mutants showed that the MIDAS is a ligand-competent site through which Mn(2+) stimulates ligand binding, whereas the LIMBS is a stimulatory Ca(2+)-binding site, occupancy of which increases the affinity of Mg(2+) for the MIDAS.

Defining the Role of Integrin Alpha 11 in Wound Healing and Fibrosis

DTIC Science & Technology

2009-09-01

Introduction Scleroderma is characterized by fibrosis, the replacement of healthy tissue with collagenous matrix. The collagen-binding integrins are...Itga11 and determine its role in the etiology of fibrosis and scleroderma . Body The heterozygous (Het) phenotype of one intact Itga11 allele and one

Cloning and characterization of cDNA encoding cardosin A, an RGD-containing plant aspartic proteinase.

PubMed

Faro, C; Ramalho-Santos, M; Vieira, M; Mendes, A; Simões, I; Andrade, R; Veríssimo, P; Lin, X; Tang, J; Pires, E

1999-10-01

Cardosin A is an abundant aspartic proteinase from pistils of Cynara cardunculus L. whose milk-clotting activity has been exploited for the manufacture of cheese. Here we report the cloning and characterization of cardosin A cDNA. The deduced amino acid sequence contains the conserved features of plant aspartic proteinases, including the plant-specific insertion (PSI), and revealed the presence of an Arg-Gly-Asp (RGD) motif, which is known to function in cell surface receptor binding by extracellular proteins. Cardosin A mRNA was detected predominantly in young flower buds but not in mature or senescent pistils, suggesting that its expression is likely to be developmentally regulated. Procardosin A, the single chain precursor, was found associated with microsomal membranes of flower buds, whereas the active two-chain enzyme generated upon removal of PSI is soluble. This result implies a role for PSI in promoting the association of plant aspartic proteinase precursors to cell membranes. To get further insights about cardosin A, the functional relevance of the RGD motif was also investigated. A 100-kDa protein that interacts specifically with the RGD sequence was isolated from octyl glucoside pollen extracts by affinity chromatography on cardosin A-Sepharose. This result suggests that the 100-kDa protein is a cardosin A receptor and indicates that the interaction between these two proteins is apparently mediated through RGD recognition. It is possible therefore that cardosin A may have a role in adhesion-mediated proteolytic mechanisms involved in pollen recognition and growth.

DISTINCT ROLES OF β1 MIDAS, ADMIDAS AND LIMBS CATION-BINDING SITES IN LIGAND RECOGNITION BY INTEGRIN α2β1*

PubMed Central

Valdramidou, Dimitra; Humphries, Martin J.; Mould, A. Paul

2012-01-01

Integrin-ligand interactions are regulated in a complex manner by divalent cations, and previous studies have identified ligand-competent, stimulatory, and inhibitory cation-binding sites. In collagen-binding integrins, such as α2β1, ligand recognition takes place exclusively at the α subunit I domain. However, activation of the αI domain depends on its interaction with a structurally similar domain in the β subunit known as the I-like or βI domain. The top face of the βI domain contains three cation-binding sites: the metal-ion dependent adhesion site (MIDAS), the ADMIDAS (adjacent to MIDAS) and LIMBS (ligand-associated metal binding site). The role of these sites in controlling ligand binding to the αI domain has yet to be elucidated. Mutation of the MIDAS or LIMBS completely blocked collagen binding to α2β1; in contrast mutation of the ADMIDAS reduced ligand recognition but this effect could be overcome by the activating mAb TS2/16. Hence, the MIDAS and LIMBS appear to be essential for the interaction between αI and βI whereas occupancy of the ADMIDAS has an allosteric effect on the conformation of βI. An activating mutation in the α2 I domain partially restored ligand binding to the MIDAS and LIMBS mutants. Analysis of the effects of Ca2+, Mg2+ and Mn2+ on ligand binding to these mutants showed that the MIDAS is a ligand-competent site through which Mn2+ stimulates ligand binding, whereas the LIMBS is a stimulatory Ca2+-binding site, occupancy of which increases the affinity of Mg2+ for the MIDAS. PMID:18820259

Melanoma cultures show different susceptibility towards E1A-, E1B-19 kDa- and fiber-modified replication-competent adenoviruses.

PubMed

Schmitz, M; Graf, C; Gut, T; Sirena, D; Peter, I; Dummer, R; Greber, U F; Hemmi, S

2006-06-01

Replicating adenovirus (Ad) vectors with tumour tissue specificity hold great promise for treatment of cancer. We have recently constructed a conditionally replicating Ad5 AdDeltaEP-TETP inducing tumour regression in a xenograft mouse model. For further improvement of this vector, we introduced four genetic modifications and analysed the viral cytotoxicity in a large panel of melanoma cell lines and patient-derived melanoma cells. (1) The antiapoptotic gene E1B-19 kDa (Delta19 mutant) was deleted increasing the cytolytic activity in 18 of 21 melanoma cells. (2) Introduction of the E1A 122-129 deletion (Delta24 mutant), suggested to attenuate viral replication in cell cycle-arrested cells, did not abrogate this activity and increased the cytolytic activity in two of 21 melanoma cells. (3) We inserted an RGD sequence into the fiber to extend viral tropism to alphav integrin-expressing cells, and (4) swapped the fiber with the Ad35 fiber (F35) enhancing the tropism to malignant melanoma cells expressing CD46. The RGD-fiber modification strongly increased cytolysis in all of the 11 CAR-low melanoma cells. The F35 fiber-chimeric vector boosted the cytotoxicity in nine of 11 cells. Our results show that rational engineering additively enhances the cytolytic potential of Ad vectors, a prerequisite for the development of patient-customized viral therapies.

Continuous sensing of tumor-targeted molecular probes with a vertical cavity surface emitting laser-based biosensor

NASA Astrophysics Data System (ADS)

Parashurama, Natesh; O'Sullivan, Thomas D.; De La Zerda, Adam; El Kalassi, Pascale; Cho, Seongjae; Liu, Hongguang; Teed, Robert; Levy, Hart; Rosenberg, Jarrett; Cheng, Zhen; Levi, Ofer; Harris, James S.; Gambhir, Sanjiv S.

2012-11-01

Molecular optical imaging is a widespread technique for interrogating molecular events in living subjects. However, current approaches preclude long-term, continuous measurements in awake, mobile subjects, a strategy crucial in several medical conditions. Consequently, we designed a novel, lightweight miniature biosensor for in vivo continuous optical sensing. The biosensor contains an enclosed vertical-cavity surface-emitting semiconductor laser and an adjacent pair of near-infrared optically filtered detectors. We employed two sensors (dual sensing) to simultaneously interrogate normal and diseased tumor sites. Having established the sensors are precise with phantom and in vivo studies, we performed dual, continuous sensing in tumor (human glioblastoma cells) bearing mice using the targeted molecular probe cRGD-Cy5.5, which targets αVβ3 cell surface integrins in both tumor neovasculature and tumor. The sensors capture the dynamic time-activity curve of the targeted molecular probe. The average tumor to background ratio after signal calibration for cRGD-Cy5.5 injection is approximately 2.43±0.95 at 1 h and 3.64±1.38 at 2 h (N=5 mice), consistent with data obtained with a cooled charge coupled device camera. We conclude that our novel, portable, precise biosensor can be used to evaluate both kinetics and steady state levels of molecular probes in various disease applications.

Continuous sensing of tumor-targeted molecular probes with a vertical cavity surface emitting laser-based biosensor

PubMed Central

Parashurama, Natesh; O’Sullivan, Thomas D.; De La Zerda, Adam; El Kalassi, Pascale; Cho, Seongjae; Liu, Hongguang; Teed, Robert; Levy, Hart; Rosenberg, Jarrett; Cheng, Zhen; Levi, Ofer; Harris, James S.

2012-01-01

Abstract. Molecular optical imaging is a widespread technique for interrogating molecular events in living subjects. However, current approaches preclude long-term, continuous measurements in awake, mobile subjects, a strategy crucial in several medical conditions. Consequently, we designed a novel, lightweight miniature biosensor for in vivo continuous optical sensing. The biosensor contains an enclosed vertical-cavity surface-emitting semiconductor laser and an adjacent pair of near-infrared optically filtered detectors. We employed two sensors (dual sensing) to simultaneously interrogate normal and diseased tumor sites. Having established the sensors are precise with phantom and in vivo studies, we performed dual, continuous sensing in tumor (human glioblastoma cells) bearing mice using the targeted molecular probe cRGD-Cy5.5, which targets αVβ3 cell surface integrins in both tumor neovasculature and tumor. The sensors capture the dynamic time-activity curve of the targeted molecular probe. The average tumor to background ratio after signal calibration for cRGD-Cy5.5 injection is approximately 2.43±0.95 at 1 h and 3.64±1.38 at 2 h (N=5 mice), consistent with data obtained with a cooled charge coupled device camera. We conclude that our novel, portable, precise biosensor can be used to evaluate both kinetics and steady state levels of molecular probes in various disease applications. PMID:23123976

A Novel Clinically Translatable Fluorescent Nanoparticle for Targeted Molecular Imaging of Tumors in Living Subjects

PubMed Central

Gao, Jinhao; Chen, Kai; Luong, Richard; Bouley, Donna M.; Mao, Hua; Qiao, Tiecheng; Gambhir, Sanjiv S.; Cheng, Zhen

2011-01-01

The use of quantum dots (QDs) in biomedical research has grown tremendously, yet successful examples of clinical applications are absent due to many clinical concerns. Here, we report on a new type of stable and biocompatible dendron-coated InP/ZnS core/shell QDs as a clinically translatable nanoprobe for molecular imaging applications. The QDs (QD710-Dendron) were demonstrated to hold several significant features: near-infrared (NIR) emission, high stability in biological media, suitable size with possible renal clearance and ability of extravasation. More importantly, a pilot mouse toxicity study confirmed that QD710-Dendron lacks significant toxicity at the doses tested. The acute tumor uptake of QD710-Dendron resulted in good contrast from the surrounding non-tumorous tissues, indicating the possibility of passive targeting of the QDs. The highly specific targeting of QD710-Dendron-RGD2 to integrin αvβ3–positive tumor cells resulted in high tumor uptake and long retention of the nanoprobe at tumor sites. In summary, QD710-Dendron and RGD modified nanoparticles demonstrate small size, high stability, biocompatibility, favorable in vivo pharmacokinetics, and successful tumor imaging properties. These features satisfy the requirements for clinical translation and should promote efforts to further investigate the possibility of using QD710-Dendron based nanoprobes in the clinical setting in the near future. PMID:22172022

A novel clinically translatable fluorescent nanoparticle for targeted molecular imaging of tumors in living subjects.

PubMed

Gao, Jinhao; Chen, Kai; Luong, Richard; Bouley, Donna M; Mao, Hua; Qiao, Tiecheng; Gambhir, Sanjiv S; Cheng, Zhen

2012-01-11

The use of quantum dots (QDs) in biomedical research has grown tremendously, yet successful examples of clinical applications are absent due to many clinical concerns. Here, we report on a new type of stable and biocompatible dendron-coated InP/ZnS core/shell QD as a clinically translatable nanoprobe for molecular imaging applications. The QDs (QD710-Dendron) were demonstrated to hold several significant features: near-infrared (NIR) emission, high stability in biological media, suitable size with possible renal clearance, and ability of extravasation. More importantly, a pilot mouse toxicity study confirmed that QD710-Dendron lacks significant toxicity at the doses tested. The acute tumor uptake of QD710-Dendron resulted in good contrast from the surrounding nontumorous tissues, indicating the possibility of passive targeting of the QDs. The highly specific targeting of QD710-Dendron-RGD(2) to integrin α(v)β(3)-positive tumor cells resulted in high tumor uptake and long retention of the nanoprobe at tumor sites. In summary, QD710-Dendron and RGD-modified nanoparticles demonstrate small size, high stability, biocompatibility, favorable in vivo pharmacokinetics, and successful tumor imaging properties. These features satisfy the requirements for clinical translation and should promote efforts to further investigate the possibility of using QD710-Dendron-based nanoprobes in the clinical setting in the near future. © 2011 American Chemical Society

Cyclic-RGD penta-peptides cRGDyK derivatized with cyclopentadienyl complexes of technetium and rhenium as radiopharmaceutical probes.

PubMed

Nadeem, Qaisar; Shen, Yunjun; Warsi, Muhammad Farooq; Nasar, Gulfam; Qadir, Muhammad Abdul; Alberto, Roger

2017-07-01

The present study reports the syntheses of half-sandwich complexes of the type [M(η 5 -C 5 H 4 CONH-R)(CO) 3 ] (M═Re, 99m Tc;R═cyclic RGD peptide (cRGDyK) for potential imaging of α v β 3 integrin expression. The 99m Tc complex was prepared directly from the reaction of [ 99m Tc(OH 2 ) 3 (CO) 3 ] + with cRGDyK, doubly conjugated to Thiele's acid [(C 5 H 5 COOH) 2 ] in water. This approach extends the viability of metal-mediated retro Diels-Alder reactions for the preparation of small molecules such as linear tripeptides to a more complex cyclic peptide carrying a [(η 5 -C 5 H 4 ) 99m Tc(CO) 3 ] tag. The Diels-Alder product [(C 5 H 5 CONH-cRGDyK) 2 ] was prepared from Thiele's acid via double peptide coupling. The Re-complex [Re(η 5 -C 5 H 4 CONH-cRGDyK)(CO) 3 ] was obtained by attaching [Re(η 5 -C 5 H 4 COOH)(CO) 3 ] directly to the N-terminus of cRGDyK. The identity of the 99m Tc-complex is confirmed by chromatographic comparison with the corresponding rhenium complex, fully characterized by spectroscopic techniques. Copyright © 2017 John Wiley & Sons, Ltd.

Kindlin-2 directly binds actin and regulates integrin outside-in signaling

PubMed Central

Bledzka, Kamila; Bialkowska, Katarzyna; Sossey-Alaoui, Khalid; Vaynberg, Julia; Pluskota, Elzbieta

2016-01-01

Reduced levels of kindlin-2 (K2) in endothelial cells derived from K2+/− mice or C2C12 myoblastoid cells treated with K2 siRNA showed disorganization of their actin cytoskeleton and decreased spreading. These marked changes led us to examine direct binding between K2 and actin. Purified K2 interacts with F-actin in cosedimentation and surface plasmon resonance analyses and induces actin aggregation. We further find that the F0 domain of K2 binds actin. A mutation, LK47/AA, within a predicted actin binding site (ABS) of F0 diminishes its interaction with actin by approximately fivefold. Wild-type K2 and K2 bearing the LK47/AA mutation were equivalent in their ability to coactivate integrin αIIbβ3 in a CHO cell system when coexpressed with talin. However, K2-LK47/AA exhibited a diminished ability to support cell spreading and actin organization compared with wild-type K2. The presence of an ABS in F0 of K2 that influences outside-in signaling across integrins establishes a new foundation for considering how kindlins might regulate cellular responses. PMID:27044892

Composite alginate gels for tunable cellular microenvironment mechanics

NASA Astrophysics Data System (ADS)

Khavari, Adele; Nydén, Magnus; Weitz, David A.; Ehrlicher, Allen J.

2016-08-01

The mechanics of the cellular microenvironment can be as critical as biochemistry in directing cell behavior. Many commonly utilized materials derived from extra-cellular-matrix create excellent scaffolds for cell growth, however, evaluating the relative mechanical and biochemical effects independently in 3D environments has been difficult in frequently used biopolymer matrices. Here we present 3D sodium alginate hydrogel microenvironments over a physiological range of stiffness (E = 1.85 to 5.29 kPa), with and without RGD binding sites or collagen fibers. We use confocal microscopy to measure the growth of multi-cellular aggregates (MCAs), of increasing metastatic potential in different elastic moduli of hydrogels, with and without binding factors. We find that the hydrogel stiffness regulates the growth and morphology of these cell clusters; MCAs grow larger and faster in the more rigid environments similar to cancerous breast tissue (E = 4-12 kPa) as compared to healthy tissue (E = 0.4-2 kpa). Adding binding factors from collagen and RGD peptides increases growth rates, and change maximum MCA sizes. These findings demonstrate the utility of these independently tunable mechanical/biochemistry gels, and that mechanical confinement in stiffer microenvironments may increase cell proliferation.

Urokinase links plasminogen activation and cell adhesion by cleavage of the RGD motif in vitronectin.

PubMed

De Lorenzi, Valentina; Sarra Ferraris, Gian Maria; Madsen, Jeppe B; Lupia, Michela; Andreasen, Peter A; Sidenius, Nicolai

2016-07-01

Components of the plasminogen activation system including urokinase (uPA), its inhibitor (PAI-1) and its cell surface receptor (uPAR) have been implicated in a wide variety of biological processes related to tissue homoeostasis. Firstly, the binding of uPA to uPAR favours extracellular proteolysis by enhancing cell surface plasminogen activation. Secondly, it promotes cell adhesion and signalling through binding of the provisional matrix protein vitronectin. We now report that uPA and plasmin induces a potent negative feedback on cell adhesion through specific cleavage of the RGD motif in vitronectin. Cleavage of vitronectin by uPA displays a remarkable receptor dependence and requires concomitant binding of both uPA and vitronectin to uPAR Moreover, we show that PAI-1 counteracts the negative feedback and behaves as a proteolysis-triggered stabilizer of uPAR-mediated cell adhesion to vitronectin. These findings identify a novel and highly specific function for the plasminogen activation system in the regulation of cell adhesion to vitronectin. The cleavage of vitronectin by uPA and plasmin results in the release of N-terminal vitronectin fragments that can be detected in vivo, underscoring the potential physiological relevance of the process. © 2016 The Authors.

Physical association and functional interaction between beta1 integrin and CD98 on human T lymphocytes

NASA Technical Reports Server (NTRS)

Miyamoto, Yuko J.; Mitchell, Jason S.; McIntyre, Bradley W.

2003-01-01

CD98 is a cell surface protein previously characterized as a cell activation marker, an amino acid transporter, and has recently been implicated in integrin-related functions. Integrins are cell surface proteins, important for homotypic cell aggregation, cell adhesion, and coactivation of T lymphocytes. We have previously shown that the anti-CD98 mAb 80A10, when coimmobilized with anti-CD3 mAb OKT3, is able to mediate human T cell coactivation that is inhibited by anti-beta1 integrin specific mAb 18D3. These results indicated a functional association of CD98 and beta1 integrin signaling but left open the question of a physical association. We now show the induction of homotypic aggregation through CD98 among human T cells and this aggregation was inhibited by anti-beta1 integrin mAb. Therefore, CD98-dependent lymphocyte proliferation and adhesion may involve integrins. Competitive binding assays and fluorescence colocalization analysis suggested that CD98 and beta1 integrin were physically associated. Differential extraction techniques and immunoprecipitations provided the first evidence that the alpha4beta1 integrin and CD98 are specifically associated on human T lymphocytes.

Selectins and chemokines use shared and distinct signals to activate β2 integrins in neutrophils

PubMed Central

Yago, Tadayuki; Zhang, Nan; Zhao, Liang; Abrams, Charles S.

2018-01-01

Rolling neutrophils receive signals while engaging P- and E-selectin and chemokines on inflamed endothelium. Selectin signaling activates β2 integrins to slow rolling velocities. Chemokine signaling activates β2 integrins to cause arrest. Despite extensive study, key aspects of these signaling cascades remain unresolved. Using complementary in vitro and in vivo assays, we found that selectin and chemokine signals in neutrophils triggered Rap1a-dependent and phosphatidylinositol-4-phosphate 5-kinase γ (PIP5Kγ90)–dependent pathways that induce integrin-dependent slow rolling and arrest. Interruption of both pathways, but not either pathway alone, blocked talin-1 recruitment to and activation of integrins. An isoform of PIP5Kγ90 lacking the talin-binding domain (PIP5Kγ87) could not activate integrins. Chemokines, but not selectins, used phosphatidylinositol-4,5-bisphosphate 3-kinase γ (PI3Kγ) in cooperation with Rap1a to mediate integrin-dependent slow rolling (at low chemokine concentrations), as well as arrest (at high chemokine concentrations). High levels of chemokines activated β2 integrins without selectin signals. When chemokines were limiting, they synergized with selectins to activate β2 integrins. PMID:29592875

Selectins and chemokines use shared and distinct signals to activate β2 integrins in neutrophils.

PubMed

Yago, Tadayuki; Zhang, Nan; Zhao, Liang; Abrams, Charles S; McEver, Rodger P

2018-04-10

Rolling neutrophils receive signals while engaging P- and E-selectin and chemokines on inflamed endothelium. Selectin signaling activates β2 integrins to slow rolling velocities. Chemokine signaling activates β2 integrins to cause arrest. Despite extensive study, key aspects of these signaling cascades remain unresolved. Using complementary in vitro and in vivo assays, we found that selectin and chemokine signals in neutrophils triggered Rap1a-dependent and phosphatidylinositol-4-phosphate 5-kinase γ (PIP5Kγ90)-dependent pathways that induce integrin-dependent slow rolling and arrest. Interruption of both pathways, but not either pathway alone, blocked talin-1 recruitment to and activation of integrins. An isoform of PIP5Kγ90 lacking the talin-binding domain (PIP5Kγ87) could not activate integrins. Chemokines, but not selectins, used phosphatidylinositol-4,5-bisphosphate 3-kinase γ (PI3Kγ) in cooperation with Rap1a to mediate integrin-dependent slow rolling (at low chemokine concentrations), as well as arrest (at high chemokine concentrations). High levels of chemokines activated β2 integrins without selectin signals. When chemokines were limiting, they synergized with selectins to activate β2 integrins. © 2018 by The American Society of Hematology.

The role of a conserved membrane proximal cysteine in altering αPS2CβPS integrin diffusion

NASA Astrophysics Data System (ADS)

Syed, Aleem; Arora, Neha; Bunch, Thomas A.; Smith, Emily A.

2016-12-01

Cysteine residues (Cys) in the membrane proximal region are common post-translational modification (PTM) sites in transmembrane proteins. Herein, the effects of a highly conserved membrane proximal α-subunit Cys1368 on the diffusion properties of αPS2CβPS integrins are reported. Sequence alignment shows that this cysteine is palmitoylated in human α3 and α6 integrin subunits. Replacing Cys1368 in wild-type integrins with valine (Val1368) putatively blocks a PTM site and alters integrins’ ligand binding and diffusion characteristics. Both fluorescence recovery after photobleaching (FRAP) and single particle tracking (SPT) diffusion measurements show Val1368 integrins are more mobile compared to wild-type integrins. Approximately 33% and 8% more Val1368 integrins are mobile as measured by FRAP and SPT, respectively. The mobile Val1368 integrins also exhibit less time-dependent diffusion, as measured by FRAP. Tandem mass spectrometry data suggest that Cys1368 contains a redox or palmitoylation PTM in αPS2CβPS integrins. This membrane proximal Cys may play an important role in the diffusion of other alpha subunits that contain this conserved residue.

β1D chain increases α7β1 integrin and laminin and protects against sarcolemmal damage in mdx mice

PubMed Central

Liu, Jianming; Milner, Derek J.; Boppart, Marni D.; Ross, Robert S.; Kaufman, Stephen J.

2012-01-01

The dystrophin–glycoprotein complex connects myofibers with extracellular matrix laminin. In Duchenne muscular dystrophy, this linkage system is absent and the integrity of muscle fibers is compromised. One potential therapy for addressing muscular dystrophy is to augment the amount of α7β1 integrin, the major laminin-binding integrin in skeletal muscle. Whereas transgenic over-expression of α7 chain may alleviate development of muscular dystrophy and extend the lifespan of severely dystrophic mdx/utrn−/− mice, further enhancing levels of α7 chain provided little additional membrane integrin and negligible additional improvement in mdx mice. We demonstrate here that normal levels of β1 chain limit formation of integrin heterodimer and that increasing β1D chain in mdx mice results in more functional integrin at the sarcolemma, more matrix laminin and decreased damage of muscle fibers. Moreover, increasing the amount of β1D chain in vitro enhances transcription of α7 integrin and α2 laminin genes and the amounts of these proteins. Thus manipulation of β1D integrin expression offers a novel approach to enhance integrin-mediated therapy for muscular dystrophy. PMID:22180459

Natalizumab in the treatment of Crohn’s disease

PubMed Central

Guagnozzi, Danila; Caprilli, Renzo

2008-01-01

The pathogenesis of Crohn’s disease (CD) is multifactorial and the activation of specific pathways of immunological system is important. In particular, the adhesion molecules (integrins) mediate the selective binding between the leukocytes and the endothelial cells regulating the migration of leukocytes into the normal and inflamed intestine. Selective adhesion molecule inhibitors interfere with the migration of leukocytes to the sites of inflammation by targeting adhesion molecules (α4-integrin or α4β7-integrin). Natalizumab is a humanized IgG4 anti-α4-integrin monoclonal antibody that inhibits both α4β7-integrin/mucosal addressin-cell adhesion molecule-1 (MadCAM-1) interaction and α4β1/vascular-cell adhesion molecule-1 (VCAM-1) binding. Pooled data from the four studies, analyzed in a Cochrane review, suggest that natalizumab is effective for induction of clinical response and remission in patients with moderately to severely active CD. In particular, natalizumab may be beneficial for patients with active inflammation or chronically active disease despite the use of conventional therapies with high level of C-reactive protein values at baseline time. Nevertheless, many problems about the utilization of natalizumab in CD remain unsolved (such as the high placebo response, the final definition of dosage and timing schedule, the definition of outcomes and the development of adverse events). PMID:19707360

Transglutaminase 2 is needed for the formation of an efficient phagocyte portal in macrophages engulfing apoptotic cells.

PubMed

Tóth, Beáta; Garabuczi, Eva; Sarang, Zsolt; Vereb, György; Vámosi, György; Aeschlimann, Daniel; Blaskó, Bernadett; Bécsi, Bálint; Erdõdi, Ferenc; Lacy-Hulbert, Adam; Zhang, Ailiang; Falasca, Laura; Birge, Raymond B; Balajthy, Zoltán; Melino, Gerry; Fésüs, László; Szondy, Zsuzsa

2009-02-15

Transglutaminase 2 (TG2), a protein cross-linking enzyme with many additional biological functions, acts as coreceptor for integrin beta(3). We have previously shown that TG2(-/-) mice develop an age-dependent autoimmunity due to defective in vivo clearance of apoptotic cells. Here we report that TG2 on the cell surface and in guanine nucleotide-bound form promotes phagocytosis. Besides being a binding partner for integrin beta(3), a receptor known to mediate the uptake of apoptotic cells via activating Rac1, we also show that TG2 binds MFG-E8 (milk fat globulin EGF factor 8), a protein known to bridge integrin beta(3) to apoptotic cells. Finally, we report that in wild-type macrophages one or two engulfing portals are formed during phagocytosis of apoptotic cells that are characterized by accumulation of integrin beta(3) and Rac1. In the absence of TG2, integrin beta(3) cannot properly recognize the apoptotic cells, is not accumulated in the phagocytic cup, and its signaling is impaired. As a result, the formation of the engulfing portals, as well as the portals formed, is much less efficient. We propose that TG2 has a novel function to stabilize efficient phagocytic portals.

SIKVAV peptide functionalized ultra-small gold nanoparticles for selective targeting of α6β1 integrin in hepatocellular carcinoma

NASA Astrophysics Data System (ADS)

Roskamp, M.; Coulter, T.; Ding, Y.; Perrins, R.; Espinosa Garcia, C.; Pace, A.; Hale, S.; Robinson, A.; Williams, P.; Aguilera Peral, U.; Patel, K.; Palmer, D.

2017-04-01

Ultra-small glycan-passivated gold nanoparticles of <2nm diameter were funtionalised with a short HS-EG(8)-COOH ligand. The nanoparticles were subsequently labelled, in a stoichiometrically controllable manner, with integrin-binding peptide SIKVAV and the maytansinoid cytotoxin DM4. In vitro assays showed significantly increased integrin-mediated uptake of SIKVAV labelled nanoparticles in HepG2 cells. SIKVAV targeted nanoparticle binding was shown to be outcompeted with free SIKVAV peptide, indicating target specific uptake. DM4 was passively attached to nanoparticles via sulfhydryl ligand exchange at the gold nanoparticle surface, which rendered them highly cytotoxic (IC50 ˜1 × 10-9M). In a rat model, pharmacokinetic studies showed that nanoparticle biodistribution was strongly altered by labelling with either peptide and DM4 moieties.

Functional integrins from normal and glycosylation-deficient baby hamster kidney cells. Terminal processing of asparagine-linked oligosaccharides is not correlated with fibronectin-binding activity.

PubMed

Koyama, T; Hughes, R C

1992-12-25

We have examined the properties of the alpha 5 beta 1 integrin of baby hamster kidney (BHK) cells, a ricin-resistant variant Ric14 lacking N-acetylglucosaminyl transferase I, and hence unable to complete assembly of hybrid- or complex-type N-glycans, and BHK cells treated with 1-deoxymannojirimycin (dMM), an inhibitor of Golgi mannosidases involved in the initial processing of N-glycan precursors. Comparable amounts of alpha 5 beta 1 integrin were isolated from these cells by chromatography of detergent extracts on a fibronectin cell-binding fragment affinity column and elution with EDTA. The alpha 5 beta 1 integrin obtained from normal BHK cells by fibronectin affinity chromatography contained mainly endoglycosidase H-resistant oligosaccharides, whereas in RicR14 cells or dMM-treated BHK cells these were entirely endoglycosidase H-sensitive. Analysis of lactoperoxidase labeled or long term biosynthetically 35S-labeled proteins from cultures of normal or glycosylation deficient cells showed similar steady state levels of alpha 5 beta 1 integrin and expression at the cell surface. Pulse-chase experiments in normal BHK cells showed rapid conversion of the alpha 5 subunit into a mature form containing oligosaccharides resistant to endoglycosidase H and slower maturation of a precursor beta 1 subunit, as in other cell types. In Ric14 cells the precursor beta 1 subunit was found to carry glycans larger than the fully processed Man5GlcNAc2 glycan of the mature subunit, indicating that the bulk precursor pool had not been translocated into the cis-Golgi compartment containing mannosidase I. We conclude that in BHK cells terminal oligosaccharide processing of alpha 5 beta 1 integrin subunits is not required for dimer formation, surface expression, and fibronectin binding, and that expression of the glycosylation defect of Ric14 cells on the alpha 5 beta 1 integrin does not account for the reduced adhesiveness of these cells on fibronectin compared with normal and dMM-treated BHK cells.

Integrin expression and integrin-mediated adhesion in vitro of human multipotent stromal cells (MSCs) to endothelial cells from various blood vessels.

PubMed

Semon, Julie A; Nagy, Lauren H; Llamas, Claire B; Tucker, H Alan; Lee, Ryang Hwa; Prockop, Darwin J

2010-07-01

Multipotent mesenchymal stromal cells (MSCs) home to damaged tissue by processes partly regulated by integrins. Integrin subunits expressed by MSCs were identified by flow cytometry (FC), immunocytochemistry (IC), and a panel of integrin-binding antibodies. In subconfluent cultures, over 80% of MSCs expressed integrin subunits beta1, beta2, and alpha3, 20%-55% expressed alpha1, alpha2, alpha4, alpha5, alpha6, and alphaV, and about 10% expressed beta3 when assayed by FC. None of the cells expressed significant levels of 13 other integrins as assayed by FC, but seven of the 13 integrins were detected by IC: beta5, alpha7, alpha8, alpha9, alpha11, alphaX, and alphaD. Expression of some integrins changed with MSC confluency: integrins beta3, alpha1, alpha3, alpha5, and alphaV increased, and alpha6 decreased. Furthermore, alpha4 was the only integrin to vary among preparations of MSCs from different donors. The results resolved some discrepancies in the literature concerning integrin expression by MSCs. We also investigated the role of specific integrins in MSC adhesion to endothelial cells (ECs) from the pulmonary artery (HPAEC), cardiac-derived microvasculature (HMVEC-C), and umbilical veins (HUVEC). In experiments with blocking antibodies to beta integrins, anti-beta5 reduced MSC adhesion to all types of ECs, anti-beta1 to both HUVEC and HPAEC, anti-beta3 to HUVEC, and anti-beta2 to HMVEC-C. With blocking antibodies to alpha integrins, anti-alphaX reduced adhesion to HPAEC and HMVEC-C, anti-alphaV to HPAEC, and both anti-alpha7 and anti-alphaD to HMVEC-C. Thus, MSCs use diverse integrins to adhere to EC from various blood vessels in vitro.

Probing conformational changes in the I-like domain and the cysteine-rich repeat of human beta 3 integrins following disulfide bond disruption by cysteine mutations: identification of cysteine 598 involved in alphaIIbbeta3 activation.

PubMed

Chen, P; Melchior, C; Brons, N H; Schlegel, N; Caen, J; Kieffer, N

2001-10-19

We have investigated receptor function and epitope expression of recombinant alpha(IIb)beta(3) mutated at Cys(177) or Cys(273) in the I-like domain as well as Cys(598), located in the fourth repeat of the membrane-proximal cysteine-rich region and mutated in a Glanzmann's thrombasthenia type II patient. The beta(3) mutants beta(3)C177A, beta(3)C273A, and beta(3)C598Y exhibited a decreased electrophoretic mobility in SDS-polyacrylamide gel electrophoresis under nonreducing conditions, confirming the disruption of the respective disulfide loops. Despite reduced surface expression, the alpha(IIb)beta(3)C177A, alpha(IIb)beta(3)C273A, and alpha(IIb)beta(3)C598Y receptors mediated cell adhesion to immobilized fibrinogen and translocated into focal adhesion plaques. The beta(3)C598Y mutation, but not the beta(3)C177A or beta(3)C273A mutations, induced spontaneous binding of the ligand mimetic monoclonal antibody PAC-1, while the beta(3)C177A and beta(3)C273A mutants exhibited reduced complex stability in the absence of Ca(2+). Epitope mapping of function-blocking monoclonal antibodies (mAbs) allowed the identification of two distinct subgroups; mAbs A2A9, pl2-46, 10E5, and P256 did not interact with alpha(IIb)beta(3)C273A and bound only weakly to alpha(IIb)beta(3)C177A, while mAbs AP2, LM609 and 7E3 bound normally to mutant alpha(IIb)beta(3)C273A, but interacted only weakly with mutant alpha(IIb)beta(3)C177A. Furthermore, a cryptic epitope recognized by mAb 4D10G3 and not exposed on wild type alpha(IIb)beta(3) became accessible only on mutant alpha(IIb)beta(3)C177A and was mapped to the 60-kDa chymotrypsin fragment of beta(3). Finally, the ligand-induced binding site (LIBS) epitopes AP5, D3, LIBS1, and LIBS2 were spontaneously expressed on all three mutants independent of RGDS or dithiothreitol treatment. Our results provide evidence that disruption of a single cysteine disulfide bond in the cysteine-rich repeat domain, but not in the I-like domain, activates integrin alpha(IIb)beta(3). In contrast, disruption of each of the disulfide bonds in the two long insertions of the I-like domain predicted to be in close contact with the alpha subunit beta-propeller domain affect the stability of the alpha(IIb)beta(3) heterodimer and inhibit complex-specific mAb binding without affecting the RGD binding capacity of the metal ion-dependent adhesion site-like domain.

Mechanisms of integrin-vascular endothelial growth factor receptor cross-activation in angiogenesis.

PubMed

Mahabeleshwar, Ganapati H; Feng, Weiyi; Reddy, Kumar; Plow, Edward F; Byzova, Tatiana V

2007-09-14

The functional responses of endothelial cells are dependent on signaling from peptide growth factors and the cellular adhesion receptors, integrins. These include cell adhesion, migration, and proliferation, which, in turn, are essential for more complex processes such as formation of the endothelial tube network during angiogenesis. This study identifies the molecular requirements for the cross-activation between beta3 integrin and tyrosine kinase receptor 2 for vascular endothelial growth factor (VEGF) receptor (VEGFR-2) on endothelium. The relationship between VEGFR-2 and beta3 integrin appears to be synergistic, because VEGFR-2 activation induces beta3 integrin tyrosine phosphorylation, which, in turn, is crucial for VEGF-induced tyrosine phosphorylation of VEGFR-2. We demonstrate here that adhesion- and growth factor-induced beta3 integrin tyrosine phosphorylation are directly mediated by c-Src. VEGF-stimulated recruitment and activation of c-Src and subsequent beta3 integrin tyrosine phosphorylation are critical for interaction between VEGFR-2 and beta3 integrin. Moreover, c-Src mediates growth factor-induced beta3 integrin activation, ligand binding, beta3 integrin-dependent cell adhesion, directional migration of endothelial cells, and initiation of angiogenic programming in endothelial cells. Thus, the present study determines the molecular mechanisms and consequences of the synergism between 2 cell surface receptor systems, growth factor receptor and integrins, and opens new avenues for the development of pro- and antiangiogenic strategies.

Assessing activation of hepatic stellate cells by (99m)Tc-3PRGD2 scintigraphy targeting integrin αvβ3: a feasibility study.

PubMed

Zhang, Xin; Xin, Jun; Shi, Yu; Xu, Weina; Yu, Shupeng; Yang, Zhiguang; Liu, Changping; Cao, Li; Guo, Qiyong

2015-03-01

Hepatic stellate cell (HSC) activation, which is accompanied by increased expression of integrin αvβ3, is an important factor in liver fibrogenesis. Molecular imaging targeting the integrin αvβ3 could provide a non-invasive method for evaluating the expression and the function of the integrin αvβ3 on the activated HSCs (aHSCs) in the injured liver, and then provide important prognostic information. (99m)Tc-3PRGD2 is such a radiotracer specific for integrin αvβ3. In this study, we aimed to compare the differences in liver uptake and retention of the (99m)Tc-3PRGD2 between normal liver and injured liver to evaluate the feasibility of (99m)Tc-3PRGD2 scintigraphy for this purpose. We used planar scintigraphy to assess changes in integrin αvβ3 binding of intravenously-administered (99m)Tc-3PRGD2 in the livers of rats with thioacetamide (TAA)-induced liver fibrosis compared with the controls. We co-injected cold c(RGDyK) with (99m)Tc-3PRGD2 to assess the specific binding of the radiotracer. We performed Sirius red staining to assess liver fibrosis, immunofluorescent colocalization to identify the location of integrin αvβ3 expressed in the fibrotic liver, and we measured protein and messenger RNA expression of integrin αvβ3 and alpha smooth muscle actin (α-SMA) in the control and fibrotic livers. The fibrotic livers showed enhanced (99m)Tc-3PRGD2 uptake and retention. The radiotracer was demonstrated to bind specifically with the integrin αvβ3 mainly expressed on the aHSCs. The liver-to-heart ratio at 30 min post-injection was higher in the fibrotic livers than in the control livers (TAA, 1.98±0.08 vs. control, 1.50±0.12, p<0.01). The liver t1/2 was longer than in the controls (TAA, 27.07±10.69 min vs. control, 12.67±4.10 min, p<0.01). The difference of heart t1/2 between the two groups was not statistically significant (TAA, 3.13±0.63 min vs. control, 3.41±0.77 min, p=0.94). (99m)Tc-3PRGD2 molecular imaging can provide a non-invasive method for assessing activation of HSCs. Copyright © 2014 Elsevier Inc. All rights reserved.

Oncofetal Chondroitin Sulfate Glycosaminoglycans Are Key Players in Integrin Signaling and Tumor Cell Motility.

PubMed

Clausen, Thomas Mandel; Pereira, Marina Ayres; Al Nakouzi, Nader; Oo, Htoo Zarni; Agerbæk, Mette Ø; Lee, Sherry; Ørum-Madsen, Maj Sofie; Kristensen, Anders Riis; El-Naggar, Amal; Grandgenett, Paul M; Grem, Jean L; Hollingsworth, Michael A; Holst, Peter J; Theander, Thor; Sorensen, Poul H; Daugaard, Mads; Salanti, Ali

2016-12-01

Many tumors express proteoglycans modified with oncofetal chondroitin sulfate glycosaminoglycan chains (ofCS), which are normally restricted to the placenta. However, the role of ofCS in cancer is largely unknown. The function of ofCS in cancer was analyzed using the recombinant ofCS-binding VAR2CSA protein (rVAR2) derived from the malaria parasite, Plasmodium falciparum We demonstrate that ofCS plays a key role in tumor cell motility by affecting canonical integrin signaling pathways. Binding of rVAR2 to tumor cells inhibited the interaction of cells with extracellular matrix (ECM) components, which correlated with decreased phosphorylation of Src kinase. Moreover, rVAR2 binding decreased migration, invasion, and anchorage-independent growth of tumor cells in vitro Mass spectrometry of ofCS-modified proteoglycan complexes affinity purified from tumor cell lines on rVAR2 columns revealed an overrepresentation of proteins involved in cell motility and integrin signaling, such as integrin-β1 (ITGB1) and integrin-α4 (ITGA4). Saturating concentrations of rVAR2 inhibited downstream integrin signaling, which was mimicked by knockdown of the core chondroitin sulfate synthesis enzymes β-1,3-glucuronyltransferase 1 (B3GAT1) and chondroitin sulfate N-acetylgalactosaminyltransferase 1 (CSGALNACT1). The ofCS modification was highly expressed in both human and murine metastatic lesions in situ and preincubation or early intravenous treatment of tumor cells with rVAR2 inhibited seeding and spreading of tumor cells in mice. This was associated with a significant increase in survival of the animals. These data functionally link ofCS modifications with cancer cell motility and further highlights ofCS as a novel therapeutic cancer target. The cancer-specific expression of ofCS aids in metastatic phenotypes and is a candidate target for therapy. Mol Cancer Res; 14(12); 1288-99. ©2016 AACR. ©2016 American Association for Cancer Research.

The relative influence of metal ion binding sites in the I-like domain and the interface with the hybrid domain on rolling and firm adhesion by integrin alpha4beta7.

PubMed

Chen, JianFeng; Takagi, Junichi; Xie, Can; Xiao, Tsan; Luo, Bing-Hao; Springer, Timothy A

2004-12-31

We examined the effect of conformational change at the beta(7) I-like/hybrid domain interface on regulating the transition between rolling and firm adhesion by integrin alpha(4)beta(7). An N-glycosylation site was introduced into the I-like/hybrid domain interface to act as a wedge and to stabilize the open conformation of this interface and hence the open conformation of the alpha(4) beta(7) headpiece. Wild-type alpha(4)beta(7) mediates rolling adhesion in Ca(2+) and Ca(2+)/Mg(2+) but firm adhesion in Mg(2+) and Mn(2+). Stabilizing the open headpiece resulted in firm adhesion in all divalent cations. The interaction between metal binding sites in the I-like domain and the interface with the hybrid domain was examined in double mutants. Changes at these two sites can either counterbalance one another or be additive, emphasizing mutuality and the importance of multiple interfaces in integrin regulation. A double mutant with counterbalancing deactivating ligand-induced metal ion binding site (LIMBS) and activating wedge mutations could still be activated by Mn(2+), confirming the importance of the adjacent to metal ion-dependent adhesion site (ADMIDAS) in integrin activation by Mn(2+). Overall, the results demonstrate the importance of headpiece allostery in the conversion of rolling to firm adhesion.

Synthesis and evaluation of diastereoisomers of 1,4,7-triazacyclononane-1,4,7-tris-(glutaric acid) (NOTGA) for multimeric radiopharmaceuticals of gallium.

PubMed

Guerra Gomez, Francisco L; Uehara, Tomoya; Rokugawa, Takemi; Higaki, Yusuke; Suzuki, Hiroyuki; Hanaoka, Hirofumi; Akizawa, Hiromichi; Arano, Yasushi

2012-11-21

In the conventional synthesis of 1,4,7-tris-(glutaric acid)-1,4,7-triazacyclononane (NOTGA), four isomeric species are usually generated by the alkylation of 1,4,7-triazacyclononane with α-bromoglutaric acid diester. To estimate their biological efficacies as well as their stability and radiochemistry, the RRR/SSS and RRS/SSR NOTGA-(t)Bu prochelators were isolated and the corresponding cyclic RGDfK (RGD) conjugates with triethylene glycol linkages were prepared. The RRR/SSS and RRS/SSR diastereomers were obtained in 69% and 17% yields, respectively. In the complexation reaction with (67)GaCl(3), both diastereomers provided >98% radiochemical yields at pH 5 within 10 min when the reaction was conducted at room temperature. However, the RRR/SSS diastereomer exhibited more pH-sensitive radiochemical yields between pH 3.5 to 4.5. Despite their diasteromeric nature, both (67)Ga-labeled RGD-NOTGA remained stable during the apo-transferrin challenge, exhibiting similar affinity for integrin α(v)β(3) and biodistribution with predominant renal excretion. Similar tumor uptake was also observed in mice bearing U87MG tumor xenograft, which resulted in impressively high contrast SPECT/CT images. These findings indicate that the RGD-NOTGA conjugates of both diastereomers presented here possess equivalent biological efficacies and their combined usage would be feasible. It is worth noting that specific properties of a given biomolecule, cell expression levels of the corresponding target molecule, and presence or absence of a pharmacokinetic modifier would affect the structural differences between diastereomers on the ligand-receptor interactions and pharmacokinetics. Thus, the preparation of corresponding conjugates and evaluation of their chemical and biological performances still remains important for applying NOTGA to other biomolecules of interest using the diastereomerically pure NOTGA-(t)Bu prochelator.

Increasing α7β1-integrin promotes muscle cell proliferation, adhesion, and resistance to apoptosis without changing gene expression

PubMed Central

Liu, Jianming; Burkin, Dean J.; Kaufman, Stephen J.

2008-01-01

The dystrophin-glycoprotein complex maintains the integrity of skeletal muscle by associating laminin in the extracellular matrix with the actin cytoskeleton. Several human muscular dystrophies arise from defects in the components of this complex. The α7β1-integrin also binds laminin and links the extracellular matrix with the cytoskeleton. Enhancement of α7-integrin levels alleviates pathology in mdx/utrn−/− mice, a model of Duchenne muscular dystrophy, and thus the integrin may functionally compensate for the absence of dystrophin. To test whether increasing α7-integrin levels affects transcription and cellular functions, we generated α7-integrin-inducible C2C12 cells and transgenic mice that overexpress the integrin in skeletal muscle. C2C12 myoblasts with elevated levels of integrin exhibited increased adhesion to laminin, faster proliferation when serum was limited, resistance to staurosporine-induced apoptosis, and normal differentiation. Transgenic expression of eightfold more integrin in skeletal muscle did not result in notable toxic effects in vivo. Moreover, high levels of α7-integrin in both myoblasts and in skeletal muscle did not disrupt global gene expression profiles. Thus increasing integrin levels can compensate for defects in the extracellular matrix and cytoskeleton linkage caused by compromises in the dystrophin-glycoprotein complex without triggering apparent overt negative side effects. These results support the use of integrin enhancement as a therapy for muscular dystrophy. PMID:18045857

Volatile anesthetics, not intravenous anesthetic propofol bind to and attenuate the activation of platelet receptor integrin αIIbβ3.

PubMed

Yuki, Koichi; Bu, Weiming; Shimaoka, Motomu; Eckenhoff, Roderic

2013-01-01

In clinical reports, the usage of isoflurane and sevoflurane was associated with more surgical field bleeding in endoscopic sinus surgeries as compared to propofol. The activation of platelet receptor αIIbβ3 is a crucial event for platelet aggregation and clot stability. Here we studied the effect of isoflurane, sevoflurane, and propofol on the activation of αIIbβ3. The effect of anesthetics on the activation of αIIbβ3 was probed using the activation sensitive antibody PAC-1 in both cell-based (platelets and αIIbβ3 transfectants) and cell-free assays. The binding sites of isoflurane on αIIbβ3 were explored using photoactivatable isoflurane (azi-isoflurane). The functional implication of revealed isoflurane binding sites were studied using alanine-scanning mutagenesis. Isoflurane and sevoflurane diminished the binding of PAC-1 to wild-type αIIbβ3 transfectants, but not to the high-affinity mutant, β3-N305T. Both anesthetics also impaired PAC-1 binding in a cell-free assay. In contrast, propofol did not affect the activation of αIIbβ3. Residues adducted by azi-isoflurane were near the calcium binding site (an important regulatory site termed SyMBS) just outside of the ligand binding site. The mutagenesis experiments demonstrated that these adducted residues were important in regulating integrin activation. Isoflurane and sevoflurane, but not propofol, impaired the activation of αIIbβ3. Azi-isoflurane binds to the regulatory site of integrin αIIbβ3, thereby suggesting that isoflurane blocks ligand binding of αIIbβ3 in not a competitive, but an allosteric manner.

Stimulatory effects of advanced glycation endproducts (AGEs) on fibronectin matrix assembly.

PubMed

Pastino, Alexandra K; Greco, Todd M; Mathias, Rommel A; Cristea, Ileana M; Schwarzbauer, Jean E

2017-05-01

Advanced glycation endproducts (AGEs) are a heterogeneous group of compounds that form via non-enzymatic glycation of proteins throughout our lifespan and at a higher rate in certain chronic diseases such as diabetes. AGEs contribute to the progression of fibrosis, in part by stimulating cellular pathways that affect gene expression. Long-lived ECM proteins are targets for non-enzymatic glycation but the question of whether the AGE-modified ECM leads to excess ECM accumulation and fibrosis remains unanswered. In this study, cellular changes due to AGE accretion in the ECM were investigated. Non-enzymatic glycation of proteins in a decellularized fibroblast ECM was achieved by incubating the ECM in a solution of methylglyoxal (MGO). Mass spectrometry of fibronectin (FN) isolated from the glycated matrix identified twenty-eight previously unidentified MGO-derived AGE modification sites including functional sites such as the RGD integrin-binding sequence. Mesangial cells grown on the glycated, decellularized matrix assembled increased amounts of FN matrix. Soluble AGE-modified bovine serum albumin (BSA) also stimulated FN matrix assembly and this effect was reduced by function-blocking antibodies against the receptor for AGE (RAGE). These results indicate that cells respond to AGEs by increasing matrix assembly and that RAGE is involved in this response. This raises the possibility that the accumulation of ECM during the progression of fibrosis may be enhanced by cell interactions with AGEs on a glycated ECM. Copyright © 2016 Elsevier B.V. All rights reserved.

A recombinant tail-less integrin beta 4 subunit disrupts hemidesmosomes, but does not suppress alpha 6 beta 4-mediated cell adhesion to laminins

PubMed Central

1995-01-01

To examine the function of the alpha 6 beta 4 integrin we have determined its ligand-binding ability and overexpressed two potentially dominant negative mutant beta 4 subunits, lacking either the cytoplasmic or extracellular domain, in bladder epithelial 804G cells. The results of cell adhesion and radioligand-binding assays showed that alpha 6 beta 4 is a receptor for several laminin isoforms, including laminin 1, 2, 4, and 5. Overexpression of the tail-less or head-less mutant beta 4 subunit did not suppress alpha 6 beta 4-mediated adhesion to laminins, as both types of transfectants adhered to these ligands in the presence of blocking anti-beta 1 antibodies as well as the controls. However, immunofluorescence experiments indicated that the endogenous alpha 6 beta 4 integrin and other hemidesmosomal markers were not concentrated in hemidesmosomes in cells overexpressing tail- less beta 4, while the distribution of these molecules was not altered in cells overexpressing the head-less subunit. Electron microscopic studies confirmed that cells overexpressing tail-less beta 4 had a drastically reduced number of hemidesmosomes, while cells expressing the head-less subunit had a normal number of these structures. Thus, expression of a tail-less, but not a head-less mutant beta 4 subunit leads to a dominant negative effect on hemidesmosome assembly without suppressing initial adhesion to laminins. We conclude that the alpha 6 beta 4 integrin binds to several laminins and plays an essential role in the assembly and/or stability of hemidesmosomes, that alpha 6 beta 4- mediated adhesion and hemidesmosome assembly have distinct requirements, and that it is possible to use a dominant negative approach to selectively interfere with a specific function of an integrin. PMID:7721947

Monocyte-endothelial adhesion in chronic rheumatoid arthritis. In situ detection of selectin and integrin-dependent interactions.

PubMed Central

Grober, J S; Bowen, B L; Ebling, H; Athey, B; Thompson, C B; Fox, D A; Stoolman, L M

1993-01-01

Blood monocytes are the principal reservoir for tissue macrophages in rheumatoid synovitis. Receptor-mediated adhesive interactions between circulating cells and the synovial venules initiate recruitment. These interactions have been studied primarily in cultured endothelial cells. Thus the functional activities of specific adhesion receptors, such as the endothelial selectins and the leukocytic integrins, have not been evaluated directly in diseased tissues. We therefore examined monocyte-microvascular interactions in rheumatoid synovitis by modifying the Stamper-Woodruff frozen section binding assay initially developed to study lymphocyte homing. Specific binding of monocytes to venules lined by low or high endothelium occurred at concentrations as low as 5 x 10(5) cells/ml. mAbs specific for P-selectin (CD62, GMP-140/PADGEM) blocked adhesion by > 90% in all synovitis specimens examined. In contrast, P-selectin-mediated adhesion to the microvasculature was either lower or absent in frozen sections of normal foreskin and placenta. mAbs specific for E-selectin (ELAM-1) blocked 20-50% of monocyte attachment in several RA synovial specimens but had no effect in others. mAbs specific for LFA-1, Mo1/Mac 1, the integrin beta 2-chain, and L-selectin individually inhibited 30-40% of adhesion. An mAb specific for the integrin beta 1-chain inhibited the attachment of elutriated monocytes up to 20%. We conclude that P-selectin associated with the synovial microvasculature initiates shear-resistant adhesion of monocytes in the Stamper-Woodruff assay and stabilizes bonds formed by other selectins and the integrins. Thus the frozen section binding assay permits direct evaluation of leukocyte-microvascular adhesive interactions in inflamed tissues and suggests a prominent role for P-selectin in monocyte recruitment in vivo. Images PMID:7685772

Intravital Imaging of Vascular Transmigration by the Lyme Spirochete: Requirement for the Integrin Binding Residues of the B. burgdorferi P66 Protein.

PubMed

Kumar, Devender; Ristow, Laura C; Shi, Meiqing; Mukherjee, Priyanka; Caine, Jennifer A; Lee, Woo-Yong; Kubes, Paul; Coburn, Jenifer; Chaconas, George

2015-12-01

Vascular extravasation, a key step in systemic infection by hematogenous microbial pathogens, is poorly understood, but has been postulated to encompass features similar to vascular transmigration by leukocytes. The Lyme disease spirochete can cause a variety of clinical manifestations, including arthritis, upon hematogenous dissemination. This pathogen encodes numerous surface adhesive proteins (adhesins) that may promote extravasation, but none have yet been implicated in this process. In this work we report the novel use of intravital microscopy of the peripheral knee vasculature to study transmigration of the Lyme spirochete in living Cd1d-/-mice. In the absence of iNKT cells, major immune modulators in the mouse joint, spirochetes that have extravasated into joint-proximal tissue remain in the local milieu and can be enumerated accurately. We show that BBK32, a fibronectin and glycosaminoglycan adhesin of B. burgdorferi involved in early steps of endothelial adhesion, is not required for extravasation from the peripheral knee vasculature. In contrast, almost no transmigration occurs in the absence of P66, an outer membrane protein that has porin and integrin adhesin functions. Importantly, P66 mutants specifically defective in integrin binding were incapable of promoting extravasation. P66 itself does not promote detectable microvascular interactions, suggesting that vascular adhesion of B. burgdorferi mediated by other adhesins, sets the stage for P66-integrin interactions leading to transmigration. Although integrin-binding proteins with diverse functions are encoded by a variety of bacterial pathogens, P66 is the first to have a documented and direct role in vascular transmigration. The emerging picture of vascular escape by the Lyme spirochete shows similarities, but distinct differences from leukocyte transmigration.

Intravital Imaging of Vascular Transmigration by the Lyme Spirochete: Requirement for the Integrin Binding Residues of the B. burgdorferi P66 Protein

PubMed Central

Kumar, Devender; Ristow, Laura C.; Shi, Meiqing; Mukherjee, Priyanka; Caine, Jennifer A.; Lee, Woo-Yong; Kubes, Paul; Coburn, Jenifer; Chaconas, George

2015-01-01

Vascular extravasation, a key step in systemic infection by hematogenous microbial pathogens, is poorly understood, but has been postulated to encompass features similar to vascular transmigration by leukocytes. The Lyme disease spirochete can cause a variety of clinical manifestations, including arthritis, upon hematogenous dissemination. This pathogen encodes numerous surface adhesive proteins (adhesins) that may promote extravasation, but none have yet been implicated in this process. In this work we report the novel use of intravital microscopy of the peripheral knee vasculature to study transmigration of the Lyme spirochete in living Cd1d -/-mice. In the absence of iNKT cells, major immune modulators in the mouse joint, spirochetes that have extravasated into joint-proximal tissue remain in the local milieu and can be enumerated accurately. We show that BBK32, a fibronectin and glycosaminoglycan adhesin of B. burgdorferi involved in early steps of endothelial adhesion, is not required for extravasation from the peripheral knee vasculature. In contrast, almost no transmigration occurs in the absence of P66, an outer membrane protein that has porin and integrin adhesin functions. Importantly, P66 mutants specifically defective in integrin binding were incapable of promoting extravasation. P66 itself does not promote detectable microvascular interactions, suggesting that vascular adhesion of B. burgdorferi mediated by other adhesins, sets the stage for P66-integrin interactions leading to transmigration. Although integrin-binding proteins with diverse functions are encoded by a variety of bacterial pathogens, P66 is the first to have a documented and direct role in vascular transmigration. The emerging picture of vascular escape by the Lyme spirochete shows similarities, but distinct differences from leukocyte transmigration. PMID:26684456

Direct Interactions with the Integrin β1 Cytoplasmic Tail Activate the Abl2/Arg Kinase*

PubMed Central

Simpson, Mark A.; Bradley, William D.; Harburger, David; Parsons, Maddy; Calderwood, David A.; Koleske, Anthony J.

2015-01-01

Integrins are heterodimeric α/β extracellular matrix adhesion receptors that couple physically to the actin cytoskeleton and regulate kinase signaling pathways to control cytoskeletal remodeling and adhesion complex formation and disassembly. β1 integrins signal through the Abl2/Arg (Abl-related gene) nonreceptor tyrosine kinase to control fibroblast cell motility, neuronal dendrite morphogenesis and stability, and cancer cell invasiveness, but the molecular mechanisms by which integrin β1 activates Arg are unknown. We report here that the Arg kinase domain interacts directly with a lysine-rich membrane-proximal segment in the integrin β1 cytoplasmic tail, that Arg phosphorylates the membrane-proximal Tyr-783 in the β1 tail, and that the Arg Src homology domain then engages this phosphorylated region in the tail. We show that these interactions mediate direct binding between integrin β1 and Arg in vitro and in cells and activate Arg kinase activity. These findings provide a model for understanding how β1-containing integrins interact with and activate Abl family kinases. PMID:25694433

Homology modeling, molecular dynamics, and docking studies of pattern-recognition transmembrane protein-lipopolysaccharide and β-1,3 glucan-binding protein from Fenneropenaeus indicus.

PubMed

Sivakamavalli, Jeyachandran; Tripathi, Sunil Kumar; Singh, Sanjeev Kumar; Vaseeharan, Baskaralingam

2015-01-01

Lipopolysaccharide and β-1,3 glucan-binding protein (LGBP) is a family of pattern-recognition transmembrane proteins (PRPs) which plays a vital role in the immune mechanism of crustaceans in adverse conditions. Fenneropenaeus indicus LGBP-deduced amino acid has conserved potential recognition motif for β-1,3 linkages of polysaccharides and putative RGD (Arg-Gly-Asp) cell adhesion sites for the activation of innate defense mechanism. In order to understand the stimulating activity of β-1,3 glucan (β-glucan) and its interaction with LGBP, a 3D model of LGBP is generated. Molecular docking is performed with this model, and the results indicate Arg71 with strong hydrogen bond from RGD domain of LGBP. Moreover, from the docking studies, we also suggest that Arg34, Lys68, Val135, and Ala146 in LGBP are important amino acid residues in binding as they have strong bonding interaction in the active site of LGBP. In our in vitro studies, yeast agglutination results suggest that shrimp F. indicus LGBP possesses sugar binding and recognition sites in its structure, which is responsible for agglutination reaction. Our results were synchronized with the already reported evidence both in vivo and in vitro experiments. This investigation may be valuable for further experimental investigation in the synthesis of novel immunomodulator.

Exosome Adherence and Internalization by Hepatic Stellate Cells Triggers Sphingosine 1-Phosphate-dependent Migration*

PubMed Central

Wang, Ruisi; Ding, Qian; Yaqoob, Usman; de Assuncao, Thiago M.; Verma, Vikas K.; Hirsova, Petra; Cao, Sheng; Mukhopadhyay, Debabrata; Huebert, Robert C.; Shah, Vijay H.

2015-01-01

Exosomes are cell-derived extracellular vesicles thought to promote intercellular communication by delivering specific content to target cells. The aim of this study was to determine whether endothelial cell (EC)-derived exosomes could regulate the phenotype of hepatic stellate cells (HSCs). Initial microarray studies showed that fibroblast growth factor 2 induced a 2.4-fold increase in mRNA levels of sphingosine kinase 1 (SK1). Exosomes derived from an SK1-overexpressing EC line increased HSC migration 3.2-fold. Migration was not conferred by the dominant negative SK1 exosome. Incubation of HSCs with exosomes was also associated with an 8.3-fold increase in phosphorylation of AKT and 2.5-fold increase in migration. Exosomes were found to express the matrix protein and integrin ligand fibronectin (FN) by Western blot analysis and transmission electron microscopy. Blockade of the FN-integrin interaction with a CD29 neutralizing antibody or the RGD peptide attenuated exosome-induced HSC AKT phosphorylation and migration. Inhibition of endocytosis with transfection of dynamin siRNA, the dominant negative dynamin GTPase construct Dyn2K44A, or the pharmacological inhibitor Dynasore significantly attenuated exosome-induced AKT phosphorylation. SK1 levels were increased in serum exosomes derived from mice with experimental liver fibrosis, and SK1 mRNA levels were up-regulated 2.5-fold in human liver cirrhosis patient samples. Finally, S1PR2 inhibition protected mice from CCl4-induced liver fibrosis. Therefore, EC-derived SK1-containing exosomes regulate HSC signaling and migration through FN-integrin-dependent exosome adherence and dynamin-dependent exosome internalization. These findings advance our understanding of EC/HSC cross-talk and identify exosomes as a potential target to attenuate pathobiology signals. PMID:26534962

Exosome Adherence and Internalization by Hepatic Stellate Cells Triggers Sphingosine 1-Phosphate-dependent Migration.

PubMed

Wang, Ruisi; Ding, Qian; Yaqoob, Usman; de Assuncao, Thiago M; Verma, Vikas K; Hirsova, Petra; Cao, Sheng; Mukhopadhyay, Debabrata; Huebert, Robert C; Shah, Vijay H

2015-12-25

Exosomes are cell-derived extracellular vesicles thought to promote intercellular communication by delivering specific content to target cells. The aim of this study was to determine whether endothelial cell (EC)-derived exosomes could regulate the phenotype of hepatic stellate cells (HSCs). Initial microarray studies showed that fibroblast growth factor 2 induced a 2.4-fold increase in mRNA levels of sphingosine kinase 1 (SK1). Exosomes derived from an SK1-overexpressing EC line increased HSC migration 3.2-fold. Migration was not conferred by the dominant negative SK1 exosome. Incubation of HSCs with exosomes was also associated with an 8.3-fold increase in phosphorylation of AKT and 2.5-fold increase in migration. Exosomes were found to express the matrix protein and integrin ligand fibronectin (FN) by Western blot analysis and transmission electron microscopy. Blockade of the FN-integrin interaction with a CD29 neutralizing antibody or the RGD peptide attenuated exosome-induced HSC AKT phosphorylation and migration. Inhibition of endocytosis with transfection of dynamin siRNA, the dominant negative dynamin GTPase construct Dyn2K44A, or the pharmacological inhibitor Dynasore significantly attenuated exosome-induced AKT phosphorylation. SK1 levels were increased in serum exosomes derived from mice with experimental liver fibrosis, and SK1 mRNA levels were up-regulated 2.5-fold in human liver cirrhosis patient samples. Finally, S1PR2 inhibition protected mice from CCl4-induced liver fibrosis. Therefore, EC-derived SK1-containing exosomes regulate HSC signaling and migration through FN-integrin-dependent exosome adherence and dynamin-dependent exosome internalization. These findings advance our understanding of EC/HSC cross-talk and identify exosomes as a potential target to attenuate pathobiology signals. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

The feasibility of 18F-AlF-NOTA-PRGD2 PET/CT for monitoring early response of Endostar antiangiogenic therapy in human nasopharyngeal carcinoma xenograft model compared with 18F-FDG

PubMed Central

Liang, Sheng; Zhang, Caiyuan; Cheng, Weiwei; Hai, Wangxi; Yin, Bing; Wang, Dengbin

2016-01-01

Purpose Radiolabeled arginine-glycine-aspartic acid (RGD) peptides have been developed for PET imaging of integrin avβ3 in the tumor vasculature, leading to great potential for noninvasively evaluating tumor angiogenesis and monitoring antiangiogenic treatment. The aim of this study was to investigate a novel one-step labeled integrin-targeted tracer, 18F-AlF-NOTA-PRGD2, for PET/CT for detecting tumor angiogenesis and monitoring the early therapeutic efficacy of antiangiogenic agent Endostar in human nasopharyngeal carcinoma (NPC) xenograft model. Experimental design and results Mice bearing NPC underwent 18F-AlF-NOTA-PRGD2 PET/CT at baseline and after 2, 4, 7, and 14 days of consecutive treatment with Endostar or PBS, compared with 18F-FDG PET/CT. Tumors were harvested at all imaging time points for histopathological analysis with H & E and microvessel density (MVD) and integrin avβ3 immunostaining. The maximum percent injected dose per gram of body weight (%ID/gmax) tumor uptake of 18F-AlF-NOTA-PRGD2 PET/CT was significantly lower than that in the control group starting from day 2 (p < 0.01), much earlier and more accurately than that of 18F-FDG PET/CT. Moreover, a moderate linear correlation was observed between tumor MVD and the corresponding tumor uptake of 18F-AlF-NOTA-PRGD2 PET/CT (r = 0.853, p < 0.01). Conclusions 18F-AlF-NOTA-PRGD2 PET/CT can be used for in vivo angiogenesis imaging and monitoring early response to Endostar antiangiogenic treatment in NPC xenograft model, favoring its potential clinical translation. PMID:27029065

Comparison study of [18F]FAl-NOTA-PRGD2, [18F]FPPRGD2, and [68Ga]Ga-NOTA-PRGD2 for PET imaging of U87MG tumors in mice.

PubMed

Lang, Lixin; Li, Weihua; Guo, Ning; Ma, Ying; Zhu, Lei; Kiesewetter, Dale O; Shen, Baozhong; Niu, Gang; Chen, Xiaoyuan

2011-12-21

[(18)F]FPPRGD2, an F-18 labeled dimeric cyclic RGDyK peptide, has favorable properties for PET imaging of angiogenesis by targeting the α(v)β(3) integrin receptor. This radiotracer has been approved by the FDA for use in clinical trials. However, the time-consuming multiple-step synthetic procedure required for its preparation may hinder the widespread usage of this tracer. The recent development of a method using an F-18 fluoride-aluminum complex to radiolabel peptides provides a strategy for simplifying the labeling procedure. On the other hand, the easy-to-prepare [(68)Ga]-labeled NOTA-RGD derivatives have also been reported to have promising properties for imaging α(v)β(3) integrin receptors. The purpose of this study was to prepare [(18)F]FPPRGD2 [corrected] , [(18)F]FAl-NOTA-PRGD2, and [(68)Ga]Ga-NOTA-PRGD2 and to compare their pharmacokinetics and tumor imaging properties using small animal PET. All three compounds showed rapid and high tracer uptake in U87MG tumors with high target-to-background ratios. The uptake in the liver, kidneys, and muscle were similar for all three tracers, and they all showed predominant renal clearance. In conclusion, [(18)F]FAl-NOTA-PRGD2 and [(68)Ga]Ga-NOTA-PRGD2 have imaging properties and pharmacokinetics comparable to those of [(18)F]FPPRGD2. Considering their ease of preparation and good imaging qualities, [(18)F]FAl-NOTA-PRGD2 and [(68)Ga]NOTA-PRGD2 are promising alternatives to [(18)F]FPPRGD2 for PET imaging of tumor α(v)β(3) integrin expression.

SU-F-SPS-08: Measuring the Interaction Of DDR Cell Receptors and Extracellular Matrix Collagen in Prostate Cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dong, J; Sarkar, A; Hoffmann, P

Purpose: Discoidin domain receptors (DDR) have recently been recognized as important players in cancer progression. DDRs are cell receptors that interact with collagen, an extracellular matrix (ECM) protein. However the detailed mechanism of their interaction is unclear. Here we attempted to examine their interaction in terms of structural (surface topography), mechanical (rupture force), and kinetic (binding probability) information on the single molecular scale with the use of atomic force microscopy (AFM). Methods: The Quantitative Nano-mechanical property Mapping (QNM) mode of AFM allowed to assess the cells in liquid growth media at their optimal physiological while being viable. Human benign prostatemore » hyperplasia (BPH-1) cell line was genetically regulated to suppress DDR expression (DDR- cells) and was compared with naturally DDR expressing cells (DDR+). Results: Binding force measurements (n = 1000) were obtained before and after the two groups were treated with fibronectin (FN), an integrin-inhibiting antibody to block the binding of integrin. The quantification indicates that cells containing DDR bind with collagen at a most probable force of 80.3–83.0 ±7.6 pN. The probability of them binding is 0.167 when other interactions (mainly due to integrin-collagen binding) are minimized. Conclusion: Together with further force measurements at different pulling speeds will determine dissociation rate, binding distance and activation barrier. These parameters in benign cells provides some groundwork in understanding DDR’s behavior in various cell microenvironments such as in malignant tumor cells. Funding supported by Richard Barber Interdisciplinary Research Program of Wayne State University.« less

Dwarfism in mice lacking collagen-binding integrins α2β1 and α11β1 is caused by severely diminished IGF-1 levels.

PubMed

Blumbach, Katrin; Niehoff, Anja; Belgardt, Bengt F; Ehlen, Harald W A; Schmitz, Markus; Hallinger, Ralf; Schulz, Jan-Niklas; Brüning, Jens C; Krieg, Thomas; Schubert, Markus; Gullberg, Donald; Eckes, Beate

2012-02-24

Mice with a combined deficiency in the α2β1 and α11β1 integrins lack the major receptors for collagen I. These mutants are born with inconspicuous differences in size but develop dwarfism within the first 4 weeks of life. Dwarfism correlates with shorter, less mineralized and functionally weaker bones that do not result from growth plate abnormalities or osteoblast dysfunction. Besides skeletal dwarfism, internal organs are correspondingly smaller, indicating proportional dwarfism and suggesting a systemic cause for the overall size reduction. In accordance with a critical role of insulin-like growth factor (IGF)-1 in growth control and bone mineralization, circulating IGF-1 levels in the sera of mice lacking either α2β1 or α11β1 or both integrins were sharply reduced by 39%, 64%, or 81% of normal levels, respectively. Low hepatic IGF-1 production resulted from diminished growth hormone-releasing hormone expression in the hypothalamus and, subsequently, reduced growth hormone expression in the pituitary glands of these mice. These findings point out a novel role of collagen-binding integrin receptors in the control of growth hormone/IGF-1-dependent biological activities. Thus, coupling hormone secretion to extracellular matrix signaling via integrins represents a novel concept in the control of endocrine homeostasis.

Dwarfism in Mice Lacking Collagen-binding Integrins α2β1 and α11β1 Is Caused by Severely Diminished IGF-1 Levels*

PubMed Central

Blumbach, Katrin; Niehoff, Anja; Belgardt, Bengt F.; Ehlen, Harald W. A.; Schmitz, Markus; Hallinger, Ralf; Schulz, Jan-Niklas; Brüning, Jens C.; Krieg, Thomas; Schubert, Markus; Gullberg, Donald; Eckes, Beate

2012-01-01

Mice with a combined deficiency in the α2β1 and α11β1 integrins lack the major receptors for collagen I. These mutants are born with inconspicuous differences in size but develop dwarfism within the first 4 weeks of life. Dwarfism correlates with shorter, less mineralized and functionally weaker bones that do not result from growth plate abnormalities or osteoblast dysfunction. Besides skeletal dwarfism, internal organs are correspondingly smaller, indicating proportional dwarfism and suggesting a systemic cause for the overall size reduction. In accordance with a critical role of insulin-like growth factor (IGF)-1 in growth control and bone mineralization, circulating IGF-1 levels in the sera of mice lacking either α2β1 or α11β1 or both integrins were sharply reduced by 39%, 64%, or 81% of normal levels, respectively. Low hepatic IGF-1 production resulted from diminished growth hormone-releasing hormone expression in the hypothalamus and, subsequently, reduced growth hormone expression in the pituitary glands of these mice. These findings point out a novel role of collagen-binding integrin receptors in the control of growth hormone/IGF-1-dependent biological activities. Thus, coupling hormone secretion to extracellular matrix signaling via integrins represents a novel concept in the control of endocrine homeostasis. PMID:22210772

Pro-metastatic NEDD9 regulates individual cell migration via caveolin-1-dependent trafficking of integrins

PubMed Central

Kozyulina, Polina Y.; Loskutov, Yuriy V.; Kozyreva, Varvara K.; Rajulapati, Anuradha; Ice, Ryan J.; Jones, Brandon. C.; Pugacheva, Elena N.

2014-01-01

The dissemination of tumor cells relies on efficient cell adhesion and migration, which in turn depends upon endocytic trafficking of integrins. In the current work, it was found that depletion of pro-metastatic protein, NEDD9, in breast cancer (BC) cells results in a significant decrease in individual cell migration due to impaired trafficking of ligand-bound integrins. NEDD9 deficiency does not affect the expression or internalization of integrins but heightens caveolae-dependent trafficking of ligand-bound integrins to early endosomes. Increase in mobility of ligand-bound integrins is concomitant with an increase in tyrosine phosphorylation of caveolin-1 (CAV1) and volume of CAV1-vesicles. NEDD9 directly binds to CAV1 and co-localizes within CAV1 vesicles. In the absence of NEDD9, the trafficking of ligand-bound integrins from early to late endosomes is impaired, resulting in a significant decrease in degradation of ligand/integrin complexes and an increase in recycling of ligand-bound integrins from early endosomes back to the plasma membrane without ligand disengagement, thus leading to low adhesion and migration. Re-expression of NEDD9 or decrease in the amount of active, tyrosine 14 phosphorylated (Tyr14) CAV1 in NEDD9 depleted cells rescues the integrin trafficking deficiency and restores cellular adhesion and migration capacity. Collectively, these findings indicate that NEDD9 orchestrates trafficking of ligand-bound integrins through the attenuation of CAV1 activity. PMID:25319010

Integrins protect cardiomyocytes from ischemia/reperfusion injury

PubMed Central

Okada, Hideshi; Lai, N. Chin; Kawaraguchi, Yoshitaka; Liao, Peter; Copps, Jeffrey; Sugano, Yasuo; Okada-Maeda, Sunaho; Banerjee, Indroneal; Schilling, Jan M.; Gingras, Alexandre R.; Asfaw, Elizabeth K.; Suarez, Jorge; Kang, Seok-Min; Perkins, Guy A.; Au, Carol G.; Israeli-Rosenberg, Sharon; Manso, Ana Maria; Liu, Zheng; Milner, Derek J.; Kaufman, Stephen J.; Patel, Hemal H.; Roth, David M.; Hammond, H. Kirk; Taylor, Susan S.; Dillmann, Wolfgang H.; Goldhaber, Joshua I.; Ross, Robert S.

2013-01-01

Ischemic damage is recognized to cause cardiomyocyte (CM) death and myocardial dysfunction, but the role of cell-matrix interactions and integrins in this process has not been extensively studied. Expression of α7β1D integrin, the dominant integrin in normal adult CMs, increases during ischemia/reperfusion (I/R), while deficiency of β1 integrins increases ischemic damage. We hypothesized that the forced overexpression of integrins on the CM would offer protection from I/R injury. Tg mice with CM-specific overexpression of integrin α7β1D exposed to I/R had a substantial reduction in infarct size compared with that of α5β1D-overexpressing mice and WT littermate controls. Using isolated CMs, we found that α7β1D preserved mitochondrial membrane potential during hypoxia/reoxygenation (H/R) injury via inhibition of mitochondrial Ca2+ overload but did not alter H/R effects on oxidative stress. Therefore, we assessed Ca2+ handling proteins in the CM and found that β1D integrin colocalized with ryanodine receptor 2 (RyR2) in CM T-tubules, complexed with RyR2 in human and rat heart, and specifically bound to RyR2 amino acids 165–175. Integrins stabilized the RyR2 interdomain interaction, and this stabilization required integrin receptor binding to its ECM ligand. These data suggest that α7β1D integrin modifies Ca2+ regulatory pathways and offers a means to protect the myocardium from ischemic injury. PMID:24091324

A novel leukocyte adhesion deficiency caused by expressed but nonfunctional β2 integrins Mac-1 and LFA-1

PubMed Central

Hogg, Nancy; Stewart, Mairi P.; Scarth, Sarah L.; Newton, Rebecca; Shaw, Jacqueline M.; Law, S.K. Alex; Klein, Nigel

1999-01-01

In the leukocyte adhesion deficiency (LAD)-1 syndrome, there is diminished expression of β2(CD18) integrins. This is caused by lesions in the β2-subunit gene and gives rise to recurrent bacterial infections, impaired pus formation, and poor wound healing. We describe a patient with clinical features compatible with a moderately severe phenotype of LAD-1 but who expresses the β2 integrins lymphocyte function– associated molecule (LFA)-1 and Mac-1 at 40%–60% of normal levels. This level of expression should be adequate for normal integrin function, but both the patient's Mac-1 on neutrophils and LFA-1 on T cells failed to bind ligands such as fibrinogen and intercellular adhesion molecule (ICAM)-1, respectively, or to display a β2-integrin activation epitope after adhesion-inducing stimuli. Unexpectedly, divalent cation treatment induced the patient's T cells to bind to ICAM-2 and ICAM-3. Sequencing of the patient's two CD18 alleles revealed the mutations S138P and G273R. Both mutations are in the β2-subunit conserved domain, with S138P a putative divalent cation coordinating residue in the metal ion–dependent adhesion site (MIDAS) motif. After K562 cell transfection with α subunits, the mutated S138P β subunit was coexpressed but did not support function, whereas the G273R mutant was not expressed. In summary, the patient described here exhibits failure of the β2 integrins to function despite adequate levels of cell-surface expression. PMID:9884339

Rheostat regulation of integrin-mediated leukocyte adhesion

PubMed Central

Douglas, Ivor S.; Dassopoulos, Themistocles

2007-01-01

The homing of activated T lymphocytes to the gut in inflammatory bowel diseases is dependent on their coordinated, integrin-mediated adhesion and de-adhesion to substrates and blood vessel walls. In this issue of the JCI, Park and colleagues reveal a key modulatory role of a binding site within β integrins, known as the ADMIDAS domain, in controlling integrin de-adhesion in mice (see the related article beginning on page 2526). These observations add to our growing understanding of how integrin adhesiveness is regulated and raise the notion of the existence of a biological rheostat for lymphocyte homing. Disturbed migratory rheostat tone could account for variations in interindividual immune responses observed in patients with inflammatory bowel disease or other lymphocyte-mediated inflammatory disorders. These findings will inform future strategies to design small molecules for the treatment of a spectrum of chronic inflammatory conditions. PMID:17786236

R-Ras Regulates Migration through an Interaction with Filamin A in Melanoma Cells

PubMed Central

Gawecka, Joanna E.; Griffiths, Genevieve S.; Ek-Rylander, Barbro; Ramos, Joe W.; Matter, Michelle L.

2010-01-01

Background Changes in cell adhesion and migration in the tumor microenvironment are key in the initiation and progression of metastasis. R-Ras is one of several small GTPases that regulate cell adhesion and migration on the extracellular matrix, however the mechanism has not been completely elucidated. Using a yeast two-hybrid approach we sought to identify novel R-Ras binding proteins that might mediate its effects on integrins. Methods and Findings We identified Filamin A (FLNa) as a candidate interacting protein. FLNa is an actin-binding scaffold protein that also binds to integrin β1, β2 and β7 tails and is associated with diverse cell processes including cell migration. Indeed, M2 melanoma cells require FLNa for motility. We further show that R-Ras and FLNa interact in co-immunoprecipitations and pull-down assays. Deletion of FLNa repeat 3 (FLNaΔ3) abrogated this interaction. In M2 melanoma cells active R-Ras co-localized with FLNa but did not co-localize with FLNa lacking repeat 3. Thus, activated R-Ras binds repeat 3 of FLNa. The functional consequence of this interaction was that active R-Ras and FLNa coordinately increased cell migration. In contrast, co-expression of R-Ras and FLNaΔ3 had a significantly reduced effect on migration. While there was enhancement of integrin activation and fibronectin matrix assembly, cell adhesion was not altered. Finally, siRNA knockdown of endogenous R-Ras impaired FLNa-dependent fibronectin matrix assembly. Conclusions These data support a model in which R-Ras functionally associates with FLNa and thereby regulates integrin-dependent migration. Thus in melanoma cells R-Ras and FLNa may cooperatively promote metastasis by enhancing cell migration. PMID:20585650

An Overview of the Mechanism of Action of the Monoclonal Antibody Vedolizumab.

PubMed

Wyant, Tim; Fedyk, Eric; Abhyankar, Brihad

2016-12-01

Vedolizumab is a novel therapeutic monoclonal antibody recently approved for the treatment of moderately to severely active ulcerative colitis and Crohn's disease in adults who have failed at least one conventional therapy. An integrin antagonist, vedolizumab binds to the α 4 β 7 integrin which is expressed specifically by a subset of gastrointestinal-homing T lymphocytes. The binding of α 4 β 7 integrin to mucosal addressin cell adhesion molecule-1 expressed on the surface of mucosal endothelial cells is a crucial component of the gut-selective homing mechanism for lymphocytes.In contrast, other monoclonal antibodies approved for the treatment of inflammatory bowel diseases, such as tumour necrosis factor α antagonists and the integrin antagonist natalizumab, act systemically or on multiple targets to reduce inflammation.The unique gut selectivity of vedolizumab may contribute to the favourable benefit-risk profile observed in vedolizumab clinical trials. In this review, we summarise data from the preclinical development of vedolizumab and describe the current understanding of the mechanism of action as it relates to other biological therapies for inflammatory bowel disease. Copyright © 2016 European Crohn’s and Colitis Organisation (ECCO). Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.

The Relative Influence of Metal Ion Binding Sites in the I-like Domain and the Interface with the Hybrid Domain on Rolling and Firm Adhesion by Integrin α4β7*

PubMed Central

Chen, JianFeng; Takagi, Junichi; Xie, Can; Xiao, Tsan; Luo, Bing-Hao; Springer, Timothy A.

2015-01-01

We examined the effect of conformational change at the β7 I-like/hybrid domain interface on regulating the transition between rolling and firm adhesion by integrin α4β7. An N-glycosylation site was introduced into the I-like/hybrid domain interface to act as a wedge and to stabilize the open conformation of this interface and hence the open conformation of the α4β7 headpiece. Wild-type α4β7 mediates rolling adhesion in Ca2+ and Ca2+/Mg2+ but firm adhesion in Mg2+ and Mn2+. Stabilizing the open headpiece resulted in firm adhesion in all divalent cations. The interaction between metal binding sites in the I-like domain and the interface with the hybrid domain was examined in double mutants. Changes at these two sites can either counterbalance one another or be additive, emphasizing mutuality and the importance of multiple interfaces in integrin regulation. A double mutant with counterbalancing deactivating ligand-induced metal ion binding site (LIMBS) and activating wedge mutations could still be activated by Mn2+, confirming the importance of the adjacent to metal ion-dependent adhesion site (ADMIDAS) in integrin activation by Mn2+. Overall, the results demonstrate the importance of headpiece allostery in the conversion of rolling to firm adhesion. PMID:15448154

Secondary reduction of alpha7B integrin in laminin alpha2 deficient congenital muscular dystrophy supports an additional transmembrane link in skeletal muscle.

PubMed

Cohn, R D; Mayer, U; Saher, G; Herrmann, R; van der Flier, A; Sonnenberg, A; Sorokin, L; Voit, T

1999-03-01

The integrins are a large family of heterodimeric transmembrane cellular receptors which mediate the association between the extracellular matrix (ECM) and cytoskeletal proteins. The alpha7beta1 integrin is a major laminin binding integrin in skeletal and cardiac muscle and is thought to be involved in myogenic differentiation and migration processes. The main binding partners of the alpha7 integrin are laminin-1 (alpha1-beta1-gamma1), laminin-2 (alpha2-beta1-gamma1) and laminin-4 (alpha2-beta2-gamma1). Targeted deletion of the gene for the alpha7 integrin subunit (ITGA7) in mice leads to a novel form of muscular dystrophy. In the present study we have investigated the expression of two alternative splice variants, the alpha7B and beta1D integrin subunits, in normal human skeletal muscle, as well as in various forms of muscular dystrophy. In normal human skeletal muscle the expression of the alpha7 integrin subunit appeared to be developmentally regulated: it was first detected at 2 years of age. In contrast, the beta1D integrin could be detected in immature and mature muscle in the sarcolemma of normal fetal skeletal muscle at 18 weeks gestation. The expression of alpha7B integrin was significantly reduced at the sarcolemma in six patients with laminin alpha2 chain deficient congenital muscular dystrophy (CMD) (age >2 years). However, this reduction was not correlated with the amount of laminin alpha2 chain expressed. In contrast, the expression of the laminin alpha2 chain was not altered in the skeletal muscle of the alpha7 knock-out mice. These data argue in favor that there is not a tight correlation between the expression of the alpha7 integrin subunit and that of the laminin alpha2 chain in either human or murine dystrophic muscle. Interestingly, in dystrophinopathies (Duchenne and Becker muscular dystrophy; DMD/BMD) expression of alpha7B was upregulated irrespective of the level of dystrophin expression as shown by a strong sarcolemmal staining pattern even in young boys (age <2 years). The expression of the beta1D integrin subunit was not altered in any of our patients with different types of muscular dystrophy. In contrast, sarcolemmal expression of beta1D integrin was significantly reduced in the alpha7 integrin knock-out mice, whereas the expression of the components of the DGC was not altered. The secondary loss of alpha7B in laminin alpha2 chain deficiency defines a biochemical change in the composition of the plasma membrane resulting from a primary protein deficiency in the basal lamina. These findings, in addition to the occurrence of a muscular dystrophy in alpha7 deficient mice, implies that the alpha7B integrin is an important laminin receptor within the plasma membrane which plays a significant role in skeletal muscle function and stability.

Galectin-3 modulates the polarized surface delivery of β1-integrin in epithelial cells.

PubMed

Hönig, Ellena; Ringer, Karina; Dewes, Jenny; von Mach, Tobias; Kamm, Natalia; Kreitzer, Geri; Jacob, Ralf

2018-05-10

Epithelial cells require a precise intracellular transport and sorting machinery in order to establish and maintain their polarized architecture. This machinery includes beta-galactoside binding galectins for glycoprotein targeting to the apical membrane. Galectin-3 sorts cargo destined for the apical plasma membrane into vesicular carriers. After delivery of cargo to the apical milieu, galectin-3 recycles back into sorting organelles. We analyzed the role of galectin-3 in the polarized distribution of β1-integrin in MDCK cells. Integrins are located primarily at the basolateral domain of epithelial cells. We demonstrate that a minor pool of β1-integrin interacts with galectin-3 at the apical plasma membrane. Knockdown of galectin-3 decreases apical delivery of β1-integrin. This loss is restored by supplementation with recombinant galectin-3 and galectin-3 overexpression. Our data suggest that galectin-3 targets newly synthesized β1-integrin to the apical membrane and promotes apical delivery of β1-integrin internalized from the basolateral membrane. In parallel, galectin-3 knockout results in a reduction in cell proliferation and an impairment in proper cyst development. Our results suggest that galectin-3 modulates the surface distribution of β1-integrin and affects the morphogenesis of polarized cells. © 2018. Published by The Company of Biologists Ltd.

Mapping structural landmarks, ligand binding sites and missense mutations to the collagen IV heterotrimers predicts major functional domains, novel interactions and variation in phenotypes in inherited diseases affecting basement membranes

PubMed Central

Des Parkin, J.; San Antonio, James D.; Pedchenko, Vadim; Hudson, Billy; Jensen, Shane T.; Savige, Judy

2016-01-01

Collagen IV is the major protein found in basement membranes. It comprises 3 heterotrimers (α1α1α2, α3α4α5, and α5α5α6) that form distinct networks, and are responsible for membrane strength and integrity. We constructed linear maps of the collagen IV heterotrimers (‘interactomes’) that indicated major structural landmarks, known and predicted ligand-binding sites, and missense mutations, in order to identify functional and disease-associated domains, potential interactions between ligands, and genotype-phenotype relationships. The maps documented more than 30 known ligand-binding sites as well as motifs for integrins, heparin, von Willebrand factor (VWF), decorin and bone morphogenetic protein (BMP). They predicted functional domains for angiogenesis and haemostasis, and disease domains for autoimmunity, tumor growth and inhibition, infection and glycation. Cooperative ligand interactions were indicated by binding site proximity, for example, between integrins, matrix metalloproteinases and heparin. The maps indicated that mutations affecting major ligand-binding sites, for example for Von Hippel Lindau (VHL) protein in the α1 chain or integrins in the α5 chain, resulted in distinctive phenotypes (Hereditary Angiopathy, Nephropathy, Aneurysms and muscle Cramps (HANAC) syndrome, and early onset Alport syndrome respectively). These maps further our understanding of basement membrane biology and disease, and suggest novel membrane interactions, functions, and therapeutic targets. PMID:21280145

The N terminus of SKAP55 enables T cell adhesion to TCR and integrin ligands via distinct mechanisms

PubMed Central

Ophir, Michael J.; Liu, Beiyun C.

2013-01-01

The T cell receptor (TCR) triggers the assembly of “SLP-76 microclusters,” which mediate signals required for T cell activation. In addition to regulating integrin activation, we show that Src kinase–associated phosphoprotein of 55 kD (SKAP55) is required for microcluster persistence and movement, junctional stabilization, and integrin-independent adhesion via the TCR. These functions require the dimerization of SKAP55 and its interaction with the adaptor adhesion and degranulation-promoting adaptor protein (ADAP). A “tandem dimer” containing two ADAP-binding SKAP55 Src homology 3 (SH3) domains stabilized SLP-76 microclusters and promoted T cell adhesion via the TCR, but could not support adhesion to integrin ligands. Finally, the SKAP55 dimerization motif (DM) enabled the coimmunoprecipitation of the Rap1-dependent integrin regulator Rap1-GTP–interacting adaptor molecule (RIAM), the recruitment of talin into TCR-induced adhesive junctions, and “inside-out” signaling to β1 integrins. Our data indicate that SKAP55 dimers stabilize SLP-76 microclusters, couple SLP-76 to the force-generating systems responsible for microcluster movement, and enable adhesion via the TCR by mechanisms independent of RIAM, talin, and β1 integrins. PMID:24368808

Age Increases Monocyte Adhesion on Collagen

NASA Astrophysics Data System (ADS)

Khalaji, Samira; Zondler, Lisa; Kleinjan, Fenneke; Nolte, Ulla; Mulaw, Medhanie A.; Danzer, Karin M.; Weishaupt, Jochen H.; Gottschalk, Kay-E.

2017-05-01

Adhesion of monocytes to micro-injuries on arterial walls is an important early step in the occurrence and development of degenerative atherosclerotic lesions. At these injuries, collagen is exposed to the blood stream. We are interested whether age influences monocyte adhesion to collagen under flow, and hence influences the susceptibility to arteriosclerotic lesions. Therefore, we studied adhesion and rolling of human peripheral blood monocytes from old and young individuals on collagen type I coated surface under shear flow. We find that firm adhesion of monocytes to collagen type I is elevated in old individuals. Pre-stimulation by lipopolysaccharide increases the firm adhesion of monocytes homogeneously in older individuals, but heterogeneously in young individuals. Blocking integrin αx showed that adhesion of monocytes to collagen type I is specific to the main collagen binding integrin αxβ2. Surprisingly, we find no significant age-dependent difference in gene expression of integrin αx or integrin β2. However, if all integrins are activated from the outside, no differences exist between the age groups. Altered integrin activation therefore causes the increased adhesion. Our results show that the basal increase in integrin activation in monocytes from old individuals increases monocyte adhesion to collagen and therefore the risk for arteriosclerotic plaques.