Sample records for ciliary muscle cells

  1. Ciliary Muscle Thickness in Anisometropia

    PubMed Central

    Kuchem, Mallory K; Sinnott, Loraine T; Kao, Chiu-Yen; Bailey, Melissa D

    2014-01-01

    Purpose The purpose of this study was to investigate the relationships between ciliary muscle thickness (CMT), refractive error, and axial length both across subjects and between the more and less myopic eyes of adults with anisometropia. Methods Both eyes of 29 adult subjects with at least 1.00 D of anisometropia were measured. Ciliary muscle thickness was measured at the maximum thickness (CMTMAX) and at 1.0 mm (CMT1), 2.0 mm (CMT2), and 3.0 mm (CMT3) posterior to the scleral spur, and also at the apical region (Apical CMTMAX = CMTMAX – CMT2, and Apical CMT1 = CMT1 – CMT2). Multilevel regression models were used to determine the relationship between the various CMT measures and cycloplegic refractive error or axial length, and to assess whether there are CMT differences between the more and less myopic eyes of an anisometropic adult. Results CMTMAX, CMT1, CMT2 and CMT3 were negatively associated with mean refractive error (all p ≤ 0.03), and the strongest association was in the posterior region (CMT2 and CMT3). Apical CMTMAX and Apical CMT1, however, were positively associated with mean refractive error (both p < 0.0001) across subjects. Within a subject, i.e., comparing the two anisometropic eyes, there was no statistically significant difference in CMT in any region. Conclusions Similar to previous studies, across anisometropic subjects, a thicker posterior region of the ciliary muscle (CMT2 and CMT3) was associated with increased myopic refractive error. Conversely, shorter, more hyperopic eyes tended to have thicker anterior, apical fiber portions of their ciliary muscle (Apical CMTMAX and Apical CMT1). There was no difference between the two eyes for any CMT measurement, indicating that in anisometropia, an eye can grow longer and more myopic than its fellow eye without resulting in an increase in CMT. PMID:24100479

  2. Neural control of the ciliary muscle.

    DOT National Transportation Integrated Search

    1963-03-01

    The cat ciliary muscle was shown to respond to both parasympathetic and sympathetic nerve impulses in vitro. Sympathetic responses were directionally opposite to parasympathetic responses and are interpreted as active contractions of the radial muscl...

  3. Variability of manual ciliary muscle segmentation in optical coherence tomography images.

    PubMed

    Chang, Yu-Cherng; Liu, Keke; Cabot, Florence; Yoo, Sonia H; Ruggeri, Marco; Ho, Arthur; Parel, Jean-Marie; Manns, Fabrice

    2018-02-01

    Optical coherence tomography (OCT) offers new options for imaging the ciliary muscle allowing direct in vivo visualization. However, variation in image quality along the length of the muscle prevents accurate delineation and quantification of the muscle. Quantitative analyses of the muscle are accompanied by variability in segmentation between examiners and between sessions for the same examiner. In processes such as accommodation where changes in muscle thickness may be tens of microns- the equivalent of a small number of image pixels, differences in segmentation can influence the magnitude and potentially the direction of thickness change. A detailed analysis of variability in ciliary muscle thickness measurements was performed to serve as a benchmark for the extent of this variability in studies on the ciliary muscle. Variation between sessions and examiners were found to be insignificant but the magnitude of variation should be considered when interpreting ciliary muscle results.

  4. Region-Specific Relationships Between Refractive Error and Ciliary Muscle Thickness in Children

    PubMed Central

    Pucker, Andrew D.; Sinnott, Loraine T.; Kao, Chiu-Yen; Bailey, Melissa D.

    2013-01-01

    Purpose. To determine if there is a relationship between refractive error and ciliary muscle thickness in different muscle regions. Methods. An anterior segment optical coherence tomographer was used to measure cycloplegic ciliary muscle thicknesses at 1 mm (CMT1), 2 mm (CMT2), and 3 mm (CMT3) posterior to the scleral spur; maximum (CMTMAX) thickness was also assessed. An autorefractor was used to determine cycloplegic spherical equivalent refractive error (SPHEQ). Apical ciliary muscle fibers were obtained by subtracting corresponding CMT2 values from CMT1 and CMTMAX. Multilevel regression models were used to determine the relationship between ciliary muscle thickness in various regions of the muscle and refractive error. Results. Subjects included 269 children with a mean age of 8.71 ± 1.51 years and a mean refractive error of +0.41 ± 1.29 diopters. In linear models with ciliary muscle thicknesses and SPHEQ, SPHEQ was significantly associated only with CMT2 (β = −11.34, P = 0.0008) and CMT 3 (β = −6.97, P = 0.007). When corresponding values of CMT2 were subtracted from CMT1 and CMTMAX, apical fibers at CMT1 (β = 14.75, P < 0.0001) and CMTMAX (β = 18.16, P < 0.0001) had a significant relationship with SPHEQ. Conclusions. These data indicated that in children the posterior ciliary muscle fibers are thicker in myopia (CMT2 and CMT3), but paradoxically, the apical ciliary muscle fibers are thicker in hyperopia (CMTMAX and CMT1). This may be the first evidence that hyperopia is associated with a thicker apical ciliary muscle region. PMID:23761093

  5. Semi-Automatic Extraction Algorithm for Images of the Ciliary Muscle

    PubMed Central

    Kao, Chiu-Yen; Richdale, Kathryn; Sinnott, Loraine T.; Ernst, Lauren E.; Bailey, Melissa D.

    2011-01-01

    Purpose To development and evaluate a semi-automatic algorithm for segmentation and morphological assessment of the dimensions of the ciliary muscle in Visante™ Anterior Segment Optical Coherence Tomography images. Methods Geometric distortions in Visante images analyzed as binary files were assessed by imaging an optical flat and human donor tissue. The appropriate pixel/mm conversion factor to use for air (n = 1) was estimated by imaging calibration spheres. A semi-automatic algorithm was developed to extract the dimensions of the ciliary muscle from Visante images. Measurements were also made manually using Visante software calipers. Interclass correlation coefficients (ICC) and Bland-Altman analyses were used to compare the methods. A multilevel model was fitted to estimate the variance of algorithm measurements that was due to differences within- and between-examiners in scleral spur selection versus biological variability. Results The optical flat and the human donor tissue were imaged and appeared without geometric distortions in binary file format. Bland-Altman analyses revealed that caliper measurements tended to underestimate ciliary muscle thickness at 3 mm posterior to the scleral spur in subjects with the thickest ciliary muscles (t = 3.6, p < 0.001). The percent variance due to within- or between-examiner differences in scleral spur selection was found to be small (6%) when compared to the variance due to biological difference across subjects (80%). Using the mean of measurements from three images achieved an estimated ICC of 0.85. Conclusions The semi-automatic algorithm successfully segmented the ciliary muscle for further measurement. Using the algorithm to follow the scleral curvature to locate more posterior measurements is critical to avoid underestimating thickness measurements. This semi-automatic algorithm will allow for repeatable, efficient, and masked ciliary muscle measurements in large datasets. PMID:21169877

  6. The accommodative ciliary muscle function is preserved in older humans

    NASA Astrophysics Data System (ADS)

    Tabernero, Juan; Chirre, Emmanuel; Hervella, Lucia; Prieto, Pedro; Artal, Pablo

    2016-05-01

    Presbyopia, the loss of the eye’s accommodation capability, affects all humans aged above 45-50 years old. The two main reasons for this to happen are a hardening of the crystalline lens and a reduction of the ciliary muscle functionality with age. While there seems to be at least some partial accommodating functionality of the ciliary muscle at early presbyopic ages, it is not yet clear whether the muscle is still active at more advanced ages. Previous techniques used to visualize the accommodation mechanism of the ciliary muscle are complicated to apply in the older subjects, as they typically require fixation stability during long measurement times and/or to have an ultrasound probe directly in contact with the eye. Instead, we used our own developed method based on high-speed recording of lens wobbling to study the ciliary muscle activity in a small group of pseudophakic subjects (around 80 years old). There was a significant activity of the muscle, clearly able to contract under binocular stimulation of accommodation. This supports a purely lenticular-based theory of presbyopia and it might stimulate the search for new solutions to presbyopia by making use of the remaining contraction force still presented in the aging eye.

  7. Ciliary muscle contraction force and trapezius muscle activity during manual tracking of a moving visual target.

    PubMed

    Domkin, Dmitry; Forsman, Mikael; Richter, Hans O

    2016-06-01

    Previous studies have shown an association of visual demands during near work and increased activity of the trapezius muscle. Those studies were conducted under stationary postural conditions with fixed gaze and artificial visual load. The present study investigated the relationship between ciliary muscle contraction force and trapezius muscle activity across individuals during performance of a natural dynamic motor task under free gaze conditions. Participants (N=11) tracked a moving visual target with a digital pen on a computer screen. Tracking performance, eye refraction and trapezius muscle activity were continuously measured. Ciliary muscle contraction force was computed from eye accommodative response. There was a significant Pearson correlation between ciliary muscle contraction force and trapezius muscle activity on the tracking side (0.78, p<0.01) and passive side (0.64, p<0.05). The study supports the hypothesis that high visual demands, leading to an increased ciliary muscle contraction during continuous eye-hand coordination, may increase trapezius muscle tension and thus contribute to the development of musculoskeletal complaints in the neck-shoulder area. Further experimental studies are required to clarify whether the relationship is valid within each individual or may represent a general personal trait, when individuals with higher eye accommodative response tend to have higher trapezius muscle activity. Copyright © 2015 Elsevier Ltd. All rights reserved.

  8. Quantification of the ciliary muscle and crystalline lens interaction during accommodation with synchronous OCT imaging

    PubMed Central

    Ruggeri, Marco; de Freitas, Carolina; Williams, Siobhan; Hernandez, Victor M.; Cabot, Florence; Yesilirmak, Nilufer; Alawa, Karam; Chang, Yu-Cherng; Yoo, Sonia H.; Gregori, Giovanni; Parel, Jean-Marie; Manns, Fabrice

    2016-01-01

    Abstract: Two SD-OCT systems and a dual channel accommodation target were combined and precisely synchronized to simultaneously image the anterior segment and the ciliary muscle during dynamic accommodation. The imaging system simultaneously generates two synchronized OCT image sequences of the anterior segment and ciliary muscle with an imaging speed of 13 frames per second. The system was used to acquire OCT image sequences of a non-presbyopic and a pre-presbyopic subject accommodating in response to step changes in vergence. The image sequences were processed to extract dynamic morphological data from the crystalline lens and the ciliary muscle. The synchronization between the OCT systems allowed the precise correlation of anatomical changes occurring in the crystalline lens and ciliary muscle at identical time points during accommodation. To describe the dynamic interaction between the crystalline lens and ciliary muscle, we introduce accommodation state diagrams that display the relation between anatomical changes occurring in the accommodating crystalline lens and ciliary muscle. PMID:27446660

  9. Quantification of the ciliary muscle and crystalline lens interaction during accommodation with synchronous OCT imaging.

    PubMed

    Ruggeri, Marco; de Freitas, Carolina; Williams, Siobhan; Hernandez, Victor M; Cabot, Florence; Yesilirmak, Nilufer; Alawa, Karam; Chang, Yu-Cherng; Yoo, Sonia H; Gregori, Giovanni; Parel, Jean-Marie; Manns, Fabrice

    2016-04-01

    Two SD-OCT systems and a dual channel accommodation target were combined and precisely synchronized to simultaneously image the anterior segment and the ciliary muscle during dynamic accommodation. The imaging system simultaneously generates two synchronized OCT image sequences of the anterior segment and ciliary muscle with an imaging speed of 13 frames per second. The system was used to acquire OCT image sequences of a non-presbyopic and a pre-presbyopic subject accommodating in response to step changes in vergence. The image sequences were processed to extract dynamic morphological data from the crystalline lens and the ciliary muscle. The synchronization between the OCT systems allowed the precise correlation of anatomical changes occurring in the crystalline lens and ciliary muscle at identical time points during accommodation. To describe the dynamic interaction between the crystalline lens and ciliary muscle, we introduce accommodation state diagrams that display the relation between anatomical changes occurring in the accommodating crystalline lens and ciliary muscle.

  10. Measuring Changes in Ciliary Muscle Thickness with Accommodation in Young Adults

    PubMed Central

    Lossing, Laura Ashley; Sinnott, Loraine T.; Kao, Chiu-Yen; Richdale, Kathryn; Bailey, Melissa D.

    2012-01-01

    Purpose To develop a measurement protocol for changes in the shape and size of the ciliary muscle with accommodation using the Zeiss Visante™ Anterior Segment Optical Coherence Tomographer (AS-OCT) and to determine the test-retest repeatability of these measurements. Methods Subjects were 25 adults ages 23–28 years. The ciliary muscle was imaged at two visits with the Visante™ while accommodative response was monitored during imaging using the PowerRefractor. Ciliary muscle thickness was measured at 1 mm (CMT1), 2 mm (CMT2), and 3 mm (CMT3) posterior to the scleral spur and at the point of maximal thickness (CMTMAX). Thickness was measured at these locations while subjects viewed a target at distance and at a 4.00-D accommodative stimulus. Outcome measures were the change in thickness between distance and the 4.00-D stimulus and the change in thickness per diopter of accommodative response (PowerRefractor). Finally, the repeatability measurements between visit 1 and visit 2 were determined with a Bland-Altman analysis. Results The statistically significant modeled changes in ciliary muscle thickness were as follows: CMTMAX = 69.2 μm (4.00-D stimulus) and 18.1 μm (per diopter of accommodation); CMT1 = 45.2 μm (4.00-D stimulus) and 12.3 μm (per diopter of accommodation); and CMT3 = −45.9 μm (4.00-D stimulus) and −12.0 μm (per diopter of accommodation); p < 0.0001 for all. Conclusions The combination of the Visante™ and the PowerRefractor is a feasible tool for measuring thickening of ciliary muscle at more anterior locations and thinning at more posterior locations during accommodation. We noted a wide range of accommodative responses during the time of image capture in this study indicating that the most accurate estimates of the change in ciliary muscle dimensions with accommodation may be obtained by using accommodative response rather than stimulus values and by using measurements taken simultaneously with image capture. PMID:22504328

  11. Magnetic resonance imaging of aging, accommodating, phakic, and pseudophakic ciliary muscle diameters

    PubMed Central

    Strenk, Susan A.; Strenk, Lawrence M.; Guo, Suqin

    2012-01-01

    PURPOSE To quantify in vivo accommodative changes in the aging human ciliary muscle diameter in phakic and pseudophakic eyes. SETTING Department of Surgery/Bioengineering, UMDNJ–Robert Wood Johnson Medical School, Piscataway, and the Institute of Ophthalmology and Visual Science UMDNJ–New Jersey Medical School, Newark, New Jersey, USA. METHODS Images were acquired from 48 eyes of 40 people between the ages of 22 and 91 years, 1 eye of 32 phakic volunteers and both eyes of 8 patients who had monocular implantation of a single-piece AcrySof intraocular lens (IOL) (Alcon Laboratories). Images were acquired during physiological accommodation and with accommodation at rest, and the diameter of the ciliary muscle ring was measured. RESULTS Results show the ciliary muscle remains active throughout life. The accommodative change in its diameter (mean 0.64 mm) (P<.00001) was undiminished by age or IOL implantation. Preliminary data showed that the accommodative decrease in muscle diameter in phakic and pseudophakic eyes was statistically identical. The phakic eyes had a marked decrease in ciliary muscle diameter with advancing age for both accommodative states (P<.000001 and P<.000001), which did not appear to be altered by IOL implantation. The lens equator was constant with age in the unaccommodated human eye, resulting in decreased circumlental space with advancing age in the phakic eyes. CONCLUSION Although the undiminished ability of the ciliary muscle to decrease its diameter with accommodation can be relied on in strategies for presbyopia correction, even in advanced presbyopia, the decreasing circumlental space and its potential effects on zonular tension must also be considered. PMID:17081859

  12. Synchronous imaging of the pulse response of the ciliary muscle and lens with SD-OCT (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Chang, Yu-Cherng; Pham, Alex; Williams, Siobhan; Alawa, Karam A.; de Freitas, Carolina; Ruggeri, Marco; Parel, Jean-Marie A.; Manns, Fabrice

    2017-02-01

    Purpose: To determine the dynamic interaction between ciliary muscle and lens during accommodation and disaccommodation through synchronous imaging of ciliary muscle and lens response to pulse stimulus Methods: The ciliary muscle and lens were imaged simultaneously in a 33 year old subject responding to a 4D pulse stimulus (accommodative stimulus at 1.7 s, disaccommodative stimulus at 7.7 s) using an existing imaging system (Ruggeri et al, 2016) consisting of an Anterior Segment Optical Coherence Tomography system, Ciliary Muscle Optical Coherence Tomography system, and custom-built accommodation module. OCT images were recorded at an effective frame rate of 13.0 frames per second for a total scan time of 11.5 s. An automated segmentation algorithm was applied to images of the anterior segment to detect the boundaries of the cornea and lens, from which lens thickness was extracted. Segmentation of the ciliary muscle was performed manually and then corrected for distortion due to refraction of the beam to obtain measurements of thicknesses at the apex and fixed distances from the scleral spur. Results: The dynamic biometric response to a pulse stimulus at 4D was determined for both the ciliary muscle and lens, suggesting the ciliary muscle and lens interact differently in accommodation and disaccommodation. Conclusions: The study introduces new data and analyses of the ciliary muscle and lens interaction during a complete accommodative response from the relaxed to the accommodated state and back, providing insight into the interplay between individual elements in the accommodative system and how their relationships may change with age.

  13. Apical localization of glutamate in GLAST-1, glutamine synthetase positive ciliary body nonpigmented epithelial cells

    PubMed Central

    Langford, Marlyn P; Gosslee, Jeffrey M; Liang, Chanping; Chen, Dequan; Redens, Thomas B.; Welbourne, Tomas C

    2007-01-01

    The distribution of glutamate (Glu), the Glu transporter GLAST-1, and glutamine synthetase (GS) in human and monkey anterior uveal tissue, as well as serum (S) to aqueous humor (AH) Glu and glutamine (Gln) gradients were investigated. Cross-linked Glu (xGlu), GLAST-1, and GS were detected using the immunofluorescent antibody technique. S/AH Glu, Gln, and alanine (Ala) concentrations were quantified by high performance liquid chromatography. xGlu immunoreactivity was detected in melanocytes, posterior pigmented epithelial/dilator muscle cells, vascular endothelial cells, and lymphocytes of the iris, as well as the pigmented (PE) and nonpigmented epithelial (NPE) cells and muscle cells of ciliary body. xGlu immunoreactivity was highly concentrated at the apices of GLAST-1, GS positive ciliary body NPE cells, and in GLAST-1 positive iris melanocytes and iris dilator muscle cells. AH Glu concentrations were lower (p < 0.001), while Gln was higher in monkey (p = 0.01) and human cataractous (p = 0.15) AH than serum. The results indicate that Glu is concentrated within GLAST-1, GS positive NPE cells and are consistent with the suggestion that Glu and Gln concentrations in AH may be due in part to GLAST-1 and GS activity in iris and ciliary body epithelial cells. PMID:19668465

  14. [Mesectodermal leiomyoma. Unusual tumor of the ciliary body].

    PubMed

    Rentería-Ruiz, Nancy Paulina; de Wit-Carter, Guillermo; Villaseñor-Diez, Jaime; Flores-Estrada, José Javier; Rodríguez-Reyes, Abelardo Antonio

    2014-01-01

    Mesectodermal leiomyoma is a benign tumor of smooth muscle of the ciliary body, which is derived from the neural crest. We report the case of a 35-year-old Mexican woman with visually impaired and blurred vision of the right eye of 2 months duration. The clinical and imaging presuntional diagnosis was adenoma of the non pigmented epithelium of the ciliary body and it was surgically resected. Microscopically, the tumor was composed of cells with round nuclei and scant cytoplasm without atypia or mitosis, arranged in a fibrillary background. The immunohistochemical markers for vimentin, muscle specific actin, smooth muscle actin and calponin were strongly positive in the cytoplasm of the neoplastic cells, while for glial fibrillary acidic protein and S-100 protein were negative in the same cellular population. Mesectodermal leiomyoma of the ciliary body is benign tumor of smooth muscle extremely rare in this location. Until now, there are just 25 previous reported cases in the literature and, the main differential diagnosis is uveal malignant melanoma, therefore some eyes were enucleated. The ultrabiomicroscopy, A and B-scan imaging studies are useful in the evaluation, however, is mandatory the microsocpic examination with routine and histochemical stains as well as the use of immunohistochemical markers such as vimentin, specific muscle actin, smooth muscle actin andcalponin to stablish the smooth muscle origin of this neoplasm, and rule out other malignant neoplams such as malignant melanoma.

  15. Age-related posterior ciliary muscle restriction - A link between trabecular meshwork and optic nerve head pathophysiology.

    PubMed

    Croft, Mary Ann; Lütjen-Drecoll, Elke; Kaufman, Paul L

    2017-05-01

    The ciliary muscle plays a major role in controlling both accommodation and outflow facility in primates. The ciliary muscle and the choroid functionally form an elastic network that extends from the trabecular meshwork all the way to the back of the eye and ultimately attaches to the elastic fiber ring that surrounds the optic nerve and to the lamina cribrosa through which the nerve passes. The ciliary muscle governs the accommodative movement of the elastic network. With age ciliary muscle mobility is restricted by progressively inelastic posterior attachments and the posterior restriction makes the contraction progressively isometric; placing increased tension on the optic nerve region. In addition, outflow facility also declines with age and limbal corneoscleral contour bows inward. Age-related loss in muscle movement and altered limbal corneoscleral contour could both compromise the basal function of the trabecular meshwork. Further, recent studies in non-human primates show that the central vitreous moves posteriorly all the way back to the optic nerve region, suggesting a fluid current and a pressure gradient toward the optic nerve. Thus, there may be pressure and tension spikes on the optic nerve region during accommodation and these pressure and tension spikes may increase with age. This constellation of events could be relevant to glaucomatous optic neuropathy. In summary, our hypothesis is that glaucoma and presbyopia may be literally linked to each other, via the choroid, and that damage to the optic nerve may be inflicted by accommodative intraocular pressure and choroidal tension "spikes", which may increase with age. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. Negative regulation of ciliary length by ciliary male germ cell-associated kinase (Mak) is required for retinal photoreceptor survival.

    PubMed

    Omori, Yoshihiro; Chaya, Taro; Katoh, Kimiko; Kajimura, Naoko; Sato, Shigeru; Muraoka, Koichiro; Ueno, Shinji; Koyasu, Toshiyuki; Kondo, Mineo; Furukawa, Takahisa

    2010-12-28

    Cilia function as cell sensors in many organs, and their disorders are referred to as "ciliopathies." Although ciliary components and transport machinery have been well studied, regulatory mechanisms of ciliary formation and maintenance are poorly understood. Here we show that male germ cell-associated kinase (Mak) regulates retinal photoreceptor ciliary length and subcompartmentalization. Mak was localized both in the connecting cilia and outer-segment axonemes of photoreceptor cells. In the Mak-null retina, photoreceptors exhibit elongated cilia and progressive degeneration. We observed accumulation of intraflagellar transport 88 (IFT88) and IFT57, expansion of kinesin family member 3A (Kif3a), and acetylated α-tubulin signals in the Mak-null photoreceptor cilia. We found abnormal rhodopsin accumulation in the Mak-null photoreceptor cell bodies at postnatal day 14. In addition, overexpression of retinitis pigmentosa 1 (RP1), a microtubule-associated protein localized in outer-segment axonemes, induced ciliary elongation, and Mak coexpression rescued excessive ciliary elongation by RP1. The RP1 N-terminal portion induces ciliary elongation and increased intensity of acetylated α-tubulin labeling in the cells and is phosphorylated by Mak. These results suggest that Mak is essential for the regulation of ciliary length and is required for the long-term survival of photoreceptors.

  17. Negative regulation of ciliary length by ciliary male germ cell-associated kinase (Mak) is required for retinal photoreceptor survival

    PubMed Central

    Omori, Yoshihiro; Chaya, Taro; Katoh, Kimiko; Kajimura, Naoko; Sato, Shigeru; Muraoka, Koichiro; Ueno, Shinji; Koyasu, Toshiyuki; Kondo, Mineo; Furukawa, Takahisa

    2010-01-01

    Cilia function as cell sensors in many organs, and their disorders are referred to as “ciliopathies.” Although ciliary components and transport machinery have been well studied, regulatory mechanisms of ciliary formation and maintenance are poorly understood. Here we show that male germ cell-associated kinase (Mak) regulates retinal photoreceptor ciliary length and subcompartmentalization. Mak was localized both in the connecting cilia and outer-segment axonemes of photoreceptor cells. In the Mak-null retina, photoreceptors exhibit elongated cilia and progressive degeneration. We observed accumulation of intraflagellar transport 88 (IFT88) and IFT57, expansion of kinesin family member 3A (Kif3a), and acetylated α-tubulin signals in the Mak-null photoreceptor cilia. We found abnormal rhodopsin accumulation in the Mak-null photoreceptor cell bodies at postnatal day 14. In addition, overexpression of retinitis pigmentosa 1 (RP1), a microtubule-associated protein localized in outer-segment axonemes, induced ciliary elongation, and Mak coexpression rescued excessive ciliary elongation by RP1. The RP1 N-terminal portion induces ciliary elongation and increased intensity of acetylated α-tubulin labeling in the cells and is phosphorylated by Mak. These results suggest that Mak is essential for the regulation of ciliary length and is required for the long-term survival of photoreceptors. PMID:21148103

  18. Age-related posterior ciliary muscle restriction – A link between trabecular meshwork and optic nerve head pathophysiology

    PubMed Central

    Lütjen-Drecoll, Elke; Kaufman, Paul L.

    2016-01-01

    The ciliary muscle plays a major role in controlling both accommodation and outflow facility in primates. The ciliary muscle and the choroid functionally form an elastic network that extends from the trabecular meshwork all the way to the back of the eye and ultimately attaches to the elastic fiber ring that surrounds the optic nerve and to the lamina cribrosa through which the nerve passes. The ciliary muscle governs the accommodative movement of the elastic network. With age ciliary muscle mobility is restricted by progressively inelastic posterior attachments and the posterior restriction makes the contraction progressively isometric; placing increased tension on the optic nerve region. In addition, outflow facility also declines with age and limbal corneoscleral contour bows inward. Age-related loss in muscle movement and altered limbal corneoscleral contour could both compromise the basal function of the trabecular meshwork. Further, recent studies in non-human primates show that the central vitreous moves posteriorly all the way back to the optic nerve region, suggesting a fluid current and a pressure gradient toward the optic nerve. Thus, there may be pressure and tension spikes on the optic nerve region during accommodation and these pressure and tension spikes may increase with age. This constellation of events could be relevant to glaucomatous optic neuropathy. In summary, our hypothesis is that glaucoma and presbyopia may be literally linked to each other, via the choroid, and that damage to the optic nerve may be inflicted by accommodative intraocular pressure and choroidal tension “spikes”, which may increase with age. PMID:27453343

  19. BMP signaling is required for development of the ciliary body.

    PubMed

    Zhao, Shulei; Chen, Qin; Hung, Fang-Cheng; Overbeek, Paul A

    2002-10-01

    The ciliary body in the eye secretes aqueous humor and glycoproteins of the vitreous body and maintains the intraocular pressure. The ciliary muscle controls the shape of the lens through the ciliary zonules to focus the image onto the retina. During embryonic development, the ciliary epithelium is derived from the optic vesicle, but the molecular signals that control morphogenesis of the ciliary body are unknown. We report that lens-specific expression of a transgenic protein, Noggin, can block BMP signaling in the mouse eye and result in failure in formation of the ciliary processes. Co-expression of transgenic BMP7 restores normal development of the ciliary epithelium. Ectopic expression of Noggin also promotes differentiation of retinal ganglion cells. These results indicate that BMP signaling is required for development of the ciliary body and may also play a role in regulation of neuronal differentiation in the developing eye.

  20. [Regeneration of the ciliary beat of human ciliated cells].

    PubMed

    Wolf, G; Koidl, B; Pelzmann, B

    1991-10-01

    The influence of an isotonic, alkaline saline solution (diluted "Emser Sole" or brine from the spa of Bad Ems) on the ciliary beat of isolated cultured human ciliated cells of the upper respiratory tract was investigated. The ciliary beat was observed via an inverted phase contrast microscope (Zeiss Axiomat IDPC) and measured microphotometrically under physiological conditions and after the damaging influence of 1% propanal solution. Under physiological conditions the saline solution had a positive, although statistically not significant influence on the frequency of the ciliary beat. After damage of the cultivated cells by 1% propanal solution, the saline solution had a significant better influence on the regeneration of the cultured cells than a physiological sodium chloride solution. It is concluded that diluted brine from Bad Ems has a positive effect on the ciliary beat of the respiratory epithelium and accelerates its regeneration after damage by viral and bacterial infections, surgery or inhaled noxae.

  1. Comparative anatomy of the accessory ciliary ganglion in mammals.

    PubMed

    Kuchiiwa, S; Kuchiiwa, T; Suzuki, T

    1989-01-01

    The orbits of 13 mammalian species (pig, sika deer, domestic sheep, horse, cat, fox, racoon dog, marten, rat, rabbit, crab-eating macaque, japanese macaque and man) were stained with silver nitrate and dissected under a dissecting microscope with special attention to the presence and location of the accessory ciliary ganglion. Some preparations were stained with thionin and examined as whole-mounts in a transmission microscope. The accessory ciliary ganglion was present in all 13 species, although the number and degree of development varied greatly from species to species. The accessory ciliary ganglion could be readily differentiated from the main ciliary ganglion in the following respects: it was located on the short ciliary nerve, and it had no root derived directly from the inferior trunk of the oculomotor nerve and it never attaches to this nerve. In many species, ganglion cells were also scattered in the short ciliary nerves in the stained whole preparations. In a few species, there were one or more small ganglia on the nerve to the inferior oblique muscle.

  2. Culture of Primary Ciliary Dyskinesia Epithelial Cells at Air-Liquid Interface Can Alter Ciliary Phenotype but Remains a Robust and Informative Diagnostic Aid

    PubMed Central

    Coles, Janice L.; Williams, Gwyneth; Rutman, Andrew; Goggin, Patricia M.; Adam, Elizabeth C.; Page, Anthony; Evans, Hazel J.; Lackie, Peter M.; O’Callaghan, Christopher; Lucas, Jane S.

    2014-01-01

    Background The diagnosis of primary ciliary dyskinesia (PCD) requires the analysis of ciliary function and ultrastructure. Diagnosis can be complicated by secondary effects on cilia such as damage during sampling, local inflammation or recent infection. To differentiate primary from secondary abnormalities, re-analysis of cilia following culture and re-differentiation of epithelial cells at an air-liquid interface (ALI) aids the diagnosis of PCD. However changes in ciliary beat pattern of cilia following epithelial cell culture has previously been described, which has brought the robustness of this method into question. This is the first systematic study to evaluate ALI culture as an aid to diagnosis of PCD in the light of these concerns. Methods We retrospectively studied changes associated with ALI-culture in 158 subjects referred for diagnostic testing at two PCD centres. Ciliated nasal epithelium (PCD n = 54; non-PCD n = 111) was analysed by high-speed digital video microscopy and transmission electron microscopy before and after culture. Results Ciliary function was abnormal before and after culture in all subjects with PCD; 21 PCD subjects had a combination of static and uncoordinated twitching cilia, which became completely static following culture, a further 9 demonstrated a decreased ciliary beat frequency after culture. In subjects without PCD, secondary ciliary dyskinesia was reduced. Conclusions The change to ciliary phenotype in PCD samples following cell culture does not affect the diagnosis, and in certain cases can assist the ability to identify PCD cilia. PMID:24586956

  3. Guinea Pig Ciliary Muscle Development

    PubMed Central

    Pucker, Andrew D.; Carpenter, Ashley R.; McHugh, Kirk M.; Mutti, Donald O.

    2014-01-01

    Purpose The purpose of this study was to develop a method for quantifying guinea pig ciliary muscle volume (CMV) and to determine its relationship to age and ocular biometric measurements. Methods Six albino guinea pigs eyes were collected at each of five ages (n=30 eyes). Retinoscopy and photography were used to document refractive error, eye size, and eye shape. Serial sections through the excised eyes were made and then labeled with an α-smooth muscle actin antibody. The CM was then visualized with an Olympus BX51 microscope, reconstructed with Stereo Investigator (MBF Bioscience) and analyzed using Neurolucida Explorer (MBF Bioscience). Full (using all sections) and partial (using a subset of sections) reconstruction methods were used to determine CMV. Results There was no significant difference between the full and partial volume determination methods (P = 0.86). The mean CMV of the 1, 10, 20, 30, and 90-day old eyes was 0.40 ± 0.16 mm3, 0.48 ± 0.13 mm3, 0.67 ± 0.15 mm3, 0.86 ± 0.35 mm3, and 1.09 ± 0.63 mm3, respectively. CMV was significantly correlated with log age (P = 0.001), ocular length (P = 0.003), limbal circumference (P = 0.01), and equatorial diameter (P = 0.003). It was not correlated with refractive error (P = 0.73) or eye shape (P = 0.60). Multivariate regression determined that biometric variables were not significantly associated with CMV after adjustment for age. Conclusions Three-dimensional reconstruction was an effective means of determining CMV. These data provide evidence that CM growth occurs with age in tandem with eye size in normal albino guinea pigs. Additional work is needed to determine the relationship between CMV and abnormal ocular growth. PMID:24901488

  4. Expression of a chemokine by ciliary body epithelium in horses with naturally occurring recurrent uveitis and in cultured ciliary body epithelial cells.

    PubMed

    Gilger, Brian C; Yang, Ping; Salmon, Jacklyn H; Jaffe, Glenn J; Allen, Janice B

    2002-07-01

    To determine whether a chemokine (RANTES)-like protein expressed by ciliary epithelium plays a role in uveitis. 3 clinically normal horses intradermal, 5 eyes from 5 horses with recurrent uveitis, and 10 normal eyes from 5 age- and sex-matched horses. Cross-reactivity and sensitivity of recombinant human (rh)-regulated upon activation, normal T-cell expressed and secreted (RANTES) protein were evaluated in horses by use of intradermal hypersensitivity reactions and a chemotaxis assay. Aqueous humor and ciliary body of eyes from clinically normal horses and horses with uveitis were examined for RANTES expression by use of an ELISA and reverse transcription-polymerase chain reaction (RT-PCR). Expression of RANTES mRNA and protein content of primary cultures of equine ciliary pigmented epithelial cells (RT-PCR) and culture supernatant (ELISA) were measured 6 or 24 hours, respectively, after cultures were stimulated with interleukin-1beta and tumor necrosis factor-alpha. Strong reactions to intradermal hypersensitivity testing and significant chemotaxis of equine leukocytes to rh-RANTES wereas observed. Aqueous humor of eyes from horses with uveitis contained increased concentrations of rh-RANTES-like protein (mean +/- SD, 45.9+/-31.7 pg/ml), compared with aqueous humor from clinically normal horses (0 pg/ml). Ciliary body from horses with uveitis expressed RANTES mRNA, whereas ciliary body from clinically normal horses had low mRNA expression. Stimulated ciliary pigmented epithelial cells expressed increased amounts of rh-RANTES-like protein (506.1+/-298.3 pg/ml) and mRNA, compared with unstimulated samples. Ciliary epithelium may play a role in recruitment and activation of leukocytes through expression of RANTES.

  5. Rapid diagnosis of primary ciliary dyskinesia: cell culture and soft computing analysis.

    PubMed

    Pifferi, Massimo; Bush, Andrew; Montemurro, Francesca; Pioggia, Giovanni; Piras, Martina; Tartarisco, Gennaro; Di Cicco, Maria; Chinellato, Iolanda; Cangiotti, Angela M; Boner, Attilio L

    2013-04-01

    Diagnosis of primary ciliary dyskinesia (PCD) sometimes requires repeated nasal brushing to exclude secondary ciliary alterations. Our aim was to evaluate whether the use of a new method of nasal epithelial cell culture can speed PCD diagnosis in doubtful cases and to identify which are the most informative parameters by means of a multilayer artificial neural network (ANN). A cross-sectional study was performed in patients with suspected PCD. All patients underwent nasal brushing for ciliary motion analysis, ultrastructural assessment and evaluation of ciliary function after ciliogenesis in culture by ANN. 151 subjects were studied. A diagnostic suspension cell culture was obtained in 117 nasal brushings. A diagnosis of PCD was made in 36 subjects (29 of whom were children). In nine out of the 36 patients the diagnosis was made only after a second brushing, because of equivocal results of both tests at first examination. In each of these subjects diagnosis of PCD was confirmed by cell culture results. Cell culture in suspension evaluated by means of ANN allows the separation of PCD from secondary ciliary dyskinesia patients after only 5 days of culture and allows diagnosis to be reached in doubtful cases, thus avoiding the necessity of a second sample.

  6. The pupillary and ciliary components of the cat Edinger-Westphal nucleus: a transsynaptic transport investigation.

    PubMed

    Erichsen, Jonathan T; May, Paul J

    2002-01-01

    The distribution of preganglionic motoneurons supplying the ciliary ganglion in the cat was defined both qualitatively and quantitatively. These cells were retrogradely labeled directly, following injections of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) into the ciliary ganglion, or were transsynaptically labeled following injections of WGA into the vitreous chamber. Almost half of the cells are distributed rostral to the oculomotor nucleus, both in and lateral to the anteromedian nucleus. Of the remaining preganglionic motoneurons, roughly 20% of the total are located dorsal to the oculomotor nucleus. Strikingly few of these neurons are actually found within the Edinger-Westphal nucleus proper. Instead, the majority are found in the adjacent supraoculomotor area or along the midline between the two somatic nuclei. An additional population, roughly 30% of the total, is located ventral to the oculomotor nucleus. This study also provides evidence for a functional subdivision of this preganglionic population. Pupil-related preganglionic motoneurons were transsynaptically labeled by injecting WGA into the anterior chamber, while lens-related preganglionic motoneurons were transsynaptically labeled by injecting WGA into the ciliary muscle. The results suggest that the pupil-related preganglionic motoneurons, that is, those controlling the iris sphincter pupillae muscle, are located rostrally, in and lateral to the anteromedian nucleus. In contrast, lens-related preganglionic motoneurons, that is, those controlling the ciliary muscle are particularly prevalent caudally, both dorsal and ventral to the oculomotor nucleus. Thus, the cat intraocular muscle preganglionic innervation is spatially organized with respect to function, despite the dispersed nature of its distribution.

  7. ICK is essential for cell type-specific ciliogenesis and the regulation of ciliary transport.

    PubMed

    Chaya, Taro; Omori, Yoshihiro; Kuwahara, Ryusuke; Furukawa, Takahisa

    2014-06-02

    Cilia and flagella are formed and maintained by intraflagellar transport (IFT) and play important roles in sensing and moving across species. At the distal tip of the cilia/flagella, IFT complexes turn around to switch from anterograde to retrograde transport; however, the underlying regulatory mechanism is unclear. Here, we identified ICK localization at the tip of cilia as a regulator of ciliary transport. In ICK-deficient mice, we found ciliary defects in neuronal progenitor cells with Hedgehog signal defects. ICK-deficient cells formed cilia with mislocalized Hedgehog signaling components. Loss of ICK caused the accumulation of IFT-A, IFT-B, and BBSome components at the ciliary tips. In contrast, overexpression of ICK induced the strong accumulation of IFT-B, but not IFT-A or BBSome components at ciliary tips. In addition, ICK directly phosphorylated Kif3a, while inhibition of this Kif3a phosphorylation affected ciliary formation. Our results suggest that ICK is a Kif3a kinase and essential for proper ciliogenesis in development by regulating ciliary transport at the tip of cilia. © 2014 The Authors.

  8. ICK is essential for cell type-specific ciliogenesis and the regulation of ciliary transport

    PubMed Central

    Chaya, Taro; Omori, Yoshihiro; Kuwahara, Ryusuke; Furukawa, Takahisa

    2014-01-01

    Cilia and flagella are formed and maintained by intraflagellar transport (IFT) and play important roles in sensing and moving across species. At the distal tip of the cilia/flagella, IFT complexes turn around to switch from anterograde to retrograde transport; however, the underlying regulatory mechanism is unclear. Here, we identified ICK localization at the tip of cilia as a regulator of ciliary transport. In ICK-deficient mice, we found ciliary defects in neuronal progenitor cells with Hedgehog signal defects. ICK-deficient cells formed cilia with mislocalized Hedgehog signaling components. Loss of ICK caused the accumulation of IFT-A, IFT-B, and BBSome components at the ciliary tips. In contrast, overexpression of ICK induced the strong accumulation of IFT-B, but not IFT-A or BBSome components at ciliary tips. In addition, ICK directly phosphorylated Kif3a, while inhibition of this Kif3a phosphorylation affected ciliary formation. Our results suggest that ICK is a Kif3a kinase and essential for proper ciliogenesis in development by regulating ciliary transport at the tip of cilia. PMID:24797473

  9. Ick Ciliary Kinase Is Essential for Planar Cell Polarity Formation in Inner Ear Hair Cells and Hearing Function.

    PubMed

    Okamoto, Shio; Chaya, Taro; Omori, Yoshihiro; Kuwahara, Ryusuke; Kubo, Shun; Sakaguchi, Hirofumi; Furukawa, Takahisa

    2017-02-22

    Cellular asymmetries play crucial roles in development and organ function. The planar cell polarity (PCP) signaling pathway is involved in the establishment of cellular asymmetry within the plane of a cell sheet. Inner ear sensory hair cells (HCs), which have several rows of staircase-like stereocilia and one kinocilium located at the vertex of the stereocilia protruding from the apical surface of each HC, exhibit a typical form of PCP. Although connections between cilia and PCP signaling in vertebrate development have been reported, their precise nature is not well understood. During inner ear development, several ciliary proteins are known to play a role in PCP formation. In the current study, we investigated a functional role for intestinal cell kinase (Ick), which regulates intraflagellar transport (IFT) at the tip of cilia, in the mouse inner ear. A lack of Ick in the developing inner ear resulted in PCP defects in the cochlea, including misorientation or misshaping of stereocilia and aberrant localization of the kinocilium and basal body in the apical and middle turns, leading to auditory dysfunction. We also observed abnormal ciliary localization of Ift88 in both HCs and supporting cells. Together, our results show that Ick ciliary kinase is essential for PCP formation in inner ear HCs, suggesting that ciliary transport regulation is important for PCP signaling. SIGNIFICANCE STATEMENT The cochlea in the inner ear is the hearing organ. Planar cell polarity (PCP) in hair cells (HCs) in the cochlea is essential for mechanotransduction and refers to the asymmetric structure consisting of stereociliary bundles and the kinocilium on the apical surface of the cell body. We reported previously that a ciliary kinase, Ick, regulates intraflagellar transport (IFT). Here, we found that loss of Ick leads to abnormal localization of the IFT component in kinocilia, PCP defects in HCs, and hearing dysfunction. Our study defines the association of ciliary transport

  10. Analysis of ciliary beat frequency and ovum transport ability in the mouse oviduct.

    PubMed

    Shi, Dongbo; Komatsu, Kouji; Uemura, Tadashi; Fujimori, Toshihiko

    2011-03-01

    The oviduct is important in reproduction where fertilization occurs, and the fertilized eggs are conveyed to the uterus. Multi-ciliated cells of the oviductal epithelium and muscle contractions are believed to generate this unidirectional flow. Although there are many studies in human oviducts, there are few reports on mouse oviductal ciliary movements where we can dissect underlying genetic programs. To study ciliary movements in the mouse oviduct, we exposed the ovary-side of the oviduct (infundibulum) longitudinally and recorded the ciliary beatings in a hanging drop preparation. We calculated the ciliary beat frequency (CBF) by automated image analysis and found that the average CBF was 10.9 ± 3.3 and 8.5 ± 2.5 Hz (±standard deviation) during the diestrus and estrus stages, respectively. Mapping of the CBF to multiple locations in the epithelium showed that the cilia beat regularly at a local level, but have a range of frequencies within the entire plane. We also observed ova with cumulus cells were transported to the uterus side by the opened oviduct at the diestrus and estrus stages. These results suggest that the ciliated cells of the infundibulum can generate unidirectional flows and are able to deliver ova by their ciliary activities despite their discordance in beating periodicity. © 2011 The Authors. Journal compilation © 2011 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.

  11. Characterization of primary cilia in human airway smooth muscle cells.

    PubMed

    Wu, Jun; Du, Hui; Wang, Xiangling; Mei, Changlin; Sieck, Gary C; Qian, Qi

    2009-08-01

    Considerable evidence indicates a key role for primary cilia of mammalian cells in mechanochemical sensing. Dysfunctions of primary cilia have been linked to the pathogenesis of several human diseases. However, cilia-related research has been limited to a few cell and tissue types; to our knowledge, no literature exists on primary cilia in airway smooth muscle (ASM). The aim of this study was to characterize primary cilia in human ASM. Primary cilia of human bronchial smooth muscle cells (HBSMCs) were examined using immunofluorescence confocal microscopy, and scanning and transmission electron microscopy. HBSMC migration and injury repair were examined by scratch-wound and epidermal growth factor (EGF)-induced migration assays. Cross-sectional images of normal human bronchi revealed that primary cilia of HBSMCs within each ASM bundle aggregated at the same horizontal level, forming a "cilium layer." Individual cilia of HBSMCs projected into extracellular matrix and exhibited varying degrees of deflection. Mechanochemical sensing molecules, polycystins, and alpha2-, alpha5-, and beta1-integrins were enriched in cilia, as was EGF receptor, known to activate jointly with integrins during cell migration. Migration assays demonstrated a ciliary contribution to HBSMC migration and wound repair. The primary cilia of ASM cells exert a role in sensing and transducing extracellular mechanochemical signals and in ASM injury repair. Defects in ASM ciliary function could potentially affect airway wall maintenance and/or remodeling, possibly relating to the genesis of bronchiectasis in autosomal dominant polycystic kidney disease, a disease of ciliopathy.

  12. Cell- and subunit-specific mechanisms of CNG channel ciliary trafficking and localization in C. elegans.

    PubMed

    Wojtyniak, Martin; Brear, Andrea G; O'Halloran, Damien M; Sengupta, Piali

    2013-10-01

    Primary cilia are ubiquitous sensory organelles that concentrate transmembrane signaling proteins essential for sensing environmental cues. Mislocalization of crucial ciliary signaling proteins, such as the tetrameric cyclic nucleotide-gated (CNG) channels, can lead to cellular dysfunction and disease. Although several cis- and trans-acting factors required for ciliary protein trafficking and localization have been identified, whether these mechanisms act in a protein- and cell-specific manner is largely unknown. Here, we show that CNG channel subunits can be localized to discrete ciliary compartments in individual sensory neurons in C. elegans, suggesting that channel composition is heterogeneous across the cilium. We demonstrate that ciliary localization of CNG channel subunits is interdependent on different channel subunits in specific cells, and identify sequences required for efficient ciliary targeting and localization of the TAX-2 CNGB and TAX-4 CNGA subunits. Using a candidate gene approach, we show that Inversin, transition zone proteins, intraflagellar transport motors and a MYND-domain protein are required to traffic and/or localize CNG channel subunits in both a cell- and channel subunit-specific manner. We further find that TAX-2 and TAX-4 are relatively immobile in specific sensory cilia subcompartments, suggesting that these proteins undergo minimal turnover in these domains in mature cilia. Our results uncover unexpected diversity in the mechanisms that traffic and localize CNG channel subunits to cilia both within and across cell types, highlighting the essential contribution of this process to cellular functions.

  13. Cell- and subunit-specific mechanisms of CNG channel ciliary trafficking and localization in C. elegans

    PubMed Central

    Wojtyniak, Martin; Brear, Andrea G.; O'Halloran, Damien M.; Sengupta, Piali

    2013-01-01

    Summary Primary cilia are ubiquitous sensory organelles that concentrate transmembrane signaling proteins essential for sensing environmental cues. Mislocalization of crucial ciliary signaling proteins, such as the tetrameric cyclic nucleotide-gated (CNG) channels, can lead to cellular dysfunction and disease. Although several cis- and trans-acting factors required for ciliary protein trafficking and localization have been identified, whether these mechanisms act in a protein- and cell-specific manner is largely unknown. Here, we show that CNG channel subunits can be localized to discrete ciliary compartments in individual sensory neurons in C. elegans, suggesting that channel composition is heterogeneous across the cilium. We demonstrate that ciliary localization of CNG channel subunits is interdependent on different channel subunits in specific cells, and identify sequences required for efficient ciliary targeting and localization of the TAX-2 CNGB and TAX-4 CNGA subunits. Using a candidate gene approach, we show that Inversin, transition zone proteins, intraflagellar transport motors and a MYND-domain protein are required to traffic and/or localize CNG channel subunits in both a cell- and channel subunit-specific manner. We further find that TAX-2 and TAX-4 are relatively immobile in specific sensory cilia subcompartments, suggesting that these proteins undergo minimal turnover in these domains in mature cilia. Our results uncover unexpected diversity in the mechanisms that traffic and localize CNG channel subunits to cilia both within and across cell types, highlighting the essential contribution of this process to cellular functions. PMID:23886944

  14. Prostaglandin F2 alpha and its analogs induce release of endogenous prostaglandins in iris and ciliary muscles isolated from cat and other mammalian species.

    PubMed

    Yousufzai, S Y; Ye, Z; Abdel-Latif, A A

    1996-09-01

    Prostaglandin F2 alpha (PGF 2 alpha) and its analog latanoprost are effective in lowering intraocular pressure (IOP) in both animal and human subjects. There is mounting experimental evidence now which indicates that the IOP-lowering effect of these PGs occurs through an increased uveoscleral outflow of aqueous humor. The ciliary muscle constitutes the main resistance in this pathway. Work from several laboratories, including our own, has shown that in this smooth muscle PGF 2 alpha has little effect on cAMP accumulation or on Ca2+ mobilization. In the present study, we hypothesized that some of the effects of PGF2 alpha and its analogs may be mediated through the release of endogenous PGs. The purpose of this work was to determine whether or not PGF2 alpha and its analogs can enhance the release of endogenous PGs in iris and ciliary muscles isolated from different species. This report documents for the first time that exogenous PGF2 alpha and its analogs, PhXA85 and latanoprost, stimulate the formation of PGE2, PGD2 and PGF2 alpha in iris and ciliary muscles isolated from cat, bovine, rabbit, dog, rhesus monkey and human. PG-induced PG release was demonstrated by means of both radioimmunoassay and radiochromatography. Kinetic studies on cat iris revealed that PGF2 alpha-induced PGE2 release is time (t 1/2 = 1.7 min) and dose-dependent (EC50 = 45 nM). The increase in PGE2 release was blocked by indomethacin (Indo) and by dexamethasone in a dose-dependent manner with IC50 s of 9.2 nM and 2.6 microM, respectively. Furthermore, dexamethasone inhibited arachidonic acid (AA) release, suggesting the involvement of phospholipase A2 in PGF2 alpha-induced PG release. The data presented demonstrate that PGF2 alpha and its analogs interact with the PG receptor to stimulate phospholipase A2 and release AA for PG synthesis. Relaxation of ciliary muscle by PGF2 alpha and its analogs, via release of endogenous PGE2, a potent activator of the adenylate cyclase system, could in

  15. Ciliary metachronal wave propagation on the compliant surface of Paramecium cells.

    PubMed

    Narematsu, Naoki; Quek, Raymond; Chiam, Keng-Hwee; Iwadate, Yoshiaki

    2015-12-01

    Ciliary movements in protozoa exhibit metachronal wave-like coordination, in which a constant phase difference is maintained between adjacent cilia. It is at present generally thought that metachronal waves require hydrodynamic coupling between adjacent cilia and the extracellular fluid. To test this hypothesis, we aspirated a Paramecium cell using a micropipette which completely sealed the surface of the cell such that no fluid could pass through the micropipette. Thus, the anterior and the posterior regions of the cell were hydrodynamically decoupled. Nevertheless, we still observed that metachronal waves continued to propagate from the anterior to the posterior ends of the cell, suggesting that in addition to hydrodynamic coupling, there are other mechanisms that can also transmit the metachronal waves. Such transmission was also observed in computational modeling where the fluid was fully decoupled between two partitions of a beating ciliary array. We also imposed cyclic stretching on the surface of live Paramecium cells and found that metachronal waves persisted in the presence of cyclic stretching. This demonstrated that, in addition to hydrodynamic coupling, a compliant substrate can also play a critical role in mediating the propagation of metachronal waves. © 2015 Wiley Periodicals, Inc.

  16. Untangling ciliary access and enrichment of two rhodopsin-like receptors using quantitative fluorescence microscopy reveals cell-specific sorting pathways

    PubMed Central

    Geneva, Ivayla I.; Tan, Han Yen; Calvert, Peter D.

    2017-01-01

    Resolution limitations of optical systems are major obstacles for determining whether proteins are enriched within cell compartments. Here we use an approach to determine the degree of membrane protein ciliary enrichment that quantitatively accounts for the differences in sampling of the ciliary and apical membranes inherent to confocal microscopes. Theory shows that cilia will appear more than threefold brighter than the surrounding apical membrane when the densities of fluorescently labeled proteins are the same, thus providing a benchmark for ciliary enrichment. Using this benchmark, we examined the ciliary enrichment signals of two G protein–coupled receptors (GPCRs)—the somatostatin receptor 3 and rhodopsin. Remarkably, we found that the C-terminal VxPx motif, required for efficient enrichment of rhodopsin within rod photoreceptor sensory cilia, inhibited enrichment of the somatostatin receptor in primary cilia. Similarly, VxPx inhibited primary cilium enrichment of a chimera of rhodopsin and somatostatin receptor 3, where the dual Ax(S/A)xQ ciliary targeting motifs within the third intracellular loop of the somatostatin receptor replaced the third intracellular loop of rhodopsin. Rhodopsin was depleted from primary cilia but gained access, without being enriched, with the dual Ax(S/A)xQ motifs. Ciliary enrichment of these GPCRs thus operates via distinct mechanisms in different cells. PMID:27974638

  17. Non-vascular smooth muscle cells in the human choroid: distribution, development and further characterization

    PubMed Central

    May, Christian Albrecht

    2005-01-01

    To characterize further non-vascular smooth muscle cells (NVSMC) in the choroid of the human eye, extensive morphological studies were performed including a three-dimensional distribution of NVSMC in the adult human eye and their appearance during development. Whole mounts and sections through the choroid and sclera of eyes of 42 human donors (between the 13th week of gestation and 89 years of age) were stained with antibodies against smooth muscle actin and other markers for smooth muscle cells. On the basis of their morphological localization, three groups of NVSMC could be distinguished in the adult eyes: (a) a semicircular arrangement of NVSMC in the suprachoroid and inner sclera, around the entry of posterior ciliary arteries and nerves; (b) NVSMC parallel to the vessels in the posterior eye segment between the point of entry of the posterior ciliary arteries and the point of exit of the vortex veins; and (c) a dense plaque-like arrangement of NVSMC in the suprachoroid, overlying the foveal region. The last of these groups showed most pronounced interindividual differences. During development, the first NVSMC to be observed at the 20th week of gestation belonged to group b. A complete NVSMC network was first observed in a 6-year-old donor eye. All three groups stained positive for smoothelin, caldesmon and calponin in all localizations. The NVSMC show a distinct distribution that might reflect different aspects of their function in the choroid and suprachoroid. All cells could be histochemically characterized as truly contractile. PMID:16191166

  18. Adjustable Augmented Rectus Muscle Transposition Surgery with or Without Ciliary Vessel Sparing for Abduction Deficiencies

    PubMed Central

    Hendler, Karen; Pineles, Stacy L.; Demer, Joseph L.; Yang, Dawn; Velez, Federico G.

    2014-01-01

    Background Vertical rectus transposition (VRT) is useful in abduction deficiencies. Posterior fixation sutures enhance the effect of VRT, but usually preclude the use of adjustable sutures. Augmentation of VRT by resection of the transposed muscles allows for an adjustable technique that can reduce induced vertical deviations and overcorrections. Methods We retrospectively reviewed the records of all patients undergoing adjustable partial or full tendon VRT augmented by resection of the transposed muscles. Ciliary vessels were preserved in most of the patients by either splitting the transposed muscle or by dragging the transposed muscle without disrupting the muscle insertion. Results Seven patients with abducens palsy and one with esotropic Duane syndrome were included. Both vertical rectus muscles were symmetrically resected by 3–5 mm. Preoperative central gaze esotropia of 30.6 ± 12.9Δ (range, 17–50Δ) decreased to 10.6 ± 8.8Δ (range, 0–25Δ) at the final visit (p = 0.003). Three patients required postoperative adjustment by recession of one of the transposed muscles due to an induced vertical deviation (mean 9.3Δ reduced to 0Δ), coupled with overcorrection (mean exotropia 11.3Δ reduced to 0 in two patients and exophoria 2Δ in one patient). At the final follow-up visit 3.8 ± 2.6 months postoperatively, one patient had a vertical deviation <4Δ, and none had overcorrection or anterior segment ischemia. Three patients required further surgery for recurrent esotropia. Conclusions Augmentation of VRT by resection of the transposed muscles can be performed with adjustable sutures and vessel-sparing technique. This allows for postoperative control of overcorrections and induced vertical deviations as well as less risk of anterior segment ischemia. PMID:24738948

  19. Adjustable augmented rectus muscle transposition surgery with or without ciliary vessel sparing for abduction deficiencies.

    PubMed

    Hendler, Karen; Pineles, Stacy L; Demer, Joseph L; Yang, Dawn; Velez, Federico G

    2014-06-01

    Vertical rectus transposition (VRT) is useful in abduction deficiencies. Posterior fixation sutures enhance the effect of VRT, but usually preclude the use of adjustable sutures. Augmentation of VRT by resection of the transposed muscles allows for an adjustable technique that can reduce induced vertical deviations and overcorrections. We retrospectively reviewed the records of all patients undergoing adjustable partial or full tendon VRT augmented by resection of the transposed muscles. Ciliary vessels were preserved in most of the patients by either splitting the transposed muscle or by dragging the transposed muscle without disrupting the muscle insertion. Seven patients with abducens palsy and one with esotropic Duane syndrome were included. Both vertical rectus muscles were symmetrically resected by 3-5 mm. Preoperative central gaze esotropia of 30.6 ± 12.9Δ (range, 17-50Δ) decreased to 10.6 ± 8.8Δ (range, 0-25Δ) at the final visit (p = 0.003). Three patients required postoperative adjustment by recession of one of the transposed muscles due to an induced vertical deviation (mean 9.3Δ reduced to 0Δ), coupled with overcorrection (mean exotropia 11.3Δ reduced to 0 in two patients and exophoria 2Δ in one patient). At the final follow-up visit 3.8 ± 2.6 months postoperatively, one patient had a vertical deviation <4Δ, and none had overcorrection or anterior segment ischemia. Three patients required further surgery for recurrent esotropia. Augmentation of VRT by resection of the transposed muscles can be performed with adjustable sutures and vessel-sparing technique. This allows for postoperative control of overcorrections and induced vertical deviations as well as less risk of anterior segment ischemia.

  20. Untangling ciliary access and enrichment of two rhodopsin-like receptors using quantitative fluorescence microscopy reveals cell-specific sorting pathways.

    PubMed

    Geneva, Ivayla I; Tan, Han Yen; Calvert, Peter D

    2017-02-15

    Resolution limitations of optical systems are major obstacles for determining whether proteins are enriched within cell compartments. Here we use an approach to determine the degree of membrane protein ciliary enrichment that quantitatively accounts for the differences in sampling of the ciliary and apical membranes inherent to confocal microscopes. Theory shows that cilia will appear more than threefold brighter than the surrounding apical membrane when the densities of fluorescently labeled proteins are the same, thus providing a benchmark for ciliary enrichment. Using this benchmark, we examined the ciliary enrichment signals of two G protein-coupled receptors (GPCRs)-the somatostatin receptor 3 and rhodopsin. Remarkably, we found that the C-terminal VxPx motif, required for efficient enrichment of rhodopsin within rod photoreceptor sensory cilia, inhibited enrichment of the somatostatin receptor in primary cilia. Similarly, VxPx inhibited primary cilium enrichment of a chimera of rhodopsin and somatostatin receptor 3, where the dual Ax(S/A)xQ ciliary targeting motifs within the third intracellular loop of the somatostatin receptor replaced the third intracellular loop of rhodopsin. Rhodopsin was depleted from primary cilia but gained access, without being enriched, with the dual Ax(S/A)xQ motifs. Ciliary enrichment of these GPCRs thus operates via distinct mechanisms in different cells. © 2017 Geneva et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  1. Ciliary photoreceptors in the cerebral eyes of a protostome larva

    PubMed Central

    2011-01-01

    Background Eyes in bilaterian metazoans have been described as being composed of either ciliary or rhabdomeric photoreceptors. Phylogenetic distribution, as well as distinct morphologies and characteristic deployment of different photopigments (ciliary vs. rhabdomeric opsins) and transduction pathways argue for the co-existence of both of these two photoreceptor types in the last common bilaterian ancestor. Both receptor types exist throughout the Bilateria, but only vertebrates are thought to use ciliary photoreceptors for directional light detection in cerebral eyes, while all other invertebrate bilaterians studied utilize rhabdomeric photoreceptors for this purpose. In protostomes, ciliary photoreceptors that express c-opsin have been described only from a non-visual deep-brain photoreceptor. Their homology with vertebrate rods and cones of the human eye has been hypothesized to represent a unique functional transition from non-visual to visual roles in the vertebrate lineage. Results To test the hypothesis that protostome cerebral eyes employ exclusively rhabdomeric photoreceptors, we investigated the ultrastructure of the larval eyes in the brachiopod Terebratalia transversa. We show that these pigment-cup eyes consist of a lens cell and a shading pigment cell, both of which are putative photoreceptors, deploying a modified, enlarged cilium for light perception, and have axonal connections to the larval brain. Our investigation of the gene expression patterns of c-opsin, Pax6 and otx in these eyes confirms that the larval eye spots of brachiopods are cerebral eyes that deploy ciliary type photoreceptors for directional light detection. Interestingly, c-opsin is also expressed during early embryogenesis in all potential apical neural cells, becoming restricted to the anterior neuroectoderm, before expression is initiated in the photoreceptor cells of the eyes. Coincident with the expression of c-opsin in the presumptive neuroectoderm, we found that middle

  2. Clinical and histopathological features of adenomas of the ciliary pigment epithelium.

    PubMed

    Chang, Ying; Wei, Wen Bin; Shi, Ji Tong; Xian, Jun Fang; Yang, Wen Li; Xu, Xiao Lin; Bai, Hai Xia; Li, Bin; Jonas, Jost B

    2016-11-01

    Adenomas of the ciliary pigment epithelium (CPE) are rare benign tumours which have mainly to be differentiated from malignant ciliary body melanomas. Here we report on a consecutive series of patients with CPE adenomas and describe their characteristics. The retrospective hospital-based case series study included all patients who were consecutively operated for CPE adenomas. Of the 110 patients treated for ciliary body tumours, five patients (4.5%) had a CPE adenoma. Mean age was 59.0 ± 9.9 years (range: 46-72 years). Mean tumour apical thickness was 6.6 ± 1.7 mm. Tumour colour was mostly homogenously brown to black, and the tumour surface was smooth. The tumour masses pushed the iris tissue forward without infiltrating iris or anterior chamber angle. Sonography revealed an irregular echogram with sharp lesion borders and signs of blood flow in Color Doppler flow imaging. Ultrasonographic biomicroscopy demonstrated medium-low internal reflectivity and acoustic attenuation. In magnetic resonance imaging (MRI), the tumours as compared to brain were hyperintense on T1-weighted images and hypointense on T2-weighted images. Tumour tissue consisted of cords and nests of pigment epithelium cells separated by septa of vascularized fibrous connective tissue, leading to a pseudo-glandular appearance. The melanin granules in the cytoplasm were large and mostly spherical in shape. In four patients, the tumours were hyperpigmented. Tumour cells were large with round or oval nuclei and clearly detectable nucleoli. These clinical characteristics of CPE adenomas, such as homogenous dark brown colour, smooth surface, iris dislocation and anterior chamber angle narrowing but no iris infiltration, segmental cataract, pigment dispersion, and, as compared to brain tissue, hypointensity and, as compared to extraocular muscles or lacrimal gland, hyperintensity on T2-weighted MRI images, may be helpful for the differentiation from ciliary body malignant melanomas. © 2016 Acta

  3. Force-response considerations in ciliary mechanosensation.

    PubMed

    Resnick, Andrew; Hopfer, Ulrich

    2007-08-15

    Considerable experimental evidence indicates that the primary, nonmotile cilium is a mechanosensory organelle in several epithelial cell types. As the relationship between cellular responses and nature and magnitude of applied forces is not well understood, we have investigated the effects of exposure of monolayers of renal collecting duct chief cells to orbital shaking and quantified the forces incident on cilia. An exposure of 24 h of these cells to orbital shaking resulted in a decrease of amiloride-sensitive sodium current by approximately 60% and ciliary length by approximately 30%. The sensitivity of the sodium current to shaking was dependent on intact cilia. The drag force on cilia due to induced fluid flow during orbital shaking was estimated at maximally 5.2x10(-3) pN at 2 Hz, approximately 4 times that of thermal noise. The major structural feature of cilia contributing to their sensitivity appears to be ciliary length. As more than half of the total drag force is exerted on the ciliary cap, one function of the slender stalk may be to expose the cap to greater drag force. Regardless, the findings indicate that the cilium is a mechanosensory organelle with a sensitivity much lower than previously recognized.

  4. Automated software for analysis of ciliary beat frequency and metachronal wave orientation in primary ciliary dyskinesia.

    PubMed

    Mantovani, Giulia; Pifferi, Massimo; Vozzi, Giovanni

    2010-06-01

    Patients with primary ciliary dyskinesia (PCD) have structural and/or functional alterations of cilia that imply deficits in mucociliary clearance and different respiratory pathologies. A useful indicator for the difficult diagnosis is the ciliary beat frequency (CBF) that is significantly lower in pathological cases than in physiological ones. The CBF computation is not rapid, therefore, the aim of this study is to propose an automated method to evaluate it directly from videos of ciliated cells. The cells are taken from inferior nasal turbinates and videos of ciliary movements are registered and eventually processed by the developed software. The software consists in the extraction of features from videos (written with C++ language) and the computation of the frequency (written with Matlab language). This system was tested both on the samples of nasal cavity and software models, and the results were really promising because in a few seconds, it can compute a reliable frequency if compared with that measured with visual methods. It is to be noticed that the reliability of the computation increases with the quality of acquisition system and especially with the sampling frequency. It is concluded that the developed software could be a useful mean for PCD diagnosis.

  5. Ciliary Body Thickness and Refractive Error in Children

    PubMed Central

    Bailey, Melissa D.; Sinnott, Loraine T.; Mutti, Donald O.

    2010-01-01

    Purpose To determine whether ciliary body thickness (CBT) is related to refractive error in school-age children. Methods Fifty-three children, 8 to 15 years of age, were recruited. CBT was measured from anterior segment OCT images (Visante; Carl Zeiss Meditec, Inc., Dublin, CA) at 1 (CBT1), 2 (CBT2) and 3 (CBT3) mm posterior to the scleral spur. Cycloplegic refractive error was measured with an autorefractor, and axial length was measured with an optical biometer. Multilevel regression models determined the relationship between CBT measurements and refractive error or axial length. A Bland-Altman analysis was used to assess the between-visit repeatability of the ciliary body measurements. Results The between-visits coefficients of repeatability for CBT1, -2, and -3 were 148.04, 165.68, and 110.90, respectively. Thicker measurements at CBT2 (r = −0.29, P = 0.03) and CBT3 (r = −0.38, P = 0.005) were associated with increasingly myopic refractive errors (multilevel model: P < 0.001). Thicker measurements at CBT2 (r = 0.40, P = 0.003) and CBT3 (r = 0.51, P < 0.001) were associated with longer axial lengths (multilevel model: P < 0.001). Conclusions Thicker ciliary body measurements were associated with myopia and a longer axial length. Future studies should determine whether this relationship is also present in animal models of myopia and determine the temporal relationship between thickening of the ciliary muscle and the onset of myopia. PMID:18566470

  6. Formation of the transition zone by Mks5/Rpgrip1L establishes a ciliary zone of exclusion (CIZE) that compartmentalises ciliary signalling proteins and controls PIP2 ciliary abundance

    PubMed Central

    Jensen, Victor L; Li, Chunmei; Bowie, Rachel V; Clarke, Lara; Mohan, Swetha; Blacque, Oliver E; Leroux, Michel R

    2015-01-01

    Cilia are thought to harbour a membrane diffusion barrier within their transition zone (TZ) that compartmentalises signalling proteins. How this “ciliary gate” assembles and functions remains largely unknown. Contrary to current models, we present evidence that Caenorhabditis elegans MKS-5 (orthologue of mammalian Mks5/Rpgrip1L/Nphp8 and Rpgrip1) may not be a simple structural scaffold for anchoring > 10 different proteins at the TZ, but instead, functions as an assembly factor. This activity is needed to form TZ ultrastructure, which comprises Y-shaped axoneme-to-membrane connectors. Coiled-coil and C2 domains within MKS-5 enable TZ localisation and functional interactions with two TZ modules, consisting of Meckel syndrome (MKS) and nephronophthisis (NPHP) proteins. Discrete roles for these modules at basal body-associated transition fibres and TZ explain their redundant functions in making essential membrane connections and thus sealing the ciliary compartment. Furthermore, MKS-5 establishes a ciliary zone of exclusion (CIZE) at the TZ that confines signalling proteins, including GPCRs and NPHP-2/inversin, to distal ciliary subdomains. The TZ/CIZE, potentially acting as a lipid gate, limits the abundance of the phosphoinositide PIP2 within cilia and is required for cell signalling. Together, our findings suggest a new model for Mks5/Rpgrip1L in TZ assembly and function that is essential for establishing the ciliary signalling compartment. PMID:26392567

  7. An autocrine ATP release mechanism regulates basal ciliary activity in airway epithelium.

    PubMed

    Droguett, Karla; Rios, Mariana; Carreño, Daniela V; Navarrete, Camilo; Fuentes, Christian; Villalón, Manuel; Barrera, Nelson P

    2017-07-15

    Extracellular ATP, in association with [Ca 2+ ] i regulation, is required to maintain basal ciliary beat frequency. Increasing extracellular ATP levels increases ciliary beating in airway epithelial cells, maintaining a sustained response by inducing the release of additional ATP. Extracellular ATP levels in the millimolar range, previously associated with pathophysiological conditions of the airway epithelium, produce a transient arrest of ciliary activity. The regulation of ciliary beat frequency is dependent on ATP release by hemichannels (connexin/pannexin) and P2X receptor activation, the blockage of which may even stop ciliary movement. The force exerted by cilia, measured by atomic force microscopy, is reduced following extracellular ATP hydrolysis. This result complements the current understanding of the ciliary beating regulatory mechanism, with special relevance to inflammatory diseases of the airway epithelium that affect mucociliary clearance. Extracellular nucleotides, including ATP, are locally released by the airway epithelium and stimulate ciliary activity in a [Ca 2+ ] i -dependent manner after mechanical stimulation of ciliated cells. However, it is unclear whether the ATP released is involved in regulating basal ciliary activity and mediating changes in ciliary activity in response to chemical stimulation. In the present study, we evaluated ciliary beat frequency (CBF) and ciliary beating forces in primary cultures from mouse tracheal epithelium, using videomicroscopy and atomic force microscopy (AFM), respectively. Extracellular ATP levels and [Ca 2+ ] i were measured by luminometric and fluorimetric assays, respectively. Uptake of ethidium bromide was measured to evaluate hemichannel functionality. We show that hydrolysis of constitutive extracellular ATP levels with apyrase (50 U ml -1 ) reduced basal CBF by 45% and ciliary force by 67%. The apyrase effect on CBF was potentiated by carbenoxolone, a hemichannel inhibitor, and oxidized ATP, an

  8. Flagellar Synchronization Is a Simple Alternative to Cell Cycle Synchronization for Ciliary and Flagellar Studies

    PubMed Central

    Dutta, Soumita

    2017-01-01

    ABSTRACT The unicellular green alga Chlamydomonas reinhardtii is an ideal model organism for studies of ciliary function and assembly. In assays for biological and biochemical effects of various factors on flagellar structure and function, synchronous culture is advantageous for minimizing variability. Here, we have characterized a method in which 100% synchronization is achieved with respect to flagellar length but not with respect to the cell cycle. The method requires inducing flagellar regeneration by amputation of the entire cell population and limiting regeneration time. This results in a maximally homogeneous distribution of flagellar lengths at 3 h postamputation. We found that time-limiting new protein synthesis during flagellar synchronization limits variability in the unassembled pool of limiting flagellar protein and variability in flagellar length without affecting the range of cell volumes. We also found that long- and short-flagella mutants that regenerate normally require longer and shorter synchronization times, respectively. By minimizing flagellar length variability using a simple method requiring only hours and no changes in media, flagellar synchronization facilitates the detection of small changes in flagellar length resulting from both chemical and genetic perturbations in Chlamydomonas. This method increases our ability to probe the basic biology of ciliary size regulation and related disease etiologies. IMPORTANCE Cilia and flagella are highly conserved antenna-like organelles that found in nearly all mammalian cell types. They perform sensory and motile functions contributing to numerous physiological and developmental processes. Defects in their assembly and function are implicated in a wide range of human diseases ranging from retinal degeneration to cancer. Chlamydomonas reinhardtii is an algal model system for studying mammalian cilium formation and function. Here, we report a simple synchronization method that allows detection of

  9. Flagellar Synchronization Is a Simple Alternative to Cell Cycle Synchronization for Ciliary and Flagellar Studies.

    PubMed

    Dutta, Soumita; Avasthi, Prachee

    2017-01-01

    The unicellular green alga Chlamydomonas reinhardtii is an ideal model organism for studies of ciliary function and assembly. In assays for biological and biochemical effects of various factors on flagellar structure and function, synchronous culture is advantageous for minimizing variability. Here, we have characterized a method in which 100% synchronization is achieved with respect to flagellar length but not with respect to the cell cycle. The method requires inducing flagellar regeneration by amputation of the entire cell population and limiting regeneration time. This results in a maximally homogeneous distribution of flagellar lengths at 3 h postamputation. We found that time-limiting new protein synthesis during flagellar synchronization limits variability in the unassembled pool of limiting flagellar protein and variability in flagellar length without affecting the range of cell volumes. We also found that long- and short-flagella mutants that regenerate normally require longer and shorter synchronization times, respectively. By minimizing flagellar length variability using a simple method requiring only hours and no changes in media, flagellar synchronization facilitates the detection of small changes in flagellar length resulting from both chemical and genetic perturbations in Chlamydomonas . This method increases our ability to probe the basic biology of ciliary size regulation and related disease etiologies. IMPORTANCE Cilia and flagella are highly conserved antenna-like organelles that found in nearly all mammalian cell types. They perform sensory and motile functions contributing to numerous physiological and developmental processes. Defects in their assembly and function are implicated in a wide range of human diseases ranging from retinal degeneration to cancer. Chlamydomonas reinhardtii is an algal model system for studying mammalian cilium formation and function. Here, we report a simple synchronization method that allows detection of small

  10. A pilot study exploring the impact of cardiac medications on ciliary beat frequency: possible implications for clinical management.

    PubMed

    Loomba, Rohit S; Bhushan, Abhinav; Afolayan, Adeleye J

    2018-05-03

    Cilia are involved in several physiologic processes, and at least a single primary cilium can be found on nearly every cell in the human body. Various factors, such as pH, temperature, exposure to medications and toxins can impact ciliary function as is manifested by changes in the ciliary beat frequency. Those with ciliary dyskinesia may also have congenital cardiac malformations and may require care in a cardiac intensive care unit. This study investigates the effect on the ciliary beat frequency of medications frequently used in a cardiac intensive care unit. The ciliated epithelial cells were obtained via nasal swab from a relatively healthy individual. These cells were cultured for 24 h. Video microscopy was then employed to determine the ciliary beat frequency at baseline and then at 15, 30, 60 and 90 min after exposure to either normal saline (control) or one of several medications. The ciliary beat frequency at each time point was then compared to the ciliary beat frequency at the same time point in the control sample as well as the baseline value for that particular sample. Epinephrine increased the ciliary beat frequency compared to the baseline and the controls up to 30 min and then subsequently led to a significant decrease in ciliary beat frequency at 90 min. On the one hand, norepinephrine, dexmedetomidine, procainamide, propranolol and enalapril all decreased ciliary beat frequency significantly throughout the 90-min observation period. On the other hand, Milrinone significantly increased the ciliary beat frequency throughout the observation period, while heparin had no impact on ciliary beat frequency. The medications frequently used in cardiac intensive care unit impact ciliary function, with most being ciliodepressant. Further investigation is needed to determine the clinical impacts and whether these effects are exaggerated in those with ciliary dyskinesia.

  11. Cyanide levels found in infected cystic fibrosis sputum inhibit airway ciliary function.

    PubMed

    Nair, Chandrika; Shoemark, Amelia; Chan, Mario; Ollosson, Sarah; Dixon, Mellissa; Hogg, Claire; Alton, Eric W F W; Davies, Jane C; Williams, Huw D

    2014-11-01

    We have previously reported cyanide at concentrations of up to 150 μM in the sputum of cystic fibrosis patients infected with Pseudomonas aeruginosa and a negative correlation with lung function. Our aim was to investigate possible mechanisms for this association, focusing on the effect of pathophysiologically relevant cyanide levels on human respiratory cell function. Ciliary beat frequency measurements were performed on nasal brushings and nasal air-liquid interface (ALI) cultures obtained from healthy volunteers and cystic fibrosis patients. Potassium cyanide decreased ciliary beat frequency in healthy nasal brushings (n = 6) after 60 min (150 μM: 47% fall, p<0.0012; 75 μM: 32% fall, p<0.0001). Samples from cystic fibrosis patients (n = 3) showed similar results (150 μM: 55% fall, p = 0.001). Ciliary beat frequency inhibition was not due to loss of cell viability and was reversible. The inhibitory mechanism was independent of ATP levels. KCN also significantly inhibited ciliary beat frequency in ALI cultures, albeit to a lesser extent. Ciliary beat frequency measurements on ALI cultures treated with culture supernatants from P. aeruginosa mutants defective in virulence factor production implicated cyanide as a key component inhibiting the ciliary beat frequency. If cyanide production similarly impairs mucocilliary clearance in vivo, it could explain the link with increased disease severity observed in cystic fibrosis patients with detectable cyanide in their airway. ©ERS 2014.

  12. KIAA0556 is a novel ciliary basal body component mutated in Joubert syndrome.

    PubMed

    Sanders, Anna A W M; de Vrieze, Erik; Alazami, Anas M; Alzahrani, Fatema; Malarkey, Erik B; Sorusch, Nasrin; Tebbe, Lars; Kuhns, Stefanie; van Dam, Teunis J P; Alhashem, Amal; Tabarki, Brahim; Lu, Qianhao; Lambacher, Nils J; Kennedy, Julie E; Bowie, Rachel V; Hetterschijt, Lisette; van Beersum, Sylvia; van Reeuwijk, Jeroen; Boldt, Karsten; Kremer, Hannie; Kesterson, Robert A; Monies, Dorota; Abouelhoda, Mohamed; Roepman, Ronald; Huynen, Martijn H; Ueffing, Marius; Russell, Rob B; Wolfrum, Uwe; Yoder, Bradley K; van Wijk, Erwin; Alkuraya, Fowzan S; Blacque, Oliver E

    2015-12-29

    Joubert syndrome (JBTS) and related disorders are defined by cerebellar malformation (molar tooth sign), together with neurological symptoms of variable expressivity. The ciliary basis of Joubert syndrome related disorders frequently extends the phenotype to tissues such as the eye, kidney, skeleton and craniofacial structures. Using autozygome and exome analyses, we identified a null mutation in KIAA0556 in a multiplex consanguineous family with hallmark features of mild Joubert syndrome. Patient-derived fibroblasts displayed reduced ciliogenesis potential and abnormally elongated cilia. Investigation of disease pathophysiology revealed that Kiaa0556 (-/-) null mice possess a Joubert syndrome-associated brain-restricted phenotype. Functional studies in Caenorhabditis elegans nematodes and cultured human cells support a conserved ciliary role for KIAA0556 linked to microtubule regulation. First, nematode KIAA0556 is expressed almost exclusively in ciliated cells, and the worm and human KIAA0556 proteins are enriched at the ciliary base. Second, C. elegans KIAA0056 regulates ciliary A-tubule number and genetically interacts with an ARL13B (JBTS8) orthologue to control cilium integrity. Third, human KIAA0556 binds to microtubules in vitro and appears to stabilise microtubule networks when overexpressed. Finally, human KIAA0556 biochemically interacts with ciliary proteins and p60/p80 katanins. The latter form a microtubule-severing enzyme complex that regulates microtubule dynamics as well as ciliary functions. We have identified KIAA0556 as a novel microtubule-associated ciliary base protein mutated in Joubert syndrome. Consistent with the mild patient phenotype, our nematode, mice and human cell data support the notion that KIAA0556 has a relatively subtle and variable cilia-related function, which we propose is related to microtubule regulation.

  13. Diagnosis of primary ciliary dyskinesia*

    PubMed Central

    Olm, Mary Anne Kowal; Caldini, Elia Garcia; Mauad, Thais

    2015-01-01

    Primary ciliary dyskinesia (PCD) is a genetic disorder of ciliary structure or function. It results in mucus accumulation and bacterial colonization of the respiratory tract which leads to chronic upper and lower airway infections, organ laterality defects, and fertility problems. We review the respiratory signs and symptoms of PCD, as well as the screening tests for and diagnostic investigation of the disease, together with details related to ciliary function, ciliary ultrastructure, and genetic studies. In addition, we describe the difficulties in diagnosing PCD by means of transmission electron microscopy, as well as describing patient follow-up procedures. PMID:26176524

  14. Seeing cilia: imaging modalities for ciliary motion and clinical connections.

    PubMed

    Peabody, Jacelyn E; Shei, Ren-Jay; Bermingham, Brent M; Phillips, Scott E; Turner, Brett; Rowe, Steven M; Solomon, George M

    2018-06-01

    The respiratory tract is lined with multiciliated epithelial cells that function to move mucus and trapped particles via the mucociliary transport apparatus. Genetic and acquired ciliopathies result in diminished mucociliary clearance, contributing to disease pathogenesis. Recent innovations in imaging technology have advanced our understanding of ciliary motion in health and disease states. Application of imaging modalities including transmission electron microscopy, high-speed video microscopy, and micron-optical coherence tomography could improve diagnostics and be applied for precision medicine. In this review, we provide an overview of ciliary motion, imaging modalities, and ciliopathic diseases of the respiratory system including primary ciliary dyskinesia, cystic fibrosis, chronic obstructive pulmonary disease, and idiopathic pulmonary fibrosis.

  15. Impact and benefit of A(2B)-adenosine receptor agonists for the respiratory tract: mucociliary clearance, ciliary beat frequency, trachea muscle tonus and cytokine release.

    PubMed

    Walaschewski, Robin; Begrow, Frank; Verspohl, Eugen J

    2013-01-01

    Adenosine is known to induce a bronchospasm in asthma- and COPD patients. The role of A(2B) receptors was investigated with respect to several parameters of the respiratory tract: tonus of smooth muscle, ciliary beat frequency as measured by high-speed video camera connected to a microscope (both in rats) and mucociliary clearance (MCC; transport of a fluorescent dye using a microdialysis procedure) in mice.  NECA (5'-N-ethylcarboxamidoadenosine) (a non-selective adenosine receptor agonist) was able to acutely induce a contraction, which was reversed to a relaxation after repeated dosing. This relaxation was completely abolished by PSB-1115, an A(2B) receptor antagonist. IL-13 (cytokine) was not involved mediating acute contractility effects. MCC was increased by BAY 60-6583 (A(2B) receptor agonist) and NECA (counteracted by the A(2B) receptor antagonist PSB-1115). Activation of A(2B) adenosine receptors by BAY 60-6583 induced an increase of the ciliary beat frequency, which could be reduced by administration of PSB-1115. Several cytokines were increased by NECA although only some are relevant because they are not blocked by A(2B) receptor antagonism. The A(2B) receptors are involved in airway relaxation, MCC improvement and ciliary beat frequency. A(2B) receptor agonists may be of therapeutic value and should be developed. © 2012 The Authors. JPP © 2012 Royal Pharmaceutical Society.

  16. Effect of azelastine on sulphur dioxide induced impairment of ciliary motility in airway epithelium.

    PubMed Central

    Tamaoki, J; Chiyotani, A; Sakai, N; Takeyama, K; Konno, K

    1993-01-01

    OBJECTIVE--The effect of azelastine on airway mucociliary transport function was studied by measuring ciliary motility of human bronchial epithelium in vitro with a photoelectric method. METHOD--Bronchial epithelial cells were obtained by fibreoptic bronchoscopy, mounted in a Rose chamber, and perfused with Krebs-Henseleit solution. The preparations were placed on a microscope stage equipped with an illuminator, and the variations of light intensity caused by ciliary beating were detected by a photometer. RESULTS--The addition of azelastine to the perfusate increased ciliary beat frequency (CBF) in a dose dependent manner without ciliary discoordination. The mean (SE) maximal increase from the baseline value and the concentration required to produce a half maximal effect were 27.0 (4.2)% and 9.2 x 10(-6) mol/l, respectively. Exposure of the cells to the perfusate containing 3 ppm sulphur dioxide rapidly decreased CBF by 59.2 (5.0)%, and was accompanied by a reduction in intracellular cyclic AMP levels from 38.1 (4.3) to 10.1 (2.4) pmol/mg protein. This effect was prevented by pretreatment of cells with azelastine in a dose dependent manner. CONCLUSIONS--Azelastine not only stimulates ciliary motility of airway epithelium and hence mucociliary transport function, but may also protect against sulphur dioxide induced ciliary dysfunction, probably by inhibiting intracellular cyclic AMP loss. PMID:8322244

  17. [The protective effect of dexpanthenol in nasal sprays. First results of cytotoxic and ciliary-toxic studies in vitro].

    PubMed

    Klöcker, N; Verse, T; Rudolph, P

    2003-03-01

    In Germany more than 60 million units of nasal decongestants are prescribed or sold over the counter. The cytotoxic and ciliary-toxic potential of alpha-sympathomimetic decongestants is well established. Furthermore, in many of the marketed products preservatives are added, predominantly benzalchonium-chloride, which can lead to a further alteration of cell- and ciliary function. Recently a protective effect of dexpanthenol was found for the human nasal mucosa. The objective of the present studies was to prove the hypothesis that dexpanthenol is able to neutralise the toxic effects of both alpha-sympathomimetic decongestants, in particular those of xylometazoline, and those of benzalconium-chloride. Therefore, systematic cytotoxic and ex vivo in vitro ciliary-toxic studies were performed. After exposition to xylometazoline in concentrations of 0.1 % and 0.05 %, the influence of dexpanthenol (5 %) and benzalconium-chloride (0,01 %) was assessed by determination of a) cell growth of FL-cells of human amnion origin, and b) ciliary beat frequency of human nasal mucosa. All tests were performed placebo-controlled. Both hypotheses were confirmed. Dexpanthenol (5 %) reduces statistically significantly the concentration-dependent toxic effects of xylometazoline, and benzalchonium-cloride regarding cell growth and ciliary beat frequency (p < 0.001). The combination of xylometazoline with dexpanthenol, while benzalconium-chloride is eliminated, resulted in a further significant increase of cell growth and ciliary beat frequency (p < 0.001), similar to control. The additive application of dexpanthenol (5 %) with nasal decongestants and/or with preserved nasal sprays seems to be able to reduce the cell- and ciliary-toxic effects of these substances.

  18. Ciliary behavior of a negatively phototactic Chlamydomonas reinhardtii.

    PubMed

    Josef, Keith; Saranak, Jureepan; Foster, Kenneth W

    2005-06-01

    With an instrument that can record the motion of both cilia of the unicellular alga Chlamydomonas reinhardtii for many hours, the behavioral differences of its two cilia have been studied to determine their specific role in phototaxis. The organism was held on a fixed micropipette with the plane of ciliary beating rotated into the imaging plane of a quadrant photodetector. The responses to square-wave light patterns of a wide range of temporal frequencies were used to characterize the responses of each cilium. Eighty-one cells were examined showing an unexpectedly diverse range of responses. Plausible common signals for the linear and nonlinear signals from the cell body are suggested. Three independent ciliary measures--the beat frequency, stroke velocity, and phasing of the two cilia--have been identified. The cell body communicates to the cilia the direction of phototaxis the cell desires to go, the absolute light intensity, and the appropriate graded transient response for tracking the light source. The complexity revealed by each measure of the ciliary response indicates many independent variables are involved in the net phototactic response. In spite of their morphological similarity, the two cilia of Chlamydomonas respond uniquely. Probably the signals from the cell body fan out to independent pathways in the cilia. Each cilium modifies the input in its own way. The change in the pattern of the effective and recovery strokes of each cilium associated with negative phototaxis has been demonstrated and its involvement in phototactic turning is described. Copyright (c) 2005 Wiley-Liss, Inc.

  19. Comparative Proteomics Reveals Timely Transport into Cilia of Regulators or Effectors as a Mechanism Underlying Ciliary Disassembly.

    PubMed

    Wang, Limei; Gu, Lixiao; Meng, Dan; Wu, Qiong; Deng, Haiteng; Pan, Junmin

    2017-07-07

    Primary cilia are assembled and disassembled during cell cycle progression. During ciliary disassembly, ciliary axonemal microtubules (MTs) are depolymerized accompanied by extensive posttranslational protein modifications of ciliary proteins including protein phosphorylation, methylation, and ubiquitination. These events are hypothesized to involve transport of effectors or regulators into cilia at the time of ciliary disassembly from the cell body. To prove this hypothesis and identify new proteins involved in ciliary disassembly, we analyzed disassembling flagella in Chlamydomonas using comparative proteomics with TMT labeling. Ninety-one proteins were found to increase more than 1.4-fold in four replicates. The proteins of the IFT machinery not only increase but also exhibit stoichiometric changes. The other proteins that increase include signaling molecules, chaperones, and proteins involved in microtubule dynamics or stability. In particular, we have identified a ciliopathy protein C21orf2, the AAA-ATPase CDC48, that is involved in segregating polypeptides from large assemblies or cellular structures, FAP203 and FAP236, which are homologous to stabilizers of axonemal microtubules. Our data demonstrate that ciliary transport of effectors or regulators is one of the mechanisms underlying ciliary disassembly. Further characterization of the proteins identified will provide new insights into our understanding of ciliary disassembly and likely ciliopathy.

  20. CCDC65 Mutation Causes Primary Ciliary Dyskinesia with Normal Ultrastructure and Hyperkinetic Cilia

    PubMed Central

    Horani, Amjad; Brody, Steven L.; Ferkol, Thomas W.; Shoseyov, David; Wasserman, Mollie G.; Ta-shma, Asaf; Wilson, Kate S.; Bayly, Philip V.; Amirav, Israel; Cohen-Cymberknoh, Malena; Dutcher, Susan K.; Elpeleg, Orly; Kerem, Eitan

    2013-01-01

    Background Primary ciliary dyskinesia (PCD) is a genetic disorder characterized by impaired ciliary function, leading to chronic sinopulmonary disease. The genetic causes of PCD are still evolving, while the diagnosis is often dependent on finding a ciliary ultrastructural abnormality and immotile cilia. Here we report a novel gene associated with PCD but without ciliary ultrastructural abnormalities evident by transmission electron microscopy, but with dyskinetic cilia beating. Methods Genetic linkage analysis was performed in a family with a PCD subject. Gene expression was studied in Chlamydomonas reinhardtii and human airway epithelial cells, using RNA assays and immunostaining. The phenotypic effects of candidate gene mutations were determined in primary culture human tracheobronchial epithelial cells transduced with gene targeted shRNA sequences. Video-microscopy was used to evaluate cilia motion. Results A single novel mutation in CCDC65, which created a termination codon at position 293, was identified in a subject with typical clinical features of PCD. CCDC65, an orthologue of the Chlamydomonas nexin-dynein regulatory complex protein DRC2, was localized to the cilia of normal nasal epithelial cells but was absent in those from the proband. CCDC65 expression was up-regulated during ciliogenesis in cultured airway epithelial cells, as was DRC2 in C. reinhardtii following deflagellation. Nasal epithelial cells from the affected individual and CCDC65-specific shRNA transduced normal airway epithelial cells had stiff and dyskinetic cilia beating patterns compared to control cells. Moreover, Gas8, a nexin-dynein regulatory complex component previously identified to associate with CCDC65, was absent in airway cells from the PCD subject and CCDC65-silenced cells. Conclusion Mutation in CCDC65, a nexin-dynein regulatory complex member, resulted in a frameshift mutation and PCD. The affected individual had altered cilia beating patterns, and no detectable

  1. The ciliary margin zone of the mammalian retina generates retinal ganglion cells

    PubMed Central

    Marcucci, Florencia; Murcia-Belmonte, Veronica; Coca, Yaiza; Ferreiro-Galve, Susana; Wang, Qing; Kuwajima, Takaaki; Khalid, Sania; Ross, M. Elizabeth; Herrera, Eloisa; Mason, Carol

    2016-01-01

    Summary The retina of lower vertebrates grows continuously by integrating new neurons generated from progenitors in the ciliary margin zone (CMZ). Whether the mammalian CMZ provides the neural retina with retinal cells is controversial. Live-imaging of embryonic retina expressing eGFP in the CMZ shows that cells migrate laterally from the CMZ to the neural retina where differentiated retinal ganglion cells (RGCs) reside. As Cyclin D2, a cell-cycle regulator, is enriched in ventral CMZ, we analyzed Cyclin D2−/− mice to test whether the CMZ is a source of retinal cells. Neurogenesis is diminished in Cyclin D2 mutants, leading to a reduction of RGCs in the ventral retina. In line with these findings, in the albino retina, the decreased production of ipsilateral RGCs is correlated with fewer Cyclin D2+ cells. Together, these results implicate the mammalian CMZ as a neurogenic site that produces RGCs and whose proper generation depends on Cyclin D2 activity. PMID:28009286

  2. Non-invasive measurements of the dynamic changes in the ciliary muscle, crystalline lens morphology, and anterior chamber during accommodation with a high-resolution OCT.

    PubMed

    Esteve-Taboada, José J; Domínguez-Vicent, Alberto; Monsálvez-Romín, Daniel; Del Águila-Carrasco, Antonio J; Montés-Micó, Robert

    2017-07-01

    The purpose of the study was to assess non-invasively the changes in the anterior chamber eye, crystalline lens morphology, and ciliary muscle during accommodation by means of an anterior chamber optical coherence tomographer (OCT), and correlate them with vergence. Twenty-five eyes of twenty-five healthy subjects, whose mean age was 29.9±7.1 years, were included and measured with an anterior chamber OCT. The central corneal thickness (CCT), anterior chamber depth (ACD), anterior crystalline lens radius of curvature (ALRC), crystalline lens thickness (CLT), and ciliary muscle area (CMA) were measured for each participant at 0, -1, -2, and -3 D of target vergence. A linear model was used to assess the correlation of each eye parameter with the vergence demand. The mean CCT showed no change for all the accommodative stimuli. The mean ACD and ALRC decreased with the vergence, about 4.5 and 30 % at -3 D, respectively. On the contrary, the CLT and CMA showed an opposite tendency, where the mean CLT was increased by 4.0 % and the mean CMA was done by 26% at -3 D. Statistical significant differences (p < 0.001) were obtained among all vergences for each eye metric, except for the CCT (p = 0.76). The ACD and ALRC decreased about 2 and 10 % per dioptre of accommodation, respectively; whereas the CLT and CMA increased about 2 and 9 %, respectively. These results add knowledge regarding the understanding of accommodation and give new perspectives for biomechanics and biometry.

  3. Primary cilia proteins: ciliary and extraciliary sites and functions.

    PubMed

    Hua, Kiet; Ferland, Russell J

    2018-05-01

    Primary cilia are immotile organelles known for their roles in development and cell signaling. Defects in primary cilia result in a range of disorders named ciliopathies. Because this organelle can be found singularly on almost all cell types, its importance extends to most organ systems. As such, elucidating the importance of the primary cilium has attracted researchers from all biological disciplines. As the primary cilia field expands, caution is warranted in attributing biological defects solely to the function of this organelle, since many of these "ciliary" proteins are found at other sites in cells and likely have non-ciliary functions. Indeed, many, if not all, cilia proteins have locations and functions outside the primary cilium. Extraciliary functions are known to include cell cycle regulation, cytoskeletal regulation, and trafficking. Cilia proteins have been observed in the nucleus, at the Golgi apparatus, and even in immune synapses of T cells (interestingly, a non-ciliated cell). Given the abundance of extraciliary sites and functions, it can be difficult to definitively attribute an observed phenotype solely to defective cilia rather than to some defective extraciliary function or a combination of both. Thus, extraciliary sites and functions of cilia proteins need to be considered, as well as experimentally determined. Through such consideration, we will understand the true role of the primary cilium in disease as compared to other cellular processes' influences in mediating disease (or through a combination of both). Here, we review a compilation of known extraciliary sites and functions of "cilia" proteins as a means to demonstrate the potential non-ciliary roles for these proteins.

  4. Sensory Regulation of Network Components Underlying Ciliary Locomotion in Hermissenda

    PubMed Central

    Crow, Terry; Tian, Lian-Ming

    2008-01-01

    Ciliary locomotion in the nudibranch mollusk Hermissenda is modulated by the visual and graviceptive systems. Components of the neural network mediating ciliary locomotion have been identified including aggregates of polysensory interneurons that receive monosynaptic input from identified photoreceptors and efferent neurons that activate cilia. Illumination produces an inhibition of type Ii (off-cell) spike activity, excitation of type Ie (on-cell) spike activity, decreased spike activity in type IIIi inhibitory interneurons, and increased spike activity of ciliary efferent neurons. Here we show that pairs of type Ii interneurons and pairs of type Ie interneurons are electrically coupled. Neither electrical coupling or synaptic connections were observed between Ie and Ii interneurons. Coupling is effective in synchronizing dark-adapted spontaneous firing between pairs of Ie and pairs of Ii interneurons. Out-of-phase burst activity, occasionally observed in dark-adapted and light-adapted pairs of Ie and Ii interneurons, suggests that they receive synaptic input from a common presynaptic source or sources. Rhythmic activity is typically not a characteristic of dark-adapted, light-adapted, or light-evoked firing of type I interneurons. However, burst activity in Ie and Ii interneurons may be elicited by electrical stimulation of pedal nerves or generated at the offset of light. Our results indicate that type I interneurons can support the generation of both rhythmic activity and changes in tonic firing depending on sensory input. This suggests that the neural network supporting ciliary locomotion may be multifunctional. However, consistent with the nonmuscular and nonrhythmic characteristics of visually modulated ciliary locomotion, type I interneurons exhibit changes in tonic activity evoked by illumination. PMID:18768639

  5. The effects of the stem cell on ciliary regeneration of injured rabbit sinonasal epithelium.

    PubMed

    Kavuzlu, Ali; Tatar, Emel Çadallı; Karagöz, Tuğba; Pınarlı, Ferda Alpaslan; Tatar, İlkan; Bayır, Ömer; Korkmaz, Mehmet Hakan

    2017-08-01

    Defects in mucosal healing after sinonasal surgery cause infection, scar formation causing obstruction, relapse of the disease within a shorter period and revision surgery. The present study aimed to create a functional ciliated epithelium using a stem cell and stem cell sheet of adipose tissue origin and to show such regeneration ultra-structurally on experimentally injured rabbit nasal epithelium. This was an experimental animal study and basic research. A total of 18 white New Zealand rabbits were divided into three groups. The medial wall of the maxillary sinus of the subjects was peeled off bilaterally. No additional procedure was applied to the subjects in Group 1. In Group 2, adipose tissue-derived mesenchymal stem cell was implanted on the wound edges of the subjects. In Group 3, a stem cell sheet of three layers was laid onto the defect area. All subjects were killed after 3 weeks. The presence of the stem cell stained with bromo-deoxyuridine was assessed with a light microscope, whereas cilia density, ciliated orientation and cilia structure were evaluated with a scanning electron microscope. Ciliary densities in Group 2 and Group 3 were statistically superior compared to the control group (p < 0.001, p = 0.007). Cilia morphology in Group 2 and Group 3 was also better than the control group (p < 0.01, p = 0.048). Ciliary orientation in Group 2 was scored highest (p < 0.01). The ratio of BrDu-stained cells was observed to be 27% in Group 3 and 8% in Group 2. Sub-epithelial recovery was observed to be better in Group 3. Adipose tissue-derived mesenchymal stem cell increased the healing of the injured maxillary sinus mucosa of the rabbits in terms of cilia presence, density and morphology regardless of the implementation technique. Level of evidence NA.

  6. A muscle stem cell for every muscle: variability of satellite cell biology among different muscle groups

    PubMed Central

    Randolph, Matthew E.; Pavlath, Grace K.

    2015-01-01

    The human body contains approximately 640 individual skeletal muscles. Despite the fact that all of these muscles are composed of striated muscle tissue, the biology of these muscles and their associated muscle stem cell populations are quite diverse. Skeletal muscles are affected differentially by various muscular dystrophies (MDs), such that certain genetic mutations specifically alter muscle function in only a subset of muscles. Additionally, defective muscle stem cells have been implicated in the pathology of some MDs. The biology of muscle stem cells varies depending on the muscles with which they are associated. Here we review the biology of skeletal muscle stem cell populations of eight different muscle groups. Understanding the biological variation of skeletal muscles and their resident stem cells could provide valuable insight into mechanisms underlying the susceptibility of certain muscles to myopathic disease. PMID:26500547

  7. Circumferential Ciliary Body Cysts Presenting as Acute Pigment Dispersion and Ocular Hypertension.

    PubMed

    Sarıgül Sezenöz, Almila; Güngör, Sirel Gür; Kıratlı, Hayyam; Akman, Ahmet

    2017-09-15

    To report a case of circumferential neuroepithelial cyst of the ciliary body presenting with pigment dispersion (PD) and ocular hypertension. 48-year-old female patient presented with a complaint of pain in the left eye. On examination, visual acuity of the left eye was 0.9, and the intraocular pressure was 48 mmHg. Biomicroscopic anterior segment examination of the left eye revealed 4+ pigmented cells in the anterior chamber. Active PD from the pupillary region at 11 o'clock was noticed at the time of the examination. Ultrasound biomicroscopy demonstrated 360º cystic lesions of the ciliary body in the left eye. The patient was diagnosed as neuroepithelial cyst of the ciliary body. Our case is unique as it is the first case of circumferential neuroepithelial ciliary body cyst presenting with acute PD and ocular hypertension.

  8. Efficient mucociliary transport relies on efficient regulation of ciliary beating.

    PubMed

    Braiman, Alex; Priel, Zvi

    2008-11-30

    The respiratory mucociliary epithelium is a synchronized and highly effective waste-disposal system. It uses mucus as a vehicle, driven by beating cilia, to transport unwanted particles, trapped in the mucus, away from the respiratory system. The ciliary machinery can function in at least two different modes: a low rate of beating that requires only ATP, and a high rate of beating regulated by second messengers. The mucus propelling velocity is linearly dependent on ciliary beat frequency (CBF). The linear dependence implies that a substantial increase in transport efficiency requires an equally substantial rise in CBF. The ability to enhance beating in response to various physiological cues is a hallmark of mucociliary cells. An intricate signaling network controls ciliary activity, which relies on interplay between calcium and cyclic nucleotide pathways.

  9. The Challenges of Diagnosing Primary Ciliary Dyskinesia

    PubMed Central

    O'Callaghan, Christopher; Knowles, Michael R.

    2011-01-01

    Primary ciliary dyskinesia (PCD) is a rare genetic disorder of ciliary structure and function. The diagnosis can be challenging, particularly when using nongenetic assays. The “gold standard” diagnostic test is ultrastructural analysis of respiratory cilia obtained by nasal scrape or brush biopsy. A few specialized centers use high-speed videomicroscopy to examine ciliary beat. Certain beat patterns correlate with ultrastructural defects, and, in some cases, subtle alterations in beat pattern can be seen when ultrastructure is normal. Recent studies have shown that nasal nitric oxide (NO) is very low in patients with PCD compared with healthy control subjects; therefore, this assay may be a useful screening or adjunctive test for PCD. Because acute respiratory illnesses may yield alterations in ciliary ultrastructure, ciliary beat, and nasal NO values, these tests should be performed during a stable baseline period. Identification of an array of PCD genes has provided the opportunity for making a definitive genetic diagnosis for PCD in some cases. All of these approaches have a role in diagnosing PCD. For example, PCD has been confirmed by identifying disease-causing mutations in a heavy dynein chain gene in individuals with normal ciliary ultrastructure but subtle defects in ciliary beat and low nasal NO. Priorities to improve nongenetic diagnostic capability include standardization of nasal NO as a screening test and the development of specialized centers using uniform approaches for the analysis of ciliary ultrastructure and ciliary beat pattern. Another chapter in this issue (see Zariwala and colleagues, pp. 430) addresses the progress toward improved capabilities for definitive genetic testing PMID:21926395

  10. Paramecium swimming and ciliary beating patterns: a study on four RNA interference mutations.

    PubMed

    Funfak, Anette; Fisch, Cathy; Abdel Motaal, Hatem T; Diener, Julien; Combettes, Laurent; Baroud, Charles N; Dupuis-Williams, Pascale

    2015-01-01

    Paramecium cells swim and feed by beating their thousands of cilia in coordinated patterns. The organization of these patterns and its relationship with cell motility has been the subject of a large body of work, particularly as a model for ciliary beating in human organs where similar organization is seen. However the rapid motion of the cells makes quantitative measurements very challenging. Here we provide detailed measurements of the swimming of Paramecium cells from high-speed video at high magnification, as they move in microfluidic channels. An image analysis protocol allows us to decouple the cell movement from the motion of the cilia, thus allowing us to measure the ciliary beat frequency (CBF) and the spatio-temporal organization into metachronal waves along the cell periphery. Two distinct values of the CBF appear at different regions of the cell: most of the cilia beat in the range of 15 to 45 Hz, while the cilia in the peristomal region beat at almost double the frequency. The body and peristomal CBF display a nearly linear relation with the swimming velocity. Moreover the measurements do not display a measurable correlation between the swimming velocity and the metachronal wave velocity on the cell periphery. These measurements are repeated for four RNAi silenced mutants, where proteins specific to the cilia or to their connection to the cell base are depleted. We find that the mutants whose ciliary structure is affected display similar swimming to the control cells albeit with a reduced efficiency, while the mutations that affect the cilia's anchoring to the cell lead to strongly reduced ability to swim. This reduction in motility can be related to a loss of coordination between the ciliary beating in different parts of the cell.

  11. Enzymes of the γ-Glutamyl Cycle in the Ciliary Body and Lens

    PubMed Central

    Ross, Leonard L.; Barber, Lee; Tate, Suresh S.; Meister, Alton

    1973-01-01

    The enzymes of the γ-glutamyl cycle have been found in rabbit ciliary body and, except for 5-oxoprolinase, also in the ocular lens. Histochemical studies show that γ-glutamyl transpeptidase is localized mainly in the basal portions of the epithelial cells of the ciliary body; the findings are similar to those observed in the chloroid plexuses. The histochemical staining reaction in the ciliary epithelium is more intense than in the chloroid plexus, intestine, and kidney. γ-Glutamyl transpeptidase staining activity in the epithelium of the intestinal and renal proximal convoluted tubules is confined to the microvillus border. Moderate transpeptidase activity was found in the cytoplasm of nonpigmented epithelial cells of the iris at the posterior pupillary margin. The histochemical and enzyme activity studies are consistent with the thesis that the γ-glutamyl cycle functions in transport of amino acids across the blood-aqueous humor barrier. Images PMID:4152058

  12. LRRC6 Mutation Causes Primary Ciliary Dyskinesia with Dynein Arm Defects

    PubMed Central

    Horani, Amjad; Ferkol, Thomas W.; Shoseyov, David; Wasserman, Mollie G.; Oren, Yifat S.; Kerem, Batsheva; Amirav, Israel; Cohen-Cymberknoh, Malena; Dutcher, Susan K.; Brody, Steven L.; Elpeleg, Orly; Kerem, Eitan

    2013-01-01

    Despite recent progress in defining the ciliome, the genetic basis for many cases of primary ciliary dyskinesia (PCD) remains elusive. We evaluated five children from two unrelated, consanguineous Palestinian families who had PCD with typical clinical features, reduced nasal nitric oxide concentrations, and absent dynein arms. Linkage analyses revealed a single common homozygous region on chromosome 8 and one candidate was conserved in organisms with motile cilia. Sequencing revealed a single novel mutation in LRRC6 (Leucine-rich repeat containing protein 6) that fit the model of autosomal recessive genetic transmission, leading to a change of a highly conserved amino acid from aspartic acid to histidine (Asp146His). LRRC6 was localized to the cytoplasm and was up-regulated during ciliogenesis in human airway epithelial cells in a Foxj1-dependent fashion. Nasal epithelial cells isolated from affected individuals and shRNA-mediated silencing in human airway epithelial cells, showed reduced LRRC6 expression, absent dynein arms, and slowed cilia beat frequency. Dynein arm proteins were either absent or mislocalized to the cytoplasm in airway epithelial cells from a primary ciliary dyskinesia subject. These findings suggest that LRRC6 plays a role in dynein arm assembly or trafficking and when mutated leads to primary ciliary dyskinesia with laterality defects. PMID:23527195

  13. Bone Marrow Stromal Cells Generate Muscle Cells and Repair Muscle Degeneration

    NASA Astrophysics Data System (ADS)

    Dezawa, Mari; Ishikawa, Hiroto; Itokazu, Yutaka; Yoshihara, Tomoyuki; Hoshino, Mikio; Takeda, Shin-ichi; Ide, Chizuka; Nabeshima, Yo-ichi

    2005-07-01

    Bone marrow stromal cells (MSCs) have great potential as therapeutic agents. We report a method for inducing skeletal muscle lineage cells from human and rat general adherent MSCs with an efficiency of 89%. Induced cells differentiated into muscle fibers upon transplantation into degenerated muscles of rats and mdx-nude mice. The induced population contained Pax7-positive cells that contributed to subsequent regeneration of muscle upon repetitive damage without additional transplantation of cells. These MSCs represent a more ready supply of myogenic cells than do the rare myogenic stem cells normally found in muscle and bone marrow.

  14. Proliferation and differentiation of direct co-culture of bone marrow mesenchymal stem cells and pigmented cells from the ciliary margin

    PubMed Central

    Li, Yan; He, Xinzheng; Li, Jun; Ni, Fangfang; Sun, Qingqing; Zhou, Yan

    2017-01-01

    Damage of retinal ganglion cells (RGCs) is the major consequence of glaucoma and regeneration of RGCs is extremely difficult once the damage has occurred. Retinal stem cells (RSCs) are considered an ideal choice for RGC regeneration. Pigmented cells from the ciliary margin (PCMs) have great retinal differentiation potential and may be an ideal RSC candidate. However, the ciliary margin is too small, so the number of cells that can be obtained is limited. Bone marrow-derived mesenchymal stem cells (BMMSCs) are another type of stem cell that have been previously investigated for RGC regeneration. BMMSCs expand sufficiently, whereas the retinal differentiation of BMMSCs is insufficient. The aim of the present study was to investigate whether the co-culture of PCMs and BMMSCs may combine the advantages of both cell types to establish a novel and effective stem cell source for RGC regeneration. Primary rat PCMs and BMMSCs were isolated and co-cultured. Cell growth was observed by an inverted microscope and proliferation was monitored by an MTT assay. Cell cycle analysis was performed by using a flow cytometer, while the expression of the photoreceptor-specific homeobox gene (cone-rod homeobox, Crx) was determined by reverse transcription-quantitative polymerase chain reaction and western blot analysis. In addition, retinal differentiation was confirmed by immunofluorescence staining of major markers of retinal differentiation, including rhodopsin, visual system homeobox 2 and heparin sulfate. The co-cultured cells expanded successfully, in a similar way to BMMSCs. In addition, the expression of Crx and retinal markers were significantly upregulated following BMMSC and PCM co-culture. The results of the present study demonstrated that the co-culture of BMMSCs and PCMs may be used as a source of RSCs. PMID:28440470

  15. Regulation of ciliary retrograde protein trafficking by the Joubert syndrome proteins ARL13B and INPP5E.

    PubMed

    Nozaki, Shohei; Katoh, Yohei; Terada, Masaya; Michisaka, Saki; Funabashi, Teruki; Takahashi, Senye; Kontani, Kenji; Nakayama, Kazuhisa

    2017-02-01

    ARL13B (a small GTPase) and INPP5E (a phosphoinositide 5-phosphatase) are ciliary proteins encoded by causative genes of Joubert syndrome. We here showed, by taking advantage of a visible immunoprecipitation assay, that ARL13B interacts with the IFT46 -: IFT56 (IFT56 is also known as TTC26) dimer of the intraflagellar transport (IFT)-B complex, which mediates anterograde ciliary protein trafficking. However, the ciliary localization of ARL13B was found to be independent of its interaction with IFT-B, but dependent on the ciliary-targeting sequence RVEP in its C-terminal region. ARL13B-knockout cells had shorter cilia than control cells and exhibited aberrant localization of ciliary proteins, including INPP5E. In particular, in ARL13B-knockout cells, the IFT-A and IFT-B complexes accumulated at ciliary tips, and GPR161 (a negative regulator of Hedgehog signaling) could not exit cilia in response to stimulation with Smoothened agonist. This abnormal phenotype was rescued by the exogenous expression of wild-type ARL13B, as well as by its mutant defective in the interaction with IFT-B, but not by its mutants defective in INPP5E binding or in ciliary localization. Thus, ARL13B regulates IFT-A-mediated retrograde protein trafficking within cilia through its interaction with INPP5E. © 2017. Published by The Company of Biologists Ltd.

  16. The Ciliary Margin Zone of the Mammalian Retina Generates Retinal Ganglion Cells.

    PubMed

    Marcucci, Florencia; Murcia-Belmonte, Veronica; Wang, Qing; Coca, Yaiza; Ferreiro-Galve, Susana; Kuwajima, Takaaki; Khalid, Sania; Ross, M Elizabeth; Mason, Carol; Herrera, Eloisa

    2016-12-20

    The retina of lower vertebrates grows continuously by integrating new neurons generated from progenitors in the ciliary margin zone (CMZ). Whether the mammalian CMZ provides the neural retina with retinal cells is controversial. Live imaging of embryonic retina expressing eGFP in the CMZ shows that cells migrate laterally from the CMZ to the neural retina where differentiated retinal ganglion cells (RGCs) reside. Because Cyclin D2, a cell-cycle regulator, is enriched in ventral CMZ, we analyzed Cyclin D2 -/- mice to test whether the CMZ is a source of retinal cells. Neurogenesis is diminished in Cyclin D2 mutants, leading to a reduction of RGCs in the ventral retina. In line with these findings, in the albino retina, the decreased production of ipsilateral RGCs is correlated with fewer Cyclin D2 + cells. Together, these results implicate the mammalian CMZ as a neurogenic site that produces RGCs and whose proper generation depends on Cyclin D2 activity. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  17. Loss and gain of cone types in vertebrate ciliary photoreceptor evolution.

    PubMed

    Musser, Jacob M; Arendt, Detlev

    2017-11-01

    Ciliary photoreceptors are a diverse cell type family that comprises the rods and cones of the retina and other related cell types such as pineal photoreceptors. Ciliary photoreceptor evolution has been dynamic during vertebrate evolution with numerous gains and losses of opsin and phototransduction genes, and changes in their expression. For example, early mammals lost all but two cone opsins, indicating loss of cone receptor types in response to nocturnal lifestyle. Our review focuses on the comparison of specifying transcription factors and cell type-specific transcriptome data in vertebrate retinae to build and test hypotheses on ciliary photoreceptor evolution. Regarding cones, recent data reveal that a combination of factors specific for long-wavelength sensitive opsin (Lws)- cones in non-mammalian vertebrates (Thrb and Rxrg) is found across all differentiating cone photoreceptors in mice. This suggests that mammalian ancestors lost all but one ancestral cone type, the Lws-cone. We test this hypothesis by a correlation analysis of cone transcriptomes in mouse and chick, and find that, indeed, transcriptomes of all mouse cones are most highly correlated to avian Lws-cones. These findings underscore the importance of specifying transcription factors in tracking cell type evolution, and shed new light on the mechanisms of cell type loss and gain in retina evolution. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  18. Exogenous ciliary neurotrophic factor (CNTF) reduces synaptic depression during repetitive stimulation.

    PubMed

    Garcia, Neus; Santafé, Manel M; Tomàs, Marta; Priego, Mercedes; Obis, Teresa; Lanuza, Maria A; Besalduch, Nuria; Tomàs, Josep

    2012-09-01

    It has been shown that ciliary neurotrophic factor (CNTF) has trophic and maintenance effects on several types of peripheral and central neurons, glia, and cells outside the nervous system. Both CNTF and its receptor, CNTF-Rα, are expressed in the muscle. We use confocal immunocytochemistry to show that the trophic cytokine and its receptor are present in the pre- and post-synaptic sites of the neuromuscular junctions (NMJs). Applied CNTF (7.5-200 ng/ml, 60 min-3 h) does not acutely affect spontaneous potentials (size or frequency) or quantal content of the evoked acetylcholine release from post-natal (in weak or strong axonal inputs on dually innervated end plates or in the most mature singly innervated synapses at P6) or adult (P30) NMJ of Levator auris longus muscle of the mice. However, CNTF reduces roughly 50% the depression produced by repetitive stimulation (40 Hz, 2 min) on the adult NMJs. Our findings indicate that, unlike neurotrophins, exogenous CNTF does not acutely modulate transmitter release locally at the mammalian neuromuscular synapse but can protect mature end plates from activity-induced synaptic depression. © 2012 Peripheral Nerve Society.

  19. A novel ICK mutation causes ciliary disruption and lethal endocrine-cerebro-osteodysplasia syndrome.

    PubMed

    Oud, Machteld M; Bonnard, Carine; Mans, Dorus A; Altunoglu, Umut; Tohari, Sumanty; Ng, Alvin Yu Jin; Eskin, Ascia; Lee, Hane; Rupar, C Anthony; de Wagenaar, Nathalie P; Wu, Ka Man; Lahiry, Piya; Pazour, Gregory J; Nelson, Stanley F; Hegele, Robert A; Roepman, Ronald; Kayserili, Hülya; Venkatesh, Byrappa; Siu, Victoria M; Reversade, Bruno; Arts, Heleen H

    2016-01-01

    Endocrine-cerebro-osteodysplasia (ECO) syndrome [MIM:612651] caused by a recessive mutation (p.R272Q) in Intestinal cell kinase (ICK) shows significant clinical overlap with ciliary disorders. Similarities are strongest between ECO syndrome, the Majewski and Mohr-Majewski short-rib thoracic dysplasia (SRTD) with polydactyly syndromes, and hydrolethalus syndrome. In this study, we present a novel homozygous ICK mutation in a fetus with ECO syndrome and compare the effect of this mutation with the previously reported ICK variant on ciliogenesis and cilium morphology. Through homozygosity mapping and whole-exome sequencing, we identified a second variant (c.358G > T; p.G120C) in ICK in a Turkish fetus presenting with ECO syndrome. In vitro studies of wild-type and mutant mRFP-ICK (p.G120C and p.R272Q) revealed that, in contrast to the wild-type protein that localizes along the ciliary axoneme and/or is present in the ciliary base, mutant proteins rather enrich in the ciliary tip. In addition, immunocytochemistry revealed a decreased number of cilia in ICK p.R272Q-affected cells. Through identification of a novel ICK mutation, we confirm that disruption of ICK causes ECO syndrome, which clinically overlaps with the spectrum of ciliopathies. Expression of ICK-mutated proteins result in an abnormal ciliary localization compared to wild-type protein. Primary fibroblasts derived from an individual with ECO syndrome display ciliogenesis defects. In aggregate, our findings are consistent with recent reports that show that ICK regulates ciliary biology in vitro and in mice, confirming that ECO syndrome is a severe ciliopathy.

  20. Genetics Home Reference: primary ciliary dyskinesia

    MedlinePlus

    ... mutations explain only 2% of primary ciliary dykinesia. Respiration. 2008;76(2):198-204. doi: 10.1159/ ... MR. Genetic causes of bronchiectasis: primary ciliary dyskinesia. Respiration. 2007;74(3):252-63. Review. Citation on ...

  1. Methods for Studying Ciliary-Mediated Chemoresponse in Paramecium.

    PubMed

    Valentine, Megan Smith; Van Houten, Judith L

    2016-01-01

    Paramecium is a useful model organism for the study of ciliary-mediated chemical sensing and response. Here we describe ways to take advantage of Paramecium to study chemoresponse.Unicellular organisms like the ciliated protozoan Paramecium sense and respond to chemicals in their environment (Van Houten, Ann Rev Physiol 54:639-663, 1992; Van Houten, Trends Neurosci 17:62-71, 1994). A thousand or more cilia that cover Paramecium cells serve as antennae for chemical signals, similar to ciliary function in a large variety of metazoan cell types that have primary or motile cilia (Berbari et al., Curr Biol 19(13):R526-R535, 2009; Singla V, Reiter J, Science 313:629-633, 2006). The Paramecium cilia also produce the motor output of the detection of chemical cues by controlling swimming behavior. Therefore, in Paramecium the cilia serve multiple roles of detection and response.We present this chapter in three sections to describe the methods for (1) assaying populations of cells for their behavioral responses to chemicals (attraction and repulsion), (2) characterization of the chemoreceptors and associated channels of the cilia using proteomics and binding assays, and (3) electrophysiological analysis of individual cells' responses to chemicals. These methods are applied to wild type cells, mutants, transformed cells that express tagged proteins, and cells depleted of gene products by RNA Interference (RNAi).

  2. cAMP Stimulates Transepithelial Short-Circuit Current and Fluid Transport Across Porcine Ciliary Epithelium.

    PubMed

    Cheng, Angela King-Wah; Civan, Mortimer M; To, Chi-Ho; Do, Chi-Wai

    2016-12-01

    To investigate the effects of cAMP on transepithelial electrical parameters and fluid transport across porcine ciliary epithelium. Transepithelial electrical parameters were determined by mounting freshly isolated porcine ciliary epithelium in a modified Ussing chamber. Similarly, fluid movement across intact ciliary body was measured with a custom-made fluid flow chamber. Addition of 1, 10, and 100 μM 8-Br-cAMP (cAMP) to the aqueous side (nonpigmented ciliary epithelium, NPE) induced a sustained increase in short-circuit current (Isc). Addition of niflumic acid (NFA) to the aqueous surface effectively blocked the cAMP-induced Isc stimulation. The administration of cAMP to the stromal side (pigmented ciliary epithelium, PE) triggered a significant stimulation of Isc only at 100 μM. No additive effect was observed with bilateral application of cAMP. Likewise, forskolin caused a significant stimulation of Isc when applied to the aqueous side. Concomitantly, cAMP and forskolin increased fluid transport across porcine ciliary epithelium, and this stimulation was effectively inhibited by aqueous NFA. Depleting Cl- in the bathing solution abolished the baseline Isc and inhibited the subsequent stimulation by cAMP. Pretreatment with protein kinase A (PKA) blockers (H89/KT5720) significantly inhibited the cAMP- and forskolin-induced Isc responses. Our results suggest that cAMP triggers a sustained stimulation of Cl- and fluid transport across porcine ciliary epithelium; Cl- channels in the NPE cells are potentially a cellular site for this PKA-sensitive cAMP-mediated response.

  3. Potential of laryngeal muscle regeneration using induced pluripotent stem cell-derived skeletal muscle cells.

    PubMed

    Dirja, Bayu Tirta; Yoshie, Susumu; Ikeda, Masakazu; Imaizumi, Mitsuyoshi; Nakamura, Ryosuke; Otsuki, Koshi; Nomoto, Yukio; Wada, Ikuo; Hazama, Akihiro; Omori, Koichi

    2016-01-01

    Conclusion Induced pluripotent stem (iPS) cells may be a new potential cell source for laryngeal muscle regeneration in the treatment of vocal fold atrophy after recurrent laryngeal nerve paralysis. Objectives Unilateral vocal fold paralysis can lead to degeneration, atrophy, and loss of force of the thyroarytenoid muscle. At present, there are some treatments such as thyroplasty, arytenoid adduction, and vocal fold injection. However, such treatments cannot restore reduced mass of the thyroarytenoid muscle. iPS cells have been recognized as supplying a potential resource for cell transplantation. The aim of this study was to assess the effectiveness of the use of iPS cells for the regeneration of laryngeal muscle through the evaluation of both in vitro and in vivo experiments. Methods Skeletal muscle cells were generated from tdTomato-labeled iPS cells using embryoid body formation. Differentiation into skeletal muscle cells was analyzed by gene expression and immunocytochemistry. The tdTomato-labeled iPS cell-derived skeletal muscle cells were transplanted into the left atrophied thyroarytenoid muscle. To evaluate the engraftment of these cells after transplantation, immunohistochemistry was performed. Results The tdTomato-labeled iPS cells were successfully differentiated into skeletal muscle cells through an in vitro experiment. These cells survived in the atrophied thyroarytenoid muscle after transplantation.

  4. [Non-ciliary functions of cilia proteins].

    PubMed

    Taulet, Nicolas; Delaval, Bénédicte

    2014-11-01

    Cilia proteins have long been characterized for their role in cilia formation and function, and their implications in ciliopathies. However, several cellular defects induced by cilia proteins deregulation suggest that they could have non-ciliary roles. Indeed, several non-ciliary functions have been recently characterized for cilia proteins including roles in intra-cellular and in vesicular transport, in spindle orientation or in the maintenance of genomic stability. These observations thus raise the crucial question of the contribution of non-ciliary functions of cilia proteins to the pathological manifestations associated with ciliopathies such as polycystic kidney disease. © 2014 médecine/sciences – Inserm.

  5. The effects of VEGF-A-inhibitors aflibercept and ranibizumab on the ciliary body and iris of monkeys.

    PubMed

    Ludinsky, Maximilian; Christner, Sarah; Su, Nan; Taubitz, Tatjana; Tschulakow, Alexander; Biesemeier, Antje; Julien-Schraermeyer, Sylvie; Schraermeyer, Ulrich

    2016-06-01

    To investigate the effects of intravitreal ranibizumab (Lucentis®) and aflibercept (Eylea®) on the ciliary body and the iris of 12 cynomolgus monkeys with regard to the fenestrations of their blood vessels. Structural changes in the ciliary body and in the iris were investigated with light, fluorescent, and transmission electron microscopy (TEM). The latter was used to specifically quantify fenestrations of the endothelium of blood vessels after treatment with aflibercept and ranibizumab. Each of the two ciliary bodies treated with aflibercept and the two treated with ranibizumab and their controls were examined after 1 and 7 days respectively. Ophthalmological investigations including funduscopy and intraocular pressure measurements were also applied. Ophthalmological investigations did not reveal any changes within the groups. Both drugs reduced the VEGF concentration in the ciliary body pigmented epithelium. The structure of the ciliary body was not influenced, while the posterior pigmented epithelium of the iris showed vacuoles after aflibercept treatment. Ranibizumab was mainly concentrated on the surface layer of the ciliary epithelium, in the blood vessel walls and the lumen of some of the blood vessels, and in the cells of the epithelium of the ciliary body. Aflibercept was more concentrated in the stroma and not in the cells of the epithelium, but as with ranibizumab, also in the blood vessel walls and some of their lumina, and again on the surface layer of the epithelium. Both aflibercept-and ranibizumab-treated eyes showed a decreased number of fenestrations of the capillaries in the ciliary body compared to the untreated controls. On day 1 and day 7, aflibercept had fewer fenestrations than the ranibizumab samples of the same day. Both aflibercept and ranibizumab were found to reach the blood vessel walls of the ciliary body, and effectively reduced their fenestrations. Aflibercept might eliminate VEGF to a greater extent, possibly due to a higher

  6. PRIMARY CILIARY DYSKINESIA: DIAGNOSTIC AND PHENOTYPIC FEATURES

    EPA Science Inventory

    Primary ciliary dyskinesia (PCD) is a genetic disease characterized by abnormalities in ciliary structure/function. We hypothesized that the major clinical and biologic phenotypic markers of the disease could be evaluated by studying a cohort of subjects suspected of having PCD. ...

  7. Variation in the ciliary neurotrophic factor gene and muscle strength in older Caucasian women.

    PubMed

    Arking, Dan E; Fallin, Daniele M; Fried, Linda P; Li, Tao; Beamer, Brock A; Xue, Qian Li; Chakravarti, Aravinda; Walston, Jeremy

    2006-05-01

    To determine whether genetic variants in the ciliary neurotrophic factor (CNTF) gene are associated with muscle strength in older women. Cross-sectional analysis of baseline data from the Women's Health and Aging Studies I (1992) and II (1994), complementary population-based studies. Twelve contiguous ZIP code areas in Baltimore, Maryland. Three hundred sixty-three Caucasian, community-dwelling women aged 70 to 79. Participants were genotyped at the CNTF locus for eight single nucleotide polymorphisms (SNPs), including the null allele rs1800169. The dependent variables were grip strength and the frailty syndrome, identified as presence of three or more of five frailty indicators (weakness, slowness, weight loss, low physical activity, exhaustion). In addition to genotypes, independent variables of body mass index (BMI) and osteoarthritis of the hands were included. Using multivariate linear regression, single SNP analysis identified five SNPs significantly associated with grip strength (P<.05), after adjusting for age, BMI, and osteoarthritis. Haplotype analysis was performed, and a single haplotype associated with grip strength was identified (P<.01). The rs1800169 null allele fully explained the association between this haplotype and grip strength under a recessive model, with individuals homozygous for the null allele exhibiting a 3.80-kg lower (95% confidence interval=1.01-6.58) grip strength. No association was seen between the CNTF null allele and frailty. Individuals homozygous for the CNTF null allele had significantly lower grip strength but did not exhibit overt frailty. Larger prospective studies are needed to confirm this finding and extend it to additional populations.

  8. Super-Resolution Imaging Reveals TCTN2 Depletion-Induced IFT88 Lumen Leakage and Ciliary Weakening.

    PubMed

    Weng, Rueyhung Roc; Yang, T Tony; Huang, Chia-En; Chang, Chih-Wei; Wang, Won-Jing; Liao, Jung-Chi

    2018-06-01

    The primary cilium is an essential organelle mediating key signaling activities, such as sonic hedgehog signaling. The molecular composition of the ciliary compartment is distinct from that of the cytosol, with the transition zone (TZ) gated the ciliary base. The TZ is a packed and organized protein complex containing multiple ciliopathy-associated protein species. Tectonic 2 (TCTN2) is one of the TZ proteins in the vicinity of the ciliary membrane, and its mutation is associated with Meckel syndrome. Despite its importance in ciliopathies, the role of TCTN2 in ciliary structure and molecules remains unclear. Here, we created a CRISPR/Cas9 TCTN2 knockout human retinal pigment epithelial cell line and conducted quantitative analysis of geometric localization using both wide-field and super-resolution microscopy techniques. We found that TCTN2 depletion resulted in partial TZ damage, loss of ciliary membrane proteins, leakage of intraflagellar transport protein IFT88 toward the basal body lumen, and cilium shortening and curving. The basal body lumen occupancy of IFT88 was also observed in si-RPGRIP1L cells and cytochalasin-D-treated wild-type cells, suggesting varying lumen accessibility for intraflagellar transport proteins under different perturbed conditions. Our findings support two possible models for the lumen leakage of IFT88, i.e., a tip leakage model and a misregulation model. Together, our quantitative image analysis augmented by super-resolution microscopy facilitates the observation of structural destruction and molecular redistribution in TCTN2 -/- cilia, shedding light on mechanistic understanding of TZ-protein-associated ciliopathies. Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  9. Clinical and genetic aspects of primary ciliary dyskinesia/Kartagener syndrome.

    PubMed

    Leigh, Margaret W; Pittman, Jessica E; Carson, Johnny L; Ferkol, Thomas W; Dell, Sharon D; Davis, Stephanie D; Knowles, Michael R; Zariwala, Maimoona A

    2009-07-01

    Primary ciliary dyskinesia is a genetically heterogeneous disorder of motile cilia. Most of the disease-causing mutations identified to date involve the heavy (dynein axonemal heavy chain 5) or intermediate(dynein axonemal intermediate chain 1) chain dynein genes in ciliary outer dynein arms, although a few mutations have been noted in other genes. Clinical molecular genetic testing for primary ciliary dyskinesia is available for the most common mutations. The respiratory manifestations of primary ciliary dyskinesia (chronic bronchitis leading to bronchiectasis, chronic rhino-sinusitis, and chronic otitis media)reflect impaired mucociliary clearance owing to defective axonemal structure. Ciliary ultrastructural analysis in most patients (>80%) reveals defective dynein arms, although defects in other axonemal components have also been observed. Approximately 50% of patients with primary ciliary dyskinesia have laterality defects (including situs inversus totalis and, less commonly, heterotaxy, and congenital heart disease),reflecting dysfunction of embryological nodal cilia. Male infertility is common and reflects defects in sperm tail axonemes. Most patients with primary ciliary dyskinesia have a history of neonatal respiratory distress, suggesting that motile cilia play a role in fluid clearance during the transition from a fetal to neonatal lung. Ciliopathies involving sensory cilia, including autosomal dominant or recessive polycystic kidney disease, Bardet-Biedl syndrome, and Alstrom syndrome, may have chronic respiratory symptoms and even bronchiectasis suggesting clinical overlap with primary ciliary dyskinesia.

  10. Regulation of anterior chamber drainage by bicarbonate-sensitive soluble adenylyl cyclase in the ciliary body.

    PubMed

    Lee, Yong S; Tresguerres, Martin; Hess, Kenneth; Marmorstein, Lihua Y; Levin, Lonny R; Buck, Jochen; Marmorstein, Alan D

    2011-12-02

    Glaucoma is a leading cause of blindness affecting as many as 2.2 million Americans. All current glaucoma treatment strategies aim to reduce intraocular pressure (IOP). IOP results from the resistance to drainage of aqueous humor (AH) produced by the ciliary body in a process requiring bicarbonate. Once secreted into the anterior chamber, AH drains from the eye via two pathways: uveoscleral and pressure-dependent or conventional outflow (C(t)). Modulation of "inflow" and "outflow" pathways is thought to occur via distinct, local mechanisms. Mice deficient in the bicarbonate channel bestrophin-2 (Best2), however, exhibit a lower IOP despite an increase in AH production. Best2 is expressed uniquely in nonpigmented ciliary epithelial (NPE) cells providing evidence for a bicarbonate-dependent communicative pathway linking inflow and outflow. Here, we show that bicarbonate-sensitive soluble adenylyl cyclase (sAC) is highly expressed in the ciliary body in NPE cells, but appears to be absent from drainage tissues. Pharmacologic inhibition of sAC in mice causes a significant increase in IOP due to a decrease in C(t) with no effect on inflow. In mice deficient in sAC IOP is elevated, and C(t) is decreased relative to wild-type mice. Pharmacologic inhibition of sAC did not alter IOP or C(t) in sAC-deficient mice. Based on these data we propose that the ciliary body can regulate C(t) and that sAC serves as a critical sensor of bicarbonate in the ciliary body regulating the secretion of substances into the AH that govern outflow facility independent of pressure.

  11. Regulation of ciliary motility: conserved protein kinases and phosphatases are targeted and anchored in the ciliary axoneme

    PubMed Central

    Wirschell, Maureen; Yamamoto, Ryosuke; Alford, Lea; Gokhale, Avanti; Gaillard, Anne; Sale, Winfield S.

    2011-01-01

    Recent evidence has revealed that the dynein motors and highly conserved signaling proteins are localized within the ciliary 9 + 2 axoneme. One key mechanism for regulation of motility is phosphorylation. Here, we review diverse evidence, from multiple experimental organisms, that ciliary motility is regulated by phosphorylation / dephosphorylation of the dynein arms through kinases and phosphatases that are anchored immediately adjacent to their axonemal substrates. PMID:21513695

  12. Bug22p, a conserved centrosomal/ciliary protein also present in higher plants, is required for an effective ciliary stroke in Paramecium.

    PubMed

    Laligné, C; Klotz, C; de Loubresse, N Garreau; Lemullois, M; Hori, M; Laurent, F X; Papon, J F; Louis, B; Cohen, J; Koll, F

    2010-04-01

    Centrioles, cilia, and flagella are ancestral conserved organelles of eukaryotic cells. Among the proteins identified in the proteomics of ciliary proteins in Paramecium, we focus here on a protein, Bug22p, previously detected by cilia and basal-body high-throughput studies but never analyzed per se. Remarkably, this protein is also present in plants, which lack centrioles and cilia. Bug22p sequence alignments revealed consensus positions that distinguish species with centrioles/cilia from plants. In Paramecium, antibody and green fluorescent protein (GFP) fusion labeling localized Bug22p in basal bodies and cilia, and electron microscopy immunolabeling refined the localization to the terminal plate of the basal bodies, the transition zone, and spots along the axoneme, preferentially between the membrane and the microtubules. RNA interference (RNAi) depletion of Bug22p provoked a strong decrease in swimming speed, followed by cell death after a few days. High-speed video microscopy and morphological analysis of Bug22p-depleted cells showed that the protein plays an important role in the efficiency of ciliary movement by participating in the stroke shape and rigidity of cilia. The defects in cell swimming and growth provoked by RNAi can be complemented by expression of human Bug22p. This is the first reported case of complementation by a human gene in a ciliate.

  13. Satellite Cells and the Muscle Stem Cell Niche

    PubMed Central

    Yin, Hang; Price, Feodor

    2013-01-01

    Adult skeletal muscle in mammals is a stable tissue under normal circumstances but has remarkable ability to repair after injury. Skeletal muscle regeneration is a highly orchestrated process involving the activation of various cellular and molecular responses. As skeletal muscle stem cells, satellite cells play an indispensible role in this process. The self-renewing proliferation of satellite cells not only maintains the stem cell population but also provides numerous myogenic cells, which proliferate, differentiate, fuse, and lead to new myofiber formation and reconstitution of a functional contractile apparatus. The complex behavior of satellite cells during skeletal muscle regeneration is tightly regulated through the dynamic interplay between intrinsic factors within satellite cells and extrinsic factors constituting the muscle stem cell niche/microenvironment. For the last half century, the advance of molecular biology, cell biology, and genetics has greatly improved our understanding of skeletal muscle biology. Here, we review some recent advances, with focuses on functions of satellite cells and their niche during the process of skeletal muscle regeneration. PMID:23303905

  14. Value of transmission electron microscopy for primary ciliary dyskinesia diagnosis in the era of molecular medicine: Genetic defects with normal and non-diagnostic ciliary ultrastructure.

    PubMed

    Shapiro, Adam J; Leigh, Margaret W

    2017-01-01

    Primary ciliary dyskinesia (PCD) is a genetic disorder causing chronic oto-sino-pulmonary disease. No single diagnostic test will detect all PCD cases. Transmission electron microscopy (TEM) of respiratory cilia was previously considered the gold standard diagnostic test for PCD, but 30% of all PCD cases have either normal ciliary ultrastructure or subtle changes which are non-diagnostic. These cases are identified through alternate diagnostic tests, including nasal nitric oxide measurement, high-speed videomicroscopy analysis, immunofluorescent staining of axonemal proteins, and/or mutation analysis of various PCD causing genes. Autosomal recessive mutations in DNAH11 and HYDIN produce normal TEM ciliary ultrastructure, while mutations in genes encoding for radial spoke head proteins result in some cross-sections with non-diagnostic alterations in the central apparatus interspersed with normal ciliary cross-sections. Mutations in nexin link and dynein regulatory complex genes lead to a collection of different ciliary ultrastructures; mutations in CCDC65, CCDC164, and GAS8 produce normal ciliary ultrastructure, while mutations in CCDC39 and CCDC40 cause absent inner dynein arms and microtubule disorganization in some ciliary cross-sections. Mutations in CCNO and MCIDAS cause near complete absence of respiratory cilia due to defects in generation of multiple cellular basal bodies; however, the scant cilia generated may have normal ultrastructure. Lastly, a syndromic form of PCD with retinal degeneration results in normal ciliary ultrastructure through mutations in the RPGR gene. Clinicians must be aware of these genetic causes of PCD resulting in non-diagnostic TEM ciliary ultrastructure and refrain from using TEM of respiratory cilia as a test to rule out PCD.

  15. High-frequency ultrasound measurements of the normal ciliary body and iris.

    PubMed

    Garcia, Julian P S; Spielberg, Leigh; Finger, Paul T

    2011-01-01

    To determine the normal ultrasonographic thickness of the iris and ciliary body. This prospective 35-MHz ultrasonographic study included 80 normal eyes of 40 healthy volunteers. The images were obtained at the 12-, 3-, 6-, and 9-o'clock radial meridians, measured at three locations along the radial length of the iris and at the thickest section of the ciliary body. Mixed model was used to estimate eye site-adjusted means and standard errors and to test the statistical difference of adjusted results. Parameters included mean thickness, standard deviation, and range. Mean thicknesses at the iris root, midway along the radial length of the iris, and at the juxtapupillary margin were 0.4 ± 0.1, 0.5 ± 0.1, and 0.6 ± 0.1 mm, respectively. Those of the ciliary body, ciliary processes, and ciliary body + ciliary processes were 0.7 ± 0.1, 0.6 ± 0.1, and 1.3 ± 0.2 mm, respectively. This study provides standard, normative thickness data for the iris and ciliary body in healthy adults using ultrasonographic imaging. Copyright 2011, SLACK Incorporated.

  16. Vasodilatory effects of nipradilol, an alpha- and beta-adrenergic blocker with nitric oxide releasing action, in rabbit ciliary artery.

    PubMed

    Yoshitomi, Takeshi; Yamaji, Kazutsuna; Ishikawa, Hitoshi; Ohnishi, Yoshitaka

    2002-12-01

    Nipradilol is a new antiglaucoma ophthalmic agent used in Japan. Topical application of nipradilol is reported to increase ocular blood flow. To investigate the action of this drug, we studied the effect of nipradilol on the isolated rabbit ciliary artery. Under the dissecting microscope, ciliary arteries were prepared from rabbit eyes and mounted on a myograph system. The effects of nipradilol on the isolated rabbit ciliary artery were investigated using isometric tension recording methods. Nipradilol provoked a dose-dependent (10 microM-1m M) relaxation in ciliary arteries that were pre-contracted with high-K solutions (K(+): 100.7 m M). It also inhibited the amplitude of smooth muscle contraction evoked by field stimulation. Nipradilol was more effective in relaxing phenylephrine-induced contraction (EC(50): 21.6+/-16.3 microM) compared to high-K solution-induced contractions (EC(50): 230+/-130 microM). Application of N(w)-nitro- L -arginine methylester (300 microM), a nitric oxide (NO) synthase inhibitor, or denudations of endothelium by rubbing the inner surface with a scalp hair did not affect this relaxation. However, NO scavenger carboxy-PTIO (1m M) or methylene blue (10 microM), a guanylate cyclase inhibitor, inhibited the nipradilol-induced relaxation. These results indicate that nipradilol relaxes the rabbit ciliary artery by two different mechanisms. First, the relaxation is due to the NO produced by denitrification of nipradilol itself. Second, nipradilol may act as an alpha-adrenergic antagonist. These actions of nipradilol may explain the mechanisms of increased ocular blood flow in vivo.

  17. Potential roles for BMP and Pax genes in the development of iris smooth muscle.

    PubMed

    Jensen, Abbie M

    2005-02-01

    The embryonic optic cup generates four types of tissue: neural retina, pigmented epithelium, ciliary epithelium, and iris smooth muscle. Remarkably little attention has focused on the development of the iris smooth muscle since Lewis ([1903] J. Am. Anat. 2:405-416) described its origins from the anterior rim of the optic cup neuroepithelium. As an initial step toward understanding iris smooth muscle development, I first determined the spatial and temporal pattern of the development of the iris smooth muscle in the chick by using the HNK1 antibody, which labels developing iris smooth muscle. HNK1 labeling shows that iris smooth muscle development is correlated in time and space with the development of the ciliary epithelial folds. Second, because neural crest is the only other neural tissue that has been shown to generate smooth muscle (Le Lievre and Le Douarin [1975] J. Embryo. Exp. Morphol. 34:125-154), I sought to determine whether iris smooth muscle development shares similarities with neural crest development. Two members of the BMP superfamily, BMP4 and BMP7, which may regulate neural crest development, are highly expressed by cells at the site of iris smooth muscle generation. Third, because humans and mice that are heterozygous for Pax6 mutations have no irides (Hill et al. [1991] Nature 354:522-525; Hanson et al. [1994] Nat. Genet. 6:168-173), I determined the expression of Pax6. I also examined the expression of Pax3 in the developing anterior optic cup. The developing iris smooth muscle coexpresses Pax6 and Pax3. I suggest that some of the eye defects caused by mutations in Pax6, BMP4, and BMP7 may be due to abnormal iris smooth muscle. Copyright 2004 Wiley-Liss, Inc.

  18. Safety assessment of thiolated polymers: effect on ciliary beat frequency in human nasal epithelial cells.

    PubMed

    Palmberger, Thomas F; Augustijns, Patrick; Vetter, Anja; Bernkop-Schnürch, Andreas

    2011-12-01

    The aim of this study was to investigate the nasal safety of gel formulations of thiolated polymers (thiomers) by assessing their effect on ciliary beat frequency (CBF) in human nasal epithelial cells. Poly(acrylic acid) 450 kDa-cysteine (PAA-cys) and alginate-cysteine (alg-cys) were synthesized by covalent attachment of L-cysteine to the polymeric backbone. The cationic polymer chitosan-thiobutylamidine (chito-TBA) was synthesized by attaching iminothiolane to chitosan. CBF using was measured by a photometric system. CBF was measured before incubating the cells with test gels, during incubation and after washing out the polymeric test gels to evaluate reversibility of cilio-inhibition. The influence of viscosity on CBF was determined by using hydroxyethylcellulose (HEC)-gels of various concentrations. Ciliary beating was observed to be affected by viscosity, but cilia were still beating in the presence of a HEC-gel displaying an apparent viscosity of 25 Pa.s. In case of thiolated polymers and their unmodified control, a concentration-dependent decrease in CBF could be observed. PAA-cys, alg-cys, chito-TBA and their corresponding unmodified controls exhibited a moderate cilio-inhibitory effect, followed by a partial recovery of CBF when used at a concentration of 1%. Alg-cys 2% and chito-TBA 2% (m/v) gels exhibited severe cilio-inhibition, which was partially reversible. L-cysteine and reduced glutathione led to mild cilio-inhibition at concentrations of 3% (m/v). Taking into account that dilution after application and cilio-modifying effects is usually more pronounced under in vitro conditions, thiomers can be considered as suitable excipients for nasal drug delivery systems.

  19. Bug22p, a Conserved Centrosomal/Ciliary Protein Also Present in Higher Plants, Is Required for an Effective Ciliary Stroke in Paramecium ▿ †

    PubMed Central

    Laligné, C.; Klotz, C.; Garreau de Loubresse, N.; Lemullois, M.; Hori, M.; Laurent, F. X.; Papon, J. F.; Louis, B.; Cohen, J.; Koll, F.

    2010-01-01

    Centrioles, cilia, and flagella are ancestral conserved organelles of eukaryotic cells. Among the proteins identified in the proteomics of ciliary proteins in Paramecium, we focus here on a protein, Bug22p, previously detected by cilia and basal-body high-throughput studies but never analyzed per se. Remarkably, this protein is also present in plants, which lack centrioles and cilia. Bug22p sequence alignments revealed consensus positions that distinguish species with centrioles/cilia from plants. In Paramecium, antibody and green fluorescent protein (GFP) fusion labeling localized Bug22p in basal bodies and cilia, and electron microscopy immunolabeling refined the localization to the terminal plate of the basal bodies, the transition zone, and spots along the axoneme, preferentially between the membrane and the microtubules. RNA interference (RNAi) depletion of Bug22p provoked a strong decrease in swimming speed, followed by cell death after a few days. High-speed video microscopy and morphological analysis of Bug22p-depleted cells showed that the protein plays an important role in the efficiency of ciliary movement by participating in the stroke shape and rigidity of cilia. The defects in cell swimming and growth provoked by RNAi can be complemented by expression of human Bug22p. This is the first reported case of complementation by a human gene in a ciliate. PMID:20118210

  20. Hierarchical signaling transduction of the immune and muscle cell crosstalk in muscle regeneration.

    PubMed

    Yang, Wenjun; Hu, Ping

    2018-04-01

    The muscle regeneration is a complicated bioprocess that involved in many cell types, including necrotic muscle cells, satellite cells, mesenchymal cells, pericytes, immune cells, and other cell types present at the injury site. Immune cells involved in both innate and adaptive immune responses regulate the progress of muscle regeneration. In this review, we discussed the roles of different immune cells in muscle regeneration. The immune cells regulate muscle regeneration through cytokine production, cell-cell contacts, and general immune environment regulation. We also describe the current known mechanism of how immune cells regulating muscle regeneration. Copyright © 2017. Published by Elsevier Inc.

  1. A novel mouse model of anterior segment dysgenesis (ASD): conditional deletion of Tsc1 disrupts ciliary body and iris development.

    PubMed

    Hägglund, Anna-Carin; Jones, Iwan; Carlsson, Leif

    2017-03-01

    Development of the cornea, lens, ciliary body and iris within the anterior segment of the eye involves coordinated interaction between cells originating from the ciliary margin of the optic cup, the overlying periocular mesenchyme and the lens epithelium. Anterior segment dysgenesis (ASD) encompasses a spectrum of developmental syndromes that affect these anterior segment tissues. ASD conditions arise as a result of dominantly inherited genetic mutations and result in both ocular-specific and systemic forms of dysgenesis that are best exemplified by aniridia and Axenfeld-Rieger syndrome, respectively. Extensive clinical overlap in disease presentation amongst ASD syndromes creates challenges for correct diagnosis and classification. The use of animal models has therefore proved to be a robust approach for unravelling this complex genotypic and phenotypic heterogeneity. However, despite these successes, it is clear that additional genes that underlie several ASD syndromes remain unidentified. Here, we report the characterisation of a novel mouse model of ASD. Conditional deletion of Tsc1 during eye development leads to a premature upregulation of mTORC1 activity within the ciliary margin, periocular mesenchyme and lens epithelium. This aberrant mTORC1 signalling within the ciliary margin in particular leads to a reduction in the number of cells that express Pax6, Bmp4 and Msx1 Sustained mTORC1 signalling also induces a decrease in ciliary margin progenitor cell proliferation and a consequent failure of ciliary body and iris development in postnatal animals. Our study therefore identifies Tsc1 as a novel candidate ASD gene. Furthermore, the Tsc1 -ablated mouse model also provides a valuable resource for future studies concerning the molecular mechanisms underlying ASD and acts as a platform for evaluating therapeutic approaches for the treatment of visual disorders. © 2017. Published by The Company of Biologists Ltd.

  2. A novel mouse model of anterior segment dysgenesis (ASD): conditional deletion of Tsc1 disrupts ciliary body and iris development

    PubMed Central

    Hägglund, Anna-Carin; Jones, Iwan

    2017-01-01

    ABSTRACT Development of the cornea, lens, ciliary body and iris within the anterior segment of the eye involves coordinated interaction between cells originating from the ciliary margin of the optic cup, the overlying periocular mesenchyme and the lens epithelium. Anterior segment dysgenesis (ASD) encompasses a spectrum of developmental syndromes that affect these anterior segment tissues. ASD conditions arise as a result of dominantly inherited genetic mutations and result in both ocular-specific and systemic forms of dysgenesis that are best exemplified by aniridia and Axenfeld–Rieger syndrome, respectively. Extensive clinical overlap in disease presentation amongst ASD syndromes creates challenges for correct diagnosis and classification. The use of animal models has therefore proved to be a robust approach for unravelling this complex genotypic and phenotypic heterogeneity. However, despite these successes, it is clear that additional genes that underlie several ASD syndromes remain unidentified. Here, we report the characterisation of a novel mouse model of ASD. Conditional deletion of Tsc1 during eye development leads to a premature upregulation of mTORC1 activity within the ciliary margin, periocular mesenchyme and lens epithelium. This aberrant mTORC1 signalling within the ciliary margin in particular leads to a reduction in the number of cells that express Pax6, Bmp4 and Msx1. Sustained mTORC1 signalling also induces a decrease in ciliary margin progenitor cell proliferation and a consequent failure of ciliary body and iris development in postnatal animals. Our study therefore identifies Tsc1 as a novel candidate ASD gene. Furthermore, the Tsc1-ablated mouse model also provides a valuable resource for future studies concerning the molecular mechanisms underlying ASD and acts as a platform for evaluating therapeutic approaches for the treatment of visual disorders. PMID:28250050

  3. Muscle Satellite Cell Protein Teneurin‐4 Regulates Differentiation During Muscle Regeneration

    PubMed Central

    Ishii, Kana; Suzuki, Nobuharu; Mabuchi, Yo; Ito, Naoki; Kikura, Naomi; Fukada, So‐ichiro; Okano, Hideyuki; Takeda, Shin'ichi

    2015-01-01

    Abstract Satellite cells are maintained in an undifferentiated quiescent state, but during muscle regeneration they acquire an activated stage, and initiate to proliferate and differentiate as myoblasts. The transmembrane protein teneurin‐4 (Ten‐4) is specifically expressed in the quiescent satellite cells; however, its cellular and molecular functions remain unknown. We therefore aimed to elucidate the function of Ten‐4 in muscle satellite cells. In the tibialis anterior (TA) muscle of Ten‐4‐deficient mice, the number and the size of myofibers, as well as the population of satellite cells, were reduced with/without induction of muscle regeneration. Furthermore, we found an accelerated activation of satellite cells in the regenerated Ten‐4‐deficient TA muscle. The cell culture analysis using primary satellite cells showed that Ten‐4 suppressed the progression of myogenic differentiation. Together, our findings revealed that Ten‐4 functions as a crucial player in maintaining the quiescence of muscle satellite cells. Stem Cells 2015;33:3017–3027 PMID:26013034

  4. Muscle satellite cell heterogeneity and self-renewal

    PubMed Central

    Motohashi, Norio; Asakura, Atsushi

    2014-01-01

    Adult skeletal muscle possesses extraordinary regeneration capacities. After muscle injury or exercise, large numbers of newly formed muscle fibers are generated within a week as a result of expansion and differentiation of a self-renewing pool of muscle stem cells termed muscle satellite cells. Normally, satellite cells are mitotically quiescent and reside beneath the basal lamina of muscle fibers. Upon regeneration, satellite cells are activated, and give rise to daughter myogenic precursor cells. After several rounds of proliferation, these myogenic precursor cells contribute to the formation of new muscle fibers. During cell division, a minor population of myogenic precursor cells returns to quiescent satellite cells as a self-renewal process. Currently, accumulating evidence has revealed the essential roles of satellite cells in muscle regeneration and the regulatory mechanisms, while it still remains to be elucidated how satellite cell self-renewal is molecularly regulated and how satellite cells are important in aging and diseased muscle. The number of satellite cells is decreased due to the changing niche during ageing, resulting in attenuation of muscle regeneration capacity. Additionally, in Duchenne muscular dystrophy (DMD) patients, the loss of satellite cell regenerative capacity and decreased satellite cell number due to continuous needs for satellite cells lead to progressive muscle weakness with chronic degeneration. Thus, it is necessary to replenish muscle satellite cells continuously. This review outlines recent findings regarding satellite cell heterogeneity, asymmetric division and molecular mechanisms in satellite cell self-renewal which is crucial for maintenance of satellite cells as a muscle stem cell pool throughout life. In addition, we discuss roles in the stem cell niche for satellite cell maintenance, as well as related cell therapies for approaching treatment of DMD. PMID:25364710

  5. Flow Field Analysis of Micromixer Powered by Ciliary Motion of Vorticella

    NASA Astrophysics Data System (ADS)

    Hayasaka, Yo; Nagai, Moeto; Matsumoto, Nobuyoshi; Kawashima, Takahiro; Shibata, Takayuki

    We demonstrate the observation of a flow field generated by ciliary motion of Vorticella in a microfluidic chamber. We applied the property that Vorticella vibrates its cilia and create a flow field to a micromixer. The stability and mixing performance of Vorticella were measured by PIV (Particle Image Velocimetry). One cell of Vorticella mixed the half area of the microchamber. We revealed that the flow field of a single cell in a chamber was more stable than that of multiple cells.

  6. Primary ciliary dyskinesia.

    PubMed

    Lobo, L J; Zariwala, M A; Noone, P G

    2014-09-01

    Primary ciliary dyskinesia (PCD) is an autosomal recessive disorder of cilia structure and function, leading to chronic infections of the respiratory tract, fertility problems and disorders of organ laterality. Making a definitive diagnosis is challenging, utilizing characteristic phenotypes, ciliary functional and ultra-structural defects in addition to newer screening tools such as nasal nitric oxide and genetic testing. There are 21 known PCD causing genes and in the future, comprehensive genetic testing may help diagnosis young infants prior to developing symptoms thus improving survival. Therapy includes surveillance of pulmonary function and microbiology in addition to, airway clearance, antibiotics and early referral to bronchiectasis centers. Standardized care at specialized centers using a multidisciplinary approach is likely to improve outcomes. In conjunction with the PCD foundation and lead investigators and clinicians are developing a network of PCD clinical centers to coordinate the effort in North America and Europe. As the network grows, care and knowledge will undoubtedly improve. © The Author 2014. Published by Oxford University Press on behalf of the Association of Physicians. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  7. [Histological structure of the trabecular meshwork in the eyeball: challenging the traditional concept and preliminary findings in rabbits, rats and mice].

    PubMed

    Shi, Yun; Zhou, Fan-Qi; Luo, Zhou-Cai; Chen, Ying-Hua; Chen, Yu; Dong, Wei-Ren

    2017-10-20

    To verify that the trabecular meshwork (TM) in the wall of the eyeball consists of smooth muscle fibers instead of collagen fibers or endothelial cells. Eighteen fresh eyeballs from 3 rabbits, 3 SD rats and 3 mice were sectioned along the sagittal plane and sliced after paraffin embedding for HE staining, VG staining, Masson staining, α-SMA immunohistochemistry or CD31 immunohistochemistry. These slices were observed under microscope and the structure of the TM was compared with those of scleral collagen fibers, ciliary muscles and endothelial cells. HE staining of the eyeball slices from the 3 animal species resulted in purplish red staining of the TM, which was highly consistent with ciliary muscle fibers. The cell?like structures on the surface of the TM were not clearly outlined, with flat nuclei showing a dark purple staining; these structures did not show obvious boundaries from the TM. Ciliary muscle fibers, which were smooth muscle cells in nature, were aligned in bundles in various directions. The longitudinally sectioned cells were flat and contained purplish cytoplasm and highly flattened nuclei. Scleral collagen fibers were stained dark red with a few fibroblasts sandwiched among them. The long axis of the fibroblasts was in parallel with that of the collagen fibers. The outline of the fibroblast was not clear and the nucleus was flat in dark blue. The vascular endothelial cells presented with different morphologies and contained light purplish cytoplasm and dark nuclei, protruding into the vascular cavity. VG staining of the TM revealed a pale red filamentous structure, and the collagen fibers were stained bright red. Masson staining of the TM showed a reticular structure consisting mainly of dark red fibers intermingled with thin green fibers. Scleral collagen fibers presented with a cord?like green wavy structure. The endothelial cells were green and flat, while the ciliary smooth muscle fibers were purple. In immunohistochemistry for α?SMA, the TM

  8. The Skeletal Muscle Satellite Cell

    PubMed Central

    2011-01-01

    The skeletal muscle satellite cell was first described and named based on its anatomic location between the myofiber plasma and basement membranes. In 1961, two independent studies by Alexander Mauro and Bernard Katz provided the first electron microscopic descriptions of satellite cells in frog and rat muscles. These cells were soon detected in other vertebrates and acquired candidacy as the source of myogenic cells needed for myofiber growth and repair throughout life. Cultures of isolated myofibers and, subsequently, transplantation of single myofibers demonstrated that satellite cells were myogenic progenitors. More recently, satellite cells were redefined as myogenic stem cells given their ability to self-renew in addition to producing differentiated progeny. Identification of distinctively expressed molecular markers, in particular Pax7, has facilitated detection of satellite cells using light microscopy. Notwithstanding the remarkable progress made since the discovery of satellite cells, researchers have looked for alternative cells with myogenic capacity that can potentially be used for whole body cell-based therapy of skeletal muscle. Yet, new studies show that inducible ablation of satellite cells in adult muscle impairs myofiber regeneration. Thus, on the 50th anniversary since its discovery, the satellite cell’s indispensable role in muscle repair has been reaffirmed. PMID:22147605

  9. Morphometric assessment of normal human ciliary body using ultrasound biomicroscopy.

    PubMed

    Okamoto, Yoshifumi; Okamoto, Fumiki; Nakano, Shinichiro; Oshika, Tetsuro

    2017-12-01

    To quantitatively assess the biometry of the ciliary body in normal human eyes using ultrasound biomicroscopy. We evaluated 85 eyes of 85 normal subjects (35 men and 50 women), whose age ranged from 11 to 86 years (mean ± SD, 56.8 ± 20.4 years). The eyes were assessed along the 3-, 6-, 9-, and 12-o'clock meridians relative to the center of the cornea. Clinical data were collected, including age, axial length, ciliary body length (CBL), ciliary body thickness (CBT), anterior chamber depth, iris root thickness, trabecular-iris angle, and scleral-ciliary process angle. Axial length was measured using A-scan ultrasonography. CBL and CBT tended to be larger in the superior than in the inferior quadrant, but the differences among the four quadrants were not statistically significant. The average CBL showed a significant positive correlation with the average CBT (r = 0.40, P < 0.001). Average CBL and CBT were significantly correlated with axial length (r = 0.33, P = 0.031; r = 0.46, P < 0.01 respectively). In addition, the average CBL was significantly correlated with anterior chamber depth (r = 0.23, P < 0.05), trabecular-iris angle (r = 0.29, P = 0.01), and scleral-ciliary process angle (r = 0.40, P < 0.001). Ultrasound biomicroscopic imaging demonstrated that the ciliary body is similar in size in all circumferences, and eyes with longer axial length have an elongated and thicker ciliary body. The values obtained in the present study may serve as standard clinical references.

  10. Ciliary dysfunction and obesity.

    PubMed

    Mok, C A; Héon, E; Zhen, M

    2010-01-01

    Obesity associates with increased health risks such as heart disease, stroke and diabetes. The steady rise in the obese population worldwide poses an increasing burden on health systems. Genetic factors contribute to the development of obesity, and the elucidation of their physiological functions helps to understand the cause, and improve the prevention, diagnosis and treatment for this disorder. Primary cilia are evolutionarily conserved organelles whose dysfunctions lead to human disorders now defined as ciliopathies. Human ciliopathies present pleiotropic and overlapping phenotypes that often include retinal degeneration, cystic renal anomalies and obesity. Increasing evidence implicates an intriguing involvement of cilia in lipid/energy homeostasis. Here we discuss recent studies in support of the key roles of ciliary genes in the development and pathology of obesity in various animal models. Genes affecting ciliary development and function may pose promising candidate underlying genetic factors that contribute to the development of common obesity.

  11. The mammalian iris-ciliary complex affects organization and synthesis of cytoskeletal proteins of organ and tissue cultured lens epithelial cells.

    PubMed

    Banerjee, A; Emanuel, K; Parafina, J; Bagchi, M

    1992-10-01

    A water soluble growth inhibitor was isolated from the mammalian ocular iris-ciliary complex. The molecular weight of this protein is 10 kD or lower as determined by ultrafiltration fractionation. The iris-ciliary (IC) complex water soluble protein(s) significantly inhibits synthesis of lower molecular weight proteins of the epithelial cells of the organ cultured mammalian ocular lens. It was also found that this inhibitory effect of IC is mediated via the structural organization of the lens. Monolayer cultures of the lens epithelial cells exposed to IC did not manifest any inhibition of their protein synthesis. Moreover, these tissue cultured lens epithelial (TCLE) cells showed a significant increase in their protein synthetic activities in response to the presence of IC factors in the culture medium. It is postulated that the IC activity is modulated via either the lens capsule, an extracellular matrix, or due to the specific organization of the intact lens. The specific effects of IC on the cytoskeletal organization and synthesis in the organ cultured lens epithelial (OCLE) and TCLE cells were also examined. Both groups, treated with IC factors, manifested significant alterations in their protein synthetic activities and cytoskeletal architecture. The 3H-leucine incorporation experiments showed that alpha-actin and alpha-tubulin synthesis is partially inhibited by IC factors in OCLE cells but vimentin synthesis is not, whereas in TCLE cells all of them showed increased synthesis in response to IC factors. Turnover rates of these proteins in both OCLE and TCLE cells were also computed. The immunofluorescence and microscopic evaluation of OCLE and TCLE cells exposed to IC factors illustrated significant alteration in the cytoarchitecture of the filaments. We demonstrate that an inhibitor(s) molecule of 10 kD or lower size isolated from IC inhibited protein synthesis of OCLE cells and stimulated protein synthesis in TCLE cells. The IC factor also affects the

  12. Decellularised skeletal muscles allow functional muscle regeneration by promoting host cell migration.

    PubMed

    Urciuolo, Anna; Urbani, Luca; Perin, Silvia; Maghsoudlou, Panagiotis; Scottoni, Federico; Gjinovci, Asllan; Collins-Hooper, Henry; Loukogeorgakis, Stavros; Tyraskis, Athanasios; Torelli, Silvia; Germinario, Elena; Fallas, Mario Enrique Alvarez; Julia-Vilella, Carla; Eaton, Simon; Blaauw, Bert; Patel, Ketan; De Coppi, Paolo

    2018-05-30

    Pathological conditions affecting skeletal muscle function may lead to irreversible volumetric muscle loss (VML). Therapeutic approaches involving acellular matrices represent an emerging and promising strategy to promote regeneration of skeletal muscle following injury. Here we investigated the ability of three different decellularised skeletal muscle scaffolds to support muscle regeneration in a xenogeneic immune-competent model of VML, in which the EDL muscle was surgically resected. All implanted acellular matrices, used to replace the resected muscles, were able to generate functional artificial muscles by promoting host myogenic cell migration and differentiation, as well as nervous fibres, vascular networks, and satellite cell (SC) homing. However, acellular tissue mainly composed of extracellular matrix (ECM) allowed better myofibre three-dimensional (3D) organization and the restoration of SC pool, when compared to scaffolds which also preserved muscular cytoskeletal structures. Finally, we showed that fibroblasts are indispensable to promote efficient migration and myogenesis by muscle stem cells across the scaffolds in vitro. This data strongly support the use of xenogeneic acellular muscles as device to treat VML conditions in absence of donor cell implementation, as well as in vitro model for studying cell interplay during myogenesis.

  13. Satellite cells in human skeletal muscle plasticity

    PubMed Central

    Snijders, Tim; Nederveen, Joshua P.; McKay, Bryon R.; Joanisse, Sophie; Verdijk, Lex B.; van Loon, Luc J. C.; Parise, Gianni

    2015-01-01

    Skeletal muscle satellite cells are considered to play a crucial role in muscle fiber maintenance, repair and remodeling. Our knowledge of the role of satellite cells in muscle fiber adaptation has traditionally relied on in vitro cell and in vivo animal models. Over the past decade, a genuine effort has been made to translate these results to humans under physiological conditions. Findings from in vivo human studies suggest that satellite cells play a key role in skeletal muscle fiber repair/remodeling in response to exercise. Mounting evidence indicates that aging has a profound impact on the regulation of satellite cells in human skeletal muscle. Yet, the precise role of satellite cells in the development of muscle fiber atrophy with age remains unresolved. This review seeks to integrate recent results from in vivo human studies on satellite cell function in muscle fiber repair/remodeling in the wider context of satellite cell biology whose literature is largely based on animal and cell models. PMID:26557092

  14. The influence of thapsigargin on Na,K-ATPase activity in cultured nonpigmented ciliary epithelial cells.

    PubMed

    Mito, T; Kuwahara, S; Delamere, N A

    1995-08-01

    Experiments were conducted to test the influence of thapsigargin on the NaK-ATPase activity of cultured cells (ODM2) derived from human nonpigmented ciliary epithelium. The rate of ouabain-sensitive ATP hydrolysis (Na,K-ATPase activity) was diminished in cells that had been pretreated with thapsigargin then permeabilized. Following 20 min exposure of intact cells to thapsigargin, the cells were permeabilized with digitonin and the rate of ouabain-sensitive ATP hydrolysis (Na,K-ATPase activity) was measured immediately in a calcium-free buffer. In permeabilized cells that had been pretreated with 1 microM thapsigargin for 20 min, the rate of ouabain-sensitive ATP hydrolysis (Na,K-ATPase activity) was reduced by 38%. Pretreatment with lesser concentrations of thapsigargin caused smaller changes of Na,K-ATPase activity. The decrease of Na,K-ATPase activity was the same whether or not calmodulin antagonists W7 or trifluoperazine were present during the thapsigargin pretreatment period. This inhibitory effect upon the Na,K-ATPase may serve to limit the extent of sodium pump activation that takes place in intact cells when thapsigargin causes sodium pump stimulation by a mechanism that appears to involve changes in cytoplasmic ion levels when potassium channels open.

  15. Regulatory T cells and skeletal muscle regeneration.

    PubMed

    Schiaffino, Stefano; Pereira, Marcelo G; Ciciliot, Stefano; Rovere-Querini, Patrizia

    2017-02-01

    Skeletal muscle regeneration results from the activation and differentiation of myogenic stem cells, called satellite cells, located beneath the basal lamina of the muscle fibers. Inflammatory and immune cells have a crucial role in the regeneration process. Acute muscle injury causes an immediate transient wave of neutrophils followed by a more persistent infiltration of M1 (proinflammatory) and M2 (anti-inflammatory/proregenerative) macrophages. New studies show that injured muscle is also infiltrated by a specialized population of regulatory T (Treg) cells, which control both the inflammatory response, by promoting the M1-to-M2 switch, and the activation of satellite cells. Treg cells accumulate in injured muscle in response to specific cytokines, such as IL-33, and promote muscle growth by releasing growth factors, such as amphiregulin. Muscle repair during aging is impaired due to reduced number of Treg cells and can be enhanced by IL-33 supplementation. Migration of Treg cells could also contribute to explain the effect of heterochronic parabiosis, whereby muscle regeneration of aged mice can be improved by a parabiotically linked young partners. In mdx dystrophin-deficient mice, a model of human Duchenne muscular dystrophy, muscle injury, and inflammation is mitigated by expansion of the Treg-cell population but exacerbated by Treg-cell depletion. These findings support the notion that immunological mechanisms are not only essential in the response to pathogenic microbes and tumor cells but also have a wider homeostatic role in tissue repair, and open new perspectives for boosting muscle growth in chronic muscle disease and during aging. © 2016 Federation of European Biochemical Societies.

  16. Ciliary locomotion in presence of boundaries

    NASA Astrophysics Data System (ADS)

    Jana, Saikat; Um, Soong Ho; Jung, Sunghwan

    2010-11-01

    Micro-organisms in nature navigate through a variety of fluidic geometries and chemical conditions. We investigate the effect of confined spaces in nature by introducing Paramecium Multimicronucleatum in two different configurations: a capillary tube & a wavy PDMS channel. Paramecium swims by creating the metachronal waves due to ciliary beating. The influence of the walls on Paramecia is characterized by measuring the velocity and observing the ciliary beating pattern. Theoretically, we also model the system by solving the stream-function with a pressure gradient. The theoretical and experimental observations are compared and conclusions are drawn about the change in the swimming characteristics as compared to free swimming without the boundaries.

  17. Loss of niche-satellite cell interactions in syndecan-3 null mice alters muscle progenitor cell homeostasis improving muscle regeneration.

    PubMed

    Pisconti, Addolorata; Banks, Glen B; Babaeijandaghi, Farshad; Betta, Nicole Dalla; Rossi, Fabio M V; Chamberlain, Jeffrey S; Olwin, Bradley B

    2016-01-01

    The skeletal muscle stem cell niche provides an environment that maintains quiescent satellite cells, required for skeletal muscle homeostasis and regeneration. Syndecan-3, a transmembrane proteoglycan expressed in satellite cells, supports communication with the niche, providing cell interactions and signals to maintain quiescent satellite cells. Syndecan-3 ablation unexpectedly improves regeneration in repeatedly injured muscle and in dystrophic mice, accompanied by the persistence of sublaminar and interstitial, proliferating myoblasts. Additionally, muscle aging is improved in syndecan-3 null mice. Since syndecan-3 null myofiber-associated satellite cells downregulate Pax7 and migrate away from the niche more readily than wild type cells, syxndecan-3 appears to regulate satellite cell homeostasis and satellite cell homing to the niche. Manipulating syndecan-3 provides a promising target for development of therapies to enhance muscle regeneration in muscular dystrophies and in aged muscle.

  18. Arrestin in ciliary invertebrate photoreceptors: molecular identification and functional analysis in vivo.

    PubMed

    Gomez, Maria Del Pilar; Espinosa, Lady; Ramirez, Nelson; Nasi, Enrico

    2011-02-02

    Arrestin was identified in ciliary photoreceptors of Pecten irradians, and its role in terminating the light response was established electrophysiologically. Downstream effectors in these unusual visual cells diverge from both microvillar photoreceptors and rods and cones; the finding that key regulatory mechanisms of the early steps of visual excitation are conserved across such distant lineages of photoreceptors underscores that a common blueprint for phototransduction exists across metazoa. Arrestin was detected by Western blot analysis of retinal lysates, and localized in ciliary photoreceptors by immunostaining of whole-eye cryosections and dissociated cells. Two arrestin isoforms were molecularly identified by PCR; these present the canonical N- and C-arrestin domains, and are identical at the nucleotide level over much of their sequence. A high degree of homology to various β-arrestins (up to 70% amino acid identity) was found. In situ hybridization localized the two transcripts within the retina, but failed to reveal finer spatial segregation, possibly because of insufficient differences between the riboprobes. Intracellular dialysis of anti arrestin antibodies into voltage-clamped ciliary photoreceptors produced a gradual slow-down of the photocurrent falling phase, leaving a tail that decayed over many seconds after light termination. The antibodies also caused spectrally neutral flashes to elicit prolonged aftercurrents in the absence of large metarhodopsin accumulation; such aftercurrents could be quenched by chromatic illumination that photoconverts metarhodopsin back to rhodopsin. These observations indicate that the antibodies depleted functionally available arrestin, and implicate this molecule in the deactivation of the photoresponse at the rhodopsin level.

  19. DisAp-dependent striated fiber elongation is required to organize ciliary arrays

    PubMed Central

    Galati, Domenico F.; Bonney, Stephanie; Kronenberg, Zev; Clarissa, Christina; Yandell, Mark; Elde, Nels C.; Jerka-Dziadosz, Maria; Giddings, Thomas H.; Frankel, Joseph

    2014-01-01

    Cilia-organizing basal bodies (BBs) are microtubule scaffolds that are visibly asymmetrical because they have attached auxiliary structures, such as striated fibers. In multiciliated cells, BB orientation aligns to ensure coherent ciliary beating, but the mechanisms that maintain BB orientation are unclear. For the first time in Tetrahymena thermophila, we use comparative whole-genome sequencing to identify the mutation in the BB disorientation mutant disA-1. disA-1 abolishes the localization of the novel protein DisAp to T. thermophila striated fibers (kinetodesmal fibers; KFs), which is consistent with DisAp’s similarity to the striated fiber protein SF-assemblin. We demonstrate that DisAp is required for KFs to elongate and to resist BB disorientation in response to ciliary forces. Newly formed BBs move along KFs as they approach their cortical attachment sites. However, because they contain short KFs that are rotated, BBs in disA-1 cells display aberrant spacing and disorientation. Therefore, DisAp is a novel KF component that is essential for force-dependent KF elongation and BB orientation in multiciliary arrays. PMID:25533842

  20. Immunolocalization of ciliary neurotrophic factor receptor alpha (CNTFRalpha) in mammalian photoreceptor cells.

    PubMed

    Beltran, William A; Rohrer, Hermann; Aguirre, Gustavo D

    2005-04-01

    To characterize the site of expression of the alpha subunit of the receptor for ciliary neurotrophic factor (CNTFRalpha) in the retina of a variety of mammalian species, and determine whether CNTFRalpha is localized to photoreceptor cells. The cellular distribution of CNTFRalpha(protein) was examined by immunocytochemistry in the adult retinas of several mammalian species that included mouse, rat, dog, cat, sheep, pig, horse, monkey, and human. Developing retinas from 3-day-old and 6-day-old rats were also included in this study. The molecular weight of CNTFRalpha in rat, dog, cat, pig, and human retinas was determined by immunoblotting. CNTFRalpha immunolabeling was present in the retina of all species. A common pattern was observed in all species, and represented labeling of the nerve fiber layer (NFL), ganglion cell layer (GCL), inner plexiform layer (IPL), inner nuclear layer (INL), and outer plexiform layer (OPL). CNTFRalpha did not immunolocalize to photoreceptor cells in both adult and developing rodent retinas, but was consistently observed in both rods and cones of non-rodent species. The molecular weight of CNTFRalpha in mammalian retinas was approximately 61-64 kDa. These findings highlight a significant difference in the expression of CNTFRalpha in the retina of rodent and non-rodent mammalian species. The expression of CNTFRalpha by rods and cones in non-rodent species may suggest a direct mechanism of action if CNTF administration results in photoreceptor rescue.

  1. Tubby family proteins are adapters for ciliary trafficking of integral membrane proteins

    PubMed Central

    Shimada, Issei S.; Loriot, Evan

    2017-01-01

    The primary cilium is a paradigmatic organelle for studying compartmentalized signaling; however, unlike soluble protein trafficking, processes targeting integral membrane proteins to cilia are poorly understood. In this study, we determine that the tubby family protein TULP3 functions as a general adapter for ciliary trafficking of structurally diverse integral membrane cargo, including multiple reported and novel rhodopsin family G protein–coupled receptors (GPCRs) and the polycystic kidney disease–causing polycystin 1/2 complex. The founding tubby family member TUB also localizes to cilia similar to TULP3 and determines trafficking of a subset of these GPCRs to neuronal cilia. Using minimal ciliary localization sequences from GPCRs and fibrocystin (also implicated in polycystic kidney disease), we demonstrate these motifs to be sufficient and TULP3 dependent for ciliary trafficking. We propose a three-step model for TULP3/TUB-mediated ciliary trafficking, including the capture of diverse membrane cargo by the tubby domain in a phosphoinositide 4,5-bisphosphate (PI(4,5)P2)-dependent manner, ciliary delivery by intraflagellar transport complex A binding to the TULP3/TUB N terminus, and subsequent release into PI(4,5)P2-deficient ciliary membrane. PMID:28154160

  2. The role of satellite cells in muscle hypertrophy.

    PubMed

    Blaauw, Bert; Reggiani, Carlo

    2014-02-01

    The role of satellite cells in muscle hypertrophy has long been a debated issue. In the late 1980s it was shown that proteins remain close to the myonucleus responsible for its synthesis, giving rise to the idea of a nuclear domain. This, together with the observation that during various models of muscle hypertrophy there is an activation of the muscle stem cells, i.e. satellite cells, lead to the idea that satellite cell activation is required for muscle hypertrophy. Thus, satellite cells are not only responsible for muscle repair and regeneration, but also for hypertrophic growth. Further support for this line of thinking was obtained after studies showing that irradiation of skeletal muscle, and therefore elimination of all satellite cells, completely prevented overload-induced hypertrophy. Recently however, using different transgenic approaches, it has become clear that muscle hypertrophy can occur without a contribution of satellite cells, even though in most situations of muscle hypertrophy satellite cells are activated. In this review we will discuss the contribution of satellite cells, and other muscle-resident stem cells, to muscle hypertrophy both in mice as well as in humans.

  3. The roles of muscle stem cells in muscle injury, atrophy and hypertrophy.

    PubMed

    Fukada, So-Ichiro

    2018-05-01

    Skeletal muscle is composed of multinuclear cells called myofibers. Muscular dystrophy (a genetic muscle disorder) induces instability in the cell membrane of myofibers and eventually causes myofibre damage. Non-genetic muscle disorders, including sarcopenia, diabetes, bedridden immobility and cancer cachexia, lead to atrophy of myofibres. In contrast, resistance training induces myofibre hypertrophy. Thus, myofibres exhibit a plasticity that is strongly affected by both intrinsic and extrinsic factors. There is no doubt that muscle stem cells (MuSCs, also known as muscle satellite cells) are indispensable for muscle repair/regeneration, but their contributions to atrophy and hypertrophy are still controversial. The present review focuses on the relevance of MuSCs to (i) muscle diseases and (ii) hypertrophy. Further, this review addresses fundamental questions about MuSCs to clarify the onset or progression of these diseases and which might lead to development of a MuSC-based therapy.

  4. Skeletal muscle satellite cells

    NASA Technical Reports Server (NTRS)

    Schultz, E.; McCormick, K. M.

    1994-01-01

    Evidence now suggests that satellite cells constitute a class of myogenic cells that differ distinctly from other embryonic myoblasts. Satellite cells arise from somites and first appear as a distinct myoblast type well before birth. Satellite cells from different muscles cannot be functionally distinguished from one another and are able to provide nuclei to all fibers without regard to phenotype. Thus, it is difficult to ascribe any significant function to establishing or stabilizing fiber type, even during regeneration. Within a muscle, satellite cells exhibit marked heterogeneity with respect to their proliferative behavior. The satellite cell population on a fiber can be partitioned into those that function as stem cells and those which are readily available for fusion. Recent studies have shown that the cells are not simply spindle shaped, but are very diverse in their morphology and have multiple branches emanating from the poles of the cells. This finding is consistent with other studies indicating that the cells have the capacity for extensive migration within, and perhaps between, muscles. Complexity of cell shape usually reflects increased cytoplasmic volume and organelles including a well developed Golgi, and is usually associated with growing postnatal muscle or muscles undergoing some form of induced adaptive change or repair. The appearance of activated satellite cells suggests some function of the cells in the adaptive process through elaboration and secretion of a product. Significant advances have been made in determining the potential secretion products that satellite cells make. The manner in which satellite cell proliferative and fusion behavior is controlled has also been studied. There seems to be little doubt that cellcell coupling is not how satellite cells and myofibers communicate. Rather satellite cell regulation is through a number of potential growth factors that arise from a number of sources. Critical to the understanding of this form

  5. Asymmetrically localized proteins stabilize basal bodies against ciliary beating forces

    PubMed Central

    Galati, Domenico F.

    2016-01-01

    Basal bodies are radially symmetric, microtubule-rich structures that nucleate and anchor motile cilia. Ciliary beating produces asymmetric mechanical forces that are resisted by basal bodies. To resist these forces, distinct regions within the basal body ultrastructure and the microtubules themselves must be stable. However, the molecular components that stabilize basal bodies remain poorly defined. Here, we determine that Fop1 functionally interacts with the established basal body stability components Bld10 and Poc1. We find that Fop1 and microtubule glutamylation incorporate into basal bodies at distinct stages of assembly, culminating in their asymmetric enrichment at specific triplet microtubule regions that are predicted to experience the greatest mechanical force from ciliary beating. Both Fop1 and microtubule glutamylation are required to stabilize basal bodies against ciliary beating forces. Our studies reveal that microtubule glutamylation and Bld10, Poc1, and Fop1 stabilize basal bodies against the forces produced by ciliary beating via distinct yet interdependent mechanisms. PMID:27807131

  6. Muscle Stem Cells: A Model System for Adult Stem Cell Biology.

    PubMed

    Cornelison, Ddw; Perdiguero, Eusebio

    2017-01-01

    Skeletal muscle stem cells, originally termed satellite cells for their position adjacent to differentiated muscle fibers, are absolutely required for the process of skeletal muscle repair and regeneration. In the last decade, satellite cells have become one of the most studied adult stem cell systems and have emerged as a standard model not only in the field of stem cell-driven tissue regeneration but also in stem cell dysfunction and aging. Here, we provide background in the field and discuss recent advances in our understanding of muscle stem cell function and dysfunction, particularly in the case of aging, and the potential involvement of muscle stem cells in genetic diseases such as the muscular dystrophies.

  7. Inactivation of Ca2+-induced ciliary reversal by high-salt extraction in the cilia of Paramecium.

    PubMed

    Kutomi, Osamu; Seki, Makoto; Nakamura, Shogo; Kamachi, Hiroyuki; Noguchi, Munenori

    2013-10-01

    Intracellular Ca(2+) induces ciliary reversal and backward swimming in Paramecium. However, it is not known how the Ca(2+) signal controls the motor machinery to induce ciliary reversal. We found that demembranated cilia on the ciliated cortical sheets from Paramecium caudatum lost the ability to undergo ciliary reversal after brief extraction with a solution containing 0.5 M KCl. KNO(3), which is similar to KCl with respect to chaotropic effect; it had the same effect as that of KCl on ciliary response. Cyclic AMP antagonizes Ca(2+)-induced ciliary reversal. Limited trypsin digestion prevents endogenous A-kinase and cAMP-dependent phosphorylation of an outer arm dynein light chain and induces ciliary reversal. However, the trypsin digestion prior to the high-salt extraction did not affect the inhibition of Ca(2+)-induced ciliary reversal caused by the high-salt extraction. Furthermore, during the course of the high-salt extraction, some axonemal proteins were extracted from ciliary axonemes, suggesting that they may be responsible for Ca(2+)-induced ciliary reversal.

  8. Interstitial Cells: Regulators of Smooth Muscle Function

    PubMed Central

    Sanders, Kenton M.; Ward, Sean M.; Koh, Sang Don

    2014-01-01

    Smooth muscles are complex tissues containing a variety of cells in addition to muscle cells. Interstitial cells of mesenchymal origin interact with and form electrical connectivity with smooth muscle cells in many organs, and these cells provide important regulatory functions. For example, in the gastrointestinal tract, interstitial cells of Cajal (ICC) and PDGFRα+ cells have been described, in detail, and represent distinct classes of cells with unique ultrastructure, molecular phenotypes, and functions. Smooth muscle cells are electrically coupled to ICC and PDGFRα+ cells, forming an integrated unit called the SIP syncytium. SIP cells express a variety of receptors and ion channels, and conductance changes in any type of SIP cell affect the excitability and responses of the syncytium. SIP cells are known to provide pacemaker activity, propagation pathways for slow waves, transduction of inputs from motor neurons, and mechanosensitivity. Loss of interstitial cells has been associated with motor disorders of the gut. Interstitial cells are also found in a variety of other smooth muscles; however, in most cases, the physiological and pathophysiological roles for these cells have not been clearly defined. This review describes structural, functional, and molecular features of interstitial cells and discusses their contributions in determining the behaviors of smooth muscle tissues. PMID:24987007

  9. Cell-specific paracrine actions of IL-6 family cytokines from bone, marrow and muscle that control bone formation and resorption.

    PubMed

    Sims, Natalie A

    2016-10-01

    Bone renews itself and changes shape throughout life to account for the changing needs of the body; this requires co-ordinated activities of bone resorbing cells (osteoclasts), bone forming cells (osteoblasts) and bone's internal cellular network (osteocytes). This review focuses on paracrine signaling by the IL-6 family of cytokines between bone cells, bone marrow, and skeletal muscle in normal physiology and in pathological states where their levels may be locally or systemically elevated. These functions include the support of osteoclast formation by osteoblast lineage cells in response to interleukin 6 (IL-6), interleukin 11 (IL-11), oncostatin M (OSM) and cardiotrophin 1 (CT-1). In addition it will discuss how bone-resorbing osteoclasts promote osteoblast activity by secreting CT-1, which acts as a "coupling factor" on osteocytes, osteoblasts, and their precursors to promote bone formation. OSM, produced by osteoblast lineage cells and macrophages, stimulates bone formation via osteocytes. IL-6 family cytokines also mediate actions of other bone formation stimuli like parathyroid hormone (PTH) and mechanical loading. CT-1, OSM and LIF suppress marrow adipogenesis by shifting commitment of pluripotent precursors towards osteoblast differentiation. Ciliary neurotrophic factor (CNTF) is released as a myokine from skeletal muscle and suppresses osteoblast differentiation and bone formation on the periosteum (outer bone surface in apposition to muscle). Finally, IL-6 acts directly on marrow-derived osteoclasts to stimulate release of "osteotransmitters" that act through the cortical osteocyte network to stimulate bone formation on the periosteum. Each will be discussed as illustrations of how the extended family of IL-6 cytokines acts within the skeleton in physiology and may be altered in pathological conditions or by targeted therapies. Copyright © 2016 Elsevier Ltd. All rights reserved.

  10. Influence of essential and fatty oils on ciliary beat frequency of human nasal epithelial cells.

    PubMed

    Neher, Andreas; Gstöttner, Michaela; Thaurer, Michael; Augustijns, Patrick; Reinelt, Monika; Schobersberger, Wolfgang

    2008-01-01

    In alternative and complementary medicine, the use of essential and fatty oils has become more and more popular. In addition to conventional medical therapies, self-medication is showing increasing popularity, using agents with unclear compounds and poorly controlled dosages. Among other disorders, these alternative treatments are used in bronchitis and rhinitis, including some topical applications. Thus, the influence on ciliated epithelia should be evaluated, because a disturbance of the ciliary function can lead to recurrent sinusitis and chronic rhinosinusitis. The aim of this study was to test the influence of fatty and essential oils on the ciliary beat frequency (CBF) of nasal mucosa in vivo. The influence of sesame oil, soy oil, peanut oil, Miglyol 840, thyme oil, lavender oil, eucalyptus oil, and menthol on the ciliary activity of nasal brushings was evaluated by digital high-speed imaging. The presence of most fatty oils resulted in an increase in CBF, the effect being highest for peanut oil. Miglyol 840 had no significant influence on CBF. The essential oils were tested at a concentration of 0.2 and 2%. Thyme oil did not affect CBF, whereas the presence of all other essentials oils resulted in an increase in CBF; the effect was higher at 0.2% than at 2%. Except thyme oil and Miglyol 840, all tested oils caused an increase in CBF. Interestingly, the 0.2% concentrations of essential oils resulted in stronger effects when compared with the 2% concentrations.

  11. Replication of Muscle Cell Using Bioimprint

    NASA Astrophysics Data System (ADS)

    Samsuri, Fahmi; Mitchell, John S.; Alkaisi, Maan M.; Evans, John J.

    2009-07-01

    In our earlier study a heat-curable PDMS or a UV curable elastomer, was used as the replicating material to introduce Bioimprint methodology to facilitate cell imaging [1-2] But, replicating conditions for thermal polymerization is known to cause cell dehydration during curing. In this study, a new type of polymer was developed for use in living cell replica formation, and it was tested on human muscle cells. The cells were incubated and cultured according to standard biological culturing procedures, and they were grown for about 10 days. The replicas were then separated from the muscle cells and taken for analysis under an Atomic Force Microscope (AFM). The new polymer was designed to be biocompatible with higher resolution and fast curing process compared to other types of silicon-based organic polymers such as polydimethylsiloxane (PDMS). Muscle cell imprints were achieved and higher resolution images were able to show the micro structures of the muscle cells, including the cellular fibers and cell membranes. The AFM is able to image features at nanoscale resolution. This capacity enables a number of characteristics of biological cells to be visualized in a unique manner. Polymer and muscle cells preparations were developed at Hamilton, in collaboration between Plant and Food Research and the Department of Electrical and Computer Engineering, University of Canterbury. Tapping mode was used for the AFM image analysis as it has low tip-sample forces and non-destructive imaging capability. We will be presenting the bioimprinting processes of muscle cells, their AFM imaging and characterization of the newly developed polymer.

  12. Increased adipogenicity of cells from regenerating skeletal muscle.

    PubMed

    Yamanouchi, Keitaro; Yada, Erica; Ishiguro, Naomi; Hosoyama, Tohru; Nishihara, Masugi

    2006-09-10

    Adipose tissue development is observed in some muscle pathologies, however, mechanisms that induce accumulation of this tissue as well as its cellular origin are unknown. The adipogenicity of cells from bupivacaine hydrochloride (BPVC)-treated and untreated muscle was compared in vitro. Culturing cells from both BPVC-treated and untreated muscles in adipogenic differentiation medium (ADM) for 10 days resulted in the appearance of mature adipocytes, but their number was 3.5-fold higher in cells from BPVC-treated muscle. Temporal expressions of PPARgamma and the presence of lipid droplets during adipogenic differentiation were examined. On day 2 of culture in ADM, only cells from BPVC-treated muscle were positive both for PPARgamma and lipid droplets. Pref-1 was expressed in cells from untreated muscle, whereas its expression was absent in cells from BPVC-treated muscle. In ADM, the presence of insulin, which negates an inhibitory effect of Pref-1 on adipogenic differentiation, was required for PPARgamma2 expression in cells from untreated muscle, but not for cells from BPVC-treated muscle. These results indicate that BPVC-induced degenerative/regenerative changes in muscle lead to increased adipogenicity of cells, and suggest that this increased adipogenicity not only involves an increase in the number of cells having adipogenic potential, but also contributes to the progression of these cells toward adipogenic differentiation.

  13. Molecular characterization and differential gene induction of the neuroendocrine-specific genes neurotensin, neurotensin receptor, PC1, PC2, and 7B2 in the human ocular ciliary epithelium.

    PubMed

    Ortego, J; Coca-Prados, M

    1997-11-01

    The ocular ciliary epithelium is a bilayer of neuroepithelial cells specialized in the secretion of aqueous humor fluid and the regulation of intraocular pressure. In this study, we report on the expression of the regulatory peptide neurotensin (NT) and a set of differentiated neuroendocrine markers including neurotensin receptors (NTrs), the prohormone convertases furin, PC1, and PC2, and the neuroendocrine polypeptide 7B2 in the ciliary epithelium. Using a human cell line, ODM-2, derived from the nonpigmented ciliary epithelium, we demonstrate that (1) NT expression is highly activated by nerve growth factor, glucocorticoid, and activators of adenylate cyclase; (2) NTr expression is up-regulated by selective ligand-activated beta2-adrenergic receptor; and (3) PC1 and PC2 expression are up-regulated via distinct signaling transduction pathways. PC1 gene expression is activated by phorbol ester, and PC2 by the same inducers as those of NT expression. A radioimmunoassay for NT detected an NT-like immunoreactivity in human ciliary epithelium and ODM-2 cell extracts, in aqueous humor, and in conditioned culture medium. The results support the view that the entire ciliary epithelium functions as a neuroendocrine tissue, synthesizing, processing, and releasing NT into the aqueous humor where it may exert important physiological functions through autocrine and/or paracrine mechanisms.

  14. Selective Expansion of Skeletal Muscle Stem Cells from Bulk Muscle Cells in Soft Three‐Dimensional Fibrin Gel

    PubMed Central

    Zhu, Pei; Zhou, Yalu; Wu, Furen; Hong, Yuanfan; Wang, Xin; Shekhawat, Gajendra; Mosenson, Jeffrey

    2017-01-01

    Abstract Muscle stem cells (MuSCs) exhibit robust myogenic potential in vivo, thus providing a promising curative treatment for muscle disorders. Ex vivo expansion of adult MuSCs is highly desired to achieve a therapeutic cell dose because of their scarcity in limited muscle biopsies. Sorting of pure MuSCs is generally required for all the current culture systems. Here we developed a soft three‐dimensional (3D) salmon fibrin gel culture system that can selectively expand mouse MuSCs from bulk skeletal muscle preparations without cell sorting and faithfully maintain their regenerative capacity in culture. Our study established a novel platform for convenient ex vivo expansion of MuSCs, thus greatly advancing stem cell‐based therapies for various muscle disorders. Stem Cells Translational Medicine 2017;6:1412–1423 PMID:28244269

  15. Loss of the ciliary kinase Nek8 causes left-right asymmetry defects.

    PubMed

    Manning, Danielle K; Sergeev, Mikhail; van Heesbeen, Roy G; Wong, Michael D; Oh, Jin-Hee; Liu, Yan; Henkelman, R Mark; Drummond, Iain; Shah, Jagesh V; Beier, David R

    2013-01-01

    A missense mutation in mouse Nek8, which encodes a ciliary kinase, produces the juvenile cystic kidneys (jck) model of polycystic kidney disease, but the functions of Nek8 are incompletely understood. Here, we generated a Nek8-null allele and found that homozygous mutant mice die at birth and exhibit randomization of left-right asymmetry, cardiac anomalies, and glomerular kidney cysts. The requirement for Nek8 in left-right patterning is conserved, as knockdown of the zebrafish ortholog caused randomized heart looping. Ciliogenesis was intact in Nek8-deficient embryos and cells, but we observed misexpression of left-sided marker genes early in development, suggesting that nodal ciliary signaling was perturbed. We also generated jck/Nek8 compound heterozygotes; these mutants developed less severe cystic disease than jck homozygotes and provided genetic evidence that the jck allele may encode a gain-of-function protein. Notably, NEK8 and polycystin-2 (PC2) proteins interact, and we found that Nek8(-/-) and Pkd2(-/-) embryonic phenotypes are strikingly similar. Nek8-deficient embryos and cells did express PC2 normally, which localized properly to the cilia. However, similar to cells lacking PC2, NEK8-depleted inner medullary collecting duct cells exhibited a defective response to fluid shear, suggesting that NEK8 may play a role in mediating PC2-dependent signaling.

  16. CCDC103 mutations cause primary ciliary dyskinesia by disrupting assembly of ciliary dynein arms

    PubMed Central

    Panizzi, Jennifer R.; Becker-Heck, Anita; Castleman, Victoria H.; Al-Mutairi, Dalal; Liu, Yan; Loges, Niki T.; Pathak, Narendra; Austin-Tse, Christina; Sheridan, Eamonn; Schmidts, Miriam; Olbrich, Heike; Werner, Claudius; Häffner, Karsten; Hellman, Nathan; Chodhari, Rahul; Gupta, Amar; Kramer-Zucker, Albrecht; Olale, Felix; Burdine, Rebecca D.; Schier, Alexander F.; O’Callaghan, Christopher; Chung, Eddie MK; Reinhardt, Richard; Mitchison, Hannah M.; King, Stephen M.; Omran, Heymut; Drummond, Iain A.

    2012-01-01

    Cilia are essential for fertilization, respiratory clearance, cerebrospinal fluid circulation, and to establish laterality1. Cilia motility defects cause Primary Ciliary Dyskinesia (PCD, MIM 242650), a disorder affecting 1:15-30,000 births. Cilia motility requires the assembly of multisubunit dynein arms that drive cilia bending2. Despite progress in understanding the genetic basis of PCD, mutations remain to be identified for several PCD linked loci3. Here we show that the zebrafish cilia paralysis mutant schmalhanstn222 (smh) mutant encodes the coiled-coil domain containing 103 protein (Ccdc103), a foxj1a regulated gene. Screening 146 unrelated PCD families identified patients in six families with reduced outer dynein arms, carrying mutations in CCDC103. Dynein arm assembly in smh mutant zebrafish was rescued by wild-type but not mutant human CCDC103. Chlamydomonas Ccdc103 functions as a tightly bound, axoneme-associated protein. The results identify Ccdc103 as a novel dynein arm attachment factor that when mutated causes Primary Ciliary Dyskinesia. PMID:22581229

  17. Extracellular matrix components direct porcine muscle stem cell behavior

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

    Wilschut, Karlijn J.; Haagsman, Henk P.; Roelen, Bernard A.J., E-mail: b.a.j.roelen@uu.nl

    2010-02-01

    In muscle tissue, extracellular matrix proteins, together with the vasculature system, muscle-residence cells and muscle fibers, create the niche for muscle stem cells. The niche is important in controlling proliferation and directing differentiation of muscle stem cells to sustain muscle tissue. Mimicking the extracellular muscle environment improves tools exploring the behavior of primary muscle cells. Optimizing cell culture conditions to maintain muscle commitment is important in stem cell-based studies concerning toxicology screening, ex vivo skeletal muscle tissue engineering and in the enhancement of clinical efficiency. We used the muscle extracellular matrix proteins collagen type I, fibronectin, laminin, and also gelatinmore » and Matrigel as surface coatings of tissue culture plastic to resemble the muscle extracellular matrix. Several important factors that determine myogenic commitment of the primary muscle cells were characterized by quantitative real-time RT-PCR and immunofluorescence. Adhesion of high PAX7 expressing satellite cells was improved if the cells were cultured on fibronectin or laminin coatings. Cells cultured on Matrigel and laminin coatings showed dominant integrin expression levels and exhibited an activated Wnt pathway. Under these conditions both stem cell proliferation and myogenic differentiation capacity were superior if compared to cells cultured on collagen type I, fibronectin and gelatin. In conclusion, Matrigel and laminin are the preferred coatings to sustain the proliferation and myogenic differentiation capacity of the primary porcine muscle stem cells, when cells are removed from their natural environment for in vitro culture.« less

  18. Catechins activate muscle stem cells by Myf5 induction and stimulate muscle regeneration.

    PubMed

    Kim, A Rum; Kim, Kyung Min; Byun, Mi Ran; Hwang, Jun-Ha; Park, Jung Il; Oh, Ho Taek; Kim, Hyo Kyeong; Jeong, Mi Gyeong; Hwang, Eun Sook; Hong, Jeong-Ho

    2017-07-22

    Muscle weakness is one of the most common symptoms in aged individuals and increases risk of mortality. Thus, maintenance of muscle mass is important for inhibiting aging. In this study, we investigated the effect of catechins, polyphenol compounds in green tea, on muscle regeneration. We found that (-)-epicatechin gallate (ECG) and (-)-epigallocatechin-3-gallate (EGCG) activate satellite cells by induction of Myf5 transcription factors. For satellite cell activation, Akt kinase was significantly induced after ECG treatment and ECG-induced satellite cell activation was blocked in the presence of Akt inhibitor. ECG also promotes myogenic differentiation through the induction of myogenic markers, including Myogenin and Muscle creatine kinase (MCK), in satellite and C2C12 myoblast cells. Finally, EGCG administration to mice significantly increased muscle fiber size for regeneration. Taken together, the results suggest that catechins stimulate muscle stem cell activation and differentiation for muscle regeneration. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Particulate matter in cigarette smoke increases ciliary axoneme beating through mechanical stimulation.

    PubMed

    Navarrette, Chelsea R; Sisson, Joseph H; Nance, Elizabeth; Allen-Gipson, Diane; Hanes, Justin; Wyatt, Todd A

    2012-06-01

    The lung's ability to trap and clear foreign particles via the mucociliary elevator is an important mechanism for protecting the lung against respirable irritants and microorganisms. Although cigarette smoke (CS) exposure and particulate inhalation are known to alter mucociliary clearance, little is known about how CS and nanoparticles (NPs) modify cilia beating at the cytoskeletal infrastructure, or axonemal, level. We used a cell-free model to introduce cigarette smoke extract (CSE) and NPs with variant size and surface chemistry to isolated axonemes and measured changes in ciliary motility. We hypothesized that CSE would alter cilia beating and that alterations in ciliary beat frequency (CBF) due to particulate matter would be size- and surface chemistry-dependent. Demembranated axonemes were isolated from ciliated bovine tracheas and exposed to adenosine triphosphate (ATP) to initiate motility. CBF was measured in response to 5% CSE, CSE filtrate, and carboxyl-modified (COOH), sulphate (SO(4))-modified (sulfonated), or PEG-coated polystyrene (PS) latex NPs ranging in size from 40 nm to 500 nm. CSE concentrations as low as 5% resulted in rapid, significant stimulation of CBF (p<0.05 vs. baseline control). Filtering CSE through a 0.2-μm filter attenuated this effect. Introduction of sulphate-modified PS beads ~300 nm in diameter resulted in a similar increase in CBF above baseline ATP levels. Uncharged, PEG-coated beads had no effect on CBF regardless of size. Similarly, COOH-coated particles less than 200 nm in diameter did not alter ciliary motility. However, COOH-coated PS particles larger than 300 nm increased CBF significantly and increased the number of motile points. These data show that NPs, including those found in CSE, mechanically stimulate axonemes in a size- and surface chemistry-dependent manner. Alterations in ciliary motility due to physicochemical properties of NPs may be important for inhalational lung injury and efficient drug delivery of

  20. Recent advances in primary ciliary dyskinesia genetics

    PubMed Central

    Kurkowiak, Małgorzata; Ziętkiewicz, Ewa; Witt, Michał

    2015-01-01

    Primary ciliary dyskinesia (PCD) is a rare genetically heterogeneous disorder caused by the abnormal structure and/or function of motile cilia. The PCD diagnosis is challenging and requires a well-described clinical phenotype combined with the identification of abnormalities in ciliary ultrastructure and/or beating pattern as well as the recognition of genetic cause of the disease. Regarding the pace of identification of PCD-related genes, a rapid acceleration during the last 2–3 years is notable. This is the result of new technologies, such as whole-exome sequencing, that have been recently applied in genetic research. To date, PCD-causative mutations in 29 genes are known and the number of causative genes is bound to rise. Even though the genetic causes of approximately one-third of PCD cases still remain to be found, the current knowledge can already be used to create new, accurate genetic tests for PCD that can accelerate the correct diagnosis and reduce the proportion of unexplained cases. This review aims to present the latest data on the relations between ciliary structure aberrations and their genetic basis. PMID:25351953

  1. [THE STRUCTURE OF LYMPHATIC CAPILLARIES OF THE CILIARY BODY OF THE HUMAN EYE].

    PubMed

    Borodin, Yu I; Bgatova, N P; Chernykh, V V; Trunov, A N; Pozhidayeva, A A; Konenkov, V I

    2015-01-01

    Using light microscopy, immunohistochemistry and electron microscopy, the structural organization of interstitial spaces and vessels of the ciliary body of the human eye (n = 5) were studied. The ciliary body was found to contain wide interstitial spaces--tissue clefts bound by collagen fibers and fibroblasts. Organ-specific lymphatic capillaries were also demonstrated in the ciliary body. According to the present findings and the lymphatic region concept, the first 2 elements of the lymphatic region of the eye were described: tissue clefts--prelymphatics and lymphatic capillaries of the ciliary body. The third element of the lymphatic region are the lymph nodes of the head and neck.

  2. Systematic discovery of novel ciliary genes through functional genomics in the zebrafish

    PubMed Central

    Choksi, Semil P.; Babu, Deepak; Lau, Doreen; Yu, Xianwen; Roy, Sudipto

    2014-01-01

    Cilia are microtubule-based hair-like organelles that play many important roles in development and physiology, and are implicated in a rapidly expanding spectrum of human diseases, collectively termed ciliopathies. Primary ciliary dyskinesia (PCD), one of the most prevalent of ciliopathies, arises from abnormalities in the differentiation or motility of the motile cilia. Despite their biomedical importance, a methodical functional screen for ciliary genes has not been carried out in any vertebrate at the organismal level. We sought to systematically discover novel motile cilia genes by identifying the genes induced by Foxj1, a winged-helix transcription factor that has an evolutionarily conserved role as the master regulator of motile cilia biogenesis. Unexpectedly, we find that the majority of the Foxj1-induced genes have not been associated with cilia before. To characterize these novel putative ciliary genes, we subjected 50 randomly selected candidates to a systematic functional phenotypic screen in zebrafish embryos. Remarkably, we find that over 60% are required for ciliary differentiation or function, whereas 30% of the proteins encoded by these genes localize to motile cilia. We also show that these genes regulate the proper differentiation and beating of motile cilia. This collection of Foxj1-induced genes will be invaluable for furthering our understanding of ciliary biology, and in the identification of new mutations underlying ciliary disorders in humans. PMID:25139857

  3. Functional heterogeneity of side population cells in skeletal muscle

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

    Uezumi, Akiyoshi; Ojima, Koichi; Fukada, So-ichiro

    2006-03-17

    Skeletal muscle regeneration has been exclusively attributed to myogenic precursors, satellite cells. A stem cell-rich fraction referred to as side population (SP) cells also resides in skeletal muscle, but its roles in muscle regeneration remain unclear. We found that muscle SP cells could be subdivided into three sub-fractions using CD31 and CD45 markers. The majority of SP cells in normal non-regenerating muscle expressed CD31 and had endothelial characteristics. However, CD31{sup -}CD45{sup -} SP cells, which are a minor subpopulation in normal muscle, actively proliferated upon muscle injury and expressed not only several regulatory genes for muscle regeneration but also somemore » mesenchymal lineage markers. CD31{sup -}CD45{sup -} SP cells showed the greatest myogenic potential among three SP sub-fractions, but indeed revealed mesenchymal potentials in vitro. These SP cells preferentially differentiated into myofibers after intramuscular transplantation in vivo. Our results revealed the heterogeneity of muscle SP cells and suggest that CD31{sup -}CD45{sup -} SP cells participate in muscle regeneration.« less

  4. Quantifying Ciliary Dynamics during Assembly Reveals Step-wise Waveform Maturation in Airway Cells.

    PubMed

    Oltean, Alina; Schaffer, Andrew J; Bayly, Philip V; Brody, Steven L

    2018-05-31

    Motile cilia are essential for clearance of particulates and pathogens from airways. For effective transport, ciliary motor proteins and axonemal structures interact to generate the rhythmic, propulsive bending, but the mechanisms that produce a dynamic waveform remain incompletely understood. Biomechanical measures of human cilia motion and their relationships to cilia assembly are needed to illuminate the biophysics of normal cilia function, and to quantify dysfunction in ciliopathies. To these ends, we analyzed cilia motion from high-speed video microscopy of ciliated cells sampled from human lung airways compared to primary-culture cells that undergo ciliogenesis in vitro. Quantitative assessment of waveform parameters showed variations in waveform shape between individual cilia; however, general trends in waveform parameters emerged, associated with progression of cilia length and stage of differentiation. When cilia emerged from cultured cells, beat frequency was initially elevated, then fell and remained stable as cilia lengthened. In contrast, the average bending amplitude and the ability to generate force gradually increased and eventually approached values observed in ex vivo samples. Dynein arm motor proteins DNAH5, DNAH9, DNAH11, and DNAH6 were localized within specific regions of the axoneme in the ex vivo cells; however distinct stages of in vitro waveform development identified by biomechanical features were associated with the progressive movement of dyneins to the appropriate proximal or distal sections of the cilium. These observations suggest that the step-wise variation in waveform development during ciliogenesis is dependent on cilia length and potentially outer dynein arm assembly.

  5. Use of a Novel Cell Adhesion Method and Digital Measurement to Show Stimulus-dependent Variation in Somatic and Oral Ciliary Beat Frequency in Paramecium

    PubMed Central

    Bell, Wade E.; Hallworth, Richard; Wyatt, Todd A.; Sisson, Joseph H.

    2015-01-01

    When Paramecium encounters positive stimuli, the membrane hyperpolarizes and ciliary beat frequency increases. We adapted an established immobilization protocol using a biological adhesive and a novel digital analysis system to quantify beat frequency in immobilized Paramecium. Cells showed low mortality and demonstrated beat frequencies consistent with previous studies. Chemoattractant molecules, reduction in external potassium, and posterior stimulation all increased somatic beat frequency. In all cases, the oral groove cilia maintained a higher beat frequency than mid-body cilia, but only oral cilia from cells stimulated with chemoattactants showed an increase from basal levels. PMID:25066640

  6. Defining the proteome of human iris, ciliary body, retinal pigment epithelium, and choroid.

    PubMed

    Zhang, Pingbo; Kirby, David; Dufresne, Craig; Chen, Yan; Turner, Randi; Ferri, Sara; Edward, Deepak P; Van Eyk, Jennifer E; Semba, Richard D

    2016-04-01

    The iris is a fine structure that controls the amount of light that enters the eye. The ciliary body controls the shape of the lens and produces aqueous humor. The retinal pigment epithelium and choroid (RPE/choroid) are essential in supporting the retina and absorbing light energy that enters the eye. Proteins were extracted from iris, ciliary body, and RPE/choroid tissues of eyes from five individuals and fractionated using SDS-PAGE. After in-gel digestion, peptides were analyzed using LC-MS/MS on an Orbitrap Elite mass spectrometer. In iris, ciliary body, and RPE/choroid, we identified 2959, 2867, and 2755 nonredundant proteins with peptide and protein false-positive rates of <0.1% and <1%, respectively. Forty-three unambiguous protein isoforms were identified in iris, ciliary body, and RPE/choroid. Four "missing proteins" were identified in ciliary body based on ≥2 proteotypic peptides. The mass spectrometric proteome database of the human iris, ciliary body, and RPE/choroid may serve as a valuable resource for future investigations of the eye in health and disease. The MS proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifiers PXD001424 and PXD002194. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. The role of molecular genetic analysis in the diagnosis of primary ciliary dyskinesia.

    PubMed

    Kim, Raymond H; A Hall, David; Cutz, Ernest; Knowles, Michael R; Nelligan, Kathleen A; Nykamp, Keith; Zariwala, Maimoona A; Dell, Sharon D

    2014-03-01

    Primary ciliary dyskinesia (PCD) is an autosomal recessive genetic disorder of motile cilia. The diagnosis of PCD has previously relied on ciliary analysis with transmission electron microscopy or video microscopy. However, patients with PCD may have normal ultrastructural appearance, and ciliary analysis has limited accessibility. Alternatively, PCD can be diagnosed by demonstrating biallelic mutations in known PCD genes. Genetic testing is emerging as a diagnostic tool to complement ciliary analysis where interpretation and access may delay diagnosis. To determine the diagnostic yield of genetic testing of patients with a confirmed or suspected diagnosis of PCD in a multiethnic urban center. Twenty-eight individuals with confirmed PCD on transmission electron microscopy of ciliary ultrastructure and 24 individuals with a probable diagnosis of PCD based on a classical PCD phenotype and low nasal nitric oxide had molecular analysis of 12 genes associated with PCD. Of 49 subjects who underwent ciliary biopsy, 28 (57%) were diagnosed with PCD through an ultrastructural defect. Of the 52 individuals who underwent molecular genetic analysis, 22 (42%) individuals had two mutations in known PCD genes. Twenty-four previously unreported mutations in known PCD genes were observed. Combining both diagnostic modalities of biopsy and molecular genetics, the diagnostic yield increased to 69% compared with 57% based on biopsy alone. The diagnosis of PCD is challenging and has traditionally relied on ciliary biopsy, which is unreliable as the sole criterion for a definitive diagnosis. Molecular genetic analysis can be used as a complementary test to increase the diagnostic yield.

  8. Neuroprotective properties of ciliary neurotrophic factor on retinoic acid (RA)-predifferentiated SH-SY5Y neuroblastoma cells.

    PubMed

    Wang, Ke; Zhou, Fanfan; Zhu, Xue; Zhang, Kai; Huang, Biao; Zhu, Lan; Zhu, Ling

    2014-01-01

    Ciliary neurotrophic factor (CNTF) is a neurocytokine, which could promote survival and/or differentiation in many cell types. In this study, the biological effects of CNTF on retinoic acid (RA)-predifferentiated SH-SY5Y neuroblastoma cells and the underlying molecular mechanism of this effect were investigated for the first time. The results showed that RA was able to increase cells susceptibility to CNTF via regulating the expression levels of CNTF receptors. A further study revealed that CNTF could induce phosphorylation of STAT3, Akt and ERK1/2 in RA-predifferentiated SH-SY5Y neuroblastoma cells, while the promoting activity of CNTF on survival and neurite growth of cells was attenuated by co-treatment with JAK2 inhibitor AG490 (25 μM), STAT3 inhibitor Curcumin (50 μM), PI3K inhibitor LY-294002 (50 µM), but not by co-treatment with MEK inhibitor PD98059 (50 μM). These findings suggested that JAK2/STAT3, as well as PI3K/Akt, play important roles in mediating the survival and neurite growth response of RA-predifferentiated cells to CNTF. Our study may be useful to further understand the functional role of CNTF and offer a convenient model to explore the therapeutic potential of CNTF in neurodegenerative diseases.

  9. Primary ciliary dyskinesia: improving the diagnostic approach

    PubMed Central

    Leigh, Margaret W.; Zariwala, Maimoona A.; Knowles, Michael R.

    2009-01-01

    Purpose of review The diagnosis of primary ciliary dyskinesia (PCD) has relied on analysis of ciliary motility and ultrastructure; however, these tests are not readily available and have not been standardized. Consequently, the diagnosis of PCD may be delayed or missed or made incorrectly. This review outlines the potential utility of new diagnostic tests, including measurement of nasal nitric oxide (NO) production and systematic analysis for mutations in gene encoding ciliary proteins. Recent findings Clinical manifestations of PCD have been expanded to include neonatal respiratory distress and heterotaxy. Measurement of nasal NO has emerged as a useful screening test for PCD based on the very low levels in PCD (approximately 1/10 of normal values). Genetic testing is emerging for PCD and demonstrates extensive genetic heterogeneity. Some genes and gene mutations involved in PCD have been defined. Approximately one third of PCD cases have identifiable gene mutations in one of 6 different genes. An international effort is focused on defining PCD-causing defects in other genes. Summary The incorporation of nasal NO measurement as a screening test to define probable PCD cases and gene mutation analysis to make a definitive diagnosis of PCD should enhance diagnostic evaluation of PCD. PMID:19300264

  10. Study of muscle cell dedifferentiation after skeletal muscle injury of mice with a Cre-Lox system.

    PubMed

    Mu, Xiaodong; Peng, Hairong; Pan, Haiying; Huard, Johnny; Li, Yong

    2011-02-03

    Dedifferentiation of muscle cells in the tissue of mammals has yet to be observed. One of the challenges facing the study of skeletal muscle cell dedifferentiation is the availability of a reliable model that can confidentially distinguish differentiated cell populations of myotubes and non-fused mononuclear cells, including stem cells that can coexist within the population of cells being studied. In the current study, we created a Cre/Lox-β-galactosidase system, which can specifically tag differentiated multinuclear myotubes and myotube-generated mononuclear cells based on the activation of the marker gene, β-galactosidase. By using this system in an adult mouse model, we found that β-galactosidase positive mononuclear cells were generated from β-galactosidase positive multinuclear myofibers upon muscle injury. We also demonstrated that these mononuclear cells can develop into a variety of different muscle cell lineages, i.e., myoblasts, satellite cells, and muscle derived stem cells. These novel findings demonstrated, for the first time, that cellular dedifferentiation of skeletal muscle cells actually occurs in mammalian skeletal muscle following traumatic injury in vivo.

  11. Tissue-specific stem cells: Lessons from the skeletal muscle satellite cell

    PubMed Central

    Brack, Andrew S.; Rando, Thomas A.

    2012-01-01

    In 1961, the satellite cell was first identified when electron microscopic examination of skeletal muscle demonstrated a cell wedged between the plasma membrane of the muscle fiber and the basement membrane. In recent years it has been conclusively demonstrated that the satellite cell is the primary cellular source for muscle regeneration and is equipped with the potential to self renew, thus functioning as a bone fide skeletal muscle stem cell (MuSC). As we move past the 50th anniversary of the satellite cell, we take this opportunity to discuss the current state of the art and dissect the unknowns in the MuSC field. PMID:22560074

  12. Ciliary body toxicities of systemic oxcarbazepine and valproic acid treatments: electron microscopic study.

    PubMed

    Göktaş, Güleser; Aktaş, Zeynep; Erdoğan, Deniz; Seymen, Cemile Merve; Karaca, Emine Esra; Cansu, Ali; Serdaroğlu, Ayşe; Kaplanoğlu, Gülnur Take

    2015-01-01

    Ciliary body is responsible for humour aqueous production in posterior chamber. Valproic acid (VPA) has been widely used for the treatment of epilepsy and other neuropsychiatric diseases such as bipolar disease and major depression. Oxcarbazepine (OXC) is a new anti-epileptic agent that has been used recently for childhood epilepsies such as VPA. In this study, we aimed to investigate the effects of VPA and OXC treatments used as antiepileptic in ciliary body by electron microscopy. In our study, 40 Wistar rats (21 days old) were divided equally into four groups which were applied saline (group 1), VPA (group 2), OXC (group 3) and VPA + OXC (group 4). The as-prepared ocular tissues were characterized by transmission electron microscopy (TEM) technique in scanning and transmission electron microscopy (SEM-TEM) (Carl Zeiss EVO LS10). The results confirmed that VPA caused dense ciliary body degeneration. Additionally, ciliary body degeneration in group 4 was supposed to be due to VPA treatment. Ciliary body damage and secondary outcomes should be considered in patients with long-term VPA therapy.

  13. Transplantated mesenchymal stem cells derived from embryonic stem cells promote muscle regeneration and accelerate functional recovery of injured skeletal muscle.

    PubMed

    Ninagawa, Nana Takenaka; Isobe, Eri; Hirayama, Yuri; Murakami, Rumi; Komatsu, Kazumi; Nagai, Masataka; Kobayashi, Mami; Kawabata, Yuka; Torihashi, Shigeko

    2013-08-01

    We previously established that mesenchymal stem cells originating from mouse embryonic stem (ES) cells (E-MSCs) showed markedly higher potential for differentiation into skeletal muscles in vitro than common mesenchymal stem cells (MSCs). Further, the E-MSCs exhibited a low risk for teratoma formation. Here we evaluate the potential of E-MSCs for differentiation into skeletal muscles in vivo and reveal the regeneration and functional recovery of injured muscle by transplantation. E-MSCs were transplanted into the tibialis anterior (TA) muscle 24 h following direct clamping. After transplantation, the myogenic differentiation of E-MSCs, TA muscle regeneration, and re-innervation were morphologically analyzed. In addition, footprints and gaits of each leg under spontaneous walking were measured by CatWalk XT, and motor functions of injured TA muscles were precisely analyzed. Results indicate that >60% of transplanted E-MSCs differentiated into skeletal muscles. The cross-sectional area of the injured TA muscles of E-MSC-transplanted animals increased earlier than that of control animals. E-MSCs also promotes re-innervation of the peripheral nerves of injured muscles. Concerning function of the TA muscles, we reveal that transplantation of E-MSCs promotes the recovery of muscles. This is the first report to demonstrate by analysis of spontaneous walking that transplanted cells can accelerate the functional recovery of injured muscles. Taken together, the results show that E-MSCs have a high potential for differentiation into skeletal muscles in vivo as well as in vitro. The transplantation of E-MSCs facilitated the functional recovery of injured muscles. Therefore, E-MSCs are an efficient cell source in transplantation.

  14. Transplantated Mesenchymal Stem Cells Derived from Embryonic Stem Cells Promote Muscle Regeneration and Accelerate Functional Recovery of Injured Skeletal Muscle

    PubMed Central

    Ninagawa, Nana Takenaka; Isobe, Eri; Hirayama, Yuri; Murakami, Rumi; Komatsu, Kazumi; Nagai, Masataka; Kobayashi, Mami; Kawabata, Yuka

    2013-01-01

    Abstract We previously established that mesenchymal stem cells originating from mouse embryonic stem (ES) cells (E-MSCs) showed markedly higher potential for differentiation into skeletal muscles in vitro than common mesenchymal stem cells (MSCs). Further, the E-MSCs exhibited a low risk for teratoma formation. Here we evaluate the potential of E-MSCs for differentiation into skeletal muscles in vivo and reveal the regeneration and functional recovery of injured muscle by transplantation. E-MSCs were transplanted into the tibialis anterior (TA) muscle 24 h following direct clamping. After transplantation, the myogenic differentiation of E-MSCs, TA muscle regeneration, and re-innervation were morphologically analyzed. In addition, footprints and gaits of each leg under spontaneous walking were measured by CatWalk XT, and motor functions of injured TA muscles were precisely analyzed. Results indicate that >60% of transplanted E-MSCs differentiated into skeletal muscles. The cross-sectional area of the injured TA muscles of E-MSC–transplanted animals increased earlier than that of control animals. E-MSCs also promotes re-innervation of the peripheral nerves of injured muscles. Concerning function of the TA muscles, we reveal that transplantation of E-MSCs promotes the recovery of muscles. This is the first report to demonstrate by analysis of spontaneous walking that transplanted cells can accelerate the functional recovery of injured muscles. Taken together, the results show that E-MSCs have a high potential for differentiation into skeletal muscles in vivo as well as in vitro. The transplantation of E-MSCs facilitated the functional recovery of injured muscles. Therefore, E-MSCs are an efficient cell source in transplantation. PMID:23914336

  15. A solid-state control system for dynein-based ciliary/flagellar motility

    PubMed Central

    2013-01-01

    Ciliary and flagellar beating requires the coordinated action of multiple dyneins with different enzymatic and motor properties. In this issue, Yamamoto et al. (2013. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201211048) identify the MIA (modifier of inner arms) complex within the Chlamydomonas reinhardtii axoneme that physically links to a known regulatory structure and provides a signaling conduit from the radial spokes to an inner arm dynein essential for waveform determination. PMID:23569213

  16. 3D Cell Printing of Functional Skeletal Muscle Constructs Using Skeletal Muscle-Derived Bioink.

    PubMed

    Choi, Yeong-Jin; Kim, Taek Gyoung; Jeong, Jonghyeon; Yi, Hee-Gyeong; Park, Ji Won; Hwang, Woonbong; Cho, Dong-Woo

    2016-10-01

    Engineered skeletal muscle tissues that mimic the structure and function of native muscle have been considered as an alternative strategy for the treatment of various muscular diseases and injuries. Here, it is demonstrated that 3D cell-printing of decellularized skeletal muscle extracellular matrix (mdECM)-based bioink facilitates the fabrication of functional skeletal muscle constructs. The cellular alignment and the shape of the tissue constructs are controlled by 3D cell-printing technology. mdECM bioink provides the 3D cell-printed muscle constructs with a myogenic environment that supports high viability and contractility as well as myotube formation, differentiation, and maturation. More interestingly, the preservation of agrin is confirmed in the mdECM, and significant increases in the formation of acetylcholine receptor clusters are exhibited in the 3D cell-printed muscle constructs. In conclusion, mdECM bioink and 3D cell-printing technology facilitate the mimicking of both the structural and functional properties of native muscle and hold great promise for producing clinically relevant engineered muscle for the treatment of muscular injuries. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Muscle satellite cells adopt divergent fates

    PubMed Central

    Zammit, Peter S.; Golding, Jon P.; Nagata, Yosuke; Hudon, Valérie; Partridge, Terence A.; Beauchamp, Jonathan R.

    2004-01-01

    Growth, repair, and regeneration of adult skeletal muscle depends on the persistence of satellite cells: muscle stem cells resident beneath the basal lamina that surrounds each myofiber. However, how the satellite cell compartment is maintained is unclear. Here, we use cultured myofibers to model muscle regeneration and show that satellite cells adopt divergent fates. Quiescent satellite cells are synchronously activated to coexpress the transcription factors Pax7 and MyoD. Most then proliferate, down-regulate Pax7, and differentiate. In contrast, other proliferating cells maintain Pax7 but lose MyoD and withdraw from immediate differentiation. These cells are typically located in clusters, together with Pax7−ve progeny destined for differentiation. Some of the Pax7+ve/MyoD−ve cells then leave the cell cycle, thus regaining the quiescent satellite cell phenotype. Significantly, noncycling cells contained within a cluster can be stimulated to proliferate again. These observations suggest that satellite cells either differentiate or switch from terminal myogenesis to maintain the satellite cell pool. PMID:15277541

  18. Sonic hedgehog acts cell-autonomously on muscle precursor cells to generate limb muscle diversity

    PubMed Central

    Anderson, Claire; Williams, Victoria C.; Moyon, Benjamin; Daubas, Philippe; Tajbakhsh, Shahragim; Buckingham, Margaret E.; Shiroishi, Toshihiko; Hughes, Simon M.; Borycki, Anne-Gaëlle

    2012-01-01

    How muscle diversity is generated in the vertebrate body is poorly understood. In the limb, dorsal and ventral muscle masses constitute the first myogenic diversification, as each gives rise to distinct muscles. Myogenesis initiates after muscle precursor cells (MPCs) have migrated from the somites to the limb bud and populated the prospective muscle masses. Here, we show that Sonic hedgehog (Shh) from the zone of polarizing activity (ZPA) drives myogenesis specifically within the ventral muscle mass. Shh directly induces ventral MPCs to initiate Myf5 transcription and myogenesis through essential Gli-binding sites located in the Myf5 limb enhancer. In the absence of Shh signaling, myogenesis is delayed, MPCs fail to migrate distally, and ventral paw muscles fail to form. Thus, Shh production in the limb ZPA is essential for the spatiotemporal control of myogenesis and coordinates muscle and skeletal development by acting directly to regulate the formation of specific ventral muscles. PMID:22987640

  19. Molecular and functional identification of a novel photopigment in Pecten ciliary photoreceptors.

    PubMed

    Arenas, Oscar; Osorno, Tomás; Malagón, Gerardo; Pulido, Camila; Gomez, María Del Pilar; Nasi, Enrico

    2018-01-26

    The two basic animal photoreceptor types, ciliary and microvillar, use different light-transduction schemes: their photopigments couple to G t versus G q proteins, respectively, to either mobilize cyclic nucleotides or trigger a lipid signaling cascade. A third class of photoreceptors has been described in the dual retina of some marine invertebrates; these present a ciliary morphology but operate via radically divergent mechanisms, prompting the suggestion that they comprise a novel lineage of light sensors. In one of these organisms, an uncommon putative opsin was uncovered that was proposed to signal through G o Orthologues subsequently emerged in diverse phyla, including mollusks, echinoderms, and chordates, but the cells in which they express have not been identified, and no studies corroborated their function as visual pigments or their suggested signaling mode. Conversely, in only one invertebrate species, Pecten irradians , have the ciliary photoreceptors been physiologically characterized, but their photopigment has not been identified molecularly. We used the transcriptome of Pecten retina to guide the cloning by polymerase chain reaction (PCR) and rapid amplification of cDNA ends (RACE) extensions of a new member of this group of putative opsins. In situ hybridization shows selective transcription in the distal retina, and specific antibodies identify a single band of the expected molecular mass in Western blots and distinctly label ciliary photoreceptors in retina sections. RNA interference knockdown resulted in a reduction in the early receptor current-the first manifestation of light transduction-and prevented the prolonged aftercurrent, which requires a large buildup of activated rhodopsin. We also obtained a full-length clone of the α-subunit of a G o from Pecten retina complementary DNA and localized it by in situ hybridization to the distal photoreceptors. Small interfering RNA targeting this G o caused a specific depression of the photocurrent

  20. Brain and muscle Arnt-like 1 promotes skeletal muscle regeneration through satellite cell expansion

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

    Chatterjee, Somik; Yin, Hongshan; Department of Cardiovascular Medicine, Third Affiliated Hospital, Hebei Medical University, Shijiazhuang 050051, Hebei

    Circadian clock is an evolutionarily conserved timing mechanism governing diverse biological processes and the skeletal muscle possesses intrinsic functional clocks. Interestingly, although the essential clock transcription activator, Brain and muscle Arnt-like 1 (Bmal1), participates in maintenance of muscle mass, little is known regarding its role in muscle growth and repair. In this report, we investigate the in vivo function of Bmal1 in skeletal muscle regeneration using two muscle injury models. Bmal1 is highly up-regulated by cardiotoxin injury, and its genetic ablation significantly impairs regeneration with markedly suppressed new myofiber formation and attenuated myogenic induction. A similarly defective regenerative response ismore » observed in Bmal1-null mice as compared to wild-type controls upon freeze injury. Lack of satellite cell expansion accounts for the regeneration defect, as Bmal1{sup −/−} mice display significantly lower satellite cell number with nearly abolished induction of the satellite cell marker, Pax7. Furthermore, satellite cell-derived primary myoblasts devoid of Bmal1 display reduced growth and proliferation ex vivo. Collectively, our results demonstrate, for the first time, that Bmal1 is an integral component of the pro-myogenic response that is required for muscle repair. This mechanism may underlie its role in preserving adult muscle mass and could be targeted therapeutically to prevent muscle-wasting diseases. - Highlights: • Bmal1 is highly inducible by muscle injury and myogenic stimuli. • Genetic ablation of Bmal1 significantly impairs muscle regeneration. • Bmal1 promotes satellite cell expansion during muscle regeneration. • Bmal1-deficient primary myoblasts display attenuated growth and proliferation.« less

  1. Muscle-derived stem cells isolated as non-adherent population give rise to cardiac, skeletal muscle and neural lineages.

    PubMed

    Arsic, Nikola; Mamaeva, Daria; Lamb, Ned J; Fernandez, Anne

    2008-04-01

    Stem cells with the ability to differentiate in specialized cell types can be extracted from a wide array of adult tissues including skeletal muscle. Here we have analyzed a population of cells isolated from skeletal muscle on the basis of their poor adherence on uncoated or collagen-coated dishes that show multi-lineage differentiation in vitro. When analysed under proliferative conditions, these cells express stem cell surface markers Sca-1 (65%) and Bcrp-1 (80%) but also MyoD (15%), Neuronal beta III-tubulin (25%), GFAP (30%) or Nkx2.5 (1%). Although capable of growing as non-attached spheres for months, when given an appropriate matrix, these cells adhere giving rise to skeletal muscle, neuronal and cardiac muscle cell lineages. A similar cell population could not be isolated from either bone marrow or cardiac tissue suggesting their specificity to skeletal muscle. When injected into damaged muscle, these non-adherent muscle-derived cells are retrieved expressing Pax7, in a sublaminar position characterizing satellite cells and participate in forming new myofibers. These data show that a non-adherent stem cell population can be specifically isolated and expanded from skeletal muscle and upon attachment to a matrix spontaneously differentiate into muscle, cardiac and neuronal lineages in vitro. Although competing with resident satellite cells, these cells are shown to significantly contribute to repair of injured muscle in vivo supporting that a similar muscle-derived non-adherent cell population from human muscle may be useful in treatment of neuromuscular disorders.

  2. Effective fiber hypertrophy in satellite cell-depleted skeletal muscle

    PubMed Central

    McCarthy, John J.; Mula, Jyothi; Miyazaki, Mitsunori; Erfani, Rod; Garrison, Kelcye; Farooqui, Amreen B.; Srikuea, Ratchakrit; Lawson, Benjamin A.; Grimes, Barry; Keller, Charles; Van Zant, Gary; Campbell, Kenneth S.; Esser, Karyn A.; Dupont-Versteegden, Esther E.; Peterson, Charlotte A.

    2011-01-01

    An important unresolved question in skeletal muscle plasticity is whether satellite cells are necessary for muscle fiber hypertrophy. To address this issue, a novel mouse strain (Pax7-DTA) was created which enabled the conditional ablation of >90% of satellite cells in mature skeletal muscle following tamoxifen administration. To test the hypothesis that satellite cells are necessary for skeletal muscle hypertrophy, the plantaris muscle of adult Pax7-DTA mice was subjected to mechanical overload by surgical removal of the synergist muscle. Following two weeks of overload, satellite cell-depleted muscle showed the same increases in muscle mass (approximately twofold) and fiber cross-sectional area with hypertrophy as observed in the vehicle-treated group. The typical increase in myonuclei with hypertrophy was absent in satellite cell-depleted fibers, resulting in expansion of the myonuclear domain. Consistent with lack of nuclear addition to enlarged fibers, long-term BrdU labeling showed a significant reduction in the number of BrdU-positive myonuclei in satellite cell-depleted muscle compared with vehicle-treated muscle. Single fiber functional analyses showed no difference in specific force, Ca2+ sensitivity, rate of cross-bridge cycling and cooperativity between hypertrophied fibers from vehicle and tamoxifen-treated groups. Although a small component of the hypertrophic response, both fiber hyperplasia and regeneration were significantly blunted following satellite cell depletion, indicating a distinct requirement for satellite cells during these processes. These results provide convincing evidence that skeletal muscle fibers are capable of mounting a robust hypertrophic response to mechanical overload that is not dependent on satellite cells. PMID:21828094

  3. Isolation of skeletal muscle stem cells by fluorescence-activated cell sorting.

    PubMed

    Liu, Ling; Cheung, Tom H; Charville, Gregory W; Rando, Thomas A

    2015-10-01

    The prospective isolation of purified stem cell populations has dramatically altered the field of stem cell biology, and it has been a major focus of research across tissues in different organisms. Muscle stem cells (MuSCs) are now among the most intensely studied stem cell populations in mammalian systems, and the prospective isolation of these cells has allowed cellular and molecular characterizations that were not dreamed of a decade ago. In this protocol, we describe how to isolate MuSCs from limb muscles of adult mice by fluorescence-activated cell sorting (FACS). We provide a detailed description of the physical and enzymatic dissociation of mononucleated cells from limb muscles, a procedure that is essential in order to maximize cell yield. We also describe a FACS-based method that is used subsequently to obtain highly pure populations of either quiescent or activated MuSCs (VCAM(+)CD31(-)CD45(-)Sca1(-)). The isolation process takes ∼5-6 h to complete. The protocol also allows for the isolation of endothelial cells, hematopoietic cells and mesenchymal stem cells from muscle tissue.

  4. Rejuvenation of the aged muscle stem cell population restores strength to injured aged muscles

    PubMed Central

    Cosgrove, Benjamin D.; Gilbert, Penney M.; Porpiglia, Ermelinda; Mourkioti, Foteini; Lee, Steven P.; Corbel, Stephane Y.; Llewellyn, Michael E.; Delp, Scott L.; Blau, Helen M.

    2014-01-01

    The aged suffer from progressive muscle weakness and regenerative failure. We demonstrate that muscle regeneration is impaired with aging due in part to a cell-autonomous functional decline in skeletal muscle stem cells (MuSCs). Two-thirds of aged MuSCs are intrinsically defective relative to young MuSCs, with reduced capacity to repair myofibers and repopulate the stem cell reservoir in vivo following transplantation due to a higher incidence of cells that express senescence markers and that have elevated p38α/β MAPK activity. We show that these limitations cannot be overcome by transplantation into the microenvironment of young recipient muscles. In contrast, subjecting the aged MuSC population to transient inhibition of p38α/β in conjunction with culture on soft hydrogel substrates rapidly expands the residual functional aged MuSC population, rejuvenating its potential for regeneration, serial transplantation, and strengthening damaged muscles of aged mice. These findings reveal a synergy between biophysical and biochemical cues that provides a paradigm for a localized autologous muscle stem cell therapy in aged individuals. PMID:24531378

  5. Detection of Leishmania spp. and associated inflammation in ocular-associated smooth and striated muscles in dogs with patent leishmaniosis.

    PubMed

    Naranjo, Carolina; Fondevila, Dolors; Leiva, Marta; Roura, Xavier; Peña, Teresa

    2010-05-01

    Canine leishmaniosis is a disease characterized by the wide distribution of the parasite throughout the tissues of the host. The purpose of this study was to describe the presence of Leishmania spp. and associated inflammation in ocular-associated muscles of dogs with patent leishmaniosis. Smooth muscles (iris dilator muscle, iris sphincter muscle, ciliary muscle, Müller muscle, smooth muscle of the periorbita and smooth muscle of the nictitating membrane) and striated muscles (orbicularis oculi muscle, obliquus dorsalis muscle and dorsal rectus muscle) were evaluated. Routine staining with hematoxylin and eosin and immunohistochemistry to detect Leishmania spp. were performed on tissue sections. Granulomatous inflammation was seen surrounding muscular fibers and was composed mainly of macrophages with scattered lymphocytes and plasma cells. This infiltrate could be seen in 52/473 (10.99%) samples of smooth muscle and 36/142 (25.35%) samples of striated muscle. Parasites were detected in 43/473 (9.09%) samples of smooth muscle and in 28/142 (19.71%) samples of striated muscle. To the authors' knowledge, this is the first report assessing the presence of Leishmania spp. and associated infiltrate in intraocular, extraocular and adnexal smooth and striated muscles. The inflammation present in those muscles could contribute to clinical signs already described, such as blepharitis, uveitis, and orbital cellulitis.

  6. In vitro myotoxic effects of bupivacaine on rhabdomyosarcoma cells, immortalized and primary muscle cells.

    PubMed

    Metterlein, Thomas; Hoffmann, Petra; Späth, Ruth; Gruber, Michael; Graf, Bernhard M; Zink, Wolfgang

    2015-01-01

    Rhabdomyosarcoma is a rare malignant skeletal muscle tumor. It mainly occurs in children and young adults and has an unsatisfactory prognosis. Prior studies showed a direct myotoxic effect of bupivacaine on differentiated muscle cells in vitro and in vivo. Exact mechanisms of this myotoxicity are still not fully understood, but a myotoxic effect on malignant muscle tumor cells has not been examined so far. Thus, the aim of this study was to examine if bupivacaine has cytotoxic effects on rhabdomyosarcoma cells, immortalized muscle cells and differentiated muscle cells. Cell lines of rhabdomyosarcoma cells, immortalized muscle cells and differentiated muscle cells were established. After microscopic identification, cells were exposed to various concentrations of bupivacaine (500, 1,000, 1,750, 2,500 and 5,000 ppm) for 1 and 2 h, respectively. 24 and 28 h after incubation the cultures were stained with propidium iodid and analyzed by flow cytometry. The fraction of dead cells was calculated for each experiment and the concentration with 50% cell survival (IC50) was computed. Cell groups as well as incubation and recovery time were compared (ANOVA/Bonferroni p < 0.01). The total number of cultured cells was similar for the different local anesthetics and examined concentrations. Increasing concentrations of bupivacaine led to a decrease in survival of muscle cells. IC50 was highest for immortalized cells, followed by rhabdomyosarcoma cells and differentiated cells. Exposure time, but not recovery time, had an influence on survival. Bupivacaine has clear but different cytotoxic effects on various muscle cell types in vitro. Differentiated primary cells seem to be more vulnerable than tumor cells possibly because of more differentiated intracellular structures.

  7. Identification and functional characterization of muscle satellite cells in Drosophila

    PubMed Central

    Reichert, Heinrich

    2017-01-01

    Work on genetic model systems such as Drosophila and mouse has shown that the fundamental mechanisms of myogenesis are remarkably similar in vertebrates and invertebrates. Strikingly, however, satellite cells, the adult muscle stem cells that are essential for the regeneration of damaged muscles in vertebrates, have not been reported in invertebrates. In this study, we show that lineal descendants of muscle stem cells are present in adult muscle of Drosophila as small, unfused cells observed at the surface and in close proximity to the mature muscle fibers. Normally quiescent, following muscle fiber injury, we show that these cells express Zfh1 and engage in Notch-Delta-dependent proliferative activity and generate lineal descendant populations, which fuse with the injured muscle fiber. In view of strikingly similar morphological and functional features, we consider these novel cells to be the Drosophila equivalent of vertebrate muscle satellite cells. PMID:29072161

  8. Stem Cells for Skeletal Muscle Tissue Engineering.

    PubMed

    Pantelic, Molly N; Larkin, Lisa M

    2018-04-19

    Volumetric muscle loss (VML) is a debilitating condition wherein muscle loss overwhelms the body's normal physiological repair mechanism. VML is particularly common among military service members who have sustained war injuries. Because of the high social and medical cost associated with VML and suboptimal current surgical treatments, there is great interest in developing better VML therapies. Skeletal muscle tissue engineering (SMTE) is a promising alternative to traditional VML surgical treatments that use autogenic tissue grafts, and rather uses isolated stem cells with myogenic potential to generate de novo skeletal muscle tissues to treat VML. Satellite cells are the native precursors to skeletal muscle tissue, and are thus the most commonly studied starting source for SMTE. However, satellite cells are difficult to isolate and purify, and it is presently unknown whether they would be a practical source in clinical SMTE applications. Alternative myogenic stem cells, including adipose-derived stem cells, bone marrow-derived mesenchymal stem cells, perivascular stem cells, umbilical cord mesenchymal stem cells, induced pluripotent stem cells, and embryonic stem cells, each have myogenic potential and have been identified as possible starting sources for SMTE, although they have yet to be studied in detail for this purpose. These alternative stem cell varieties offer unique advantages and disadvantages that are worth exploring further to advance the SMTE field toward highly functional, safe, and practical VML treatments. The following review summarizes the current state of satellite cell-based SMTE, details the properties and practical advantages of alternative myogenic stem cells, and offers guidance to tissue engineers on how alternative myogenic stem cells can be incorporated into SMTE research.

  9. Bioenergetics mechanisms regulating muscle stem cell self-renewal commitment and function.

    PubMed

    Abreu, Phablo

    2018-04-16

    Muscle stem cells or satellite cells are crucial for muscle maintenance and repair. These cells are mitotically quiescent and uniformly express the transcription factor Pax7, intermittently entering the cell cycle to give rise to daughter myogenic precursors cells and fuse with neighboring myofibers or self-renew, replenishing the stem cell pool in adult skeletal muscle. Pivotal roles of muscle stem cells in muscle repair have been uncovered, but it still remains unclear how muscle stem cell self-renewal is molecularly regulated and how muscle stem cells maintain muscle tissue homeostasis. Defects in muscle stem cell regulation to maintain/return to quiescence and self-renew are observed in degenerative conditions such as aging and neuromuscular disease. Recent works has suggested the existence of metabolic regulation and mitochondrial alterations in muscle stem cells, influencing the self-renewal commitment and function. Here I present a brief overview of recent understanding of how metabolic reprogramming governs self-renewal commitment, which is essential for conservation of muscle satellite cell pools throughout life, as well as the implications for regenerative medicine. Copyright © 2018. Published by Elsevier Masson SAS.

  10. Skeletal muscle satellite cells cultured in simulated microgravity

    NASA Technical Reports Server (NTRS)

    Molnar, Greg; Hartzell, Charles R.; Schroedl, Nancy A.; Gonda, Steve R.

    1993-01-01

    Satellite cells are postnatal myoblasts responsible for providing additional nuclei to growing or regenerating muscle cells. Satellite cells retain the capacity to proliferate and differentiate in vitro and therefore provide a useful model to study postnatal muscle development. Most culture systems used to study postnatal muscle development are limited by the two-dimensional (2-D) confines of the culture dish. Limiting proliferation and differentiation of satellite cells in 2-D could potentially limit cell-cell contacts important for developing the level of organization in skeletal muscle obtained in vivo. Culturing satellite cells on microcarrier beads suspended in the High-Aspect-Ratio-Vessel (HARV) designed by NASA provides a low shear, three-dimensional (3-D) environment to study muscle development. Primary cultures established from anterior tibialis muscles of growing rats (approximately 200 gm) were used for all studies and were composed of greater than 75 % satellite cells. Different inoculation densities did not affect the proliferative potential of satellite cells in the HARV. Plating efficiency, proliferation, and glucose utilization were compared between 2-D flat culture and 3-D HARV culture. Plating efficiency (cells attached - cells plated x 100) was similar between the two culture systems. Proliferation was reduced in HARV cultures and this reduction was apparent for both satellite cells and non-satellite cells. Furthermore, reduction in proliferation within the HARV could not be attributed to reduced substrate availability since glucose levels in media from HARV and 2-D cell culture were similar. Morphologically, microcarrier beads within the HARVS were joined together by cells into three-dimensional aggregates composed of greater than 10 beads/aggregate. Aggregation of beads did not occur in the absence of cells. Myotubes were often seen on individual beads or spanning the surface of two beads. In summary, proliferation and differentiation of

  11. Development of the larval nervous system of the sand dollar, Dendraster excentricus.

    PubMed

    Burke, R D

    1983-01-01

    Transformation of the gastrula to the pluteus includes development of the ability of the larva to control the direction of ciliary beat and coordinate activities of the ciliary band with activities of the esophageal muscles (48-60 h, 15 degrees C). Glyoxylic acid-induced fluorescence shows several cells of the animal plate to contain catecholamines in the 36-h gastrula. As the ectoderm thickens to form the ciliary band (36 48 h), the catecholamine-containing cells increase in number and occur dispersed throughout the band. Tissues with the ultrastructural characteristics of nerves first became apparent associated with the ciliary band in 60-h larvae. The coincident development of coordinated behaviour and the appearance of cells with ultrastructural and histochemical characteristics of nerves suggests that the larval nervous system is derived at least in part from cells of the animal plate and develops in association with the ciliary bands.

  12. Use of a novel cell adhesion method and digital measurement to show stimulus-dependent variation in somatic and oral ciliary beat frequency in Paramecium.

    PubMed

    Bell, Wade E; Hallworth, Richard; Wyatt, Todd A; Sisson, Joseph H

    2015-01-01

    When Paramecium encounters positive stimuli, the membrane hyperpolarizes and ciliary beat frequency increases. We adapted an established immobilization protocol using a biological adhesive and a novel digital analysis system to quantify beat frequency in immobilized Paramecium. Cells showed low mortality and demonstrated beat frequencies consistent with previous studies. Chemoattractant molecules, reduction in external potassium, and posterior stimulation all increased somatic beat frequency. In all cases, the oral groove cilia maintained a higher beat frequency than mid-body cilia, but only oral cilia from cells stimulated with chemoattactants showed an increase from basal levels. © 2014 The Author(s) Journal of Eukaryotic Microbiology © 2014 International Society of Protistologists.

  13. PEDF-derived peptide promotes skeletal muscle regeneration through its mitogenic effect on muscle progenitor cells.

    PubMed

    Ho, Tsung-Chuan; Chiang, Yi-Pin; Chuang, Chih-Kuang; Chen, Show-Li; Hsieh, Jui-Wen; Lan, Yu-Wen; Tsao, Yeou-Ping

    2015-08-01

    In response injury, intrinsic repair mechanisms are activated in skeletal muscle to replace the damaged muscle fibers with new muscle fibers. The regeneration process starts with the proliferation of satellite cells to give rise to myoblasts, which subsequently differentiate terminally into myofibers. Here, we investigated the promotion effect of pigment epithelial-derived factor (PEDF) on muscle regeneration. We report that PEDF and a synthetic PEDF-derived short peptide (PSP; residues Ser(93)-Leu(112)) induce satellite cell proliferation in vitro and promote muscle regeneration in vivo. Extensively, soleus muscle necrosis was induced in rats by bupivacaine, and an injectable alginate gel was used to release the PSP in the injured muscle. PSP delivery was found to stimulate satellite cell proliferation in damaged muscle and enhance the growth of regenerating myofibers, with complete regeneration of normal muscle mass by 2 wk. In cell culture, PEDF/PSP stimulated C2C12 myoblast proliferation, together with a rise in cyclin D1 expression. PEDF induced the phosphorylation of ERK1/2, Akt, and STAT3 in C2C12 myoblasts. Blocking the activity of ERK, Akt, or STAT3 with pharmacological inhibitors attenuated the effects of PEDF/PSP on the induction of C2C12 cell proliferation and cyclin D1 expression. Moreover, 5-bromo-2'-deoxyuridine pulse-labeling demonstrated that PEDF/PSP stimulated primary rat satellite cell proliferation in myofibers in vitro. In summary, we report for the first time that PSP is capable of promoting the regeneration of skeletal muscle. The signaling mechanism involves the ERK, AKT, and STAT3 pathways. These results show the potential utility of this PEDF peptide for muscle regeneration. Copyright © 2015 the American Physiological Society.

  14. Monitoring Autophagy in Muscle Stem Cells.

    PubMed

    García-Prat, Laura; Muñoz-Cánoves, Pura; Martínez-Vicente, Marta

    2017-01-01

    Autophagy is critical not only for the cell's adaptive response to starvation but also for cellular homeostasis, by acting as quality-control machinery for cytoplasmic components. This basal autophagic activity is particularly needed in postmitotic cells for survival maintenance. Recently, basal autophagic activity was reported in skeletal muscle stem cells (satellite cells) in their dormant quiescent state. Satellite cells are responsible for growth as well as for regeneration of muscle in response to stresses such as injury or disease. In the absence of stress, quiescence is the stem cell state of these cells throughout life, although which mechanisms maintain long-life quiescence remains largely unknown. Our recent findings showed that autophagy is necessary for quiescence maintenance in satellite cells and for retention of their regenerative functions. Importantly, damaged organelles and proteins accumulated in these cells with aging and this was connected to age-associated defective autophagy. Refueling of autophagy through genetic and pharmacological strategies restored aged satellite cell functions, and these finding have biomedical implications. In this chapter, we describe different experimental strategies to evaluate autophagic activity in satellite cells in resting muscle of mice. They should facilitate our competence to investigate stem cell functions both during tissue homeostasis as in pathological conditions.

  15. European Respiratory Society guidelines for the diagnosis of primary ciliary dyskinesia.

    PubMed

    Lucas, Jane S; Barbato, Angelo; Collins, Samuel A; Goutaki, Myrofora; Behan, Laura; Caudri, Daan; Dell, Sharon; Eber, Ernst; Escudier, Estelle; Hirst, Robert A; Hogg, Claire; Jorissen, Mark; Latzin, Philipp; Legendre, Marie; Leigh, Margaret W; Midulla, Fabio; Nielsen, Kim G; Omran, Heymut; Papon, Jean-Francois; Pohunek, Petr; Redfern, Beatrice; Rigau, David; Rindlisbacher, Bernhard; Santamaria, Francesca; Shoemark, Amelia; Snijders, Deborah; Tonia, Thomy; Titieni, Andrea; Walker, Woolf T; Werner, Claudius; Bush, Andrew; Kuehni, Claudia E

    2017-01-01

    The diagnosis of primary ciliary dyskinesia is often confirmed with standard, albeit complex and expensive, tests. In many cases, however, the diagnosis remains difficult despite the array of sophisticated diagnostic tests. There is no "gold standard" reference test. Hence, a Task Force supported by the European Respiratory Society has developed this guideline to provide evidence-based recommendations on diagnostic testing, especially in light of new developments in such tests, and the need for robust diagnoses of patients who might enter randomised controlled trials of treatments. The guideline is based on pre-defined questions relevant for clinical care, a systematic review of the literature, and assessment of the evidence using the GRADE (Grading of Recommendations, Assessment, Development and Evaluation) approach. It focuses on clinical presentation, nasal nitric oxide, analysis of ciliary beat frequency and pattern by high-speed video-microscopy analysis, transmission electron microscopy, genotyping and immunofluorescence. It then used a modified Delphi survey to develop an algorithm for the use of diagnostic tests to definitively confirm and exclude the diagnosis of primary ciliary dyskinesia; and to provide advice when the diagnosis was not conclusive. Finally, this guideline proposes a set of quality criteria for future research on the validity of diagnostic methods for primary ciliary dyskinesia. Copyright ©ERS 2017.

  16. CD133+ cells derived from skeletal muscles of Duchenne muscular dystrophy patients have a compromised myogenic and muscle regenerative capability.

    PubMed

    Meng, Jinhong; Muntoni, Francesco; Morgan, Jennifer

    2018-05-12

    Cell-mediated gene therapy is a possible means to treat muscular dystrophies like Duchenne muscular dystrophy. Autologous patient stem cells can be genetically-corrected and transplanted back into the patient, without causing immunorejection problems. Regenerated muscle fibres derived from these cells will express the missing dystrophin protein, thus improving muscle function. CD133+ cells derived from normal human skeletal muscle contribute to regenerated muscle fibres and form muscle stem cells after their intra-muscular transplantation into an immunodeficient mouse model. But it is not known whether CD133+ cells derived from DMD patient muscles have compromised muscle regenerative function. To test this, we compared CD133+ cells derived from DMD and normal human muscles. DMD CD133+ cells had a reduced capacity to undergo myogenic differentiation in vitro compared with CD133+ cells derived from normal muscle. In contrast to CD133+ cells derived from normal human muscle, those derived from DMD muscle formed no satellite cells and gave rise to significantly fewer muscle fibres of donor origin, after their intra-muscular transplantation into an immunodeficient, non-dystrophic, mouse muscle. DMD CD133+ cells gave rise to more clones of smaller size and more clones that were less myogenic than did CD133+ cells derived from normal muscle. The heterogeneity of the progeny of CD133+ cells, combined with the reduced proliferation and myogenicity of DMD compared to normal CD133+ cells, may explain the reduced regenerative capacity of DMD CD133+ cells. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

  17. Development and characterization of a complete set of Triticum aestivum-Roegneria ciliaris disomic addition lines.

    PubMed

    Kong, Lingna; Song, Xinying; Xiao, Jin; Sun, Haojie; Dai, Keli; Lan, Caixia; Singh, Pawan; Yuan, Chunxia; Zhang, Shouzhong; Singh, Ravi; Wang, Haiyan; Wang, Xiue

    2018-05-31

    A complete set wheat-R. ciliaris disomic addition lines (DALs) were characterized and the homoeologous groups and genome affinities of R. ciliaris chromosomes were determined. Wild relatives are rich gene resources for cultivated wheat. The development of alien addition chromosome lines not only greatly broadens the genetic diversity, but also provides genetic stocks for comparative genomics studies. Roegneria ciliaris (genome S c S c Y c Y c ), a tetraploid wild relative of wheat, is tolerant or resistant to many abiotic and biotic stresses. To develop a complete set of wheat-R. ciliaris disomic addition lines (DALs), we undertook a euplasmic backcrossing program to overcome allocytoplasmic effects and preferential chromosome transmission. To improve the efficiency of identifying chromosomes from S c and Y c , we established techniques including sequential genomic in situ hybridization/fluorescence in situ hybridization (FISH) and molecular marker analysis. Fourteen DALs of wheat, each containing one pair of R. ciliaris chromosomes pairs, were characterized by FISH using four repetitive sequences [pTa794, pTa71, RcAfa and (GAA) 10 ] as probes. One hundred and sixty-two R. ciliaris-specific markers were developed. FISH and marker analysis enabled us to assign the homoeologous groups and genome affinities of R. ciliaris chromosomes. FHB resistance evaluation in successive five growth seasons showed that the amphiploid, DA2Y c , DA5Y c and DA6S c had improved FHB resistance, indicating their potential value in wheat improvement. The 14 DALs are likely new gene resources and will be phenotyped for more agronomic performances traits.

  18. A Special Population of Regulatory T Cells Potentiates Muscle Repair

    PubMed Central

    Burzyn, Dalia; Kuswanto, Wilson; Kolodin, Dmitriy; Shadrach, Jennifer L.; Cerletti, Massimiliano; Jang, Young; Sefik, Esen; Tan, Tze Guan; Wagers, Amy J.; Benoist, Christophe; Mathis, Diane

    2014-01-01

    SUMMARY Long recognized to be potent suppressors of immune responses, Foxp3+CD4+ regulatory T (Treg) cells are being rediscovered as regulators of nonimmunological processes. We describe a phenotypically and functionally distinct population of Treg cells that rapidly accumulated in the acutely injured skeletal muscle of mice, just as invading myeloidlineage cells switched from a proinflammatory to a proregenerative state. A Treg population of similar phenotype accumulated in muscles of genetically dystrophic mice. Punctual depletion of Treg cells during the repair process prolonged the proinflammatory infiltrate and impaired muscle repair, while treatments that increased or decreased Treg activities diminished or enhanced (respectively) muscle damage in a dystrophy model. Muscle Treg cells expressed the growth factor Amphiregulin, which acted directly on muscle satellite cells in vitro and improved muscle repair in vivo. Thus, Treg cells and their products may provide new therapeutic opportunities for wound repair and muscular dystrophies. PMID:24315098

  19. Beta-Adrenergic Receptor Expression in Muscle Cells

    NASA Technical Reports Server (NTRS)

    Young, Ronald B.; Bridge, K.; Vaughn, J. R.

    1999-01-01

    beta-adrenergic receptor (bAR) agonists presumably exert their physiological action on skeletal muscle cells through the bAR. Since the signal generated by the bAR is cyclic AMP (cAMP), experiments were initiated in primary chicken muscle cell cultures to determine if artificial elevation of intracellular cAMP by treatment with forskolin would alter the population of bAR expressed on the surface of muscle cells. Chicken skeletal muscle cells after 7 days in culture were employed for the experiments because muscle cells have attained a steady state with respect to muscle protein metabolism at this stage. Cells were treated with 0-10 uM forskolin for a total of three days. At the end of the 1, 2, and 3 day treatment intervals, the concentration of cAMP and the bAR population were measured. Receptor population was measured in intact muscle cell cultures as the difference between total binding of [H-3]CGP-12177 and non-specific binding of [H-3]CGP-12177 in the presence of 1 uM propranolol. Intracellular cAMP concentration was measured by radioimmunoassay. The concentration of cAMP in forskolin-treated cells increased up to 10-fold in a dose dependent manner. Increasing concentrations of forskolin also led to an increase in (beta)AR population, with a maximum increase of approximately 50% at 10 uM. This increase in (beta)AR population was apparent after only 1 day of treatment, and the pattern of increase was maintained for all 3 days of the treatment period. Thus, increasing the intracellular concentration of cAMP leads to up-regulation of (beta)AR population. Clenbuterol and isoproterenol gave similar effects on bAR population. The effect of forskolin on the quantity and apparent synthesis rate of the heavy chain of myosin (mhc) were also investigated. A maximum increase of 50% in the quantity of mhc was observed at 0.2 UM forskolin, but higher concentrations of forskolin reduced the quantity of mhc back to control levels.

  20. Calcium stone lithoptysis in promary ciliary dyskinesia

    EPA Science Inventory

    BACKGROUND: An association between lithoptysis and primary ciliary dyskinesia (PCD) has not been previously reported. However, reports of lithoptysis from 2 older patients (>60 yr) prompted a study of this association. METHODS: We performed a prospective study of all PCD patients...

  1. Electrical stimulation as a biomimicry tool for regulating muscle cell behavior

    PubMed Central

    Ahadian, Samad; Ostrovidov, Serge; Hosseini, Vahid; Kaji, Hirokazu; Ramalingam, Murugan; Bae, Hojae; Khademhosseini, Ali

    2013-01-01

    There is a growing need to understand muscle cell behaviors and to engineer muscle tissues to replace defective tissues in the body. Despite a long history of the clinical use of electric fields for muscle tissues in vivo, electrical stimulation (ES) has recently gained significant attention as a powerful tool for regulating muscle cell behaviors in vitro. ES aims to mimic the electrical environment of electroactive muscle cells (e.g., cardiac or skeletal muscle cells) by helping to regulate cell-cell and cell-extracellular matrix (ECM) interactions. As a result, it can be used to enhance the alignment and differentiation of skeletal or cardiac muscle cells and to aid in engineering of functional muscle tissues. Additionally, ES can be used to control and monitor force generation and electrophysiological activity of muscle tissues for bio-actuation and drug-screening applications in a simple, high-throughput, and reproducible manner. In this review paper, we briefly describe the importance of ES in regulating muscle cell behaviors in vitro, as well as the major challenges and prospective potential associated with ES in the context of muscle tissue engineering. PMID:23823664

  2. Electrical stimulation as a biomimicry tool for regulating muscle cell behavior.

    PubMed

    Ahadian, Samad; Ostrovidov, Serge; Hosseini, Vahid; Kaji, Hirokazu; Ramalingam, Murugan; Bae, Hojae; Khademhosseini, Ali

    2013-01-01

    There is a growing need to understand muscle cell behaviors and to engineer muscle tissues to replace defective tissues in the body. Despite a long history of the clinical use of electric fields for muscle tissues in vivo, electrical stimulation (ES) has recently gained significant attention as a powerful tool for regulating muscle cell behaviors in vitro. ES aims to mimic the electrical environment of electroactive muscle cells (e.g., cardiac or skeletal muscle cells) by helping to regulate cell-cell and cell-extracellular matrix (ECM) interactions. As a result, it can be used to enhance the alignment and differentiation of skeletal or cardiac muscle cells and to aid in engineering of functional muscle tissues. Additionally, ES can be used to control and monitor force generation and electrophysiological activity of muscle tissues for bio-actuation and drug-screening applications in a simple, high-throughput, and reproducible manner. In this review paper, we briefly describe the importance of ES in regulating muscle cell behaviors in vitro, as well as the major challenges and prospective potential associated with ES in the context of muscle tissue engineering.

  3. Comparative anatomy of the ciliary body of the West Indian manatee (Trichechus manatus) and selected species.

    PubMed

    Natiello, Michelle; Lewis, Patricia; Samuelson, Don

    2005-01-01

    To examine the anatomy of the ciliary body in the West Indian manatee (Trichechus manatus), paying close attention to its vascularization and to compare to those of its distant relative, the African elephant (Loxodonta africana), the amphibious hippopotamus (Hippopotamus amphibius) and the aquatic short-finned pilot whale (Globicephala macrorhynchus). Specimens from each species were preserved in 10% buffered formalin, and observed stereomicroscopically before being embedded in paraffin, sectioned and stained by Masson trichrome, hematoxylin and eosin, and periodic acid-Schiff for light microscopic evaluation. The network of blood vessels in the ciliary processes of the West Indian manatee appear to have an intricate pattern, especially with regard to venous outflow. Those of the elephant are slightly less complex, while those of the hippopotamus and whale have different vascular patterns within the ciliary body. Musculature within the ciliary body is absent within the manatee and pilot whale. In general, there appears to be a direct relationship between the increased development of vasculature and the loss of musculature within the ciliary bodies of the aquatic and amphibious mammals presently studied. Specifically, the ciliary body of the West Indian manatee has a comparatively unique construction, especially with regard to its vasculature.

  4. Primary Ciliary Dyskinesia.

    PubMed

    Knowles, Michael R; Zariwala, Maimoona; Leigh, Margaret

    2016-09-01

    Primary ciliary dyskinesia (PCD) is a recessive genetically heterogeneous disorder of motile cilia with chronic otosinopulmonary disease and organ laterality defects in ∼50% of cases. The prevalence of PCD is difficult to determine. Recent diagnostic advances through measurement of nasal nitric oxide and genetic testing has allowed rigorous diagnoses and determination of a robust clinical phenotype, which includes neonatal respiratory distress, daily nasal congestion, and wet cough starting early in life, along with organ laterality defects. There is early onset of lung disease in PCD with abnormal airflow mechanics and radiographic abnormalities detected in infancy and early childhood. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. Proton irradiation of malignant melanoma of the ciliary body.

    PubMed Central

    Gragoudas, E S; Goitein, M; Koehler, A; Wagner, M S; Verhey, L; Tepper, J; Suit, H D; Schneider, R J; Johnson, K N

    1979-01-01

    This is our first case of malignant melanoma of the ciliary body treated with proton beam irradiation, a technique that we developed for irradiating choroidal melanomas. After 21 months of follow-up no growth of the tumour has been observed, and shrinkage of the tumour was noted on the follow-up photographs and by ultrasonography. The 32P uptake test, which was positive before treatment, turned negative 14 months after irradiation. The described technique of proton beam irradiation might offer an alternative for the treatment of ciliary body melanomas when the present techniques of iridocyclectomy cannot be applied because of the size of the lesion. Images PMID:106873

  6. Cellular dynamics in the muscle satellite cell niche

    PubMed Central

    Bentzinger, C Florian; Wang, Yu Xin; Dumont, Nicolas A; Rudnicki, Michael A

    2013-01-01

    Satellite cells, the quintessential skeletal muscle stem cells, reside in a specialized local environment whose anatomy changes dynamically during tissue regeneration. The plasticity of this niche is attributable to regulation by the stem cells themselves and to a multitude of functionally diverse cell types. In particular, immune cells, fibrogenic cells, vessel-associated cells and committed and differentiated cells of the myogenic lineage have emerged as important constituents of the satellite cell niche. Here, we discuss the cellular dynamics during muscle regeneration and how disease can lead to perturbation of these mechanisms. To define the role of cellular components in the muscle stem cell niche is imperative for the development of cell-based therapies, as well as to better understand the pathobiology of degenerative conditions of the skeletal musculature. PMID:24232182

  7. Application of cell co-culture system to study fat and muscle cells.

    PubMed

    Pandurangan, Muthuraman; Hwang, Inho

    2014-09-01

    Animal cell culture is a highly complex process, in which cells are grown under specific conditions. The growth and development of these cells is a highly unnatural process in vitro condition. Cells are removed from animal tissues and artificially cultured in various culture vessels. Vitamins, minerals, and serum growth factors are supplied to maintain cell viability. Obtaining result homogeneity of in vitro and in vivo experiments is rare, because their structure and function are different. Living tissues have highly ordered complex architecture and are three-dimensional (3D) in structure. The interaction between adjacent cell types is quite distinct from the in vitro cell culture, which is usually two-dimensional (2D). Co-culture systems are studied to analyze the interactions between the two different cell types. The muscle and fat co-culture system is useful in addressing several questions related to muscle modeling, muscle degeneration, apoptosis, and muscle regeneration. Co-culture of C2C12 and 3T3-L1 cells could be a useful diagnostic tool to understand the muscle and fat formation in animals. Even though, co-culture systems have certain limitations, they provide a more realistic 3D view and information than the individual cell culture system. It is suggested that co-culture systems are useful in evaluating the intercellular communication and composition of two different cell types.

  8. Molecular basis for CPAP-tubulin interaction in controlling centriolar and ciliary length

    PubMed Central

    Zheng, Xiangdong; Ramani, Anand; Soni, Komal; Gottardo, Marco; Zheng, Shuangping; Ming Gooi, Li; Li, Wenjing; Feng, Shan; Mariappan, Aruljothi; Wason, Arpit; Widlund, Per; Pozniakovsky, Andrei; Poser, Ina; Deng, Haiteng; Ou, Guangshuo; Riparbelli, Maria; Giuliano, Callaini; Hyman, Anthony A.; Sattler, Michael; Gopalakrishnan, Jay; Li, Haitao

    2016-01-01

    Centrioles and cilia are microtubule-based structures, whose precise formation requires controlled cytoplasmic tubulin incorporation. How cytoplasmic tubulin is recognized for centriolar/ciliary-microtubule construction remains poorly understood. Centrosomal-P4.1-associated-protein (CPAP) binds tubulin via its PN2-3 domain. Here, we show that a C-terminal loop-helix in PN2-3 targets β-tubulin at the microtubule outer surface, while an N-terminal helical motif caps microtubule's α-β surface of β-tubulin. Through this, PN2-3 forms a high-affinity complex with GTP-tubulin, crucial for defining numbers and lengths of centriolar/ciliary-microtubules. Surprisingly, two distinct mutations in PN2-3 exhibit opposite effects on centriolar/ciliary-microtubule lengths. CPAPF375A, with strongly reduced tubulin interaction, causes shorter centrioles and cilia exhibiting doublet- instead of triplet-microtubules. CPAPEE343RR that unmasks the β-tubulin polymerization surface displays slightly reduced tubulin-binding affinity inducing over-elongation of newly forming centriolar/ciliary-microtubules by enhanced dynamic release of its bound tubulin. Thus CPAP regulates delivery of its bound-tubulin to define the size of microtubule-based cellular structures using a ‘clutch-like' mechanism. PMID:27306797

  9. Trichinella spiralis: nurse cell formation with emphasis on analogy to muscle cell repair

    PubMed Central

    Wu, Zhiliang; Sofronic-Milosavljevic, Lj; Nagano, Isao; Takahashi, Yuzo

    2008-01-01

    Trichinella infection results in formation of a capsule in infected muscles. The capsule is a residence of the parasite which is composed of the nurse cell and fibrous wall. The process of nurse cell formation is complex and includes infected muscle cell response (de-differentiation, cell cycle re-entry and arrest) and satellite cell responses (activation, proliferation and differentiation). Some events that occur during the nurse cell formation are analogous to those occurring during muscle cell regeneration/repair. This article reviews capsule formation with emphasis on this analogy. PMID:18710582

  10. Comparison of tight junction protein expression in the ciliary epithelia of mouse, rabbit, cat and human eyes.

    PubMed

    Karim, M J; Biswas, S; Bhattacherjee, P; Paterson, C A

    2011-06-01

    Tight junctions in the nonpigmented epithelium (NPE) of the ciliary processes and the iris vascular endothelium form the ocular blood aqueous barrier that prevents leakage of proteins, immune cells and non-immune cells of blood into the anterior chamber. We attempted to determine whether ultrastructural differences in tight junctions reported in earlier studies are reflected in the expression pattern of tight junction proteins (TJP) and whether the TJP in mice, rabbits and cats resemble those of humans. For immunohistochemistry, 10 μm thick cryosections were rehydrated in PBS and fixed in 50 mM ammonium chloride at room temperature. After rinses in PBS, the sections were incubated twice in 0.1% Triton X-100, 10% goat serum, specific primary antibody or in PBS. After rinses in PBS, the sections were incubated in FITC-conjugated secondary antibody. After rinses in PBS, the sections were mounted with Vectashield mounting medium with propidium iodide, examined and photographed using a confocal microscope. The expression patterns of TJP in ocular ciliary epithelium of human, rabbit, cat and mouse were similar. Occludin immunoreactivity was observed as a sharp line along the junction between pigmented epithelium (PE) and NPE, and along the apico-lateral surfaces of NPE. Very light staining of the ciliary stroma was observed in cat and mouse. Claudin-1 was expressed along the entire boundaries of NPE and was more distinct between PE and NPE in rabbit. The ciliary stroma showed faint staining in cat and mouse. ZO-1 showed staining between PE and NPE, and at the adjacent membrane. Moderate staining was seen in PE in cat and mouse, which suggests that claudin-1, occludin and ZO-1 are expressed along the junction between PE and NPE, and the apico-lateral border of NPE. Lack of major difference in the expression patterns among the different species is important for validating the use of rabbit, mouse and cat in studies of intraocular inflammation in humans.

  11. Apoptosis in capillary endothelial cells in ageing skeletal muscle

    PubMed Central

    Wang, Huijuan; Listrat, Anne; Meunier, Bruno; Gueugneau, Marine; Coudy-Gandilhon, Cécile; Combaret, Lydie; Taillandier, Daniel; Polge, Cécile; Attaix, Didier; Lethias, Claire; Lee, Kijoon; Goh, Kheng Lim; Béchet, Daniel

    2014-01-01

    The age-related loss of skeletal muscle mass and function (sarcopenia) is a consistent hallmark of ageing. Apoptosis plays an important role in muscle atrophy, and the intent of this study was to specify whether apoptosis is restricted to myofibre nuclei (myonuclei) or occurs in satellite cells or stromal cells of extracellular matrix (ECM). Sarcopenia in mouse gastrocnemius muscle was characterized by myofibre atrophy, oxidative type grouping, delocalization of myonuclei and ECM fibrosis. Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL) indicated a sharp rise in apoptosis during ageing. TUNEL coupled with immunostaining for dystrophin, paired box protein-7 (Pax7) or laminin-2α, respectively, was used to identify apoptosis in myonuclei, satellite cells and stromal cells. In adult muscle, apoptosis was not detected in myofibres, but was restricted to stromal cells. Moreover, the age-related rise in apoptotic nuclei was essentially due to stromal cells. Myofibre-associated apoptosis nevertheless occurred in old muscle, but represented < 20% of the total muscle apoptosis. Specifically, apoptosis in old muscle affected a small proportion (0.8%) of the myonuclei, but a large part (46%) of the Pax7+ satellite cells. TUNEL coupled with CD31 immunostaining further attributed stromal apoptosis to capillary endothelial cells. Age-dependent rise in apoptotic capillary endothelial cells was concomitant with altered levels of key angiogenic regulators, perlecan and a perlecan domain V (endorepellin) proteolytic product. Collectively, our results indicate that sarcopenia is associated with apoptosis of satellite cells and impairment of capillary functions, which is likely to contribute to the decline in muscle mass and functionality during ageing. PMID:24245531

  12. Skeletal muscle cells express ICAM-1 after muscle overload and ICAM-1 contributes to the ensuing hypertrophic response.

    PubMed

    Dearth, Christopher L; Goh, Qingnian; Marino, Joseph S; Cicinelli, Peter A; Torres-Palsa, Maria J; Pierre, Philippe; Worth, Randall G; Pizza, Francis X

    2013-01-01

    We previously reported that leukocyte specific β2 integrins contribute to hypertrophy after muscle overload in mice. Because intercellular adhesion molecule-1 (ICAM-1) is an important ligand for β2 integrins, we examined ICAM-1 expression by murine skeletal muscle cells after muscle overload and its contribution to the ensuing hypertrophic response. Myofibers in control muscles of wild type mice and cultures of skeletal muscle cells (primary and C2C12) did not express ICAM-1. Overload of wild type plantaris muscles caused myofibers and satellite cells/myoblasts to express ICAM-1. Increased expression of ICAM-1 after muscle overload occurred via a β2 integrin independent mechanism as indicated by similar gene and protein expression of ICAM-1 between wild type and β2 integrin deficient (CD18-/-) mice. ICAM-1 contributed to muscle hypertrophy as demonstrated by greater (p<0.05) overload-induced elevations in muscle protein synthesis, mass, total protein, and myofiber size in wild type compared to ICAM-1-/- mice. Furthermore, expression of ICAM-1 altered (p<0.05) the temporal pattern of Pax7 expression, a marker of satellite cells/myoblasts, and regenerating myofiber formation in overloaded muscles. In conclusion, ICAM-1 expression by myofibers and satellite cells/myoblasts after muscle overload could serve as a mechanism by which ICAM-1 promotes hypertrophy by providing a means for cell-to-cell communication with β2 integrin expressing myeloid cells.

  13. Skeletal Muscle Cells Express ICAM-1 after Muscle Overload and ICAM-1 Contributes to the Ensuing Hypertrophic Response

    PubMed Central

    Dearth, Christopher L.; Goh, Qingnian; Marino, Joseph S.; Cicinelli, Peter A.; Torres-Palsa, Maria J.; Pierre, Philippe; Worth, Randall G.; Pizza, Francis X.

    2013-01-01

    We previously reported that leukocyte specific β2 integrins contribute to hypertrophy after muscle overload in mice. Because intercellular adhesion molecule-1 (ICAM-1) is an important ligand for β2 integrins, we examined ICAM-1 expression by murine skeletal muscle cells after muscle overload and its contribution to the ensuing hypertrophic response. Myofibers in control muscles of wild type mice and cultures of skeletal muscle cells (primary and C2C12) did not express ICAM-1. Overload of wild type plantaris muscles caused myofibers and satellite cells/myoblasts to express ICAM-1. Increased expression of ICAM-1 after muscle overload occurred via a β2 integrin independent mechanism as indicated by similar gene and protein expression of ICAM-1 between wild type and β2 integrin deficient (CD18-/-) mice. ICAM-1 contributed to muscle hypertrophy as demonstrated by greater (p<0.05) overload-induced elevations in muscle protein synthesis, mass, total protein, and myofiber size in wild type compared to ICAM-1-/- mice. Furthermore, expression of ICAM-1 altered (p<0.05) the temporal pattern of Pax7 expression, a marker of satellite cells/myoblasts, and regenerating myofiber formation in overloaded muscles. In conclusion, ICAM-1 expression by myofibers and satellite cells/myoblasts after muscle overload could serve as a mechanism by which ICAM-1 promotes hypertrophy by providing a means for cell-to-cell communication with β2 integrin expressing myeloid cells. PMID:23505517

  14. Isolation, characterization, and molecular regulation of muscle stem cells

    PubMed Central

    Fukada, So-ichiro; Ma, Yuran; Ohtani, Takuji; Watanabe, Yoko; Murakami, Satoshi; Yamaguchi, Masahiko

    2013-01-01

    Skeletal muscle has great regenerative capacity which is dependent on muscle stem cells, also known as satellite cells. A loss of satellite cells and/or their function impairs skeletal muscle regeneration and leads to a loss of skeletal muscle power; therefore, the molecular mechanisms for maintaining satellite cells in a quiescent and undifferentiated state are of great interest in skeletal muscle biology. Many studies have demonstrated proteins expressed by satellite cells, including Pax7, M-cadherin, Cxcr4, syndecan3/4, and c-met. To further characterize satellite cells, we established a method to directly isolate satellite cells using a monoclonal antibody, SM/C-2.6. Using SM/C-2.6 and microarrays, we measured the genes expressed in quiescent satellite cells and demonstrated that Hesr3 may complement Hesr1 in generating quiescent satellite cells. Although Hesr1- or Hesr3-single knockout mice show a normal skeletal muscle phenotype, including satellite cells, Hesr1/Hesr3-double knockout mice show a gradual decrease in the number of satellite cells and increase in regenerative defects dependent on satellite cell numbers. We also observed that a mouse's genetic background affects the regenerative capacity of its skeletal muscle and have established a line of DBA/2-background mdx mice that has a much more severe phenotype than the frequently used C57BL/10-mdx mice. The phenotype of DBA/2-mdx mice also seems to depend on the function of satellite cells. In this review, we summarize the methodology of direct isolation, characterization, and molecular regulation of satellite cells based on our results. The relationship between the regenerative capacity of satellite cells and progression of muscular disorders is also summarized. In the last part, we discuss application of the accumulating scientific information on satellite cells to treatment of patients with muscular disorders. PMID:24273513

  15. Effects of glaucoma medications and preservatives on cultured human trabecular meshwork and non-pigmented ciliary epithelial cell lines.

    PubMed

    Ammar, David A; Kahook, Malik Y

    2011-10-01

    We investigated the potential cytotoxicity of various topical ophthalmic glaucoma formulations containing different preservatives in cultured human trabecular meshwork (TM) and non-pigmented ciliary epithelial (NPCE) cell lines. We tested 0.004% travoprost preserved with either 0.015% benzalkonium chloride (BAK), sofZia or 0.001% Polyquad (PQ); and 0.005% latanoprost preserved with 0.020% BAK. We also tested a range of BAK concentrations in balanced salt solution (BSS). TM cells were treated for 10 min at 37°C with solutions diluted 1:10 to mimic the reduced penetration of topical preparations to the anterior chamber. Viability was determined by the uptake of the fluorescent vital dye calcein-AM (n = 6). BAK solutions (diluted 1:10) demonstrated a dose-dependent reduction in cell viability in both cell types (TM and NPCE). With a 1:10 dilution of 0.020% BAK, there were significantly more living NPCE cells (89 ± 6%) than TM cells (57 ± 6%; p < 0.001). In TM cells, travoprost + BAK had statistically fewer live cells (83 ± 5%) than both travoprost + sofZia (97 ± 5%) and travoprost + PQ (97 ± 6%; p < 0.05). Compared with BSS-treated NPCE cells, travoprost had statistically fewer live cells (p < 0.05) when preserved with BAK (85 ± 16%), sofZia (91 ± 6%) or PQ (94 ± 2%). These results demonstrate that substitution of BAK from topical ophthalmic drugs results in greater viability of cultured TM cells, the cells involved in the conventional outflow pathway. Cultured NPCE, responsible for aqueous inflow, appear more resilient to BAK.

  16. Injectable biomimetic liquid crystalline scaffolds enhance muscle stem cell transplantation

    PubMed Central

    Sleep, Eduard; McClendon, Mark T.; Preslar, Adam T.; Chen, Charlotte H.; Sangji, M. Hussain; Pérez, Charles M. Rubert; Haynes, Russell D.; Meade, Thomas J.; Blau, Helen M.; Stupp, Samuel I.

    2017-01-01

    Muscle stem cells are a potent cell population dedicated to efficacious skeletal muscle regeneration, but their therapeutic utility is currently limited by mode of delivery. We developed a cell delivery strategy based on a supramolecular liquid crystal formed by peptide amphiphiles (PAs) that encapsulates cells and growth factors within a muscle-like unidirectionally ordered environment of nanofibers. The stiffness of the PA scaffolds, dependent on amino acid sequence, was found to determine the macroscopic degree of cell alignment templated by the nanofibers in vitro. Furthermore, these PA scaffolds support myogenic progenitor cell survival and proliferation and they can be optimized to induce cell differentiation and maturation. We engineered an in vivo delivery system to assemble scaffolds by injection of a PA solution that enabled coalignment of scaffold nanofibers with endogenous myofibers. These scaffolds locally retained growth factors, displayed degradation rates matching the time course of muscle tissue regeneration, and markedly enhanced the engraftment of muscle stem cells in injured and noninjured muscles in mice. PMID:28874575

  17. Unique among ciliopathies: primary ciliary dyskinesia, a motile cilia disorder.

    PubMed

    Praveen, Kavita; Davis, Erica E; Katsanis, Nicholas

    2015-01-01

    Primary ciliary dyskinesia (PCD) is a ciliopathy, but represents the sole entity from this class of disorders that results from the dysfunction of motile cilia. Characterized by respiratory problems appearing in childhood, infertility, and situs defects in ~50% of individuals, PCD has an estimated prevalence of approximately 1 in 10,000 live births. The diagnosis of PCD can be prolonged due to a lack of disease awareness, coupled with the fact that symptoms can be confused with other more common genetic disorders, such as cystic fibrosis, or environmental insults that result in frequent respiratory infections. A primarily autosomal recessive disorder, PCD is genetically heterogeneous with >30 causal genes identified, posing significant challenges to genetic diagnosis. Here, we provide an overview of PCD as a disorder underscored by impaired ciliary motility; we discuss the recent advances towards uncovering the genetic basis of PCD; we discuss the molecular knowledge gained from PCD gene discovery, which has improved our understanding of motile ciliary assembly; and we speculate on how accelerated diagnosis, together with detailed phenotypic data, will shape the genetic and functional architecture of this disorder.

  18. Muscle stem cell dysfunction impairs muscle regeneration in a mouse model of Down syndrome.

    PubMed

    Pawlikowski, Bradley; Betta, Nicole Dalla; Elston, Tiffany; Williams, Darian A; Olwin, Bradley B

    2018-03-09

    Down syndrome, caused by trisomy 21, is characterized by a variety of medical conditions including intellectual impairments, cardiovascular defects, blood cell disorders and pre-mature aging phenotypes. Several somatic stem cell populations are dysfunctional in Down syndrome and their deficiencies may contribute to multiple Down syndrome phenotypes. Down syndrome is associated with muscle weakness but skeletal muscle stem cells or satellite cells in Down syndrome have not been investigated. We find that a failure in satellite cell expansion impairs muscle regeneration in the Ts65Dn mouse model of Down syndrome. Ts65Dn satellite cells accumulate DNA damage and over express Usp16, a histone de-ubiquitinating enzyme that regulates the DNA damage response. Impairment of satellite cell function, which further declines as Ts65Dn mice age, underscores stem cell deficiencies as an important contributor to Down syndrome pathologies.

  19. Nuclear fusion-independent smooth muscle differentiation of human adipose-derived stem cells induced by a smooth muscle environment.

    PubMed

    Zhang, Rong; Jack, Gregory S; Rao, Nagesh; Zuk, Patricia; Ignarro, Louis J; Wu, Benjamin; Rodríguez, Larissa V

    2012-03-01

    Human adipose-derived stem cells hASC have been isolated and were shown to have multilineage differentiation capacity. Although both plasticity and cell fusion have been suggested as mechanisms for cell differentiation in vivo, the effect of the local in vivo environment on the differentiation of adipose-derived stem cells has not been evaluated. We previously reported the in vitro capacity of smooth muscle differentiation of these cells. In this study, we evaluate the effect of an in vivo smooth muscle environment in the differentiation of hASC. We studied this by two experimental designs: (a) in vivo evaluation of smooth muscle differentiation of hASC injected into a smooth muscle environment and (b) in vitro evaluation of smooth muscle differentiation capacity of hASC exposed to bladder smooth muscle cells. Our results indicate a time-dependent differentiation of hASC into mature smooth muscle cells when these cells are injected into the smooth musculature of the urinary bladder. Similar findings were seen when the cells were cocultured in vitro with primary bladder smooth muscle cells. Chromosomal analysis demonstrated that microenvironment cues rather than nuclear fusion are responsible for this differentiation. We conclude that cell plasticity is present in hASCs, and their differentiation is accomplished in the absence of nuclear fusion. Copyright © 2011 AlphaMed Press.

  20. Xin, an actin binding protein, is expressed within muscle satellite cells and newly regenerated skeletal muscle fibers.

    PubMed

    Hawke, Thomas J; Atkinson, Daniel J; Kanatous, Shane B; Van der Ven, Peter F M; Goetsch, Sean C; Garry, Daniel J

    2007-11-01

    Xin is a muscle-specific actin binding protein of which its role and regulation within skeletal muscle is not well understood. Here we demonstrate that Xin mRNA is robustly upregulated (>16-fold) within 12 h of skeletal muscle injury and is localized to the muscle satellite cell population. RT-PCR confirmed the expression pattern of Xin during regeneration, as well as within primary muscle myoblast cultures, but not other known stem cell populations. Immunohistochemical staining of single myofibers demonstrate Xin expression colocalized with the satellite cell marker Syndecan-4 further supporting the mRNA expression of Xin in satellite cells. In situ hybridization of regenerating muscle 5-7 days postinjury illustrates Xin expression within newly regenerated myofibers. Promoter-reporter assays demonstrate that known myogenic transcription factors [myocyte enhancer factor-2 (MEF2), myogenic differentiation-1 (MyoD), and myogenic factor-5 (Myf-5)] transactivate Xin promoter constructs supporting the muscle-specific expression of Xin. To determine the role of Xin within muscle precursor cells, proliferation, migration, and differentiation analysis using Xin, short hairpin RNA (shRNA) were undertaken in C2C12 myoblasts. Reducing endogenous Xin expression resulted in a 26% increase (P < 0.05) in cell proliferation and a 20% increase (P < 0.05) in myoblast migratory capacity. Skeletal muscle myosin heavy chain protein levels were increased (P < 0.05) with Xin shRNA administration; however, this was not accompanied by changes in myoglobin protein (another marker of differentiation) nor overt morphological differences relative to differentiating control cells. Taken together, the present findings support the hypothesis that Xin is expressed within muscle satellite cells during skeletal muscle regeneration and is involved in the regulation of myoblast function.

  1. ciliaFA: a research tool for automated, high-throughput measurement of ciliary beat frequency using freely available software

    PubMed Central

    2012-01-01

    Background Analysis of ciliary function for assessment of patients suspected of primary ciliary dyskinesia (PCD) and for research studies of respiratory and ependymal cilia requires assessment of both ciliary beat pattern and beat frequency. While direct measurement of beat frequency from high-speed video recordings is the most accurate and reproducible technique it is extremely time consuming. The aim of this study was to develop a freely available automated method of ciliary beat frequency analysis from digital video (AVI) files that runs on open-source software (ImageJ) coupled to Microsoft Excel, and to validate this by comparison to the direct measuring high-speed video recordings of respiratory and ependymal cilia. These models allowed comparison to cilia beating between 3 and 52 Hz. Methods Digital video files of motile ciliated ependymal (frequency range 34 to 52 Hz) and respiratory epithelial cells (frequency 3 to 18 Hz) were captured using a high-speed digital video recorder. To cover the range above between 18 and 37 Hz the frequency of ependymal cilia were slowed by the addition of the pneumococcal toxin pneumolysin. Measurements made directly by timing a given number of individual ciliary beat cycles were compared with those obtained using the automated ciliaFA system. Results The overall mean difference (± SD) between the ciliaFA and direct measurement high-speed digital imaging methods was −0.05 ± 1.25 Hz, the correlation coefficient was shown to be 0.991 and the Bland-Altman limits of agreement were from −1.99 to 1.49 Hz for respiratory and from −2.55 to 3.25 Hz for ependymal cilia. Conclusions A plugin for ImageJ was developed that extracts pixel intensities and performs fast Fourier transformation (FFT) using Microsoft Excel. The ciliaFA software allowed automated, high throughput measurement of respiratory and ependymal ciliary beat frequency (range 3 to 52 Hz) and avoids operator error due to selection bias. We have

  2. Skeletal Muscle Satellite Cell Activation Following Cutaneous Burn in Rats

    DTIC Science & Technology

    2013-12-01

    satellite cell activation and survival during oxidative stress. J Muscle Res Cell Motil 2011;32(2):99–109. [33] Rathbone CR, Booth FW, Lees SJ. Sirt1 ...Skeletal muscle satellite cell activation following cutaneous burn in rats Xiaowu Wu*, Thomas J. Walters, Christopher R. Rathbone Extremity Trauma...f o Article history: Accepted 15 October 2012 Keywords: Muscle precursor cell Thermal injury Atrophy Skeletal muscle Activation a b s t r a c t

  3. Accuracy of Immunofluorescence in the Diagnosis of Primary Ciliary Dyskinesia

    PubMed Central

    Frost, Emily; Dixon, Mellisa; Ollosson, Sarah; Kilpin, Kate; Patel, Mitali; Scully, Juliet; Rogers, Andrew V.; Mitchison, Hannah M.; Bush, Andrew; Hogg, Claire

    2017-01-01

    Rationale: The standard approach to diagnosis of primary ciliary dyskinesia (PCD) in the United Kingdom consists of assessing ciliary function by high-speed microscopy and ultrastructure by election microscopy, but equipment and expertise is not widely available internationally. The identification of biallelic disease-causing mutations is also diagnostic, but many disease-causing genes are unknown, and testing is not widely available outside the United States. Fluorescent antibodies to ciliary proteins are used to validate research genetic studies, but diagnostic utility in this disease has not been systematically evaluated. Objectives: To determine utility of a panel of six fluorescent labeled antibodies as a diagnostic tool for PCD. Methods: The study used immunofluorescent labeling of nasal brushings from a discovery cohort of 35 patients diagnosed with PCD by ciliary ultrastructure, and a diagnostic accuracy cohort of 386 patients referred with symptoms suggestive of disease. The results were compared with diagnostic outcome. Measurements and Main Results: Immunofluorescence correctly identified mislocalized or absent staining in 100% of the discovery cohort. In the diagnostic cohort immunofluorescence successfully identified 22 of 25 patients with PCD and normal staining in all 252 in whom PCD was considered highly unlikely. In addition, immunofluorescence provided a result in 55% (39) of cases that were previously inconclusive. Immunofluorescence results were available within 14 days, costing $187 per sample compared with electron microscopy (27 days; cost $1,452). Conclusions: Immunofluorescence is a highly specific diagnostic test for PCD, and it improves the speed and availability of diagnostic testing. However, sensitivity is limited and immunofluorescence is not suitable as a stand-alone test. PMID:28199173

  4. Overexpression of Striated Muscle Activator of Rho Signaling (STARS) Increases C2C12 Skeletal Muscle Cell Differentiation.

    PubMed

    Wallace, Marita A; Della Gatta, Paul A; Ahmad Mir, Bilal; Kowalski, Greg M; Kloehn, Joachim; McConville, Malcom J; Russell, Aaron P; Lamon, Séverine

    2016-01-01

    Skeletal muscle growth and regeneration depend on the activation of satellite cells, which leads to myocyte proliferation, differentiation and fusion with existing muscle fibers. Skeletal muscle cell proliferation and differentiation are tightly coordinated by a continuum of molecular signaling pathways. The striated muscle activator of Rho signaling (STARS) is an actin binding protein that regulates the transcription of genes involved in muscle cell growth, structure and function via the stimulation of actin polymerization and activation of serum-response factor (SRF) signaling. STARS mediates cell proliferation in smooth and cardiac muscle models; however, whether STARS overexpression enhances cell proliferation and differentiation has not been investigated in skeletal muscle cells. We demonstrate for the first time that STARS overexpression enhances differentiation but not proliferation in C2C12 mouse skeletal muscle cells. Increased differentiation was associated with an increase in the gene levels of the myogenic differentiation markers Ckm, Ckmt2 and Myh4, the differentiation factor Igf2 and the myogenic regulatory factors (MRFs) Myf5 and Myf6. Exposing C2C12 cells to CCG-1423, a pharmacological inhibitor of SRF preventing the nuclear translocation of its co-factor MRTF-A, had no effect on myotube differentiation rate, suggesting that STARS regulates differentiation via a MRTF-A independent mechanism. These findings position STARS as an important regulator of skeletal muscle growth and regeneration.

  5. Transcriptome analysis of cattle muscle identifies potential markers for skeletal muscle growth rate and major cell types.

    PubMed

    Guo, Bing; Greenwood, Paul L; Cafe, Linda M; Zhou, Guanghong; Zhang, Wangang; Dalrymple, Brian P

    2015-03-13

    This study aimed to identify markers for muscle growth rate and the different cellular contributors to cattle muscle and to link the muscle growth rate markers to specific cell types. The expression of two groups of genes in the longissimus muscle (LM) of 48 Brahman steers of similar age, significantly enriched for "cell cycle" and "ECM (extracellular matrix) organization" Gene Ontology (GO) terms was correlated with average daily gain/kg liveweight (ADG/kg) of the animals. However, expression of the same genes was only partly related to growth rate across a time course of postnatal LM development in two cattle genotypes, Piedmontese x Hereford (high muscling) and Wagyu x Hereford (high marbling). The deposition of intramuscular fat (IMF) altered the relationship between the expression of these genes and growth rate. K-means clustering across the development time course with a large set of genes (5,596) with similar expression profiles to the ECM genes was undertaken. The locations in the clusters of published markers of different cell types in muscle were identified and used to link clusters of genes to the cell type most likely to be expressing them. Overall correspondence between published cell type expression of markers and predicted major cell types of expression in cattle LM was high. However, some exceptions were identified: expression of SOX8 previously attributed to muscle satellite cells was correlated with angiogenesis. Analysis of the clusters and cell types suggested that the "cell cycle" and "ECM" signals were from the fibro/adipogenic lineage. Significant contributions to these signals from the muscle satellite cells, angiogenic cells and adipocytes themselves were not as strongly supported. Based on the clusters and cell type markers, sets of five genes predicted to be representative of fibro/adipogenic precursors (FAPs) and endothelial cells, and/or ECM remodelling and angiogenesis were identified. Gene sets and gene markers for the analysis of

  6. Fiber optic laser light scattering measurement of ciliary function of the fallopian tube

    NASA Astrophysics Data System (ADS)

    Halbert, Sheridan A.; Lim, Kap; Lee, Wylie I.

    1990-07-01

    A fiber-optic laser light-scattering system (FLS) for measuring ciliary function was evaluated by means of three sets of in vitro experiments. First, FLS performance was compared to that of a previously proven benchtop laser system (BLS). Using tissue excised from rabbit fallopian tubes, ciliary beat frequency (CBF) of each sample was measured with FLS and BLS. Paired CBF measurements showed excellent correlation between the two systems (r =0.93). Second, the FLS was used to evaluate the dependency of CBF on temperature (T) by using tissue sampies of rabbit oviductal fimbna. Regression analysis of CBF vs T showed a linear relationship over the range of 18-37°C for both individual samples (r =0.98) and pooled data from all experiments (r = 0.84). Fmally, the relalionship between CBF and ciliary ovum transport rate (TR) was tested by using T to modulate CBF of rabbit fimbria, in vitro. The relationship was linear over the range of CBF from 10 to 30 Hz (r2 = 0.83). At 37°C, CBF = 31+/-1 Hz, and TR = O.12+/-.02 mm/sec. equal to ovum transport rate in situ. The FLS is a valuable tool for characterizing ciliary activity and thus ovum transport function. Owing to the fact that ciliary dyskinesia resulting from disease of the fallopian tube is associated with infeitility, the FLS may be useful to acquire data important to the clinical evaluation of fallopian tube function and female infertility.

  7. Effects of dorzolamide on choroidal blood flow, ciliary blood flow, and aqueous production in rabbits.

    PubMed

    Reitsamer, Herbert A; Bogner, Barbara; Tockner, Birgit; Kiel, Jeffrey W

    2009-05-01

    To determine the effects of topical dorzolamide (a carbonic anhydrase inhibitor) on choroidal and ciliary blood flow and the relationship between ciliary blood flow and aqueous flow. The experiments were performed in four groups of pentobarbital-anesthetized rabbits treated with topical dorzolamide (2%, 50 microL). In all groups, intraocular pressure (IOP) and mean arterial pressure (MAP) at the eye level were measured continuously by direct cannulation. In group 1, aqueous flow was measured by fluorophotometry before and after dorzolamide treatment. In group 2, aqueous flow was measured after dorzolamide at normal MAP and while MAP was held constant at 80, 55, or 40 mm Hg with occluders on the aorta and vena cava. In group 3, the same MAP levels were used, and ciliary blood flow was measured transsclerally by laser Doppler flowmetry (LDF). In group 4, choroidal blood flow was measured by LDF with the probe tip positioned in the vitreous over the posterior pole during ramp increases and decreases in MAP before and after dorzolamide. Dorzolamide lowered IOP by 19% (P < 0.01) and aqueous flow by 17% (P < 0.01), and increased ciliary blood flow by 18% (P < 0.01), which was associated with a significant reduction in ciliary vasculature resistance (-7%, P < 0.01). Dorzolamide shifted the relationship between ciliary blood flow and aqueous flow downward relative to the previously determined control relationship in the rabbit. Dorzolamide did not alter choroidal blood flow, choroidal vascular resistance, or the choroidal pressure flow relationship. Acute topical dorzolamide is a ciliary vasodilator and has a direct inhibitory effect on aqueous production, but it does not have a detectable effect on choroidal hemodynamics at the posterior pole in the rabbit.

  8. Protein Interaction Analysis Provides a Map of the Spatial and Temporal Organization of the Ciliary Gating Zone.

    PubMed

    Takao, Daisuke; Wang, Liang; Boss, Allison; Verhey, Kristen J

    2017-08-07

    The motility and signaling functions of the primary cilium require a unique protein and lipid composition that is determined by gating mechanisms localized at the base of the cilium. Several protein complexes localize to the gating zone and may regulate ciliary protein composition; however, the mechanisms of ciliary gating and the dynamics of the gating components are largely unknown. Here, we used the BiFC (bimolecular fluorescence complementation) assay and report for the first time on the protein-protein interactions that occur between ciliary gating components and transiting cargoes during ciliary entry. We find that the nucleoporin Nup62 and the C termini of the nephronophthisis (NPHP) proteins NPHP4 and NPHP5 interact with the axoneme-associated kinesin-2 motor KIF17 and thus spatially map to the inner region of the ciliary gating zone. Nup62 and NPHP4 exhibit rapid turnover at the transition zone and thus define dynamic components of the gate. We find that B9D1, AHI1, and the N termini of NPHP4 and NPHP5 interact with the transmembrane protein SSTR3 and thus spatially map to the outer region of the ciliary gating zone. B9D1, AHI1, and NPHP5 exhibit little to no turnover at the transition zone and thus define components of a stable gating structure. These data provide the first comprehensive map of the molecular orientations of gating zone components along the inner-to-outer axis of the ciliary gating zone. These results advance our understanding of the functional roles of gating zone components in regulating ciliary protein composition. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. Measurement of ciliary beat frequency using ultra-high resolution optical coherence tomography

    NASA Astrophysics Data System (ADS)

    Chen, Jason J.; Jing, Joseph C.; Su, Erica; Badger, Christopher; Coughlan, Carolyn A.; Chen, Zhongping; Wong, Brian J. F.

    2016-02-01

    Ciliated epithelial cells populate up to 80% of the surface area of the human airway and are responsible for mucociliary transport, which is the key protective mechanism that provides the first line of defense in the respiratory tract. Cilia beat in a rhythmic pattern and may be easily affected by allergens, pollutants, and pathogens, altering ciliary beat frequency (CBF) subsequently. Diseases including cystic fibrosis, chronic obstructive pulmonary disease, and primary ciliary dyskinesia may also decrease CBF. CBF is therefore a critical component of respiratory health. The current clinical method of measuring CBF is phase-contrast microscopy, which involves a tissue biopsy obtained via brushing of the nasal cavity. While this method is minimally invasive, the tissue sample must be oriented to display its profile view, making the visualization of a single layer of cilia challenging. In addition, the conventional method requires subjective analysis of CBF, e.g., manually counting by visual inspection. On the contrary, optical coherence tomography (OCT) has been used to study the retina in ophthalmology as well as vasculature in cardiology, and offers higher resolution than conventional computed tomography and magnetic resonance imaging. Based on this technology, our lab specifically developed an ultra-high resolution OCT system to image the microstructure of the ciliated epithelial cells. Doppler analysis was also performed to determine CBF. Lastly, we also developed a program that utilizes fast Fourier transform to determine CBF under phase-contrast microscopy, providing a more objective method compared to the current method.

  10. Circular flow patterns induced by ciliary activity in reconstituted human bronchial epithelium

    NASA Astrophysics Data System (ADS)

    Viallat, Annie; Khelloufi, Kamel; Gras, Delphine; Chanez, Pascal; Aix Marseille Univ., CNRS, CINaM, Marseille, France Team; Aix Marseille Univ., CNRS, Inserm, LAI, Marseille, France Team

    2016-11-01

    Mucociliary clearance is the transport at the surface of airways of a complex fluid layer, the mucus, moved by the beats of microscopic cilia present on epithelial ciliated cells. We explored the coupling between the spatial organisation and the activity of cilia and the transport of surface fluids on reconstituted cultures of human bronchial epithelium at air-liquid interface, obtained by human biopsies. We reveal the existence of stable local circular surface flow patterns of mucus or Newtonian fluid at the epithelium surface. We find a power law over more than 3 orders of magnitude showing that the average ciliated cell density controls the size of these flow patterns, and, therefore the distance over which mucus can be transported. We show that these circular flow patterns result from the radial linear increase of the local propelling forces (due to ciliary beats) on each flow domain. This linear increase of local forces is induced by a fine self-regulation of both cilia density and orientation of ciliary beats. Local flow domains grow and merge during ciliogenesis to provide macroscopic mucus transport. This is possible only when the viscoelastic mucus continuously exerts a shear stress on beating cilia, revealing a mechanosensitive function of cilia. M. K. Khelloufi thanks the society MedBioMed for financial support. This work was supported by the ANR MUCOCIL project, Grant ANR-13-BSV5-0015 of the French Agence Nationale de la Recherche.

  11. Single Cell Gene Expression Profiling of Skeletal Muscle-Derived Cells.

    PubMed

    Gatto, Sole; Puri, Pier Lorenzo; Malecova, Barbora

    2017-01-01

    Single cell gene expression profiling is a fundamental tool for studying the heterogeneity of a cell population by addressing the phenotypic and functional characteristics of each cell. Technological advances that have coupled microfluidic technologies with high-throughput quantitative RT-PCR analyses have enabled detailed analyses of single cells in various biological contexts. In this chapter, we describe the procedure for isolating the skeletal muscle interstitial cells termed Fibro-Adipogenic Progenitors (FAPs ) and their gene expression profiling at the single cell level. Moreover, we accompany our bench protocol with bioinformatics analysis designed to process raw data as well as to visualize single cell gene expression data. Single cell gene expression profiling is therefore a useful tool in the investigation of FAPs heterogeneity and their contribution to muscle homeostasis.

  12. Ciliary neurotrophic factor promotes the activation of corneal epithelial stem/progenitor cells and accelerates corneal epithelial wound healing.

    PubMed

    Zhou, Qingjun; Chen, Peng; Di, Guohu; Zhang, Yangyang; Wang, Yao; Qi, Xia; Duan, Haoyun; Xie, Lixin

    2015-05-01

    Ciliary neurotrophic factor (CNTF), a well-known neuroprotective cytokine, has been found to play an important role in neurogenesis and functional regulations of neural stem cells. As one of the most innervated tissue, however, the role of CNTF in cornea epithelium remains unclear. This study was to explore the roles and mechanisms of CNTF in the activation of corneal epithelial stem/progenitor cells and wound healing of both normal and diabetic mouse corneal epithelium. In mice subjecting to mechanical removal of corneal epithelium, the corneal epithelial stem/progenitor cell activation and wound healing were promoted by exogenous CNTF application, while delayed by CNTF neutralizing antibody. In cultured corneal epithelial stem/progenitor cells, CNTF enhanced the colony-forming efficiency, stimulated the mitogenic proliferation, and upregulated the expression levels of corneal epithelial stem/progenitor cell-associated transcription factors. Furthermore, the promotion of CNTF on the corneal epithelial stem/progenitor cell activation and wound healing was mediated by the activation of STAT3. Moreover, in diabetic mice, the content of CNTF in corneal epithelium decreased significantly when compared with that of normal mice, and the supplement of CNTF promoted the diabetic corneal epithelial wound healing, accompanied with the advanced activation of corneal epithelial stem/progenitor cells and the regeneration of corneal nerve fibers. Thus, the capability of expanding corneal epithelial stem/progenitor cells and promoting corneal epithelial wound healing and nerve regeneration indicates the potential application of CNTF in ameliorating limbal stem cell deficiency and treating diabetic keratopathy. © 2014 AlphaMed Press.

  13. Laser light-scattering spectroscopy: a new application in the study of ciliary activity.

    PubMed Central

    Lee, W I; Verdugo, P

    1976-01-01

    A uniquely precise and simple method to study ciliary activity by laser light-scattering spectroscopy has been developed and validated. A concurrent study of the effect of Ca2+ on ciliary activity in vitro by laser scattering spectroscopy and high speed cinematography has demonstrated that this new method is simpler and as accurate and reproducible as the high speed film technique. PMID:963208

  14. Bioinformatics and reanalysis of subtracted expressed sequence tags from the human ciliary body: Identification of novel biological functions.

    PubMed

    Escribano, Julio; Coca-Prados, Miguel

    2002-08-28

    The ciliary body is largely known for its major roles in the regulation of aqueous humor secretion, intraocular pressure, and accommodation of the lens. In this review article we applied bioinformatics to re-examine hundreds of expressed sequence tags (ESTs) previously isolated by subtractive hybridization from a human ciliary body library [1]. The DNA sequences of these clones have been recently added to the web site of NEIBank. DNA sequence comparisons of subtracted ESTs were performed against all entries in the last available release of the non-redundant database containing GenBank, EMBL, DDBJ and PDB sequences using the BlastN program accessed through NCBI's BLAST services on the internet (NCBI). Sequences were also compared and mapped using the Blast search program provided through the Internet by the Human Genome Project (UCSC). A total number of 284 independent ESTs were classified in 17 functional groups. Analysis of their relationships allowed to define the expression of five major groups of known genes: (i) protein synthesis, folding, secretion and degradation (20%); (ii) energy supply and biosynthesis (12%); (iii) contractility and cytoskeleton structure (6%); (iv) cellular signaling and cell cycle regulation (7%); and (v) nerve cell related tasks (2%), including neuropeptide processing and putative non-visual phototransduction and circadian rhythm control. The largest group contain unidentified sequences, a total of 105 sequences, accounting for 37% of ESTs. The unidentified sequences show similarity to genomic non-coding regions, or genes of unknown function. The most highly represented EST, correspond to myocilin, a gene involved in glaucoma. The data also confirms the secretory functions of the ciliary epithelium, and its high metabolism; the presence of a neuroendocrine peptidergic system presumably involved in the regulation of the intraocular pressure and/or aqueous humor secretion. Additional genes may be related to a non-visual phototransduction

  15. Isolation of Primary Human Skeletal Muscle Cells

    PubMed Central

    Spinazzola, Janelle M.; Gussoni, Emanuela

    2017-01-01

    Primary myoblast culture is a valuable tool in research of muscle disease, pathophysiology, and pharmacology. This protocol describes techniques for dissociation of cells from human skeletal muscle biopsies and enrichment for a highly myogenic population by fluorescence-activated cell sorting (FACS). We also describe methods for assessing myogenicity and population expansion for subsequent in vitro study. PMID:29152538

  16. Swimming Speed of Larval Snail Does Not Correlate with Size and Ciliary Beat Frequency

    PubMed Central

    Chan, Kit Yu Karen; Jiang, Houshuo; Padilla, Dianna K.

    2013-01-01

    Many marine invertebrates have planktonic larvae with cilia used for both propulsion and capturing of food particles. Hence, changes in ciliary activity have implications for larval nutrition and ability to navigate the water column, which in turn affect survival and dispersal. Using high-speed high-resolution microvideography, we examined the relationship between swimming speed, velar arrangements, and ciliary beat frequency of freely swimming veliger larvae of the gastropod Crepidula fornicata over the course of larval development. Average swimming speed was greatest 6 days post hatching, suggesting a reduction in swimming speed towards settlement. At a given age, veliger larvae have highly variable speeds (0.8–4 body lengths s−1) that are independent of shell size. Contrary to the hypothesis that an increase in ciliary beat frequency increases work done, and therefore speed, there was no significant correlation between swimming speed and ciliary beat frequency. Instead, there are significant correlations between swimming speed and visible area of the velar lobe, and distance between centroids of velum and larval shell. These observations suggest an alternative hypothesis that, instead of modifying ciliary beat frequency, larval C. fornicata modify swimming through adjustment of velum extension or orientation. The ability to adjust velum position could influence particle capture efficiency and fluid disturbance and help promote survival in the plankton. PMID:24367554

  17. The Impact of Aerobic Exercise on the Muscle Stem Cell Response.

    PubMed

    Joanisse, Sophie; Snijders, Tim; Nederveen, Joshua P; Parise, Gianni

    2018-04-16

    Satellite cells are indispensable for skeletal muscle repair and regeneration and are associated with muscle growth in humans. Aerobic exercise training results in improved skeletal muscle health also translating to an increase in satellite cell pool activation. We postulate that aerobic exercise improves satellite cell function in skeletal muscle.

  18. Morphological Alterations Within the Peripheral Fixation of the Iris Dilator Muscle in Eyes With Pigmentary Glaucoma

    PubMed Central

    Flügel-Koch, Cassandra M.; Tektas, Ozan Y.; Kaufman, Paul L.; Paulsen, Friedrich P.; Lütjen-Drecoll, Elke

    2014-01-01

    Purpose. To analyze the peripheral fixation of the iris dilator muscle in normal eyes and in eyes with pigmentary glaucoma (PG). Methods. Using 63 control eyes (age 18 months–99 years), the peripheral iris dilator was investigated by light microscopy, immunohistochemistry, and electron microscopy. Development was studied using 18 differently aged fetal eyes stained immunohistochemically against α-smooth muscle (SM) actin. The peripheral iris dilator muscle in PG was analyzed using semithin and ultrathin sections of six glutaraldehyde-fixed eyes from three donors aged 38, 62, and 74 years. Results. In normal eyes, the peripheral end of the iris dilator muscle is arranged in a sphincter-like manner. Arcade-shaped tendinous connections associated with myofibroblasts (iridial strands) anchor the iris dilator within the elastic–fibromuscular ciliary meshwork that also serves as fixation area for the elastic tendons of the inner ciliary muscle portions. The iridial strands are innervated and can adapt their length during accommodation. The PG eyes show incomplete circular bundles and iridial strands that are mainly anchored to the iris stroma and the flexible uveal parts of the trabecular meshwork. Conclusions. The normal anchorage of the peripheral iris dilator and its presumably neuronally regulated length adaptation stabilize the peripheral iris during accommodation. Insufficient fixation in PG could promote posterior bowing of the iris with rubbing against the zonular fibers and pigment liberation from the iris pigmented epithelium. PMID:24938519

  19. Development of a nitric oxide-releasing analogue of the muscle relaxant guaifenesin for skeletal muscle satellite cell myogenesis.

    PubMed

    Wang, Guqi; Burczynski, Frank J; Hasinoff, Brian B; Zhang, Kaidong; Lu, Qilong; Anderson, Judy E

    2009-01-01

    Nitric oxide (NO) mediates activation of satellite precursor cells to enter the cell cycle. This provides new precursor cells for skeletal muscle growth and muscle repair from injury or disease. Targeting a new drug that specifically delivers NO to muscle has the potential to promote normal function and treat neuromuscular disease, and would also help to avoid side effects of NO from other treatment modalities. In this research, we examined the effectiveness of the NO donor, iosorbide dinitrate (ISDN), and a muscle relaxant, methocarbamol, in promoting satellite cell activation assayed by muscle cell DNA synthesis in normal adult mice. The work led to the development of guaifenesin dinitrate (GDN) as a new NO donor for delivering nitric oxide to muscle. The results revealed that there was a strong increase in muscle satellite cell activation and proliferation, demonstrated by a significant 38% rise in DNA synthesis after a single transdermal treatment with the new compound for 24 h. Western blot and immunohistochemistry analyses showed that the markers of satellite cell myogenesis, expression of myf5, myogenin, and follistatin, were increased after 24 h oral administration of the compound in adult mice. This research extends our understanding of the outcomes of NO-based treatments aimed at promoting muscle regeneration in normal tissue. The potential use of such treatment for conditions such as muscle atrophy in disuse and aging, and for the promotion of muscle tissue repair as required after injury or in neuromuscular diseases such as muscular dystrophy, is highlighted.

  20. Stimulation of aortic smooth muscle cell mitogenesis by serotonin

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

    Nemecek, G.M.; Coughlin, S.R.; Handley, D.A.

    1986-02-01

    Bovine aortic smooth muscle cells in vitro responded to 1 nM to 10 ..mu..M serotonin with increased incorporation of (/sup 3/H)thymidine into DNA. The mitogenic effect of serotonin was half-maximal at 80 nM and maximal above 1 ..mu..M. At a concentration of 1 ..mu..M, serotonin stimulated smooth muscle cell mitogenesis to the same extent as human platelet-derived growth factor (PDGF) at 12 ng/ml. Tryptamine was approx. = 1/10th as potent as serotonin as a mitogen for smooth muscle cells. Other indoles that are structurally related to serotonin (D- and L-tryptophan, 5-hydroxy-L-tryptophan, N-acetyl-5-hydroxytryptamine, melatonin, 5-hydroxyindoleacetic acid, and 5-hydroxytryptophol) and quipazine weremore » inactive. The stimulatory effect of serotonin on smooth muscle cell DNA synthesis required prolonged (20-24 hr) exposure to the agonist and was attenuated in the presence of serotonin D receptor antagonists. When smooth muscle cells were incubated with submaximal concentrations of serotonin and PDGF, synergistic rather than additive mitogenic responses were observed. These data indicate that serotonin has a significant mitogenic effect on smooth muscle cells in vitro, which appears to be mediated by specific plasma membrane receptors.« less

  1. Efficient Generation of iPS Cells from Skeletal Muscle Stem Cells

    PubMed Central

    Tan, Kah Yong; Eminli, Sarah; Hettmer, Simone; Hochedlinger, Konrad; Wagers, Amy J.

    2011-01-01

    Reprogramming of somatic cells into inducible pluripotent stem cells generally occurs at low efficiency, although what limits reprogramming of particular cell types is poorly understood. Recent data suggest that the differentiation status of the cell targeted for reprogramming may influence its susceptibility to reprogramming as well as the differentiation potential of the induced pluripotent stem (iPS) cells that are derived from it. To assess directly the influence of lineage commitment on iPS cell derivation and differentiation, we evaluated reprogramming in adult stem cell and mature cell populations residing in skeletal muscle. Our data using clonal assays and a second-generation inducible reprogramming system indicate that stem cells found in mouse muscle, including resident satellite cells and mesenchymal progenitors, reprogram with significantly greater efficiency than their more differentiated daughters (myoblasts and fibroblasts). However, in contrast to previous reports, we find no evidence of biased differentiation potential among iPS cells derived from myogenically committed cells. These data support the notion that adult stem cells reprogram more efficiently than terminally differentiated cells, and argue against the suggestion that “epigenetic memory” significantly influences the differentiation potential of iPS cells derived from distinct somatic cell lineages in skeletal muscle. PMID:22028872

  2. Human Satellite Cell Transplantation and Regeneration from Diverse Skeletal Muscles

    PubMed Central

    Xu, Xiaoti; Wilschut, Karlijn J.; Kouklis, Gayle; Tian, Hua; Hesse, Robert; Garland, Catharine; Sbitany, Hani; Hansen, Scott; Seth, Rahul; Knott, P. Daniel; Hoffman, William Y.; Pomerantz, Jason H.

    2015-01-01

    Summary Identification of human satellite cells that fulfill muscle stem cell criteria is an unmet need in regenerative medicine. This hurdle limits understanding how closely muscle stem cell properties are conserved among mice and humans and hampers translational efforts in muscle regeneration. Here, we report that PAX7 satellite cells exist at a consistent frequency of 2–4 cells/mm of fiber in muscles of the human trunk, limbs, and head. Xenotransplantation into mice of 50–70 fiber-associated, or 1,000–5,000 FACS-enriched CD56+/CD29+ human satellite cells led to stable engraftment and formation of human-derived myofibers. Human cells with characteristic PAX7, CD56, and CD29 expression patterns populated the satellite cell niche beneath the basal lamina on the periphery of regenerated fibers. After additional injury, transplanted satellite cells robustly regenerated to form hundreds of human-derived fibers. Together, these findings conclusively delineate a source of bona-fide endogenous human muscle stem cells that will aid development of clinical applications. PMID:26352798

  3. Matrix metalloproteinase inhibition negatively affects muscle stem cell behavior

    PubMed Central

    Bellayr, Ian; Holden, Kyle; Mu, Xiaodong; Pan, Haiying; Li, Yong

    2013-01-01

    Skeletal muscle is a large and complex system that is crucial for structural support, movement and function. When injured, the repair of skeletal muscle undergoes three phases: inflammation and degeneration, regeneration and fibrosis formation in severe injuries. During fibrosis formation, muscle healing is impaired because of the accumulation of excess collagen. A group of zinc-dependent endopeptidases that have been found to aid in the repair of skeletal muscle are matrix metalloproteinases (MMPs). MMPs are able to assist in tissue remodeling through the regulation of extracellular matrix (ECM) components, as well as contributing to cell migration, proliferation, differentiation and angiogenesis. In the present study, the effect of GM6001, a broad-spectrum MMP inhibitor, on muscle-derived stem cells (MDSCs) is investigated. We find that MMP inhibition negatively impacts skeletal muscle healing by impairing MDSCs in migratory and multiple differentiation abilities. These results indicate that MMP signaling plays an essential role in the wound healing of muscle tissue because their inhibition is detrimental to stem cells residing in skeletal muscle. PMID:23329998

  4. Effect of cAMP on short-circuit current in isolated human ciliary body.

    PubMed

    Wu, Ren-yi; Ma, Ning; Hu, Qian-qian

    2013-07-01

    Cyclic adenosine monophosphate (cAMP) could activate chloride channels in bovine ciliary body and trigger an increase in the ionic current (short-circuit current, Isc) across the ciliary processes in pigs. The purpose of this study was to investigate how cAMP modulates Isc in isolated human ciliary processes and the possible involvement of chloride transport across the tissue in cAMP-induced Isc change. In an Ussing-type chamber system, the Isc changes induced by the cAMP analogue 8-bromo-cAMP and an adenylyl cyclase activator forskolin in isolated human ciliary processes were assessed. The involvement of Cl(-) component in the bath solution was investigated. The effect of Cl(-) channel (10 µmol/L niflumic acid and 1 mmol/L 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS)), K(+) channel (10 mmol/L tetraethylammonium chloride (TEA)), or Na(+) channel blockers (1 mmol/L amiloride) on 8-bromo-cAMP-induced Isc change was also studied. Dose-dependently, 8-bromo-cAMP (10 nmol/L-30 µmol/L) or forskolin (10 nmol/L-3 µmol/L) increased Isc across the ciliary processes with an increase in negative potential difference on the non-pigmented epithelium (NPE) side of the tissue. Isc increase induced by 8-bromo-cAMP was more pronounced when the drug was applied on the NPE side than on the pigmented epithelium side. When the tissue was bathed in low Cl(-) solutions, the Isc increase was significantly inhibited. Finally, niflumic acid and DIDS, but not TEA or amiloride, significantly prevented the Isc increase induced by 8-bromo-cAMP. cAMP stimulates stroma-to-aqueous anionic transport in isolated human ciliary processes. Chloride is likely to be among the ions, the transportation of which across the tissue is triggered by cAMP, suggesting the potential role of cAMP in the process of aqueous humor formation in human eyes.

  5. Differential regulation of gene expression of neurotensin and prohormone convertases PC1 and PC2 in the bovine ocular ciliary epithelium: possible implications on neurotensin processing.

    PubMed

    Ortego, Javier; Wollmann, Guido; Coca-Prados, Miguel

    2002-11-15

    Prohormone convertases PC1 and PC2 are enzymes involved in the intracellular processing of pro-neurotensin/neuromedin N (pro-NT/NN) through the regulated secretory pathway. In this study, we present evidence of the differential gene expression of pro-NT/NN, pro-PC1 and pro-PC2 in two cell lines established from the neuroendocrine ocular ciliary epithelium. Dexamethasone and forskolin were found to synergistically up-regulate NT/NN mRNA expression in both cell types. The pigmented cells released NT, and this release was enhanced by agents that induced its biosynthesis. In contrast, nonpigmented cells exhibited a significantly reduced neurotensin secretion in response to inducers, leading to an accumulation of the peptide. PC1 and PC2 mRNA expression was induced in a cell-specific manner by the same agents that enhanced pro-NT/NN biosynthesis. These results demonstrate cell-specific processing of pro-NT/NN by the ciliary epithelium. Copyright 2002 Elsevier Science Ireland Ltd.

  6. Requirement of myomaker-mediated stem cell fusion for skeletal muscle hypertrophy.

    PubMed

    Goh, Qingnian; Millay, Douglas P

    2017-02-10

    Fusion of skeletal muscle stem/progenitor cells is required for proper development and regeneration, however the significance of this process during adult muscle hypertrophy has not been explored. In response to muscle overload after synergist ablation in mice, we show that myomaker, a muscle specific membrane protein essential for myoblast fusion, is activated mainly in muscle progenitors and not myofibers. We rendered muscle progenitors fusion-incompetent through genetic deletion of myomaker in muscle stem cells and observed a complete reduction of overload-induced hypertrophy. This blunted hypertrophic response was associated with a reduction in Akt and p70s6k signaling and protein synthesis, suggesting a link between myonuclear accretion and activation of pro-hypertrophic pathways. Furthermore, fusion-incompetent muscle exhibited increased fibrosis after muscle overload, indicating a protective role for normal stem cell activity in reducing myofiber strain associated with hypertrophy. These findings reveal an essential contribution of myomaker-mediated stem cell fusion during physiological adult muscle hypertrophy.

  7. Msx1-Positive Progenitors in the Retinal Ciliary Margin Give Rise to Both Neural and Non-neural Progenies in Mammals.

    PubMed

    Bélanger, Marie-Claude; Robert, Benoit; Cayouette, Michel

    2017-01-23

    In lower vertebrates, stem/progenitor cells located in a peripheral domain of the retina, called the ciliary margin zone (CMZ), cooperate with retinal domain progenitors to build the mature neural retina. In mammals, it is believed that the CMZ lacks neurogenic potential and that the retina develops from one pool of multipotent retinal progenitor cells (RPCs). Here we identify a population of Msx1-expressing progenitors in the mouse CMZ that is both molecularly and functionally distinct from RPCs. Using genetic lineage tracing, we report that Msx1 progenitors have unique developmental properties compared with RPCs. Msx1 lineages contain both neural retina and non-neural ciliary epithelial progenies and overall generate fewer photoreceptors than classical RPC lineages. Furthermore, we show that the endocytic adaptor protein Numb regulates the balance between neural and non-neural fates in Msx1 progenitors. These results uncover a population of CMZ progenitors, distinct from classical RPCs, that also contributes to mammalian retinogenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. Design of a muscle cell-specific expression vector utilising human vascular smooth muscle alpha-actin regulatory elements.

    PubMed

    Keogh, M C; Chen, D; Schmitt, J F; Dennehy, U; Kakkar, V V; Lemoine, N R

    1999-04-01

    The facility to direct tissue-specific expression of therapeutic gene constructs is desirable for many gene therapy applications. We describe the creation of a muscle-selective expression vector which supports transcription in vascular smooth muscle, cardiac muscle and skeletal muscle, while it is essentially silent in other cell types such as endothelial cells, hepatocytes and fibroblasts. Specific transcriptional regulatory elements have been identified in the human vascular smooth muscle cell (VSMC) alpha-actin gene, and used to create an expression vector which directs the expression of genes in cis to muscle cells. The vector contains an enhancer element we have identified in the 5' flanking region of the human VSMC alpha-actin gene involved in mediating VSMC expression. Heterologous pairing experiments have shown that the enhancer does not interact with the basal transcription complex recruited at the minimal SV40 early promoter. Such a vector has direct application in the modulation of VSMC proliferation associated with intimal hyperplasia/restenosis.

  9. Handedness and situs inversus in primary ciliary dyskinesia.

    PubMed Central

    McManus, I. C.; Martin, N.; Stubbings, G. F.; Chung, E. M. K.; Mitchison, H. M.

    2004-01-01

    ...The limbs on the right side are stronger. [The] cause may be ... [that] ... motion, and abilities of moving, are somewhat holpen from the liver, which lieth on the right side. (Sir Francis Bacon, Sylva sylvarum (1627).)Fifty per cent of people with primary ciliary dyskinesia (PCD) (also known as immotile cilia syndrome or Siewert-Kartagener syndrome) have situs inversus, which is thought to result from absent nodal ciliary rotation and failure of normal symmetry breaking. In a study of 88 people with PCD, only 15.2% of 46 individuals with situs inversus, and 14.3% of 42 individuals with situs solitus, were left handed. Because cerebral lateralization is therefore still present, the nodal cilia cannot be the primary mechanism responsible for symmetry breaking in the vertebrate body. Intriguingly, one behavioural lateralization, wearing a wrist-watch on the right wrist, did correlate with situs inversus. PMID:15615683

  10. Handedness and situs inversus in primary ciliary dyskinesia.

    PubMed

    McManus, I C; Martin, N; Stubbings, G F; Chung, E M K; Mitchison, H M

    2004-12-22

    ... The limbs on the right side are stronger. [The] cause may be ... [that] ... motion, and abilities of moving, are somewhat holpen from the liver, which lieth on the right side. (Sir Francis Bacon, Sylva sylvarum (1627).)Fifty per cent of people with primary ciliary dyskinesia (PCD) (also known as immotile cilia syndrome or Siewert-Kartagener syndrome) have situs inversus, which is thought to result from absent nodal ciliary rotation and failure of normal symmetry breaking. In a study of 88 people with PCD, only 15.2% of 46 individuals with situs inversus, and 14.3% of 42 individuals with situs solitus, were left handed. Because cerebral lateralization is therefore still present, the nodal cilia cannot be the primary mechanism responsible for symmetry breaking in the vertebrate body. Intriguingly, one behavioural lateralization, wearing a wrist-watch on the right wrist, did correlate with situs inversus.

  11. Skeletal muscle stem cells from animals I. Basic cell biology

    USDA-ARS?s Scientific Manuscript database

    Skeletal muscle stem cells from food-producing animals have been of interest to agricultural life scientists seeking to develop a better understanding of the molecular regulation of lean tissue (skeletal muscle protein hypertrophy) and intramuscular fat (marbling) development. Enhanced understanding...

  12. A new ex vivo method for the study of nasal drops on ciliary function.

    PubMed

    Levrier, J; Molon-Noblot, S; Duval, D; Lloyd, K G

    1989-01-01

    Any pharmaceutical nasal preparation should respect the physiological function of the mucociliary transport system and should undergo testing to this effect. An experimental protocol has been developed using the guinea pig in order to assess the effects of commercial nasal drop preparations on mucociliary function. The method presented here consists of applying in vivo the test solution on the nasal respiratory epithelium. After a specified contact time and following rapid sacrifice of the animal, the mucosa is removed; the beating frequency of the cilia is then recorded ex vivo by micro-photo-oscillography. The method is sensitive to compounds known to diminish mucociliary function as sodium mercurothiolate inhibits ciliary movement of the nasal epithelium ex vivo. This inhibition of ciliary movement is long-lasting, although reversible. This method can be used to test the action of intranasally administered pharmaceutical preparations on mucociliary function. Commercially available solutions of the nasal vasoconstrictors tymazoline, fenoxazoline or oxymetazoline do not alter ciliary movement ex vivo at dose levels equal to or greater than those clinically utilized. ATP significantly enhances nasal ciliary frequency in instances where a low basal rate occurred. Thus, this method can be used for the testing of the maintenance of nasal ciliary function in the presence of compounds and preparations which will be applied into the nostrils. The advantages over previous techniques include a closer approach to the therapeutic utilization and the maintained physiological conditions of the mucosa during drug administration.

  13. Protein Availability and Satellite Cell Dynamics in Skeletal Muscle.

    PubMed

    Shamim, Baubak; Hawley, John A; Camera, Donny M

    2018-06-01

    Human skeletal muscle satellite cells are activated in response to both resistance and endurance exercise. It was initially proposed that satellite cell proliferation and differentiation were only required to support resistance exercise-induced hypertrophy. However, satellite cells may also play a role in muscle fibre remodelling after endurance-based exercise and extracellular matrix regulation. Given the importance of dietary protein, particularly branched chain amino acids, in supporting myofibrillar and mitochondrial adaptations to both resistance and endurance-based training, a greater understanding of how protein intake impacts satellite cell activity would provide further insight into the mechanisms governing skeletal muscle remodelling with exercise. While many studies have investigated the capacity for protein ingestion to increase post-exercise rates of muscle protein synthesis, few investigations have examined the role for protein ingestion to modulate satellite cell activity. Here we review the molecular mechanisms controlling the activation of satellite cells in response to mechanical stress and protein intake in both in vitro and in vivo models. We provide a mechanistic framework that describes how protein ingestion may enhance satellite activity and promote exercise adaptations in human skeletal muscle.

  14. hemingway is required for sperm flagella assembly and ciliary motility in Drosophila.

    PubMed

    Soulavie, Fabien; Piepenbrock, David; Thomas, Joëlle; Vieillard, Jennifer; Duteyrat, Jean-Luc; Cortier, Elisabeth; Laurençon, Anne; Göpfert, Martin C; Durand, Bénédicte

    2014-04-01

    Cilia play major functions in physiology and development, and ciliary dysfunctions are responsible for several diseases in humans called ciliopathies. Cilia motility is required for cell and fluid propulsion in organisms. In humans, cilia motility deficiencies lead to primary ciliary dyskinesia, with upper-airways recurrent infections, left-right asymmetry perturbations, and fertility defects. In Drosophila, we identified hemingway (hmw) as a novel component required for motile cilia function. hmw encodes a 604-amino acid protein characterized by a highly conserved coiled-coil domain also found in the human orthologue, KIAA1430. We show that HMW is conserved in species with motile cilia and that, in Drosophila, hmw is expressed in ciliated sensory neurons and spermatozoa. We created hmw-knockout flies and found that they are hearing impaired and male sterile. hmw is implicated in the motility of ciliated auditory sensory neurons and, in the testis, is required for elongation and maintenance of sperm flagella. Because HMW is absent from mature flagella, we propose that HMW is not a structural component of the motile axoneme but is required for proper acquisition of motile properties. This identifies HMW as a novel, evolutionarily conserved component necessary for motile cilium function and flagella assembly.

  15. Development and epithelial organisation of muscle cells in the sea anemone Nematostella vectensis.

    PubMed

    Jahnel, Stefan M; Walzl, Manfred; Technau, Ulrich

    2014-01-01

    Nematostella vectensis, a member of the cnidarian class Anthozoa, has been established as a promising model system in developmental biology, but while information about the genetic regulation of embryonic development is rapidly increasing, little is known about the cellular organization of the various cell types in the adult. Here, we studied the anatomy and development of the muscular system of N. vectensis to obtain further insights into the evolution of muscle cells. The muscular system of N. vectensis is comprised of five distinct muscle groups, which are differentiated into a tentacle and a body column system. Both systems house longitudinal as well as circular portions. With the exception of the ectodermal tentacle longitudinal muscle, all muscle groups are of endodermal origin. The shape and epithelial organization of muscle cells vary considerably between different muscle groups. Ring muscle cells are formed as epitheliomuscular cells in which the myofilaments are housed in the basal part of the cell, while the apical part is connected to neighboring cells by apical cell-cell junctions. In the longitudinal muscles of the column, the muscular part at the basal side is connected to the apical part by a long and narrow cytoplasmic bridge. The organization of these cells, however, remains epitheliomuscular. A third type of muscle cell is represented in the longitudinal muscle of the tentacle. Using transgenic animals we show that the apical cell-cell junctions are lost during differentiation, resulting in a detachment of the muscle cells to a basiepithelial position. These muscle cells are still located within the epithelium and outside of the basal matrix, therefore constituting basiepithelial myocytes. We demonstrate that all muscle cells, including the longitudinal basiepithelial muscle cells of the tentacle, initially differentiate from regular epithelial cells before they alter their epithelial organisation. A wide range of different muscle cell

  16. Centrosomal protein Dzip1l binds Cby, promotes ciliary bud formation, and acts redundantly with Bromi to regulate ciliogenesis in the mouse.

    PubMed

    Wang, Chengbing; Li, Jia; Takemaru, Ken-Ichi; Jiang, Xiaogang; Xu, Guoqiang; Wang, Baolin

    2018-03-15

    The primary cilium is a microtubule-based organelle required for Hedgehog (Hh) signaling and consists of a basal body, a ciliary axoneme and a compartment between the first two structures, called the transition zone (TZ). The TZ serves as a gatekeeper to control protein composition in cilia, but less is known about its role in ciliary bud formation. Here, we show that centrosomal protein Dzip1l is required for Hh signaling between Smoothened and Sufu. Dzip1l colocalizes with basal body appendage proteins and Rpgrip1l, a TZ protein. Loss of Dzip1l results in reduced ciliogenesis and dysmorphic cilia in vivo Dzip1l interacts with, and acts upstream of, Cby, an appendage protein, in ciliogenesis. Dzip1l also has overlapping functions with Bromi (Tbc1d32) in ciliogenesis, cilia morphogenesis and neural tube patterning. Loss of Dzip1l arrests ciliogenesis at the stage of ciliary bud formation from the TZ. Consistent with this, Dzip1l mutant cells fail to remove the capping protein Cp110 (Ccp110) from the distal end of mother centrioles and to recruit Rpgrip1l to the TZ. Therefore, Dzip1l promotes ciliary bud formation and is required for the integrity of the TZ. © 2018. Published by The Company of Biologists Ltd.

  17. Requirement of myomaker-mediated stem cell fusion for skeletal muscle hypertrophy

    PubMed Central

    Goh, Qingnian; Millay, Douglas P

    2017-01-01

    Fusion of skeletal muscle stem/progenitor cells is required for proper development and regeneration, however the significance of this process during adult muscle hypertrophy has not been explored. In response to muscle overload after synergist ablation in mice, we show that myomaker, a muscle specific membrane protein essential for myoblast fusion, is activated mainly in muscle progenitors and not myofibers. We rendered muscle progenitors fusion-incompetent through genetic deletion of myomaker in muscle stem cells and observed a complete reduction of overload-induced hypertrophy. This blunted hypertrophic response was associated with a reduction in Akt and p70s6k signaling and protein synthesis, suggesting a link between myonuclear accretion and activation of pro-hypertrophic pathways. Furthermore, fusion-incompetent muscle exhibited increased fibrosis after muscle overload, indicating a protective role for normal stem cell activity in reducing myofiber strain associated with hypertrophy. These findings reveal an essential contribution of myomaker-mediated stem cell fusion during physiological adult muscle hypertrophy. DOI: http://dx.doi.org/10.7554/eLife.20007.001 PMID:28186492

  18. Clinical and Genetic Aspects of Primary Ciliary Dyskinesia / Kartagener Syndrome

    PubMed Central

    Leigh, Margaret W.; Pittman, Jessica E.; Carson, Johnny L.; Ferkol, Thomas W.; Dell, Sharon D.; Davis, Stephanie D.; Knowles, Michael R.; Zariwala, Maimoona A.

    2013-01-01

    Primary ciliary dyskinesia (PCD) is a genetically heterogeneous disorder of motile cilia. Most of the disease-causing mutations identified to date involve the heavy (DNAH5) or intermediate (DNAI1) chain dynein genes in ciliary outer dynein arms, although a few mutations have been noted in other genes. Clinical molecular genetic testing for PCD is available for the most common mutations. The respiratory manifestations of PCD (chronic bronchitis leading to bronchiectasis, chronic rhino-sinusitis and chronic otitis media) reflect impaired mucociliary clearance owing to defective axonemal structure. Ciliary ultrastructural analysis in most patients (>80%) reveals defective dynein arms, although defects in other axonemal components have also been observed. Approximately 50% of PCD patients have laterality defects (including situs inversus totalis and, less commonly, heterotaxy and congenital heart disease), reflecting dysfunction of embryological nodal cilia. Male infertility is common and reflects defects in sperm tail axonemes. Most PCD patients have a history of neonatal respiratory distress, suggesting that motile cilia play a role in fluid clearance during the transition from a fetal to neonatal lung. Ciliopathies involving sensory cilia, including autosomal dominant or recessive polycystic kidney disease, Bardet-Biedl syndrome, and Alstrom syndrome, may have chronic respiratory symptoms and even bronchiectasis suggesting clinical overlap with PCD. PMID:19606528

  19. Stem cell antigen-1 in skeletal muscle function.

    PubMed

    Bernstein, Harold S; Samad, Tahmina; Cholsiripunlert, Sompob; Khalifian, Saami; Gong, Wenhui; Ritner, Carissa; Aurigui, Julian; Ling, Vivian; Wilschut, Karlijn J; Bennett, Stephen; Hoffman, Julien; Oishi, Peter

    2013-08-15

    Stem cell antigen-1 (Sca-1) is a member of the Ly-6 multigene family encoding highly homologous, glycosyl-phosphatidylinositol-anchored membrane proteins. Sca-1 is expressed on muscle-derived stem cells and myogenic precursors recruited to sites of muscle injury. We previously reported that inhibition of Sca-1 expression stimulated myoblast proliferation in vitro and regulated the tempo of muscle repair in vivo. Despite its function in myoblast expansion during muscle repair, a role for Sca-1 in normal, post-natal muscle has not been thoroughly investigated. We systematically compared Sca-1-/- (KO) and Sca-1+/+ (WT) mice and hindlimb muscles to elucidate the tissue, contractile, and functional effects of Sca-1 in young and aging animals. Comparison of muscle volume, fibrosis, myofiber cross-sectional area, and Pax7+ myoblast number showed little differences between ages or genotypes. Exercise protocols, however, demonstrated decreased stamina in KO versus WT mice, with young KO mice achieving results similar to aging WT animals. In addition, KO mice did not improve with practice, while WT animals demonstrated conditioning over time. Surprisingly, myomechanical analysis of isolated muscles showed that KO young muscle generated more force and experienced less fatigue. However, KO muscle also demonstrated incomplete relaxation with fatigue. These findings suggest that Sca-1 is necessary for muscle conditioning with exercise, and that deficient conditioning in Sca-1 KO animals becomes more pronounced with age.

  20. Chloral hydrate alters the organization of the ciliary basal apparatus and cell organelles in sea urchin embryos

    NASA Technical Reports Server (NTRS)

    Chakrabarti, A.; Schatten, H.; Mitchell, K. D.; Crosser, M.; Taylor, M.

    1998-01-01

    The mitotic inhibitor, chloral hydrate, induces ciliary loss in the early embryo phase of Lytechinus pictus. It causes a breakdown of cilia at the junction of the cilium and the basal body known as the basal plate. This leaves the plasma membrane temporarily unsealed. The basal apparatus accessory structures, consisting of the basal body, basal foot, basal foot cap, striated side arm, and striated rootlet, are either misaligned or disintegrated by treatment with chloral hydrate. Furthermore, microtubules which are associated with the basal apparatus are disassembled. Mitochondria accumulate at the base of cilia - underneath the plasma membrane - and show alterations in their structural organization. The accumulation of mitochondria is observed in 40% of all electron micrograph sections while 60% show the areas mostly devoid of mitochondria. The microvilli surrounding a cilium and striated rootlet remain intact in the presence of chloral hydrate. These results suggest that deciliation in early sea urchin embryos by chloral hydrate is caused by combined effects on the ciliary membrane and on microtubules in the cilia. Furthermore, it is suggested that chloral hydrate can serve as a tool to explore the cytoskeletal mechanisms that are involved in cilia motility in the developing sea urchin embryo.

  1. Regulation of polycystin-1 ciliary trafficking by motifs at its C-terminus and polycystin-2 but not by cleavage at the GPS site

    PubMed Central

    Su, Xuefeng; Wu, Maoqing; Yao, Gang; El-Jouni, Wassim; Luo, Chong; Tabari, Azadeh; Zhou, Jing

    2015-01-01

    ABSTRACT Failure to localize membrane proteins to the primary cilium causes a group of diseases collectively named ciliopathies. Polycystin-1 (PC1, also known as PKD1) is a large ciliary membrane protein defective in autosomal dominant polycystic kidney disease (ADPKD). Here, we developed a large set of PC1 expression constructs and identified multiple sequences, including a coiled-coil motif in the C-terminal tail of PC1, regulating full-length PC1 trafficking to the primary cilium. Ciliary trafficking of wild-type and mutant PC1 depends on the dose of polycystin-2 (PC2, also known as PKD2), and the formation of a PC1–PC2 complex. Modulation of the ciliary trafficking module mediated by the VxP ciliary-targeting sequence and Arf4 and Asap1 does not affect the ciliary localization of full-length PC1. PC1 also promotes PC2 ciliary trafficking. PC2 mutations truncating its C-terminal tail but not those changing the VxP sequence to AxA or impairing the pore of the channel, leading to a dead channel, affect PC1 ciliary trafficking. Cleavage at the GPCR proteolytic site (GPS) of PC1 is not required for PC1 trafficking to cilia. We propose a mutually dependent model for the ciliary trafficking of PC1 and PC2, and that PC1 ciliary trafficking is regulated by multiple cis-acting elements. As all pathogenic PC1 mutations tested here are defective in ciliary trafficking, ciliary trafficking might serve as a functional read-out for ADPKD. PMID:26430213

  2. Primary ciliary dyskinesia: current state of the art

    PubMed Central

    Bush, Andrew; Chodhari, Rahul; Collins, Nicola; Copeland, Fiona; Hall, Pippa; Harcourt, Jonny; Hariri, Mohamed; Hogg, Claire; Lucas, Jane; Mitchison, Hannah M; O'Callaghan, Christopher; Phillips, Gill

    2007-01-01

    Primary ciliary dyskinesia (PCD) is usually inherited as an autosomal recessive disorder and presents with upper and lower respiratory tract infection, and mirror image arrangement in around 50% of cases. Cilia dysfunction is also implicated in a wider spectrum of disease, including polycystic liver and kidney disease, central nervous system problems including retinopathy and hydrocephalus, and biliary atresia. Cilia are complex structures, containing more than 250 proteins; recent studies have begun to locate PCD genes scattered throughout the genome. Screening tests for PCD include nasal nitric oxide and in vivo tests of ciliary motility such as the saccharin test. Specific diagnosis requires examination of cilia by light and electron microscopy, with epithelial culture in doubtful cases. This is only available in supra‐regional centres, recently centrally funded by the National Commissioning Group. Treatment is not evidence based and recommendations are largely extrapolated from cystic fibrosis and other suppurative lung diseases. PMID:17634184

  3. Diagnosis of primary ciliary dyskinesia: summary of the ERS Task Force report

    PubMed Central

    Lucas, Jane S.

    2017-01-01

    Key points Primary ciliary dyskinesia (PCD) is a genetically and clinically heterogeneous disease characterised by abnormal motile ciliary function. There is no “gold standard” diagnostic test for PCD. The European Respiratory Society (ERS) Task Force Guidelines for diagnosing PCD recommend that patients should be referred for diagnostic testing if they have several of the following features: persistent wet cough; situs anomalies; congenital cardiac defects; persistent rhinitis; chronic middle ear disease with or without hearing loss; or a history, in term infants, of neonatal upper and lower respiratory symptoms or neonatal intensive care admission. The ERS Task Force recommends that patients should be investigated in a specialist PCD centre with access to a range of complementary tests: nasal nitric oxide, high-speed video microscopy analysis and transmission electron microscopy. Additional tests including immunofluorescence labelling of ciliary proteins and genetic testing may also help determine the diagnosis. Educational aims This article is intended for primary and secondary care physicians interested in primary ciliary dyskinesia (PCD), i.e. those who identify patients for testing, and those involved in diagnosing and managing PCD patients. It aims: to inform readers about the new European Respiratory Society Task Force Guidelines for diagnosing patients with PCDto enable primary and secondary care physicians to: identify patients who need diagnostic testing; understand the diagnostic tests that their patients will undergo, the results of the tests and their limitations; and ensure that appropriate care is subsequently delivered. PMID:28894478

  4. Satellite cell depletion prevents fiber hypertrophy in skeletal muscle.

    PubMed

    Egner, Ingrid M; Bruusgaard, Jo C; Gundersen, Kristian

    2016-08-15

    The largest mammalian cells are the muscle fibers, and they have multiple nuclei to support their large cytoplasmic volumes. During hypertrophic growth, new myonuclei are recruited from satellite stem cells into the fiber syncytia, but it was recently suggested that such recruitment is not obligatory: overload hypertrophy after synergist ablation of the plantaris muscle appeared normal in transgenic mice in which most of the satellite cells were abolished. When we essentially repeated these experiments analyzing the muscles by immunohistochemistry and in vivo and ex vivo imaging, we found that overload hypertrophy was prevented in the satellite cell-deficient mice, in both the plantaris and the extensor digitorum longus muscles. We attribute the previous findings to a reliance on muscle mass as a proxy for fiber hypertrophy, and to the inclusion of a significant number of regenerating fibers in the analysis. We discuss that there is currently no model in which functional, sustainable hypertrophy has been unequivocally demonstrated in the absence of satellite cells; an exception is re-growth, which can occur using previously recruited myonuclei without addition of new myonuclei. © 2016. Published by The Company of Biologists Ltd.

  5. Human skeletal muscle-derived stem cells retain stem cell properties after expansion in myosphere culture

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

    Wei, Yan; Department of Otolaryngology, Head and Neck Surgery, The Third Affiliated Hospital of Sun Yat-sen University, Guang Zhou; Li, Yuan

    2011-04-15

    Human skeletal muscle contains an accessible adult stem-cell compartment in which differentiated myofibers are maintained and replaced by a self-renewing stem cell pool. Previously, studies using mouse models have established a critical role for resident stem cells in skeletal muscle, but little is known about this paradigm in human muscle. Here, we report the reproducible isolation of a population of cells from human skeletal muscle that is able to proliferate for extended periods of time as floating clusters of rounded cells, termed 'myospheres' or myosphere-derived progenitor cells (MDPCs). The phenotypic characteristics and functional properties of these cells were determined usingmore » reverse transcription-polymerase chain reaction (RT-PCR), flow cytometry and immunocytochemistry. Our results showed that these cells are clonogenic, express skeletal progenitor cell markers Pax7, ALDH1, Myod, and Desmin and the stem cell markers Nanog, Sox2, and Oct3/4 significantly elevated over controls. They could be maintained proliferatively active in vitro for more than 20 weeks and passaged at least 18 times, despite an average donor-age of 63 years. Individual clones (4.2%) derived from single cells were successfully expanded showing clonogenic potential and sustained proliferation of a subpopulation in the myospheres. Myosphere-derived cells were capable of spontaneous differentiation into myotubes in differentiation media and into other mesodermal cell lineages in induction media. We demonstrate here that direct culture and expansion of stem cells from human skeletal muscle is straightforward and reproducible with the appropriate technique. These cells may provide a viable resource of adult stem cells for future therapies of disease affecting skeletal muscle or mesenchymal lineage derived cell types.« less

  6. Effect of Cilia Beat Frequency on Muco-ciliary Clearance

    PubMed Central

    Sedaghat, M.H.; Shahmardan, M.M.; Norouzi, M.; Heydari, M.

    2016-01-01

    Background: The airway surface liquid (ASL), which is a fluid layer coating the interior epithelial surface of the bronchi and bronchiolesis, plays an important defensive role against foreign particles and chemicals entering lungs. Objective: Numerical investigation has been employed to solve two-layer model consisting of mucus layer as a viscoelastic fluid and periciliary liquid layer as a Newtonian fluid to study the effects of cilia beat frequency (CBF) at various amounts of mucus properties on muco-ciliary transport problem. Methods: Hybrid finite difference-lattice Boltzmann-method (FB-LBM) has been used to solve the momentum equations and to simulate cilia forces, and also the PCL-mucus interface more accurately, immersed boundary method (IBM) has been employed. The main contribution of the current study is to use an Oldroyd-B model as the constitutive equation of mucus. Results: Our results show that increasing CBF and decreasing mucus viscosity ratio have great effects on mucus flow, but the effect of viscosity ratio is more significant. The results also illustrate that the relation between cilia beat frequency and mean mucus velocity is almost linear and it has similar behavior at different values of viscosity ratio. Conclusion: Numerical investigation based on hybrid IB-FD-LBM has been used to study the effect of CBF at various mounts of mucus viscosity ratio on the muco-ciliary clearance. The results showed that the effect of viscosity ratio on the muco-ciliary transport process is more significant compared with CBF. PMID:28144596

  7. Morphological alterations within the peripheral fixation of the iris dilator muscle in eyes with pigmentary glaucoma.

    PubMed

    Flügel-Koch, Cassandra M; Tektas, Ozan Y; Kaufman, Paul L; Paulsen, Friedrich P; Lütjen-Drecoll, Elke

    2014-06-17

    To analyze the peripheral fixation of the iris dilator muscle in normal eyes and in eyes with pigmentary glaucoma (PG). Using 63 control eyes (age 18 months-99 years), the peripheral iris dilator was investigated by light microscopy, immunohistochemistry, and electron microscopy. Development was studied using 18 differently aged fetal eyes stained immunohistochemically against α-smooth muscle (SM) actin. The peripheral iris dilator muscle in PG was analyzed using semithin and ultrathin sections of six glutaraldehyde-fixed eyes from three donors aged 38, 62, and 74 years. In normal eyes, the peripheral end of the iris dilator muscle is arranged in a sphincter-like manner. Arcade-shaped tendinous connections associated with myofibroblasts (iridial strands) anchor the iris dilator within the elastic-fibromuscular ciliary meshwork that also serves as fixation area for the elastic tendons of the inner ciliary muscle portions. The iridial strands are innervated and can adapt their length during accommodation. The PG eyes show incomplete circular bundles and iridial strands that are mainly anchored to the iris stroma and the flexible uveal parts of the trabecular meshwork. The normal anchorage of the peripheral iris dilator and its presumably neuronally regulated length adaptation stabilize the peripheral iris during accommodation. Insufficient fixation in PG could promote posterior bowing of the iris with rubbing against the zonular fibers and pigment liberation from the iris pigmented epithelium. Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.

  8. Volumetric muscle loss injury repair using in situ fibrin gel cast seeded with muscle-derived stem cells (MDSCs)

    PubMed Central

    Matthias, Nadine; Hunt, Samuel D.; Wu, Jianbo; Lo, Jonathan; Smith Callahan, Laura A.; Li, Yong; Huard, Johnny; Darabi, Radbod

    2018-01-01

    Volumetric muscle defect, caused by trauma or combat injuries, is a major health concern leading to severe morbidity. It is characterized by partial or full thickness loss of muscle and its bio-scaffold, resulting in extensive fibrosis and scar formation. Therefore, the ideal therapeutic option is to use stem cells combined with bio-scaffolds to restore muscle. For this purpose, muscle-derived stem cells (MDSCs) are a great candidate due to their unique multi-lineage differentiation potential. In this study, we evaluated the regeneration potential of MDSCs for muscle loss repair using a novel in situ fibrin gel casting. Muscle defect was created by a partial thickness wedge resection in the tibialis anterior (TA)muscles of NSG mice which created an average of 25% mass loss. If untreated, this defect leads to severe muscle fibrosis. Next, MDSCs were delivered using a novel in situ fibrin gel casting method. Our results demonstrated MDSCs are able to engraft and form new myofibers in the defect when casted along with fibrin gel. LacZ labeled MDSCs were able to differentiate efficiently into new myofibers and significantly increase muscle mass. This was also accompanied by significant reduction of fibrotic tissue in the engrafted muscles. Furthermore, transplanted cells also contributed to new vessel formation and satellite cell seeding. These results confirmed the therapeutic potential of MDSCs and feasibility of direct in situ casting of fibrin/MDSC mixture to repair muscle mass defects. PMID:29331939

  9. Vinpocetine Attenuates the Osteoblastic Differentiation of Vascular Smooth Muscle Cells

    PubMed Central

    Chen, Xiu-Juan; Wang, Na; Yi, Peng-Fei; Song, Min; Zhang, Bo; Wang, Yu-Zhong; Liang, Qiu-Hua

    2016-01-01

    Vascular calcification is an active process of osteoblastic differentiation of vascular smooth muscle cells; however, its definite mechanism remains unknown. Vinpocetine, a derivative of the alkaloid vincamine, has been demonstrated to inhibit the high glucose-induced proliferation of vascular smooth muscle cells; however, it remains unknown whether vinpocetine can affect the osteoblastic differentiation of vascular smooth muscle cells. We hereby investigated the effect of vinpocetine on vascular calcification using a beta-glycerophosphate-induced cell model. Our results showed that vinpocetine significantly reduced the osteoblast-like phenotypes of vascular smooth muscle cells including ALP activity, osteocalcin, collagen type I, Runx2 and BMP-2 expression as well as the formation of mineralized nodule. Vinpocetine, binding to translocation protein, induced phosphorylation of extracellular signal-related kinase and Akt and thus inhibited the translocation of nuclear factor-kappa B into the nucleus. Silencing of translocator protein significantly attenuated the inhibitory effect of vinpocetine on osteoblastic differentiation of vascular smooth muscle cells. Taken together, vinpocetine may be a promising candidate for the clinical therapy of vascular calcification. PMID:27589055

  10. Vinpocetine Attenuates the Osteoblastic Differentiation of Vascular Smooth Muscle Cells.

    PubMed

    Ma, Yun-Yun; Sun, Lin; Chen, Xiu-Juan; Wang, Na; Yi, Peng-Fei; Song, Min; Zhang, Bo; Wang, Yu-Zhong; Liang, Qiu-Hua

    2016-01-01

    Vascular calcification is an active process of osteoblastic differentiation of vascular smooth muscle cells; however, its definite mechanism remains unknown. Vinpocetine, a derivative of the alkaloid vincamine, has been demonstrated to inhibit the high glucose-induced proliferation of vascular smooth muscle cells; however, it remains unknown whether vinpocetine can affect the osteoblastic differentiation of vascular smooth muscle cells. We hereby investigated the effect of vinpocetine on vascular calcification using a beta-glycerophosphate-induced cell model. Our results showed that vinpocetine significantly reduced the osteoblast-like phenotypes of vascular smooth muscle cells including ALP activity, osteocalcin, collagen type I, Runx2 and BMP-2 expression as well as the formation of mineralized nodule. Vinpocetine, binding to translocation protein, induced phosphorylation of extracellular signal-related kinase and Akt and thus inhibited the translocation of nuclear factor-kappa B into the nucleus. Silencing of translocator protein significantly attenuated the inhibitory effect of vinpocetine on osteoblastic differentiation of vascular smooth muscle cells. Taken together, vinpocetine may be a promising candidate for the clinical therapy of vascular calcification.

  11. Whole-Organism Developmental Expression Profiling Identifies RAB-28 as a Novel Ciliary GTPase Associated with the BBSome and Intraflagellar Transport

    PubMed Central

    Sanders, Anna A. W. M.; Li, Chunmei; Kennedy, Julie; Cai, Jerry; Scheidel, Noemie; Kennedy, Breandán N.; Morin, Ryan D.; Leroux, Michel R.; Blacque, Oliver E.

    2016-01-01

    Primary cilia are specialised sensory and developmental signalling devices extending from the surface of most eukaryotic cells. Defects in these organelles cause inherited human disorders (ciliopathies) such as retinitis pigmentosa and Bardet-Biedl syndrome (BBS), frequently affecting many physiological and developmental processes across multiple organs. Cilium formation, maintenance and function depend on intracellular transport systems such as intraflagellar transport (IFT), which is driven by kinesin-2 and IFT-dynein motors and regulated by the Bardet-Biedl syndrome (BBS) cargo-adaptor protein complex, or BBSome. To identify new cilium-associated genes, we employed the nematode C. elegans, where ciliogenesis occurs within a short timespan during late embryogenesis when most sensory neurons differentiate. Using whole-organism RNA-Seq libraries, we discovered a signature expression profile highly enriched for transcripts of known ciliary proteins, including FAM-161 (FAM161A orthologue), CCDC-104 (CCDC104), and RPI-1 (RP1/RP1L1), which we confirm are cilium-localised in worms. From a list of 185 candidate ciliary genes, we uncover orthologues of human MAP9, YAP, CCDC149, and RAB28 as conserved cilium-associated components. Further analyses of C. elegans RAB-28, recently associated with autosomal-recessive cone-rod dystrophy, reveal that this small GTPase is exclusively expressed in ciliated neurons where it dynamically associates with IFT trains. Whereas inactive GDP-bound RAB-28 displays no IFT movement and diffuse localisation, GTP-bound (activated) RAB-28 concentrates at the periciliary membrane in a BBSome-dependent manner and undergoes bidirectional IFT. Functional analyses reveal that whilst cilium structure, sensory function and IFT are seemingly normal in a rab-28 null allele, overexpression of predicted GDP or GTP locked variants of RAB-28 perturbs cilium and sensory pore morphogenesis and function. Collectively, our findings present a new approach for

  12. Whole-Organism Developmental Expression Profiling Identifies RAB-28 as a Novel Ciliary GTPase Associated with the BBSome and Intraflagellar Transport.

    PubMed

    Jensen, Victor L; Carter, Stephen; Sanders, Anna A W M; Li, Chunmei; Kennedy, Julie; Timbers, Tiffany A; Cai, Jerry; Scheidel, Noemie; Kennedy, Breandán N; Morin, Ryan D; Leroux, Michel R; Blacque, Oliver E

    2016-12-01

    Primary cilia are specialised sensory and developmental signalling devices extending from the surface of most eukaryotic cells. Defects in these organelles cause inherited human disorders (ciliopathies) such as retinitis pigmentosa and Bardet-Biedl syndrome (BBS), frequently affecting many physiological and developmental processes across multiple organs. Cilium formation, maintenance and function depend on intracellular transport systems such as intraflagellar transport (IFT), which is driven by kinesin-2 and IFT-dynein motors and regulated by the Bardet-Biedl syndrome (BBS) cargo-adaptor protein complex, or BBSome. To identify new cilium-associated genes, we employed the nematode C. elegans, where ciliogenesis occurs within a short timespan during late embryogenesis when most sensory neurons differentiate. Using whole-organism RNA-Seq libraries, we discovered a signature expression profile highly enriched for transcripts of known ciliary proteins, including FAM-161 (FAM161A orthologue), CCDC-104 (CCDC104), and RPI-1 (RP1/RP1L1), which we confirm are cilium-localised in worms. From a list of 185 candidate ciliary genes, we uncover orthologues of human MAP9, YAP, CCDC149, and RAB28 as conserved cilium-associated components. Further analyses of C. elegans RAB-28, recently associated with autosomal-recessive cone-rod dystrophy, reveal that this small GTPase is exclusively expressed in ciliated neurons where it dynamically associates with IFT trains. Whereas inactive GDP-bound RAB-28 displays no IFT movement and diffuse localisation, GTP-bound (activated) RAB-28 concentrates at the periciliary membrane in a BBSome-dependent manner and undergoes bidirectional IFT. Functional analyses reveal that whilst cilium structure, sensory function and IFT are seemingly normal in a rab-28 null allele, overexpression of predicted GDP or GTP locked variants of RAB-28 perturbs cilium and sensory pore morphogenesis and function. Collectively, our findings present a new approach for

  13. Variation of Ciliary Beat Pattern in Three Different Beating Planes in Healthy Subjects.

    PubMed

    Kempeneers, Celine; Seaton, Claire; Chilvers, Mark A

    2017-05-01

    Digital high-speed video microscopy (DHSV) allows analysis of ciliary beat frequency (CBF) and ciliary beat pattern (CBP) of respiratory cilia in three planes. Normal reference data use a sideways edge to evaluate ciliary dyskinesia and calculate CBF using the time needed for a cilium to complete 10 beat cycles. Variability in CBF within the respiratory epithelium has been described, but data concerning variation of CBP is limited in healthy epithelium. This study aimed to document variability of CBP in normal samples, to compare ciliary function in three profiles, and to compare CBF calculated over five or 10 beat cycles. Nasal brushing samples from 13 healthy subjects were recorded using DHSV in three profiles. CBP and CBF over a 10-beat cycle were evaluated in all profiles, and CBF was reevaluated over five-beat cycles in the sideways edges. A uniform CBP was seen in 82.1% of edges. In the sideways profile, uniformity within the edge was lower (uniform normal CBP, 69.1% [sideways profile]; 97.1% [toward the observer], 92.0% [from above]), and dyskinesia was higher. Interobserver agreement for dyskinesia was poor. CBF was not different between profiles (P = .8097) or between 10 and five beat cycles (P = .1126). Our study demonstrates a lack of uniformity and consistency in manual CBP analysis of samples from healthy subjects, emphasizing the risk of automated CBP analysis in limited regions of interest and of single and limited manual CBP analysis. The toward the observer and from above profiles may be used to calculate CBF but may be less sensitive for evaluation of ciliary dyskinesia and CBP. CBF can be measured reliably by evaluation of only five-beat cycles. Copyright © 2016 American College of Chest Physicians. Published by Elsevier Inc. All rights reserved.

  14. Regulation of polycystin-1 ciliary trafficking by motifs at its C-terminus and polycystin-2 but not by cleavage at the GPS site.

    PubMed

    Su, Xuefeng; Wu, Maoqing; Yao, Gang; El-Jouni, Wassim; Luo, Chong; Tabari, Azadeh; Zhou, Jing

    2015-11-15

    Failure to localize membrane proteins to the primary cilium causes a group of diseases collectively named ciliopathies. Polycystin-1 (PC1, also known as PKD1) is a large ciliary membrane protein defective in autosomal dominant polycystic kidney disease (ADPKD). Here, we developed a large set of PC1 expression constructs and identified multiple sequences, including a coiled-coil motif in the C-terminal tail of PC1, regulating full-length PC1 trafficking to the primary cilium. Ciliary trafficking of wild-type and mutant PC1 depends on the dose of polycystin-2 (PC2, also known as PKD2), and the formation of a PC1-PC2 complex. Modulation of the ciliary trafficking module mediated by the VxP ciliary-targeting sequence and Arf4 and Asap1 does not affect the ciliary localization of full-length PC1. PC1 also promotes PC2 ciliary trafficking. PC2 mutations truncating its C-terminal tail but not those changing the VxP sequence to AxA or impairing the pore of the channel, leading to a dead channel, affect PC1 ciliary trafficking. Cleavage at the GPCR proteolytic site (GPS) of PC1 is not required for PC1 trafficking to cilia. We propose a mutually dependent model for the ciliary trafficking of PC1 and PC2, and that PC1 ciliary trafficking is regulated by multiple cis-acting elements. As all pathogenic PC1 mutations tested here are defective in ciliary trafficking, ciliary trafficking might serve as a functional read-out for ADPKD. © 2015. Published by The Company of Biologists Ltd.

  15. Interactions between neutrophils and macrophages promote macrophage killing of rat muscle cells in vitro

    NASA Technical Reports Server (NTRS)

    Nguyen, Hal X.; Tidball, James G.

    2003-01-01

    Current evidence indicates that the physiological functions of inflammatory cells are highly sensitive to their microenvironment, which is partially determined by the inflammatory cells and their potential targets. In the present investigation, interactions between neutrophils, macrophages and muscle cells that may influence muscle cell death are examined. Findings show that in the absence of macrophages, neutrophils kill muscle cells in vitro by superoxide-dependent mechanisms, and that low concentrations of nitric oxide (NO) protect against neutrophil-mediated killing. In the absence of neutrophils, macrophages kill muscle cells through a NO-dependent mechanism, and the presence of target muscle cells causes a three-fold increase in NO production by macrophages, with no change in the concentration of inducible nitric oxide synthase. Muscle cells that are co-cultured with both neutrophils and macrophages in proportions that are observed in injured muscle show cytotoxicity through a NO-dependent, superoxide-independent mechanism. Furthermore, the concentration of myeloid cells that is necessary for muscle killing is greatly reduced in assays that use mixed myeloid cell populations, rather than uniform populations of neutrophils or macrophages. These findings collectively show that the magnitude and mechanism of muscle cell killing by myeloid cells are modified by interactions between muscle cells and neutrophils, between muscle cells and macrophages and between macrophages and neutrophils.

  16. The molecular signature of muscle stem cells is driven by nutrient availability and innate cell metabolism.

    PubMed

    Ryall, James G; Lynch, Gordon S

    2018-07-01

    To discuss how innate muscle stem-cell metabolism and nutrient availability can provide temporal regulation of chromatin accessibility and transcription. Fluorescence-activated cell sorting coupled with whole transcriptome sequencing revealed for the first time that quiescent and proliferating skeletal muscle stem cells exhibit a process of metabolic reprogramming, from fatty-acid oxidation during quiescence to glycolysis during proliferation. Using a combination of immunofluorescence and chromatin immunoprecipitation sequencing, this shift in metabolism has been linked to altered availability of key metabolites essential for histone (de)acetylation and (de)methylation, including acetyl-CoA, s-adenosylmethionine and α-ketoglutarate. Importantly, these changes in metabolite availability have been linked to muscle stem-cell function. Together, these results provide greater insight into how muscle stem cells interact with their local environment, with important implications for metabolic diseases, skeletal muscle regeneration and cell-transplantation therapies.

  17. Nutrition and muscle catabolism in maintenance hemodialysis: does feeding make muscle cells selective self-eaters?

    PubMed

    Franch, Harold A

    2009-01-01

    Efforts to build muscle by increased protein feeding in hemodialysis patients have been thwarted by parallel increases in both muscle protein synthesis and degradation. The evidence suggests that muscle cells replace older proteins in response to feeding rather than using new proteins to drive muscle cell hypertrophy. This review presents the hypothesis that protein feeding provides an opportunity for muscle to accelerate proteolysis of proteins that have been damaged by oxidation, nitrosylation, and/or glycosylation and to replace damaged mitochondria that contribute to oxidative stress. Increases in proteolysis with feeding are driven by insulin resistance and the increased oxidative stress of mitochondrial respiration. Oxidized proteins and organelles are excellent substrates for degradation by the proteasome, macroautophagy, and chaperone-mediated autophagy: these systems of proteolysis seem to be activated by oxydatiative stress. Replacement of oxidized and other damaged proteins may be a benefit of protein feeding in hemodialysis, but alternative strategies, including exercise, will be required to build muscle.

  18. Nutrition and Muscle Catabolism in Maintenance Hemodialysis: Does Feeding Make Muscle Cells Selective Self-Eaters?

    PubMed Central

    Franch, Harold A.

    2009-01-01

    Efforts to build muscle by increased protein feeding in hemodialysis patients have been thwarted by parallel increases in both muscle protein synthesis and degradation. The evidence suggests that muscle cells replace older proteins in response to feeding rather than using new proteins to drive muscle cell hypertrophy. This review presents the hypothesis that protein feeding provides an opportunity for muscle to accelerate proteolysis of proteins which have been damaged by oxidation, nitrosylation and/or glycosylation and to replace damaged mitochondria that contribute to oxidative stress. Increases in proteolysis with feeding are driven by insulin resistance and the increased oxidative stress of mitochondrial respiration. Oxidized proteins and organelles are excellent substrates for degradation by the proteasome, macroautophagy, and chaperone-mediated autophagy: these systems of proteolysis seem to be activated by oxydatiative stress. Replacement of oxidized and other damaged proteins may be a benefit of protein feeding in hemodialysis, but alternative strategies, including exercise, will be required to build muscle. PMID:19121779

  19. Gene therapy and tissue engineering based on muscle-derived stem cells.

    PubMed

    Deasy, Bridget M; Huard, Johnny

    2002-08-01

    Skeletal muscle represents a convenient source of stem cells for cell-based tissue and genetic engineering. Muscle-derived stem cells (MDSCs) exhibit both multipotentiality and self-renewal capabilities, and are considered to be distinct from the well-studied satellite cell, another type of muscle stem cell that is capable of self-renewal and myogenic lineage differentiation. The MDSC appears to have less restricted differentiation capabilities as compared with the satellite cell, and may be a precursor of the satellite cell. This review considers the evidence for the existence of MDSCs as well as their origin. We will discuss recent investigations highlighting the potential of stem cell transplantation for the treatment of skeletal, cardiac and smooth muscle injuries and disease. We will highlight challenges in bridging the gap between understanding basic stem cell biology and clinical utilization for cell therapy.

  20. Induction of apoptosis by pyrrolidinedithiocarbamate and N-acetylcysteine in vascular smooth muscle cells.

    PubMed

    Tsai, J C; Jain, M; Hsieh, C M; Lee, W S; Yoshizumi, M; Patterson, C; Perrella, M A; Cooke, C; Wang, H; Haber, E; Schlegel, R; Lee, M E

    1996-02-16

    Pyrrolidinedithiocarbamate (PDTC) and N-acetylcysteine (NAC) have been used as antioxidants to prevent apoptosis in lymphocytes, neurons, and vascular endothelial cells. We report here that PDTC and NAC induce apoptosis in rat and human smooth muscle cells. In rat aortic smooth muscle cells, PDTC induced cell shrinkage, chromatin condensation, and DNA strand breaks consistent with apoptosis. In addition, overexpression of Bcl-2 suppressed vascular smooth muscle cell death caused by PDTC and NAC. The viability of rat aortic smooth muscle cells decreased within 3 h of treatment with PDTC and was reduced to 30% at 12 h. The effect of PDTC and NAC on smooth muscle cells was not species specific because PDTC and NAC both caused dose-dependent reductions in viability in rat and human aortic smooth muscle cells. In contrast, neither PDTC nor NAC reduced viability in human aortic endothelial cells. The use of antioxidants to induce apoptosis in vascular smooth muscle cells may help prevent their proliferation in arteriosclerotic lesions.

  1. Embryonic stem cells improve skeletal muscle recovery after extreme atrophy in mice.

    PubMed

    Artioli, Guilherme Giannini; De Oliveira Silvestre, João Guilherme; Guilherme, João Paulo Limongi França; Baptista, Igor Luchini; Ramos, Gracielle Vieira; Da Silva, Willian José; Miyabara, Elen Haruka; Moriscot, Anselmo Sigari

    2015-03-01

    We injected embryonic stem cells into mouse tibialis anterior muscles subjected to botulinum toxin injections as a model for reversible neurogenic atrophy. Muscles were exposed to botulinum toxin for 4 weeks and allowed to recover for up to 6 weeks. At the onset of recovery, a single muscle injection of embryonic stem cells was administered. The myofiber cross-sectional area, single twitch force, peak tetanic force, time-to-peak force, and half-relaxation time were determined. Although the stem cell injection did not affect the myofiber cross-sectional area gain in recovering muscles, most functional parameters improved significantly compared with those of recovering muscles that did not receive the stem cell injection. Muscle function recovery was accelerated by embryonic stem cell delivery in this durable neurogenic atrophy model. We conclude that stem cells should be considered a potential therapeutic tool for recovery after extreme skeletal muscle atrophy. © 2014 Wiley Periodicals, Inc.

  2. Molecular circuitry of stem cell fate in skeletal muscle regeneration, ageing, and disease

    PubMed Central

    Almada, Albert E.; Wagers, Amy J.

    2016-01-01

    Satellite cells are adult myogenic stem cells that function to repair damaged muscle. The enduring capacity for muscle regeneration requires efficient satellite cell expansion after injury, differentiation to produce myoblasts that can reconstitute damaged fibers, and self-renewal to replenish the muscle stem cell pool for subsequent rounds of injury and repair. Emerging studies indicate that misregulations of satellite cell fate and function contribute to age-associated muscle dysfunction and influence the severity of muscle diseases, including Duchenne Muscular Dystrophy (DMD). It has also become apparent that satellite cell fate during muscle regeneration, aging, and in the context of DMD is governed by an intricate network of intrinsic and extrinsic regulators. Targeted manipulation of this network may offer unique opportunities for muscle regenerative medicine. PMID:26956195

  3. Regulation of human airway ciliary beat frequency by intracellular pH

    PubMed Central

    Sutto, Zoltan; Conner, Gregory E; Salathe, Matthias

    2004-01-01

    pHi affects a number of cellular functions, but the influence of pHi on mammalian ciliary beat frequency (CBF) is not known. CBF and pHi of single human tracheobronchial epithelial cells in submerged culture were measured simultaneously using video microscopy (for CBF) and epifluorescence microscopy with the pH-sensitive dye BCECF. Baseline CBF and pHi values in bicarbonate-free medium were 7.2 ± 0.2 Hz and 7.49 ± 0.02, respectively (n = 63). Alkalization by ammonium pre-pulse to pHi 7.78 ± 0.02 resulted in a 2.2 ± 0.1 Hz CBF increase (P < 0.05). Following removal of NH4Cl, pHi decreased to 7.24 ± 0.02 and CBF to 5.8 ± 0.1 Hz (P < 0.05). Removal of extracellular CO2 to change pHi resulted in similar CBF changes. Pre-activation of cAMP-dependent protein kinase (10 μm forskolin), broad inhibition of protein kinases (100 μm H-7), inhibition of PKA (10 μm H-89), nor inhibition of phosphatases (10 μm cyclosporin + 1.5 μm okadaic acid) changed pHi-mediated changes in CBF, nor were they due to [Ca2+]i changes. CBF of basolaterally permeabilized human tracheobronchial cells, re-differentiated at the air–liquid interface, was 3.9 ± 0.3, 5.7 ± 0.4, 7.0 ± 0.3 and 7.3 ± 0.3 Hz at basolateral i.e., intracellular pH of 6.8, 7.2, 7.6 and 8.0, respectively (n = 18). Thus, intracellular alkalization stimulates, while intracellular acidification attenuates human airway CBF. Since phosphorylation and [Ca2+]i changes did not seem to mediate pHi-induced CBF changes, pHi may directly act on the ciliary motile machinery. PMID:15308676

  4. Regulation of the muscle fiber microenvironment by activated satellite cells during hypertrophy

    PubMed Central

    Fry, Christopher S.; Lee, Jonah D.; Jackson, Janna R.; Kirby, Tyler J.; Stasko, Shawn A.; Liu, Honglu; Dupont-Versteegden, Esther E.; McCarthy, John J.; Peterson, Charlotte A.

    2014-01-01

    Our aim in the current study was to determine the necessity of satellite cells for long-term muscle growth and maintenance. We utilized a transgenic Pax7-DTA mouse model, allowing for the conditional depletion of > 90% of satellite cells with tamoxifen treatment. Synergist ablation surgery, where removal of synergist muscles places functional overload on the plantaris, was used to stimulate robust hypertrophy. Following 8 wk of overload, satellite cell-depleted muscle demonstrated an accumulation of extracellular matrix (ECM) and fibroblast expansion that resulted in reduced specific force of the plantaris. Although the early growth response was normal, an attenuation of hypertrophy measured by both muscle wet weight and fiber cross-sectional area occurred in satellite cell-depleted muscle. Isolated primary myogenic progenitor cells (MPCs) negatively regulated fibroblast ECM mRNA expression in vitro, suggesting a novel role for activated satellite cells/MPCs in muscle adaptation. These results provide evidence that satellite cells regulate the muscle environment during growth.—Fry, C. S., Lee, J. D., Jackson, J. R., Kirby, T. J., Stasko, S. A., Liu, H., Dupont-Versteegden, E. E., McCarthy, J. J., Peterson, C. A. Regulation of the muscle fiber microenvironment by activated satellite cells during hypertrophy. PMID:24376025

  5. Injected Human Muscle Precursor Cells Overexpressing PGC-1α Enhance Functional Muscle Regeneration after Trauma

    PubMed Central

    Haralampieva, Deana; Salemi, Souzan; Betzel, Thomas; Dinulovic, Ivana; Krämer, Stefanie D.; Schibli, Roger; Sulser, Tullio; Ametamey, Simon M.

    2018-01-01

    While many groups demonstrated new muscle tissue formation after muscle precursor cell (MPC) injection, the capacity of these cells to heal muscle damage, for example, sphincter in stress urinary incontinence, in long-term is still limited. Therefore, the first goal of our project was to optimize the functional regenerative potential of hMPC by genetic modification to overexpress human peroxisome proliferator-activated receptor gamma coactivator 1-alpha (hPGC-1α), key regulator of exercise-mediated adaptation. Moreover, we aimed at establishing a feasible methodology for noninvasive PET visualization of implanted cells and their microenvironment in muscle crush injury model. PGC-1α-bioengineered muscles showed enhanced marker expression for myogenesis (α-actinin, MyHC, and Desmin), vascularization (VEGF), neuronal (ACHE), and mitochondrial (COXIV) activity. Consistently, use of hPGC-1α_hMPCs produced significantly increased contractile force one to three weeks postinjury. PET imaging showed distinct differences in radiotracer signals ([18F]Fallypride and [11C]Raclopride (both targeting dopamine 2 receptors (D2R)) and [64Cu]NODAGA-RGD (targeting neovascularization)) between GFP_hMPCs and hD2R_hPGC-1α_hMPCs. After muscle harvesting, inflammation levels were in parallel to radiotracer uptake amount, with significantly lower uptake in hPGC-1α overexpressing samples. In summary, we facilitated early functional muscle tissue regeneration, introducing a novel approach to improve skeletal muscle regeneration. Besides successful tracking of hMPCs in muscle crush injuries, we showed that in high-inflammation areas, the specificity of radioligands might be significantly reduced, addressing a possible bottleneck of neovascularization PET imaging. PMID:29531537

  6. Volumetric muscle loss injury repair using in situ fibrin gel cast seeded with muscle-derived stem cells (MDSCs).

    PubMed

    Matthias, Nadine; Hunt, Samuel D; Wu, Jianbo; Lo, Jonathan; Smith Callahan, Laura A; Li, Yong; Huard, Johnny; Darabi, Radbod

    2018-03-01

    Volumetric muscle defect, caused by trauma or combat injuries, is a major health concern leading to severe morbidity. It is characterized by partial or full thickness loss of muscle and its bio-scaffold, resulting in extensive fibrosis and scar formation. Therefore, the ideal therapeutic option is to use stem cells combined with bio-scaffolds to restore muscle. For this purpose, muscle-derived stem cells (MDSCs) are a great candidate due to their unique multi-lineage differentiation potential. In this study, we evaluated the regeneration potential of MDSCs for muscle loss repair using a novel in situ fibrin gel casting. Muscle defect was created by a partial thickness wedge resection in the tibialis anterior (TA) muscles of NSG mice which created an average of 25% mass loss. If untreated, this defect leads to severe muscle fibrosis. Next, MDSCs were delivered using a novel in situ fibrin gel casting method. Our results demonstrated MDSCs are able to engraft and form new myofibers in the defect when casted along with fibrin gel. LacZ labeled MDSCs were able to differentiate efficiently into new myofibers and significantly increase muscle mass. This was also accompanied by significant reduction of fibrotic tissue in the engrafted muscles. Furthermore, transplanted cells also contributed to new vessel formation and satellite cell seeding. These results confirmed the therapeutic potential of MDSCs and feasibility of direct in situ casting of fibrin/MDSC mixture to repair muscle mass defects. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

  7. Smooth muscle-like tissue constructs with circumferentially oriented cells formed by the cell fiber technology.

    PubMed

    Hsiao, Amy Y; Okitsu, Teru; Onoe, Hiroaki; Kiyosawa, Mahiro; Teramae, Hiroki; Iwanaga, Shintaroh; Kazama, Tomohiko; Matsumoto, Taro; Takeuchi, Shoji

    2015-01-01

    The proper functioning of many organs and tissues containing smooth muscles greatly depends on the intricate organization of the smooth muscle cells oriented in appropriate directions. Consequently controlling the cellular orientation in three-dimensional (3D) cellular constructs is an important issue in engineering tissues of smooth muscles. However, the ability to precisely control the cellular orientation at the microscale cannot be achieved by various commonly used 3D tissue engineering building blocks such as spheroids. This paper presents the formation of coiled spring-shaped 3D cellular constructs containing circumferentially oriented smooth muscle-like cells differentiated from dedifferentiated fat (DFAT) cells. By using the cell fiber technology, DFAT cells suspended in a mixture of extracellular proteins possessing an optimized stiffness were encapsulated in the core region of alginate shell microfibers and uniformly aligned to the longitudinal direction. Upon differentiation induction to the smooth muscle lineage, DFAT cell fibers self-assembled to coiled spring structures where the cells became circumferentially oriented. By changing the initial core-shell microfiber diameter, we demonstrated that the spring pitch and diameter could be controlled. 21 days after differentiation induction, the cell fibers contained high percentages of ASMA-positive and calponin-positive cells. Our technology to create these smooth muscle-like spring constructs enabled precise control of cellular alignment and orientation in 3D. These constructs can further serve as tissue engineering building blocks for larger organs and cellular implants used in clinical treatments.

  8. Smooth Muscle-Like Tissue Constructs with Circumferentially Oriented Cells Formed by the Cell Fiber Technology

    PubMed Central

    Hsiao, Amy Y.; Okitsu, Teru; Onoe, Hiroaki; Kiyosawa, Mahiro; Teramae, Hiroki; Iwanaga, Shintaroh; Kazama, Tomohiko; Matsumoto, Taro; Takeuchi, Shoji

    2015-01-01

    The proper functioning of many organs and tissues containing smooth muscles greatly depends on the intricate organization of the smooth muscle cells oriented in appropriate directions. Consequently controlling the cellular orientation in three-dimensional (3D) cellular constructs is an important issue in engineering tissues of smooth muscles. However, the ability to precisely control the cellular orientation at the microscale cannot be achieved by various commonly used 3D tissue engineering building blocks such as spheroids. This paper presents the formation of coiled spring-shaped 3D cellular constructs containing circumferentially oriented smooth muscle-like cells differentiated from dedifferentiated fat (DFAT) cells. By using the cell fiber technology, DFAT cells suspended in a mixture of extracellular proteins possessing an optimized stiffness were encapsulated in the core region of alginate shell microfibers and uniformly aligned to the longitudinal direction. Upon differentiation induction to the smooth muscle lineage, DFAT cell fibers self-assembled to coiled spring structures where the cells became circumferentially oriented. By changing the initial core-shell microfiber diameter, we demonstrated that the spring pitch and diameter could be controlled. 21 days after differentiation induction, the cell fibers contained high percentages of ASMA-positive and calponin-positive cells. Our technology to create these smooth muscle-like spring constructs enabled precise control of cellular alignment and orientation in 3D. These constructs can further serve as tissue engineering building blocks for larger organs and cellular implants used in clinical treatments. PMID:25734774

  9. Fetal adenoma of the pigmented ciliary epithelium associated with persistent hyperplastic primary vitreous.

    PubMed

    Doro, S; Werblin, T P; Haas, B; Iwamoto, T; Jakobiec, F A

    1986-10-01

    A 1.5-year-old girl presented with a peripheral iris mass. When the girl was 3 years old, the lesion was excised after it had manifested significant growth. A stalk of fibrovascular tissue was noted to extend from the lesion to the optic disc. Histopathologically, the tumor was a well-circumscribed, pigmented ciliary body adenoma. Electron microscopy revealed characteristic neuroepithelial melanosomes, distinct from those of choroidal melanocytes, and occasional annulate lamellae. A fibrovascular membrane extended over the tumor surface and was adherent to lens capsule. The association of this adenoma with a persistent stalk of primary vitreous indicates a congenital origin of this tumor. Both adenoma and adenocarcinoma of the pigmented and nonpigmented ciliary epithelium tend to be disorders of adults. The authors report the youngest presentation of a pigment epithelium adenoma, the only well-documented case associated with persistent hyperplastic primary vitreous, and the only documentation of annulate lamellae in a ciliary body tumor.

  10. Lymphatic Muscle Cells in Rat Mesenteric Lymphatic Vessels of Various Ages

    PubMed Central

    Bridenbaugh, Eric A.; Nizamutdinova, Irina Tsoy; Jupiter, Daniel; Nagai, Takashi; Thangaswamy, Sangeetha; Chatterjee, Victor

    2013-01-01

    Abstract Background Recent studies on aging-associated changes in mesenteric lymph flow in situ demonstrated predominance of the severe negative chronotropic effect of aging on the contractility of aged mesenteric lymphatic vessels (MLV). At the same time, contraction amplitude of the aged vessels was only slightly diminished by aging and can be rapidly stimulated within 5–15 minutes. However, the detailed quantitative evaluation of potential aging-associated changes in muscle cells investiture in MLV has never been performed. Methods and Results In this study we, for the first time, performed detailed evaluation of muscle cells investiture in MLV in reference to the position of lymphatic valve in different zones of lymphangion within various age groups (3-mo, 9-mo and 24-mo Fischer-344 rats). Using visual and quantitative analyses of the images of MLV immunohistochemically labeled for actin, we confirmed that the zones located close upstream (pre-valve zones) and above lymphatic valves (valve zones) possess the lowest investiture of lymphatic muscle cells. Most of the high muscle cells investiture zones exist downstream to the lymphatic valve (post-valve zones). The muscle cells investiture of these zones is not affected by aging, while pre-valve and valve zones demonstrate significant aging-associated decrease in muscle cells investiture. Conclusions The low muscle cells investiture zones in lymphatic vessels consist of predominantly longitudinally oriented muscle cells which are positioned in pre-valve and valve zones and connect adjacent lymphangions. These cells may provide important functional impact on the biomechanics of the lymphatic valve gating and electrical coupling between lymphangions, while their aging-associated changes may delimit adaptive reserves of aged lymphatic vessels. PMID:23531183

  11. Quantitative optical coherence tomography imaging of intermediate flow defect phenotypes in ciliary physiology and pathophysiology

    NASA Astrophysics Data System (ADS)

    Huang, Brendan K.; Gamm, Ute A.; Jonas, Stephan; Khokha, Mustafa K.; Choma, Michael A.

    2015-03-01

    Cilia-driven fluid flow is a critical yet poorly understood aspect of pulmonary physiology. Here, we demonstrate that optical coherence tomography-based particle tracking velocimetry can be used to quantify subtle variability in cilia-driven flow performance in Xenopus, an important animal model of ciliary biology. Changes in flow performance were quantified in the setting of normal development, as well as in response to three types of perturbations: mechanical (increased fluid viscosity), pharmacological (disrupted serotonin signaling), and genetic (diminished ciliary motor protein expression). Of note, we demonstrate decreased flow secondary to gene knockdown of kif3a, a protein involved in ciliogenesis, as well as a dose-response decrease in flow secondary to knockdown of dnah9, an important ciliary motor protein.

  12. Aging, metabolism and stem cells: Spotlight on muscle stem cells.

    PubMed

    García-Prat, Laura; Muñoz-Cánoves, Pura

    2017-04-15

    All tissues and organs undergo a progressive regenerative decline as they age. This decline has been mainly attributed to loss of stem cell number and/or function, and both stem cell-intrinsic changes and alterations in local niches and/or systemic environment over time are known to contribute to the stem cell aging phenotype. Advancing in the molecular understanding of the deterioration of stem cell cells with aging is key for targeting the specific causes of tissue regenerative dysfunction at advanced stages of life. Here, we revise exciting recent findings on why stem cells age and the consequences on tissue regeneration, with a special focus on regeneration of skeletal muscle. We also highlight newly identified common molecular pathways affecting diverse types of aging stem cells, such as altered proteostasis, metabolism, or senescence entry, and discuss the questions raised by these findings. Finally, we comment on emerging stem cell rejuvenation strategies, principally emanating from studies on muscle stem cells, which will surely burst tissue regeneration research for future benefit of the increasing human aging population. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  13. Insights into the Structure and Function of Ciliary and Flagellar Doublet Microtubules

    PubMed Central

    Linck, Richard; Fu, Xiaofeng; Lin, Jianfeng; Ouch, Christna; Schefter, Alexandra; Steffen, Walter; Warren, Peter; Nicastro, Daniela

    2014-01-01

    Cilia and flagella are conserved, motile, and sensory cell organelles involved in signal transduction and human disease. Their scaffold consists of a 9-fold array of remarkably stable doublet microtubules (DMTs), along which motor proteins transmit force for ciliary motility and intraflagellar transport. DMTs possess Ribbons of three to four hyper-stable protofilaments whose location, organization, and specialized functions have been elusive. We performed a comprehensive analysis of the distribution and structural arrangements of Ribbon proteins from sea urchin sperm flagella, using quantitative immunobiochemistry, proteomics, immuno-cryo-electron microscopy, and tomography. Isolated Ribbons contain acetylated α-tubulin, β-tubulin, conserved protein Rib45, >95% of the axonemal tektins, and >95% of the calcium-binding proteins, Rib74 and Rib85.5, whose human homologues are related to the cause of juvenile myoclonic epilepsy. DMTs contain only one type of Ribbon, corresponding to protofilaments A11-12-13-1 of the A-tubule. Rib74 and Rib85.5 are associated with the Ribbon in the lumen of the A-tubule. Ribbons contain a single ∼5-nm wide filament, composed of equimolar tektins A, B, and C, which interact with the nexin-dynein regulatory complex. A summary of findings is presented, and the functions of Ribbon proteins are discussed in terms of the assembly and stability of DMTs, ciliary motility, and other microtubule systems. PMID:24794867

  14. Exhaustive exercise--a near death experience for skeletal muscle cells?

    PubMed

    Behringer, Michael; Montag, Johannes; Franz, Alexander; McCourt, Molly L; Mester, Joachim; Nosaka, Kazunori Ken

    2014-12-01

    In sports medicine, muscle enzymes in the blood are frequently used as an indicator of muscle damage. It is commonly assumed that mechanical stress disrupts plasma membrane to an extent that allows large molecules, such as enzymes, to leak into the extracellular space. However, this does not appear to fully explain changes in muscle enzyme activity in the blood after exercise. Apart from this mechanically induced membrane damage, we hypothesize that, under critical metabolic conditions, ATP consuming enzymes like creatine kinase (CK) are "volitionally" expulsed by muscle cells in order to prevent cell death. This would put themselves into a situation comparable to that of CK deficient muscle fibers, which have been shown in animal experiments to be virtually infatigable at the expense of muscle strength. Additionally we expand on this hypothesis with the idea that membrane blebbing is a way for the muscle fibers to store CK in fringe areas of the muscle fiber or to expulse CK from the cytosol by detaching the blebs from the plasma membrane. The blebbing has been shown to occur in heart muscle cells under ischaemic conditions and has been speculated to be an alternative pathway for the expulsion of troponin. The blebbing has also been seen skeletal muscle cells when intracellular calcium concentration increases. Cytoskeletal damage, induced by reactive oxygen species (ROS) or by calcium activated proteases in concert with increasing intracellular pressure, seems to provoke this type of membrane reaction. If these hypotheses are confirmed by future investigations, our current understanding of CK as a blood muscle damage marker will be fundamentally affected. Copyright © 2014 Elsevier Ltd. All rights reserved.

  15. ALS-related misfolded protein management in motor neurons and muscle cells.

    PubMed

    Galbiati, Mariarita; Crippa, Valeria; Rusmini, Paola; Cristofani, Riccardo; Cicardi, Maria Elena; Giorgetti, Elisa; Onesto, Elisa; Messi, Elio; Poletti, Angelo

    2014-12-01

    Amyotrophic Lateral Sclerosis (ALS) is the most common form of adult-onset motor neuron disease. It is now considered a multi-factorial and multi-systemic disorder in which alterations of the crosstalk between neuronal and non-neuronal cell types might influence the course of the disease. In this review, we will provide evidence that dysfunctions of affected muscle cells are not only a marginal consequence of denervation associated to motor neurons loss, but a direct consequence of cell muscle toxicity of mutant SOD1. In muscle, the misfolded state of mutant SOD1 protein, unlike in motor neurons, does not appear to have direct effects on protein aggregation and mitochondrial functionality. Muscle cells are, in fact, more capable than motor neurons to handle misfolded proteins, suggesting that mutant SOD1 toxicity in muscle is not mediated by classical mechanisms of intracellular misfolded proteins accumulation. Several recent works indicate that a higher activation of molecular chaperones and degradative systems is present in muscle cells, which for this reason are possibly able to better manage misfolded mutant SOD1. However, several alterations in gene expression and regenerative potential of skeletal muscles have also been reported as a consequence of the expression of mutant SOD1 in muscle. Whether these changes in muscle cells are causative of ALS or a consequence of motor neuron alterations is not yet clear, but their elucidation is very important, since the understanding of the mechanisms involved in mutant SOD1 toxicity in muscle may facilitate the design of treatments directed toward this specific tissue to treat ALS or at least to delay disease progression. Copyright © 2014 Elsevier Ltd. All rights reserved.

  16. Polymorphisms of alpha-actinin-3 and ciliary neurotrophic factor in national-level Italian athletes.

    PubMed

    Persi, A; Maltese, P E; Bertelli, M; Cecchin, S; Ciaghi, M; Guarnieri, M C; Agnello, L; Maggioni, M A; Merati, G; Veicsteinas, A

    2013-06-01

    The R577X polymorphism of the alpha-actinin-3 (ACTN3) gene and the IVS1-6G>A polymorphism of the ciliary neurotrophic factor (CNTF) gene have been associated with a favourable muscle phenotype (more muscle fibres with high glycolytic activity), reduced predisposition for congenital dystrophy and resistance to sarcopenia in old age. The aim of this study was to look for evidence of selective pressure towards genotypes favourable for strong muscle activity in a sample of national-level Italian athletes. We analysed two stop codon polymorphisms in the DNA of 50 Italian athletes, specialised in power or endurance sports, and compared their genotypic distribution with those of a population of 50 controls. In a representative sub-group of athletes (N.=42) we then compared the genetic data with anaerobic threshold, assessed by an incremental exercise test up to exhaustion. The athlete group showed an allelic distribution of ACTN3 (R/R:64%, R/X:16%, X/X:20%) and CNTF (G/G:72%, G/A:26%, A/A:2%), significantly imbalanced towards alleles R/R and G/G, respectively, compared to controls (ACTN3=R/R:40% R/X:22% X/X:38% and CNTF=G/G:52%, G/A:24%, A/A:24%) (p=0.0024 and p=0.0001, respectively). Only the ACTN3 577X/X polymorphism showed a significant association with the anaerobic threshold of athletes (F-ratio= 4.037; p=0.025). Factorial ANOVA demonstrated a non significant interaction between favourable allelic patterns of ACTN3 and CNTF genes on aerobic performance in the athlete group. The relationship found between favourable muscle phenotype and this genetic profile may have interesting implications in sport performance and training, athlete selection and different clinical activities, such as physical rehabilitation and modifying phenotypes associated with neuromuscular diseases.

  17. Does the disruption of horizontal anterior ciliary vessels affect the blood-aqueous barrier function?

    PubMed

    Pan, Meihua; Yang, Mei; Xie, Renyi; Zhao, Zhimin; Huang, Xingxing

    2017-12-01

    To investigate the significance of the anterior ciliary vessels (ACVs) preservation during the conventional horizontal strabismus surgery. Patients (≥ 8 years) with horizontal strabismus were randomly allocated into group 1 (with ACV preservation) and group 2 (without ACV preservation). The surgical eyes in group 1 were further divided into group A (one rectus muscle operated) and group B (two rectus muscles operated). Similarly, eyes in group 2 were divided into group C (one rectus muscle operated) and group D (two rectus muscles operated). The success rate of ACV preservation was calculated. The anterior chamber flare measurements of each eye by laser flare photometry were recorded on the day prior to and after operation. The flare values between groups and between pre- and post-operation in each group were compared by one-way analysis of variance and a paired t-test respectively. In groups A and B, the success rate of ACV preservation was 82% (27/33) and 70% (28/40)respectively, and the flare values between pre- and post-operation showed no significant differences(4.378 ± 1.527, 4.544 ± 1.452, P = 0.526; 4.625 ± 1.090, 4.989 ± 1.468, P = 0.101 respectively). However, the postoperative values were significantly increased in group C and group D(4.661 ± 1.031, 5.039 ± 1.310, P = 0.025; 4.933 ± 1.691, 5.502 ± 1.430, P = 0.000 respectively). The postoperative flare readings of group D were significantly higher than group B, while group A and group C had no significant variation. ACV preservation probably has clinical significance in reducing the undesirable influence on the blood-aqueous barrier.

  18. Peripheral sensory processing in mammalian gravity receptors - Observations of ciliary tuft configurations

    NASA Technical Reports Server (NTRS)

    Ross, Muriel D.; Donovan, Kathleen; Rogers, Charles

    1987-01-01

    Scanning electron microscopy was used to study dynamic polarizations of clustered cells of the anterior part of rat saccular macula and to shed light on the possible roles of two types of hair cells integrated into the same neural circuitry: those with short stereocilia and long kinocilium (ss/lk), and those with long stereocilia and still longer kinocilium (ls/lk). It was found that the ss/lk-type cells could be further subdivided into two types, whereas the ls/lk cells consisted of four major kinds. It was also found that the kinocilium was most often fixed in a recovery stroke position (curved basally, and the upper portion projected back over the tuft) and that the kinocilia were not aligned in parallel in any given part of a macula, even though each cilium pointed in the proper direction relative to the striola line. The possibility of a relationship between the ciliary tuft morphology and the function of the hair cell of which it is a part is discussed.

  19. PACS-1 Mediates Phosphorylation-Dependent Ciliary Trafficking of the CNG Channel in Olfactory Sensory Neurons

    PubMed Central

    Jenkins, Paul M.; Zhang, Lian; Thomas, Gary; Martens, Jeffrey R.

    2009-01-01

    Impaired ciliary protein transport in olfactory sensory neurons (OSNs) leads to anosmia, and is a newly recognized clinical manifestation of a class of human disorders called ciliopathies. Surprisingly little is known regarding the mechanisms controlling trafficking to this unique neuronal compartment. Here, we show a novel role for phosphofurin acidic cluster-sorting protein 1 (PACS-1) in the ciliary trafficking of the olfactory CNG channel. PACS-1 is an intracellular sorting protein that mediates its effects through the binding of acidic clusters on cargo protein. This interaction is dependent on CK2 phosphorylation of both PACS-1 and its cargo. We show that CNGB1b contains two putative PACS-1 binding sites, which are phosphorylated by the serine/threonine protein kinase, CK2. Additionally, we show that PACS-1 is expressed in OSNs and interacts in complex with the CNG channel. CK2 inhibition in native OSNs causes a loss of CNG channel from cilia and subsequent olfactory dysfunction, while adenoviral expression of mutant PACS-1 causes similar mislocalization. These results provide a mechanism for the subunit-dependent ciliary trafficking of the CNG channel and offer insight into the mechanisms of ciliary transport. PMID:19710307

  20. Sepsis induces long-term metabolic and mitochondrial muscle stem cell dysfunction amenable by mesenchymal stem cell therapy

    PubMed Central

    Rocheteau, P.; Chatre, L.; Briand, D.; Mebarki, M.; Jouvion, G.; Bardon, J.; Crochemore, C.; Serrani, P.; Lecci, P. P.; Latil, M.; Matot, B.; Carlier, P. G.; Latronico, N.; Huchet, C.; Lafoux, A.; Sharshar, T.; Ricchetti, M.; Chrétien, F.

    2015-01-01

    Sepsis, or systemic inflammatory response syndrome, is the major cause of critical illness resulting in admission to intensive care units. Sepsis is caused by severe infection and is associated with mortality in 60% of cases. Morbidity due to sepsis is complicated by neuromyopathy, and patients face long-term disability due to muscle weakness, energetic dysfunction, proteolysis and muscle wasting. These processes are triggered by pro-inflammatory cytokines and metabolic imbalances and are aggravated by malnutrition and drugs. Skeletal muscle regeneration depends on stem (satellite) cells. Herein we show that mitochondrial and metabolic alterations underlie the sepsis-induced long-term impairment of satellite cells and lead to inefficient muscle regeneration. Engrafting mesenchymal stem cells improves the septic status by decreasing cytokine levels, restoring mitochondrial and metabolic function in satellite cells, and improving muscle strength. These findings indicate that sepsis affects quiescent muscle stem cells and that mesenchymal stem cells might act as a preventive therapeutic approach for sepsis-related morbidity. PMID:26666572

  1. Sepsis induces long-term metabolic and mitochondrial muscle stem cell dysfunction amenable by mesenchymal stem cell therapy.

    PubMed

    Rocheteau, P; Chatre, L; Briand, D; Mebarki, M; Jouvion, G; Bardon, J; Crochemore, C; Serrani, P; Lecci, P P; Latil, M; Matot, B; Carlier, P G; Latronico, N; Huchet, C; Lafoux, A; Sharshar, T; Ricchetti, M; Chrétien, F

    2015-12-15

    Sepsis, or systemic inflammatory response syndrome, is the major cause of critical illness resulting in admission to intensive care units. Sepsis is caused by severe infection and is associated with mortality in 60% of cases. Morbidity due to sepsis is complicated by neuromyopathy, and patients face long-term disability due to muscle weakness, energetic dysfunction, proteolysis and muscle wasting. These processes are triggered by pro-inflammatory cytokines and metabolic imbalances and are aggravated by malnutrition and drugs. Skeletal muscle regeneration depends on stem (satellite) cells. Herein we show that mitochondrial and metabolic alterations underlie the sepsis-induced long-term impairment of satellite cells and lead to inefficient muscle regeneration. Engrafting mesenchymal stem cells improves the septic status by decreasing cytokine levels, restoring mitochondrial and metabolic function in satellite cells, and improving muscle strength. These findings indicate that sepsis affects quiescent muscle stem cells and that mesenchymal stem cells might act as a preventive therapeutic approach for sepsis-related morbidity.

  2. The central role of muscle stem cells in regenerative failure with aging

    PubMed Central

    Blau, Helen M; Cosgrove, Benjamin D; Ho, Andrew T V

    2016-01-01

    Skeletal muscle mass, function, and repair capacity all progressively decline with aging, restricting mobility, voluntary function, and quality of life. Skeletal muscle repair is facilitated by a population of dedicated muscle stem cells (MuSCs), also known as satellite cells, that reside in anatomically defined niches within muscle tissues. In adult tissues, MuSCs are retained in a quiescent state until they are primed to regenerate damaged muscle through cycles of self-renewal divisions. With aging, muscle tissue homeostasis is progressively disrupted and the ability of MuSCs to repair injured muscle markedly declines. Until recently, this decline has been largely attributed to extrinsic age-related alterations in the microenvironment to which MuSCs are exposed. However, as highlighted in this Perspective, recent reports show that MuSCs also progressively undergo cell-intrinsic alterations that profoundly affect stem cell regenerative function with aging. A more comprehensive understanding of the interplay of stem cell–intrinsic and extrinsic factors will set the stage for improving cell therapies capable of restoring tissue homeostasis and enhancing muscle repair in the aged. PMID:26248268

  3. Fusogenic micropeptide Myomixer is essential for satellite cell fusion and muscle regeneration.

    PubMed

    Bi, Pengpeng; McAnally, John R; Shelton, John M; Sánchez-Ortiz, Efrain; Bassel-Duby, Rhonda; Olson, Eric N

    2018-04-10

    Regeneration of skeletal muscle in response to injury occurs through fusion of a population of stem cells, known as satellite cells, with injured myofibers. Myomixer, a muscle-specific membrane micropeptide, cooperates with the transmembrane protein Myomaker to regulate embryonic myoblast fusion and muscle formation. To investigate the role of Myomixer in muscle regeneration, we used CRISPR/Cas9-mediated genome editing to generate conditional knockout Myomixer alleles in mice. We show that genetic deletion of Myomixer in satellite cells using a tamoxifen-regulated Cre recombinase transgene under control of the Pax7 promoter abolishes satellite cell fusion and prevents muscle regeneration, resulting in severe muscle degeneration after injury. Satellite cells devoid of Myomixer maintain expression of Myomaker, demonstrating that Myomaker alone is insufficient to drive myoblast fusion. These findings, together with prior studies demonstrating the essentiality of Myomaker for muscle regeneration, highlight the obligatory partnership of Myomixer and Myomaker for myofiber formation throughout embryogenesis and adulthood.

  4. Age-Related Changes in Centripetal Ciliary Body Movement Relative to Centripetal Lens Movement in Monkeys

    PubMed Central

    Croft, Mary Ann; McDonald, Jared P.; Nadkarni, Nivedita V.; Lin, Ting-Li; Kaufman, Paul L.

    2009-01-01

    The goal was to determine the age-related changes in accommodative movements of the lens and ciliary body in rhesus monkeys. Varying levels of accommodation were stimulated via the Edinger-Westphal (E-W) nucleus in 26 rhesus monkeys, aged 6-27 years, and the refractive changes were measured by coincidence refractometry. Centripetal ciliary process (CP) and lens movements were measured by computerized image analysis of goniovideographic images. Ultrasound biomicroscopy (UBM) at 50 MHz was used to visualize and measure accommodative forward movements of the ciliary body in relation to age, accommodative amplitude, and centripetal CP and lens movements. At ∼3 diopters of accommodation, the amount of centripetal lens movement required did not significantly change with age (p=0.10; n=18 monkeys); however, the amount of centripetal CP movement required significantly increased with age (p=0.01; n=18 monkeys), while the amount of forward ciliary body movement significantly decreased with age (p=0.007; n=11 monkeys). In the middle-aged animals (12-16.5 years), a greater amount of centripetal CP movement was required to induce a given level of lens movement and thereby a given level of accommodation (p=0.01), compared to the young animals (6-10 yrs). Collectively, the data suggests that, with age, the accommodative system may be attempting to compensate for the loss of forward ciliary body movement by increasing the amount of centripetal CP movement. This, in turn, would allow enough zonular relaxation to achieve the magnitude of centripetal lens movement necessary for a given amplitude of accommodation. PMID:19635475

  5. Lsd1 regulates skeletal muscle regeneration and directs the fate of satellite cells.

    PubMed

    Tosic, Milica; Allen, Anita; Willmann, Dominica; Lepper, Christoph; Kim, Johnny; Duteil, Delphine; Schüle, Roland

    2018-01-25

    Satellite cells are muscle stem cells required for muscle regeneration upon damage. Of note, satellite cells are bipotent and have the capacity to differentiate not only into skeletal myocytes, but also into brown adipocytes. Epigenetic mechanisms regulating fate decision and differentiation of satellite cells during muscle regeneration are not yet fully understood. Here, we show that elevated levels of lysine-specific demethylase 1 (Kdm1a, also known as Lsd1) have a beneficial effect on muscle regeneration and recovery after injury, since Lsd1 directly regulates key myogenic transcription factor genes. Importantly, selective Lsd1 ablation or inhibition in Pax7-positive satellite cells, not only delays muscle regeneration, but changes cell fate towards brown adipocytes. Lsd1 prevents brown adipocyte differentiation of satellite cells by repressing expression of the novel pro-adipogenic transcription factor Glis1. Together, downregulation of Glis1 and upregulation of the muscle-specific transcription program ensure physiological muscle regeneration.

  6. Chronic inflammation in skeletal muscle impairs satellite cells function during regeneration: can physical exercise restore the satellite cell niche?

    PubMed

    Perandini, Luiz Augusto; Chimin, Patricia; Lutkemeyer, Diego da Silva; Câmara, Niels Olsen Saraiva

    2018-06-01

    Chronic inflammation impairs skeletal muscle regeneration. Although many cells are involved in chronic inflammation, macrophages seem to play an important role in impaired muscle regeneration since these cells are associated with skeletal muscle stem cell (namely, satellite cells) activation and fibro-adipogenic progenitor cell (FAP) survival. Specifically, an imbalance of M1 and M2 macrophages seems to lead to impaired satellite cell activation, and these are the main cells that function during skeletal muscle regeneration, after muscle damage. Additionally, this imbalance leads to the accumulation of FAPs in skeletal muscle, with aberrant production of pro-fibrotic factors (e.g., extracellular matrix components), impairing the niche for proper satellite cell activation and differentiation. Treatments aiming to block the inflammatory pro-fibrotic response are partially effective due to their side effects. Therefore, strategies reverting chronic inflammation into a pro-regenerative pattern are required. In this review, we first describe skeletal muscle resident macrophage ontogeny and homeostasis, and explain how macrophages are replenished after muscle injury. We next discuss the potential role of chronic physical activity and exercise in restoring the M1 and M2 macrophage balance and consequently, the satellite cell niche to improve skeletal muscle regeneration after injury. © 2018 Federation of European Biochemical Societies.

  7. Possible Role of Non-Muscle Alpha-Actinins in Muscle Cell Mechanosensitivity

    PubMed Central

    Ogneva, Irina V.; Biryukov, Nikolay S.; Leinsoo, Toomas A.; Larina, Irina M.

    2014-01-01

    The main hypothesis suggested that changes in the external mechanical load would lead to different deformations of the submembranous cytoskeleton and, as a result, dissociation of different proteins from its structure (induced by increased/decreased mechanical stress). The study subjects were fibers of the soleus muscle and cardiomyocytes of Wistar rats. Changes in external mechanical conditions were reconstructed by means of antiorthostatic suspension of the animals by their tails for 6, 12, 18, 24 and 72 hours. Transversal stiffness was measured by atomic force microscopy imaging; beta-, gamma-actin, alpha-actinin 1 and alpha-actinin 4 levels in membranous and cytoplasmic fractions were quantified by Western blot analysis; expression rates of the corresponding genes were studied using RT-PCR. Results: In 6 hours, alpha-actinin 1 and alpha-actinin 4 levels decreased in the membranous fraction of proteins of cardiomyocytes and soleus muscle fibers, respectively, but increased in the cytoplasmic fraction of the abovementioned cells. After 6–12 hours of suspension, the expression rates of beta-, gamma-actin, alpha-actinin 1 and alpha-actinin 4 were elevated in the soleus muscle fibers, but the alpha-actinin 1 expression rate returned to the reference level in 72 hours. After 18–24 hours, the expression rates of beta-actin and alpha-actinin 4 increased in cardiomyocytes, while the alpha-actinin 1 expression rate decreased in soleus muscle fibers. After 12 hours, the beta- and gamma-actin content dropped in the membranous fraction and increased in the cytoplasmic protein fractions from both cardiomyocytes and soleus muscle fibers. The stiffness of both cell types decreased after the same period of time. Further, during the unloading period the concentration of nonmuscle actin and different isoforms of alpha-actinins increased in the membranous fraction from cardiomyocytes. At the same time, the concentration of the abovementioned proteins decreased in the soleus

  8. Intercellular adhesion molecule-1 expression by skeletal muscle cells augments myogenesis

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

    Goh, Qingnian; Dearth, Christopher L.; Corbett, Jacob T.

    We previously demonstrated that the expression of intercellular adhesion molecule-1 (ICAM-1) by skeletal muscle cells after muscle overload contributes to ensuing regenerative and hypertrophic processes in skeletal muscle. The objective of the present study is to reveal mechanisms through which skeletal muscle cell expression of ICAM-1 augments regenerative and hypertrophic processes of myogenesis. This was accomplished by genetically engineering C2C12 myoblasts to stably express ICAM-1, and by inhibiting the adhesive and signaling functions of ICAM-1 through the use of a neutralizing antibody or cell penetrating peptide, respectively. Expression of ICAM-1 by cultured skeletal muscle cells augmented myoblast–myoblast adhesion, myotube formation,more » myonuclear number, myotube alignment, myotube–myotube fusion, and myotube size without influencing the ability of myoblasts to proliferate or differentiate. ICAM-1 augmented myotube formation, myonuclear accretion, and myotube alignment through a mechanism involving adhesion-induced activation of ICAM-1 signaling, as these dependent measures were reduced via antibody and peptide inhibition of ICAM-1. The adhesive and signaling functions of ICAM-1 also facilitated myotube hypertrophy through a mechanism involving myotube–myotube fusion, protein synthesis, and Akt/p70s6k signaling. Our findings demonstrate that ICAM-1 expression by skeletal muscle cells augments myogenesis, and establish a novel mechanism through which the inflammatory response facilitates growth processes in skeletal muscle. - Highlights: • We examined mechanisms through which skeletal muscle cell expression of ICAM-1 facilitates events of in vitro myogenesis. • Expression of ICAM-1 by cultured myoblasts did not influence their ability to proliferate or differentiate. • Skeletal muscle cell expression of ICAM-1 augmented myoblast fusion, myotube alignment, myotube–myotube fusion, and myotube size. • ICAM-1 augmented myogenic processes

  9. Mechanical positioning of multiple nuclei in muscle cells.

    PubMed

    Manhart, Angelika; Windner, Stefanie; Baylies, Mary; Mogilner, Alex

    2018-06-01

    Many types of large cells have multiple nuclei. In skeletal muscle fibers, the nuclei are distributed along the cell to maximize their internuclear distances. This myonuclear positioning is crucial for cell function. Although microtubules, microtubule associated proteins, and motors have been implicated, mechanisms responsible for myonuclear positioning remain unclear. We used a combination of rough interacting particle and detailed agent-based modeling to examine computationally the hypothesis that a force balance generated by microtubules positions the muscle nuclei. Rather than assuming the nature and identity of the forces, we simulated various types of forces between the pairs of nuclei and between the nuclei and cell boundary to position the myonuclei according to the laws of mechanics. We started with a large number of potential interacting particle models and computationally screened these models for their ability to fit biological data on nuclear positions in hundreds of Drosophila larval muscle cells. This reverse engineering approach resulted in a small number of feasible models, the one with the best fit suggests that the nuclei repel each other and the cell boundary with forces that decrease with distance. The model makes nontrivial predictions about the increased nuclear density near the cell poles, the zigzag patterns of the nuclear positions in wider cells, and about correlations between the cell width and elongated nuclear shapes, all of which we confirm by image analysis of the biological data. We support the predictions of the interacting particle model with simulations of an agent-based mechanical model. Taken together, our data suggest that microtubules growing from nuclear envelopes push on the neighboring nuclei and the cell boundaries, which is sufficient to establish the nearly-uniform nuclear spreading observed in muscle fibers.

  10. Cultured smooth muscle cells of the human vesical sphincter are more sensitive to histamine than are detrusor smooth muscle cells.

    PubMed

    Neuhaus, Jochen; Oberbach, Andreas; Schwalenberg, Thilo; Stolzenburg, Jens-Uwe

    2006-05-01

    To compare histamine receptor expression in cultured smooth muscle cells from the human detrusor and internal sphincter using receptor-specific agonists. Smooth muscle cells from the bladder dome and internal sphincter were cultured from 5 male patients undergoing cystectomy for bladder cancer therapy. Calcium transients in cells stimulated with carbachol, histamine, histamine receptor 1 (H1R)-specific heptanecarboxamide (HTMT), dimaprit (H2R), and R-(alpha)-methylhistamine (H3R) were measured by calcium imaging. Histamine receptor proteins were detected by Western blot analysis and immunocytochemistry. H1R, H2R, and H3R expression was found in tissue and cultured cells. Carbachol stimulated equal numbers of detrusor and sphincter cells (60% and 51%, respectively). Histamine stimulated significantly more cells than carbachol in detrusor (100%) and sphincter (99.34%) cells. Calcium responses to carbachol in detrusor and sphincter cells were comparable and did not differ from those to histamine in detrusor cells. However, histamine and specific agonists stimulated more sphincter cells than did carbachol (P <0.001), and the calcium increase was greater in sphincter cells than in detrusor cells. Single cell analysis revealed comparable H2R responses in detrusor and sphincter cells, but H1R and H3R-mediated calcium reactions were significantly greater in sphincter cells. Histamine very effectively induces calcium release in smooth muscle cells. In sphincter cells, histamine is even more effective than carbachol regarding the number of reacting cells and the intracellular calcium increase. Some of the variability in the outcome of antihistaminic interstitial cystitis therapies might be caused by the ineffectiveness of the chosen antihistaminic or unintentional weakening of sphincteric function.

  11. Local myogenic pulp-derived cell injection enhances craniofacial muscle regeneration in vivo.

    PubMed

    Jung, J E; Song, M J; Shin, S; Choi, Y J; Kim, K H; Chung, C J

    2017-02-01

    To enhance myogenic differentiation in pulp cells isolated from extracted premolars by epigenetic modification using a DNA demethylation agent, 5-aza-2'-deoxycytidine (5-Aza), and to evaluate the potent stimulatory effect of 5-Aza-treated pulp cell injection for craniofacial muscle regeneration in vivo. Pulp cells were isolated from premolars extracted for orthodontic purposes from four adults (age range, 18-22.1 years). Levels of myogenic differentiation and functional contraction response in vitro were compared between pulp cells with or without pre-treatment of 5-Aza. Changes in muscle regeneration in response to green fluorescent protein (GFP)-labelled myogenic pulp cell injection in vivo were evaluated using a cardiotoxin (CTX)-induced muscle injury model of the gastrocnemius as well as the masseter muscle in mice. Pre-treatment of 5-Aza in pulp cells stimulated myotube formation, myogenic differentiation in terms of desmin and myogenin expression, and the level of collagen gel contraction. The local injection of 5-Aza pre-treated myogenic pulp cells was engrafted into the host tissue and indicated signs of enhanced muscle regeneration in both the gastrocnemius and the masseter muscles. The epigenetic modification of pulp cells from extracted premolars and the local injection of myogenic pulp cells may stimulate craniofacial muscles regeneration in vivo. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  12. Dense-body aggregates as plastic structures supporting tension in smooth muscle cells.

    PubMed

    Zhang, Jie; Herrera, Ana M; Paré, Peter D; Seow, Chun Y

    2010-11-01

    The wall of hollow organs of vertebrates is a unique structure able to generate active tension and maintain a nearly constant passive stiffness over a large volume range. These properties are predominantly attributable to the smooth muscle cells that line the organ wall. Although smooth muscle is known to possess plasticity (i.e., the ability to adapt to large changes in cell length through structural remodeling of contractile apparatus and cytoskeleton), the detailed structural basis for the plasticity is largely unknown. Dense bodies, one of the most prominent structures in smooth muscle cells, have been regarded as the anchoring sites for actin filaments, similar to the Z-disks in striated muscle. Here, we show that the dense bodies and intermediate filaments formed cable-like structures inside airway smooth muscle cells and were able to adjust the cable length according to cell length and tension. Stretching the muscle cell bundle in the relaxed state caused the cables to straighten, indicating that these intracellular structures were connected to the extracellular matrix and could support passive tension. These plastic structures may be responsible for the ability of smooth muscle to maintain a nearly constant tensile stiffness over a large length range. The finding suggests that the structural plasticity of hollow organs may originate from the dense-body cables within the smooth muscle cells.

  13. Effects of voluntary wheel running on satellite cells in the rat plantaris muscle.

    PubMed

    Kurosaka, Mitsutoshi; Naito, Hisashi; Ogura, Yuji; Kojima, Atsushi; Goto, Katsumasa; Katamoto, Shizuo

    2009-01-01

    This study investigated the effects of voluntary wheel running on satellite cells in the rat plantaris muscle. Seventeen 5-week-old male Wistar rats were assigned to a control (n = 5) or training (n = 12) group. Each rat in the training group ran voluntarily in a running-wheel cage for 8 weeks. After the training period, the animals were anesthetized, and the plantaris muscles were removed, weighed, and analyzed immunohistochemically and biochemically. Although there were no significant differences in muscle weight or fiber area between the groups, the numbers of satellite cells and myonuclei per muscle fiber, percentage of satellite cells, and citrate synthase activity were significantly higher in the training group compared with the control group (p < 0.05). The percentage of satellite cells was also positively correlated with distance run in the training group (r = 0.61, p < 0.05). Voluntary running can induce an increase in the number of satellite cells without changing the mean fiber area in the rat plantaris muscle; this increase in satellite cell content is a function of distance run. Key pointsThere is no study about the effect of voluntary running on satellite cells in the rat plantaris muscle.Voluntary running training causes an increase of citrate synthase activity in the rat plantaris muscle but does not affect muscle weight and mean fiber area in the rat plantaris muscle.Voluntary running can induce an increase in the number of satellite cells without hypertrophy of the rat plantaris muscle.

  14. Role of smooth muscle cells on endothelial cell cytosolic free calcium in porcine coronary arteries.

    PubMed

    Budel, S; Schuster, A; Stergiopoulos, N; Meister, J J; Bény, J L

    2001-09-01

    We tested the hypothesis that the cytosolic free calcium concentration in endothelial cells is under the influence of the smooth muscle cells in the coronary circulation. In the left descending branch of porcine coronary arteries, cytosolic free calcium concentration ([Ca(2+)](i)) was estimated by determining the fluorescence ratio of two calcium probes, fluo 4 and fura red, in smooth muscle and endothelial cells using confocal microscopy. Acetylcholine and potassium, which act directly on smooth muscle cells to increase [Ca(2+)](i), were found to indirectly elevate [Ca(2+)](i) in endothelial cells; in primary cultures of endothelial cells, neither stimulus affected [Ca(2+)](i), yet substance P increased the fluorescence ratio twofold. In response to acetylcholine and potassium, isometric tension developed by arterial strips with intact endothelium was attenuated by up to 22% (P < 0.05) compared with strips without endothelium. These findings suggest that stimuli that increase smooth muscle [Ca(2+)](i) can indirectly influence endothelial cell function in porcine coronary arteries. Such a pathway for negative feedback can moderate vasoconstriction and diminish the potential for vasospasm in the coronary circulation.

  15. MOR23 promotes muscle regeneration and regulates cell adhesion and migration

    PubMed Central

    Griffin, Christine A.; Kafadar, Kimberly A.; Pavlath, Grace K.

    2009-01-01

    Summary Odorant receptors (ORs) in the olfactory epithelium bind to volatile small molecules leading to the perception of smell. ORs are expressed in many tissues but their functions are largely unknown. We show multiple ORs display distinct mRNA expression patterns during myogenesis in vitro and muscle regeneration in vivo. Mouse OR23 (MOR23) expression is induced during muscle regeneration when muscle cells are extensively fusing and plays a key role in regulating migration and adhesion of muscle cells in vitro, two processes common during tissue repair. A soluble ligand for MOR23 is secreted by muscle cells in vitro and muscle tissue in vivo. MOR23 is necessary for proper skeletal muscle regeneration as loss of MOR23 leads to increased myofiber branching, commonly associated with muscular dystrophy. Together these data identify a functional role for an OR outside of the nose and suggest a larger role for ORs during tissue repair. PMID:19922870

  16. Nandrolone decanoate increases satellite cell numbers in the chicken pectoralis muscle.

    PubMed

    Allouh, Mohammed Z; Rosser, Benjamin W C

    2010-02-01

    The anabolic androgenic steroid nandrolone decanoate has minimal androgenic effects and, thus, is widely used to induce muscle hypertrophy in both patients and athletes. Although increases in satellite cell numbers and satellite cells giving rise to new myonuclei are associated with hypertrophy in many experimental models, the relationship between nandrolone and satellite cells is poorly understood. Here we test the hypothesis that nandrolone administration is associated with an increase in satellite cell numbers in muscle. Nandrolone was injected at weekly intervals for four weeks into the right pectoralis muscle of female white leghorn chickens aged 63 days post hatch. Age/size/sex matched control birds received saline injections. The contralateral pectoralis was excised for study from each control and nandrolone treated bird. An antibody against Pax7 and immunocytochemical techniques were used to identify satellite cells. Nandrolone significantly increased mean pectoralis mass by approximately 22%, and mean fiber diameter by about 24%. All satellite cell indices that were quantified increased significantly in chicken pectoralis with administration of nandrolone. Nandrolone injected birds had on average higher satellite cell frequencies (#SC nuclei/all nuclei within basal lamina), number of satellite cells per millimeter of fiber, and satellite cell concentrations (closer together). Myonuclei were further apart (less concentrated) in nandrolone injected muscle. However, an overall increase in myonuclear numbers was revealed by a significantly greater mean number of myonuclei per millimeter of fiber in nandrolone injected muscle. Our results suggest that satellite cells may be key cellular vectors for nandrolone induced muscle fiber hypertrophy.

  17. Engineered Muscle Actuators: Cells and Tissues

    DTIC Science & Technology

    2007-01-10

    tissue culture perfusion bioreactors The UNC group led the development of the final version of the integrated cell culture bioreactor . The system was...construct engineered in vitro from primary mammalian cells (C) The first demonstration of developmental improvements in engineered tendon constitutive...2007 Final Performance Report 1 Nov 2004 - 31 Oct 2006 4. TITLE AND SUBTITLE 5.. CONTRACT NUMBER Engineered Muscle Actuators: Cells and Tissues FA9550

  18. Identification and characterization of a non-satellite cell muscle resident progenitor during postnatal development.

    PubMed

    Mitchell, Kathryn J; Pannérec, Alice; Cadot, Bruno; Parlakian, Ara; Besson, Vanessa; Gomes, Edgar R; Marazzi, Giovanna; Sassoon, David A

    2010-03-01

    Satellite cells are resident myogenic progenitors in postnatal skeletal muscle involved in muscle postnatal growth and adult regenerative capacity. Here, we identify and describe a population of muscle-resident stem cells, which are located in the interstitium, that express the cell stress mediator PW1 but do not express other markers of muscle stem cells such as Pax7. PW1(+)/Pax7(-) interstitial cells (PICs) are myogenic in vitro and efficiently contribute to skeletal muscle regeneration in vivo as well as generating satellite cells and PICs. Whereas Pax7 mutant satellite cells show robust myogenic potential, Pax7 mutant PICs are unable to participate in myogenesis and accumulate during postnatal growth. Furthermore, we found that PICs are not derived from a satellite cell lineage. Taken together, our findings uncover a new and anatomically identifiable population of muscle progenitors and define a key role for Pax7 in a non-satellite cell population during postnatal muscle growth.

  19. Comparison and analysis of Wuding and avian chicken skeletal muscle satellite cells.

    PubMed

    Tong, H Q; Jiang, Z Q; Dou, T F; Li, Q H; Xu, Z Q; Liu, L X; Gu, D H; Rong, H; Huang, Y; Chen, X B; Jois, M; Te Pas, M F W; Ge, C R; Jia, J J

    2016-10-05

    Chicken skeletal muscle satellite cells are located between the basement membrane and the sarcolemma of mature muscle fibers. Avian broilers have been genetically selected based on their high growth velocity and large muscle mass. The Wuding chicken is a famous local chicken in Yunnan Province that undergoes non-selection breeding and is slow growing. In this study, we aimed to explore differences in the proliferation and differentiation properties of satellite cells isolated from the two chicken breeds. Using immunofluorescence, hematoxylin-eosin staining and real-time polymerase chain reaction analysis, we analyzed the in vitro characteristics of proliferating and differentiating satellite cells isolated from the two chicken breeds. The growth curve of satellite cells was S-shaped, and cells from Wuding chickens entered the logarithmic phase and plateau phase 1 day later than those from Avian chicken. The results also showed that the two skeletal muscle satellite cell lines were positive for Pax7, MyoD and IGF-1. The expression of Pax7 followed a downward trend, whereas that of MyoD and IGF-1 first increased and subsequently decreased in cells isolated from the two chickens. These data indicated that the skeletal muscle satellite cells of Avian chicken grow and differentiate faster than did those of Wuding chickens. We suggest that the methods of breeding selection applied to these breeds regulate the characteristics of skeletal muscle satellite cells to influence muscle growth.

  20. Ureter smooth muscle cell orientation in rat is predominantly longitudinal.

    PubMed

    Spronck, Bart; Merken, Jort J; Reesink, Koen D; Kroon, Wilco; Delhaas, Tammo

    2014-01-01

    In ureter peristalsis, the orientation of the contracting smooth muscle cells is essential, yet current descriptions of orientation and composition of the smooth muscle layer in human as well as in rat ureter are inconsistent. The present study aims to improve quantification of smooth muscle orientation in rat ureters as a basis for mechanistic understanding of peristalsis. A crucial step in our approach is to use two-photon laser scanning microscopy and image analysis providing objective, quantitative data on smooth muscle cell orientation in intact ureters, avoiding the usual sectioning artifacts. In 36 rat ureter segments, originating from a proximal, middle or distal site and from a left or right ureter, we found close to the adventitia a well-defined longitudinal smooth muscle orientation. Towards the lamina propria, the orientation gradually became slightly more disperse, yet the main orientation remained longitudinal. We conclude that smooth muscle cell orientation in rat ureter is predominantly longitudinal, though the orientation gradually becomes more disperse towards the proprial side. These findings do not support identification of separate layers. The observed longitudinal orientation suggests that smooth muscle contraction would rather cause local shortening of the ureter, than cause luminal constriction. However, the net-like connective tissue of the ureter wall may translate local longitudinal shortening into co-local luminal constriction, facilitating peristalsis. Our quantitative, minimally invasive approach is a crucial step towards more mechanistic insight into ureter peristalsis, and may also be used to study smooth muscle cell orientation in other tube-like structures like gut and blood vessels.

  1. Heterologous live infectious bronchitis virus vaccination in day-old commercial broiler chicks: clinical signs, ciliary health, immune responses and protection against variant infectious bronchitis viruses.

    PubMed

    Awad, Faez; Hutton, Sally; Forrester, Anne; Baylis, Matthew; Ganapathy, Kannan

    2016-01-01

    Groups of one-day-old broiler chicks were vaccinated via the oculo-nasal route with different live infectious bronchitis virus (IBV) vaccines: Massachusetts (Mass), 793B, D274 or Arkansas (Ark). Clinical signs and gross lesions were evaluated. Five chicks from each group were humanely killed at intervals and their tracheas collected for ciliary activity assessment and for the detection of CD4+, CD8+ and IgA-bearing B cells by immunohistochemistry (IHC). Blood samples were collected at intervals for the detection of anti-IBV antibodies. At 21 days post-vaccination (dpv), protection conferred by different vaccination regimes against virulent M41, QX and 793B was assessed. All vaccination programmes were able to induce high levels of CD4+, CD8+ and IgA-bearing B cells in the trachea. Significantly higher levels of CD4+ and CD8+ expression were observed in the Mass2 + 793B2-vaccinated group compared to the other groups (subscripts indicate different manufacturers). Protection studies showed that the group of chicks vaccinated with Mass2 + 793B2 produced 92% ciliary protection against QX challenge; compared to 53%, 68% and 73% ciliary protection against the same challenge virus by Mass1 + D274, Mass1 + 793B1 and Mass3 + Ark, respectively. All vaccination programmes produced more than 85% ciliary protection against M41 and 793B challenges. It appears that the variable levels of protection provided by different heterologous live IBV vaccinations are dependent on the levels of local tracheal immunity induced by the respective vaccine combination. The Mass2 + 793B2 group showed the worst clinical signs, higher mortality and severe lesions following vaccination, but had the highest tracheal immune responses and demonstrated the best protection against all three challenge viruses.

  2. Skeletal Muscle Regeneration, Repair and Remodelling in Aging: The Importance of Muscle Stem Cells and Vascularization.

    PubMed

    Joanisse, Sophie; Nederveen, Joshua P; Snijders, Tim; McKay, Bryon R; Parise, Gianni

    2017-01-01

    Sarcopenia is the age-related loss of skeletal muscle mass and strength. Ultimately, sarcopenia results in the loss of independence, which imposes a large financial burden on healthcare systems worldwide. A critical facet of sarcopenia is the diminished ability for aged muscle to regenerate, repair and remodel. Over the years, research has focused on elucidating underlying mechanisms of sarcopenia and the impaired ability of muscle to respond to stimuli with aging. Muscle-specific stem cells, termed satellite cells (SC), play an important role in maintaining muscle health throughout the lifespan. It is well established that SC are essential in skeletal muscle regeneration, and it has been hypothesized that a reduction and/or dysregulation of the SC pool, may contribute to accelerated loss of skeletal muscle mass that is observed with advancing age. The preservation of skeletal muscle tissue and its ability to respond to stimuli may be impacted by reduced SC content and impaired function observed with aging. Aging is also associated with a reduction in capillarization of skeletal muscle. We have recently demonstrated that the distance between type II fibre-associated SC and capillaries is greater in older compared to younger adults. The greater distance between SC and capillaries in older adults may contribute to the dysregulation in SC activation ultimately impairing muscle's ability to remodel and, in extreme circumstances, regenerate. This viewpoint will highlight the importance of optimal SC activation in addition to skeletal muscle capillarization to maximize the regenerative potential of skeletal muscle in older adults. © 2016 S. Karger AG, Basel.

  3. Intercellular adhesion molecule-1 expression by skeletal muscle cells augments myogenesis.

    PubMed

    Goh, Qingnian; Dearth, Christopher L; Corbett, Jacob T; Pierre, Philippe; Chadee, Deborah N; Pizza, Francis X

    2015-02-15

    We previously demonstrated that the expression of intercellular adhesion molecule-1 (ICAM-1) by skeletal muscle cells after muscle overload contributes to ensuing regenerative and hypertrophic processes in skeletal muscle. The objective of the present study is to reveal mechanisms through which skeletal muscle cell expression of ICAM-1 augments regenerative and hypertrophic processes of myogenesis. This was accomplished by genetically engineering C2C12 myoblasts to stably express ICAM-1, and by inhibiting the adhesive and signaling functions of ICAM-1 through the use of a neutralizing antibody or cell penetrating peptide, respectively. Expression of ICAM-1 by cultured skeletal muscle cells augmented myoblast-myoblast adhesion, myotube formation, myonuclear number, myotube alignment, myotube-myotube fusion, and myotube size without influencing the ability of myoblasts to proliferate or differentiate. ICAM-1 augmented myotube formation, myonuclear accretion, and myotube alignment through a mechanism involving adhesion-induced activation of ICAM-1 signaling, as these dependent measures were reduced via antibody and peptide inhibition of ICAM-1. The adhesive and signaling functions of ICAM-1 also facilitated myotube hypertrophy through a mechanism involving myotube-myotube fusion, protein synthesis, and Akt/p70s6k signaling. Our findings demonstrate that ICAM-1 expression by skeletal muscle cells augments myogenesis, and establish a novel mechanism through which the inflammatory response facilitates growth processes in skeletal muscle. Copyright © 2014 Elsevier Inc. All rights reserved.

  4. Intercellular Adhesion Molecule-1 Expression by Skeletal Muscle Cells Augments Myogenesis

    PubMed Central

    Goh, Qingnian; Dearth, Christopher L.; Corbett, Jacob T.; Pierre, Philippe; Chadee, Deborah N.; Pizza, Francis X.

    2014-01-01

    We previously demonstrated that the expression of intercellular adhesion molecule-1 (ICAM-1) by skeletal muscle cells after muscle overload contributes to ensuing regenerative and hypertrophic processes in skeletal muscle. The objective of the present study is to reveal mechanisms through which skeletal muscle cell expression of ICAM-1 augments regenerative and hypertrophic processes of myogenesis. This was accomplished by genetically engineering C2C12 myoblasts to stably express ICAM-1, and by inhibiting the adhesive and signaling functions of ICAM-1 through the use of a neutralizing antibody or cell penetrating peptide, respectively. Expression of ICAM-1 by cultured skeletal muscle cells augmented myoblast-myoblast adhesion, myotube formation, myonuclear number, myotube alignment, myotube-myotube fusion, and myotube size without influencing the ability of myoblasts to proliferate or differentiate. ICAM-1 augmented myotube formation, myonuclear accretion, and myotube alignment through a mechanism involving adhesion-induced activation of ICAM-1 signaling, as these dependent measures were reduced via antibody and peptide inhibition of ICAM-1. The adhesive and signaling functions of ICAM-1 also facilitated myotube hypertrophy through a mechanism involving myotube-myotube fusion, protein synthesis, and Akt/p70s6k signaling. Our findings demonstrate that ICAM-1 expression by skeletal muscle cells augments myogenesis, and establish a novel mechanism through which the inflammatory response facilitates growth processes in skeletal muscle. PMID:25281303

  5. Plasticity of the Muscle Stem Cell Microenvironment.

    PubMed

    Dinulovic, Ivana; Furrer, Regula; Handschin, Christoph

    2017-01-01

    Satellite cells (SCs) are adult muscle stem cells capable of repairing damaged and creating new muscle tissue throughout life. Their functionality is tightly controlled by a microenvironment composed of a wide variety of factors, such as numerous secreted molecules and different cell types, including blood vessels, oxygen, hormones, motor neurons, immune cells, cytokines, fibroblasts, growth factors, myofibers, myofiber metabolism, the extracellular matrix and tissue stiffness. This complex niche controls SC biology-quiescence, activation, proliferation, differentiation or renewal and return to quiescence. In this review, we attempt to give a brief overview of the most important players in the niche and their mutual interaction with SCs. We address the importance of the niche to SC behavior under physiological and pathological conditions, and finally survey the significance of an artificial niche both for basic and translational research purposes.

  6. Plasticity of the Muscle Stem Cell Microenvironment

    PubMed Central

    Dinulovic, Ivana; Furrer, Regula; Handschin, Christoph

    2018-01-01

    Satellite cells (SCs) are adult muscle stem cells capable of repairing damaged and creating new muscle tissue throughout life. Their functionality is tightly controlled by a microenvironment composed of a wide variety of factors, such as numerous secreted molecules and different cell types, including blood vessels, oxygen, hormones, motor neurons, immune cells, cytokines, fibroblasts, growth factors, myofibers, myofiber metabolism, the extracellular matrix and tissue stiffness. This complex niche controls SC biology – quiescence, activation, proliferation, differentiation or renewal and return to quiescence. In this review, we attempt to give a brief overview of the most important players in the niche and their mutual interaction with SCs. We address the importance of the niche to SC behavior under physiological and pathological conditions, and finally survey the significance of an artificial niche both for basic and translational research purposes. PMID:29204832

  7. Akt1 deficiency diminishes skeletal muscle hypertrophy by reducing satellite cell proliferation.

    PubMed

    Moriya, Nobuki; Miyazaki, Mitsunori

    2018-05-01

    Skeletal muscle mass is determined by the net dynamic balance between protein synthesis and degradation. Although the Akt/mechanistic target of rapamycin (mTOR)-dependent pathway plays an important role in promoting protein synthesis and subsequent skeletal muscle hypertrophy, the precise molecular regulation of mTOR activity by the upstream protein kinase Akt is largely unknown. In addition, the activation of satellite cells has been indicated as a key regulator of muscle mass. However, the requirement of satellite cells for load-induced skeletal muscle hypertrophy is still under intense debate. In this study, female germline Akt1 knockout (KO) mice were used to examine whether Akt1 deficiency attenuates load-induced skeletal muscle hypertrophy through suppressing mTOR-dependent signaling and satellite cell proliferation. Akt1 KO mice showed a blunted hypertrophic response of skeletal muscle, with a diminished rate of satellite cell proliferation following mechanical overload. In contrast, Akt1 deficiency did not affect the load-induced activation of mTOR signaling and the subsequent enhanced rate of protein synthesis in skeletal muscle. These observations suggest that the load-induced activation of mTOR signaling occurs independently of Akt1 regulation and that Akt1 plays a critical role in regulating satellite cell proliferation during load-induced muscle hypertrophy.

  8. Ciliary contact interactions dominate surface scattering of swimming eukaryotes

    PubMed Central

    Kantsler, Vasily; Dunkel, Jörn; Polin, Marco; Goldstein, Raymond E.

    2013-01-01

    Interactions between swimming cells and surfaces are essential to many microbiological processes, from bacterial biofilm formation to human fertilization. However, despite their fundamental importance, relatively little is known about the physical mechanisms that govern the scattering of flagellated or ciliated cells from solid surfaces. A more detailed understanding of these interactions promises not only new biological insights into structure and dynamics of flagella and cilia but may also lead to new microfluidic techniques for controlling cell motility and microbial locomotion, with potential applications ranging from diagnostic tools to therapeutic protein synthesis and photosynthetic biofuel production. Due to fundamental differences in physiology and swimming strategies, it is an open question of whether microfluidic transport and rectification schemes that have recently been demonstrated for pusher-type microswimmers such as bacteria and sperm cells, can be transferred to puller-type algae and other motile eukaryotes, because it is not known whether long-range hydrodynamic or short-range mechanical forces dominate the surface interactions of these microorganisms. Here, using high-speed microscopic imaging, we present direct experimental evidence that the surface scattering of both mammalian sperm cells and unicellular green algae is primarily governed by direct ciliary contact interactions. Building on this insight, we predict and experimentally verify the existence of optimal microfluidic ratchets that maximize rectification of initially uniform Chlamydomonas reinhardtii suspensions. Because mechano-elastic properties of cilia are conserved across eukaryotic species, we expect that our results apply to a wide range of swimming microorganisms. PMID:23297240

  9. Rules of tissue packing involving different cell types: human muscle organization

    PubMed Central

    Sánchez-Gutiérrez, Daniel; Sáez, Aurora; Gómez-Gálvez, Pedro; Paradas, Carmen; Escudero, Luis M.

    2017-01-01

    Natural packed tissues are assembled as tessellations of polygonal cells. These include skeletal muscles and epithelial sheets. Skeletal muscles appear as a mosaic composed of two different types of cells: the “slow” and “fast” fibres. Their relative distribution is important for the muscle function but little is known about how the fibre arrangement is established and maintained. In this work we capture the organizational pattern in two different healthy muscles: biceps brachii and quadriceps. Here we show that the biceps brachii muscle presents a particular arrangement, based on the different sizes of slow and fast fibres. By contrast, in the quadriceps muscle an unbiased distribution exists. Our results indicate that the relative size of each cellular type imposes an intrinsic organization into natural tessellations. These findings establish a new framework for the analysis of any packed tissue where two or more cell types exist. PMID:28071729

  10. Rules of tissue packing involving different cell types: human muscle organization.

    PubMed

    Sánchez-Gutiérrez, Daniel; Sáez, Aurora; Gómez-Gálvez, Pedro; Paradas, Carmen; Escudero, Luis M

    2017-01-10

    Natural packed tissues are assembled as tessellations of polygonal cells. These include skeletal muscles and epithelial sheets. Skeletal muscles appear as a mosaic composed of two different types of cells: the "slow" and "fast" fibres. Their relative distribution is important for the muscle function but little is known about how the fibre arrangement is established and maintained. In this work we capture the organizational pattern in two different healthy muscles: biceps brachii and quadriceps. Here we show that the biceps brachii muscle presents a particular arrangement, based on the different sizes of slow and fast fibres. By contrast, in the quadriceps muscle an unbiased distribution exists. Our results indicate that the relative size of each cellular type imposes an intrinsic organization into natural tessellations. These findings establish a new framework for the analysis of any packed tissue where two or more cell types exist.

  11. Vessel-associated stem cells from skeletal muscle: From biology to future uses in cell therapy.

    PubMed

    Sancricca, Cristina; Mirabella, Massimiliano; Gliubizzi, Carla; Broccolini, Aldobrando; Gidaro, Teresa; Morosetti, Roberta

    2010-06-26

    Over the last years, the existence of different stem cells with myogenic potential has been widely investigated. Besides the classical skeletal muscle progenitors represented by satellite cells, numerous multipotent and embryologically unrelated progenitors with a potential role in muscle differentiation and repair have been identified. In order to conceive a therapeutic approach for degenerative muscle disorders, it is of primary importance to identify an ideal stem cell endowed with all the features for a possible use in vivo. Among all emerging populations, vessel-associated stem cells are a novel and promising class of multipotent progenitors of mesodermal origin and with high myogenic potential which seem to best fit all the requirements for a possible cell therapy. In vitro and in vivostudies have already tested the effectiveness and safety of vessel-associated stem cells in animal models. This leads to the concrete possibility in the future to start pilot human clinical trials, hopefully opening the way to a turning point in the treatment of genetic and acquired muscle disorders.

  12. Vessel-associated stem cells from skeletal muscle: From biology to future uses in cell therapy

    PubMed Central

    Sancricca, Cristina; Mirabella, Massimiliano; Gliubizzi, Carla; Broccolini, Aldobrando; Gidaro, Teresa; Morosetti, Roberta

    2010-01-01

    Over the last years, the existence of different stem cells with myogenic potential has been widely investigated. Besides the classical skeletal muscle progenitors represented by satellite cells, numerous multipotent and embryologically unrelated progenitors with a potential role in muscle differentiation and repair have been identified. In order to conceive a therapeutic approach for degenerative muscle disorders, it is of primary importance to identify an ideal stem cell endowed with all the features for a possible use in vivo. Among all emerging populations, vessel-associated stem cells are a novel and promising class of multipotent progenitors of mesodermal origin and with high myogenic potential which seem to best fit all the requirements for a possible cell therapy. In vitro and in vivo studies have already tested the effectiveness and safety of vessel-associated stem cells in animal models. This leads to the concrete possibility in the future to start pilot human clinical trials, hopefully opening the way to a turning point in the treatment of genetic and acquired muscle disorders. PMID:21607121

  13. Signaling via the transcriptionally regulated activin receptor 2B is a novel mediator of neuronal cell death during chicken ciliary ganglion development.

    PubMed

    Koszinowski, S; Buss, K; Kaehlcke, K; Krieglstein, K

    2015-04-01

    The TGF-β ligand superfamily members activin A and BMP control important aspects of embryonic neuronal development and differentiation. Both are known to bind to activin receptor subtypes IIA (ActRIIA) and IIB, while in the avian ciliary ganglion (CG), so far only ActRIIA-expression has been described. We show that the expression of ACVR2B, coding for the ActRIIB, is tightly regulated during CG development and the knockdown of ACVR2B expression leads to a deregulation in the execution of neuronal apoptosis and therefore affects ontogenetic programmed cell death in vivo. While the differentiation of choroid neurons was impeded in the knockdown, pointing toward a reduction in activin A-mediated neural differentiation signaling, naturally occurring neuronal cell death in the CG was not prevented by follistatin treatment. Systemic injections of the BMP antagonist noggin, on the other hand, reduced the number of apoptotic neurons to a similar extent as ACVR2B knockdown. We therefore propose a novel pathway in the regulation of CG neuron ontogenetic programmed cell death, which could be mediated by BMP and signals via the ActRIIB. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  14. Short- and long-term effects on the ciliary body and the aqueous outflow pathways of high-intensity focused ultrasound cyclocoagulation.

    PubMed

    Aptel, Florent; Béglé, Aurélie; Razavi, Arash; Romano, Fabrice; Charrel, Thomas; Chapelon, Jean-Yves; Denis, Philippe; Lafon, Cyril

    2014-09-01

    Several physical methods can be used to coagulate the ciliary body and decrease intra-ocular pressure in patients with glaucoma. The study described here investigated the short- and long-term effects of high-intensity focused ultrasound (HIFU) cyclocoagulation on the aqueous humor production structures and outflow pathways. Thirty-four rabbit eyes were sonicated with a ring-shaped probe containing six miniaturized HIFU transducers. Light, scanning electron and transmission electron microscopy and corrosion casts were performed. In the affected regions, the epithelium of the ciliary processes was degenerated or necrotic and sloughed off. Examinations performed several months afterward revealed involution of the ciliary processes. Vascular corrosion cast revealed focal interruption of the ciliary body microvasculature. In most animals, a sustained fluid space was seen between the sclera, the ciliary body and the choroid, likely indicating an increase in the aqueous outflow by the uveoscleral pathway. These results suggest that HIFU cyclocoagulation has a dual effect on aqueous humor dynamics. Copyright © 2014 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.

  15. Qualitative evaluation of the iris and ciliary body by ultrasound biomicroscopy in subjects with angle closure.

    PubMed

    Ku, Judy Y; Nongpiur, Monisha E; Park, Judy; Narayanaswamy, Arun K; Perera, Shamira A; Tun, Tin A; Kumar, Rajesh S; Baskaran, Mani; Aung, Tin

    2014-12-01

    To qualitatively analyze anterior chamber structures imaged by ultrasound biomicroscopy (UBM) in primary angle-closure patients. Subjects diagnosed as primary angle-closure suspect (PACS), primary angle-closure glaucoma (PACG), and previous acute primary angle closure (APAC) were recruited prospectively along with a group of normal controls. UBM was performed under standardized dark room conditions and qualitative assessment was carried out using a set of reference photographs of standard UBM images to categorize the various anatomic features related to angle configuration. These included overall and basal iris thicknesses, iris convexity, iris angulation, ciliary body size, and ciliary sulcus. A total of 60 PACS, 114 PACG, 41 APAC, and 33 normal controls were included. Patients were predominantly older Chinese females. After controlling the confounding effect of age and sex, eyes with overall thicker irides [medium odds ratio (OR) 3.58, thick OR 2.84] when compared with thin irides have a significantly higher likelihood of having PACS/PACG/APAC versus controls. Thicker basal iris component (medium OR 4.13, thick OR 3.39) also have higher likelihood of having angle closure when compared with thin basal iris thickness. Subjects with basal iris insertion, mild iris angulation, and large ciliary body have a higher OR of having angle closure. In contrast, the presence/absence of a ciliary sulcus did not influence the likelihood of angle closure. Eyes with thicker overall and basal iris thicknesses are more likely to have angle closure than controls. Other features that increase the likelihood of angle closure include basal iris insertion, mild iris angulation, and large ciliary body.

  16. Unexpected genetic heterogeneity for primary ciliary dyskinesia in the Irish Traveller population.

    PubMed

    Casey, Jillian P; McGettigan, Paul A; Healy, Fiona; Hogg, Claire; Reynolds, Alison; Kennedy, Breandan N; Ennis, Sean; Slattery, Dubhfeasa; Lynch, Sally A

    2015-02-01

    We present a study of five children from three unrelated Irish Traveller families presenting with primary ciliary dyskinesia (PCD). As previously characterized disorders in the Irish Traveller population are caused by common homozygous mutations, we hypothesised that all three PCD families shared the same recessive mutation. However, exome sequencing showed that there was no pathogenic homozygous mutation common to all families. This finding was supported by histology, which showed that each family has a different type of ciliary defect; transposition defect (family A), nude epithelium (family B) and absence of inner and outer dynein arms (family C). Therefore, each family was analysed independently using homozygosity mapping and exome sequencing. The affected siblings in family A share a novel 1 bp duplication in RSPH4A (NM_001161664.1:c.166dup; p.Arg56Profs*11), a radial-spoke head protein involved in ciliary movement. In family B, we identified three candidate genes (CCNO, KCNN3 and CDKN1C), with a 5-bp duplication in CCNO (NM_021147.3:c.258_262dup; p.Gln88Argfs*8) being the most likely cause of ciliary aplasia. This is the first study to implicate CCNO, a DNA repair gene reported to be involved in multiciliogenesis, in PCD. In family C, we identified a ∼3.5-kb deletion in DYX1C1, a neuronal migration gene previously associated with PCD. This is the first report of a disorder in the relatively small Irish Traveller population to be caused by >1 disease gene. Our study identified at least three different PCD genes in the Irish Traveller population, highlighting that one cannot always assume genetic homogeneity, even in small consanguineous populations.

  17. Amylin competes for binding sites of CGRP in the chamber angle and uvea of monkey, cat, and pig eye.

    PubMed

    Alajuuma, Päivi; Oksala, Olli; Uusitalo, Hannu

    2003-12-01

    Calcitonin gene-related peptide (CGRP) binding sites have been identified previously in the eyes of monkey, cat, pig, and guinea pig. In this study, the ability of cat, human, and rat amylins to displace the binding of CGRP in the anterior part of the eye of monkey, cat, and pig was studied. The location and displacement of 125I-hCGRPalpha by amylins as concentrations of 1-1000 nM were studied in cryosections by autoradiography. In the monkey eye, cat and rat amylins were able to compete for the binding sites of CGRP in ciliary muscle and ciliary processes. In the cat eye, cat and human amylins clearly displaced CGRP binding from ciliary muscle, ciliary processes, iris, and chamber angle. Furthermore, rat amylin clearly displaced CGRP binding from ciliary muscle and ciliary processes. In the pig eye, cat, human, and rat amylins competed for the binding sites of CGRP in ciliary muscle, ciliary processes, iris, and limbal conjunctiva. Specific amylin receptors or the possible physiological role of amylin in the eye have not hitherto been reported. It seems, however, that amylin can bind to ocular CGRP receptors and thus probably plays a role in the regulation of the same functions as CGRP, (e.g., aqueous humor outflow).

  18. Neural cell adhesion molecule mediates initial interactions between spinal cord neurons and muscle cells in culture

    PubMed Central

    1983-01-01

    Previous studies in this laboratory have described a cell surface glycoprotein, called neural cell adhesion molecule or N-CAM, that appears to be a ligand in the adhesion between neural membranes. N-CAM antigenic determinants were also shown to be present on embryonic muscle and an N-CAM-dependent adhesion was demonstrated between retinal cell membranes and muscle cells in short-term assays. The present studies indicate that these antigenic determinants are associated with the N-CAM polypeptide, and that rapid adhesion mediated by this molecule occurs between spinal cord membranes and muscle cells. Detailed examination of the effects of anti-(N-CAM) Fab' fragments in cultures of spinal cord with skeletal muscle showed that the Fab' fragments specifically block adhesion of spinal cord neurites and cells to myotubes. The Fab' did not affect binding of neurites to fibroblasts and collagen substrate, and did not alter myotube morphology. These results indicate that N-CAM adhesion is essential for the in vitro establishment of physical associations between nerve and muscle, and suggest that binding involving N-CAM may be an important early step in synaptogenesis. PMID:6863388

  19. Isolation and Purification of Satellite Cells for Skeletal Muscle Tissue Engineering

    PubMed Central

    Syverud, Brian C; Lee, Jonah D; VanDusen, Keith W; Larkin, Lisa M

    2015-01-01

    Engineered skeletal muscle holds promise as a source of graft tissue for the repair of traumatic injuries such as volumetric muscle loss. The resident skeletal muscle stem cell, the satellite cell, has been identified as an ideal progenitor for tissue engineering due to its role as an essential player in the potent skeletal muscle regeneration mechanism. A significant challenge facing tissue engineers, however, is the isolation of sufficiently large satellite cell populations with high purity. The two common isolation techniques, single fiber explant culture and enzymatic dissociation, can yield either a highly pure satellite cell population or a suitably large number or cells but fail to do both simultaneously. As a result, it is often necessary to use a purification technique such as pre-plating or cell sorting to enrich the satellite cell population post-isolation. Furthermore, the absence of complex chemical and biophysical cues influencing the in vivo satellite cell “niche” complicates the culture of isolated satellite cells. Techniques under investigation to maximize myogenic proliferation and differentiation in vitro are described in this article, along with current methods for isolating and purifying satellite cells. PMID:26413555

  20. Muscle progenitor cells proliferation doesn't sufficiently contribute to maintaining stretched soleus muscle mass during gravitational unloading

    NASA Astrophysics Data System (ADS)

    Tarakina, M. V.; Turtikova, O. V.; Nemirovskaya, T. L.; Kokontcev, A. A.; Shenkman, B. S.

    Skeletal muscle work hypertrophy is usually connected with muscle progenitor satellite cells (SC) activation with subsequent incorporation of their nuclei into myofibers. Passive stretch of unloaded muscle was earlier established to prevent atrophic processes and is accompanied by enhanced protein synthesis. We hypothesized that elimination of SC proliferation capacity by γ-irradiation would partly avert stretched muscle fiber capability to maintain their size under the conditions of gravitational unloading. To assess the role of muscle progenitor (satellite) cells in development of passive stretch preventive effect SC proliferation was suppressed by local exposing to ionized radiation (2500 rad), subsequent hindlimb suspension or hindlimb suspension with concomitant passive stretch were carried out. Reduction of myofiber cross-sectional area and decrease in myonuclei number accompanying unloaded muscle atrophy were completely abolished by passive stretch both in irradiated and sham-treated animals. We conclude that SC did not make essential contribution to passive stretch preventive action under the conditions of simulated weightlessness.

  1. Satellite cell proliferation in adult skeletal muscle

    NASA Technical Reports Server (NTRS)

    Morrison, Paul R. (Inventor); Thomason, Donald B. (Inventor); Stancel, George M. (Inventor); Booth, Frank W. (Inventor)

    1995-01-01

    Novel methods of retroviral-mediated gene transfer for the in vivo corporation and stable expression of eukaryotic or prokaryotic foreign genes in tissues of living animals is described. More specifically, methods of incorporating foreign genes into mitotically active cells are disclosed. The constitutive and stable expression of E. coli .beta.-galactosidase gene under the promoter control of the Moloney murine leukemia virus long terminal repeat is employed as a particularly preferred embodiment, by way of example, establishes the model upon which the incorporation of a foreign gene into a mitotically-active living eukaryotic tissue is based. Use of the described methods in therapeutic treatments for genetic diseases, such as those muscular degenerative diseases, is also presented. In muscle tissue, the described processes result in genetically-altered satellite cells which proliferate daughter myoblasts which preferentially fuse to form a single undamaged muscle fiber replacing damaged muscle tissue in a treated animal. The retroviral vector, by way of example, includes a dystrophin gene construct for use in treating muscular dystrophy. The present invention also comprises an experimental model utilizable in the study of the physiological regulation of skeletal muscle gene expression in intact animals.

  2. Satellite-like cells contribute to pax7-dependent skeletal muscle repair in adult zebrafish

    PubMed Central

    Berberoglu, Michael A.; Gallagher, Thomas L.; Morrow, Zachary T.; Talbot, Jared C.; Hromowyk, Kimberly J.; Tenente, Inês M.; Langenau, David M.; Amacher, Sharon L.

    2017-01-01

    Satellite cells, also known as muscle stem cells, are responsible for skeletal muscle growth and repair in mammals. Pax7 and Pax3 transcription factors are established satellite cell markers required for muscle development and regeneration, and there is great interest in identifying additional factors that regulate satellite cell proliferation, differentiation, and/or skeletal muscle regeneration. Due to the powerful regenerative capacity of many zebrafish tissues, even in adults, we are exploring the regenerative potential of adult zebrafish skeletal muscle. Here, we show that adult zebrafish skeletal muscle contains cells similar to mammalian satellite cells. Adult zebrafish satellite-like cells have dense heterochromatin, express Pax7 and Pax3, proliferate in response to injury, and show peak myogenic responses 4–5 days post-injury (dpi). Furthermore, using a pax7a-driven GFP reporter, we present evidence implicating satellite-like cells as a possible source of new muscle. In lieu of central nucleation, which distinguishes regenerating myofibers in mammals, we describe several characteristics that robustly identify newly-forming myofibers from surrounding fibers in injured adult zebrafish muscle. These characteristics include partially overlapping expression in satellite cells and regenerating myofibers of two RNA-binding proteins Rbfox2 and Rbfoxl1, known to regulate embryonic muscle development and function. Finally, by analyzing pax7a; pax7b double mutant zebrafish, we show that Pax7 is required for adult skeletal muscle repair, as it is in the mouse. PMID:28279710

  3. The physiopathologic interplay between stem cells and tissue niche in muscle regeneration and the role of IL-6 on muscle homeostasis and diseases.

    PubMed

    Forcina, Laura; Miano, Carmen; Musarò, Antonio

    2018-06-01

    Skeletal muscle is a complex, dynamic tissue characterized by an elevated plasticity. Although the adult muscle is mainly composed of multinucleated fibers with post mitotic nuclei, it retains a remarkable ability to regenerate in response to traumatic events. The regenerative potential of the adult skeletal muscle relies in the activity of satellite cells, mononucleated cells residing within the muscle in intimate association with myofibers. Satellite cells normally remain quiescent in their sublaminar position, sporadically entering the cell cycle to guarantee an efficient cellular turnover, by fusing with pre-existing myofibers, and to maintain the stem cell pool. However, after muscle injury satellite cells undergo an extensive increase of their activity in response to environmental stimuli, thereby participating to the regeneration of a functional muscle tissue. Nevertheless, regeneration is affected in several pathologic conditions and by a wide range of environmental signals that are highly variable, not only through time, but also depending on the physiological or pathological conditions of the musculature. Among these factors, the interleukin-6 (IL-6) plays a critical physiopathologic role on muscle homeostasis and diseases. The basis of muscle regeneration and the impact of IL-6 on the physiopathology of skeletal muscle will be discussed. Copyright © 2018 Elsevier Ltd. All rights reserved.

  4. Vitamin K2 improves proliferation and migration of bovine skeletal muscle cells in vitro.

    PubMed

    Rønning, Sissel Beate; Pedersen, Mona Elisabeth; Berg, Ragnhild Stenberg; Kirkhus, Bente; Rødbotten, Rune

    2018-01-01

    Skeletal muscle function is highly dependent on the ability to regenerate, however, during ageing or disease, the proliferative capacity is reduced, leading to loss of muscle function. We have previously demonstrated the presence of vitamin K2 in bovine skeletal muscles, but whether vitamin K has a role in muscle regulation and function is unknown. In this study, we used primary bovine skeletal muscle cells, cultured in monolayers in vitro, to assess a potential effect of vitamin K2 (MK-4) during myogenesis of muscle cells. Cell viability experiments demonstrate that the amount of ATP produced by the cells was unchanged when MK-4 was added, indicating viable cells. Cytotoxicity analysis show that MK-4 reduced the lactate dehydrogenase (LDH) released into the media, suggesting that MK-4 was beneficial to the muscle cells. Cell migration, proliferation and differentiation was characterised after MK-4 incubation using wound scratch analysis, immunocytochemistry and real-time PCR analysis. Adding MK-4 to the cells led to an increased muscle proliferation, increased gene expression of the myogenic transcription factor myod as well as increased cell migration. In addition, we observed a reduction in the fusion index and relative gene expression of muscle differentiation markers, with fewer complex myotubes formed in MK-4 stimulated cells compared to control cells, indicating that the MK-4 plays a significant role during the early phases of muscle proliferation. Likewise, we see the same pattern for the relative gene expression of collagen 1A, showing increased gene expression in proliferating cells, and reduced expression in differentiating cells. Our results also suggest that MK-4 incubation affect low density lipoprotein receptor-related protein 1 (LRP1) and the low-density lipoprotein receptor (LDLR) with a peak in gene expression after 45 min of MK-4 incubation. Altogether, our experiments show that MK-4 has a positive effect on muscle cell migration and

  5. Expression of an insulin-regulatable glucose carrier in muscle and fat endothelial cells

    NASA Astrophysics Data System (ADS)

    Vilaró, Senen; Palacín, Manuel; Pilch, Paul F.; Testar, Xavier; Zorzano, Antonio

    1989-12-01

    INSULIN rapidly stimulates glucose use in the major target tissues, muscle and fat, by modulating a tissue-specific glucose transporter isoform1-6. Access of glucose to the target tissue is restricted by endothelial cells which line the walls of nonfenestrated capillaries of fat and muscle7. Thus, we examined whether the capillary endothelial cells are actively involved in the modulation of glucose availability by these tissues. We report here the abundant expression of the muscle/fat glucose transporter isoform in endothelial cells, using an immunocytochemical analysis with a monoclonal antibody specific for this isoform1. This expression is restricted to endothelial cells from the major insulin target tissues, and it is not detected in brain and liver where insulin does not activate glucose transport. The expression of the muscle/fat transporter isoform in endothelial cells is significantly greater than in the neighbouring muscle and fat cells. Following administration of insulin to animals in vivo, there occurs a rapid increase in the number of muscle/fat transporters present in the lumenal plasma membrane of the capillary endothelial cells. These results document that insulin promotes the translocation of the muscle/fat glucose transporter in endothelial cells. It is therefore likely that endothelial cells play an important role in the regulation of glucose use by the major insulin target tissues in normal and diseased states.

  6. Smooth Muscle-Like Cells Generated from Human Mesenchymal Stromal Cells Display Marker Gene Expression and Electrophysiological Competence Comparable to Bladder Smooth Muscle Cells.

    PubMed

    Brun, Juliane; Lutz, Katrin A; Neumayer, Katharina M H; Klein, Gerd; Seeger, Tanja; Uynuk-Ool, Tatiana; Wörgötter, Katharina; Schmid, Sandra; Kraushaar, Udo; Guenther, Elke; Rolauffs, Bernd; Aicher, Wilhelm K; Hart, Melanie L

    2015-01-01

    The use of mesenchymal stromal cells (MSCs) differentiated toward a smooth muscle cell (SMC) phenotype may provide an alternative for investigators interested in regenerating urinary tract organs such as the bladder where autologous smooth muscle cells cannot be used or are unavailable. In this study we measured the effects of good manufacturing practice (GMP)-compliant expansion followed by myogenic differentiation of human MSCs on the expression of a range of contractile (from early to late) myogenic markers in relation to the electrophysiological parameters to assess the functional role of the differentiated MSCs and found that differentiation of MSCs associated with electrophysiological competence comparable to bladder SMCs. Within 1-2 weeks of myogenic differentiation, differentiating MSCs significantly expressed alpha smooth muscle actin (αSMA; ACTA2), transgelin (TAGLN), calponin (CNN1), and smooth muscle myosin heavy chain (SM-MHC; MYH11) according to qRT-PCR and/or immunofluorescence and Western blot. Voltage-gated Na+ current levels also increased within the same time period following myogenic differentiation. In contrast to undifferentiated MSCs, differentiated MSCs and bladder SMCs exhibited elevated cytosolic Ca2+ transients in response to K+-induced depolarization and contracted in response to K+ indicating functional maturation of differentiated MSCs. Depolarization was suppressed by Cd2+, an inhibitor of voltage-gated Ca2+-channels. The expression of Na+-channels was pharmacologically identified as the Nav1.4 subtype, while the K+ and Ca2+ ion channels were identified by gene expression of KCNMA1, CACNA1C and CACNA1H which encode for the large conductance Ca2+-activated K+ channel BKCa channels, Cav1.2 L-type Ca2+ channels and Cav3.2 T-type Ca2+ channels, respectively. This protocol may be used to differentiate adult MSCs into smooth muscle-like cells with an intermediate-to-late SMC contractile phenotype exhibiting voltage-gated ion channel

  7. Fibronectin Matrix Polymerization Regulates Smooth Muscle Cell Phenotype through a Rac1 Dependent Mechanism

    PubMed Central

    Shi, Feng; Long, Xiaochun; Hendershot, Allison; Miano, Joseph M.; Sottile, Jane

    2014-01-01

    Smooth muscle cells are maintained in a differentiated state in the vessel wall, but can be modulated to a synthetic phenotype following injury. Smooth muscle phenotypic modulation is thought to play an important role in the pathology of vascular occlusive diseases. Phenotypically modulated smooth muscle cells exhibit increased proliferative and migratory properties that accompany the downregulation of smooth muscle cell marker proteins. Extracellular matrix proteins, including fibronectin, can regulate the smooth muscle phenotype when used as adhesive substrates. However, cells produce and organize a 3-dimensional fibrillar extracellular matrix, which can affect cell behavior in distinct ways from the protomeric 2-dimensional matrix proteins that are used as adhesive substrates. We previously showed that the deposition/polymerization of fibronectin into the extracellular matrix can regulate the deposition and organization of other extracellular matrix molecules in vitro. Further, our published data show that the presence of a fibronectin polymerization inhibitor results in increased expression of smooth muscle cell differentiation proteins and inhibits vascular remodeling in vivo. In this manuscript, we used an in vitro cell culture system to determine the mechanism by which fibronectin polymerization affects smooth muscle phenotypic modulation. Our data show that fibronectin polymerization decreases the mRNA levels of multiple smooth muscle differentiation genes, and downregulates the levels of smooth muscle α-actin and calponin proteins by a Rac1-dependent mechanism. The expression of smooth muscle genes is transcriptionally regulated by fibronectin polymerization, as evidenced by the increased activity of luciferase reporter constructs in the presence of a fibronectin polymerization inhibitor. Fibronectin polymerization also promotes smooth muscle cell growth, and decreases the levels of actin stress fibers. These data define a Rac1-dependent pathway wherein

  8. Osteogenic differentiation capacity of human skeletal muscle-derived progenitor cells.

    PubMed

    Oishi, Teruyo; Uezumi, Akiyoshi; Kanaji, Arihiko; Yamamoto, Naoki; Yamaguchi, Asami; Yamada, Harumoto; Tsuchida, Kunihiro

    2013-01-01

    Heterotopic ossification (HO) is defined as the formation of ectopic bone in soft tissue outside the skeletal tissue. HO is thought to result from aberrant differentiation of osteogenic progenitors within skeletal muscle. However, the precise origin of HO is still unclear. Skeletal muscle contains two kinds of progenitor cells, myogenic progenitors and mesenchymal progenitors. Myogenic and mesenchymal progenitors in human skeletal muscle can be identified as CD56(+) and PDGFRα(+) cells, respectively. The purpose of this study was to investigate the osteogenic differentiation potential of human skeletal muscle-derived progenitors. Both CD56(+) cells and PDGFRα(+) cells showed comparable osteogenic differentiation potential in vitro. However, in an in vivo ectopic bone formation model, PDGFRα(+) cells formed bone-like tissue and showed successful engraftment, while CD56(+) cells did not form bone-like tissue and did not adapt to an osteogenic environment. Immunohistological analysis of human HO sample revealed that many PDGFRα(+) cells were localized in proximity to ectopic bone formed in skeletal muscle. MicroRNAs (miRNAs) are known to regulate many biological processes including osteogenic differentiation. We investigated the participation of miRNAs in the osteogenic differentiation of PDGFRα(+) cells by using microarray. We identified miRNAs that had not been known to be involved in osteogenesis but showed dramatic changes during osteogenic differentiation of PDGFRα(+) cells. Upregulation of miR-146b-5p and -424 and downregulation of miR-7 during osteogenic differentiation of PDGFRα(+) cells were confirmed by quantitative real-time RT-PCR. Inhibition of upregulated miRNAs, miR-146b-5p and -424, resulted in the suppression of osteocyte maturation, suggesting that these two miRNAs have the positive role in the osteogenesis of PDGFRα(+) cells. Our results suggest that PDGFRα(+) cells may be the major source of HO and that the newly identified miRNAs may

  9. Using Transgenic Zebrafish to Study Muscle Stem/Progenitor Cells.

    PubMed

    Nguyen, Phong D; Currie, Peter D

    2017-01-01

    Understanding muscle stem cell behaviors can potentially provide insights into how these cells act and respond during normal growth and diseased contexts. The zebrafish is an ideal model organism to examine these behaviors in vivo where it would normally be technically challenging in other mammalian models. This chapter will describe the procedures required to successfully conduct live imaging of zebrafish transgenics that has specifically been adapted for skeletal muscle.

  10. Mutations in RSPH1 cause primary ciliary dyskinesia with a unique clinical and ciliary phenotype.

    PubMed

    Knowles, Michael R; Ostrowski, Lawrence E; Leigh, Margaret W; Sears, Patrick R; Davis, Stephanie D; Wolf, Whitney E; Hazucha, Milan J; Carson, Johnny L; Olivier, Kenneth N; Sagel, Scott D; Rosenfeld, Margaret; Ferkol, Thomas W; Dell, Sharon D; Milla, Carlos E; Randell, Scott H; Yin, Weining; Sannuti, Aruna; Metjian, Hilda M; Noone, Peadar G; Noone, Peter J; Olson, Christina A; Patrone, Michael V; Dang, Hong; Lee, Hye-Seung; Hurd, Toby W; Gee, Heon Yung; Otto, Edgar A; Halbritter, Jan; Kohl, Stefan; Kircher, Martin; Krischer, Jeffrey; Bamshad, Michael J; Nickerson, Deborah A; Hildebrandt, Friedhelm; Shendure, Jay; Zariwala, Maimoona A

    2014-03-15

    Primary ciliary dyskinesia (PCD) is a genetically heterogeneous recessive disorder of motile cilia, but the genetic cause is not defined for all patients with PCD. To identify disease-causing mutations in novel genes, we performed exome sequencing, follow-up characterization, mutation scanning, and genotype-phenotype studies in patients with PCD. Whole-exome sequencing was performed using NimbleGen capture and Illumina HiSeq sequencing. Sanger-based sequencing was used for mutation scanning, validation, and segregation analysis. We performed exome sequencing on an affected sib-pair with normal ultrastructure in more than 85% of cilia. A homozygous splice-site mutation was detected in RSPH1 in both siblings; parents were carriers. Screening RSPH1 in 413 unrelated probands, including 325 with PCD and 88 with idiopathic bronchiectasis, revealed biallelic loss-of-function mutations in nine additional probands. Five affected siblings of probands in RSPH1 families harbored the familial mutations. The 16 individuals with RSPH1 mutations had some features of PCD; however, nasal nitric oxide levels were higher than in patients with PCD with other gene mutations (98.3 vs. 20.7 nl/min; P < 0.0003). Additionally, individuals with RSPH1 mutations had a lower prevalence (8 of 16) of neonatal respiratory distress, and later onset of daily wet cough than typical for PCD, and better lung function (FEV1), compared with 75 age- and sex-matched PCD cases (73.0 vs. 61.8, FEV1 % predicted; P = 0.043). Cilia from individuals with RSPH1 mutations had normal beat frequency (6.1 ± Hz at 25°C), but an abnormal, circular beat pattern. The milder clinical disease and higher nasal nitric oxide in individuals with biallelic mutations in RSPH1 provides evidence of a unique genotype-phenotype relationship in PCD, and suggests that mutations in RSPH1 may be associated with residual ciliary function.

  11. Ciliary neurotrophic factor is an endogenous pyrogen.

    PubMed

    Shapiro, L; Zhang, X X; Rupp, R G; Wolff, S M; Dinarello, C A

    1993-09-15

    Fever is initiated by the action of polypeptide cytokines called endogenous pyrogens, which are produced by the host during inflammation, trauma, or infection and which elevate the thermoregulatory set point in the hypothalamus. Ciliary neurotrophic factor (CNTF) supports the differentiation and survival of central and peripheral neurons. We describe the activity of CNTF as intrinsically pyrogenic in the rabbit. CNTF induced a monophasic fever which rose rapidly (within the first 12 min) following intravenous injection; CNTF fever was blocked by pretreatment with indomethacin. The fever induced by CNTF was not due to contaminating endotoxins. Increasing doses of CNTF resulted in prolongation of the fever, suggesting the subsequent induction of additional endogenous pyrogenic activity. After passive transfer of plasma obtained during CNTF-induced fever, endogenous pyrogen activity was not present in the circulation; CNTF also did not induce the endogenous pyrogens interleukin 1, tumor necrosis factor, or interleukin 6 in vitro. Nevertheless, a second endogenous pyrogen may originate within the central nervous system following the systemic injection of CNTF. Of the four endogenous pyrogens described to date (interleukin 1, tumor necrosis factor, interferon, and interleukin 6), CNTF, like interleukin 6, utilizes the cell-surface gp 130 signal-transduction apparatus.

  12. AMP-activated Protein Kinase Stimulates Warburg-like Glycolysis and Activation of Satellite Cells during Muscle Regeneration*

    PubMed Central

    Fu, Xing; Zhu, Mei-Jun; Dodson, Mike V.; Du, Min

    2015-01-01

    Satellite cells are the major myogenic stem cells residing inside skeletal muscle and are indispensable for muscle regeneration. Satellite cells remain largely quiescent but are rapidly activated in response to muscle injury, and the derived myogenic cells then fuse to repair damaged muscle fibers or form new muscle fibers. However, mechanisms eliciting metabolic activation, an inseparable step for satellite cell activation following muscle injury, have not been defined. We found that a noncanonical Sonic Hedgehog (Shh) pathway is rapidly activated in response to muscle injury, which activates AMPK and induces a Warburg-like glycolysis in satellite cells. AMPKα1 is the dominant AMPKα isoform expressed in satellite cells, and AMPKα1 deficiency in satellite cells impairs their activation and myogenic differentiation during muscle regeneration. Drugs activating noncanonical Shh promote proliferation of satellite cells, which is abolished because of satellite cell-specific AMPKα1 knock-out. Taken together, AMPKα1 is a critical mediator linking noncanonical Shh pathway to Warburg-like glycolysis in satellite cells, which is required for satellite activation and muscle regeneration. PMID:26370082

  13. Variability in Beta-Adrenergic Receptor Population in Cultured Chicken Muscle Cells

    NASA Technical Reports Server (NTRS)

    Young, Ronald B; Bridge, Kristin Y.; Vaughn, Jeffrey R.

    1998-01-01

    Investigations into expression of the beta-adrenergic receptor (bAR) in chicken skeletal muscle cells in culture were initiated because several beta-adrenergic receptor agonists are known to increase skeletal muscle protein deposition in avian and mammalian species. During initial attempts to study the bAR population on the surface of chicken skeletal muscle cells, we observed a high degree of variability that was later found to be the result of using different batches of horse serum in the cell culture media. The separation between total binding and nonspecific binding in cells grown in two serum samples was approximately two-fold The number of nuclei within multinucleated myotubes was not significantly different in cells grown in the two serum samples. To investigate whether these two sera had an effect on coupling efficiency between bAR population and cAMP production, the ability of these cells to synthesize cAMP was also assessed. Despite the two-fold difference in receptor population, the ability of these cells to synthesize cAMP was not significantly different. Because of the possible link between bAR population and muscle protein, we also determined if the quantity of the major skeletal muscle protein, myosin, was affected by conditions that so drastically affected the bAR population. The quantity of myosin heavy chain was not significantly different.

  14. Vortex arrays and ciliary tangles underlie the feeding-swimming trade-off in starfish larvae

    NASA Astrophysics Data System (ADS)

    Gilpin, William; Prakash, Vivek N.; Prakash, Manu

    2017-04-01

    Many marine invertebrates have larval stages covered in linear arrays of beating cilia, which propel the animal while simultaneously entraining planktonic prey. These bands are strongly conserved across taxa spanning four major superphyla, and they are responsible for the unusual morphologies of many invertebrate larvae. However, few studies have investigated their underlying hydrodynamics. Here, we study the ciliary bands of starfish larvae, and discover a beautiful pattern of slowly evolving vortices that surrounds the swimming animals. Closer inspection of the bands reveals unusual ciliary `tangles' analogous to topological defects that break up and re-form as the animal adjusts its swimming stroke. Quantitative experiments and modelling demonstrate that these vortices create a physical trade-off between feeding and swimming in heterogeneous environments, which manifests as distinct flow patterns or `eigenstrokes' representing each behaviour--potentially implicating neuronal control of cilia. This quantitative interplay between larval form and hydrodynamic function may generalize to other invertebrates with ciliary bands, and illustrates the potential effects of active boundary conditions in other biological and synthetic systems.

  15. BRE facilitates skeletal muscle regeneration by promoting satellite cell motility and differentiation

    PubMed Central

    Xiao, Lihai; Lee, Kenneth Ka Ho

    2016-01-01

    ABSTRACT The function of the Bre gene in satellite cells was investigated during skeletal muscle regeneration. The tibialis anterior leg muscle was experimentally injured in Bre knockout mutant (BRE-KO) mice. It was established that the accompanying muscle regeneration was impaired as compared with their normal wild-type counterparts (BRE-WT). There were significantly fewer pax7+ satellite cells and smaller newly formed myofibers present in the injury sites of BRE-KO mice. Bre was required for satellite cell fusion and myofiber formation. The cell fusion index and average length of newly-formed BRE-KO myofibers were found to be significantly reduced as compared with BRE-WT myofibers. It is well established that satellite cells are highly invasive which confers on them the homing ability to reach the muscle injury sites. Hence, we tracked the migratory behavior of these cells using time-lapse microscopy. Image analysis revealed no difference in directionality of movement between BRE-KO and BRE-WT satellite cells but there was a significant decrease in the velocity of BRE-KO cell movement. Moreover, chemotactic migration assays indicated that BRE-KO satellite cells were significantly less responsive to chemoattractant SDF-1α than BRE-WT satellite cells. We also established that BRE normally protects CXCR4 from SDF-1α-induced degradation. In sum, BRE facilitates skeletal muscle regeneration by enhancing satellite cell motility, homing and fusion. PMID:26740569

  16. Fate of 3H-thymidine labelled myogenic cells in regeneration of muscle isografts.

    PubMed

    Gutmann, E; Mares, V; Stichová, J

    1976-03-05

    Intact and denervated extensor digitorum longus (EDL) muscles of 20-day-old inbred Lewis-Wistar rats were labelled with 3H-thymidine. Ninety minutes after the injection of the isotope 4.0% of the nuclei were labelled in the intact (i.e. innervated) and 9.6% in the muscles, denervated 3 days before administration of the isotope. The labelled EDL muscles were grafted into the bed of the previously removed EDL muscles of inbred animals and these isografts were studied 30 days later. In the EDL muscles, regenerated from innervated isografts only occasionally labelled endothelial cells were found whereas in the muscles regenerated from denervated isografts also parenchymal muscle nuclei were regularly labelled. The incidence of labelled nuclei in the regenerated EDL muscles was, however, about 20 times lower than in the donor EDL muscles. The presen experiments provide a direct proof of utilization of donor satelite cell nuclei for regeneration in grafted muscle tissue. With respect to the low incidence of labelled nuclei in regenerated EDL muscles, other sources of cells apparently also contribute to the regeneration process.

  17. MASTR directs MyoD-dependent satellite cell differentiation during skeletal muscle regeneration

    PubMed Central

    Mokalled, Mayssa H.; Johnson, Aaron N.; Creemers, Esther E.; Olson, Eric N.

    2012-01-01

    In response to skeletal muscle injury, satellite cells, which function as a myogenic stem cell population, become activated, expand through proliferation, and ultimately fuse with each other and with damaged myofibers to promote muscle regeneration. Here, we show that members of the Myocardin family of transcriptional coactivators, MASTR and MRTF-A, are up-regulated in satellite cells in response to skeletal muscle injury and muscular dystrophy. Global and satellite cell-specific deletion of MASTR in mice impairs skeletal muscle regeneration. This impairment is substantially greater when MRTF-A is also deleted and is due to aberrant differentiation and excessive proliferation of satellite cells. These abnormalities mimic those associated with genetic deletion of MyoD, a master regulator of myogenesis, which is down-regulated in the absence of MASTR and MRTF-A. Consistent with an essential role of MASTR in transcriptional regulation of MyoD expression, MASTR activates a muscle-specific postnatal MyoD enhancer through associations with MEF2 and members of the Myocardin family. Our results provide new insights into the genetic circuitry of muscle regeneration and identify MASTR as a central regulator of this process. PMID:22279050

  18. PACS-1 mediates phosphorylation-dependent ciliary trafficking of the cyclic-nucleotide-gated channel in olfactory sensory neurons.

    PubMed

    Jenkins, Paul M; Zhang, Lian; Thomas, Gary; Martens, Jeffrey R

    2009-08-26

    Impaired ciliary protein transport in olfactory sensory neurons (OSNs) leads to anosmia, and is a newly recognized clinical manifestation of a class of human disorders called ciliopathies. Surprisingly little is known regarding the mechanisms controlling trafficking to this unique neuronal compartment. Here, we show a novel role for phosphofurin acidic cluster-sorting protein 1 (PACS-1) in the ciliary trafficking of the olfactory cyclic-nucleotide-gated (CNG) channel. PACS-1 is an intracellular sorting protein that mediates its effects through the binding of acidic clusters on cargo protein. This interaction is dependent on CK2 phosphorylation of both PACS-1 and its cargo. We show that CNGB1b contains two putative PACS-1 binding sites, which are phosphorylated by the serine/threonine protein kinase, CK2. Additionally, we show that PACS-1 is expressed in OSNs and interacts in complex with the CNG channel. CK2 inhibition in native OSNs causes a loss of CNG channel from cilia and subsequent olfactory dysfunction, while adenoviral expression of mutant PACS-1 causes similar mislocalization. These results provide a mechanism for the subunit-dependent ciliary trafficking of the CNG channel and offer insight into the mechanisms of ciliary transport.

  19. Automated image analysis reveals the dynamic 3-dimensional organization of multi-ciliary arrays

    PubMed Central

    Galati, Domenico F.; Abuin, David S.; Tauber, Gabriel A.; Pham, Andrew T.; Pearson, Chad G.

    2016-01-01

    ABSTRACT Multi-ciliated cells (MCCs) use polarized fields of undulating cilia (ciliary array) to produce fluid flow that is essential for many biological processes. Cilia are positioned by microtubule scaffolds called basal bodies (BBs) that are arranged within a spatially complex 3-dimensional geometry (3D). Here, we develop a robust and automated computational image analysis routine to quantify 3D BB organization in the ciliate, Tetrahymena thermophila. Using this routine, we generate the first morphologically constrained 3D reconstructions of Tetrahymena cells and elucidate rules that govern the kinetics of MCC organization. We demonstrate the interplay between BB duplication and cell size expansion through the cell cycle. In mutant cells, we identify a potential BB surveillance mechanism that balances large gaps in BB spacing by increasing the frequency of closely spaced BBs in other regions of the cell. Finally, by taking advantage of a mutant predisposed to BB disorganization, we locate the spatial domains that are most prone to disorganization by environmental stimuli. Collectively, our analyses reveal the importance of quantitative image analysis to understand the principles that guide the 3D organization of MCCs. PMID:26700722

  20. Retained Myogenic Potency of Human Satellite Cells from Torn Rotator Cuff Muscles Despite Fatty Infiltration.

    PubMed

    Koide, Masashi; Hagiwara, Yoshihiro; Tsuchiya, Masahiro; Kanzaki, Makoto; Hatakeyama, Hiroyasu; Tanaka, Yukinori; Minowa, Takashi; Takemura, Taro; Ando, Akira; Sekiguchi, Takuya; Yabe, Yutaka; Itoi, Eiji

    2018-01-01

    Rotator cuff tears (RCTs) are a common shoulder problem in the elderly that can lead to both muscle atrophy and fatty infiltration due to less physical load. Satellite cells, quiescent cells under the basal lamina of skeletal muscle fibers, play a major role in muscle regeneration. However, the myogenic potency of human satellite cells in muscles with fatty infiltration is unclear due to the difficulty in isolating from small samples, and the mechanism of the progression of fatty infiltration has not been elucidated. The purpose of this study was to analyze the population of myogenic and adipogenic cells in disused supraspinatus (SSP) and intact subscapularis (SSC) muscles of the RCTs from the same patients using fluorescence-activated cell sorting. The microstructure of the muscle with fatty infiltration was observed as a whole mount condition under multi-photon microscopy. Myogenic differentiation potential and gene expression were evaluated in satellite cells. The results showed that the SSP muscle with greater fatty infiltration surrounded by collagen fibers compared with the SSC muscle under multi-photon microscopy. A positive correlation was observed between the ratio of muscle volume to fat volume and the ratio of myogenic precursor to adipogenic precursor. Although no difference was observed in the myogenic potential between the two groups in cell culture, satellite cells in the disused SSP muscle showed higher intrinsic myogenic gene expression than those in the intact SSC muscle. Our results indicate that satellite cells from the disused SSP retain sufficient potential of muscle growth despite the fatty infiltration.

  1. Improved Cell Culture Method for Growing Contracting Skeletal Muscle Models

    NASA Technical Reports Server (NTRS)

    Marquette, Michele L.; Sognier, Marguerite A.

    2013-01-01

    An improved method for culturing immature muscle cells (myoblasts) into a mature skeletal muscle overcomes some of the notable limitations of prior culture methods. The development of the method is a major advance in tissue engineering in that, for the first time, a cell-based model spontaneously fuses and differentiates into masses of highly aligned, contracting myotubes. This method enables (1) the construction of improved two-dimensional (monolayer) skeletal muscle test beds; (2) development of contracting three-dimensional tissue models; and (3) improved transplantable tissues for biomedical and regenerative medicine applications. With adaptation, this method also offers potential application for production of other tissue types (i.e., bone and cardiac) from corresponding precursor cells.

  2. Prosurvival Factors Improve Functional Engraftment of Myogenically Converted Dermal Cells into Dystrophic Skeletal Muscle

    PubMed Central

    Muir, Lindsey A.; Murry, Charles E.

    2016-01-01

    In Duchenne muscular dystrophy (DMD) and other muscle wasting disorders, cell therapies are a promising route for promoting muscle regeneration by supplying a functional copy of the missing dystrophin gene and contributing new muscle fibers. The clinical application of cell-based therapies is resource intensive, and it will therefore be necessary to address key limitations that reduce cell engraftment into muscle tissue. A pressing issue is poor donor cell survival following transplantation, which in preclinical studies limits the ability to effectively test the impact of cell-based therapy on whole muscle function. We, therefore, sought to improve engraftment and the functional impact of in vivo myogenically converted dermal fibroblasts (dFbs) using a prosurvival cocktail (PSC) that includes heat shock followed by treatment with insulin-like growth factor-1, a caspase inhibitor, a Bcl-XL peptide, a KATP channel opener, basic fibroblast growth factor, Matrigel, and cyclosporine A. Advantages of dFbs include compatibility with the autologous setting, ease of isolation, and greater proliferative potential than DMD satellite cells. dFbs expressed tamoxifen-inducible MyoD and carried a mini-dystrophin gene driven by a muscle-specific promoter. After transplantation into muscles of mdx mice, a 70% reduction in donor cells was observed by day 5, and a 94% reduction by day 28. However, treatment with PSC gave a nearly three-fold increase in donor cells in early engraftment, and greatly increased the number of donor-contributed muscle fibers and total engrafted area in transplanted muscles. Furthermore, dystrophic muscles that received dFbs with PSC displayed reduced injury with eccentric contractions and an increase in maximum isometric force. Thus, enhancing survival of myogenic cells increases engraftment and improves structure and function of dystrophic muscle. PMID:27503462

  3. Prosurvival Factors Improve Functional Engraftment of Myogenically Converted Dermal Cells into Dystrophic Skeletal Muscle.

    PubMed

    Muir, Lindsey A; Murry, Charles E; Chamberlain, Jeffrey S

    2016-09-07

    In Duchenne muscular dystrophy (DMD) and other muscle wasting disorders, cell therapies are a promising route for promoting muscle regeneration by supplying a functional copy of the missing dystrophin gene and contributing new muscle fibers. The clinical application of cell-based therapies is resource intensive, and it will therefore be necessary to address key limitations that reduce cell engraftment into muscle tissue. A pressing issue is poor donor cell survival following transplantation, which in preclinical studies limits the ability to effectively test the impact of cell-based therapy on whole muscle function. We, therefore, sought to improve engraftment and the functional impact of in vivo myogenically converted dermal fibroblasts (dFbs) using a prosurvival cocktail (PSC) that includes heat shock followed by treatment with insulin-like growth factor-1, a caspase inhibitor, a Bcl-XL peptide, a K ATP channel opener, basic fibroblast growth factor, Matrigel, and cyclosporine A. Advantages of dFbs include compatibility with the autologous setting, ease of isolation, and greater proliferative potential than DMD satellite cells. dFbs expressed tamoxifen-inducible MyoD and carried a mini-dystrophin gene driven by a muscle-specific promoter. After transplantation into muscles of mdx mice, a 70% reduction in donor cells was observed by day 5, and a 94% reduction by day 28. However, treatment with PSC gave a nearly three-fold increase in donor cells in early engraftment, and greatly increased the number of donor-contributed muscle fibers and total engrafted area in transplanted muscles. Furthermore, dystrophic muscles that received dFbs with PSC displayed reduced injury with eccentric contractions and an increase in maximum isometric force. Thus, enhancing survival of myogenic cells increases engraftment and improves structure and function of dystrophic muscle.

  4. Exome sequencing for the differential diagnosis of ciliary chondrodysplasias: Example of a WDR35 mutation case and review of the literature.

    PubMed

    Antony, Dinu; Nampoory, Narayanan; Bacchelli, Chiara; Melhem, Motasem; Wu, Kaman; James, Chela T; Beales, Philip L; Hubank, Mike; Thomas, Daisy; Mashankar, Anant; Behbehani, Kazem; Schmidts, Miriam; Alsmadi, Osama

    2017-12-01

    Exome sequencing is becoming widely popular and affordable, making it one of the most desirable methods for the identification of rare genetic variants for clinical diagnosis. Here, we report the clinical application of whole exome sequencing for the ultimate diagnosis of a ciliary chondrodysplasia case presented with an initial clinical diagnosis of Asphyxiating Thoracic Dystrophy (ATD, Jeune Syndrome). We have identified a novel homozygous missense mutation in WDR35 (c.206G > A), a gene previously associated with Sensenbrenner Syndrome, Ellis-van Creveld syndrome and Short-rib polydactyly syndrome type V. The genetic findings in this family led to the re-evaluation of the initial diagnosis and a differential diagnosis of Sensenbrenner Syndrome was made after cautious re-examination of the patient. Cell culture studies revealed normal subcellular localization of the mutant WDR35 protein in comparison to wildtype protein, pointing towards impaired protein-protein interaction and/or altered cell signaling pathways as a consequence of the mutated allele. This research study highlights the importance of including pathogenic variant identification in the diagnosis pipeline of ciliary chondrodysplasias, especially for clinically not fully defined phenotypes. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  5. Functional dysregulation of stem cells during aging: a focus on skeletal muscle stem cells.

    PubMed

    García-Prat, Laura; Sousa-Victor, Pedro; Muñoz-Cánoves, Pura

    2013-09-01

    Aging of an organism is associated with the functional decline of tissues and organs, as well as a sharp decline in the regenerative capacity of stem cells. A prevailing view holds that the aging rate of an individual depends on the ratio of tissue attrition to tissue regeneration. Therefore, manipulations that favor the balance towards regeneration may prevent or delay aging. Skeletal muscle is a specialized tissue composed of postmitotic myofibers that contract to generate force. Satellite cells are the adult stem cells responsible for skeletal muscle regeneration. Recent studies on the biology of skeletal muscle and satellite cells in aging have uncovered the critical impact of systemic and niche factors on stem cell functionality and demonstrated the capacity of aged satellite cells to rejuvenate and increase their regenerative potential when exposed to a youthful environment. Here we review the current literature on the coordinated relationship between cell extrinsic and intrinsic factors that regulate the function of satellite cells, and ultimately determine tissue homeostasis and repair during aging, and which encourage the search for new anti-aging strategies. © 2013 The Authors Journal compilation © 2013 FEBS.

  6. Further considerations on in vitro skeletal muscle cell death

    PubMed Central

    Battistelli, Michela; Salucci, Sara; Burattini, Sabrina; Falcieri, Elisabetta

    2013-01-01

    Summary The present review discusses the apoptotic behavior induced by chemical and physical triggers in C2C12 skeletal muscle cells, comparing myoblast to myotube sensitivity, and investigating it by means of morphological, biochemical and cytofluorimetric analyses. After all treatments, myotubes, differently from myoblasts, showed a poor sensitivity to cell death. Intriguingly, in cells exposed to staurosporine, etoposide and UVB radiation, apoptotic and normal nuclei within the same fibercould be revealed. The presence of nuclear-dependent “territorial” death domains in the syncytium could explain a delayed cell death of myotubes compared to mononucleated cells. Moreover, autophagic granules abundantly appeared in myotubes after each treatment. Autophagy could protect muscle cell integrity against chemical and physical stimuli, making C2C12 myotubes, more resistant to cell death induction. PMID:24596689

  7. Localization of multidrug resistance-associated protein 2 in the nonpigmented ciliary epithelium of the eye.

    PubMed

    Pelis, Ryan M; Shahidullah, Mohammad; Ghosh, Sikha; Coca-Prados, Miguel; Wright, Stephen H; Delamere, Nicholas A

    2009-05-01

    The nonpigmented epithelium (NPE) of the ciliary body represents an important component of the blood-aqueous barrier of the eye. Many therapeutic drugs penetrate poorly across the NPE into the aqueous humor of the eye interior. Several of these therapeutic drugs, such as methotrexate, vincristine, and etoposide, are substrates of the multidrug resistance-associated protein 2 (MRP2). Abundant MRP2 protein was detected by Western blot in homogenates of human ciliary body and freshly dissected porcine NPE. In cultured porcine NPE, the intracellular accumulation of the MRP2 substrates calcein (1.8-fold), 5-(and-6)-carboxy-2',7'-dichlorofluorescein (22.1-fold), and doxorubicin (1.9-fold) was significantly increased in the presence of 50 microM MK571 ((E)-3-[[[3-[2-(7-chloro-2-quinolinyl)-ethenyl]phenyl]-[[3-dimethylamino)-3-oxopropyl]thio]methyl]thio]-propanoic acid), an MRP inhibitor. In addition, the intracellular accumulation of the MRP2 substrate glutathione methylfluorescein was increased by 50 microM MK571 (4.3-fold), 500 microM indomethacin (2.6-fold), and 50 microM cyclosporin A (2.1-fold) but not by 500 microM sulfinpyrazone. These data are consistent with MRP2-mediated transport activity in cultured NPE, and MRP2 mRNA (reverse transcriptase-polymerase chain reaction) and protein (Western blot) were detected in the cultured cells. Immunolocalization studies in native human and porcine eyes showed MRP2 protein at the apical interface of the NPE and pigmented cell layers. Close examination of MRP2 immunoreactivity supported the conclusion that MRP2 is localized in the apical membrane of the NPE. MRP2 at the apical membrane of NPE cells may be involved in protecting intraocular tissues from exposure to potentially harmful toxins.

  8. The microprotein Minion controls cell fusion and muscle formation

    PubMed Central

    Zhang, Qiao; Vashisht, Ajay A.; O'Rourke, Jason; Corbel, Stéphane Y; Moran, Rita; Romero, Angelica; Miraglia, Loren; Zhang, Jia; Durrant, Eric; Schmedt, Christian; Sampath, Srinath C.; Sampath, Srihari C.

    2017-01-01

    Although recent evidence has pointed to the existence of small open reading frame (smORF)-encoded microproteins in mammals, their function remains to be determined. Skeletal muscle development requires fusion of mononuclear progenitors to form multinucleated myotubes, a critical but poorly understood process. Here we report the identification of Minion (microprotein inducer of fusion), a smORF encoding an essential skeletal muscle specific microprotein. Myogenic progenitors lacking Minion differentiate normally but fail to form syncytial myotubes, and Minion-deficient mice die perinatally and demonstrate a marked reduction in fused muscle fibres. The fusogenic activity of Minion is conserved in the human orthologue, and co-expression of Minion and the transmembrane protein Myomaker is sufficient to induce cellular fusion accompanied by rapid cytoskeletal rearrangement, even in non-muscle cells. These findings establish Minion as a novel microprotein required for muscle development, and define a two-component programme for the induction of mammalian cell fusion. Moreover, these data also significantly expand the known functions of smORF-encoded microproteins. PMID:28569745

  9. Ex vivo bupivacaine treatment results in increased adipogenesis of skeletal muscle cells in the rat.

    PubMed

    Yamanouchi, Keitaro; Nakamura, Katsuyuki; Takegahara, Yuki; Nakano, Shin-ichi; Nishihara, Masugi

    2013-11-01

    Intramuscular adipose tissue (IMAT) is observed in some skeletal muscle pathologies. IMAT is implicated not only in the disorders of muscle contraction, but also of metabolism and insulin sensitivity due to its nature as a secretary organ. Several studies indicate the presence of cells with adipogenic potential in skeletal muscle. However, the mechanism of fate specification that triggers these cells to enter an adipogenic program in vivo remains to be solved. In the present study, we examined whether activation of the adipogenic program of muscle-resident cells precedes their proliferation upon muscle injury. For this purpose, muscle injury was induced by injecting bupivacaine (BPVC) to excised skeletal muscle ex vivo. Cells isolated from ex vivo BPVC-treated muscle exhibited higher adipogenic potential than those from saline-treated muscle. Pre-plating exposure of skeletal muscle cells to basic fibroblast growth factor (bFGF) mimicked the effect of ex vivo BPVC-treatment, suggesting that bFGF released from extracellular matrix in response to muscle injury activates their adipogenic program. Interestingly, the number of myotubes were significantly reduced in the culture from BPVC-treated muscle, suggesting that adipocytes negatively regulate myogenesis. © 2013 Japanese Society of Animal Science.

  10. Membrane segregation and downregulation of raft markers during sarcolemmal differentiation in skeletal muscle cells.

    PubMed

    Draeger, A; Monastyrskaya, K; Burkhard, F C; Wobus, A M; Moss, S E; Babiychuk, E B

    2003-10-15

    Muscle contraction implies flexibility in combination with force resistance and requires a high degree of sarcolemmal organization. Smooth muscle cells differentiate largely from mesenchymal precursor cells and gradually assume a highly periodic sarcolemmal organization. Skeletal muscle undergoes an even more striking differentiation programme, leading to cell fusion and alignment into myofibrils. The lipid bilayer of each cell type is further segregated into raft and non-raft microdomains of distinct lipid composition. Considering the extent of developmental rearrangement in skeletal muscle, we investigated sarcolemmal microdomain organization in skeletal and smooth muscle cells. The rafts in both muscle types are characterized by marker proteins belonging to the annexin family which localize to the inner membrane leaflet, as well as glycosyl-phosphatidyl-inositol (GPI)-anchored enzymes attached to the outer leaflet. We demonstrate that the profound structural rearrangements that occur during skeletal muscle maturation coincide with a striking decrease in membrane lipid segregation, downregulation of annexins 2 and 6, and a significant decrease in raft-associated 5'-nucleotidase activity. The relative paucity of lipid rafts in mature skeletal in contrast to smooth muscle suggests that the organization of sarcolemmal microdomains contributes to the muscle-specific differences in stimulatory responses and contractile properties.

  11. Adenosine receptor distribution in Rhesus monkey ocular tissue.

    PubMed

    Beach, Krista M; Hung, Li-Fang; Arumugam, Baskar; Smith, Earl L; Ostrin, Lisa A

    2018-05-21

    Adenosine receptor (ADOR) antagonists, such as 7-methylxanthine (7-MX), have been shown to slow myopia progression in humans and animal models. Adenosine receptors are found throughout the body, and regulate the release of neurotransmitters such as dopamine and glutamate. However, the role of adenosine in eye growth is unclear. Evidence suggests that 7-MX increases scleral collagen fibril diameter, hence preventing axial elongation. This study used immunohistochemistry (IHC) and reverse-transcription quantitative polymerase chain reaction (RT-qPCR) to examine the distribution of the four ADORs in the normal monkey eye to help elucidate potential mechanisms of action. Eyes were enucleated from six Rhesus monkeys. Anterior segments and eyecups were separated into components and flash-frozen for RNA extraction or fixed in 4% paraformaldehyde and processed for immunohistochemistry against ADORA1, ADORA2a, ADORA2b, and ADORA3. RNA was reverse-transcribed, and qPCR was performed using custom primers. Relative gene expression was calculated using the ΔΔCt method normalizing to liver expression, and statistical analysis was performed using Relative Expression Software Tool. ADORA1 immunostaining was highest in the iris sphincter muscle, trabecular meshwork, ciliary epithelium, and retinal nerve fiber layer. ADORA2a immunostaining was highest in the corneal epithelium, trabecular meshwork, ciliary epithelium, retinal nerve fiber layer, and scleral fibroblasts. ADORA2b immunostaining was highest in corneal basal epithelium, limbal stem cells, iris sphincter, ciliary muscle, ciliary epithelium, choroid, isolated retinal ganglion cells and scattered scleral fibroblasts. ADORA3 immunostaining was highest in the iris sphincter, ciliary muscle, ciliary epithelium, choroid, isolated retinal ganglion cells, and scleral fibroblasts. Compared to liver mRNA, ADORA1 mRNA was significantly higher in the brain, retina and choroid, and significantly lower in the iris/ciliary body. ADORA

  12. Loss of myogenic potential and fusion capacity of muscle stem cells isolated from contractured muscle in children with cerebral palsy.

    PubMed

    Domenighetti, Andrea A; Mathewson, Margie A; Pichika, Rajeswari; Sibley, Lydia A; Zhao, Leyna; Chambers, Henry G; Lieber, Richard L

    2018-04-25

    Cerebral palsy (CP) is the most common cause of pediatric neurodevelopmental and physical disability in the United States. It is defined as a group of motor disorders caused by a non-progressive perinatal insult to the brain. While the brain lesion is non-progressive, there is a progressive, lifelong impact on skeletal muscles, which are shorter, spastic, and may develop debilitating contractures. Satellite cells are resident muscle stem cells that are indispensable for postnatal growth and regeneration of skeletal muscles. Here we measured the myogenic potential of satellite cells isolated from contractured muscles in children with CP. When compared to typically developing (TD) children, satellite cell-derived myoblasts from CP differentiated more slowly (Slope: 0.013{plus minus}0.013 CP vs. 0.091{plus minus}0.024 TD over 24 hours, P<0.001) and fused less (Fusion Index: 21.3{plus minus}8.6 CP vs. 81.3{plus minus}7.7 TD after 48 hours, P<0.001) after exposure to low-serum conditions that stimulated myotube formation. This impairment was associated with downregulation of several markers important for myoblast fusion and myotube formation, including DNA methylation-dependent inhibition of pro-myogenic Integrin Beta 1D (ITGB1D) protein expression levels (-50% at 42 hours), and ~25% loss of integrin-mediated FAK kinase phosphorylation. The cytidine analog 5-Azacytidine (5-AZA), a demethylating agent, restored ITGB1D levels and promoted myogenesis in CP cultures. Our data demonstrate that muscle contractures in CP are associated with loss of satellite cell myogenic potential that is dependent on DNA methylation patterns affecting expression of genetic programs associated with muscle stem cell differentiation and muscle fiber formation.

  13. Establishment and cryopreservation of a giant panda skeletal muscle-derived cell line.

    PubMed

    Yu, Fang-Jian; Zeng, Chang-Jun; Zhang, Yan; Wang, Cheng-Dong; Xiong, Tie-Yi; Fang, Sheng-Guo; Zhang, He-Min

    2015-06-01

    The giant panda Ailuropoda melanoleuca is an endangered species and is a symbol for wildlife conservation. Although efforts have been made to protect this rare and endangered species through breeding and conservative biology, the long-term preservation of giant panda genome resources (gametes, tissues, organs, genomic libraries, etc.) is still a practical option. In this study, the giant panda skeletal muscle-derived cell line was successfully established via primary explants culture and cryopreservation techniques. The population doubling time of giant panda skeletal cells was approximately 33.8 h, and this population maintained a high cell viability before and after cryopreservation (95.6% and 90.7%, respectively). The two skeletal muscle-specific genes SMYD1 and MYF6 were expressed and detected by RT-PCR in the giant panda skeletal muscle-derived cell line. Karyotyping analysis revealed that the frequencies of giant panda skeletal muscle cells showing a chromosome number of 2n=42 ranged from 90.6∼94.2%. Thus, the giant panda skeletal muscle-derived cell line provides a vital resource and material platform for further studies and is likely to be useful for the protection of this rare and endangered species.

  14. Action of obestatin in skeletal muscle repair: stem cell expansion, muscle growth, and microenvironment remodeling.

    PubMed

    Gurriarán-Rodríguez, Uxía; Santos-Zas, Icía; González-Sánchez, Jessica; Beiroa, Daniel; Moresi, Viviana; Mosteiro, Carlos S; Lin, Wei; Viñuela, Juan E; Señarís, José; García-Caballero, Tomás; Casanueva, Felipe F; Nogueiras, Rubén; Gallego, Rosalía; Renaud, Jean-Marc; Adamo, Sergio; Pazos, Yolanda; Camiña, Jesús P

    2015-06-01

    The development of therapeutic strategies for skeletal muscle diseases, such as physical injuries and myopathies, depends on the knowledge of regulatory signals that control the myogenic process. The obestatin/GPR39 system operates as an autocrine signal in the regulation of skeletal myogenesis. Using a mouse model of skeletal muscle regeneration after injury and several cellular strategies, we explored the potential use of obestatin as a therapeutic agent for the treatment of trauma-induced muscle injuries. Our results evidenced that the overexpression of the preproghrelin, and thus obestatin, and GPR39 in skeletal muscle increased regeneration after muscle injury. More importantly, the intramuscular injection of obestatin significantly enhanced muscle regeneration by simulating satellite stem cell expansion as well as myofiber hypertrophy through a kinase hierarchy. Added to the myogenic action, the obestatin administration resulted in an increased expression of vascular endothelial growth factor (VEGF)/vascular endothelial growth factor receptor 2 (VEGFR2) and the consequent microvascularization, with no effect on collagen deposition in skeletal muscle. Furthermore, the potential inhibition of myostatin during obestatin treatment might contribute to its myogenic action improving muscle growth and regeneration. Overall, our data demonstrate successful improvement of muscle regeneration, indicating obestatin is a potential therapeutic agent for skeletal muscle injury and would benefit other myopathies related to muscle regeneration.

  15. Neuronal differentiation of stem cells isolated from adult muscle.

    PubMed

    Romero-Ramos, Marina; Vourc'h, Patrick; Young, Henry E; Lucas, Paul A; Wu, Young; Chivatakarn, Onanong; Zaman, Rumina; Dunkelman, Noushin; el-Kalay, Mohammad A; Chesselet, Marie-Françoise

    2002-09-15

    Lineage uncommitted pluripotent stem cells reside in the connective tissue of skeletal muscle. The present study was carried out with pluripotent stem cells (PPSCs) isolated from 6-month old rat muscle. Before differentiation, these cells were vimentin+, CD90+, CD45-, and varied in their expression of CD34. The PPSCs were expanded as non-adherent aggregates under similar conditions to those used to generate neurospheres from embryonic or neural stem cells. The PPSC-derived neurospheres were positive for nestin, an early marker present in neuronal precursors, and expressed the two alternative mRNA forms of the neuroectodermal marker Pax-6, as well as mRNA for Oct-4, a gene related to the pluripotentiality of stem cells. To confirm their neural potential, PPSC-derived neurospheres were plated on coated coverslips under varying conditions: Neurobasal medium with N2 or B27, and either NT3 or BDNF. After 4-6 days the cells expressed neuronal (Tuj1+, NF68), astrocytic (GFAP) and oligodendrocytic (MOSP+, MBP+) markers, both by immunocytochemistry and RT-PCR. In addition, PPSCs were cultured as monolayers under adherent conditions, exposed to growth factors and defined differentiating conditions for 5 hr, and subsequently kept for 2 days in a maturation medium. At this point they gave rise to a mixed population of early neural progenitors (Nestin+ or NG2+), immature and mature neurons (Tuj1+ and NF145+) and myelin producing oligodendrocytes (CNPase + and MOSP+). Our study shows that PPSCs present in adult muscle can overcome germ lineage restrictions and express the molecular characteristics of brain cells. Therefore, PPSCs isolated from adult muscle could provide a novel source for autologous cell replacement in neurodegenerative and demyelinating diseases. Copyright 2002 Wiley-Liss, Inc.

  16. Myostatin inhibition induces muscle fibre hypertrophy prior to satellite cell activation.

    PubMed

    Wang, Qian; McPherron, Alexandra C

    2012-05-01

    Muscle fibres are multinucleated post-mitotic cells that can change dramatically in size during adulthood. It has been debated whether muscle fibre hypertrophy requires activation and fusion of muscle stem cells, the satellite cells. Myostatin (MSTN) is a negative regulator of skeletal muscle growth during development and in the adult, and MSTN inhibition is therefore a potential therapy for muscle wasting diseases, some of which are associated with a depletion of satellite cells. Conflicting results have been obtained in previous analyses of the role of MSTN on satellite cell quiescence. Here, we inhibited MSTN in adult mice with a soluble activin receptor type IIB and analysed the incorporation of new nuclei using 5-bromo-2-deoxyuridine (BrdU) labelling by isolating individual myofibres. We found that satellite cells are activated by MSTN inhibition. By varying the dose and time course for MSTN inhibition, however, we found that myofibre hypertrophy precedes the incorporation of new nuclei, and that the overall number of new nuclei is relatively low compared to the number of total myonuclei. These results reconcile some of the previous work obtained by other methods. In contrast with previous reports, we also found that Mstn null mice do not have increased satellite cell numbers during adulthood and are not resistant to sarcopaenia. Our results support a previously proposed model of hypertrophy in which hypertrophy can precede satellite cell activation. Studies of the metabolic and functional effects of postnatal MSTN inhibition are needed to determine the consequences of increasing the cytoplasm/myonuclear ratio after MSTN inhibition.

  17. Influence of exercise and aging on extracellular matrix composition in the skeletal muscle stem cell niche.

    PubMed

    Garg, Koyal; Boppart, Marni D

    2016-11-01

    Skeletal muscle is endowed with a remarkable capacity for regeneration, primarily due to the reserve pool of muscle resident satellite cells. The satellite cell is the physiologically quiescent muscle stem cell that resides beneath the basal lamina and adjacent to the sarcolemma. The anatomic location of satellite cells is in close proximity to vasculature where they interact with other muscle resident stem/stromal cells (e.g., mesenchymal stem cells and pericytes) through paracrine mechanisms. This mini-review describes the components of the muscle stem cell niche, as well as the influence of exercise and aging on the muscle stem cell niche. Although exercise promotes ECM reorganization and stem cell accumulation, aging is associated with dense ECM deposition and loss of stem cell function resulting in reduced regenerative capacity and strength. An improved understanding of the niche elements will be valuable to inform the development of therapeutic interventions aimed at improving skeletal muscle regeneration and adaptation over the life span. Copyright © 2016 the American Physiological Society.

  18. Superparamagnetic iron oxide nanoparticles regulate smooth muscle cell phenotype

    PubMed Central

    Angelopoulos, Ioannis; Southern, Paul; Pankhurst, Quentin A.

    2016-01-01

    Abstract Superparamagnetic iron oxide nanoparticles (SPION) are used for an increasing range of biomedical applications, from imaging to mechanical actuation of cells and tissue. The aim of this study was to investigate the loading of smooth muscle cells (SMC) with SPION and to explore what effect this has on the phenotype of the cells. Adherent human SMC were loaded with ∼17 pg of unconjugated, negatively charged, 50 nm SPION. Clusters of the internalized SPION particles were held in discrete cytoplasmic vesicles. Internalized SPION did not cause any change in cell morphology, proliferation, metabolic activity, or staining pattern of actin and calponin, two of the muscle contractile proteins involved in force generation. However, internalized SPION inhibited the increased gene expression of actin and calponin normally observed when cells are incubated under differentiation conditions. The observed change in the control of gene expression of muscle contractile apparatus by SPION has not previously been described. This finding could offer novel approaches for regulating the phenotype of SMC and warrants further investigation. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2412–2419, 2016. PMID:27176658

  19. Evaluation of hematopoietic potential generated by transplantation of muscle-derived stem cells in mice.

    PubMed

    Farace, Francoise; Prestoz, Laetitita; Badaoui, Sabrina; Guillier, Martine; Haond, Celine; Opolon, Paule; Thomas, Jean-Leon; Zalc, Bernard; Vainchenker, William; Turhan, Ali G

    2004-02-01

    Muscle tissue of adult mice has been shown to contain stem cells with hematopoietic repopulation ability in vivo. To determine the functional characteristics of stem cells giving rise to this hematopoietic activity, we have performed hematopoietic reconstitution experiments by the use of muscle versus marrow transplantation in lethally irradiated mice and followed the fate of transplanted cells by Y-chimerism using PCR and fluorescence in situ hybridization (FISH) analysis. We report here that transplantation of murine muscle generate a major hematopoietic chimerism at the level of CFU-C, CFU-S, and terminally-differentiated cells in three generations of lethally irradiated mice followed up to 1 year after transplantation. This potential is totally abolished when muscle grafts were performed by the use of muscle from previously irradiated mice. As compared to marrow transplantation, muscle transplants were able to generate similar potencies to give rise to myeloid, T, B, and natural killer (NK) cells. Interestingly, marrow stem cells that have been generated in primary and then in secondary recipients were able to contribute efficiently to myofibers in the muscle tissue of tertiary recipients. Altogether, our data demonstrate that muscle-derived stem cells present a major hematopoietic repopulating ability with evidence of self-replication in vivo. They are radiation-sensitive and similar to marrow-derived stem cells in terms of their ability to generate multilineage hematopoiesis. Finally, our data demonstrate that muscle-derived hematopoietic stem cells do not lose their ability to contribute to myofiber generation after at least two rounds of serial transplantation, suggesting a potential that is probably equivalent to that generated by marrow transplantation.

  20. Defining the role of mesenchymal stromal cells on the regulation of matrix metalloproteinases in skeletal muscle cells

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

    Sassoli, Chiara; Nosi, Daniele; Tani, Alessia

    Recent studies indicate that mesenchymal stromal cell (MSC) transplantation improves healing of injured and diseased skeletal muscle, although the mechanisms of benefit are poorly understood. In the present study, we investigated whether MSCs and/or their trophic factors were able to regulate matrix metalloproteinase (MMP) expression and activity in different cells of the muscle tissue. MSCs in co-culture with C2C12 cells or their conditioned medium (MSC-CM) up-regulated MMP-2 and MMP-9 expression and function in the myoblastic cells; these effects were concomitant with the down-regulation of the tissue inhibitor of metalloproteinases (TIMP)-1 and -2 and with increased cell motility. In the singlemore » muscle fiber experiments, MSC-CM administration increased MMP-2/9 expression in Pax-7{sup +} satellite cells and stimulated their mobilization, differentiation and fusion. The anti-fibrotic properties of MSC-CM involved also the regulation of MMPs by skeletal fibroblasts and the inhibition of their differentiation into myofibroblasts. The treatment with SB-3CT, a potent MMP inhibitor, prevented in these cells, the decrease of α-smooth actin and type-I collagen expression induced by MSC-CM, suggesting that MSC-CM could attenuate the fibrogenic response through mechanisms mediated by MMPs. Our results indicate that growth factors and cytokines released by these cells may modulate the fibrotic response and improve the endogenous mechanisms of muscle repair/regeneration. - Highlights: • MSC-CM contains paracrine factors that up-regulate MMP expression and function in different skeletal muscle cells. • MSC-CM promotes myoblast and satellite cell migration, proliferation and differentiation. • MSC-CM negatively interferes with fibroblast-myoblast transition in primary skeletal fibroblasts. • Paracrine factors from MSCs modulate the fibrotic response and improve the endogenous mechanisms of muscle regeneration.« less

  1. Fabrication and Manipulation of Ciliary Microrobots with Non-reciprocal Magnetic Actuation

    PubMed Central

    Kim, Sangwon; Lee, Seungmin; Lee, Jeonghun; Nelson, Bradley J.; Zhang, Li; Choi, Hongsoo

    2016-01-01

    Magnetically actuated ciliary microrobots were designed, fabricated, and manipulated to mimic cilia-based microorganisms such as paramecia. Full three-dimensional (3D) microrobot structures were fabricated using 3D laser lithography to form a polymer base structure. A nickel/titanium bilayer was sputtered onto the cilia part of the microrobot to ensure magnetic actuation and biocompatibility. The microrobots were manipulated by an electromagnetic coil system, which generated a stepping magnetic field to actuate the cilia with non-reciprocal motion. The cilia beating motion produced a net propulsive force, resulting in movement of the microrobot. The magnetic forces on individual cilia were calculated with various input parameters including magnetic field strength, cilium length, applied field angle, actual cilium angle, etc., and the translational velocity was measured experimentally. The position and orientation of the ciliary microrobots were precisely controlled, and targeted particle transportation was demonstrated experimentally. PMID:27470077

  2. Epithelial Cell Culture from Human Adenoids: A Functional Study Model for Ciliated and Secretory Cells

    PubMed Central

    González, Claudia; Espinosa, Marisol; Sánchez, María Trinidad; Droguett, Karla; Ríos, Mariana; Fonseca, Ximena; Villalón, Manuel

    2013-01-01

    Background. Mucociliary transport (MCT) is a defense mechanism of the airway. To study the underlying mechanisms of MCT, we have both developed an experimental model of cultures, from human adenoid tissue of ciliated and secretory cells, and characterized the response to local chemical signals that control ciliary activity and the secretion of respiratory mucins in vitro. Materials and Methods. In ciliated cell cultures, ciliary beat frequency (CBF) and intracellular Ca2+ levels were measured in response to ATP, UTP, and adenosine. In secretory cultures, mucin synthesis and secretion were identified by using immunodetection. Mucin content was taken from conditioned medium and analyzed in the presence or absence of UTP. Results. Enriched ciliated cell monolayers and secretory cells were obtained. Ciliated cells showed a basal CBF of 10.7 Hz that increased significantly after exposure to ATP, UTP, or adenosine. Mature secretory cells showed active secretion of granules containing different glycoproteins, including MUC5AC. Conclusion. Culture of ciliated and secretory cells grown from adenoid epithelium is a reproducible and feasible experimental model, in which it is possible to observe ciliary and secretory activities, with a potential use as a model to understand mucociliary transport control mechanisms. PMID:23484122

  3. Muscle regeneration potential and satellite cell activation profile during recovery following hindlimb immobilization in mice.

    PubMed

    Guitart, Maria; Lloreta, Josep; Mañas-Garcia, Laura; Barreiro, Esther

    2018-05-01

    Reduced muscle activity leads to muscle atrophy and function loss in patients and animal models. Satellite cells (SCs) are postnatal muscle stem cells that play a pivotal role in skeletal muscle regeneration following injury. The regenerative potential, satellite cell numbers, and markers during recovery following immobilization of the hindlimb for 7 days were explored. In mice exposed to 7 days of hindlimb immobilization, in those exposed to recovery (7 days, splint removal), and in contralateral control muscles, muscle precursor cells were isolated from all hindlimb muscles (fluorescence-activated cell sorting, FACS) and SCs, and muscle regeneration were identified using immunofluorescence (gastrocnemius and soleus) and electron microscopy (EM, gastrocnemius). Expression of ki67, pax7, myoD, and myogenin was quantified (RT-PCR) from SC FACS yields. Body and grip strength were determined. Following 7 day hindlimb immobilization, a decline in SCs (FACS, immunofluorescence) was observed together with an upregulation of SC activation markers and signs of muscle regeneration including fusion to existing myofibers (EM). Recovery following hindlimb immobilization was characterized by a program of muscle regeneration events. Hindlimb immobilization induced a decline in SCs together with an upregulation of markers of SC activation, suggesting that fusion to existing myofibers takes place during unloading. Muscle recovery induced a significant rise in muscle precursor cells and regeneration events along with reduced SC activation expression markers and a concomitant rise in terminal muscle differentiation expression. These are novel findings of potential applicability for the treatment of disuse muscle atrophy, which is commonly associated with severe chronic and acute conditions. © 2017 Wiley Periodicals, Inc.

  4. Muscle Satellite Cells: Exploring the Basic Biology to Rule Them.

    PubMed

    Almeida, Camila F; Fernandes, Stephanie A; Ribeiro Junior, Antonio F; Keith Okamoto, Oswaldo; Vainzof, Mariz

    2016-01-01

    Adult skeletal muscle is a postmitotic tissue with an enormous capacity to regenerate upon injury. This is accomplished by resident stem cells, named satellite cells, which were identified more than 50 years ago. Since their discovery, many researchers have been concentrating efforts to answer questions about their origin and role in muscle development, the way they contribute to muscle regeneration, and their potential to cell-based therapies. Satellite cells are maintained in a quiescent state and upon requirement are activated, proliferating, and fusing with other cells to form or repair myofibers. In addition, they are able to self-renew and replenish the stem pool. Every phase of satellite cell activity is highly regulated and orchestrated by many molecules and signaling pathways; the elucidation of players and mechanisms involved in satellite cell biology is of extreme importance, being the first step to expose the crucial points that could be modulated to extract the optimal response from these cells in therapeutic strategies. Here, we review the basic aspects about satellite cells biology and briefly discuss recent findings about therapeutic attempts, trying to raise questions about how basic biology could provide a solid scaffold to more successful use of these cells in clinics.

  5. Intracerebral transplants of primary muscle cells: a potential 'platform' for transgene expression in the brain

    NASA Technical Reports Server (NTRS)

    Jiao, S.; Schultz, E.; Wolff, J. A.

    1992-01-01

    After the transplantation of rat primary muscle cells into the caudate or cortex of recipient rats, the muscle cells were able to persist for at least 6 months. Muscle cells transfected with expression plasmids prior to transplantation were able to express reporter genes in the brains for at least 2 months. These results suggest that muscle cells might be a useful 'platform' for transgene expression in the brain.

  6. Muscle glycogen and cell function--Location, location, location.

    PubMed

    Ørtenblad, N; Nielsen, J

    2015-12-01

    The importance of glycogen, as a fuel during exercise, is a fundamental concept in exercise physiology. The use of electron microscopy has revealed that glycogen is not evenly distributed in skeletal muscle fibers, but rather localized in distinct pools. In this review, we present the available evidence regarding the subcellular localization of glycogen in skeletal muscle and discuss this from the perspective of skeletal muscle fiber function. The distribution of glycogen in the defined pools within the skeletal muscle varies depending on exercise intensity, fiber phenotype, training status, and immobilization. Furthermore, these defined pools may serve specific functions in the cell. Specifically, reduced levels of these pools of glycogen are associated with reduced SR Ca(2+) release, muscle relaxation rate, and membrane excitability. Collectively, the available literature strongly demonstrates that the subcellular localization of glycogen has to be considered to fully understand the role of glycogen metabolism and signaling in skeletal muscle function. Here, we propose that the effect of low muscle glycogen on excitation-contraction coupling may serve as a built-in mechanism, which links the energetic state of the muscle fiber to energy utilization. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  7. Imbalance between pSmad3 and Notch induces CDK inhibitors in old muscle stem cells.

    PubMed

    Carlson, Morgan E; Hsu, Michael; Conboy, Irina M

    2008-07-24

    Adult skeletal muscle robustly regenerates throughout an organism's life, but as the muscle ages, its ability to repair diminishes and eventually fails. Previous work suggests that the regenerative potential of muscle stem cells (satellite cells) is not triggered in the old muscle because of a decline in Notch activation, and that it can be rejuvenated by forced local activation of Notch. Here we report that, in addition to the loss of Notch activation, old muscle produces excessive transforming growth factor (TGF)-beta (but not myostatin), which induces unusually high levels of TGF-beta pSmad3 in resident satellite cells and interferes with their regenerative capacity. Importantly, endogenous Notch and pSmad3 antagonize each other in the control of satellite-cell proliferation, such that activation of Notch blocks the TGF-beta-dependent upregulation of the cyclin-dependent kinase (CDK) inhibitors p15, p16, p21 and p27, whereas inhibition of Notch induces them. Furthermore, in muscle stem cells, Notch activity determines the binding of pSmad3 to the promoters of these negative regulators of cell-cycle progression. Attenuation of TGF-beta/pSmad3 in old, injured muscle restores regeneration to satellite cells in vivo. Thus a balance between endogenous pSmad3 and active Notch controls the regenerative competence of muscle stem cells, and deregulation of this balance in the old muscle microniche interferes with regeneration.

  8. A Method for the Direct Identification of Differentiating Muscle Cells by a Fluorescent Mitochondrial Dye

    PubMed Central

    Miyake, Tetsuaki; McDermott, John C.; Gramolini, Anthony O.

    2011-01-01

    Identification of differentiating muscle cells generally requires fixation, antibodies directed against muscle specific proteins, and lengthy staining processes or, alternatively, transfection of muscle specific reporter genes driving GFP expression. In this study, we examined the possibility of using the robust mitochondrial network seen in maturing muscle cells as a marker of cellular differentiation. The mitochondrial fluorescent tracking dye, MitoTracker, which is a cell-permeable, low toxicity, fluorescent dye, allowed us to distinguish and track living differentiating muscle cells visually by epi-fluorescence microscopy. MitoTracker staining provides a robust and simple detection strategy for living differentiating cells in culture without the need for fixation or biochemical processing. PMID:22174849

  9. A home away from home: challenges and opportunities in engineering in vitro muscle satellite cell niches

    PubMed Central

    Cosgrove, Benjamin D.; Sacco, Alessandra; Gilbert, Penney M.; Blau, Helen M.

    2009-01-01

    Satellite cells are skeletal muscle stem cells with a principal role in postnatal skeletal muscle regeneration. Satellite cells, like many tissue-specific adult stem cells, reside in a quiescent state in an instructive, anatomically defined niche. The satellite cell niche constitutes a distinct membrane-enclosed compartment within the muscle fiber, containing a diversity of biochemical and biophysical signals that influence satellite cell function. A major limitation to the study and clinical utility of satellite cells is that upon removal from the muscle fiber and plating in traditional plastic tissue culture platforms, their muscle stem cell properties are rapidly lost. Clearly, the maintenance of stem cell function is critically dependent on in vivo niche signals, highlighting the need to create novel in vitro microenvironments that allow for the maintenance and propagation of satellite cells while retaining their potential to function as muscle stem cells. Here, we discuss how emerging biomaterials technologies offer great promise for engineering in vitro microenvironments to meet these challenges. In engineered biomaterials, signaling molecules can be presented in a manner that more closely mimics cell-cell and cell-matrix interactions and matrices can be fabricated with diverse rigidities that approximate in vivo tissues. The development of in vitro microenvironments in which niche features can be systematically modulated will be instrumental not only to future insights into muscle stem cell biology and therapeutic approaches to muscle diseases and muscle wasting with aging, but also will provide a paradigm for the analysis of numerous adult tissue-specific stem cells. PMID:19751902

  10. Action of Obestatin in Skeletal Muscle Repair: Stem Cell Expansion, Muscle Growth, and Microenvironment Remodeling

    PubMed Central

    Gurriarán-Rodríguez, Uxía; Santos-Zas, Icía; González-Sánchez, Jessica; Beiroa, Daniel; Moresi, Viviana; Mosteiro, Carlos S; Lin, Wei; Viñuela, Juan E; Señarís, José; García-Caballero, Tomás; Casanueva, Felipe F; Nogueiras, Rubén; Gallego, Rosalía; Renaud, Jean-Marc; Adamo, Sergio; Pazos, Yolanda; Camiña, Jesús P

    2015-01-01

    The development of therapeutic strategies for skeletal muscle diseases, such as physical injuries and myopathies, depends on the knowledge of regulatory signals that control the myogenic process. The obestatin/GPR39 system operates as an autocrine signal in the regulation of skeletal myogenesis. Using a mouse model of skeletal muscle regeneration after injury and several cellular strategies, we explored the potential use of obestatin as a therapeutic agent for the treatment of trauma-induced muscle injuries. Our results evidenced that the overexpression of the preproghrelin, and thus obestatin, and GPR39 in skeletal muscle increased regeneration after muscle injury. More importantly, the intramuscular injection of obestatin significantly enhanced muscle regeneration by simulating satellite stem cell expansion as well as myofiber hypertrophy through a kinase hierarchy. Added to the myogenic action, the obestatin administration resulted in an increased expression of vascular endothelial growth factor (VEGF)/vascular endothelial growth factor receptor 2 (VEGFR2) and the consequent microvascularization, with no effect on collagen deposition in skeletal muscle. Furthermore, the potential inhibition of myostatin during obestatin treatment might contribute to its myogenic action improving muscle growth and regeneration. Overall, our data demonstrate successful improvement of muscle regeneration, indicating obestatin is a potential therapeutic agent for skeletal muscle injury and would benefit other myopathies related to muscle regeneration. PMID:25762009

  11. Transplanted hematopoietic stem cells demonstrate impaired sarcoglycan expression after engraftment into cardiac and skeletal muscle.

    PubMed

    Lapidos, Karen A; Chen, Yiyin E; Earley, Judy U; Heydemann, Ahlke; Huber, Jill M; Chien, Marcia; Ma, Averil; McNally, Elizabeth M

    2004-12-01

    Pluripotent bone marrow-derived side population (BM-SP) stem cells have been shown to repopulate the hematopoietic system and to contribute to skeletal and cardiac muscle regeneration after transplantation. We tested BM-SP cells for their ability to regenerate heart and skeletal muscle using a model of cardiomyopathy and muscular dystrophy that lacks delta-sarcoglycan. The absence of delta-sarcoglycan produces microinfarcts in heart and skeletal muscle that should recruit regenerative stem cells. Additionally, sarcoglycan expression after transplantation should mark successful stem cell maturation into cardiac and skeletal muscle lineages. BM-SP cells from normal male mice were transplanted into female delta-sarcoglycan-null mice. We detected engraftment of donor-derived stem cells into skeletal muscle, with the majority of donor-derived cells incorporated within myofibers. In the heart, donor-derived nuclei were detected inside cardiomyocytes. Skeletal muscle myofibers containing donor-derived nuclei generally failed to express sarcoglycan, with only 2 sarcoglycan-positive fibers detected in the quadriceps muscle from all 14 mice analyzed. Moreover, all cardiomyocytes with donor-derived nuclei were sarcoglycan-negative. The absence of sarcoglycan expression in cardiomyocytes and skeletal myofibers after transplantation indicates impaired differentiation and/or maturation of bone marrow-derived stem cells. The inability of BM-SP cells to express this protein severely limits their utility for cardiac and skeletal muscle regeneration.

  12. PITX2 Enhances the Regenerative Potential of Dystrophic Skeletal Muscle Stem Cells.

    PubMed

    Vallejo, Daniel; Hernández-Torres, Francisco; Lozano-Velasco, Estefanía; Rodriguez-Outeiriño, Lara; Carvajal, Alejandra; Creus, Carlota; Franco, Diego; Aránega, Amelia Eva

    2018-04-10

    Duchenne muscular dystrophy (DMD), one of the most lethal genetic disorders, involves progressive muscle degeneration resulting from the absence of DYSTROPHIN. Lack of DYSTROPHIN expression in DMD has critical consequences in muscle satellite stem cells including a reduced capacity to generate myogenic precursors. Here, we demonstrate that the c-isoform of PITX2 transcription factor modifies the myogenic potential of dystrophic-deficient satellite cells. We further show that PITX2c enhances the regenerative capability of mouse DYSTROPHIN-deficient satellite cells by increasing cell proliferation and the number of myogenic committed cells, but importantly also increasing dystrophin-positive (revertant) myofibers by regulating miR-31. These PITX2-mediated effects finally lead to improved muscle function in dystrophic (DMD/mdx) mice. Our studies reveal a critical role for PITX2 in skeletal muscle repair and may help to develop therapeutic strategies for muscular disorders. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

  13. Pharyngeal satellite cells undergo myogenesis under basal conditions and are required for pharyngeal muscle maintenance

    PubMed Central

    Randolph, Matthew E.; Phillips, Brittany L.; Choo, Hyo-Jung; Vest, Katherine E.; Vera, Yandery; Pavlath, Grace K.

    2015-01-01

    The pharyngeal muscles of the nasal, oral, and laryngeal pharynxes are required for swallowing. Pharyngeal muscles are preferentially affected in some muscular dystrophies yet spared in others. Muscle stem cells, called satellite cells, may be critical factors in the development of pharyngeal muscle disorders; however, very little is known about pharyngeal satellite cells (PSC) and their role in pharyngeal muscles. We show that PSC are distinct from the commonly studied hindlimb satellite cells both transcriptionally and biologically. Under basal conditions PSC proliferate, progress through myogenesis, and fuse with pharyngeal myofibers. Furthermore, PSC exhibit biologic differences dependent on anatomic location in the pharynx. Importantly, PSC are required to maintain myofiber size and myonuclear number in pharyngeal myofibers. Together, these results demonstrate that PSC are critical for pharyngeal muscle maintenance and suggest that satellite cell impairment could contribute to pharyngeal muscle pathology associated with various muscular dystrophies and aging. PMID:26178867

  14. Improved adductor function after canine recurrent laryngeal nerve injury and repair using muscle progenitor cells.

    PubMed

    Paniello, Randal C; Brookes, Sarah; Bhatt, Neel K; Bijangi-Vishehsaraei, Khadijeh; Zhang, Hongji; Halum, Stacey

    2017-12-08

    Muscle progenitor cells (MPCs) can be isolated from muscle samples and grown to a critical mass in culture. They have been shown to survive and integrate when implanted into rat laryngeal muscles. In this study, the ability of MPC implants to enhance adductor function of reinnervated thyroarytenoid muscles was tested in a canine model. Animal study. Sternocleidomastoid muscle samples were harvested from three canines. Muscle progenitor cells were isolated and cultured to 10 7 cells over 4 to 5 weeks, then implanted into right thyroarytenoid muscles after ipsilateral recurrent laryngeal nerve transection and repair. The left sides underwent the same nerve injury, but no cells were implanted. Laryngeal adductor force was measured pretreatment and again 6 months later, and the muscles were harvested for histology. Muscle progenitor cells were successfully cultured from all dogs. Laryngeal adductor force measurements averaged 60% of their baseline pretreatment values in nonimplanted controls, 98% after implantation with MPCs, and 128% after implantation with motor endplate-enhanced MPCs. Histology confirmed that the implanted MPCs survived, became integrated into thyroarytenoid muscle fibers, and were in close contact with nerve endings, suggesting functional innervation. Muscle progenitor cells were shown to significantly enhance adductor function in this pilot canine study. Patient-specific MPC implantation could potentially be used to improve laryngeal function in patients with vocal fold paresis/paralysis, atrophy, and other conditions. Further experiments are planned. NA. Laryngoscope, 2017. © 2017 The American Laryngological, Rhinological and Otological Society, Inc.

  15. Ciliary neurotrophic factor is an endogenous pyrogen.

    PubMed Central

    Shapiro, L; Zhang, X X; Rupp, R G; Wolff, S M; Dinarello, C A

    1993-01-01

    Fever is initiated by the action of polypeptide cytokines called endogenous pyrogens, which are produced by the host during inflammation, trauma, or infection and which elevate the thermoregulatory set point in the hypothalamus. Ciliary neurotrophic factor (CNTF) supports the differentiation and survival of central and peripheral neurons. We describe the activity of CNTF as intrinsically pyrogenic in the rabbit. CNTF induced a monophasic fever which rose rapidly (within the first 12 min) following intravenous injection; CNTF fever was blocked by pretreatment with indomethacin. The fever induced by CNTF was not due to contaminating endotoxins. Increasing doses of CNTF resulted in prolongation of the fever, suggesting the subsequent induction of additional endogenous pyrogenic activity. After passive transfer of plasma obtained during CNTF-induced fever, endogenous pyrogen activity was not present in the circulation; CNTF also did not induce the endogenous pyrogens interleukin 1, tumor necrosis factor, or interleukin 6 in vitro. Nevertheless, a second endogenous pyrogen may originate within the central nervous system following the systemic injection of CNTF. Of the four endogenous pyrogens described to date (interleukin 1, tumor necrosis factor, interferon, and interleukin 6), CNTF, like interleukin 6, utilizes the cell-surface gp 130 signal-transduction apparatus. PMID:8378338

  16. miR-378 attenuates muscle regeneration by delaying satellite cell activation and differentiation in mice.

    PubMed

    Zeng, Ping; Han, Wanhong; Li, Changyin; Li, Hu; Zhu, Dahai; Zhang, Yong; Liu, Xiaohong

    2016-09-01

    Skeletal muscle mass and homeostasis during postnatal muscle development and regeneration largely depend on adult muscle stem cells (satellite cells). We recently showed that global overexpression of miR-378 significantly reduced skeletal muscle mass in mice. In the current study, we used miR-378 transgenic (Tg) mice to assess the in vivo functional effects of miR-378 on skeletal muscle growth and regeneration. Cross-sectional analysis of skeletal muscle tissues showed that the number and size of myofibers were significantly lower in miR-378 Tg mice than in wild-type mice. Attenuated cardiotoxin-induced muscle regeneration in miR-378 Tg mice was found to be associated with delayed satellite cell activation and differentiation. Mechanistically, miR-378 was found to directly target Igf1r in muscle cells both in vitro and in vivo These miR-378 Tg mice may provide a model for investigating the physiological and pathological roles of skeletal muscle in muscle-associated diseases in humans, particularly in sarcopenia. © The Author 2016. Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  17. Notch Signaling in Vascular Smooth Muscle Cells

    PubMed Central

    Baeten, J.T.; Lilly, B.

    2018-01-01

    The Notch signaling pathway is a highly conserved pathway involved in cell fate determination in embryonic development and also functions in the regulation of physiological processes in several systems. It plays an especially important role in vascular development and physiology by influencing angiogenesis, vessel patterning, arterial/venous specification, and vascular smooth muscle biology. Aberrant or dysregulated Notch signaling is the cause of or a contributing factor to many vascular disorders, including inherited vascular diseases, such as cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy, associated with degeneration of the smooth muscle layer in cerebral arteries. Like most signaling pathways, the Notch signaling axis is influenced by complex interactions with mediators of other signaling pathways. This complexity is also compounded by different members of the Notch family having both overlapping and unique functions. Thus, it is vital to fully understand the roles and interactions of each Notch family member in order to effectively and specifically target their exact contributions to vascular disease. In this chapter, we will review the Notch signaling pathway in vascular smooth muscle cells as it relates to vascular development and human disease. PMID:28212801

  18. Differential Effects of RET and TRKB on Axonal Branching and Survival of Parasympathetic Neurons

    PubMed Central

    Simpson, Julie; Keefe, Julie; Nishi, Rae

    2014-01-01

    Interactions between neurons and their targets of innervation influence many aspects of neural development. To examine how synaptic activity interacts with neurotrophic signaling, we determined the effects of blocking neuromuscular transmission on survival and axonal outgrowth of ciliary neurons from the embryonic chicken ciliary ganglion. Ciliary neurons undergo a period of cell loss due to programmed cell death between embryonic Days (E) 8 and 14 and they innervate the striated muscle of the iris. The nicotinic antagonist d-tubocurarine (dTC) induces an increase in branching measured by counting neurofilament-positive voxels (NF-VU) in the iris between E14–17 while reducing ciliary neuron survival. Blocking ganglionic transmission with dihyro-β-erythroidin and α-methyllycacontine does not mimic dTC. At E8, many trophic factors stimulate neurite outgrowth and branching of neurons placed in cell culture; however, at E13, only GDNF stimulates branching selectively in cultured ciliary neurons. The GDNF-induced branching at E13 could be inhibited by BDNF. Blocking ret signaling in vivo with a dominant negative (dn)ret decreases survival of ciliary and choroid neurons at E14 and prevents dTC induced increases in NF-VU in the iris at E17. Blocking TRKB signaling with dn TRKB increases NF-VU in the iris at E17 and decreases neuronal survival at E17, but not at E14. Thus, RET promotes survival during programmed cell death in the ciliary ganglion and contributes to promoting branching when synaptic transmission is blocked while TRKB inhibits branching and promotes maintenance of neuronal survival. These studies highlight the multifunctional nature of trophic molecule function during neuronal development. PMID:22648743

  19. Sparing of extraocular muscle in aging and muscular dystrophies: A myogenic precursor cell hypothesis

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

    Kallestad, Kristen M.; Hebert, Sadie L.; McDonald, Abby A.

    2011-04-01

    The extraocular muscles (EOM) are spared from pathology in aging and many forms of muscular dystrophy. Despite many studies, this sparing remains an enigma. The EOM have a distinct embryonic lineage compared to somite-derived muscles, and we have shown that they continuously remodel throughout life, maintaining a population of activated satellite cells even in aging. These data suggested the hypothesis that there is a population of myogenic precursor cells (mpcs) in EOM that is different from those in limb, with either elevated numbers of stem cells and/or mpcs with superior proliferative capacity compared to mpcs in limb. Using flow cytometry,more » EOM and limb muscle mononuclear cells were compared, and a number of differences were seen. Using two different cell isolation methods, EOM have significantly more mpcs per mg muscle than limb skeletal muscle. One specific subpopulation significantly increased in EOM compared to limb was positive for CD34 and negative for Sca-1, M-cadherin, CD31, and CD45. We named these the EOMCD34 cells. Similar percentages of EOMCD34 cells were present in both newborn EOM and limb muscle. They were retained in aged EOM, whereas the population decreased significantly in adult limb muscle and were extremely scarce in aged limb muscle. Most importantly, the percentage of EOMCD34 cells was elevated in the EOM from both the mdx and the mdx/utrophin{sup -/-} (DKO) mouse models of DMD and extremely scarce in the limb muscles of these mice. In vitro, the EOMCD34 cells had myogenic potential, forming myotubes in differentiation media. After determining a media better able to induce proliferation in these cells, a fusion index was calculated. The cells isolated from EOM had a 40% higher fusion index compared to the same cells isolated from limb muscle. The EOMCD34 cells were resistant to both oxidative stress and mechanical injury. These data support our hypothesis that the EOM may be spared in aging and in muscular dystrophies due to a

  20. Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

    PubMed Central

    Agley, Chibeza C.; Rowlerson, Anthea M.; Velloso, Cristiana P.; Lazarus, Norman L.; Harridge, Stephen D. R.

    2015-01-01

    The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated from human muscle biopsy samples using enzymatic digestion and their myogenic properties studied in culture. Quantitatively, the two main adherent cell types obtained from enzymatic digestion are: (i) the satellite cells (termed myogenic cells or muscle precursor cells), identified initially as CD56+ and later as CD56+/desmin+ cells and (ii) muscle-derived fibroblasts, identified as CD56– and TE-7+. Fibroblasts proliferate very efficiently in culture and in mixed cell populations these cells may overrun myogenic cells to dominate the culture. The isolation and purification of different cell types from human muscle is thus an important methodological consideration when trying to investigate the innate behavior of either cell type in culture. Here we describe a system of sorting based on the gentle enzymatic digestion of cells using collagenase and dispase followed by magnetic activated cell sorting (MACS) which gives both a high purity (>95% myogenic cells) and good yield (~2.8 x 106 ± 8.87 x 105 cells/g tissue after 7 days in vitro) for experiments in culture. This approach is based on incubating the mixed muscle-derived cell population with magnetic microbeads beads conjugated to an antibody against CD56 and then passing cells though a magnetic field. CD56+ cells bound to microbeads are retained by the field whereas CD56– cells pass unimpeded through the column. Cell suspensions from any stage of the sorting process can be plated and cultured. Following a given intervention, cell morphology, and the expression and localization of proteins including nuclear transcription factors can be quantified using immunofluorescent labeling with specific antibodies and an image processing and analysis package. PMID:25650991

  1. Effects of antiglaucoma drugs on [32P]orthophosphate incorporation into phospholipids of cat iris and ciliary process.

    PubMed

    Yorio, T; DeLoach, G; Satumtira, N

    1985-01-01

    The effects of antiglaucoma drugs on [32P]-orthophosphate incorporation into phospholipids of iris and ciliary process were investigated. Both iris and ciliary process rapidly incorporated 32Pi into the major phospholipids, with the acidic phosphoinositides demonstrating a greater labelling than phosphatidylcholine, indicating a greater turnover. The muscarinic agonists, carbachol and pilocarpine, stimulated 32Pi-labelling of phosphatidylinositol (PI) and phosphatidic acid (PA) in both iris and ciliary process. These effects were blocked by atropine, suggesting that the response was mediated through muscarinic receptors. The beta blocking ocular hypotensive drugs, propranolol, timolol and atenolol, produced varying effects on 32P incorporation into phospholipids of iris and ciliary process. Propranolol stimulated 32Pi-labelling into phosphatidylinositol 4', 5' bisphosphate (PIP2), phosphatidylinositol 4' phosphate (PIP), PI and PA. Timolol decreased 32Pi-incorporation into PIP2 and PI, whereas atenolol, a selective beta 1 antagonist, had no significant effect on 32Pi-labelling of phospholipids. The above findings on propranolol agree with previous observations which demonstrated that propranolol redirects glycerolipid metabolism through multiple effects on the enzymes in phospholipid biosynthesis, particularly in stimulating phosphatidylinositol kinases. The results with timolol suggest that this drug may decrease phosphoinositide hydrolysis. The effects of these ocular hypotensive, non-selective beta blocking drugs on phospholipid turnover may ultimately limit the accumulation of breakdown products which could serve as cellular messengers.

  2. Heterogeneity of adult masseter muscle satellite cells with cardiomyocyte differentiation potential.

    PubMed

    Huang, Wei; Liang, Jialiang; Feng, Yuliang; Jia, Zhanfeng; Jiang, Lin; Cai, Wenfeng; Paul, Christian; Gu, Jianguo G; Stambrook, Peter J; Millard, Ronald W; Zhu, Xiao-Lan; Zhu, Ping; Wang, Yigang

    2018-05-26

    Although resident cardiac stem cells have been reported, regeneration of functional cardiomyocytes (CMs) remains a challenge. The present study identifies an alternative progenitor source for CM regeneration without the need for genetic manipulation or invasive heart biopsy procedures. Unlike limb skeletal muscles, masseter muscles (MM) in the mouse head are developed from Nkx2-5 mesodermal progenitors. Adult masseter muscle satellite cells (MMSCs) display heterogeneity in developmental origin and cell phenotypes. The heterogeneous MMSCs that can be characterized by cell sorting based on stem cell antigen-1 (Sca1) show different lineage potential. While cardiogenic potential is preserved in Sca1 + MMSCs as shown by expression of cardiac progenitor genes (including Nkx2-5), skeletal myogenic capacity is maintained in Sca1 - MMSCs with Pax7 expression. Sca1 + MMSC-derived beating cells express cardiac genes and exhibit CM-like morphology. Electrophysiological properties of MMSC-derived CMs are demonstrated by calcium transients and action potentials. These findings show that MMSCs could serve as a novel cell source for cardiomyocyte replacement. Copyright © 2018. Published by Elsevier Inc.

  3. Long-term clearance from small airways in subjects with ciliary dysfunction.

    PubMed

    Lindström, Maria; Falk, Rolf; Hjelte, Lena; Philipson, Klas; Svartengren, Magnus

    2006-05-20

    The objective of this study was to investigate if long-term clearance from small airways is dependent on normal ciliary function. Six young adults with primary ciliary dyskinesia (PCD) inhaled 111 Indium labelled Teflon particles of 4.2 microm geometric and 6.2 microm aerodynamic diameter with an extremely slow inhalation flow, 0.05 L/s. The inhalation method deposits particles mainly in the small conducting airways. Lung retention was measured immediately after inhalation and at four occasions up to 21 days after inhalation. Results were compared with data from ten healthy controls. For additional comparison three of the PCD subjects also inhaled the test particles with normal inhalation flow, 0.5 L/s, providing a more central deposition. The lung retention at 24 h in % of lung deposition (Ret24) was higher (p < 0.001) in the PCD subjects, 79 % (95% Confidence Interval, 67.6;90.6), compared to 49% (42.3;55.5) in the healthy controls. There was a significant clearance after 24 h both in the PCD subjects and in the healthy controls with equivalent clearance. The mean Ret24 with slow inhalation flow was 73.9 +/- 1.9% compared to 68.9 +/- 7.5% with normal inhalation flow in the three PCD subjects exposed twice. During day 7-21 the three PCD subjects exposed twice cleared 9% with normal flow, probably representing predominantly alveolar clearance, compared to 19% with slow inhalation flow, probably representing mainly small airway clearance. This study shows that despite ciliary dysfunction, clearance continues in the small airways beyond 24 h. There are apparently additional clearance mechanisms present in the small airways.

  4. Immunohistochemical and ultrastructural properties of the larval ciliary band-associated strand in the sea urchin Hemicentrotus pulcherrimus.

    PubMed

    Katow, Hideki; Katow, Tomoko; Yoshida, Hiromi; Kiyomoto, Masato; Uemura, Isao

    2016-01-01

    The swimming activity of sea urchin larvae is dependent on the ciliary band (CB) on the larval surface and is regulated by several neurotransmitters, including serotonin (5HT), dopamine, and γ-aminobutyric acid (GABA). However, the CB signal transmission mechanism remains unknown. The present study investigated the structural relationship between the CB and external signal receptors by immunohistochemical and transmission electron microscopic analyses of sea urchin, Hemicentrotus pulcherrimus, larvae. Glutamate decarboxylase (GAD; GABA synthetase) was detected in a strand of multiple cells along the circumoral CB in 6-arm plutei. The GAD-expressing strand was closely associated with the CB on the oral ectoderm side. The ciliary band-associated strand (CBAS) also expressed the 5HT receptor (5HThpr) and encephalopsin (ECPN) throughout the cytoplasm and comprised 1- to 2-μm diameter axon-like long stretched regions and sporadic 6- to 7-μm diameter bulbous nucleated regions (perikarya) that protruded into the oral ectoderm side. Besides the laterally polarized morphology of the CBAS cells, Epith-2, which is the epithelial lateral cell surface-specific protein of the sea urchin embryo and larva, was expressed exclusively by perikarya but not by the axon-like regions. The CBAS exposed its narrow apical surface on the larval epithelium between the CB and squamous cells and formed adherens junctions (AJs) on the apical side between them. Despite the presence of the CBAS axon-like regions, tubulins, such as α-, β-, and acetylated α-tubulins, were not detected. However, the neuroendocrine cell marker protein synaptophysin was detected in the axon-like regions and in bouton-like protrusions that contained numerous small ultrastructural vesicles. The unique morphology of the CBAS in the sea urchin larva epithelium had not been reported. The CBAS expresses a remarkable number of receptors to environmental stimuli and proteins that are probably involved in signal

  5. Embryonic and post-embryonic development of the polyclad flatworm Maritigrella crozieri; implications for the evolution of spiralian life history traits

    PubMed Central

    2010-01-01

    ciliary band muscle and flask-shaped epidermal serotonergic cells of the apical organ. Based on current phylogenies, the distribution of ciliary bands and apical organs between polyclads and other spiralians is not congruent with a hypothesis of homology. However, some similarities exist, and this study sets an anatomical framework from which to investigate cellular and molecular mechanisms that will help to distinguish between parallelism, convergence and homology of these features. PMID:20426837

  6. Differential gene expression profiling of human adipose stem cells differentiating into smooth muscle-like cells by TGFβ1/BMP4

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

    Elçin, Ayşe Eser; Parmaksiz, Mahmut; Dogan, Arin

    Regenerative repair of the vascular system is challenging from the perspectives of translational medicine and tissue engineering. There are fundamental hurdles in front of creating bioartificial arteries, which involve recaputilation of the three-layered structure under laboratory settings. Obtaining and maintaining smooth muscle characteristics is an important limitation, as the transdifferentiated cells fail to display mature phenotype. This study aims to shed light on the smooth muscle differentiation of human adipose stem cells (hASCs). To this end, we first acquired hASCs from lipoaspirate samples. Upon characterization, the cells were induced to differentiate into smooth muscle (SM)-like cells using a variety ofmore » inducer combinations. Among all, TGFβ1/BMP4 combination had the highest differentiation efficiency, based on immunohistochemical analyses. hSM-like cell samples were compared to hASCs and to the positive control, human coronary artery-smooth muscle cells (hCA-SMCs) through gene transcription profiling. Microarray findings revealed the activation of gene groups that function in smooth muscle differentiation, signaling pathways, extracellular modeling and cell proliferation. Our results underline the effectiveness of the growth factors and suggest some potential variables for detecting the SM-like cell characteristics. Evidence in transcriptome level was used to evaluate the TGFβ1/BMP4 combination as a previously unexplored effector for the smooth muscle differentiation of adipose stem cells. - Highlights: • Human adipose stem cells (hASCs) were isolated, characterized and cultured. • Growth factor combinations were evaluated for their effectiveness in differentiation using IHC. • hASCs were differentiated into smooth muscle (SM)-like cells using TGF-β1 and BMP4 combination. • Microarray analysis was performed for hASCs, SM-like cells and coronary artery-SMCs. • Microarray data was used to perform hierarchical clustering and

  7. Fibronectin promotes differentiation of neural crest progenitors endowed with smooth muscle cell potential

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

    Costa-Silva, Bruno; Programa de Pos-graduacao em Neurociencias, Centro de Ciencias Biologicas, Universidade Federal de Santa Catarina, Campus Universitario - Trindade, 88040-900, Florianopolis, S.C.; Coelho da Costa, Meline

    The neural crest (NC) is a model system used to investigate multipotency during vertebrate development. Environmental factors control NC cell fate decisions. Despite the well-known influence of extracellular matrix molecules in NC cell migration, the issue of whether they also influence NC cell differentiation has not been addressed at the single cell level. By analyzing mass and clonal cultures of mouse cephalic and quail trunk NC cells, we show for the first time that fibronectin (FN) promotes differentiation into the smooth muscle cell phenotype without affecting differentiation into glia, neurons, and melanocytes. Time course analysis indicated that the FN-induced effectmore » was not related to massive cell death or proliferation of smooth muscle cells. Finally, by comparing clonal cultures of quail trunk NC cells grown on FN and collagen type IV (CLIV), we found that FN strongly increased both NC cell survival and the proportion of unipotent and oligopotent NC progenitors endowed with smooth muscle potential. In contrast, melanocytic progenitors were prominent in clonogenic NC cells grown on CLIV. Taken together, these results show that FN promotes NC cell differentiation along the smooth muscle lineage, and therefore plays an important role in fate decisions of NC progenitor cells.« less

  8. Voltage-dependent inward currents in smooth muscle cells of skeletal muscle arterioles

    PubMed Central

    Shirokov, Roman E.

    2018-01-01

    Voltage-dependent inward currents responsible for the depolarizing phase of action potentials were characterized in smooth muscle cells of 4th order arterioles in mouse skeletal muscle. Currents through L-type Ca2+ channels were expected to be dominant; however, action potentials were not eliminated in nominally Ca2+-free bathing solution or by addition of L-type Ca2+ channel blocker nifedipine (10 μM). Instead, Na+ channel blocker tetrodotoxin (TTX, 1 μM) reduced the maximal velocity of the upstroke at low, but not at normal (2 mM), Ca2+ in the bath. The magnitude of TTX-sensitive currents recorded with 140 mM Na+ was about 20 pA/pF. TTX-sensitive currents decreased five-fold when Ca2+ increased from 2 to 10 mM. The currents reduced three-fold in the presence of 10 mM caffeine, but remained unaltered by 1 mM of isobutylmethylxanthine (IBMX). In addition to L-type Ca2+ currents (15 pA/pF in 20 mM Ca2+), we also found Ca2+ currents that are resistant to 10 μM nifedipine (5 pA/pF in 20 mM Ca2+). Based on their biophysical properties, these Ca2+ currents are likely to be through voltage-gated T-type Ca2+ channels. Our results suggest that Na+ and at least two types (T- and L-) of Ca2+ voltage-gated channels contribute to depolarization of smooth muscle cells in skeletal muscle arterioles. Voltage-gated Na+ channels appear to be under a tight control by Ca2+ signaling. PMID:29694371

  9. Observing planar cell polarity in multiciliated mouse airway epithelial cells.

    PubMed

    Vladar, Eszter K; Lee, Yin Loon; Stearns, Tim; Axelrod, Jeffrey D

    2015-01-01

    The concerted movement of cilia propels inhaled contaminants out of the lungs, safeguarding the respiratory system from toxins, pathogens, pollutants, and allergens. Motile cilia on the multiciliated cells (MCCs) of the airway epithelium are physically oriented along the tissue axis for directional motility, which depends on the planar cell polarity (PCP) signaling pathway. The MCCs of the mouse respiratory epithelium have emerged as an important model for the study of motile ciliogenesis and the PCP signaling mechanism. Unlike other motile ciliated or planar polarized tissues, airway epithelial cells are relatively easily accessible and primary cultures faithfully model many of the essential features of the in vivo tissue. There is growing interest in understanding how cells acquire and polarize motile cilia due to the impact of mucociliary clearance on respiratory health. Here, we present methods for observing and quantifying the planar polarized orientation of motile cilia both in vivo and in primary culture airway epithelial cells. We describe how to acquire and evaluate electron and light microscopy images of ciliary ultrastructural features that reveal planar polarized orientation. Furthermore, we describe the immunofluorescence localization of PCP pathway components as a simple readout for airway epithelial planar polarization and ciliary orientation. These methods can be adapted to observe ciliary orientation in other multi- and monociliated cells and to detect PCP pathway activity in any tissue or cell type. Copyright © 2015 Elsevier Inc. All rights reserved.

  10. Automatic analysis of ciliary beat frequency using optical flow

    NASA Astrophysics Data System (ADS)

    Figl, Michael; Lechner, Manuel; Werther, Tobias; Horak, Fritz; Hummel, Johann; Birkfellner, Wolfgang

    2012-02-01

    Ciliary beat frequency (CBF) can be a useful parameter for diagnosis of several diseases, as e.g. primary ciliary dyskinesia. (PCD). CBF computation is usually done using manual evaluation of high speed video sequences, a tedious, observer dependent, and not very accurate procedure. We used the OpenCV's pyramidal implementation of the Lukas-Kanade algorithm for optical flow computation and applied this to certain objects to follow the movements. The objects were chosen by their contrast applying the corner detection by Shi and Tomasi. Discrimination between background/noise and cilia by a frequency histogram allowed to compute the CBF. Frequency analysis was done using the Fourier transform in matlab. The correct number of Fourier summands was found by the slope in an approximation curve. The method showed to be usable to distinguish between healthy and diseased samples. However there remain difficulties in automatically identifying the cilia, and also in finding enough high contrast cilia in the image. Furthermore the some of the higher contrast cilia are lost (and sometimes found) by the method, an easy way to distinguish the correct sub-path of a point's path have yet to be found in the case where the slope methods doesn't work.

  11. Differential Gene Expression Profiling of Dystrophic Dog Muscle after MuStem Cell Transplantation

    PubMed Central

    Babarit, Candice; Larcher, Thibaut; Dubreil, Laurence; Leroux, Isabelle; Zuber, Céline; Ledevin, Mireille; Deschamps, Jack-Yves; Fromes, Yves; Cherel, Yan; Guevel, Laetitia; Rouger, Karl

    2015-01-01

    Background Several adult stem cell populations exhibit myogenic regenerative potential, thus representing attractive candidates for therapeutic approaches of neuromuscular diseases such as Duchenne Muscular Dystrophy (DMD). We have recently shown that systemic delivery of MuStem cells, skeletal muscle-resident stem cells isolated in healthy dog, generates the remodelling of muscle tissue and gives rise to striking clinical benefits in Golden Retriever Muscular Dystrophy (GRMD) dog. This global effect, which is observed in the clinically relevant DMD animal model, leads us to question here the molecular pathways that are impacted by MuStem cell transplantation. To address this issue, we compare the global gene expression profile between healthy, GRMD and MuStem cell treated GRMD dog muscle, four months after allogenic MuStem cell transplantation. Results In the dystrophic context of the GRMD dog, disease-related deregulation is observed in the case of 282 genes related to various processes such as inflammatory response, regeneration, calcium ion binding, extracellular matrix organization, metabolism and apoptosis regulation. Importantly, we reveal the impact of MuStem cell transplantation on several molecular and cellular pathways based on a selection of 31 genes displaying signals specifically modulated by the treatment. Concomitant with a diffuse dystrophin expression, a histological remodelling and a stabilization of GRMD dog clinical status, we show that cell delivery is associated with an up-regulation of genes reflecting a sustained enhancement of muscle regeneration. We also identify a decreased mRNA expression of a set of genes having metabolic functions associated with lipid homeostasis and energy. Interestingly, ubiquitin-mediated protein degradation is highly enhanced in GRMD dog muscle after systemic delivery of MuStem cells. Conclusions Overall, our results provide the first high-throughput characterization of GRMD dog muscle and throw new light on the

  12. Species variation in biology and physiology of the ciliary epithelium: similarities and differences.

    PubMed

    Do, Chi Wai; Civan, Mortimer M

    2009-04-01

    Glaucoma is a leading cause of irreversible blindness worldwide. Lowering intraocular pressure (IOP) is the only strategy documented to delay the appearance and retard the progression of vision loss. One major approach for lowering IOP is to slow the rate of aqueous humor formation by the ciliary epithelium. As discussed in the present review, the transport basis for this secretion is largely understood. However, several substantive issues are yet to be resolved, including the integrated regulation of secretion, the functional topography of the ciliary epithelium, and the degree and significance of species variation in aqueous humor inflow. This review discusses species differences in net secretion, particularly of Cl(-) and HCO(3)(-) secretion. Identifying animal models most accurately mimicking aqueous humor formation in the human will facilitate development of future novel initiatives to lower IOP.

  13. Serum Proteases Potentiate BMP-Induced Cell Cycle Re-entry of Dedifferentiating Muscle Cells during Newt Limb Regeneration.

    PubMed

    Wagner, Ines; Wang, Heng; Weissert, Philipp M; Straube, Werner L; Shevchenko, Anna; Gentzel, Marc; Brito, Goncalo; Tazaki, Akira; Oliveira, Catarina; Sugiura, Takuji; Shevchenko, Andrej; Simon, András; Drechsel, David N; Tanaka, Elly M

    2017-03-27

    Limb amputation in the newt induces myofibers to dedifferentiate and re-enter the cell cycle to generate proliferative myogenic precursors in the regeneration blastema. Here we show that bone morphogenetic proteins (BMPs) and mature BMPs that have been further cleaved by serum proteases induce cell cycle entry by dedifferentiating newt muscle cells. Protease-activated BMP4/7 heterodimers that are present in serum strongly induced myotube cell cycle re-entry with protease cleavage yielding a 30-fold potency increase of BMP4/7 compared with canonical BMP4/7. Inhibition of BMP signaling via muscle-specific dominant-negative receptor expression reduced cell cycle entry in vitro and in vivo. In vivo inhibition of serine protease activity depressed cell cycle re-entry, which in turn was rescued by cleaved-mimic BMP. This work identifies a mechanism of BMP activation that generates blastema cells from differentiated muscle. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Three-dimensional reconstruction of the angioarchitecture of the ciliary body of the West Indian manatee (Trichechus manatus).

    PubMed

    Natiello, Michelle; Samuelson, Don

    2005-01-01

    To examine the angioarchitecture of the ciliary body in the West Indian manatee (Trichechus manatus), through the use of three-dimensional reconstruction. Specimens from West Indian manatee were preserved in 10% buffered formalin, embedded in paraffin, serial sectioned and stained by Masson trichrome for light microscopic three-dimensional reconstruction and evaluation. The network of blood vessels in the ciliary processes of the West Indian manatee is fed by the major arterial circle that lies mostly near the base of the iris. The branching arterioles give rise to a capillary-sinusoidal bed that extends internally along each process, emptying into two sets of veins, one being elevated. The elevated and nonelevated veins join posteriorly before emptying into the choroidal venous system. The angioarchitecture of the ciliary body of the West Indian manatee is clearly unique when compared to those previously examined in land mammals. Three-dimensional reconstruction of paraffin sections is an effective means to evaluate vascular patterns in ocular specimens, especially those unavailable for corrosion casting.

  15. Skeletal Muscle Satellite Cells Are Committed to Myogenesis and Do Not Spontaneously Adopt Nonmyogenic Fates

    PubMed Central

    Starkey, Jessica D.; Yamamoto, Masakazu; Yamamoto, Shoko; Goldhamer, David J.

    2011-01-01

    The developmental potential of skeletal muscle stem cells (satellite cells) remains controversial. The authors investigated satellite cell developmental potential in single fiber and clonal cultures derived from MyoDiCre/+;R26REYFP/+ muscle, in which essentially all satellite cells are permanently labeled. Approximately 60% of the clones derived from cells that co-purified with muscle fibers spontaneously underwent adipogenic differentiation. These adipocytes stained with Oil-Red-O and expressed the terminal differentiation markers, adipsin and fatty acid binding protein 4, but did not express EYFP and were therefore not of satellite cell origin. Satellite cells mutant for either MyoD or Myf-5 also maintained myogenic programming in culture and did not adopt an adipogenic fate. Incorporation of additional wash steps prior to muscle fiber plating virtually eliminated the non-myogenic cells but did not reduce the number of adherent Pax7+ satellite cells. More than half of the adipocytes observed in cultures from Tie2-Cre mice were recombined, further demonstrating a non-satellite cell origin. Under adipogenesis-inducing conditions, satellite cells accumulated cytoplasmic lipid but maintained myogenic protein expression and did not fully execute the adipogenic differentiation program, distinguishing them from adipocytes observed in muscle fiber cultures. The authors conclude that skeletal muscle satellite cells are committed to myogenesis and do not spontaneously adopt an adipogenic fate. PMID:21339173

  16. Three distinct cell populations express extracellular matrix proteins and increase in number during skeletal muscle fibrosis.

    PubMed

    Chapman, Mark A; Mukund, Kavitha; Subramaniam, Shankar; Brenner, David; Lieber, Richard L

    2017-02-01

    Tissue extracellular matrix (ECM) provides structural support and creates unique environments for resident cells (Bateman JF, Boot-Handford RP, Lamandé SR. Nat Rev Genet 10: 173-183, 2009; Kjaer M. Physiol Rev 84: 649-98, 2004). However, the identities of cells responsible for creating specific ECM components have not been determined. In striated muscle, the identity of these cells becomes important in disease when ECM changes result in fibrosis and subsequent increased tissue stiffness and dysfunction. Here we describe a novel approach to isolate and identify cells that maintain the ECM in both healthy and fibrotic muscle. Using a collagen I reporter mouse, we show that there are three distinct cell populations that express collagen I in both healthy and fibrotic skeletal muscle. Interestingly, the number of collagen I-expressing cells in all three cell populations increases proportionally in fibrotic muscle, indicating that all cell types participate in the fibrosis process. Furthermore, while some profibrotic ECM and ECM-associated genes are significantly upregulated in fibrotic muscle, the fibrillar collagen gene expression profile is not qualitatively altered. This suggests that muscle fibrosis in this model results from an increased number of collagen I-expressing cells and not the initiation of a specific fibrotic collagen gene expression program. Finally, in fibrotic muscle, we show that these collagen I-expressing cell populations differentially express distinct ECM proteins-fibroblasts express the fibrillar components of ECM, fibro/adipogenic progenitors cells differentially express basal laminar proteins, and skeletal muscle progenitor cells differentially express genes important for the satellite cell. Copyright © 2017 the American Physiological Society.

  17. Three distinct cell populations express extracellular matrix proteins and increase in number during skeletal muscle fibrosis

    PubMed Central

    Chapman, Mark A.; Mukund, Kavitha; Subramaniam, Shankar; Brenner, David

    2017-01-01

    Tissue extracellular matrix (ECM) provides structural support and creates unique environments for resident cells (Bateman JF, Boot-Handford RP, Lamandé SR. Nat Rev Genet 10: 173–183, 2009; Kjaer M. Physiol Rev 84: 649–98, 2004). However, the identities of cells responsible for creating specific ECM components have not been determined. In striated muscle, the identity of these cells becomes important in disease when ECM changes result in fibrosis and subsequent increased tissue stiffness and dysfunction. Here we describe a novel approach to isolate and identify cells that maintain the ECM in both healthy and fibrotic muscle. Using a collagen I reporter mouse, we show that there are three distinct cell populations that express collagen I in both healthy and fibrotic skeletal muscle. Interestingly, the number of collagen I-expressing cells in all three cell populations increases proportionally in fibrotic muscle, indicating that all cell types participate in the fibrosis process. Furthermore, while some profibrotic ECM and ECM-associated genes are significantly upregulated in fibrotic muscle, the fibrillar collagen gene expression profile is not qualitatively altered. This suggests that muscle fibrosis in this model results from an increased number of collagen I-expressing cells and not the initiation of a specific fibrotic collagen gene expression program. Finally, in fibrotic muscle, we show that these collagen I-expressing cell populations differentially express distinct ECM proteins—fibroblasts express the fibrillar components of ECM, fibro/adipogenic progenitors cells differentially express basal laminar proteins, and skeletal muscle progenitor cells differentially express genes important for the satellite cell. PMID:27881411

  18. Immortalized human myotonic dystrophy muscle cell lines to assess therapeutic compounds.

    PubMed

    Arandel, Ludovic; Polay Espinoza, Micaela; Matloka, Magdalena; Bazinet, Audrey; De Dea Diniz, Damily; Naouar, Naïra; Rau, Frédérique; Jollet, Arnaud; Edom-Vovard, Frédérique; Mamchaoui, Kamel; Tarnopolsky, Mark; Puymirat, Jack; Battail, Christophe; Boland, Anne; Deleuze, Jean-Francois; Mouly, Vincent; Klein, Arnaud F; Furling, Denis

    2017-04-01

    Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are autosomal dominant neuromuscular diseases caused by microsatellite expansions and belong to the family of RNA-dominant disorders. Availability of cellular models in which the DM mutation is expressed within its natural context is essential to facilitate efforts to identify new therapeutic compounds. Here, we generated immortalized DM1 and DM2 human muscle cell lines that display nuclear RNA aggregates of expanded repeats, a hallmark of myotonic dystrophy. Selected clones of DM1 and DM2 immortalized myoblasts behave as parental primary myoblasts with a reduced fusion capacity of immortalized DM1 myoblasts when compared with control and DM2 cells. Alternative splicing defects were observed in differentiated DM1 muscle cell lines, but not in DM2 lines. Splicing alterations did not result from differentiation delay because similar changes were found in immortalized DM1 transdifferentiated fibroblasts in which myogenic differentiation has been forced by overexpression of MYOD1. As a proof-of-concept, we show that antisense approaches alleviate disease-associated defects, and an RNA-seq analysis confirmed that the vast majority of mis-spliced events in immortalized DM1 muscle cells were affected by antisense treatment, with half of them significantly rescued in treated DM1 cells. Immortalized DM1 muscle cell lines displaying characteristic disease-associated molecular features such as nuclear RNA aggregates and splicing defects can be used as robust readouts for the screening of therapeutic compounds. Therefore, immortalized DM1 and DM2 muscle cell lines represent new models and tools to investigate molecular pathophysiological mechanisms and evaluate the in vitro effects of compounds on RNA toxicity associated with myotonic dystrophy mutations. © 2017. Published by The Company of Biologists Ltd.

  19. Immortalized human myotonic dystrophy muscle cell lines to assess therapeutic compounds

    PubMed Central

    Arandel, Ludovic; Polay Espinoza, Micaela; Matloka, Magdalena; Bazinet, Audrey; De Dea Diniz, Damily; Naouar, Naïra; Rau, Frédérique; Jollet, Arnaud; Edom-Vovard, Frédérique; Mamchaoui, Kamel; Tarnopolsky, Mark; Puymirat, Jack; Battail, Christophe; Boland, Anne; Deleuze, Jean-Francois; Mouly, Vincent; Klein, Arnaud F.

    2017-01-01

    ABSTRACT Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are autosomal dominant neuromuscular diseases caused by microsatellite expansions and belong to the family of RNA-dominant disorders. Availability of cellular models in which the DM mutation is expressed within its natural context is essential to facilitate efforts to identify new therapeutic compounds. Here, we generated immortalized DM1 and DM2 human muscle cell lines that display nuclear RNA aggregates of expanded repeats, a hallmark of myotonic dystrophy. Selected clones of DM1 and DM2 immortalized myoblasts behave as parental primary myoblasts with a reduced fusion capacity of immortalized DM1 myoblasts when compared with control and DM2 cells. Alternative splicing defects were observed in differentiated DM1 muscle cell lines, but not in DM2 lines. Splicing alterations did not result from differentiation delay because similar changes were found in immortalized DM1 transdifferentiated fibroblasts in which myogenic differentiation has been forced by overexpression of MYOD1. As a proof-of-concept, we show that antisense approaches alleviate disease-associated defects, and an RNA-seq analysis confirmed that the vast majority of mis-spliced events in immortalized DM1 muscle cells were affected by antisense treatment, with half of them significantly rescued in treated DM1 cells. Immortalized DM1 muscle cell lines displaying characteristic disease-associated molecular features such as nuclear RNA aggregates and splicing defects can be used as robust readouts for the screening of therapeutic compounds. Therefore, immortalized DM1 and DM2 muscle cell lines represent new models and tools to investigate molecular pathophysiological mechanisms and evaluate the in vitro effects of compounds on RNA toxicity associated with myotonic dystrophy mutations. PMID:28188264

  20. Continuous Release of Tumor-Derived Factors Improves the Modeling of Cachexia in Muscle Cell Culture.

    PubMed

    Jackman, Robert W; Floro, Jess; Yoshimine, Rei; Zitin, Brian; Eiampikul, Maythita; El-Jack, Kahlid; Seto, Danielle N; Kandarian, Susan C

    2017-01-01

    Cachexia is strongly associated with a poor prognosis in cancer patients but the biological trigger is unknown and therefore no therapeutics exist. The loss of skeletal muscle is the most deleterious aspect of cachexia and it appears to depend on secretions from tumor cells. Models for studying wasting in cell culture consist of experiments where skeletal muscle cells are incubated with medium conditioned by tumor cells. This has led to candidates for cachectic factors but some of the features of cachexia in vivo are not yet well-modeled in cell culture experiments. Mouse myotube atrophy measured by myotube diameter in response to medium conditioned by mouse colon carcinoma cells (C26) is consistently less than what is seen in muscles of mice bearing C26 tumors with moderate to severe cachexia. One possible reason for this discrepancy is that in vivo the C26 tumor and skeletal muscle share a circulatory system exposing the muscle to tumor factors in a constant and increasing way. We have applied Transwell®-adapted cell culture conditions to more closely simulate conditions found in vivo where muscle is exposed to the ongoing kinetics of constant tumor secretion of active factors. C26 cells were incubated on a microporous membrane (a Transwell® insert) that constitutes the upper compartment of wells containing plated myotubes. In this model, myotubes are exposed to a constant supply of cancer cell secretions in the medium but without direct contact with the cancer cells, analogous to a shared circulation of muscle and cancer cells in tumor-bearing animals. The results for myotube diameter support the idea that the use of Transwell® inserts serves as a more physiological model of the muscle wasting associated with cancer cachexia than the bolus addition of cancer cell conditioned medium. The Transwell® model supports the notion that the dose and kinetics of cachectic factor delivery to muscle play a significant role in the extent of pathology.

  1. FOXP3+ T Cells Recruited to Sites of Sterile Skeletal Muscle Injury Regulate the Fate of Satellite Cells and Guide Effective Tissue Regeneration

    PubMed Central

    Castiglioni, Alessandra; Basso, Veronica; Vezzoli, Michela; Monno, Antonella; Almada, Albert E.; Mondino, Anna; Wagers, Amy J.; Manfredi, Angelo A.; Rovere-Querini, Patrizia

    2015-01-01

    Muscle injury induces a classical inflammatory response in which cells of the innate immune system rapidly invade the tissue. Macrophages are prominently involved in this response and required for proper healing, as they are known to be important for clearing cellular debris and supporting satellite cell differentiation. Here, we sought to assess the role of the adaptive immune system in muscle regeneration after acute damage. We show that T lymphocytes are transiently recruited into the muscle after damage and appear to exert a pro-myogenic effect on muscle repair. We observed a decrease in the cross-sectional area of regenerating myofibers after injury in Rag2-/- γ-chain-/- mice, as compared to WT controls, suggesting that T cell recruitment promotes muscle regeneration. Skeletal muscle infiltrating T lymphocytes were enriched in CD4+CD25+FOXP3+ cells. Direct exposure of muscle satellite cells to in vitro induced Treg cells effectively enhanced their expansion, and concurrently inhibited their myogenic differentiation. In vivo, the recruitment of Tregs to acutely injured muscle was limited to the time period of satellite expansion, with possibly important implications for situations in which inflammatory conditions persist, such as muscular dystrophies and inflammatory myopathies. We conclude that the adaptive immune system, in particular T regulatory cells, is critically involved in effective skeletal muscle regeneration. Thus, in addition to their well-established role as regulators of the immune/inflammatory response, T regulatory cells also regulate the activity of skeletal muscle precursor cells, and are instrumental for the proper regeneration of this tissue. PMID:26039259

  2. Notch signal reception is required in vascular smooth muscle cells for ductus arteriosus closure

    PubMed Central

    Krebs, Luke T.; Norton, Christine R.; Gridley, Thomas

    2017-01-01

    Summary The ductus arteriosus is an arterial vessel that shunts blood flow away from the lungs during fetal life, but normally occludes after birth to establish the adult circulation pattern. Failure of the ductus arteriosus to close after birth is termed patent ductus arteriosus, and is one of the most common congenital heart defects. Our previous work demonstrated that vascular smooth muscle cell expression of the Jag1 gene, which encodes a ligand for Notch family receptors, is essential for postnatal closure of the ductus arteriosus in mice. However, it was not known what cell population was responsible for receiving the Jag1-mediated signal. Here we show, using smooth muscle cell-specific deletion of the Rbpj gene, which encodes a transcription factor that mediates all canonical Notch signaling, that Notch signal reception in the vascular smooth muscle cell compartment is required for ductus arteriosus closure. These data indicate that homotypic vascular smooth muscle cell interactions are required for proper contractile smooth muscle cell differentiation and postnatal closure of the ductus arteriosus in mice. PMID:26742650

  3. Normal Muscle Oxygen Consumption and Fatigability in Sickle Cell Patients Despite Reduced Microvascular Oxygenation and Hemorheological Abnormalities

    PubMed Central

    Waltz, Xavier; Pichon, Aurélien; Lemonne, Nathalie; Mougenel, Danièle; Lalanne-Mistrih, Marie-Laure; Lamarre, Yann; Tarer, Vanessa; Tressières, Benoit; Etienne-Julan, Maryse; Hardy-Dessources, Marie-Dominique; Hue, Olivier; Connes, Philippe

    2012-01-01

    Background/Aim Although it has been hypothesized that muscle metabolism and fatigability could be impaired in sickle cell patients, no study has addressed this issue. Methods We compared muscle metabolism and function (muscle microvascular oxygenation, microvascular blood flow, muscle oxygen consumption and muscle microvascular oxygenation variability, which reflects vasomotion activity, maximal muscle force and local muscle fatigability) and the hemorheological profile at rest between 16 healthy subjects (AA), 20 sickle cell-hemoglobin C disease (SC) patients and 16 sickle cell anemia (SS) patients. Results Muscle microvascular oxygenation was reduced in SS patients compared to the SC and AA groups and this reduction was not related to hemorhelogical abnormalities. No difference was observed between the three groups for oxygen consumption and vasomotion activity. Muscle microvascular blood flow was higher in SS patients compared to the AA group, and tended to be higher compared to the SC group. Multivariate analysis revealed that muscle oxygen consumption was independently associated with muscle microvascular blood flow in the two sickle cell groups (SC and SS). Finally, despite reduced muscle force in sickle cell patients, their local muscle fatigability was similar to that of the healthy subjects. Conclusions Sickle cell patients have normal resting muscle oxygen consumption and fatigability despite hemorheological alterations and, for SS patients only, reduced muscle microvascular oxygenation and increased microvascular blood flow. Two alternative mechanisms can be proposed for SS patients: 1) the increased muscle microvascular blood flow is a way to compensate for the lower muscle microvascular oxygenation to maintain muscle oxygen consumption to normal values or 2) the reduced microvascular oxygenation coupled with a normal resting muscle oxygen consumption could indicate that there is slight hypoxia within the muscle which is not sufficient to limit

  4. Normal muscle oxygen consumption and fatigability in sickle cell patients despite reduced microvascular oxygenation and hemorheological abnormalities.

    PubMed

    Waltz, Xavier; Pichon, Aurélien; Lemonne, Nathalie; Mougenel, Danièle; Lalanne-Mistrih, Marie-Laure; Lamarre, Yann; Tarer, Vanessa; Tressières, Benoit; Etienne-Julan, Maryse; Hardy-Dessources, Marie-Dominique; Hue, Olivier; Connes, Philippe

    2012-01-01

    Although it has been hypothesized that muscle metabolism and fatigability could be impaired in sickle cell patients, no study has addressed this issue. We compared muscle metabolism and function (muscle microvascular oxygenation, microvascular blood flow, muscle oxygen consumption and muscle microvascular oxygenation variability, which reflects vasomotion activity, maximal muscle force and local muscle fatigability) and the hemorheological profile at rest between 16 healthy subjects (AA), 20 sickle cell-hemoglobin C disease (SC) patients and 16 sickle cell anemia (SS) patients. Muscle microvascular oxygenation was reduced in SS patients compared to the SC and AA groups and this reduction was not related to hemorhelogical abnormalities. No difference was observed between the three groups for oxygen consumption and vasomotion activity. Muscle microvascular blood flow was higher in SS patients compared to the AA group, and tended to be higher compared to the SC group. Multivariate analysis revealed that muscle oxygen consumption was independently associated with muscle microvascular blood flow in the two sickle cell groups (SC and SS). Finally, despite reduced muscle force in sickle cell patients, their local muscle fatigability was similar to that of the healthy subjects. Sickle cell patients have normal resting muscle oxygen consumption and fatigability despite hemorheological alterations and, for SS patients only, reduced muscle microvascular oxygenation and increased microvascular blood flow. Two alternative mechanisms can be proposed for SS patients: 1) the increased muscle microvascular blood flow is a way to compensate for the lower muscle microvascular oxygenation to maintain muscle oxygen consumption to normal values or 2) the reduced microvascular oxygenation coupled with a normal resting muscle oxygen consumption could indicate that there is slight hypoxia within the muscle which is not sufficient to limit mitochondrial respiration but increases muscle

  5. Defining the transcriptional signature of skeletal muscle stem cells.

    PubMed

    Yablonka-Reuveni, Z; Day, K; Vine, A; Shefer, G

    2008-04-01

    Satellite cells, the main source of myoblasts in postnatal muscle, are located beneath the myofiber basal lamina. The myogenic potential of satellite cells was initially documented based on their capacity to produce progeny that fused into myotubes. More recently, molecular markers of resident satellite cells were identified, further contributing to defining these cells as myogenic stem cells that produce differentiating progeny and self-renew. Herein, we discuss aspects of the satellite cell transcriptional milieu that have been intensively investigated in our research. We elaborate on the expression patterns of the paired box (Pax) transcription factors Pax3 and Pax7, and on the myogenic regulatory factors myogenic factor 5 (Myf5), myogenic determination factor 1 (MyoD), and myogenin. We also introduce original data on MyoD upregulation in newly activated satellite cells, which precedes the first round of cell proliferation. Such MyoD upregulation occurred even when parent myofibers with their associated satellite cells were exposed to pharmacological inhibitors of hepatocyte growth factor and fibroblast growth factor receptors, which are typically involved in promoting satellite cell proliferation. These observations support the hypothesis that most satellite cells in adult muscle are committed to rapidly entering myogenesis. We also detected expression of serum response factor in resident satellite cells prior to MyoD expression, which may facilitate the rapid upregulation of MyoD. Aspects of satellite cell self-renewal based on the reemergence of cells expressing Pax7, but not MyoD, in myogenic cultures are discussed further herein. We conclude by describing our recent studies using transgenic mice in which satellite cells are traced and isolated based on their expression of green fluorescence protein driven by regulatory elements of the nestin promoter (nestin-green fluorescence protein). This feature provides us with a novel means of studying satellite cell

  6. Melatonin synthesis in the human ciliary body triggered by TRPV4 activation: Involvement of AANAT phosphorylation.

    PubMed

    Alkozi, Hanan Awad; Perez de Lara, María J; Pintor, Jesús

    2017-09-01

    Melatonin is a substance synthesized in the pineal gland as well as in other organs. This substance is involved in many ocular functions, giving its synthesis in numerous eye structures. Melatonin is synthesized from serotonin through two enzymes, the first limiting step into the synthesis of melatonin being aralkylamine N-acetyltransferase (AANAT). In this current study, AANAT phosphorylation after the activation of TRPV4 was studied using human non-pigmented epithelial ciliary body cells. Firstly, it was necessary to determine the adequate time and dose of the TRPV4 agonist GSK1016790A to reach the maximal phosphorylation of AANAT. An increase of 72% was observed after 5 min incubation with 10 nM GSK (**p < 0.05, n = 6) with a concomitant rise in N-acetyl serotonin and melatonin synthesis. The involvement of a TRPV4 channel in melatonin synthesis was verified by antagonist and siRNA studies as a previous step to studying intracellular signalling. Studies performed on the second messengers involved in GSK induced AANAT phosphorylation were carried out by inhibiting several pathways. In conclusion, the activation of calmodulin and calmodulin-dependent protein kinase II was confirmed, as shown by the cascade seen in AANAT phosphorylation (***p < 0.001, n = 4). This mechanism was also established by measuring N-acetyl serotonin and melatonin levels. In conclusion, the activation of a TRPV4 present in human ciliary body epithelial cells produced an increase in AANAT phosphorylation and a further melatonin increase by a mechanism in which Ca-calmodulin and the calmodulin-dependent protein kinase II are involved. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. Differential requirement for satellite cells during overload-induced muscle hypertrophy in growing versus mature mice.

    PubMed

    Murach, Kevin A; White, Sarah H; Wen, Yuan; Ho, Angel; Dupont-Versteegden, Esther E; McCarthy, John J; Peterson, Charlotte A

    2017-07-10

    Pax7+ satellite cells are required for skeletal muscle fiber growth during post-natal development in mice. Satellite cell-mediated myonuclear accretion also appears to persist into early adulthood. Given the important role of satellite cells during muscle development, we hypothesized that the necessity of satellite cells for adaptation to an imposed hypertrophic stimulus depends on maturational age. Pax7 CreER -R26R DTA mice were treated for 5 days with vehicle (satellite cell-replete, SC+) or tamoxifen (satellite cell-depleted, SC-) at 2 months (young) and 4 months (mature) of age. Following a 2-week washout, mice were subjected to sham surgery or 10 day synergist ablation overload of the plantaris (n = 6-9 per group). The surgical approach minimized regeneration, de novo fiber formation, and fiber splitting while promoting muscle fiber growth. Satellite cell density (Pax7+ cells/fiber), embryonic myosin heavy chain expression (eMyHC), and muscle fiber cross sectional area (CSA) were evaluated via immunohistochemistry. Myonuclei (myonuclei/100 mm) were counted on isolated single muscle fibers. Tamoxifen treatment depleted satellite cells by ≥90% and prevented myonuclear accretion with overload in young and mature mice (p < 0.05). Satellite cells did not recover in SC- mice after overload. Average muscle fiber CSA increased ~20% in young SC+ (p = 0.07), mature SC+ (p < 0.05), and mature SC- mice (p < 0.05). In contrast, muscle fiber hypertrophy was prevented in young SC- mice. Muscle fiber number increased only in mature mice after overload (p < 0.05), and eMyHC expression was variable, specifically in mature SC+ mice. Reliance on satellite cells for overload-induced hypertrophy is dependent on maturational age, and global responses to overload differ in young versus mature mice.

  8. Contractile activity of human skeletal muscle cells prevents insulin resistance by inhibiting pro-inflammatory signalling pathways.

    PubMed

    Lambernd, S; Taube, A; Schober, A; Platzbecker, B; Görgens, S W; Schlich, R; Jeruschke, K; Weiss, J; Eckardt, K; Eckel, J

    2012-04-01

    Obesity is closely associated with muscle insulin resistance and is a major risk factor for the pathogenesis of type 2 diabetes. Regular physical activity not only prevents obesity, but also considerably improves insulin sensitivity and skeletal muscle metabolism. We sought to establish and characterise an in vitro model of human skeletal muscle contraction, with a view to directly studying the signalling pathways and mechanisms that are involved in the beneficial effects of muscle activity. Contracting human skeletal muscle cell cultures were established by applying electrical pulse stimulation. To induce insulin resistance, skeletal muscle cells were incubated with human adipocyte-derived conditioned medium, monocyte chemotactic protein (MCP)-1 and chemerin. Similarly to in exercising skeletal muscle in vivo, electrical pulse stimulation induced contractile activity in human skeletal muscle cells, combined with the formation of sarcomeres, activation of AMP-activated protein kinase (AMPK) and increased IL-6 secretion. Insulin-stimulated glucose uptake was substantially elevated in contracting cells compared with control. The incubation of skeletal muscle cells with adipocyte-conditioned media, chemerin and MCP-1 significantly reduced the insulin-stimulated phosphorylation of Akt. This effect was abrogated by concomitant pulse stimulation of the cells. Additionally, pro-inflammatory signalling by adipocyte-derived factors was completely prevented by electrical pulse stimulation of the myotubes. We showed that the effects of electrical pulse stimulation on skeletal muscle cells were similar to the effect of exercise on skeletal muscle in vivo in terms of enhanced AMPK activation and IL-6 secretion. In our model, muscle contractile activity eliminates insulin resistance by blocking pro-inflammatory signalling pathways. This novel model therefore provides a unique tool for investigating the molecular mechanisms that mediate the beneficial effects of muscle

  9. Bioenergetic Impairment in Congenital Muscular Dystrophy Type 1A and Leigh Syndrome Muscle Cells

    PubMed Central

    Fontes-Oliveira, Cibely C.; Steinz, Maarten; Schneiderat, Peter; Mulder, Hindrik; Durbeej, Madeleine

    2017-01-01

    Skeletal muscle has high energy requirement and alterations in metabolism are associated with pathological conditions causing muscle wasting and impaired regeneration. Congenital muscular dystrophy type 1A (MDC1A) is a severe muscle disorder caused by mutations in the LAMA2 gene. Leigh syndrome (LS) is a neurometabolic disease caused by mutations in genes related to mitochondrial function. Skeletal muscle is severely affected in both diseases and a common feature is muscle weakness that leads to hypotonia and respiratory problems. Here, we have investigated the bioenergetic profile in myogenic cells from MDC1A and LS patients. We found dysregulated expression of genes related to energy production, apoptosis and proteasome in myoblasts and myotubes. Moreover, impaired mitochondrial function and a compensatory upregulation of glycolysis were observed when monitored in real-time. Also, alterations in cell cycle populations in myoblasts and enhanced caspase-3 activity in myotubes were observed. Thus, we have for the first time demonstrated an impairment of the bioenergetic status in human MDC1A and LS muscle cells, which could contribute to cell cycle disturbance and increased apoptosis. Our findings suggest that skeletal muscle metabolism might be a promising pharmacological target in order to improve muscle function, energy efficiency and tissue maintenance of MDC1A and LS patients. PMID:28367954

  10. Bioengineered constructs combined with exercise enhance stem cell-mediated treatment of volumetric muscle loss

    PubMed Central

    Quarta, Marco; Cromie, Melinda; Chacon, Robert; Blonigan, Justin; Garcia, Victor; Akimenko, Igor; Hamer, Mark; Paine, Patrick; Stok, Merel; Shrager, Joseph B.; Rando, Thomas A.

    2017-01-01

    Volumetric muscle loss (VML) is associated with loss of skeletal muscle function, and current treatments show limited efficacy. Here we show that bioconstructs suffused with genetically-labelled muscle stem cells (MuSCs) and other muscle resident cells (MRCs) are effective to treat VML injuries in mice. Imaging of bioconstructs implanted in damaged muscles indicates MuSCs survival and growth, and ex vivo analyses show force restoration of treated muscles. Histological analysis highlights myofibre formation, neovascularisation, but insufficient innervation. Both innervation and in vivo force production are enhanced when implantation of bioconstructs is followed by an exercise regimen. Significant improvements are also observed when bioconstructs are used to treat chronic VML injury models. Finally, we demonstrate that bioconstructs made with human MuSCs and MRCs can generate functional muscle tissue in our VML model. These data suggest that stem cell-based therapies aimed to engineer tissue in vivo may be effective to treat acute and chronic VML. PMID:28631758

  11. Distribution of CD163-positive cell and MHC class II-positive cell in the normal equine uveal tract.

    PubMed

    Sano, Yuto; Matsuda, Kazuya; Okamoto, Minoru; Takehana, Kazushige; Hirayama, Kazuko; Taniyama, Hiroyuki

    2016-02-01

    Antigen-presenting cells (APCs) in the uveal tract participate in ocular immunity including immune homeostasis and the pathogenesis of uveitis. In horses, although uveitis is the most common ocular disorder, little is known about ocular immunity, such as the distribution of APCs. In this study, we investigated the distribution of CD163-positive and MHC II-positive cells in the normal equine uveal tract using an immunofluorescence technique. Eleven eyes from 10 Thoroughbred horses aged 1 to 24 years old were used. Indirect immunofluorescence was performed using the primary antibodies CD163, MHC class II (MHC II) and CD20. To demonstrate the site of their greatest distribution, positive cells were manually counted in 3 different parts of the uveal tract (ciliary body, iris and choroid), and their average number was assessed by statistical analysis. The distribution of pleomorphic CD163- and MHC II-expressed cells was detected throughout the equine uveal tract, but no CD20-expressed cells were detected. The statistical analysis demonstrated the distribution of CD163- and MHC II-positive cells focusing on the ciliary body. These results demonstrated that the ciliary body is the largest site of their distribution in the normal equine uveal tract, and the ciliary body is considered to play important roles in uveal and/or ocular immune homeostasis. The data provided in this study will help further understanding of equine ocular immunity in the normal state and might be beneficial for understanding of mechanisms of ocular disorders, such as equine uveitis.

  12. Sprouty1 Regulates Reversible Quiescence of a Self-Renewing Adult Muscle Stem Cell Pool during Regeneration

    PubMed Central

    Shea, Kelly L.; Xiang, Wanyi; LaPorta, Vincent S.; Licht, Jonathan D.; Keller, Charles; Basson, M. Albert; Brack, Andrew S.

    2010-01-01

    Summary Satellite cells are a heterogeneous population of skeletal muscle specific stem cells capable of self-renewal and differentiation after transplantation. Whether quiescent satellite cells can self-renew and contribute to muscle fiber repair in their endogenous environment in normal regenerating muscle has remained unknown. The transcription factor Pax7 is expressed in satellite cells and is critical for establishing the adult satellite cell pool. Using a temporally-inducible genetic lineage tracing approach (Pax7-CreERtm; R26R-lacZ) to fate-map adult satellite cells, we show that in response to injury quiescent adult Pax7+ cells enter the cell cycle; a subpopulation return to quiescence to fully replenish the satellite cell compartment and the others contribute to de novo muscle fiber formation. We demonstrate that Sprouty1 (Spry1), an inhibitor of receptor tyrosine kinase signaling, is robustly expressed in quiescent Pax7+ satellite cells in uninjured adult muscle, down-regulated in proliferating myogenic cells in injured muscles, and re-induced as Pax7+ cells return to quiescence in regenerated muscles. We show through deletion of Spry1 specifically in cycling adult Pax7+ satellite cells, that Spry1 is required for the return to quiescence and homeostasis of the self-renewing Pax7+ satellite cell pool during repair. Satellite cells unable to return to quiescence succumb to apoptosis leading to a diminished self-renewing Pax7-derived satellite cell pool. Our results define a novel role for Spry1 in adult stem cell biology and tissue repair. PMID:20144785

  13. Optimisation of isolation of richly pure and homogeneous primary human colonic smooth muscle cells.

    PubMed

    Tattoli, I; Corleto, V D; Taffuri, M; Campanini, N; Rindi, G; Caprilli, R; Delle Fave, G; Severi, C

    2004-11-01

    Inherent properties of gastrointestinal smooth muscle can be assessed using isolated cell suspensions. Currently available isolation techniques, based on short 2-h enzymatic digestion, however, present the disadvantage of low cellular yield with brief viability. These features are an important limiting factor especially in studies in humans in which tissue may not be available daily and mixing of samples is not recommended. To optimise the isolation procedure of cells from human colon to obtain a richly pure primary smooth muscle cell preparation. Slices of circular muscle layer, obtained from surgical specimens of human colon, were incubated overnight in Dulbecco's modified eagle's medium supplemented with antibiotics, foetal bovine serum, an ATP-regenerating system and collagenase. On the following day, digested muscle strips were suspended in HEPES buffer, and spontaneously dissociated smooth muscle cells were harvested and used either immediately or maintained in suspension for up to 72 h. Cell yield, purity, viability, contractile responses, associated intracellular calcium signals and RNA and protein extraction were evaluated and compared to cell suspensions obtained with the current short digestion protocol. The overnight isolation protocol offers the advantage of obtaining a pure, homogeneous, long-life viable cell suspension that maintains a fully differentiated smooth muscle phenotype unchanged for at least 72 h and that allows multiple functional/biochemical studies and efficient RNA extraction from a single human specimen.

  14. Differentiation of mammalian skeletal muscle cells cultured on microcarrier beads in a rotating cell culture system

    NASA Technical Reports Server (NTRS)

    Torgan, C. E.; Burge, S. S.; Collinsworth, A. M.; Truskey, G. A.; Kraus, W. E.

    2000-01-01

    The growth and repair of adult skeletal muscle are due in part to activation of muscle precursor cells, commonly known as satellite cells or myoblasts. These cells are responsive to a variety of environmental cues, including mechanical stimuli. The overall goal of the research is to examine the role of mechanical signalling mechanisms in muscle growth and plasticity through utilisation of cell culture systems where other potential signalling pathways (i.e. chemical and electrical stimuli) are controlled. To explore the effects of decreased mechanical loading on muscle differentiation, mammalian myoblasts are cultured in a bioreactor (rotating cell culture system), a model that has been utilised to simulate microgravity. C2C12 murine myoblasts are cultured on microcarrier beads in a bioreactor and followed throughout differentiation as they form a network of multinucleated myotubes. In comparison with three-dimensional control cultures that consist of myoblasts cultured on microcarrier beads in teflon bags, myoblasts cultured in the bioreactor exhibit an attenuation in differentiation. This is demonstrated by reduced immunohistochemical staining for myogenin and alpha-actinin. Western analysis shows a decrease, in bioreactor cultures compared with control cultures, in levels of the contractile proteins myosin (47% decrease, p < 0.01) and tropomyosin (63% decrease, p < 0.01). Hydrodynamic measurements indicate that the decrease in differentiation may be due, at least in part, to fluid stresses acting on the myotubes. In addition, constraints on aggregate size imposed by the action of fluid forces in the bioreactor affect differentiation. These results may have implications for muscle growth and repair during spaceflight.

  15. Preventive Effects of Poloxamer 188 on Muscle Cell Damage Mechanics Under Oxidative Stress.

    PubMed

    Wong, Sing Wan; Yao, Yifei; Hong, Ye; Ma, Zhiyao; Kok, Stanton H L; Sun, Shan; Cho, Michael; Lee, Kenneth K H; Mak, Arthur F T

    2017-04-01

    High oxidative stress can occur during ischemic reperfusion and chronic inflammation. It has been hypothesized that such oxidative challenges could contribute to clinical risks such as deep tissue pressure ulcers. Skeletal muscles can be challenged by inflammation-induced or reperfusion-induced oxidative stress. Oxidative stress reportedly can lower the compressive damage threshold of skeletal muscles cells, causing actin filament depolymerization, and reduce membrane sealing ability. Skeletal muscles thus become easier to be damaged by mechanical loading under prolonged oxidative exposure. In this study, we investigated the preventive effect of poloxamer 188 (P188) on skeletal muscle cells against extrinsic oxidative challenges (H 2 O 2 ). It was found that with 1 mM P188 pre-treatment for 1 h, skeletal muscle cells could maintain their compressive damage threshold. The actin polymerization dynamics largely remained stable in term of the expression of cofilin, thymosin beta 4 and profilin. Laser photoporation demonstrated that membrane sealing ability was preserved even as the cells were challenged by H 2 O 2 . These findings suggest that P188 pre-treatment can help skeletal muscle cells retain their normal mechanical integrity in oxidative environments, adding a potential clinical use of P188 against the combined challenge of mechanical-oxidative stresses. Such effect may help to prevent deep tissue ulcer development.

  16. Dietary tributyrin, an HDAC inhibitor, promotes muscle growth through enhanced terminal differentiation of satellite cells.

    PubMed

    Murray, Robert L; Zhang, Wei; Iwaniuk, Marie; Grilli, Ester; Stahl, Chad H

    2018-05-01

    Muscle growth and repair rely on two main mechanisms - myonuclear accretion and subsequent protein accumulation. Altering the ability of muscle resident stem cells (satellite cells) to progress through their myogenic lineage can have a profound effect on lifetime muscle growth and repair. The use of the histone deacetylase (HDAC) inhibitor, butyrate, has had positive outcomes on the in vitro promotion of satellite cell myogenesis. In animal models, the use of butyrate has had promising results in treating myopathic conditions as well as improving growth efficiency, but the impact of dietary butyrate on satellite cells and muscle growth has not been elucidated. We investigated the impact of tributyrin, a butyrate prodrug, on satellite cell activity and muscle growth in a piglet model. Satellite cells from tributyrin-treated piglets had altered myogenic potential, and piglets receiving tributyrin had a ~40% increase in DNA:protein ratio after 21 days, indicating the potential for enhanced muscle growth. To assess muscle growth potential, piglets were supplemented tributyrin (0.5%) during either the neonatal phase (d1-d21) and/or the nursery phase (d21-d58) in a 2 × 2 factorial design. Piglets who received tributyrin during the neonatal phase had improved growth performance at the end of the study and had a ~10% larger loin eye area and muscle fiber cross-sectional area. Tributyrin treatment in the nursery phase alone did not have a significant effect on muscle growth or feed efficiency. These findings suggest that tributyrin is a potent promoter of muscle growth via altered satellite cell myogenesis. © 2018 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.

  17. Recombination within the apospory specific genomic region leads to the uncoupling of apomixis components in Cenchrus ciliaris.

    PubMed

    Conner, Joann A; Gunawan, Gunawati; Ozias-Akins, Peggy

    2013-07-01

    Apomixis enables the clonal propagation of maternal genotypes through seed. If apomixis could be harnessed via genetic engineering or introgression, it would have a major economic impact for agricultural crops. In the grass species Pennisetum squamulatum and Cenchrus ciliaris (syn. P. ciliare), apomixis is controlled by a single dominant "locus", the apospory-specific genomic region (ASGR). For P. squamulatum, 18 published sequenced characterized amplified region (SCAR) markers have been identified which always co-segregate with apospory. Six of these markers are conserved SCARs in the closely related species, C. ciliaris and co-segregate with the trait. A screen of progeny from a cross of sexual × apomictic C. ciliaris genotypes identified a plant, A8, retaining two of the six ASGR-linked SCAR markers. Additional and newly identified ASGR-linked markers were generated to help identify the extent of recombination within the ASGR. Based on analysis of missing markers, the A8 recombinant plant has lost a significant portion of the ASGR but continues to form aposporous embryo sacs. Seedlings produced from aposporous embryo sacs are 6× in ploidy level and hence the A8 recombinant does not express parthenogenesis. The recombinant A8 plant represents a step forward in reducing the complexity of the ASGR locus to determine the factor(s) required for aposporous embryo sac formation and documents the separation of expression of the two components of apomixis in C. ciliaris.

  18. Identification of Skeletal Muscle Satellite Cells by Immunofluorescence with Pax7 and Laminin Antibodies.

    PubMed

    Feng, Xuesong; Naz, Faiza; Juan, Aster H; Dell'Orso, Stefania; Sartorelli, Vittorio

    2018-04-19

    Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, antibodies for specific cell markers are applied on tissue sections. In adult skeletal muscle, satellite cells (SCs) are stem cells that contribute to muscle repair and regeneration. Therefore, it is important to visualize and trace the satellite cell population under different physiological conditions. In resting skeletal muscle, SCs reside between the basal lamina and myofiber plasma membrane. A commonly used marker for identifying SCs on the myofibers or in cell culture is the paired box protein Pax7. In this article, an optimized Pax7 immunofluorescence protocol on skeletal muscle sections is presented that minimizes non-specific staining and background. Another antibody that recognizes a protein (laminin) of the basal lamina was also added to help identify SCs. Similar protocols can also be used to perform double or triple labeling with Pax7 and antibodies for additional proteins of interest.

  19. Functional short- and long-term effects of nasal CPAP with and without humidification on the ciliary function of the nasal respiratory epithelium.

    PubMed

    Sommer, J Ulrich; Kraus, Marius; Birk, Richard; Schultz, Johannes D; Hörmann, Karl; Stuck, Boris A

    2014-03-01

    Continuous positive airway pressure (CPAP) is the gold standard in the treatment of obstructive sleep apnea (OSA), but its impact on ciliary function is unclear to date. Furthermore, CPAP is associated with numerous side effects related to the nose and upper airway. Humidified CPAP is used to relieve these symptoms, but again, little is known regarding its effect on ciliary function of the nasal respiratory epithelium. In this prospective, randomized, crossover trial, 31 patients with OSA (AHI >15/h) were randomized to two treatment arms: nasal continuous positive airway pressure (nCPAP) with humidification or nCPAP without humidification for one night in each modality to assess short-term effects of ciliary beat frequency (CBF) and mucus transport time (MTT) and consecutively for 8 weeks in each modality to assess long-term effects in a crossover fashion. The baseline CBF was 4.8 ± 0.6 Hz, and baseline MTT was 540 ± 221 s. After one night of CPAP with and without humidification, ciliary function increased moderately yet with statistical significance (p <0.05). The short-term groups with and without humidification did not differ statistically significant. Regarding long-term effects of CPAP, a statistically significant increase in ciliary function above the baseline level and above the short-term level was shown without humidification (7.2 ± 0.4 Hz; 402 ± 176 s; p <0.01). The increase above baseline level was even more pronounced with humidification (9.3 ± 0.7 Hz; 313 ± 95 s; p <0.01). There was a statistically significant difference between both groups at long-term assessment with regard to CBF (p <0.01). Independent of airway humidification, nCPAP has moderate effects on short-term ciliary function of the nasal respiratory epithelium. However, a significant increase in ciliary function-both in terms of an increased CBF and a decreased MTT-was detected after long-term use. The effect was more pronounced when humidification was used during nCPAP.

  20. A Novel Method for Differentiation of Human Mesenchymal Stem Cells into Smooth Muscle-Like Cells on Clinically Deliverable Thermally Induced Phase Separation Microspheres

    PubMed Central

    Parmar, Nina; Ahmadi, Raheleh

    2015-01-01

    Muscle degeneration is a prevalent disease, particularly in aging societies where it has a huge impact on quality of life and incurs colossal health costs. Suitable donor sources of smooth muscle cells are limited and minimally invasive therapeutic approaches are sought that will augment muscle volume by delivering cells to damaged or degenerated areas of muscle. For the first time, we report the use of highly porous microcarriers produced using thermally induced phase separation (TIPS) to expand and differentiate adipose-derived mesenchymal stem cells (AdMSCs) into smooth muscle-like cells in a format that requires minimal manipulation before clinical delivery. AdMSCs readily attached to the surface of TIPS microcarriers and proliferated while maintained in suspension culture for 12 days. Switching the incubation medium to a differentiation medium containing 2 ng/mL transforming growth factor beta-1 resulted in a significant increase in both the mRNA and protein expression of cell contractile apparatus components caldesmon, calponin, and myosin heavy chains, indicative of a smooth muscle cell-like phenotype. Growth of smooth muscle cells on the surface of the microcarriers caused no change to the integrity of the polymer microspheres making them suitable for a cell-delivery vehicle. Our results indicate that TIPS microspheres provide an ideal substrate for the expansion and differentiation of AdMSCs into smooth muscle-like cells as well as a microcarrier delivery vehicle for the attached cells ready for therapeutic applications. PMID:25205072

  1. Comparative responses of the Savanna grasses Cenchrus ciliaris and Themeda triandra to defoliation.

    PubMed

    Hodgkinson, K C; Ludlow, M M; Mott, J J; Baruch, Z

    1989-04-01

    Two perennial tussock grasses of savannas were compared in a glasshouse study to determine why they differed in their ability to withstand frequent, heavy grazing; Cenchrus ciliaris is tolerant and Themeda triandra is intolerant of heavy grazing. Frequent defoliation at weekly intervals for six weeks reduced shoot biomass production over a subsequent 42 day regrowth period compared with previously undefoliated plants (infrequent) in T. triandra, but not in C. ciliaris. Leaf area of T. triandra expanded rapidly following defoliation but high initial relative growth rates of shoots were not sustained after 14 days of regrowth because of reducing light utilising efficiency of leaves. Frequently defoliated plants were slower in rate of leaf area expansion and this was associated with reduced photosynthetic capacity of newly formed leaves, lower allocation of photosynthate to leaves but not lower tiller numbers. T. triandra appears well adapted to a regime where defoliation is sufficiently infrequent to allow carbon to be fixed to replace that used in initial leaf area expansion. In contrast, C. ciliaris is better adapted to frequent defoliation than is T. triandra, because horizontally orientated nodal tillers are produced below the defoliation level. This morphological adaptation resulted in a 10-fold higher leaf area remaining after defoliation compared with similarly defoliated T. triandra, which together with the maintenance of moderate levels of light utilising efficiency, contributed to the higher leaf area and shoot weight throughout the regrowth period.

  2. Daikenchuto ameliorates muscle hypercontractility in a murine T-cell-mediated persistent gut motor dysfunction model.

    PubMed

    Akiho, Hirotada; Nakamura, Kazuhiko

    2011-01-01

    Low-grade inflammation and immunological alterations are evident in functional gastrointestinal disorders such as irritable bowel syndrome (IBS). We evaluated the effects of daikenchuto (DKT), a pharmaceutical grade Japanese herbal medicine, on the hypercontractility of intestinal smooth muscle persisting after acute inflammation induced by a T-cell-activating anti-CD3 antibody (αCD3). BALB/c mice were injected with αCD3 (12.5 μg, i.p.), and DKT (2.7 g/kg) was administered orally once daily for 1 week. The contraction of isolated small intestinal muscle strips and muscle cells was examined on day 7 after αCD3 injection. The gene and protein expressions in the small intestines were evaluated by real-time PCR and multiplex immunoassays, respectively, on days 1, 3 and 7 after αCD3 injection. αCD3 injection resulted in significant increases in carbachol-evoked contractility in the muscle strips and isolated smooth muscle cells on day 7. DKT ameliorated the αCD3-induced muscle hypercontractility on day 7 in both the muscle strips and smooth muscle cells. αCD3 injection rapidly up- and downregulated the mRNA and protein expressions of pro- and anti-inflammatory cytokines, respectively. Although the influence of DKT on the mRNA expressions was moderate, the protein expressions of IL-13 and IL-17 were significantly decreased. We observed changes in the intestinal muscle contractility in muscle strips and muscle cells following resolution of inflammation in a T-cell-mediated model of enteropathy. The observed modulation of cytokine expression and function by DKT may lead to the development of new pharmacotherapeutic strategies aimed at a wide variety of gut motor dysfunction disorders. Copyright © 2011 S. Karger AG, Basel.

  3. Major histocompatibility complex class II molecule expression on muscle cells is regulated by differentiation: implications for the immunopathogenesis of muscle autoimmune diseases.

    PubMed

    Mantegazza, R; Gebbia, M; Mora, M; Barresi, R; Bernasconi, P; Baggi, F; Cornelio, F

    1996-08-01

    Major histocompatibility complex (MHC) class II molecules are expressed on myoblasts after interferon-gamma (IFN-gamma) treatment, suggesting a muscle cell involvement in antigen presentation in inflammatory myopathies. However, they were not observed on normal or pathological myofibers. This discrepancy might be related to different responsiveness of developmentally differentiated muscle cells to IFN-gamma. Myoblasts expressed class II transcripts and proteins after IFN-gamma, while myotubes and innervated contracting muscle cells did not show staining for class II molecules. At all cell stages no loss of IFN-gamma receptor was detected indicating that myofiber maturation blocks their capacity to express MHC class II molecules. This suggests that completely differentiated myofibers cannot participate in class II restricted immunological reactions.

  4. Myogenic Maturation by Optical-Training in Cultured Skeletal Muscle Cells.

    PubMed

    Asano, Toshifumi; Ishizuka, Toru; Yawo, Hiromu

    2017-01-01

    Optogenetic techniques are powerful tools for manipulating biological processes in identified cells using light under high temporal and spatial resolutions. Here, we describe an optogenetic training strategy to promote morphological maturation and functional development of skeletal muscle cells in vitro. Optical stimulation with a rhythmical frequency facilitates specific structural alignment of sarcomeric proteins. Optical stimulation also depolarizes the membrane potential, and induces contractile responses in synchrony with the given pattern of light pulses. These results suggest that optogenetic techniques can be employed to manipulate activity-dependent processes during myogenic development and control contraction of photosensitive skeletal muscle cells with high temporal and special precision.

  5. Aging of the skeletal muscle extracellular matrix drives a stem cell fibrogenic conversion.

    PubMed

    Stearns-Reider, Kristen M; D'Amore, Antonio; Beezhold, Kevin; Rothrauff, Benjamin; Cavalli, Loredana; Wagner, William R; Vorp, David A; Tsamis, Alkiviadis; Shinde, Sunita; Zhang, Changqing; Barchowsky, Aaron; Rando, Thomas A; Tuan, Rocky S; Ambrosio, Fabrisia

    2017-06-01

    Age-related declines in skeletal muscle regeneration have been attributed to muscle stem cell (MuSC) dysfunction. Aged MuSCs display a fibrogenic conversion, leading to fibrosis and impaired recovery after injury. Although studies have demonstrated the influence of in vitro substrate characteristics on stem cell fate, whether and how aging of the extracellular matrix (ECM) affects stem cell behavior has not been investigated. Here, we investigated the direct effect of the aged muscle ECM on MuSC lineage specification. Quantification of ECM topology and muscle mechanical properties reveals decreased collagen tortuosity and muscle stiffening with increasing age. Age-related ECM alterations directly disrupt MuSC responses, and MuSCs seeded ex vivo onto decellularized ECM constructs derived from aged muscle display increased expression of fibrogenic markers and decreased myogenicity, compared to MuSCs seeded onto young ECM. This fibrogenic conversion is recapitulated in vitro when MuSCs are seeded directly onto matrices elaborated by aged fibroblasts. When compared to young fibroblasts, fibroblasts isolated from aged muscle display increased nuclear levels of the mechanosensors, Yes-associated protein (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ), consistent with exposure to a stiff microenvironment in vivo. Accordingly, preconditioning of young fibroblasts by seeding them onto a substrate engineered to mimic the stiffness of aged muscle increases YAP/TAZ nuclear translocation and promotes secretion of a matrix that favors MuSC fibrogenesis. The findings here suggest that an age-related increase in muscle stiffness drives YAP/TAZ-mediated pathogenic expression of matricellular proteins by fibroblasts, ultimately disrupting MuSC fate. © 2017 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

  6. The Satellite Cell in Male and Female, Developing and Adult Mouse Muscle: Distinct Stem Cells for Growth and Regeneration

    PubMed Central

    Neal, Alice; Boldrin, Luisa; Morgan, Jennifer Elizabeth

    2012-01-01

    Satellite cells are myogenic cells found between the basal lamina and the sarcolemma of the muscle fibre. Satellite cells are the source of new myofibres; as such, satellite cell transplantation holds promise as a treatment for muscular dystrophies. We have investigated age and sex differences between mouse satellite cells in vitro and assessed the importance of these factors as mediators of donor cell engraftment in an in vivo model of satellite cell transplantation. We found that satellite cell numbers are increased in growing compared to adult and in male compared to female adult mice. We saw no difference in the expression of the myogenic regulatory factors between male and female mice, but distinct profiles were observed according to developmental stage. We show that, in contrast to adult mice, the majority of satellite cells from two week old mice are proliferating to facilitate myofibre growth; however a small proportion of these cells are quiescent and not contributing to this growth programme. Despite observed changes in satellite cell populations, there is no difference in engraftment efficiency either between satellite cells derived from adult or pre-weaned donor mice, male or female donor cells, or between male and female host muscle environments. We suggest there exist two distinct satellite cell populations: one for muscle growth and maintenance and one for muscle regeneration. PMID:22662253

  7. * Tissue-Specific Extracellular Matrix Enhances Skeletal Muscle Precursor Cell Expansion and Differentiation for Potential Application in Cell Therapy.

    PubMed

    Zhang, Deying; Zhang, Yong; Zhang, Yuanyuan; Yi, Hualin; Wang, Zhan; Wu, Rongpei; He, Dawei; Wei, Guanghui; Wei, Shicheng; Hu, Yun; Deng, Junhong; Criswell, Tracy; Yoo, James; Zhou, Yu; Atala, Anthony

    2017-08-01

    Skeletal muscle precursor cells (MPCs) are considered a key candidate for cell therapy in the treatment of skeletal muscle dysfunction due to injury, disease, or age. However, expansion of a sufficient number of functional skeletal muscle cells in vitro from a small tissue biopsy has been challenging due to changes in phenotypic expression of these cells under traditional culture conditions. Thus, the aim of the study was to develop a better culture system for the expansion and myo-differentiation of MPCs that could further be used for therapy. For this purpose, we developed an ideal method of tissue decellularization and compared the ability of different matrices to support MPC growth and differentiation. Porcine-derived skeletal muscle and liver and kidney extracellular matrix (ECM) were generated by decellularization methods consisting of distilled water, 0.2 mg/mL DNase, or 5% fetal bovine serum. Acellular matrices were further homogenized, dissolved, and combined with a hyaluronic acid-based hydrogel decorated with heparin (ECM-HA-HP). The cell proliferation and myogenic differentiation capacity of human MPCs were assessed when grown on gel alone, ECM, or each ECM-HA-HP substrate. Human MPC proliferation was significantly enhanced when cultured on the ECM-HA-HP substrates compared to the other substrates tested, with the greatest proliferation on the muscle ECM-HA-HP (mECM-HA-HP) substrate. The number of differentiated myotubes was significantly increased on the mECM-HA-HP substrate compared to the other gel-ECM substrates, as well as the numbers of MPCs expressing specific myogenic cell markers (i.e., myosin, desmin, myoD, and myf5). In conclusion, skeletal mECM-HA-HP as a culture substrate provided an optimal culture microenvironment potentially due to its similarity to the in vivo environment. These data suggest a potential use of skeletal muscle-derived ECM gel for the expansion and differentiation of human MPCs for cell-based therapy for skeletal muscle

  8. beta. -adrenergic relaxation of smooth muscle: differences between cells and tissues

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

    Scheid, C.R.

    1987-09-01

    The present studies were carried out in an attempt to resolve the controversy about the Na/sup +/ dependence of ..beta..-adrenergic relaxation in smooth muscle. Previous studies on isolated smooth muscle cells from the toad stomach had suggested that at least some of the actions of ..beta..-adrenergic agents, including a stimulatory effect on /sup 45/Ca efflux, were dependent on the presence of a normal transmembrane Na/sup +/ gradient. Studies by other investigators using tissues derived from mammalian sources had suggested that the relaxing effect of ..beta..-adrenergic agents was Na/sup +/ independent. Uncertainty remained as to whether these discrepancies reflected differences betweenmore » cells and tissues or differences between species. Thus, in the present studies, the authors utilized both tissues and cells from the same source, the stomach muscle of the toad Bufo marinus, and assessed the Na/sup +/ dependence of ..beta..-adrenergic relaxation. They found that elimination of a normal Na/sup +/ gradient abolished ..beta..-adrenergic relaxation of isolated cells. In tissues, however, similar manipulations had no effect on relaxation. The reasons for this discrepancy are unclear but do not appear to be attributable to changes in smooth muscle function following enzymatic dispersion. Thus the controversy concerning the mechanisms of ..beta..-adrenergic relaxation may reflect inherent differences between tissues and cells.« less

  9. Two different roles of purified CD45+c-Kit+Sca-1+Lin- cells after transplantation in muscles.

    PubMed

    Yoshimoto, Momoko; Chang, Hsi; Shiota, Mitsutaka; Kobayashi, Hirohiko; Umeda, Katsutsugu; Kawakami, Atsushi; Heike, Toshio; Nakahata, Tatsutoshi

    2005-05-01

    Recent studies have indicated that bone marrow cells can regenerate damaged muscles and that they can adopt phenotypes of other cells by cell fusion. Our direct visualization system gave evidence of massive muscle regeneration by green fluorescent protein (GFP)-labeled CD45+c-Kit+Sca-1+Lin- cells (KSL cells), and we investigated the role of KSL cells in muscle regeneration after transplantation with or without lethal irradiation. In the early phase, GFP signals were clearly observed in all the muscles of only irradiated mice. Transverse cryostat sections showed GFP+myosin+ muscle fibers, along with numerous GFP+ hematopoietic cells in damaged muscle. These phenomena were temporary, and GFP signals had dramatically reduced 30 days after transplantation. After 6 months, GFP+ fibers could hardly be detected, but GFP+c-Met+ mononuclear cells were located beneath the basal lamina where satellite cells usually exist in both conditioned mice. Immunostaining of isolated single fibers revealed GFP+PAX7+, GFP+MyoD+, and GFP+Myf5+ satellite-like cells on the fibers. Single-fiber cultures from these mice showed proliferation of GFP+ fibers. These results indicate two different roles of KSL cells: one leading to regeneration of damaged muscles in the early phase and the other to conversion into satellite cells in the late phase.

  10. Smooth muscle architecture within cell-dense vascular tissues influences functional contractility.

    PubMed

    Win, Zaw; Vrla, Geoffrey D; Steucke, Kerianne E; Sevcik, Emily N; Hald, Eric S; Alford, Patrick W

    2014-12-01

    The role of vascular smooth muscle architecture in the function of healthy and dysfunctional vessels is poorly understood. We aimed at determining the relationship between vascular smooth muscle architecture and contractile output using engineered vascular tissues. We utilized microcontact printing and a microfluidic cell seeding technique to provide three different initial seeding conditions, with the aim of influencing the cellular architecture within the tissue. Cells seeded in each condition formed confluent and aligned tissues but within the tissues, the cellular architecture varied. Tissues with a more elongated cellular architecture had significantly elevated basal stress and produced more contractile stress in response to endothelin-1 stimulation. We also found a correlation between the contractile phenotype marker expression and the cellular architecture, contrary to our previous findings in non-confluent tissues. Taken with previous results, these data suggest that within cell-dense vascular tissues, smooth muscle contractility is strongly influenced by cell and tissue architectures.

  11. Identification of myogenic-endothelial progenitor cells in the interstitial spaces of skeletal muscle.

    PubMed

    Tamaki, Tetsuro; Akatsuka, Akira; Ando, Kiyoshi; Nakamura, Yoshihiko; Matsuzawa, Hideyuki; Hotta, Tomomitsu; Roy, Roland R; Edgerton, V Reggie

    2002-05-13

    Putative myogenic and endothelial (myo-endothelial) cell progenitors were identified in the interstitial spaces of murine skeletal muscle by immunohistochemistry and immunoelectron microscopy using CD34 antigen. Enzymatically isolated cells were characterized by fluorescence-activated cell sorting on the basis of cell surface antigen expression, and were sorted as a CD34+ and CD45- fraction. Cells in this fraction were approximately 94% positive for Sca-1, and mostly negative (<3% positive) for CD14, 31, 49, 144, c-kit, and FLK-1. The CD34+/45- cells formed colonies in clonal cell cultures and colony-forming units displayed the potential to differentiate into adipocytes, endothelial, and myogenic cells. The CD34+/45- cells fully differentiated into vascular endothelial cells and skeletal muscle fibers in vivo after transplantation. Immediately after sorting, CD34+/45- cells expressed only c-met mRNA, and did not express any other myogenic cell-related markers such as MyoD, myf-5, myf-6, myogenin, M-cadherin, Pax-3, and Pax-7. However, after 3 d of culture, these cells expressed mRNA for all myogenic markers. CD34+/45- cells were distinct from satellite cells, as they expressed Bcrp1/ABCG2 gene mRNA (Zhou et al., 2001). These findings suggest that myo-endothelial progenitors reside in the interstitial spaces of mammalian skeletal muscles, and that they can potentially contribute to postnatal skeletal muscle growth.

  12. Differentiation of Human Adipose Derived Stem Cells into Smooth Muscle Cells Is Modulated by CaMKIIγ

    PubMed Central

    Aji, Kaisaier; Maimaijiang, Munila; Aimaiti, Abudusaimi; Rexiati, Mulati; Azhati, Baihetiya; Tusong, Hamulati

    2016-01-01

    The multifunctional Ca2+/calmodulin-dependent protein kinase II (CaMKII) is known to participate in maintenance and switches of smooth muscle cell (SMC) phenotypes. However, which isoform of CaMKII is involved in differentiation of adult mesenchymal stem cells into contractile SMCs remains unclear. In the present study, we detected γ isoform of CaMKII in differentiation of human adipose derived stem cells (hASCs) into SMCs that resulted from treatment with TGF-β1 and BMP4 in combination for 7 days. The results showed that CaMKIIγ increased gradually during differentiation of hASCs as determined by real-time PCR and western blot analysis. The siRNA-mediated knockdown of CaMKIIγ decreased the protein levels and transcriptional levels of smooth muscle contractile markers (a-SMA, SM22a, calponin, and SM-MHC), while CaMKIIγ overexpression increases the transcriptional and protein levels of smooth muscle contractile markers. These results suggested that γ isoform of CaMKII plays a significant role in smooth muscle differentiation of hASCs. PMID:27493668

  13. Intraocular gene transfer of ciliary neurotrophic factor rescues photoreceptor degeneration in RCS rats.

    PubMed

    Huang, Shun-Ping; Lin, Po-Kang; Liu, Jorn-Hon; Khor, Chin-Ni; Lee, Yih-Jing

    2004-01-01

    Ciliary neurotrophic factor (CNTF) is known as an important factor in the regulation of retinal cell growth. We used both recombinant CNTF and an adenovirus carrying the CNTF gene to regulate retinal photoreceptor expression in a retinal degenerative animal, Royal College of Surgeons (RCS) rats. Cells in the outer nuclear layer of the retinae from recombinant-CNTF-treated, adenoviral-CNTF-treated, saline-operated, and contralateral untreated preparations were examined for those exhibiting CNTF photoreceptor protective effects. Cell apoptosis in the outer nuclear layer of the retinae was also detected. It was found that CNTF had a potent effect on delaying the photoreceptor degeneration process in RCS rats. Furthermore, adenovirus CNTF gene transfer was proven to be better at rescuing photoreceptors than that when using recombinant CNTF, since adenoviral CNTF prolonged the photoreceptor protection effect. The function of the photoreceptors was also examined by taking electroretinograms of different animals. Adenoviral-CNTF-treated eyes showed better retinal function than did the contralateral control eyes. This study indicates that adenoviral CNTF effectively rescues degenerating photoreceptors in RCS rats. Copyright 2004 National Science Council, ROC and S. Karger AG, Basel

  14. A 310-bp minimal promoter mediates smooth muscle cell-specific expression of telokin.

    PubMed

    Smith, A F; Bigsby, R M; Word, R A; Herring, B P

    1998-05-01

    A cell-specific promoter located in an intron of the smooth muscle myosin light chain kinase gene directs transcription of telokin exclusively in smooth muscle cells. Transgenic mice were generated in which a 310-bp rabbit telokin promoter fragment, extending from -163 to +147, was used to drive expression of simian virus 40 large T antigen. Smooth muscle-specific expression of the T-antigen transgene paralleled that of the endogenous telokin gene in all smooth muscle tissues except uterus. The 310-bp promoter fragment resulted in very low levels of transgene expression in uterus; in contrast, a transgene driven by a 2.4-kb fragment (-2250 to +147) resulted in high levels of transgene expression in uterine smooth muscle. Telokin expression levels correlate with the estrogen status of human myometrial tissues, suggesting that deletion of an estrogen response element (ERE) may account for the low levels of transgene expression driven by the 310-bp rabbit telokin promoter in uterine smooth muscle. Experiments in A10 smooth muscle cells directly showed that reporter gene expression driven by the 2.4-kb, but not 310-bp, promoter fragment could be stimulated two- to threefold by estrogen. This stimulation was mediated through an ERE located between -1447 and -1474. Addition of the ERE to the 310-bp fragment restored estrogen responsiveness in A10 cells. These data demonstrate that in addition to a minimal 310-bp proximal promoter at least one distal cis-acting regulatory element is required for telokin expression in uterine smooth muscle. The distal element may include an ERE between -1447 and -1474.

  15. Proteolysis inhibition by hibernating bear serum leads to increased protein content in human muscle cells.

    PubMed

    Chanon, Stéphanie; Chazarin, Blandine; Toubhans, Benoit; Durand, Christine; Chery, Isabelle; Robert, Maud; Vieille-Marchiset, Aurélie; Swenson, Jon E; Zedrosser, Andreas; Evans, Alina L; Brunberg, Sven; Arnemo, Jon M; Gauquelin-Koch, Guillemette; Storey, Kenneth B; Simon, Chantal; Blanc, Stéphane; Bertile, Fabrice; Lefai, Etienne

    2018-04-03

    Muscle atrophy is one of the main characteristics of human ageing and physical inactivity, with resulting adverse health outcomes. To date, there are still no efficient therapeutic strategies for its prevention and/or treatment. However, during hibernation, bears exhibit a unique ability for preserving muscle in conditions where muscle atrophy would be expected in humans. Therefore, our objective was to determine whether there are components of bear serum which can control protein balance in human muscles. In this study, we exposed cultured human differentiated muscle cells to bear serum collected during winter and summer periods, and measured the impact on cell protein content and turnover. In addition, we explored the signalling pathways that control rates of protein synthesis and degradation. We show that the protein turnover of human myotubes is reduced when incubated with winter bear serum, with a dramatic inhibition of proteolysis involving both proteasomal and lysosomal systems, and resulting in an increase in muscle cell protein content. By modulating intracellular signalling pathways and inducing a protein sparing phenotype in human muscle cells, winter bear serum therefore holds potential for developing new tools to fight human muscle atrophy and related metabolic disorders.

  16. Slow-Adhering Stem Cells Derived from Injured Skeletal Muscle Have Improved Regenerative Capacity

    DTIC Science & Technology

    2011-08-01

    images. Flow Cytometry Assay of Stem Cell Markers SASCs (1 105) isolated from noninjured or injured muscle were collected and washed twice with...muscle. Results of flow cytometry further verified Sca-1 and CD34 expression in isolated SASCs, and a greater percentage of cells were positive for Sca-1...from both injured and control noninjured muscle were analyzed using flow cytometry for the immunofluorescent signal of Sca-1 and CD34. Results

  17. AICAR inhibits oxygen consumption by intact skeletal muscle cells in culture.

    PubMed

    Spangenburg, Espen E; Jackson, Kathryn C; Schuh, Rosemary A

    2013-12-01

    Activation of 5' adenosine monophosphate-activated protein kinase (AMPK) with aminoimidazole carboxamide ribonucleotide (AICAR) increases skeletal muscle glucose uptake and fatty acid oxidation. The purpose of these experiments was to utilize AICAR to enhance palmitate consumption by mitochondria in cultured skeletal muscle cells. In these experiments, we treated C2C12 myotubes or adult single skeletal muscle fibers with varying concentrations of AICAR for different lengths of time. Surprisingly, acute AICAR exposure at most concentrations (0.25-1.5 mM), but not all (0.1 mM), modestly inhibited oxygen consumption even though AICAR increased AMPK phosphorylation. The data suggest that AICAR inhibited oxygen consumption by the cultured muscle in a non-specific manner. The results of these experiments are expected to provide valuable information to investigators interested in using AICAR in cell culture studies.

  18. Myogenic Progenitor Cells Control Extracellular Matrix Production by Fibroblasts during Skeletal Muscle Hypertrophy.

    PubMed

    Fry, Christopher S; Kirby, Tyler J; Kosmac, Kate; McCarthy, John J; Peterson, Charlotte A

    2017-01-05

    Satellite cells, the predominant stem cell population in adult skeletal muscle, are activated in response to hypertrophic stimuli and give rise to myogenic progenitor cells (MPCs) within the extracellular matrix (ECM) that surrounds myofibers. This ECM is composed largely of collagens secreted by interstitial fibrogenic cells, which influence satellite cell activity and muscle repair during hypertrophy and aging. Here we show that MPCs interact with interstitial fibrogenic cells to ensure proper ECM deposition and optimal muscle remodeling in response to hypertrophic stimuli. MPC-dependent ECM remodeling during the first week of a growth stimulus is sufficient to ensure long-term myofiber hypertrophy. MPCs secrete exosomes containing miR-206, which represses Rrbp1, a master regulator of collagen biosynthesis, in fibrogenic cells to prevent excessive ECM deposition. These findings provide insights into how skeletal stem and progenitor cells interact with other cell types to actively regulate their extracellular environments for tissue maintenance and adaptation. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Induction of muscle stem cell quiescence by the secreted niche factor Oncostatin M.

    PubMed

    Sampath, Srinath C; Sampath, Srihari C; Ho, Andrew T V; Corbel, Stéphane Y; Millstone, Joshua D; Lamb, John; Walker, John; Kinzel, Bernd; Schmedt, Christian; Blau, Helen M

    2018-04-18

    The balance between stem cell quiescence and proliferation in skeletal muscle is tightly controlled, but perturbed in a variety of disease states. Despite progress in identifying activators of stem cell proliferation, the niche factor(s) responsible for quiescence induction remain unclear. Here we report an in vivo imaging-based screen which identifies Oncostatin M (OSM), a member of the interleukin-6 family of cytokines, as a potent inducer of muscle stem cell (MuSC, satellite cell) quiescence. OSM is produced by muscle fibers, induces reversible MuSC cell cycle exit, and maintains stem cell regenerative capacity as judged by serial transplantation. Conditional OSM receptor deletion in satellite cells leads to stem cell depletion and impaired regeneration following injury. These results identify Oncostatin M as a secreted niche factor responsible for quiescence induction, and for the first time establish a direct connection between induction of quiescence, stemness, and transplantation potential in solid organ stem cells.

  20. Aggregate Mesenchymal Stem Cell Delivery Ameliorates the Regenerative Niche for Muscle Repair.

    PubMed

    Ruehle, Marissa A; Stevens, Hazel Y; Beedle, Aaron M; Guldberg, Robert E; Call, Jarrod A

    2018-05-18

    Duchenne muscular dystrophy (DMD) is a severe muscle wasting disease due to the absence of the dystrophin protein from the muscle cell membrane which renders the muscle susceptible to continuous damage. In DMD patients, muscle weakness, together with cycles of degeneration/regeneration and replacement with non-contractile tissue, limit mobility and lifespan. Since the loss of dystrophin result in loss of polarity and a reduction in the number of self-renewing satellite cells, it is postulated that these patients could achieve an improved quality of life if delivered cells could restore satellite cell function. In this study we used both an established myotoxic injury model in wildtype (WT) mice and mdx mice alone (spontaneous muscle damage). Single (SC) and aggregated (AGG) mesenchymal stem cells (MSCs) were injected into the gastrocnemius muscles 4 hours after injury (WT mice). The recovery of peak isometric torque was longitudinally assessed over 5 weeks, with earlier takedowns for histological assessment of healing (fiber cross section area and central nucleation) and MSC retention. AGG-treated WT mice had significantly greater torque recovery at day 14 than SC or saline-treated mice and a greater CSA at day 10, compared to SC/saline. AGG-treated mdx mice had a greater peak isometric torque compared to SC/saline. In vitro immunomodulatory factor secretion of AGG-MSCs was higher than SC-MSCs for all tested growth factors with the largest difference observed in hepatocyte growth factor (HGF). Future studies are necessary to pair immunomodulatory factor secretion with functional attributes, to better predict the potential therapeutic value of MSC treatment modalities. This article is protected by copyright. All rights reserved.

  1. Bladder Smooth Muscle Cells Differentiation from Dental Pulp Stem Cells: Future Potential for Bladder Tissue Engineering

    PubMed Central

    Song, Bing; Jiang, Wenkai; Alraies, Amr; Liu, Qian; Gudla, Vijay; Oni, Julia; Wei, Xiaoqing; Sloan, Alastair; Ni, Longxing; Agarwal, Meena

    2016-01-01

    Dental pulp stem cells (DPSCs) are multipotent cells capable of differentiating into multiple cell lines, thus providing an alternative source of cell for tissue engineering. Smooth muscle cell (SMC) regeneration is a crucial step in tissue engineering of the urinary bladder. It is known that DPSCs have the potential to differentiate into a smooth muscle phenotype in vitro with differentiation agents. However, most of these studies are focused on the vascular SMCs. The optimal approaches to induce human DPSCs to differentiate into bladder SMCs are still under investigation. We demonstrate in this study the ability of human DPSCs to differentiate into bladder SMCs in a growth environment containing bladder SMCs-conditioned medium with the addition of the transforming growth factor beta 1 (TGF-β1). After 14 days of exposure to this medium, the gene and protein expression of SMC-specific marker (α-SMA, desmin, and calponin) increased over time. In particular, myosin was present in differentiated cells after 11 days of induction, which indicated that the cells differentiated into the mature SMCs. These data suggested that human DPSCs could be used as an alternative and less invasive source of stem cells for smooth muscle regeneration, a technology that has applications for bladder tissue engineering. PMID:26880982

  2. Effect of ionizing radiation on human skeletal muscle precursor cells

    PubMed Central

    Jurdana, Mihaela; Cemazar, Maja; Pegan, Katarina; Mars, Tomaz

    2013-01-01

    Background Long term effects of different doses of ionizing radiation on human skeletal muscle myoblast proliferation, cytokine signalling and stress response capacity were studied in primary cell cultures. Materials and methods Human skeletal muscle myoblasts obtained from muscle biopsies were cultured and irradiated with a Darpac 2000 X-ray unit at doses of 4, 6 and 8 Gy. Acute effects of radiation were studied by interleukin – 6 (IL-6) release and stress response detected by the heat shock protein (HSP) level, while long term effects were followed by proliferation capacity and cell death. Results Compared with non-irradiated control and cells treated with inhibitor of cell proliferation Ara C, myoblast proliferation decreased 72 h post-irradiation, this effect was more pronounced with increasing doses. Post-irradiation myoblast survival determined by measurement of released LDH enzyme activity revealed increased activity after exposure to irradiation. The acute response of myoblasts to lower doses of irradiation (4 and 6 Gy) was decreased secretion of constitutive IL-6. Higher doses of irradiation triggered a stress response in myoblasts, determined by increased levels of stress markers (HSPs 27 and 70). Conclusions Our results show that myoblasts are sensitive to irradiation in terms of their proliferation capacity and capacity to secret IL-6. Since myoblast proliferation and differentiation are a key stage in muscle regeneration, this effect of irradiation needs to be taken in account, particularly in certain clinical conditions. PMID:24294183

  3. Regulation of myogenesis and skeletal muscle regeneration: effects of oxygen levels on satellite cell activity.

    PubMed

    Chaillou, Thomas; Lanner, Johanna T

    2016-12-01

    Reduced oxygen (O 2 ) levels (hypoxia) are present during embryogenesis and exposure to altitude and in pathologic conditions. During embryogenesis, myogenic progenitor cells reside in a hypoxic microenvironment, which may regulate their activity. Satellite cells are myogenic progenitor cells localized in a local environment, suggesting that the O 2 level could affect their activity during muscle regeneration. In this review, we present the idea that O 2 levels regulate myogenesis and muscle regeneration, we elucidate the molecular mechanisms underlying myogenesis and muscle regeneration in hypoxia and depict therapeutic strategies using changes in O 2 levels to promote muscle regeneration. Severe hypoxia (≤1% O 2 ) appears detrimental for myogenic differentiation in vitro, whereas a 3-6% O 2 level could promote myogenesis. Hypoxia impairs the regenerative capacity of injured muscles. Although it remains to be explored, hypoxia may contribute to the muscle damage observed in patients with pathologies associated with hypoxia (chronic obstructive pulmonary disease, and peripheral arterial disease). Hypoxia affects satellite cell activity and myogenesis through mechanisms dependent and independent of hypoxia-inducible factor-1α. Finally, hyperbaric oxygen therapy and transplantation of hypoxia-conditioned myoblasts are beneficial procedures to enhance muscle regeneration in animals. These therapies may be clinically relevant to treatment of patients with severe muscle damage.-Chaillou, T. Lanner, J. T. Regulation of myogenesis and skeletal muscle regeneration: effects of oxygen levels on satellite cell activity. © FASEB.

  4. Doublecortin marks a new population of transiently amplifying muscle progenitor cells and is required for myofiber maturation during skeletal muscle regeneration.

    PubMed

    Ogawa, Ryo; Ma, Yuran; Yamaguchi, Masahiko; Ito, Takahito; Watanabe, Yoko; Ohtani, Takuji; Murakami, Satoshi; Uchida, Shizuka; De Gaspari, Piera; Uezumi, Akiyoshi; Nakamura, Miki; Miyagoe-Suzuki, Yuko; Tsujikawa, Kazutake; Hashimoto, Naohiro; Braun, Thomas; Tanaka, Teruyuki; Takeda, Shin'ichi; Yamamoto, Hiroshi; Fukada, So-Ichiro

    2015-01-01

    Muscle satellite cells are indispensable for muscle regeneration, but the functional diversity of their daughter cells is unknown. Here, we show that many Pax7(+)MyoD(-) cells locate both beneath and outside the basal lamina during myofiber maturation. A large majority of these Pax7(+)MyoD(-) cells are not self-renewed satellite cells, but have different potentials for both proliferation and differentiation from Pax7(+)MyoD(+) myoblasts (classical daughter cells), and are specifically marked by expression of the doublecortin (Dcx) gene. Transplantation and lineage-tracing experiments demonstrated that Dcx-expressing cells originate from quiescent satellite cells and that the microenvironment induces Dcx in myoblasts. Expression of Dcx seems to be necessary for myofiber maturation because Dcx-deficient mice exhibited impaired myofiber maturation resulting from a decrease in the number of myonuclei. Furthermore, in vitro and in vivo studies suggest that one function of Dcx in myogenic cells is acceleration of cell motility. These results indicate that Dcx is a new marker for the Pax7(+)MyoD(-) subpopulation, which contributes to myofiber maturation during muscle regeneration. © 2015. Published by The Company of Biologists Ltd.

  5. Comparative Physical Mapping of the Apospory-Specific Genomic Region in Two Apomictic Grasses: Pennisetum squamulatum and Cenchrus ciliaris

    PubMed Central

    Goel, Shailendra; Chen, Zhenbang; Akiyama, Yukio; Conner, Joann A.; Basu, Manojit; Gualtieri, Gustavo; Hanna, Wayne W.; Ozias-Akins, Peggy

    2006-01-01

    In gametophytic apomicts of the aposporous type, each cell of the embryo sac is genetically identical to somatic cells of the ovule because they are products of mitosis, not of meiosis. The egg of the aposporous embryo sac follows parthenogenetic development into an embryo; therefore, uniform progeny result even from heterozygous plants, a trait that would be valuable for many crop species. Attempts to introgress apomixis from wild relatives into major crops through traditional breeding have been hindered by low or no recombination within the chromosomal region governing this trait (the apospory-specific genomic region or ASGR). The lack of recombination also has been a major obstacle to positional cloning of key genes. To further delineate and characterize the nonrecombinant ASGR, we have identified eight new ASGR-linked, AFLP-based molecular markers, only one of which showed recombination with the trait for aposporous embryo sac development. Bacterial artificial chromosome (BAC) clones identified with the ASGR-linked AFLPs or previously mapped markers, when mapped by fluorescence in situ hybridization in Pennisetum squamulatum and Cenchrus ciliaris, showed almost complete macrosynteny between the two apomictic grasses throughout the ASGR, although with an inverted order. A BAC identified with the recombinant AFLP marker mapped most proximal to the centromere of the ASGR-carrier chromosome in P. squamulatum but was not located on the ASGR-carrier chromosome in C. ciliaris. Exceptional regions where synteny was disrupted probably are nonessential for expression of the aposporous trait. The ASGR appears to be maintained as a haplotype even though its position in the genome can be variable. PMID:16547108

  6. Phenytoin preferentially inhibits L-type calcium currents in whole-cell patch-clamped cardiac and skeletal muscle cells.

    PubMed

    Rivet, M; Bois, P; Cognard, C; Raymond, G

    1990-10-01

    The effect of the anticonvulsant diphenylhydantoin (phenytoin) was tested on the inward calcium currents of whole-cell patch-clamped cells from rat and human muscles and from frog atrium. A concentration of 10 microM phenytoin was required to obtain a threshold inhibitory effect and, even with high concentrations (100 microM), the inhibition was not complete. In skeletal muscle (rat and human cells in culture), phenytoin (30 microM) exerted a more potent effect on the high-threshold calcium current (ICa,L inhibition: 53 +/- 6% mean +/- SDn-1) rather than on the low-threshold one (ICa,T inhibition: 16 +/- 10%). Similar results were obtained on dissociated frog atrial cells. These data are to be contrasted with those previously reported on neuronal cells, where specific inhibition of ICa,T was reported. Thus, the action of phenytoin appears to be different in muscle and nerve so that phenytoin does not appear to be a specific inhibitor of ICa,T.

  7. Neutral endopeptidase inhibitor potentiates the tachykinin-induced increase in ciliary beat frequency in rabbit trachea.

    PubMed

    Kondo, M; Tamaoki, J; Takizawa, T

    1990-08-01

    We used cultured rabbit tracheal epithelium to determine the effect of mammalian-derived tachykinin on airway ciliary activity and its modulation by neutral endopeptidase EC 3.4.24.11 (NEP). Neurokinin A (NKA) caused dose-dependent increases in ciliary beat frequency (CBF), as measured by a photoelectric method, with the maximal increase from the baseline 15.7 +/- 1.7% (mean +/- SEM, p less than 0.01), whereas substance P (SP) had no effect. The NKA-induced increase in CBF was not inhibited by phentolamine, propranolol, or atropine, but it was abolished by the tachykinin antagonist [D-Pro2, D-Trp7,9]SP. Pretreatment of tissue with thiorphan (10(-5) M), a NEP inhibitor, had little effect on CBF responses to NKA; however, it significantly potentiated the responses to SP (14.9 +/- 3.0%, p less than 0.01). Other peptidase inhibitors, including captopril, bestatin, and leupeptin, did not alter the tachykinin-induced CBF response, suggesting that angiotensin converting enzyme, aminopeptidases, and serine proteinases do not modulate ciliary activity in response to tachykinins. These results suggest that NKA increases CBF by acting directly on tachykinin receptors and that NEP may play a role in modulating the tachykinin-induced stimulatory effects on CBF.

  8. Reduced anaerobic and aerobic performance in children with primary ciliary dyskinesia.

    PubMed

    Simsek, Senem; Inal-Ince, Deniz; Cakmak, Aslihan; Emiralioglu, Nagehan; Calik-Kutukcu, Ebru; Saglam, Melda; Vardar-Yagli, Naciye; Ozcelik, Hayriye Ugur; Sonbahar-Ulu, Hazal; Bozdemir-Ozel, Cemile; Kiper, Nural; Arikan, Hulya

    2018-05-01

    Primary ciliary dyskinesia (PCD) restricts lifestyle and increases morbidity. The aim of the study was to investigate anaerobic and aerobic performance in children with PCD and their healthy counterparts. Thirty-one children with PCD and 29 age- and sex-matched healthy subjects were studied. Pulmonary function, hand grip strength (HGS), quadriceps strength (QMS), physical activity, anaerobic capacity (muscle power sprint test), and aerobic performance (modified shuttle walk test (MSWT)) were determined. Pulmonary function, HGS, QMS, mean anaerobic power (MAP), and MSWT distance in PCD were significantly lower than those of healthy subjects (p < 0.05). In PCD, the MAP was significantly correlated with age, FEV 1 , and the mean kcal for 3 days (p < 0.05), and age was its independent predictor (p < 0.05). The MSWT distance was significantly related to gender and weight (p < 0.05), and gender was selected as its independent predictor (p < 0.05). In healthy controls, the MAP was significantly associated with age, gender, FVC, FEV 1 , HGS, QMS, and the mean kcal for three days (p < 0.05). The MSWT distance was significantly related to weight and body mass index in healthy group (p < 0.05). Anaerobic and aerobic performance is impaired in PCD from the early stages. Age determines anaerobic performance. Gender is the determinant of aerobic performance. Whether skeletal muscle characteristics and sex-related changes in body composition affect anaerobic and aerobic capacity in PCD children warrants further study. What is Known: • Exercise performance is determined by anaerobic and aerobic power. • Few studies have shown that PCD patients have lower aerobic performance which is associated with impaired lung function. What is New: • The present research indicated that both anaerobic and aerobic exercise capacity determined using field testing is impaired in PCD from the early stages. • Anaerobic capacity was found to be independently

  9. Circulating smooth muscle progenitor cells in atherosclerosis and plaque rupture: current perspective and methods of analysis.

    PubMed

    Bentzon, Jacob F; Falk, Erling

    2010-01-01

    Smooth muscle cells play a critical role in the development of atherosclerosis and its clinical complications. They were long thought to derive entirely from preexisting smooth muscle cells in the arterial wall, but this understanding has been challenged by the claim that circulating bone marrow-derived smooth muscle progenitor cells are an important source of plaque smooth muscle cells in human and experimental atherosclerosis. This theory is today accepted by many cardiovascular researchers and authors of contemporary review articles. Recently, however, we and others have refuted the existence of bone marrow-derived smooth muscle cells in animal models of atherosclerosis and other arterial diseases based on new experiments with high-resolution microscopy and improved techniques for smooth muscle cell identification and tracking. These studies have also pointed to a number of methodological deficiencies in some of the seminal papers in the field. For those unaccustomed with the methods used in this research area, it must be difficult to decide what to believe and why to do so. In this review, we summarize current knowledge about the origin of smooth muscle cells in atherosclerosis and direct the reader's attention to the methodological challenges that have contributed to the confusion in the field. 2009 Elsevier Inc. All rights reserved.

  10. Transplantation of Embryonic Spinal Cord Derived Cells Helps to Prevent Muscle Atrophy after Peripheral Nerve Injury

    PubMed Central

    Ruven, Carolin; Li, Wen; Li, Heng; Wong, Wai-Man; Wu, Wutian

    2017-01-01

    Injuries to peripheral nerves are frequent in serious traumas and spinal cord injuries. In addition to surgical approaches, other interventions, such as cell transplantation, should be considered to keep the muscles in good condition until the axons regenerate. In this study, E14.5 rat embryonic spinal cord fetal cells and cultured neural progenitor cells from different spinal cord segments were injected into transected musculocutaneous nerve of 200–300 g female Sprague Dawley (SD) rats, and atrophy in biceps brachii was assessed. Both kinds of cells were able to survive, extend their axons towards the muscle and form neuromuscular junctions that were functional in electromyographic studies. As a result, muscle endplates were preserved and atrophy was reduced. Furthermore, we observed that the fetal cells had a better effect in reducing the muscle atrophy compared to the pure neural progenitor cells, whereas lumbar cells were more beneficial compared to thoracic and cervical cells. In addition, fetal lumbar cells were used to supplement six weeks delayed surgical repair after the nerve transection. Cell transplantation helped to preserve the muscle endplates, which in turn lead to earlier functional recovery seen in behavioral test and electromyography. In conclusion, we were able to show that embryonic spinal cord derived cells, especially the lumbar fetal cells, are beneficial in the treatment of peripheral nerve injuries due to their ability to prevent the muscle atrophy. PMID:28264437

  11. Comparative analysis of rat mesenchymal stem cells derived from slow and fast skeletal muscle in vitro.

    PubMed

    Okumachi, Etsuko; Lee, Sang Yang; Niikura, Takahiro; Iwakura, Takashi; Dogaki, Yoshihiro; Waki, Takahiro; Takahara, Shunsuke; Ueha, Takeshi; Sakai, Yoshitada; Kuroda, Ryosuke; Kurosaka, Masahiro

    2015-03-01

    Skeletal muscle comprises different kinds of muscle fibres that can be classified as slow and fast fibres. The purpose of this study was to compare the yield, proliferation, and multi-potentiality of rat mesenchymal stem cells (MSCs) from the tibialis anterior (TA; fast muscle) and soleus (SO; slow muscle) in vitro. The TA and SO muscles were harvested, and isolated cells were plated. After two hours, the cells were washed extensively to remove any cell that did not adhere to the cell culture plate. The adherent cells, namely MSCs, were then cultured. Both types of MSCs were differentiated toward the osteogenic, chondrogenic and adipogenic lineages using lineage specific induction factors. The colony-forming unit fibroblast (CFU-F) assay revealed that the SO contained significantly higher quantities of MSCs than the TA. The self-renewal capacity of MSCs derived from the TA was significantly higher at later passages (passage 9-11). Both types of MSCs exhibited similar cell surface antigens to bone marrow (BM)-derived MSCs and were positive for CD29, CD44, and CD90 and negative for CD11b, CD34, and CD45. TA-derived MSCs were superior in terms of osteogenic differentiation capacity, but there was no significant difference in chondrogenic and adipogenic differentiation capacity. Our results demonstrated significant differences in the properties of muscle-derived MSCs from different muscle types (i.e. fast or slow muscles). The greater expandability and osteogenic differentiation ability of TA-derived MSCs suggests that fast muscle may be a better source for generating large numbers of MSCs for bone regeneration.

  12. TAK1 modulates satellite stem cell homeostasis and skeletal muscle repair

    PubMed Central

    Ogura, Yuji; Hindi, Sajedah M.; Sato, Shuichi; Xiong, Guangyan; Akira, Shizuo; Kumar, Ashok

    2015-01-01

    Satellite cells are resident adult stem cells that are required for regeneration of skeletal muscle. However, signalling mechanisms that regulate satellite cell function are less understood. Here we demonstrate that transforming growth factor-β-activated kinase 1 (TAK1) is important in satellite stem cell homeostasis and function. Inactivation of TAK1 in satellite cells inhibits muscle regeneration in adult mice. TAK1 is essential for satellite cell proliferation and its inactivation causes precocious differentiation. Moreover, TAK1-deficient satellite cells exhibit increased oxidative stress and undergo spontaneous cell death, primarily through necroptosis. TAK1 is required for the activation of NF-κB and JNK in satellite cells. Forced activation of NF-κB improves survival and proliferation of TAK1-deficient satellite cells. Furthermore, TAK1-mediated activation of JNK is essential to prevent oxidative stress and precocious differentiation of satellite cells. Collectively, our study suggests that TAK1 is required for maintaining the pool of satellite stem cells and for regenerative myogenesis. PMID:26648529

  13. Positive and Negative Regulation of Muscle Cell Identity by Members of the hedgehog and TGF-β Gene Families

    PubMed Central

    Du, Shao Jun; Devoto, Stephen H.; Westerfield, Monte; Moon, Randall T.

    1997-01-01

    We have examined whether the development of embryonic muscle fiber type is regulated by competing influences between Hedgehog and TGF-β signals, as previously shown for development of neuronal cell identity in the neural tube. We found that ectopic expression of Hedgehogs or inhibition of protein kinase A in zebrafish embryos induces slow muscle precursors throughout the somite but muscle pioneer cells only in the middle of the somite. Ectopic expression in the notochord of Dorsalin-1, a member of the TGF-β superfamily, inhibits the formation of muscle pioneer cells, demonstrating that TGF-β signals can antagonize the induction of muscle pioneer cells by Hedgehog. We propose that a Hedgehog signal first induces the formation of slow muscle precursor cells, and subsequent Hedgehog and TGF-β signals exert competing positive and negative influences on the development of muscle pioneer cells. PMID:9314535

  14. Pathogenesis of Congenital Rubella Virus Infection in Human Fetuses: Viral Infection in the Ciliary Body Could Play an Important Role in Cataractogenesis.

    PubMed

    Nguyen, Thong Van; Pham, Van Hung; Abe, Kenji

    2015-01-01

    Development of congenital rubella syndrome associated with rubella virus infection during pregnancy is clinically important, but the pathogenicity of the virus remains unclear. Pathological examination was conducted on 3 aborted fetuses with congenital rubella infection. At autopsy, all 3 aborted fetuses showed congenital cataract confirmed by gross observation. Rubella virus infection occurred via systemic organs including circulating hematopoietic stem cells confirmed by immunohistochemical and molecular investigations, and major histopathogical changes were found in the liver. It is noteworthy that the virus infected the ciliary body of the eye, suggesting a possible cause of cataracts. Our study based on the pathological examination demonstrated that the rubella virus infection occurred via systemic organs of human fetuses. This fact was confirmed by immunohistochemistry and direct detection of viral RNA in multiple organs. To the best of our knowledge, this study is the first report demonstrating that the rubella virus infection occurred via systemic organs of the human body. Importantly, virus infection of the ciliary body could play an important role in cataractogenesis.

  15. NG2 Proteoglycan Ablation Reduces Foam Cell Formation and Atherogenesis via Decreased Low-Density Lipoprotein Retention by Synthetic Smooth Muscle Cells.

    PubMed

    She, Zhi-Gang; Chang, Yunchao; Pang, Hong-Bo; Han, Wenlong; Chen, Hou-Zao; Smith, Jeffrey W; Stallcup, William B

    2016-01-01

    Obesity and hyperlipidemia are critical risk factors for atherosclerosis. Because ablation of NG2 proteoglycan in mice leads to hyperlipidemia and obesity, we investigated the impact of NG2 ablation on atherosclerosis in apoE null mice. Immunostaining indicates that NG2 expression in plaque, primarily by synthetic smooth muscle cells, increases during atherogenesis. NG2 ablation unexpectedly results in decreased (30%) plaque development, despite aggravated obesity and hyperlipidemia. Mechanistic studies reveal that NG2-positive plaque synthetic smooth muscle cells in culture can sequester low-density lipoprotein to enhance foam-cell formation, processes in which NG2 itself plays direct roles. In agreement with these observations, low-density lipoprotein retention and lipid accumulation in the NG2/ApoE knockout aorta is 30% less than that seen in the control aorta. These results indicate that synthetic smooth muscle cell-dependent low-density lipoprotein retention and foam cell formation outweigh obesity and hyperlipidemia in promoting mouse atherogenesis. Our study sheds new light on the role of synthetic smooth muscle cells during atherogenesis. Blocking plaque NG2 or altering synthetic smooth muscle cells function may be promising therapeutic strategies for atherosclerosis. © 2015 American Heart Association, Inc.

  16. Interaction of Vascular Smooth Muscle Cells Under Low Shear Stress

    NASA Technical Reports Server (NTRS)

    Seidel, Charles L.

    1998-01-01

    The blood vessel wall consists of three cellular layers, an outer adventitial, a middle medial and an inner intimal layer. When the blood vessel forms in the embryo it begins as a tube composed of a single cell type called endothelial cells. Over time, other cells are recruited from the surrounding tissue to form additional layers on the outer surface of the endothelial tube. The cells that are recruited are called mesenchymal cells. Mesenchymal cells are responsible for the production of connective tissue that holds the blood vessel together and for developing into vascular smooth muscle cells that are responsible for regulating the diameter of the vessel (1) and therefore, blood flow. In a fully developed blood vessel, the endothelial cells make- up the majority of cells in the intimal layer while the mesenchymal cells make-up the majority of cells in the medial and adventitial layers. Within the medial layer of a mature vessel, cells are organized into multiple circular layers of alternating bands of connective tissue and cells. The cell layer is composed of a mixture of mesenchymal cells that have not developed into smooth muscle cells and fully developed smooth muscle cells (2). The assembly and organization of complex tissues is directed in part by a signaling system composed of proteins on the cell surface called adhesion molecules. Adhesion molecules enable cells to recognize each other as well as the composition of the connective tissue in which they reside (3). It was hypothesized that the different cell types that compose the vascular wall possess different adhesion molecules that enable them to recognize each other and through this recognition system, form the complex layered organization of the vascular wall. In other words, the layered organization is an intrinsic property of the cells. If this hypothesis is correct then the different cells that make up the vessel wall, when mixed together, should organize themselves into a layered structure

  17. Barx2 is Expressed in Satellite Cells and is Required for Normal Muscle Growth and Regeneration

    PubMed Central

    Meech, Robyn; Gonzalez, Katie N.; Barro, Marietta; Gromova, Anastasia; Zhuang, Lizhe; Hulin, Julie-Ann; Makarenkova, Helen P.

    2015-01-01

    Muscle growth and regeneration are regulated through a series of spatiotemporally dependent signaling and transcriptional cascades. Although the transcriptional program controlling myogenesis has been extensively investigated, the full repertoire of transcriptional regulators involved in this process is far from defined. Various homeodomain transcription factors have been shown to play important roles in both muscle development and muscle satellite cell-dependent repair. Here, we show that the homeodomain factor Barx2 is a new marker for embryonic and adult myoblasts and is required for normal postnatal muscle growth and repair. Barx2 is coexpressed with Pax7, which is the canonical marker of satellite cells, and is upregulated in satellite cells after muscle injury. Mice lacking the Barx2 gene show reduced postnatal muscle growth, muscle atrophy, and defective muscle repair. Moreover, loss of Barx2 delays the expression of genes that control proliferation and differentiation in regenerating muscle. Consistent with the in vivo observations, satellite cell-derived myoblasts cultured from Barx2−/− mice show decreased proliferation and ability to differentiate relative to those from wild-type or Barx2+/− mice. Barx2−/− myoblasts show reduced expression of the differentiation-associated factor myogenin as well as cell adhesion and matrix molecules. Finally, we find that mice lacking both Barx2 and dystrophin gene expression have severe early onset myopathy. Together, these data indicate that Barx2 is an important regulator of muscle growth and repair that acts via the control of satellite cell proliferation and differentiation. PMID:22076929

  18. mTOR is necessary for proper satellite cell activity and skeletal muscle regeneration

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

    Zhang, Pengpeng; Department of Animal Sciences, Purdue University, West Lafayette, IN 47907; Liang, Xinrong

    The serine/threonine kinase mammalian target of rapamycin (mTOR) is a key regulator of protein synthesis, cell proliferation and energy metabolism. As constitutive deletion of Mtor gene results in embryonic lethality, the function of mTOR in muscle stem cells (satellite cells) and skeletal muscle regeneration remains to be determined. In this study, we established a satellite cell specific Mtor conditional knockout (cKO) mouse model by crossing Pax7{sup CreER} and Mtor{sup flox/flox} mice. Skeletal muscle regeneration after injury was severely compromised in the absence of Mtor, indicated by increased number of necrotic myofibers infiltrated by Evans blue dye, and reduced number andmore » size of regenerated myofibers in the Mtor cKO mice compared to wild type (WT) littermates. To dissect the cellular mechanism, we analyzed satellite cell-derived primary myoblasts grown on single myofibers or adhered to culture plates. The Mtor cKO myoblasts exhibited defective proliferation and differentiation kinetics when compared to myoblasts derived from WT littermates. At the mRNA and protein levels, the Mtor cKO myoblasts expressed lower levels of key myogenic determinant genes Pax7, Myf5, Myod, Myog than did the WT myoblasts. These results suggest that mTOR is essential for satellite cell function and skeletal muscle regeneration through controlling the expression of myogenic genes. - Highlights: • Pax7{sup CreER} was used to delete Mtor gene in satellite cells. • Satellite cell specific deletion of Mtor impairs muscle regeneration. • mTOR is necessary for satellite cell proliferation and differentiation. • Deletion of Mtor leads to reduced expression of key myogenic genes.« less

  19. Inflammatory cells in rat skeletal muscle are elevated after electrically stimulated contractions.

    PubMed

    McLoughlin, Thomas J; Mylona, Eleni; Hornberger, Troy A; Esser, Karyn A; Pizza, Francis X

    2003-03-01

    We determined the effect of muscle contractions resulting from high-frequency electrical stimulation (HFES) on inflammatory cells in rat tibialis anterior (TA), plantaris (Pln), and soleus (Sol) muscles at 6, 24, and 72 h post-HFES. A minimum of four and a maximum of seven rats were analyzed at each time point. HFES, applied to the sciatic nerve, caused the Sol and Pln to contract concentrically and the TA to contract eccentrically. Neutrophils were higher (P < 0.05) at 6 and 24 h after HFES in the Sol, Pln, and TA muscles relative to control muscles. ED1(+) macrophages in the Pln were elevated at 6 and 24 h after HFES and were also elevated in the Sol and TA after HFES relative to controls. ED2(+) macrophages in the Sol and TA were elevated at 24 and 72 h after HFES, respectively, and were also elevated in the Pln after HFES relative to controls. In contrast to the TA muscles, the Pln and Sol muscles showed no gross histological abnormalities. Collectively, these data indicate that both eccentric and concentric contractions can increase inflammatory cells in muscle, regardless of whether overt histological signs of injury are apparent.

  20. [Complications of a voluminous congenital ciliary body cyst].

    PubMed

    Kottler, Ulrike B; Schwenn, Oliver

    2002-09-01

    Multiple cysts of iris and ciliary body may cause many complications such as acute or chronic angle closure glaucoma. We present a boy with multiple congenital cysts of the iris pigment epithelium and a voluminous cyst of the unpigmented ciliary body epithelium of the right eye. This resulted in lenticular astigmatism, concomitant strabismus divergens and subsequently to anisometropia. The voluminous cyst (8 x 12 mm in diameter) was folded around the lens, reached the optic axis and resulted in displacement of the lens and contact between the iris and the corneal endothelium from 6.30 to 11. After puncture and partial resection of the cyst at the age of 8 months the boy developed a subcapsular multivesicular cataractic clouding of the temporal lens and a progredient myopia (up to - 14.0/- 2.0/0 degrees ); in contrast the left eye was hyperopic (+ 3.5/- 3.75/0 degrees ). The lens was subluxated superonasally due to congenital damage of the zonular fibres. Because development of visual acuity seemed limited by these determinants (20/200 at the right eye), cataract surgery with posterior capsulorhexis, anterior vitrectomy, and implantation of a capsular tension ring and posterior chamber intraocular lens was performed at the age of nearly five. Actually, there is an orthotropia, best corrected visual acuity in the distance of 20/32 in the right and 20/20 in the left eye; binocular vision is somewhat restricted. Usually congenital cysts are clinically not very relevant; occasionally surgical intervention is required to ensure adequate development of visual acuity.

  1. Cytokine Response of Cultured Skeletal Muscle Cells Stimulated with Proinflammatory Factors Depends on Differentiation Stage

    PubMed Central

    Podbregar, Matej; Lainscak, Mitja; Prelovsek, Oja; Mars, Tomaz

    2013-01-01

    Myoblast proliferation and myotube formation are critical early events in skeletal muscle regeneration. The attending inflammation and cytokine signaling are involved in regulation of skeletal muscle cell proliferation and differentiation. Secretion of muscle-derived cytokines upon exposure to inflammatory factors may depend on the differentiation stage of regenerating muscle cells. Cultured human myoblasts and myotubes were exposed to 24-hour treatment with tumor necrosis factor (TNF)-α or lipopolysaccharide (LPS). Secretion of interleukin 6 (IL-6), a major muscle-derived cytokine, and interleukin 1 (IL-1), an important regulator of inflammatory response, was measured 24 hours after termination of TNF-α or LPS treatment. Myoblasts pretreated with TNF-α or LPS displayed robustly increased IL-6 secretion during the 24-hour period after removal of treatments, while IL-1 secretion remained unaltered. IL-6 secretion was also increased in myotubes, but the response was less pronounced compared with myoblasts. In contrast to myoblasts, IL-1 secretion was markedly stimulated in LPS-pretreated myotubes. We demonstrate that preceding exposure to inflammatory factors stimulates a prolonged upregulation of muscle-derived IL-6 and/or IL-1 in cultured skeletal muscle cells. Our findings also indicate that cytokine response to inflammatory factors in regenerating skeletal muscle partially depends on the differentiation stage of myogenic cells. PMID:23509435

  2. Functional vascular smooth muscle cells derived from human induced pluripotent stem cells via mesenchymal stem cell intermediates

    PubMed Central

    Bajpai, Vivek K.; Mistriotis, Panagiotis; Loh, Yuin-Han; Daley, George Q.; Andreadis, Stelios T.

    2012-01-01

    Aims Smooth muscle cells (SMC) play an important role in vascular homeostasis and disease. Although adult mesenchymal stem cells (MSC) have been used as a source of contractile SMC, they suffer from limited proliferation potential and culture senescence, particularly when originating from older donors. By comparison, human induced pluripotent stem cells (hiPSC) can provide an unlimited source of functional SMC for autologous cell-based therapies and for creating models of vascular disease. Our goal was to develop an efficient strategy to derive functional, contractile SMC from hiPSC. Methods and results We developed a robust, stage-wise, feeder-free strategy for hiPSC differentiation into functional SMC through an intermediate stage of multipotent MSC, which could be coaxed to differentiate into fat, bone, cartilage, and muscle. At this stage, the cells were highly proliferative and displayed higher clonogenic potential and reduced senescence when compared with parental hair follicle mesenchymal stem cells. In addition, when exposed to differentiation medium, the myogenic proteins such as α-smooth muscle actin, calponin, and myosin heavy chain were significantly upregulated and displayed robust fibrillar organization, suggesting the development of a contractile phenotype. Indeed, tissue constructs prepared from these cells exhibited high levels of contractility in response to receptor- and non-receptor-mediated agonists. Conclusion We developed an efficient stage-wise strategy that enabled hiPSC differentiation into contractile SMC through an intermediate population of clonogenic and multipotent MSC. The high yield of MSC and SMC derivation suggests that our strategy may facilitate an acquisition of the large numbers of cells required for regenerative medicine or for studying vascular disease pathophysiology. PMID:22941255

  3. Isoform variants of troponin in skeletal and cardiac muscle cells cultured with and without nerves.

    PubMed

    Toyota, N; Shimada, Y

    1983-05-01

    Immunofluorescence microscopy shows that cultured skeletal and cardiac muscle cells of chicken embryos exhibit the same stainabilities with antibodies against skeletal and cardiac troponin components as do those in embryos. Muscle cells of each type cultured with motor or sympathetic nerves or in medium containing the nerve extract exhibit the same reactivities as do those in adult animals. Cardiac muscle cells incubated in the nerve-conditioned medium also change the form of troponin components to the adult type. It appears that the differentiation of individual muscle fibers to specific types is induced by nerves, and especially by the neurohumoral effect.

  4. Muscle Stem Cells Undergo Extensive Clonal Drift during Tissue Growth via Meox1-Mediated Induction of G2 Cell-Cycle Arrest.

    PubMed

    Nguyen, Phong Dang; Gurevich, David Baruch; Sonntag, Carmen; Hersey, Lucy; Alaei, Sara; Nim, Hieu Tri; Siegel, Ashley; Hall, Thomas Edward; Rossello, Fernando Jaime; Boyd, Sarah Elizabeth; Polo, Jose Maria; Currie, Peter David

    2017-07-06

    Organ growth requires a careful balance between stem cell self-renewal and lineage commitment to ensure proper tissue expansion. The cellular and molecular mechanisms that mediate this balance are unresolved in most organs, including skeletal muscle. Here we identify a long-lived stem cell pool that mediates growth of the zebrafish myotome. This population exhibits extensive clonal drift, shifting from random deployment of stem cells during development to reliance on a small number of dominant clones to fuel the vast majority of muscle growth. This clonal drift requires Meox1, a homeobox protein that directly inhibits the cell-cycle checkpoint gene ccnb1. Meox1 initiates G 2 cell-cycle arrest within muscle stem cells, and disrupting this G 2 arrest causes premature lineage commitment and the resulting defects in muscle growth. These findings reveal that distinct regulatory mechanisms orchestrate stem cell dynamics during organ growth, beyond the G 0 /G 1 cell-cycle inhibition traditionally associated with maintaining tissue-resident stem cells. Copyright © 2017. Published by Elsevier Inc.

  5. Primary ciliary dyskinesia. Recent advances in diagnostics, genetics, and characterization of clinical disease.

    PubMed

    Knowles, Michael R; Daniels, Leigh Anne; Davis, Stephanie D; Zariwala, Maimoona A; Leigh, Margaret W

    2013-10-15

    Primary ciliary dyskinesia (PCD) is a genetically heterogeneous recessive disorder of motile cilia that leads to oto-sino-pulmonary diseases and organ laterality defects in approximately 50% of cases. The estimated incidence of PCD is approximately 1 per 15,000 births, but the prevalence of PCD is difficult to determine, primarily because of limitations in diagnostic methods that focus on testing ciliary ultrastructure and function. Diagnostic capabilities have recently benefitted from (1) documentation of low nasal nitric oxide production in PCD and (2) discovery of biallelic mutations in multiple PCD-causing genes. The use of these complementary diagnostic approaches shows that at least 30% of patients with PCD have normal ciliary ultrastructure. More accurate identification of patients with PCD has also allowed definition of a strong clinical phenotype, which includes neonatal respiratory distress in >80% of cases, daily nasal congestion and wet cough starting soon after birth, and early development of recurrent/chronic middle-ear and sinus disease. Recent studies, using advanced imaging and pulmonary physiologic assessments, clearly demonstrate early onset of lung disease in PCD, with abnormal air flow mechanics by age 6-8 years that is similar to cystic fibrosis, and age-dependent onset of bronchiectasis. The treatment of PCD is not standardized, and there are no validated PCD-specific therapies. Most patients with PCD receive suboptimal management, which should include airway clearance, regular surveillance of pulmonary function and respiratory microbiology, and use of antibiotics targeted to pathogens. The PCD Foundation is developing a network of clinical centers, which should improve diagnosis and management of PCD.

  6. Primary Ciliary Dyskinesia. Recent Advances in Diagnostics, Genetics, and Characterization of Clinical Disease

    PubMed Central

    Daniels, Leigh Anne; Davis, Stephanie D.; Zariwala, Maimoona A.; Leigh, Margaret W.

    2013-01-01

    Primary ciliary dyskinesia (PCD) is a genetically heterogeneous recessive disorder of motile cilia that leads to oto-sino-pulmonary diseases and organ laterality defects in approximately 50% of cases. The estimated incidence of PCD is approximately 1 per 15,000 births, but the prevalence of PCD is difficult to determine, primarily because of limitations in diagnostic methods that focus on testing ciliary ultrastructure and function. Diagnostic capabilities have recently benefitted from (1) documentation of low nasal nitric oxide production in PCD and (2) discovery of biallelic mutations in multiple PCD-causing genes. The use of these complementary diagnostic approaches shows that at least 30% of patients with PCD have normal ciliary ultrastructure. More accurate identification of patients with PCD has also allowed definition of a strong clinical phenotype, which includes neonatal respiratory distress in >80% of cases, daily nasal congestion and wet cough starting soon after birth, and early development of recurrent/chronic middle-ear and sinus disease. Recent studies, using advanced imaging and pulmonary physiologic assessments, clearly demonstrate early onset of lung disease in PCD, with abnormal air flow mechanics by age 6–8 years that is similar to cystic fibrosis, and age-dependent onset of bronchiectasis. The treatment of PCD is not standardized, and there are no validated PCD-specific therapies. Most patients with PCD receive suboptimal management, which should include airway clearance, regular surveillance of pulmonary function and respiratory microbiology, and use of antibiotics targeted to pathogens. The PCD Foundation is developing a network of clinical centers, which should improve diagnosis and management of PCD. PMID:23796196

  7. Characteristics of the Localization of Connexin 43 in Satellite Cells during Skeletal Muscle Regeneration In Vivo

    PubMed Central

    Ishido, Minenori; Kasuga, Norikatsu

    2015-01-01

    For myogenesis, new myotubes are formed by the fusion of differentiated myoblasts. In the sequence of events for myotube formation, intercellular communication through gap junctions composed of connexin 43 (Cx43) plays critical roles in regulating the alignment and fusion of myoblasts in advances of myotube formation in vitro. On the other hand, the relationship between the expression patterns of Cx43 and the process of myotube formation in satellite cells during muscle regeneration in vivo remains poorly understood. The present study investigated the relationship between Cx43 and satellite cells in muscle regeneration in vivo. The expression of Cx43 was detected in skeletal muscles on day 1 post-muscle injury, but not in control muscles. Interestingly, the expression of Cx43 was not localized on the inside of the basement membrane of myofibers in the regenerating muscles. Moreover, although the clusters of differentiated satellite cells, which represent a more advanced stage of myotube formation, were observed on the inside of the basement membrane of myofibers in regenerating muscles, the expression of Cx43 was not localized in the clusters of these satellite cells. Therefore, in the present study, it was suggested that Cx43 may not directly contribute to muscle regeneration via satellite cells. PMID:26019374

  8. Regeneration of the oesophageal muscle layer from oesophagus acellular matrix scaffold using adipose-derived stem cells.

    PubMed

    Wang, Fang; Maeda, Yasuko; Zachar, Vladimir; Ansari, Tahera; Emmersen, Jeppe

    2018-06-14

    This study explored the feasibility of constructing a tissue engineered muscle layer in the oesophagus using oesophageal acellular matrix (OAM) scaffolds and human aortic smooth muscle cells (hASMCs) or human adipose-derived stem cells (hASCs). The second objective was to investigate the effect of hypoxic preconditioning of seeding cells on cell viability and migration depth. Our results demonstrated that hASMCs and hASCs could attach and adhere to the decellularized OAM scaffold and survive and proliferate for at least 7 days depending on the growth conditions. This indicates adipose-derived stem cells (ASCs) have the potential to substitute for smooth muscle cells (SMCs) in the construction of tissue engineered oesophageal muscle layers. Copyright © 2018 Elsevier Inc. All rights reserved.

  9. Robust generation and expansion of skeletal muscle progenitors and myocytes from human pluripotent stem cells.

    PubMed

    Shelton, Michael; Kocharyan, Avetik; Liu, Jun; Skerjanc, Ilona S; Stanford, William L

    2016-05-15

    Human pluripotent stem cells provide a developmental model to study early embryonic and tissue development, tease apart human disease processes, perform drug screens to identify potential molecular effectors of in situ regeneration, and provide a source for cell and tissue based transplantation. Highly efficient differentiation protocols have been established for many cell types and tissues; however, until very recently robust differentiation into skeletal muscle cells had not been possible unless driven by transgenic expression of master regulators of myogenesis. Nevertheless, several breakthrough protocols have been published in the past two years that efficiently generate cells of the skeletal muscle lineage from pluripotent stem cells. Here, we present an updated version of our recently described 50-day protocol in detail, whereby chemically defined media are used to drive and support muscle lineage development from initial CHIR99021-induced mesoderm through to PAX7-expressing skeletal muscle progenitors and mature skeletal myocytes. Furthermore, we report an optional method to passage and expand differentiating skeletal muscle progenitors approximately 3-fold every 2weeks using Collagenase IV and continued FGF2 supplementation. Both protocols have been optimized using a variety of human pluripotent stem cell lines including patient-derived induced pluripotent stem cells. Taken together, our differentiation and expansion protocols provide sufficient quantities of skeletal muscle progenitors and myocytes that could be used for a variety of studies. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  10. Molecular and cell-based therapies for muscle degenerations: a road under construction.

    PubMed

    Berardi, Emanuele; Annibali, Daniela; Cassano, Marco; Crippa, Stefania; Sampaolesi, Maurilio

    2014-01-01

    Despite the advances achieved in understanding the molecular biology of muscle cells in the past decades, there is still need for effective treatments of muscular degeneration caused by muscular dystrophies and for counteracting the muscle wasting caused by cachexia or sarcopenia. The corticosteroid medications currently in use for dystrophic patients merely help to control the inflammatory state and only slightly delay the progression of the disease. Unfortunately, walkers and wheel chairs are the only options for such patients to maintain independence and walking capabilities until the respiratory muscles become weak and the mechanical ventilation is needed. On the other hand, myostatin inhibition, IL-6 antagonism and synthetic ghrelin administration are examples of promising treatments in cachexia animal models. In both dystrophies and cachectic syndrome the muscular degeneration is extremely relevant and the translational therapeutic attempts to find a possible cure are well defined. In particular, molecular-based therapies are common options to be explored in order to exploit beneficial treatments for cachexia, while gene/cell therapies are mostly used in the attempt to induce a substantial improvement of the dystrophic muscular phenotype. This review focuses on the description of the use of molecular administrations and gene/stem cell therapy to treat muscular degenerations. It reviews previous trials using cell delivery protocols in mice and patients starting with the use of donor myoblasts, outlining the likely causes for their poor results and briefly focusing on satellite cell studies that raise new hope. Then it proceeds to describe recently identified stem/progenitor cells, including pluripotent stem cells and in relationship to their ability to home within a dystrophic muscle and to differentiate into skeletal muscle cells. Different known features of various stem cells are compared in this perspective, and the few available examples of their use in

  11. KCl cotransport regulation and protein kinase G in cultured vascular smooth muscle cells.

    PubMed

    Adragna, N C; Zhang, J; Di Fulvio, M; Lincoln, T M; Lauf, P K

    2002-05-15

    K-Cl cotransport is activated by vasodilators in erythrocytes and vascular smooth muscle cells and its regulation involves putative kinase/phosphatase cascades. N-ethylmaleimide (NEM) activates the system presumably by inhibiting a protein kinase. Nitrovasodilators relax smooth muscle via cGMP-dependent activation of protein kinase G (PKG), a regulator of membrane channels and transporters. We investigated whether PKG regulates K-Cl cotransport activity or mRNA expression in normal, PKG-deficient-vector-only-transfected (PKG-) and PKG-catalytic-domain-transfected (PKG+) rat aortic smooth muscle cells. K-Cl cotransport was calculated as the Cl-dependent Rb influx, and mRNA was determined by semiquantitative RT-PCR. Baseline K-Cl cotransport was higher in PKG+ than in PKG- cells (p <0.01). At 0.5 mM, NEM stimulated K-Cl cotransport by 5-fold in PKG- but not in PKG+ cells. However, NEM was more potent although less effective to activate K-Cl cotransport in normal (passage 1-3) and PKG+ than in PKG- cells. In PKG- cells, [(dihydroindenyl) oxy] alkanoic acid (300 mM) but not furosemide (1 mM) inhibited K-Cl cotransport. Furthermore, no difference in K-Cl cotransport mRNA expression was observed between these cells. In conclusion, this study shows that manipulation of PKG expression in vascular smooth muscle cells affects K-Cl cotransport activity and its activation by NEM.

  12. Sphingosine-1-phosphate mediates epidermal growth factor-induced muscle satellite cell activation

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

    Nagata, Yosuke, E-mail: cynagata@mail.ecc.u-tokyo.ac.jp; Ohashi, Kazuya; Wada, Eiji

    2014-08-01

    Skeletal muscle can regenerate repeatedly due to the presence of resident stem cells, called satellite cells. Because satellite cells are usually quiescent, they must be activated before participating in muscle regeneration in response to stimuli such as injury, overloading, and stretch. Although satellite cell activation is a crucial step in muscle regeneration, little is known of the molecular mechanisms controlling this process. Recent work showed that the bioactive lipid sphingosine-1-phosphate (S1P) plays crucial roles in the activation, proliferation, and differentiation of muscle satellite cells. We investigated the role of growth factors in S1P-mediated satellite cell activation. We found that epidermalmore » growth factor (EGF) in combination with insulin induced proliferation of quiescent undifferentiated mouse myoblast C2C12 cells, which are also known as reserve cells, in serum-free conditions. Sphingosine kinase activity increased when reserve cells were stimulated with EGF. Treatment of reserve cells with the D-erythro-N,N-dimethylsphingosine, Sphingosine Kinase Inhibitor, or siRNA duplexes specific for sphingosine kinase 1, suppressed EGF-induced C2C12 activation. We also present the evidence showing the S1P receptor S1P2 is involved in EGF-induced reserve cell activation. Moreover, we demonstrated a combination of insulin and EGF promoted activation of satellite cells on single myofibers in a manner dependent on SPHK and S1P2. Taken together, our observations show that EGF-induced satellite cell activation is mediated by S1P and its receptor. - Highlights: • EGF in combination with insulin induces proliferation of quiescent C2C12 cells. • Sphingosine kinase activity increases when reserve cells are stimulated with EGF. • EGF-induced activation of reserve cells is dependent on sphingosine kinase and ERK. • The S1P receptor S1P2 is involved in EGF-induced reserve cell activation. • EGF-induced reserve cell activation is mediated by S1P and

  13. The number of satellite cells in slow and fast fibres from human vastus lateralis muscle.

    PubMed

    Kadi, Fawzi; Charifi, Nadia; Henriksson, Jan

    2006-07-01

    The aim of this investigation was to study the distribution of satellite cells in slow (type I fibres) and fast (type II fibres) fibres from human vastus lateralis muscle. This muscle is characterised by a mixed fibre type composition and is considered as the site of choice for biopsies in research work and for clinical diagnosis. Biopsy samples were obtained from five healthy young volunteers and a total of 1,747 type I fibres and 1,760 type II fibres were assessed. Satellite cells and fibre type composition were studied on serial muscle cross-sections stained with specific monoclonal antibodies. From a total of 218 satellite cells, 116 satellite cells were found in contact with type I fibres (53.6+/-8% of the satellite cells associated to type I fibres) and 102 satellite cells in contact with type II fibres (46.4+/-8% of the satellite cells associated to type II fibres). There was no significant difference (P=0.4) between the percentages of satellite cells in contact with type I and with type II fibres. Additionally, there was no relationship between the mean number of satellite cells per fibre and the mean cross-sectional area of muscle fibres. In conclusion, our results show that there is no fibre type-specific distribution of satellite cells in a human skeletal muscle with mixed fibre type composition.

  14. Identification of a new stem cell population that generates Drosophila flight muscles.

    PubMed

    Gunage, Rajesh D; Reichert, Heinrich; VijayRaghavan, K

    2014-08-18

    How myoblast populations are regulated for the formation of muscles of different sizes is an essentially unanswered question. The large flight muscles of Drosophila develop from adult muscle progenitor (AMP) cells set-aside embryonically. The thoracic segments are all allotted the same small AMP number, while those associated with the wing-disc proliferate extensively to give rise to over 2500 myoblasts. An initial amplification occurs through symmetric divisions and is followed by a switch to asymmetric divisions in which the AMPs self-renew and generate post-mitotic myoblasts. Notch signaling controls the initial amplification of AMPs, while the switch to asymmetric division additionally requires Wingless, which regulates Numb expression in the AMP lineage. In both cases, the epidermal tissue of the wing imaginal disc acts as a niche expressing the ligands Serrate and Wingless. The disc-associated AMPs are a novel muscle stem cell population that orchestrates the early phases of adult flight muscle development.

  15. Components of Torpedo electric organ and muscle that cause aggregation of acetylcholine receptors on cultured muscle cells

    PubMed Central

    1984-01-01

    The synaptic portion of a muscle fiber's basal lamina sheath has molecules tightly bound to it that cause aggregation of acetylcholine receptors (AChRs) on regenerating myofibers. Since basal lamina and other extracellular matrix constituents are insoluble in isotonic saline and detergent solutions, insoluble detergent-extracted fractions of tissues receiving cholinergic input may provide an enriched source of the AChR-aggregating molecules for detailed characterization. Here we demonstrate that such an insoluble fraction from Torpedo electric organ, a tissue with a high concentration of cholinergic synapses, causes AChRs on cultured chick muscle cells to aggregate. We have partially characterized the insoluble fraction, examined the response of muscle cells to it, and devised ways of extracting the active components with a view toward purifying them and learning whether they are similar to those in the basal lamina at the neuromuscular junction. The insoluble fraction from the electric organ was rich in extracellular matrix constituents; it contained structures resembling basal lamina sheaths and had a high density of collagen fibrils. It caused a 3- to 20-fold increase in the number of AChR clusters on cultured myotubes without significantly affecting the number or size of the myotubes. The increase was first seen 2-4 h after the fraction was added to cultures and it was maximal by 24 h. The AChR-aggregating effect was dose dependent and was due, at least in part, to lateral migration of AChRs present in the muscle cell plasma membrane at the time the fraction was applied. Activity was destroyed by heat and by trypsin. The active component(s) was extracted from the insoluble fraction with high ionic strength or pH 5.5 buffers. The extracts increased the number of AChR clusters on cultured myotubes without affecting the number or degradation rate of surface AChRs. Antiserum against the solubilized material blocked its effect on AChR distribution and bound to the

  16. Treatment of Tourniquet-Induced Ischemia Reperfusion Injury with Muscle Progenitor Cells

    DTIC Science & Technology

    2011-09-01

    application. Muscle mass, isometric contractile properties, and selected histologic properties were evaluated at 2 wk after ischemia. Results. IRI...results showed that a small number of trans- planted cells differentiated and formed muscle fibers , which could potentially contribute to force genera...the wet weight of the muscle (in g); q is the angle of fiber pinnation (12.8 for TA); Lf is the mean fiber length (57% of TAmuscle length); and r is

  17. Epigenetic stress responses induce muscle stem-cell ageing by Hoxa9 developmental signals.

    PubMed

    Schwörer, Simon; Becker, Friedrich; Feller, Christian; Baig, Ali H; Köber, Ute; Henze, Henriette; Kraus, Johann M; Xin, Beibei; Lechel, André; Lipka, Daniel B; Varghese, Christy S; Schmidt, Manuel; Rohs, Remo; Aebersold, Ruedi; Medina, Kay L; Kestler, Hans A; Neri, Francesco; von Maltzahn, Julia; Tümpel, Stefan; Rudolph, K Lenhard

    2016-12-15

    The functionality of stem cells declines during ageing, and this decline contributes to ageing-associated impairments in tissue regeneration and function. Alterations in developmental pathways have been associated with declines in stem-cell function during ageing, but the nature of this process remains poorly understood. Hox genes are key regulators of stem cells and tissue patterning during embryogenesis with an unknown role in ageing. Here we show that the epigenetic stress response in muscle stem cells (also known as satellite cells) differs between aged and young mice. The alteration includes aberrant global and site-specific induction of active chromatin marks in activated satellite cells from aged mice, resulting in the specific induction of Hoxa9 but not other Hox genes. Hoxa9 in turn activates several developmental pathways and represents a decisive factor that separates satellite cell gene expression in aged mice from that in young mice. The activated pathways include most of the currently known inhibitors of satellite cell function in ageing muscle, including Wnt, TGFβ, JAK/STAT and senescence signalling. Inhibition of aberrant chromatin activation or deletion of Hoxa9 improves satellite cell function and muscle regeneration in aged mice, whereas overexpression of Hoxa9 mimics ageing-associated defects in satellite cells from young mice, which can be rescued by the inhibition of Hoxa9-targeted developmental pathways. Together, these data delineate an altered epigenetic stress response in activated satellite cells from aged mice, which limits satellite cell function and muscle regeneration by Hoxa9-dependent activation of developmental pathways.

  18. Growth, biomass production and photosynthesis of Cenchrus ciliaris L. under Acacia tortilis (Forssk.) Hayne based silvopastoral systems in semi arid tropics.

    PubMed

    Mishra, A K; Tiwari, H S; Bhatt, R K

    2010-11-01

    The growth, biomass production and photosynthesis of Cenchrus ciliaris was studied under the canopies of 17 yr old Acacia tortilis trees in semi arid tropical environment. On an average the full grown canopy of A. tortilis at the spacing of 4 x 4 m allowed 55% of total Photosynthetically Active Radiation (PAR) which in turn increased Relative Humidity (RH) and reduced under canopy temperature to -1.75 degrees C over the open air temperature. C. ciliaris attained higher height under the shade of A. tortilis. The tiller production and leaf area index decreased marginally under the shade of tree canopies as compared to the open grown grasses. C. ciliaris accumulated higher chlorophyll a and b under the shade of tree canopies indicating its shade adaptation potential. The assimilatory functions such as rate of photosynthesis, transpiration, stomatal conductance, photosynthetic water use efficiency (PN/TR) and carboxylation efficiency (PN/CINT) decreased under the tree canopies due to low availability of PAR. The total biomass production in term of fresh and dry weight decreased under the tree canopies. On average of 2 yr C. ciliaris had produced 12.78 t ha(-1) green and 3.72 -t ha(-1) dry biomass under the tree canopies of A. tortilis. The dry matter yield reduced to 38% under the tree canopies over the open grown grasses. The A. tortilis + C. ciliaris maintained higher soil moisture, organic carbon content and available N P K for sustainable biomass production for the longer period. The higher accumulation of crude protein, starch, sugar and nitrogen in leaves and stem of C. ciliaris indicates that this grass species also maintained its quality under A. tortilis based silvopastoral system. The photosynthesis and dry matter accumulation are closely associated with available PAR indicating that for sustainable production of this grass species in the silvopasture systems for longer period about 55% or more PAR is required.

  19. Nitric oxide inhibits calpain-mediated proteolysis of talin in skeletal muscle cells

    NASA Technical Reports Server (NTRS)

    Koh, T. J.; Tidball, J. G.

    2000-01-01

    We tested the hypothesis that nitric oxide can inhibit cytoskeletal breakdown in skeletal muscle cells by inhibiting calpain cleavage of talin. The nitric oxide donor sodium nitroprusside prevented many of the effects of calcium ionophore on C(2)C(12) muscle cells, including preventing talin proteolysis and release into the cytosol and reducing loss of vinculin, cell detachment, and loss of cellular protein. These results indicate that nitric oxide inhibition of calpain protected the cells from ionophore-induced proteolysis. Calpain inhibitor I and a cell-permeable calpastatin peptide also protected the cells from proteolysis, confirming that ionophore-induced proteolysis was primarily calpain mediated. The activity of m-calpain in a casein zymogram was inhibited by sodium nitroprusside, and this inhibition was reversed by dithiothreitol. Previous incubation with the active site-targeted calpain inhibitor I prevented most of the sodium nitroprusside-induced inhibition of m-calpain activity. These data suggest that nitric oxide inhibited m-calpain activity via S-nitrosylation of the active site cysteine. The results of this study indicate that nitric oxide produced endogenously by skeletal muscle and other cell types has the potential to inhibit m-calpain activity and cytoskeletal proteolysis.

  20. Pericytes: multitasking cells in the regeneration of injured, diseased, and aged skeletal muscle

    PubMed Central

    Birbrair, Alexander; Zhang, Tan; Wang, Zhong-Min; Messi, Maria L.; Mintz, Akiva; Delbono, Osvaldo

    2014-01-01

    Pericytes are perivascular cells that envelop and make intimate connections with adjacent capillary endothelial cells. Recent studies show that they may have a profound impact in skeletal muscle regeneration, innervation, vessel formation, fibrosis, fat accumulation, and ectopic bone formation throughout life. In this review, we summarize and evaluate recent advances in our understanding of pericytes' influence on adult skeletal muscle pathophysiology. We also discuss how further elucidating their biology may offer new approaches to the treatment of conditions characterized by muscle wasting. PMID:25278877

  1. Akirin1 (Mighty), a novel promyogenic factor regulates muscle regeneration and cell chemotaxis

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

    Salerno, Monica Senna; Dyer, Kelly; Bracegirdle, Jeremy

    2009-07-15

    Akirin1 (Mighty) is a downstream target gene of myostatin and has been shown to be a promyogenic factor. Although expressed in many tissues, akirin1 is negatively regulated by myostatin specifically in skeletal muscle tissue. In this manuscript we have characterized the possible function of akirin1 in postnatal muscle growth. Molecular and immunohistological analyses indicated that while low levels of akirin1 are associated with quiescent satellite cells (SC), higher levels of akirin1 are detected in activated proliferating SC indicating that akirin1 could be associated with satellite cell activation. In addition to SC, macrophages also express akirin1, and increased expression of akirin1more » resulted in more efficient chemotaxis of both macrophages and myoblasts. Akirin1 appears to regulate chemotaxis of both macrophages and myoblasts by reorganising actin cytoskeleton, leading to more efficient lamellipodia formation via a PI3 kinase dependent pathway. Expression analysis during muscle regeneration also indicated that akirin1 expression is detected very early (day 2) in regenerating muscle, and expression gradually peaks to coincide the nascent myotube formation stage of muscle regeneration. Based on these results we propose that akirin1 could be acting as a transducer of early signals of muscle regeneration. Thus, we speculate that myostatin regulates key steps of muscle regeneration including chemotaxis of inflammatory cells, SC activation and migration through akirin1.« less

  2. A new index for characterizing micro-bead motion in a flow induced by ciliary beating: Part I, experimental analysis.

    PubMed

    Bottier, Mathieu; Blanchon, Sylvain; Pelle, Gabriel; Bequignon, Emilie; Isabey, Daniel; Coste, André; Escudier, Estelle; Grotberg, James B; Papon, Jean-François; Filoche, Marcel; Louis, Bruno

    2017-07-01

    Mucociliary clearance is one of the major lines of defense of the respiratory system. The mucus layer coating the pulmonary airways is moved along and out of the lung by the activity of motile cilia, thus expelling the particles trapped in it. Here we compare ex vivo measurements of a Newtonian flow induced by cilia beating (using micro-beads as tracers) and a mathematical model of this fluid flow, presented in greater detail in a second companion article. Samples of nasal epithelial cells placed in water are recorded by high-speed video-microscopy and ciliary beat pattern is inferred. Automatic tracking of micro-beads, used as markers of the flow generated by cilia motion, enables us also to assess the velocity profile as a function of the distance above the cilia. This profile is shown to be essentially parabolic. The obtained experimental data are used to feed a 2D mathematical and numerical model of the coupling between cilia, fluid, and micro-bead motion. From the model and the experimental measurements, the shear stress exerted by the cilia is deduced. Finally, this shear stress, which can easily be measured in the clinical setting, is proposed as a new index for characterizing the efficiency of ciliary beating.

  3. A new index for characterizing micro-bead motion in a flow induced by ciliary beating: Part I, experimental analysis

    PubMed Central

    Bottier, Mathieu; Blanchon, Sylvain; Pelle, Gabriel; Bequignon, Emilie; Coste, André; Escudier, Estelle; Grotberg, James B.; Papon, Jean-François

    2017-01-01

    Mucociliary clearance is one of the major lines of defense of the respiratory system. The mucus layer coating the pulmonary airways is moved along and out of the lung by the activity of motile cilia, thus expelling the particles trapped in it. Here we compare ex vivo measurements of a Newtonian flow induced by cilia beating (using micro-beads as tracers) and a mathematical model of this fluid flow, presented in greater detail in a second companion article. Samples of nasal epithelial cells placed in water are recorded by high-speed video-microscopy and ciliary beat pattern is inferred. Automatic tracking of micro-beads, used as markers of the flow generated by cilia motion, enables us also to assess the velocity profile as a function of the distance above the cilia. This profile is shown to be essentially parabolic. The obtained experimental data are used to feed a 2D mathematical and numerical model of the coupling between cilia, fluid, and micro-bead motion. From the model and the experimental measurements, the shear stress exerted by the cilia is deduced. Finally, this shear stress, which can easily be measured in the clinical setting, is proposed as a new index for characterizing the efficiency of ciliary beating. PMID:28708889

  4. DNA methylation and differentiation: HOX genes in muscle cells

    PubMed Central

    2013-01-01

    Background Tight regulation of homeobox genes is essential for vertebrate development. In a study of genome-wide differential methylation, we recently found that homeobox genes, including those in the HOX gene clusters, were highly overrepresented among the genes with hypermethylation in the skeletal muscle lineage. Methylation was analyzed by reduced representation bisulfite sequencing (RRBS) of postnatal myoblasts, myotubes and adult skeletal muscle tissue and 30 types of non-muscle-cell cultures or tissues. Results In this study, we found that myogenic hypermethylation was present in specific subregions of all four HOX gene clusters and was associated with various chromatin epigenetic features. Although the 3′ half of the HOXD cluster was silenced and enriched in polycomb repression-associated H3 lysine 27 trimethylation in most examined cell types, including myoblasts and myotubes, myogenic samples were unusual in also displaying much DNA methylation in this region. In contrast, both HOXA and HOXC clusters displayed myogenic hypermethylation bordering a central region containing many genes preferentially expressed in myogenic progenitor cells and consisting largely of chromatin with modifications typical of promoters and enhancers in these cells. A particularly interesting example of myogenic hypermethylation was HOTAIR, a HOXC noncoding RNA gene, which can silence HOXD genes in trans via recruitment of polycomb proteins. In myogenic progenitor cells, the preferential expression of HOTAIR was associated with hypermethylation immediately downstream of the gene. Other HOX gene regions also displayed myogenic DNA hypermethylation despite being moderately expressed in myogenic cells. Analysis of representative myogenic hypermethylated sites for 5-hydroxymethylcytosine revealed little or none of this base, except for an intragenic site in HOXB5 which was specifically enriched in this base in skeletal muscle tissue, whereas myoblasts had predominantly 5

  5. Differential Effect of Zoledronic Acid on Human Vascular Smooth Muscle Cells

    PubMed Central

    Albadawi, Hassan; Haurani, Mounir J.; Oklu, Rahmi; Trubiano, Jordan P.; Laub, Peter J.; Yoo, Hyung-Jin; Watkins, Michael T.

    2012-01-01

    Introduction The activation of human vascular smooth muscle cell proliferation, adhesion and migration is essential for intimal hyperplasia formation. These experiments were designed to test whether Zoledronic Acid (ZA) would modulate indices of human smooth muscle cell activation, exert differential effects on proliferating vs. quiescent cells and determine whether these effects were dependent on GTPase binding proteins prenylation. ZA was chosen for testing in these experiments because it is clinically used in humans with cancer, and has been shown to modulate rat smooth muscle cell proliferation and migration. Methods Human aortic smooth muscle cells (HASMC) were cultured under either proliferating or growth arrest (quiescent) conditions in the presence or absence of ZA for 48 hours, whereupon the effect of ZA on HASMC proliferation, cellular viability, metabolic activity and membrane integrity were compared. In addition, the effect of ZA on adhesion and migration were assessed in proliferating cells. The effect of increased concentration of ZA on the mevalonate pathway and genomic/cellular stress related poly ADP Ribose polymerase (PARP) enzyme activity were assessed using the relative prenylation of Rap-1A/B protein and the formation of poly ADP- ribosylated proteins (PAR) respectively. Results There was a dose dependent inhibition of cellular proliferation, adhesion and migration following ZA treatment. ZA treatment decreased indices of cellular viability and significantly increased membrane injury in proliferating vs. quiescent cells. This was correlated with the appearance of unprenylated Rap-1A protein and dose dependent down regulation of PARP activity. Conclusions These data suggest that ZA is effective in inhibiting HASMC proliferation, adhesion and migration which coincide with the appearance of unprenylated RAP-1A/B protein, thereby suggesting that the mevalonate pathway may play a role in the inhibition of HASMC activation. PMID:23164362

  6. The quasi-parallel lives of satellite cells and atrophying muscle

    PubMed Central

    Biressi, Stefano; Gopinath, Suchitra D.

    2015-01-01

    Skeletal muscle atrophy or wasting accompanies various chronic illnesses and the aging process, thereby reducing muscle function. One of the most important components contributing to effective muscle repair in postnatal organisms, the satellite cells (SCs), have recently become the focus of several studies examining factors participating in the atrophic process. We critically examine here the experimental evidence linking SC function with muscle loss in connection with various diseases as well as aging, and in the subsequent recovery process. Several recent reports have investigated the changes in SCs in terms of their differentiation and proliferative capacity in response to various atrophic stimuli. In this regard, we review the molecular changes within SCs that contribute to their dysfunctional status in atrophy, with the intention of shedding light on novel potential pharmacological targets to counteract the loss of muscle mass. PMID:26257645

  7. Inadequate satellite cell replication compromises muscle regrowth following postnatal nutrient restriction

    USDA-ARS?s Scientific Manuscript database

    Perinatal growth impairment permanently compromises skeletal muscle mass. The present study assessed the contribution of muscle satellite cell replicative capacity to this deficit. Mouse dams were fed either a low protein (LP, n=7) or control (C, n=6) diet during lactation. Pups were weaned at 21 d ...

  8. Palladium-103 plaque radiation therapy for ciliary body melanoma through a functioning glaucoma filtering bleb.

    PubMed

    Pathan, Arif H K; Barash, Alexander; Tena, Lawrence B; Finger, Paul T

    2018-01-01

    To provide a clinical description of the long-term outcome of a 103 Pd plaque-irradiated ciliary body melanoma with extrascleral extension while attempting to preserve a subadjacent glaucoma filtering bleb. A 75-year-old woman with pseudoexfoliative glaucoma for 17 years, 16 years status post argon laser trabeculoplasty, and 15 years status post trabeculectomy in the left eye, was diagnosed with an ipsilateral ciliary body melanoma with visible extrascleral extension. Treatment involved insertion of a 103 Pd radioactive plaque over the functioning trabeculectomy, with removal 7 days later. At plaque insertion, amniotic membrane grafts were used to cover the plaque and protect the filtering site. The tumor was successfully treated without clinical evidence of harm to the filtering bleb, with resultant stable intraocular pressure. However, the patient developed blebitis 1.5 years later. Though it resolved with topical antibiotic therapy, the bleb became less succulent. Two years postoperatively, she developed a spontaneous hyphema that resolved after one injection of transscleral bevacizumab 1.25 mg. Her tumor continually regressed in thickness. Without additional glaucoma surgery, her intraocular pressure remained well-controlled on topical medications for 6 years. Ciliary body melanoma with minimal extrascleral extension beneath a functioning filtering bleb can be treated using radioactive plaque therapy. In this case, we were able to achieve both tumor regression and glaucoma control by covering the plaque with an amniotic membrane graft.

  9. Vortex arrays and ciliary tangles underlie the feeding-swimming tradeoff in starfish larvae

    NASA Astrophysics Data System (ADS)

    Gilpin, William; Prakash, Vivek N.; Prakash, Manu

    2016-11-01

    Many marine invertebrates have larval stages covered in linear arrays of beating cilia, which propel the animal while simultaneously entraining planktonic prey. These bands are strongly conserved across taxa spanning four major superphyla, and they are responsible for the unusual morphologies of many invertebrates. However, few studies have investigated their underlying hydrodynamics. Here, we study the ciliary bands of starfish larvae, and discover a beautiful pattern of slowly-evolving vortices that surrounds the swimming animals. Closer inspection of the bands reveals unusual ciliary "tangles" analogous to topological defects that break-up and re-form as the animal adjusts its swimming stroke. Quantitative experiments and modeling demonstrate that these vortices create a physical tradeoff between feeding and swimming in heterogenous environments, which manifests as distinct flow patterns or "eigenstrokes" representing each behavior-potentially implicating neuronal control of cilia. This quantitative interplay between larval form and hydrodynamic function generalizes to other invertebrates, and illustrates the potential effects of active boundary conditions in other biological and synthetic systems.

  10. Comparison of Different Muscle-Relaxant Anesthetics on Growth, Migration and Invasion of Gastric Cancer Cells.

    PubMed

    Jiang, Aihua; Zhao, Huishan; Liu, Xiaofei; Yu, Mingwei; Chen, Jian; Jiang, Wen G

    2017-08-01

    Muscle relaxants, also known as neuromuscular blocking agents, can block nerve impulses to the muscles and are always used in surgery for general anesthesia. However, the effect of muscle-relaxant anesthetics on cell activity in gastric cancer is currently unknown. The present study aimed to examine and compare the role of three different muscle-relaxant anesthetics in gastric cancer cells. Gastric cancer cells (SGC7901 and BGC 823) were treated with a different dose of muscle-relaxant anesthetics, Rocuronium bromide (Rb), Vecuronium bromide (Vb) and Cisatracurium Besilate (CB). Using in vitro models, the effects on gastric cancer cell invasion, growth and migration of various anesthetics were subsequently investigated. We found that Rb increased the growth, invasion and migration of gastric cancer cells SGC7901 and BGC823. However, Vb and CB, as relatively mitigative anesthetics, did not significantly affect gastric cancer cell malignant phenotype at their regular blood concentration. Our results are important in selecting the type and dose of anesthetic used for surgery of gastric cancer patients. An understanding of the effect of muscle-relaxant anesthetics and their impact on tumor metastasis is critical, since it provides insight into the appropriate anesthetic strategy that could improve long-term survival in some patients with gastric cancer. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  11. Optimal ciliary beating patterns

    NASA Astrophysics Data System (ADS)

    Vilfan, Andrej; Osterman, Natan

    2011-11-01

    We introduce a measure for energetic efficiency of single or collective biological cilia. We define the efficiency of a single cilium as Q2 / P , where Q is the volume flow rate of the pumped fluid and P is the dissipated power. For ciliary arrays, we define it as (ρQ) 2 / (ρP) , with ρ denoting the surface density of cilia. We then numerically determine the optimal beating patterns according to this criterion. For a single cilium optimization leads to curly, somewhat counterintuitive patterns. But when looking at a densely ciliated surface, the optimal patterns become remarkably similar to what is observed in microorganisms like Paramecium. The optimal beating pattern then consists of a fast effective stroke and a slow sweeping recovery stroke. Metachronal waves lead to a significantly higher efficiency than synchronous beating. Efficiency also increases with an increasing density of cilia up to the point where crowding becomes a problem. We finally relate the pumping efficiency of cilia to the swimming efficiency of a spherical microorganism and show that the experimentally estimated efficiency of Paramecium is surprisingly close to the theoretically possible optimum.

  12. Muscle stem cell intramuscular delivery within hyaluronan methylcellulose improves engraftment efficiency and dispersion.

    PubMed

    Davoudi, Sadegh; Chin, Chih-Ying; Cooke, Michael J; Tam, Roger Y; Shoichet, Molly S; Gilbert, Penney M

    2018-04-26

    Adult skeletal muscle tissue harbors the capacity for self-repair due to the presence of tissue resident muscle stem cells (MuSCs). Advances in the area of prospective MuSC isolation demonstrated the potential of cell transplantation therapy as a regenerative medicine strategy to restore strength and long-term regenerative capacity to aged, injured, or diseased skeletal muscle tissue. However, cell loss during ejection, limits to post-injection proliferation, and poor donor cell dispersion distal to the injection site are amongst hurdles to overcome to maximize MuSC transplant impact. Here, we assess a physical blend of hyaluronan and methylcellulose (HAMC) as a bioactive, shear thinning hydrogel cell delivery system to improve MuSC transplantation efficiency. Using in vivo transplantation studies, we found that the HAMC delivery system results in a >45% increase in the number of donor-derived fibers as compared to saline delivery. We demonstrate that increases in donor-derived fibers when using HAMC are attributed to increased MuSC proliferation via a CD44-independent mechanism, preventing injected cell active clearance, and supporting in vivo expansion by delaying differentiation. Furthermore, we observed a significant improvement in donor fiber dispersion when MuSCs were delivered in HAMC. Our study results suggest that HAMC is a promising muscle stem cell delivery vehicle. Copyright © 2018 Elsevier Ltd. All rights reserved.

  13. Inflammation induced by mast cell deficiency rather than the loss of interstitial cells of Cajal causes smooth muscle dysfunction in W/Wv mice

    PubMed Central

    Winston, John H.; Chen, Jinghong; Shi, Xuan-Zheng; Sarna, Sushil K.

    2014-01-01

    The initial hypothesis suggested that the interstitial cells of Cajal (ICC) played an essential role in mediating enteric neuronal input to smooth muscle cells. Much information for this hypothesis came from studies in W/Wv mice lacking ICC. However, mast cells, which play critical roles in regulating inflammation in their microenvironment, are also absent in W/Wv mice. We tested the hypothesis that the depletion of mast cells in W/Wv mice generates inflammation in fundus muscularis externa (ME) that impairs smooth muscle reactivity to Ach, independent of the depletion of ICC. We performed experiments on the fundus ME from wild type (WT) and W/Wv mice before and after reconstitution of mast cells by bone marrow transplant. We found that mast cell deficiency in W/Wv mice significantly increased COX-2 and iNOS expression and decreased smooth muscle reactivity to Ach. Mast cell reconstitution or concurrent blockade of COX-2 and iNOS restored smooth muscle contractility without affecting the suppression of c-kit in W/Wv mice. The expression of nNOS and ChAT were suppressed in W/Wv mice; mast cell reconstitution did not restore them. We conclude that innate inflammation induced by mast cell deficiency in W/Wv mice impairs smooth muscle contractility independent of ICC deficiency. The impairment of smooth muscle contractility and the suppression of the enzymes regulating the synthesis of Ach and NO in W/Wv mice need to be considered in evaluating the role of ICC in regulating smooth muscle and enteric neuronal function in W/Wv mice. PMID:24550836

  14. Asynchronous inflammation and myogenic cell migration limit muscle tissue regeneration mediated by a cellular scaffolds

    PubMed Central

    Garg, Koyal; Ward, Catherine L.; Corona, Benjamin T.

    2016-01-01

    Volumetric muscle loss (VML) following orthopaedic trauma results in chronic loss of strength and can contribute to disability. Tissue engineering and regenerative medicine approaches to regenerate the lost skeletal muscle and improve functional outcomes are currently under development. At the forefront of these efforts, decellularized extracellular matrices (ECMs) have reached clinical testing and provide the foundation for other approaches that include stem/progenitor cell delivery. ECMs have been demonstrated to possess many qualities to initiate regeneration, to include stem cell chemotaxis and pro-regenerative macrophage polarization. However, the majority of observations indicate that ECM-repair of VML does not promote appreciable muscle fiber regeneration. In a recent study, ECM-repair of VML was compared to classical muscle fiber regeneration (Garg et al., 2014, Cell & Tissue Research) mediated by autologous minced grafts. The most salient findings of this study were: 1) Satellite cells did not migrate into the scaffold beyond ~0.5 mm from the remaining host tissue, although other migratory stem cells (Sca-1+) were observed throughout the scaffold;2) Macrophage migration to the scaffold was over two-times that observed with muscle grafts, but they appeared to be less active, as gene expression of pro- and anti-inflammatory cytokines (TNF-α, IL-12, IL-4, IL-10, VEGF, and TGF-β1) was significantly reduced in scaffold-repaired muscles; And, 3) scaffolds did not promote appreciable muscle fiber regeneration. Collectively, these data suggest that the events following ECM transplantation in VML are either incongruous or asynchronous with classical muscle fiber regeneration. PMID:26949720

  15. Regulatory T cells suppress muscle inflammation and injury in muscular dystrophy

    PubMed Central

    Villalta, S. Armando; Rosenthal, Wendy; Martinez, Leonel; Kaur, Amanjot; Sparwasser, Tim; Tidball, James G.; Margeta, Marta; Spencer, Melissa J.; Bluestone, Jeffrey A.

    2016-01-01

    We examined the hypothesis that regulatory T cells (Tregs) modulate muscle injury and inflammation in the mdx mouse model of Duchenne muscular dystrophy (DMD). Although Tregs were largely absent in the muscle of wildtype mice and normal human muscle, they were present in necrotic lesions, displayed an activated phenotype and showed increased expression of interleukin (IL)-10 in dystrophic muscle from mdx mice. Depletion of Tregs exacerbated muscle injury and the severity of muscle inflammation, which was characterized by an enhanced interferon-gamma (IFNγ) response and activation of M1 macrophages. To test the therapeutic value of targeting Tregs in muscular dystrophy, we treated mdx mice with IL-2/anti-IL-2 complexes (IL-2c), and found that Tregs and IL-10 concentrations were increased in muscle, resulting in reduced expression of cyclooygenase-2 and decreased myofiber injury. These findings suggest that Tregs modulate the progression of muscular dystrophy by suppressing type 1 inflammation in muscle associated with muscle fiber injury, and highlight the potential of Treg-modulating agents as therapeutics for DMD. PMID:25320234

  16. Laminin-111 improves skeletal muscle stem cell quantity and function following eccentric exercise.

    PubMed

    Zou, Kai; De Lisio, Michael; Huntsman, Heather D; Pincu, Yair; Mahmassani, Ziad; Miller, Matthew; Olatunbosun, Dami; Jensen, Tor; Boppart, Marni D

    2014-09-01

    Laminin-111 (α1, β1, γ1; LM-111) is an important component of the extracellular matrix that is required for formation of skeletal muscle during embryonic development. Recent studies suggest that LM-111 supplementation can enhance satellite cell proliferation and muscle function in mouse models of muscular dystrophy. The purpose of this study was to determine the extent to which LM-111 can alter satellite and nonsatellite stem cell quantity following eccentric exercise-induced damage in young adult, healthy mice. One week following injection of LM-111 or saline, mice either remained sedentary or were subjected to a single bout of downhill running (EX). While one muscle was preserved for evaluation of satellite cell number, the other muscle was processed for isolation of mesenchymal stem cells (MSCs; Sca-1+CD45-) via FACS at 24 hours postexercise. Satellite cell number was approximately twofold higher in LM-111/EX compared with all other groups (p<.05), and the number of satellite cells expressing the proliferation marker Ki67 was 50% to threefold higher in LM-111/EX compared with all other groups (p<.05). LM-111 also increased the quantity of embryonic myosin heavy chain-positive (eMHC+) fibers in young mice after eccentric exercise (p<.05). Although MSC percentage and number were not altered, MSC proinflammatory gene expression was decreased, and hepatocyte growth factor gene expression was increased in the presence of LM-111 (p<.05). Together, these data suggest that LM-111 supplementation provides a viable solution for increasing skeletal muscle stem cell number and/or function, ultimately allowing for improvements in the regenerative response to eccentric exercise. ©AlphaMed Press.

  17. Triptolide inhibits TGF-β1-induced cell proliferation in rat airway smooth muscle cells by suppressing Smad signaling

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

    Chen, Ming; Lv, Zhiqiang; Huang, Linjie

    Background: We have reported that triptolide can inhibit airway remodeling in a murine model of asthma via TGF-β1/Smad signaling. In the present study, we aimed to investigate the effect of triptolide on airway smooth muscle cells (ASMCs) proliferation and the possible mechanism. Methods: Rat airway smooth muscle cells were cultured and made synchronized, then pretreated with different concentration of triptolide before stimulated by TGF-β1. Cell proliferation was evaluated by MTT assay. Flow cytometry was used to study the influence of triptolide on cell cycle and apoptosis. Signal proteins (Smad2, Smad3 and Smad7) were detected by western blotting analysis. Results: Triptolidemore » significantly inhibited TGF-β1-induced ASMC proliferation (P<0.05). The cell cycle was blocked at G1/S-interphase by triptolide dose dependently. No pro-apoptotic effects were detected under the concentration of triptolide we used. Western blotting analysis showed TGF-β1 induced Smad2 and Smad3 phosphorylation was inhibited by triptolide pretreatment, and the level of Smad7 was increased by triptolide pretreatment. Conclusions: Triptolide may function as an inhibitor of asthma airway remodeling by suppressing ASMCs proliferation via negative regulation of Smad signaling pathway. - Highlights: • In this study, rat airway smooth muscle cells were cultured and made synchronized. • Triptolide inhibited TGF-β1-induced airway smooth muscle cells proliferation. • Triptolide inhibited ASMCs proliferation via negative regulation of Smad signaling pathway.« less

  18. Monocyte activation by smooth muscle cell-derived matrices.

    PubMed

    Kaufmann, J; Jorgensen, R W; Martin, B M; Franzblau, C

    1990-12-01

    Mononuclear phagocytes adhere to and penetrate the vessel wall endothelium and contact the subendothelial space prior to the development of the atherosclerotic plaque. In an attempt to model the early events of plaque development we used an elastin-rich, multicomponent, cell-derived matrix from neonatal rat aortic smooth muscle cells as a substratum for monocytes. Using this model, we show that human monocyte morphology and metabolism are markedly altered by the matrix substratum. When a mixed mononuclear cell population is seeded on matrix or plastic, only monocytes adhere to the matrix surface. In contrast, lymphocytes as well as monocytes adhere to the plastic surface. The matrix-adherent monocytes develop large intracellular granules and form extensive clusters of individual cells. Metabolically, these cells develop sodium fluoride resistant non-specific esterase activity and their media contain more growth factor activity and PGE2. Although total protein synthesis is equivalent in both cultures, the matrix contact induces an increase in specific proteins in the media. We also show that a purified alpha-elastin substratum induces some, but not all, of the monocyte changes seen when using the matrix substratum. Using the alpha-elastin substratum, there is selective adhesion of monocytes and increased growth factor activity, however, the cells are morphologically different from the matrix-adherent cells. Thus, the use of the smooth muscle cell-derived matrix, in conjunction with purified matrix components, serves as a model that can provide insight into the mechanisms of monocyte adhesion and stimulation by the matrix environment that exists in vivo. Such mechanisms may be particularly important in atherogenesis.

  19. Immortalization of cat iris sphincter smooth muscle cells by SV40 virus: growth, morphological, biochemical and pharmacological characteristics.

    PubMed

    Ocklind, A; Yousufzai, S Y; Ghosh, S; Coca-Prados, M; St Jernschantz, J; Abdel-Latif, A A

    1995-11-01

    The purpose of this study was to establish immortalized cell cultures of cat iris sphincter smooth muscle cells for a model investigating ocular receptors and their signal transduction pathways. Cultured cat iris sphincter muscle cells were immortalized by viral transformation with SV40 virus and the morphological and immunocytochemical properties of the normal and immortalized cells were investigated. The transformed cell clone, SV-CISM-2, was further characterized biochemically and pharmacologically. The normal muscle cells showed characteristics of smooth muscle cells, as judged by their growth and the presence of smooth muscle alpha-actin and desmin. After seven passages the normal cells ceased to proliferate. In contrast, the immortalized cells retained their proliferative ability for more than 220 population doublings over 55 passages. The transformation phenotype in these cells was confirmed by their expression of the large T-antigen, the incorporation of viral DNA into cellular DNA, growth in agarose and in low-serum medium, and complete loss of contact inhibition. The immortalized cells expressed smooth muscle alpha-actin, desmin and MLC protein. Biochemical and pharmacological studies on the SV-CISM cells revealed the presence of several functional receptors including muscarinic cholinergic, beta-adrenergic, peptidergic (substance P and endothelin). Platelet-activating factor, and prostaglandin (PG). Muscarinic stimulation of these cells resulted in: (a) a dose-dependent increase in the release of arachidonic acid (AA) and (PGs) and enhancement in the production of inositol trisphosphate (IP3); and (b) a substantial increase in MLC phosphorylation (118%), an indicator of smooth muscle contractility. The stimulatory effects of carbachol on these responses were completely blocked by atropine, a muscarinic receptor antagonist. This study constitutes the first successful immortalization of iris sphincter smooth muscle cells. The SV-CISM-2 cells can serve as

  20. Restoration of heart functions using human embryonic stem cells derived heart muscle cells.

    PubMed

    Gepstein, Lior; Kehat, Izhak

    2005-02-01

    Extract: Recent advances in molecular and cellular biology and specifically in the areas of stem cell biology and tissue engineering have paved the way for the development of a new field in biomedicine, regenerative medicine. This exciting approach seeks to develop new biological solutions, using the mobilization of endogenous stem cells or delivery of exogenous cells to replace or modify the function of diseased, absent, or malfunctioning tissue. The adult heart represents an attractive candidate for these emerging technologies, since adult cardiomyocytes have limited regenerative capacity. Thus, any significant heart cell loss or dysfunction, such as occurs during heart attack, is mostly irreversible and may lead to the development of progressive heart failure, one of the leading causes of world-wide morbidity and mortality. Similarly, dysfunction of the specialized electrical conduction system within the heart may result in inefficient rhythm initiation or impulse conduction, leading to significant slowing of the heart rate, usually requiring the implantation of a permanent electronic pacemaker. Replacement of the dysfunctional myocardium (heart muscle) by implantation of external heart muscle cells is emerging as a novel paradigm for restoration of the myocardial electromechanical properties, but has been significantly hampered by the paucity of cell sources for human heart cells and by the relatively limited evidence for functional integration between grafted and host cells. The recently described human embryonic stem cell (hESC) lines may provide a possible solution for the aforementioned cell sourcing problem.