L-type Ca2+ channel function is linked to dystrophin expression in mammalian muscle

Background

In dystrophic mdx skeletal muscle, aberrant Ca²⁺ homeostasis and fibre degeneration are found. The absence of dystrophin in models of Duchenne muscular dystrophy (DMD) has been connected to altered ion channel properties e.g. impaired L-type Ca²⁺ currents. In regenerating mdx muscle, ‘revertant’ fibres restore dystrophin expression. Their functionality involving DHPR-Ca²⁺-channels is elusive.

Methods and Results

We developed a novel ‘in-situ’ confocal immuno-fluorescence and imaging technique that allows, for the first time, quantitative subcellular dystrophin-DHPR colocalization in individual, non-fixed, muscle fibres. Tubular DHPR signals alternated with second harmonic generation signals originating from myosin. Dystrophin-DHPR colocalization was substantial in wt fibres, but diminished in most mdx fibres. Mini-dystrophin (MinD) expressing fibres successfully restored colocalization. Interestingly, in some aged mdx fibres, colocalization was similar to wt fibres. Most mdx fibres showed very weak membrane dystrophin staining and were classified ‘mdx-like’. Some mdx fibres, however, had strong ‘wt-like’ dystrophin signals and were identified as ‘revertants’. Split mdx fibres were mostly ‘mdx-like’ and are not generally ‘revertants’. Correlations between membrane dystrophin and DHPR colocalization suggest a restored putative link in ‘revertants’. Using the two-micro-electrode-voltage clamp technique, Ca²⁺-current amplitudes (i_max) showed very similar behaviours: reduced amplitudes in most aged mdx fibres (as seen exclusively in young mdx mice) and a few mdx fibres, most likely ‘revertants’, with amplitudes similar to wt or MinD fibres. Ca²⁺ current activation curves were similar in ‘wt-like’ and ‘mdx-like’ aged mdx fibres and are not the cause for the differences in current amplitudes. i_max amplitudes were fully restored in MinD fibres.

Conclusions

We present evidence for a direct/indirect DHPR-dystrophin interaction present in wt, MinD and ‘revertant’ mdx fibres but absent in remaining mdx fibres. Our imaging technique reliably detects single isolated ‘revertant’ fibres that could be used for subsequent physiological experiments to study mechanisms and therapy concepts in DMD.     

Hmgb3 Is Regulated by MicroRNA-206 during Muscle Regeneration

Background

MicroRNAs (miRNAs) have been recently involved in most of human diseases as targets for potential strategies to rescue the pathological phenotype. Since the skeletal muscle is a spread-wide highly differentiated and organized tissue, rescue of severely compromised muscle still remains distant from nowadays. For this reason, we aimed to identify a subset of miRNAs major involved in muscle remodelling and regeneration by analysing the miRNA-profile of single fibres isolated from dystrophic muscle, which was here considered as a model of chronic damage.

Methodology/Principal Findings

The miRNA-signature associated to regenerating (newly formed) and remodelling (resting) fibres was investigated in animal models of muscular dystrophies and acute damage, in order to distinguish which miRNAs are primary related to muscle regeneration. In this study we identify fourteen miRNAs associated to dystrophic fibres responsible for muscle regeneration and remodelling, and confirm over-expression of the previously identified regeneration-associated myomiR-206. In particular, a functional binding site for myomiR-206 was identified and validated in the 3′untranslated region (3′UTR) of an X-linked member of a family of sequence independent chromatin-binding proteins (Hmgb3) that is preferentially expressed in hematopoietic stem cells. During regeneration of single muscle fibres, Hmgb3 messenger RNA (mRNA) and protein expression was gradually reduced, concurrent with the up-regulation of miR-206.

Conclusion/Significance

Our results elucidate a negative feedback circuit in which myomiR-206 represses Hmgb3 expression to modulate the regeneration of single muscle fibres after acute and chronic muscle damage. These findings suggest that myomiR-206 may be a potential therapeutic target in muscle diseases.     

Resistance training inhibits the elevation of skeletal muscle derived-BDNF level concomitant with improvement of muscle strength in zucker diabetic rat

[Purpose]

In the present study, we investigated the effects of 8 weeks of progressive resistance training on the level of skeletal muscle derived BDNF as well as glucose intolerance in Zucker diabetic rats.

[Methods]

Six week-old male Zucker diabetic fatty (ZDF) and Zucker lean control (ZLC) rats were randomly divided into 3 groups: sedentary ZLC (ZLC-Con), sedentary ZDF (ZDF-Con), and exercised ZDF (ZDF-Ex). Progressive resistance training using a ladder and tail weights was performed for 8 weeks (3 days/week).

[Results]

After 8 weeks of resistance training, substantial reduction in body weight was observed in ZDF-Ex compared to ZDF-Con. Though the skeletal muscle volume did not change, grip strength grip strength was significantly higher in ZDF-Ex compared to ZDF-Con. In the soleus, the level of BDNF was increased in ZDF-Con, but was significantly decreased (p<0.05) in ZDF-Ex, showing a training effect. Moreover, we found that there was a negative correlation (r=-0.657; p=0.004) between grip strength and BDNF level whereas there was a positive correlation (r=0.612; p=0.008) between plasma glucose level and BDNF level in skeletal muscle.

[Conclusion]

Based upon our results, we demonstrated that resistance training inhibited the elevation of skeletal muscle derived-BDNF expression concomitant with the improvement of muscle strength in zucker diabetic rats. In addition, muscle-derived BDNF might be a potential mediator for the preventive effect of resistance training on the progress of type 2 diabetes.     

The Vitamin D Receptor Agonist BXL-01-0029 as a Potential New Pharmacological Tool for the Treatment of Inflammatory Myopathies

Objective

This study aims to investigate in vitro the effect of the VDR agonist BXL-01-0029 onto IFNγ/TNFα-induced CXCL10 secretion by human skeletal muscle cells compared to elocalcitol (VDR agonist), methylprednisolone, methotrexate, cyclosporin A, infliximab and leflunomide; to assess in vivo circulating CXCL10 level in subjects at time of diagnosis with IMs, before therapy, together with TNFα, IFNγ, IL-8, IL-6, MCP-1, MIP-1β and IL-10, vs. healthy subjects.

Methods

Human fetal skeletal muscle cells were used for in vitro studies; ELISA and Bio-Plex were used to measure cell supernatant and IC₅₀ determination or serum cytokines; Western blot and Bio-Plex were for cell signaling analysis.

Results

BXL-01-0029 decreased with the highest potency IFNγ/TNFα-induced CXCL10 protein secretion and targeted cell signaling downstream of TNFα in human skeletal muscle cells; CXCL10 level was the highest in sera of subjects diagnosed with IMs before therapy and the only one significantly different vs. healthy controls.

Conclusions

Our in vitro and in vivo data, while confirm the relevance of CXCL10 in IMs, suggested BXL-01-0029 as a novel pharmacological tool for IM treatment, hypothetically to be used in combination with the current immunosuppressants to minimize side effects.     

Poor regenerative outcome after skeletal muscle necrosis induced by Bothrops asper venom: alterations in microvasculature and nerves

Background

Viperid snakebite envenoming is characterized by prominent local tissue damage, including muscle necrosis. A frequent outcome of such local pathology is deficient skeletal muscle regeneration, which causes muscle dysfunction, muscle loss and fibrosis, thus provoking permanent sequelae that greatly affect the quality of life of patients. The causes of such poor regenerative outcome of skeletal muscle after viperid snakebites are not fully understood.

Methodology/Principal Findings

A murine model of muscle necrosis and regeneration was adapted to study the effects of the venom and isolated toxins of Bothrops asper, the medically most important snake in Central America. Gastrocnemius muscle was injected with either B. asper venom, a myotoxic phospholipase A₂ (Mtx), a hemorrhagic metalloproteinase (SVMP), or saline solution. At various time intervals, during one month, tissue samples were collected and analyzed by histology, and by immunocytochemical and immunohistochemical techniques aimed at detecting muscle fibers, collagen, endothelial cells, myoblasts, myotubes, macrophages, TUNEL-positive nuclei, and axons. A successful regenerative response was observed in muscle injected with Mtx, which induces myonecrosis but does not affect the microvasculature. In contrast, poor regeneration, with fibrosis and atrophic fibers, occurred when muscle was injected with venom or SVMP, both of which provoke necrosis, microvascular damage leading to hemorrhage, and poor axonal regeneration.

Conclusions/Significance

The deficient skeletal muscle regeneration after injection of B. asper venom is likely to depend on the widespread damage to the microvasculature, which affects the removal of necrotic debris by phagocytes, and the provision of nutrients and oxygen required for regeneration. In addition, deficient axonal regeneration is likely to contribute to the poor regenerative outcome in this model.     

Expression and functional roles of angiopoietin-2 in skeletal muscles

Background

Angiopoietin-1 (ANGPT1) and angiopoietin-2 (ANGPT2) are angiogenesis factors that modulate endothelial cell differentiation, survival and stability. Recent studies have suggested that skeletal muscle precursor cells constitutively express ANGPT1 and adhere to recombinant ANGPT1 and ANGPT2 proteins. It remains unclear whether or not they also express ANGPT2, or if ANGPT2 regulates the myogenesis program of muscle precursors. In this study, ANGPT2 regulatory factors and the effects of ANGPT2 on proliferation, migration, differentiation and survival were identified in cultured primary skeletal myoblasts. The cellular networks involved in the actions of ANGPT2 on skeletal muscle cells were also analyzed.

Methodology/Principal Findings

Primary skeletal myoblasts were isolated from human and mouse muscles. Skeletal myoblast survival, proliferation, migration and differentiation were measured in-vitro in response to recombinant ANGPT2 protein and to enhanced ANGPT2 expression delivered with adenoviruses. Real-time PCR and ELISA measurements revealed the presence of constitutive ANGPT2 expression in these cells. This expression increased significantly during myoblast differentiation into myotubes. In human myoblasts, ANGPT2 expression was induced by H₂O₂, but not by TNFα, IL1β or IL6. ANGPT2 significantly enhanced myoblast differentiation and survival, but had no influence on proliferation or migration. ANGPT2-induced survival was mediated through activation of the ERK1/2 and PI-3 kinase/AKT pathways. Microarray analysis revealed that ANGPT2 upregulates genes involved in the regulation of cell survival, protein synthesis, glucose uptake and free fatty oxidation.

Conclusion/Significance

Skeletal muscle precursors constitutively express ANGPT2 and this expression is upregulated during differentiation into myotubes. Reactive oxygen species exert a strong stimulatory influence on muscle ANGPT2 expression while pro-inflammatory cytokines do not. ANGPT2 promotes skeletal myoblast survival and differentiation. These results suggest that muscle-derived ANGPT2 production may play a positive role in skeletal muscle fiber repair.     

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

Background

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.

Methodology/Principal Findings

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.

Conclusions/Significance

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.     

Participation of myosin Va and Pka type I in the regeneration of neuromuscular junctions

Background

The unconventional motor protein, myosin Va, is crucial for the development of the mouse neuromuscular junction (NMJ) in the early postnatal phase. Furthermore, the cooperative action of protein kinase A (PKA) and myosin Va is essential to maintain the adult NMJ. We here assessed the involvement of myosin Va and PKA in NMJ recovery during muscle regeneration.

Methodology/Principal Findings

To address a putative role of myosin Va and PKA in the process of muscle regeneration, we used two experimental models the dystrophic mdx mouse and Notexin-induced muscle degeneration/regeneration. We found that in both systems myosin Va and PKA type I accumulate beneath the NMJs in a fiber maturation-dependent manner. Morphologically intact NMJs were found to express stable nicotinic acetylcholine receptors and to accumulate myosin Va and PKA type I in the subsynaptic region. Subsynaptic cAMP signaling was strongly altered in dystrophic muscle, particularly in fibers with severely subverted NMJ morphology.

Conclusions/Significance

Our data show a correlation between the subsynaptic accumulation of myosin Va and PKA type I on the one hand and NMJ regeneration status and morphology, AChR stability and specificity of subsynaptic cAMP handling on the other hand. This suggests an important role of myosin Va and PKA type I for the maturation of NMJs in regenerating muscle.     

Direct isolation, culture and transplant of mouse skeletal muscle derived endothelial cells with angiogenic potential

Background

Although diseases associated with microvascular endothelial dysfunction are among the most prevalent illnesses to date, currently no method exists to isolate pure endothelial cells (EC) from skeletal muscle for in vivo or in vitro study.

Methodology

By utilizing multicolor fluorescent-activated cell sorting (FACS), we have isolated a distinct population of Sca-1⁺, CD31⁺, CD34^dim and CD45⁻ cells from skeletal muscles of C57BL6 mice. Characterization of this population revealed these cells are functional EC that can be expanded several times in culture without losing their phenotype or capabilities to uptake acetylated low-density lipoprotein (ac-LDL), produce nitric oxide (NO) and form vascular tubes. When transplanted subcutaneously or intramuscularly into the tibialis anterior muscle, EC formed microvessels and integrated with existing vasculature.

Conclusion

This method, which is highly reproducible, can be used to study the biology and role of EC in diseases such as peripheral vascular disease. In addition this method allows us to isolate large quantities of skeletal muscle derived EC with potential for therapeutic angiogenic applications.     

Mural Cell Associated VEGF Is Required for Organotypic Vessel Formation

Background

Blood vessels comprise endothelial cells, mural cells (pericytes/vascular smooth muscle cells) and basement membrane. During angiogenesis, mural cells are recruited to sprouting endothelial cells and define a stabilizing context, comprising cell-cell contacts, secreted growth factors and extracellular matrix components, that drives vessel maturation and resistance to anti-angiogenic therapeutics.

Methods and Findings

To better understand the basis for mural cell regulation of angiogenesis, we conducted high content imaging analysis on a microtiter plate format in vitro organotypic blood vessel system comprising primary human endothelial cells co-cultured with primary human mural cells. We show that endothelial cells co-cultured with mural cells undergo an extensive series of phenotypic changes reflective of several facets of blood vessel formation and maturation: Loss of cell proliferation, pathfinding-like cell migration, branching morphogenesis, basement membrane extracellular matrix protein deposition, lumen formation, anastamosis and development of a stabilized capillary-like network. This phenotypic sequence required endothelial-mural cell-cell contact, mural cell-derived VEGF and endothelial VEGFR2 signaling. Inhibiting formation of adherens junctions or basement membrane structures abrogated network formation. Notably, inhibition of mural cell VEGF expression could not be rescued by exogenous VEGF.

Conclusions

These results suggest a unique role for mural cell-associated VEGF in driving vessel formation and maturation.     

Increased neointimal thickening in dystrophin-deficient mdx mice

Background

The dystrophin gene, which is mutated in Duchenne muscular dystrophy (DMD), encodes a large cytoskeletal protein present in muscle fibers. While dystrophin in skeletal muscle has been extensively studied, the function of dystrophin in vascular smooth muscle is less clear. Here, we have analyzed the role of dystrophin in injury-induced arterial neointima formation.

Methodology/Principal Findings

We detected a down-regulation of dystrophin, dystroglycan and β-sarcoglycan mRNA expression when vascular smooth muscle cells de-differentiate in vitro. To further mimic development of intimal lesions, we performed a collar-induced injury of the carotid artery in the mdx mouse, a model for DMD. As compared with control mice, mdx mice develop larger lesions with increased numbers of proliferating cells. In vitro experiments demonstrate increased migration of vascular smooth muscle cells from mdx mice whereas the rate of proliferation was similar in cells isolated from wild-type and mdx mice.

Conclusions/Significance

These results show that dystrophin deficiency stimulates neointima formation and suggest that expression of dystrophin in vascular smooth muscle cells may protect the artery wall against injury-induced intimal thickening.     

Murine and human myogenic cells identified by elevated aldehyde dehydrogenase activity: implications for muscle regeneration and repair

Background

Despite the initial promise of myoblast transfer therapy to restore dystrophin in Duchenne muscular dystrophy patients, clinical efficacy has been limited, primarily by poor cell survival post-transplantation. Murine muscle derived stem cells (MDSCs) isolated from slowly adhering cells (SACs) via the preplate technique, induce greater muscle regeneration than murine myoblasts, primarily due to improved post-transplantation survival, which is conferred by their increased stress resistance capacity. Aldehyde dehydrogenase (ALDH) represents a family of enzymes with important morphogenic as well as oxidative damage mitigating roles and has been found to be a marker of stem cells in both normal and malignant tissue. In this study, we hypothesized that elevated ALDH levels could identify murine and human muscle derived cell (hMDC) progenitors, endowed with enhanced stress resistance and muscle regeneration capacity.

Methodology/Principal Findings

Skeletal muscle progenitors were isolated from murine and human skeletal muscle by a modified preplate technique and unfractionated enzymatic digestion, respectively. ALDH^hi subpopulations isolated by fluorescence activate cell sorting demonstrated increased proliferation and myogenic differentiation capacities compared to their ALDH^lo counterparts when cultivated in oxidative and inflammatory stress media conditions. This behavior correlated with increased intracellular levels of reduced glutathione and superoxide dismutase. ALDH^hi murine myoblasts were observed to exhibit an increased muscle regenerative potential compared to ALDH^lo myoblasts, undergo multipotent differentiation (osteogenic and chondrogenic), and were found predominately in the SAC fraction, characteristics that are also observed in murine MDSCs. Likewise, human ALDH^hi hMDCs demonstrated superior muscle regenerative capacity compared to ALDH^lo hMDCs.

Conclusions

The methodology of isolating myogenic cells on the basis of elevated ALDH activity yielded cells with increased stress resistance, a behavior that conferred increased regenerative capacity of dystrophic murine skeletal muscle. This result demonstrates the critical role of stress resistance in myogenic cell therapy as well as confirms the role of ALDH as a marker for rapid isolation of murine and human myogenic progenitors for cell therapy.     

Simple and high yielding method for preparing tissue specific extracellular matrix coatings for cell culture

Background

The native extracellular matrix (ECM) consists of a highly complex, tissue-specific network of proteins and polysaccharides, which help regulate many cellular functions. Despite the complex nature of the ECM, in vitro cell-based studies traditionally assess cell behavior on single ECM component substrates, which do not adequately mimic the in vivo extracellular milieu.

Methodology/Principal Findings

We present a simple approach for developing naturally derived ECM coatings for cell culture that provide important tissue-specific cues unlike traditional cell culture coatings, thereby enabling the maturation of committed C2C12 skeletal myoblast progenitors and human embryonic stem cells differentiated into cardiomyocytes. Here we show that natural muscle-specific coatings can (i) be derived from decellularized, solubilized adult porcine muscle, (ii) contain a complex mixture of ECM components including polysaccharides, (iii) adsorb onto tissue culture plastic and (iv) promote cell maturation of committed muscle progenitor and stem cells.

Conclusions

This versatile method can create tissue-specific ECM coatings, which offer a promising platform for cell culture to more closely mimic the mature in vivo ECM microenvironment.     

Notch Signalling Is Required for the Formation of Structurally Stable Muscle Fibres in Zebrafish

Background

Accurate regulation of Notch signalling is central for developmental processes in a variety of tissues, but its function in pectoral fin development in zebrafish is still unknown.

Methodology/Principal Findings

Here we show that core elements necessary for a functional Notch pathway are expressed in developing pectoral fins in or near prospective muscle territories. Blocking Notch signalling at different levels of the pathway consistently leads to the formation of thin, wavy, fragmented and mechanically weak muscles fibres and loss of stress fibres in endoskeletal disc cells in pectoral fins. Although the structural muscle genes encoding Desmin and Vinculin are normally transcribed in Notch-disrupted pectoral fins, their proteins levels are severely reduced, suggesting that weak mechanical forces produced by the muscle fibres are unable to stabilize/localize these proteins. Moreover, in Notch signalling disrupted pectoral fins there is a decrease in the number of Pax7-positive cells indicative of a defect in myogenesis.

Conclusions/Significance

We propose that by controlling the differentiation of myogenic progenitor cells, Notch signalling might secure the formation of structurally stable muscle fibres in the zebrafish pectoral fin.     

Regenerative defect in vastus lateralis muscle of patients with chronic obstructive pulmonary disease

Background

Impaired skeletal muscle regeneration could contribute to the progression of muscle atrophy in patients with chronic obstructive pulmonary disease (COPD).

Methods

Satellite cells and myogenesis-related proteins were compared between healthy subjects and patients with COPD, with or without muscle atrophy. Satellite cells were isolated and cultured to assess their proliferative and differentiation aptitudes.

Results

Although satellite cell numbers in muscle samples were similar between groups, the proportion of muscle fibers with central nuclei was increased in COPD. In muscle homogenates, increased expression of MyoD and decreased expression of myogenin and MRF4 were observed in COPD. In cultured satellite cells of patients with COPD, increased protein content was observed for Pax7, Myf5 (proliferation phase) and myogenin (differentiation phase) while myosin heavy chain protein content was significantly lower during differentiation.

Conclusion

In COPD, the number of central nuclei was increased in muscle fibers suggesting a greater number of attempts to regenerate muscle tissue than in healthy subjects. Myogenesis signaling was also altered in muscle homogenates in patients with COPD and there was a profound reduction in the differentiation potential in this population as indicated by a reduced ability to incorporate myosin heavy chain into newly formed myotubes. Collectively, these results indicate that skeletal muscle regenerative capacity termination is impaired in COPD and could contribute to the progression of muscle atrophy progression in this population.     

Osteoprotegerin,Pericytes and Bone-Like Vascular Calcification Are Associated with Carotid Plaque Stability

Background and Purpose

Vascular calcification, recapitulating bone formation, has a profound impact on plaque stability. The aim of the present study was to determine the influence of bone-like vascular calcification (named osteoid metaplasia = OM) and of osteoprotegerin on plaque stability.

Methods

Tissue from carotid endarterectomies were analysed for the presence of calcification and signs of vulnerability according to AHA grading system. Osteoprotegerin (OPG), pericytes and endothelial cells were sought using immuno-histochemistry. Symptoms and preoperative imaging findings (CT-scan, MRI and Doppler-scan) were analyzed. Human pericytes were cultured to evaluate their ability to secrete OPG and to influence mineralization in the plaque.

Results

Seventy-three carotid plaques (49 asymptomatic and 24 symptomatic) were harvested. A significantly higher presence of OM (18.4% vs 0%, p<0.01), OPG (10.2% of ROI vs 3.4% of ROI, p<0.05) and pericytes (19% of ROI vs 3.8% of ROI, p<0.05) were noted in asymptomatic compared to symptomatic plaques. Consistently, circulating OPG levels were higher in the plasma of asymptomatic patients (3.2 ng/mL vs 2.5 ng/mL, p = 0.05). In vitro, human vascular pericytes secreted considerable amounts of OPG and underwent osteoblastic differentiation. Pericytes also inhibited the osteoclastic differentiation of CD14+ cells through their secretion of OPG.

Conclusions

OPG (intraplaque an plasmatic) and OM are associated with carotid plaque stability. Pericytes may be involved in the secretion of intraplaque OPG and in the formation of OM.     

Perivascular Mural Cells of the Mouse Choroid Demonstrate Morphological Diversity That Is Correlated to Vasoregulatory Function

Objective

Perivascular mural cells of the choroid have been implicated in physiological functioning as well as in retinal disease pathogenesis. However details regarding their form and function are not well understood. We aim to characterize choroidal mural cells in the adult mouse choroid in terms of their distribution and morphology, and correlate these to their contractile behavior.

Methods

Sclerochoroidal flat-mounted explants were prepared from albino transgenic mice in which the α-smooth muscle actin (α-SMA) promoter drives the expression of green fluorescent protein (GFP). α-SMA-expressing smooth muscle cells and pericytes in the living choroid were thereby rendered fluorescent and imaged with confocal microscopy and live-cell imaging in situ.

Results

Choroidal perivascular mural cells demonstrate significant diversity in terms of their distribution and morphology at different levels of the vasculature. They range from densely-packed circumferentially-oriented cells that provide complete vascular coverage in primary arteries to widely-spaced stellate-shaped cells that are distributed sparsely over terminal arterioles. Mural cells at each level are immunopositive for contractile proteins α-SMA and desmin and demonstrate vasoconstrictory contractile movements in response to endothelin-1 and the calcium ionophore, A23187, and vasodilation in response to the calcium chelator, BAPTA. The prominence of vasoregulatory contractile responses varies with mural cell morphology and density, and is greater in vessels with dense coverage of mural cells with circumferential cellular morphologies. In the choriocapillaris, pericytes demonstrate a sparse, horizontal distribution and are selectively distributed only to the scleral surface of the choriocapillaris.

Conclusions

Diversity and regional specialization of perivascular mural cells may subserve varying requirements for vasoregulation in the choroid. The model of the α-SMA-GFP transgenic albino mouse provides a useful and intact system for the morphological and functional study of choroidal mural cells.