Majority Of Smooth Muscle Cells Research Articles

LIM homeodomain transcription factor Isl1 directs normal pyloric development by targeting Gata3

BackgroundAbnormalities in pyloric development or in contractile function of the pylorus cause reflux of duodenal contents into the stomach and increase the risk of gastric metaplasia and cancer. Abnormalities of the pyloric region are also linked to congenital defects such as the relatively common neonatal hypertrophic pyloric stenosis, and primary duodenogastric reflux. Therefore, understanding pyloric development is of great clinical relevance. Here, we investigated the role of the LIM homeodomain transcription factor Isl1 in pyloric development.ResultsExamination of Isl1 expression in developing mouse stomach by immunohistochemistry, whole mount in situ hybridization and real-time quantitative PCR demonstrated that Isl1 is highly expressed in developing mouse stomach, principally in the smooth muscle layer of the pylorus. Isl1 expression was also examined by immunofluorescence in human hypertrophic pyloric stenosis where the majority of smooth muscle cells were found to express Isl1. Isl1 function in embryonic stomach development was investigated utilizing a tamoxifen-inducible Isl1 knockout mouse model. Isl1 deficiency led to nearly complete absence of the pyloric outer longitudinal muscle layer at embryonic day 18.5, which is consistent with Gata3 null mouse phenotype. Chromatin immunoprecipitation, luciferase assays, and electrophoretic mobility shift assays revealed that Isl1 ensures normal pyloric development by directly targeting Gata3.ConclusionsThis study demonstrates that the Isl1-Gata3 transcription regulatory axis is essential for normal pyloric development. These findings are highly clinically relevant and may help to better understand pathways leading to pyloric disease.

Abstract 9922: The Induction of YAP Expression Following Arterial Injury is Crucial for Smooth Muscle Phenotypic Modulation and Neointima Formation

Rationale: Abnormal proliferation and migration of vascular smooth muscle cells (SMCs) are the key events in the progression of neointimal formation in response to vascular injury. Previous studies have shown that YAP is a potent oncogene promoting cell migration and proliferation, while the role of YAP in vascular diseases remain unknown. Objective: The goal of this study is to investigate the functional role of YAP in smooth muscle phenotypic modulation in vitro and in vivo. Methods and Results: In vitro in cell culture and in vivo in both mouse and rat arterial injury models YAP expression is significantly induced and correlated with the vascular SMC synthetic phenotype. YAP is also highly expressed in human carotid atherosclerotic plaque and localized in the majority of SMCs. Over-expression of YAP promotes SMC migration and proliferation while attenuating smooth muscle contractile gene expression. Conversely, knocking-down endogenous YAP in SMCs up-regulates smooth muscle gene expression but attenuates SMC proliferation and migration. Consistent with this, knocking-down YAP expression in a rat carotid balloon injury model attenuates injury-induced smooth muscle dedifferentiation and neointima formation. Furthermore, carotid artery ligation in mice with genetic deletion of YAP specifically in SMCs leads to increase smooth muscle-specific gene expression and reduction of neointima formation. Conclusion: YAP plays a novel integrative role in smooth muscle phenotypic modulation by inhibiting smooth muscle-specific gene expression while promoting smooth muscle proliferation and migration in vitro and in vivo. Blocking the induction of YAP would be a potential therapeutic approach for ameliorating vascular occlusive diseases.

Mitochondrial Motility and Vascular Smooth Muscle Proliferation

Mitochondria are widely described as being highly dynamic and adaptable organelles, and their movement is thought to be vital for cell function. Yet, in various native cells, including those of heart and smooth muscle, mitochondria are stationary and rigidly structured. The significance of the differences in mitochondrial behavior to the physiological function of cells is unclear and was studied in single myocytes and intact resistance-sized cerebral arteries. We hypothesized that mitochondrial dynamics is controlled by the proliferative status of the cells. High-speed fluorescence imaging of mitochondria in live vascular smooth muscle cells shows that the organelle undergoes significant reorganization as cells become proliferative. In nonproliferative cells, mitochondria are individual (≈ 2 μm by 0.5 μm), stationary, randomly dispersed, fixed structures. However, on entering the proliferative state, mitochondria take on a more diverse architecture and become small spheres, short rod-shaped structures, long filamentous entities, and networks. When cells proliferate, mitochondria also continuously move and change shape. In the intact pressurized resistance artery, mitochondria are largely immobile structures, except in a small number of cells in which motility occurred. When proliferation of smooth muscle was encouraged in the intact resistance artery, in organ culture, the majority of mitochondria became motile and the majority of smooth muscle cells contained moving mitochondria. Significantly, restriction of mitochondrial motility using the fission blocker mitochondrial division inhibitor prevented vascular smooth muscle proliferation in both single cells and the intact resistance artery. These results show that mitochondria are adaptable and exist in intact tissue as both stationary and highly dynamic entities. This mitochondrial plasticity is an essential mechanism for the development of smooth muscle proliferation and therefore presents a novel therapeutic target against vascular disease.

Knock out of neuronal nitric oxide synthase exacerbates intestinal ischemia/reperfusion injury in mice

Recent investigation of the intestine following ischemia and reperfusion (I/R) has revealed that nitric oxide synthase (NOS) neurons are more strongly affected than other neuron types. This implies that NO originating from NOS neurons contributes to neuronal damage. However, there is also evidence of the neuroprotective effects of NO. In this study, we compared the effects of I/R on the intestines of neuronal NOS knockout (nNOS(-/-)) mice and wild-type mice. I/R caused histological damage to the mucosa and muscle and infiltration of neutrophils into the external muscle layers. Damage to the mucosa and muscle was more severe and greater infiltration by neutrophils occurred in the first 24h in nNOS(-/-) mice. Immunohistochemistry for the contractile protein, α-smooth muscle actin, was used to evaluate muscle damage. Smooth muscle actin occurred in the majority of smooth muscle cells in the external musculature of normal mice but was absent from most cells and was reduced in the cytoplasm of other cells following I/R. The loss was greater in nNOS(-/-) mice. Basal contractile activity of the longitudinal muscle and contractile responses to nerve stimulation or a muscarinic agonist were reduced in regions subjected to I/R and the effects were greater in nNOS(-/-) mice. Reductions in responsiveness also occurred in regions of operated mice not subjected to I/R. This is attributed to post-operative ileus that is not significantly affected by knockout of nNOS. The results indicate that deleterious effects are greater in regions subjected to I/R in mice lacking nNOS compared with normal mice, implying that NO produced by nNOS has protective effects that outweigh any damaging effect of this free radical produced by enteric neurons.

Enhanced attachment, growth and migration of smooth muscle cells on microcarriers produced using thermally induced phase separation

Microcarriers are widely used for the expansion of cells in vitro, but also offer an approach for combining cell transplantation and tissue bulking for regenerative medicine in a minimally invasive manner. This could be beneficial in conditions associated with muscle damage or atrophy, such as faecal incontinence, where the use of bulking materials or cell transplantation alone has proven to be ineffective. Microcarriers currently available have not been designed for this purpose and are likely to be suboptimal due to their physical and biochemical properties. The aim of this study was to investigate macroporous microspheres of polylactide–co-glycolide (PLGA), prepared using a thermally induced phase separation technique, for their suitability as cell microcarriers for the transplantation of smooth muscle cells. Cell attachment, growth and migration were studied and compared with commercially available porcine gelatin microcarriers (Cultispher-S) in suspension culture. Smooth muscle cells attached more rapidly to the PLGA microcarriers, which also significantly enhanced the rate of cell growth compared with Cultispher-S microcarriers. The majority of smooth muscle cells attached to the PLGA microcarriers in suspension culture were able to migrate away over a 15day period of static culture, unlike Cultispher-S microcarriers which retained the majority of cells. The ability of PLGA microcarriers to enhance cell growth combined with their capacity to release cells at the sites of delivery are features that make them ideally suited for use as a cell transplantation delivery device in tissue engineering and regenerative medicine.

Origin of Neointimal Smooth Muscle

The origin of smooth muscle cells (SMCs) in atherosclerotic lesions has been a hotly debated topic for more than four decades. Until recently, it was widely accepted that the majority of smooth muscle cells in atherosclerotic lesions originate from the medial layer of the vessel wall, with local SMC proliferation and migration leading to remodeling of the vessel wall and vessel lumen.1,2 Recent reports in the literature, however, claimed that SMCs in atherosclerotic lesions originate primarily from circulating bone marrow progenitor cells.3,4 Where these cells originate from is not merely academic, because the cells that give rise to SMCs within atherosclerotic lesions would likely serve as targets for current and future therapeutic interventions. The bone marrow–derived origin suggested by these articles would indicate that attempts to control SMC phenotype in in-stent restenosis, transplant atherosclerosis, or fibrous cap rupture should be targeted toward these cells from the marrow. However, we cautioned in 2004 that if anything, the bone marrow–derived intimal SMC paradigm was “Lost in Transdifferentiation”,5 and further studies were needed to clearly resolve this critically important issue. See page 2696 In this issue of Arteriosclerosis, Thrombosis, and Vascular Biology , Bentzon and colleagues provide excellent evidence that bone marrow–derived cells do not give rise to vascular SMCs in hyperlipidemia-induced atherosclerotic plaques in the apoE knockout mouse.6 These investigators studied plaques in apoE−/− mice that had their bone marrow reconstituted from sex-mismatched apoE−/−/eGFP+ animals, and then performed meticulous high-resolution z -axis sectioning of atherosclerotic lesions induced by Western diet to try to identify transdifferentiated cells staining for both the eGFP/Y chromosome lineage tags and the SMC marker smooth muscle α-actin (SMαA). Of critical importance, and in contrast to many previous studies in this area where investigators examined relatively small numbers of intimal SMCs …

EARLY ACTIVATION OF INTERNAL MEDIAL SMOOTH MUSCLE CELLS IN THE RABBIT AORTA AFTER MECHANICAL INJURY: RELATIONSHIP WITH INTIMAL THICKENING AND PHARMACOLOGICAL APPLICATIONS

1. Smooth muscle cells (SMC) participate in both inflammatory and dedifferentiation processes during atherosclerosis, as well as during mechanical injury following angioplasty. In the latter, we studied medial SMC differentiation and inflammation processes implicated early after de-endothelialization in relation to mechanical stresses. We hypothesized that activation of a subpopulation of SMC within the media plays a crucial role in the early phase of neointimal formation. 2. For this purpose, we used a rabbit model of balloon injury to study activation and differentiation of medial SMC in the early time after denudation and just before neointima thickening. Inflammation was evaluated by the expression of vascular cell adhesion molecule (VCAM)-1, integrin alpha4beta1 and nuclear factor (NF)-kB. Myosin isoforms and 2P1A2 antigen, a membrane protein expressed by rabbit dedifferentiated SMC, were used as markers of differentiation. 3. On day 2 after de-endothelialization, VCAM-1, alpha4beta1 and NF-kB were coexpressed by a well-defined subpopulation of SMC of the internal part of the media, in the vicinity of the blood stream. At the same time, the majority of SMC throughout the media expressed non-muscle myosin heavy chain-B (nm-MHC-B) and 2P1A2 antigen. On day 7, when intimal thickening appeared, SMC of the media were no longer activated, whereas some intimal SMC expressed the activation markers. Thus, after de-endothelialization, early dedifferentiation occurs in most of the medial SMC, whereas activation concerned only a subpopulation of SMC located in the internal media. Using the T-type voltage-operated calcium channel blocker mibefradil (0.1-1 micromol/L) in SMC culture, we showed that this agent exhibited an antiproliferative effect in a dose-dependent manner only on undifferentiated cells. 4. In conclusion, the results suggest that the activated SMC represent cells that are potentially able to migrate and participate in the intimal thickening process. Thus, the medial SMC inflammatory process, without any contribution of inflammatory cells, may represent a major mechanism underlying the development of intimal thickening following mechanical stress. In humans, inhibition of T-type calcium channels may be a tool to prevent the early proliferation step leading to neointimal formation.

骨髄由来血管前駆細胞と血管リモデリング

Exuberant accumulation of smooth muscle cells plays a principal role in the pathogenesis of vascular diseases. It has been assumed that smooth muscle cells derived from the adjacent medial layer migrate, proliferate and synthesize extracellular matrix. Although a lot of effort has been devoted targeting migration and proliferation of medial smooth muscle cells, no effective therapy to prevent occlusive vascular remodeling has been established. Here, we show that bone marrow cells give rise to the majority of smooth muscle cells that contribute to arterial remodeling in models of post-angioplasty restenosis, graft vasculopathy and hyperlipidemia-induced atherosclerosis. Notably, purified hematopoietic stem cells differentiated into smooth muscle cells in vitro and in vivo. Our findings suggest that somatic stem cells contribute to pathological remodeling of remote organs. Our study provides the basis for the development of a new therapeutic strategies for vascular diseases, targeting mobilization, homing, differentiation and proliferation of bone marrow-derived vascular progenitor cells.

Specialised pacemaking cells in the rabbit urethra.

1. Collagenase dispersal of strips of rabbit urethra yielded, in addition to normal spindle-shaped smooth muscle cells, a small proportion of branched cells which resembled the interstitial cells of Cajal dispersed from canine colon. These were clearly distinguishable from smooth muscle in their appearance under the phase-contrast microscope, their immunohistochemistry and their ultrastructure. They had abundant vimentin filaments but no myosin, a discontinuous basal lamina, sparse rough endoplasmic reticulum, many mitochondria and a well-developed smooth endoplasmic reticulum. 2. Interstitial cells were non-contractile but exhibited regular spontaneous depolarisations in current clamp. These could be increased in frequency by noradrenaline and blocked by perfusion with calcium-free solution. In voltage clamp they showed abundant calcium-activated chloride current and spontaneous transient inward currents which could be blocked by chloride channel blockers. 3. The majority of smooth muscle cells were vigorously contractile when stimulated but did not show spontaneous electrical activity in current clamp. In voltage clamp, smooth muscle cells showed very little calcium-activated chloride current. 4. We conclude that there are specialised pacemaking cells in the rabbit urethra that may be responsible for initiating the slow waves recorded from smooth muscle cells in the intact syncitium.

Immunohistological and ultrastructural analysis of the intimal thickening in coarctation of human aorta.

The histological nature and characteristics of aortic coarctation are not clearly defined, the aim of this study is to analyse intimal thickening in aortic coarctation. In order to characterize the components of intimal thickening in coarctation, narrowed segments of aorta obtained after surgery from ten children were examined immunocytochemically and by electron microscopy. Histological analysis of aortic coarctation demonstrated a widened subendothelial region with separation of endothelial cells from the internal elastic lamina. Masson's trichrome staining showed a marked increase in extracellular matrix and cell numbers in the intimal thickening compared with normal aorta. Cellular component analysis demonstrated invagination of the intima by smooth muscle actin-positive cells, with a fragmentation of the internal elastic lamina. No proliferating smooth muscle and inflammatory cells were identified in the intima. In order to characterize the smooth muscle cell phenotypes, various smooth muscle cell markers were sought using specific monoclonal antibodies: alpha-smooth muscle actin, smooth muscle-myosin heavy chain, heavy caldesmon, desmin. In moderate coarcted aorta, at least two distinct smooth muscle phenotypes were identified. In the juxtamedial part of the intima smooth muscle, cells were differentiated and expressed all smooth muscle markers; in the subendothelial part of the intimal thickening, the majority of smooth muscle cells expressed only alpha-smooth muscle actin and appeared dedifferentiated. In regions of marked stenosis, a strong expression of smooth muscle-myosin heavy chain, and heavy caldesmon in the intimal thickening pointed to the presence of redifferentiated smooth muscle cells, not still expressing desmin. Electron microscopic examination also revealed a variety of smooth muscle cell phenotypes in the intimal thickening. In the superficial layer, smooth muscle cells appeared to be in the synthetic state, while in the deeper part, both synthetic and contractile components were identified. These observations indicated that human coarctation was characterized by intimal recruitment of non-proliferating smooth muscle cells with dedifferentiated phenotype. However, the presence of smooth muscle cells with an intermediate phenotype in the narrowest part of the coarctation suggest that the redifferentiation process could participate in the pathogenesis of aortic coarctation.

Differentiation of smooth muscle cells in human blood vessels as defined by smoothelin, a novel marker for the contractile phenotype.

Smoothelin is a constituent of the cytoskeleton specific for smooth muscle cells (SMCs) in a broad range of species. It has been postulated that smoothelin represents a marker of highly differentiated, contractile SMCs. Here, we present data on the presence of smoothelin in the human vascular system that support this hypothesis. For this purpose, smoothelin distribution was studied (1) during vasculogenesis of the placenta, (2) in normal adult blood vessels, and (3) in atherosclerotic lesions. Smoothelin was first observed in placental tissue at approximately week 10 to 11 of gestation. In full-term placenta, it was found in the SMCs of vessels in the large stem villi and in the chorionic plate. Furthermore, it was present in the fetal arteries of smaller stem villi, but it was not found in the veins. In adult blood vessels, a small population of aortic (approximately 10%) and large muscular artery (approximately 30% to 50%) SMCs was positive for smoothelin. In general, smoothelin and desmin were coexpressed in the same SMCs, but expression of desmin appeared to be less abundant. However, the majority of SMCs in these blood vessels were smoothelin- and desmin negative but expressed vimentin, whereas alpha-smooth muscle actin (alpha-SMA) was present in all SMCs. The SMCs in the media of small muscular arteries were positive for smoothelin and desmin (> 95%), whereas the vimentin-positive SMC type was scarce. Smoothelin was absent in capillaries, pericytic venules, and small veins but was occasionally observed in the SMCs of large veins. Thus, the distribution of smoothelin in the SMCs of the vascular system appears to be limited to blood vessels that are capable of pulsatile contraction. In atherosclerotic femoral arteries, smoothelin-positive cells were detected in the media, the atheromatous plaque, and the intimal thickening. Smoothelin-positive cells were present primarily at the luminal portion of advanced lesions. The presence of a considerable number of such smoothelin-positive cells at that location may indicate that these plaques are no longer expanding.

Expression rate of vinculin isoforms in human aortocoronary saphenous vein grafts

Aortocoronary bypass conduits derived from saphenous veins usually develop diffuse intimal thickening, one of the major causes of haemodynamically relevant graft stenosis. To elucidate the role of smooth muscle cell proliferation in late graft failure, specimens from highly stenotic or occluded vein grafts implanted into the arterial circulation for more than 5 years were tested for their expression rate of meta-vinculin. Since the cytoskeletal protein meta-vinculin is present exclusively in contractile smooth muscle cells, the determination of the relative amounts of meta-vinculin (150 kDa) and its low-molecular weight isoform vinculin (130 kDa) allows characterization of the phenotypic status of smooth muscle cells. Using immunoblotting techniques, the quantitative relation of meta-vinculin in tissue samples obtained from autoptic vein grafts ( n=10) was measured and compared with those of native saphenous veins ( n=6). In vein grafts, the fractional meta-vinculin content of the total vinculin immunoreactivity ranged from 32%–46% (mean 39.6%), whereas the range was 39%–53% (mean 46.7%) in native veins. By applying Student's t-test, a statistical significance was not demonstrated suggesting that the majority of smooth muscle cells in intimal thickenings consisted of a contractile phenotype. Immunohistochemically, the vinculin immunoreactivity in the intimal layer of vein grafts was reduced as compared to native saphenous veins. The distribution of vinculin in grafted veins closely resembled that in arteriosclerotic coronary arteries with intimal thickening. Hence, our biochemical data demonstrate parallels between the pathogenesis of late vein graft stenosis and degenerative arteriosclerotic lesions.

Morphological variability of smooth muscle cells in human nasal swell bodies

The complex functional behavior of nasal swell bodies is still not completely understood. In the present study the histology of the vessels involved in the swelling mechanism is examined and the ultrastructural appearances described of the different types of smooth muscle cells located in the vascular wall of swell bodies in the human inferior turbinate. Even though the majority of smooth muscle cells of the nasal swell bodies showed a normal, elongated appearance comparable to other smooth muscle cells elsewhere in the body, a variety of cells with atypical shapes could be detected that have not been described previously in vessels of the nasal mucosa. The diameters of the smooth muscle cells in general were strikingly variable. The individual smooth muscle cells were surrounded by a basal lamina that was occasionally disrupted or doubled. Myoblasts were separated by a connective tissue space containing collagen fibrils, mature elastin fibers and bundles of microfibrils. The latter two types of fibers and fibrils occurred mainly in the outer parts of the muscular coat. The endowment of cytoplasmic components was similar in all smooth muscle cells of the vascular wall in the swell bodies. These findings indicate that the specific feature of smooth musculature presumably resides in the unusual morphological variability of the single cells present, as well as in the striking heterogeneity of the arrangement of bundles of these cells in the vascular wall.

Suppression of pseudointimal hyperplasia by a novel prostacyclin analogue: beraprost.

The aim of this study was to evaluate the effect of a novel prostaglandin I2 analogue, beraprost (BPT), on the pseudointimal hyperplasia (PIH) of polytetrafluoroethylene (PTFE) prostheses. A total of 12 rabbits were equally divided into three groups. The control group was given a placebo daily, group 1 was given BPT orally 2 mg/kg per day, and group 2 was given BPT orally 4 mg/kg b.i.d. Exactly 1 cm of the inferior vena cava was resected and replaced by a 3-cm PTFE tube graft. All the grafts were patent when harvested 4 weeks after implantation, but the lumens were narrowed to various extents by PIH. PIH, determined by the dry weight of the intraluminal tissue deposit, was significantly (P < 0.01) suppressed in groups 1 and 2 compared with the control group. High-magnification light microscopy with various staining methods revealed the PIH to be composed mainly of smooth muscle cells (SMCs) and collagen fibrils in all three groups. Transmission electron microscopy revealed that the majority of SMCs in groups 1 and 2 were contractile in form, in contrast with the synthetic form seen in the control group. In conclusion, BPT attenuated the PIH of PTFE grafts by inhibiting the phenotype change in the SMCs.

Ultrastructural, immunochemical and electrophoretic study of smooth muscle cells in internal mammary arteries of patients undergoing coronary bypass surgery.

The internal mammary artery (IMA) is used widely in bypass grafting for coronary artery disease because of its resistance to atherosclerotic obstruction. Since there are no data on the ultrastructure of IMA or the phenotype of its smooth muscle cells (SMC), we studied the distal parts of left IMA obtained at the time of surgery from 14 coronary bypass patients, aged 43-67 years. Eight IMA were examined by transmission electron microscopy. The distribution of the cytoskeletal proteins actin, vimentin, and desmin in the intima-media of 6 IMA was studied by immunofluorescence microscopy, polyacrylamide gel electrophoresis, and two-dimensional gel electrophoresis. The intimas were very thin, from 3 to 32 microns. The thinnest regions contained no cells. Most intimal cells had the ultrastructural features of SMC; no foam cells were found. The majority of both intimal and medial SMC had a myofilament-rich phenotype. Cells reacting to antibodies of vimentin, desmin and alpha-actin were found in both intima and media. alpha-Actin formed 67% of all actin isoforms in the intima-medial extracts. Our study confirms ultrastructurally the reported scarcity of atherosclerosis in the human IMA and shows that the majority of SMC in the IMA of even severely atherosclerotic coronary bypass patients are both ultrastructurally and biochemically in a differentiated state, which agrees with their resistance to atherosclerosis.

Smooth muscle phenotypic expression in human carotid arteries. II. Atherosclerosis-free diffuse intimal thickenings compared with the media.

The volume fraction of synthetic organelles in smooth muscle cells from areas of atherosclerosis-free, diffuse intimal thickenings (DIT) and media of human carotid artery has been determined morphometrically. Tissue was obtained at autopsy from men and women aged 35 to 80 years, at 6 to 12 hours after death. With both sexes, the volume fraction of synthetic organelles in smooth muscle cells from the DIT was not significantly different from the volume fraction of synthetic organelles in cells of the subjacent media (0.25 and 0.21, respectively). This is in marked contrast to our earlier study in which the volume fraction of synthetic organelles in smooth muscle cells of DIT from a similar region of artery, but adjacent to atheromatous plaques, was highly significantly different from cells of the subjacent media (0.48 and 0.23, respectively). Only in regions near atherosclerotic plaques do the majority of smooth muscle cells of DIT express a different phenotype from cells of the media.