Subpopulation Of Smooth Muscle Cells Research Articles

CAMP Response Element-binding Protein Content Is a Molecular Determinant of Smooth Muscle Cell Proliferation and Migration

We hypothesized that cAMP response element-binding protein (CREB) could function as a molecular determinant of smooth muscle cell fate. In arterial sections from the systemic and pulmonary circulation, CREB content was high in proliferation-resistant medial subpopulations of smooth muscle cells and low in proliferation-prone regions. In vessels from neonatal calves exposed to chronic hypoxia, CREB content was depleted and smooth muscle cell (SMC) proliferation was accelerated. Induction of quiescence by serum deprivation in culture led to increased CREB content. Highly proliferative SMC in culture were observed to have low CREB content. Exposure to proliferative stimuli such as hypoxia or platelet-derived growth factor decreased SMC CREB content. Assessment of CREB gene transcription by nuclear run-on analysis and transcription from a CREB promoter-luciferase construct indicate that CREB levels in SMC are in part controlled at the level of transcription. Overexpression of wild type or constitutively active CREB in primary cultures of SMC arrested cell cycle progression. Additionally, expression of constitutively active CREB decreased both proliferation and chemokinesis. Consistent with these functional properties, active CREB decreased the expression of multiple cell cycle regulatory genes, as well as genes encoding growth factors, growth factor receptors, and cytokines. Our data suggest a unique mode of cellular phenotype determination at the level of the nuclear content of CREB.

Regional differentiation of desmin, connexin43, and connexin45 expression patterns in rat aortic smooth muscle.

The gap-junctional protein, connexin43, is differentially expressed in vascular smooth muscle cells (SMCs) according to phenotype. Previous studies suggest that desmin-negative SMCs are characterized by high levels of connexin43, whereas desmin-positive SMCs (of a more contractile phenotype) typically have low connexin43 levels. In this study, we examine systematically the inverse relationship between connexin43 and desmin in SMCs of defined regions of the rat aortic media and determine whether additional connexin isotypes are expressed and contribute to this relationship. Immunoconfocal microscopy demonstrated that (1) the inverse relationship between connexin43 and desmin expression holds true for the media of sequential aortic zones, with 1 exception, the ascending aorta, and (2) an additional vascular connexin, connexin45, is expressed by aortic SMCs. Examination of connexin43, connexin45, and desmin expression in sequential aortic zones reveals 3 SMC subpopulations. The first, predominating in the aortic arch and thoracic aorta, is desmin negative and contains high connexin43 levels; the second, predominating in the abdominal aorta and iliac artery, is desmin positive and contains low connexin43 levels; and the third, which is restricted to the ascending aorta, is desmin positive and expresses high connexin43 levels. Connexin45 levels are high in the ascending aorta but low in the other aortic segments. In para-aortic veins, a fourth SMC subpopulation appears, one that is desmin positive and contains connexin45 but not connexin43. These results demonstrate that a diversity of connexin expression patterns characterizes distinctive subpopulations of medial SMCs in situ with a potential to contribute to regional differentiation of vascular function.

Smooth muscle cells of injured rat and rabbit arteries in culture: contractile and cytoskeletal proteins

The aim of this study is to determine whether subpopulations of smooth muscle cells (SMC), as distinguished by variations in contractile and cytoskeletal proteins, appear in the neointima at different times after vascular injury, and/or whether subpopulations develop during serial passaging of these cells. Rat aortae and rabbit carotid arteries were injured with a 2F Fogarty balloon catheter and cultures established from the resulting neointima and the media 2, 6, 12, 16 and 24 weeks later. Cultures were examined at passages 1–5 and subpopulations of SMC categorised by intensity of staining for each protein by immunohistochemistry. Two populations of SMC with different staining intensities (‘++’, ‘+’) were observed for each of the following proteins: α-SM actin, SM-myosin, desmin and vimentin. Populations without these proteins were also found. Changes in the percentages of cells expressing these proteins were transitory, indicating that the populations were not limited to a particular tissue (neointima or media), time after injury or passage number. One exception was found in rabbit cultures where the number of desmin-expressing cells quickly decreased with both time after injury and time in culture. Subpopulations of SMC were found at all times after injury in the media and neointima of rat and rabbit arteries, and after multiple passage of these cells. There was no pattern of development of one population suggesting that either no subpopulation has a proliferative or migratory advantage over others, or that only one population exists that is capable of diverse phenotypic changes.

Adrenomedullin expression and growth inhibitory effects in distinct pulmonary artery smooth muscle cell subpopulations.

The vasodilator peptide adrenomedullin is elevated in patients with pulmonary hypertension and has been implicated in the inhibition of vascular remodeling. We questioned whether adrenomedullin is released by human pulmonary artery smooth muscle cells (PASMCs) and inhibits PASMC growth and release of endothelin, a known smooth muscle cell mitogen. The majority of PASMCs isolated from proximal pulmonary arteries and all PASMCs from distal pulmonary arteries released adrenomedullin, although at differing rates (mean, 177 +/- 28 and 62 +/- 11 fmol/10(5) cells/24 h, respectively). These cells were designated ADM+. However, some proximal PASMC isolates did not release adrenomedullin, designated ADM-. Northern blot analysis confirmed adrenomedullin expression in proximal ADM+ but not ADM- isolates. ADM- and distal ADM+ PASMCs proliferated faster in serum than did proximal ADM+ cells. Adrenomedullin potently and dose-dependently (mean EC(50) = 2.2 +/- 0.5 nM) increased intracellular cyclic adenosine monophosphate (cAMP) in ADM- isolates via specific adrenomedullin receptors. In contrast, both adrenomedullin and calcitonin gene-related peptide modestly elevated cAMP in 50% of ADM+ isolates. Adrenomedullin dose-dependently inhibited platelet-derived growth factor-stimulated [3H]thymidine incorporation and endothelin release in ADM- cells but did not affect [3H]thymidine uptake in ADM+ isolates. We conclude that distinct subpopulations of human PASMCs release and respond to adrenomedullin. The heterogeneity of adrenomedullin release and the inhibition of PASMC DNA synthesis and endothelin release suggest that adrenomedullin may function as a paracrine mediator in the inhibition of pulmonary vascular remodeling.

α5β1 Integrin Expression and Luminal Edge Fibronectin Matrix Assembly by Smooth Muscle Cells after Arterial Injury

Fibronectin is secreted from the cell as a soluble protein that must then polymerize to regulate cell function. To elucidate the process of fibronectin matrix assembly in vascular disease, we immunostained sections of balloon-injured rat carotid artery for the fibronectin-binding alpha5beta1 integrin. Whereas alpha5beta1 integrin was not evident in the normal carotid artery, its expression was induced after a vascular injury. By 14 days, the alpha5beta1 integrin was localized exclusively to the less differentiated smooth muscle cells (SMCs) at the luminal surface of the neointima. Platelet-derived growth factor-BB, dominant in neointimal formation, selectively increased the expression of the alpha5beta1 integrin by human SMCs in culture. To track the assembly of fibronectin fibers, fluorescence-labeled soluble fibronectin protomers were added to cultured SMCs and to fresh segments of normal and balloon-injured rat carotid arteries. Fibronectin fiber formation in cultured SMCs could be detected within 10 minutes, and was blocked by an RGD peptide, an anti-beta1 integrin antibody, and an anti-alpha5beta1 integrin antibody, but not by an anti-beta3 integrin antibody. En face confocal microscopy of arterial segments revealed that soluble fibronectin had polymerized on the alpha5beta1 integrin-expressing SMCs of the luminal surface of the injured arterial neointima, but not on the alpha5beta1 integrin-negative neointimal SMCs below this or on the endothelial cells of uninjured arteries. Furthermore, in situ fibronectin assembly by the neointimal SMCs was inhibited by an RGD peptide and by an anti-beta1 integrin antibody. These studies indicate that a subpopulation of SMCs in the repairing artery wall orchestrates integrin-mediated fibronectin assembly.

Differential expression of connexin43 and desmin defines two subpopulations of medial smooth muscle cells in the human internal mammary artery.

Upregulation of connexin43-gap junctions is associated with transition of contractile vascular smooth muscle cells (SMCs) to the synthetic state. To determine whether phenotypically distinct subpopulations of medial SMCs differentially express connexin43, we investigated the human distal internal mammary artery, a structurally heterogeneous vessel with features ranging from elastic to elastomuscular to muscular. Immunoconfocal microscopy combined with quantitative analysis and complemented by in situ hybridization showed that SMCs in the elastic medial regions expressed high levels of connexin43 but low levels of desmin, whereas those of muscular medial regions expressed low levels of connexin43 but high levels of desmin. Ultrastructurally, SMCs of both regions were of the contractile phenotype, but the former cells were irregular in shape with relatively prominent synthetic organelles whereas the latter were spindle shaped with fewer synthetic organelles. Vimentin, smooth muscle alpha-actin, calponin, h-caldesmon, and myosin heavy chains (SM1 and SM2) were equally highly expressed by most cells in both subpopulations. The connexin43/desmin expression pattern of SMCs in regions of intimal thickening resembled those of elastic medial regions. These findings refine the view suggested from previous studies that high levels of connexin43 expression are associated with SMCs of a less contractile/more synthetic phenotype. In the internal mammary artery, the 2 subpopulations of SMCs with markedly different connexin43 expression levels both represent a differentiated contractile phenotype, but the subpopulation showing high levels of connexin43-gap junctions is characterized by low levels of desmin and structural features that reflect a more synthetic tendency.

Differential cholesteryl ester accumulation in two human vascular smooth muscle cell subpopulations exposed to aggregated LDL: effect of PDGF-stimulation and HMG-CoA reductase inhibition

Vascular smooth muscle cells (VSMC) are a major component of atheromatous plaque and they exhibit a high heterogeneity in morphology and proliferative activity. Two cell subpopulations from the media of human pulmonary artery were isolated according to the kinetics of outgrowth from the explants; the first wave of cell outgrowth (VSMC-I) and the second wave (VSMC-II) were separately cultured. They were characterized by premitotic DNA synthesis ([ 3H]thymidine incorporation) and cholesterol synthesis ([ 14C]acetate incorporation). DNA and cholesterol synthesis were approximately 13- and 5-fold, respectively, higher in VSMC-I than in VSMC-II. When these subpopulations were exposed to 100 μg/ml of aggregated low density lipoproteins (agLDL), their cholesteryl ester (CE) content increased 4.3-fold over that induced by native LDL. The increase in CE induced by native or agLDL was approximately 2.7-fold higher in VSMC-I than in VSMC-II. These results suggest that agLDL uptake is related, at least in part, to the cellular proliferative status. Platelet derived growth factor (PDGF) did not increase agLDL uptake in any subpopulation, although it efficiently promoted proliferative activity in both cell types and increased native LDL uptake and cholesterol synthesis in VSMC-II. Simvastatin strongly inhibited CE accumulation from agLDL in VSMC-I, either unstimulated or PDGF-stimulated (>80% inhibition). In contrast, it only blocked agLDL uptake in PDGF stimulated VSMC-II (50% inhibition). Our results indicate that the quantitative effect of simvastatin on CE accumulation from agLDL is dependent on phenotypic cell characteristics and it can be modulated in response to mitogenic stimulus.

Hypoxia induces cell-specific changes in gene expression in vascular wall cells: implications for pulmonary hypertension.

Mammals respond to reduced oxygen concentrations (hypoxia) in many different ways at the systemic, local, cellular and molecular levels. Within the pulmonary circulation, exposure to chronic hypoxia has been demonstrated to illicit increases in pulmonary artery pressure as well as dramatic structural changes in both large and small vessels. It has become increasingly clear that the response to hypoxia in vivo is differentially regulated at the level of specific cell types within the vessel wall. For instance, in large pulmonary blood vessels there is now convincing evidence to suggest that the medial layer is made up of many different subpopulations of smooth muscle cells. In response to hypoxia there are remarkable differences in the proliferative and matrix producing responses of these cells to the hypoxic environment. Some cell populations proliferate and increase matrix protein synthesis, while in other cell populations no apparent change in the proliferative or differentiation state of the cell takes place. In more peripheral vessels, the predominant proliferative changes in response to hypoxia in the pulmonary circulation occur in the adventitial layer rather than in the medial layer. Here again, specific increases in proliferation and matrix protein synthesis take place. Accumulating evidence suggests that the unique responses exhibited by specific cell types of hypoxia in vivo can be modeled in vitro. We have isolated, in culture, specific medial cell populations which demonstrate significant increases in proliferation in response to hypoxia, and others which exhibit no change or, in fact, a decrease in proliferation under hypoxic conditions. We have also isolated and cloned several unique populations of adventitial fibroblasts. There is good evidence that only certain fibroblast populations are capable of responding to hypoxia with an increase in proliferation. We have begun to elucidate the signaling pathways which are activated in those cell populations that exhibit proliferative responses to hypoxia. We show that hypoxia, in the absence of serum or mitogens, specifically activates select members of the protein kinase C isozyme family, as well as members of the mitogen-activated protein kinase (MAPK) family of proteins. This selective activation appears to take place in response to hypoxia only in those cells exhibiting a proliferative response, and antagonists of this pathway inhibit the response. Thus, there appear to be cells within each organ that demonstrate unique responses to hypoxia. A better understanding of why these cells exist and how they specifically transduce hypoxia-mediated signals will lead to a better understanding of how the changes in the pulmonary circulation take place under conditions of chronic hypoxia.

Pathogenesis of abdominal aortic aneurysms: Possible role of differential production of proteoglycans by smooth muscle cells

Purpose: In vivo and in vitro observations strongly suggest that marked differences exist in the phenotype, growth, and matrix-producing capabilities of distinct smooth muscle cell subpopulations. An earlier study from our laboratory showed differences in matrix metalloproteinase expression patterns in cultures of medial smooth muscle cells from tissue affected by abdominal aortic aneurysm (AAA) or atherosclerotic occlusive disease and from normal arterial tissue. In this study we were interested in ascertaining whether smooth muscle cells from the same sample groups also synthesized different proteoglycan profiles that correlated with vascular disease. Methods: Proteoglycans from smooth muscle cell monolayer cultures from tissue affected by AAA or atherosclerotic occlusive disease and from normal arterial tissue were examined by means of immunoblotting and affinity-blotting composite agarose polyacrylamide gel electrophoresis (CAPAGE) and sodium dodecyl sulphate PAGE. Enzyme-linked immunosorbent assay (ELISA) was used to quantitate perlecan levels in smooth muscle cell monolayer media samples. Results: Versican, perlecan, and biglycan levels were significantly elevated in AAA smooth muscle cell cultures. Two populations of smooth muscle cell versican were identified by means of CAPAGE-immunoblotting and by means of a novel affinity-blotting technique with biotinylated hyaluronan. A small keratan sulfate–substituted proteoglycan was present in similar levels in all smooth muscle cell cultures. This proteoglycan had a free core protein of about 55 kd after keratanase digestion and had a relatively high charge-to-mass ratio, as was evident from its electrophoretic mobility in CAPAGE; this proteoglycan was tentatively identified as keratocan. Immunoblotting with monoclonal antibodies 3-G-10 (anti- D heparan sulfate, heparan sulfate stubs generated by heparitinase treatment) and 10-E-4 (anti–native heparan sulfate chains) helped identify several smooth muscle cell heparan sulfate–substituted proteoglycans. Elevated levels of intact and processed perlecan core protein were identified in AAA cultures by means of immunoblotting with a monoclonal antibody to perlecan core protein (A76). ELISA measurements confirmed that perlecan levels were significantly higher in AAA smooth muscle cell cultures compared with the normal arterial tissue and tissue affected by atherosclerotic occlusive disease. Conclusions: Because heparan sulfate proteoglycans can bind growth factors, their elevated synthesis by AAA smooth muscle cells in combination with an increased expression of matrix metalloproteinases may at least partly explain the differential proliferative capacity of the AAA smooth muscle cells examined and may govern the pattern of abnormal cellular proliferation and matrix protein synthesis observed in the pathogenesis of vascular disease. (J Vasc Surg 1998;28:676-86.)

Smooth Muscle Cell Heterogeneity: Role of Specific Smooth Muscle Cell Subpopulations in Pulmonary Vascular Disease

The severity of chronic forms of pulmonary hypertension is determined, at least in part, by the extent of structural changes in the pulmonary arterial wall. Depending on the stimulus causing pulmonary hypertension, and on the location within the lung of the vessel examined, variable degrees of intimal, medial, and/or adventitial thickening have been observed. Intimal and medial changes are due, in large part, to smooth muscle cell (SMC) proliferation, hypertrophy, and matrix deposition. Blood-borne and locally produced mitogens, hypoxia, and mechanical stress are the stimuli that collectively drive these cellular responses by activating a cascade of intracellular signaling mechanisms, including tyrosine kinases, calcium (Ca2+), mitogen-activated protein kinase (MAPK), and protein kinase C (PKC), that affect SMC growth, differentiation, and/or matrix protein synthesis.

Plasminogen activator expression in rat arterial smooth muscle cells depends on their phenotype and is modulated by cytokines.

Cultured rat aortic smooth muscle cells (SMCs) exhibit at least 2 phenotypic variants: (1) a spindle-shaped phenotype, obtained from normal adult media, and (2) an epithelioid phenotype, obtained from intimal thickening 15 days after endothelial injury. Both phenotypes can be cloned from each location, with normal media yielding a majority of spindle-shaped clones and intimal thickening yielding a majority of epithelioid clones. These findings suggest that intimal thickening develops essentially from a subpopulation of medial SMCs exhibiting epithelioid features in vitro. Using zymographic and Northern blot analyses, we have studied plasminogen activator (PA) expression by these SMCs. Our results show that epithelioid SMCs, cultured as whole SMC populations or as clones, display higher PA activity than do spindle-shaped SMCs, irrespective of their origin. This is mainly due to differences in the expression of tissue PA and, to a lesser extent, urokinase PA and is accompanied by a decrease in PA inhibitor 1. Tissue PA activity is increased by basic fibroblast growth factor and platelet-derived growth factor-BB, particularly in epithelioid SMCs. Taken together, these results indicate that SMCs are heterogeneous with respect to their proteolytic profile, at least as far as the PA system is concerned. Proteolytic activity of the different SMC populations is modulated by cytokines that play a role in intimal thickening. Our results are in agreement with the suggestion that epithelioid SMCs are mainly responsible for intimal thickening.

Smooth muscle-specific expression of the smooth muscle myosin heavy chain gene in transgenic mice requires 5'-flanking and first intronic DNA sequence.

The smooth muscle myosin heavy chain (SM-MHC) gene encodes a major contractile protein whose expression exclusively marks the smooth muscle cell (SMC) lineage. To better understand smooth muscle differentiation at the transcriptional level, we have initiated studies to identify those DNA sequences critical for expression of the SM-MHC gene. Here we report the identification of an SM-MHC promoter-intronic DNA fragment that directs smooth muscle-specific expression in transgenic mice. Transgenic mice harboring an SM-MHC-lacZ reporter construct containing approximately 16 kb of the SM-MHC genomic region from -4.2 to + 11.6 kb (within the first intron) expressed the lacZ transgene in all smooth muscle tissue types. The inclusion of the intronic sequence was required for transgene expression, since 4.2 kb of the 5'-flanking region alone was not sufficient for expression. In the adult mouse, transgene expression was observed in both arterial and venous smooth muscle, in airway smooth muscle of the trachea and bronchi, and in the smooth muscle layers of all abdominal organs, including the stomach, intestine, ureters, and bladder. During development, transgene expression was first detected in airway SMCs at embryonic day 12.5 and in vascular and visceral SMC tissues by embryonic day 14.5. Of interest, expression of the SM-MHC transgene was markedly reduced or absent in some SMC tissues, including the pulmonary circulation. Moreover, the transgene exhibited a heterogeneous pattern between individual SMCs within a given tissue, suggesting the possibility of the existence of different SM-MHC gene regulatory programs between SMC subpopulations and/or of episodic rather than continuous expression of the SM-MHC gene. To our knowledge, results of these studies are the first to identify a promoter region that confers complete SMC specificity in vivo, thus providing a system with which to define SMC-specific transcriptional regulatory mechanisms and to design vectors for SMC-specific gene targeting.

Smooth muscle cells isolated from discrete compartments of the mature vascular media exhibit unique phenotypes and distinct growth capabilities.

Heterogeneity of smooth muscle cell (SMC) phenotype and function is rapidly emerging as an important concept. We have recently described that phenotypically distinct SMC subpopulations in bovine pulmonary arteries exhibit unique proliferative and matrix-producing responses to hypoxic pulmonary hypertension. To provide better understanding of the molecular mechanisms contributing to this phenomenon, experimental studies will require a reliable in vitro model. The purpose of the present study was first to determine if distinct SMC subpopulations, similar to those observed in vivo, could be selectively isolated from the mature arterial media, and then to evaluate whether select SMC subpopulations would exhibit heightened responses to growth-promoting stimuli and hypoxia. We were able to reproducibly isolate at least four phenotypically unique cell subpopulations from the inner, middle, and outer compartments of the arterial media. Differences in cell phenotype were demonstrated by morphological appearance and differential expression of muscle-specific proteins. The isolated cell subpopulations exhibited markedly different growth capabilities. Two SMC subpopulations grew slowly in 10% serum and were quiescent in plasma-based medium. The other two cell subpopulations, exhibiting nonmuscle characteristics, grew rapidly in 10% serum and proliferated in plasma-based medium and in response to hypoxia. Certain colonies of the nonmuscle-like cell subpopulations were found to grow autonomously under serum-deprived conditions and to secrete mitogenic factors. Our data, demonstrating that phenotypically distinct cells with enhanced growth potential exist within the normal arterial media, support the idea that these unique cells could contribute selectively to the pathogenesis of vascular disease.

Heterogeneity of airway smooth muscle at tissue and cellular levels

Heterogeneity of smooth muscle behavior is rapidly emerging as an important phenomenon. However, it is only recently that investigators have begun to explore its physiological and pathogenic importance. Stenmark's group recently reported the existence of four subpopulations of smooth muscle cells in pulmonary artery, and noted that only a specific group of cells was likely responsible for the hyperplasia of smooth muscle in the hypertensive artery. Our studies reported here were focused on determining whether heterogeneity of contractile properties existed in airway smooth muscle cells. Firstly, mechanical properties of muscle strips, free of cartilage and epithelium, were compared among the different generations of canine airway from trachea down to bronchial generation 6. Two distinct groups of smooth muscle were detected from these airways: (i) an extrapulmonary group, consisting of muscle strips from the trachea, and bronchial generation 1 and 2, characterized by higher maximum shortening capacity (delta Lmax) and zero-load velocity (V0) of shortening; (ii) an intrapulmonary group, containing bronchi from generation 3 to 6, with lower delta Lmax and V0. This led to the implication that smooth muscles from these two groups consisted of different types of cells. Secondly, single smooth muscle cells were isolated from trachea and bronchus (from generation 3 to 6) enzymatically, and the unloaded shortening capacity of fresh isolated cells was measured employing electrical field stimulation at room temperature. Two types of cells were identified from these preparations based on the length of muscle cells: type I, with a mean length of 110 +/- 3 microns (SE), predominated in trachea (up to 84%); type II, with a mean length of 200 +/- 9 microns (SE), accounted for 58% in bronchus. The mechanical properties of these two types of cells were also different. Type I cells shortened by 28% of their original lengths in response to maximal stimulation, while most type II cells (90% of total type II cells) shortened 16% (type IIA), and the remainder shortened 58% (type IIB). Because of the paucity of information about the orientation and arrangement of these different types of smooth muscle cells in the airway tree, it is difficult to apply these data directly for interpretation of differences in mechanical properties of smooth muscle cells obtained from strips from extrapulmonary and intrapulmonary airways. Existence of great numbers of cells with lower shortening capacity in intrapulmonary airways may be the major factor responsible for lower mechanical performance of these airway smooth muscle strips. The relatively lower mechanical performance of intrapulmonary airway smooth muscle may be of importance in preventing excessive narrowing of these airways during muscle contraction and optimizing airflow.

Phenotypic heterogeneity of rat arterial smooth muscle cell clones. Implications for the development of experimental intimal thickening.

It is well accepted that smooth muscle cells (SMCs) cultured from normal rat arterial media have different morphological and biological features compared with SMCs cultured from experimental intimal thickening (IT) 15 days after endothelial injury. It is not known, however, whether the phenotypic modulation producing IT cells occurs in any medial SMCs or only in a particular SMC subpopulation. To distinguish among these possibilities, the phenotypic features of SMC clones derived from normal adult media and the IT 15 days after endothelial lesion were analyzed according to morphological appearance, replicative activity in the presence and absence of fetal calf serum, and [3H]thymidine incorporation and motile activity; these features were compared with those of the respective SMC parental populations. Two categories of SMC clones predominated: spindle clones, with morphological features similar to those of the parental population from the normal media, and epithelioid clones, with morphological features similar to those of the IT parental population. Both categories were present among clones produced from normal media and IT; however, spindle was more common among normal media clones, and epithelioid, among IT clones. The behavior in vitro was distinct for each category of clones and did not depend on their origin. Our results are compatible with the possibility that the SMC population of IT in vivo derives mainly from SMCs belonging to the category exhibiting epithelioid features in vitro.

Expression and localization of tropoelastin mRNA in the developing bovine pulmonary artery is dependent on vascular cell phenotype.

During vascular development, the expression of tropoelastin (TE) messenger ribonucleic acid (mRNA) has been shown to be time dependent and to form complex patterns along the longitudinal and radial arterial axes. The factors contributing to these patterns of TE expression are not known, but it has been suggested that they reflect phenotypic changes in developing smooth muscle cells (SMC). In order to examine a possible correlation between the developmental state of the SMC and TE expression during lung vascular development, we localized and assessed relative TE mRNA expression in the developing bovine main pulmonary artery (PA), and correlated the observed patterns of TE expression to changes in SMC phenotype as determined by the expression of various developmentally related SMC proteins. Further, because TE expression can be modulated by physical forces such as pressure, fetal PA TE expression was evaluated with regard to changes in fetal arterial pressure. We found that expression of TE mRNA exhibited a biphasic pattern during fetal development. In early gestation, expression was noted throughout the entire PA wall; at midgestation, expression was markedly decreased in the outer wall but maintained in the inner vascular media; at late gestation, reexpression was observed throughout the entire PA wall, albeit in a heterogeneous pattern. Immunohistochemical studies showed that the decrease in SMC TE expression during midgestation coincided with the acquisition of SMC-specific proteins such as smooth muscle myosin heavy chains and desmin. The reexpression of TE late in gestation occurred in these "differentiated" SMC and was temporally associated with a large increase in arterial pressure shown to occur in late gestation. In addition, we identified an SMC population defined by its immunoreactivity to the muscle-specific cytoskeletal protein meta-vinculin that did not express TE mRNA either during fetal PA development or postnatally when PA hypertension was induced. We conclude that both the developmental state of the SMC and hemodynamic forces correlate with the pattern of PA TE mRNA expression during pulmonary vascular development. Further, a subpopulation of SMC defined by meta-vinculin expression exists in the fetal and neonatal bovine vascular wall and does not express detectable levels of TE mRNA regardless of vascular pressure.

Differential processes of vascular smooth muscle cell differentiation within elastic and muscular arteries of rats and rabbits: an immunofluorescence study of desmin and vimentin distribution.

Two main populations of smooth muscle cells exist in the arterial media of adult mammals with respect to expression of two intermediate filament proteins: vimentin-positive/desmin-negative cells (V+/D-) and vimentin-positive/desmin-positive ones (V+/D+). However, it is still not understood how this phenotypic diversity is established. Since the proportion and the distribution patterns of the two muscle cell populations depend both on the type of blood vessel and the species examined, the aim of the present study was to determine and to compare their developmental origin in various artery segments from two different species. Using confocal scanning laser microscopy and sections stained by means of immunofluorescence, the distribution patterns of desmin and vimentin were compared in transverse sections of thoracic and abdominal aortas (elastic arteries) and of the femoral artery (muscular artery) of newborn and adult rats (n = 12) and rabbits (n = 12). The comparison of sections labelled with specific antibodies showed the existence of a subpopulation of smooth muscle cells in the aortas, but not in the femoral artery, which expressed desmin in newborns but not in adults. These data suggest that the phenotype of smooth muscle cells in elastic arteries but not in muscular arteries is modulated during development.

Hypoxia selectively induces proliferation in a specific subpopulation of smooth muscle cells in the bovine neonatal pulmonary arterial media.

Medial thickening of the pulmonary arterial wall, secondary to smooth muscle cell (SMC) hyperplasia, is commonly observed in neonatal hypoxic pulmonary hypertension. Because recent studies have demonstrated the existence of multiple phenotypically distinct SMC populations within the arterial media, we hypothesized that these SMC subpopulations would differ in their proliferative responses to hypoxic pulmonary hypertension and thus contribute in selective ways to the vascular remodeling process. Expression of meta-vinculin, a muscle-specific cytoskeletal protein, has been shown to reliably distinguish two unique SMC subpopulations within the bovine pulmonary arterial media. Therefore, to assess the proliferative responses of phenotypically distinct SMC subpopulations in the setting of neonatal pulmonary hypertension, we performed double immunofluorescence staining on pulmonary artery cryosections from control and hypertensive calves with antibodies against meta-vinculin and the proliferation-associated nuclear antigen, Ki-67. We found that, although neonatal pulmonary hypertension caused significant increases in overall cell replication, proliferation occurred almost exclusively in one, the meta-vinculin-negative SMC population, but not the other SMC population expressing meta-vinculin. We also examined fetal pulmonary arteries, where proliferative rates were high and meta-vinculin expression again reliably distinguished two SMC subpopulations. In contrast to the hypertensive neonate, we found in the fetus that the relative proliferative rates of both SMC subpopulations were equal, thus suggesting the existence of different mechanisms controlling proliferation and expression of cytoskeletal proteins in the fetus and neonate. We conclude that phenotypically distinct SMC populations in the bovine arterial media exhibit specific and selective proliferative responses to neonatal pulmonary hypertension. Distinct SMC subpopulations may, thus, contribute in unique ways to vascular homeostasis under both normal and pathologic conditions.

Correlation between the presence of an immature smooth muscle cell population in tunica media and the development of atherosclerotic lesion. A study on different-sized rabbit arteries from cholesterol-fed and Watanabe heritable hyperlipemic rabbits

Mapping the distribution of an immature smooth muscle cell (SMC) subpopulation in large- and small-sized arterial vessels was carried out in normocholesterolemic rabbits and compared with the mapping atherosclerotic lesions in endogenously (Watanabe heritable hyperlipemic, WHHL) and exogenously derived (cholesterol-fed, CT) hypercholesterolemic rabbits. This cell subset is identified by a specific myosin isoform content and displays an intermediate degree of differentiation between fetal- and adult-type SMC. Monoclonal anti-myosin antibodies, immunofluorescence procedures, and different arterial segments of a rabbit vessel tree, i.e. from aorta to dental pulp (common carotid, external carotid, lingual, facial, maxillary, inferior alveolar arteries, and dental branches of alveolar arteries) were studied. WHHL of different ages (3 to 12 months), and two different concentrations of CT (2% and 0.2%) in the diet for 3 and 12 months, respectively, were used. The results of the present study indicate that: (1) using a diet with a higher percentage of CT (rabbits fed 2% CT-diet for 3 months) there is maximum expansion of atherosclerotic lesions from the aorta up to the maxillary artery; (2) localization of atherosclerotic lesions with a lower CT content in the diet is dependent on the duration of feeding and may involve the aorta up to the external carotid artery; (3) the development of the atherosclerotic lesion in hypercholesterolemic rabbit is strictly related to the appearance of an intermediate SMC subtype; (4) atherosclerotic lesions occur only in those arterial sites which, in corresponding normocholesterolemic rabbit, contain intermediate-type SMC; and (5) no differences can be found in the distribution of SMC subpopulations present in the lesions from WHHL, CT-fed animals, or at various arterial levels, whereas some discrepancies can be shown in aortic atherogenesis.

A Subpopulation of Smooth Muscle Cells in Injured Rat Arteries Expresses Platelet-Derived Growth Factor–B Chain mRNA

Proliferation of smooth muscle cells (SMCs) and formation of a neointima are characteristics of the response of rat carotid arteries to balloon injury. Rat platelet-derived growth factor (PDGF)-B was cloned, thus allowing us to use species-specific probes to carry out in situ hybridization on the surface of injured arteries. A distinct population of luminal SMCs (7% to 10%) in the developing neointima expressed PDGF-B mRNA, but very few luminal SMCs still expressed PDGF-B (0.5%) when the lesion had stopped growing. Primary SMC cultures revealed expression of PDGF-B mRNA in 1.6% of SMCs derived from normal tunica media and in 11% of SMCs derived from the neointima. These data demonstrate that SMCs in the injured vessel wall are heterogeneous with regard to PDGF-B expression and that subculturing of these cells may give rise to cultures that are either positive or negative for PDGF-B expression. Furthermore, with abundant expression of the PDGF receptor beta-subunit expressed by intimal SMCs, our findings provide evidence that PDGF-B synthesized by these cells may be involved in intimal lesion formation via a paracrine or autocrine mechanism.