Microvascular pericytes contain muscle and nonmuscle actins

We have affinity-fractionated rabbit antiactin immunoglobulins (IgG) into classes that bind preferentially to either muscle or nonmuscle actins. The pools of muscle- and nonmuscle-specific actin antibodies were used in conjunction with fluorescence microscopy to characterize the actin in vascular pericytes, endothelial cells (EC), and smooth muscle cells (SMC) in vitro and in situ. Nonmuscle-specific antiactin IgG stained the stress fibers of cultured EC and pericytes but did not stain the stress fibers of cultured SMC, although the cortical cytoplasm associated with the plasma membrane of SMC did react with nonmuscle-specific antiactin. Whereas the muscle-specific antiactin IgG failed to stain EC stress fibers and only faintly stained their cortical cytoplasm, these antibodies reacted strongly with the fiber bundles of cultured SMC and pericytes. Similar results were obtained in situ. The muscle-specific antiactin reacted strongly with the vascular SMC of arteries and arterioles as well as with the perivascular cells (pericytes) associated with capillaries and post-capillary venules. The non-muscle-specific antiactin stained the endothelium and the pericytes but did not react with SMC. These findings indicate that pericytes in culture and in situ possess both muscle and nonmuscle isoactins and support the hypothesis that the pericyte may represent the capillary and venular correlate of the SMC.     

Smooth muscle-specific alpha tropomyosin is a marker of fully differentiated smooth muscle in lung.

Tropomyosin (Tm) is one of the major components of smooth muscle. Currently it is impossible to easily distinguish the two major smooth muscle (sm) forms of Tm at a protein level by immunohistochemistry due to lack of specific antibodies. Alpha-sm Tm contains a unique 2a exon not found in any other Tm. We have produced a polyclonal antibody to this exon that specifically detects alpha-sm Tm. We demonstrate here the utility of this antibody for the study of smooth muscle. The tissue distribution of alpha-sm Tm was shown to be highly specific to smooth muscle. Alpha-sm Tm showed an identical profile and tissue colocalization with alpha-sm actin both by Western blotting and immunohistochemistry. Using lung as a model organ system, we examined the developmental appearance of alpha-sm Tm in comparison to alpha-sm actin in both the mouse and human. Alpha-sm Tm is a late-onset protein, appearing much later than actin in both species. There were some differences in onset of appearance in vascular and airway smooth muscle with airway appearing earlier. Alpha-sm Tm can therefore be used as a good marker of mature differentiated smooth muscle cells. Along with alpha-sm actin and sm-myosin antibodies, alpha-sm Tm is a valuable tool for the study of smooth muscle.     

A function for filamentous alpha-smooth muscle actin: retardation of motility in fibroblasts

Actins are known to comprise six mammalian isoforms of which beta- and gamma-nonmuscle actins are present in all cells, whereas alpha-smooth muscle (alpha-sm) actin is normally restricted to cells of the smooth muscle lineages. alpha-Sm actin has been found also to be expressed transiently in certain nonmuscle cells, in particular fibroblasts, which are referred to as myofibroblasts. The functional significance of alpha-sm actin in fibroblasts is unknown. However, myofibroblasts appear to play a prominent role in stromal reaction in breast cancer, at the site of wound repair, and in fibrotic reactions. Here, we show that the presence of alpha-sm actin is a signal for retardation of migratory behavior in fibroblasts. Comparison in a migration assay of fibroblast cell strains with and without alpha-sm actin revealed migratory restraint in alpha-sm actin-positive fibroblasts. Electroporation of monoclonal antibody (mAb) 1A4, which recognizes specifically the NH2-terminal Ac-EEED sequence of alpha-sm actin, significantly increased the frequency of migrating cells over that obtained with an unrelated antibody or a mAb against beta-actin. Time- lapse video microscopy revealed migratory rates of 4.8 and 3.0 microns/h, respectively. To knock out the alpha-sm actin protein, several antisense phosphorothioate oligodeoxynucleotide (ODNs) were tested. One of these, 3'UTI, which is complementary to a highly evolutionary conserved 3' untranslated (3'UT) sequence of alpha-sm actin mRNA, was found to block alpha-sm actin synthesis completely without affecting the synthesis of any other proteins as analyzed by two-dimensional gel electrophoresis. Targeting by antisense 3'UTI significantly increased motility compared with the corresponding sense ODN. alpha-Sm actin inhibition also led to the formation of less prominent focal adhesions as revealed by immunofluorescence staining against vinculin, talin, and beta1-integrin. We propose that an important function of filamentous alpha-sm actin is to immobilize the cells.     

Coexpression of α-sarcomeric actin, α-smooth muscle actin and desmin during myogenesis in rat and mouse embryos I. Skeletal muscle

Expression of vimentin, desmin, alpha-sarcomeric and alpha-smooth muscle actins in embryonic tissues of rat and mice was examined using an immunohistochemical approach. The results showed a similarity in the expression of desmin and alpha-actin isoforms (alpha-sr and alpha-sm) in skeletal muscle cells during murine feto-embryonic development. In the two species, coexpression of alpha-sr and alpha-sm actins has been observed in cardiomyoblasts, myotomal myoblasts and myotubes. The intensity of alpha-sm actin expression decreased during the terminal steps of myogenesis and disappeared completely in mature cardiomyocytes and myofibres. Desmin was expressed in all prefusion myoblasts (type 1 and 2 myoblasts), myotubes, and in myofibres. The appearance of desmin in myoblasts of somites preceded by a few hours the expression of the alpha-actins (alpha-sr and alpha-sm). Our study on vimentin expression, limited to rat embryos, revealed that somite premyoblasts expressed only vimentin, type 1 myoblasts expressed vimentin and desmin, and type 2 myoblasts (rhabdomyoblasts) expressed desmin and alpha-actins (alpha-sr and alpha-sm). Our study demonstrates the resemblance between feto-embryonic myogenesis and myogenic neoplastic differentiation: desmin appears before the alpha-actins in embryonic myoblasts, and can be considered as a marker of an initial step in myogenic differentiation. alpha-sm actin, considered as a striated muscle cell feto-embryonic actin, is expressed transiently in skeletal myoblasts and cardiomyoblasts during development and reappears during neoplastic transformation of skeletal muscle.     

Appearance of alpha-smooth muscle actin in human eye lens cells of anterior capsular cataract and in cultured bovine lens-forming cells

Using light and electron-microscopic immunolocalization techniques, and gel electrophoresis combined with immunoblotting, we have examined the expression of cytoskeletal proteins in normal human fetal, child and adult lenses, in human anterior capsular cataract and in bovine lens cells in vivo and in vitro. In this report, we focus our observations on the pattern of actin-isoform expression during normal and pathological situations in vivo and culture conditions. We have noted that cells of developing and mature human lenses as well as bovine lens cells in situ contain only beta- and gamma-actins. In contrast, alpha-smooth muscle (alpha-sm) actin, an isoform typical of smooth muscle differentiation, was demonstrated in bovine lens cells at different times of culture. Moreover, the multilayered cells observed in the subcapsular zone of human anterior capsular cataract were characterized by the presence of alpha-sm actin. Thus, extensive changes in actin-isoform expression take place in lens cells growing in culture and may also occur during cataractogenesis. The biological meaning of the appearance of a marker of myoid differentiation in the ectodermally derived lens-forming cells is discussed.     

Regulation of smooth muscle actin expression and contraction in adult human mesenchymal stem cells

Prior studies have demonstrated the expression of a contractile actin isoform, alpha-smooth muscle actin, in bone marrow stromal cells. One objective of the current study was to correlate contractility with alpha-smooth muscle actin expression in human bone marrow stroma-derived mesenchymal stem cells. A second objective was to determine the effects of transforming growth factor-beta1, platelet derived growth factor-BB, and a microfilament-modifying agent on alpha-smooth muscle actin expression and alpha-smooth muscle actin-enabled contraction. Adult human bone marrow stromal cells were passaged in monolayer and their inducibility to chondrocytic, osteoblastic, and adipogenic phenotypes was demonstrated. Western blot analysis was employed along with densitometry to quantify the alpha-smooth muscle actin content of the cells and their contractility was evaluated by their contraction of a type I collagen-glycosaminoglycan sponge-like matrix into which they were seeded. Transforming growth factor-beta1 (1 ng/ml) significantly increased and platelet-derived growth factor-BB (10 ng/ml) decreased alpha-smooth muscle actin expression and the contractility of the cells. Cytochalasin D also blocked cell contraction. There was a notably high correlation of cell-mediated contraction normalized to the DNA content of the matrices with alpha-smooth muscle actin content of the cells by linear regression analysis (R(2) = 0.88). These findings lay the groundwork for considering the role of alpha-smooth muscle actin-enabled contraction in mesenchymal stem cells and in their connective tissue cell progeny.     

The mammalian anti-α-smooth muscle actin monoclonal antibody recognizes an α-actin-like protein in planaria (Dugesia lugubris s.l.)

The presence of an alpha-smooth muscle (alpha-sm) actin-like protein in planaria (Dugesia lugubris s.l.) is reported. The protein shows a 42 kDa molecular weight determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis and is specifically recognized by the mammalian anti alpha-sm actin monoclonal antibody. When a planarian is induced to regenerate by head amputation, the immunostaining of the alpha-sm actin-like molecule becomes important in the area of growing blastema, reaching a maximum between 70-120 hours after injury. Conventional electron microscopy at the 4-day-regeneration stage shows that blastema-forming cells are a homogeneous population whose morphological features resemble those of migrating mesenchyme-like cells; only the myoblasts show a recognizable phenotype. The immunocytochemical localization of alpha-sm actin-like molecule by immunoperoxidase (light microscopy) and immunogold stains (electron microscopy) was carried out on both intact and injured worms. The antigen was localized mainly at the basal portion of the epidermal cells and in the undifferentiated mesenchyme-like cells. Myoblasts, but not differentiated myofibers, were also labelled by this antibody. The results indicate that in the lower Eumetazoan planarians, as well as in vertebrates, the alpha-sm actin can be considered to be a marker for myoid differentiation. The suggestion that alpha-sm actin can be used as a marker for mesenchyme-like cells in vertebrates and in invertebrates is also discussed.     

Chemically induced rhabdomyosarcomas in rats. Ultrastructural, immunohistochemical, biochemical features and expression of alpha-actin isoforms

A series of 14 primary and two metastatic rat rhabdomyosarcomas (RMS) induced with nickel sulfide was studied by light microscopy, transmission electron microscopy, indirect immunofluorescence, avidin-biotin-peroxidase immunohistochemistry and two-dimensional gel electrophoresis. Monoclonal or affinity-purified polyclonal antibodies were used for the immunohistochemical demonstration of vimentin, desmin, alpha-smooth muscle (alpha-sm) actin and alpha-sarcomeric (alpha-sr) actin. By histological and ultrastructural studies, four categories of RMS were diagnosed on the basis of the neoplastic cell types. These were: (1) well-differentiated RMS (n = 2), (2) pleomorphic RMS (n = 8), (3) embryonal RMS (n = 4), and (4) embryonal myosarcomas (n = 2). Immunohistochemically, all these neoplasms expressed desmin and alpha-sr actin, reflecting their rhabdomyoblastic origin. Two dimensional gel electrophoresis performed on five neoplasms demonstrated alpha, beta and gamma actins spots in all cases. This study demonstrates that the alpha-sr actin antibody represents a good marker for rhabdomyoblastic differentiation is useful in the diagnosis of RMS since it was present in all morphologically confirmed RMS and in two ultrastructurally undifferentiated sarcomas positive for desmin. Neoplastic cells positive for alpha-sm actin were noted in 11 confirmed RMS. Double indirect immunofluorescence showed that all alpha-sm and alpha-sr positive cells also contained desmin. Co-expression of alpha-sr and alpha-sm actins was studied in serial sections of formalin-fixed, paraffin-embedded tumor tissue. Both alpha-sm and alpha-sr actins were localized in some rhabdomyoblasts. This study confirms our previous observations in human tumors and shows, for the first time, that alpha-sr and alpha-sm actins can be present in the same neoplastic cell in vivo.     

Contractile proteins in pericytes at the blood-brain and blood-retinal barriers

Evidence from a variety of sources suggests that pericytes have contractile properties and may therefore function in the regulation of capillary blood flow. However, it has been suggested that contractility is not a ubiquitous function of pericytes, and that pericytes surrounding true capillaries apparently lack the machinery for contraction. The present study used a variety of techniques to investigate the expression of contractile proteins in the pericytes of the CNS. The results of immunocytochemistry on cryosections of brain and retina, retinal whole-mounts and immunoblotting of isolated brain capillaries indicate strong expression of the smooth muscle isoform of actin (α-SM actin) in a significant number of mid-capillary pericytes. Immunogold labelling at the ultrastructural level showed that α-SM actin expression in capillaries was exclusive to pericytes, and endothelial cells were negative. Compared to α-SM actin, non-muscle myosin was present in lower concentrations. By contrast, smooth muscle myosin isoforms, were absent. Pericytes were strongly positive for the intermediate filament protein vimentin, but lacked desmin which was consistently found in vascular smooth muscle cells. These results add support for a contractile role in pericytes of the CNS microvasculature, similar to that of vascular smooth muscle cells.     

Collagen gel contraction serves to rapidly distinguish epithelial- and mesenchymal-derived cells irrespective of alpha-smooth muscle actin expression

Mesenchymal-like cells in the stroma of breast cancer may arise as a consequence of plasticity within the epithelial compartment, also referred to as epithelial-mesenchymal transition, or by recruitment of genuine mesenchymal cells from the peritumoral stroma. Cells of both the epithelial compartment and the stromal compartment express alpha smooth muscle actin (alpha-sm actin) as part of a myoepithelial or a myofibroblastic differentiation program, respectively. Moreover, because both epithelial- and mesenchymal-derived cells are nontumorigenic, other means of discrimination are warranted. Here, we describe the contraction of hydrated collagen gels as a rapid functional assay for the distinction between epithelial- and mesenchymal-derived stromal-like cells irrespective of the status of alpha-sm actin expression. Three epithelial-derived cell lines and three genuine mesenchymal-derived breast cell lines were plated on top of hydrated collagen lattices. Reduction in gel height was measured every hour for 6 h and after 22 h using an x-y-z automated position table. Significantly, the epithelial-derived cells, irrespective of a high alpha-sm actin expression, had a fivefold lower contractility (10.0% reduction in gel height) than their true mesenchymal counterparts (53.1% reduction in gel height). To test whether at all force generation could be induced in the nonmesenchymal cells by alpha-sm actin, transductions were performed to obtain a tetracycline-dependent expression. Expression under these conditions did not augment contractility. It is concluded that epithelial-derived mesenchymal-like cells are functionally defective within a connective tissue environment irrespective of an apparent contractile phenotype.     

Regulation of alpha-smooth muscle actin and other polypeptides in proliferating and density-arrested vascular smooth muscle cells

We have examined alpha-smooth muscle actin (alpha-SM actin) protein and mRNA levels in proliferating and density-arrested rabbit vascular smooth muscle cells (SMC) and also studied overall polypeptide synthesis in these cells by two-dimensional (2-D) gel electrophoresis. Of the approximately 1,000 cellular polypeptides resolved by 2-D gel analysis, we consistently detected increased expression of 12 polypeptides in growth-arrested SMC. These polypeptides, with apparent molecular weights of 24,000 to 55,000 exhibited relative increases of between fourfold to greater than tenfold. Three of these polypeptides were expressed at undetectable levels in proliferating SMC. We also detected 12 secreted polypeptides that were expressed at higher levels in growth-arrested SMC. More changes were associated with the secreted polypeptides, since they represented approximately 4% of the total resolved secreted polypeptides, while only 1% of the cellular polypeptides were increased in high-density growth-arrested cells. Under these conditions we observed no change in relative alpha-SM actin protein content as determined by 2-D gel analysis and Western blots. This was corroborated by high levels of alpha-SM actin mRNA levels in both proliferating and high-density growth-arrested SMC. These results indicate rabbit vascular SMC maintain a high level of expression of a smooth muscle differentiation marker (alpha-SM actin) in a proliferation- and density-independent manner. We also examined polypeptide synthesis in SMC isolated by enzymatic digestion of the aorta vs. cells isolated by the explant method. We found that although overall protein patterns were remarkably similar, several differences were observed. These differences were not due to increased contamination by fibroblasts, since both enzymatically- and explant-derived SMC contained high levels of alpha-SM actin as determined by immunofluorescence and by Northern analysis.     

Transforming growth factor-beta1 promotes the morphological and functional differentiation of the myofibroblast

The myofibroblast is responsible for the generation of contractile force associated with wound contraction and pathological contractures and is characterized by the presence of alpha-smooth muscle (alpha-sm) actin-containing stress fibers, vinculin-containing fibronexus adhesion complexes, and fibronectin fibrils containing the ED-A splice variant. Transforming growth factor-beta1 (TGF-beta1) can promote the expression of alpha-sm actin in myofibroblasts, but the functional significance of this increased expression is unclear. In this study, we demonstrate, using the stress-relaxed collagen lattice contraction assay, that TGF-beta1 promoted a dose-dependent increase in the generation of contractile force in myofibroblasts and a concomitant increase in the expression of alpha-sm actin. We also demonstrate that TGF-beta1 enhanced the formation of the structural elements important in myofibroblast contractile force generation and transmission, including stress fibers, vinculin-containing fibronexus adhesion complexes, and fibronectin fibrils, and that this enhancement occurred prior to, and independent of, alpha-sm actin expression. This differentiated myofibroblast phenotype was not stable. Removal of TGF-beta1 resulted in reduced expression of alpha-sm actin as well as a decreased assembly of stress fibers and vinculin-containing adhesion complexes; however, there was no reduction in fibronectin fibrils. We conclude that TGF-beta1 promotes the morphological and functional differentiation of the myofibroblast by first enhancing the formation of the structural elements characteristic of the myofibroblast followed by increased expression of alpha-sm actin and contractile force generation.     

Differentiation of the smooth muscle cell phenotypes during embryonic development of coronary vessels in the rat

Smooth muscle cell (SMC) maturation during embryonic development of coronary arteries and veins was studied in rats using different markers of the contractile phenotypes. The spatio-temporal pattern of distribution of these markers compared with the developing tunica media was examined. Alpha-smooth muscle actin (alpha-SMA) was the first marker of the SMC in the tunica media of coronary arteries found in ED16 hearts, followed by smooth muscle myosin heavy chain isoform which occurred on ED17. Subsequently 1E12 antigen was expressed in coronary artery wall in ED18 hearts, and finally smoothelin. The markers occur within the proximal part of the coronary arteries and deploy toward the apex. They are also found within the great vessels. None of the markers except for the alpha-SMA were found in coronary veins during embryonic life. We conclude that the SMC population of the developing tunica media of coronary vessels differentiates by the acquisition of particular markers and this process lasts till the end of the prenatal and early postnatal life.     

Immunolocalization of muscle and nonmuscle isoforms of actin in myogenic cells and adult skeletal muscle

In vertebrate skeletal muscle, the proliferating myoblasts synthesize nonmuscle isoforms of actin, and the cells begin to express muscle-specific actin isoforms during their myogenic differentiation. To study the distributions of the actin isoforms in myogenic cells and fully differentiated skeletal muscle, we prepared a peptide antibody specific for the skeletal alpha isoform of actin and used this antibody along with an antibody specifically reactive with nonmuscle gamma actin to stain cultured myotubes and adult skeletal myofibrils by double-indirect immunofluorescence. At this level of resolution, no differences in isoform localization were seen: Both muscle and nonmuscle actins were detected in the myotubes and in the striations of mature myofibrils. Myotubes were also double-stained using immunogold electron microscopy, and the isoform distributions were determined quantitatively by counting the two sizes of gold particles that corresponded to labeling with each antibody. A quantitative analysis of immunoreactivity revealed that, although both forms were present in all actin-containing structures, nonmuscle actin was relatively more prevalent along the edges (cortical microfilaments) of the myotubes, whereas the muscle isoform predominated in the interior regions (containing forming myofibrils). Thus, we have found evidence of a heterogeneous distribution of muscle and nonmuscle actin isoforms in differentiating myogenic cells, and we have demonstrated that a nonmuscle actin isoform is a component of the muscle contractile apparatus.     

Pericyte growth and contractile phenotype: Modulation by endothelial-synthesized matrix and comparison with aortic smooth muscle

We compared the effects of endothelial-synthesized matrix and purified matrix molecules on pericyte (PC) and aortic smooth muscle cell (SMC) growth, heparin sensitivity, and contractile phenotype in vitro. When PC are plated on endothelial-synthesized (EC) matrix, cell number is, on average, 3.1-fold higher than identical populations grown on plastic. Under the same conditions, SMC proliferation is stimulated 1.6-fold. Purified matrix molecules, such as collagen type IV (COLL) or fibronectin (FN), both major components of the EC matrix, stimulate PC/SMC growth 1.2–1.7-fold. Heparin (100 μg/ml), which inhibits the growth of early passage SMC by 60%, inhibits PC growth ～50%, when cells were plated on plastic. However, PC plated on EC matrix in the presence of heparin (100 μg/ml) grow as well as parallel cultures grown on plastic (in the absence of heparin). Concomitant with matrix-stimulated proliferation, we observed a marked reduction in PC containing alpha vascular smooth muscle actin (αVSMA), as seen by immunofluorescence using affinity-purified antibodies (173/615 positive pericytes on DOC matrix (28%) vs. 221/285 (77%) positive on glass). SMC respond similarly. Whereas αVSMA protein is markedly altered when PC and SMC are cultured on EC matrix, similar reductions in mRNA are not observed. However, Northern blotting does reveal that PC contain 17–30 times the steady-state levels of αVSMA mRNA compared to SMC. When SMC and PC cultures on plastic are treated with heparin, the steady-state levels of vascular smooth muscle actin mRNA increase 5 and 1.5 fold, respectively. Similarly, heparin treatment of PC grown on plastic induces a 1.8 fold increase in nonmuscle actin mRNA. These heparin-induced alterations in isoactin mRNA levels are not seen when PC are cultured on EC matrix. We also observed reductions in αVSMA and β actin mRNA levels when PC are plated on FN, where they maintain a ratio of 13:1 (α:β). Similar ratios are found in SMC present in rat and bovine aortae in vivo. These steady-state isoactin mRNA ratios are slightly different from those seen in cultured PC (8–10:1; α:β). These results suggest that selective synthesis and remodelling of the endothelial basal lamina may signal alterations in pericyte growth and contractile phenotype during normal vascular morphogenesis, angiogenesis, or during the microvascular remodelling that accompanies hypertensive onset. © 1993 Wiley-Liss, Inc.     

Cell cycle versus density dependence of smooth muscle alpha actin expression in cultured rat aortic smooth muscle cells

Cultured smooth muscle cells (SMC) undergo induction of smooth muscle (SM) alpha actin at confluency. Since confluent cells exhibit contact inhibition of growth, this finding suggests that induction of SM alpha actin may be associated with cell cycle withdrawal. This issue was further examined in the present study using fluorescence-activated cell sorting of SMC undergoing induction at confluency and by examination of the effects of FBS and platelet-derived growth factor (PDGF) on SM alpha actin expression in postconfluent SMC cultures that had already undergone induction. Cell sorting was based on DNA content or differential incorporation of bromodeoxyuridine (Budr). The fractional synthesis of SM alpha actin in confluent cells was increased two- to threefold compared with subconfluent log phase cells, but no differences were observed between confluent cycling (Budr+) and noncycling (Budr-) cells. In cultures not exposed to Budr, confluent cycling S + G2 cells exhibited similar induction. These data indicate that cell cycle withdrawal is not a prerequisite for the induction of SM alpha actin synthesis in SMC at confluency. Growth stimulation of postconfluent cultures with either FBS or PDGF resulted in marked repression of SM alpha actin synthesis but the level of repression was not directly related to entry into S phase in that PDGF was a more potent repressor of SM alpha actin synthesis than was FBS despite a lesser mitogenic effect. This differential effect of FBS versus PDGF did not appear to be due to transforming growth factor-beta present in FBS since addition of transforming growth factor-beta had no effect on PDGF-induced repression. Likewise, FBS (0.1-10.0%) failed to inhibit PDGF-induced repression. Taken together these data demonstrate that factors other than replicative frequency govern differentiation of cultured SMC and suggest that an important function of potent growth factors such as PDGF may be the repression of muscle-specific characteristics.     

Heterogeneity of microvascular pericytes for smooth muscle type alpha- actin

Microvascular pericytes are believed to be involved in various functions such as regulation of capillary blood flow and endothelial proliferation. Since pericytes represent a morphologically heterogeneous cell population ranging from circular smooth musclelike to elongated fibroblast-like morphology it is possible that regulation of blood flow (via contractility) and control of endothelial proliferation (as well as other metabolic functions) may be accomplished by different subsets of pericytes. In the present study we provide evidence for heterogeneity of pericytes at the molecular level by using two novel technical approaches. These are (a) immunostaining of whole mounts of the microvascular beds of the rat mesentery and bovine retina and (b) immunoblotting studies of microdissected retinal microvessels. We show that pericytes of true capillaries (midcapillaries) apparently lack the smooth muscle isoform of alpha-actin whereas transitional pericytes of pre- and postcapillary microvascular segments do express this isoform. Thus, regulation of capillary blood flow may be accomplished by the smooth muscle-related pre- and postcapillary pericytes whereas the nonmuscle pericytes of true capillaries may play a role in other functions.     

Arterial smooth muscle cells in vivo: relationship between actin isoform expression and mitogenesis and their modulation by heparin

Quiescent smooth muscle cells (SMC) in normal artery express a pattern of actin isoforms with alpha-smooth muscle (alpha SM) predominance that switches to beta predominance when the cells are proliferating. We have examined the relationship between the change in actin isoforms and entry of SMC into the growth cycle in an in vivo model of SMC proliferation (balloon injured rat carotid artery). alpha SM actin mRNA declined and cytoplasmic (beta + gamma) actin mRNAs increased in early G0/G1 (between 1 and 8 h after injury). In vivo synthesis and in vitro translation experiments demonstrated that functional alpha SM mRNA is decreased 24 h after injury and is proportional to the amount of mRNA present. At 36 h after injury, SMC prepared by enzymatic digestion were sorted into G0/G1 and S/G2 populations; only the SMC committed to proliferate (S/G2 fraction) showed a relative slight decrease in alpha SM actin and, more importantly, a large decrease in alpha SM actin mRNA. A switch from alpha SM predominance to beta predominance was present in the whole SMC population 5 d after injury. To determine if the change in actin isoforms was associated with proliferation, we inhibited SMC proliferation by approximately 80% with heparin, which has previously been shown to block SMC in late G0/G1 and to reduce the growth fraction. The switch in actin mRNAs and synthesis at 24 h was not prevented; however, alpha SM mRNA and protein were reinduced at 5 d in the heparin-treated animals compared to saline-treated controls. These results suggest that in vivo the synthesis of actin isoforms in arterial SMC depends on the mRNA levels and changes after injury in early G0/G1 whether or not the cells subsequently proliferate. The early changes in actin isoforms are not prevented by heparin, but they are eventually reversed if the SMC are kept in the resting state by the heparin treatment.     

Cultured aortic smooth muscle cells from newborn and adult rats show distinct cytoskeletal features

It is well known that arterial smooth muscle cells (SMC) of adult rats, cultured in a medium containing fetal calf serum (FCS), replicate actively and lose the expression of differentiation markers, such as desmin, smooth muscle (SM) myosin and alpha-SM actin. We report here that compared to freshly isolated cells, primary cultures of SMC from newborn animals show no change in the number of alpha-SM actin containing cells and a less important decrease in the number of desmin and SM myosin containing cells than that seen in primary cultures of SMC from adult animals; moreover, contrary to what is seen in SMC cultured from adult animals, they show an increase of alpha-SM actin mRNA level, alpha-SM actin synthesis and expression per cell. These features are partially maintained at the 5th passage, when the cytoskeletal equipment of adult SMC has further evolved toward dedifferentiation. Cloned newborn rat SMC continue to express alpha-SM actin, desmin and SM myosin at the 5th passage. Thus, newborn SMC maintain, at least in part, the potential to express differentiated features in culture. Heparin has been proposed to control proliferation and differentiation of arterial SMC. When cultured in the presence of heparin, newborn SMC show an increase of alpha-SM actin synthesis and content but no modification of the proportion of alpha-SM actin total (measured by Northern blots) and functional (measured by in vitro translation in a reticulocyte lysate) mRNAs compared to control cells cultured for the same time in FCS containing medium. This suggests that heparin action is exerted at a translational or post-translational level. Cultured newborn rat aortic SMC furnish an in vitro model for the study of several aspects of SMC differentiation and possibly of mechanisms leading to the establishment and prevention of atheromatous plaques.