Stem cell marker expression,proliferation and apoptosis of vascular smooth muscle cells

Vascular endothelial Flt-1 and other stem cell markers are variably expressed in vascular smooth muscle cells (SMCs) during normal and pathological conditions, but their biological role remains uncertain. In normal rat aorta, rare flt-1+ and c-kit+ SMCs were detected. Fifteen days after injury, 61.8+3.8, 45.7+3% of the intimal cells resulted flt-1+ and c-kit+ and expressed low level of alpha-smooth muscle actin; CD133+ cells were 5.6+0.7%. BrDU+/flt-1+ largely predominated in the neointima, whereas BrDU+/CD133+ cells were rare. Forty-five and sixty days after injury, intimal proliferation such as BrDU+ cells was greatly reduced. After sixty days, intimal stem marker expression had almost disappeared whereas alpha-smooth muscle actin was restored. Flk-1 and Oct-4 SMC immunodection was consistently negative. In vitro, intimal cells obtained fifteen days after injury exhibited an epithelioid phenotype and increased flt-1 and c-kit protein and mRNA and low smooth muscle markers compared to spindle-shaped medial and intimal SMCs obtained after sixty days. Epithelioid clones, independently from layer of origin, were similar in stem cell marker expression. The anti-flt-1 blocking antibody added to epithelioid SMC cultures reduced serum-deprived apoptosis and migration but not PDGF-BB-induced proliferation, and increased cell-populated collagen lattice contraction. In conclusion, stem marker expression in vascular SMCs was variable, chronologically regulated and prevailed in epithelioid populations and clones; among stem markers, flt-1 expression critically regulates intimal SMC response to microenviromental changes.     

IL-10 inhibits vascular smooth muscle cell activation in vitro and in vivo

The anti-inflammatory cytokine IL-10 inhibits intimal hyperplasia after stent implantation via a powerful inactivation of monocytes. We tested the hypothesis that IL-10 may also inhibit vascular smooth muscle cell (SMC) activation via the inhibition of the NF-kappaB/I-kappaB system. The IL-10 receptor was detected in rat SMCs in vitro and in vivo. In LPS-stimulated rat SMCs, 1 ng/ml recombinant murine IL-10 (mIL-10) reduced I-kappaBalpha and I-kappaBbeta degradation, NF-kappaB activation, as well as the expression of the NF-kappaB-dependent gene IL-6 by 32%, 31%, 75%, and 19%, respectively (P < 0.05 for all). Similar results were obtained in vivo 6 h and 4 days after balloon abrasion of the rat aorta, a model in which intimal hyperplasia results essentially from SMC activation. Moreover, mIL-10 reduced SMC proliferation and migration in vitro (by 60% for both, P < 0.0001), resulting in reduced SMC proliferation and intimal growth 14 days after balloon abrasion of the rat aorta (by 76% and 75%, respectively; P < 0.005). In conclusion, mIL-10 has a direct inhibitory effect on SMCs in vitro and in vivo. This effect is mediated in part by NF-kappaB inactivation and may participate in the overall protective effect of IL-10 on postangioplasty restenosis.     

Increased production of prostacyclin (PGI2) by vascular intimal smooth muscle cells from atherosclerotic rabbit aorta

In vitro PGI2 synthesis by aortic strips obtained from thoracic aorta of rabbits fed a high cholesterol diet was examined and compared with that of control rabbits fed a normal diet. In this report, the amounts of PGI2 produced were shown as 6-keto-PGF1 alpha per microgram of aortic tissue DNA instead of per mg wet weight. We also investigated PGI2 synthesis by cultured smooth muscle cells (SMC) obtained from atherosclerotic intima. Basal PGI2 production by aortic strips from atherosclerotic rabbit aorta was significantly augmented compared with that of controls. Arachidonic acid (AA)-induced PGI2 production by atherosclerotic aorta was also significantly higher than that of controls. PGI2 producing capacities of intimal and medial layers, separated from atherosclerotic aorta, were examined and the intimal layer was found to elicit a significantly greater PGI2 production than the medial layer. Furthermore, cultured intimal SMC obtained from atherosclerotic rabbit aorta produced a greater amount of PGI2 than medial SMC from normal rabbit aorta at various cultured conditions. These results suggest that the possibility of enhanced PGI2 production by atherosclerotic aorta may well be considered as a defence mechanism of the vessel wall against damaging stimuli.     

Coculture of human endothelium and smooth muscle cells: functional heterogeneity of endothelial cells from zones with different susceptibility to atherosclerosis

Using co-culture technique and 3H-thymidine radioautography we have studied effects of human aortic endothelial cells (EC), isolated separately from zones of low (LP) and high (HP) probability of atherosclerosis of grossly normal and atherosclerotic aortas, on 3H-thymidine incorporation by human intimal smooth muscle cells (SMCs). It was found that EC activity depended on a zone of probability, from which the cells were isolated, and on the degree of atherosclerotic lesion. The first-passage ECs from grossly normal LP zones inhibited 3H-thymidine incorporation, compared to control, incubated without Ecs, SMCs (63.5 +/- 27.5%). Cells from HP zones of the same vessels were less active or stimulated SMC proliferation (99.4 +/- 42.9%). EC cultures obtained from both LP and HP zones of atherosclerotic vessels had, as a rule, no effect or increased 3H-thymidine incorporation by SMCs (100.3 +/- 19.8 and 124.1 +/- 20.1%). In contrast to morphologically heterogeneous primary and first-passage cultures obtained from high seeding density, EC monolayers obtained with a split 1:10 were composed predominantly of small mononuclear cells. These cultures effectively inhibited SMC DNA synthesis independently of a zone of probability and a degree of atherosclerotic lesion of aorta (60.4 +/- 10.0 and 51.5 +/- 12.7%). The obtained data suggest that EC morphological heterogeneity is accompanied by functional changes of cells and may be involved in atherosclerotic plaque formation.     

Oxidized alginate-cross-linked alginate/gelatin hydrogel fibers for fabricating tubular constructs with layered smooth muscle cells and endothelial cells in collagen gels

Hydrogel fibers that possessed a cell-adhesive surface and were degradable via enzymatic reactions were developed for fabricating tubular constructs with smooth muscle cell (SMC) and endothelial cell (EC) layers, similar to native blood vessels, in collagen gels. The fibers were prepared by soaking hydrogel fibers prepared from a solution of sodium alginate and gelatin containing bovine ECs (BECs) in medium containing oxidized alginate (AO). BECs soaked in 8.0% (w/v) AO showed no reduction in viability within 3 h of soaking. Furthermore, mouse SMCs (MSMCs) adhered and proliferated on the AO-cross-linked hydrogels. Based on these results, we prepared AO-cross-linked hydrogel fibers containing BECs, covered their surface with MSMCs, and embedded them in collagen gels. We then degraded the fibers using alginate lyase to obtain channels in the collagen gels. Histological analysis of the released ECs using a specific fluorescent dye revealed the formation of tubular structures with layered BECs and MSMCs.     

Vascular smooth muscle cell glycocalyx influences shear stress-mediated contractile response.

This study addressed the influence of the rate of shear stress application on aortic smooth muscle cell (SMC) contraction and the role of specific glycosaminoglycans in this mechanotransduction. Rat aortic SMCs were exposed to either a step increase in shear stress (0 to 25 dyn/cm(2)) or a ramp increase in shear stress (0 to 25 dyn/cm(2) over 5 min) in a parallel plate flow chamber, and cell contraction was characterized by cell area reduction. SMCs contracted at levels similar to those reported previously and equally in response to both a step and ramp increase in shear stress. When the cells were pretreated with heparinase III or chondroitinase ABC to remove the glycosaminoglycans heparan sulfate and chondroitin sulfate, respectively, from the glycocalyx, the contraction response to increases in shear stress was significantly inhibited. These studies indicate that specific components of the SMC glycocalyx play an important role in the mechanotransduction of shear stress into a contractile response and that the rate of application of shear stress does not affect the SMC contraction.     

Increased production of prostacyclin (PGI2) by vascular intimal smooth muscle cells from atherosclerotic rabbit aorta

PGI₂ synthesis by aortic strips obtained from thoracic aorta of rabbits fed a high cholesterol diet was examined and compared with that of control rabbits fed a normal diet. In this report, the amounts of PGI₂ produced were shown as 6-keto-PGF_1α per μg of aortic tissue DNA instead of per mg wet weight. We also investigated PGI₂ synthesis by cultured smooth muscle cells (SMC) obtained from atherosclerotic intima.Basal PGI₂ production by aortic strips from atherosclerotic rabbit aorta was significantly augmented compared with that of controls. Arachidonic acid (AA)-induced PGI₂ production by atherosclerotic aorta was also significantly higher than that of controls. PGI₂ producing capacities of intimal and medial layers, separated from atherosclerotic aorta, were examined and the intimal layer was found to elicit a significantly greater PGI₂ production than the medial layer.Furthermore, cultured intimal SMC obtained from atherosclerotic rabbit aorta produced a greater amount of PGI₂ than medial SMC from normal rabbit aorta at various cultured conditions. These results suggest that the possibility of enhanced PGI₂ production by atherosclerotic aorta may well be considered as a defence mechanism of the vessel wall against damaging stimuli.     

Generation of human vascular smooth muscle subtypes provides insight into embryological origin-dependent disease susceptibility

Heterogeneity of embryological origins is a hallmark of vascular smooth muscle cells (SMCs) and may influence the development of vascular disease. Differentiation of human pluripotent stem cells (hPSCs) into developmental origin-specific SMC subtypes remains elusive. Here we describe a chemically defined protocol in which hPSCs were initially induced to form neuroectoderm, lateral plate mesoderm or paraxial mesoderm. These intermediate populations were further differentiated toward SMCs (>80% MYH11(+) and ACTA2(+)), which displayed contractile ability in response to vasoconstrictors and invested perivascular regions in vivo. Derived SMC subtypes recapitulated the unique proliferative and secretory responses to cytokines previously documented in studies using aortic SMCs of distinct origins. Notably, this system predicted increased extracellular matrix degradation by SMCs derived from lateral plate mesoderm, which was confirmed using rat aortic SMCs from corresponding origins. This differentiation approach will have broad applications in modeling origin-dependent disease susceptibility and in developing bioengineered vascular grafts for regenerative medicine.     

Expression of fibronectin variants in vascular and visceral smooth muscle cells in development

Monoclonal antibodies recognizing extra domain A (ED-A) and extra domain B (ED-B) fibronectin (FN) sequences were used to characterize FN variants expressed in human vascular smooth muscle cells (SMC) during fetal and postnatal development and to compare spectrum of FN variants produced by vascular and visceral SMC. In 8- to 12-week-old fetuses both ED-A-containing FN (A-FN) and ED-B-containing FN (B-FN) were found in all smooth muscles studied--aorta, esophagus, stomach, and jejunum. By 20-25 weeks of gestation relative amounts of both A-FN and B-FN were reduced significantly in the aortic media (fivefold for A-FN and twofold for B-FN), while in visceral SMC only B-FN content was decreased. All the adult visceral smooth muscles examined contained A-FN rather than B-FN. Therefore, the cells from adult aortic media appear to be the only SMC so far known to produce FN that contains neither ED-A nor ED-B. Moreover, the data obtained show that, unlike other cells, medial SMC are embedded in vivo in the extracellular matrix that contains FN lacking both ED-A and ED-B. SMC from the minor intimal thickenings in the human child aorta as well as those from the atherosclerotic plaques produce A-FN rather than B-FN. We conclude that (1) vascular SMC change the spectrum of produced FN variants at least twice--during prenatal development between 12 and 20 weeks of gestation, and during the postnatal period, when they are recruited into the intimal cell population; (2) the production of FN variants in visceral SMC is also developmentally regulated; (3) all visceral SMC unlike the cells from adult aortic media produce A-FN; (4) the presence of ED-A and ED-B sequences in the FN molecule is not necessary for the extracellular matrix assembly in vivo.     

Phenotypic changes of human smooth muscle cells during development: late expression of heavy caldesmon and calponin.

Expression of the regulatory contractile proteins, heavy caldesmon (h-caldesmon) and calponin was studied in human aortic smooth muscle cells (SMCs) during development and compared with the expression of alpha-SM-actin and smooth muscle-myosin heavy chain (SM-MHCs). For this study, novel monoclonal antibodies specific to SM-MHCs, h-caldesmon, and calponin were developed and characterized. Aortic SMCs from fetuses of 8-10 and 20-22 weeks of gestation express alpha-SM-actin and SM-MHCs, but neither h-caldesmon nor calponin were expressed as demonstrated by immunoblotting and immunofluorescence techniques. In the adult aortic tunica media, SMCs contain all four markers. Thus, the expression of calponin, similar to the expression of alpha-SM-actin, SM-MHCs, and h-caldesmon, is developmentally regulated in aortic SMCs. In the adult aortic subendothelial (preluminal) part of tunica intima, numerous cells containing SM-MHCs, but lacking h-caldesmon and calponin, were found. These results illustrate the similarity of SMCs from intimal thickenings and immature (fetal) SMCs. Expression of contractile proteins in the developing SMCs is coordinately regulated; however, distinct groups of proteins appear to exist whose expression is regulated differently. Actin and myosin, being major contractile proteins, also play a structural role and appear rather early in development, whereas caldesmon and calponin, being involved in regulation of contraction, can serve as markers of higher SMC differentiation steps. In contrast, h-caldesmon and calponin were already present in visceral SMCs (trachea, esophagus) of the 10-week-old fetus. These results demonstrate that the time course of maturation of visceral SMCs is different from that of vascular SMCs.     

Enzyme histochemical expressions of smooth muscle cell modulation in arterial development, hypertension and remodeling

In order to define metabolic profiles of smooth muscle cell (SMC) modulation, 16 enzyme activities linked to nucleotide hydrolysis, lipolysis, lysosomal reactivity and intermediate glucose catabolism were compared in four rat arterial models, exhibiting four metabolic phenotypes of modulated smooth muscle cells: (i) "primary synthetic" statein immature aorta; (ii) "contractile" state in adult aorta; (iii) "hypertensive" state in aorta of hypertensive rat, SHR; (iiii) "secondary synthetic" state in diffuse intimal thickening of ligated carotid artery. Contractile SMC presented strong activities of enzymes linked to nucleotide ester hydrolysis and contractility (ATP-A-Ca, ATP-A-Mg, ATP-A-Ca/Mg, 5'nucleotidase) and to lipolytic process (butyryl cholinesterase, acid esterase). These enzyme activities were more pronounced in "hypertensive SMC". Incontrast, the same enzymes were weakly active or not expressed in "synthetic SMC". Increased lysosomal enzyme reactivity was a particular expression of "secondary synthetic SMC". The observed enzyme abnormalities in reactively modulated SMC (proliferative-synthetic phenotype) might be related to the loss of contractility and to the enhanced cell proliferation and lipid accumulation, characteristic features of modulated SMC in atherogenesis.     

Different alpha-adrenoceptors mediate migration of vascular smooth muscle cells and adventitial fibroblasts in vitro

Norepinephrine directly induces growth of the vascular wall, which may involve not only proliferation of smooth muscle cells (SMCs) and adventitial fibroblasts (AFBs) but also augmentation of their migration. To test this hypothesis, growth-arrested SMCs and AFBs from rat aorta were exposed to norepinephrine. Norepinephrine caused dose-dependent migration of both cell types that was dependent on chemotaxis. In contrast, platelet-derived growth factor (PDGF)-BB, used as a positive control, stimulated both chemotaxis and chemokinesis. Only alpha(1D)-adrenoceptors (AR) and alpha(2)-AR antagonists inhibited norepinephrine migration of SMCs, whereas norepinephrine migration of AFBs was only inhibited by alpha(1A)-AR and alpha(1B)-AR antagonists; beta-AR blockade was without effect. Norepinephrine and PDGF-BB were additive for AFB, but not SMC, migration. Stimulation of migration was reversed at high norepinephrine concentrations (10 microM); this inhibition was mediated by alpha(2)- and beta-ARs in AFBs but not in SMCs. Thus norepinephrine induces migration of SMCs and AFBs via different alpha-ARs. This action may participate in wall remodeling and norepinephrine potentiation of injury-induced intimal lesion growth.     

Adult vascular smooth muscle cells in culture express neural stem cell markers typical of resident multipotent vascular stem cells

Differentiation of resident multipotent vascular stem cells (MVSCs) or de-differentiation of vascular smooth muscle cells (vSMCs) might be responsible for the SMC phenotype that plays a major role in vascular diseases such as arteriosclerosis and restenosis. We examined vSMCs from three different species (rat, murine and bovine) to establish whether they exhibit neural stem cell characteristics typical of MVSCs. We determined their SMC differentiation, neural stem cell marker expression and multipotency following induction in vitro by using immunocytochemistry, confocal microscopy, fluorescence-activated cell sorting analysis and quantitative real-time polymerase chain reaction. MVSCs isolated from rat aortic explants, enzymatically dispersed rat SMCs and rat bone-marrow-derived mesenchymal stem cells served as controls. Murine carotid artery lysates and primary rat aortic vSMCs were both myosin-heavy-chain-positive but weakly expressed the neural crest stem cell marker, Sox10. Each vSMC line examined expressed SMC differentiation markers (smooth muscle α–actin, myosin heavy chain and calponin), neural crest stem cell markers (Sox10⁺, Sox17⁺) and a glia marker (S100β⁺). Serum deprivation significantly increased calponin and myosin heavy chain expression and decreased stem cell marker expression, when compared with serum-rich conditions. vSMCs did not differentiate to adipocytes or osteoblasts following adipogenic or osteogenic inductive stimulation, respectively, or respond to transforming growth factor-β1 or Notch following γ-secretase inhibition. Thus, vascular SMCs in culture express neural stem cell markers typical of MVSCs, concomitant with SMC differentiation markers, but do not retain their multipotency. The ultimate origin of these cells might have important implications for their use in investigations of vascular proliferative disease in vitro.     

CeReS-18, a cell regulatory sialoglycopeptide, inhibits proliferation and migration of rat vascular smooth muscle cells

CeReS-18, a cell regulatory sialoglycopeptide, has been shown to inhibit proliferation of a wide array of target cells. In the present study, the effect of CeReS-18 on vascular smooth muscle cell (SMC) proliferation was characterized in cultured rat aorta SMCs (A7r5). More extensively, the effect of CeReS-18 on platelet-derived growth factor (PDGF)-induced SMC migration was examined using a modified Boyden's chamber assay. CeReS-18 inhibits both SMC proliferation and migration in a concentration-dependent, calcium-sensitive, and reversible manner. Furthermore, cells preincubated with the inhibitor had an increased sensitivity to CeReS-18-mediated inhibition of SMC migration. Immunoprecipitation and in vitro phosphorylation assays demonstrated that MAP kinase activity was inhibited in the CeReS-18-treated cells and pretreatment with CeReS-18 suppressed the activation of MAP kinase stimulated by PDGF. However, it is not likely that the suppression of the MAP kinase pathway was directly responsible for the ability of CeReS-18 to inhibit migration of the rat aorta smooth muscle cells since a MEK-specific inhibitor, PD98059, did not influence A7r5 cell migration.     

Shear stress inhibits smooth muscle cell migration via nitric oxide-mediated downregulation of matrix metalloproteinase-2 activity

Vascular smooth muscle cell (SMC) migration is a hallmark of intimal hyperplasia (IH), the progression of which is affected by hemodynamic conditions at the diseased site. The realization that SMCs are exposed to blood flow in both denuded vessels (direct blood flow) and intact vessels (interstitial blood flow) motivated this study of the effects of fluid flow shear stress (SS) on SMC migration. Rat aortic SMCs were seeded onto Matrigel-coated cell culture inserts, and their migratory activity toward PDGF-BB when exposed to SS in a rotating disk apparatus was quantified. Four hours of either 10 or 20 dyn/cm2 SS significantly inhibited SMC migration to the bottom side of the insert. This inhibition was associated with downregulation of SMC matrix metalloproteinase (MMP)-2 activation. Four hours of 10 dyn/cm2 SS also drastically increased SMC production of NO. A NO synthase inhibitor (N(G)-nitro-L-arginine methyl ester; 100 microM) abolished the shear-induced increase in SMC NO production as well as the inhibition of migration and MMP-2 activity. A NO donor (S-nitroso-N-acetyl-penicillamine; 500 microM) suppressed SMC migration via the reduction of both total and active MMP-2 levels. Addition of 10 microM MMP-2 inhibitor I to inserts significantly reduced SMC migration. Western blots showed no effect of 4 h of 20 dyn/cm2 SS on SMC production of PDGF-AA, another chemical known to suppress SMC migration. Thus it appears that SS acts to suppress SMC migration by upregulating the cellular production of NO, which in turn inhibits MMP-2 activity.     

Dendroaspis natriuretic peptide-like immunoreactivity and its regulation in rat aortic vascular smooth muscle

Dendroaspis natriuretic peptide (DNP) is a recently isolated 38 amino acid peptide that shares structural and functional properties with the other members of the natriuretic peptide family. The present study demonstrates the presence of DNP-like immunoreactivity in sections of rat aorta, carotid artery and renal vasculature and tubules. DNP-like immunoreactivity was detected in culture aortic vascular smooth muscle cells and medium and is regulated by endothelin-1, angiotensin II and sodium nitroprusside but not by transforming growth factor-beta. Our observations indicate that DNP elicits a marked inhibitory effect on DNA synthesis in culture rat aortic vascular smooth muscle cells.     

Deformation-induced endothelin B receptor-mediated smooth muscle cell apoptosis is matrix-dependent

To maintain normal blood flow, pressure overload in both arteries and veins requires a structural adaptation of the vessel wall (remodelling) that involves smooth muscle cell (SMC) hypertrophy and/or hyperplasia. Due to its potent vasoconstrictor and growth-promoting effects, endothelin-1 (ET-1) is a likely candidate to initiate and/or promote remodelling in blood vessels exposed to a chronic increase in blood pressure. To test this hypothesis, isolated segments of the rabbit carotid artery and jugular vein were perfused at different levels of intraluminal pressure. In both types of segments, pressure overload (160 and 20 mmHg, respectively) resulted in an increase in endothelial prepro-ET-1 and SMC endothelin B receptor (ETB-R) expression. Moreover, in pressurised segments from the carotid artery an ETB-R antagonist-sensitive increase in SMC apoptosis in the media was observed, while in the vein medial SMC started to proliferate. Isolated SMC from these rabbit blood vessels as well as from the aorta and vena cava of the rat, when cultured on a collagen or laminin matrix, uniformly revealed an ETB-R-mediated increase in apoptosis upon exposure to mechanical deformation plus exogenous ET-1 (10 nmol/L). However, when grown on a fibronectin matrix, the cultured SMC did not respond with an increase in apoptosis under otherwise identical experimental conditions. These findings suggest that deformation-induced activation of the endothelin system in the vessel wall not only plays a crucial role in remodelling, but that the structural components of the vessel wall, in particular the cell-matrix interaction, determine how SMC respond phenotypically to these changes in gene expression.     

Pitavastatin attenuates the PDGF-induced LR11/uPA receptor-mediated migration of smooth muscle cells

Statins, inhibitors of HMG-CoA reductase, elicit various actions on vascular cells including the modulation of proliferation and migration of smooth muscle cells (SMCs). Here, we have elucidated the mechanism by which statins, in particular pitavastatin, attenuate the migration activity of SMCs. The expression of LR11, a member of the LDL receptor family and an enhancer of cell surface localization of urokinase-type plasminogen activator receptor (uPAR), is increased in cultured SMCs by treatment with PDGF-BB. Pitavastatin attenuates the PDGF-BB -induced surface expression of LR11 and uPAR. The increased migration of SMCs observed both upon overexpression of LR11 and via stimulation of secretion of soluble LR11 is not reversed by pitavastatin. In vivo studies showed that the SMCs expressing LR11 in plaques are almost congruent with intimal cells expressing nonmuscle myosin heavy chain (SMemb). Pitavastatin reduced the expression of LR11 and SMemb, and the levels of LR11, uPAR, and SMemb in cultured intimal SMCs were reduced to those seen in medial SMCs. We propose that this statin reduces PDGF-induced migration through the attenuation of the LR11/uPAR system in SMCs. Modulation of the LR11/uPAR system with statins suggests a novel treatment strategy for atherogenesis based on suppression of intimal SMC migration.