Quiescent Smooth Muscle Cells Research Articles

CREG promotes a mature smooth muscle cell phenotype and reduces neointimal formation in balloon-injured rat carotid artery

We previously showed that cellular repressor of E1A-stimulated genes (CREG) is up-regulated during serum starvation-induced vascular smooth muscle cell (SMC) differentiation. The aim of this study was to determine the role of CREG in maintaining the quiescent, differentiated phenotype of SMCs both in culture and in balloon-injured rat carotid artery. In cultured SMCs recombinant virus-mediated CREG expression enhanced cellular differentiation, inhibited proliferation, and reduced synthesis of extracellular matrix component fibronectin. In contrast, CREG knockdown via retroviral transfer of short hairpin RNAs abrogated serum starvation-induced SMC differentiation and growth arrest. Both immunostaining and Western analysis demonstrated marked down-regulation of CREG in the vascular media after balloon injury to the rat carotid artery. Retrovirus-mediated CREG transfer to the injured artery inhibited SMC dedifferentiation and proliferation, and reduced neointimal hyperplasia. These results suggest that CREG participates in the maintenance of quiescent mature SMC phenotype in the arterial media by promoting SMC differentiation and growth arrest and that CREG gene transfer has therapeutic potential for vascular diseases associated with neointimal hyperplasia.

Smooth muscle cell rigidity and extracellular matrix organization influence endothelial cell spreading and adhesion formation in coculture

Efforts to develop functional tissue-engineered blood vessels have focused on improving the strength and mechanical properties of the vessel wall, while the functional status of the endothelium within these vessels has received less attention. Endothelial cell (EC) function is influenced by interactions between its basal surface and the underlying extracellular matrix. In this study, we utilized a coculture model of a tissue-engineered blood vessel to evaluate EC attachment, spreading, and adhesion formation to the extracellular matrix on the surface of quiescent smooth muscle cells (SMCs). ECs attached to and spread on SMCs primarily through the alpha(5)beta(1)-integrin complex, whereas ECs used either alpha(5)beta(1)- or alpha(v)beta(3)-integrin to spread on fibronectin (FN) adsorbed to plastic. ECs in coculture lacked focal adhesions, but EC alpha(5)beta(1)-integrin bound to fibrillar FN on the SMC surface, promoting rapid fibrillar adhesion formation. As assessed by both Western blot analysis and quantitative real-time RT-PCR, coculture suppressed the expression of focal adhesion proteins and mRNA, whereas tensin protein and mRNA expression were elevated. When attached to polyacrylamide gels with similar elastic moduli as SMCs, focal adhesion formation and the rate of cell spreading increased relative to ECs in coculture. Thus, the elastic properties are only one factor contributing to EC spreading and focal adhesion formation in coculture. The results suggest that the softness of the SMCs and the fibrillar organization of FN inhibit focal adhesions and reduce cell spreading while promoting fibrillar adhesion formation. These changes in the type of adhesions may alter EC signaling pathways in tissue-engineered blood vessels.

Bladder smooth muscle cell differentiation and induction of mesenchymal stem cell differentiation in a three dimensional hydrogel matrix

Purpose To allow cell attachment and proliferation an adequate temporary scaffold must be provided, while synthesis of extra-cellular matrix proteins and tissue re-organisation take place. Bladder tissue engineering suffers from the formation of scar tissue partly due to the phenotypic switch of smooth muscle cells from a quiescent contractile to a synthetic proliferative phenotype. Material and methods Human bladder smooth muscle and mesenchymal stem cells were successfully cultured three-dimensionally in enzymatically degradable polyethylene-glycol hydrogels, modified with integrin binding peptides (e.g. RGD peptide sequence). Cell viability was analyzed using the the live/dead staining method (Rodamin reaction). Results On zymography (FACS) the smooth muscle cells showed an up-regulation of matrix metalloproteinase 2 (MMP-2) speaking in favour of differentiation into the quiescent, contractile type of smooth muscle cells. 3-dimensional growth of mesenchymal stem cells in the hydrogel induced up-regulation of smooth muscle alpha-actin and myosin and a down-regulation of CD 90, a marker for un-differentiated mesenchymal stem cells. All these findings are in favour of stem cell differentiation into smooth muscle cells. Furthermore an up-regulation of alpha1 beta1 integrin subunits was noted in favour of differentiation into quiescent contractile smooth muscle cells. Conclusions We have demonstrated the feasibility of a model for three-dimensional growth of human bladder smooth muscle cells, differentiating into the quiescent contractile type and of mesenchymal stem cells differentiating into functional smooth muscle cells. This model can be used to study reconstructive processes and can also contribute to the understanding of underlying basic mechanisms of human smooth muscle cell differentiation.

Pyeloureteric peristalsis: role of atypical smooth muscle cells and interstitial cells of Cajal‐like cells as pacemakers

Pyeloureteric peristalsis has long been considered to be triggered by pacemaker atypical smooth muscle cells (SMC) located in the proximal regions of the renal pelvis. However, interstitial cells with many of the morphological features and c-Kit immuno-reactivity of interstitial cells of Cajal (ICC), the established pacemaker cells in the intestine, have been demonstrated to be present in small numbers within the ureteropelvic junction (UPJ) of many mammals. Freshly isolated ICC-like cells (ICC-LC) of the mouse UPJ also display autorhyhmicity. This review discusses the notion that ureteric peristalsis depends on the presence of both atypical SMC and ICC-LC which form separate but interconnected networks that drive electrically quiescent typical SMC. In contrast to the intestine or prostate, all regenerative potential discharge in the mouse UPJ is abolished by the L-type Ca(2+) channel blocker nifedipine revealing a fundamental pacemaker signal. Whether these pacemaker transients arise from atypical SMC or ICC-LC or both has yet to be established. We speculate that the presence of spontaneously active ICC-LC in the distal regions of the UPJ maintains rudimentary peristaltic waves and movement of urine towards the bladder after pyeloureteral obstruction or pyeloplasty and disconnection from the proximal pacemaker drive.

Mdm2, but not Mdm4, protects terminally differentiated smooth muscle cells from p53-mediated caspase-3-independent cell death

p53 is a potent inhibitor of cell growth and an inducer of apoptosis. During embryonic development, Mdm2 and Mdm4 inhibit the growth suppressive activities of p53. However, whether tight surveillance of p53 activity is required in quiescent cells is unknown. To test this, conditional inactivation of mdm2 and mdm4 was carried out in smooth muscle cells (SMCs). Upon SMC-specific inactivation of mdm2, and not of mdm4, mice rapidly became ill and died. Necropsy showed small intestinal dilation, and histological analyses indicated a severe reduction in the number of intestinal SMCs. Increased p53 levels and activity were detected in the remaining SMCs, and the phenotype was completely rescued on a p53-null background. Interestingly, intestinal SMCs are caspase-3-negative and therefore did not undergo caspase-3-dependent apoptotic cell death. Together, Mdm2, but not Mdm4, prevents accumulation of active p53 in quiescent SMCs and thereby the induction of p53-mediated caspase-3-independent cell death.

A system for the direct co-culture of endothelium on smooth muscle cells

The development of a functional, adherent endothelium is one of the major factors limiting the successful development of tissue engineered vascular grafts (TEVGs). The adhesion and function of endothelial cells (ECs) on smooth muscle cells (SMCs) are poorly understood. The goal of this research was to optimize conditions for the direct culture of endothelium on SMCs, and to develop an initial assessment of co-culture on EC function. The co-culture consisted of a culture substrate, a basal adhesion protein, a layer of porcine SMCs, a medial adhesion protein, and a layer of porcine ECs. Conditions that led to successful co-culture were: a polystyrene culture substrate, a quiescent state for SMCs, subconfluent density for SMC seeding and confluent density for EC seeding, and fibronectin (FN) for the basal adhesion protein. EC adhesion was not enhanced by addition of FN, collagen I, collagen IV or laminin (LN) to the medial layer. 3-D image reconstruction by confocal microscopy indicated that SMCs did not migrate over ECs and the cells were present in two distinct layers. Co-cultures could be consistently maintained for as long as 10 days. After exposure to 5 dyne/cm 2 for 7.5 h, ECs remained adherent to SMCs. PECAM staining indicated junction formation between ECs, but at a lower level than that observed with EC monocultures. Co-culturing ECs with SMCs did not change the growth rate of ECs, but EC DiI-Ac-LDL uptake was increased. Thus, a confluent and adherent layer of endothelium can be directly cultured on quiescent SMCs.

Control of smooth muscle cell proliferation in vascular disease.

Smooth muscle cell proliferation has previously been regarded as a central feature in vascular disease. The role of this process has recently been substantially re-evaluated, and we have reconsidered the functional importance of smooth muscle cell proliferation, the origin of proliferating smooth muscle cells in lesions, and the mechanisms whereby smooth muscle cell proliferation is controlled. In this review, we summarize recent progress in the understanding of smooth muscle cell proliferation, with a particular focus on how interactions between the extracellular matrix, smooth muscle cells, and mitogens control critical steps in this process. Irrespective of the origin of smooth muscle cells in vascular lesions, fundamental interactions between the extracellular matrix and cell surface integrins are necessary in order to initiate a proliferative response in a quiescent smooth muscle cell, in a similar manner to any non-malignant cell. These interactions trigger intracellular signaling and cell cycle entry, which facilitate cell cycle progression and proliferation by mitogens. In addition, extracellular matrix interactions may also control the availability and activity of growth factors such as heparin-binding mitogens, which can be sequestered by heparan sulfate containing extracellular matrix components and regulate smooth muscle cell proliferation. New insights into mechanisms whereby the extracellular matrix takes part in the control of smooth muscle cell proliferation suggest a number of putative targets for future therapies that can be applied to increase plaque stability, prevent the clinical consequences of atherosclerosis and improve outcomes after interventional procedures and organ transplantation.

INHIBITION OF MITOGENIC SIGNALING AND INDUCTION OF APOPTOSIS IN HUMAN BLADDER SMOOTH MUSCLE CELLS TREATED WITH DOXAZOSIN

The alpha1 antagonist doxazosin is used to treat lower urinary tract symptoms and is believed to function primarily as a smooth muscle relaxant. However, doxazosin has been shown to inhibit proliferation and induce apoptosis in nonbladder smooth muscle. Consequently, we examined the effects of doxazosin on human bladder smooth muscle cell (SMC) proliferation and apoptosis. Primary human bladder SMCs were cultured in M199 with 10% fetal bovine serum (FBS) until they reached 65% confluency and then they were made quiescent by serum starvation in M199 with 0.4% FBS for 24 hours. The quiescent bladder SMCs were pretreated for 30 minutes with doxazosin or vehicle (dimethyl sulfoxide) and then stimulated with 10% FBS for 24 hours. Measurement of 5'-bromo-2'-deoxyuridine (BrdU) uptake by flow cytometry was used to determine the effect of doxazosin on cell cycle progression. Western immunoblot was used to examine cell cycle protein expression and phosphorylation of the retinoblastoma protein (Rb) and cyclin A, both of which regulate cell cycle progression. Cellular proliferation was inhibited in a dose dependent manner by doxazosin. There was nearly a 50% decrease in BrdU uptake at 10 microM doxazosin and an approximately 90% decrease in BrdU at 25 microM doxazosin. Notably, doxazosin inhibited phosphorylation of Rb and expression of cyclin A, both of which are necessary for cell cycle progression. At concentrations of 25 microM doxazosin or greater apoptosis was induced in the bladder SMCs, as indicated by an increase in subG1 DNA content. Our study demonstrates that doxazosin inhibits mitogen induced proliferation of human bladder SMC by blocking cell cycle progression at the of G1/S border. Doxazosin induced cell cycle inhibition appears to be at least in part due to an inhibition of mitogen induced Rb phosphorylation and cyclin A expression. At higher concentrations doxazosin induces apoptosis in human bladder SMCs.

Mechanosensitive p27Kip1 regulation and cell cycle entry in vascular smooth muscle cells.

Cyclic stretch plays an important role in the homeostasis of vessel structure. Increased forces might, however, contribute to remodeling processes, resulting in vascular proliferative diseases. The initial molecular events necessary for mechanosensitive cell cycle entry of quiescent smooth muscle cells are poorly understood. In this study, we demonstrate that mechanical strain resulted in a rapid, integrin-dependent but mitogen-independent activation of phosphoinositide 3-kinase (PI3-K)/protein kinase B (Akt) in quiescent vascular smooth muscle cells. Subsequently, downstream ALL 1 fused gene from chromosome X (AFX)-like forkhead transcription factors were inactivated, leading to transcriptional downregulation of p27Kip1. This contrasted with the posttranscriptional protein reduction of p27Kip1 in cells stimulated with serum mitogens. Stretch-mediated p27Kip1 downregulation was accompanied by activation of cyclin-dependent kinase 2, hyperphosphorylation of retinoblastoma protein, and proliferation. Forkhead transcription factor inactivation and p27Kip1 downregulation were prevented by the PI3-K inhibitors wortmannin and LY294002. Pharmacological blockade of other kinases, such as p42/44, p38, and protein kinase A or C, did not influence the mechanosensitive gene regulation. p27Kip1 downregulation and cell cycle entry were, however, prevented by overexpression of a constitutively inactive form of Akt or constitutively active forms of forkhead transcription factors. Our data demonstrate that the earliest cell cycle events can occur in a solely mechanosensitive fashion. Vascular smooth muscle cells are, furthermore, able to use transcriptional or posttranscriptional mechanisms to regulate p27Kip1, depending on the stimulus to which they are exposed. This observation has novel implications for understanding of vascular proliferative diseases.

PGE 2 stimulates vascular smooth muscle cell proliferation via the EP2 receptor

Prostaglandins released by injured vascular tissue can modulate smooth muscle cell (SMC) proliferation. The mechanism of action of PGE 2 was investigated with porcine coronary artery SMCs obtained by explant culture. DNA and RNA syntheses exhibited a concentration-dependent increase following treatment of quiescent SMCs with PGE 2, while PGI 2 had no effect. PGE 2 also elevated PCNA expression, bromodeoxyuridine incorporation, and cell number, indicative of a hyperplastic growth response. Furthermore, induction of c- fos expression required activation of both phosphatidylinositol 3-kinase and mitogen-activated protein kinase. Contrary to these data, treatment of proliferating cells with PGE 2 caused a reduction in DNA synthesis. A role for PKA in either growth stimulation or inhibition was excluded. Interestingly, only quiescent SMCs expressed EP2 receptors, and the selective EP2 receptor agonist butaprost confirmed that this receptor was essential for growth stimulation and possibly inhibition. These data suggest that the growth state-dependent actions of PGE 2 on SMC proliferation may be mediated via the EP2 receptor.

Adventitial Myofibroblasts Play no Major Role in Neointima Formation After Angioplasty

Objective—Myofibroblasts migrating from adventitia have been suggested to constitute a majority of neointimal cells after angioplasty. We sought to examine this hypothesis by use of smoothelin, which is a marker for the quiescent smooth muscle cell (SMC) phenotype while not expressed by myofibroblasts.Design—Balloon angioplasty was performed in left iliac arteries of 25 rabbits that were killed after 3-56 days. Arterial cross-sections were immunostained for (X-actin (general marker), smoothelin (quiescent SMC phenotype), and Ki-67 (proliferative phenotype).Results—Adventitial cells became transiently actinpositive (myofibroblasts) but did not express smoothelin at any time point. In media, angioplasty induced transient proliferation and coinciding transient decrease in smoothelin expression. Neointimal cells, present 7 days after angioplasty, were initially proliferating and smoothelin-negative but changed to non-proliferating, smoothelin-positive cells after 56 days where 82 ± 10% of cells stained positive for smoothelin. This phenotypic modulation of medial and intimal cells began in media and moved gradually towards the lumen.Conclusion—At late follow-up, the majority of intimal cells are smoothelin-positive indicating that adventitial myofibroblasts play no major role for neointima formation.

Cardiac ankyrin repeat protein (CARP) expression in human and murine atherosclerotic lesions: activin induces CARP in smooth muscle cells.

Cardiac ankyrin repeat protein (CARP) is a transcription factor-related protein that has been studied most extensively in the heart. In the present study, we investigated the expression and the potential function of CARP in human and murine atherosclerosis. CARP expression was observed by in situ hybridization in endothelial cells lining human atherosclerotic plaques, whereas lesion macrophages were devoid of CARP. Furthermore, we established that CARP mRNA and smooth muscle (SM) alpha-actin antigen both colocalized in a subset of intimal smooth muscle cells (SMCs), whereas no CARP mRNA was encountered in quiescent SMCs in the media. The CARP mRNA-expressing intimal SMCs were distinct from intimal SMCs that synthesized the activation marker osteopontin or proliferating cell nuclear antigen. In addition, we showed that activin A, a member of the TGFbeta superfamily that prevents SMC-rich lesion formation, induced CARP mRNA expression in cultured SMCs. Based on our data and the knowledge that CARP reduces the proliferation of cultured SMCs, we propose that CARP is involved in inhibition of vascular lesion formation.

Differential effect of simvastatin on activation of Rac 1 vs. activation of the heat shock protein 27-mediated pathway upon oxidative stress, in human smooth muscle cells

In the present study, we have analyzed the response of human smooth muscle cell (SMC)s to oxidative stress, in terms of recruitment of key elements of the stress-activated protein kinase (SAPK) pathway, such as Rac 1, p38, and the small heat shock protein (HSP)27. The level of expression of three small HSPs, αB-crystallin, HSP20, HSP27, as well as the phosphorylation levels of HSP27 and p38, were higher in cultured, asynchronously growing SMCs originating from left interior mammary artery (LIMA) than those originating from aorta, saphenous vein, and umbilical vein, validating the choice of SMCs from LIMA as a model system in our study. In synchronized, quiescent SMCs from LIMA, oxidative stress (H 2O 2 stimulation)-induced membrane translocation of Rac 1, p38 phosphorylation, membrane translocation, and phosphorylation of HSP27. In these cells, simvastatin (S), an HMG-CoA reductase inhibitor, blocked, in a mevalonate-dependent way, oxidative stress-induced membrane translocation of Rac 1. However, S pretreatment prior to oxidative stress increased the levels of p38 phosphorylation, HSP27 membrane translocation/phosphorylation, actin polymerization, and apoptosis in these cells, in a mevalonate-dependent way. These results establish that S pretreatment has a stimulatory effect on the stress-activated p38/HSP27 pathway, despite its blocking effect on Rac 1 activation.

The vascular NADPH oxidase subunit p47phox is involved in redox-mediated gene expression

An NADPH oxidase is thought to be a main source of vascular superoxide (O2−) production. The functional role of this oxidase, however, and the contribution of the different subunits of the enzyme to cellular signaling are still incompletely understood. We determined the role of the p47phox subunit of the oxidase in O2− generation and signaling in aortic rings and cultured smooth muscle cells (SMC) from wild-type (WT) and p47phox-deficient (p47phox -/-) mice. Basal O2− levels in aortae of p47phox -/- mice were lower than those in WT aortae. Infusion of [val5]-angiotensin II increased O2− levels in aortae from WT more than in aortae from p47phox -/- mice. O2− generation was similar in quiescent SMC from WT and p47phox -/- mice. However, exposure to thrombin selectively increased O2− generation in VSMC from WT, but not from p47phox -/- mice. Thrombin-activated redox-mediated signal transduction and gene expression was attenuated in VSMC from p47phox -/- compared to cells from WT mice as determined by p38 MAP kinase activation and VEGF gene expression. We conclude that p47phox is important for vascular ROS production and redox-modulated signaling and gene expression in VSMC.

Influence of Class I interferons on performance of vascular cells on stent material in vitro

Purpose: Numerous reports suggest that Class 1 interferons (IFNs), particularly IFN-γ, inhibit migration and proliferation of different types of human cells. The objective of the present study was to determine the effect of Class I IFNs on viability and growth characteristics of human aortic endothelial cells (ECs), smooth muscle cells (SMC) and fibroblasts (FBs) in vitro. Methods: Stainless-steel (316-l) disks were coated with fibrin meshwork containing IFN-γ or IFN-α. The discs and IFN embedded meshwork were incubated with human EC, SMC and FB, and then cultured, whereas control cells were seeded onto uncoated surfaces or plain fibrin meshwork. Concentrations of recombinant IFN varied from 5 to 20 ng/cm 2. Assessment of effect on cell viability, growth and attachment was performed utilizing Alamar Blue (AB) assay. Cell morphology was assessed by scanning electron microscopy (SEM). Results: We have now shown inhibitory capacity of IFN-γ on all three types of unstimulated cells. The growth-inhibitory effect was maximal with SMC, while it was minimal with FB and EC. IFN-γ abrogated mitogenic responses of SMC but not EC and partially FB to VEGF and FGF stimulation. IFN-α was able to inhibit EC growth and, to a lesser extent, FB, and did influence growth rates of SMC. Biochemical analysis of lactate dehydrogenase activity suggested that IFN was not toxic to vascular cells. We also measured the expression of cell adhesive molecules: P- and E-selections, PECAM and ICAM-1. These molecules were upregulated by IFN in EC. Media derived from quiescent human SMC displayed low immunoreactive elastase activity, while conditional media after IFN-γ treatment but not IFN-α treatment had approximately a threefold greater activity. Conclusion: These data suggest that IFN-γ significantly inhibits SMC growth in the absence of significant endothelial toxicity and is dose-dependent; however, animal experiments are needed to further explore the antirestenotic effects of IFNs.

Cyclic nucleotide phosphodiesterase 1C promotes human arterial smooth muscle cell proliferation.

Proliferation of arterial smooth muscle cells (SMCs) is a key event in the formation of advanced atherosclerotic lesions and restenosis after angioplasty. Cyclic nucleotides (cAMP and cGMP) inhibit arterial SMC proliferation, and elevation of cyclic nucleotides reduces neointimal formation after angioplasty in animal models. Degradation of cAMP and cGMP is catalyzed by cyclic nucleotide phosphodiesterases (PDEs). One of these, PDE1C, hydrolyzes cAMP and cGMP and is expressed in proliferating human SMCs but is absent in quiescent human aorta. Thus, PDE1C expression is low in cultured human SMCs made quiescent by attaching to fibrillar collagen type I. After release from the fibrillar collagen, PDE1C expression is induced and associated with traverse through S-phase of the cell cycle. Further, PDE1C is expressed in vivo in human fetal aorta containing proliferating SMCs, but not in newborn aorta in which SMC proliferation has ceased. Inhibition of PDE1C in SMCs isolated from normal aorta or from lesions of atherosclerosis using antisense oligonucleotides or a PDE1 inhibitor results in suppression of SMC proliferation. In conclusion, PDE1C expression is a marker of human SMC proliferation ex vivo and in vivo. Inhibition of PDE1C leads to inhibition of human SMC proliferation. Because PDE1C is absent in quiescent SMCs, PDE1C inhibitors may target proliferating SMCs in lesions of atherosclerosis or restenosis.

Low density lipoprotein receptor-related protein mediates apolipoprotein E inhibition of smooth muscle cell migration.

This research was undertaken to identify the cell surface receptor responsible for mediating apolipoprotein E (apoE) inhibition of platelet-derived growth factor (PDGF)-directed smooth muscle cell migration. Initial studies revealed the expression of the low density lipoprotein receptor (LDLR), the LDL receptor-related protein (LRP), the very low density lipoprotein receptor (VLDL), and apoE receptor-2 in mouse aortic smooth muscle cells. Smooth muscle cells isolated from LDLR-null, VLDL-null, and apoE receptor-2-null mice were responsive to apoE inhibition of PDGF-directed smooth muscle cell migration, suggesting that these receptors were not involved. An antisense RNA expression knockdown strategy, utilizing morpholino antisense RNA against LRP, was used to reduce LRP expression in smooth muscle cells to assess the role of this receptor in apoE inhibition of cell migration. Results showed that apoE was unable to inhibit PDGF-directed migration of LRP-deficient smooth muscle cells. The role of LRP in mediating apoE inhibition of PDGF-directed smooth muscle cell migration was confirmed by experiments showing that antibodies against LRP effectively suppressed apoE inhibition of PDGF-directed smooth muscle cell migration. Taken together, these results document that apoE binding to LRP is required for its inhibition of PDGF-directed smooth muscle cell migration.

Diltiazem, a calcium antagonist, inhibits matrix metalloproteinase-1 (tissue collagenase) production and collagenolytic activity in human vascular smooth muscle cells.

Calcium antagonists (CAs) are widely prescribed for patients with cardiovascular diseases. CAs have been reported to inhibit smooth muscle cell (SMC) proliferation in addition to their effects on vascular tone. To determine whether CAs potentially affect vascular remodeling, we measured the expression of matrix-degrading enzymes in growth factor-stimulated SMC. Human cultured SMC were stimulated with 10 ng/ml of platelet-derived growth factor (PDGF)-BB with or without a calcium antagonist, diltiazem. In the cell counting assay, diltiazem (10-5 M) alone had no effect on the proliferation of quiescent SMC, however 10-6-10-5 M of diltiazem dose-dependently inhibited PDGF-stimulated SMC proliferation. The inhibitory effects of diltiazem on SMC proliferation were further confirmed by a 5-bromo-2'-deoxyuridine (BrdU) incorporation assay and flow cytometry. In Western blotting, matrix metalloproteinase (MMP)-1 (tissue collagenase) but not MMP-2 (72-kDa gelatinase) expression was upregulated by PDGF and phorbol ester (TPA), which were reduced by diltiazem in a dose-dependent manner. The downregulation of MMP-1 expression was consistent with the reduction of collagenolytic activity measured by a FITC-conjugated type I collagen breakdown assay. PDGF-stimulated c-Jun/AP-1 expression, a major transcriptional factor for MMP-1, was not affected by diltiazem. In contrast, intracellular calcium ions measured with a fluorometric assay of Fluo-3AM-loaded cells revealed that the PDGF-stimulated increase in the intracellular calcium content was dose-dependently reduced by diltiazem. Our data suggest that diltiazem inhibits not only proliferation but also MMP-1 expression and collagenolytic activity in PDGF-stimulated SMC. The administration of CAs potentially influences the process of vascular remodeling, and this possibility should be further verified in vivo.

Endothelium-independent conversion of angiotensin I by vascular smooth muscle cells.

The conversion of angiotensin I (AT-I) to angiotensin II (AT-II) by angiotensin I-converting enzyme (ACE) is a key step in the action of angiotensins. ACE is constitutively expressed in endothelial cells, but can also be detected at low levels in smooth muscle cells (SMC). Furthermore, in rats the ACE activity can be induced in SMC in vivo by experimental hypertension or vascular injury and in vivo by corticoid treatment. This study was therefore undertaken to evaluate the conversion of AT-I and its subsequent effects in SMC in basal conditions and after stimulation by dexamethasone. Using rat and human SMC, showed that dexamethasone induced ACE expression and that this enzyme was functional, leading to AT-II-dependent intracellular signaling. A fourfold increase in phospholipase C activity in response to AT-I was observed in dexamethasone-activated SMC compared with quiescent SMC. This effect of dexamethasone on signal transduction is dependent on ACE activity, whereas AT-II receptor parameters remain unchanged. The action of AT-I was blocked by an AT1 receptor antagonist, suggesting that it was mediated by AT-II. Similarly, dexamethasone-induced ACE expression was present in human SMC, and calcium signaling was mobilized in response to AT-I in activated human cells. Experiments performed with cocultures of endothelial cells and SMC in a Transwell system showed that the response to AT-I was limited to the compartment where AT-I was localized, suggesting that AT-I does not pass through the endothelial cell barrier to interact with underlying SMC. Our data suggest that in rat, as in human SMC, the conversion of AT-I into AT-II and the signal transduction in response to AT-I are ACE expression-dependent. In addition, the present findings show that this SMC response to AT-I is endothelium-independent, supporting the idea of a local generation of AT-II in the vascular wall.

Effects of advanced glycation end products on the proliferation and fibronectin production of smooth muscle cells.

The aim of this study was to evaluate the effects of advanced glycation end-products (AGEs) on the proliferative activity and fibronectin production of smooth muscle cells (SMCs). AGE-bovine serum albumin (AGE-BSA) was prepared by incubation with D-glucose at 37 degrees C for 60 days. Cultured SMCs were obtained from explants isolated from porcine abdominal aorta and used between passages 3 and 10. The proliferative activity of SMCs was examined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay and by incorporation of 3H-thymidine into DNA. Fibronectin production was assessed by competitive ELISA assay for both fibronectin secreted into the culture medium (M-FN) and cell-associated fibronectin (C-FN), i.e., both intra- and peri-cellular fibronectin. Theassay revealed that AGE-BSA did not produce any change in optical density (A570) of SMCs at concentrations of up to 20 microg/ml, but decreased that of SMCs at a concentration of 40 microg/ml. The addition of PDGF (5 ng/ml) induced an increase in 3H-thymidine incorporation into DNA of quiescent SMCs, while the addition of AGE-BSA (20 microg/ml) had no effect. In contrast, AGE-BSA significantly increased C-FN of SMCs (30.8+/-8.58 ng/microg TP), compared to unmodified BSA (16.5+/-4.19 ng/microg TP). However, no difference in M-FN levels was observed between cells treated with AGE-BSA and unmodified BSA. The addition of anti-transforming growth factor (TGF)-beta antibody restored the levels of C-FN in SMCs cultured in 20 microg/ml of AGE-BSA, suggesting that TGF-beta might act as an intermediate factor in AGE-induced fibronectin production by SMCs. Our results suggest that interaction of AGE-modified proteins with SMCs may play a role in the development of atherosclerosis in diabetic and non-diabetic patients.