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PubMed1996

The fumagillin analogue TNP-470 inhibits DNA synthesis of vascular smooth muscle cells stimulated by platelet-derived growth factor and insulin-like growth factor-I. Possible involvement of cyclin-dependent kinase 2.

The effect of an angiogenesis inhibitor, TNP-470, on DNA synthesis and its underlying signaling cascades stimulated by platelet-derived growth factor (PDGF)-BB and insulin-like growth factor (IGF)-I were examined in bovine vascular smooth muscle cells (SMCs). PDGF-BB (10 ng/mL)- and IGF-I (100 ng/mL)-stimulated increase in DNA synthesis was completely abolished by simultaneous treatment with TNP-470 (1.0 ng/mL). TNP-470 had no effects on PDGF receptor autophosphorylation or early signal transduction, such as activation of mitogen-activated protein kinase and immediate early gene expression. PDGF-BB induced an increase in mRNA levels of cyclin D1, cyclin-dependent kinase (cdk) 4, and cdk2, as well as the activity of cdk2, which preceded the G1/S boundary, as estimated by the kinetics of DNA synthesis. The PDGF-BB-induced activation of cdk2 was inhibited by TNP-470, which was correlated with decreased cdk2 mRNA levels. In contrast, TNP-470 had no or less marked effect on cyclin D1 and cdk4 mRNA levels induced by PDGF-BB. TNP-470 also inhibited a much smaller increase in cdk2 mRNA levels and activation stimulated by IGF-I. In conclusion, TNP-470 potently inhibits DNA synthesis of SMCs, and this inhibition is associated with decreased levels of cdk2 mRNA and activity.

Animals↗
PubMed1998

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

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

Animals↗
PubMed1998

Synergistic inhibition of vascular smooth muscle cell migration by phosphodiesterase 3 and phosphodiesterase 4 inhibitors.

Cyclic nucleotide phosphodiesterases (PDEs) hydrolyze cAMP or cGMP and terminate their signaling. Two important families of PDEs that regulate cAMP signaling in cardiovascular tissues are the cGMP-inhibited PDEs (PDE3) and the cAMP-specific PDEs (PDE4). In this study, we have used a combination of an in vitro motility assay and a sensitive method for the measurement of cAMP in order to determine the relative roles of PDE3 and of PDE4 in the regulation of cAMP-mediated inhibition of VSMC migration. Our data demonstrate that forskolin, an activator of adenylyl cyclases, causes concentration-dependent inhibition of platelet-derived growth factor-induced VSMC migration. Incubation of cultured VSMCs with a PDE4-selective inhibitor, Ro 20-1724, markedly potentiated both the antimigratory effect and the increase in cAMP caused by forskolin. Cilostamide, a PDE3-selective compound, did not affect either the antimigratory activity of forskolin or its ability to increase cAMP. Cilostamide and Ro 20-1724 interacted synergistically to potentiate the inhibition of VSMC migration by forskolin and caused a supra-additive increase in cAMP. These data are consistent with an important role for both PDE3 and PDE4 in the regulation of cAMP-mediated inhibition of VSMC migration.

1-Methyl-3-isobutylxanthine↗
PubMed1998

Lipid lowering promotes accumulation of mature smooth muscle cells expressing smooth muscle myosin heavy chain isoforms in rabbit atheroma.

Smooth muscle cells (SMCs) in the atherosclerotic intima characteristically differ from those in the arterial media, for example, by reduced expression of SMC differentiation/maturation markers such as smooth muscle myosin heavy chain isoforms (SM1 and SM2). This study tested the hypothesis that lipid lowering promotes maturation of intimal SMCs in 33 rabbits subjected to balloon injury and cholesterol feeding (0.3%) for 4 months (Baseline group, n=15); some of which then were switched to a low-cholesterol diet for 8 months (Low group at 8 months, n=3) or 16 months (Low group at 16 months, n=10). The remaining rabbits continued to consume a high-cholesterol diet for 16 months (High group, n=5). We monitored SMC phenotype by expression of immunoreactive alpha-smooth muscle actin, SM1, and SM2. alpha-Actin is an early marker, and SM1 and SM2 are late markers for SMC differentiation/maturation. Only fully differentiated or mature SMCs express SM2. Data are reported as the percentage of the alpha-actin-positive intimal area occupied by smooth muscle myosin-positive SMCs determined by color image analysis of immunostained sections. Levels of SM1 and SM2, highly expressed by SMCs in the normal aortic media (n=5) decreased in the aortic intima of the Baseline and High groups, indicating a less mature phenotype. In contrast, SM1 and SM2 increased in the Low (16 months) group, indicating that intimal SMCs exhibit a more mature phenotype after lipid lowering. Electron microscopy also showed the presence of mature intimal SMCs with abundant myofilaments. Furthermore, lipid lowering reduced levels of platelet-derived growth factor-B in the arterial intima, a factor known to suppress smooth muscle myosin expression. These data demonstrate that lipid lowering favors accumulation of mature SMCs in the atherosclerotic intima in association with reduced levels of platelet-derived growth factor-B expression. Intimal SMCs in the Low group also displayed reduced expression of matrix metalloproteinases-3 and -9 compared with the Baseline and High groups. These findings shed new light on the effects of lipid lowering at the level of the vascular wall, which may influence the biology of the atheroma.

Actin Cytoskeleton↗
PubMed1998

Peroxisome proliferator-activated receptor gamma activators inhibit gene expression and migration in human vascular smooth muscle cells.

Migration of vascular smooth muscle cells (VSMCs) plays an important role in atherogenesis and restenosis after arterial interventions. The expression of matrix metalloproteinases (MMPs), particularly MMP-9, contributes to VSMC migration. This process requires degradation of basal laminae and other components of the arterial extracellular matrix. Peroxisome proliferator-activated receptors (PPARs), members of the nuclear receptor family, regulate gene expression after activation by various ligands. Recent studies have suggested opposing effects of PPAR gamma (PPARgamma) activation on atherogenesis. The present study tested the hypotheses that human VSMCs express PPAR alpha (PPARalpha) and PPARgamma and that PPAR agonists in VSMCs modulate MMP-9 expression and activity, as well as VSMC migration. Human VSMCs expressed PPARalpha and PPARgamma mRNA and protein. Treatment of VSMCs with the PPARgamma ligands troglitazone and the naturally occurring 15-deoxy-Delta12, 14-prostaglandin J2 (15d-PGJ2) decreased phorbol 12-myristate 13-acetate-induced MMP-9 mRNA and protein levels, as well as MMP-9 gelatinolytic activity in the supernatants in a concentration-dependent manner. Six different PPARalpha activators lacked such effects. Addition of prostaglandin F2alpha, known to limit PPARgamma activity, diminished the MMP-9 inhibition seen with either troglitazone or 15d-PGJ2, further implicating PPARgamma in these effects. Finally, troglitazone and 15d-PGJ2 inhibited the platelet-derived growth factor-BB-induced migration of VSMCs in vitro in a concentration-dependent manner. PPARgamma activation may regulate VSMC migration and expression and activity of MMP-9. Thus, PPARgamma activation in VSMCs, via the antidiabetic agent troglitazone or naturally occurring ligands, may act to counterbalance other potentially proatherosclerotic PPARgamma effects.

Becaplermin↗
PubMed1998

Heparan sulfate proteoglycans mediate a potent inhibitory signal for migration of vascular smooth muscle cells.

Migration of vascular smooth muscle cells (SMCs) is a key step in vascular remodeling and formation of pathological lesions in diseased arteries and may be controlled by extracellular matrix (ECM) and by factors that regulate ECM composition, such as platelet-derived growth factor (PDGF). In culture, PDGF-AB and -BB enhance but PDGF-AA (although having no effect alone) suppresses SMC migration stimulated by other PDGF isoforms. To determine whether the migration-inhibitory mechanism of PDGF-AA was mediated by ECM composition, we examined baboon SMC migration in a Boyden chamber assay using filters coated with different ECM proteins. PDGF-AA suppressed the PDGF-BB-induced migration of baboon SMCs on a filter coated with basement membrane proteins (Matrigel) and fibronectin but failed to inhibit cell migration on a type I collagen (Vitrogen)-coated filter. Fibronectin and fibronectin fragments that contain heparin-binding domains permitted PDGF-AA inhibition of cell migration, but a fragment lacking heparin-binding domains did not. Treatment of SMCs with heparin lyases II and III, but not with chondroitin ABC lyase, diminished the PDGF-AA-mediated inhibition of migration. PDGF-AA stimulated accumulation of proteoglycan (PG) in the cell layer more potently than did PDGF-BB, whereas the turnover of cell layer PG was unaffected by either PDGF-AA or -BB. Northern blot analysis revealed that PDGF-AA increased syndecan-1 mRNA expression more than did PDGF-BB, whereas both PDGF isoforms decreased perlecan expression. The changes in cell migration and PG synthesis induced by PDGF-AA were accompanied by changes in the morphology of SMCs. PDGF-AA dramatically induced the spreading of SMCs, whereas the heparin lyase treatment of PDGF-AA-stimulated cultures diminished cell spreading. The data suggest that PDGF-AA selectively modifies heparan sulfate PG accumulation on SMCs and thereby influences the interactions of SMCs with heparin-binding ECM proteins. These interactions, in turn, generate signals that suppress SMC migration.

Animals↗
PubMed1999

Endothelial cells modulate the proliferation of mural cell precursors via platelet-derived growth factor-BB and heterotypic cell contact.

Embryological data suggest that endothelial cells (ECs) direct the recruitment and differentiation of mural cell precursors. We have developed in vitro coculture systems to model some of these events and have shown that ECs direct the migration of undifferentiated mesenchymal cells (10T1/2 cells) and induce their differentiation toward a smooth muscle cell/pericyte lineage. The present study was undertaken to investigate cell proliferation in these cocultures. ECs and 10T1/2 cells were cocultured in an underagarose assay in the absence of contact. There was a 2-fold increase in bromodeoxyuridine labeling of 10T1/2 cells in response to ECs, which was completely inhibited by the inclusion of neutralizing antiserum against platelet-derived growth factor (PDGF)-B. Antisera against PDGF-A, basic fibroblast growth factor, or transforming growth factor (TGF)-beta had no effect on EC-stimulated 10T1/2 cell proliferation. EC proliferation was not influenced by coculture with 10T1/2 cells in the absence of contact. The cells were then cocultured so that contact was permitted. Double labeling and fluorescence-activated cell sorter analysis revealed that ECs and 10T1/2 cells were growth-inhibited by 43% and 47%, respectively. Conditioned media from contacting EC-10T1/2 cell cocultures inhibited the growth of both cell types by 61% and 48%, respectively. Although we have previously shown a role for TGF-beta in coculture-induced mural cell differentiation, growth inhibition resulting from contacting cocultures or conditioned media was not suppressed by the presence of neutralizing antiserum against TGF-beta. Furthermore, the decreased proliferation of 10T1/2 cells in the direct cocultures could not be attributed to downregulation of the PDGF-B in ECs or the PDGF receptor-beta in the 10T1/2 cells. Our data suggest that modulation of proliferation occurs during EC recruitment of mesenchymal cells and that heterotypic cell-cell contact and soluble factors play a role in growth control during vessel assembly.

Animals↗
PubMed1999

A dual inhibitor of platelet-derived growth factor beta-receptor and Src kinase activity potently interferes with motogenic and mitogenic responses to PDGF in vascular smooth muscle cells. A novel candidate for prevention of vascular remodeling.

PP1 has previously been described as an inhibitor of the Src-family kinases p56(Lck) and FynT. We have therefore decided to use PP1 to determine the functional role of Src in platelet-derived growth factor (PDGF)-induced proliferation and migration of human coronary artery smooth muscle cells (HCASMCs). A synthetic protocol for PP1/AGL1872 has been developed, and the inhibitory activity of PP1/AGL1872 against Src was examined. PP1/AGL1872 potently inhibited recombinant p60(c-src) in vitro and Src-dependent tyrosine phosphorylation in p60(c-srcF572)-transformed NIH3T3 cells. PP1/AGL1872 also potently inhibited PDGF-stimulated migration of HCASMCs, as determined in the modified Boyden chamber, as well as PDGF-stimulated proliferation of HCASMCs. Surprisingly, in addition to inhibition of Src kinase, PP1/AGL1872 was found to inhibit PDGF receptor kinase in cell-free assays and in various types of intact cells, including HCASMCs. PP1/AGL1872 did not inhibit phosphorylation of the vascular endothelial growth factor receptor KDR (VEGF receptor-2; kinase-insert domain containing receptor) in cell-free assays as well as in intact human coronary artery endothelial cells. In line with the insensitivity of KDR, PP1/AGL1872 had only a weak effect on vascular endothelial growth factor-stimulated migration of human coronary artery endothelial cells. On treatment of cells expressing different receptor tyrosine kinases, the activities of the epidermal growth factor receptor, fibroblast growth factor receptor-1, and insulin-like growth factor-1 receptor were resistant to PP1/AGL1872, whereas PDGF alpha-receptor was susceptible, albeit to a lesser extent than PDGF beta-receptor. These data suggest that the previously described tyrosine kinase inhibitor PP1/AGL1872 is not selective for the Src family of tyrosine kinases. It is also a potent inhibitor of the PDGF beta-receptor kinase but is not a ubiquitous tyrosine kinase inhibitor. PP1/AGL1872 inhibits migration and proliferation of HCASMCs probably by interference with 2 distinct tyrosine phosphorylation events, creating a novel and potent inhibitory principle with possible relevance for the treatment of pathological HCASMC activity, such as vascular remodeling and restenosis.

3T3 Cells↗
PubMed2000

Kallistatin stimulates vascular smooth muscle cell proliferation and migration in vitro and neointima formation in balloon-injured rat artery.

Kallistatin, a serine proteinase inhibitor (serpin), is expressed in the endothelial and smooth muscle cells of blood vessels. The potential function of kallistatin in vascular biology was investigated by studying its role in the proliferation and migration of cultured primary aortic vascular smooth muscle cells (VSMCs) in vitro and in neointima formation in rat artery after balloon angioplasty in vivo. Exogenous kallistatin induced a >2-fold increase of VSMC proliferation and cell growth as measured by [(3)H]thymidine incorporation and cell counts and a 2.3-fold increase of cell migration in modified Boyden chambers. In balloon-injured vessels, endogenous kallistatin mRNA and protein levels increased up to 10-fold as determined by competitive polymerase chain reaction and by ELISA. Intense staining of kallistatin mRNA was identified in the proliferating VSMCs of balloon-injured arteries during cell migration from media to neointima by in situ hybridization histochemistry and immunohistochemistry. We observed an induction of kallistatin expression by platelet-derived growth factor (PDGF) and upregulation of p42/44 mitogen-activated protein kinase (MAPK) activity by kallistatin in cultured VSMCs. Conversely, adenovirus-mediated transfer of kallistatin antisense cDNA into cultured VSMCs inhibited PDGF-induced p42/44 MAPK activity and cell proliferation. Furthermore, local delivery of adenovirus carrying kallistatin antisense cDNA significantly downregulated kallistatin mRNA levels and attenuated neointima formation in balloon-injured rat arteries in vivo. These results indicate that kallistatin may play an important role in mediating PDGF-induced MAPK pathway on VSMC proliferation and in neointima formation after balloon angioplasty.

Adenoviridae↗
PubMed2000

Heparin blockade of thrombin-induced smooth muscle cell migration involves inhibition of epidermal growth factor (EGF) receptor transactivation by heparin-binding EGF-like growth factor.

Agonists of G protein-coupled receptors, such as thrombin, act in part by transactivating the epidermal growth factor (EGF) receptor (EGFR). Although at first a ligand-independent mechanism for EGFR transactivation was postulated, it has recently been shown that this transactivation by various G protein-coupled receptor agonists can involve heparin-binding EGF-like growth factor (HB-EGF). Because thrombin stimulation of vascular smooth muscle cell migration is blocked by heparin and because heparin can displace HB-EGF, we investigated the possibility that thrombin stimulation of smooth muscle cells (SMCs) depends on EGFR activation by HB-EGF. In rat SMCs, EGFR phosphorylation and extracellular signal-regulated kinase (ERK) activation in response to thrombin are inhibited not only by the EGFR inhibitor AG1478 and by EGFR blocking antibody but also by heparin and by neutralizing HB-EGF antibody. HB-EGF-dependent signaling induced by thrombin is inhibited by batimastat, which suggests a requirement for pro-HB-EGF shedding by a metalloproteinase. We further demonstrate that this novel pathway is required for the migration of rat and baboon SMCs in response to thrombin. We conclude from these data that the inhibitory effect of heparin on SMC migration induced by thrombin relies, at least in part, on a blockade of HB-EGF-mediated EGFR transactivation.

Animals↗
PubMed1998

Differences in cellular responses to mitogens in arterial smooth muscle cells derived from patients with moyamoya disease.

BACKGROUND AND PURPOSE: Moyamoya disease is a progressive cerebrovascular occlusive disease affecting primarily children. The etiology remains unknown. We examined the chemotactic and proliferative activities of inflammatory cell products from arterial smooth muscle cells (SMCs) derived from moyamoya patients and compared them with those from control subjects. METHODS: We used 12 SMC strains from moyamoya patients and eight from control subjects. SMC migration was examined in a micro chemotaxis chamber. DNA synthesis was measured by an immunoperoxidase technique. RESULTS: Platelet-derived growth factor (PDGF)-BB markedly stimulated cell migration and DNA synthesis in control SMCs. PDGF-AA stimulated only DNA synthesis in control SMCs. In moyamoya SMCs, PDGF-AA and PDGF-BB stimulated cell migration but not DNA synthesis. Basic fibroblast growth factor had little migratory activity but stimulated DNA synthesis in moyamoya SMCs and control SMCs. Conversely, hepatocyte growth factor stimulated cell migration but not DNA synthesis in moyamoya SMCs and control SMCs. In contrast, interleukin-1 beta (IL-1 beta) significantly stimulated the migration and DNA synthesis of control SMCs, while it inhibited moyamoya SMC migration. The levels of IL-1 beta-induced nitric oxide production did not differ between moyamoya SMCs and control SMCs, suggesting that IL-1 beta inhibits the migration of moyamoya SMCs through a nitric oxide-independent pathway. CONCLUSIONS: The differences in responses to PDGF and IL-1 in moyamoya SMCs are involved in the mechanism by which intimal thickening develops in moyamoya disease.

Anticoagulants↗
PubMed2000

Growth factors stimulate neointimal cells in vitro and increase the thickness of the neointima formed at the neck of porcine aneurysms treated by embolization.

BACKGROUND AND PURPOSE: Growth factors (GFs) may favor the healing of aneurysms treated with endovascular techniques by stimulating neointima formation. METHODS: Bilateral carotid aneurysms were constructed with venous pouches in 50 pigs and embolized intraoperatively with collagen sponges with and without GFs (platelet-derived growth factor-BB [PDGF-BB] 0.15 or 1.5 microg or transforming growth factor-beta(1) [TGF-beta(1)] 60 or 600 ng) in each animal. DNA synthesis, cell proliferation, and collagen secretion assays were performed to assess the in vitro effects of GFs on neointimal cells harvested from the treated aneurysms. (125)I-PDGF-BB was used to study in vivo GF release from sponges. The thickness of the neointima at the surface of the sponges was measured 2 weeks after surgery. Since porcine aneurysms tend to heal after collagen sponge embolization, this experiment was repeated in dogs, which have shown a propensity for recurrence with the same technique, with 600 ng TGF-beta(1) or platelet extracts. RESULTS: PDGF-BB stimulated DNA synthesis and cell proliferation, while TGF-beta(1) strongly increased collagen synthesis of neointimal cells in vitro. Clearance of (125)I-PDGF-BB from the sponges followed a biphasic curve, with 1.5% of exogenous PDGF-BB remaining at 1 week. The local delivery of PDGF-BB (0.15 or 1.5 microg) and TGF-beta(1) (600 ng) significantly increased neointimal thickness at the neck of porcine aneurysms, while 60 ng of TGF-beta(1) had no demonstrable effect. TGF-beta(1) (600 ng) or platelet extracts had no influence on canine aneurysms. CONCLUSIONS: PDGF-BB and TGF-beta(1) can stimulate neointimal cells in vitro and neointima formation in vivo, but TGF-beta(1) and platelet extracts do not compensate for deficient thrombosis in canine aneurysms. Effects on the long-term results of embolization remain speculative.

Aneurysm↗
PubMed2001

Broad-spectrum and selective serine protease inhibitors prevent expression of platelet-derived growth factor-BB and cerebral vasospasm after subarachnoid hemorrhage: vasospasm caused by cisternal injection of recombinant platelet-derived growth factor-BB.

BACKGROUND AND PURPOSE: Plasma serine protease cascade, including the complement system and thrombin, is activated in the subarachnoid space during the acute phase after subarachnoid hemorrhage (SAH). To examine the effect of protease cascade-based inflammation and subsequent vascular repair in the development of cerebral vasospasm, we examined the effect of 2 synthetic serine protease inhibitors-FUT-175, an inhibitor of thrombin and the complement system, and argatroban, a selective inhibitor of thrombin-on the development of cerebral vasospasm in a rabbit SAH model. METHODS: One hundred Japanese White male rabbits were used in the study. The SAH was simulated by a single injection of autologous arterial blood into the cisterna magna. To evaluate the development of cerebral vasospasm, the caliber of the basilar artery was measured on x-ray film before and at 2 days after SAH. Nine groups of rabbits (n=6 each) were treated with continuous intravenous injection of FUT-175 (2.5, 5, 10, or 20 mg/d), argatroban (1.25, 2.5, or 5 mg/d), or the same amount of saline (vehicle) for 48 hours, starting 40 minutes after SAH. Two days after SAH, the expression of homodimer of platelet-derived growth factor-BB (PDGF-BB) in the basilar artery was examined with immunohistochemical techniques. In 20 normal rabbits, 5 microg of recombinant PDGF-BB or vehicle was injected into the cisterna magna, and the basilar arteries were examined on angiograms for 48 hours. RESULTS: Significant differences were observed in the caliber of the basilar arteries between the vehicle group and the groups with the 3 larger doses of FUT-175 (vehicle, 52+/-5.0%; 5 mg, 79+/-5.7%; 10 mg, 80+/-2.5%; 20 mg, 80+/-3.7%) and between the vehicle group and the groups with the 2 larger doses of argatroban (vehicle, 52+/-6.4%; 2.5 mg, 81+/-9.0%; 5 mg, 85+/-4.1%) (P<0.05). In the histological examination, administration of effective doses of FUT-175 or argatroban suppressed the expression of PDGF-BB in the endothelial and medial smooth muscle cell layers. Exogenous PDGF-BB caused delayed and prolonged vasoconstriction on normal basilar arteries. CONCLUSIONS: Activation of the serine protease cascade and/or thrombin after SAH was demonstrated to play an essential role in the development of cerebral vasospasm. The expression of PDGF-BB-like protein in the arterial walls correlated with the development of cerebral vasospasm. Elevated PDGF-BB level in the subarachnoid space was found to induce delayed and chronic vasoconstriction.

Animals↗
PubMed2005

Prednisolone inhibits proliferation of cultured pulmonary artery smooth muscle cells of patients with idiopathic pulmonary arterial hypertension.

BACKGROUND: Idiopathic pulmonary arterial hypertension (IPAH) is associated with proliferation of smooth muscle cells (SMCs) in small pulmonary arteries. There is no therapy that specifically inhibits SMC proliferation. Recent studies reported that prednisolone (PSL) inhibits the postangioplasty proliferation of SMCs in atherosclerotic arteries. In this study, we tested the hypothesis that PSL has antiproliferative effects on pulmonary artery SMCs of patients with IPAH. METHODS AND RESULTS: Pulmonary artery SMCs were harvested from the pulmonary arteries of 6 patients with IPAH who underwent lung transplantation. Control SMCs were obtained from 5 patients with bronchogenic carcinoma who underwent lung lobectomy. After incubation in the presence of platelet-derived growth factor (PDGF), PSL was added at different concentrations and cell proliferation was assessed by 3H-thymidine incorporation. PSL (2x10(-4) and 2x10(-3) mol/L) significantly inhibited PDGF-stimulated proliferation (P<0.05) of SMCs from patients with IPAH but did not affect cell viability of SMCs, as confirmed by trypan blue staining. In cell cycle analysis using a microscope-based multiparameter laser scanning cytometer, PSL inhibited the progression of SMCs from G(0)/G1 to the S phase. This inhibition was associated with increased p27 expression level. PSL (2x10(-4) mol/L) also inhibited PDGF-induced SMC migration. CONCLUSIONS: Our results indicate that PSL has an antiproliferative effect on cultured SMCs of pulmonary arteries from patients with IPAH and suggest that PSL may be potentially useful therapeutically in patients with IPAH.

Becaplermin↗
PubMed2001

Innate diversity of adult human arterial smooth muscle cells: cloning of distinct subtypes from the internal thoracic artery.

Vascular smooth muscle cells (SMCs) perform diverse functions and this functional heterogeneity could be based on differential recruitment of distinct SMC subsets. In humans, however, there is little support for such a paradigm, partly because isolation of pure human SMC subsets has proven difficult. We report the cloning of 12 SMC lines from a single fragment of human internal thoracic artery and the elucidation of 2 distinct cellular profiles. Epithelioid clones (n=9) were polygonal at confluence, 105+/-9 micrometer in length, and had a doubling time of 39+/-2 hours. Spindle-shaped clones (n=3) were larger (267+/-18 micrometer long, P<0.01) and grew slower (doubling time 65+/-4 hours, P<0.01). Both types of clones expressed smooth muscle (SM) alpha-actin, SM-myosin heavy chains, h-caldesmon, and calponin, but only spindle-shaped clones expressed metavinculin. Epithelioid clones displayed greater proliferation in response to platelet-derived growth factor-BB and fibroblast growth factor-2 and were more responsive to the migratory effect of platelet-derived growth factor-BB. Spindle-shaped clones showed more robust Ca(2+) transients in response to angiotensin II, histamine, and norepinephrine, crawled more quickly, and expressed more type I collagen. On serum withdrawal, spindle-shaped clones differentiated into a contraction-competent cell. A regional basis for diversity among SMCs was suggested by stepwise arterial digestion, which liberated small, SM alpha-actin-positive cells from the abluminal medial layers and larger SMCs from all layers. These results identify inherent SMC diversity in the media of the adult internal thoracic artery and suggest differential participation of SMC subsets in the regulation of human arterial behavior.

Actins↗
PubMed2001

Phosphoinositide 3-kinase isoforms selectively couple receptors to vascular L-type Ca(2+) channels.

Heterodimeric class I phosphoinositide 3-kinase (PI3K) has been shown to be involved in the stimulation of voltage-gated Ca(2+) channels by various mediators. In this study, we bring evidences that vascular L-type Ca(2+) channels can be modulated by both tyrosine kinase-regulated class Ia and G protein-regulated class Ib PI3Ks. Purified recombinant PI3Ks increased the peak Ca(2+) channel current density when applied intracellularly. Furthermore, PI3Kalpha-, beta-, and delta-mediated stimulations of Ca(2+) channel currents were increased by preactivation by a phosphotyrosyl peptide, whereas PI3Kgamma- and beta-mediated effects were increased by Gbetagamma. In freshly isolated and cultured vascular myocytes, angiotensin II and Gbetagamma stimulated L-type Ca(2+) channel current. In contrast, platelet-derived growth factor (PDGF)-BB and the phosphotyrosyl peptide did not stimulate Ca(2+) channel current in freshly isolated cells despite the presence of endogenous PDGF receptors and PI3Kalpha and PI3Kgamma. Interestingly, when endogenous PI3Kbeta expression arose in cultured myocytes, both PDGF and phosphotyrosyl peptide stimulated Ca(2+) channels through PI3Kbeta, as revealed by the inhibitory effect of an anti-PI3Kbeta antibody. These results suggest that endogenous PI3Kbeta but not PI3Kalpha is specifically involved in PDGF receptor-induced stimulation of Ca(2+) channels and that different isoforms of PI3K regulate physiological increases of Ca(2+) influx in vascular myocytes stimulated by vasoconstrictor or growth factor.

Angiotensin II↗
PubMed2002

Effects of dominant-negative c-Jun on platelet-derived growth factor-induced vascular smooth muscle cell proliferation.

Although platelet-derived growth factor (PDGF)-BB is thought to participate in vascular disorders, the mechanism of PDGF-induced vascular smooth muscle cell (SMC) proliferation is not fully understood. This study was undertaken to examine the role of c-Jun in PDGF-BB-induced proliferation of rat aortic SMCs. PDGF-BB (10 ng/mL) significantly increased activator protein (AP)-1 DNA binding activity in SMCs, followed by the increase in [(3)H]thymidine incorporation and cell number. SMCs were infected with recombinant adenovirus containing TAM67, a dominant-negative c-Jun lacking the transactivation domain of wild c-Jun (Ad-DN-c-Jun), to inhibit endogenous AP-1. Ad-DN-c-Jun, which specifically blocked AP-1 transcriptional activity, significantly inhibited PDGF-BB-induced increases in [(3)H]thymidine incorporation or cell number. As shown by flow cytometric analysis, Ad-DN-c-Jun inhibited PDGF-BB-induced entrance of SMCs into S phase, leading to a G(1) arrest. Ad-DN-c-Jun attenuated PDGF-BB-induced downregulation of p27(Kip1), as shown by Western blot analysis, and the prevented PDGF-BB-induced decrease in cyclin E/cyclin-dependent kinase 2 complex-associated p27(Kip1), as shown by immunoprecipitation study. Furthermore, protein kinase assay showed that Ad-DN-c-Jun blocked PDGF-BB-induced activation of cyclin-dependent kinase 2. Our results provide the first evidence that dominant-negative c-Jun inhibits PDGF-BB-induced vascular SMC proliferation by preventing the downregulation of p27(Kip1), thereby supporting the important role of c-Jun in vascular SMC proliferation.

Animals↗
PubMed2002

Different effects of high and low shear stress on platelet-derived growth factor isoform release by endothelial cells: consequences for smooth muscle cell migration.

In the present study, we analyzed the effect of conditioned media (CM) from bovine aortic endothelial cells exposed to laminar shear stress (SS) of 5 dyne/cm2 (SS5) or 15 dyne/cm2 (SS15) for 16 hours on smooth muscle cell (SMC) migration. In response to CM from bovine aortic endothelial cells exposed to SS5 (CMSS5) and SS15 (CMSS15), migration was 45 +/- 5.5 and 30 +/- 1.5 cells per field, respectively (P<0.05). Similar results were obtained with SS of 2 versus 20 dyne/cm2 and also when SS of 5 and 15 dyne/cm2 lasted 24 hours. Platelet-derived growth factor (PDGF)-AA levels in CMSS5 and CMSS15 were 9 +/- 7 and 18 +/- 5 ng/10(6) cells for 16 hours, respectively (P<0.05); PDGF-BB levels in CMSS5 and CMSS15 were 38 +/- 10 and 53 +/- 10 ng/10(6) cells for 16 hours, respectively (P<0.05). PDGF receptor alpha (PDGFRalpha) and PDGF receptor beta (PDGFRbeta) in SMCs were phosphorylated by CMSS15>CMSS5. In response to CMSS15, a neutralizing antibody against PDGF-AA enhanced SMC migration to a level comparable to that of CMSS5; in contrast, antibodies against PDGF-BB abolished SMC migration. Transfection of SMCs with a dominant-negative PDGFRalpha or PDGFRbeta increased or inhibited, respectively, SMC migration in response to CMSS15. Overexpression of wild-type PDGFRalpha inhibited SMC migration in response to CMSS5, CMSS15, or recombinant PDGF-BB (P<0.001). These results suggest that the ability of high SS to inhibit arterial wall thickening in vivo may be related to enhanced activation of PDGFRalpha in SMCs by PDGF isoforms secreted by the endothelium.

Animals↗