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ABSTRACT: Peroxisome proliferator-activated receptor {gamma} (PPARgamma), the nuclear receptor that binds the insulin-sensitizing thiazolidinediones (TZDs), is prominently upregulated in intimal vascular smooth muscle cells (VSMC) after mechanical injury to the vessel wall. Several TZD PPARgamma ligands have been shown to inhibit neointima formation in both normal and insulin-resistant vasculature. The suppression of intimal hyperplasia by TZD PPARgamma ligands probably results from their activity to inhibit VSMC growth and promote apoptosis. TZDs prevent VSMC proliferation by blocking the activity of regulatory proteins, such as phosphorylation of the retinoblastoma protein (Rb). Rb functions as a G(1) gatekeeper by controlling S phase gene expression mediated by the E2F transcription factor. Consistent with their effect on Rb phosphorylation, PPARgamma ligands inhibit the mitogenic induction of minichromosome maintenance (MCM) proteins 6 and 7, two E2F-regulated S phase genes essential for DNA replication. PPARgamma ligands also induced apoptosis in VSMC, which correlated with a potent induction of GADD45, a gene implicated in controlling cell growth and survival. A constitutively active form of PPARgamma targeted the same cell cycle regulators as did PPARgamma ligands, consistent with a nuclear-receptor-dependent mechanism of action. This review will summarize mechanisms through which PPARgamma modulates VSMC proliferation and apoptosis suggesting that PPARgamma itself is a novel important regulator of cell cycle and apoptosis and may provide a new therapeutic approach to prevent restenosis.
International Journal of Obesity 04/2005; 29 Suppl 1:S26-30. · 4.69 Impact Factor
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ABSTRACT: The steroid dexamethasone inhibits neointimal hyperplasia development in rats but not in humans. This study investigates the differential effects of dexamethasone on rat and human smooth muscle cell migration and matrix metalloproteinase (MMP) activity.
Rat aortic smooth muscle cells were harvested from Sprague-Dawley rats. Human aortic smooth muscle cells were obtained from Clonetics. Boyden chamber migration assays were performed with chemoattractant (platelet-derived growth factor) and varying concentrations of dexamethasone (10(-9) to 10(-5) mol/L). Zymography of culture media was used to assess MMP activity, and Western blot analysis was used for quantification of MMP-2 and tissue inhibitor of MMP-2 (TIMP-2) secretion.
Dexamethasone inhibits rat aortic smooth muscle cell migration in a dose-dependent fashion. An increase in concentrations of dexamethasone does not effect human aortic smooth muscle cell migration. Rat aortic smooth muscle cell MMP-2 activity is inhibited with dexamethasone in a dose-dependent fashion, and human aortic smooth muscle cell MMP-2 activity is unchanged with dexamethasone. MMP-2 secretion is inhibited with dexamethasone in rat aortic smooth muscle cells but remains unaltered in human aortic smooth muscle cells. Dexamethasone increases rat aortic smooth muscle cell TIMP-2 secretion, and human aortic smooth muscle cell TIMP-2 secretion remains constant.
Dexamethasone inhibits rat aortic smooth muscle cell migration, MMP-2 activity, and MMP-2 secretion and increases TIMP-2 secretion. These effects are not observed in human aortic smooth muscle cells. These findings may explain why dexamethasone inhibits neointimal hyperplasia in animal models but is ineffective in humans. Inhibition of human smooth muscle cell migration in vitro may be useful in predicting the effectiveness of future therapeutic agents for treatment of neointimal hyperplasia in humans.
Journal of Vascular Surgery 07/2002; 35(6):1253-9. · 3.21 Impact Factor
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ABSTRACT: Dexamethasone (DEX) has been shown to inhibit development of neointimal hyperplasia in rats. We hypothesize that DEX inhibits neointimal hyperplasia by altering matrix metalloproteinase (MMP) activity, resulting in inhibition of smooth muscle cell migration.
Rat aortic smooth muscle cells (RASMC) were harvested and cultured for two to four passages. A migration assay was performed in a Boyden chamber with chemoattractant (platelet-derived growth factor) and varying concentrations of DEX (10(-9) to 10(-5) M). The number of migrated cells was counted under light microscopy. Zymography was performed on culture media to assess MMP activity, and Western blotting was performed to assay MMP and levels of tissue inhibitors of MMPs (TIMPs).
DEX progressively inhibited RASMC migration in a dose-dependent fashion. This effect was statistically significant for concentrations of 10(-7) to 10(-5) M (P < 0.0005). Zymography showed that DEX inhibits MMP-2 activity in a dose-dependent manner. Western blots indicated that total MMP-2 secretion was inhibited and that TIMP-2 secretion was increased by DEX.
DEX inhibits platelet-derived growth factor-induced migration of RASMCs and MMP-2 activity in vitro. Our data suggest that DEX suppresses MMP activity and secretion, resulting in the inhibition of smooth muscle cell migration. This may explain the mechanism by which DEX inhibits neointimal hyperplasia.
Journal of Surgical Research 02/2002; 102(2):57-62. · 2.25 Impact Factor
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ABSTRACT: Atherosclerosis is a major vascular complication of diabetes and the primary cause of mortality in persons with this disease. Metabolic abnormalities related to the Insulin Resistance Syndrome or Metabolic Syndrome may importantly contribute to the increased risk of atherosclerosis associated with diabetes. Thiazolidinediones (TZDs) are oral insulin sensitizers in broad clinical use that enhance insulin-stimulated glucose uptake into skeletal muscle. TZDs can also improve cardiovascular risk factors and exert direct effects on vascular cells to potentially retard the atherosclerotic process. Direct vascular effects of TZDs likely result from their activity as ligands for the nuclear receptor, PPARgamma. All of the major cell types in the vasculature express PPARgamma, including intimal macrophages and vascular smooth muscle cells (VSMCs) in human atheroma. TZDs block VSMC growth by inducing cell cycle arrest in G1 through an inhibition of retinoblastoma protein phosphorylation. Migration of monocytes and VSMCs is also inhibited by TZDs, possibly through decreased matrix metalloproteinase production. Activation of PPARgamma by TZDs in macrophages induces ABCA1 transporter expression to promote reverse cholesterol transport. These antiatherogenic activities may also occur in vivo because TZDs have been shown to inhibit lesion formation in several animal models. Thus, TZD activation of PPARgamma may protect against atherosclerosis both by normalizing proatherogenic metabolic abnormalities of the insulin resistance/diabetes milieu and through an inhibition of vascular cell growth and movement.
Arteriosclerosis Thrombosis and Vascular Biology 01/2002; 21(12):1891-5. · 6.37 Impact Factor
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ABSTRACT: Vascular smooth muscle cell (VSMC) migration involves adhesion, locomotion, and invasion regulated by various signaling molecules, among which the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinases (MAPK) play a critical role. We have shown that the peroxisome proliferator-activated receptor-gamma (PPAR-gamma) ligands troglitazone and rosiglitazone inhibit VSMC migration downstream of ERK MAPK. The purpose of the current study was to more specifically determine which step(s) in VSMC migration are targeted by inhibition of the ERK MAPK pathway or activation of PPAR-gamma. VSMC adhesion was not affected by the ERK MAPK pathway inhibitor PD98059 or PPAR-gamma ligands. Phosphorylation and activation of myosin light chain kinase (MLCK) play important roles in cell locomotion. Platelet-derived growth factor (PDGF)-induced MLCK phosphorylation (1.7-fold) was completely blocked by PD98059 at 30 microM (p < 0.05), but not by troglitazone or rosiglitazone. PDGF-directed migration (5.8-fold) was inhibited by PD98059 (-88% at 30 microM) and the MLCK inhibitor ML9 (0.1-1 microM, -84% at 1 microM) (all p < 0.05). The transcription factor Ets-1 mediates matrix metalloproteinase induction required for tissue invasion by VSMC. PDGF (20 ng/ml) stimulated an Ets-1 protein expression (14-fold at 60 min) in VSMC, which was inhibited by PD98059 (-72% at 30 microM), troglitazone (-69% at 20 microM), and rosiglitazone (-54% at 10 microM) (all p < 0.05). Immunohistochemistry of rat aortae 2 h after balloon injury showed a dramatic upregulation of Ets-1, which was markedly inhibited in animals that had received troglitazone treatment. In contrast, phosphorylated ERK MAPK was not affected by troglitazone. These data are consistent with PPAR-gamma ligands exerting their anti-migratory effects downstream of ERK MAPK activation by blocking nuclear events, such as Ets-1 expression, required for cell invasion in response to arterial injury.
Journal of Cardiovascular Pharmacology 01/2002; 38(6):909-21. · 2.29 Impact Factor
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ABSTRACT: The cyclin-dependent kinase inhibitor p21(Cip1) is up-regulated in response to mitogenic stimulation in various cells. PPARgamma ligands troglitazone (TRO, 10 microm) and rosiglitazone (RSG, 10 microm) attenuated the induction of p21(Cip1) protein by platelet-derived growth factor (PDGF) and insulin without affecting cognate mRNA levels in rat aortic smooth muscle cells (RASMC). The protein kinase Cdelta (PKCdelta) inhibitor rottlerin also blocked the induction of p21(Cip1) protein, whereas the conventional PKC isotype inhibitor Gö 6976 had no effect. Kinetic studies using the protein synthesis inhibitor cycloheximide showed that TRO, RSG, and rottlerin shortened the half-life of p21(Cip1) protein. TRO, RSG, and rottlerin inhibited PDGF-induced expression of p21(Cip1), but they did not affect insulin-induced expression of p21(Cip1). Both ligands inhibited PKCdelta enzymatic activity in PDGF-stimulated RASMC but not in insulin-stimulated cells. Adenovirus-mediated overexpression of PKCdelta rescued the down-regulation of p21(Cip1) expression both by TRO and RSG in PDGF-treated RASMC. These data suggested that the PKCdelta pathway plays a critical role in PDGF-induced expression of p21(Cip1) in RASMC and may be the potential target for PPARgamma ligand effects. Src kinase-dependent tyrosine phosphorylation of PKCdelta was decreased substantially by TRO and RSG. Tyrosine phosphorylation and activation of c-Src in response to PDGF were unaffected by either PPARgamma ligand. Protein-tyrosine-phosphatase inhibitors sodium orthovanadate and dephostatin prevented PPARgamma ligand effects on PKCdelta tyrosine phosphorylation and enzymatic activity. Both inhibitors also reversed PPARgamma ligand effects on p21(Cip1) expression in PDGF-treated RASMC. PPARgamma ligands enhanced protein-tyrosine-phosphatase activity in RASMC, which may be the mechanism for decreased PKCdelta tyrosine phosphorylation and activity. PPARgamma ligands regulate p21(Cip1) at a post-translational level by blocking PKCdelta signaling and accelerating p21(Cip1) turnover.
Journal of Biological Chemistry 01/2002; 276(50):47650-7. · 4.77 Impact Factor
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ABSTRACT: Migration, proliferation and differentiation of vascular smooth muscle cells (VSMC) and macrophages are important pathological responses that contribute to the development and progression of vascular lesions. Cytokines such as TNFalpha are present at sites of vascular injury and regulate a variety of cellular functions of inflammatory cells and VSMC. Cell migration, proliferation and differentiation require de novo gene transcription resulting from extracellular signals being transduced to the nucleus, where multiple genes are regulated to participate in lesion formation. In VSMC and macrophages, TNFalpha induces activation of the extracellular signal-regulated kinases 1/2 (ERK 1/2), which transmit signals from the cytosol to the nucleus. Potential nuclear targets of TNFalpha-activated ERK 1/2 include the transcription factors Ets-1, Egr-1, and c-fos, which are known to regulate cellular growth, differentiation, and migration. The aim of this study was to investigate the expression of the transcription factors Ets-1, Egr-1 and c-fos in different types of vascular lesions, their regulation by TNFalpha and the role of ERK 1/2 in these signaling events. Atherosclerotic lesions from fructose-fed LDL-receptor deficient mice and neointimal lesions from rat aortae 2 weeks post balloon injury demonstrated the presence and colocalization of TNFalpha, phosphorylated and activated ERK 1/2, and transcription factors Ets-1, Egr-1 and c-fos. Neointimal lesions consisted primarily of VSMC, whereas atherosclerotic lesions predominantly contained macrophages. In cultured rat aortic VSMC, TNFalpha (100 U/ml) stimulated a rapid and transient expression of Ets-1, Egr-1 and c-fos with a maximal induction 1 h after stimulation. In cultured RAW 264.7 mouse macrophages, TNFalpha similarly induced the expression of Ets-1, Egr-1, and c-fos. Induction of these transcription factors was mediated via ERK 1/2 activation, since the ERK 1/2-pathway inhibitor PD98059 (10-30 microM) significantly inhibited their TNFalpha-induced expression. TNFalpha induced ERK 1/2 activation in both cell types. These findings underscore the importance of the ERK 1/2 pathway in the expression of TNFalpha-regulated transcription factors, which may participate in different forms of vascular lesion formation.
Atherosclerosis 12/2001; 159(1):93-101. · 3.79 Impact Factor
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ABSTRACT: Tumor necrosis factor alpha (TNFalpha) interferes with insulin signaling in adipose tissue and may promote insulin resistance. Insulin resistance is associated with vascular injury, but little is known about the interaction of TNFalpha and insulin in the vasculature. By activating the Insulin receptor (IR) --> IRS-1 --> phosphatidylinositol-3-kinase (PI3K) --> Akt-pathway, insulin protects vascular smooth muscle cells (VSMC) from undergoing apoptosis. We therefore investigated the effect of TNFalpha on insulin's antiapoptotic signaling in rat aortic VSMC. Insulin induced rapid tyrosine-phosphorylation of the IR and IRS-1 and caused a 2.8-fold increase of IRS-1-bound PI3K. TNFalpha had no effect on insulin-induced tyrosine-phosphorylation of IR or IRS-1, but inhibited insulin-stimulated IRS-1/PI3K-association by 84%. Insulin-induced phosphorylation of Akt downstream of PI3K was inhibited by TNFalpha in a similar pattern. We next examined the effect of TNFalpha on insulin's protective actions on H(2)O(2)-induced apoptosis. Insulin alone prevented 72.8% of H(2)O(2)-induced apoptosis, which was significantly inhibited by TNFalpha. TNFalpha alone did not induce apoptosis. In contrast, TNFalpha had no effect on PDGF-induced antiapoptotic signal transduction via Akt. Thus, TNFalpha selectively interferes with insulin's antiapoptotic signaling in VSMC by inhibiting the association of IRS-1/PI3K and the downstream activation of Akt.
Biochemical and Biophysical Research Communications 09/2001; 287(3):662-70. · 2.48 Impact Factor
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ABSTRACT: Peroxisome proliferator activated receptor gamma (PPARgamma) is a member of the nuclear receptor superfamily. Ligand activation of PPARgamma has been shown to cause growth arrest in several human tumor cell types, but the underlying molecular mechanism has not been elucidated. We report here that the PPARgamma ligand troglitazone (TRO) inhibited MCF-7 cell proliferation by blocking events critical for G1 --> S progression. Flow cytometry demonstrated that TRO at 20 microM increased the percentage of cells in G1 from 51 to 69% after 24 h. Accumulation of cells in G1 was accompanied by an attenuation of Rb protein phosphorylation associated with decreased CDK4 and CDK2 activities. Inhibition of CDK activity by TRO correlates with decreased protein levels for several G1 regulators of Rb phosphorylation (cyclin D1, and CDKs 2, 4, and 6). Overexpression of cyclin D1 partially rescued MCF-7 cells from TRO-mediated G1 arrest. Targeting of G1 regulatory proteins, particularly cyclin D1, and the resulting induction of G1 arrest by TRO may provide a novel antiproliferative therapy for human breast cancer.
Biochemical and Biophysical Research Communications 09/2001; 286(5):916-22. · 2.48 Impact Factor
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ABSTRACT: Monocyte chemotactic protein 1 (MCP-1)-directed transendothelial migration of monocytes plays a key role in the early development of atherosclerosis. Migration of monocytes requires degradation of extracellular matrices, a process that involves matrix metalloproteinases (MMP) and tissue inhibitors of MMPs (TIMP). Recent studies suggest that the alpha1-adrenergic receptor antagonist doxazosin (Dox) might have antiatherosclerotic effects, although the underlying mechanisms are poorly understood. The purpose of the present study was to determine the effects of Dox on MCP-1-directed monocyte migration, MMP-9 activity, and TIMP-1 expression. MCP-1 (50 ng/ml) stimulated migration of human peripheral blood monocytes (HPBM) 2.7+/-0.42-fold and THP-1 human monocytes 5.9+/-0.83-fold compared with unstimulated control. Dox inhibited MCP-1-induced migration in a dose-dependent manner, with a maximal reduction at 10 microM of 69.5+/-5.9% in HPBM and 72.2+/-3.2% in THP-1 cells. Dox blocked migration even after pretreatment with phenoxybenzamine, an irreversible alpha1-adrenergic receptor antagonist (HPBM: phenoxybenzamine 1 microM + Dox 10 microM, 71.9+/-2.2% inhibition; THP-1 cells: phenoxybenzamine 1 microM + Dox 10 microM: 78+/-7.7% inhibition), suggesting that the antimigratory activity of Dox is mediated through a novel mechanism unrelated to its blocking of the alpha1-adrenergic receptor. Dox (10 microM) inhibited MMP-9 activity by 67.6+/-10.5%, whereas MMP-9 protein levels were not affected. Also, Dox increased PMA-induced-tissue inhibitor of MMPs-1 (TIMP-1) expression by 134.4+/-6.6%. Dox 10 microM. The present study demonstrates a potential novel antiatherosclerotic action of Dox by blocking MCP-1-directed monocyte migration, which might be partly mediated by inhibition of MMP-9 activity.
Journal of Cardiovascular Pharmacology 06/2001; 37(5):532-9. · 2.29 Impact Factor
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ABSTRACT: Retinoids inhibit rat vascular smooth muscle cell (VSMC) proliferation in vitro and intimal hyperplasia in vivo. We examined the mechanism of the antiproliferative effect of retinoids on human coronary artery smooth muscle cells (human CASMCs). The RAR ligands all-trans-retinoic acid (atRA) and ethyl-p-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthyl)-l-propenyl]-benzoic acid (TTNPB); a pan-RXR/RAR agonist, 9-cis-retinoic acid (9cRA); and the RXR-selective ligand AGN4204 all inhibited DNA synthesis stimulated with platelet-derived growth factor and insulin (IC(50): TTNPB 63 nmol/L, atRA 120 nmol/L, AGN4204 460 nmol/L, 9cRA 1.5 micromol/L). All retinoids blocked cell cycle progression as determined by flow cytometry and inhibited retinoblastoma protein (Rb) phosphorylation. TTNPB, atRA, and AGN4204 inhibited the mitogenic induction of cyclin D1, whereas 9cRA had no effect. None of the retinoids affected the expression of CDK 2, 4, or 6 or cyclin E. All retinoids attenuated mitogen-induced downregulation of CDKI p27(Kip1), a major negative regulator of Rb phosphorylation, partly through stabilizing p27(Kip1) turnover. These data demonstrate that retinoids have antiproliferative activity by modulating G(1) --> S cell cycle regulators in human CASMCs through inhibition of Rb phosphorylation and elevation of p27(Kip1) levels.
Arteriosclerosis Thrombosis and Vascular Biology 05/2001; 21(5):746-51. · 6.37 Impact Factor
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ABSTRACT: To determine the effect of thiazolidinediones (TZDs) on experimental retinal neovascularization.
The ability of the TZDs troglitazone and rosiglitazone maleate (1-20 micromol/L) to inhibit retinal endothelial cell (REC) proliferation, migration, tube formation, and signaling was determined in response to vascular endothelial growth factor (VEGF). In vivo studies were performed using the oxygen-induced ischemia model of retinal neovascularization. Neonatal mice were treated with intravitreous injection of 0.5 microL of troglitazone (100 micromol/L) or rosiglitazone maleate (100 micromol/L), or vehicle, and retinal neovascularization was assayed qualitatively and quantitatively by means of angiography and histological examination.
Expression of the TZD receptor, peroxisome proliferator-activated receptor gamma, was confirmed in RECs by means of Western immunoblotting. Rosiglitazone and troglitazone inhibited VEGF-induced migration (P< .05), proliferation (P< .05), and tube formation (P< .01) by RECs in vitro beginning at 10 micromol/L. Rosiglitazone and troglitazone inhibited phosphorylation of extracellular signal-regulated mitogen-activated protein kinase 1 in RECs. Intravitreous injection of rosiglitazone or troglitazone inhibited development of retinal neovascularization (P< .01) but did not significantly inhibit VEGF overexpression in the ganglion cell layer of the ischemic retina.
The TZDs inhibit experimental retinal neovascularization with an effect that is primarily downstream of VEGF expression.
The TZDs are widely prescribed and should be evaluated for their potential to inhibit the progression of diabetic retinopathy.
Archives of Ophthalmology 05/2001; 119(5):709-17. · 3.71 Impact Factor
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ABSTRACT: Peroxisome proliferator-activated receptor-gamma (PPARgamma) is a ligand-activated nuclear receptor expressed in all of the major cell types found in atherosclerotic lesions: monocytes/macrophages, endothelial cells, and smooth muscle cells. In vitro, PPARgamma ligands inhibit cell proliferation and migration, 2 processes critical for vascular lesion formation. In contrast to these putative antiatherogenic activities, PPARgamma has been shown in vitro to upregulate the CD36 scavenger receptor, which could promote foam cell formation. Thus, it is unclear what impact PPARgamma activation will have on the development and progression of atherosclerosis. This issue is important because thiazolidinediones, which are ligands for PPARgamma, have recently been approved for the treatment of type 2 diabetes, a state of accelerated atherosclerosis. We report herein that the PPARgamma ligand, troglitazone, inhibited lesion formation in male low density lipoprotein receptor-deficient mice fed either a high-fat diet, which also induces type 2 diabetes, or a high-fructose diet. Troglitazone decreased the accumulation of macrophages in intimal xanthomas, consistent with our in vitro observation that troglitazone and another thiazolidinedione, rosiglitazone, inhibited monocyte chemoattractant protein-1-directed transendothelial migration of monocytes. Although troglitazone had some beneficial effects on metabolic risk factors (in particular, a reduction of insulin levels in the diabetic model), none of the systemic cardiovascular risk factors was consistently improved in either model. These observations suggest that the inhibition of early atherosclerotic lesion formation by troglitazone may result, at least in part, from direct effects of PPARgamma activation in the artery wall.
Arteriosclerosis Thrombosis and Vascular Biology 04/2001; 21(3):365-71. · 6.37 Impact Factor
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ABSTRACT: Angiotensin (Ang) II has been shown to enhance the development of atherosclerotic lesions. Migration of monocytes is an early critical step in the atherosclerotic process. To elucidate mechanisms by which Ang II promotes atherogenesis, we investigated its effects on human monocyte migration. Ang II induced migration of human peripheral blood monocytes (HPBM) and human THP-1 monocytes at concentrations between 0.01 and 1 micromol/L, with a 3.6+/-0.6-fold induction in HPBM and a 4.8+/-0.9-fold induction in THP-1 cells at 1 micromol/L Ang II (both P<0.01 versus unstimulated cells). Addition of the Ang II receptor type 1 (AT1-R) antagonist losartan (1 to 100 micromol/L) suppressed Ang II-induced migration of HPBM and THP-1 monocytes in a dose-dependent manner, demonstrating an AT1-R-mediated mechanism. Ang II-directed migration was also blocked by the Src kinase inhibitor PP2 (10 micromol/L), by the extracellular-regulated protein kinase (ERK 1/2) inhibitor PD98059 (30 micromol/L), and by the p38-MAPK inhibitor SB203580 (10 micromol/L), indicating that Src, ERK 1/2, and p38 are all involved in Ang II-induced migration of HPBM and human THP-1 monocytes. The proline-rich tyrosine kinase 2 (Pyk2) and paxillin are 2 cytoskeleton-associated proteins involved in cell movement, phosphorylated by Ang II in other cell types, and abundantly expressed in monocytes. Ang II (1 micromol/L) induced Pyk2 and paxillin phosphorylation in human THP-1 monocytes, peaking after 10 minutes for Pyk2 with a 6.7+/-0.9-fold induction and after 2 minutes for paxillin with a 3.2+/-0.4-fold induction. Ang II-induced phosphorylation of both proteins was suppressed by losartan and the Src inhibitor PP2, whereas no effect was observed with PD98059 and SB203580. This study demonstrates a novel proatherogenic action of Ang II on human monocytes by stimulating their migration, through an AT1-R-dependent process, involving signaling through Src, ERK 1/2, and p38. Furthermore, the promigratory actions of Ang II in human monocytes are associated with the phosphorylation of 2 cytoskeleton-associated proteins, Pyk2 and paxillin.
Hypertension 03/2001; 37(2 Part 2):587-93. · 6.21 Impact Factor
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ABSTRACT: Compared with nondiabetic subjects, type 2 diabetic individuals are at an increased risk for coronary artery disease and coronary restenosis after angioplasty or stenting. Increased proliferation and migration of vascular smooth muscle cells (VSMCs) contribute importantly to the formation of both atherosclerotic and restenotic lesions. Therefore, pharmaceutical interventions targeting proteins that regulate VSMC growth or movement are a promising new approach to treat diabetes-associated cardiovascular disease. Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is a member of the nuclear receptor superfamily that, when activated by thiazolidinedione (TZD) insulin sensitizers, regulates a host of target genes. All of the major cells in the vasculature express PPAR-gamma, including endothelial cells, VSMCs, and monocytes/macrophages. PPAR-gamma is present in intimal macrophages and VSMCs in early human atheromas. In an animal model of vascular injury; PPAR-gamma levels are substantially elevated in the neointima that forms after mechanical injury of the endothelium. Recent experimental studies provide evidence that PPAR-gamma may function to protect the vasculature from injury. Cell culture studies have shown that TZD PPAR-gamma ligands inhibit both the proliferation and migration of VSMCs. These antiatherogenic activities of PPAR-gamma may also occur in vivo, because TZDs inhibit lesion formation in several animal models. PPAR-gamma ligands may also protect the vasculature indirectly by normalizing metabolic abnormalities of the diabetic milieu that increase cardiovascular risk. Activation of PPAR-gamma, newly defined in vascular cells, may be a useful approach to protect the vasculature in diabetes.
Diabetes Care 03/2001; 24(2):392-7. · 8.09 Impact Factor
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ABSTRACT: Neointima formation involves tissue expression of matrix proteins and growth factors. The role of alphavbeta3, but not alphavbeta5 integrin in vascular cells has been sufficiently investigated. The aim of the present study was to determine and compare the function of alphavbeta3 and alphavbeta5 integrins in rat aortic (RASMC) and human coronary vascular smooth muscle cells (HCSMC) and to characterize their expression accompanying neointima formation in vivo. RASMC and HCSMC express alphavbeta3 and alphavbeta5 integrin subunits. The alphavbeta5 integrin predominantly mediated adhesion of RASMCs to vitronectin and spreading on vitronectin via RGD-binding sequences. In contrast, the alphavbeta3 integrin did not contribute to the adhesion and spreading on fibronectin, vitronectin, gelatin or collagen I coated layers. PDGF-directed migration through gelatin coated membranes involved both alphavbeta3 and alphavbeta5 integrins. Selective blocking antibodies for alphavbeta3 and alphavbeta5 inhibited migration of RASMC and HCSMC by more than 60 % (p < 0.01). Integrin expression was studied in vivo in thoracic aorta of Sprague Dawley rats before and after balloon injury. In situ hybridization demonstrated low signals for alphav, beta3 and beta5 mRNA in uninjured aorta, which increased significantly at 14 days, localized predominantly in the neointima. Northern analysis of aorta after 14 days of injury also demonstrated an upregulation of alphav, beta3 and beta5 mRNA compared to uninjured aorta. Consistent with the increase in message levels, increased integrin protein expression was seen in the neointima after 7 and 14 days. This study provides evidence that alphavbeta3 and alphavbeta5 are elevated during neointima formation in the rat and indicates a novel role for alphavbeta5 participating in mechanisms regulating smooth muscle cell migration.
Archiv für Kreislaufforschung 03/2001; 96(1):42-9. · 7.35 Impact Factor
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ABSTRACT: Monocyte chemotactic protein-1 (MCP-1)-directed transendothelial migration of monocytes plays a key role in the development of inflammatory diseases. Infiltration of tissues by monocytes requires degradation of extracellular matrices, a process that involves matrix metalloproteinases. We studied the effects of peroxisome proliferator-activated receptor (PPAR) gamma, alpha, and retinoid X receptor alpha (RXRalpha) ligands on MCP-1-directed migration and matrix metalloproteinase expression of a human acute monocytic leukemia cell line (THP-1). PPARgamma ligands attenuated MCP-1-induced migration, with 50% inhibition (IC(50)) at 2.8 microM for troglitazone and 4.8 microM for rosiglitazone. PPARalpha ligands WY-14643 (IC(50): 0.9 microM) and 5,8,11,14-eicosatetranoic acid (IC(50): 9.9 microM), and the potent RXRalpha ligand AGN 4204 (IC(50): 3.6 nM) also blocked monocyte migration. Troglitazone, rosiglitazone, or AGN 4204 inhibited phorbol 12-myristate 13-acetate (PMA)-induced matrix metalloproteinase-9 expression. PPARalpha activators WY-14643 and 5,8,11,14-eicosatetraynoic acid, however, had no inhibitory effect. AGN 4204 increased PMA-induced tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) expression, whereas all PPAR ligands showed no effect. All PPAR and RXRalpha ligands blocked chemotaxis of THP-1 monocytes in the absence of a matrix barrier. This study demonstrates that activated PPARs and RXRalpha, block MCP-1-directed monocyte migration, mediated, at least in part, through their effects on matrix metalloproteinase-9 or TIMP-1 production, or chemotaxis.
European Journal of Pharmacology 09/2000; 401(3):259-70. · 2.52 Impact Factor
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ABSTRACT: To determine the antiangiogenic effects of peroxisome proliferator-activated receptor (PPAR)-gamma agonists on ocular cells involved in the pathogenesis of choroidal neovascularization (CNV) in vitro and on experimental laser photocoagulation-induced CNV in vivo.
PPAR-gamma expression in human retinal pigment epithelial (RPE) cells and bovine choroidal endothelial cells (CECs) was determined using an RNase protection assay and Western blot analysis. Two PPAR-gamma ligands, troglitazone (TRO) and rosiglitazone (RSG; 0.1-20 microM), were used to assess effects on RPE and CEC proliferation and migration and CEC tube formation in response to vascular endothelial growth factor (VEGF). The effects of intravitreal injection of TRO on laser photocoagulation-induced CNV lesions in rat eyes (15 experimental, 15 control, nine burns per eye) and cynomolgus monkey eyes (two experimental, two control, seven paramacular burns per eye) was assessed by fluorescein angiography and histologic evaluation. RESULTS. PPAR-gamma1 was expressed in both RPE and CEC. PPAR-gamma ligands significantly inhibited VEGF-induced migration and proliferation in both cell types and tube formation of CEC in a dose-response manner. CNV in rats was markedly inhibited by intravitreous injection of TRO (P < 0.001). Lesions showed significantly less fluorescein leakage and were histologically thinner in the TRO-treated animals. Similar findings were present in the TRO-treated lesions in two monkey eyes. The drug showed no apparent adverse effects in the adjacent retina or in control eyes.
The inhibition of VEGF-induced choroidal angiogenesis in vitro, and CNV in vivo by PPAR-gamma ligands suggests the potential application of these agents in the large group of patients with age-related macular degeneration complicated by CNV.
Investigative Ophthalmology & Visual Science 08/2000; 41(8):2309-17. · 3.60 Impact Factor
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ABSTRACT: Tumor necrosis factor alpha (TNFalpha) interferes with insulin signaling in adipose tissue and may promote insulin resistance. Insulin binding to the insulin receptor (IR) triggers its autophosphorylation, resulting in phosphorylation of Shc and the downstream activation of p42/p44 extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase (ERK1/2), which mediates insulin-induced proliferation in vascular smooth muscle cells (VSMC). Since insulin resistance is a risk factor for vascular disease, we examined the effects of TNFalpha on mitogenic signaling by insulin. In rat aortic VSMC, insulin induced rapid phosphorylation of the IR and Shc and caused a 5.3-fold increase in activated, phosphorylated ERK1/2 at 10 min. Insulin induced a biphasic ERK1/2 activation with a transient peak at 10 min and a sustained late phase after 2 h. Preincubation (30-120 min) with TNFalpha had no effect on insulin-induced IR phosphorylation. In contrast, TNFalpha transiently suppressed insulin-induced ERK1/2 activation. Insulin-induced phosphorylation of Shc was inhibited by TNFalpha in a similar pattern. Since mitogenic signaling by insulin in VSMC requires ERK1/2 activation, we examined the effect of TNFalpha on insulin-induced proliferation. Insulin alone induced a 3.4-fold increase in DNA synthesis, which TNFalpha inhibited by 48%. TNFalpha alone was not mitogenic. Inhibition of ERK1/2 activation with PD98059 also inhibited insulin-stimulated DNA synthesis by 57%. TNFalpha did not inhibit platelet-derived growth factor-induced ERK1/2 activation or DNA synthesis in VSMC. Thus, TNFalpha selectively interferes with insulin-induced mitogenic signaling by inhibiting the phosphorylation of Shc and the downstream activation of ERK1/2.
Journal of Biological Chemistry 07/2000; 275(24):18279-83. · 4.77 Impact Factor
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ABSTRACT: Peroxisome proliferator-activated receptor gamma (PPARgamma) is a member of the nuclear receptor superfamily that is activated by binding certain fatty acids, eicosanoids, and insulin-sensitizing thiazolidinediones (TZD). The TZD troglitazone (TRO) inhibits vascular smooth muscle cell proliferation and migration both in vitro and in vivo. The precise mechanism of its antiproliferative activity, however, has not been elucidated. We report here that PPARgamma ligands inhibit rat aortic vascular smooth muscle cell proliferation by blocking the events critical for G(1) --> S progression. Flow cytometry demonstrated that both TRO and another TZD, rosiglitazone, prevented G(1) --> S progression induced by platelet-derived growth factor and insulin. Movement of cells from G(1) --> S was also inhibited by the non-TZD, natural PPARgamma ligand 15-deoxy-(12,14)Delta prostaglandin J(2) (15d-PGJ(2)), and the mitogen-activated protein kinase pathway inhibitor PD98059. Inhibition of G(1) --> S exit by these compounds was accompanied by a substantial blockade of retinoblastoma protein phosphorylation. TRO and rosiglitazone attenuated both the mitogen-induced degradation of p27(kip1) and the mitogenic induction of p21(cip1). 15d-PGJ(2) and PD98059 inhibited both the degradation of p27(kip1) and the induction of cyclin D1 in response to mitogens. These effects resulted in the inhibition of mitogenic stimulation of cyclin-dependent kinases activated by cyclins D1 and E. These data demonstrate that PPARgamma ligands are antiproliferative drugs that act by modulating cyclin-dependent kinase inhibitors; they may provide a new therapeutic approach for proliferative vascular diseases.
Journal of Biological Chemistry 07/2000; 275(29):22435-41. · 4.77 Impact Factor
