Arginase promotes neointima formation in rat injured carotid arteries.

OBJECTIVE: Arginase stimulates the proliferation of cultured vascular smooth muscle cells (VSMCs); however, the influence of arginase on VSMC growth in vivo is not known. This study investigated the impact of arginase on cell cycle progression and neointima formation after experimental arterial injury. METHODS AND RESULTS: Balloon injury of rat carotid arteries resulted in a sustained increase in arginase activity in the vessel wall and the induction of arginase I protein in both the media and neointima of injured vessels. Furthermore, local perivascular application of the potent and selective arginase inhibitors S-(2-boronoethyl)-L-cysteine (BEC) or N(G)-hydroxy-nor-L-arginine (L-OHNA) immediately after injury markedly attenuated medial and neointimal DNA synthesis and neointima formation. Substantial arginase I protein and arginase activity was also detected in rat cultured aortic VSMCs. Moreover, treatment of VSMCs with BEC or L-OHNA, or knockdown of arginase I protein, arrested cells in the G(0)/G(1) phase of the cell cycle and induced the expression of the cyclin-dependent protein kinase inhibitor, p21. CONCLUSIONS: This study demonstrates that arginase is essential for VSMCs to enter the cell cycle and that arginase I contributes to the remodeling response after arterial injury. Arginase I represents a potentially new therapeutic target for the treatment of vasculoproliferative disorders.

Nitric oxide stimulates heme oxygenase-1 gene transcription via the Nrf2/ARE complex to promote vascular smooth muscle cell survival.

OBJECTIVE: Previous studies from our laboratory and others found that NO is a potent inducer of heme oxygenase-1 (HO-1) gene transcription in vascular smooth muscle cells (SMC), however, the mechanism responsible for the induction of HO-1 gene expression has not been elucidated. In the present study, we determined the signaling pathway responsible for the induction of HO-1 and its biological significance. METHODS: Cultured rat aortic SMC were exposed to nitrosative stress by treating cells with various NO donors or with inflammatory cytokines. RESULTS: Nitrosative stress stimulated an increase in HO-1 mRNA expression and promoter activity in vascular SMC. However, mutation of the antioxidant response element (ARE) in the HO-1 promoter or overexpression of a dominant-negative mutant of NF-E2-related factor-2 (Nrf2) abrogated the activation by NO. Electromobility shift assays using an ARE probe detected a complex that was significantly increased in intensity by NO. In addition, the migration of this complex was retarded by using an antibody directed against Nrf2. NO also increased Nrf2 mRNA expression, total and nuclear Nrf2 levels, and the binding of Nrf2 to the HO-1 promoter. Finally, treatment of SMC with NO stimulated apoptosis that was increased by HO-1 inhibition. CONCLUSIONS: These results demonstrate that nitrosative stress induces HO-1 gene transcription through the activation of the Nrf2/ARE complex to counteract NO-induced apoptosis of vascular SMC. The capacity of nitrosative stress to activate Nrf2 and stimulate HO-1 gene transcription may represent a critical adaptive response to maintain cell viability at sites of vascular inflammation and atherosclerosis.

Butylated hydroxyanisole stimulates heme oxygenase-1 gene expression and inhibits neointima formation in rat arteries.

OBJECTIVE: Butylated hydroxyanisole (BHA) is a synthetic phenolic compound that is a potent inducer of phase II genes. Since heme oxygenase-1 (HO-1) is a vasoprotective protein that is upregulated by phase II inducers, the present study examined the effects of BHA on HO-1 gene expression and vascular smooth muscle cell proliferation. METHODS: The regulation of HO-1 gene expression and vascular cell growth by BHA was studied in cultured rat aortic smooth muscle cells and in balloon injured rat carotid arteries. RESULTS: Treatment of cultured smooth muscle cells with BHA stimulated the expression of HO-1 protein, mRNA and promoter activity in a time- and concentration-dependent manner. BHA-mediated HO-1 expression was dependent on the activation of NF-E2-related factor-2 by p38 mitogen-activated protein kinase. BHA also inhibited cell cycle progression and DNA synthesis in an HO-1-dependent manner. In addition, the local perivascular delivery of BHA immediately after arterial injury of rat carotid arteries induced HO-1 protein expression and markedly attenuated neointima formation. CONCLUSIONS: These studies demonstrate that BHA stimulates HO-1 gene expression in vascular smooth muscle cells, and that the induction of HO-1 contributes to the antiproliferative actions of this phenolic antioxidant. BHA represents a potentially novel therapeutic agent in treating or preventing vasculoproliferative disease.

Single perivascular delivery of mitomycin C stimulates p21 expression and inhibits neointima formation in rat arteries.

OBJECTIVE: Mitomycin C (MMc) is an antibiotic that exerts a potent antiproliferative effect in tumor cells. Because the proliferation of vascular smooth muscle cells (VSMCs) plays a prominent role in the development of restenosis after percutaneous coronary interventions, the present study examined the effect of MMc on VSMC proliferation and on neointima formation after arterial balloon injury. METHODS AND RESULTS: Treatment of cultured rat aortic VSMCs with MMc (1 nmol to 30 micromol/L) inhibited VSMC proliferation in a concentration-dependent manner. Whereas high concentrations of MMc (1 to 30 micromol/L) induced VSMC apoptosis, as reflected by DNA laddering and caspase-3 activation, lower concentrations of MMc (1 to 300 nmol/L) directly inhibited VSMC growth by arresting cells in the G2/M phase of the cell cycle. The antiproliferative action of MMc was associated with a selective increase in the expression of the cyclin-dependent kinase inhibitor p21, and with a decrease in cyclin B1-cyclin-dependent kinase-1 complex activity. Finally, the local perivascular delivery of MMc immediately after balloon injury of rat carotid arteries induced p21 expression and markedly attenuated neointima formation. CONCLUSIONS: These studies demonstrate that MMc exerts a potent inhibitory effect on VSMC proliferation and neointima formation after arterial injury. MMc represents a potentially new therapeutic agent in treating and preventing vasculoproliferative disease.

Endoplasmic reticulum stress stimulates heme oxygenase-1 gene expression in vascular smooth muscle. Role in cell survival.

Heme oxygenase-1 (HO-1) is a cytoprotective protein that catalyzes the degradation of heme to biliverdin, iron, and carbon monoxide (CO). In the present study, we found that endoplasmic reticulum (ER) stress induced by a variety of experimental agents stimulated a time- and concentration-dependent increase in HO-1 mRNA and protein in vascular smooth muscle cells (SMC). The induction of HO-1 by ER stress was blocked by actinomycin D or cycloheximide and was independent of any changes in HO-1 mRNA stability. Luciferase reporter assays indicated that ER stress stimulated HO-1 promoter activity via the antioxidant response element. Moreover, ER stress induced the nuclear import of Nrf2 and the binding of Nrf2 to the HO-1 antioxidant response element. Interestingly, ER stress stimulated SMC apoptosis, as demonstrated by annexin V binding, caspase-3 activation, and DNA laddering. The induction of apoptosis by ER stress was potentiated by HO inhibition, whereas it was prevented by addition of HO substrate. In addition, exposure of SMC to exogenously administered CO inhibited ER stress-mediated apoptosis, and this was associated with a decrease in the expression of the proapoptotic protein, GADD153. In contrast, the other HO-1 products failed to block apoptosis or GADD153 expression during ER stress. These results demonstrated that ER stress is an inducer of HO-1 gene expression in vascular SMC and that HO-1-derived CO acts in an autocrine fashion to inhibit SMC apoptosis. The capacity of ER stress to stimulate the HO-1/CO system provides a novel mechanism by which this organelle regulates cell survival.

Cyclic strain stimulates L-proline transport in vascular smooth muscle cells.

BACKGROUND: The increase in vessel wall strain in hypertension contributes to arterial remodeling by stimulating vascular smooth muscle cell (SMC) proliferation and collagen synthesis. Because L-proline is essential for the synthesis of collagen and cell growth, we examined whether cyclic strain regulates the transcellular transport of L-proline by vascular SMC. METHODS: Cultured rat aortic SMCs were subjected to mechanical strain using the Flexercell 3000 Strain Unit. RESULTS: Cyclic strain increased L-proline transport in a time- and strain-degree-dependent manner that was inhibited by cycloheximide or actinomycin D. Kinetic studies indicated that cyclic strain-induced L-proline uptake was mediated by an increase in transport capacity independent of any change in the affinity for L-proline. Cyclic strain stimulated the expression of system A amino acid transporter 2 mRNA in a time-dependent fashion that paralleled the increase in L-proline transport. Cyclic strain also induced the release of transforming growth factor-beta1 in a time- and strain-dependent manner. Moreover, conditioned media from SMCs exposed to cyclic strain stimulated the transport of L-proline in control, static SMCs and this was significantly attenuated by a transforming growth factor-beta1 neutralizing antibody. CONCLUSIONS: These results demonstrate that cyclic strain stimulates L-proline transport by inducing system A amino acid transporter 2 gene expression through the autocrine release of transforming growth factor-beta1. The ability of cyclic strain to induce system A amino acid transporter 2 expression may promote arterial remodeling in hypertension by providing vascular SMCs with the necessary intracellular levels of L-proline required for collagen synthesis and cell growth.

Platelet-derived growth factor stimulates LAT1 gene expression in vascular smooth muscle: role in cell growth.

Platelet-derived growth factor (PDGF) contributes to vascular disease by stimulating the growth of vascular smooth muscle cells (SMCs). Since amino acids are required for cell growth, the present study examined the effect of PDGF on system L amino acid transport, which is the predominant cellular pathway for the uptake of essential amino acids. System L amino acid transport was monitored by measuring the uptake of L-leucine. Treatment of SMCs with PDGF stimulated L-leucine transport in a concentration- and time-dependent manner, and this was associated with a selective increase in LAT1 mRNA and protein. PDGF failed to induce the expression of the other system L transport proteins, LAT2 and the heavy chain of the 4F2 cell surface antigen. The induction of LAT1 by PDGF was dependent on de novo RNA and protein synthesis and on mTOR activity. Serum, thrombin, and angiotensin II likewise stimulated L-leucine transport by inducing LAT1 expression. Inhibition of system L amino acid transport by the model substrate 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid blocked growth factor-mediated SMC proliferation and induced SMC apoptosis, whereas it had no effect on quiescent cells. These results demonstrate that growth factors stimulate system L amino acid transport by inducing LAT1 gene expression and that system L amino acid transport is essential for SMC proliferation and survival. The capacity of vascular mitogens to induce LAT1 expression may represent a basic mechanism by which tho acid transport * apoptosis

Physiologic cyclic stretch inhibits apoptosis in vascular endothelium.

Since apoptosis of endothelial cells (ECs) plays an important role in the pathogenesis of atherosclerosis, we investigated the effect of cyclic stretch on EC apoptosis. Application of moderate, physiologic levels of cyclic stretch (6-10% at 1 Hz) inhibited EC apoptosis. This anti-apoptotic effect was dependent on the activation of phosphatidylinositol 3-kinase and associated with the activation of Akt and the phosphorylation of Bad. Interestingly, a higher potentially pathologic level of cyclic stretch (20% at 1 Hz) stimulated EC apoptosis. The ability of physiologic cyclic stretch to inhibit EC apoptosis may provide a previously unrecognized mechanism by which hemodynamic forces exert an anti-atherogenic effect.

Heme oxygenase-1-derived carbon monoxide is an autocrine inhibitor of vascular smooth muscle cell growth.

Vascular smooth muscle cells (SMCs) generate carbon monoxide (CO) via the catabolism of heme by the enzyme heme oxygenase (HO). In the present study, we found that serum stimulated a time- and concentration-dependent increase in the levels of HO-1 messenger RNA (mRNA) and protein in vascular SMCs. The induction of HO-1 expression by serum was inhibited by actinomycin D or cycloheximide. In addition, serum stimulated HO activity, as reflected by an increase in the concentration of bilirubin in the culture media. Treatment of vascular SMCs with serum stimulated DNA synthesis and this was potentiated by the HO inhibitors, zinc and tin protoporphyrin-IX as well as by the CO scavenger, hemoglobin. The iron chelator desferrioxamine had no effect on DNA synthesis. However, exposure of vascular SMCs to exogenous CO inhibited serum-stimulated SMC proliferation and the phosphorylation of retinoblastoma protein. In addition, CO arrested SMCs at the G(1)/S transition phase of the cell cycle and selectively blocked the serum-stimulated expression of cyclin A mRNA and protein without affecting the expression of cyclin D1 and E. CO also inhibited the serum-stimulated activation of cyclin A-associated kinase activity and cyclin-dependent kinase 2 activity. These results demonstrate that serum stimulates HO-1 gene expression and CO synthesis. Furthermore, they show that CO acts in a negative feedback fashion to inhibit vascular SMC growth by regulating specific components of the cell cycle machinery. The capacity of vascular mitogens to induce CO synthesis may provide a novel mechanism by which these agents modulate cell growth.