Regulation of vascular smooth muscle cell growth by aldose reductase

Aldose reductase (AR) is a broad-specificity aldo-keto reductase with wide species and tissue distribution. The enzyme has been implicated in the development of pleiotropic complications of long-term diabetes. However, the euglycemic function of the enzyme remains unclear. To examine its potential role in cell growth, changes in AR mRNA and protein were measured in human aortic smooth muscle cells exposed in culture to serum or thrombin. Stimulation by these mitogens led to an increase in the abundance of AR mRNA and protein. Furthermore, inhibition of the AR by tolrestat and sorbinil diminished DNA synthesis and cell proliferation in response to serum. Immunohistochemical staining with anti-AR antibodies revealed no significant expression of AR in the smooth muscle cells of rat carotid arteries. However, 10 and 21 days after balloon injury, intense staining was associated with the proliferating cells of the neointima. Treatment of these animals with 40 mg/kg/day sorbinil diminished the ratio of neointima to the media. Together, these observations suggest that, in vascular smooth muscle cells (VSMC), AR is a growth-responsive gene product and that inhibition of AR prevents VSMC growth and decreases intimal hyperplasia and restenosis.     

Differential regulation of Na+/H+ antiporter gene expression in vascular smooth muscle cells by hypertrophic and hyperplastic stimuli

The Na+/H+ antiporter is a ubiquitous transmembrane protein that plays a vital role in cell growth via regulation of intracellular Na+ and H+. In vascular smooth muscle cells (VSMC), vasoconstrictors and mitogens rapidly activate the antiporter, suggesting that both should have growth promoting effects. Indeed, angiotensin II increases VSMC protein and volume (hypertrophy), but does not increase cell number (hyperplasia). In the present work we investigated whether alterations in the steady state levels of Na+/H+ antiporter mRNA might differentiate these VSMC growth responses. Differences in function of the Na+/H+ antiporter appeared likely because exposure of growth-arrested VSMC for 24 h to 100 nM angiotensin II decreased intracellular pH from 7.08 to 6.99, while exposure to 10% calf serum caused an increase to 7.18. Simultaneous measurement of Na+/H+ antiporter mRNA levels, using the human c28 cDNA, revealed a 25-fold increase in response to serum (as well as to platelet-derived and fibroblast growth factors), but no change in response to angiotensin II. All agonists increased mRNA levels of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase approximately 3-fold. The increase in Na+/H+ antiporter mRNA induced by serum was first apparent within 2 h and peaked 24 h after treatment. These results demonstrate that expression of Na+/H+ antiporter mRNA in VSMC is dependent on growth state: hyperplastic agonists (serum, platelet-derived and fibroblast growth factor) increase the steady state levels of Na+/H+ antiporter mRNA while a hypertrophic agonist (angiotensin II) does not.     

Heparin suppresses specific second messenger pathways for protooncogene expression in rat vascular smooth muscle cells.

We investigated the molecular mechanisms underlying the ability of heparin to inhibit vascular smooth muscle cell (VSMC) growth. Previous experiments have shown that heparin inhibits induction of c-fos and c-myc protooncogene mRNA in rat VSMC stimulated by phorbol 12-myristate 13-acetate (PMA) but not when stimulated by epidermal growth factor (EGF) (Pukac, L. A., Castellot, J. J., Wright, T. C., Caleb, B. L., and Karnovsky, M. J. (1990) Cell Regul. 1, 435-443). The present experiments show that these mitogens activate distinct second messenger pathways in VSMC, because PMA but not EGF induction of c-fos and c-myc mRNA was suppressed in protein kinase C (PKC) down-regulated VSMC; this suggests that EGF does not act through a PKC-dependent pathway for induction of these genes. Heparin inhibited serum stimulation of c-fos mRNA in control VSMC, but heparin did not inhibit the smaller but significant serum stimulation of c-fos mRNA in PKC down-regulated VSMC, indicating that heparin may selectively inhibit PKC-dependent, but not PKC-independent, stimulation of gene expression. To further determine if heparin inhibits non-PKC pathways, VSMC were treated with dibutyryl cAMP, 3-isobutyl-1-methyl-xanthine, and Ca2+ ionophore A23187; stimulation of c-fos mRNA by this treatment was not inhibited by heparin. DNA synthesis and cell proliferation were inhibited in rat VSMC exposed briefly to heparin during the G0/G1 phase of the cell cycle. These experiments indicate heparin can act early in the cell cycle and suggest PKC-dependent but not PKC-independent signaling pathways for gene expression are selectively sensitive to heparin inhibition.     

Chronic insulin treatment amplifies PDGF-induced motility in differentiated aortic smooth muscle cells by suppressing the expression and function of PTP1B

Hyperinsulinemia plays a major role in the pathogenesis of vascular disease. Restenosis occurs at an accelerated rate in hyperinsulinemia and is dependent on increased vascular smooth muscle cell movement from media to neointima. PDGF plays a critical role in mediating neointima formation in models of vascular injury. We have reported that PDGF increases the levels of protein tyrosine phosphatase PTP1B and that PTP1B suppresses PDGF-induced motility in cultured cells and that it attenuates neointima formation in injured carotid arteries. Others have reported that insulin enhances the mitogenic and motogenic effects of PDGF in cultured smooth muscle cells and that hyperinsulinemia promotes vascular remodeling. In the present study, we tested the hypothesis that insulin amplifies PDGF-induced cell motility by suppressing the expression and function of PTP1B. We found that chronic but not acute treatment of cells with insulin enhances PDGF-induced motility in differentiated cultured primary rat aortic smooth muscle cells and that it suppresses PDGF-induced upregulation of PTP1B protein. Moreover, insulin suppresses PDGF-induced upregulation of PTP1B mRNA levels, PTP1B enzyme activity, and binding of PTP1B to the PDGF receptor-beta, and it enhances PDGF-induced PDGF receptor phosphotyrosylation. Treatment with insulin induces time-dependent upregulation of phosphatidylinositol 3-kinase (PI3-kinase)-delta and activation of Akt, an enzyme downstream of PI3-kinase. Finally, inhibition of PI3-kinase activity, or its function, by pharmacological or genetic means rescues PTP1B activity in insulin-treated cells. These observations uncover novel mechanisms that explain how insulin amplifies the motogenic capacity of the pivotal growth factor PDGF.     

Cyclic strain modulates resistance to oxidant stress by increasing G6PDH expression in smooth muscle cells

Vascular smooth muscle cells (VSMC) may be subjected to mechanical forces, such as cyclic strain, that promote the formation of reactive oxygen species (ROS). We hypothesized that VSMC modulate this adverse milieu by increasing the expression of glucose-6-phosphate dehydrogenase (G6PDH) to maintain or restore intracellular glutathione (GSH) levels. Cyclic strain increased superoxide formation, which resulted in diminished GSH because of an increase in oxidized glutathione formation; there was also an increase in glutathione peroxidase and glutathione reductase activities. G6PDH activity and protein expression were enhanced concomitant with decreases in GSH levels and remained elevated until intracellular GSH levels were restored. To confirm the role of G6PDH in repleting GSH stores, we inhibited G6PDH activity with DHEA or inhibited enzyme expression with an antisense oligodeoxynucleotide. Diminished G6PDH activity or expression was associated with persistently depleted GSH levels and inhibition of the cyclic strain-mediated increase in glutathione reductase activity. These observations demonstrate that cyclic strain promotes oxidant stress in VSMC, which, in turn, induces G6PDH expression. When G6PDH is inhibited, GSH levels are not restored because of impaired glutathione reductase activity. These data suggest that G6PDH is a critical determinant of the response to oxidant stress in VSMC.     

USP10 exacerbates neointima formation by stabilizing Skp2 protein in vascular smooth muscle cells

The underlying mechanism of neointima formation remains unclear. Ubiquitin-specific peptidase 10 (USP10) is a deubiquitinase that plays a major role in cancer development and progression. However, the function of USP10 in arterial restenosis is unknown. Herein, USP10 expression was detected in mouse arteries and increased after carotid ligation. The inhibition of USP10 exhibited thinner neointima in the model of mouse carotid ligation. In vitro data showed that USP10 deficiency reduced proliferation and migration of rat thoracic aorta smooth muscle cells (A7r5) and human aortic smooth muscle cells (HASMCs). Mechanically, USP10 can bind to Skp2 and stabilize its protein level by removing polyubiquitin on Skp2 in the cytoplasm. The overexpression of Skp2 abrogated cell cycle arrest induced by USP10 inhibition. Overall, the current study demonstrated that USP10 is involved in vascular remodeling by directly promoting VSMC proliferation and migration via stabilization of Skp2 protein expression.     

Ceramide-induced inhibition of Akt is mediated through protein kinase Czeta: implications for growth arrest.

We recently demonstrated that ceramide-coated balloon catheters limit vascular smooth muscle cell (VSMC) growth after stretch injury in vivo. In that study, inhibition of VSMC growth was correlated with a decrease in phosphorylation of the cell survival kinase Akt (protein kinase B). Utilizing cultured A7r5 VSMCs, we have now examined the mechanism by which ceramide inhibits Akt phosphorylation/activation. Our initial studies showed that ceramide-induced inhibition of Akt phosphorylation was not mediated through diminution in phosphoinositide 3-kinase activity. As we have previously demonstrated that protein kinase Czeta (PKCzeta) is a target of ceramide, we proposed an alternative signaling mechanism by which ceramide induces inhibition of Akt through activation of PKCzeta. We demonstrate that C(6)-ceramide (but not the inactive analog dihydro-C(6)-ceramide) induced PKCzeta activity and also caused a selective increase in the association between Akt and PKCzeta, without affecting PKCepsilon, in A7r5 cells. In addition, the ability of ceramide to significantly decrease platelet-derived growth factor-induced Akt phosphorylation or cell proliferation was abrogated in A7r5 cells overexpressing a dominant-negative mutant of PKCzeta. Taken together, these data suggest that ceramide-mediated activation of PKCzeta leads to diminished Akt activation and consequent growth arrest in VSMCs. The therapeutic potential for ceramide to limit dysregulated VSMC growth has direct applicability to vascular diseases such as restenosis and atherosclerosis.     

MAPKK-dependent growth factor-induced upregulation of P2Y2 receptors in vascular smooth muscle cells.

The ATP- and UTP-sensitive P2Y2 receptor which mediates both contractile and mitogenic effects has recently been shown to be upregulated in the synthetic phenotype of the vascular smooth muscle cell (VSMC). Using a competitive RT-PCR we demonstrate that the P2Y2 receptor mRNA is increased by fetal calf serum and other growth factors in a MAPKK-dependent way. This was confirmed at the functional level by examining UTP-stimulated release of intracellular Ca2+. Furthermore, the P2Y2 receptor mRNA is positively autoregulated by ATP and the mRNA is rapidly degraded with only 26% remaining after 1 h in the presence of actinomycin D. Our results indicate growth factor regulation and rapid turnover of the P2Y2 receptor mRNA, which may be of importance in atherosclerosis and neointima formation after balloon angioplasty.     

Metabolic regulation of aldose reductase activity by nitric oxide donors

Regulation of aldose reductase (AR), a member of the aldo-keto reductase superfamily, by nitric oxide (NO) donors was examined. Incubation of human recombinant AR with S-nitrosoglutathione (GSNO) led to inactivation of the enzyme and the formation of an AR-glutathione adduct. In contrast, incubation with S-nitroso-N-acetyl penicillamine (SNAP) or N-(beta-D-glucopyranosyl)-SNAP (GlycoSNAP) led to an increase in enzyme activity which was accompanied by the direct nitrosation of the enzyme and the formation of a mixed disulfide with the NO-donor. To examine in vivo modification, red blood cells (RBC) and rat aortic vascular smooth muscle cells (VSMC) were incubated with 1 mM GSNO or SNAP. Exposure of VSMC to SNAP and GSNO for 2 h at 37 degrees C led to approximately 71% decrease in the enzyme activity with DL-glyceraldehyde as the substrate. Similarly, exposure of RBC in 5 mM glucose to NO-donors for 30 min at room temperature, followed by increasing the glucose concentration to 40 mM, resulted in >75% decrease in the formation of sorbitol. These investigations indicate that NO and/or its bioactive metabolites can regulate cellular AR, leading to either activation (by nitrosation) or inactivation (by S-thiolation).     

C-type natriuretic peptide for reduction of restenosis: gene transfer is superior over single peptide administration

BACKGROUND: Restenosis is still a significant clinical problem limiting the long-term therapeutic success following balloon dilation or stent implantation. New approaches are necessary inhibiting neointima formation and simultaneously promoting re-endothelialization. Therefore, long-term therapeutic effects of adventitial liposome-mediated C-type natriuretic protein (CNP) gene and CNP peptide applications in a porcine model for restenosis post-angioplasty were investigated. METHODS: For in vitro applications, primary cultures of porcine vascular smooth muscle cells (VSMCs) and endothelial cells (ECs) were used. Gene transfer was performed with cationic lipid DOCSPER [1,3-dioleoyloxy-2-(N5-carbamoylspermine)propane]. In vivo treatment of pig femoral arteries was adventitial using a needle injection catheter following balloon angioplasty. Arteries were investigated by angiography, Evan's blue staining, histomorphometry, immunohistochemistry, PCR and RT-PCR. RESULTS: Using CNP gene transfer in vitro, 29.4+/-7.2% reduction of cell proliferation in VSMCs was observed. In ECs, the CNP gene did not compromise cellular growth. For the CNP peptide the optimal concentration was 1 mM with 50.7+/-11.3% reduction of VSMC proliferation and 12.1+/-5.3% enhancement of growth of ECs. Three weeks following application in vivo complete re-endothelialization was observed in all treated groups. At 3 months significant reduction of neointima formation was observed using CNP gene vs. CNP peptide (85.9+/-7.8% vs. 63.3+/-27.6% reduction, P<0.05) compared to control treatment. CONCLUSION: Periadventitial liposome-mediated CNP gene transfer in vivo resulted in a significant long-term reduction of neointima formation without compromising endothelial repair and was superior over single CNP peptide administration. Advantages of CNP are its physiological origin and simultaneous inhibition of VSMC proliferation and promotion of EC growth.     

c-Ski inhibits the proliferation of vascular smooth muscle cells via suppressing Smad3 signaling but stimulating p38 pathway

Proliferation of vascular smooth muscle cells (VSMCs) plays key roles in the progression of intimal hyperplasia, but the molecular mechanisms that trigger VSMC proliferation after vascular injury remain unclear. c-Ski, a co-repressor of transforming growth factor β (TGF-β)/Smad signaling, was detected to express in VSMC of rat artery. During the course of arterial VSMC proliferation induced by balloon injury in rat, the endogenous protein expressions of c-Ski decreased markedly in a time-dependent manner. In vivo c-Ski gene delivery was found to significantly suppress balloon injury-induced VSMC proliferation and neointima formation. Further investigation in A10 rat aortic smooth muscle cells demonstrated that overexpression of c-Ski gene inhibited TGF-β1 (1 ng/ml)-induced A10 cell proliferation while knockdown of c-Ski by RNAi enhanced the stimulatory effect of TGF-β1 on A10 cell growth. Western blot for signaling detection showed that suppression of Smad3 phosphorylation while stimulating p38 signaling associated with upregulation of cyclin-dependent kinase inhibitors p21 and p27 was responsible for the inhibitory effect of c-Ski on TGF-β1-induced VSMC proliferation. These data suggest that the decrease of endogenous c-Ski expression is implicated in the progression of VSMC proliferation after arterial injury and c-Ski administration represents a promising role for treating intimal hyperplasia via inhibiting the proliferation of VSMC.