Endothelin stimulates c-fos and c-myc expression and proliferation of vascular smooth muscle cells

Recently, a potent vasoconstrictor peptide, endothelin (EDT), was isolated from vascular endothelial cells. We examined its effect on rat vascular smooth muscle cells (VSMCs). EDT induced the elevation of intracellular calcium, which was dependent on extracellular calcium and inhibited by a calcium-channel antagonist in a competitive manner. EDT caused a rapid and transient increase in the c-fos and c-myc mRNA levels and stimulated the DNA synthesis of VSMCs in a dose-dependent manner. This effect of EDT on the proliferation of VSMCs might be related to the development of atherosclerosis.     

Interleukin-6 stimulates c-myc expression and proliferation of cultured vascular smooth muscle cells

The effects of interleukin-6 (IL-6), a cytokine recently found to be secreted by monocytes and macrophages, on c-myc expression and proliferation of cultured vascular smooth muscle cells (VSMC) were investigated. Treatment with IL-6 caused rapid increase in the c-myc mRNA level of VSMC. It also stimulated DNA synthesis and proliferation of the cells significantly and dose-dependently at concentrations of more than 10 U/ml. These results suggest that IL-6 may be important in the proliferation of VSMC, which is a key event in the development of arteriosclerosis, as a factor mediating immune cell-VSMC interaction.     

Heparin increases mRNA levels of thrombospondin but not fibronectin in human vascular smooth muscle cells

The effects of heparin (180 micrograms/ml) on steady state mRNA levels for fibronectin, thrombospondin, actin and collagen types I and III were investigated in human umbilical artery smooth muscle cells. Heparin caused a 120% increase in thrombospondin mRNA levels and a 60% and 180% increase in the mRNA levels of procollagen chains alpha 2(I) and alpha 1(III), respectively. No change in fibronectin or actin mRNA levels resulted from heparin treatment. We reported earlier (Biochem. Biophys. Res. Comm. 148:1264, 1987) that heparin increases smooth muscle cell synthesis of both fibronectin and thrombospondin. These data show that heparin coordinately regulates thrombospondin mRNA and protein levels. The heparin induced increase in fibronectin biosynthesis apparently reflects control at the translational or post-translational level.     

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.     

Heparin inhibits proliferation of fetal vascular smooth muscle cells in the absence of platelet-derived growth factor

Proliferation of smooth muscle cells from the pulmonary arteries and aortas of fetal calves is inhibited by heparin in vitro. This effect is reversible and dose dependent. Comparisons with effects of other polysaccharides indicate that only extensively sulfated polysaccharides inhibit proliferation of smooth muscle cells but that specific structural features of heparin are required to achieve maximum effect. Heparin-Sepharose chromatography of medium containing fetal calf serum reduces the ability of that medium to promote growth of smooth muscle cells from fetal pulmonary arteries, suggesting that heparin may remove soluble growth factors in serum. However, inhibition of fetal pulmonary artery smooth muscle cell proliferation by heparin is identical in media supplemented either with serum prepared from fetal calf plasma, in which platelet-derived growth factor (PDGF) is not detectable, or with fetal calf serum, which contains relatively abundant PDGF (114 pg/ml). Thus, inhibition of fetal pulmonary artery smooth muscle cell proliferation by heparin is not mediated solely by decreased availability or activity of exogenous PDGF. These studies suggest that morphogenesis of the smooth muscle investment of the pulmonary arteries could be regulated by local production of heparin-like inhibitors of smooth muscle cell growth.     

Heparin interactions with cultured human vascular endothelial and smooth muscle cells: incidence on vascular smooth muscle cell proliferation

The binding, internalization, and metabolism of [3H]-heparin by human umbilical vein endothelial cells (HUVEC) and human umbilical arterial smooth muscle cells (HUASMC) have been characterized using size-exclusion HPLC. Incubation of HUVEC with [3H]-heparin demonstrated selective binding of high-molecular-weight (MW) components (MW = 21 kd), which was followed by rapid, temperature-dependent internalization. Over the next 3 hours, this internalized [3H]-heparin was degraded to low-MW fragments (MW = 0.9 kd). Primary cultures of HUASMC selectively bound extremely high-MW components (MW = 40 kd) and also smaller components whose MW (0.9 kd) corresponded to that of the heparin metabolite(s) formed by HUVEC. Subcultured HUASMC bound only the 40-kd components. Internalization of heparin by smooth muscle cells (SMC) was significantly slower than that determined for HUVEC, and even after 4 hours there was no evidence of the heparin being metabolized. However, when incubating primary rabbit aortic SMC with purified low-MW heparin fragment(s) produced in culture by HUVEC, a significantly lower proliferative response of these cells (IC50 = 18.4 micrograms/ml) was obtained. Virtually no effect was observed with subcultured SMC in the range of the tested concentrations (0-20 micrograms/ml). These fragments were 10- to 15-fold more effective in inhibiting primary SMC growth than was standard heparin. Furthermore, heparin fractions in the same range of molecular weights, purified either after nitrous acid or heparinase depolymerization of standard heparin, showed no activity on primary SMC growth, thus indicating a high degree of selectivity of the heparin metabolite(s) produced by HUVEC in culture.     

Ectopic expression of Smad7 inhibits transforming growth factor-beta responses in vascular smooth muscle cells.

Vascular injury stimulates the cytokine-growth factor network in the vascular wall, including transforming growth factor-beta (TGF-beta). Reportedly, the intracellular signaling of TGF-beta is mediated by Smad proteins. We tested the effects of the ectopic expression of inhibitory Smads in cultured rat smooth muscle cells (SMC) to identify the role of TGF-beta/Smad signaling on the phenotypic modulation of SMC. The cells exposed to human recombinant TGF-beta1 (10 ng/ml) were stimulated Smad2 phosphorylation. Infection with the replication-deficient adenovirus vector expressing Smad7, but not bacterial beta-galactosidase or Smad6, was found to inhibit TGF-beta-induced Smad2 phosphorylation in a dose-dependent manner. TGF-beta suppressed the serum-induced proliferation of SMC from 36.3% to 51.0% (p<0.01), as measured by hand-counting, and this inhibition was attenuated by the ectopic expression of Smad7 (from 30.7% to 74.8% of the reduction of TGF-beta-response, p<0.05), but not Smad6. A BrdU incorporation assay also showed that TGF-beta-mediated growth inhibition was attenuated by exogenous Smad7 and that this inhibition can be reversed by an additional expression of exogenous Smad2. TGF-beta increased the expression of alpha-smooth muscle actin and myosin heavy chain by 1.3-fold and 1.6-fold in comparison to the control, respectively, and these increases were attenuated by exogenous Smad7, but not Smad6. Our data indicate that Smads mediate TGF-beta responses on SMC phenotypes. Smad7, but not Smad6, may specifically act as an inhibitor of TGF-beta responses.     

Hydrogen peroxide inhibits insulin signaling in vascular smooth muscle cells

Both insulin resistance and reactive oxygen species (ROS) have been reported to play essential pathophysiological roles in cardiovascular diseases, such as hypertension and atherosclerosis. However, the mechanistic link between ROS, such as H2O2 and insulin resistance in the vasculature, remains undetermined. Akt, a Ser/Thr kinase, mediates various biological responses induced by insulin. In this study, we examined the effects of H2O2 on Akt activation in the insulin-signaling pathway in vascular smooth muscle cells (VSMCs). In VSMCs, insulin stimulates Akt phosphorylation at Ser473. Pretreatment with H2O2 concentration- and time-dependently inhibited insulin-induced Akt phosphorylation with significant inhibition observed at 50 microM for 10 min. A ROS inducer, diamide, also inhibited insulin-induced Akt phosphorylation. In addition, H2O2 inhibited insulin receptor binding partially and inhibited insulin receptor autophosphorylation almost completely. However, pretreatment with a protein kinase C inhibitor, GF109203X (2 microM), for 30 min did not block the inhibitory effects of H2O2 on insulin-induced Akt phosphorylation, suggesting that protein kinase C is not involved in the inhibition by H2O2. We conclude that ROS inhibit a critical insulin signal transduction component required for Akt activation in VSMCs, suggesting potential cellular mechanisms of insulin resistance, which would require verification in vivo.     

Rho expression and activation in vascular smooth muscle cells

Phenotypic modulation of smooth muscle cells (SMC) involves dramatic changes in expression and organization of contractile and cytoskeletal proteins, but little is known of how this process is regulated. The present study used a cell culture model to investigate the possible involvement of RhoA, a known regulator of the actin cytoskeleton. In rabbit aortic SMC seeded into primary culture at moderate density, Rho activation was high at two functionally distinct time-points, first as cells modulated to the synthetic phenotype, and again upon confluence and return to the contractile phenotype. Rho expression increased with time, such that maximal expression occurred upon return to the contractile state. Transient transfection of synthetic state cells with constitutively active RhoA (Val14RhoA) caused a reduction in cell size and reorganization of cytoskeletal proteins to resemble that of the contractile phenotype. Actin and myosin filaments were tightly packed and highly organised while vimentin localised to the perinuclear region; focal adhesions were enlarged and concentrated at the cell periphery. Conversely, inhibition of endogenous Rho by C3 exoenzyme resulted in complete loss of contractile filaments without affecting vimentin distribution; focal adhesions were reduced in size and number. Treatment of synthetic state SMC with known regulators of SMC phenotype, heparin and thrombin, caused a modest increase in Rho activation. Long-term confluence and serum deprivation induced cells to return to a more contractile phenotype and this was augmented by heparin and thrombin. The results implicate RhoA for a role in regulating SMC phenotype and further show that activation of Rho by heparin and thrombin correlates with the ability of these factors to promote the contractile phenotype.     

NONOates regulate KCl cotransporter-1 and -3 mRNA expression in vascular smooth muscle cells

Nitric oxide (NO) donors regulate KCl cotransport (KCC) activity and cotransporter-1 and -3 (KCC1 and KCC3) mRNA expression in sheep erythrocytes and in primary cultures of rat vascular smooth muscle cells (VSMCs), respectively. In this study, we used NONOates as rapid and slow NO releasers to provide direct evidence implicating NO as a regulator of KCC3 gene expression at the mRNA level. In addition, we used the expression of KCC3 mRNA to further investigate the mechanism of action of these NO donors at the cellular level. Treatment of VSMCs with rapid NO releasers, like NOC-5 and NOC-9, as well as with the direct NO-independent soluble guanylyl cyclase (sGC) stimulator YC-1, acutely increased KCC3 mRNA expression in a concentration- and time-dependent manner. The slow NO releaser NOC-18 had no effect on KCC3 gene expression. A specific NO scavenger completely prevented the NONOate-induced KCC3 mRNA expression. Inhibition of sGC with LY-83583 blocked the NONOate- and YC-1-induced KCC3 mRNA expression. This study shows that in primary cultures of rat VSMCs, the fast NO releasers NOC-9 and NOC-5, but not the slow NO releaser NOC-18, acutely upregulate KCC3 mRNA expression in a NO/sGC-dependent manner.     

Heparin rapidly and selectively regulates protein tyrosine phosphorylation in vascular smooth muscle cells

Aberrant vascular smooth muscle cell (VSMC) hyperplasia is the hallmark of atherosclerosis and restenosis seen after vascular surgery. Heparin inhibits VSMC proliferation in animal models and in cell culture. To test our hypothesis that heparin mediates its antiproliferative effect by altering phosphorylation of key mitogenic signaling proteins in VSMC, we examined tyrosine phosphorylation of cellular proteins in quiescent VSMC stimulated with serum in the presence or absence of heparin. Western blot analysis with anti-phosphotyrosine antibodies shows that heparin specifically alters the tyrosine phosphorylation of only two proteins (42 kDa and 200 kDa). The 200 kDa protein (p200) is dephosphorylated within 2.5 min after heparin treatment with an IC50 that closely parallels the IC50 for growth inhibition. Studies using the tyrosine phosphatase inhibitor, sodium orthovanadate, indicate that heparin blocks p200 phosphorylation by inhibiting a kinase. Phosphorylation of p200 is not altered in heparin-resistant cells, supporting a role for p200 in mediating the antiproliferative effect of heparin. Purification and sequence analysis indicate that p200 exhibits very high homology to the heavy chain of nonmuscle myosin IIA. The 42 kDa protein, identified as mitogen activated protein kinase (MAPK), undergoes dephosphorylation within 15 min after heparin treatment, and this effect is also not seen in heparin-resistant cells. The identification of only two heparin-regulated tyrosine phosphoproteins suggests that they may be key mediators of the antiproliferative effect of heparin.     

Ribozyme to human TGF-beta1 mRNA inhibits the proliferation of human vascular smooth muscle cells

Transforming growth factor-beta (TGF-beta) has been reported to be involved in the pathogenesis of cardiovascular proliferative diseases such as hypertensive vascular disease, atherosclerosis, and arterial restenosis after angioplasty. We designed a 38-base DNA-RNA chimeric hammerhead ribozyme to cleave human TGF-beta1 mRNA as a gene therapy for human arterial proliferative diseases. In the presence of MgCl(2), synthetic ribozyme to human TGF-beta1 mRNA cleaved the synthetic target RNA into two RNA fragments of predicted size. A control mismatch ribozyme, with one different base in the catalytic loop region, was inactive. DNA-RNA chimeric ribozyme (0. 01-1.0 microM) significantly inhibited angiotensin II (Ang II)-stimulated DNA synthesis in a dose-dependent manner in human vascular smooth muscle cells (VSMC). The mismatch ribozyme did not affect Ang II-stimulated DNA synthesis in the cells. DNA-RNA chimeric ribozyme (1.0 microM) inhibited the proliferation of human VSMC in the presence of Ang II. DNA-RNA chimeric ribozyme (1.0 microM) significantly inhibited Ang II-stimulated TGF-beta1 mRNA and protein expression in human VSMC. These results indicate that the designed DNA-RNA chimeric hammerhead ribozyme targeted to human TGF-beta1 mRNA can effectively and potentially inhibit growth of human VSMC by cleaving the TGF-beta1 mRNA. This finding suggests that this ribozyme will be useful in the gene therapy of arterial proliferative diseases.     

TNFalpha inhibits insulin's antiapoptotic signaling in vascular smooth muscle cells

Mutations in TIGR/MYOC (myocilin), a secretory protein of unknown function, have been recently linked to glaucoma. Most known mutations map to the C-terminus, an olfactomedin-like domain. We have previously shown that, in contrast to the wild-type, a truncated form of myocilin lacking the olfactomedin domain is not secreted. In this study, we present evidence that the mutant protein is not correctly processed in the endoplasmic reticulum (ER) and accumulates into insoluble aggregates. In addition, we show that the presence of increasing amounts of mutant protein induces a fraction of the soluble, native myocilin to move to the insoluble fraction. Given the importance of such protein aggregates in the etiology of several aging-related diseases, we propose that olfactomedin-defective mutants might contribute to the pathology of glaucoma through a mechanism involving intracellular accumulation of misfolded proteins.     

Protein kinase C inhibits insulin-induced Akt activation in vascular smooth muscle cells.

Protein kinase C (PKC) activation, enhanced by hyperglycemia, is associated with many tissue abnormalities observed in diabetes. Akt is a serine/threonine kinase that mediates various biological responses induced by insulin. We hypothesized that the negative regulation of Akt in the vasculature by PKC could contribute to insulin resistant states and, may therefore play a role in the pathogenesis of cardiovascular disease. In this study, we specifically looked at the ability of PKC to inhibit Akt activation induced by insulin in cultured rat aortic vascular smooth muscle cells (VSMCs). Activation of Akt was determined by immunoblotting with a phospho-Akt antibody that selectively recognizes Ser473 phosphorylated Akt. A PKC activator, phorbol 12-myristate 13-acetate (PMA), inhibited insulin-dependent Akt phosphorylation. However, PMA did not inhibit platelet-derived growth factor (PDGF)-induced activation of Akt. We further showed that the PKC inhibitor, G06983, blocked the PMA-induced inhibition of Akt phosphorylation by insulin. In addition, we demonstrated that PMA inhibited the insulin-induced tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1). From these data, we conclude that PKC is a potent negative regulator of the insulin signal in the vasculature, which indicate an important role of PKC in the development of insulin resistance in cardiovascular disease.