Phorbol ester effects on alpha 1-adrenoceptor binding and phosphatidylinositol metabolism in cultured vascular smooth muscle cells

Tumor promoting phorbol esters stimulate Ca++ phospholipid-dependent protein kinase C. It has been suggested that this enzyme regulates the functional properties of different cell membrane receptors. In this study we investigated the effect of phorbol esters on alpha 1-adrenoceptor binding and phosphatidylinositol metabolism in cultured smooth muscle cells derived from rabbit aorta. Treatment of these cells with biologically active phorbol esters for 15 min. to 2 hours caused a marked decrease of norepinephrine stimulation of inositol phospholipid metabolism and a 3 fold decrease in agonist affinity for 125I-HEAT binding to alpha 1-adrenoceptors in the intact smooth muscle cells. The ability of phorbol esters to modulate alpha 1-adrenoceptor responsiveness suggests that activation of protein kinase C may represent an important mechanism regulating alpha 1-adrenergic receptor functional properties.     

Juxtacrine effects of IL-1 alpha precursor promote iNOS expression in vascular smooth muscle cells

After injury to the blood vessel wall, vascular smooth muscle cells (SMC) synthesize interleukin (IL)-1 and inducible nitric oxide (NO) synthase (iNOS). The present study tested whether endogenous production of IL-1 alpha stimulates iNOS expression in vascular SMC, and assessed whether IL-1 alpha exerts autocrine effects on the cells producing IL-1 alpha or juxtacrine effects on cells that contact the IL-1 alpha producing cells. Rat aortic SMC were transiently transfected with expression plasmids encoding either IL-1 alpha precursor, which localizes to the plasma membrane, or mature IL-1 alpha, which remains cytosolic. iNOS mRNA levels, determined by RT-PCR, and production of nitrite, a stable oxidation product of NO, were markedly elevated in SMC overexpressing IL-1 alpha precursor, and modestly elevated in SMC overexpressing mature IL-1 alpha, relative to SMC transfected with vector alone. Exposure to exogenous IL-1 beta or TNF-alpha further stimulated iNOS gene expression in SMC producing IL-1 alpha; low levels of IL-1 beta (20 pg/ml) were effective in SMC transfected with IL-1 alpha precursor plasmid, whereas SMC transfected with mature IL-1 alpha plasmid or vector alone required higher concentrations of IL-1 beta (200 and 2,000 pg/ml, respectively). The increases in iNOS mRNA levels and NO production in SMC overexpressing IL-1 alpha precursor were prevented by exogenous IL-1 receptor antagonist, suggesting that these effects were mediated by the type I IL-1 receptor. Immunostaining studies indicated that IL-1 alpha precursor stimulates iNOS gene expression via cell-cell contact. Expression of iNOS was enhanced in cells that were in contact with a cell overexpressing IL-1 alpha precursor (identified by coexpression of green fluorescent protein), and in cells that were overexpressing IL-1 alpha themselves, but only when the cell contacted another cell. Together these results indicate that IL-1 alpha precursor acts by cell-cell contact as an autocrine and juxtacrine enhancer of iNOS gene expression, inducing moderate iNOS expression on its own, and markedly augmenting the responsiveness of rat aortic SMC to exogenous cytokines.     

Caveolin-1 is required for contractile phenotype expression by airway smooth muscle cells

Airway smooth muscle cells exhibit phenotype plasticity that underpins their ability to contribute both to acute bronchospasm and to the features of airway remodelling in chronic asthma. A feature of mature, contractile smooth muscle cells is the presence of abundant caveolae, plasma membrane invaginations that develop from the association of lipid rafts with caveolin-1, but the functional role of caveolae and caveolin-1 in smooth muscle phenotype plasticity is unknown. Here, we report a key role for caveolin-1 in promoting phenotype maturation of differentiated airway smooth muscle induced by transforming growth factor (TGF)-β(1). As assessed by Western analysis and laser scanning cytometry, caveolin-1 protein expression was selectively enriched in contractile phenotype airway myocytes. Treatment with TGF-β(1) induced profound increases in the contractile phenotype markers sm-α-actin and calponin in cells that also accumulated abundant caveolin-1; however, siRNA or shRNAi inhibition of caveolin-1 expression largely prevented the induction of these contractile phenotype marker proteins by TGF-β(1). The failure by TGF-β(1) to adequately induce the expression of these smooth muscle specific proteins was accompanied by a strongly impaired induction of eukaryotic initiation factor-4E binding protein(4E-BP)1 phosphorylation with caveolin-1 knockdown, indicating that caveolin-1 expression promotes TGF-β(1) signalling associated with myocyte maturation and hypertrophy. Furthermore, we observed increased expression of caveolin-1 within the airway smooth muscle bundle of guinea pigs repeatedly challenged with allergen, which was associated with increased contractile protein expression, thus providing in vivo evidence linking caveolin-1 expression with accumulation of contractile phenotype myocytes. Collectively, we identify a new function for caveolin-1 in controlling smooth muscle phenotype; this mechanism could contribute to allergic asthma.     

Postsynaptic alpha adrenoceptors on vascular smooth muscle

A heterogeneous population of alpha adrenoceptors mediates vasoconstriction in the canine saphenous vein (CSV). Studies with isolated strips of venous smooth muscle incubated with selective alpha-adrenoceptor agonists and antagonists revealed that both alpha 1 and alpha 2 adrenoceptors exist independently in this tissue and both subtypes mediate a contractile response. Measurement of contractile responses in reduced or zero external calcium conditions indicates that stimulation of alpha 1 adrenoceptors induces contractions by influx of extracellular calcium and release of calcium from internal stores. In contrast, 45Ca uptake studies suggest that activation of the postsynaptic alpha 2 adrenoceptor produces vasoconstriction dependent only on influx of extracellular calcium. The influx of calcium produced by the selective alpha 2-adrenoceptor agonist BHT-920 is inhibited by calcium entry blockers. Measurements of transmembrane potentials from smooth muscle cells of the CSV suggest that alpha 1-adrenoceptor activation produces depolarization and contraction (electromechanical coupling) whereas alpha 2-adrenoceptor stimulation does not result in concentration-dependent depolarization of the smooth muscle cells (pharmacomechanical coupling).     

Endothelin-1 decreases CD36 protein expression in vascular smooth muscle cells

Recent studies have shown that CD36 plays important roles as a major scavenger receptor for oxidized low-density lipoproteins and as a crucial transporter for long-chain fatty acids. CD36 deficiency might be associated with insulin resistance and abnormal dynamics of long-chain fatty acids. Endothelin-1 (ET-1), which is synthesized and secreted by vascular endothelial cells, is the most potent endogenous vasoconstrictor known and also stimulates the proliferation of vascular smooth muscle cells (VSMCs) and thus is believed to play an important role in the development of various circulatory disorders, including hypertension and atherosclerosis. The aim of the present study was to investigate the regulatory effect of ET-1 on CD36 expression in cultured VSMCs. VSMCs were treated for different times (0-24 h) with a fixed concentration (100 nM) of ET-1 or with different concentrations (0-100 nM) for a fixed time (24 h); then CD36 expression was determined using Western blots. CD36 expression was significantly decreased by ET in a time- and dose-dependent manner. This inhibitory effect was prevented by the ET(A) receptor antagonist BQ-610 (10 microM) but not the ET(B) receptor antagonist BQ-788 (10 microM). To further explore the underlying mechanisms of ET-1 action, we examined the involvement of the tyrosine kinase-mediated and MAPK-mediated pathways. The inhibitory effect of ET-1 on CD36 protein expression was blocked by inhibition of tyrosine kinase activation by use of genistein (100 microM) and by the ERK inhibitor PD-98059 (75 microM) but not by the p38 MAPK inhibitor SB-203580 (20 microM). In conclusion, we have demonstrated that ET-1, acting via the ET(A) receptor, suppresses CD36 protein expression in VSMCs by activation of the tyrosine kinase and ERK pathways.     

Tensile properties of fibroblasts and vascular smooth muscle cells

Tensile properties of fibroblasts (FBs) and vascular smooth muscle cells (VSMCs) of synthetic and contractile phenotypes were studied using a newly developed micro-tensile tester. FBs were obtained from the rabbit patellar tendon. Synthetic and contractile VSMCs were isolated from the rabbit thoracic aorta with an explant and an enzymatic digestion method, respectively. Each cell was attached to the fine tips of a pair of micropipettes with a cell adhesive and, then, stretched at the speed of 6 microm/sec. Load and length were obtained using a cantilever-type load cell and a VDA, respectively.FBs were broken at the load of 0.9 microN and the elongation to failure of 86 microm, and had the stiffness of 0.02 N/m. VSMCs were not broken even at 2.4 microN. The stiffness of synthetic and contractile VSMCs were 0.09 and 0.17 N/m, respectively. Such large different tensile properties among the three cells are attributable to the differences in components and cytoskeletal structures.     

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.     

Induction of vascular smooth muscle alpha-isoactin expression in BC3H1 cells

An isoactin analysis was performed on L-[35S]cysteine labeled BC3H1 cells to determine if these smooth muscle-like cells synthesize vascular smooth muscle actin. Three different NH2-terminal peptides were identified on thin layer electrophoretograms of DNase I-purified and trypsin-digested BC3H1 cell actin. Results obtained from secondary digestion with thermolysin or Staphylococcus aureus V8 protease showed that the most acidic NH2-terminal peptide was derived from vascular smooth muscle alpha-isoactin. Treatment of cell monolayers with serum-free medium caused a 3-fold increase in the level of alpha-isoactin expression and a concomitant decrease in the level of non-muscle beta- and gamma-isoactin. Cell-cell contact was required for induction of alpha-isoactin, and the effects of serum depletion on isoactin expression and cell growth were reversible. The intensity of about 11 out of 500 polypeptide spots on two-dimensional gels of BC3H1 cell polypeptides also was influenced by the culture conditions. The finding that smooth muscle isoactin expression was coupled to cell growth conditions indicate the potential usefulness of BC3H1 cells in studies of isoactin expression and utilization during vascular smooth muscle development.     

Regulation of SM22 alpha expression by arginine vasopressin and PDGF-BB in vascular smooth muscle cells

Vascular smooth muscle (SM) cells (VSMC) undergo phenotypic modulation in vivo and in vitro. This process involves coordinated changes in expression of multiple SM-specific genes. In cultured VSMC, arginine vasopressin (AVP) increases and PDGF decreases expression of SM alpha-actin (SMA), the earliest marker of SM cells (SMC). However, it is unknown whether these agents regulate other SM genes in a similar fashion. SM22 alpha appears secondary to SMA during development and is also a marker for SMC. This study examined the regulation of SM22 alpha expression by AVP and PDGF in cultured VSMC. Levels of SM22 alpha mRNA and protein were increased by AVP and suppressed by PDGF. Consistent with these changes, AVP increased SM22 alpha promoter activity, whereas PDGF inhibited basal promoter activity and blocked AVP-induced increase. Activation of both JNK and p38 MAPK pathways was necessary for AVP-mediated induction of SM22 alpha promoter. Expression of constitutively active Ras produced similar suppressions on SM22 alpha promoter activity as PDGF. Signaling relayed from PDGF/Ras activation involved Raf, or a protein that competes for this site, Ral-GDS, and phosphatidylinositol 3-kinase activation. Truncational analysis showed that the proximal location of three CArG boxes in the promoter was sufficient for AVP stimulation. Mutations in this CArG box reduced basal and AVP-stimulated promoter activity without effecting PDGF suppression. Overexpression of serum response factor enhanced basal and AVP-stimulated promoter activity but had no effect on PDGF-BB-induced suppression. These data indicate that AVP and PDGF initiate specific signaling pathways that control expression of multiple SM genes leading to phenotypic modulation.     

Functional expression of NOS 1 in vascular smooth muscle

Substances that increase intracellular calcium concentration ([Ca(2+)](i)), such as serotonin, are known to induce vascular smooth muscle (VSM) contraction. However, increases in [Ca(2+)](i) also activate Ca(2+)/calmodulin-dependent nitric oxide synthases (NOS), which leads to increases in cGMP and activation of cGMP-dependent protein kinase (PKG). One recently identified substrate protein of PKG is the small heat shock protein, HSP20. The purpose of this study was to determine if serotonin activates a Ca(2+)-dependent NOS in VSM. Strips of bovine carotid arterial smooth muscle denuded of endothelium were stimulated with serotonin in the presence and absence of the nonspecific NOS inhibitor N-monomethyl-L-arginine (L-NMMA). Activation of NOS was determined by increases in cGMP and in the phosphorylation of HSP20. Immunohistochemical and Western blotting techniques were performed to identify specific NOS isoforms in bovine carotid arterial smooth muscle preparations. Serotonin stimulation led to significant increases in cGMP and in the phosphorylation of HSP20, which were inhibited by pretreatment with L-NMMA. Antibodies against NOS 1 stained the media of bovine carotid and human renal arteries, whereas antibodies against NOS 3 stained only the endothelium. Additionally, the conversion of radiolabeled L-arginine to L-citrulline NOS activity demonstrated a consistent amount of activity present in the endothelium-denuded smooth muscle preparations that was reduced by 99% with an NOS 1 specific inhibitor. Finally, an NOS 1 specific inhibitor, 7-nitroindazole, augmented contractions induced by high extracellular KCl. This study demonstrates that NOS 1 is present in VSM and may effect physiological contractile responses.     

alpha 1-Proteinase inhibitor, alpha 1-antichymotrypsin, and alpha 2-macroglobulin are the antiapoptotic factors of vascular smooth muscle cells

Serum depletion induces cell death. Whereas serum contains growth factors and adhesion molecules that are important for survival, serum is also likely to have antiapoptotic factor(s). We show here that the plasma proteinase inhibitors alpha1-proteinase inhibitor, alpha1-antichymotrypsin, and alpha2-macroglobulin function as critical antiapoptotic factors for human vascular smooth muscle cells. Cell survival was assured when serum-free medium was supplemented with any one or all of the above serine proteinase inhibitors. In contrast, the cells were sensitive to apoptosis when cultured in medium containing serum from which the proteinase inhibitors were removed. The antiapoptotic effect conferred by the proteinase inhibitors was proportional to proteinase inhibitory activity. Without proteinase inhibitors, the extracellular matrix was degraded, and cells could not attach to the matrix. Cell survival was dependent on the intact extracellular matrix. In the presence of the caspase inhibitor z-VAD, the cells detached but did not die. The activity of caspases was elevated without proteinase inhibitors; in contrast, caspases were not activated when medium was supplemented with one of the proteinase inhibitors. In conclusion, the plasma proteinase inhibitors prevent degradation of extracellular matrix by proteinases derived from cells. Presumably an intact cell-matrix interaction inhibits caspase activation and supports cell survival.     

Platelet derived growth factor regulates ABCA1 expression in vascular smooth muscle cells

The ATP-binding cassette transporter A1 (ABCA1) regulates lipid efflux from peripheral cells to High-density lipoprotein. The platelet-derived growth factor (PDGF) is a potent mitogen that enables vascular smooth muscle cells to participate in atherosclerosis. In this report, we showed that PDGF suppressed endogenous expression of ABCA1 in cultured vascular smooth muscle cells. Exposure of CRL-208 cells to PDGF elicited a rapid phosphorylation of a kinase downstream from PI3-K, Akt. The constitutively active form of both p110, a subunit of PI3-K, and Akt inhibited activity of the ABCA1 promoter. In conclusion, PI3-K-Akt pathways participate in PDGF-suppression of ABCA1 expression.     

Glucose upregulates plasminogen activator inhibitor-1 gene expression in vascular smooth muscle cells

We investigated the effects of high concentrations of glucose on plasminogen activator inhibitor-1 (PAI-1) gene expression in cultured rat vascular smooth muscle cells (VSMC). In response to a high glucose concentration (27.5 mM), PAI-1 mRNA increased within 2 h, peaked at 4 h, remained elevated for another 4 h, then decreased to basal levels at 24 h. On the other hand, mannose at the same concentration (22.5 mM mannose plus 5.5 mM glucose) as an osmotic control had little effect on PAI-1 mRNA expression. The expression of PAI-1 mRNA that was also increased by H(2)O(2), angiotensin II, or phorbol myristate acetate, was reversed by the MAPK kinase (MEK) inhibitor PD98059 or the specific protein kinase C (PKC) inhibitor GF109203X. High glucose appeared to activate MAPK and PKC in VSMC judging from Elk-1 and AP-1 activation, respectively. PD98059 inhibited and GF109203X prevented subsequent PAI-1 induction by glucose. These results suggest that glucose at high concentrations induces PAI-1 gene expression in VSMC at least partially via MAPK and PKC activation. This direct effect of glucose might have important implications for the increased plasma concentrations of PAI-1 and possibly atherosclerosis that are associated with diabetes.     

Basic fibroblast growth factor regulates extracellular matrix and contractile protein expression independent of proliferation in vascular smooth muscle cells

Summary Basic fibroblast growth factor (bFGF) can influence proliferation and differentiation in vascular smooth muscle cells. Basic FGF promotes some features of the synthetic phenotype (proliferation) but is known to inhibit others (collagen synthesis). Whether bFGF availability influences smooth muscle cell phenotype independent of proliferation is not known. The purpose of this study was to determine if the effects of bFGF on extracellular matrix and contractile protein expression are dependent on changes in proliferation. Basic FGF availability was manipulated by adding bFGF to cultured cells or by inhibiting bFGF expression using antisense RNA, and adjusting culture conditions such that proliferation was held constant. Compared to cells cultured in serum alone, smooth muscle α-actin and myosin heavy chain expression was markedly reduced by added bFGF, but was not influenced by antisense inhibition of bFGF expression. Under the same conditions, collagen synthesis was inhibited by added bFGF, and was stimulated by reduced bFGF expression. These consequences of altering bFGF availability were not associated with changes in FGF receptor expression. These findings demonstrate that alterations in bFGF availability can regulate smooth muscle cell phenotype independent of proliferation, which may be related to the regulation of smooth muscle cell phenotype in vivo.     

Endothelin-1 induces the expression of C-reactive protein in rat vascular smooth muscle cells

Numerous studies have shown that both vasoconstrictive peptide endothelin-1 (ET-1) and inflammatory marker C-reactive protein (CRP) are implicated in the inflammatory process of atherosclerosis. The purpose of the present study was to observe effect of ET-1 on CRP production and the molecular mechanisms in rat vascular smooth muscle cells (VSMCs). The results showed that ET-1 was capable of stimulating VSMCs to produce CRP both in protein and in mRNA levels in vitro and in vivo. ET_A receptor antagonist BQ123, but not ET_B receptor antagonist BQ788, inhibited CRP production in VSMCs. In addition, ET-1 was able to elicit reactive oxygen species (ROS) generation and mitogen-activated protein kinase (MAPK) activation, and antioxidant pyrrolidine dithiocarbamate and p38MAPK inhibitor SB203580 inhibited ET-1-induced CRP expression. The results demonstrate that ET-1 induces CPR production in VSMCs via ET_A receptor followed by ROS and MAPK signal pathway, which may contribute to better understanding of the role of ET-1 in inflammatory activation of the vessel wall during atherogenesis.