Cell cycle versus density dependence of smooth muscle alpha actin expression in cultured rat aortic smooth muscle cells

Cultured smooth muscle cells (SMC) undergo induction of smooth muscle (SM) alpha actin at confluency. Since confluent cells exhibit contact inhibition of growth, this finding suggests that induction of SM alpha actin may be associated with cell cycle withdrawal. This issue was further examined in the present study using fluorescence-activated cell sorting of SMC undergoing induction at confluency and by examination of the effects of FBS and platelet-derived growth factor (PDGF) on SM alpha actin expression in postconfluent SMC cultures that had already undergone induction. Cell sorting was based on DNA content or differential incorporation of bromodeoxyuridine (Budr). The fractional synthesis of SM alpha actin in confluent cells was increased two- to threefold compared with subconfluent log phase cells, but no differences were observed between confluent cycling (Budr+) and noncycling (Budr-) cells. In cultures not exposed to Budr, confluent cycling S + G2 cells exhibited similar induction. These data indicate that cell cycle withdrawal is not a prerequisite for the induction of SM alpha actin synthesis in SMC at confluency. Growth stimulation of postconfluent cultures with either FBS or PDGF resulted in marked repression of SM alpha actin synthesis but the level of repression was not directly related to entry into S phase in that PDGF was a more potent repressor of SM alpha actin synthesis than was FBS despite a lesser mitogenic effect. This differential effect of FBS versus PDGF did not appear to be due to transforming growth factor-beta present in FBS since addition of transforming growth factor-beta had no effect on PDGF-induced repression. Likewise, FBS (0.1-10.0%) failed to inhibit PDGF-induced repression. Taken together these data demonstrate that factors other than replicative frequency govern differentiation of cultured SMC and suggest that an important function of potent growth factors such as PDGF may be the repression of muscle-specific characteristics.     

ATP-induced calcium transient in cultured rat aortic smooth muscle cells

To characterize the excitatory purinoceptors in vascular smooth muscle cells and the biochemical mechanisms of their actions, the effects of ATP and other nucleotides on Ca2+ mobilization in cultured smooth muscle cells mainly from rat aorta were investigated. ATP induced a transient and dose-dependent increase in the cytosolic Ca2+ concentration. ATP also induced a rapid production of inositol trisphosphate (IP3). The agonist form of ATP was metal-free ATP and its half-maximal effect was obtained at about 0.1 microM. 4-beta-Phorbol 12-myristate 13-acetate (PMA) or 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) inhibited both Ca2+ response and IP3 production. In addition, TMB-8 but not PMA, significantly decreased the amount of releasable Ca2+ presumably in the sarcoplasmic reticulum. Pertussis toxin also inhibited the Ca2+ response. Based on the dose-dependent effects of various nucleotides and adenosine on the Ca2+ response, it was concluded that the P2 subclass of purinoceptor is involved in the observed ATP effects. In addition, the observed absence or very weak effect of alpha, beta-methylene ATP relative to the effect of ATP suggests that the excitatory P2-purinoceptors in vascular smooth muscle cells do not form a homogeneous group, because the opposite order of potency for these two nucleotides was reported previously for the P2 purinoceptors involved in contraction of some isolated blood vessels.     

Homocysteine potentiates calcification of cultured rat aortic smooth muscle cells

Aortic calcification was demonstrated in experimental animal models of hyperhomocysteinemia. Mild hyperhomocysteinemia was associated with aortic calcification, suggesting a relationship between homocysteine (HCY) and the pathogenesis of aortic calcification. In the present study, the effect of HCY on vascular calcification was examined in calcifying and non-calcifying vascular smooth muscle cells (VSMCs). Cell calcification was induced by incubation of VSMCs with beta-glycerophosphate. Proliferation of VSMCs was studied by cell counting, 3H-thymidine (3H-TdR) and 3H-leucine (3H-Leu) incorporation. 45Ca accumulation, cell calcium content, and alkaline phosphatase (ALP) activity were measured as indices of calcification. The results showed that the proliferation of calcifying VSMCs, which was indicated by cell counting, 3H-TdR and 3H-Leu incorporation in calcifying VSMCs, was enhanced as compared with that of non-calcifying VSMCs. HCY promoted increases in cell number, 3H-TdR and 3H-Leu incorporation in both calcifying and non-calcifying VSMCs, but with more prominent effect in calcifying VSMCs. The stimulating effects of HCY on the three parameters in calcifying VSMCs were antagonized by PD98059, a specific inhibitor of mitogen activated protein kinase kinase (MAPKK). The ALP activity, 45Ca uptake, and calcium deposition in the calcifying VSMCs were greater than those in non-calcifying VSMCs. PD98059 had no effect on ALP activity, 45Ca uptake, and calcium deposition in calcifying VSMCs. HCY caused marked increases in 45Ca uptake and calcium deposition both in calcifying and non-calcifying VSMCs. HCY, however, enhanced ALP activity in the calcified VSMCs but not in the non-calcifying VSMCs. The non-calcifying VSMCs treated with HCY showed the same low ALP activity, as did the control VSMCs. In calcifying VSMCs, the HCY-induced increases in 45Ca uptake, calcium deposition, and ALP activity were also attenuated by PD98059. The results demonstrated that HCY potentiated VSMC calcification probably through the mechanisms by which HCY promotes atherosclerosis.     

Pure atmospheric pressure promotes an expression of osteopontin in human aortic smooth muscle cells

Although the types of pathophysiological stimulation that initiate an overexpression of OPN have yet to be determined, we hypothesized that mechanical stress is one of the candidates which initiates OPN expression in vascular smooth muscle cells. Cell proliferation assay indicated that a pure atmospheric pressure of 160 mmHg activated cell proliferation by 11% in human aortic smooth muscle cells (HASMC) compared to nonpressurized controls. Immunoblot analysis probed with an anti-OPN antibody demonstrated a 50% increase in OPN. Dual-luciferase reporter assay demonstrated that OPN promoter, corresponding to the -771 through -1 region of OPN gene, was highly responsive to pure atmospheric pressure by ten times that of the control. From these observations, we concluded that pure atmospheric pressure directly promotes an expression of OPN in HASMC, with these results also suggesting that high blood pressure-mediated mechanical compression is involved in the process of atherosclerosis and remodeling via OPN expression in HASMC.     

Sesquiterpene lactones inhibit inducible nitric oxide synthase gene expression in cultured rat aortic smooth muscle cells.

Nitric oxide (NO) is an important regulator and effector molecule in various inflammatory disease states. High output of NO during inflammation is generated by the inducible isoform of nitric oxide synthase (iNOS). Sesquiterpene lactones are derived from Mexican-Indian medicinal plants and are known to have potent anti-inflammatory properties. The mechanisms by which sesquiterpene lactones exert their anti-inflammatory effects are not fully understood. In the current studies we determined if the sesquiterpene lactones, parthenolide and isohelenin, modulate iNOS gene expression in cultured rat aortic smooth muscle cells (RASMC) treated with lipopolysaccharide and interferon-gamma. Treatment with parthenolide or isohelenin inhibited NO production and iNOS mRNA expression in a concentration-dependent manner. Transient transfection studies with an iNOS promoter-luciferase reporter plasmid demonstrated that parthenolide and isohelenin also inhibited activation of the iNOS promoter. Inhibition of iNOS promoter activation was associated with inhibition of both I-kappaBalpha degradation and nuclear translocation of NF-kappaB. Neither parthenolide nor isohelenin induced the heat shock response in RASMC. We conclude that sesquiterpene lactones inhibit iNOS gene expression by a mechanism involving stabilization of the I-kappaBalpha/NF-kappaB complex. This effect is not related to induction of the heat shock response. The ability of sesquiterpene lactones to inhibit iNOS gene expression may account, in part, for their anti-inflammatory effects.     

培养大鼠主动脉血管平滑肌细胞产生巨噬细胞集落刺激因子及其受体的表达

本研究用培养大鼠主动脉血管平滑肌细胞（ＶＳＭＣｓ）,结果如下：（１）用生物活性检测法发现ＶＳＭＣｓ无血清条件培养液可刺激巨噬细胞集落形成,其作用能被抗巨噬细胞集落刺激因子（ＭＣＳＦ）抗体抑制;（２）用免疫细胞化学技术证明ＶＳＭＣｓ存在ＭＣＳＦ受体;（３）用Ｎｏｒｔｈｅｒｎ ｂｌｏｔ技术证明ＶＳＭＣｓ有ＭＣＳＦ及其受本的ｍＲＮＡ表达,血清刺激使两者表达明显增强。本研究首次报道了培养大鼠主动脉ＶＳＭＣ     

Regulation of fibronectin by platelet-derived growth factors in cultured rat thoracic aortic smooth muscle cells

Induction of Fibronectin (FN) gene expression by platelet-derived growth factor (PDGF) isoforms in rat thoracic aortic smooth muscle cells (SMC) was examined. PDGF-BB enhances FN levels in SMC cultures in a time- and concentration-response fashion. PDGF-AA and PDGF-AB show no effect on FN levels. The effects of insulin and insulin-like growth factor-I (IGF-I) on PDGF-BB-induced FN levels were examined. No additivity of FN levels is observed between PDGF-BB and insulin and/or IGF-I. Experiments also show that PDGF-BB enhances FN mRNA levels, implying that acquisition of additional FN mRNA units accounts for the increase in FN levels. Induction of FN and FN mRNA levels by PDGF-BB could be one of the initial events in vascular SMC proliferation and extracellular matrix expansion, leading to atherosclerosis and hypertension.     

Regulation of NaK-ATPase by platelet-derived growth factors in cultured rat thoracic aortic smooth muscle cells

Regulation of (Na⁺ + K⁺)-adenosine triphosphatase (NaK-ATPase) by platelet-derived growth factor (PDGF) in cultured rat thoracic aortic smooth muscle cells (SMC) was examined. PDGF-BB enhances SMC proliferation and NaK-ATPase activity. Ouabain, an inhibitor of NaK-ATPase activity, prevents PDGF-BB-induced SMC proliferation. As shown by Western blot and immunochemiluminescence analysis, PDGF-BB also enhances ₁, truncated ₁, and ₁ NaK-ATPase subunit levels. PDGF-AA and PDGF-AB show no effect on ₁ and truncated ₁ levels in slot blot analysis. Induction of NaK-ATPase subunit levels by PDGF-BB could be one of the initial events in vascular SMC proliferation.     

Thromboxane A2 receptors are influenced by cell density in cultured rat aortic smooth muscle cells.

The influence of cell density on the binding characteristics of thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptors in rat aortic vascular smooth muscle cells in culture were determined using [1S- (1 alpha, 2 beta (5Z), 3a (1E, 3R*), 4 alpha)]- 7 -[3- (3-hydroxy -4- (4'-iodophenoxy)-1-butenyl)-7-oxabicyclo-[2.2.1]heptan- 2yl]-5-heptenoic acid (125I-BOP). The Bmax for 125I-BOP was 5,430 +/- 139 sites/cell (26.9 +/- 5.7 fmoles/mg protein) for cells cultured in 1% fetal calf serum and 2809 +/- 830 sites/cell (13.1 +/- 2.2 fmoles/mg protein) for cells cultured in 10% fetal calf serum. Cells were allowed to grow to varying densities and then harvested for assay. There was a negative correlation between the Bmax and the cell density per flask. The Kd for I-BOP did not significantly vary in any of the studies. The results demonstrate that cell density plays an important role in influencing the expression of vascular TXA2/PGH2 receptors.     

Density-related expression of caldesmon and vinculin in cultured rabbit aortic smooth muscle cells

Quantitative immunoblotting techniques were used to study the effects of seeding density on the expression of caldesmon and vinculin variants, which are sensitive markers of vascular smooth muscle cell (SMC) phenotypic modulation in culture. Rabbit aortic SMC were seeded at different densities: 13 x 10(4) cells/cm2 (high density), 3 x 10(4) cells/cm2 (medium density), and 0.2 x 10(4) cells/cm2 (low density) and cultured in the presence of 5% fetal calf serum. Irrespective of cell density and growth phase, caldesmon150 was gradually and irreversibly substituted by caldesmon77, but at high seeding density this substitution proceeded at a slower rate. The fraction of meta-vinculin (smooth muscle variant of vinculin) was reduced after seeding SMC in culture, but was reestablished when the cells reached confluency. Thus, high SMC seeding density is essential but not sufficient to keep vascular SMC cultured in the presence of serum in the contractile phenotype.     

Complex regulation of collagen gene expression in cultured bovine aortic smooth muscle cells

Cultured bovine aortic smooth muscle cells display an increase in production of type I and type III collagen as a function of the number of days in second passage (Beldekas, J. C., Gerstenfeld, L. C., Sonenshein, G. E., and Franzblau, C. (1982) J. Biol. Chem. 257, 12552-12556). In this study, we report that the regulation of these events is highly complex and relates to the growth state of the cells. Cultures, seeded at 1.5 X 10(6) cells/75-cm2 flask, produced very little collagenous protein early when the cells were proliferating rapidly. As they approached confluence at day 6, collagen synthesis began to increase. Maximal collagen synthesis was observed at day 14. In contrast, the levels of the mRNAs for type I and type III collagen increased only up to the 10th day and thereafter decreased. Cell-free translation analyses indicated that the translational activity of the collagen mRNAs was increasing over the time course. These results suggest that both translational and pretranslational sites are involved in the control of collagen production by aortic smooth muscle cells, and that collagen synthesis is inversely related to the proliferative state of the cells in culture.     

Platelet-derived growth factor regulates actin isoform expression and growth state in cultured rat aortic smooth muscle cells

The role of platelet-derived growth factor (PDGF) in the control of smooth muscle cell (SMC) differentiation was explored in vitro by examining its effects on expression of the smooth muscle (SM) specific contractile protein SM alpha actin in cultured rat aortic SMC. Quiescent, postconfluent SMC express maximal levels of alpha actin and responded to human platelet-derived growth factor (partially purified from platelets) by entering the cell cycle and undergoing approximately one synchronous round of DNA synthesis. Concomitantly, these cultures exhibited a marked reduction in alpha actin synthesis. Chronic treatment with PDGF (72 hours at 8 or 12 hour intervals) was associated with a transient increase in thymidine labeling index and a decrease in alpha actin expression. Interestingly, between 48 and 72 hours following initial treatment, thymidine labeling indices returned to near control levels while SM alpha actin expression remained depressed. This effect was reversible; fractional alpha actin synthesis increased immediately after PDGF removal. When subsequently stimulated with 10% fetal bovine serum (FBS), cells chronically pretreated with PDGF entered S phase approximately 4 hours earlier than cells pretreated with PDGF vehicle, consistent with the idea that the maintained suppression of alpha actin synthesis in SMC subjected to chronic PDGF treatment was associated with partial cell cycle transit. Chronic treatment with highly purified recombinant PDGF-BB elicited similar effects on alpha actin synthesis and partial cell cycle transit. Flow cytometric analysis of chronic PDGF-treated SMC demonstrated a 25% increase in forward angle light scatter, an index of cell size. These data implicate a possible role for PDGF in regulation of SMC differentiation and suggest a potentially important role for this mitogen in the phenotypic modulation accompanying SMC growth and in mediation of the cellular hypertrophy associated with cell cycle progression.     

Inverse relationship between connexin43 and desmin expression in cultured porcine aortic smooth muscle cells.

Our previous work has shown that in vascular tissues the elastic medial regions express high levels of the gap junctional protein, connexin43, but low levels of desmin, while the muscular medial regions express low levels of connexin43 but high levels of desmin. It is uncertain, however, whether this regional difference at the tissue level extends down to the level of the individual cell, or reflects an averaged relationship of groups of cells of different connexin43 and desmin expression. The present study has addressed this question using cultured porcine aortic smooth muscle cells. Immunoconfocal microscopic analysis of single-labeled cells showed that while smooth muscle alpha-actin, calponin and vimentin were positively labeled in the majority of medial smooth muscle cells both in intact porcine aorta and corresponding cultured cells, desmin and connexin43 labeling was highly heterogeneous. In the cultured cells, 0.3-0.5% of cells were found to be desmin-positive, and quantitative analysis after double labeling for desmin and connexin43 revealed that the desmin-positive cells were smaller, and contained significantly lower numbers and smaller sizes of connexin43 gap-junctional spots than did desmin-negative cells. Our findings demonstrate that an inverse expression pattern of connexin43 and desmin holds true at the level of the individual cell. This suggests a close relationship between intrinsic phenotypic control and the regulation of connexin43 expression in the arterial smooth muscle cell.     

Metabolism of cytoplasmic triacylglycerol in cultured aortic smooth muscle cells

A turnover of cytoplasmic triacylglycerol was studied in cultured rat, rabbit, and bovine aortic smooth muscle cells. Cytoplasmic triacylglycerol was labeled with [3H]glycerol in the presence of oleic acid in the medium and its loss from the cell was studied in the presence of carrier glycerol. Multiple additions of Isuprel or dibutyryl cyclic AMP during the chase period did not enhance the loss of labeled triacylglycerol. The rate of hydrolysis of cellular triacylglycerol was unchanged in the absence or in the presence of 100 microM chloroquine. Modulation of cellular cholesterol content by addition of low density lipoprotein or high density apolipoprotein--sphingomyelin liposomes did not affect the residence time of the cellular triacylglycerol. We conclude that cytoplasmic triacylglycerol in cultured aortic smooth muscle cells is metabolized by an extralysosomal enzyme which is neither catecholamine responsive nor affected by modulation of cellular cholesterol.     

Regulation of glucose transport by angiotensin II and glucose in cultured vascular smooth muscle cells

Glucose transport in response to angiotensin II (AII) was assessed in cultured vascular smooth muscle (VSM) cells by measuring the uptake of [³H]-2-deoxyglucose, a radiolabeled non-metabolizable glucose analog. Significant stimulation occurred by 2 hr of exposure with the maximum effect being observed between 6 and 8 hr. AII effects were concentration dependent with a threshold response being detected at 0.1 nM. AII-stimulated transport was blocked by saralasin, an AII receptor antagonist, indicating that AII binding to a specific receptor is required for AII to elicit the transport response. AII-stimulated transport was also blocked when cells were incubated with cycloheximide for 6 hr, suggesting that protein synthesis is required for the long-term effects of AII on glucose transport. A specific protein synthesized in response to AII stimulation was the GLUT 1 glucose transporter as assessed by western blot analysis. Inhibition of protein kinase C (PKC) by bisindolylmaleimide and staurosporine did not affect VSM responsiveness to AII, suggesting that AII is capable of stimulating glucose transport through a PKC-independent mechanism; however, VSM responsiveness to AII did appear to be dependent upon the presence of extracellular calcium. The importance of calmodulin in mediating the response of VSM cells to AII was indicated by the inhibition of AII-stimulated glucose transport when VSM cells were incubated in the presence of the calmodulin inhibitors, calmidazolium and W7. Finally, glucose uptake increased with decreasing levels of glucose in the incubation medium. This was accompanied by a corresponding decrease in the relative effectiveness of AII in stimulating glucose uptake. J. Cell. Physiol. 177:94–102, 1998. © 1998 Wiley-Liss, Inc.     

Internalization and processing of human angiogenin by cultured aortic smooth muscle cells

Human angiogenin is a 14-kDa plasma protein with angiogenic and ribonucleolytic activities. Angiogenin binds specifically to aortic smooth muscle cells, activates second messenger pathways, and inhibits their proliferation. Human and bovine aortic smooth muscle cells were used to study the internalization and intracellular fate of human angiogenin at 37 degrees C. Using a specific antibody against angiogenin, we found that the internalized native protein was localized in the perinuclear region at 30 min and then dispersed throughout the cytoplasm. In conditions favoring receptor-mediated endocytosis, internalization of iodinated angiogenin showed a first peak at 5 min and then further increased for up to 24 h. The half-life of the molecule, calculated as 12 h in chase experiments, could contribute to its intracellular accumulation. In cell extracts, in addition to the 14-kDa protein, a 8.7-kDa fragment was observed at 24 h, and three fragments with molecular mass of 10.5, 8.7, and 6. 1 kDa were detected at 48 h. Our data point to a specific internalization and processing of human angiogenin by aortic smooth muscle cells.