Angiotensin II induces hypertrophy, not hyperplasia, of cultured rat aortic smooth muscle cells

We have explored the hypothesis that contractile agonists are important regulators of smooth muscle cell growth by examining the effects of one potent contractile agonist, angiotensin II (AII), on both cell proliferation and cellular hypertrophy. AII neither stimulated proliferation of cells made quiescent in a defined serum-free media nor augmented cell proliferation induced by serum or platelet-derived growth factor. However, AII did induce cellular hypertrophy of postconfluent quiescent cultures following 4 days of treatment, increasing smooth muscle cell protein content by 20% as compared with vehicle-treated controls. AII-induced hypertrophy was maximal at 1 microM, had an ED50 of 5 nM, and was blocked by the specific AII receptor antagonist Sar1,Ile8 AII. The cellular hypertrophy was due to an increase in protein synthesis, which was elevated within 6-9 hours following AII treatment, while no changes in protein degradation were apparent. AII was even more effective in inducing hypertrophy of subconfluent cultures, causing a 38% increase in protein content after 4 days of treatment (1 microM) and showing a maximal response at concentrations as low as 0.1 nM. Interestingly, in subconfluent cultures, AII treatment (1 microM, 4 days) was associated with a 50% increase in the fraction of cells with 4C DNA content with the virtual absence of cells in S-phase of the cell cycle, consistent with either arrest of cells in the G2 phase of the cell cycle or development of tetraploidy.(ABSTRACT TRUNCATED AT 250 WORDS)     

氧化型胆固醇对血管平滑肌细胞的损伤作用

目的　以胆固醇为对照 ,观察 3 β 5α 6β 三羟胆固烷 (cholestane 3 β,5α,6β triol)、2 5 羟胆固醇 ( 2 5 hydroxycholesterol)、7 酮胆固醇 ( 7 ketocholesterol)及环氧胆固醇 (cholesterol 5α,6α epoxide)四种氧化型胆固醇对大鼠主动脉平滑肌细胞的损伤作用。方法　取 6～ 10代细胞 ,测定细胞存活率、细胞培养液乳酸脱氢酶活力 ,用电子自旋共振自旋标记检测膜脂流动性和膜蛋白构象。结果　氧化型胆固醇呈时间和剂量依赖性降低细胞存活率、增加培养液乳酸脱氢酶活力 ,以 3 β 5α 6β 三羟胆固烷损伤最重。氧化型胆固醇还使膜脂流动性降低、膜蛋白构象改变及运动减慢。胆固醇除改变膜脂流动性外 ,在相同剂量及相同作用时间的情况下 ,对细胞无损伤作用。结论　氧化型胆固醇对血管平滑肌细胞有损伤作用 ,其中以 3 β 5α 6β 三羟胆固烷损伤最重 ,胆固醇对细胞没有损伤作用。氧化型胆固醇的细胞损伤与膜物理性质改变有关。     

Glucose-induced hyperproliferation of cultured rat aortic smooth muscle cells through polyol pathway hyperactivity

Aims/hypothesis. The protein kinase C (PKC), platelet-derived growth factor (PDGF) and polyol pathway play important parts in the hyperproliferation of smooth muscle cells, a characteristic feature of diabetic macroangiopathy. The precise mechanism, however, remains unclear. This study investigated the relation between polyol pathway, protein kinase C and platelet-derived growth factor in the development of diabetic macroangiopathy. Methods. Smooth muscle cells were cultured with 5.5 or 20 mmol/l glucose with or without an aldose reductase inhibitor, epalrestat, or a PKC-β specific inhibitor, LY333 531. Protein kinase C activities, the expression of PKC-βII isoform and PDGF-β receptor protein, free cytosolic NAD⁺:NADH ratio, the contents of reduced glutathione, and proliferation activities were measured. Results. Smooth muscle cells cultured with 20 mmol/l glucose showed statistically significant increases in protein kinase C activities, the expression of PKC-βII isoform and PDGF-β receptor protein, and proliferation activities, compared with smooth muscle cells cultured with 5.5 mmol/l glucose. Although epalrestat and LY333 531 inhibited protein kinase C activation induced by glucose to the same degree, the effects of epalrestat on proliferation activities and expression of the PDGF-β receptor were more prominent than those of LY333 531. Epalrestat improved the glucose-induced decrease in free cytosolic NAD⁺:NADH ratio and reduced glutathione content, but LY333 531 did not. The increased expression of membranous PKC-βII isoform was normalized by epalrestat. Conclusion/interpretation. These observations suggest that polyol pathway hyperactivity contributes to the development of diabetic macroangiopathy through protein kinase C, PDGF-β receptor, and oxidative stress, and that an aldose reductase inhibitor has a therapeutic value for this complication. [Diabetologia (2001) 44: 480–487] Received: 2 October 2000 and in revised form: 13 December 2000     

Mechanisms of angiotensin II- and arginine vasopressin-induced increases in protein synthesis and content in cultured rat aortic smooth muscle cells. Evidence for selective increases in smooth muscle isoactin expression

Previous studies from this laboratory have demonstrated that angiotensin II (Ang II) and arginine vasopressin (AVP) are potent hypertrophic agents in cultured rat aortic smooth muscle cells. The present study identified major proteins that accumulate in Ang II-induced and AVP-induced hypertrophic cells and initiated studies of the mechanisms that contribute to their accumulation. Smooth muscle cell hypertrophy induced by Ang II and/or AVP (1 microM each) was associated with widespread increases in the content of many cellular proteins that were resolved by one- and two-dimensional gel electrophoresis. However, increases were also selective in nature, with increases in certain individual proteins, including actin (twofold to threefold), vimentin (2.5-fold to sevenfold), tropomyosin (threefold to sixfold), and myosin heavy chain, far exceeding overall increases in cellular protein content (20-40%). Increases in actin content were due largely to increased expression of smooth muscle alpha-actin (3.6- to 7.5-fold), as opposed to nonmuscle beta-actin (1.7- to 2.5-fold). Increases in smooth muscle alpha-actin were accompanied by a fivefold to eightfold increases in smooth muscle alpha-actin mRNA, indicating that these changes were not due exclusively to translational controls. Results demonstrate that contractile agonist-induced hypertrophy in cultured smooth muscle cells is due, in part, to increased expression of smooth muscle contractile proteins. Furthermore, the fact that Ang II and AVP induced selective increases in smooth muscle alpha-actin suggests that these agonists may not only regulate growth of vascular smooth muscle but may also promote expression of smooth muscle-specific contractile proteins during differentiation of vascular smooth muscle.     

Relative insensitivity to glucagon of sterol synthesis in cultured rat aortic smooth muscle cells

Summary The smooth muscle cell plays an important role in the process of atherogenesis. In these experiments the effect of glucagon and dibutyryl cyclic AMP on sterol synthesis in cultured rat arterial smooth muscle cells was studied. Glucagon in concentrations of 1×10⁻⁹ mol/l inhibited the incorporation of sodium (2−¹⁴C)acetate into non-saponifiable lipids and digitonin precipitable sterols but lower concentrations of glucagon had no effect. In cells which were exposed to serum, dibutyryl cyclic AMP also resulted in a decrease in the incorporation of labelled acetate into sterols but when the cells were grown in serum free medium, dibutyryl cyclic AMP had no inhibitory effect on sterol synthesis. These results provide further evidence that sterol metabolism in arterial smooth cells may be influenced by hormones but suggest that glucagon is relatively less important than insulin in this respect.     

Testosterone increases thromboxane A2 receptors in cultured rat aortic smooth muscle cells.

Previous studies have demonstrated increased contractile responses to thromboxane A2 (TXA2) mimetics in aortas obtained from male rats compared with those obtained from females. This study was designed to determine the effects of testosterone and 17 beta-estradiol treatment on TXA2 receptors in cultured rat aortic smooth muscle cells (RASMCs). TXA2 receptor affinity and density were determined through equilibrium binding experiments using the TXA2/prostaglandin H2 mimetic [1S-(1 alpha,2 beta(5Z),3 alpha(1E,3R*),4 alpha)]-7-[3-(3- hydroxy-4-(4'-125iodophenoxy)-1-butenyl)-7-oxabicyclo[2.2.1]hep tan-2-yl]- 5-heptenoic acid (125I-BOP). Incubation with testosterone (100 nM) for 24 or 48 hours resulted in a significant (p less than 0.05) 31% and 48% increase in TXA2 receptor density without any change in affinity. 17 beta-Estradiol (100 nM) had no significant effect on either the density or affinity of TXA2 receptors. Coincubation with the testosterone receptor antagonist hydroxyflutamide (1 microM) blocked the testosterone-induced increase in TXA2 receptor density. The maximum increase in intracellular free calcium induced by I-BOP was significantly (p less than 0.05) greater in testosterone-treated RASMCs than controls. Similarly, increases in inositol trisphosphate induced by the TXA2/prostaglandin H2 mimetic U46619 were significantly (p less than 0.05) greater in testosterone-treated RASMCs compared with controls. The results demonstrate that testosterone increases vascular TXA2 receptor density and support the notion that sex steroid hormones modulate the expression of this receptor.     

Uptake and release of adenosine by cultured rat aortic smooth muscle

We wanted to determine whether CO2, H+ and K+ affect the adenosine metabolism of vascular smooth muscle in a way that could account for the effects of these substances on vascular reactivity and their ability to modulate adenosine-induced vascular relaxation. Accordingly, 1-week-old cultures of rat aortic smooth muscle were incubated in phosphate-buffered saline with various [K+]'s and pH's and aerated in an incubation chamber with gases containing various proportions of CO2. Uptake was measured as 14C incorporation into cellular constituents during exposure to 2 microM [14C]adenosine. Release was measured as net extracellular adenosine accumulation. Uptake of adenosine was not significantly affected by any of the experimental maneuvers, except that it was greatly attenuated by dipyridamole (10(-5) and 10(-4) M) and transiently enhanced by the low CO2 levels. Adenosine release, however, was depressed by lowering atmospheric CO2 (0% vs 5%) and also by normocapnic acidosis (pH 6.8 vs pH 7.4). We conclude that vascular smooth muscle in culture releases adenosine at a rate that might have vasoactive significance in vivo. Furthermore, some of the vascular actions of CO2 and H+, but not those of K+, may be partially explained by their effects on vascular smooth muscle's adenosine metabolism.     

Human proinsulin C-peptide prevents proliferation of rat aortic smooth muscle cells cultured in high-glucose conditions

Aims/hypothesis Proinsulin C-peptide is involved in several biological activities. However, the role of C-peptide in vascular smooth muscle cells is unclear. We therefore investigated its effects, in vascular smooth muscle cells in high-glucose conditions.Methods Rat aortic smooth muscle cells were cultured with 5.5 or 20 mmol/l glucose with or without C-peptide (1 to 100 nmol/l) for 3 weeks. Proliferation activities, the protein expression of platelet-derived growth factor (PDGF)-beta receptor, the phosphorylation of p42/p44 mitogen-activated protein (MAP) kinases, and glucose uptake were measured.Results The proliferation activities increased approximately three-fold under high-glucose conditions (p<0.05). C-peptide suppressed hyperproliferation activities that were induced by high glucose. This happened in a dose-dependent manner from 1 to 100 nmol/l of C-peptide. C-peptide (10 and 100 nmol/l) inhibited the increased protein expression of PDGF-beta receptor and the phosphorylation of p42/p44 MAP kinases that had been induced by high glucose (p<0.05). Furthermore, 100 nmol/l of C-peptide augmented the impaired glucose uptake in the high-glucose conditions.Conclusions/interpretation These observations suggest that C-peptide could prevent diabetic macroangiopathy by inhibiting smooth muscle cell growth and ameliorating glucose utilisation in smooth muscle cells. C-peptide may thus be a novel agent for treating diabetic macroangiopathy in patients with type 1 and type 2 diabetes.     

体外培养高糖、高脂喂养大鼠主动脉平滑肌细胞钙化的研究

目的 检测高糖、高脂喂养Goto-Kakizaki（GK）糖尿病大鼠主动脉平滑肌细胞（SMCs）的血管钙化指标,探讨糖尿病血管钙化的相关机制.方法 高糖、高脂喂养GK及Wistar大鼠2周,同时分离培养两组大鼠的主动脉SMCs,Wistar大鼠SMCs作为对照.通过细胞计数法观察细胞生长状况,以甲基百里香酚蓝比色法测定两组大鼠细胞层及培养上清中钙的含量,实时定量PCR检测两组细胞碱性磷酸酶（ALP）、骨桥蛋白（OPN）、核心结合因子α-1（Cbfα-1）、α-平滑肌肌动蛋白（α-SMA）的基因表达.结果 与Wistar大鼠SMCs相比,GK大鼠SMCs生长速度明显缓慢（F =363.392,P＜0.05）;细胞层钙含量[（0.56±0.22） vs.（0.39±0.09）,t=2.47,P＜0.05]明显增加,培养上清中钙含量[（0.82±0.22）vs.（1.20±0.17）,t=-22.573,P＜0.05]明显减少.GK大鼠SMCs中ALP （t=12.963,P＜0.05）、OPN（t=8.305,P＜0.05）及Cbfα-1（t=10.109,P＜0.05）的基因表达增加,同时α-SMA（t=-8.219,P＜ 0.05）的基因表达减少.结论 高糖、高脂喂养的GK糖尿病大鼠的主动脉平滑肌细胞易发生钙化.     

Hematoporphyrin binding to cultured aortic smooth muscle cells from spontaneously atherosclerotic turkey

Porphyrins are known to be accumulated in vivo by tumors and atherosclerotic plaques. We studied the interaction of cultured aortic smooth muscle cells (SMC) from spontaneously atherosclerotic Broad Breasted White Turkeys (BBWT) with free hematoporphyrin (Hp) and low density lipoprotein (LDL)-Hp complexes. A significantly higher binding of LDL-Hp to SMC as compared to free Hp was observed. These data indicate that porphyrin binding to vascular SMC represents a possible mechanism for porphyrin accumulation by atherosclerotic plaques. This process is mediated, at least in part, by LDL.     

Studies of hypertension-induced vascular hypertrophy in cultured smooth muscle cells from spontaneously hypertensive rats

Mechanisms of vascular hypertrophy induced by hypertension were studied in cultured aortic smooth muscle cells from spontaneously hypertensive rats (SHR) and stroke-prone SHR (SHRSP) and compared with those from normotensive Wistar-Kyoto (WKY) rats. Fetal calf serum-stimulated ornithine decarboxylase (ODC) activity of cultured smooth muscle cells was greater in SHR and SHRSP than in WKY. Beta- but not alpha-adrenergic agonist stimulated ODC activity acutely in cultured smooth muscle cells from WKY, and isoprenaline-induced activation was blocked by the beta-blocker, propranolol, and enhanced by the phosphodiesterase inhibitor, 1-methyl-3-isobutylxanthine. These results indicate that cultured vascular smooth muscle cells from SHR and SHRSP are more prone to increase the protein synthesis than those from WKY through the trophic induction of ODC activity and that the regulation of ODC activity by catecholamines is mediated through beta-agonistic effect in cultured smooth muscle cells.     

Turnover of phospholipids in rat aortic smooth muscle cells in culture

Smooth muscle cells derived from rat aortic media were explanted and grown in culture for 14 to 60 days. During that time they formed a confluent multilayer and deposited extracellular material resembling newly formed elastin. The lipid composition of the cells in culture differed slightly from the parent cells in the intact aorta with respect to a higher phospholipid/DNA ratio and a higher lecithin content. The cholesterol content resembled that of parent cells. After incubation with labeled precursor the cultured cells show an active lipid synthesis; choline is incorporated mainly into lecithin, whereas glycerol and palmitate appear in phospholipids and to a lesser extent in neutral lipids. After a 2 hour pulse and up to 96 hour chase there is a linear fall in the specific activity of lecithin with a half-time of 28 to 30 hours. The rate of fall in specific activity of glycerol- or choline-labeled lecithin was found to be similar, indicating that choline does not turn over by an exchange reaction and is a suitable marker for studying phospholipid turnover in cultured cells. The results provide a basis for investigation of the effect of increasing cellular cholesterol content on phospholipid turnover.     

β-磷酸甘油诱导体外培养大鼠主动脉平滑肌细胞的钙化

目的:研究β磷酸甘油对体外培养的大鼠主动脉平滑肌细胞的钙化作用。方法:采用组织块培养法体外培养大鼠主动脉平滑肌细胞。细胞分为钙化组和正常组。钙化组加入10mmol/Lβ磷酸甘油,0.1μmol/L胰岛素及50μg/L维生素C（钙化培养基）诱导细胞钙化,继续培养14d。茜素红S染色观察钙结节计数并测定细胞钙沉积含量。采用四唑盐比色法（MTT）测量细胞增殖。结果:钙化组钙结节计数为（7.4±3.8）%,较正常组（4.3±1.9）%显著增加（P<0.05）;细胞钙沉积含量为（5.4±0.4）mmol/g蛋白,较正常组（1.2±0.5）mmol/g蛋白显著增加（P<0.05）。MTT检测细胞增殖,钙化组细胞增殖为0.071±0.011,较正常组0.040±0.009明显增加（P<0.01）。结论:β磷酸甘油能诱导体外培养的大鼠主动脉平滑肌细胞的钙化,钙化能促进平滑肌细胞的增殖。     

Effect of hypoxia on cholesterol accumulation in cultured rabbit aortic smooth muscle cells

We studied the effect of hypoxia on cholesterol accumulation in cultured rabbit aortic smooth muscle cells, which were incubated in a medium with normolipemic rabbit serum (NRS) or hyperlipemic rabbit serum (HRS). The cells were incubated in a humidified atmosphere of either 20% O2, 75% N2 and 5% CO2 (control cells) or 2% O2, 93% N2 and 5% CO2 (hypoxic cells). In a medium containing 20% NRS, the free cholesterol level of hypoxic cells was only a little higher than that of control cells, and there was no significant difference in esterified cholesterol content. On the other hand, in a medium containing 20% HRS, the free cholesterol level was slightly higher and the esterified cholesterol level was markedly higher in hypoxic cells compared with control cells. These results show that hypoxia promotes the accumulation of cholesterol, especially as ester, in smooth muscle cells cultured with hyperlipemic serum. These in vitro experiments indicate that hypoxia in the arterial wall associated with hyperlipidemia may play an important role in atherogenesis, although the precise mechanism remains unclear.     

Antiproliferative action of cyclic GMP-elevating vasodilators in cultured rabbit aortic smooth muscle cells

In cultured rabbit aortic smooth muscle cells (SMCs), sodium nitroprusside (SNP) (10(-7) to 10(-4) M), atrial natriuretic peptide (ANP) (10(-9) to 10(-6) M) and 8-bromo-cyclic GMP (10(-6) to 10(-3) M) inhibited the whole blood serum (WBS)-induced DNA synthesis by about 30%. The doses of SNP and ANP necessary for the inhibition of the WBS-induced DNA synthesis were similar to those necessary for the formation of cellular cyclic GMP (cGMP). These agents were effective even when added 6 h after stimulation of the cells with WBS. These results suggest that cGMP inhibits the proliferation of rabbit aortic SMCs by inhibiting the progression from the G1 into S phase of the cell cycle and raise the possibility that cGMP-elevating vasodilators may suppress the atherogenic process by inhibiting vascular SMC proliferation.     

Molecular mechanism of progesterone-induced antiproliferation in rat aortic smooth muscle cells

Previously we demonstrated that progesterone at physiologic levels dose dependently inhibited cell proliferation in cultured rat aortic smooth muscle cells (RASMCs). However, the molecular mechanism underlying of progesterone-induced antiproliferation was not clear. Here we demonstrated that progesterone induced a reduction of the [(3)H]thymidine incorporation into RASMCs during the S-phase of the cell cycle. Western blotting analysis revealed that the protein levels of cyclin A, cyclin E, and cyclin-dependent-kinase (CDK) 2 but not cyclin D1 and CDK4 decreased after progesterone treatment, but those of CDK-inhibitory proteins, p21 and p27, increased. Immunoprecipitation showed that the formations of the CDK2-p21 and CDK2-p27 complex were increased and the assayable CDK2 kinase activity was decreased in the progesterone-treated RASMCs. In contrast, the formations of the CDK4-p21 and CDK4-p27 complex and the assayable CDK4 kinase activity were not changed significantly by progesterone treatment. Pretreatment of RASMCs with a p21 or p27 antisense oligonucleotide reduced the progesterone-induced inhibition of [(3)H]thymidine incorporation into RASMCs. In conclusion, these data suggest that progesterone inhibits RASMCs proliferation by increasing the levels of p21 and p27 protein, which in turn inhibit CDK2 kinase activity, and finally interrupt the cell cycle.     

Induction of polyploidy in cultures of neonatal rat aortic smooth muscle cells

Arterial smooth muscle cells become tetraploid with age and hypertension. To further study this phenomenon, neonatal rat aortic smooth muscle cells were placed in cell culture and studied over time. Numerous cells with tetraploid and even octaploid DNA content appeared beginning in primary cultures. These increases in DNA content per cell were determined by quantitative fluorescence microscopy and flow cytometry, and true polyploidy was confirmed by chromosome counts. In contrast, cells from adult rat aortas failed to produce significant polyploid cells over time in culture. In vitro culture of neonatal aortic cells may therefore be a model system for studying the initiation of polyploidy in arterial smooth muscle.     

Influence of animal age on the beta-adrenergic system in cultured rat aortic and mesenteric artery smooth muscle cells.

It has previously been reported that aortic smooth muscle cells cultured from old rats have a marked decline in beta-adrenergic stimulated cAMP accumulation. We wished to confirm this observation and determine whether this decline was secondary to loss of beta-adrenergic receptors (BAR). Primary cultures of aortic and mesenteric artery smooth muscle cells were obtained by enzymatic digestion from young and old male Fischer 344 rats. In aortic cells from old animals, there was a decline in beta-adrenergic receptor density and a rightward shift in the dose response curve to isoproterenol without a change in maximal cAMP accumulation. In mesenteric artery cells, there were no age changes in these parameters. Beta-adrenergic receptor subtype distribution was determined and was similar between all age groups and vessel types. These findings differ from whole tissue studies and suggest that cultured smooth muscle cells have limitations as a model for the aging adrenergic system.     

Phosphorylation of hydroxylysine residues in collagen synthesized by cultured aortic smooth muscle cells.

O5-Phosphohydroxylysine was chemically synthesized and techniques were established for its identification by combined use of cation-exchange chromatography, thin-layer electrophoresis at pH 1.9 and 3.5, and thin-layer chromatography. Clean separation of phosphohydroxylysine from the other phospho amino acids, phosphoethanolamine, and phosphocholine was achieved. Conditions were also determined to permit hydrolysis of proteins in 2 M HCl without loss of the phosphono group of phosphohydroxylysine residues. Experiments were then performed showing that 32P was incorporated into the hydroxylysine residues of cell-associated collagens when cultured calf aorta medial smooth muscle cells were incubated with [32P]orthophosphate. In other experiments, the cells incorporated [3H]lysine into hydroxylysine residues of cell-associated collagen and then 32P into phosphohydroxylysine residues. The doubly labeled phosphohydroxylysine subsequently isolated showed nearly 1:1 stoichiometry with respect to incorporation of precursor lysine and phosphorus. Finally, in preliminary experiments done with a cell-free extract of the smooth muscle cells, 32P was transferred from [gamma-32P]ATP to hydroxylysine residues in several kinds of collagenous substrates. Thus, this work shows that smooth muscle cells have the capacity to phosphorylate hydroxylysine residues in their cell-associated collagens and provides preliminary evidence that a protein kinase is involved.