Vasoactive factors and growth factors alter vascular smooth muscle cell EC-SOD expression

Oxygen free radicals have been suggested to play important roles in atherogenesis and other pathological processes in the blood vessel wall. The vascular wall contains large amounts of extracellular superoxide dismutase (EC-SOD), which is produced and secreted to the extracellular space by smooth muscle cells. In this study, we investigated the influence of factors regulating tension and proliferation of vascular smooth muscle cells and of some interstitial matrix components on EC-SOD expression. The expression and secretion of EC-SOD were upregulated by histamine, vasopressin, oxytocin, endothelin-1, angiotensin II, serotonin, heparin, and heparan sulfate and were downregulated by platelet-derived growth factors-AA and -BB, acidic and basic fibroblast growth factors, and epidermal growth factor. The responses were slow and developed over several days. The findings suggest that various physiological and pathological conditions might markedly influence EC-SOD expression, significantly altering the susceptibility of the vascular wall to effects of the superoxide radical.     

Plant sterols regulate rat vascular smooth muscle cell growth and prostacyclin release in culture

Cardiovascular disease (CVD) is the leading cause of death in the USA and other industrialized countries. A large number of epidemiological studies have established a direct correlation between diet and the development and progression of atherosclerosis. Several studies have shown the incidence of CVD to be lower in populations consuming a predominantly plant-based diet, as compared to meat-based diets. Besides being low in fat and cholesterol, vegetarian and Asian diets contain a large variety of phytochemicals, which may function in the body. For example, phytosterols (PS) are plant sterols that interfere with the absorption of cholesterol from the intestine when present in adequate amounts. Although PS may also function at a cellular level in the body, there are few studies examining the action of PS on cells involved in atherosclerosis. The purpose of this study was to examine the effect of dietary PS on vascular smooth muscle cell (VSMC) growth and function, since VSMC play a central role in the development of atherosclerosis. VSMC were treated with 16 microM cholesterol, 25-hydroxycholesterol, campesterol and beta-sitosterol (SIT) using an ethanol as a vehicle. Cell growth was determined by cell counting and cell proliferation by DNA synthesis, which was measured by [(3)H]-thymidine incorporation. Cholesterol supplementation had no effect on cell growth and proliferation. 25-Hydroxycholesterol decreased cell growth by 68% and DNA synthesis by 99%. SIT was found to inhibit VSMC growth more effectively than campesterol. Of the two PS, campesterol decreased cell growth by 16% and SIT decreased cell growth by 30%. DNA synthesis was decreased 25% by SIT supplementation but was not influenced by campesterol or cholesterol supplementation. Cholesterol, campesterol and SIT were not cytotoxic to VSMC and did not significantly alter cell viability. 25-Hydroxycholesterol, however, was cytotoxic and decreased cell viability by 45% as determined by lactate dehydrogenase release and a trypan blue dye exclusion test. De novo cholesterol synthesis was decreased 28% by campesterol, 49% by SIT and 23% by cholesterol. Beta-sitosterol exhibited a greater effect on cholesterol synthesis than campesterol or cholesterol supplementation. Measurement of cell sterol content demonstrated incorporation of PS into VSMC at the expense of cholesterol. Campesterol decreased VSMC cholesterol content by 36%, representing 40% of the total sterol content following treatment. Beta-sitosterol decreased VSMC cholesterol by 41% following supplementation and represented 49% of the total sterol amount. Cholesterol treatment did not alter the cholesterol content of the cells. Prostacyclin production was significantly altered by PS treatment. Basal prostacyclin release was increased 43% by campesterol and 81% by SIT. A23187 stimulated prostacyclin release was increased 25% by campesterol and 54% by SIT. SIT supplementation induced a greater effect on prostacyclin release from VSMC than cholesterol or campesterol supplementation. The in vitro results presented here suggest that dietary PS, especially SIT, may offer protection from the VSMC hyperproliferation found in atherosclerosis. Further in vivo research is needed to support these observations.     

Beta-type transforming growth factor specifies organizational behavior in vascular smooth muscle cell cultures

In culture, vascular smooth muscle cells (SMC) grow in a "hill-and-valley" (multilayered) pattern of organization. We have studied the growth, behavioral organization, and biosynthetic phenotype of rat aortic SMC exposed to purified platelet-derived growth regulatory molecules. We show that multilayered growth is not a constitutive feature of cultured SMC, and that beta-type transforming growth factor (TGF-beta) is the primary determinant of multilayered growth and the hill-and-valley pattern of organization diagnostic for SMC in culture. TGF-beta inhibited, in a dose-dependent manner, the serum- or platelet-derived growth factor-mediated proliferation of these cells in two-dimensional culture, but only when cells were plated at subconfluent densities. The ability of TGF-beta to inhibit SMC growth was inversely correlated to plating cell density. When SMC were plated at monolayer density (5 X 10(4) cells/cm2) to allow maximal cell-to-cell contact, TGF-beta potentiated cell growth. This differential response of SMC to TGF-beta may contribute to the hill-and-valley pattern of organization. Unlike its effect on other cell types, TGF-beta did not enhance the synthesis of fibronectin or its incorporation into the extracellular matrix. However, the synthesis of a number of other secreted proteins was altered by TGF-beta treatment. SMC treated with TGF-beta for 4 or 8 h secreted markedly enhanced amounts of an Mr 38,000-D protein doublet whose synthesis is known to be increased by heparin (another inhibitor of SMC growth), suggesting metabolic similarities between heparin- and TGF-beta-mediated SMC growth inhibition. The data suggest that TGF-beta may play an important and complex regulatory role in SMC proliferation and organization during development and after vascular injury.     

Purification of a 190 kDa protein from smooth muscle: relationship to talin

Several studies of vinculin-binding proteins have described a 190 kDa protein in chicken gizzard smooth muscle which binds radioiodinated vinculin. We have purified and studied the 190 kDa protein from chicken gizzard smooth muscle. By indirect immunofluorescence, an antiserum raised against the 190 kDa protein stains adhesion plaques (focal contacts), ruffling membranes, and fibrillar streaks on the dorsal and ventral surfaces of fibroblasts. Both the binding to vinculin and the location of the protein in fibroblasts are properties shared with talin, a 215 kDa protein in smooth muscle and fibroblasts. Because antisera against talin and the 190 kDa cross-react the relationship of these two proteins has been investigated further. Upon prolonged storage at 4 degrees C, purified talin degrades into a 190 kDa fragment. A 190 kDa fragment is also generated from talin by the Staphylococcus aureus V-8 proteinase and by trypsin. Comparison of partial peptide maps of talin and the 190 kDa protein reveal that the proteins are very similar and when the 190 kDa fragment of talin is compared with the purified 190 kDa protein by partial proteolytic digestion no differences are found in the pattern of peptides generated. In addition, the amount of 190 kDa protein detected in muscle tissues excised from chick embryos can be drastically reduced if proteinase inhibitors are added to the tissue homogenates. We conclude that the purified 190 kDa dalton protein is a proteolytic fragment of talin. Although markedly reduced by proteinase inhibitors, detection of the 190 kDa protein is not completely abolished, suggesting that some talin may already be cleaved within living cells.     

The effect of beta-aminopropionitrile on elastin gene expression in smooth muscle cell cultures.

When beta-aminopropionitrile (BAPN) is added to neonatal rat aortic smooth muscle cell cultures there is a decrease in insoluble elastin accumulation with a concomitant increase in tropoelastin and tropoelastin fragments in the culture medium. The experiments described here examine the biological significance of this fragmentation. BAPN, as well as purified tropoelastin fragments isolated from spent medium of cells grown in the presence of BAPN, were added to cultures. A decrease in elastin mRNA was observed in cultures grown in the presence of BAPN and also in those cultures to which the purified tropoelastin moieties were added. These studies indicate that the inhibition of lysyl oxidase by BAPN prevents elastin crosslinking which results in an increase in tropoelastin moieties, thus leading to a down regulation of the steady state levels of elastin mRNA.     

Vascular smooth muscle cell growth and insulin regulation of mitogen-activated protein kinase in hypertension

Hyperinsulinemia (HI) and insulin resistance (IR) are frequentlyassociated with hypertension and atherosclerosis. However, the exactroles of HI and IR in the development of hypertension are unclear.Mitogen-activated protein kinases (MAPK) are well-characterized intracellular mediators of cell proliferation. In this study, weexamined the contribution of MAPK pathway in insulin-stimulated mitogenesis using primary vascular smooth muscle cells (VSMCs) isolatedfrom aortas of normotensive Wistar-Kyoto rats (WKY) and spontaneoushypertensive rats (SHR). VSMCs were grown to confluence in culture,serum starved, and examined for DNA synthesis {using [³H]thymidine (TDR),immunoprecipitated MAPK activity, and MAPK phosphatase (MKP-1)induction}. Basal rate of TDR incorporation into DNA was twofoldhigher in SHR compared with WKY (P < 0.005). Insulin caused a dose-dependent increase in TDR incorporation (150% over basal levels with 100 nM in 12 h). Stimulation was sustained for 24 h with a decline toward basal in 36 h. Pretreatment with insulin-like growth factor I (IGF-I) receptor antibody did notabolish mitogenesis mediated by 10-100 nM insulin, suggesting thatinsulin effect is mediated via its own receptors. Insulin had a smallmitogenic effect in WKY (33% over basal). Insulin-stimulated mitogenesis was accompanied by a dose-dependent increase in MAPK activity in SHR, with a peak activation (>2-fold over basal) between 5 and 10 min with 100 nM insulin. Insulin had very small effects onMAPK activity in WKY. In contrast, serum-stimulated MAPK activation wascomparable in WKY and SHR. Pretreatment with MEK inhibitor, PD-98059,completely blocked insulin's effect on MAPK activation andmitogenesis. Inhibition of phosphatidylinositol 3-kinase with wortmannin also prevented insulin's effects on MAPK activation andmitogenesis. In WKY, insulin and IGF-I treatment resulted in a rapidinduction of MKP-1, the dual-specificity MAPK phosphatase. Incontrast, VSMCs from SHR were resistant to insulin with respect toMPK-1 expression. We conclude that insulin is mitogenic in SHR, and theeffect appears to be mediated by sustained MAPK activation due toimpaired insulin-mediated MKP-1 mRNA expression, which may act asan inhibitory feedback loop in attenuating MAPK signaling.

     

Sugars regulate cold-induced gene expression and freezing-tolerance in barley cell cultures

The hypothesis that the extracellular concentration of sugars helps regulate the acclimation of plant cells to cold was tested in this work. Suspension cultures were used to control the concentration of sugars in the medium supplied to barley cell cultures (Hordeum vulgare L. cv. Igri), replacing the medium daily to help maintain the concentration. Freezing tolerance and the levels of mRNA expression of the stress-response genes blt4.9 (coding for a non- specific lipid transfer protein) and dhn1 (coding for a dehydrin) were measured. Similar levels of freezing-tolerance and gene expression were obtained in the experiments as occur during cold-acclimation in the crown of the whole plant. In the cell cultures, cold (6/2 degrees C) did not induce an increase in freezing tolerance or in the expression of detectable levels of blt4.9 or dhn1 mRNAs when only 1 g l-1 sucrose was supplied. However, the cells in this low sucrose medium in the cold were not sugar-starved, indicating that this did not explain the failure of the cells to acclimate when grown in the cold environment. Ten g l-1 sucrose supplied to cells grown in the warm (25 degrees C) induced acclimation to freezing and up-regulation of expression of blt4.9 and dhn1 mRNAs. Osmolality of the medium did not explain this. Thirty g l-1 sucrose induced yet higher levels of freezing tolerance and of blt4.9 and dhn1 mRNAs in cultures grown in either the cold or the warm environment. The results implicate sugars in the regulation of cold acclimation     

Interaction of circulating cell-derived and plasma growth factors in stimulating cultured smooth muscle cell replication

Multiple growth factors that circulate in plasma have been shown to stimulate cellular growth in vitro. The plasma growth factors appear to stimulate DNA synthesis in cultured fibroblasts only after prior exposure of cell growth factors derived from circulating cell types, such as platelets and macrophages. The purpose of these studies was to investigate the role of the plasma growth factors in stimulating smooth muscle cell replication following exposure to platelet-derived growth factor (PDGF). Following transient exposure to PDGF, insulin stimulated smooth muscle cell replication but only when supraphysiologic concentrations were used (i.e., greater than 1.0 μg/ml). Somatomedin-C (Sm-C), in contrast, was found to stimulate a 320% increase in [³H]thymidine incorporation when concentrations that are present in extracellular fluids were used (i.e., 0.5–10 ng/ml). Epidermal growth factor (EGF), an important mitogen for multiple cell types, caused a 70% increase in [³H]thymidine incorporation when added to quiescent cells following PDGF exposure, and EGF caused a substantial increase in the absolute level of [³H]thymidine incorporation when coincubated with Sm-C. When EGF (1 ng/ml) was incubated simultaneously with concentrations of Sm-C between 1 and 10 ng/ml plus Sm-C-deficient plasma, maximal [³H]thymidine incorporation was 2.1-fold greater in the presence of EGF. In contrast, insulin (20 ng/ml), when coincubated with Sm-C under similar conditions, had no enhancing effect on the cellular response to Sm-C. None of the plasma factors tested was an effective stimultant of replication when incubated either in serum-free medium or in the presence of Sm-C-deficient plasma without prior PDGF exposure. Hydrocortisone was shown to inhibit smooth muscle cell replication in concentrations between 10^?7 and 10^?5M. In summary, multiple plasma growth factors can stimulate the smooth muscle cell replication, and Sm-C appears to be most effective of those tested. Insulin and EGF appear to work by different mechanisms; that is, EGF can facilitate the cellular response to Sm-C, whereas insulin is effective only at supraphysiologic concentrations at which it will directly bind to Sm-C receptors.     

Mechanical stretching stimulates smooth muscle cell growth, nuclear protein import, and nuclear pore expression through mitogen-activated protein kinase activation

Although it is known that mechanical stretching of cells can induce significant increases in cell growth and shape, the intracellular signaling pathways that induce this response at the level of the cell nucleus is unknown. The transport of molecules from the cell cytoplasm to the nucleoplasm through the nuclear pore is a key pathway through which gene expression can be controlled in some conditions. It is presently unknown if mechanical stimuli can induce changes in nuclear pore expression and/or function. The purpose of the present investigation was to determine if mechanical stretching of a cell will alter nuclear protein import and the mechanisms that may be responsible. Vascular smooth muscle cells that were mechanically stretched exhibited an increase in proliferating cell nuclear antigen expression, cell number, and cell size within 24-48 h. Cells were microinjected with marker proteins for nuclear import. Nuclear protein import was significantly stimulated in stretched cells when compared with control. This was associated with an increase in the expression of nuclear pore proteins as detected by Western blots. Inhibition of the MAPK pathway blocked the stretch-induced stimulation of both cell proliferation and nuclear protein import. We conclude that nuclear protein import and nuclear pore density can adapt to mechanical stimuli during the process of cell growth through a MAPK-mediated mechanism.     

Congo red binding of elastin in aortic smooth muscle cell cultures

These studies demonstrate that the strong binding capacity of elastin for Congo red can be used to advantage in aortic smooth muscle cell cultures. A fibrous elastin network fluoresces when Congo red is added. Congo red does not alter accumulation of elastin or of total protein, even when the cells are grown in the presence of the dye for long periods of time, indicating that it is not toxic. Porcine pancreatic elastase was used to solubilize elastin in these cultures, to determine the molar ratio of Congo red to elastin, thus making it possible to estimate the amount of elastin solubilized when the cultures are injured. Congo red binding to elastin will be useful in studying elastin accumulation and/or degradation in vitro and in vivo.     

Modulation of vascular smooth muscle cell growth by magnesium-role of mitogen-activated protein kinases

We tested the hypothesis that Mg(2+) influences growth of vascular smooth muscle cells (VSMCs) by modulating cell cycle activation through mitogen-activated protein (MAP) kinase-dependent pathways. Rat VSMCs were grown in culture medium containing normal Mg(2+) (1.02 mmol/L, control) and increasing concentrations of Mg(2+) (2-4 mmol/L) for 1-8 days. Effects of varying extracellular Mg(2+) concentration ([Mg(2+)](e)) on intracellular free Mg(2+) concentration ([Mg(2+)](i)) were assessed using mag-fura. Growth actions of Mg(2+) were evaluated by measuring cell cycle activation, DNA synthesis, and protein synthesis. Expression of cell cycle promoters, cyclin D1, cyclin E, Cdk2, and Cdk4 was assessed by immunoblotting. Phosphorylation of cell cycle inhibitors p21(cip1) and p27(kip1) and MAP kinases, ERK1/2, p38MAP kinase, and JNK was evaluated using phospho-specific antibodies. [Mg(2+)](i) increased in a dose-dependent manner in response to increasing [Mg(2+)](e). These effects were evident within 2 days and maximal responses were obtained after 6 days. High [Mg(2+)](e) induced cell cycle activation with a lower proportion of cells in G(1) phase (75 +/- 1.0%) and a higher fraction of cells in S phase (12 +/- 0.7%) versus control (G(1), 88.5 +/- 1.4%; S, 6.8 +/- 1.2%; P < 0.05). This was associated with increased protein content of cyclin D1 and Cdk4 and decreased activation of p21(cip1) and p27(kip1). In cells exposed to 2 mmol/L Mg(2+), DNA and protein synthesis was increased approximately threefold. Phosphorylation of MEK1/2 and ERK1/2 was enhanced two to threefold in cells grown in 2 mmol/L Mg(2+). These effects were rapid, occurring within 2 days. Phosphorylation of MEK3/6, p38 MAP kinase, and JNK was unaltered by increasing [Mg2](e). PD98059 (10(-5) mol/L), specific MEK1/2 inhibitor, but not SB202190 (10(-5) mol/L) (specific p38 MAP kinase inhibitor), attenuated Mg(2+)-induced growth actions. These data demonstrate the novel findings that cell cycle activation and growth regulation by Mg(2+) occurs via ERK1/2-dependent, p38 MAP kinase-independent pathways.     

Collagen synthesis and accumulation in long-term rabbit aortic smooth muscle cell cultures

Summary This study describes the ability of aortic smooth muscle cells to synthesize and accumulate collagen with time in culture. Inasmuch as smooth muscle cell cultures multilayer and continue to divide, albeit slowly, and can be maintained in the same vessels where seeded for extended periods of time, a long-term aging study from a single subcultivated population of cells was carried out. This is different from the usual cell-culture aging achieved by an increase in cell population doublings obtained by repeated subcultivations. The latter process, which is trypsin induced, involves a changing cellular environment including the extracellular matrix that is produced by the cells in culture. Second subcultures of weanling rabbit, aortic media, smooth muscle cells maintained for different periods of time up to 14 wk displayed decreasing hydroxyproline formation with time. Proline hydroxylation was determined by pulsing these second-passage cells with [¹⁴C]proline for 24 h at various times during the 14 wk period. The cell layer and medium were evaluated separately for radioactive proline and hydroxyproline and the medium for bacterial collagenase-susceptible protein as well. The percent of hydroxylation in the medium decreased from >31% within 1 wk after plating to 15.2% after 14 wk in culture. The percent of collagenase-susceptible protein in the medium decreased in a comparable manner. The DNA levels increased during the entire period although initially somewhat more rapidly. Accumulation of protein in the extracellular matrix continued during the 14-wk span. The accumulation of hydroxyproline in the extracellular matrix also continued to increase throughout the culture period, but it did slow down significantly. Yet the cells appear not to have lost their ability to accumulate connective tissue and protein in the insoluble cell layer. The data suggest clearly that the percent collagen synthesis relative to total protein synthesis decreases in the older cultures; total protein synthesis also decreases as expected. This study was supported by NIH Program Projects AG00001 and HL 13262.     

Role of PDGF-A expression in the control of vascular smooth muscle cell growth by transforming growth factor-beta

Transforming growth factor-beta (TGF-beta) is a multifunctional regulatory peptide that can inhibit or promote the proliferation of cultured vascular smooth muscle cells (SMCs), depending on cell density (Majack, R. A. 1987. J. Cell Biol. 105:465-471). In this study, we have examined the mechanisms underlying the growth-promoting effects of TGF-beta in confluent SMC cultures. In mitogenesis assays using confluent cells, TGF-beta was found to potentiate the stimulatory effects of serum, PDGF, and basic fibroblast growth factor (bFGF), and was shown to act individually as a mitogen for SMC. In gene and protein expression experiments, TGF-beta was found to regulate the expression of PDGF-A and thrombospondin, two potential mediators of SMC proliferative events. The induction of thrombospondin protein and mRNA was density-dependent, delayed relative to its induction by PDGF and, based on cycloheximide experiments, appeared to depend on the de novo synthesis of an intermediary protein (probably PDGF-A). The relationship between PDGF-A expression and TGF-beta-mediated mitogenesis was investigated, and it was determined that a PDGF-like activity (probably PDGF-A) was the biological mediator of the growth-stimulatory effects of TGF-beta on confluent SMC. The effects of purified homodimers of PDGF-A on SMC replication were investigated, and it was determined that PDGF-AA was mitogenic for cultured SMC, particularly when used in combination with other growth factors such as bFGF and PDGF-BB. The data suggest several molecular mechanisms that may account for the ability of TGF-beta to promote the growth of confluent SMC in culture.     

Diffusable growth factors induce bladder smooth muscle differentiation

Bladder smooth muscle differentiation is dependent on the presence of bladder epithelium. Previously, we have shown that direct contact between the epithelium and bladder mesenchyme (BLM) is necessary for this interaction. In this study, we tested the hypothesis that bladder smooth muscle can be induced via diffusable growth factors. Fourteen-day embryonic rat bladders were separated into bladder mesenchyme (prior to smooth muscle differentiation) and epithelium by enzymatic digestion and microdissection. Six in vitro experiments were performed with either direct cellular contact or no contact (1) 14-d embryonic bladder mesenchyme (BLM) alone (control), (Contact) (2) 14-d embryonic bladders intact (control), (3) 14-d embryonic bladder mesenchyme combined with BPH-1 cells (an epithelial prostate cell line) in direct contact, (4) 14-d embryonic bladder mesenchyme with recombined bladder epithelium (BLE) in direct contact, (No Contact) (5) 14-d embryonic bladder mesenchyme with BPH-1 prostatic epithelial cells cocultured in type 1 collagen gel on the bottom of the well, and (6) 14-d embryonic bladder mesenchyme with BPH-1 epithelium cultured in a monolayer on a transwell filter. In each case the bladder tissue was cultured on Millicell-CM 0.4-microm membranes for 7 d in plastic wells using serum free medium. Growth was assessed by observing the size of the bladder organoids in histologic cross section as well as the vertical height obtained in vitro. Immunohistochemical analysis of the tissue explants was performed to assess cellular differentiation with markers for smooth muscle alpha-actin and pancytokeratin to detect epithelial cells. Control (1) bladder mesenchyme grown alone did not exhibit growth or smooth muscle and epithelial differentiation. Contact experiments (2) intact embryonic bladder, (3) embryonic bladder mesenchyme recombined with BPH-1 cells, and (4) embryonic bladder mesenchyme recombined with urothelium each exhibited excellent growth and bladder smooth muscle and epithelial differentiation. Both noncontact experiments (5) and (6) exhibited growth as well as bladder smooth muscle and epithelial differentiation but to a subjectively lesser degree than the contact experiments. Direct contact of the epithelium with bladder mesenchyme provides the optimal environment for growth and smooth muscle differentiation. Smooth muscle growth and differentiation can also occur without direct cell to cell contact and is not specific to urothelium. This data supports the hypothesis that epithelium produces diffusable growth factors that induce bladder smooth muscle.     

影响主动脉平滑肌细胞迁移的因素

平滑肌细胞(vascular smooth muscle cell,VSMC)的迁移对血管发育、动脉粥样硬化和术后再狭窄等起到关键性的作用。主要从激发VSMC迁移的关键炎性细胞因子、细胞间相互作用的核心成员、microRNA、细胞骨架和上述各因素的迁移信号通路这几方面来综述VSMC的迁移。     

Integrin-linked kinase functions as a downstream signal of platelet-derived growth factor to regulate actin polymerization and vascular smooth muscle cell migration

Background  

Vascular smooth muscle cell migration and accumulation in response to growth factors extensively contribute to the development of intimal thickening within the vessel wall. Cumulative evidence has shown that actin cytoskeleton polymerization and rearrangement are critical steps during cellular spreading and migration. Integrin-linked kinase, an intracellular serine/threonine kinase, is a cytoplasmic interactor of integrin beta-1 and beta-3 receptors regulating cell-cell and/or cell-extracellular matrix interaction, cell contraction, extracellular matrix modification, and cell spreading and migration in response to various stimuli. However, the regulatory role of ILK during vascular smooth muscle cell migration and the importance of integrin signaling in occlusive vascular diseases are not yet fully elucidated.