Modulation of high affinity phenylalkylamine binding sites on cultured human embryonal vascular smooth muscle cells.

Two phenylalkylamine Ca2+ channel ligands, (+/-)-[3H]verapamil ((+/-)-[3H]V) (-)-[3H]desmethoxyverapamil ((-)-[3H]DV), were employed in whole cell binding assays to characterize the specific high affinity binding sites on Ca2+ channels, their cooperativity and modulations induced on cultured human embryonal vascular smooth muscle preparation (VSM) by: 1) Beta-adrenergic stimulation of the cell, 2) exposure to high K+ concentration, 3) exposure to high concentration of Mg2+ ions, 4) the presence of a benzothiazepine Ca2+ channel antagonist and modulator d-cis-diltiazem, and 5) guanylylimidodiphosphate. The total amounts of specific (+/-)-[3H]V and (-)-[3H]DV binding sites present on VSM cells increased significantly after beta-adrenergic receptor activation, following cell membrane depolarization induced by high concentrations of K+, in the presence of Ca2+ chelator Na3EDTA, and after incubation of VSM cells with a benzothiazine-type Ca2+ channel blocker d-cis-diltiazem. A marked reduction of (-)-[3H]DV binding was observed after permanent G-protein activation by a nonhydrolyzable analog of guanylylimidodiphosphate, after incubation of the cells with norepinephrine, and after incubation of VSM cells with millimolar concentration of Mg2+. The results suggest the existence of multiple modulations of specific (-)-[3H]DV binding sites on Ca2+ channel corresponding to the way of activation of the cell and also to the immediate "state" of the membrane bound Ca2+ channels present on VSM cells, the positive heterotropic interaction after beta-adrenergic stimulation, the homotropic positive allosteric interaction induced by d-cis-diltiazem and pure noncompetitive inhibition induced by guanylylimidodiphosphate. The presence of high concentrations of Mg2+ inhibited whereas the presence of Ca2+ chelator, of ethylenediamine-tetraacetic acid sodium salt, significantly increased the total number of specific high affinity (-)-[3H]DV binding sites on VSM cells.     

Arterial smooth muscle cell proteoglycans synthesized in the presence of glucosamine demonstrate reduced binding to LDL.

Atherosclerosis is the main cause of morbidity and mortality in diabetes, yet the underlying mechanisms remain unclear. Retention of atherogenic lipoproteins by vascular proteoglycans is thought to play a key role in the development of atherosclerotic lesions. High glucose levels cause a variety of diabetic complications by several mechanisms, including upregulation of the hexosamine pathway. Glucosamine, a component of the hexosamine pathway, is a precursor for the synthesis of glycosaminoglycan components of proteoglycans. This study evaluated whether high glucose or glucosamine supplementation of vascular smooth muscle cells would increase proteoglycan synthesis, leading to increased lipoprotein retention. Aortic smooth muscle cells were exposed to physiologic (5.6 mM) or high (25 mM) glucose levels, such as seen in diabetes, or to glucosamine (12 mM). Extracellular proteoglycans were characterized by sulfate incorporation, molecular sieve chromatography, and SDS-PAGE. LDL interactions were assessed by affinity chromatography and gel mobility shift assay. Proteoglycans synthesized in the presence of high glucose demonstrated no differences in size, sulfate incorporation, or LDL binding affinity compared with proteoglycans synthesized under physiological glucose conditions. However, proteoglycans synthesized in the presence of glucosamine had smaller glycosaminoglycan chains than control proteoglycans with a corresponding decrease in lipoprotein retention.Thus, glucose and glucosamine have different effects on proteoglycan biosynthesis and different effects on lipoprotein retention.     

High binding affinity of electronegative LDL to human aortic proteoglycans depends on its aggregation level

Electronegative LDL [LDL(-)] is an atherogenic subfraction of plasma LDL that has increased apolipoprotein E (apoE) and apoC-III content, high density, and increased susceptibility to aggregation. These characteristics suggest that LDL(-) could bind to proteoglycans (PGs); therefore, our aim was to evaluate its affinity to PGs. Binding of LDL(-) and native LDL [LDL(+)] to human aortic PGs was determined by precipitation of LDL-glycosaminoglycan complexes, LDL incubation in PG-coated microtiter wells, and affinity chromatography on PG column. All methods showed that LDL(-) had higher binding affinity to PGs than did LDL(+). PG capacity to bind LDL(-) was increased approximately 4-fold compared with LDL(+) in precipitation and microtiter assays. Chromatography on PG column showed LDL(-) to consist of two subpopulations, one with higher and one with lower PG binding affinity than LDL(+). Unexpectedly, the lower PG affinity subpopulation had increased apoE and apoC-III content. In contrast, the high PG affinity subpopulation presented phospholipase C (PLC)-like activity and increased aggregation. These results suggest that PLC-like activity could alter LDL lipid composition, thereby promoting particle aggregation and binding to PGs. This propensity of a subpopulation of LDL(-) to bind to PGs could facilitate its retention in the extracellular matrix of arterial intima and contribute to atherosclerosis progression.     

Fibrinogen is chemotactic for vascular smooth muscle cells

We studied the effect of fibrinogen on the migration of bovine aortic smooth muscle cells in culture, using a Neuro Probe 48-well micro chemotaxis chamber. Fibrinogen stimulated the migration of the cells dose-dependently at concentrations from 30 to 1000 micrograms/ml. A modified checkerboard analysis of the response demonstrated that the effect was largely chemotactic in nature. The present results suggest that fibrinogen may play an important role in the pathogenesis of arterial intimal thickening and atherosclerosis.     

Cholesterol is necessary both for the toxic effect of Abeta peptides on vascular smooth muscle cells and for Abeta binding to vascular smooth muscle cell membranes

Accumulation of beta amyloid (Abeta) in the brain is central to the pathogenesis of Alzheimer's disease. Abeta can bind to membrane lipids and this binding may have detrimental effects on cell function. In this study, surface plasmon resonance technology was used to study Abeta binding to membranes. Abeta peptides bound to synthetic lipid mixtures and to an intact plasma membrane preparation isolated from vascular smooth muscle cells. Abeta peptides were also toxic to vascular smooth muscle cells. There was a good correlation between the toxic effect of Abeta peptides and their membrane binding. 'Ageing' the Abeta peptides by incubation for 5 days increased the proportion of oligomeric species, and also increased toxicity and the amount of binding to lipids. The toxicities of various Abeta analogs correlated with their lipid binding. Significantly, binding was influenced by the concentration of cholesterol in the lipid mixture. Reduction of cholesterol in vascular smooth muscle cells not only reduced the binding of Abeta to purified plasma membrane preparations but also reduced Abeta toxicity. The results support the view that Abeta toxicity is a direct consequence of binding to lipids in the membrane. Reduction of membrane cholesterol using cholesterol-lowering drugs may be of therapeutic benefit because it reduces Abeta-membrane binding.     

Mechanical strain induces specific changes in the synthesis and organization of proteoglycans by vascular smooth muscle cells

In the mechanically active environment of the artery, cells sense mechanical stimuli and regulate extracellular matrix structure. In this study, we explored the changes in synthesis of proteoglycans by vascular smooth muscle cells in response to precisely controlled mechanical strains. Strain increased mRNA for versican (3.2-fold), biglycan (2.0-fold), and perlecan (2.0-fold), whereas decorin mRNA levels decreased to a third of control levels. Strain also increased versican, biglycan, and perlecan core proteins, with a concomitant decrease in decorin core protein. Deformation did not alter the hydrodynamic size of proteoglycans as evidenced by molecular sieve chromatography but increased sulfate incorporation in both chondroitin/dermatan sulfate proteoglycans and heparan sulfate proteoglycans (p < 0.05 for both). Using DNA microarrays, we also identified the gene for the hyaluronan-linking protein TSG6 as mechanically induced in smooth muscle cells. Northern analysis confirmed a 4.0-fold increase in steady state mRNA for TSG6 following deformation. Size exclusion chromatography under associative conditions showed that versican-hyaluronan aggregation was enhanced following deformation. These data demonstrate that mechanical deformation increases specific vascular smooth muscle cell proteoglycan synthesis and aggregation, indicating a highly coordinated extracellular matrix response to biomechanical stimulation.     

Dysregulation of CREB binding protein triggers thrombin-induced proliferation of vascular smooth muscle cells

Thrombin is a potent mitogen for vascular smooth muscle cells (VSMCs). CBP has been regarded as a potential therapeutic target on the basis of its ability to affect cell growth. Therefore we hypothesized that CBP mediates thrombin-induced proliferation of VSMCs. We constructed recombinant adenoviral vector that expresses four short hairpin RNA (shRNA) targeting rat CBP mRNA (CBP-shRNA/Ad). VSMCs were infected with CBP-shRNA/Ad and treated with thrombin. CBP level were analyzed by quantitative real-time PCR and Western blot. To evaluate VSMC proliferation, the cell cycle and DNA synthesis were analyzed by flow cytometry and (3)H-thymidine incorporation, respectively. CBP-shRNA/Ad infection inhibited thrombin-induced CBP expression in a dose-dependent manner concomitant with a decrease in the percentage of cells in the S phase and in DNA synthesis. These findings suggest that CBP plays a pivotal role in the S phase progression of VSMCs.     

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

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

Sulphate turnover of surface proteoglycans in cultured rat smooth muscle cells

Turnover of radioactive sulphate-labelled proteoglycans in cultured rat smooth muscle cells was detected by pulse chase techniques. The degradation appeared to take the form of desulphation of sulphated macromolecules, with a loss in total sulphate of approximately 50% in 5 days. The desulphation process occurred in the pericellular/matrix compartment of the culture system and was unaffected by inhibition of matrix formation by beta-aminopropionitrile, or by incubation of cells with lysomotropic inhibitors. There was no evidence for further degradation of desulphated species even when exogenous, radio-labelled proteoglycans were added to fresh cultures and incubated for four days. Labelled macromolecules initiated on xyloside acceptors were desulphated by rat smooth muscle cell cultures more slowly than intact proteoglycans.     

Vascular smooth muscle cells synthesize,secrete and express coagulation factor V

Expression of cellular procoagulant activity may be one of the more important responses to vascular injury. Because factor V, a coagulation cofactor in the prothrombinase complex, catalyzes the conversion of prothrombin to thrombin, it may be a key to understanding this response. Therefore, we have investigated the synthesis, secretion and expression of factor V by vascular smooth muscle cells, which proliferate at sites of vascular injury. Cultured aortic vascular smooth muscle cells constitutively secreted Factor V activity, as determined by a functional assay. Labeled factor V was immunoprecipitated from conditioned medium of [³⁵S]methionine-labeled cells, indicating that the secreted factor V was synthesized by vascular smooth muscle cells. Treatment of vascular smooth muscle cells with tunicamycin prevented secretion of factor V, suggesting that its secretion was dependent on the presence of N-linked carbohydrate. Factor V activity was also expressed on the vascular smooth muscle cell surface, as indicated by the ability of cultured cells to promote factor X_a-catalyzed prothrombin activation. These data suggest that the proliferation of smooth muscle cells in response to vascular injury may be one mechanism that links vascular disease with thrombosis.     

Small GTP binding protein Ras contributes to norepinephrine-induced mitogenesis of vascular smooth muscle cells

Norepinephrine stimulates release of arachidonic acid from tissue lipids. Arachidonic acid metabolites generated through the lipoxygenase and cytochrome P-450 pathways but not cyclooxygenase stimulate mitogen activated protein (MAP) kinase activity and proliferation of vascular smooth muscle cells (VSMC). Moreover, norepinephrine has been shown to activate the Ras/MAP kinase pathway through generation of cytochrome P450 metabolite of arachidonic acid, 20-hydroxyeicosatetraenoic acid (20-HETE). The purpose of this study was to investigate the contribution of Ras in norepinephrine-induced mitogenesis in aortic VSMC. Farnesylation of Ras by farnesyl transferase is required for its full activation. Norepinephrine-induced DNA synthesis, as measured by [3H]-thymidine incorporation, was attenuated by inhibitors of Ras farnesyl transferase FPT III and BMS-191563. These agents also inhibited 20-HETE-stimulated [3H]-thymidine incorporation. In cells transiently transfected with dominant negative Ras (RasN17), norepinephrine, and 20-HETE-induced proliferation of VSMC was attenuated. Both norepinephrine and 20-HETE increased localization of Ras to plasma membrane and MAP kinase activity; FPT III attenuated these effects. These data suggest that VSMC proliferation induced by norepinephrine and 20-HETE is mediated by Ras/MAP kinase pathway.     

Stem cells, vascular smooth muscle cells and atherosclerosis

Stem cells have the ability to differentiate into a variety of cells to replace dead cells or to repair tissue. Recently, accumulating evidence indicates that mechanical forces, cytokines and other factors can influence stem cell differentiation into vascular smooth muscle cells (SMCs). In developmental process, SMCs originate from several sources, which show a great heterogenicity in different vessel walls. In adult vessels, SMCs display a less proliferative nature, but are altered in response to risk factors for atherosclerosis. Traditional view on SMC origins in atherosclerotic lesions is challenged by the recent findings that stem cells and smooth muscle progenitors contribute to the development of atherosclerotic lesions. Vascular progenitor cells circulating in human blood and the presence of adventitia in animals are recent discoveries, but the source of these cells is still unknown. The present review gives an update on the progress of stem cell and SMC research in atherosclerosis, and discusses possible mechanisms of stem/progenitor cell differentiation that contribute to the disease process.     

A membrane potential-sensitive dye for vascular smooth muscle cells assays

Changes in membrane potential of rat aorta smooth muscle cells were investigated using the bis-oxonol sensitive probe DIBAC2(3). We compared the changes in membrane potential induced by a high external KCl concentration in aorta smooth muscle cells from normotensive 2 kidney (2K) and from renal hypertensive 2 kidney-1 clip (2K-1C) rats. The spectral properties of the membrane potential were first characterized in aqueous buffers and in cultured smooth muscle cells from 2K and 2K-1C rat aortas. Fluorescence emission and the images were recorded using a laser scanning confocal microscope. The relationship between fluorescence intensity (FI) and membrane potential (psi(m)) as a function of the increasing extracellular KCl concentration was linear in the 5-40 mmol/L KCl range in both 2K and 2K-1C rat aorta cells. Cell membranes from 2K-1C rat aorta cells were more depolarized (-55 mV) than 2K rat aorta cells (-65 mV). The results show that in 2K-1C aorta cells only 10 mmol/L KCl was needed to induce complete membrane depolarization while in 2K cells 40 mmol/L KCl was needed to induce a similar effect. This study clearly shows that the method is suitable to measure the membrane potential in cultured smooth muscle cells.     

LOX-1 mediates lysophosphatidylcholine-induced oxidized LDL uptake in smooth muscle cells

To assess the role of 14-3-3 proteins in the magnesium-dependent inhibition of nitrate reductase (NR) we tested the effect of magnesium on NR binding to 14-3-3s by coimmunoprecipitation and gel filtration. The stability of the 14-3-3 complex of NR was, unlike its activity, unaffected by magnesium. We therefore conclude that binding to 14-3-3s per se does not inhibit NR. Magnesium inhibited 14-3-3-bound NR much more strongly than 14-3-3-free NR. 14-3-3s possibly reinforce NR inhibition by magnesium.     

LDL receptor-related protein LRP6 regulates proliferation and survival through the Wnt cascade in vascular smooth muscle cells

Initial studies have established expression of low-density lipoprotein (LDL) receptor-related protein 6 (LRP6) in vascular smooth muscle cells (VSMCs). We hypothesized that LRP6 is a critical mediator governing the regulation of the canonical Wnt/beta-catenin/T cell factor 4 (Tcf-4) cascade in the vasculature. This hypothesis was based on our previous work demonstrating a role for the beta-catenin/Tcf-4 pathway in vascular remodeling as well as work in other cell systems establishing a role for LRP family members in the Wnt cascade. In line with our hypothesis, LRP6 upregulation significantly increased Wnt-1-induced Tcf activation. Moreover, a dominant interfering LRP6 mutant lacking the carboxyl intracellular domain (LRP6DeltaC) abolished Tcf activity. LRP6-induced stimulation of Tcf was blocked in VSMCs harboring constitutive expression of a dominant negative Tcf-4 transgene lacking the beta-catenin binding domain, suggesting that LRP6-induced activation of Tcf was mediated through a beta-catenin-dependent signal. Expression of the dominant interfering LRP6DeltaC transgene was sufficient to abolish the Wnt-induced survival as well as cyclin D1 activity and cell cycle progression. In conclusion, these findings provide the first evidence of a role for an LDL receptor-related protein in the regulation of VSMC proliferation and survival through the evolutionary conserved Wnt signaling cascade.