Phosphatidylinositol 3-kinase and focal adhesion kinase are early signals in the growth factor-like responses to thrombospondin-1 seen in human vascular smooth muscle.

Thrombospondin-1 (TSP-1) is a matricellular protein that is expressed in negligible amounts in normal blood vessels but is markedly upregulated in vascular injury. Although TSP-1 can act as a pleiotropic regulator for human vascular smooth muscle cells (HVSMCs), the intracellular signaling pathways stimulated by this protein remain obscure. In cultured HVSMCs derived from saphenous vein, TSP-1 induces tyrosine phosphorylation of a number of cellular proteins, with a complex temporal pattern of activation. Immunoprecipitation techniques have identified the early tyrosine-phosphorylated signals as being the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI 3-K) and focal adhesion kinase (FAK). Tyrosine phosphorylation of the p85 subunit of PI 3-K showed a biphasic response to TSP-1 stimulation, which corresponded to a biphasic activation of the lipid kinase. Treatment with both wortmannin and LY294002 inhibited PI 3-K activity of HVSMCs but did not affect tyrosine phosphorylation of the p85 regulatory subunit. TSP-1-stimulated FAK phosphorylation, however, was substantially reduced by these inhibitors, as was the TSP-1-induced chemotaxis of these cells. These results suggest that activation of PI 3-K is an early signal induced by TSP-1 and is critical for chemotaxis. Activation of this kinase precedes and may occur upstream from FAK phosphorylation, although the nature of the interaction between these 2 enzymes remains obscure.

Effect of hypercholesterolaemia on voltage-operated calcium channel currents in rabbit arterial smooth muscle cells.

Cholesterol is a major component of cell membranes and influences membrane fluidity. Watanabe heritable hyperpercholesterolaemic rabbits (WHHL) possess defective receptors for low density lipoprotein leading to increased plasma cholesterol, accumulation of cholesterol in the arterial wall and atherosclerosis. In this study calcium channel currents (IBa) were compared using conventional whole cell voltage clamp techniques in ear artery cells isolated from control New Zealand White rabbits (NZ) with those from WHHL. IBa were larger in cells isolated from NZ than from WHHL, however cell capacitance was also greater in NZ cells. Consequently, there was no significant difference in current density between NZ and WHHL cells either in the absence of drug or in the presence of the calcium channel agonist (+)202 791. Current voltage-relationships, kinetics of fast inactivation and steady-state inactivation of IBa also did not differ significantly between WHHL and NZ. These findings suggest that hypercholesterolaemia in WHHL has no direct effect on calcium channel current density or voltage-modulation in arterial smooth muscle cells.

Role of intracellular calcium ([Ca2+]i) and tyrosine phosphorylation in adhesion of cultured vascular smooth muscle cells to fibrinogen.

OBJECTIVE: Fibrinogen is an independent risk factor for cardiovascular disease. This study has investigated the role of intracellular Ca2+ ([Ca2+]i) and tyrosine phosphorylation in the attachment of human and rat-derived cultured vascular smooth muscle cells to fibrinogen. METHODS: Cells were cultured from human saphenous vein segments (HVSMC) and from an established rat aortic cell line (A7r5). [Ca2+]i was measured using fura-2 and adhesion was studied using pre-coated 96 well polystyrene plates. RESULTS: Fibrinogen increased [Ca2+]i in both cell types. In A7r5 cells fibrinogen-induced increases in [Ca2+]i were partially inhibited by a peptide containing the amino acid sequence Arg-Gly-Asp (RGD) which interferes with binding to integrins. In contrast RGD increased [Ca2+]i in HVSMC, but did not inhibit responses to fibrinogen. Ni2+, an inorganic calcium channel blocker largely abolished the rise in [Ca2+]i, but blockers of voltage-operated calcium channels failed to affect [Ca2+]i responses to fibrinogen in either cell type. Genistein, an inhibitor of tyrosine kinase inhibited fibrinogen-induced rises in [Ca2+]i, while daidzein, an inactive analogue, was without effect. Adhesion of cells to fibrinogen was concentration- and time-dependent. Cell adhesion to fibrinogen was partially inhibited by RGD peptide in both cell types. Adhesion of cell to fibrinogen was inhibited by chelation of [Ca2+]i with BAPTA-AM, inhibition of Ca2+ entry by Ni2+, and inhibition of tyrosine kinases by genistein, but heparin had no effect on adhesion. CONCLUSIONS: Vascular smooth muscle cells attach to fibrinogen in part through RGD-type interactions. Activation of tyrosine kinase(s) and a subsequent rise in [Ca2+]i appear to be important signals mediating the response to fibrinogen.

Platelet-derived growth factor beta-receptors can both promote and inhibit chemotaxis in human vascular smooth muscle cells.

The effect of the three platelet-derived growth factor (PDGF) isoforms AA, AB, and BB on migration was investigated in cultured human saphenous vein smooth muscle cells. The modified Boyden chamber technique yielded efficacies BB >> AB, AA = 0. However, the BB concentration-response relationship displayed a pronounced peak, occurring between 1 and 10 ng/mL, with no response above this range. Checkerboard analysis showed that the promotion of migration at low concentrations was chemotactic in nature but that the downturn was independent of gradient. Furthermore, at high concentrations BB was able to prevent chemotaxis induced by fetal calf serum and epidermal growth factor (EGF). Experiments using low concentrations of BB in combination with high concentrations of AA to saturate PDGF alpha-receptors in the presence and absence of a neutralizing antibody to alpha-receptors revealed that alpha-receptor activation induced partial inhibition of chemotaxis but this did not account for the inhibition of migration by high concentrations of BB. Despite possessing no significant chemotactic action itself, high concentrations of the AB isoform completely inhibited BB induced chemotaxis. Taken together these results suggest that the chemotactic signal induced by PDGF is dominated by PDGF beta-receptors and switches from positive at low concentrations to negative at higher concentrations. Stimulation of DNA synthesis by the three isoforms (as measured by [3H] thymidine incorporation) yielded saturable responses for the AB and BB isoforms, with similar efficacy and weak or no response for the AA isoform. Concentration-dependent patterns of tyrosine phosphorylation of certain proteins mirrored the form of the chemotactic response and suggest one possible underlying regulatory mechanism to account for the disparity between PDGF-induced chemotaxis and DNA synthesis.

Thrombospondin-1 is a potent mitogen and chemoattractant for human vascular smooth muscle cells.

Thrombospondin-1 (TSP-1) is a matricellular protein that is present in negligible amounts in normal human vasculature but occurs in significant amounts in diseased vessels. In this study, we examined the effect of TSP-1 on DNA synthesis, proliferation, and migration in human vascular smooth muscle cells grown from saphenous vein. TSP-1 (0.1 to 30 micrograms/mL) elicited a concentration-dependent increase in DNA synthesis under serum-free conditions. In combination with platelet-derived growth factor, TSP-1 induced a synergistic effect on DNA synthesis that was significantly higher than the additive effect of both agents. In proliferation assays, TSP-1 increased cell numbers by 50% relative to the serum-free controls over 14 days. In migration assays, conducted using modified Boyden chambers, TSP-1 (> or = 10 micrograms/mL) elicited marked chemotaxis to a degree equivalent to platelet-derived growth factor. The chemotactic response to TSP-1 (10 micrograms/mL) was abolished by the GRGDSP peptide but unaffected by the control GRGESP peptide, whereas neither peptide inhibited DNA synthesis stimulated by TSP-1. Inhibition of tyrosine kinase activity with genistein or tyrphostin A23 abolished DNA synthesis induced by TSP-1, and a neutralizing antibody to platelet-derived growth factor had no effect on DNA synthesis. Similarly, migration in response to TSP-1 was largely inhibited by these tyrosine kinase inhibitors. TSP-1 is a strong mitogen and chemoattractant for human vascular smooth muscle cells under serum-free conditions. The novel finding that TSP-1 is mitogenic for human cells contrasts with previous studies that have not shown any significant effect of TSP-1 itself on the growth of animal-derived smooth muscle cells. TSP-1 may play an important modulatory role in the local regulation of vascular smooth muscle function in vascular pathologies in humans.

Differential effects of lovastatin on mitogen induced calcium influx in human cultured vascular smooth muscle cells.

1. In this study the effect of lovastatin, an inhibitor of cholesterol and isoprenoid synthesis, on the rises in intracellular calcium concentration ([Ca2+]i) induced by platelet derived growth factor BB (PDGF-BB), angiotensin II (AII), low density lipoproteins (LDL) and foetal calf serum (FCS) was examined in human cultured vascular smooth muscle cells (VSMC) from saphenous vein. Changes in [Ca2+]i were measured in cell suspensions by the Ca2+ sensitive probe, fura 2. 2. Incubation with lovastatin for 24-26 h markedly reduced the peak rise and sustained phase of [Ca2+]i elevation in response to PDGF-BB but the responses to AII, LDL and FCS were unaffected. Further experiments showed that lovastatin pretreatment inhibited PDGF-BB induced Ca2+ influx but not intracellular Ca2+ release. This inhibition could be overcome by co-incubation with mevalonic acid. 3. Pretreatment of cells with the heterotrimeric G protein inhibitor pertussis toxin for up to 24 h completely abolished AII-induced [Ca2+]i rises but the response to PDGF-BB was unaffected. 4. The tyrosine kinase inhibitor genistein largely abolished PDGF-BB-induced [Ca2+]i elevation but had no significant effect on AII-induced responses. 5. Pre-incubation with lovastatin had no effect on the level of tyrosine phosphorylation of PDGF-beta receptors (as measured by Western blot) in response to the PDGF-BB ligand. 6. PDGF-BB elicits Ca2+ influx via a tyrosine kinase-dependent mechanism distinct from the heterotrimeric G protein coupled pathway utilized by AII. Lovastatin most likely acts by inhibition of isoprenylation (via blockade of isoprenoid synthesis) of an intermediate molecule involved in PDGF-BB-induced Ca2+ influx.

Platelet-derived growth factor (PDGF): actions and mechanisms in vascular smooth muscle.

1. PDGF is a highly hydrophilic cationic glycoprotein (M(r) 28-35kDa) produced by platelets, monocyte/macrophages, endothelial cells and vascular smooth muscle cells under some conditions. 2. Since its original description, PDGF has attracted much attention and it is currently believed to play a role in atherosclerosis and other vascular pathologies. 3. This review describes the vascular biology of PDGF. It particularly focuses on recent findings regarding the intracellular signals activated by PDGF in the context of vascular smooth muscle cell proliferation, migration and, contraction.

Effect of angiotension II on the expression of the early growth response gene c-fos and DNA synthesis in human vascular smooth muscle cells.

OBJECTIVES: The aims of this study were to characterize the angiotensin II receptor subtype present on vascular smooth muscle cells from human saphenous vein and to assess the effect of angiotensin II on the expression of the early growth response gene c-fos and on DNA synthesis. METHODS AND RESULTS: Using radioligand binding studies, we have defined the angiotensin II receptors present on these cells as being predominantly of the AT1 subtype. Angiotensin II increased peak intracellular calcium levels by 126 +/- 16 nmol/l (mean +/- SEM) in 17/49 cultures. Angiotensin II induced c-fos expression in a concentration-dependent manner only in cultures that exhibited an intracellular calcium transient in response to stimulation with angiotensin II. The induction of c-fos was inhibited by the selective AT1 antagonist losartan in accordance with the binding studies. Angiotensin II stimulated DNA synthesis with a maximal increase of 66.4% +/- 20.5% over serum-free levels at 1 nmol/l (mean +/- SEM, n = 6, P < 0.05). DNA synthesis declined with increasing angiotensin II concentration, falling to control values at 1 mumol/l, suggesting that a growth-inhibitory influence may counter-balance the stimulatory effect that is observed at lower concentrations. CONCLUSION: Vascular smooth muscle cells from human saphenous vein possess predominantly AT1 receptors and in response to angiotensin II show an induction of c-fos and a modest increase in DNA synthesis.