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±16nmol/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 µmol/l, suggesting that a growth-inhibitory influence may counterbalance 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.
Thrombospondin-1 is a large matricellular protein that acts as a pleiotropic growth factor for human vascular smooth muscle cells, and may play a role in the progression of vascular disease. Although we have previously demonstrated the dependence of both thrombospondin-1-stimulated cell chemotaxis and proliferation on tyrosine kinases, the receptor mechanisms involved remain obscure. This investigation aims to determine the nature of the receptor(s) involved in the cellular responses to thrombospondin-1. Cellular signals were identified by western blotting following cell stimulation, while cellular responses were assessed by measuring DNA synthesis and chemotaxis. These data demonstrate that thrombospondin-1-induced cell chemotaxis can be inhibited by a peptide containing the Arg-Gly-Asp motif, a function-blocking αvβ3 antibody, a function-blocking integrin-associated protein (IAP) antibody and pertussis toxin, while thrombospondin-1-stimulated DNA synthesis is inhibited by a function-blocking α3β1 antibody. Similarly the Arg-Gly-Asp-containing peptide inhibits tyrosine phosphorylation of focal adhesion kinase and the p85 regulatory subunit of phosphatidylinositol 3-kinase, but does not significantly affect tyrosine phosphorylation, or activation, of extracellular-regulated kinase. These data suggest that soluble thrombospondin-1 interacts with human vascular smooth muscle cells via two independent and separable receptor-binding sites, to differentially stimulate cell chemotaxis and DNA synthesis.
Abstract 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 α-receptors in the presence and absence of a neutralizing antibody to α-receptors revealed that α-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 β-receptors and switches from positive at low concentrations to negative at higher concentrations. Stimulation of DNA synthesis by the three isoforms (as measured by [ 3 H] 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.
Abstract 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 μg/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 (≥10 μg/mL) elicited marked chemotaxis to a degree equivalent to platelet-derived growth factor. The chemotactic response to TSP-1 (10 μg/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.
Abstract Restenosis remains the largest single obstacle to the long‐term success of invasive vascular interventions. Lovastatin, an HMG‐CoA reductase inhibitor, has been shown to reduce myointimal hyperplasia in animal models of restenosis and in one clinical coronary restenosis trial. We have assessed the effect of lovastatin on the growth of cultured human vascular smooth muscle cells derived from saphenous vein and vascular graft stenoses. Lovastatin (2 μM) inhibited proliferation over 14 days in saphenous vein (and graft stenoses) derived vascular smooth muscle cells by 42% and 32%, respectively: this was not significantly different. Lovastatin (10 μM) reduced [methyl 3H]‐thymidine uptake by 51% in saphenous vein‐derived cells. These concentrations were significantly higher than those achieved in plasma during therapeutic dosage. Lovastatin‐induced inhibition of vascular smooth muscle cell proliferation and [methyl 3 H]‐thymidine uptake was completely reversed by adding mevalonate (100 μM) but cholesterol (10–40 μl ‐1 ) had no effect. Isopentenyl adenine (25–50 μM) did not affect the inhibition of [methyl 3 H]‐thymidine uptake by lovastatin (10 μM), but farnesol (20 μM), another isoprenoid precursor of cholesterol synthesis, reversed the antiproliferative effect.
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