Laura J. Betteridge

Inhibition of human vascular smooth muscle cell proliferation by lovastatin: the role of isoprenoid intermediates of cholesterol synthesis

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 3H]‐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 3H]‐thymidine uptake by lovastatin (10 μM), but farnesol (20 μM), another isoprenoid precursor of cholesterol synthesis, reversed the antiproliferative effect.

Abnormal growth regulation of vascular smooth muscle cells by heparin in patients with restenosis

Proliferation of vascular smooth muscle cells (VSMC) underlies myointimal hyperplasia, which can lead to restenosis after angioplasty and vascular surgery. We propose that some individuals have an intrinsic capacity for this exaggerated response to vascular injury, partly through decreased sensitivity to the physiological growth inhibitor heparin. We investigated the effect of heparin on VSMC from restenotic lesions and from apparently normal vessels of the same patients, and VSMC from control patients undergoing primary bypass procedures. Cells from patients with restenosis (both restenotic lesion and undiseased vein) showed much lower sensitivity to growth inhibition by heparin than the controls (median inhibition 8 [95% Cl -2 to 25] vs 22 [15-44]%, p < 0.001); this finding suggests aberrant growth regulation in these cells.

Effect of angiotensin 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±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.

Effect of calcium channel blockers on the growth of human vascular smooth muscle cells derived from saphenous vein and vascular graft stenoses.

Summary: Vascular restenosis after invasive interventions is an important clinical problem for which no preventive pharmacologic therapy exists. Calcium channel blockers have been shown to inhibit myointimal hyper-plasia in animal models of restenosis and in some small and flawed clinical coronary restenosis trials. We examined the inhibitory effect of amlodipine, verapamil, and diltiazem on the growth of cultured human vascular smooth muscle cells (VSMC) derived from saphenous vein (n = 20) and graft stenoses (n = 7), in 14-day proliferation assays and [methyl 3H]thymidine uptake studies. Amlodipine and verapamil produced significant inhibition (30%) of VSMC proliferation and DNA synthesis at 10 μM but not at 500 nM-1 μM. To our knowledge, this is the first study to examine the antiproliferative effect of calcium channel blockers in VSMC derived from human graft stenoses. Growth inhibition of VSMC from graft stenoses was not significantly different from that of control saphenous vein-derived cells. We conclude, therefore, that calcium channel blockers inhibit human VSMC proliferation in vitro, regardless of whether the cells were grown from graft stenoses or saphenous vein. However, the concentrations at which these calcium channel blockers elicit antiproliferative effects may not be attainable during therapeutic dosing in humans.

Cellular biology of human intimal hyperplastic stenosis

Restenosis after angioplasty and vascular surgery remains a major unsolved clinical problem. Vascular smooth muscle cell (VSMC) hyperplasia is an invariable response, but in 20-50% of cases proceeds to compromise the vessel lumen. We sought to identify cellular characteristics of human VSMC which are associated with restenosis. Human VSMC were grown from 135 samples of vascular tissue derived from patients undergoing primary cardiovascular surgery and revision surgery for restenosis. Cells derived from normal vein and artery, atherosclerotic plaques and from stenotic lesions were studied for successful proliferation in cell culture. Furthermore, growth rates were measured in response to 15% foetal calf serum +/- inhibition with heparin (100 micrograms/ml). Significantly fewer cells from atherosclerotic plaques progress to the third passage in cell culture than those derived from stenoses and controls (p < 0.001, Chi square) and growth rates after the third passage could not be studied in these cells. Of cells that progress to this stage, growth rates do not differ between stenosis-derived and normal cells under standard conditions. VSMC from mature atherosclerotic plaques may have undergone senescent changes. Stenosis-derived cells do not grow more rapidly than normal cells, but are significantly less sensitive to heparin (p < 0.001, Mann-Witney test), which is a major physiological inhibitor of VSMC growth. Differences in biological characteristics of human VSMC, observed in cell culture, may provide important insights into human vascular disease.

Inhibition of human vascular smooth muscle cell proliferation by the novel multiple-action antihypertensive agent carvedilol.

We examined the antiproliferative effect of the novel multiple-action antihypertensive agent carvedilol on human vascular smooth muscle cells (VSMC). Carvedilol inhibited the increase in cell number induced by foetal calf serum (FCS) in 86% (18 of 21) of human VSMC grown both from saphenous vein (17.6 +/- 3.5% inhibition, mean +/- SEM, n = 15) and restenotic lesions (31.4 +/- 5.5% inhibition, mean +/- SEM, n = 5). Carvedilol had a greater antiproliferative effect than other beta-adrenoceptor antagonists. In comparison with calcium channel blockers, carvedilol (10 microM) elicited a degree of growth inhibition similar to that of verapamil, but was less effective than the dihydropyridine amlodipine at equimolar concentrations. Although carvedilol had a greater antiproliferative effect on cells derived from restenotic lesions cells than on control saphenous vein cells, the difference was not statistically significant. In the present study, the antiproliferative effect of carvedilol on human VSMC in vitro occurred at concentrations higher than those in plasma. Although this may represent a limitation to the clinical efficacy of carvedilol against proliferation of VSMC associated with hypertension and atherosclerosis, the apparent relative selectivity of carvedilol for restenosis-derived cells is a promising line of investigation.

Consistent responses of the human vascular smooth muscle cell in culture: Implications for restenosis

PURPOSE The mechanisms whereby restenoses occur at discrete sites within the vasculature remain uncertain. We have recently reported that vascular smooth muscle cells (VSMC) derived from patients with graft stenoses are resistant to growth inhibition by heparin. In this study, we have examined whether VSMC proliferation rates and responses to inhibition by heparin vary according to the individual or the anatomic site of origin. METHODS Long saphenous veins from seven patients were divided into proximal, middle, and distal portions, and VSMC were cultured separately from each. VSMC proliferation in response to 15% fetal calf serum +/- 100 micrograms/ml heparin was measured by counting triplicate samples at 0, 3, 7, 10, and 14 days. This experiment was repeated from the second to the sixth passage (n = 6) and for artery and vein pairs derived from four additional patients. RESULTS Differences between vein segment cultures of individual veins were found not to differ significantly from experimental error for either proliferation or heparin inhibition and were not altered by repeated passage (ANOVA). There were, however, significant differences in sensitivity to heparin inhibition between patients (p = 0.02) (ANOVA). There were no significant differences between paired samples of artery and vein for either proliferation or heparin inhibition (Mann-Whitney test). CONCLUSIONS VSMC growth characteristics reflect the individual patient and are maintained in cell culture.