Insulin Inhibits Migration of Vascular Smooth Muscle Cells With Inducible Nitric Oxide Synthase

Abstract

Abstract-Vascular smooth muscle cell (VSMC) migration participates in atherosclerosis and arterial restenosis after balloon angioplasty. Because these processes are enhanced in insulin-resistant states, our goal was to determine whether insulin affects VSMC migration and, if so, how. The migration of primary cultured VSMCs from canine femoral artery was measured with the use of a wound migration assay and related to cGMP levels. Insulin (1 nmol/L) did not affect migration or cGMP production in control cells. When inducible nitric oxide synthase (iNOS) was induced by 24-hour preincubation with lipopolysaccharide and interleuken-1␤, basal migration decreased, cGMP production increased, and insulin inhibited migration by Ͼ90% and stimulated cGMP production by 3-fold. The nitric oxide synthase inhibitor N G -monomethyl-L-arginine blocked the affect of insulin on the migration of VSMCs with iNOS. 8-Bromo-cGMP inhibited VSMC migration in control cells, and 1-H-1[1,2,4]oxadiazolo- [4,3a]quinoxolin-1-one, a selective inhibitor of guanylate cyclase, blocked the inhibition by insulin of migration of cells with iNOS. We conclude that insulin does not normally affect cGMP production or the migration of these VSMCs. However, after the induction of iNOS, insulin stimulates cGMP production and inhibits migration via an NOS-and a cGMP-dependent mechanism. (Hypertension. 2000;35[part 2]:303-306.)Key Words: cyclic 3Ј,5Јguanosine monophosphate Ⅲ muscle, smooth, vascular Ⅲ nitric oxide Ⅲ insulin I nsulin resistance, which occurs in obesity, non-insulindependent diabetes mellitus, and essential hypertension, is associated with increased atherosclerosis and restenosis after balloon angioplasty, 1-3 but the reasons for this are obscure. The migration of vascular smooth muscle cells (VSMCs) from the arterial media through the internal elastic membrane into the neointima is an integral part of the pathogenesis of atherosclerosis and restenosis. 4 The insulin-resistant conditions noted are marked by hyperinsulinemia as the pancreas attempts to maintain plasma glucose concentration at a normal level. 1 Because hyperinsulinemia has been thought to aggravate atherosclerosis or restenosis, 5 several investigators have studied the effects of insulin on VSMC migration. 6 -8 Although some studies have shown a small stimulation of VSMC migration with insulin, the results are controversial. In some studies, the concentrations of insulin that were used were supraphysiological, 8 such that the insulin-like growth factor-1 pathway may have been activated. Insulin-like growth factor-1 is known to stimulate VSMC migration. 9 In other studies, insulin alone had no effect on VSMC migration 6 but increased it when it was stimulated by another agent. 7 We have reported that physiological concentrations of insulin stimulated cGMP production in primary cultured VSMCs from canine femoral artery. 10,11 These cells were cultured under conditions such that they expressed the inducible isoform of nitric oxide synthase (iNOS), 11 and an inhibitor of NOS blocked ...

Insulin-Stimulated Cyclic Guanosine Monophosphate Inhibits Vascular Smooth Muscle Cell Migration by Inhibiting Ca/Calmodulin-Dependent Protein Kinase II

“…Endothelia and adventitia were stripped away; the media of the arteries were minced, incubated at 37°C in a solution containing elastase (type V, Sigma) and collagenase (type I, Worthington Biochemical); and cultured VSMCs were prepared as previously described. 6 Cultured cells of passages 3 to 10 were used in experiments. Because a permissive amount of NO is necessary for insulin to stimulate cGMP production in cultured dog and rat VSMCs (Kahn et al 14 and data not shown), most experiments were performed with cells that had been induced to express iNOS as previously described.…”

“…Because a permissive amount of NO is necessary for insulin to stimulate cGMP production in cultured dog and rat VSMCs (Kahn et al 14 and data not shown), most experiments were performed with cells that had been induced to express iNOS as previously described. 6 …”

“…The percentage of initial wound area that had closed at 9 hours was taken as an index of migration, as previously described. 6 …”

“…Immunoreactive bands were detected with horseradish peroxidase-conjugated secondary antibody and visualized on enhanced chemiluminescence film (Hyperfilm, Amersham) as described previously. 6 The membrane was stripped, reprobed with anti-␤-actin antibody (Sigma) at 1:2000 dilution, and immunoactive bands were detected as described above. …”

“…This hypothesis is consistent with our previous report, which demonstrated that if cultured canine VSMCs had been induced to express inducible nitric oxide synthase (iNOS), insulin stimulated cGMP production, which in turn inhibited cell migration in a wound closure assay. 6 Others have reported that cGMP inhibits VSMC migration, but the mechanisms are unknown. 7,8 The mechanisms by which wounding stimulates migration of cultured VSMCs have not been determined.…”