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Angiotensin II–mediated oxidative stress may play a role in the pathogenesis of coronary atherosclerosis. We examined the effects of pressure on the angiotensin II–mediated increase in oxidative stress and migration of cultured human coronary smooth muscle cells (SMCs). Increased pressure (100 mm Hg) by helium gas for 48 hours increased angiotensin II–mediated oxidative stress as evaluated by flow cytometry and SMC migration (from 15.9±2.2 to 32.0±2.4 cells per 4 high-power fields, P P N -acetylcysteine (100 mmol/L) but not PD123319 (1 μmol/L) also blocked pressure-induced increases in angiotensin II–mediated oxidative stress and SMC migration ( P

Abstract —In vitro and in vivo evidence of a decrease in vascular smooth muscle cell (SMC) migration induced by 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors has been reported. When added to SMC cultures for 6 hours, the HMG-CoA reductase inhibitors fluvastatin, simvastatin, and pravastatin at 1 μmol/L resulted in a 48%, 50%, and 16% suppression, respectively, of human coronary SMC migration; these reductions mirrored the suppression in oxidative stress induced by 1 μmol/L lysophosphatidylcholine (lyso-PC) of 50%, 53% and 19%, respectively. The hydroxylated metabolites of fluvastatin, M 2 and M 3 , at 1 μmol/L also suppressed the enhancement of SMC migration by 58% and 45% and the increase in oxidative stress induced by lyso-PC of 58% and 49%, respectively. Lyso-PC activated phospholipase D and protein kinase C (PKC), and this activation was also suppressed by HMG-CoA reductase inhibitors. The inhibition of phospholipase D and PKC was reversed by 100 μmol/L mevalonate, its isoprenoid derivative, farnesol, and geranylgeraniol but not by 10 μmol/L squalene. Antisense oligodeoxynucleotides at 5 μmol/L to PKC-α, but not those to the PKC-β isoform, suppressed the lyso-PC–mediated increases in SMC migration and oxidative stress. These findings suggest that HMG-CoA reductase inhibitors have direct antimigratory effects on the vascular wall beyond their effects on plasma lipids and that they might exert such antimigratory effects via suppression of the phospholipase D– and PKC (possibly PKC-α)-induced increase in oxidative stress, which might in turn prevent significant coronary artery disease.

Background —To elucidate the roles of vascular D 1 -like receptors in atherosclerosis, the effects of the specific D 1 -like agonists on platelet-derived growth factor (PDGF)-BB–mediated oxidative stress in vascular smooth muscle cells (VSMCs) were studied. Methods and Results —Immunohistochemical studies demonstrated the coexistence of D 1A and D 1B dopamine receptors in VSMCs. Western blotting revealed a band of ≈70 kDa for D 1A and D 1B dopamine receptors. VSMCs stimulated by PDGF-BB exhibited increased oxidative stress directly measured by flow cytometry. These effects were prevented by dopamine, SKF 38393, or YM 435, and this prevention was reversed by Sch 23390. These effects were blocked by a specific protein kinase A (PKA) inhibitor, N -(2-[ p -bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide (H 89). The PDGF-BB–mediated increase in oxidative stress of VSMCs was significantly suppressed by the indirect phospholipase D (PLD) inhibitor suramin or the specific protein kinase C (PKC) inhibitor calphostin C. Both antisense but neither sense nor scrambled oligonucleotides to D 1A and D 1B receptors inhibited dopamine-induced suppression of increase in oxidative stress of VSMCs induced by PDGF-BB. Conclusions —These findings suggest that vascular D 1 -like receptors (D 1A and D 1B receptors) inhibit any increase in oxidative stress of VSMCs, possibly through activation of PKA and suppression of PLD and PKC.

Abstract Vascular smooth muscle cell (VSMC) migration and proliferation are believed to play key roles in atherosclerosis. To elucidate the role of vascular dopamine D1-like receptors in atherosclerosis, the effects of dopamine and specific D1-like agonists SKF 38393 and YM 435 on platelet-derived growth factor (PDGF) BB-mediated VSMC migration and proliferation were studied. We observed that cells stimulated by PDGF-BB (5 ng/mL), showed increased migration and proliferation. These effects were prevented by coincubation with dopamine, SKF 38393, or YM 435 (1 to 10 μmol/L), and this prevention was reversed by Sch 23390 (1 to 10 μmol/l), a specific D1-like antagonist. These actions are mimicked by forskolin (1 to 10 μmol/L), a direct activator of adenylate cyclase and 8-bromo-cAMP at 0.1 to 1 mmol/L and are blocked by a specific protein kinase A inhibitor, N -[2-( p -bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide (H 89), but not blocked by its negative control, N -[2-( N -formyl)- p -chlorociannamylamino)ethyl]-5-isoquinoline sulfonamide (H 85). PDGF-BB (5 ng/mL)-mediated activation of phospholipase D, protein kinase C, and mitogen activated protein kinase activity were significantly suppressed by coincubation with dopamine. These results suggest that vascular D1-like receptor agonists inhibit migration and proliferation of VSMC, possibly through protein kinase A activation and suppression of activated phospholipase D, protein kinase C, and mitogen-activated protein kinase activity.

Abstract The migration of medial smooth muscle cells (SMCs) into the intima is proposed to be an important process of intimal thickening in atherosclerotic lesions. The present study examined the possible effect of a novel endothelium-derived relaxing peptide, C-type natriuretic peptide (CNP), on oxidized low-density lipoprotein (LDL)–induced migration of cultured human coronary artery SMCs by the Boyden’s chamber method. The effect of CNP was compared with that of atrial and brain natriuretic peptides (ANP and BNP, respectively). Oxidized LDL stimulates SMC migration in a concentration-dependent manner between 20 and 200 μg/mL. This stimulation was chemotactic in nature but was not chemokinetic. By contrast, native LDL was without significant activity. CNP-22 clearly inhibited SMC migration stimulated with 200 μg/mL oxidized LDL in a concentration-dependent manner between 10 −9 and 10 −6 mol/L. ANP-(1-28) and BNP-32 also inhibited oxidized LDL–induced SMC migration at concentrations of 10 −7 and 10 −6 mol/L, but these effects were weaker than the effect of CNP-22. Such inhibition by these natriuretic peptides was paralleled by an increase in the cellular level of cGMP. Oxidized LDL–induced migration was significantly inhibited by a stable analogue of cGMP, 8-bromo-cGMP, or an activator of the cytosolic guanylate cyclase, sodium nitroprusside. These natriuretic peptides did not suppress the cell adhesion either in the absence or presence of oxidized LDL. These data indicate that oxidized LDL stimulates migration of human coronary artery SMCs and that natriuretic peptides, especially CNP, inhibit this stimulated SMC migration, at least in part, through a cGMP-dependent process. Taken together with the finding that oxidized LDL is present in the intima, CNP may play a role as a local antimigration factor during the process of intimal thickening in hypercholesterolemia-induced coronary atherosclerosis.

Abstract The migration of coronary artery medial smooth muscle cells (SMCs) into the intima is proposed to be an important process of intimal thickening in coronary atherosclerotic lesions. In the current study, we examined the possible interaction of adrenomedullin, a novel vasorelaxant peptide, and angiotensin II (Ang II) on human coronary artery SMC migration using Boyden’s chamber method. Ang II stimulated SMC migration in a concentration-dependent manner between 10 −6 and 10 −8 mol/L. This stimulation was clearly blocked by the Ang II type 1 receptor antagonist losartan but not by the type 2 receptor antagonist PD 123319. The migration stimulatory effect of Ang II was chemotactic in nature for cultured human coronary artery SMCs but was not chemokinetic. Human adrenomedullin clearly inhibited Ang II–induced migration in a concentration-dependent manner. Human adrenomedullin stimulated cAMP formation in these cells. Inhibition by adrenomedullin of Ang II–induced SMC migration was paralleled by an increase in the cellular level of cAMP. 8-Bromo-cAMP, a cAMP analogue, and forskolin, an activator of adenylate cyclase, inhibited the Ang II–induced SMC migration. These results suggest that Ang II stimulates SMC migration via type 1 receptors in human coronary artery and adrenomedullin inhibits Ang II–induced migration at least partly through a cAMP-dependent mechanism. Taken together with the finding that adrenomedullin is synthesized in and secreted from vascular endothelial cells, this peptide may play a role as a local antimigration factor in certain pathological conditions.

Abstract Adrenomedullin has recently been isolated from human pheochromocytoma. We designed the present study to examine the effect of adrenomedullin on the production of the vasoconstrictive and growth-promoting peptide endothelin-1 (ET-1) after stimulation with platelet-derived growth factor (PDGF) in cultured rat glomerular mesangial cells. PDGF stimulated ET-1 production in a concentration-dependent manner. Rat adrenomedullin inhibited this stimulated ET-1 production in a concentration-dependent manner between 10 −7 and 10 −8 mol/L. Rat adrenomedullin also increased the cellular level of cAMP in a concentration-dependent manner between 10 −7 and 10 −8 mol/L. Human adrenomedullin was less effective than rat adrenomedullin with respect to inhibiting ET-1 production and increasing cAMP levels. The addition of 8-bromo-cAMP (10 −3 and 10 −4 mol/L) reduced PDGF-induced ET-1 production. Furthermore, forskolin (10 −4 and 10 −5 mol/L), an activator of adenylate cyclase, reduced PDGF-induced ET-1 production. In contrast, the basal production of ET-1 was not significantly altered by rat and human adrenomedullin. These results indicate that adrenomedullin inhibits PDGF-induced ET-1 production in cultured rat mesangial cells, probably through a cAMP-dependent process.

Abstract Adrenomedullin recently has been found to potently stimulate cAMP formation in cultured rat vascular smooth muscle cells (VSMCs). In the present study, we examined the effect of adrenomedullin on the production of a vasoconstrictive and growth-promoting peptide, endothelin-1, after stimulation with a clotting enzyme, thrombin, and a potent mitogen, platelet-derived growth factor (PDGF), in cultured rat VSMCs. Thrombin and PDGF stimulated endothelin-1 production in a dose-dependent manner. Rat adrenomedullin significantly inhibited thrombin- and PDGF-stimulated endothelin-1 production in a dose-dependent manner between 10 −7 and 10 −9 mol/L. Inhibition by rat adrenomedullin of thrombin- and PDGF-stimulated endothelin-1 production was paralleled by an increase in the cellular level of cAMP. Human adrenomedullin also inhibited thrombin- and PDGF-stimulated endothelin-1 production and increased cAMP levels. The addition of 8-bromo-cAMP, a cAMP analogue, reduced thrombin- and PDGF-induced endothelin-1 production. Furthermore, forskolin, a potent activator of adenylate cyclase, reduced thrombin- and PDGF-induced endothelin-1 production. In contrast, basal production of endothelin-1 was not altered by rat or human adrenomedullin. These results indicate that adrenomedullin inhibits not basal but thrombin- and PDGF-induced ET-1 production in cultured VSMCs probably through a cAMP-dependent process. Taken together with the finding that adrenomedullin is synthesized in and secreted from vascular endothelial cells, adrenomedullin may modulate vascular tone as a paracrine regulator partially through the inhibition of VSMC endothelin-1 production in some pathophysiological states.

We have recently shown that the porcine aorta releases immunoreactive endothelin-1 in a time-dependent way. Here, we examined the inhibition by atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) of endothelin-1 secretion after stimulation with angiotensin II (Ang II) by using porcine aorta. Ang II dose-dependently stimulated immunoreactive endothelin-1 secretion. Porcine ANP-(1-28) and porcine BNP-26 both inhibited such secretion in a dose-dependent way. The addition of a cyclic guanosine 5'-monophosphate (cGMP) analogue, 8-bromo-cGMP, reduced the immunoreactive endothelin-1 secretion after stimulation with Ang II. In cultured porcine endothelial cells the inhibition by porcine ANP-(1-28) and porcine BNP-26 of immunoreactive endothelin-1 secretion after stimulation with Ang II was paralleled by an increase in the cellular cGMP level. Rat ANP-(5-25) was weaker than porcine ANP-(1-28) in inhibiting immunoreactive endothelin-1 secretion and increasing cGMP in cultured cells. There was negative correlation between the percent decrease in immunoreactive endothelin-1 and the percent increase in cGMP. Neither porcine ANP-(1-28) nor BNP-26 affected the number or sensitivity of Ang II binding sites in cultured porcine endothelial cells. These results suggest that ANP and BNP inhibit endothelin-1 secretion after stimulation with Ang II, probably through a cGMP-dependent process.

We describe a patient with pheochromocytoma, which secretes dopamine. He was admitted to hospital because of chronic diarrhea. After surgical resection of the tumor, dramatic cessation of the diarrhea and blood pressure elevation were observed. Decreased expression of dopamine beta-hydroxylase in the tumor was considered a possible mechanism of producing a pathophysiological concentration of dopamine. This case shows that excessive excretion of dopamine, a vasodilative hormone, may affect blood pressure.

Recently, we have reported two cases with endothelin (ET)-secreting tumor presenting with hypertension and elevated plasma endothelin-1 (ET-1) levels. The present study examines the histopathology of the ET-secreting tumor in one of these cases. The neoplasm was located in the skin and microscopically characterized as a malignant hemangioendothelioma by remarkable intravascular proliferations of atypical endothelium with the expression of Factor VIII-related antigen in the tumor cells. The tumor enlarged rapidly with rising ET-1 and blood pressure levels. The ET-1 content and its messenger RNA expression in the tumor extract were higher than those from normal parts of skin. ET-1 may play a pathophysiological role in patients with ET-secreting malignant hemangioendothelioma.

The effect of glucocorticoids on the atrial natriuretic factor (ANF)-mediated formation of cyclic guanosine monophosphate (cGMP) by intact vascular smooth muscle cells (VSMC) was studied in rats. Cultured VSMC were obtained from the renal arteries of 14-week-old Wistar rats by the explant method. Micromolar concentrations of dexamethasone, given as pretreatment for 48 hours, suppressed the ANF-mediated response. The dexamethasone-induced suppression was detectable at 6 hours and reached a maximum 24 hours after administration in a dose-dependent manner. Inhibitors of protein synthesis blocked this effect of the glucocorticoid. The basal activity of guanylate cyclase in the dexamethasone-treated cells was lower than in the control cells. Other steroids having glucocorticoid action mimicked this suppression of the ANF-mediated response. This suppression was blocked by a glucocorticoid receptor antagonist. The results suggest that glucocorticoids suppress ANF-mediated cGMP formation by VSMC through glucocorticoid type II receptors and the induction of protein synthesis. Suppression of the ANF-mediated response may play a role in glucocorticoid-induced hypertension.

Heparin shows a blood pressure-lowering effect in various hypertensive rat models. This study was designed to examine the effect of heparin on vasoconstrictor endothelin-1 (ET-1) production by cultured human umbilical vein endothelial cells (HUVECs). ET-1 and cyclic GMP levels in the medium were determined by radioimmunoassay. ET-1 mRNA was quantified by densitometric Northern blot analysis. ET-1 was released into the medium in a time-dependent manner, and its release was augmented by thrombin (10 U/ml). Heparin suppressed both basal and thrombin-stimulated ET-1 secretion and its mRNA expression in a dose-dependent manner. Heparin suppressed ET-1 mRNA expression in a time-dependent fashion. Heparin did not suppress both basal and thrombin-stimulated ET-1 production in the presence of NG-monomethyl-L-arginine (L-NMMA) (10(-5) M). The production of cGMP stimulated by thrombin was significantly enhanced by heparin, but not in the presence of L-NMMA (10(-5) M). Heparin may suppress vasoconstrictor ET-1 production mediated by the enhancement of endothelium-derived nitric oxide in HUVECs.

P82 Vascular smooth muscle cell (VSMC) migration is believed to play a key role in atherosclerosis. To elucidate the roles of rat vascular D 1A and D 1B dopamine receptors in atherosclerosis, the effect of antisense oligonucleotides (AS) to D 1A (+1-+21 of rat D 1A receptors cDNA) and D 1B (-12-+6 of rat D 1B receptors cDNA) receptors on dopamine-mediated suppression of platelet-derived growth factor (PDGF) BB-mediated VSMC migration evaluated by Boyden′s chamber method was studied. To avoid the degradation, phosphothioate-modified oligodeoxynucleotides were synthesized and purified by high-performance liquid chromatography. These oligonucleotides were added to serum free medium for 24 h before the start of PDGF BB stimulation with transfection using lipofectin reagent. Immunohistochemical studies (double staining) demonstrated the coexistence of D 1A and D 1B dopamine receptors in single VSMC derived from rat renal artery. Western blotting revealed a band of approximately 70 kD for D 1A and D 1B dopamine receptors. Increased VSMC migration by PDGF BB 5 ng/ml (16-fold) was suppressed significantly by coincubation with dopamine 0.025-10 μmol/l (15-59%). This suppression by dopamine 10 μmol/l was reversed by D 1A AS 46% and D 1B AS 51%, but by neither sense (S) nor scramble (RS) oligonucleotides to these receptors. These suppression by antisense oligonucleotides (21-51%) are dose-dependent (1-10 μmol/l) and time-dependent (0-4 hrs). Dopamine 10 μmol/l-induced cyclic AMP formation is also suppressed by D 1A AS 50% and D 1B AS 58%, but by neither S nor RS to these receptors. PDGF BB (5 ng/ml)-mediated activation of phospholipase D (PLD) activity (107%) measured by [ 3 H]-ethanolamine and protein kinase C (PKC) activity (111%) activities measured by synthetic peptide phosphorylation were significantly suppressed by coincubation with dopamine 10 μmol/l (48%, 49%), which was reversed by D 1A AS 45% and D 1B AS 50%, but by neither S nor RS to these receptors. These results suggest that vascular D 1A and D 1B receptors inhibit migration of VSMC, possibly through cAMP formation and suppression of PLD and PKC activity.

The effect of glucocorticoid on the prostaglandin E1 (PGE1)-mediated cyclic AMP (cAMP) formation by vascular smooth muscle cells (VSMC) from renal arteries (RA) was studied in rats. Dexamethasone (DEX) at concentrations ranging from 10(-10) to approximately 10(-8) mol/l dose-dependently potentiates the PGE1-mediated response. This facilitation began at 6 h and reached its maximum after 24 h of DEX administration. Aldosterone (10(-6) mol/l) did not affect the dose-response curve of PGE1. Inhibitors of protein and RNA synthesis blocked this glucocorticoid effect. The basal activity of adenylate cyclase in DEX-treated cells was twice as high as in control cells. Treatment of VSMC with DEX increased cholera toxin- and pertussis toxin-stimulated adenylate cyclase activity. DEX treatment also augments forskolin-stimulated adenylate cyclase activity. These results suggest that DEX increases PGE1-mediated cAMP formation of VSMC from RA through a mechanism that involves the induction of protein synthesis, and that the activation of the catalytic unit may play some role in this facilitating process.

In seven patients with mild to moderate essential hypertension, the effect of a new angiotensin converting enzyme (ACE) inhibitor, cilazapril, on atrial natriuretic factor (ANF) was examined at rest and during exercise. The exercise protocol consisted of three fixed workloads (25, 50 and 75W) every 4 min with the use of a sitting bicycle ergometer. Cilazapril reduced blood pressure without increasing the heart rate, both at rest and during exercise. The resting pre-exercise left ventricular ejection fraction was not altered. The plasma ANF level was increased by exercise, but at the 75-W workload it was decreased by cilazapril; the resting plasma ANF value was not altered. At the 75-W workload the plasma ANF level was inversely correlated to the left ventricular ejection fraction (n = 14, r = -0.64, P less than 0.01). These results suggest that the observed decrease in the plasma ANF level during exercise induced by cilazapril is, in part, related to an improvement in the cardiac overload, and that cilazapril may be effective in physically active hypertensive patients.

Cultured porcine endothelial cells derived from aortas spontaneously released immunoreactive endothelin into the medium in a time-dependent manner. This release was completely inhibited by cycloheximide and is, therefore, directly related to de novo protein synthesis. The endothlin-induced release was further stimulated by adrenaline. Adrenaline-induced stimulation was completely inhibited by the alpha-adrenergic blocker phentolamine and was not inhibited by the beta-adrenergic blocker propranolol. Cycloheximide completely prevented the adrenaline-stimulated as well as the basal release. These results suggest that cultured endothelial cells release endothelin slowly but continuously and that this release can be stimulated by adrenaline via alpha-adrenergic receptors. We speculate that the endothelium generates vasoconstrictor signals through endothelin production, thus contributing to the regulation of vascular tone.