Hiroaki Kano

Adrenomedullin as a novel antiproliferative factor of vascular smooth muscle cells

Objective The present study was designed to examine whether adrenomedullin affects fetal calf serum (FCS) stimulated proliferation in cultured rat vascular smooth muscle cells (VSMCs). Methods Rat VSMCs were grown from explants of Sprague-Dawley rat aorta and were grown using the standard cell culture method. After incubation for 24 h with various concentrations of adrenomedullin in the presence of 5% FCS, trichloroacetic acid-insoluble tritiated thymidine was measured in a liquid scintillation counter. After incubation for 48 h, cell counts were performed. Cyclic adenosine 3,5-monophosphate (AMP) levels were determined by radioimmunoassay. Results Rat adrenomedullin exhibited concentration dependent inhibition of the FCS-stimulated increase in thymidine incorporation between 10-7 and 10-9 mol/l and of cell number at 10-7 mol/l. However, the calcitonin generelated peptide (CGRP) receptor antagonist human CGRP(8-37) abolished these antiproliferative effects of rat adrenomedullin. Inhibition by adrenomedullin of FCS-stimulated cellular proliferation was paralleled by an increase in the cellular level of cyclic AMP. 8-Bromocyclic AMP, a cyclic AMP analogue, and forskolin, an activator of adenylate cyclase, inhibited the FCS-stimulated increase in thymidine incorporation and cell number. Conclusions These results suggst that adrenomedullin inhibits FCS-stimulated proliferation in cultured rat VSMCs, probably through a cyclic AMP-dependent process. Taken together with the finding that adrenomedullin is synthesized in and secreted from vascular endothelial cells, adrenomedullin may play a role as an antiproliferative factor for VSMCs in a paracrine fashion.

Plasma Adrenomedullin Concentrations in Essential Hypertension

We designed the present study to assess any changes in plasma concentrations of the novel vasorelaxant peptide adrenomedullin in patients with essential hypertension. Plasma adrenomedullin concentrations were measured in 45 patients with untreated essential hypertension, 15 patients with borderline hypertension, and 30 normotensive control subjects. After 4 weeks of effective calcium channel blocker-based antihypertensive therapy, adrenomedullin concentrations were measured again. The concentrations were higher in hypertensive patients with increased serum creatinine levels or decreased glomerular filtration rates compared with borderline hypertensive patients and normotensive subjects, although values in normotensive and hypertensive individuals overlapped. Plasma adrenomedullin concentrations were positively correlated with serum creatinine levels and inversely correlated with glomerular filtration rates in the hypertensive patients, whereas adrenomedullin values were not correlated with blood pressure level, left ventricular mass index, or left ventricular ejection fraction. Despite blood pressure control with antihypertensive therapy, plasma adrenomedullin concentrations were not changed. Reversed-phase high-performance liquid chromatographic analysis showed that a major component of immunoreactive adrenomedullin in the plasma of normotensive subjects and hypertensive patients is human adrenomedullin-(1-52). These results indicate that plasma adrenomedullin concentrations are elevated in many hypertensive patients with renal dysfunction and its major component is human adrenomedullin-(1-52).

Adrenomedullin as a Novel Antimigration Factor of Vascular Smooth Muscle Cells

The present study investigated the effect of adrenomedullin, a novel vasorelaxant peptide, on the migration of cultured rat vascular smooth muscle cells (SMCs) by using the Boyden-chamber method. Fetal calf serum (FCS) and platelet-derived growth factor (PDGF)-BB strongly stimulated SMC migration. Adrenomedullin clearly inhibited SMC migration stimulated with 5% and 10% FCS in a concentration-dependent manner. The migration induced by 10 and 25 ng/mL PDGF-BB was also inhibited by adrenomedullin in a concentration-dependent manner. Inhibition by adrenomedullin of FCS- and PDGF-induced SMC migration was paralleled by an increase in the cellular level of cAMP. In fact, the percent increase in cAMP level was strongly correlated with the percent decrease in migration activity of SMCs after treatment with adrenomedullin. 8-Bromo cAMP, a cAMP analogue, reproduced the inhibition by adrenomedullin of FCS- and PDGF-induced SMC migration. An activator of adenylate cyclase, forskolin, also reduced FCS- and PDGF-induced SMC migration. These data indicate that adrenomedullin inhibits the migration of SMCs stimulated with FCS and PDGF, probably through a cAMP-dependent process. On the basis of these results and the finding that adrenomedullin is synthesized in and secreted from vascular endothelial cells, adrenomedullin may play a role as a local antimigration factor in some pathophysiological states.

Induction by Lysophosphatidylcholine, a Major Phospholipid Component of Atherogenic Lipoproteins, of Human Coronary Artery Smooth Muscle Cell Migration

BACKGROUND The objectives of the present study were (1) to determine whether lysophosphatidylcholine (lyso-PC), a prominent component of oxidatively modified LDL, induces migration of human coronary artery smooth muscle cells (SMCs) and, if so, to clarify the mechanism, and (2) to investigate the possible interactions of lyso-PC and platelet-derived growth factor (PDGF)-BB, endothelin- (ET-1), adrenomedullin (AM), or vitamin E on SMC migration by the Boydens chamber method. METHODS AND RESULTS Lyso-PC induced SMC migration in a concentration-dependent manner between 10(-6) and 5 x 10(-5) mol/L. By contrast, phosphatidylcholine was without significant activity, and lysophosphatidylinositol and lysophosphatidylserine were much less effective than lyso-PC. Lyso-PC increased basic fibroblast growth factor (bFGF) production in a concentration-dependent manner between 10(-6) and 5 x 10(-5) mol/L in these cells. Furthermore, lyso-PC-induced SMC migration was inhibited by neutralizing antibody to bFGF but not by neutralizing antibody to transforming growth factor-beta1. Lyso-PC-induced migration was significantly enhanced by PDGF-BB or ET-1 but was clearly inhibited by human AM and vitamin E. CONCLUSIONS These results indicate that (1) lyso-PC induces human coronary artery SMC migration at least in part through release of endogenous bFGF and (2) this lyso-PC-induced migration can be further induced by PDGF-BB and ET-1 and can be inhibited by human AM and vitamin E. Lyso-PC may recruit medial SMCs during the process of coronary atherosclerosis in part by releasing bFGF in concert with PDGF-BB or ET-1 in vascular tissues. This lyso-PC-induced SMC migration may be suppressed by AM and vitamin E under certain pathological conditions.

Inhibition of Endothelin Production by Adrenomedullin in Vascular Smooth Muscle Cells

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.

Antioxidants Improve Impaired Insulin-Mediated Glucose Uptake and Prevent Migration and Proliferation of Cultured Rabbit Coronary Smooth Muscle Cells Induced by High Glucose

BACKGROUND To explore the role of intracellular oxidative stress in high glucose-induced atherogenesis, we examined the effect of probucol and/or alpha-tocopherol on the migration and growth characteristics of cultured rabbit coronary vascular smooth muscle cells (VSMCs). METHODS AND RESULTS Chronic high-glucose-medium (22. 2 mmol/L) treatment increased platelet-derived growth factor (PDGF)-BB-mediated VSMC migration, [3H]thymidine incorporation, and cell number compared with VSMCs treated with normal-glucose medium (5.6 mmol/L+16.6 mmol/L mannose). Probucol and alpha-tocopherol significantly suppressed high glucose-induced increase in VSMC migration, cell number, and [3H]thymidine incorporation. Probucol and alpha-tocopherol suppressed high glucose-induced elevation of the cytosolic ratio of NADH/NAD+, phospholipase D, and membrane-bound protein kinase C activation. Probucol, alpha-tocopherol, and calphostin C improved the high glucose-induced suppression of insulin-mediated [3H]deoxyglucose uptake. Chronic high-glucose treatment increased the oxidative stress, which was significantly suppressed by probucol, alpha-tocopherol, suramin, and calphostin C. CONCLUSIONS These findings suggest that probucol and alpha-tocopherol may suppress high glucose-induced VSMC migration and proliferation via suppression of increases in the cytosolic ratio of free NADH/NAD+, phospholipase D, and protein kinase C activation induced by high glucose, which result in reduction in intracellular oxidative stress.

Increased Nitric Oxide Production in Patients with Hypotension during Hemodialysis

Hypotension is a major complication of hemodialysis that often requires aggressive resuscitative measures and premature termination of hemodialysis. Heparin is widely used as an anticoagulant during hemodialysis and has been shown to reduce blood pressure in hypertensive humans [1]. We have recently shown that heparin promotes vasodilator nitric oxide production and suppresses vasoconstrictor endothelin-1 production by human vascular endothelial cells in culture [2]. Elevated levels of endothelin-1 have been reported in hypertensive patients [3, 4]. Blocking of this peptide action by a selective antagonist has been reported to reduce blood pressure [5]. Nitric oxide promotes the formation of cyclic guanosine 35-monophosphate (cGMP), which causes vasodilatation and inhibits the production of endothelin-1 in aortic endothelial cells [6]. We hypothesized that nitric oxide stimulated by heparin might play a role in vasodilatation and thereby lead to hypotensive episodes during hemodialysis. To test this hypothesis, we measured plasma levels of nitrite (NO2) and nitrate (NO3), the products of nitric oxide, and endothelin-1 and cGMP levels in patients who did and did not have hypotensive episodes during hemodialysis sessions in which heparin was used. Methods Our study was approved by the institutional ethics committee of the study hospital and followed institutional ethical guidelines. Informed consent was obtained before the initiation of the study. Study participants included 13 patients with end-stage renal failure who were receiving 4-hour maintenance hemodialysis three times a week. On the basis of their blood pressure responses during hemodialysis, the patients were divided into two groups: Six patients had hypotensive episodes during hemodialysis and 7 did not. Patients who had hypotensive episodes during hemodialysis were defined as those in whom mean arterial pressure decreased more than 20 mm Hg (hypotension occurred 3.7 0.05 hours [mean SD] after initiation of dialysis). Renal failure was caused by hypotension in 6 patients, by chronic glomerulonephritis in 5 patients, and by diabetes mellitus in 2 patients. All patients were in stable condition before and after hemodialysis. None of the patients was receiving antihypertensive or other medications that could have confounded the data. The clinical characteristics of both groups are shown in Table 1. No clinical variables differed between patients who did and did not have a hypotensive episode during hemodialysis. All patients were given heparin at a bolus dose of 2000 U at the initiation of dialysis, followed by 1000 U/h. Table 1. Baseline Characteristics of Patients Who Received Maintenance Hemodialysis* In all cases, blood samples were obtained when dialysis was initiated. After 4 hours of dialysis, the plasma was quickly separated by centrifugation and was divided into three separate portions for the measurement of nitric oxide synthesis and plasma endothelin-1 and cGMP levels. Nitric oxide synthesis was determined by measuring the products of nitric oxide, NO2 and NO3. For the measurement of NO2 and NO3 levels, plasma was quickly deproteinized using 5% trichloroacetic acid and was divided into two aliquots. Because NO3 is stable in blood, one of the aliquots was evaporated and dissolved in Tris-HCl buffer. Nitrate was converted in the presence of NO3 reductase. For the measurement of NO2, Greiss reagents were added to the plasma for stabilization. The samples were then evaporated and dissolved in Tris-HCl buffer. Absorbance was measured at 543 nm by spectrophotometer as previously reported [2]. Plasma endothelin-1 levels were measured according to previously described methods [3]. A commercial kit (Amersham, Tokyo, Japan) was used to measure plasma cGMP levels after extraction by ethanol. The statistical significance of differences in the variables between patients with and those without hypotensive episodes was evaluated with the Student t-test. Differences in the mean values between the variables before and after hemodialysis in each group were analyzed with two-tailed paired t-tests. Multiple regression analysis was done to determine the contribution of several factors to mean blood pressure, the change in blood pressure, and increased nitric oxide production. Results Plasma levels of NO2 and NO3 in both groups of patients are shown in Figure 1. In the patients who had hypotensive episodes during hemodialysis, mean blood pressure decreased from 107 mm Hg to 84 mm Hg (P < 0.001; Figure 1, panel A), and plasma NO2 and NO3 levels increased from 5.8 M to 19.7 M (P < 0.01; Figure 1, panel B). In the patients who did not have hypotensive episodes and whose mean blood pressure did not change (100 14.8 mm Hg), plasma NO2 and NO3 levels did not increase. The correlation coefficient for the association between the change in blood pressure and the changes in NO2 and NO3 levels was 0.759 [P < 0.01]. In both groups of patients, plasma levels of cGMP and endothelin-1 decreased after hemodialysis (Figure 1, panels C and D). The plasma levels of NO2 and NO3 correlated inversely with the mean blood pressure after a 4-hour hemodialysis session (r = 0.85; P < 0.001). Multiple regression analysis showed that the standard coefficients for the association of nitric oxide production and plasma cGMP and endothelin-1 levels with mean blood pressure after hemodialysis were 0.743( 0.16),-0.07 ( 0.024),and 0.31 (0.47), respectively (P = 0.0016 [P < 0.01], 0.68 (P > 0.1), and 0.102 [P > 0.1], respectively). The correlation coefficient and nonstandard coefficients for the association of baseline systolic blood pressure and baseline heart rate with nitric oxide levels after hemodialysis were 0.129( 0.532)(P > 0.1) and 0.477( 4.79)(P > 0.1), respectively. This suggests that neither was substantially associated with increased nitric oxide production. Dialysate levels of NO2 and NO (3) in all patients during the observation period were lower than the detection limit of our assay (<0.5 M). Hemodialysis produced no electrocardiographic evidence of myocardial ischemia, even during hypotensive episodes. Figure 1. Mean blood pressures ( panel A ); the sum of plasma nitrite ( N0 ) and nitrate ( NO ) levels ( panel B ); plasma cyclic guanosine 35-monophosphate ( cGMP ) levels ( panel C ), and plasma endothelin-1 levels ( panel D ) in patients with hypotensive episodes ( hypotension [+]) and those without hypotensive episodes ( hypotension []) before and after a single 4-hour hemodialysis ( HD ) session. t t Discussion Our study was the first in which nitric oxide production was directly measured during hemodialysis. Nitric oxide production at the initiation of hemodialysis did not differ significantly between patients who had and those who did not have a hypotensive episode during hemodialysis. However, nitric oxide production markedly increased during hemodialysis-induced hypotensive episodes, and a strong negative correlation was seen (P = 0.0003) between nitric oxide production and mean blood pressure after hemodialysis. This suggests that enhanced nitric oxide biosynthesis may contribute to hemodialysis-induced hypotension. Beasley and Brenner [7] have proposed that hemodialysis-associated hypotension is mediated by the production of cytokine-induced nitric oxide in vascular smooth-muscle cells. Noris and colleagues [8] have reported that in uremic patients, platelets may be a source of increased nitric oxide production, thereby leading to hypotension. We have previously reported that heparin promotes nitric oxide production by human vascular endothelial cells in culture [2]. Although all patients received an equal amount of heparin, some patients showed increased plasma nitric oxide production during hypotensive episodes. Therefore, although heparin may not be involved in nitric oxide production, sensitivity to heparin in each patient should be clarified. The exact mechanism of increased nitric oxide production is still unknown. Dialysate levels of NO (2) and NO3 in all patients during the observation period were below the detection limit of our assay; thus, a reduced blood flow rate does not contribute to greater accumulation of NO2 and NO3 in patients with hypotensive episodes during hemodialysis. Plasma levels of cGMP decreased after hemodialysis, a finding similar to those in a previous report [9]. This decrease indicates that the vasodepressor effect of nitric oxide may be independent of vascular cGMP levels, although plasma cGMP levels may not necessarily reflect the levels in tissue. Converse and colleagues [10] reported that hemodialysis-induced hypotension is caused by paradoxical withdrawal of sympathetic vasoconstrictors, thereby producing vasodepressor syncope [10]. This suggests the presence of nerve firing that triggers cardioinhibitory and vasodepressor responses, which may be a mechanism independent of vascular cGMP [11]. In our study, plasma endothelin-1 levels decreased with hemodialysis; this finding is similar to findings in a previous report [12]. Because endothelin-1 is a potent vasoconstrictor peptide, the decrease in its plasma level may cause a reduction in blood pressure. However, multiple regression analysis showed that the contribution of plasma endothelin-1 levels to mean blood pressure after hemodialysis was not statistically significant (P = 0.1), suggesting that endothelin-1 in plasma does not contribute much to dialysis-induced hypotension. In conclusion, nitric oxide production was increased in patients who had hypotensive episodes during hemodialysis compared with those who had no hypotensive episodes. Nitric oxide production was negatively correlated with mean blood pressure after hemodialysis, which suggests that increased nitric oxide production contributes to hemodialysis-induced hypotension.

Mechanisms of Action of Troglitazone in the Prevention of High Glucose–Induced Migration and Proliferation of Cultured Coronary Smooth Muscle Cells

Recent findings suggest that high glucose levels may promote atherosclerosis in coronary vascular smooth muscle cells (VSMCs). To explore the intracellular mechanisms of action by which troglitazone affects this process, we examined the effect of troglitazone on the migration and growth characteristics of cultured rabbit coronary VSMCs. Treatment with chronic high glucose medium (22.2 mmol/L) for 5 days increased VSMC migration by 92%, [3H]thymidine incorporation by 135%, and cell number by 32% compared with VSMCs treated with normal glucose (5.5 mmol/L glucose + 16.6 mmol/L mannose) medium. Trolitazone at 100 nmol/L and 1 mumol/L significantly suppressed high glucose-induced VSMC migration by 34% and 42%, respectively, the proliferative effect (as measured by cell number) by 17% and 27%, and [3H]thymidine incorporation by 45% and 60% (n = 6, P < .05). The high glucose-induced impairment of insulin-mediated [3H]deoxyglucose uptake was blocked by a protein kinase C (PKC) inhibitor (calphostin C, 1 mumol/L) and was also improved by troglitazone without any change in insulin receptor number and affinity. The high glucose-induced insulin-mediated increase in cell number and in [3H]thymidine incorporation was suppressed by troglitazone. Troglitazone (1 mumol/L) also suppressed high glucose-induced phospholipase D activation, elevation of the cytosolic NADH/NAD+ ratio (as measured by the cytosolic ratio of lactate/pyruvate), and membrane-bound PKC activation. Flow cytometric DNA histogram analysis of cell cycle stage showed that high glucose-induced increase in the percentage of cells in the S phase was suppressed by 1 mumol/L troglitazone. These findings suggest that PKC may be a link between impairment of insulin-mediated glucose uptake and the increase in migration and proliferation induced by high glucose levels and that troglitazone may be clinically useful for the treatment of high glucose-induced coronary atherosclerosis.

Adrenomedullin Is a Potent Inhibitor of Angiotensin II–Induced Migration of Human Coronary Artery Smooth Muscle Cells

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 Boydens 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.

Effect of natriuretic peptide family on the oxidized LDL-induced migration of human coronary artery smooth muscle cells.

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 Boydens 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 micrograms/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 micrograms/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.