Takao Hanehira

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).

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.

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.

Association between angiotensin-converting enzyme gene polymorphisms and regression of left ventricular hypertrophy in patients treated with angiotensin-converting enzyme inhibitors

PURPOSE An insertion/deletion (ID) polymorphism of the angiotensin-converting enzyme (ACE) gene is associated with left ventricular hypertrophy. The present study examined polymorphisms of the ACE gene in patients with essential hypertension and left ventricular hypertrophy who were participants in a long-term trial of therapy with an ACE inhibitor. PATIENTS AND METHODS ACE inhibitor therapy was administered for >2 years to 54 patients with hypertension who had moderate or severe left ventricular hypertrophy. Cardiac dimensions were monitored by echocardiography before the initiation of therapy and after 1 and 2 years of treatment. Serum ACE activity and plasma concentrations of brain natriuretic peptide, a marker for left ventricular hypertrophy, were also monitored. RESULTS Eighteen patients had the II genotype for the angiotensin-converting enzyme gene, 19 had the ID genotype, and 17 had the DD genotype. Baseline (mean +/- SD) serum ACE activity was significantly greater (P <0.05) in the DD (18 +/- 7 IU/L) group than in the II (7 +/- 4 IU/L) or ID (12 +/- 6 IU/L) groups. ACE inhibitor therapy was effective in controlling blood pressure, and it reduced posterior and septal wall thickness, left ventricular mass index, and plasma brain natriuretic peptide concentration in all three groups. Despite similar blood pressure reductions, after 2 years, mean (+/- SD) regression in posterior wall thickness was significantly less (P <0.05) in the DD group (-9% +/- 5%) than in the ID (-21% +/- 7%) and II (-21% +/- 9%) groups. Similar results were seen for the reductions in brain natriuretic peptide levels. The magnitudes of regression of septal wall thickness and left ventricular mass index during therapy were less in the DD group than the II group (P <0.05). CONCLUSION Hypertensive patients with the DD genotype are less likely to have regression of left ventricular hypertrophy when treated with ACE inhibitors than are patients with other ACE genotypes.

Interaction of Adrenomedullin and Platelet-Derived Growth Factor on Rat Mesangial Cell Production of Endothelin

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.

An accelerated increase of plasma adrenomedullin in acute asthma

A novel vasorelaxant peptide, adrenomedullin (AM), has been isolated from the acid extract of human pheochromocytoma. We have recently shown that AM inhibits histamine- and acetylcholine-induced bronchoconstriction in anesthetized guinea pigs in vivo, and this bronchodilatory effect is long-lasting. Here, we measured plasma AM concentrations in nine patients with an acute attack of bronchial asthma. The results were compared with values in 30 age-matched normal control subjects and seven age-matched stable asthmatic patients. The mean AM concentrations of patients with an acute asthma attack (98 +/- 22 pg/mL) were clearly higher than those of normal control subjects (18 +/- 2 pg/mL) and stable asthmatic patients (21 +/- 3 pg/mL). Reverse-phase high-performance liquid chromatography (HPLC) showed that the major component of plasma immunoreactive AM in patients with an asthma attack and in normal subjects equally corresponded to authentic human AM(1-52). Our results suggest that plasma AM is markedly increased in many of the patients during an acute attack of bronchial asthma, but it is not observed in stable asthmatic patients. Although this report is preliminary, the observed increase of circulating AM during an acute asthma attack may represent a compensatory mechanism against the bronchoconstriction, probably through its bronchodilatory action.

Effects of Cardiac Natriuretic Peptides on Oxidized Low-Density Lipoprotein– and Lysophosphatidylcholine-Induced Human Mesangial Cell Migration

The objectives of the present study were (1) to determine whether oxidized LDL and lysophosphatidylcholine (lyso-PtdCho), a major phospholipid component of oxidized LDL, stimulate the migration of cultured human mesangial cells and (2) to investigate the possible effects on mesangial cell migration of the cardiac natriuretic peptides atrial and brain natriuretic peptide (ANP and BNP). Oxidized LDL (10 and 100 microg/mL) and lyso-PtdCho (10(-7) to 10(-5) mol/L) stimulated migration in a concentration-dependent manner. In contrast, the effects of native LDL and phosphatidylcholine were modest or nonexistent. Protein kinase C (PKC) inhibitor and downregulation of PKC activity by phorbol ester inhibited oxidized LDL- and lyso-PtdCho-induced migration. Human ANP(1-28) and human BNP-32 significantly inhibited oxidized LDL- and lyso-PtdCho-induced migration in a concentration-dependent manner. C-ANF (des-[Glu(18),Ser(19),Gly(20),Leu(21),Gly(22)]ANP(4-23)), a specific ligand for ANP clearance receptors, could not inhibit oxidized LDL- and lyso-PtdCho-induced migration. Inhibition by ANP and BNP of lyso-PtdCho-induced migration was paralleled by an increase in the cellular level of GMP. Oxidized LDL- and lyso-PtdCho-induced migrations were inhibited by 8-bromo-cGMP. The results suggest that oxidized LDL and lyso-PtdCho stimulate the migration of human mesangial cells, at least in part, through a PKC-dependent process and that ANP and BNP inhibit this stimulated migration, probably through a cGMP-dependent process.

PROMOTION OF NITRIC OXIDE FORMATION BY HEPARIN IN CULTURED AORTIC ENDOTHELIAL CELLS FROM SPONTANEOUSLY HYPERTENSIVE RATS

1. The present study examined the effect of heparin on nitric oxide (NO) formation and cyclic guanosine 3′, 5′‐monophos‐phate (cGMP) levels in cultured aortic endothelial cells (EC) from spontaneously hypertensive rats (SHR) and normoten‐sive Wistar‐Kyoto (WKY) rats.

Effect of thrombin and PDGF on endothelin production in cultured mesangial cells derived from spontaneously hypertensive rats.

1. Basal endothelin‐1 (ET‐1) production in mesangial cells of spontaneously hypertensive rats (SHR) was not different from that of Wistar‐Kyoto (WKY) rats, although a trend toward increased ET‐1 production was observed in these cells of SHR.