Atsushi Namiki

Lack of Insulin Receptor Substrate-2 Causes Progressive Neointima Formation in Response to Vessel Injury

Background Insulin resistance is associated with atherosclerosis, but its mechanism is unknown. It has been reported that insulin receptor substrate (IRS)‐1 deficient (IRS‐1‐/‐) mice showed insulin resistance without type 2 diabetes, whereas the IRS‐2 deficient (IRS‐2‐/‐) mice showed insulin resistance with type 2 diabetes. Methods and Results We investigated neointima formation in the IRS‐1‐/‐ and IRS‐2‐/‐ mice at 8 and 20 weeks. The IRS‐2‐/‐ mice showed much greater neointima formation than the IRS‐1‐/‐ and wild‐type mice at 8 weeks. At 20 weeks, the IRS‐2‐/‐ mice had greater neointima formation than the IRS‐1‐/‐ mice, which showed more enhanced neointima formation than the wild‐type mice. The IRS‐1‐/‐ and IRS‐2‐/‐ mice had dyslipidemia, hypertension, and insulin resistance. The IRS‐2‐/‐ mice had more metabolic abnormalities than the IRS‐1‐/‐ mice at 8 and 20 weeks. IRS‐2 expression was detected, but IRS‐1 expression was not detected in the vessels. Conclusions The neointima formation in the IRS‐1‐/‐ and IRS‐2‐/‐ mice appears to be related to abnormalities induced by the altered metabolic milieu in insulin‐resistant states. Moreover, because neointima formation was much greater in the IRS‐2‐/‐ mice than in the IRS‐1‐/‐ mice at 8 and 20 weeks, it is suggested that a lack of IRS‐2 renders the vasculature more susceptible to injury in the abnormal metabolic milieu, and IRS‐2 may have a protective effect on neointima formation. We conclude that IRS‐2 is protective and retards the development of neointima formation in insulin‐resistant states. (Circulation. 2003;107:3073‐3080.)

Endothelin-1- and endothelin-3-induced vasorelaxation via common generation of endothelium-derived nitric oxide

The vasorelaxant effects by endothelin-1 (ET-1) and endothelin-3 (ET-3), and their mechanisms of action were studied in isolated porcine pulmonary arterial strips. ET-1 and ET-3 dose-dependently (10(-9) - 10(-8) M) relaxed vascular strips precontracted with norepinephrine only in the presence of endothelium. The maximal vasorelaxant effect by ET-1 was about 70% of that by ET-3. The ET-1- and ET-3- induced vasorelaxation was blocked by NG-nitro-L-arginine, an inhibitor of nitric oxide synthesis, and methylene blue, an inhibitor of soluble guanylate cyclase. The present data suggest that vascular smooth muscle relaxation induced by ET-1 and ET-3 is mainly ascribed to synthesis and release of nitric oxide from L-arginine in endothelium.

Expression of parathyroid hormone-related protein in human and experimental atherosclerotic lesions: functional role in arterial intimal thickening

We investigated the expression of parathyroid hormone-related protein (PTHrP) in atherosclerotic lesions and the role of PTHrP in the development of arterial neointima formation. Immunohistochemical staining of PTHrP in the neointima of rat aorta produced by balloon injury and of rat femoral artery produced by non-obstructive polyethylene cuff placement, and in the atherosclerotic lesion of human coronary artery was performed using anti-human PTHrP-(1-34) antibody. Anti-muscle actin antibody, HHF-35, and anti-macrophage antibody, HAM-56, were used to identify smooth muscle cells and macrophages, respectively. Immunoreactivity of PTHrP was detected in the thickened intima of rat and human lesions where the predominant cell types were smooth muscle cells or macrophages dependently on the lesion type. In the next series of experiments, we examined the effect of PTHrP on the development of cuff-induced intimal thickening of rat femoral artery. Either PTHrP-(1-34) or PTHrP-(7-34), a PTH/PTHrP receptor antagonist, suspended in pluronic F-127 gel was locally applied around the rat femoral artery. Intimal thickening induced by cuff placement was evaluated 2 weeks later. PTHrP-(1-34) dose-dependently inhibited intimal thickening determined as intima/media ratio and % stenosis whereas PTHrP-(7-34) dose-dependently enhanced that. These results suggest that PTHrP, which is expressed in atherosclerotic lesions, inhibits the development of neointimal formation.

Amino-terminal fragment (1-34) of parathyroid hormone-related protein inhibits migration and proliferation of cultured vascular smooth muscle cells

We investigated the effects of amino-terminal fragment (1-34) of parathyroid hormone-related protein [PTHrP-(1-34)] on the migration and proliferation of vascular smooth muscle cells (VSMCs). Cultured VSMCs (5-9th passage) obtained from the aortas of male Wistar rats were used in this study. Migration of VSMCs was assessed using a modified Boydens chamber. Proliferation of VSMCs was evaluated by measuring [3H]thymidine incorporation and counting cell numbers. PTHrP-(1-34) inhibited 10% fetal bovine serum (FBS)-induced increase in migration of VSMCs (61% of control at 1 micromol/l) in a concentration-dependent manner. PTHrP-(1-34) also inhibited 5% FBS-induced increase in [3H]thymidine incorporation (37% of control at 1 micromol/l) and cell number of VSMCs (33% of control at 1 micromol/l) in a concentration-dependent manner. Parathyroid hormone (PTH)-(1-34) inhibited the migration and DNA synthesis of VSMCs to a similar extent. PTHrP-(7-34), a PTH/PTHrP receptor antagonist, significantly inhibited these effects of PTHrP and PTH. PTHrP-(1-34) also inhibited platelet-derived growth factor-BB (5 ng/ml)-induced migration and DNA synthesis of VSMCs. These findings suggest that PTHrP-(1-34) inhibits the migration and proliferation of VSMCs through PTH/PTHrP receptors.

Antifibrotic response of cardiac fibroblasts in hypertensive hearts through enhanced TIMP-1 expression by basic fibroblast growth factor.

BACKGROUND Cardiac fibroblasts (CFs) play a pivotal role in the development of myocardial fibrosis. We previously demonstrated that direct injection of basic fibroblast growth factor (bFGF) into the hypertensive Dahl salt-sensitive (DS) rat heart prevented systolic dysfunction and left ventricular dilation effectively. However, the precise role played by bFGF in fibrotic response of CFs remains unclear. We suggested potential effects of bFGF on the fibrotic response of CFs in vitro. METHODS AND RESULTS Histopathologic assessment of cardiac fibrosis demonstrated a marked decline in the extent of perivascular and interstitial fibrosis in bFGF-injected hypertensive DS rat hearts. CFs harvested from the hearts of noninjected DS rats demonstrated a significantly increased messenger RNA (mRNA) expression of matrix metalloproteinase (MMP)-2, MMP-9, and both collagen I and III. In contrast, bFGF treatment in the CFs induced a marked increase in tissue inhibitor of MMP (TIMP)-1 expression and a marked decline in MMP-9 activation. bFGF also induced a decline in α-smooth muscle actin and collagen I and III mRNA expression in the CFs accompanied by inhibited differentiation of CFs into myofibroblasts. Small interfering RNA targeting FGF receptor 1 confirmed a specific interference of the mRNA expression changes elicited by bFGF. In vivo examination confirmed many TIMP-1-positive CFs in perivascular spaces of bFGF-injected hearts. CONCLUSIONS Up-regulated TIMP-1 expression and down-regulated MMP-9 activation by bFGF in CFs could prevent excessive ECM degradation and collagen deposition in perivascular spaces effectively, leading to prevention of cardiac fibrosis during hypertensive heart failure. SUMMARY Cardiac fibroblasts (CFs) play a pivotal role in myocardial fibrosis. The precise role of CFs in fibrotic response played by growth factors remains unclear. Our results indicates that basic fibroblast growth factor could up-regulate TIMP-1 expression and down-regulate MMP-9 activation in CFs in perivascular spaces, leading to inhibited progression of cardiac fibrosis during hypertensive heart failure.

Effect of endothelin on angiotensin converting enzyme activity in cultured vascular smooth muscle cells

We evaluated the effect of endothelin-1 (ET) on the angiotensin converting enzyme (ACE) activity in rat aortic smooth muscle cells (VSMCs). ACE activity was determined by radioimmunoassay of the amount of angiotensin II generated after the addition of angiotensin I (500 pg/ml) to cultured VSMCs. The antibody used had less than 0.1% cross-reactivity with angiotensin I. ACE activity increased 1.9-fold 5 hr after the addition of 10(-6) M ET under serum-free conditions. This stimulatory effect of ET on ACE activity in VSMCs was completely inhibited by 10(-7) M captopril. Results suggested that the ACE present in SMCs is stimulated by ET.

Basic fibroblast growth factor inhibits ventricular remodeling in Dahl salt-sensitive hypertensive rats

Objective Basic fibroblast growth factor (bFGF) inhibits the progression of ventricular remodeling in ischemic and hypertensive heart diseases (HHDs). Recent studies have revealed that bFGF induces the transition from myofibroblasts to fibroblasts with decreased expression of α-smooth muscle actin (α-SMA). To clarify the mechanisms underlying the reduced ventricular remodeling in hypertensive heart diseases caused by bFGF, we examined the degree of interstitial fibrosis associated with α-smooth muscle actin expression and matrix metalloproteinase activity in hypertensive heart diseases. Methods Dahl salt-sensitive rats were fed with a high-salt diet from 6 to 18 weeks of age and injected with a single dose of bFGF (100 μg) into the left myocardium at 15 weeks. Others were administered PBS without bFGF. Control age-matched Dahl salt-sensitive rats were fed with a low-salt diet. Results Cardiac systolic function was well preserved and decompensation of heart failure was prevented at 18 weeks in the rats treated with bFGF at 15 weeks. The bFGF-treated rats had significantly fewer interstitial α-SMA-positive myofibroblasts and significantly decreased prolyl 4-hydroxylase expression. Increased matrix metalloproteinase-9 gelatinase activity correlated with the downregulation of transforming growth factor-β1 by bFGF, suggesting that inhibited extracellular matrix deposition is associated with a decreased number of myofibroblasts induced by bFGF. Conclusion bFGF can inhibit the progression of ventricular remodeling by inhibiting interstitial fibrosis and promoting angiogenesis without decreasing blood pressure in hypertensive heart disease.

Comparative analysis of the therapeutic effects of long-acting and short-acting loop diuretics in the treatment of chronic heart failure using 123I-metaiodobenzylguanidine scintigraphy

Loop diuretics are essential for the treatment of chronic heart failure (CHF) but short‐acting diuretics are reported to induce sympathetic nervous system (SNS) activation. This study was performed to compare therapeutic effects of two loop diuretics, long‐acting azosemide and short‐acting furosemide, using 123I‐metaiodobenzylguanidine (123I‐MIBG) scintigraphy.

Vascular Smooth Muscle Relaxation by α-Adrenoceptor Blocking Action of Dobutamine in Isolated Rabbit Aorta

We investigated the mechanism of vascular relaxation produced by dobutamine, a positive inotropic agent with beta 1-adrenergic action. Dobutamine concentration-dependently (10 nM-10 microM) relaxed ring segments of rabbit aorta partially precontracted with 1 microM phenylephrine (PE) but did not relax those precontracted with 40 mM K+ or 5 microM prostaglandin F2 alpha (PGF2 alpha). The relaxation was not completely inhibited by pretreatment with 10 microM propranolol. Dobutamine did not significantly increase tissue cyclic AMP levels concomitantly with relaxation as does isoproterenol (ISO) in rabbit aorta. Dobutamine produced a parallel rightward shift in concentration-response curves to PE. The Schild plot analysis resulted in a linear regression of a slope of 1.077 +/- 0.077, which was not significantly different from unity. The pA2 value of dobutamine as compared with PE in rabbit aorta was 6.81 +/- 0.03. Dobutamine causes arterial dilatation mediated not only through a beta-adrenergic action but also through an alpha-adrenergic blocking action in rabbit aorta.

Vascular smooth muscle relaxation induced by epidermal growth factor is endothelium-dependent

Epidermal growth factor (EGF) is released from platelets during aggregation. Because we thought that EGF played a role in vascular tone, we investigated its vascular reactivity using isolated rat aortic strips with and without the endothelium. In the presence of endothelium, EGF relaxed vascular smooth muscle precontracted with 40 mM K+, 10(-5) M prostaglandin F2 alpha or 10(-6) M norepinephrine. The relaxation induced by EGF was more prominent on the prostaglandin F2 alpha- and norepinephrine-induced contractions than on the K(+)-induced contraction. Atropine (10(-5) M) and aspirin (10(-5) M) had no effect on the EGF-induced relaxation, but methylene blue (10(-5) M) partly abolished the relaxation evoked by EGF. These results suggest that EGF relaxes vascular smooth muscle in the presence of the endothelium. They also suggest that EGF has an effect on the endothelium to produce relaxing factor independent of cyclooxygenase; the releasing factor activates soluble guanylate cyclase, resulting in relaxation of vascular smooth muscle through the production of cyclic GMP.