Keisuke Tokuda

Transforming Growth Factor-β Function Blocking Prevents Myocardial Fibrosis and Diastolic Dysfunction in Pressure-Overloaded Rats

Background—Excessive myocardial fibrosis impairs cardiac function in hypertensive hearts. Roles of transforming growth factor (TGF)-&bgr; in myocardial remodeling and cardiac dysfunction were examined in pressure-overloaded rats. Methods and Results—Pressure overload was induced by a suprarenal aortic constriction in Wistar rats. Fibroblast activation (proliferation and phenotype transition to myofibroblasts) was observed after day 3 and peaked at days 3 to 7. Thereafter, myocyte hypertrophy and myocardial fibrosis developed by day 28. At day 28, echocardiography showed normal left ventricular fractional shortening, but the decreased ratio of early to late filling velocity of the transmitral Doppler velocity and hemodynamic measurement revealed left ventricular end-diastolic pressure elevation, indicating normal systolic but abnormal diastolic function. Myocardial TGF-&bgr; mRNA expression was induced after day 3, peaked at day 7, and remained modestly increased at day 28. An anti–TGF-&bgr; neutralizing antibody, which was administered intraperitoneally daily from 1 day before operation, inhibited fibroblast activation and subsequently prevented collagen mRNA induction and myocardial fibrosis, but not myocyte hypertrophy. Neutralizing antibody reversed diastolic dysfunction without affecting blood pressure and systolic function. Conclusions—TGF-&bgr; plays a causal role in myocardial fibrosis and diastolic dysfunction through fibroblast activation in pressure-overloaded hearts. Our findings may provide an insight into a new therapeutic strategy to prevent myocardial fibrosis and diastolic dysfunction in pressure-overloaded hearts.

Hypertensive Myocardial Fibrosis and Diastolic Dysfunction: Another Model of Inflammation?

Abstract—Excessive myocardial fibrosis deteriorates diastolic function in hypertensive hearts. Involvement of macrophages is suggested in fibrotic process in various diseased situations. We sought to examine the role of macrophages in myocardial remodeling and cardiac dysfunction in pressure-overloaded hearts. In Wistar rats with suprarenal aortic constriction, pressure overload induced perivascular macrophage accumulation and fibroblast proliferation with a peak at day 3, decreasing to lower levels by day 28. Myocyte chemoattractant protein (MCP)-1 mRNA was upregulated after day 1, peaking at day 3 and returning to insignificant levels by day 28, whereas transforming growth factor (TGF)-&bgr; induction was observed after day 3, with a peak at day 7, and remained relatively elevated at day 28. After day 7, concentric left ventricular (LV) hypertrophy developed, associated with reactive fibrosis and myocyte hypertrophy. At day 28, echocardiography showed normal LV fractional shortening but decreased ratio of early to late filling wave of transmitral Doppler velocity, and hemodynamic studies revealed elevated LV end-diastolic pressure, suggesting normal systolic but impaired diastolic function. Chronic treatment with an anti-MCP-1 monoclonal neutralizing antibody inhibited not only macrophage accumulation but also fibroblast proliferation and TGF-&bgr; induction. Furthermore, the neutralizing antibody attenuated myocardial fibrosis, but not myocyte hypertrophy, and ameliorated diastolic dysfunction without affecting blood pressure and systolic function. In conclusion, roles of MCP-1-mediated macrophage accumulation are suggested in myocardial fibrosis in pressure-overloaded hearts through TGF-&bgr;-mediated process. Inhibition of inflammation may be a new strategy to prevent myocardial fibrosis and resultant diastolic dysfunction in hypertensive hearts.

Diastolic dysfunction in hypertensive hearts : Roles of perivascular inflammation and reactive myocardial fibrosis

There is increasing evidence that myocardial fibrosis plays a role in the pathogenesis of diastolic dysfunction in hypertensive heart disease. However, it has been difficult to explore the mechanisms of isolated diastolic dysfunction in hypertensive hearts because of the lack of adequate animal models. Recently, we demonstrated that Wistar rats with a suprarenal aortic constriction (AC) can be used as a model of cardiac hypertrophy associated with preserved systolic, but impaired diastolic function without overt congestive heart failure. In this model, acute pressure elevation induces reactive myocardial fibrosis (perivascular fibrosis followed by intermuscular interstitial fibrosis) and myocyte/left ventricular (LV) hypertrophy. Perivascular macrophage infiltration, which is mediated by monocyte chemoattractant protein-1 (MCP-1) and intercellular adhesion molecule-1, exerts a key role in myocardial fibrosis, but not in myocyte/LV hypertrophy. Transforming growth factor (TGF)-β is crucial for reactive fibrosis in AC rats. MCP-1 function blocking not only inhibits macrophage infiltration and TGF-β induction but also prevents reactive fibrosis and diastolic dysfunction, without affecting blood pressure, myocyte/LV hypertrophy, or systolic function. Accordingly, a substantial role of inflammation is indicated in myocardial fibrosis and diastolic dysfunction in hypertensive hearts. Currently, the precise mechanisms whereby acute pressure elevation triggers inflammation remain unknown, but it is likely that activation of the tissue angiotensin system is involved in the induction of the inflammatory process.

Pressure-independent effects of angiotensin II on hypertensive myocardial fibrosis.

Abstract—Angiotensin II (Ang II) is implicated in the proinflammatory process in various disease situations. Thus, we sought to determine the role of Ang II in early inflammation-induced fibrosis of pressure-overloaded (PO) hearts. PO was induced by suprarenal aortic constriction (AC) at day 0 in male Wistar rats, and they were orally administered 0.1 mg/kg per day candesartan every day from day −7. This was the maximum dose of candesartan that did not change arterial pressure in hypertensive rats with AC (AC rats). In AC rats, cardiac angiotensin-converting enzyme (ACE) activity was transiently enhanced after day 1 and peaked at day 3, declining to lower levels by day 14, whereas serum ACE activity was not changed. In AC rats, PO induced early fibroinflammatory changes (monocyte chemoattractant factor [MCP]-1 and transforming growth factor [TGF]-&bgr; expression, perivascular macrophage accumulation, and fibroblast proliferation), and thereafter, left ventricular hypertrophy developed, featuring myocyte hypertrophy, intramyocardial arterial wall thickening, and perivascular and interstitial fibroses. Candesartan suppressed the induction of MCP-1 and TGF-&bgr; and reduced macrophage accumulation and fibroblast proliferation in PO hearts. Candesartan significantly prevented perivascular and interstitial fibrosis. However, candesartan did not affect myocyte hypertrophy and arterial wall thickening. In conclusion, a subdepressor dose of candesartan prevented the MCP-1–mediated inflammatory process and reactive myocardial fibrosis in PO hearts. Ang II might play a key role in reactive fibrosis in hypertensive hearts, independent of arterial pressure changes.

Pressure Overload–Induced Transient Oxidative Stress Mediates Perivascular Inflammation and Cardiac Fibrosis through Angiotensin II

Oxidative stress is implicated in the pathogenesis of various cardiovascular diseases. We have shown that in Wistar rats with a suprarenal aortic constriction (AC), pressure overload–induced transient perivascular inflammation (monocyte chemoattractant protein-1 [MCP-1] induction and macrophage accumulation) in the early phase is the determinant of reactive myocardial fibrosis and resultant diastolic dysfunction in the late phase. Thus, we investigated the role of reactive oxygen species production in cardiac remodeling in AC rats. Superoxide production and the footprint of lipid peroxidation were assessed using dihydroethidium staining and immunohistostaining against 4-hydroxy-2-nonenal (4-HNE), respectively. In sham rats, dihydroethidium and 4-HNE signals were scarcely found in the heart. At day 3, AC rats showed dihydroethidium signals mainly in the intramyocardial arterial wall, whereas modest 4-HNE staining was observed diffusely in the myocardium. These signals declined to lower levels by day 14 despite sustained hypertension. Chronic administration of a subdepressor dose of an angiotensin II type 1 receptor blocker candesartan reduced the pressure overload–induced dihydroethidium and 4-HNE signals at day 3. Moreover, candesartan decreased MCP-1 induction and macrophage infiltration at day 3 and prevented myocardial fibrosis at day 14, without affecting left ventricle and myocyte hypertrophy. In conclusion, acute pressure overload induced self-limited superoxide production mainly in the vascular wall. The reactive oxygen species production would contribute to the perivascular inflammation and subsequent myocardial fibrosis. Angiotensin II was suggested to have a pressure-independent effect on the reactive oxygen species production.

Roles of Intercellular Adhesion Molecule-1 in Hypertensive Cardiac Remodeling

Abstract—Recently, we have shown that in rats with a suprarenal abdominal aortic constriction (AC), pressure overload induces early perivascular fibro-inflammatory changes (transforming growth factor [TGF]-&bgr; induction and fibroblast proliferation) within the first week after AC and then causes the development of cardiac remodeling (myocyte hypertrophy and reactive myocardial fibrosis) associated with diastolic dysfunction. Intercellular adhesion molecule (ICAM)-1 is implicated in the recruitment of leukocytes, especially macrophages, in various inflammatory situations. Thus, we sought to investigate the causal relation of ICAM-1 to macrophage recruitment and cardiac remodeling in AC rats. In AC rats, immunoreactive ICAM-1 was observed transiently on endothelial cells of the intramyocardial coronary arterioles after day 1, with a peak at day 3, returning to baseline by day 7. Also, ED1+ macrophage accumulation was found in the area adjacent to the arteries expressing ICAM-1. Chronic treatment with an anti–ICAM-1 neutralizing antibody, but not with control IgG, remarkably reduced the accumulations of macrophages and proliferative fibroblasts and inhibited the upregulation of TGF-&bgr; expression. Furthermore, the neutralizing antibody significantly prevented myocardial fibrosis without affecting arterial pressure and left ventricular and myocyte hypertrophy. In conclusion, ICAM-1 expression was induced by pressure overload in the intramyocardial arterioles, and triggered perivascular macrophage accumulation. In pressure-overloaded hearts, a crucial role in ICAM-1–mediated macrophage accumulation was suggested in the development of myocardial fibrosis, through TGF-&bgr; induction and fibroblast activation.

Rho-kinase inhibition reduces neointima formation after vascular injury by enhancing Bax expression and apoptosis.

Summary: Recently, we have shown that a specific Rho‐kinase inhibitor, Y27632 (R‐(+)‐trans‐N‐(4‐pyridyl)‐4‐(1‐aminoethyl)‐ cyclohexanecarboxamide), prevents neointima formation after vascular injury associated with increased terminal deoxynucleotidyl transferase‐mediated dUTP nickend labeling (TUNEL)+ smooth muscle cells. Because the mechanism of the action of Y27632 remains unclear, we investigated the expression changes in Bcl family proteins, apoptosis regulators of smooth muscle cells, in the rat carotid artery after balloon injury (BI). Y27632 (BI + Y group) or saline (BI group) was administered peritoneally from Day 1 to Day 14 after BI. Y27632 markedly prevented neointima formation at Day 14. In the BI group, TUNEL+ smooth muscle cells were transiently increased in the neointima, but not in the media, with a peak at Day 7, returning to a lower level by Day 14. Y27632 significantly increased TUNEL+ smooth muscle cells at Days 7 and 14. Smooth muscle cell apoptosis was confirmed by electron microscopic examination. At Day 14, although proapoptotic Bax was slightly, but not significantly, increased in the BI group, it was significantly upregulated in the BI + Y group. Antiapoptotic Bcl‐xL was upregulated in the BI group, and the upregulated Bcl‐xL was not affected by Y27632. These findings indicate that Rho‐kinase inhibition induces neointimal smooth muscle cell apoptosis through Bax upregulation, resulting in reduced neointima formation.

Sub-depressor dose of angiotensin type-1 receptor blocker inhibits transforming growth factor-β-mediated perivascular fibrosis in hypertensive rat hearts

Summary: Recently, we have shown that pressure overload transiently induces transforming growth factor‐&bgr;‐mediated fibroblast proliferation and reactive myocardial fibrosis that extends from the perivascular space. However, the upper stream event of transforming growth factor‐&bgr;induction has remained unknown. Thus, we sought to determine whether angiotensin II mediates the fibrotic process in pressure‐overloaded hearts. Male Wistar rats were administered orally everyday 0.1 mg/kg per day of candesartan, an angiotensin type‐1 receptor blocker, or the vehicle from Day 7, and underwent a suprarenal aortic constriction (AC) at Day 0. This dose was the maximum dose of candesartan that does not induce the depressor effect in AC rats. In AC + vehicle (control) rats, pressure overload induced myocardial transforming growth factor‐&bgr; expression and perivascular fibroblast proliferation at Day 3 and thereafter left ventricular hypertrophy associated with cardiomyocyte hypertrophy and perivascular fibrosis. AC + candesartan rats showed suppressed transforming growth factor‐&bgr; expression and reduced number of proliferating fibroblasts, while not changing arterial pressure. Furthermore, perivascular fibrosis, but not myocyte hypertrophy, was significantly inhibited associated with reduced collagen mRNA expression. In conclusion, angiotensin II may play a role in reactive myocardial fibrosis in pressure‐overloaded hearts, through the mechanism independent of hemodynamic change.

Perivascular Inflammation and Hypertensive Cardiovascular Remodeling

Myocardial fibrosis has drawn an attention as the pathogenesis of impaired diastolic function in hypertensive hearts. However, the mechanisms whereby hypertension provokes myocardial fibrosis are not fully understood. Recently, we have demonstrated that Wistar rat with a suprarenal aortic constriction is a model of cardiac hypertrophy associated with preserved systolic, but impaired diastolic function. In this model, a rapid blood pressure rise provokes transient upregulations of intercellular adhesion molecule-1 (ICAM-1) and monocyte chemoattractant factor-1 (MCP-1) in the intramyocardial arteries, which in turn trigger perivascular macrophage infiltration. Following the inflammatory changes, myocardial fibrosis extends from perivascular to intermuscular interstitial spaces, which resulting in typical feature of reactive fibrosis. MCP-1 function blocking not only inhibits macrophage infiltration but also prevents reactive fibrosis and diastolic dysfunction, while not affecting blood pressure, myocyte hypertrophy, and systolic function. Tissue angiotensin system is a possible activator of perivascular inflammation. Accordingly, a substantial role of inflammation is suggested in myocardial fibrosis and diastolic dysfunction in hypertensive hearts. Targeting inflammation may be a new strategy for prevention and treatment of myocardial fibrosis and diastolic dysfunction in hypertensive hearts.