Long-term treatment with valsartan improved cyclic variation of the myocardial integral backscatter signal and diastolic dysfunction in hypertensive patients: the echocardiographic assessment.

Myocardial fibrosis is the major determinant of diastolic property of the left ventricle (LV). Experimental and clinical studies have suggested that angiotensin receptor blockers attenuate myocardial fibrosis in various heart diseases. The integrated backscatter signal (IBS) represents a promising ultrasonic method for assessing the characterization of myocardial tissue: cardiac cycle-dependent variation of the IBS (IBS-CV) is negatively correlated with myocardial collagen deposition in hypertensive hearts. Using non-invasive echocardiographic techniques, we performed a prospective, multi-center trial to examine whether long-term treatment with valsartan would improve myocardial fibrosis and diastolic dysfunction in hypertensives. This study included 43 hypertensive patients who had impaired diastolic function (transmitral Doppler flow early to late filling velocity ratio [E/A ratio] <1.0) and preserved systolic function (LV ejection fraction [LVEF] >50%). Twelve-month valsartan treatment reduced blood pressure (BP) and LV mass index. Valsartan significantly increased not only IBS-CV but also E/A ratio without changing LVEF. The effects of valsartan were compared between two subgroups: one with low IBS-CV (IBS-CV <5.08 dB [the average of 43 patients at baseline]), the other with high IBS-CV (IBS-CV >5.08 dB). At baseline, BP, LV mass index, LVEF, and E/A ratio were similar in the two groups. Valsartan significantly increased IBS-CV and E/A ratio in the low IBS-CV group, but not in the high IBS-CV group, despite comparable reductions in BP and LV mass. In conclusion, long-term valsartan treatment attenuated myocardial fibrosis and improved diastolic dysfunction in hypertensives. It is suggested that in the low IBS-CV group, improvement of diastolic dysfunction by valsartan may be caused by attenuation of myocardial fibrosis, and not by regression of LV hypertrophy.

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.

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)-beta is crucial for reactive fibrosis in AC rats. MCP-1 function blocking not only inhibits macrophage infiltration and TGF-beta 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.

Hypertensive myocardial fibrosis and diastolic dysfunction: another model of inflammation?

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)-beta 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-beta 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-beta-mediated process. Inhibition of inflammation may be a new strategy to prevent myocardial fibrosis and resultant diastolic dysfunction in hypertensive hearts.

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

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]-beta 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-beta 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.

Roles of intercellular adhesion molecule-1 in hypertensive cardiac remodeling.

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]-beta 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-beta 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-beta induction and fibroblast activation.

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

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-beta-mediated perivascular fibrosis in hypertensive rat hearts.

Recently, we have shown that pressure overload transiently induces transforming growth factor-beta-mediated fibroblast proliferation and reactive myocardial fibrosis that extends from the perivascular space. However, the upper stream event of transforming growth factor-beta 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-beta 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-beta 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.

Transforming growth factor-beta 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)-beta 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-beta mRNA expression was induced after day 3, peaked at day 7, and remained modestly increased at day 28. An anti-TGF-beta 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-beta 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.

Coexistence of hypercholesterolemia and hypertension impairs adventitial vascularization.

We have shown that adventitial vasa vasorum (AVV) formation is enhanced in hypertensive rat aorta to compensate hypoxia in the thickened media and that hypercholesterolemia impairs angiogenesis in rat ischemic hindlimb. Thus, we examined the effects of coexistence of hypercholesterolemia and hypertension on AVV formation. In Wistar rats, hypercholesterolemia was established by high-cholesterol diet from Day -14 (HC rats), and hypertension was induced by a suprarenal aortic constriction at Day 0 (HT rats). At Day 28, we studied AVV density, adventitial area, and medial thickness in the ascending aorta of control (standard diet+sham operation), HC, HT, and HC+HT rats (n=5/group). In HC rats, although the adventitial area was modestly increased, the AVV density and medial thickness were unchanged versus controls. In addition to medial thickening, marked enlargement of the adventitial area accompanied by increased AVV density was observed in HT rats, compared with controls. HC+HT rats showed lower AVV density, despite larger adventitial area, than HT rats, whereas the medial thickness was similar in HT and HC+HT rats. Immunohistostaining revealed hypoxia-inducible factor-1alpha expression in the media only in HC+HT rats but not in the other 3 groups, suggesting persistent medial hypoxia in HC+HT rats. In conclusion, it is suggested that coexistence of hypercholesterolemia and hypertension impairs AVV formation, resulting in insufficient compensation for hypoxia in the thickened media. Our findings provide an insight into the mechanism of the aggravation of arteriosclerosis when both hypercholesterolemia and hypertension are present.

Hypoxia-inducible factor-1alpha/vascular endothelial growth factor pathway for adventitial vasa vasorum formation in hypertensive rat aorta.

The roles of adventitial vasa vasorum have been highlighted in vascular wall homeostasis. Vascular endothelial growth factor (VEGF) is a potent angiogenic factor in physiological and pathophysiological conditions. However, little is known regarding the changes in adventitial vasa vasorum and the mechanism of the formation in hypertensive arteries. Accordingly, endothelial cell proliferation, adventitial vasa vasorum count, and expression of VEGF signaling axis proteins were examined in the ascending aorta of hypertensive Wistar rats that underwent suprarenal aortic constriction. Hypertension not only induced medial and adventitial thickening but also significantly increased adventitial vasa vasorum count by day 28. Preceding the medial thickening, BrdU(+)-proliferative endothelial cells were observed in the adventitia but not in the media and intima after day 3; they peaked at day 7 and remained modestly increased at day 28. The BrdU(+) endothelial cells showed induction of Ets-1, a transcription factor mediating angiogenic response of VEGF. Furthermore, concomitant expression of VEGF and a hypoxia-inducible transcription factor (HIF-1alpha) was observed in the outer layers of medial smooth muscle cells at day 3 and extended to the middle layers of medial smooth muscle cells at day 7, returning to lower levels by day 28. In conclusion, adventitial vasa vasorum formation was induced by hypertension through the HIF-1alpha/VEGF/Ets-1 pathway during hypertensive remodeling.