Joseph S. Janicki

Impaired diastolic function and coronary reserve in genetic hypertension. Role of interstitial fibrosis and medial thickening of intramyocardial coronary arteries.

Left ventricular hypertrophy (LVH) in rats with genetic hypertension is accompanied by abnormal myocardial diastolic stiffness and impaired coronary reserve. Whether these functional defects are related to a structural remodeling of the myocardium that includes an interstitial and perivascular fibrosis, myocyte hypertrophy, and medial thickening of intramyocardial coronary arteries is uncertain. To address these issues, 14-week-old male spontaneously hypertensive rats with established hypertension and LVH were treated with low-dose (SLO group: 2.5 mg/kg/day, n = 11) or high-dose (SHI group: 20 mg/kg/day, n = 9) oral lisinopril for 12 weeks to sustain hypertension and LVH or to normalize arterial pressure and myocardial mass, respectively. When SHI and SLO groups were compared with age- and sex-matched 26-week-old untreated spontaneously hypertensive rats (n = 11) and normotensive Wistar-Kyoto rats (n = 9), we found 1) normalization of blood pressure (p less than 0.005) and complete regression of LVH (p less than 0.005) in the SHI group and no significant blood pressure or LVH reduction in the SLO group, 2) complete regression of morphometrically determined myocardial interstitial and perivascular fibrosis in SHI and SLO groups (p less than 0.025) associated with normalization of diastolic stiffness, measured in the isolated heart (p less than 0.025), and 3) regression of medial wall thickening of intramyocardial coronary arteries only in the SHI group (P less than 0.005), accompanied by a normalization of coronary vasodilator reserve to adenosine (p less than 0.005). Thus, interstitial fibrosis and not LVH is responsible for abnormal myocardial diastolic stiffness, whereas medical wall thickening of intramyocardial resistance vessels, influenced by arterial pressure, is associated with impaired coronary reserve.

TEMPORAL DIFFERENCES IN FIBROBLAST PROLIFERATION AND PHENOTYPE EXPRESSION IN RESPONSE TO CHRONIC ADMINISTRATION OF ANGIOTENSIN II OR ALDOSTERONE

Chronic activation of the circulating renin-angiotensin-aldosterone system (RAAS), as can occur with unilateral renal ischemia (URI), is associated with an adverse structural remodeling of the right and left ventricles characterized by reparative (i.e., microscopic scars) and reactive (i.e., perivascular/interstitial) fibrosis. The time course and cells involved in fibroplastic and fibrogenic phases of these events are unclear. Hearts were examined over the course of 8 weeks in rats infused with either angiotensin II or aldosterone, and compared to rats with URI. Tissue sections from the same heart were stained with hematoxylin and eosin, collagen specific picrosirius red, or immunolabeled with PCNA or alpha smooth muscle actin antibody. With angiotensin II or renal ischemia, fibroblast proliferation, presenting as focal accumulations at both sites of myocyte necrosis and widespread perivascular locations, was present in each ventricle on days 2 and 4, but not thereafter, alpha-Smooth muscle actin containing cells (myofibroblasts) appeared at day 2 and persisted through week 2 with renal ischemia and week 6 with angiotensin II. Macrophages, neutrophils and lymphocytes were transiently found at sites of necrosis between day 2-4 of renal ischemia. AngII-induced necrotic sites were characterized by macrophages and lymphocytes from day 2 through week 6, and neutrophils at day 2-4. Increased collagen volume fraction, presenting as immature scars associated with fibroblast clusters and interstitial/perivascular fibrosis, was evident on day 14 in both ventricles. In contrast, fibroblast proliferation during aldosterone infusion did not appear in both ventricles until week 3 and was associated with a subsequent reparative and reactive fibrosis as early as 4 weeks. Myofibroblasts became evident between 3-6 weeks; macrophages and lymphocytes were seen between 3-8 weeks. Neutrophils were not seen at any time point with aldosterone. Thus, the temporal cellular response and appearance of myocardial fibrosis associated with chronic elevations in angiotensin II and/or aldosterone differ. We conclude that separate pathogenic mechanisms are operative with these effector hormones of the RAAS.

Myocardial fibrosis: role of ventricular systolic pressure, arterial hypertension, and circulating hormones

The myocardium contains myocyte and non-myocyte cells. A disproportionate growth of the nonmyocyte cell population can alter myocardial structure and lead to pathologic hypertrophy. Myocardial fibrosis, the result of cardiac fibroblast growth or abnormal accumulation of fibrillar collagen within the interstitial space, can adversely influence myocardial stiffness and ultimately ventricular function. We have examined the relative importance of ventricular systolic and arterial pressures and the effector hormones of the renin-angiotensin--aldosterone system in mediating this reactive fibrous tissue response in the hypertensive left and normotensive right ventricles in various experimental models of arterial hypertension. To date, our findings implicate arterial hypertension, together with an elevation in plasma aldosterone, as being contributory to the fibrosis in renovascular hypertension that creates tissue heterogeneity in either ventricle and impaired diastolic function. The endocrine properties of aldosterone in this nonclassical mineralocorticoid target tissue, the myocardium, requires further investigation.

Extracellular matrix regulation of metalloproteinase and antiproteinase in human heart fibroblast cells.

Following myocardial infarction, extracellular matrix (ECM) is disrupted, which leads to the generation of collagen‐ and elastin‐derived peptides (CDPs and EDPs, respectively). To investigate whether ECM‐derived peptides (i.e., CDPs and EDPs) induce extracellular proteinases in human heart fibroblast (HHF) cells, we isolated CDP and EDP using gelfiltration and antibody affinity column chromatography. The CDP and EDP were characterized by their intrinsic fluorescence due to cross‐link structure (pyridinoline and desmosine, respectively) and by immunoblot analysis using anti‐desmosine antibody. Neutrophil elastase and cathepsin G were identified using selective chromogenic substrates and by their specific inhibition with α1‐proteinase inhibitor and α1‐antichymotrypsin, respectively. Elastase and cathepsin G were elevated in the infarcted tissue. Selective inhibition of matrix metalloproteinase (MMP) by a higher concentration of tetracycline or doxycycline in zymographic gels elicited an inhibition constant (IC50) of 278 ± 10 μM and indicated that majority of MMP in the infarcted tissue is from fibroblast cells. The HHF proliferation was measured using an acid‐phosphatase assay. The EDP and CDP induce HHF cell proliferation. After EDP treatment phenotypic (formation of pseudopodia) changes were observed in HHF cells. To measure whether phenotypic changes by EDP or CDP are associated with MMP and tissue inhibitor of metalloproteinase (TIMP) expression in HHF cells, we measured MMP and TIMP expression by zymographic and Northern blot (mRNA) analyses. The expression of MMP and TIMP were upregulated at both the protein and gene transcription levels. These results suggested that during ischemic cardiomyopathy, initially neutrophil proteinase activates latent myocardial MMP which can degrade ECM, which continuously degrades if not controlled by TIMP, leading to ventricular dilatation and dysfunction.

Prevention of angiotensin II induced myocyte necrosis and coronary vascular damage by lisinopril and losartan in the rat.

OBJECTIVEnThe aims were to determine: (1) if angiotensin converting enzyme (ACE) inhibition and angiotensin II receptor blockade can prevent angiotensin II induced coronary vascular damage; (2) if the cardioprotective properties of ACE inhibition are dose dependent; and (3) if the cardioprotective properties of ACE inhibition are independent of its ability to prevent the conversion of angiotensin I to angiotensin II.nnnMETHODSnControl rats and rats with either renovascular hypertension or continuous angiotensin II infusion (150 ng.min-1) for 14 d were subdivided into nine groups as follows: unoperated and untreated controls (n = 5); untreated renovascular hypertension (n = 8); untreated angiotensin II (n = 9); a renovascular hypertension group receiving one of the following doses of lisinopril 20 (n = 8), 2.5 (n = 4), and 0.6 (n = 6) mg.kg-1.d-1; a renovascular hypertension group receiving losartan (7.5 mg.d-1, n = 4); and an angiotensin II group receiving either the high dose of lisinopril (n = 6) or losartan (n = 4). Treatment was started one day before initiation of renovascular hypertension and angiotensin II infusion and continued throughout the study period. The number and size of necrotic areas and numbers of damaged coronary vessels were determined in sections of right and left ventricular tissue.nnnRESULTSnBoth coronary vascular injury and myocyte injury induced by angiotensin II were prevented by losartan. In renovascular hypertension, the lowest dose of lisinopril prevented vascular and attenuated myocyte damage but to a lesser degree than the higher doses. The cardioprotective ability of ACE inhibition is primarily the result of its ability to prevent the conversion of angiotensin I to angiotensin II.nnnCONCLUSIONSnAngiotensin II related cardiomyocyte necrosis and coronary vascular damage are angiotensin type 1 receptor mediated and completely preventable with the receptor antagonist losartan. The ability of ACE inhibition to prevent this damage is dose dependent and primarily related to the degree to which the inhibitor can prevent the conversion of angiotensin I to angiotensin II.

Cardioreparation with Lisinopril in the Management of Hypertension and Heart Failure

Myocyte growth is seen in all forms of myocardial hypertrophy. In certain disease states, particularly arterial hypertension, components of the hypertrophic remodelling process, other than myocyte growth, distort myocardial structure and thereby adversely alter its mechanical behaviour. Such a pathologic structural remodelling includes a perivascular and interstitial fibrosis that impairs myocardial stiffness and a medial thickening of intramyocardial coronary arteries that attenuates its vasodilator reserve to ischaemic and pharmacologic provocation. The concept of cardioreparation embodies both a regression in myocyte hypertrophy and the pathologic components of the structurally remodelled myocardium and in so doing restores structure and function to normal. Implicit in this concept is the supposition that heart failure will be reversible. The concept of reparation was tested in 14-week-old male spontaneously hypertensive rats having left ventricular hypertrophy, diastolic dysfunction with myocardial fibrosis, and impaired coronary vascular reserve to adenosine, using the angiotensin-converting enzyme inhibitor lisinopril. A regression in left ventricular hypertrophy, perivascular and interstitial fibrosis, and medial thickening of intramural vessels were obtained after 12 weeks of oral lisinopril administration. It would now seem logical to determine whether cardioreparation can be achieved with lisinopril in patients with hypertension and left ventricular hypertrophy, in whom pathologic remodelling of the myocardium is responsible for symptomatic heart failure.

The effect of non-antihypertensive doses of angiotensin converting enzyme inhibitor on myocardial necrosis and hypertrophy in young rats with renovascular hypertension

In renovascular hypertensive rats, low doses of angiotensin converting enzyme (ACE) inhibitors have been found to prevent myocardial hypertrophy independent of blood pressure level. This finding would suggest humoral rather than mechanical control of myocyte growth. The aim of this study was to examine the effect of nonantihypertensive doses of ACE inhibitor on myocardial hypertrophy and necrosis in hypertensive rats. Renovascular hypertension (RHT) was induced in four‐week‐old Wistar rats. Twenty‐eight animals were treated for four weeks with three doses of ramipril (0.01, 0.1 or 1.0u2003mg/kg/day, which are unable to lower blood pressure. Fourteen animals were not treated (RHT group). A sham operated, age/sex‐matched group was used as control (nu2003=u200310). Myocardial histology was analysed in 3u2003μm thick sections of the ventricle stained with either haematoxylin‐eosin, reticulin silver stain or Massons trichrome. There was a significant correlation between systolic blood pressure and left ventricular to body weight ratio in both sets of animals: untreated plus controls and ramipril‐treated rats. ACE inhibition prevented myocyte and perivascular necrosis and fibrosis in a dose‐dependent manner. We conclude that myocardial hypertrophy in rats with renovascular hypertension is directly related to arterial pressure, and that this relationship is not affected by nonantihypertensive doses of ACE inhibitor. Myocardial necrosis/fibrosis and coronary artery damage induced by angiotensin II are prevented by ACE inhibitor in a dose‐dependent manner, despite the presence of arterial hypertension.

Structural and Functional Consequences of Myocardial Collagen Remodeling

Myocardial fibrillar collagens provide for muscle fiber and cardiac myocyte alignment and impart a tensile strength to the myocardium that maintains ventricular shape and size, and governs tissue stiffness. This network of collagen is intimately related with the myocyte and muscle fiber, as well as the coronary vasculature. Consisting primarily of collagen types I and III, fibrillar collagen is relatively inelastic and, even though normally present in relatively small amounts, plays an important role in the behavior of the ventricle during diastole. In renovascular and genetic hypertension, the hypertrophic response of the myocardium includes a progressive remodeling of the collagen matrix. Typically, there is an increase in collagen concentration, thickening of existing fibrillar collagen, and the addition of new collagen to all components of the matrix. The consequences of this remodeling are a stiffer myocardium and left ventricular diastolic dysfunction. These pathophysiologic aspects of the hypertrophic process are independent of the concomitant remodeling of the myocyte. Thus, an abnormal accumulation of interstitial collagen is a major distinguishing factor between physiologic and pathologic hypertrophy. Removal of less than half of the normal amount of collagen following collagenase activation results in a dilated ventricle with increased compliance. Collagenase activation, collagen degradation, and a dilated, thin-walled left ventricle are evident during ischemia, in dilated cardiomyopathy, and at end-stage heart failure. Thus, chronic changes in the shape and size of the heart are the result of an inadequate interstitial collagen matrix.

Myocardial Collagen and Its Functional Role

Even though normally present in relatively small amounts, myocardial collagen strongly influences ventricular diastolic function. Removal of less than half of the normal amount results in a dilated ventricle with increased compliance. In contrast, an abnormal increase in collagen concentration results in a stiffer myocardium and ventricular diastolic dysfunction.