Yao Sun

Fibrosis of atria and great vessels in response to angiotensin II or aldosterone infusion

OBJECTIVE Myocardial fibrosis, associated with increased expression of angiotensin converting enzyme (ACE) and bradykinin (BK) receptor binding at sites of tissue repair, accompanies chronic elevations in circulating angiotensin II (AngII) and/or aldosterone (ALDO) that simulate chronic cardiac failure. A role for increased ventricular wall stress, associated with arterial hypertension, that can accompany such neurohormonal activation when ventricular function is not compromised, has been held responsible for this structural remodeling. To address this proposition, we monitored morphology of right and left atria and pulmonary artery, where stress is not increased, and compared these structures with hypertensive aorta. METHODS Experimental groups included: (1) unoperated and untreated controls; (2) intact rats receiving AngII (9 micrograms/h) for 2 weeks and which causes arterial hypertension; (3) uninephrectomized control rats on a high sodium diet for 6 weeks; and (4) uninephrectomized rats receiving ALDO (0.75 micrograms/h) and a high sodium diet for 6 weeks and which results in gradual onset arterial hypertension. Fibrosis was identified by light microscopy in sections stained with collagen specific picrosirius red, while ACE, AngII and BK receptors binding were localized and quantitated by in vitro autoradiography using 125I-351A, 125I[Sar1,Ile8]AngII, and 125I[Tyr8]BK, respectively. AngII receptor subtype was defined by the presence of excess AT1 (losartan) or AT2 (PD123177) receptor antagonists, respectively. RESULTS With either AngII or ALDO administration, and compared to controls, we found: (1) microscopic scarring that replaced lost myocytes in both left and right atria; (2) an increase in adventitial collagen of both pulmonary artery and aorta (perivascular fibrosis); (3) markedly increased ACE binding at fibrous tissue sites in both atria and great vessels; (4) unchanged atrial and great vessel AT1 receptor binding; and (5) significantly increased BK receptor binding at sites of atrial and perivascular fibrosis. CONCLUSIONS Thus, the appearance of atrial fibrosis and perivascular fibrosis of aorta and pulmonary artery, together with associated increase in ACE and BK receptor binding, in rats receiving AngII or ALDO suggests these responses are not related to altered ventricular wall stress or arterial hypertension, but rather to these effector hormones of the circulating renin-angiotensin-aldosterone system. Local BK, regulated by ACE found in fibrous tissue and BK receptor binding may play a role in structural remodeling of atria and great vessels in these rat models that stimulate chronic cardiac failure.

Myofibroblasts and local angiotensin II in rat cardiac tissue repair

Tissue repair is a fundamental property of vascularized tissue. At sites of injury, phenotypically transformed fibroblast-like cells are responsible for fibrous tissue formation, expressed principally as type I and III fibrillar collagens. These cells are termed myofibroblasts because they contain alpha-smooth muscle actin microfilaments and are contractile. In vivo studies of injured rat cardiac tissues and in vitro cell culture studies have shown that such fibroblast-like cells contain requisite components for angiotensin peptide generation and angiotensin II receptors. Such locally generated angiotensin II acts in an autocrine paracrine manner to regulate collagen turnover and thereby tissue homeostasis in injured tissue.

Angiotensin II receptor blockade and myocardial fibrosis of the infarcted rat heart

Tissue angiotensin II (AngII) is increased in the infarcted rat heart, where it may have autocrine or paracrine properties that influence cellular protein synthesis and growth and therefore tissue repair. It was our hypothesis that treatment with an AT1 receptor antagonist would attenuate fibrous tissue formation after myocardial infarction (MI). To investigate a role for local AngII in the regulation of connective tissue formation during early and late wound healing that follows MI, this study was undertaken. Animals were randomized into two groups in which rats were or were not treated with the AT1 receptor antagonist losartan (10 mg x kg(-1) daily gavage). At 1 and 4 weeks after experimental MI was induced by coronary artery ligation, rat hearts were examined. Infarct size, infarct area, and collagen volume fraction at the site of infarction and in noninfarcted myocardium were determined by picrosirius red staining with videodensitometry. Quantitative in vitro autoradiography was used to detect AngII receptor binding density ((125)I-(Sar1,Ile8)AngII). Compared with an untreated MI control group, in losartan-treated rats we found (1) infarct size was comparable in both groups at weeks 1 and 4, (2) infarct area was comparable between groups at week 1 but was significantly reduced (p < 0.05) at week 4 in losartan-treated rats, (3) a detectable reduction in collagen volume fraction at the site of MI was not found at week 1 but was reduced (p < 0.05) at remote sites at week 4, (4) AngII receptor binding density was reduced (p < 0.05) by 50% at the site of MI at both weeks 1 and 4 in keeping with delivery of losartan to this site of injury. Thus AT1 receptor antagonism appears to influence late phase wound healing at and remote to the site of MI and suggests an association between AngII and the fibrogenic response that appears in the injured rat heart. Although still speculative, an attenuation in fibrosis after MI may account for less ventricular dysfunction and geometric remodeling of right and left ventricles and ventricular arrhythmias that have been observed in such rats treated with angiotensin converting enzyme inhibitor or AT1 receptor antagonist.

Angiotensin converting enzyme and kininase-II-like activities in cultured valvular interstitial cells of the rat heart

OBJECTIVE The function of angiotensin converting enzyme (ACE) at cell sites of high collagen turnover, such as heart valves, is uncertain. The aim of this study was to assess ACE and kininase-II-like activities and collagen turnover in cultured valvular interstitial cells of the adult rat heart. METHODS The valvular interstitial cell phenotype was determined by immunolabelling (rhodamine phalloidin, desmin, and Griffonia simplicifolia lectin), and the presence of ACE mRNA and protein was confirmed by reverse transcriptase-polymerase chain reaction analysis, ACE monoclonal antibody and in vitro autoradiography, respectively. ACE and kininase-II-like activities in valvular interstitial cells were analysed by high performance liquid chromatography. Angiotensin II (AT1) and bradykinin receptors in valvular interstitial cell membranes were examined by western immunoblotting and binding assay. Type I collagen and collagenase in valvular interstitial cell culture media were determined by ELISA and zymography, respectively. Type I collagen mRNA expression in cultured valvular interstitial cells was determined by northern blot analysis and in situ hybridisation. RESULTS In intact valvular interstitial cells or their cell membrane we found: (1) actin microfilaments, but not desmin or lectin labelling; (2) ACE mRNA expression and binding activity; (3) conversion of angiotensin I to angiotensin II, which was completely inhibited by 50 microM lisinopril, while kinase-II-like activity exceeded ACE activity and was not inhibited by lisinopril; (4) AT1 and bradykinin receptors in valvular interstitial cell membrane preparations; (5) type I collagen mRNA expression and collagenase activity; and (6) angiotensin II induced increase in type I collagen synthesis and mRNA expression. CONCLUSIONS Cultured valvular interstitial cells represent a nonendothelial, non-smooth-muscle cell type that expresses mRNA for ACE and type I collagen. ACE and kininase-II-like activities in valvular interstitial cells may be involved in the regulation of peptides that influence collagen turnover. Angiotensin II stimulates type I collagen synthesis and mRNA expression in these cells.

Connective tissue: a metabolic Entity?

The heart is composed of parenchyma (cardiac myocytes) and stroma (connective tissue). Stroma is presumed inert and therefore little attention has been paid to its regulation. Contrary to this notion, evidence presented here raises the possibility that connective tissue is a metabolically active entity capable of regulating peptide hormone generation and degradation and these hormones, in an autocrine manner, regulate collagen turnover. This concept has evolved from quantitative in vitro autoradiography (using 125I-351A), which localized angiotensin converting enzyme (ACE) binding density within the heart. A heterogenous distribution was found. Low-density ACE is present within atria and ventricles. At sites of high collagen turnover, such as valve leaflets, adventitia and fibrous tissue of diverse etiologic origins. ACE binding density is high and independent of circulating angiotensin II. ACE-producing cells at these sites, identified by monoclonal ACE antibody and 125I-351A binding, include fibroblast-like alpha actin-containing cells that express the transcript for type I collagen (in situ hybridization). Receptor-ligand binding for angiotensin II and bradykinin is found in fibrous tissue, where these peptides may provide for a reciprocal regulation of fibroblast collagen turnover. Connective tissue formation is attenuated by ACE inhibition or antagonism of type I angiotensin II receptor. Thus, emerging evidence raises the possibility that stroma and its cellular constituents is a dynamic, metabolically active entity regulating its own peptide hormone composition and, in turn, its turnover of fibrillar collagen.

Cells expressing angiotensin II receptors in fibrous tissue of rat heart

OBJECTIVE Following left coronary artery ligation, markedly increased angiotensin II (AngII) receptor binding appears at the site of myocardial infarction (MI). This is also true for the fibrosed visceral pericardium in rats following pericardiotomy (with or without MI). Cells expressing AngII receptors at these sites remain unknown. In the present study, we sought to identify cells expressing AngII receptors at these sites of fibrosis in the rat heart during both early and late stages of wound healing. METHODS MI was created by left coronary artery ligation. Sham operation included thoracotomy, pericardiotomy and placement of silk ligature around the left coronary artery without MI. Hearts were collected at postoperative week 1 and 4. In serial sections: autoradiography (125I[Sar1,Ile8]AngII) was used to determine cells expressing AngII receptors; hematoxylin-eosin and alpha smooth muscle actin were used for identification of cell morphology and phenotype, respectively; and picrosirius red for identification of fibrillar collagen. RESULTS (1) at week 1, necrotic tissue at the site of MI was surrounded by granulation tissue that included macrophages, alpha smooth muscle actin fibroblast-like cells, or myofibroblasts, fibrillar collagen, and new vessels; (2) at week 4, scar tissue had formed and remaining cells were primarily myofibroblasts; (3) pericardial fibrosis was evident at weeks 1 and 4 and contained myofibroblasts, not macrophages or new vessels; (4) at week 1 and 4 myofibroblasts were the predominant cell expressing high-density AngII receptors at the site of MI, while fibroblasts, macrophages and vessels demonstrated low-density AngII receptor binding; and (5) at weeks 1 and 4, myofibroblasts express high-density AngII receptor binding in pericardial fibrosis. CONCLUSION In a rat model of tissue repair involving either MI or pericardial fibrosis, increased AngII receptor expression is primarily associated with myofibroblasts. This suggests AngII may play a role in mediating the fibrogenic response provided by this wound healing cell at sites of tissue injury in the rat heart.

Angiotensin II and extracellular matrix homeostasis

As a circulating hormone, endocrine properties of angiotensin (Ang) II are integral to circulatory homeostasis. Produced de novo its autocrine/paracrine properties contribute to biologic responses involving various connective tissues (e.g. extracellular matrix, adipose tissue, bone and its marrow). In this brief review, we develop the concept of extracellular matrix homeostasis, a self regulation of cellular composition and structure, wherein fibroblast-derived AngII regulates elaboration of TGF-beta 1, a fibrogenic cytokine responsible for connective tissue formation at normal and pathologic sites of collagen turnover.

Angiotensin-converting enzyme and wound healing in diverse tissues of the rat.

Autoradiographic binding density of angiotensin-converting enzyme (ACE), an indirect measure of ACE activity, is markedly increased at sites of fibrous tissue that appear in the injured heart. This includes myocardial infarction (MI) caused by left coronary artery ligation; endocardial fibrosis of the interventricular septum and perivascular fibrosis of intramyocardial coronary arterioles of the right ventricle, each of which appear remote to MI; and pericardial fibrosis after pericardiotomy (without MI). Expressed in fibroblast-like cells found at each site of tissue repair, ACE may be common to tissue repair in the rat heart, irrespective of the etiologic basis of injury. To address this hypothesis and to determine whether this also applies to other tissues (skin and kidney), the present study was undertaken. ACE binding density was measured by quantitative in vitro autoradiography (125I-351A) in injured rat heart, skin, and kidney. Experimental observations included foreign-body fibrosis after placement of silk ligature in skin or myocardium, endomyocardial myocyte necrosis and fibrosis that accompanied isoproterenol administration (1 mg/kg sc x 2 days), and embolic infarction of the kidney as a result of mural thrombus of the left ventricle that appeared after anterior MI. Fibrosis was identified by collagen-specific staining with picrosirius red. Hematoxylin-eosin staining and immunohistochemical labeling with alpha-smooth muscle actin (alpha-SMA) antibody were used to address cell morphology and phenotype, respectively. We found (1) endomyocardial fibrosis 2 weeks after isoproterenol; (2) fibrosis surrounding silk suture in heart and skin 1 week after placement; (3) renal infarction 1 week after left coronary artery ligation; (4) numerous fibroblast-like cells containing alpha-SMA, as well as macrophages, at sites of repair in all tissues studied; and (5) markedly increased ACE binding density at each of these sites. Thus ACE is integral to tissue repair in the heart, skin, and kidney of the rat, irrespective of the etiologic basis of injury. At these sites ACE may serve to regulate local concentrations of substances involved in tissue repair.

Connective Tissue and Repair in the Heart

The heart is composed of highly differentiated cardiac myocytes, which constitute parenchyma, and stroma or connective tissue. Fibrillar collagen turnover in the heart and its valve leaflets, in particular, is dynamic and essential to tissue repair. Emerging evidence further suggests connective tissue is a metabolically active entity, where peptide hormones are generated and degraded and, in turn, these peptides regulate collagen turnover. This concept arose from quantitative in vitro autoradiography using an iodinated derivative of lisinopril (125I-351A) as ligand to localize angiotensin converting enzyme (ACE) binding density within the heart. A heterogeneous distribution was found: low-density ACE binding within atria and ventricles; high ACE binding density at sites of high collagen turnover, such as valve leaflets, adventitia, and fibrous tissue of diverse etiologic origins. ACE-producing cells at these latter sites were identified by monoclonal ACE antibody. They included valvular interstitial cells (VIC) and fibroblast-like cells each of which also contained alpha-smooth muscle actin and the transcript for type I collagen (in situ hybridization). Substrate utilization in cultured VIC was found to include angiotensin I and bradykinin. Angiotensin II and bradykinin receptor-ligand binding was observed in VIC and at fibrous tissue sites. Connective tissue ACE is independent of circulating angiotensin II. In vivo, fibrous tissue formation is attenuated by ACE inhibition or antagonism of AT1 receptor. Angiotensin II and bradykinin are stimulatory and inhibitory, respectively, to cultured adult cardiac fibroblast collagen synthesis suggesting a paradigm of reciprocal regulation to fibroblast collagen turnover. Stroma and its cellular constituents represent a dynamic metabolic entity that regulates its own peptide hormone composition and turnover of fibrillar collagen. These findings may provide insights that could be used to advantage to either promote or forestall fibrous tissue formation depending on the nature of cardiovascular disease.

Angiotensin II receptor blockade during gestation attenuates collagen formation in the developing rat heart

OBJECTIVE Fetal cardiac development includes rapid formation of a three-dimensional collagen network, composed mainly of type I and III fibrillar collagens. Collagen fibrils have been found in cardiac jelly at very early stages of cardiac development and are thought to have structural and functional properties. In adult rat cardiac tissue, angiotensin II (AngII) via AT1 receptor binding and AngII-regulated expression of transforming growth factor beta-1 (TGF-beta 1) each upregulate collagen transcription. AT1 and AT2 receptor subtypes are developmentally regulated; both have been localized in fetal tissue where the AT2 receptor is considered a determinant of morphogenesis. We sought to determine whether blockade of either receptor would result in attenuation of collagen mRNA expression and fibrillar collagen accumulation and alter TGF-beta 1 mRNA expression in the developing fetal heart examined at birth. METHODS Pregnant rats were treated either with an AT1 receptor antagonist losartan or an AT2 receptor antagonist PD123319 and compared with untreated age-matched controls. Offspring were studied within 24 h of birth. Type I and type III collagen mRNA expression, as well as TGF-beta 1 mRNA expression, were examined by in situ hybridization. Collagen concentration was determined spectrophotometrically by picrosirius red staining and type I and III collagens were detected by immunoblotting. RESULTS We found: (1) comparable birth weights in control and PD123319-treated animals, but reduced body weight in newborn losartan-treated animals; (2) compared to untreated animals, type I collagen and TGF-beta 1 mRNA expression in cardiac tissue were each equally reduced in both losartan and PD123319-treated animals; (3) increased type III collagen mRNA expression in both PD123319- and losartan-treated groups; and (4) a significant decrease in total soluble cardiac collagen concentration in both losartan and PD123319-treated groups, confirmed by attenuated immunoreactivity of type I and III collagens in whole heart extracts by Western blotting. CONCLUSIONS The results of these pharmacologic interventions suggest AngII receptors are expressed in cardiac tissue during gestation, where both AT1 and AT2 receptors are involved in the regulation of type I and III collagen expression and structural protein accumulation. These effects appear to be mediated, in part, by attenuated cardiac TGF-beta 1 levels. The marked decrease in newborn cardiac collagen content has yet undefined functional consequences.