Angelino Calderone

Beneficial Effects of Long-Term Use of the Antioxidant Probucol in Heart Failure in the Rat

Background—Congestive heart failure (CHF) is a disease that is characterized by progressive left ventricular (LV) dysfunction and dilatation. Oxidative stress is thought to contribute to the progression of CHF, and antioxidants have been shown to have beneficial effects when started early after myocardial infarction (MI). In this study, we tested whether the powerful antioxidant probucol would attenuate progression of CHF once it was established after MI in the rat. Methods and Results—Ligation of a coronary artery was used to create an MI in rats (n=266). Survivors were then randomized 20 days after MI to either probucol 61 mg · kg−1 · d−1 or vehicle and followed up for a total of 100 days after MI. Studies of cardiac hemodynamics, LV remodeling, cardiac apoptosis and morphology, systemic neurohumoral activation, oxidative stress, and renal function were then evaluated. Probucol improved LV function (LV maximum rate of pressure rise from 3103 to 4250 mm Hg/s, P <0.05, and LV end-diastolic pressure decrease from 28 to 24 mm Hg, P <0.05), reduced pulmonary weights, prevented right ventricular systolic hypertension, and preserved renal function compared with vehicle. Probucol also prevented LV dilatation, prevented wall thinning (1.70 versus 1.42 mm, P <0.05), reduced cardiac fibrosis and cardiac apoptosis, attenuated increased myocardial cell cross-sectional area, and increased scar thickness. Conclusions—In chronic CHF, probucol exerts multiple beneficial morphological effects that result in better LV remodeling and function, reduced neurohumoral activation, and preserved renal function.

Female Rats Fed a High-Fat Diet Were Associated with Vascular Dysfunction and Cardiac Fibrosis in the Absence of Overt Obesity and Hyperlipidemia: Therapeutic Potential of Resveratrol

It remains presently unknown whether vascular reactivity is impaired and whether maladaptive cardiac remodeling occurs before the onset of overt obesity and in the absence of hyperlipidemia. Normal female rats were fed a high-fat diet for 8 weeks and were associated with a modest nonsignificant increase of body weight (standard diet, 300 ± 10, versus high-fat diet, 329 ± 14 g) and a normal plasma lipid profile. In rats fed a high-fat diet, systolic (171 ± 7 mm Hg) and diastolic blood pressures (109 ± 3) were increased compared to a standard diet (systolic blood pressure, 134 ± 8; diastolic blood pressure, 96 ± 5 mm Hg), and acetylcholine-dependent relaxation of isolated aortic rings (high-fat diet, 22 ± 5%, versus standard diet, 53 ± 8%) was significantly reduced. Furthermore, perivascular fibrosis was detected in the heart of rats fed a high-fat diet. The exogenous addition of resveratrol (trans-3,5,4′-trihydroxystilbene) (0.1 μM) to aortic rings isolated from rats fed a high-fat diet restored acetylcholine-mediated relaxation (47 ± 9%). The administration of resveratrol (20 mg/kg/day for 8 weeks) to rats fed a high-fat diet prevented the increase in blood pressure and preserved acetylcholine-dependent relaxation of isolated aortic rings. However, resveratrol therapy failed to attenuate the perivascular fibrotic response. These data have demonstrated that a high-fat diet fed to normal female rats can elicit a hypertensive response and induce perivascular fibrosis before the development of overt obesity and in the absence of hyperlipidemia. Resveratrol therapy can prevent the hypertensive response in female rats fed a high-fat diet but is without effect on the progression of perivascular fibrosis.

Lung structural remodeling and pulmonary hypertension after myocardial infarction: complete reversal with irbesartan

OBJECTIVES The severity of pulmonary hypertension associated with heart failure carries a poor prognosis. The lungs are very sensitive to the constrictive and proliferative effects of angiotensin-II and could represent a preferential target for this peptide. METHODS Rats with large myocardial infarcts or sham surgery received the angiotensin-II receptor antagonist irbesartan (40 mg/kg/day) or vehicle for 2 or 8 weeks (n=5 to 8 for each group). Hemodynamic and morphometric measurements were obtained followed by immunohistochemistry, immunofluorescence analysis and electron microscopic characterization of lung sections. RESULTS The infarct groups developed progressive pulmonary hypertension and right ventricular hypertrophy with elevated left ventricular filling pressures (all P<0.01). Despite similar infarct size, filling pressures were lower (P<0.01) while pulmonary hypertension and right ventricular hypertrophy were completely normalized by irbesartan. Isolated lungs pressure-flow relationships were identical at 2 weeks. At 8 weeks it was steepest and shifted upward in the infarct group (P<0.001), and completely normalized by irbesartan. Lung weight doubled after infarct with no evidence of pulmonary edema and was also normalized by irbesartan. Important lungs structural remodeling evidenced by collagen and reticulin deposition, thickening of the alveolar septa and proliferation of cells with ultrastructural characteristics of myofibroblasts (pericytes) were identified after infarct. CONCLUSIONS After large myocardial infarct there is important pulmonary structural remodeling in which myofibroblasts (pericytes) proliferation may play an important role. This initially protective mechanism against high filling pressures could eventually contribute to the development of pulmonary hypertension and right ventricular hypertrophy. Future studies are needed to determine if angiotensin-II directly modulates pulmonary remodeling after myocardial infarct.

Resident Nestin+ Neural-Like Cells and Fibers Are Detected in Normal and Damaged Rat Myocardium

The present study examined whether nestin+ neural-like stem cells detected in the scar tissue of rats 1 week after myocardial infarction (MI) were derived from bone marrow and/or were resident cells of the normal myocardium. Irradiated male Wistar rats transplanted with β-actin promoter-driven, green fluorescent protein (GFP)–labeled, unfractionated bone marrow cells were subjected to coronary artery ligation. Three weeks after MI, GFP-labeled bone marrow cells were detected in the infarct region, and a modest number were associated with nestin immunoreactivity. The paucity of GFP+/nestin+ cells in the scar tissue provided the impetus to explore whether neural-like stem cells were derived from cardiac tissue. Nestin mRNA and immunoreactivity were detected in normal rat myocardium, and transcript levels were increased in the damaged heart after MI. In primary-passage, cardiac tissue-derived neural cells, filamentous nestin staining was associated with a diffuse, cytoplasmic glial fibrillary acidic protein signal. Unexpectedly, in viable myocardium, numerous nestin+/glial fibrillary acidic protein+ fiberlike structures of varying length were detected and observed in close proximity to neurofilament-M+ fibers. The infarct region was likewise innervated, and the preponderance of neurofilament-M+ fibers appeared to be physically associated with nestin+ fiberlike structures. These data highlight the novel observation that the normal rat heart contained resident nestin+/glial fibrillary acidic protein+ neural-like stem cells, fiberlike structures, and nestin mRNA levels that were increased in response to myocardial ischemia. Cardiac tissue-derived neural stem cell migration to the infarct region and concomitant nestin+ fiberlike innervation represent obligatory events of reparative fibrosis in the damaged rat myocardium.

The rat heart contains a neural stem cell population; Role in sympathetic sprouting and angiogenesis

Nestin-expressing cells were identified in the normal rat heart characterized by a small cell body and numerous processes and following an ischemic insult migrated to the infarct region. The present study was undertaken to identify the phenotype, origin and biological role of nestin-expressing cells during reparative fibrosis. A neural stem cell phenotype was identified based on musashi-1 expression, growth as a neurosphere, and differentiation to a neuronal cell. Using the Wnt1-cre; Z/EG transgenic mouse model, which expresses EGFP in embryologically-derived neural crest cells, the reporter signal was detected in nestin-expressing cells residing in the heart. In infarcted human hearts, nestin-expressing cells were detected in the viable myocardium and the scar and morphologically analogous to the population identified in the rat heart. Following either an ischemic insult or the acute administration of 6-hydroxydopamine, sympathetic sprouting was dependent on the physical association of neurofilament-M immunoreactive fibres with nestin-positive processes emanating from neural stem cells. To specifically study the biological role of the subpopulation in the infarct region, neural stem cells were isolated from the scar, fluorescently labelled and transplanted in the heart of 3-day post-MI rats. Injected scar-derived neural stem cells migrated to the infarct region and were used as a substrate for de novo blood vessel formation. These data have demonstrated that the heart contains a resident population of neural stem cells derived from the neural crest and participate in reparative fibrosis. Their manipulation could provide an alternative approach to ameliorate the healing process following ischemic injury.

Nestin‐expressing neural stem cells identified in the scar following myocardial infarction

Nerve fiber innervation of the scar following myocardial damage may have occurred either via the growth of pre‐existing fibers and/or the mobilization of neural stem cells. The present study examined whether neural stem cells were recruited to the infarct region of the rat heart following coronary artery ligation. The neural stem cell marker nestin was detected in the infarct region of 1‐week post‐myocardial infarct (MI) male rats and cultured scar‐derived neural‐like cells. By contrast, nestin staining was undetected in either scar myofibroblasts or cardiac myocytes residing in the non‐infarcted left ventricle. Reactive astrocytes were isolated from the infarct region and characterized by the co‐expression of nestin, glial fibrillary acidic protein, and vimentin. Specific staining of oligodendrocytes and neurons was also detected in the infarct region and cultured scar‐derived neural‐like cells. Furthermore, neurofilament‐M positive fibers were identified in the scar and tyrosine hydroxylase immunoreactivity was observed in peripherin‐positive neurons. Neurite formation was induced in PC12 cells treated with the conditioned‐media of primary passage scar‐derived cells, highlighting the synthesis and secretion of neurotrophic factors. Nerve growth factor (NGF) and brain‐derived neurotrophic factor were detected in myofibroblasts and neural cells, and both cell types expressed the NGF receptors trkA and p75. These data highlight the novel observation that neural stem cells were recruited to the infarct region of the damaged rat heart and may contribute in part to nerve fiber growth and subsequent innervation of the scar.

Coordinated upregulation of the cardiac endothelin system in a rat model of heart failure

The potent vascular, cardiac, and renal actions of endothelin-1 (ET-1) suggest a role for this vasoconstrictor peptide in the pathophysiology of heart failure (HF). Recent studies have shown increased levels of ET-1 peptide accompanied by increased ETB receptor binding in the left ventricle during experimental HF. However, much less is known about the regulation of mRNA expression of these genes in HF. We compared the levels of mRNA expression for ET-1 and ET receptors (ETA and ETB) in the left ventricle of rats with HF induced by coronary artery ligation (n = 6) vs. sham-operated animals (n = 6). Levels of mRNA for ET-1 were determined by ribonuclease protection assay (RPA) using beta-actin as the internal control, whereas ET receptors were quantified by quantitative-competitive RT-PCR. Compared with sham animals, ET-1, ETA, and ETB receptor mRNA levels were markedly upregulated in the left ventricle by 6.6 +/- 1.8-fold (p < 0.01), 3.2 +/- 0.6-fold (p < 0.05), and 3.5 +/- 1.0-fold (p < 0.05), respectively. ET-1 mRNA levels were measured in two additional groups of rats (HF and sham; n = 6 each) treated for 4 weeks with the selective ETA receptor antagonist LU135252. This treatment had no significant effect on ET-1 mRNA expression in sham animals but reduced the upregulation of ET-1 expression in the HF group by 41 +/- 19% (p < 0.05). This study confirms the potential importance of ET-1 in HF and suggests that increased expression of ET-1 and ET receptors in the failing ventricle may contribute to alteration in basal cardiac contractility and myocardial remodeling.

Scar myofibroblasts of the infarcted rat heart express natriuretic peptides

The present study examined whether natriuretic peptide expression in the scar of post‐myocardial infarcted (MI) rats was derived at least in part by residing myofibroblasts. ANP and BNP mRNA levels were significantly increased in the non‐infarcted left ventricle and scar of 1‐week post‐MI male rats, as compared to the left ventricle of normal rats. The infarct region contained myofibroblasts and contracted cardiac myocytes residing predominantly in the epicardial border zone. In primary passage scar‐derived myofibroblasts, α‐myosin heavy chain mRNA was undetectable, whereas ANP, BNP, as well as adrenomedullin and corin mRNA expression persisted. In 1–3 day cultured primary passage myofibroblasts, prepro‐ANP, mature ANP, and BNP staining was observed in the cytoplasm/perinuclear region co‐incident with unorganized α‐smooth muscle actin. Following 4–7 days in culture, myofibroblasts expressed organized α‐smooth muscle actin filaments. However, natriuretic peptides were predominantly detected in the nucleus and cytoplasm, and thin filaments occupying the perinuclear region were positive for prepro‐ANP and BNP. Isoproterenol treatment of first passage scar myofibroblasts increased protein synthesis and induced BNP mRNA expression, whereas ANP mRNA levels remained unchanged. By contrast, neither ANP nor BNP mRNAs were induced following exposure to AII despite increased protein synthesis. These data highlight the novel observation that scar myofibroblasts synthesized ANP, BNP, adrenomedullin, and expressed the pro‐convertase corin. Constitutive and sympathetic‐driven natriuretic peptide synthesis by myofibroblasts may in part influence reparative fibrosis. J. Cell. Physiol. 207: 165–173, 2006.

Ovarian hormones induce TGF-β3 and fibronectin mRNAs but exhibit a disparate action on cardiac fibroblast proliferation

UNLABELLED Prior to menopause, women have a lower risk of cardiovascular disease compared to age-matched men. Despite the well-documented beneficial physiological effects of ovarian hormones on vascular reactivity and growth, very little is known with regard to the direct action on cardiac cells. OBJECTIVE The following study examined the pattern of ovarian hormone receptor subtype expression in cardiac fibroblasts, the modulator role of 17 beta-estradiol and progesterone on growth and their respective influence on putative molecular events of extracellular matrix remodeling. METHODS AND RESULTS Neonatal rat cardiac fibroblasts were isolated from 1- to 3-day-old Sprague--Dawley rats. Immunofluorescence and Western blot analysis revealed the presence of estrogen receptor-alpha (ER-alpha), and -beta (ER-beta) subtypes, with the ER-alpha subtype localized on the plasma membrane. Likewise, both progesterone receptor-A (PR-A), and -B (PR-B) subtypes were expressed in cardiac fibroblasts, and the PR-B appeared to be the predominant subtype associated with the plasma membrane. Despite the presence of both ER subtypes, the treatment of cardiac fibroblasts with 1 microM 17 beta-estradiol exerted a modest decrease in DNA synthesis. By contrast, progesterone treatment caused a dose-dependent decrease in [3H]thymidine uptake, without a concomitant induction of apoptosis. The progesterone-mediated decrease in DNA synthesis was associated with the upregulation of the cyclin-dependent kinase inhibitor p27(Kip1), whereas p21(cip) and proliferating cell nuclear antigen protein levels were unchanged. Lastly, despite the modest effect on DNA synthesis, 17 beta-estradiol increased the steady-state mRNA levels of transforming growth factor-beta(3) and fibronectin. Likewise, progesterone increased the expression of both transforming growth factor-beta(3), and fibronectin mRNA. CONCLUSION Collectively, these data are the first to highlight the presence of estrogen and progesterone receptor subtypes on the plasma membrane of neonatal rat cardiac fibroblasts, and further underscore the ability of ovarian hormones to directly suppress DNA synthesis, and influence putative molecular events associated with extracellular matrix remodeling.

Effects of Resveratrol (trans-3,5,4′-Trihydroxystilbene) Treatment on Cardiac Remodeling following Myocardial Infarction

Resveratrol (RES; trans-3,5,4′-trihydroxystilbene) has been shown to improve health and slow the progression of disease in various models. Several cardioprotective mechanisms have been identified including antioxidant, anti-inflammatory, and antifibrotic actions. Each of these actions is thought to have the ability to attenuate the pathophysiology underlying the deleterious cardiac structural remodeling that results from acute myocardial infarction (MI). Therefore, we evaluated the effect of resveratrol treatment on the progression of cardiac remodeling after MI. Four groups of rats (sham, n = 6; sham + RES, n = 21; MI, n = 26; MI + RES, n = 24) were treated for 13 weeks, starting 7 days before ligation of the left anterior descending coronary artery. Serial transthoracic echocardiography revealed that resveratrol had no effect on MI-induced left-ventricular and left-atrial dilatation or reduction in left-ventricular fractional shortening. Consistent with these findings, resveratrol did not improve the deterioration of hemodynamic function or reduce infarct size at 12 weeks post-MI. Resveratrol-treated animals did, however, show preserved cardiac contractile reserve in response to dobutamine administration. Radioligand binding revealed that MI reduced β-adrenergic receptor density. Resveratrol administration increased β-adrenoceptor density, so that resveratrol-treated MI rats had β-adrenoceptor densities similar to normal rats. Real-time reverse transcription-polymerase chain reaction revealed that MI-induced changes in sarcoplasmic reticulum Ca2+-ATPase 2 and transforming growth factor β-1 expression were unaltered by resveratrol, whereas MI-induced increases in atrial natriuretic factor (ANF) and connective tissue growth factor (CTGF) expression were attenuated. Resveratrol treatment does not improve cardiac remodeling and global hemodynamic function post-MI but does preserve contractile reserve and attenuate ANF and CTGF up-regulation.