Ex vivo-expanded bone marrow CD34(+) for acute myocardial infarction treatment: in vitro and in vivo studies.

BACKGROUND AIMS: Bone marrow (BM)-derived cells appear to be a promising therapeutic source for the treatment of acute myocardial infarction (AMI). However, the quantity and quality of the cells to be used, along with the appropriate time of administration, still need to be defined. We thus investigated the use of BM CD34(+)-derived cells as cells suitable for a cell therapy protocol (CTP) in the treatment of experimental AMI. METHODS: The need for a large number of cells was satisfied by the use of a previously established protocol allowing the expansion of human CD34(+) cells isolated from neonatal and adult hematopoietic tissues. We evaluated gene expression, endothelial differentiation potential and cytokine release by BM-derived cells during in vitro culture. Basal and expanded CD34(+) cells were used as a delivery product in a murine AMI model consisting of a coronary artery ligation (CAL). Cardiac function recovery was evaluated after injecting basal or expanded cells. RESULTS: Gene expression analysis of in vitro-expanded cells revealed that endothelial markers were up-regulated during culture. Moreover, expanded cells generated a CD14(+) subpopulation able to differentiate efficiently into VE-cadherin-expressing cells. In vivo, we observed a cardiac function recovery in mice sequentially treated with basal and expanded cells injected 4 h and 7 days after CAL, respectively. CONCLUSIONS: Our data suggest that combining basal and expanded BM-derived CD34(+) cells in a specific temporal pattern of administration might represent a promising strategy for a successful cell-based therapy.

Histone deacetylase inhibition enhances self renewal and cardioprotection by human cord blood-derived CD34 cells.

BACKGROUND: Use of peripheral blood- or bone marrow-derived progenitors for ischemic heart repair is a feasible option to induce neo-vascularization in ischemic tissues. These cells, named Endothelial Progenitors Cells (EPCs), have been extensively characterized phenotypically and functionally. The clinical efficacy of cardiac repair by EPCs cells remains, however, limited, due to cell autonomous defects as a consequence of risk factors. The devise of "enhancement" strategies has been therefore sought to improve repair ability of these cells and increase the clinical benefit. PRINCIPAL FINDINGS: Pharmacologic inhibition of histone deacetylases (HDACs) is known to enhance hematopoietic stem cells engraftment by improvement of self renewal and inhibition of differentiation in the presence of mitogenic stimuli in vitro. In the present study cord blood-derived CD34(+) were pre-conditioned with the HDAC inhibitor Valproic Acid. This treatment affected stem cell growth and gene expression, and improved ischemic myocardium protection in an immunodeficient mouse model of myocardial infarction. CONCLUSIONS: Our results show that HDAC blockade leads to phenotype changes in CD34(+) cells with enhanced self renewal and cardioprotection.

A mouse model for spatial and temporal expression of HGF in the heart.

In order to study the effects of Hepatocyte Growth Factor (HGF) in the heart, two transgenic mice were developed, one carrying a bidirectional HGF-TetO-GFP responder construct and the other carrying a α-MHC-tTA transactivator construct. Crosses were carried out between heterozygotes, so that litters contained bitransgenic α-MHC-tTA/HGF-TetO-GFP+, thus expressing HGF and GFP exclusively in the heart and only in the absence of Doxycycline. Our data show that the expression of HGF was indeed restricted to the heart and that the expression was limited to the timeframe of the absence of Doxycycline. Surprisingly the expression was variable even between bitransgenic littermates. In the setting of a model of ischemia-reperfusion, the expression of HGF ameliorates cardiac functionality, enhances proliferation and diminishes the scarred area, proving that this is a good model to study the beneficial influences and functional roles of HGF in the heart.

Cardioprotective function of the long pentraxin PTX3 in acute myocardial infarction.

BACKGROUND: Despite widespread clinical use as a prognostic marker in ischemic heart disorders, the actual pathogenetic role of the short pentraxin, C-reactive protein, has not undergone stringent genetic testing because of evolutionary divergence between mouse and humans. The long pentraxin PTX3 is conserved in evolution, is expressed in the heart under inflammatory conditions, and is a candidate prognostic marker in acute myocardial infarction. It was therefore important to assess whether PTX3 plays a pathogenetic role in acute myocardial infarction. METHODS AND RESULTS: In a model of acute myocardial infarction caused by coronary artery ligation and reperfusion, tissue mRNA expression and circulating levels of PTX3 increased. The interleukin-1R-MyD88 pathway plays a pivotal role in the induction of PTX3 transcript after ischemia. ptx3-deficient mice showed exacerbated heart damage (33% larger infarcts in null mice; P=0.0047). Increased myocardial damage in ptx3-deficient mice was associated with a greater no-reflow area, increased neutrophil infiltration, decreased number of capillaries, and increased number of apoptotic cardiomyocytes. In addition, ptx3-deficient mice with acute myocardial infarction showed higher circulating levels of interleukin-6 and increased C3 deposition in lesional tissue. The phenotype was reversed by exogenous PTX3. CONCLUSIONS: Thus, PTX3 plays a nonredundant, regulatory, cardioprotective role in acute myocardial infarction in mice. Our results suggest that modulation of the complement cascade contributes to the cardioprotective function of PTX3.

Effect of beta-adrenergic and renin-angiotensin system blockade on myocyte apoptosis and oxidative stress in diabetic hypertensive rats.

Diabetes aggravates the clinical severity and represents an additional independent risk factor of hypertension. Since both diseases separately concur to cardiomyocyte apoptosis, a mechanism at least partly involving unbalanced oxidative stress, we investigated whether the combination of diabetes and hypertension potentiated cardiac cell death in experimental models, compared to either disease alone. We also evaluated the short-term effects of different drugs in these models. Streptozotocin-induced diabetic normotensive (WKY) or hypertensive (SHR) rats were treated for one week with a DA(2)/alpha(2) agonist (CHF-1024), a selective beta1 adrenergic blocker (metoprolol), an angiotensin II-receptor blocker (valsartan) or a radical scavenger (tempol). In separate experiments, isolated cardiomyocytes were cultured in high glucose medium (25 mM) containing the same drugs. Although the number of apoptotic cardiomyocytes and the myocardial density of oxygen radicals were higher in non diabetic hypertensive than in normotensive controls, diabetes raised these variables to comparable absolute levels in both strains. All drugs except metoprolol significantly reduced apoptosis and oxidative stress in the diabetic animals of both strains and in the isolated myocytes cultured with high glucose. In conclusion, hypertensive rat is no more susceptible than its normotensive control to acute apoptosis induced by diabetes. Oxidative stress might be considered the common trigger for cardiac myocyte apoptosis in both conditions.

Cardiovascular oxidative stress is reduced by an ACE inhibitor in a rat model of streptozotocin-induced diabetes.

Blockade of the renin-angiotensin system (RAS) reduces cardiovascular morbidity and mortality in diabetic patients. Ang II-mediated generation of reactive oxygen species (ROS) has been suggested to be involved in several diabetic complications. We investigated whether the inhibition of Ang II production with an ACE inhibitor (ACEi) reduces oxidative stress and limits structural cardiovascular remodeling in a rat model of streptozotocin (STZ)-induced diabetes. Diabetic rats were treated for 7 weeks with an ACEi (lisinopril, 5 mg/kg/d), an antioxidant (N-acetyl-l-cysteine (NAC), 0.5 g/kg/d) and their combination. At sacrifice, ROS in the myocardium and thoracic aorta, LV myocyte number and size and aorta morphology were determined by quantitative histological methods. Superoxide and hydroxyl radical content, detected by dihydroethidium (DHE) and 8-hydroxydeoxyguanosine (8-OHdG), were 6.7 and 4.5-fold, respectively, higher in diabetic myocardium than in non-diabetic controls (p<0.001). The amount of superoxide was 5-fold higher in the thoracic aorta of diabetic rats compared to controls (p<0.001). Diabetes caused a modest increase in myocyte volume (+13%, p<0.01), a reduction of LV myocyte number (-43%, p<0.001), an accumulation of collagen around coronary arterioles (1.9-fold increase, p<0.01) and a decrease in arterial elastin/collagen ratio (-63%, p<0.001) compared to controls. Treatment with the ACEi attenuated ROS formation and prevented phenotypic changes in the heart (cardiomyocyte hypertrophy, perivascular fibrosis) and in the aorta of diabetic rats to the same extent as NAC. The absence of an additive effect, suggests a common mechanism of action, through the reduction of oxidative stress.

Junctional adhesion molecule-A-deficient polymorphonuclear cells show reduced diapedesis in peritonitis and heart ischemia-reperfusion injury.

Junctional Adhesion Molecule-A (JAM-A) is a transmembrane adhesive protein expressed at endothelial junctions and in leukocytes. Here we report that JAM-A is required for the correct infiltration of polymorphonuclear leukocytes (PMN) into an inflamed peritoneum or in the heart upon ischemia-reperfusion injury. The defect was not observed in mice with an endothelium-restricted deficiency of the protein but was still detectable in mice transplanted with bone marrow from JAM-A(-/-) donors. Microscopic examination of mesenteric and heart microvasculature of JAM-A(-/-) mice showed high numbers of PMN adherent on the endothelium or entrapped between endothelial cells and the basement membrane. In vitro, in the absence of JAM-A, PMN adhered more efficiently to endothelial cells and basement membrane proteins, and their polarized movement was strongly reduced. This paper describes a nonredundant role of JAM-A in controlling PMN diapedesis through the vessel wall.

Cardiac specific increase in aldosterone production induces coronary dysfunction in aldosterone synthase-transgenic mice.

BACKGROUND: Elevated circulating aldosterone level is associated with impaired cardiovascular function. Although the mechanisms are not fully understood, aldosterone antagonists decrease total and cardiovascular mortality in heart failure and myocardial infarction. Aldosterone induces cardiac fibrosis in experimental models, and it is synthesized locally in rat heart. These observations suggest pathological effects of aldosterone in heart that remain unclear. METHODS AND RESULTS: Transgenic mice (TG) that overexpress the terminal enzyme of aldosterone biosynthesis, aldosterone synthase (AS), in heart have been raised by gene targeting with the alpha-myosin heavy chain promoter. AS mRNA increased 100-fold and aldosterone concentration 1.7-fold in hearts of male TG mice relative to wild-type. No structural or myocardial alterations were evidenced, because ventricle/body weight, AT1 and AT2 receptor binding, and collagen content were unchanged in TG. No alteration in cardiac function was evidenced by echocardiography, isolated perfused heart, or whole-cell patch clamp experiments. In contrast, coronary function was impaired, because basal coronary flow was decreased in isolated perfused heart (-55% of baseline values), and vasodilatation to acetylcholine, bradykinin, and sodium nitroprusside was decreased by 75%, 60%, and 75%, respectively, in TG mice compared with wild-type, showing that the defect was not related to NO production. CONCLUSIONS: Increased cardiac aldosterone production in male mice induces a major coronary endothelium-independent dysfunction with no detectable alterations in cardiac structure and function. However, coronary dysfunction may be harmful for coronary adaptation to increased flow demand.

In vivo cardioprotection by N-acetylcysteine and isosorbide 5-mononitrate in a rat model of ischemia-reperfusion.

AIMS: We evaluated the effect of N-acetylcysteine (NAC, infused i.v.), isosorbide 5-mononitrate (IS5MN, by gavage), or their combination on cardiac injury in an in vivo rat model of 30-min ischemia followed by 24 hours or 7 days of reperfusion. RESULTS: When administered immediately prior to reperfusion with continuous infusion for 24 h, the combination of NAC + IS5MN reduced infarct size (29 +/- 6 vs. 59 +/- 4% area-at-risk, p < 0.01) and the infiltration of polymorphonuclear leukocytes (226 +/- 15 vs. 315 +/- 18 cells mm(-2) of area-at-risk, p = 0.002) and monocytes/macrophages (118 +/- 8 vs. 194 +/- 22 cells mm(-2), p = 0.012), compared to vehicle. NAC or IS5MN alone did not reduce infarct size at 24 hours of reperfusion. The same dose regimen of NAC and IS5MN did not reduce infarct size with permanent ischemia for 24 hours not followed by reperfusion. After 7 days of reperfusion (3 days of treatment with NAC + IS5MN or vehicle and 4 days of wash-out), infarct size was similar in the vehicle and NAC + IS5MN groups, but LV end-diastolic pressure and diastolic LV chamber wall stress were significantly lower in the animals treated with NAC + IS5MN (5 +/- 1 mmHg and 62 +/- 7 dyne mm(-2), respectively) compared to vehicle (9 +/- 1 mmHg and 123 +/- 18 dyne mm(-2), p < 0.05). CONCLUSIONS: We demonstrate in a rat model of cardiac ischemia-reperfusion treated with NAC and IS5MN, according to a regimen that mimicked a clinical situation (drugs started at time of reperfusion), that the short-term benefit seen after 24 h of reperfusion (51% reduction of infarct size) is maintained after one week, possibly through modulation of the inflammatory response to cardiac injury.

Appraisal of the role of angiotensin II and aldosterone in ventricular myocyte apoptosis in adult normotensive rat.

Despite previous observations on isolated ventricular myocytes, there are still few evidences that angiotensin II induces cardiomyocyte apoptosis in vivo. The possibility that aldosterone, the final hormone of the renin-angiotensin-aldosterone system under Ang II control, can stimulate cardiac apoptosis has not yet been explored. Angiotensin II or aldosterone (1mg/kg each) were infused in adult normotensive rats for different times, and the number of apoptotic ventricular myocyte nuclei was quantified by the TUNEL method, along with caspase-3 activation. The role of angiotensin II type 1 receptor in vivo was assessed by selective blockade with valsartan and ex vivo by binding experiments. In addition, myocytes in primary culture were incubated with Ang II or aldosterone in presence of spironolactone. Continuous infusion of Ang II induced a rapid, AT(1)-mediated increase of apoptotic cardiomyocyte nuclei (from 14+/-9 to 188+/-35 TdT-labeled nuclei/10(6) after 3h, P<0.005) and of activated caspase-3, that normalized after 24h. The normalization was associated with a down-regulation of myocardial AT(1) receptors. Aldosterone stimulated cardiomyocyte apoptosis both in vivo and in isolated cells, to a similar extent as Ang II. The maximal apoptotic rate reported here ( approximately 0.02%) and the transient effect of Ang II suggest that myocyte loss by apoptosis is limited in the present model. The data on aldosterone-induced ventricular myocyte apoptosis deserve further attention to delineate the role of aldosterone in cell death and offer possible mechanistic explanations on the benefits afforded by aldosterone receptor antagonists in heart failure.

Role of brain natriuretic peptide in the diagnosis and management of heart failure: current concepts.

BACKGROUND: Progression of heart failure is related to ventricular remodeling, a process associated to neurohormonal activation. Brain natriuretic peptide (BNP), a member of the natriuretic peptide family, has recently emerged as an important neurohormone in the pathophysiology of heart failure. METHODS: In this update, some of the recent advances on the role of BNP in heart failure are summarized. In particular, the role of BNP in diagnosis of heart disease, as a prognostic marker of cardiovascular events and as a possible guide to optimize heart failure therapy is discussed. RESULTS: Recent results from 4,300 patients enrolled in the Valsartan Heart Failure Trial (Val-HeFT) confirmed that BNP is the strongest predictor of outcome in heart failure, when compared to other neurohormones and clinical markers. The current use of BNP in the screening and diagnosis of heart failure and its possible future roles are presented. CONCLUSION: In recent years, there has been an impressive accumulation of data supporting an important role of BNP as a diagnostic and prognostic marker of heart failure. Development of rapid, accurate and affordable diagnostic methods will allow the routine monitoring of BNP in a wide spectrum of settings, from general practice to controlled clinical trials.

Comparative measurement of N-terminal pro-brain natriuretic peptide and brain natriuretic peptide in ambulatory patients with heart failure.

It is not clear whether brain natriuretic peptide (BNP) or N-terminal proBNP (NT-proBNP) is superior as a diagnostic and prognostic indicator in cardiac diseases. Here, we compare the clinical correlations of both peptides in a population of 92 ambulatory patients with heart failure, using a well-established immunoradiometric assay (IRMA) for BNP and an automated electrochemiluminescence immunoassay for NT-proBNP. The analytical correlation between the two peptides was satisfactory over a wide range of concentrations (1-686 pM for BNP) with the equation: NT-proBNP = 3.48 x BNP -19 and a correlation coefficient r2=0.94. In addition, the concentration of both peptides increased in a similar fashion according to the severity of the disease New York Heart Association (NYHA) functional class, left ventricular ejection fraction, etiology) and age; for instance, the ratios between median levels measured in NYHA class III vs. class II patients were comparable for BNP (383 vs. 16 pM, ratio 24) and NT-proBNP (1306 vs. 57 pM, ratio 23). We conclude that N-terminal proBNP, as assayed in the present study, correlates equally to BNP with clinical variables in patients with heart failure.