Marco Matteucci

Extracellular vesicles from human cardiac progenitor cells inhibit cardiomyocyte apoptosis and improve cardiac function after myocardial infarction

AIMS Recent evidence suggests that cardiac progenitor cells (CPCs) may improve cardiac function after injury. The underlying mechanisms are indirect, but their mediators remain unidentified. Exosomes and other secreted membrane vesicles, hereafter collectively referred to as extracellular vesicles (EVs), act as paracrine signalling mediators. Here, we report that EVs secreted by human CPCs are crucial cardioprotective agents. METHODS AND RESULTS CPCs were derived from atrial appendage explants from patients who underwent heart valve surgery. CPC-conditioned medium (CM) inhibited apoptosis in mouse HL-1 cardiomyocytic cells, while enhancing tube formation in human umbilical vein endothelial cells. These effects were abrogated by depleting CM of EVs. They were reproduced by EVs secreted by CPCs, but not by those secreted by human dermal fibroblasts. Transmission electron microscopy and nanoparticle tracking analysis showed most EVs to be 30-90 nm in diameter, the size of exosomes, although smaller and larger vesicles were also present. MicroRNAs most highly enriched in EVs secreted by CPCs compared with fibroblasts included miR-210, miR-132, and miR-146a-3p. miR-210 down-regulated its known targets, ephrin A3 and PTP1b, inhibiting apoptosis in cardiomyocytic cells. miR-132 down-regulated its target, RasGAP-p120, enhancing tube formation in endothelial cells. Infarcted hearts injected with EVs from CPCs, but not from fibroblasts, exhibited less cardiomyocyte apoptosis, enhanced angiogenesis, and improved LV ejection fraction (0.8 ± 6.8 vs. -21.3 ± 4.5%; P < 0.05) compared with those injected with control medium. CONCLUSION EVs are the active component of the paracrine secretion by human CPCs. As a cell-free approach, EVs could circumvent many of the limitations of cell transplantation.

Improved Myocardial Perfusion in Chronic Diabetic Mice by the Up-Regulation of pLKB1 and AMPK Signaling

Previous studies related impaired myocardial microcirculation in diabetes to oxidative stress and endothelial dysfunction. Thus, this study was aimed to determine the effect of up‐regulating pAMPK‐pAKT signaling on coronary microvascular reactivity in the isolated heart of diabetic mice. We measured coronary resistance in wild‐type and streptozotocin (STZ)‐treated mice, during perfusion pressure changes. Glucose, insulin, and adiponectin levels in plasma and superoxide formation, NOx levels and heme oxygenase (HO) activity in myocardial tissue were determined. In addition, the expression of HO‐1, 3‐nitrotyrosine, pLKB1, pAMPK, pAKT, and peNOS proteins in control and diabetic hearts were measured. Coronary response to changes in perfusion pressure diverged from control in a time‐dependent manner following STZ administration. The responses observed at 28 weeks of diabetes (the maximum time examined) were mimicked by L‐NAME administration to control animals and were associated with a decrease in serum adiponectin and myocardial pLKB1, pAMPK, pAKT, and pGSK‐3 expression. Cobalt protoporphyrin treatment to induce HO‐1 expression reversed the microvascular reactivity seen in diabetes towards that of controls. Up‐regulation of HO‐1 was associated with an increase in adiponectin, pLKB1, pAKT, pAMPK, pGSK‐3, and peNOS levels and a decrease in myocardial superoxide and 3‐nitrotyrosine levels. In the present study we describe the time course of microvascular functional changes during the development of diabetes and the existence of a unique relationship between the levels of serum adiponectin, pLKB1, pAKT, and pAMPK activation in diabetic hearts. The restoration of microvascular function suggests a new therapeutic approach to even advanced cardiac microvascular derangement in diabetes. J. Cell. Biochem. 109: 1033–1044, 2010.

Regional mapping of myocardial hibernation phenotype in idiopathic end-stage dilated cardiomyopathy

Myocardial hibernation (MH) is a well‐known feature of human ischaemic cardiomyopathy (ICM), whereas its presence in human idiopathic dilated cardiomyopathy (DCM) is still controversial. We investigated the histological and molecular features of MH in left ventricle (LV) regions of failing DCM or ICM hearts. We examined failing hearts from DCM (n = 11; 41.9 ± 5.45 years; left ventricle‐ejection fraction (LV‐EF), 18 ± 3.16%) and ICM patients (n = 12; 58.08 ± 1.7 years; LVEF, 21.5 ± 6.08%) undergoing cardiac transplantation, and normal donor hearts (N, n = 8). LV inter‐ventricular septum (IVS) and antero‐lateral free wall (FW) were transmurally (i.e. sub‐epicardial, mesocardial and sub‐endocardial layers) analysed. LV glycogen content was shown to be increased in both DCM and ICM as compared with N hearts (P < 0.001), with a U‐shaped transmural distribution (lower values in mesocardium). Capillary density was homogenously reduced in both DCM and ICM as compared with N (P < 0.05 versus N), with a lower decrease independent of the extent of fibrosis in sub‐endocardial and sub‐epicardial layers of DCM as compared with ICM. HIF1‐α and nestin, recognized ischaemic molecular hallmarks, were similarly expressed in DCM‐LV and ICM‐LV myocardium. The proteomic profile was overlapping by ~50% in DCM and ICM groups. Morphological and molecular features of MH were detected in end‐stage ICM as well as in end‐stage DCM LV, despite epicardial coronary artery patency and lower fibrosis in DCM hearts. Unravelling the presence of MH in the absence of coronary stenosis may be helpful to design a novel approach in the clinical management of DCM.

Aortic valve disease with severe ventricular dysfunction: Stentless valve for better recovery

BACKGROUND Stentless bioprostheses and homografts show better hemodynamic profiles compared with conventional stented bioprostheses and mechanical valves. Few data are available on stentless aortic valve implantation for patients with severe left ventricular dysfunction. The aim of this retrospective study was to assess the potential benefits of stentless aortic valve implantation for patients undergoing isolated aortic valve replacement with left ventricular ejection fraction < or = 35%. METHODS From November 1988 through March 2000, 53 patients (45 men and 8 women, aged 64.2 +/- 15.2 years) with a LVEF < or = 35% (mean EF, 28.7 +/- 5.4%) underwent isolated, primary aortic valve replacement for chronic aortic valve disease. Twenty patients received stentless aortic valves and 33 patients received conventional stented bioprostheses and mechanical valves. Predictive factors for LVEF recovery at echocardiographic follow-up (36.2 +/- 32.1 months) were analyzed by simple and multiple regression analysis. RESULTS There were no significant differences between groups in early and late mortality. Stentless aortic valve implantation required a longer aortic cross-clamp time (p = 0.037). The stentless aortic valve group showed a better LVEF recovery (p = 0.016). Stentless aortic valves had a larger indexed effective orifice area compared with conventional stented bioprostheses and mechanical valves (p < 0.0001). A smaller indexed effective orifice area (p = 0.0008), chronic obstructive pulmonary disease (p = 0.015), and implantation of a conventional stented bioprosthesis or mechanical valve (p = 0.016) were related to reduced LVEF recovery by univariate analysis. A larger indexed effective orifice area (p = 0.024) was an independent predictive factor for a better LVEF recovery by multivariate analysis. CONCLUSIONS Stentless aortic valve implantation for patients with severe left ventricular dysfunction, even if technically more demanding, is a safe procedure that warrants a larger indexed effective orifice area leading to an enhanced LVEF recovery.

Impact of Obesity on the Expression Profile of Natriuretic Peptide System in a Rat Experimental Model

Natriuretic peptides (NPs) play an important role in obesity and aim of this study was to evaluate, in cardiac tissue of obese Zucker rats (O, n = 29) their transcriptomic profile compared to controls (CO, n = 24) by Real-Time PCR study; CNP protein expression was evaluated by immunostaining and immunometric tests. Myocardial histology was performed, confirming no alteration of organ structure. While ANP and BNP are cardiac peptides, CNP is mainly an endothelial hormone; thus its expression, as well as that of NPR-B and NPR-C, was also evaluated in kidney and lung of an animal subgroup (n = 20). In heart, lower BNP mRNA levels in O vs CO (p = 0.02) as well as ANP and CNP (p = ns), were detected. NPR-B/NPR-A mRNA was similar in O and CO, while NPR-C was numerically lower (p = ns) in O than in CO. In kidney, CNP/NPR-B/NPR-C mRNA was similar in O and CO, while in lung CNP/NPR-C expression decreased and NPR-B increased (p = ns) in O vs CO. Subdividing into fasting and hyperglycemic rats, the pattern of mRNA expression for each gene analyzed remained unchanged. The trend observed in heart, kidney and lung for CNP protein concentrations and immunohistochemistry reflected the mRNA expression. TNF-α and IL-6 mRNA were measured in each tissue and no significant genotype effect was detected in any tissue. The main NP variations were observed at the cardiac level, suggesting a reduced release by cardiac cells. The understanding of mechanisms involved in the modulation of the NP system in obesity could be a useful starting point for future clinical study devoted to identifying new obesity treatment strategies.

Whole-Body Evaluation of MIBG Tissue Extraction in a Mouse Model of Long-Lasting Type II Diabetes and Its Relationship with Norepinephrine Transport Protein Concentration

Accelerated cardiac washout of 123I-metaiodobenzylguanidine (MIBG), which is clinically used as an index of cardiac neuropathy in diabetes, is ascribed to decreased norepinephrine reuptake into synaptic vesicles. However, accelerated washout frequently contrasts with preserved early tracer uptake, whose significance remains undetermined. The aim of this study was to investigate in a mouse model of long-lasting type II diabetes whether the mismatch between MIBG early uptake and washout is the consequence of a more generalized disorder of the autonomic nervous system. Methods: Nine mice were given low doses of streptozotocin by intraperitoneal injection for 5 consecutive days. At 7 mo after streptozotocin, MIBG kinetics were evaluated by heart and liver time–activity curves and by tracer accumulation in the bladder. Data were compared with those obtained in 10 sham mice and correlated with the cardiac and hepatic tissue expression of norepinephrine transporter (NET) as assessed with a 3H-desipramine saturation binding assay. Results: In diabetic mice, myocardial and liver MIBG retention was reduced at 2 h and was associated with both increased tracer washout and reduced NET density. The rate of myocardial washout correlated with the degree of urinary MIBG excretion. Conclusion: The paradoxic observation of preserved early uptake associated with accelerated washout of MIBG in diabetes seems to be explained by a generalized disorder in NET function leading to reduced whole-body tracer removal from the blood and increased tracer availability for early myocardial uptake.

Barley beta-glucan promotes MnSOD expression and enhances angiogenesis under oxidative microenvironment

Manganese superoxide dismutase (MnSOD), a foremost antioxidant enzyme, plays a key role in angiogenesis. Barley‐derived (1.3) β‐d‐glucan (β‐d‐glucan) is a natural water‐soluble polysaccharide with antioxidant properties. To explore the effects of β‐d‐glucan on MnSOD‐related angiogenesis under oxidative stress, we tested epigenetic mechanisms underlying modulation of MnSOD level in human umbilical vein endothelial cells (HUVECs) and angiogenesis in vitro and in vivo. Long‐term treatment of HUVECs with 3% w/v β‐d‐glucan significantly increased the level of MnSOD by 200% ± 2% compared to control and by 50% ± 4% compared to untreated H2O2‐stressed cells. β‐d‐glucan‐treated HUVECs displayed greater angiogenic ability. In vivo, 24 hrs‐treatment with 3% w/v β‐d‐glucan rescued vasculogenesis in Tg (kdrl: EGFP) s843Tg zebrafish embryos exposed to oxidative microenvironment. HUVECs overexpressing MnSOD demonstrated an increased activity of endothelial nitric oxide synthase (eNOS), reduced load of superoxide anion (O2−) and an increased survival under oxidative stress. In addition, β‐d‐glucan prevented the rise of hypoxia inducible factor (HIF)1‐α under oxidative stress. The level of histone H4 acetylation was significantly increased by β‐d‐glucan. Increasing histone acetylation by sodium butyrate, an inhibitor of class I histone deacetylases (HDACs I), did not activate MnSOD‐related angiogenesis and did not impair β‐d‐glucan effects. In conclusion, 3% w/v β‐d‐glucan activates endothelial expression of MnSOD independent of histone acetylation level, thereby leading to adequate removal of O2−, cell survival and angiogenic response to oxidative stress. The identification of dietary β‐d‐glucan as activator of MnSOD‐related angiogenesis might lead to the development of nutritional approaches for the prevention of ischemic remodelling and heart failure.

Up-regulation of heme oxygenase-1 after infarct initiation reduces mortality, infarct size and left ventricular remodeling: experimental evidence and proof of concept

BackgroundUp-regulation of HO-1 by genetic manipulation or pharmacological pre-treatment has been reported to provide benefits in several animal models of myocardial infarction (MI). However, its efficacy following MI initiation (as in clinical reality) remains to be tested. Therefore, this study investigated whether HO-1 over-expression, by cobalt protoporphyrin (CoPP) administered after LAD ligation, is still able to improve functional and structural changes in left ventricle (LV) in a rat model of 4-week MI.MethodsA total of 144 adult male Wistar rats were subjected to either left anterior coronary artery ligation or sham-operation. The effect of CoPP treatment (5 mg/kg i.p. at the end of the surgical session and, then, once a week for 4 weeks) was evaluated on the basis of survival, electro- and echocardiography, plasma levels of B-type natriuretic peptide (BNP), endothelin-1 and prostaglandin E2, coronary microvascular reactivity, MI size, LV wall thickness and vascularity. Besides, the expression of HO-1 and connexin-43 in different LV territories was assessed by western blot analysis and immunohistochemistry, respectively.ResultsCoPP induced an increased expression of HO-1 protein with >16 h delay. CoPP treatment significantly reduced mortality, MI size, BNP concentration, ECG alterations, LV dysfunction, microvascular constriction, capillary rarefaction and restored connexin-43 expression as compared to untreated MI. These functional and structural changes were paralleled by increased HO-1 expression in all LV territories. HO activity inhibition by tin-mesoporphyrin abolished the differences between CoPP-treated and untreated MI animals.ConclusionsThis is the first report demonstrating the putative role of pharmacological induction of HO-1 following coronary occlusion to benefit infarcted and remote territories, leading to better cardiac function in a 4-week MI outcome.

Cardioprotection by cardiac progenitor cell-secreted exosomes: role of pregnancy-associated plasma protein-A

Aims Cell therapy trials using cardiac-resident progenitor cells (CPCs) and bone marrow-derived mesenchymal stem/progenitor cells (BMCs) in patients after myocardial infarction have provided encouraging results. Exosomes, nanosized extracellular vesicles of endosomal origin, figure prominently in the bioactivities of these cells. However, a head-to-head comparison of exosomes from the two cell types has not been performed yet. Methods and results CPCs and BMCs were derived from cardiac atrial appendage specimens and sternal bone marrow, respectively, from patients (n = 20; age, 69.9 ± 10.9) undergoing heart surgery for aortic valve disease and/or coronary artery disease. Vesicles were purified from cell conditioned media by centrifugation/filtration and ultracentrifugation. Vesicle preparations were predominantly composed of exosomes based on particle size and marker expression (CD9, CD63, CD81, Alix, and TSG-101). CPC-secreted exosomes prevented staurosporine-induced cardiomyocyte apoptosis more effectively than BMC-secreted exosomes. In vivo, CPC-secreted exosomes reduced scar size and improved ventricular function after permanent coronary occlusion in rats more efficiently than BMC-secreted exosomes. Both types of exosomes stimulated blood vessel formation. CPC-secreted exosomes, but not BMC-derived exosomes, enhanced ventricular function after ischaemia/reperfusion. Proteomics profiling identified pregnancy-associated plasma protein-A (PAPP-A) as one of the most highly enriched proteins in CPC vs. BMC exosomes. The active form of PAPP-A was detected on CPC exosome surfaces. These vesicles released insulin-like growth factor-1 (IGF-1) via proteolytic cleavage of IGF-binding protein-4 (IGFBP-4), resulting in IGF-1 receptor activation, intracellular Akt and ERK1/2 phosphorylation, decreased caspase activation, and reduced cardiomyocyte apoptosis. PAPP-A knockdown prevented CPC exosome-mediated cardioprotection both in vitro and in vivo. Conclusion These results suggest that CPC-secreted exosomes may be more cardioprotective than BMC-secreted exosomes, and that PAPP-A-mediated IGF-1 release may explain the benefit. They illustrate a general mechanism whereby exosomes may function via an active protease on their surface, which releases a ligand in proximity to the transmembrane receptor bound by the ligand.

b-Gamma-glutamyltransferase activity in human vulnerable carotid plaques

OBJECTIVE The atherosclerotic plaque that is vulnerable to rupture and to superimposed thrombosis is mainly represented by a thin-cap fibroatheroma with or without ulceration/thrombosis and inflammatory infiltrates. Total serum gamma-glutamyltransferase (GGT) activity is an independent predictor for cardiovascular events. Four GGT fractions have been identified in plasma and only one of them (b-GGT) in atherosclerotic plaques, but the possible role of GGT in plaque pathophysiology has not been assessed yet. We investigated the relationships between plaque b-GGT activity and the histological features of plaque vulnerability. METHODS AND RESULTS Plaque GGT activity was investigated in 65 patients undergoing carotid endarterectomy; plaques were histologically characterized and immunostained for GGT. Intra-plaque total and fractional GGT activity was determined by a cost-effective test of molecular size exclusion chromatography, and compared with histological markers of plaque vulnerability. Plaque cholesterol content was also measured by chromatography. b-GGT was the only fraction detected within the atherosclerotic plaques and intra-plaque b-GGT activity correlated to plaque cholesterol content (r = 0.667, P < 0.0001), plasma b-GGT and f-GGT fractions (r = 0.249; r = 0.298, both P < 0.05). Higher b-GGT activity was found in thin-cap fibroatheromas and it was associated to histological markers of vulnerable plaques, i.e., larger necrotic areas, greater macrophage infiltration and higher cholesterol content (P < 0.05). CONCLUSIONS intra-plaque b-GGT activity correlates with the histological markers of vulnerable plaque and with plasma b-GGT in human carotid atherosclerosis; these data support the possible role of b-GGT in clinically significant atherosclerotic disease.