Giacomo Frati

Isolation and Expansion of Adult Cardiac Stem Cells From Human and Murine Heart

Cardiac myocytes have been traditionally regarded as terminally differentiated cells that adapt to increased work and compensate for disease exclusively through hypertrophy. However, in the past few years, compelling evidence has accumulated suggesting that the heart has regenerative potential. Recent studies have even surmised the existence of resident cardiac stem cells, endothelial cells generating cardiomyocytes by cell contact or extracardiac progenitors for cardiomyocytes, but these findings are still controversial. We describe the isolation of undifferentiated cells that grow as self-adherent clusters (that we have termed “cardiospheres”) from subcultures of postnatal atrial or ventricular human biopsy specimens and from murine hearts. These cells are clonogenic, express stem and endothelial progenitor cell antigens/markers, and appear to have the properties of adult cardiac stem cells. They are capable of long-term self-renewal and can differentiate in vitro and after ectopic (dorsal subcutaneous connective tissue) or orthotopic (myocardial infarction) transplantation in SCID beige mouse to yield the major specialized cell types of the heart: myocytes (ie, cells demonstrating contractile activity and/or showing cardiomyocyte markers) and vascular cells (ie, cells with endothelial or smooth muscle markers).

Cardiomyocytes induce endothelial cells to trans-differentiate into cardiac muscle: Implications for myocardium regeneration

The concept of tissue-restricted differentiation of postnatal stem cells has been challenged by recent evidence showing pluripotency for hematopoietic, mesenchymal, and neural stem cells. Furthermore, rare but well documented examples exist of already differentiated cells in developing mammals that change fate and trans-differentiate into another cell type. Here, we report that endothelial cells, either freshly isolated from embryonic vessels or established as homogenous cells in culture, differentiate into beating cardiomyocytes and express cardiac markers when cocultured with neonatal rat cardiomyocytes or when injected into postischemic adult mouse heart. Human umbilical vein endothelial cells also differentiate into cardiomyocytes under similar experimental conditions and transiently coexpress von Willebrand factor and sarcomeric myosin. In contrast, neural stem cells, which efficiently differentiate into skeletal muscle, differentiate into cardiomyocytes at a low rate. Fibroblast growth factor 2 and bone morphogenetic protein 4, which activate cardiac differentiation in embryonic cells, do not activate cardiogenesis in endothelial cells or stimulate trans-differentiation in coculture, suggesting that different signaling molecules are responsible for cardiac induction during embryogenesis and in successive periods of development. The fact that endothelial cells can generate cardiomyocytes sheds additional light on the plasticity of endothelial cells during development and opens perspectives for cell autologous replacement therapies.

Does ministernotomy improve postoperative outcome in aortic valve operation? A prospective randomized study

BACKGROUND The aim of this study was to compare the postoperative outcome obtained in patients undergoing elective aortic valve operation, either through ministernotomy or conventional sternotomy. METHODS Between January 1999 and July 2001, 80 consecutive patients undergoing elective aortic valve replacement were randomly divided into two groups: group I (n = 40 patients) undergoing a ministernotomy approach (reversed-C or reversed-L), and group II (n = 40 patients) undergoing conventional sternotomy. RESULTS The length of skin incision was significantly shorter in group I than in group II (8.2+/-1.3 cm versus 23.7+/-2.6 cm, p < 0.001). No significant differences were found in cardiopulmonary bypass duration, associated procedures, or aortic cross-clamping times. Total operating time was 3.7+/-0.46 hours in group I compared with 3.4+/-0.6 hours in group II (p = 0.014). A similar incidence of cardiac, neurologic, infective, and renal complications between groups was found. Mean mediastinal drainage and mean blood transfusions (amount of blood transfused) per patient were greater in group II (p < 0.004 and p < 0.001, respectively). Twenty-five (62.5%) patients in group II and 15 (37.5%) patients in group I required postoperative blood transfusion (p = 0.04). Mechanical ventilation time was significantly longer in group II (6.2+/-1.8 hours versus 4.4+/-0.9 hours, p = 0.006). Five days after the surgical procedure, spirometric data analysis demonstrated a significantly lower total lung capacity and maximum inspiratory and expiratory pressures in group II compared with group I (p = 0.003, p = 0.007, and p < 0.001, respectively). CONCLUSIONS Our results showed that ministernotomy had not only important cosmetic advantages but also beneficial effects in blood loss and transfusion, postoperative pain, and probably in sternal stability. Ministernotomy also improved recovery of respiratory function and allowed earlier extubation and hospital discharge.

Clinical Outcomes With Drug-Eluting and Bare-Metal Stents in Patients With ST-Segment Elevation Myocardial Infarction Evidence From a Comprehensive Network Meta-Analysis

OBJECTIVES The authors investigated the relative safety and efficacy of different drug-eluting stents (DES) and bare metal stents (BMS) in patients with ST-segment elevation myocardial infarction (STEMI) using a network meta-analysis. BACKGROUND The relative safety of DES and BMS in patients with STEMI continues to be debated, and whether advances have been made in this regard with second-generation DES is unknown. METHODS Randomized controlled trials comparing currently U.S. approved DES or DES with BMS in patients with STEMI were searched using MEDLINE, EMBASE, and Cochrane databases. Information on study design, inclusion and exclusion criteria, sample characteristics, and clinical outcomes was extracted. RESULTS Twenty-two trials including 12,453 randomized patients were analyzed. At 1-year follow-up, cobalt-chromium everolimus eluting stents (CoCr-EES) were associated with significantly lower rates of cardiac death or myocardial infarction (MI) and stent thrombosis (ST) than BMS. Differences in ST were apparent as early as 30 days and were maintained for 2 years. CoCr-EES were also associated with significantly lower rates of 1-year ST than paclitaxel-eluting stents (PES). Sirolimus-eluting stents (SES) were also associated with significantly lower rates of 1-year cardiac death/myocardial infarction than BMS. CoCr-EES, PES, and SES, but not zotarolimus-eluting stents, had significantly lower rates of 1-year target vessel revascularization (TVR) than BMS, with SES also showing lower rates of TVR than PES. CONCLUSIONS In patients with STEMI, steady improvements in outcomes have been realized with the evolution from BMS to first-generation and now second-generation DES, with the most favorable safety and efficacy profile thus far demonstrated with CoCr-EES.

Heart-targeted overexpression of caspase3 in mice increases infarct size and depresses cardiac function

Up-regulation of proapoptotic genes has been reported in heart failure and myocardial infarction. To determine whether caspase genes can affect cardiac function, a transgenic mouse was generated. Cardiac tissue-specific overexpression of the proapoptotic gene Caspase3 was induced by using the rat promoter of α-myosin heavy chain, a model that may represent a unique tool for investigating new molecules and antiapoptotic therapeutic strategies. Cardiac-specific Caspase3 expression induced transient depression of cardiac function and abnormal nuclear and myofibrillar ultrastructural damage. When subjected to myocardial ischemia–reperfusion injury, Caspase3 transgenic mice showed increased infarct size and a pronounced susceptibility to die. In this report, we document an unexpected property of the proapoptotic gene caspase3 on cardiac contractility. Despite inducing ultrastructural damage, Caspase3 does not trigger a full apoptotic response in the cardiomyocyte. We also implicate Caspase3 in determining myocardial infarct size after ischemia–reperfusion injury, because its cardiomyocyte-specific overexpression increases infarct size.

Incidence and predictors of coronary stent thrombosis: Evidence from an international collaborative meta-analysis including 30 studies, 221,066 patients, and 4276 thromboses

BACKGROUND Stent thrombosis remains among the most feared complications of percutaneous coronary intervention (PCI) with stenting. However, data on its incidence and predictors are sparse and conflicting. We thus aimed to perform a collaborative systematic review on incidence and predictors of stent thrombosis. METHODS PubMed was systematically searched for eligible studies from the drug-eluting stent (DES) era (1/2002-12/2010). Studies were selected if including ≥ 2000 patients undergoing stenting or reporting on ≥ 25 thromboses. Study features, patient characteristics, and incidence of stent thrombosis were abstracted and pooled, when appropriate, with random-effect methods (point estimate [95% confidence intervals]), and consistency of predictors was formally appraised. RESULTS A total of 30 studies were identified (221,066 patients, 4276 thromboses), with DES used in 87%. After a median of 22 months, definite, probable, or possible stent thrombosis had occurred in 2.4% (2.0%; 2.9%), with acute in 0.4% (0.2%; 0.6%), subacute in 1.1% (1.0%; 1.3%), late in 0.5% (0.4%; 0.6%), and very late in 0.6% (0.4%; 0.8%). Similar figures were computed for studies reporting only on DES. From a total of 47 candidate variables, definite/probable stent thrombosis was more commonly and consistently predicted by early antiplatelet therapy discontinuation, extent of coronary disease, and stent number/length, with acute coronary syndrome at admission, diabetes, smoking status, and bifurcation/ostial disease also proving frequent predictors, but less consistently. CONCLUSIONS Despite numerous possible risk factors, the most common and consistent predictors of stent thrombosis are early antiplatelet therapy discontinuation, extent of coronary disease, and stent number/length.

Cardiovascular Influences of α1b-Adrenergic Receptor Defect in Mice

Background—The &agr;1-adrenergic receptors (&agr;1-ARs) play a key role in cardiovascular homeostasis. However, the functional role of &agr;1-AR subtypes in vivo is still unclear. The aim of this study was to evaluate the cardiovascular influences of &agr;1b-AR. Methods and Results—In transgenic mice lacking &agr;1-AR (KO) and their wild-type controls (WT), we evaluated blood pressure profile and cardiovascular remodeling induced by the chronic administration (18 days via osmotic pumps) of norepinephrine, angiotensin II, and subpressor doses of phenylephrine. Our results indicate that norepinephrine induced an increase in blood pressure levels only in WT mice. In contrast, the hypertensive state induced by angiotensin II was comparable between WT and KO mice. Phenylephrine did not modify blood pressure levels in either WT or KO mice. The cardiac hypertrophy and eutrophic vascular remodeling evoked by norepinephrine was observed only in WT mice, and this effect was independent of the hypertensive state because it was similar to that observed during subpressor phenylephrine infusion. Finally, the cardiac hypertrophy induced by thoracic aortic constriction was comparable between WT and KO mice. Conclusions—Our data demonstrate that the lack of &agr;1b-AR protects from the chronic increase of arterial blood pressure induced by norepinephrine and concomitantly prevents cardiovascular remodeling evoked by adrenergic activation independently of blood pressure levels.

Cardiac stem cells: isolation, expansion and experimental use for myocardial regeneration

Cellular cardiomyoplasty (myogenic cell grafting) is actively being explored as a novel method to regenerate damaged myocardium. The adult human heart contains small populations of indigenous committed cardiac stem cells or multipotent cardiac progenitor cells, identified by their cell-surface expression of c-kit (the receptor for stem cell factor), P-glycoprotein (a member of the multidrug resistance protein family), and Sca-1 (stem cell antigen 1, a mouse hematopoietic stem cell marker) or a Sca-1-like protein. Cardiac stem cells represent a logical source to exploit in cardiac regeneration therapy because, unlike other adult stem cells, they are likely to be intrinsically programmed to generate cardiac tissue in vitro and to increase cardiac tissue viability in vitro. Cardiac stem cell therapy could, therefore, change the fundamental approach to the treatment of heart disease.

Differentiation of human adult cardiac stem cells exposed to extremely low-frequency electromagnetic fields

AIMS Modulation of cardiac stem cell (CSC) differentiation with minimal manipulation is one of the main goals of clinical applicability of cell therapy for heart failure. CSCs, obtained from human myocardial bioptic specimens and grown as cardiospheres (CSps) and cardiosphere-derived cells (CDCs), can engraft and partially regenerate the infarcted myocardium, as previously described. In this paper we assessed the hypothesis that exposure of CSps and CDCs to extremely low-frequency electromagnetic fields (ELF-EMFs), tuned at Ca2+ ion cyclotron energy resonance (Ca2+-ICR), may drive their differentiation towards a cardiac-specific phenotype. METHODS AND RESULTS A significant increase in the expression of cardiac markers was observed after 5 days of exposure to Ca2+-ICR in both human CSps and CDCs, as evidenced at transcriptional, translational, and phenotypical levels. Ca2+ mobilization among intracellular storages was observed and confirmed by compartmentalized analysis of Ca2+ fluorescent probes. CONCLUSIONS These results suggest that ELF-EMFs tuned at Ca2+-ICR could be used to drive cardiac-specific differentiation in adult cardiac progenitor cells without any pharmacological or genetic manipulation of the cells that will be used for therapeutic purposes.

Extracorporeal membrane oxygenation (ECMO) in patients with H1N1 influenza infection: a systematic review and meta-analysis including 8 studies and 266 patients receiving ECMO

IntroductionH1N1 influenza can cause severe acute lung injury (ALI). Extracorporeal membrane oxygenation (ECMO) can support gas exchange in patients failing conventional mechanical ventilation, but its role is still controversial. We conducted a systematic review and meta-analysis on ECMO for H1N1-associated ALI.MethodsCENTRAL, Google Scholar, MEDLINE/PubMed and Scopus (updated 2 January 2012) were systematically searched. Studies reporting on 10 or more patients with H1N1 infection treated with ECMO were included. Baseline, procedural, outcome and validity data were systematically appraised and pooled, when appropriate, with random-effect methods.ResultsFrom 1,196 initial citations, 8 studies were selected, including 1,357 patients with confirmed/suspected H1N1 infection requiring intensive care unit admission, 266 (20%) of whom were treated with ECMO. Patients had a median Sequential Organ Failure Assessment (SOFA) score of 9, and had received mechanical ventilation before ECMO implementation for a median of two days. ECMO was implanted before inter-hospital patient transfer in 72% of cases and in most patients (94%) the veno-venous configuration was used. ECMO was maintained for a median of 10 days. Outcomes were highly variable among the included studies, with in-hospital or short-term mortality ranging between 8% and 65%, mainly depending on baseline patient features. Random-effect pooled estimates suggested an overall in-hospital mortality of 28% (95% confidence interval 18% to 37%; I2 = 64%).ConclusionsECMO is feasible and effective in patients with ALI due to H1N1 infection. Despite this, prolonged support (more than one week) is required in most cases, and subjects with severe comorbidities or multiorgan failure remain at high risk of in-hospital death.