Search

Background CD 34 + stem/progenitor cells are involved in vascular homeostasis and in neovascularization of ischemic tissues. The number of circulating CD 34 + stem cells is a predictive biomarker of adverse cardiovascular outcomes in diabetic patients. Here, we provide evidence that hyperglycemia can be "memorized" by the stem cells through epigenetic changes that contribute to onset and maintenance of their dysfunction in diabetes mellitus. Methods and Results Cord-blood-derived CD 34 + stem cells exposed to high glucose displayed increased reactive oxygen species production, overexpression of p66 shc gene, and downregulation of antioxidant genes catalase and manganese superoxide dismutase when compared with normoglycemic cells. This altered oxidative state was associated with impaired migration ability toward stromal-cell-derived factor 1 alpha and reduced protein and mRNA expression of the C-X-C chemokine receptor type 4 ( CXCR 4) receptor. The methylation analysis by bisulfite Sanger sequencing of the CXCR 4 promoter revealed a significant increase in DNA methylation density in high-glucose CD 34 + stem cells that negatively correlated with mRNA expression (Pearson r=-0.76; P=0.004). Consistently, we found, by chromatin immunoprecipitation assay, a more transcriptionally inactive chromatin conformation and reduced RNA polymerase II engagement on the CXCR 4 promoter. Notably, alteration of CXCR 4 DNA methylation, as well as transcriptional and functional defects, persisted in high-glucose CD 34 + stem cells despite recovery in normoglycemic conditions. Importantly, such an epigenetic modification was thoroughly confirmed in bone marrow CD 34 + stem cells isolated from sternal biopsies of diabetic patients undergoing coronary bypass surgery. Conclusions CD 34 + stem cells "memorize" the hyperglycemic environment in the form of epigenetic modifications that collude to alter CXCR 4 receptor expression and migration.

The recruitment of bone marrow (BM)-derived progenitor cells to the lung is related to pulmonary remodelling and the pathogenesis of pulmonary hypertension (PH). Although sildenafil is a known target in PH treatment, the underlying molecular mechanism is still elusive. To test the hypothesis that the therapeutic effect of sildenafil is linked to the reduced recruitment of BM-derived progenitor cells, we induced pulmonary remodelling in rats by two-week exposure to chronic hypoxia (CH, 10% oxygen), a trigger of BM-derived progenitor cells. Rats were treated with either placebo (saline) or sildenafil (1.4 mg/kg/day ip) during CH. Control rats were kept in room air (21% oxygen) with no treatment. As expected, sildenafil attenuated the CH-induced increase in right ventricular systolic pressure and right ventricular hypertrophy. However, sildenafil suppressed the CH-induced increase in c-kit + cells in the adventitia of pulmonary arteries. Moreover, sildenafil reduced the number of c-kit + cells that colocalize with tyrosine kinase receptor 2 (VEGF-R2) and CD68 (a marker for macrophages), indicating a positive effect on moderating hypoxia-induced smooth muscle cell proliferation and inflammation without affecting the pulmonary levels of hypoxia-inducible factor (HIF)-1α. Furthermore, sildenafil depressed the number of CXCR4 + cells. Collectively, these findings indicate that the improvement in pulmonary haemodynamic by sildenafil is linked to decreased recruitment of BM-derived c-kit + cells in the pulmonary tissue. The attenuation of the recruitment of BM-derived c-kit + cells by sildenafil may provide novel therapeutic insights into the control of pulmonary remodelling., (© 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

The anthracycline doxorubicin (Dox) is widely used in oncology, but it may cause a cardiomyopathy with bleak prognosis that cannot be effectively prevented. The secretome of human amniotic fluid-derived stem cells (hAFS) has previously been demonstrated to significantly reduce ischemic cardiac damage. Here it is shown that, following hypoxic preconditioning, hAFS conditioned medium (hAFS-CM) antagonizes senescence and apoptosis of cardiomyocytes and cardiac progenitor cells, two major features of Dox cardiotoxicity. Mechanistic studies with mouse neonatal ventricular cardiomyocytes (mNVCM) reveal that hAFS-CM inhibition of Dox-elicited senescence and apoptosis is associated with decreased DNA damage, nuclear translocation of NF-kB, and upregulation of the NF-kB controlled genes, Il6 and Cxcl1, promoting mNVCM survival. Furthermore, hAFS-CM induces expression of the efflux transporter, Abcb1b, and Dox extrusion from mNVCM. The PI3K/Akt signaling cascade, upstream of NF-kB, is potently activated by hAFS-CM and pre-treatment with a PI3K inhibitor abrogates NF-kB accumulation into the nucleus, modulation of Il6, Cxcl1 and Abcb1b, and prevention of Dox-initiated senescence and apoptosis in response to hAFS-CM. These results support the concept that hAFS are a valuable source of cardioprotective factors and lay the foundations for the development of a stem cell-based paracrine treatment of chemotherapy-related cardiotoxicity.

Cardiovascular disease is the leading cause of morbidity and mortality in the developed world. Although ongoing therapeutic strategies ameliorate symptoms and prolong life for patients with cardiovascular diseases, they do not solve the critical issue related to the loss of cardiac tissue. Accordingly, stem/progenitor cell therapy has emerged as a paramount approach for cardiac repair and regeneration. In this regard, c-kit(+) cells have animated much interest and controversy. These cells are self-renewing, clonogenic, and multipotent and display a noteworthy potential to differentiate into all cardiovascular lineages. However, their functional contribution to cardiomyocyte turnover is one of the centrally debated issues concerning their regenerative potential. Regardless, plentiful preclinical and clinical studies have been conducted which provide evidence for the capacity of c-kit(+) cells to improve cardiac function. The purpose of this review is to give a comprehensive, impartial, critical description and evaluation of the literature on c-kit(+) cells from bench to bedside in order to address their true potential, benefits and controversies.

Ischemic diseases are the major cause of death and morbidity in Western countries. In the last decade, cell therapy has been suggested to be a promising treatment both in acute/chronic myocardial and peripheral ischemia. Different cell lineages have been tested, including endothelial progenitor cells. A subpopulation of bone marrow-derived immature ECPs, expressing the highly conserved stem cell glycoprotein antigen prominin-1 or CD133 marker, was shown to possess pro-angiogenic and antiapoptotic effects on ischemic tissues. The mechanisms implicated in CD133+ cells ability to contribute to neovascularization processes have been attributed to their ability to directly differentiate into newly forming vessels and to indirectly activate pro-angiogenic signaling by paracrine mechanisms. A large body of in vivo experimental evidences has demonstrated the potential of CD133+ cells to reverse ischemia. Moreover, several clinical trials have reported promising beneficial effects after infusion of autologous CD133+ into ischemic heart and limbs exploiting various delivery strategies. These trials have contributed to characterize the CD133+ manufacturing process as an advanced cell product (AMP). The aim of this review is to summarize available experimental and clinical data on CD133+ cells in the context of myocardial and peripheral ischemia, and to focus on the development of the CD133+ cell as an anti-ischemic AMP.

Human c-kit(+) cardiac progenitor cells (CPCs) are multipotent and may be used for cardiac repair. The effect of cardiovascular risk factors and medications on CPCs isolation efficiency, c-kit stem cell marker expression, and ex vivo proliferative potential is unknown and was examined in the present work. Cells from human right atrial appendages (n = 50) were expanded in culture; after ∼16 days (T0), it was established the percentage of CPCs and c-kit protein mean fluorescence intensity (MFI) by fluorescence activated cell sorting (FACS). Thereafter, CPCs were isolated by high throughput sorting; after culturing for 4 passages CPCs-derived cells were re-analyzed to assess c-kit(+) cell percentage and enrichment vs T0. The association between 19 demographic and clinical variables to CPCs number and MFI at T0, and CPCs enrichment at P4, was determined by multiple linear regression analysis with stepwise selection procedure. The results revealed that (1) at T0, the number of isolated CPCs directly correlated to β-blocker treatment; (2) at T0, c-kit protein expression directly correlated to pulmonary hypertension (PH); (3) at P4, CPC's enrichment inversely correlated to smoke, atrial fibrillation (AF), a history of myocardial infarction, whereas it directly correlated to PH and statins. Patient clinical profile and medications differently modulate CPCs isolation and amplification potential ex vivo. These results may provide new insights for the understanding of cardiac homeostasis and suggest both limitations and possible enhancing strategies for the therapeutic use of cardiac-resident progenitor cells., (Copyright © 2012 Mosby, Inc. All rights reserved.)

Adult human cardiac mesenchymal-like stromal cells (CStC) represent a relatively accessible cell type useful for therapy. In this light, their conversion into cardiovascular precursors represents a potential successful strategy for cardiac repair. The aim of the present work was to reprogram CStC into functionally competent cardiovascular precursors using epigenetically active small molecules. CStC were exposed to low serum (5% FBS) in the presence of 5 µM all-trans Retinoic Acid (ATRA), 5 µM Phenyl Butyrate (PB), and 200 µM diethylenetriamine/nitric oxide (DETA/NO), to create a novel epigenetically active cocktail (EpiC). Upon treatment the expression of markers typical of cardiac resident stem cells such as c-Kit and MDR-1 were up-regulated, together with the expression of a number of cardiovascular-associated genes including KDR, GATA6, Nkx2.5, GATA4, HCN4, NaV1.5, and α-MHC. In addition, profiling analysis revealed that a significant number of microRNA involved in cardiomyocyte biology and cell differentiation/proliferation, including miR 133a, 210 and 34a, were up-regulated. Remarkably, almost 45% of EpiC-treated cells exhibited a TTX-sensitive sodium current and, to a lower extent in a few cells, also the pacemaker I(f) current. Mechanistically, the exposure to EpiC treatment introduced global histone modifications, characterized by increased levels of H3K4Me3 and H4K16Ac, as well as reduced H4K20Me3 and H3s10P, a pattern compatible with reduced proliferation and chromatin relaxation. Consistently, ChIP experiments performed with H3K4me3 or H3s10P histone modifications revealed the presence of a specific EpiC-dependent pattern in c-Kit, MDR-1, and Nkx2.5 promoter regions, possibly contributing to their modified expression. Taken together, these data indicate that CStC may be epigenetically reprogrammed to acquire molecular and biological properties associated with competent cardiovascular precursors.

Preclinical studies performed in cell culture and animal systems have shown the outstanding ability of stem cells to repair ischemic heart and lower limbs by promoting the formation of new blood vessels and new myocytes. In contrast, clinical studies of stem cell administration in patients with myocardial ischemia have revealed only modest, although promising, results. Basic investigations have shown the feasibility of adult cells reprogramming into pluripotent cells by defined factors, thus opening the way to the devise of protocols to ex vivo derive virtually unexhausted cellular pools. In contrast, cellular and molecular studies have indicated that risk factors limit adult-derived stem cell survival, proliferation and engraftment in ischemic tissues. The use of fully reprogrammed cells raises safety concerns; therefore, adult cells remain a primary option for clinicians interested in therapeutic cardiovascular repair. Pharmacologic approaches have been devised to restore the cardiovascular repair ability of failing progenitors from patients at risk. In the present contribution, the most advanced pharmacologic approaches to (re)program, boost, and condition endothelial and cardiac progenitor cells to enhance cardiovascular regeneration are discussed., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Aims: The aim of this study was to assess the effect of granulocyte colony-stimulating factor (G-CSF) on left ventricular (LV) function and volumes in patients with anterior ST-elevation myocardial infarction (STEMI) and depressed LV ejection fraction (EF)., Methods and Results: Sixty consecutive patients with anterior STEMI, undergoing primary angioplasty percutaneous coronary intervention (PCI), with symptom-to-reperfusion time of 2-12 h and EF ≤45% after PCI, were randomized to G-CSF 5 μg/kg b.i.d. subcutaneously (n = 24) or placebo (n = 25) for 5 days, starting <12 h after PCI. The primary endpoint was an increase from baseline to 6 months of 5% in left ventricular ejection fraction (LVEF), as measured by magnetic resonance imaging (MRI). Co-primary endpoint was a ≥20 mL difference in end-diastolic volume (EDV). Infarct size and perfusion were evaluated with late gadolinium enhancement (LGE) and gated (99m)Technetium Sestamibi single-photon emission computed tomography (SPECT). Left ventricular EDV and end-systolic volume (ESV) increased from baseline to 6 months in the placebo group (81.7 ± 24.4 to 94.4 ± 26.0 mL/m(2), P < 0.00005 and 45.2 ± 20.0 to 53.2 ± 23.8 mL/m(2), P = 0.016) but were unchanged in the G-CSF group (82.2 ± 20.3 to 85.7 ± 23.7 mL/m(2), P = 0.40 and 46.0 ± 18.2 to 48.4 ± 20.8 mL/m(2), P = 0.338). There were no significant differences in EF or perfusion between groups. A significant reduction in transmural LGE segments was seen at 6 months in the G-CSF vs. placebo groups (4.38 ± 2.9 to 3.3 ± 2.6, P = 0.04 and 4.2 ± 2.6 to 3.6 ± 2.7, P = 0.301, respectively). Significantly more placebo patients had a change in left ventricular end-diastolic volume abovethe median (9.3 mL/m(2)) when reperfusion time exceeded 180 min (median time-to-reperfusion) (P = 0.0123). Severe adverse events were similar between groups., Conclusion: Early G-CSF administration attenuates ventricular remodelling in patients with anterior STEMI and EF ≤45% after successful PCI.

The aim of the present study was to develop and validate a good manufacturing practice (GMP) compliant procedure for the preparation of bone marrow (BM) derived CD133(+) cells for cardiovascular repair. Starting from available laboratory protocols to purify CD133(+) cells from human cord blood, we implemented these procedures in a GMP facility and applied quality control conditions defining purity, microbiological safety and vitality of CD133(+) cells. Validation of CD133(+) cells isolation and release process were performed according to a two-step experimental program comprising release quality checking (step 1) as well as 'proofs of principle' of their phenotypic integrity and biological function (step 2). This testing program was accomplished using in vitro culture assays and in vivo testing in an immunosuppressed mouse model of hindlimb ischemia. These criteria and procedures were successfully applied to GMP production of CD133(+) cells from the BM for an ongoing clinical trial of autologous stem cells administration into patients with ischemic cardiomyopathy. Our results show that GMP implementation of currently available protocols for CD133(+) cells selection is feasible and reproducible, and enables the production of cells having a full biological potential according to the most recent quality requirements by European Regulatory Agencies.

In diabetic patients and animal models of diabetes mellitus (DM), circulating endothelial progenitor cell (EPC) number is lower than in normoglycaemic conditions and EPC angiogenic properties are inhibited. Stromal cell derived factor-1 (SDF-1) plays a key role in bone marrow (BM) c-kit(+) stem cell mobilization into peripheral blood (PB), recruitment from PB into ischemic tissues and differentiation into endothelial cells. The aim of the present study was to examine the effect of DM in vivo and in vitro, on murine BM-derived c-kit(+) cells and on their response to SDF-1. Acute hindlimb ischemia was induced in streptozotocin-treated DM and control mice; circulating c-kit(+) cells exhibited a rapid increase followed by a return to control levels which was significantly faster in DM than in control mice. CXCR4 expression by BM c-kit(+) cells as well as SDF-1 protein levels in the plasma and in the skeletal muscle, both before and after the induction of ischemia, were similar between normoglycaemic and DM mice. However, BM-derived c-kit(+) cells from DM mice exhibited an impaired differentiation towards the endothelial phenotype in response to SDF-1; this effect was associated with diminished protein kinase phosphorylation. Interestingly, SDF-1 ability to induce differentiation of c-kit(+) cells from DM mice was restored when cells were cultured under normoglycaemic conditions whereas c-kit(+) cells from normoglycaemic mice failed to differentiate in response to SDF-1 when they were cultured in hyperglycaemic conditions. These results show that DM diminishes circulating c-kit(+) cell number following hindlimb ischemia and inhibits SDF-1-mediated AKT phosphorylation and differentiation towards the endothelial phenotype of BM-derived c-kit(+) cells.

Emerging evidences indicate that endothelial progenitor cells (EPCs) actively contribute in regulating cardiovascular homeostasis, and interest is growing for possible future diagnostic and therapeutic applications in the cardiovascular arena. In the present clinically-oriented review, special attention was given to the clinical implications of the potential of EPCs to test and strengthen the capacity of the organism to challenge atherosclerosis, vascular remodelling and ischemia. Accumulating data suggest that the vasculo-protective functions of EPCs may be used as cellular biomarkers for endothelial damage, or may be pharmacologically modulated to enhance the body's defence to atherosclerosis. Furthermore, biomedical engineering and cell transplantation open new scenarios to reverse vascular and graft remodelling and achieve therapeutic angiogenesis in limb and heart ischemia. However, a number of unsolved issues remain to be exploited, such as the identification of the true identity of EPCs and a better characterization of their role in vascular homeostasis under normal and pathologic conditions.

High Mobility Box 1 Protein (HMGB1) is a cytokine released into the extracellular space by necrotic cells and activated macrophages in response to injury. We recently demonstrated that HMGB1 administration into the mouse heart during acute myocardial infarction induces cardiac tissue regeneration by activating resident cardiac c-kit+ cells (CSCs) and significantly enhances left ventricular function. In the present study it was analyzed the hypothesis that human cardiac fibroblasts (cFbs) exposed to HMGB1 may exert a paracrine effect on mouse and human CSCs. Human cFbs expressed the HMGB1 receptor RAGE. Luminex technology and ELISA assays revealed that HMGB1 significantly enhanced VEGF, PlGF, Mip-1alpha, IFN-gamma, GM-CSF, Il-10, Il-1beta, Il-4, Il-1ra, Il-9 and TNF-alpha in cFbs cell culture medium. HMGB1-stimulated cFbs conditioned media induced CSC migration and proliferation. These effects were significantly higher to those obtained when HMGB1 was added directly to the culture medium. In conclusion, we provide evidence that HMGB1 may act in a paracrine manner stimulating growth factor, cytokine and chemokine release by cFbs which, in turn, modulate CSC function. Via this mechanism HMGB1 may contribute to cardiac tissue regeneration.

During cardiac development, the epicardium is the source of multipotent mesenchymal cells, which give rise to endothelial and smooth muscle cells in coronary vessels and also, possibly, to cardiomyocytes. The aim of the present study was to determine whether stem cells are retained in the adult human and murine epicardium and to investigate the regenerative potential of these cells following acute myocardial infarction. We show that c-kit(+) and CD34(+) cells can indeed be detected in human fetal and adult epicardium and that they represent 2 distinct populations. Both subsets of cells were negative for CD45, a cell surface marker that identifies the hematopoietic cell lineage. Immunofluorescence revealed that freshly isolated c-kit(+) and CD34(+) cells expressed early and late cardiac transcription factors and could acquire an endothelial phenotype in vitro. In the murine model of myocardial infarction, there was an increase in the absolute number and proliferation of epicardial c-kit(+) cells 3 days after coronary ligation; at this time point, epicardial c-kit(+) cells were identified in the subepicardial space and expressed GATA4. Furthermore, 1 week after myocardial infarction, cells coexpressing c-kit(+), together with endothelial or smooth muscle cell markers, were identified in the wall of subepicardial blood vessels. In summary, the postnatal epicardium contains a cell population with stem cell characteristics that retains the ability to give rise to myocardial precursors and vascular cells. These cells may play a role in the regenerative response to cardiac damage.

We report the case of a 60-year-old man with patent coronary by-pass grafts on the left anterior descending and circumflex coronary arteries, who experienced recurrent Canadian class IV angina refractory to medical and interventional treatments for a dominant right coronary artery occlusion. He underwent autologous PBSC transplant into the inferior ventricular wall through a minimally invasive approach as a stand-alone therapy, in an attempt to induce therapeutic angiogenesis. Six months after the operation, angina significantly ameliorated; scintigraphy and coronary angiography showed a marked improvement in perfusion of the target injection area. These benefits had persisted at 25 months after stem cell transplant. [ABSTRACT FROM AUTHOR]