Enhanced angiogenic and cardiomyocyte differentiation capacity of epigenetically reprogrammed mouse and human endothelial progenitor cells augments their efficacy for ischemic myocardial repair.

RATIONALE: Although bone marrow endothelial progenitor cell (EPC)-based therapies improve the symptoms in patients with ischemic heart disease, their limited plasticity and decreased function in patients with existing heart disease limit the full benefit of EPC therapy for cardiac regenerative medicine. OBJECTIVE: We hypothesized that reprogramming mouse or human EPCs, or both, using small molecules targeting key epigenetic repressive marks would lead to a global increase in active gene transcription, induce their cardiomyogenic potential, and enhance their inherent angiogenic potential. METHOD AND RESULTS: Mouse Lin-Sca1(+)CD31(+) EPCs and human CD34(+) cells were treated with inhibitors of DNA methyltransferases (5-Azacytidine), histone deacetylases (valproic acid), and G9a histone dimethyltransferase. A 48-hour treatment led to global increase in active transcriptome, including the reactivation of pluripotency-associated and cardiomyocyte-specific mRNA expression, whereas endothelial cell-specific genes were significantly upregulated. When cultured under appropriate differentiation conditions, reprogrammed EPCs showed efficient differentiation into cardiomyocytes. Treatment with epigenetic-modifying agents show marked increase in histone acetylation on cardiomyocyte and pluripotent cell-specific gene promoters. Intramyocardial transplantation of reprogrammed mouse and human EPCs in an acute myocardial infarction mouse model showed significant improvement in ventricular functions, which was histologically supported by their de novo cardiomyocyte differentiation and increased capillary density and reduced fibrosis. Importantly, cell transplantation was safe and did not form teratomas. CONCLUSIONS: Taken together, our results suggest that epigenetically reprogrammed EPCs display a safe, more plastic phenotype and improve postinfarct cardiac repair by both neocardiomyogenesis and neovascularization.

Interleukin-10 treatment attenuates pressure overload-induced hypertrophic remodeling and improves heart function via signal transducers and activators of transcription 3-dependent inhibition of nuclear factor-κB.

BACKGROUND: Inflammation plays a critical role in adverse cardiac remodeling and heart failure. Therefore, approaches geared toward inhibiting inflammation may provide therapeutic benefits. We tested the hypotheses that genetic deletion of interleukin-10 (IL-10), a potent antiinflammatory cytokine, exacerbates pressure overload-induced adverse cardiac remodeling and hypertrophy and that IL-10 therapy inhibits this pathology. METHODS AND RESULTS: Cardiac hypertrophy was induced in wild-type and IL-10 knockout mice by isoproterenol (ISO) infusion. ISO-induced left ventricular dysfunction and hypertrophic remodeling, including fibrosis and fetal gene expression, were further exaggerated in knockout mice compared with wild-type mice. Systemic recombinant mouse IL-10 administration markedly improved left ventricular function and not only inhibited but also reversed ISO-induced cardiac remodeling. Intriguingly, a very similar cardioprotective response of IL-10 was found in transverse aortic constriction-induced hypertrophy and heart failure models. In neonatal rat ventricular myocytes and H9c2 myoblasts, ISO activated nuclear factor-κB and inhibited signal transducers and activators of transcription 3 (STAT3) phosphorylation. Interestingly, IL-10 suppressed ISO-induced nuclear factor-κB activation and attenuated STAT3 inhibition. Moreover, pharmacological and genetic inhibition of STAT3 reversed the protective effects of IL-10, whereas ectopic expression of constitutively active STAT3 mimicked the IL-10 responses on the ISO effects, confirming that the IL-10-mediated inhibition of nuclear factor-κB is STAT3 dependent. CONCLUSION: Taken together, our results suggest IL-10 treatment as a potential therapeutic approach to limit the progression of pressure overload-induced adverse cardiac remodeling.

Histone deacetylase 1 deficiency impairs differentiation and electrophysiological properties of cardiomyocytes derived from induced pluripotent cells.

Epigenetic and chromatin modifications play particularly important roles in embryonic and induced pluripotent stem cells (ESCs and iPSCs) allowing for the cells to both differentiate and dedifferentiate back to a pluripotent state. We analyzed how the loss of a key chromatin-modifying enzyme, histone deacetylase 1 (HDAC1), affects early and cardiovascular differentiation of both ESCs and iPSCs. We also investigated potential differences between these two cell types when differentiation is induced. Our data indicate an essential role for HDAC1 in deacetylating regulatory regions of key pluripotency-associated genes during early differentiation. Although HDAC1 functions primarily as a HDAC, its loss also affects DNA methylation in ESCs and iPSCs both during pluripotency and differentiation. We show that HDAC1 plays a crucial, nonredundant role in cardiomyocyte differentiation and maturation. Our data also elucidate important differences between ESCs and iPSCs, when levels of this enzyme are reduced, that affect their ability to differentiate into functional cardiomyocytes. As varying levels of chromatin-modifying enzymes are likely to exist in patient-derived iPSCs, understanding the molecular circuitry of these enzymes in ESCs and iPSCs is critical for their potential use in cardiovascular therapeutic applications

Interleukin-10 deficiency impairs bone marrow-derived endothelial progenitor cell survival and function in ischemic myocardium.

RATIONALE: Endothelial progenitor cell (EPC) survival and function in the injured myocardium is adversely influenced by hostile microenvironment such as ischemia, hypoxia, and inflammatory response, thereby compromising full benefits of EPC-mediated myocardial repair. OBJECTIVE: We hypothesized that interleukin-10 (IL-10) modulates EPC biology leading to enhanced survival and function after transplantation in the ischemic myocardium. METHODS AND RESULTS: Myocardial infarction (MI)-induced mobilization of bone marrow EPC (Sca-1+Flk1+cells) into the circulation was significantly impaired in IL-10 knockout (KO) mice. Bone marrow transplantation to replace IL-10 KO marrow with wild-type (WT) marrow attenuated these effects. Impaired mobilization was associated with lower stromal cell-derived factor (SDF)-1 expression levels in the myocardium of KO mice. Interestingly, SDF-1 administration reversed mobilization defect in KO mice. In vitro, hypoxia-mediated increases in CXCR4 expression and cell survival were lower in IL-10-deficient EPCs. Furthermore, SDF-1-induced migration of WT EPCs was inhibited by AMD3100, an inhibitor of CXCR4. To further study the effect of IL-10 on in vivo EPC survival and engraftment into vascular structures, GFP-labeled EPC were injected intramyocardially after induction of MI, and the mice were treated with either saline or recombinant IL-10. The IL-10-treated group showed increased retention of transplanted EPCs in the myocardium and was associated with significantly reduced EPC apoptosis after MI. Interestingly, increased EPC retention and their association with the vascular structures was observed in IL-10-treated mice. Increased EPC survival and angiogenesis in the myocardium of IL-10-treated mice corroborated with improved left ventricular function, reduced infarct size, and fibrosis in the myocardium. In vitro, IL-10-induced increase in VEGF expression in WT EPC was abrogated by STAT3 inhibitor, suggesting IL-10 signals through STAT3 activation. CONCLUSIONS: Taken together, our studies demonstrate that MI-induced EPC mobilization was impaired in IL-10 KO mice and that IL-10 increases EPC survival and function possibly through activation of STAT3/VEGF signaling cascades, leading to attenuation of MI-induced left ventricular dysfunction and remodeling.

Ebf1-mediated down-regulation of Id2 and Id3 is essential for specification of the B cell lineage.

Gene knockout experiments in mice have suggested a hierarchical model of early B cell commitment wherein E2A proteins (E47 and E12) activate early B cell factor (Ebf1), which in turn activates expression of the B cell commitment factor, Pax5. In IL-7 receptor alpha (IL-7Ralpha) knockout mice, B cell development is blocked before B-lineage commitment at the prepro-B cell stage in adult animals. In IL-7Ralpha(-/-) prepro-B cells, E47 is expressed and yet is insufficient to transcriptionally activate the putative downstream target gene, Ebf1. In this study, we show that further increases of E47 expression in IL-7Ralpha(-/-) prepro-B cells fails to activate Ebf1, but rather leads to a dramatic induction of the E2A inhibitory factors, Id2 and Id3. In contrast, enforced expression of Ebf1 in IL-7Ralpha(-/-) bone marrow potently down-regulates Id2 and Id3 mRNA expression and restores B cell differentiation in vivo. Down-regulation of both Id2 and Id3 during B cell specification is essential in that overexpression of either Id2 or Id3 in wild-type bone marrow blocks B cell specification at the prepro-B cell stage. Collectively, these studies suggest a model where Ebf1 induction specifies the B cell fate by dramatically increasing activity of E47 at the posttranslational level.

Template requirements for recognition and copying by Sindbis virus RNA-dependent RNA polymerase.

The Sindbis virus (SIN) nonstructural protein nsP4 possesses the RNA-dependent RNA polymerase activity required for the replication of the SIN genome and transcription of a subgenomic mRNA during infection. Isolation of this protein from other viral components of the RNA synthetic complex allowed the characterization of template requirements for nsP4-mediated genome replication. The major findings of this study are: (i) in the absence of other viral proteins nsP4 is capable of copying SIN plus- and minus-strand templates, but does not transcribe subgenomic RNA; (ii) mutations in the 3' conserved sequence element and poly(A) tail of the plus-strand template prevent nsP4-mediated de novo initiation of minus-strand RNA synthesis; (iii) nsP4-dependent terminal addition of nucleotides occurs on template RNA possessing certain mutations in the 3'CSE and polyadenylate tail ; (iv) nsP4 is capable of minus-strand synthesis independent of the sequence at the 5' end of the template; (v) an A-U rich sequence in the 3'CSE represents a binding site for a replicase component, probably nsP4; (vi) plus-strand genomic RNA synthesis is dependent on the 3' end of the minus-strand template. These studies begin to define the specific interactions with the viral RNA templates mediated by individual components of the viral replication complex and suggest a model for ternary complex formation during the initiation of minus-strand RNA synthesis.