Dnmt3a-mediated inhibition of Wnt in cardiac progenitor cells improves differentiation and remote remodeling after infarction.

Adult cardiac progenitor cells (CPCs) display a low capacity to differentiate into cardiomyocytes in injured hearts, strongly limiting the regenerative capacity of the mammalian myocardium. To identify new mechanisms regulating CPC differentiation, we used primary and clonally expanded Sca-1+ CPCs from murine adult hearts in homotypic culture or coculture with cardiomyocytes. Expression kinetics analysis during homotypic culture differentiation showed downregulation of Wnt target genes concomitant with increased expression of the Wnt antagonist, Wnt inhibitory factor 1 (Wif1), which is necessary to stimulate CPC differentiation. We show that the expression of the Wif1 gene is repressed by DNA methylation and regulated by the de novo DNA methyltransferase Dnmt3a. In addition, miR-29a is upregulated early during CPC differentiation and downregulates Dnmt3a expression, thereby decreasing Wif1 gene methylation and increasing the efficiency of differentiation of Sca-1+ CPCs in vitro. Extending these findings in vivo, transient silencing of Dnmt3a in CPCs subsequently injected in the border zone of infarcted mouse hearts improved CPC differentiation in situ and remote cardiac remodeling. In conclusion, miR-29a and Dnmt3a epigenetically regulate CPC differentiation through Wnt inhibition. Remote effects on cardiac remodeling support paracrine signaling beyond the local injection site, with potential therapeutic interest for cardiac repair.

Paracrine nitric oxide induces expression of cardiac sarcomeric proteins in adult progenitor cells through soluble guanylyl cyclase/cyclic-guanosine monophosphate and Wnt/ß-catenin inhibition.

AIM: Cardiac progenitor cells (CPC) from adult hearts can differentiate to several cell types composing the myocardium but the underlying molecular pathways are poorly characterized. We examined the role of paracrine nitric oxide (NO) in the specification of CPC to the cardiac lineage, particularly through its inhibition of the canonical Wnt/ß-catenin pathway, a critical step preceding cardiac differentiation. METHODS AND RESULTS: Sca1 + CPC from adult mouse hearts were isolated by magnetic-activated cell sorting and clonally expanded. Pharmacologic NO donors increased their expression of cardiac myocyte-specific sarcomeric proteins in a concentration and time-dependent manner. The optimal time window for NO efficacy coincided with up-regulation of CPC expression of Gucy1a3 (coding the alpha1 subunit of guanylyl cyclase). The effect of paracrine NO was reproduced in vitro upon co-culture of CPC with cardiac myocytes expressing a transgenic NOS3 (endothelial nitric oxide synthase) and in vivo upon injection of CPC in infarcted hearts from cardiac-specific NOS3 transgenic mice. In mono- and co-cultures, this effect was abrogated upon inhibition of soluble guanylyl cyclase or nitric oxide synthase, and was lost in CPC genetically deficient in Gucy1a3. Mechanistically, NO inhibits the constitutive activity of the canonical Wnt/ß-catenin in CPC and in cell reporter assays in a guanylyl cyclase-dependent fashion. This was paralleled with decreased expression of ß-catenin and down-regulation of Wnt target genes in CPC and abrogated in CPC with a stabilized, non-inhibitable ß-catenin. CONCLUSIONS: Exogenous or paracrine sources of NO promote the specification towards the myocyte lineage and expression of cardiac sarcomeric proteins of adult CPC. This is contingent upon the expression and activity of the alpha1 subunit of guanylyl cyclase in CPC that is necessary for NO-mediated inhibition of the canonical Wnt/ß-catenin pathway.

Antagonistic cross-regulation between Wnt and Hedgehog signalling pathways controls post-embryonic retinal proliferation.

Continuous neurogenesis in the adult nervous system requires a delicate balance between proliferation and differentiation. Although Wnt/ß-catenin and Hedgehog signalling pathways are thought to share a mitogenic function in adult neural stem/progenitor cells, it remains unclear how they interact in this process. Adult amphibians produce retinal neurons from a pool of neural stem cells localised in the ciliary marginal zone (CMZ). Surprisingly, we found that perturbations of the Wnt and Hedgehog pathways result in opposite proliferative outcomes of neural stem/progenitor cells in the CMZ. Additionally, our study revealed that Wnt and Hedgehog morphogens are produced in mutually exclusive territories of the post-embryonic retina. Using genetic and pharmacological tools, we found that the Wnt and Hedgehog pathways exhibit reciprocal inhibition. Our data suggest that Sfrp-1 and Gli3 contribute to this negative cross-regulation. Altogether, our results reveal an unexpected antagonistic interplay of Wnt and Hedgehog signals that may tightly regulate the extent of neural stem/progenitor cell proliferation in the Xenopus retina.

Erythropoietin preserves the endothelial differentiation capacity of cardiac progenitor cells and reduces heart failure during anticancer therapies.

Anticancer therapies, such as targeting of STAT3 or the use of anthracyclins (doxorubicin), can induce cardiomyopathy. In mice prone to developing heart failure as a result of reduced cardiac STAT3 expression (cardiomyocyte-restricted deficiency of STAT3) or treatment with doxorubicin, we observed impaired endothelial differentiation capacity of Sca-1(+) cardiac progenitor cells (CPCs) in conjunction with attenuated CCL2/CCR2 activation. Mice in both models also displayed reduced erythropoietin (EPO) levels in the cardiac microenvironment. EPO binds to CPCs and seems to be responsible for maintaining an active CCL2/CCR2 system. Supplementation with the EPO derivative CERA in a hematocrit-inactive low dose was sufficient to upregulate CCL2, restore endothelial differentiation of CPCs, and preserve the cardiac microvasculature and cardiac function in both mouse models. Thus, low-dose EPO treatment could potentially be exploited as a therapeutic strategy to reduce the risk of heart failure in certain treatment regimens.

Moderate caveolin-1 downregulation prevents NADPH oxidase-dependent endothelial nitric oxide synthase uncoupling by angiotensin II in endothelial cells.

OBJECTIVE: We analyzed the role of caveolin-1 (Cav-1) in the cross-talk between NADPH oxidase and endothelial nitric oxide synthase (eNOS) signaling in endothelial caveolae. METHODS AND RESULTS: In intact endothelial cells, angiotensin II (AII) concurrently increased NO and O(2)(-·) production (to 158±12% and 209±5% of control). NO production was sensitive to inhibition of NADPH oxidase and small interfering RNA downregulation of nonreceptor tyrosine kinase cAbl. Reciprocally, N-nitro-l-arginine methyl ester, a NOS inhibitor, partly inhibited O(2)(-·) stimulated by AII (by 47±11%), indicating eNOS uncoupling, as confirmed by increased eNOS monomer/dimer ratio (by 35%). In endothelial cell fractions separated by isopycnic ultracentrifugation, AII promoted colocalization of cAbl and the NADPH oxidase subunit p47phox with eNOS to Cav-1-enriched fractions, as confirmed by proximity ligation assay. Downregulation of Cav-1 by small interfering RNA (to 50%), although it preserved eNOS confinement, inhibited AII-stimulated p47phox translocation and NADPH oxidase activity in Cav-1-enriched fractions and reversed eNOS uncoupling. AII infusion produced hypertension and decreased blood hemoglobin-NO in Cav-1(+/+) mice but not in heterozygote Cav-1(+/-) mice with similar Cav-1 reduction. CONCLUSIONS: Cav-1 critically regulates reactive oxygen species-dependent eNOS activation but also eNOS uncoupling in response to AII, underlining the possibility to treat endothelial dysfunction by modulating Cav-1 abundance.

Wnt/beta-catenin signaling is involved in the induction and maintenance of primitive hematopoiesis in the vertebrate embryo.

The formation of primitive (embryonic) blood in vertebrates is mediated by spatio-temporally restricted signaling between different tissue layers. In Xenopus, in which primitive blood originates in the ventral blood island, this involves the secretion of bone morphogenetic protein (BMP) ligands by the ectoderm that signal to the underlying mesoderm during gastrulation. Using novel transgenic reporter lines, we report that the canonical Wnt/ß-catenin pathway is also activated in the blood islands in Xenopus. Furthermore, Wnt-reporter activity was also detected in the blood islands of the mouse yolk sac. By using morpholino-mediated depletion in Xenopus, we identified Wnt4 as the ligand that is expressed in the mesoderm of the ventral blood island and is essential for the expression of hematopoietic and erythroid marker genes. Injection of an inducible Wnt-interfering construct further showed that, during gastrulation, Wnt/ß-catenin signaling is required both in the mesoderm and in the overlying ectoderm for the formation of the ventral blood island. Using recombination assays with embryonic explants, we document that ectodermal BMP4 expression is dependent on Wnt4 signals from the mesoderm. Our results thus reveal a unique role for Wnt4-mediated canonical signaling in the formation and maintenance of the ventral blood island in Xenopus.

Beta-Catenin downregulation attenuates ischemic cardiac remodeling through enhanced resident precursor cell differentiation.

We analyzed the effect of conditional, alphaMHC-dependent genetic beta-catenin depletion and stabilization on cardiac remodeling following experimental infarct. beta-Catenin depletion significantly improved 4-week survival and left ventricular (LV) function (fractional shortening: CT(Deltaex3-6): 24 +/- 1.9%; beta-cat(Deltaex3-6): 30.2 +/- 1.6%, P < 0.001). beta-Catenin stabilization had opposite effects. No significant changes in adult cardiomyocyte survival or hypertrophy were observed in either transgenic line. Associated with the functional improvement, LV scar cellularity was altered: beta-catenin-depleted mice showed a marked subendocardial and subepicardial layer of small cTnT(pos) cardiomyocytes associated with increased expression of cardiac lineage markers Tbx5 and GATA4. Using a Cre-dependent lacZ reporter gene, we identified a noncardiomyocyte cell population affected by alphaMHC-driven gene recombination localized to these tissue compartments at baseline. These cells were found to be cardiac progenitor cells since they coexpressed markers of proliferation (Ki67) and the cardiomyocyte lineage (alphaMHC, GATA4, Tbx5) but not cardiac Troponin T (cTnT). The cell population overlaps in part with both the previously described c-kit(pos) and stem cell antigen-1 (Sca-1)(pos) precursor cell population but not with the Islet-1(pos) precursor cell pool. An in vitro coculture assay of highly enriched (>95%) Sca-1(pos) cardiac precursor cells from beta-catenin-depleted mice compared to cells isolated from control littermate demonstrated increased differentiation toward alpha-actin(pos) and cTnT(pos) cardiomyocytes after 10 days (CT(Deltaex3-6): 38.0 +/- 1.0% alpha-actin(pos); beta-cat(Deltaex3-6): 49.9 +/- 2.4% alpha-actin(pos), P < 0.001). We conclude that beta-catenin depletion attenuates postinfarct LV remodeling in part through increased differentiation of GATA4(pos)/Sca-1(pos) resident cardiac progenitor cells.

Chicken beta-globin insulator overcomes variegation of transgenes in Xenopus embryos.

Chromatin structure and gene transcription regulation are intimately linked, and mosaic expression of randomly integrated transgenes into the genome is frequently observed. This variegation of transgene expression is likely due to the genomic integration site, which can affect the behavior of the integrated DNA sequence in a positive or a negative way. Insulators are a class of DNA elements that can protect genes from inappropriate signals emanating from their environment by acting as boundaries that prevent the spreading of nearby condensed chromatin that may otherwise silence expression. Here we show that transgenes escape this silencing in Xenopus laevis and Xenopus tropicalis embryos and that a stable, uniform, and heritable expression pattern is obtained when transgenes are flanked with tandem copies of the chicken beta-globin 5'HS4 insulator. Our data also indicate that the insulator confers copy-number-dependent transgene expression and can increase transgene expression from weak regulatory elements. Hence, it will be an invaluable tool for generating stable lines expressing different levels of a particular coding sequence.

Rosuvastatin increases vascular endothelial PPARgamma expression and corrects blood pressure variability in obese dyslipidaemic mice.

AIMS: Statins improve atherosclerotic diseases through cholesterol-reducing effects. Whether the latter exclusively mediate similar benefits, e.g. on hypertension, in the metabolic syndrome is unclear. We examined the effects of rosuvastatin on the components of this syndrome, as reproduced in mice doubly deficient in LDL receptors and leptin (DKO). METHODS AND RESULTS: DKO received rosuvastatin (10 mg/kg/day or 20 mg/kg/day) or saline for 12 weeks. Saline-treated DKO mice had elevated blood pressure (BP) and nitric oxide-sensitive BP variability recorded by telemetry. Compared with saline, rosuvastatin (20 mg/kg/day) had no effect on weight gain and a minor effect on plasma cholesterol. Despite incomplete correction of insulin sensitivity, rosuvastatin fully corrected BP and its variability (P = 0.01), in conjunction with upregulation of PPARgamma (but not PPARalpha) in the aortic arch. Rosuvastatin similarly increased PPARgamma (P = 0.002) and SOD1 (P = 0.01) expression in isolated endothelial cells. Both GW9662, a PPARgamma-specific antagonist, and siRNA raised against PPARgamma abrogated rosuvastatin's effect, which was reproduced in PPARgamma- (but not PPARalpha-) dependent transactivation assays. CONCLUSION: Beyond partial improvement in insulin sensitivity, rosuvastatin normalized BP homeostasis in obese dyslipidaemic mice independently of changes in body weight or plasma cholesterol. Upregulation of PPARgamma and SOD1 in the endothelium may be involved as a unique vasculoprotective effect of statin treatment.

Human high mobility group box transcription factor 1 affects thymocyte development and transgene variegation.

It has been shown previously that a human CD2 (hCD2) disabled locus control region (LCR) transgene is unable to establish an open chromatin configuration in all the T cells, and this leads to position effect variegation of the transgene. In this study we show that thymus-specific overexpression of human high mobility group box transcription factor 1 (HBP1), a transcription factor that binds a specific sequence within the hCD2 LCR, affects thymus cellularity as well as the number of CD8(+) thymocytes in two independent transgenic mouse lines and increases the proportion of T cells that fully activate the transgenic locus in hCD2 variegating mice in a sequence-specific dependent manner. This finding suggests that overexpression of HBP1 can affect lineage commitment and can relieve the suppressive influence of heterochromatin, allowing thymocytes to express the variegating target locus more efficiently. These effects could be the result of direct HBP1 action on LCR activity. Alternatively, the extra HBP1 molecules may sequester repressive elements away from the LCR, thus allowing transcription permissive states to form on the transgene locus.

Modulation of heterochromatin protein 1 dynamics in primary Mammalian cells.

Heterochromatin protein 1 (HP1beta), a key component of condensed DNA, is strongly implicated in gene silencing and centromeric cohesion. Heterochromatin has been considered a static structure, stabilizing crucial aspects of nuclear organization and prohibiting access to transcription factors. We demonstrate here, by fluorescence recovery after photobleaching, that a green fluorescent protein-HP1beta fusion protein is highly mobile within both the euchromatin and heterochromatin of ex vivo resting murine T cells. Moreover, T cell activation greatly increased this mobility, indicating that such a process may facilitate (hetero)chromatin remodeling and permit access of epigenetic modifiers and transcription factors to the many genes that are consequently derepressed.