Publications

The activity of heme oxygenase enzymes (HOs) is responsible for the endogenous source of carbon monoxide (CO). Their activities can be inhibited by tin protoporphyrin-IX (SnPPIX). Recent data indicate the involvement of HOs in the regulation of angiogenesis. Here, we investigated the role of the HO pathway in the production and angiogenic activity of vascular endothelial growth factor (VEGF) in endothelial cells treated with SnPPIX, or cultured in the presence of a CO-releasing molecule (CO-RM). Addition of CO-RM or induction of HO-1 by hemin resulted in a threefold elevation in CO production in culture medium (up to 20.3 microg/L) and was associated with a 30% increase in VEGF synthesis. Much higher levels of CO (up to 60 microg/L) and a further increase in VEGF production (by 277%) were measured in cells treated with prostaglandin-J(2), a potent activator of HO-1. SnPPIX prevented the induction of CO generation and inhibited VEGF synthesis. Moreover, SnPPIX reduced the VEGF-elicited angiogenic activities of endothelial cells by decreasing their proliferation (by 26%), migration (by 46%), formation of tubes on Matrigel (by 48%), and outgrowth of capillaries from endothelial spheroids (by 30%). In contrast, overexpression of HO-1 or incubation of cells with CO-RM led to an increase in capillary sprouting. Thus, HO activity up-regulates VEGF production and augments the capability of endothelial cells to respond to exogenous stimulation.

too low to efficiently compensate for the loss of functional myocardium in heart disease.We hypothesize that cardiomyocyte proliferation is tightly controlled by regulatory mechanisms that can be reactivated to promote endogenous repair after heart injuries.Unequivocal identification of cycling cardiomyocytes is a fundamental requirement for the investigation of these mechanisms.Methods: We used a preexisting transgenic mouse model based on the FUCCI (Fluorescent Ubiquitination-based Cell Cycle Indicator) system to discriminate cycling cardiomyocytes (S/G2/M phases, green fluorescent nuclei) from non-cycling ones (G0/G1 phases, red fluorescent nuclei).In mice ubiquitously expressing FUCCI constructs, we used the myocyte-specific perinuclear marker Pericentriolar Material 1 (PCM-1) to distinguish cardiomyocyte from non-myocyte cells in the heart tissue.Single cardiomyocyte isolations from FUCCI mice neonatal hearts followed by FACS sorting into 96-well plates were performed to obtain single cell transcriptome of cycling and non-cycling cardiomyocytes at different post-natal stages.Results: The presented transgenic strategy proved to be efficient for the identification of cycling cardiomyocytes in neonate, juvenile and adult mouse hearts in vitro and in vivo.The validity of transgenes expression was verified in both systems with immunohistochemistry by co-localization of cardiomyocytes and cell cycle markers with FUCCI fluorescence.The FUCCI system allowed us to confirm previous observations of a gradual decrease in the number of cycling cardiomyocyte during the neonatal period.P0 and P7 cardiomyocyte transcriptome were generated using single cell RNA-sequencing.The cell cycle stage of cardiomyocytes as indicated by their FUCCI fluorescence was highly correlated by their transcriptome profile.Conclusion: Strategies that allow to unambiguously identify cycling cardiomyocytes in vitro and in vivo provide important tools to study the regulation of cardiomyocyte proliferation.With the FUCCI system we seek to identify novel molecular targets that could promote cardiomyocyte proliferation, by using transcriptome profiling (RNA-seq) of isolated cycling cardiomyocytes.

<p>PDF - 55KB, Shortening of the minCMV promoter.</p>

Abstract Aims MicroRNA-378a, highly expressed in skeletal muscles, was demonstrated to affect myoblasts differentiation and to promote tumour angiogenesis. We hypothesized that miR-378a could play a pro-angiogenic role in skeletal muscle and may be involved in regeneration after ischaemic injury in mice. Methods and results Silencing of miR-378a in murine C2C12 myoblasts did not affect differentiation but impaired their secretory angiogenic potential towards endothelial cells. miR-378a knockout (miR-378a−/−) in mice resulted in a decreased number of CD31-positive blood vessels and arterioles in gastrocnemius muscle. In addition, diminished endothelial sprouting from miR-378a−/− aortic rings was shown. Interestingly, although fibroblast growth factor 1 (Fgf1) expression was decreased in miR-378a−/− muscles, this growth factor did not mediate the angiogenic effects exerted by miR-378a. In vivo, miR-378a knockout did not affect the revascularization of the ischaemic muscles in both normo- and hyperglycaemic mice subjected to femoral artery ligation (FAL). No difference in regenerating muscle fibres was detected between miR-378a−/− and miR-378+/+ mice. miR-378a expression temporarily declined in ischaemic skeletal muscles of miR-378+/+ mice already on Day 3 after FAL. At the same time, in the plasma, the level of miR-378a-3p was enhanced. Similar elevation of miR-378a-3p was reported in the plasma of patients with intermittent claudication in comparison to healthy donors. Local adeno-associated viral vectors-based miR-378a overexpression was enough to improve the revascularization of the ischaemic limb of wild-type mice on Day 7 after FAL, what was not reported after systemic delivery of vectors. In addition, the number of infiltrating CD45+ cells and macrophages (CD45+ CD11b+ F4/80+ Ly6G−) was higher in the ischaemic muscles of miR-378a−/− mice, suggesting an anti-inflammatory action of miR-378a. Conclusions Data indicate miR-378a role in the pro-angiogenic effect of myoblasts and vascularization of skeletal muscle. After the ischaemic insult, the anti-angiogenic effect of miR-378a deficiency might be compensated by enhanced inflammation.

HO-1 is necessary for a proper proangiogenic function of BMDCs. A low level of HO-1 in hyperglycemic mice decreases restoration of perfusion in ischemic muscle, which can be rescued by a local injection of conditioned media from cultured BMDCs.