Mécanismes impliqués dans la modulation de la néovascularisation post-ischémique : rôle de la rénine et des microARNs

Ischemic cardiovascular diseases remain the leading cause of morbidity and mortality in industrialized countries. The severity of ischemic damages is greatly influenced by the ability of the organism to form new blood vessels (neovascularization). Our laboratory has demonstrated in recent years that several risk factors and clinical phenotypes associated with the development of atherosclerosis can also impair post-ischemic neovascularization. The purpose of my research was to better understand the mechanisms that are involved in the modulation of post-ischemic neovascularization in pathological conditions. We first investigated the specific role of renin, a key component of the renin-angiotensin system (RAS) whose chronic stimulation is involved in atherosclerosis, endothelial cell (EC) dysfunction and the inhibition of neovascularization. Subsequently, we investigated the potential role of microRNAs (miRs) in the modulation of post-ischemic neovascularization under atherosclerotic conditions. miRs are small non-coding RNAs that negatively regulate the expression of more than 60% of genes. They are increasingly recognized as having a vital role in physiological and pathological biological processes. In this thesis, we used a mouse model of hindlimb ischemia to test the hypothesis that renin and miRs are involved in the modulation post-ischemic neovascularization during pathological conditions.     The results of my first article demonstrate for the first time that treatment with aliskiren, the only direct renin inhibitor used clinically, significantly improves post-ischemic neovascularization in vivo. Indeed, we observed an improvement of tissue perfusion recovery, capillary and arteriolar densities, and higherexpression of key angiogenic factors (VEGF, NO) in ischemic muscles of mice treated with aliskiren. In summary, our results suggest that the beneficial effect of aliskiren on neovascularization is at least in part due to an antioxidant action that enhances the functional activity of ECs and circulating angiogenic cells (PACs). My second project aimed at identifying and characterizing the role of different miRs which could be involved in post-ischemic neovascularization failure associated with atherosclerotic conditions. Using next-generation sequencing technology and qRT-PCR, we identified 2 miRs that are significantly decreased in hypercholesterolemic conditions: miR-150 (a pro-angiogenic miR) and miR-146b (an anti-inflammatory miR). We have shown that these 2 miRs are decreased in vivo in the ischemic muscles of hypercholesterolemic (HC) mice, and also in vitro in human ECs exposed to oxLDL. Decreased expression of miR-150 and miR-146b by hypercholesterolemia and oxLDL appears to inhibit post-ischemic neovascularization by two distinct mechanisms (Article 2 and 3). On one hand, decreased expression of miR-150 impairs the angiogenic functions of ECs and PACs by stimulating the expression of SRCIN1, an important endogenous inhibitor of the pro-angiogenic Src / Akt / eNOS pathway (article 2). On the other hand, decreased expression of miR146b causes endothelial dysfunction by increasing oxidative stress and inflammation, a mechanism that involves the induction of the proinflammatory TRAF6 gene (Article 3). Importantly, our results demonstrate for the first time that a local intramuscular injection of miR-150 or miR-146b can recover post-ischemic neovascularization in hypercholesterolemic conditions. We also show that forced expression of miR-150 or miR-146b can enhance the angiogenic functions of human ECs exposed to oxLDL. Finally, the number and functional activity of bone marrow PACs isolated from HC mice can also be restored by miR-150 and miR-146b. In conclusion, our work has characterized the role of miR-150 and miR-146b for the angiogenic failure associated with atherosclerotic conditions. This thesis also demonstrates for the first time that therapies based on the inhibition of renin or the modulation of specific miRs can improve post-ischemic neovascularization in pathological situations. Our results could help developing new strategies for the treatment of severe ischemic cardiovascular diseases.

Keyphrases: micro-ARN (miR), Inflammation, Neovascularization, Angiogenesis, Endothelial cell (EC), Proangiogenic cell (PAC), Atherosclerosis, Hypercholesterolemia, micro-RNA (miR), Renin, Inflammation, Oxydative stress, Ischemia