Project

ApoE knockout mouse; S100A4; Atherosclerosis; Macrophages; Smooth muscle cells; CrispR/Cas9

Lead
L’athérosclérose est une pathologie qui provoque la sténose et l’occlusion des artères; elle est responsable de la plupart des maladies cardiovasculaires. La plaque d’athérome se développe sur plusieurs décennies à cause de l’accumulation dans la partie interne de la paroi vasculaire, l’intima, de cholestérol, de cellules inflammatoires et de cellules musculaires lisses (CMLs). Nous étudions le rôle de ces CMLs dans le développement de la plaque d’athérome.
Lay summary
Contenu et objectifs du travail de recherche Lors de la formation de la plaque d’athérome, les CMLs, présentent dans un phénotype différencié dans la media, s’accumulent dans l’intima et acquièrent un phénotype synthétique. Nous avons isolé 2 populations différentes de CMLs de la coronaire du porc in vitro, typiques de ces deux phénotypes. Récemment nous avons démontré que la S100A4 recombinante sous forme multimérique, avec le PDGF-BB, agissent en synergie pour induire le phénotype synthétique et pro-inflammatoire des CMLs. De plus, la neutralisation de la S100A4 dans les souris ApoE-/- induit une stabilisation des plaques d’athérome. In vivo, nous étudierons l’influence de la neutralisation de la S100A4 sur l’évolution de la plaque d’athérome à différent stade de sa progression, en utilisant un dispositif posé autour de la carotide de la souris ApoE-/-, qui induit des plaques stables et instables. Pour investiguer le rôle de la S100A4 spécifiquement produite par les CMLs, nous développons un modèle de souris transgénique ApoE-/-, où les CMLs sont tracées avec un fluorochrome et n’expriment plus la S100A4. Dans ce but nous avons créé une souris S100A4 floxée avec la technique CrispR/Cas9. Le séquençage de l’ARN des cellules intimales dans ces différents modèles est envisagé. En utilisant notre modèle in vitro de CMLs de porc, nous allons continuer à déchiffrer les mécanismes impliqués dans la transition phénotypique des CMLs. Nous aimerions aussi clarifier le mode de libération de la S100A4 dans le milieu extracellulaire, inexpliqué à ce jour. Contexte scientifique et social du projet de recherche L’étude de la S100A4 devrait permettre d’élucider certains mécanismes d’accumulation des CMLs dans l’intima. Notre but ultime est le développement d’outils influençant l’évolution des lésions d’athérosclérose.

Direct link to Lay Summary

Last update: 23.05.2019

Smooth muscle cells (SMCs) accumulate into the intima during the process of atherosclerosis, where they switch from a contractile to a synthetic phenotype. They are classically considered as beneficial players by contributing to the fibrous cap formation and plaque stability. Recent breakthroughs have demonstrated that a large proportion of SMCs in atherosclerotic plaques are hardly detectable with the classical SMC markers and acquire pro-inflammatory macrophage-like phenotype. Therefore it is necessary to identify factors and mechanisms responsible for the detrimental SMC phenotypic changes, with the aim of developing appropriate tools to influence SMC phenotypic profile and promote plaque stabilization and/or regression.In our model, we have isolated two distinct SMC populations from the porcine coronary artery: spindle-shaped (S), typical of the contractile phenotype and rhomboid (R), representing the synthetic phenotype. We have identified S100A4 as a marker of R-SMCs in vitro and of intimal SMCs in pigs, mice and humans. Recently we have shown that extracellular S100A4, in synergy with platelet-derived growth factor-BB (PDGF-BB), induces transition toward a R-phenotype associated with pro-inflammatory properties and nuclear factor-kB (NF-kB) activation through Toll-like receptor-4 (TLR4). Noteworthy, extracellular S100A4 neutralization promotes atherosclerotic plaque stabilization in ApoE-/- mice. In the frame of this grant, we will continue our studies on the role of S100A4 in SMC phenotypic transition and atherosclerotic plaque evolution.Aim 1. We have demonstrated that neutralization of extracellular S100A4 influences the evolution of atherosclerotic plaques in ApoE-/- mice under high fat diet towards a more stable plaque phenotype. Therefore, extracellular S100A4 is causally related to atherosclerotic plaque progression. We plan to further investigate how extracellular S100A4 neutralization affects atherosclerotic plaques at different stages of atherosclerosis progression and influence the level of SMC differentiation. For this purpose, in vivo alteration of shear-stress will be induced in ApoE-/- mouse carotid artery by using a cast. This model will allow the study of stable and unstable plaques. Additionally, balloon catheter injury will be performed in ApoE-/- mouse carotid artery. In both models, we will perform systemic neutralization of extracellular S100A4.Aim 2. To investigate whether SMC-specific S100A4 expression is responsible for the SMC phenotypic transition, an experimental model where S100A4 expression is exclusively abrogated in SMCs is required. For this purpose, we will develop a SMC lineage tracing mouse model associated with SMC-specific deletion of S100A4. We have already generated the S100A4-floxed F0 mice taking advantage of the CrispR/Cas9 technology. In this new model (Myh11-CreERT2:R26-Stop-eYFP:ApoE-/-:S100A4fl/fl), different strategies will be used to induce lesion formation: high fat diet, carotid balloon-injury, and carotid cast implantation. The full S100A4 knockout mice, which will undergo carotid ligation, will be also studied. RNA sequencing of intimal SMCs will be instrumental in characterizing unidentified or misidentified SMCs in atherosclerotic plaques of our mouse models.Aim 3. By using our porcine SMC in vitro model, we plan to thoroughly decipher the mechanisms involved in S100A4-induced SMC phenotypic transition. The co-dependence between TLR4 and PDGF-receptors as well as NF-kB and Krüppel-like factor-4 will be studied with specific inhibitors or by siRNA approach. To address how S100A4 influence PDGF-BB-induced S- to R-SMC phenotypic change, we will deplete S100A4 using the CrispR/Cas9 technology. Based on data recently acquired by RNA sequencing, we will explore the role of other inflammatory factors with a particular attention for granulocyte macrophage-colony stimulating factor, which should provide new perspectives on the possible activation of monocytes by SMCs. The relevance of our findings will be evaluated in human tissue samples by immunohistochemistry. Finally, we plan to clarify the still puzzling process of S100A4 release from SMCs.Conclusion. Our in vivo mouse models should bring new insights into the role of SMC-derived S100A4 in atherosclerotic plaque progression and into the characterization of unidentified or misidentified intimal SMCs. Our in vitro porcine model should be instrumental in understanding S100A4 expression, release and regulation in SMCs and the mechanisms that allow SMC accumulation in the intima. The ultimate aim of our work is the development of tools to influence the evolution of atherosclerotic lesions and hence reduce adverse outcome of atherosclerosis on clinical events.