Microrna-214/199a Cluster Is Upregulated in Carotid Artery Stenosis Patients and Suppresses St2-Mediated Endothelial Cells Angiogenesis

Background: Carotid artery stenosis (CAS) is an important risk factor for ischemic neurological events, being responsible for ≈20% ischemic strokes. Stroke is a leading cause of death and an important source of human disability. Growing evidences suggests a potential regulatory function for several microRNAs (miRNAs) on dysfunctional endothelium, contributing to the evolution of carotid plaque toward growth, instability, and rupture. Previous studies evoke a targeted approach to miRNA analysis on tissue from the carotid plaque itself. Nevertheless, the potential role of miRNAs in CAS development is mostly unknown and a discovery-based approach to a circulating miRNA profiling in peripheral blood, as a biomarker of stenosis progression, has not yet been extensively studied. Rationale: miR-214 is expressed in all major vascular cell types, and modulation of miR-214 levels in endothelial cells (ECs) significantly influences angiogenesis. Our recently preliminary data showed that an increase in miR-214 and a decrease in miR-126 levels were observed in blood samplings from 42 CAS patients undergoing carotid stenting as compared to those of healthy controls. In order to further confirm the important role of microRNAs on regulating the endothelial dysfunction and arterial stenosis, we performed a mouse model of carotid artery injury by wire denudation. Histopathology showed that neointimal formation and intimal hyperplasia occurred in the injured arteries at 7 days after the procedure. In vitro study showed that the miR-214 expression was remarkably upregulated both in H2O2-treated ECs and culture medium. Besides, bioinformatics analysis revealed that ST2 (IL-33 receptor) is a potential target of miR-214. Accordingly, it is rationale to believe that miR-214/199a cluster expression profiles could provide a new diagnostic tool that complements the monitoring of CAS progression, improving therapeutic approaches to prevent ischemic stroke. The aims of the proposed 3-year study are: (1) to assess the PRINCIPAL role of miR- 214/199a cluster and its potential targets ST2 in wire injury-related neointimal hyperplasia formation, and cellular and molecular regulation of inflammatory response on the denuded luminal surface; (2) in vitro studies employing cultured ECs, to investigate whether miR-214/199a-ST2 axis would involve in EC function, angiogenesis and vascular leakage mainly through ST2/TRAF6-AkteNOS signaling pathway (i.e., by delivering with agomir-214/199a and antagomir-214/199a); (3) as well as to investigate a strong correlation between circulating levels of miR-214/199a and sST2 level, and severity of CAS. Significance: In this proposal, we will investigate the molecular and cellular mechanisms involved in miR- 214/199a-ST2 signaling in the setting of carotid denudation injury. miR-214/199a-ST2 signaling in the ECs could function as a new cytokine-medicated angiogenesis pathway in response to vascular injury. We attempt to test the hypothesis that guide wire to cause intraluminal injury on mouse carotid artery and hence to elicit neointimal hyperplasia via the upregulation of the expressions of miR-214/199a cluster in vessel wall, which in turn may impair IL-33 mediated ST2/PI3K/Akt/eNOS driven angiogenesis. If the result of our study is positive, it will provide extremely important pre-clinical relevant information for our future clinical practice regarding the treatment of patients with CAS and asymptomatic CAS.

Project ID:PC10608-2353
External Project ID:MOST106-2314-B182-050