Endothelial Cell-Derived Extracellular Vesicles Impair the Angiogenic Response of Coronary Artery Endothelial Cells

Cardiovascular disease (CVD) is the most prominent cause of death of adults in the United States with coronary artery disease being the most common type of CVD. Following a myocardial event, the coronary endothelium plays an important role in the recovery of the ischemic myocardium. Specifically, endothelial cells (EC) must be able to elicit a robust angiogenic response necessary for tissue revascularization and repair. However, local or distant cues may prevent effective revascularization. Extracellular vesicles (EV) are produced by all cells and endothelium is a rich source of EVs that have access to the main circulation thereby potentially impacting local and distant tissue function. Systemic inflammation associated with conditions such as obesity as well as the acute inflammatory response elicited by a cardiac event can significantly increase the EV release by endothelium and alter their miRNA, protein or lipid cargo. Our laboratory has previously shown that EVs released by adipose tissue endothelial cells exposed to chronic inflammation have angiostatic effects on naïve adipose tissue EC in vitro. Whether the observed effect is specific to EVs from adipose tissue endothelium or is a more general feature of the endothelial EVs exposed to pro-inflammatory cues is currently unclear. The objective of this study was to investigate the angiostatic effects of EVs produced by EC from the coronary artery and adipose microvasculature exposed to pro-inflammatory cytokines (PIC) on naïve coronary artery EC. We have found that EVs from both EC sources have angiostatic effects on the coronary endothelium. EVs produced by cells in a pro-inflammatory environment reduced proliferation and barrier function of EC without impacting cellular senescence. Some of these functional effects could be attributed to the miRNA cargo of EVs. Several miRNAs such as miR-451, let-7, or miR-23a impact on multiple pathways responsible for proliferation, cellular permeability and angiogenesis. Collectively, our data suggests that EVs may compete with pro-angiogenic cues in the ischemic myocardium therefore slowing down the repair response. Acute treatments with inhibitors that prevent endogenous EV release immediately after an ischemic event may contribute to better efficacy of therapeutic approaches using functionalized exogenous EVs or other pro-angiogenic approaches

STAT4 deficiency reduces the development of atherosclerosis in mice

Atherosclerosis is a chronic inflammatory process that leads to plaque formation in large and medium sized vessels. T helper 1 (Th1) cells constitute the majority of plaque infiltrating pro-atherogenic T cells and are induced via IFNγ-dependent activation of T-box (Tbet) and/or IL-12-dependent activation of signal transducer and activator of transcription 4 (STAT4). We thus aimed to define a role for STAT4 in atherosclerosis. STAT4-deficiency resulted in a ∼71% reduction (p < 0.001) in plaque burden in Stat4(-/-)Apoe(-/-) vs Apoe(-/-) mice fed chow diet and significantly attenuated atherosclerosis (∼31%, p < 0.01) in western diet fed Stat4(-/-)Apoe(-/-) mice. Surprisingly, reduced atherogenesis in Stat4(-/-)Apoe(-/-) mice was not due to attenuated IFNγ production in vivo by Th1 cells, suggesting an at least partially IFNγ-independent pro-atherogenic role of STAT4. STAT4 is expressed in T cells, but also detected in macrophages (MΦs). Stat4(-/-)Apoe(-/-)in vitro differentiated M1 or M2 MΦs had reduced cytokine production compare to Apoe(-/-) M1 and M2 MΦs that was accompanied by reduced induction of CD69, I-A(b), and CD86 in response to LPS stimulation. Stat4(-/-)Apoe(-/-) MΦs expressed attenuated levels of CCR2 and demonstrated reduced migration toward CCL2 in a transwell assay. Importantly, the percentage of aortic CD11b(+)F4/80(+)Ly6C(hi) MΦs was reduced in Stat4(-/-)Apoe(-/-) vs Apoe(-/-) mice. Thus, this study identifies for the first time a pro-atherogenic role of STAT4 that is at least partially independent of Th1 cell-derived IFNγ, and primarily involving the modulation of MΦ responses

STAT4 deficiency reduces obesity-induced insulin resistance and adipose tissue inflammation

Signal transducer and activator of transcription (STAT) 4 is one of the seven members of the STAT family. STAT4 has a prominent role in mediating interleukin-12-induced T-helper cell type 1 lineage differentiation. T cells are key players in the maintenance of adipose tissue (AT) inflammation. The role of STAT4 in obesity and AT inflammation is unknown. We sought to determine the role of STAT4 in AT inflammation in obesity-induced insulin resistance. We studied STAT4-null mice on the C57Bl6/J background. We have found that STAT4(-/-)C57Bl6/J mice develop high-fat diet-induced obesity (DIO) similar to wild-type controls, but that they have significantly improved insulin sensitivity and better glucose tolerance. Using flow cytometry and real-time PCR, we show that STAT4(-/-) mice with DIO produce significantly reduced numbers of inflammatory cytokines and chemokines in adipocytes, have reduced numbers of CD8(+) cells, and display increased alternative (M2) macrophage polarization. CD8(+) cells, but not CD4(+) cells, from STAT4(-/-) mice displayed reduced in vitro migration. Also, we found that adipocyte inflammation is reduced and insulin signaling is improved in STAT4(-/-) mice with DIO. We have identified STAT4 as a key contributor to insulin resistance and AT inflammation in DIO. Targeting STAT4 activation could be a novel approach to reducing AT inflammation and insulin resistance in obesity