Friday, May 16, 2025
5/16/2025
Regulation of angiogenesis by cardiac fibroblasts - Project Summary Activated cardiac fibroblasts (cFBs) are the primary source of extracellular matrix (ECM) following myocardial infarction (MI). However, studies suggest that cFBs also regulate neovascularization in the ischemic heart by expressing pro- and anti-angiogenic secreted ligands. We recently identified that fetal epicardial-derived fibroblasts express angiogenic factors that facilitate coronary angiogenesis during embryogenesis, which are also expressed in adult cFBs following myocardial infarction (MI). Specifically, we determined that collagen type XVIII (Col18a1) and slit guidance ligand 2 (Slit2) were enriched in cFBs compared to cardiomyocytes. Col18a1 sources the anti-angiogenic peptide endostatin, which was downregulated in the border zone of the damaged heart 7 days post-MI but upregulated in the mature scar at 28 days post-MI. Slit2, a secreted glycoprotein that binds to roundabout receptors expressed in endothelial cells and pericytes, has been shown to promote angiogenesis and vessel stabilization and limit fibrosis and inflammation. In the ischemic heart, Slit2 expression was activated in the border zone and infarct areas 7 days post-MI but quickly downregulated in the reparative and maturation phases of MI. These data suggest that cFBs coordinate the secretion of angiogenic cues in a temporal and spatial-specific manner to support cardiac repair; however, the investigation of anti- and pro- angiogenic factors in fibroblasts has yet to be elucidated. Our central hypothesis is that cardiac fibroblasts regulate neovascularization by expressing endogenous angiogenic-modifying factors following ischemia. Our overall objective for this study is to identify mechanisms that increase vascular perfusion and promote positive clinical outcomes for patients suffering from ischemic heart disease. The following two specific aims will address our hypothesis: Specific Aim 1. To evaluate the function of anti-angiogenic factors in cardiac fibroblasts following myocardial infarction. We will assess cFB activation and angiogenic functions in vitro with or without overexpression of Col18a1 using adenoviruses. For in vivo studies, we will analyze a Col18a1 knockout mouse following a time course of MI and measure cardiac function, fibrosis, angiogenesis, and coronary and collateral vasculature formation. Specific Aim 2: To determine if the expression of pro-angiogenic factors alters angiogenesis and repair following myocardial infarction. The fibroblast-specific Tcf21MCM mouse will be crossed to Slit2flox/flox mice. We will perform MI, measure cardiac function, and quantify angiogenesis. We will perform single-cell RNA sequencing on cFBs lacking Slit2 to identify fibroblast-directed programs to enhance neoangiogenesis. Additionally, we will utilize an AAV9-cTNT-mSlit2-eGFP to express Slit2 in the heart and evaluate cardiac functional recovery and vascular formation after MI. Overall, accomplishing these studies will provide an understanding of the cells and molecular signals that enhance vascular perfusion following MI and guide the development of novel vascular therapeutics.
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