Rs = SD. doi:10.1371/journal.pone.0070570.gVegfr1 Regulates Coronary AngiogenesisFigure 8. Working model shows the Vegf-Notch signaling in the ventricular endocardium that regulates coronary angiogenesis by the ventricular endocardial cells. A, Schematics showing that a balanced Vegf signaling in the endocardium (Control) is essential for the coronary angiogenesis by the ventricular endocardial cells. When the Vegf signaling in the endocardium is disrupted by deleting the proangiogenic Vegfr2 (R2 CKO) [27] or the anti-angiogenic Vegfr1 (R1 CKO; this study), the coronary angiogenesis is blocked or accelerated, respectively. B, Diagrams depicting a mode of controlled Vegf-Notch signaling that is necessary for normal coronary angiogenesis. Enhanced Vegf2 signaling in the endocardial cells by removal of the inhibitory sVegfr1 results in the increased Dll4 expression and accelerated augmented coronary angiogenesis whereas blocking Notch signaling prohibits the process (not shown in the diagrams), MedChemExpress Gracillin suggesting that balanced Vegf and Notch signals collaborate in the endocardial cells to control the coronary angiogenesis. doi:10.1371/journal.pone.0070570.gVegfr1 MedChemExpress GNF-7 suppresses the Vegf-Notch signaling in the endocardial cells thereby limiting their angiogenic differentiation during the coronary angiogenesis.DiscussionWe have recently shown that the myocardial Vegfa to endocardial Vegfr2 signaling is essential for embryonic coronary angiogenesis by the progenitor cells within the endocardium to form the coronary arteries [33]. In this study using a conditional deletion strategy, we show that the endocardially-derived Vegfr1 is also required for normal coronary angiogenesis (Fig. 8A), it negatively regulates the process, possibly limiting the proangiogenic Vegf-Notch signaling. Vegfr1 is a negative regulator of Vegf signaling through its soluble form, sVegfr1, which has no intracellular and transmembrane domain [29,30,31,32,33,34]. It binds Vegfa, Vegfb, and Pigf. In mice, sVegfr1 acts as a decoy receptor of Vegfr2 and by binding to Vegfa, it suppresses the major proangiogenic signaling of Vegfa to Vegfr2. Global deletion of Vegfr1 in mice results in early embryonic death due to endothelial overgrowth and disruptive primitive vessel formation [35]. In quails, injection of sVegfr1 into the embryonic hearts inhibits the formation of coronary plexuses likely through binding to Vegfb [28,42].The cardiac endocardial cells and vascular endothelial cells have the same embryonic origin and share most of the cellular makers and functions [43,44]. Like in the vascular endothelium where loss of sVegfr1 causes peripheral vascular defects, including the overgrowth of endothelial cells and disorganized vascular pattern [32,33,35], our in vivo deletion study shows that loss of sVegfr1 in the cardiac endocardium results in excessive formation of abnormal coronary plexuses and overexpression of endothelial genes including Dll4. Further ex vivo coronary angiogenesis assay reveals increased angiogenesis as a major underlying mechanism of the defect. Thus, the current study establishes a tissue-specific role of Vegfr1 in the endocardium required for 23977191 coronary vessel formation. Vascular angiogenesis requires angiogenic sprouting from a selected subpopulation of endothelial cells [45,46,47]. Interaction of reciprocal Vegf and Notch signaling coordinates this selection [48]. Specifically, Dll4, a transmembrane ligand in the Notch pathway, expressed by angiogenic cells, activa.Rs = SD. doi:10.1371/journal.pone.0070570.gVegfr1 Regulates Coronary AngiogenesisFigure 8. Working model shows the Vegf-Notch signaling in the ventricular endocardium that regulates coronary angiogenesis by the ventricular endocardial cells. A, Schematics showing that a balanced Vegf signaling in the endocardium (Control) is essential for the coronary angiogenesis by the ventricular endocardial cells. When the Vegf signaling in the endocardium is disrupted by deleting the proangiogenic Vegfr2 (R2 CKO) [27] or the anti-angiogenic Vegfr1 (R1 CKO; this study), the coronary angiogenesis is blocked or accelerated, respectively. B, Diagrams depicting a mode of controlled Vegf-Notch signaling that is necessary for normal coronary angiogenesis. Enhanced Vegf2 signaling in the endocardial cells by removal of the inhibitory sVegfr1 results in the increased Dll4 expression and accelerated augmented coronary angiogenesis whereas blocking Notch signaling prohibits the process (not shown in the diagrams), suggesting that balanced Vegf and Notch signals collaborate in the endocardial cells to control the coronary angiogenesis. doi:10.1371/journal.pone.0070570.gVegfr1 suppresses the Vegf-Notch signaling in the endocardial cells thereby limiting their angiogenic differentiation during the coronary angiogenesis.DiscussionWe have recently shown that the myocardial Vegfa to endocardial Vegfr2 signaling is essential for embryonic coronary angiogenesis by the progenitor cells within the endocardium to form the coronary arteries [33]. In this study using a conditional deletion strategy, we show that the endocardially-derived Vegfr1 is also required for normal coronary angiogenesis (Fig. 8A), it negatively regulates the process, possibly limiting the proangiogenic Vegf-Notch signaling. Vegfr1 is a negative regulator of Vegf signaling through its soluble form, sVegfr1, which has no intracellular and transmembrane domain [29,30,31,32,33,34]. It binds Vegfa, Vegfb, and Pigf. In mice, sVegfr1 acts as a decoy receptor of Vegfr2 and by binding to Vegfa, it suppresses the major proangiogenic signaling of Vegfa to Vegfr2. Global deletion of Vegfr1 in mice results in early embryonic death due to endothelial overgrowth and disruptive primitive vessel formation [35]. In quails, injection of sVegfr1 into the embryonic hearts inhibits the formation of coronary plexuses likely through binding to Vegfb [28,42].The cardiac endocardial cells and vascular endothelial cells have the same embryonic origin and share most of the cellular makers and functions [43,44]. Like in the vascular endothelium where loss of sVegfr1 causes peripheral vascular defects, including the overgrowth of endothelial cells and disorganized vascular pattern [32,33,35], our in vivo deletion study shows that loss of sVegfr1 in the cardiac endocardium results in excessive formation of abnormal coronary plexuses and overexpression of endothelial genes including Dll4. Further ex vivo coronary angiogenesis assay reveals increased angiogenesis as a major underlying mechanism of the defect. Thus, the current study establishes a tissue-specific role of Vegfr1 in the endocardium required for 23977191 coronary vessel formation. Vascular angiogenesis requires angiogenic sprouting from a selected subpopulation of endothelial cells [45,46,47]. Interaction of reciprocal Vegf and Notch signaling coordinates this selection [48]. Specifically, Dll4, a transmembrane ligand in the Notch pathway, expressed by angiogenic cells, activa.