Abstract 3440: A Mouse Model of Germinal Matrix Hemorrhage based on Interactions between Vascular Endothelial Growth Factor (VEGF) and Pericellular Proteases

Abstract

A Mouse Model of Germinal Matrix Hemorrhage based on Interactions between Vascular Endothelial Growth Factor (VEGF) and Pericellular Proteases Dianer Yang, Jessica Baumann, Hung-Chi Liang, Chia-Yi Kuan Division of Developmental Biology and Neurology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA. Introduction: Germinal Matrix-Intraventricular Hemorrhaging (GMH-IVH) is a major threat to premature infants with a high incidence of neonatal mortality and life-long neurological deficits. The etiology of GMH-IVH is complex and involves both vascular and extravascular factors . However, there are few animal models of GMH-IVH and none addresses the interaction between vasculature and endogenous proteases in pathogenesis. Hypothesis: We hypothesize that over-expression of the vascular endothelial growth factor (VEGF) in the germinal matrix is able to induce pericellular proteases (tissue-type plasminogen activator, matrix metalloproteinse, or cathepsins) leading to hemorrhage in mouse embryos. Methods: We used a tetracycline-controlled transcriptional activator (tTA) system (bi-transgenic GFAP-tTA: TetO-VEGF165 embryos/mice) to over-express VEGF in the cortical ventricular zone from mid-gestation, when no doxycycline was administered. In the first set of investigation, we examined the cortical vasculature in E15-18 bi-transgenic embryos and sibling controls. The focus is to evaluate the density of blood vessels and the distribution of CD31+ endothelial cells and NG2+ pericytes. Microarray and in-situ hybridization were used to explore the downstream mediators of VEGF. Biochemical assays were used to compare the activity of various proteases. In the second set of study, doxycycline was administered to bi-transgenic embryos from E8 to E18 to adjust the timing of VEGF-induced angiogenesis. Results: The majority of bi-transgenic GFAP-tTA: TetO-VEGF165 embryos die before birth. In situ hybridization confirmed increased expression of VEGF in the cortical ventricular zone in bi-transgenic embryos, which exhibited overgrowth of blood vessels, CD31+ endothelial cells, and NG2+ pericytes at E16. The abnormal vessels showed glomeruloid microvascular proliferation-like morphology. Biochemical analysis revealed significantly increased Cathepsin B activity in the cortex of E16 embryos. Microarray and in-situ hybridization confirmed elevated expression of multiple Cathepsins. By E18, the bi-transgenic embryos exhibited spontaneous hemorrhage into the dilated ventricular space. Conclusions: High expression of VEGF in the ventricular zone of developing cerebral cortex is sufficient to induce vascular overgrowth and pericellular protease activity, leading to GMH-IVH-like outcomes. These results suggest that anti-VEGF or anti-Cathepsin treatment may interrupt this pathological process.