Date of Award

5-2007

Level of Access

Campus-Only Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biochemistry and Molecular Biology

Advisor

Calvin P.H. Vary

Second Committee Member

Douglas B. Spicer

Third Committee Member

Robert E. Friesel

Abstract

Genetic studies show that TGFß superfamily members are essential for vascular development, although the mechanism through which this pathway operates is poorly understood. Endoglin (eng, CD105), the target gene in the human vascular disease hereditary hemorrhagic telangiectasia type 1 (HHT1), is a TGFß co-receptor required for angiogenesis. It is predominantly expressed in endothelial cells, however its expression has been detected in other cell types, including multiple stem cell populations, neural crest cells and vascular smooth muscle cells (vSMCs). Endoglin null embryos die by E11.5 due to impaired angiogenesis characterized by a failure of vSMC recruitment. Based on the phenotype of the endoglin null mouse it is unclear whether impaired vSMC recruitment is secondary to a lack of endoglin expression in the endothelial cells or if endoglin expression is also required in developing vSMCs. We demonstrate that endoglin is required for myogenic differentiation of neural crest stem cells (NCSCs). Overexpression of endoglin in the neural crest in vivo caused pericardial hemorrhaging, correlating with altered vascular smooth muscle cell investment in the walls of major vessels and upregulation of total smooth muscle a- actin (a-SMA) protein levels. To further investigate the role of endoglin in cell autonomous signaling, we generated a conditional transgene and subsequently overexpressed endoglin in a cell type specific manner utilizing smooth muscle (SM22acre) and endothelial (Tie2cre) drivers. In both cases, we observed hemorrhaging in the dorsal aorta. Furthermore, using a genetic complementation assay we replaced endoglin expression using the conditional transgene in null embryos in a cell type specific manner using the SM22acre and Tie2cre drivers. Our results demonstrated that vascular smooth muscle cell recruitment to the dorsal aorta can be partially rescued using the Tie2cre driver, and to a lesser extent, using the SM22acre driver. These results indicated that endoglin expression in endothelial cells alone is not sufficient to rescue the null phenotype, and suggest that endoglin expression is required in both cell types for normal angiogenesis. Furthermore, we demonstrate a role for endoglin in arteriovenous specification through regulation of the orphan nuclear receptor COUPTFII, which was recently identified as a transcription factor important for venous identity. Endoglin null embryos exhibited and increase in ectopic COUPTFII expression in arteries. Aberrant expression of COUPTFII could be rescued by conditionally replacing endoglin expression in endothelial cells in the endoglin null background. Finally we demonstrate a molecular mechanism through which endoglin signaling may occur. Endoglin phosphorylation by ALK1 was specifically enhanced in the presence of BMPRII, but not TßRII. Furthermore, endoglin was phosphorylated in response to ligand stimulation of particular type I and type II receptor pairs. BMP9 specifically induced endoglin phosphorylation through the ALK1/BMPRII complex, while TGFß1 preferentially utilized the ALK5/TßRII complex. These data suggest that BMP9 is a physiologic ligand for the ALK1/BMPRII complex. These studies provide a more thorough understanding of the function of endoglin in angiogenesis, which will potentially lead to (i) a broader understanding of vascular development and (ii) a more comprehensive strategy for treatment of HHT and other vascular diseases.

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