Date of Award

5-2014

Level of Access

Campus-Only Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Functional Genomics/Interdisciplinary

Advisor

Calvin Vary

Second Committee Member

Robert Friesel

Third Committee Member

Samuel Hess

Abstract

Endoglin is a transforming growth factor-ß (TGF-ß) superfamily co-receptor that is upregulated in endothelial cells during angiogenesis and when mutated in humans, results in the progressive vascular disease hereditary hemorrhagic telangiectasia (HHT). Mice null for endoglin expression are embryonic lethal due to defective angiogenesis. The molecular mechanisms underlying endoglin’s role in angiogenesis are still poorly understood. Bone morphogenetic protein 9 (BMP9) signaling has been implicated in HHT and vascular remodeling, acting via the HHT target genes, endoglin and activin receptor-like kinase (ALK1). Understanding how BMP9 regulates both the cellular localization and downstream signaling events involving endoglin will further our knowledge about HHT development, angiogenesis and endothelial cell function.

These studies have focused on examining BMP9 and endoglin expression and the molecular mechanisms they regulate to control endothelial cell function. To analyze the protein expression pattern in response to BMP9 and endoglin expression, mass spectrometry was utilized in combination with isotope coded affinity tag (ICAT) technology. These tools allowed me to identify novel proteins that were upregulated or downregulated in response to BMP9 stimulation and endoglin expression in endothelial cells. These methods implicated a discrete set of proteins regulated by BMP9 and endoglin. Mass spectrometry was also used to compare protein expression in wild type and endoglin heterozygous mouse (the model of HHT1) lung tissue samples, providing us with insight into the in vivo state of HHT1. The cross correlation of the in vitro and in vivo mass spectrometry analysis results identified distinct signaling pathways regulated by the BMP9 and endoglin expression.

Angiogenic signaling responses identified in these studies included the upregulation of the chemokine, stromal cell-derived factor 1 (CXCL12/SDF1), and downregulation of its receptor, C-X-C chemokine receptor type 4 (CXCR4) by BMP9 stimulation. Knockdown of endoglin and ALK1 impaired SDF1 and CXCR4 expression responses suggesting their role in propagating BMP9 signal transduction. The data implicate BMP9 in the regulation of the SDF1/CXCR4 chemokine axis in endothelial cells. A possible role was identified for BMP9 signaling via endoglin in a switch from an SDF1-responsive autocrine phenotype to an SDF1 nonresponsive paracrine state that represses endothelial cell migration and may promote vessel maturation. In vitro and in vivo studies are consistent in suggesting an important role for endoglin and BMP9 regulation of the SDF1/CXCR4 signaling axis in endothelial cells and possibly HHT pathogenesis.

Here I also suggest endoglin expression is regulating the maturation and remodeling phase of angiogenesis via its role in focal adhesion formation and extracellular matrix (ECM) regulation in response to BMP9. Endoglin deficiency alters the expression of focal adhesion proteins zyxin, integrin α5 and integrin β1 which may change their ability to transmit regulatory signals through binding the ECM. Interestingly, the BMP/TGF-P transcription factors, SMADs, and zyxin have recently been implicated in the newly emerging signaling cascade, the Hippo pathway. The Hippo pathway downstream transcription factors, YAP and TAZ, have recently been shown to regulate cell adhesion, ECM function and mechanotrasduction signaling (1-3). Expression of the target genes of the Hippo pathway, CCN1 (CYR61) and CCN2 (CTGF), are altered when endoglin is deficient in vitro and may be contributing to the defect in ECM remodeling and inflammatory response. Identification of a direct link between the Hippo and BMP signaling cascade via endoglin may elucidate their crosstalk in vascular remodeling during angiogenesis and the pathogenesis of HHT.

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