Specific Aims Transforming growth factor (TGF-?) superfamily signaling in endothelial cells regulates essential components of angiogenesis and vascular morphogenesis, including proliferation and capillary tube formation. TGF-? superfamily ligands exert their regulatory effects through the endothelial cell specific TGF-? receptor complex, ALK1 (type I receptor) and endoglin (co-receptor), along with the ubiquitous type I TGF-? receptor, ALK5, to activate the canonical Smad 1/5/8 and Smad 2/3 pathways, respectively. TGF-? ligands also signal through non-Smad pathways such as MAPKs and PI3K/Akt, although the underlying mechanisms remain obscure. A critical role for endoglin and ALK1 in TGF-? signaling in endothelial cells is supported by their mutation resulting in the human vascular disease, hereditary hemorrhagic telangiectasia (HHT1 and 2), embryonic lethal phenotype due to defects in angiogenesis when either endoglin or ALK1 is targeted for deletion in mice, and by the elevated expression of endoglin during inflammation and tumor-induced angiogenesis. While important biological roles for endoglin have been established, the molecular basis for endoglin function in vascular biology remains poorly characterized. Here in vitro angiogenesis assays were employed to investigate precisely when and how endoglin regulates endothelial capillary sprouting and tube formation. Comparison of endoglin-null and wild type endothelial cells revealed that endoglin differentially regulates the stability of capillary sprouts and tubes in response to its physiologically relevant high-affinity ligands, TGF-? and BMP-9. Specifically, TGF-? resulted in regression of the capillary sprouts and tube structures, primarily through suppression of endoglin-dependent Akt signaling. Conversely, endoglin enhanced Akt signaling in response to BMP-9 to promote capillary stability. These outcomes are attributed to the association between endoglin and the scaffolding/trafficking protein, GIPC, since disrupting their interaction abrogated such endoglin-dependent effects. Given that I recently reported the enhancement of Smad 1/5/8 signaling through GIPC and endoglin, there exists a potential crosstalk between Akt and endoglin-dependent Smad 1/5/8 signaling, which I propose to investigate. Lastly, I discovered a novel interaction between endogenous endoglin and Akt, a finding that will likely yield new facets of endoglin biology. Based upon these preliminary data, I propose the following hypothesis: Endoglin associates with GIPC to promote angiogenesis by stabilizing endothelial capillaries via BMP-9-dependent Akt activation while destabilizing capillaries via TGF-?-dependent suppression of Akt signaling and cell survival mechanisms. This hypothesis will be addressed by the objectives outlined in two specific aims.

Public Health Relevance

A distinctly innovative aspect of my application is in defining a new signaling role forendoglin in altering endothelial cell behavior. The current paradigm describes endoglinas an auxiliary TGF- co-receptor that regulates the balance of two opposing signalingpathways; Smad1/5/8 and Smad2/3; which invoke pro- or antiangiogenic responses;respectively [12-22]. However; several lines of evidence challenge this overly simplisticrole for endoglin. First; an embryonic lethal phenotype is observed in mice bearinghomozygous deletions [1;2]. Second; the significance of endoglin signaling to the Smadpathways during angiogenesis is not fully understood; since these canonical signaltransducers can be activated without endoglin expression [25]. My preliminary findingsindicate that endoglin engages other important signaling and cellular functions such asregulating Akt activation and downstream cell survival mechanisms. I have so farcharacterized the ability for endoglin to differentially regulate Akt activation in responseto its direct ligands; TGF- and BMP-9. A very intriguing aspect my research proposaltries to address; therefore; is how endoglin recognizes two structurally related ligands toelicit such divergent effects on Akt activation. Another innovative aspect of my researchproposal involves the plan to use in vitro model systems that incorporate many; if not all;of the separate components of the angiogenic process; such as endothelial cellproliferation; degradation of basement membrane; migration; alignment; and capillarytube formation. Several groups have employed cell proliferation and migration assays todissect the mechanisms by which endoglin regulates angiogenesis. However;appropriate in vitro systems that comprise most of the sequential stages of angiogenesiswould be favorable to monitor the effects of endoglin during angiogenic progression.Manipulating various conditions such as ligand treatment or effects of altering proteinlevels; would all be possible. I have begun employing such methods to study endoglinfunction. My preliminary findings based on capillary tube formation assay suggest thatendoglin plays an important role in regulating the stability of sprouted capillary tubes;and that this dynamic process is largely governed by Akt signaling and cell survivalmechanisms. How endoglin might contribute to the stability of these maturing vesselsduring angiogenesis is an entirely unexplored area of research; and the basis of myresearch plan. By employing several new in vitro angiogenesis assays including micro-carrier and co-culturing methods; the proposed work will yield significant insight intowhen and how endoglin exerts its effects during angiogenesis.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Transition Award (R00)
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Special Emphasis Panel (NSS)
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Galis, Zorina S
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Ohio State University
Schools of Pharmacy
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Kumar, S; Pan, C C; Bloodworth, J C et al. (2014) Antibody-directed coupling of endoglin and MMP-14 is a key mechanism for endoglin shedding and deregulation of TGF-? signaling. Oncogene 33:3970-9
Pan, Christopher C; Bloodworth, Jeffrey C; Mythreye, Karthikeyan et al. (2012) Endoglin inhibits ERK-induced c-Myc and cyclin D1 expression to impede endothelial cell proliferation. Biochem Biophys Res Commun 424:620-3
Lee, Nam Y; Golzio, Christelle; Gatza, Catherine E et al. (2012) Endoglin regulates PI3-kinase/Akt trafficking and signaling to alter endothelial capillary stability during angiogenesis. Mol Biol Cell 23:2412-23