Date of Award

12-2007

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Legacy Department

Microbiology

Advisor

Chen, Wen Y

Committee Member

Larcom , Lyndon L

Committee Member

Temesvari , Lesly

Committee Member

Tzeng , Tzuen-Rong

Abstract

Beginning with the development of an embryo and throughout one's adult life angiogenesis plays an essential role for organ growth and repair. The balancing of pro-angiogenic factors to anti-angiogenic factors is what regulates the process of angiogenesis. When this balance is disrupted in one direction or the other, numerous disorders can occur including ocular and inflammatory diseases and, in particular, tumor growth and metastasis. Therefore, inhibition of angiogenesis has become an effective therapy for arresting the growth of tumors.
A key regulator of angiogenesis involves vascular endothelial growth factor (VEGF) and its receptor, VEGFR-2 (KDR). The binding of VEGF to KDR activates the receptor which, in turn, initiates the signaling cascade leading to cell proliferation, migration, and survival. The extracellular portion of KDR consists of seven Ig-like domains (Ig1-7), in which Ig1-3 is believed to be responsible for the ligand binding and Ig4-7 for receptor dimerization. We hypothesized that a soluble fragment of the KDR extracellular domain (KDR-Ig4-7) that lacks a tyrosine kinase domain may serve as a KDR antagonist by binding to the membrane bound KDR to form a KDR dimer thus preventing VEGF induced signal transduction. In this study various KDR proteins (KDR-Ig4-7, KDR-Ig4-6, KDR-Ig5-7 and KDR-Ig5-6) were produced, purified and characterized for their potential anti-angiogenic ability. We demonstrated that KDR-Ig4-7 was able to specifically bind to human umbilical vein endothelial cells (HUVEC), and accumulated specifically in tumors indicating its ability to target endothelial cells. Furthermore, KDR-Ig4-7 was able to inhibit HUVEC (and various breast and leukemic cancer cells) proliferation, tube formation and migration. An apoptosis assay conducted using T47-D cells revealed that the induction of apoptosis was the likely mechanism for the inhibition of cell proliferation by KDR-Ig4-7. The inhibitory effect of KDR-Ig-4-7 was at least, in part, through the blocking of ERK phosphorylation. Among all the variants tested, KDR-Ig-4-7 was the most potent inhibitor, which suggested the involvement of domains Ig4 and Ig7 in the receptor dimerization process. Moreover, we found that the inhibitory activity of KDR-Ig4-7 was enhanced in the presence of VEGF, which suggested that VEGF might induce a conformational change which favors the interaction between KDR-Ig4-7 and KDR. In our 4T1 xenograft animal studies, however, KDR-Ig4-7 treatment was not effective. In summary, our results suggest that KDR-Ig4-7 is a functional antagonist to KDR.

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Oncology Commons

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