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Angiogenesis is the process of new blood vessel growth from preexisting vessels. This process relies on the activity of Vascular Endothelial Growth Factor Receptor-2 (VEGFR-2) and occurs in both normal and pathological tissues. Angiogenesis is often dysregulated in diseases such as cancer and many efforts have been made to treat such diseases by targeting the VEGFR-2 pathway. VEGFR-2 is activated upon ligand binding and subsequent autophosphorylation of tyrosine residues in the kinase domain, which leads to endothelial cell survival, proliferation, and growth – all of which are required for angiogenesis to occur. It was previously demonstrated that methylation of VEGFR-2 at Lys1041 enhanced its tyrosine autophosphorylation and is required for VEGFR-2 mediated angiogenesis in zebrafish and tumor growth in mouse. However, the Lysine Methyltransferase (KMT) involved in the methylation of VEGFR-2 remains unknown. This study aimed to identify the KMT involved in the methylation of VEGFR-2. We have identified Enhancer of zeste homolog 2 (EZH2) as the KMT likely responsible for catalyzing methylation of K1041 on VEGFR-2. Over-expression of EZH2 was found to increase phosphorylation of Tyr1054, one of the required phosphorylation sites for VEGFR-2 activation, in whole cell lysates and VEGFR-2 purified by immunoprecipitation. The effect of over-expression of EZH2 in the phosphorylation of VEGFR-2 at Tyr1054 was dose-dependent - increasing concentrations of EZH2 resulted in increasing phosphorylation of VEGFR-2 at Tyr1054. Moreover, we determined that EZH2 physically interacts with VEGFR-2 as demonstrated by co-immunoprecipitation in vitro GST-pulldown assays. The C-terminus of EZH2 (amino acids 371-746), physically interacted with VEGFR-2. Taken together, we have identified EZH2 as a candidate KMT involved in the methylation of Lys1041, which increases phosphorylation of VEGFR-2 at Tyr1054.

Temporal and spatial molecular imaging of disease biomarkers using non-invasive MRI with high resolution is largely limited by lack of MRI contrast agents with high sensitivity, high specificity, optimized biodistribution and pharmacokinetics. In this dissertation, I report my Ph. D. work on the development of protein-based MRI contrast agents (ProCAs) specifically targeting different cancer biomarkers, such as grastrin-releasing peptide receptor (GRPR), prostate specific membrane antigen (PSMA), and vascular endothelial growth factor receptor-2 (VEGFR-2). Similar to non-targeted ProCAs, these biomarker-targeted ProCAs exhibit 5 - 10 times higher r1 and r2 relaxivites than that of clinical MRI contrast agents. In addition, these biomarker-targeted ProCAs have high Gd3+ binding affinities and metal selectivities. The highest binding affinity of the three GRPR-targeted contrast reagents obtained by grafting a GRPR ligand binding moiety into ProCA32 for GRPR is 2.7 x 10-9 M. We further demonstrate that GRPR-targeted ProCAs were able to semi-quantitatively evaluate GRPR expression levels in xenograft mice model by MRI. In addition, we have also created a PSMA-targeted ProCA which has a binding affinity to PSMA biomarker of 5.2 x 10-7 M. Further, we developed VEGFR-targeted contrast agent which is able to image VEGFR2 in mice models using T1-weighted and T2-weighted sequences. Moreover, the relaxivities and coordination water numbers of ProCAs can be tuned by protein design of ProCA4. Since disease biomarkers are expressed in various tumors and diseases, our results may have strong preclinical and clinical implications for the diagnosis and therapeutics of cancer and other type of diseases.

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.

Anti-angiogenic therapy reduces both plaque growth and intimal neovascularization in apolipoprotein-E-deficient mice (apoE-/-). Vascular endothelial growth factor (VEGF) has been suggested as playing a role in the development of atherosclerosis. We examined the hypothesis that VEGF and VEGF receptor-2 (VEGFR-2) expression is upregulated in apoE-/- and, since it could be driven by oxidative stress, tested whether dietary supplementation with vitamins C and E could downregulate it.Two-month-old apoE-/- received vitamin C combined with alpha- or beta-tocopherol for 4 weeks. Aortic VEGF and VEGFR-2 expression were measured by RT-qPCR and western blot.ApoE-/- showed significantly higher expression of aortic VEGF and VEGFR-2 mRNA (P