Vascular endothelial growth factor inhibitors in the treatment of abnormal angiogenesis

Angiogenesis is defined as the development of blood vessels from a pre-existing vasculature and has a major role in both normal physiology and pathological functions. Vascular endothelial growth factor-A (VEGF-A) is a key pro-angiogenic factor which binds to the receptor tyrosine kinases, VEGFR1 and VEGFR2. VEGFR2 is the main pro-angiogenic receptor whilst VEGFR1 is thought to be indirectly antiangiogenic. Specifically targeting VEGFR1 or VEGFR2 could provide benefits for various disease states involving angiogenesis, for instance, increasing its activity in repairing arterial damage or in decreasing blood vessel growth to tumours. This study uses novel affinity reagents called Affimers, which are synthetic antibody-mimetic proteins. The Affimers were selected against VEGFR1 and VEGFR2 via phage display and expressed using the IPTG-induced E.coli expression system. Dosedependence studies of VEGFR-specific Affimers on proangiogenic responses within human umbilical vein endothelial cells (HUVECs) were carried out in the presence of VEGF ligands. Live cell imaging of an A431 epithelial cell line, stably transfected with the fluorescence ubiquitin cell cycle indicator (FUCCI) system, was also used to assess the effects of VEGFR Affimers on cancer cell cycle progression in the presence of VEGF and placental growth factor (PlGF). VEGFR1-specific Affimers were useful immunofluorescence tools and also promoted angiogenic responses, cell viability, proliferation, migration and endothelial tube formation. Additionally, VEGFR2-inhibitory Affimers promoted endothelial cell viability, whilst decreasing proangiogenic responses: migration. VEGFR1- and -2 specific Affimers, had differential, dosage dependent, effects on the pattern of cell growth, thereby altering the G1 and S phases cell cycle. These in vitro studies show that inhibiting VEGFR1 could stimulate angiogenesis to promote cardiovascular recovery. In contrast, VEGFR2-inhibitory Affimers show promise as useful anti-angiogenic tools. Live-cell imaging demonstrated that VEGFR Affimers are also valuable tools in assessing both endothelial and epithelial cell function. This study suggests that Affimers may provide a viable alternative for the usage of antibodies in therapeutic and diagnostic procedures as part of the treatment for both heart disease and cancer.