Application of FITC for detecting the binding of antiangiogenic peptide to HUVECs

Angiogenesis is the generation of new blood vessels from the existing vasculature. The angiogenic programme requires the degradation of the basement membrane, endothelial cell migration and invasion of the extracellular matrix, with endothelial cell proliferation and capillary lumen formation before maturation and stabilization of the new vasculature. Angiogenesis is dependent on a delicate equilibrium between endogenous angiogenic and antiangiogenic factors. However, under pathological conditions, this tight regulation becomes lost which can result in the formation of the different diseases. Endostatin, a 20 kDa fragment of collagen XVIII, is a member of a group of endogenous antiangiogenic proteins activated by proteolytic processing. Endostatin inhibits endothelial cell proliferation, migration/invasion, and tube formation. Inhibitory action by endostatin has been proposed to involve binding to the receptor. The use of a fluorescent probe as a detector and its application in imaging methods is widely used. The use of fluorescence helps us to accurately count cells, cell surface receptors, or intracellular organelles. The number of fluorescent probes having traceability in in vitro and in vivo studies is very high. One of these probes is Fluorescein isothiocyanat (FITC) with a maximum absorption at 490 nm and a maximum emission at 525 nm. In this study, 27-amino acid fragment corresponding to the N-terminal domain of endostatin was labeled with fluorescence probes, FITC. Various conditions such as concentration, time, and temperature were tested to achieve the proper conditions for marking peptides. Unlabeled FITCs separated by gel filtration Chromatography method with sephedex G10. HUVECs are harvested and treated with conjugate samples (FITC-peptide) from gel filtration chromatography. After the required time, the cells were fixed on separate lamelles and the binding of labeled peptides to their receptors on HUVECs was analyzed by Fluorescence microscope. The purpose of this study is to use Conjugated peptides with FITC for detect the binding of a peptide derived from N-terminal of endostatin to vascular endothelial cells and also confirms the function of peptide after binding to FITC. Our findings by Fluorescence microscopy confirm binding antiangiogenic peptide to its specific receptor. The connection of the fluorescent probe to the peptide can be used as a suitable molecular tool for future studies in both the in vitro and in vivo domains.