"Combined effect of anti-VEGF and chemically modified heparin. Role in the inhibition of angiogenesis in ocular diseases, an in vitro study."

• Grant number: 24/21930-0
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: March 01, 2025
• End date: February 28, 2026
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Principal Investigator: Helena Bonciani Nader
• Grantee: Ana Carolyne Izu Akamine
• Host Institution: Escola Paulista de Medicina (EPM). Universidade Federal de São Paulo (UNIFESP). Campus São Paulo. São Paulo , SP, Brazil
• Associated research grant: 23/07464-4 - Glycosaminoglycans and the cross-talk cell and extracellular matrix: a translational approach, AP.TEM

Abstract

Objective: This project aims to investigate the anti-angiogenic effects of chemically modified heparins, specifically 6-desulfated heparin, in combination with bevacizumab, an anti-VEGF monoclonal antibody. The study will be conducted on human endothelial cells, specifically the ECV304 cell line, which is widely used in angiogenesis research due to its representative characteristics of endothelial cells. Introduction: Angiogenesis is a fundamental process involving the formation of new blood vessels from an existing vasculature. This process is essential in both physiological conditions, such as wound healing and reproduction, and in pathological conditions, including tumor growth, metastasis, and degenerative diseases like age-related macular degeneration and diabetic retinopathy. Angiogenesis comprises several complex steps regulated by growth factors, cytokines, signaling molecules, and extracellular matrix (ECM) components. The ECM plays a vital role in angiogenesis, providing structural and functional support for cells and tissues. Key ECM components include glycoproteins like laminin and fibronectin, as well as glycosaminoglycans (GAGs) and proteoglycans. GAGs, such as heparan sulfate and heparin, are linear polysaccharides that interact with various molecules, including growth factors, thereby modulating biological processes like cell proliferation, migration, and differentiation. Heparin is a sulfated GAG known for its anticoagulant activity, composed of alternating disaccharides of L-iduronic acid and D-glucosamine with a high density of sulfate groups. Due to its polyanionic nature, heparin interacts with multiple proteins, influencing several biological processes, including the modulation of angiogenesis. When chemically modified, heparin can lose its anticoagulant and hemorrhagic activities without losing its anti-inflammatory and anti-angiogenic properties. Objectives: The primary goal of this study is to evaluate the anti-angiogenic activity of 6-desulfated heparin in combination with bevacizumab as anti-angiogenic agents in vitro in human endothelial cells by assessing proliferation, viability, and capillary-like structure formation in culture, as well as to understand the role of ECM components in this context. Materials and Methods: ECV304 cells, human endothelial cells derived from bladder carcinoma and widely used in angiogenesis studies due to their ability to form capillary-like structures, will be used. Cells will be cultured under specific conditions to ensure growth and viability. 6-desulfated heparin will be prepared by selectively removing sulfate groups at the 6-carbon position of glucosamine in heparin, resulting in a chemically modified form without anticoagulant activity. Bevacizumab, an anti-VEGF monoclonal antibody, will be used in combination with the modified heparin to assess its synergistic effect in inhibiting angiogenesis. Experiments will include assays for cell proliferation, cell migration, and capillary-like structure formation in Matrigel, as well as immunofluorescence analyses to assess cell interactions with ECM components. This project aims to contribute to the understanding of the anti-angiogenic mechanisms of chemically modified heparins and their interactions with the ECM, paving the way for new therapeutic strategies against angiogenic and neoplastic diseases.