Antibody neutralization of vascular endothelial growth factor inhibits wound granulation tissue formation

Objective. The goal of this work was to test the functional role of vascular endothelial growth factor (VEGF) in promoting the vigorous granulation tissue formation, wound fluid accumulation, and angiogenic responses characteristic of this wound model. Background. Formation of vessel-rich granulation tissue is central to wound repair and is thought to be regulated by locally liberated angiogenic factors. Despite the clinical importance of granulation tissue formation in the early stage of wound healing, surprisingly little is known about the molecular identity of signals leading to granulation tissue invasion of a wound space. Methods. A ventral hernia, surgically created in the abdominal wall of 15 swine, was repaired using silicone sheeting and skin closure. An osmotic minipump, inserted in a remote subcutaneous pocket, delivered saline (n = 5), an irrelevant control antibody (n = 5), or neutralizing anti-VEGF antibody (n = 5) into the wound environment. Serial ultrasonography on Days 2, 4, 7, 9, 11, and 14 was used to determine the dimensions of the subcutaneous granulation tissue and wound fluid compartment. VEGF and transforming growth factor β1 (TGF-β1) levels in serial wound fluid samples were quantitated by ELISA. On Day 14, animals were sacrificed and the abdominal wall was harvested for histologic, biochemical, and molecular analyses. Results. In animals receiving saline or an irrelevant antibody, a nearly linear 4-fold increase in granulation tissue thickness and 7-fold increase in wound fluid volume were measured over the 14-day study interval. In contrast, in animals receiving anti-VEGF neutralizing antibody, Day 14 granulation tissue thickness and wound fluid volume measurements were essentially unchanged from Day 2 values. Moreover, in the anti-VEGF animals, ultrasonography was unable to resolve the "angiogenic zone" typical of both controls, and correspondingly, wound vessel count and vascular surface area estimates derived from image analysis of histological sections were 3-fold lower in the anti-VEGF animals compared with the saline and antibody controls. Finally, VEGF levels in wound fluid detectable by ELISA analysis were strikingly (10-fold) reduced in anti-VEGF animals on Postsurgery Days 7-14. In contrast, TGF-β1 levels were unaffected by the anti-VEGF treatment. Conclusion. Functional VEGF is a key mediator in wound angiogenesis, fluid accumulation, and granulation tissue formation.