Generation of a novel proteolysis resistant vascular endothelial growth factor₁₆₅ variant by a site‐directed mutation at the plasmin sensitive cleavage site

Abstract

Vascular endothelial growth factor (VEGF) is a potent angiogenic mediator in tissue repair. In non-healing human wounds plasmin cleaves and inactivates VEGF 165 . In the present study, we generated recombinant VEGF 165 mutants resistant to plasmin proteolysis. Substitution of Arg110 with Ala110 or Gln110, and Ala111 with Pro111 yielded plasmin-resistant and biologically active VEGF 165 mutants. In addition, substitution of Ala111 with Pro111 resulted in a substantial degree of stabilization when incubated in wound £uid obtained from non-healing wounds. These results suggest that the plasmin cleavage site Arg110/Ala111 and the carboxyl-terminal domain play an important role in the mitogenic activity of VEGF 165 .

Plasmin Modulates Vascular Endothelial Growth Factor-A-Mediated Angiogenesis during Wound Repair

“…12,14,15 Loss of the carboxyl-terminal heparin binding domain through plasmin digestion significantly reduces VEGF165 mitogenic activity on human umbilical vein endothelial cells, supporting the crucial significance of the heparin-binding domains for VEGF-A function. 12,15 So far, little is known about the prevalence and biological significance of proteolytic digestion of VEGF-A in vivo.…”

“…11,12,14,15 Plasmin digestion of VEGF165 yields two fragments: an amino-terminal homodimer (VEGF1-110) containing the VEGF receptor binding site and a carboxyl-terminal polypeptide comprising the heparinbinding domains (VEGF111-165). 12,14,15 Loss of the carboxyl-terminal heparin binding domain through plasmin digestion significantly reduces VEGF165 mitogenic activity on human umbilical vein endothelial cells, supporting the crucial significance of the heparin-binding domains for VEGF-A function. 12,15 So far, little is known about the prevalence and biological significance of proteolytic digestion of VEGF-A in vivo.…”

“…15 Hence, plasmin-catalyzed VEGF165 proteolysis and inactivation may lead to reduced VEGF165 availability at the wound site and contribute to an impaired healing response. To further characterize the biological relevance of the protease sensitivity of VEGF165 during cutaneous repair, in the present study we tested the stability and activity of a locally applied VEGF165 mutant resistant to plasmin proteolysis (VEGF165 A111P ) in a genetic mouse model of impaired healing (db/db mouse).…”

RETRACTED: Gene therapy and wound healing

“…There is increasing evidence that the persisting infiltration of neutrophils and macrophages plays a major role in the generation of a protease rich and prooxidant hostile microenvironment (21)(22)(23). Recent experimental and several comprehensive clinical studies demonstrated that the proteolytic activity of matrix metalloproteinases and serine proteinases is significantly upregulated in the exudate of chronic wounds (24)(25)(26)(27), thus contributing to the degradation of extracellular matrix molecules (24,25), growth factors and their receptors (28)(29)(30). In addition, proteolytic degradation products generated by high protease activity in the chronic wound environment may exert inhibitory effects on cell function (31).…”

“…Administration of recombinant TIMP-2, a metalloproteinase inhibitor, promoted tissue repair in a rat model (128). Along these lines, our group generated a novel protease resistant VEGF165 molecule, which is characterized by an increased stability in the protease rich microenvironment of the chronic wound (28,129). This VEGF mutant is expected to exert superior angiogenic properties at the chronic wound site.…”