Multiple fragments related to angiostatin and endostatin in fluid from venous leg ulcers

To investigate whether compromised angiogenesis could contribute to the impaired healing of venous leg ulcers, we have analyzed fluids from venous leg ulcers for the presence of the angiogenesis inhibitors angiostatin and endostatin. Multiple fragments related to angiostatin were detected by Western blot analysis. One angiostatin fragment was identified by mass spectrometry as plasminogen kringle domains 1-3 containing amino acids 82-343 of plasminogen, and a fraction containing this fragment inhibited tubule formation of human umbilical vein endothelial cells in a Matrigel assay. The leg ulcer fluids also contained endogenous endostatin (20 kDa) as well as higher molecular weight endostatin-related proteins. The concentrations of endostatin in the wound fluids, which ranged from 12.8 to 65.5 ng/ml, were higher than the concentration in human serum (7.7 ng/ml). Most of the endostatin in leg ulcer fluid appeared to be bound to the proteoglycan glypican-1. These data suggest that anti-angiogenic activity is present at the site of venous leg ulcers, and at least in the case of angiostatin, is biologically active.