Angiostatic activity of human plasminogen fragments is highly dependent on glycosylation

Abstract

To assess the importance of carbohydrate moieties to the antiangiogenic activity of plasminogen fragments, we cloned the fragment corresponding to amino acids Val 79 to Thr 346 (Kint3-4) that presents the three glycosylation sites. The activity of glycosylated and unglycosylated Kint3-4 was tested in murine sponge implant model. We observed a significant decrease in the neovascularization on the sponge after treatment with Kint3-4 by histological examination and determination of the hemoglobin levels. The effects were more intense with the glycosylated than the unglycosylated protein. Technecium-labeled red blood cells confirmed the inhibition of cell infiltration in the implanted sponge. Studies using melanoma B16F1 implanted in a mouse demonstrated that treatment with glycosylated Kint3-4 (0.15 nmol ⁄ 48 h) during 14 days suppresses tumor growth by 80%. The vascular endothelial growth factor mRNA levels on the tumor were reduced after treatment. Kint3-4 is a potent plasminogen fragment that has been found to inhibit tumor growth. (Cancer Sci 2010; 101: 453-459) T he vasculature is an important organ. Angiogenesis, the formation of new blood vessels from pre-existing capillaries, is a fundamental and complex process involved in reproduction, cellular development, and pathological events, such as inflammation, diabetic retinopathy, endometriosis, adiposity, and cancer.(1,2) During tumor growth and chronic inflammation, neovascularization is required for the provision of nutrients and oxygen and to mobilize host cells, such as leukocytes. (3,4) The new vascular network demands an extensive interplay between cells, soluble factors, and extracellular components, and is tightly regulated by the expression of angiogenic and angiostatic factors.(5,6) A wide range of molecules mediates angiogenesis by acting directly or indirectly on endothelial cells via accessory cells, such as polymorphonuclear leukocytes.(7-9) Among these molecules are fragments of the plasmatic glycoprotein plasminogen, which have different angiostatic activity. Plasminogen presents five segments that, because of their specific structure, were designated as kringles (K1-K5). The antiproliferative activity of plasminogen is shared by K1, K2, K3, and K4 being the most potent angiostatin (K1-4 corresponding to amino acids Cys 84 -Cys 435 ), followed by K2-3. (10,11) The sequence numbering of plasminogen starts with Glu 1 of the mature protein or Glu 20 for human plasminogen when including signal peptide. K2 and K3 individually or in combination exhibit weak activity when compared with K2-3, and a combination of K1-3 and K4 resulted in the same activity level of K1-4.(11) It was also observed that the most potent inhibitor of endothelial cell angiogenesis of is K1-3 (amino acids Cys 84 -Cys 333 of plasminogen). (12) These results show that proteolytic processing with the exposure of antiangiogenic sites, kringle conformation, interkringle interaction, and carbohydrate moieties can alter efficiency and function. Despite the anti-angiogenic activity...

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