Endostatin binds to blood vessels in situ independent of heparan sulfate and does not compete for fibroblast growth factor-2 binding

Abstract

Endostatin is a carboxyl-terminal proteolytic fragment of collagen XVIII and a potent inhibitor of angiogenesis. The mechanism of action is unknown, but the crystal structure of endostatin predicts a prominent heparan sulfate binding site, suggesting that endostatin competitively inhibits heparin-binding angiogenic factors, such as basic fibroblast growth factor (FGF-2). The goal of the study was to map endostatin binding sites in intact human tissues and to determine whether this binding is heparan sulfate dependent. In situ binding was performed with recombinant epitope-tagged murine endostatin. Endostatin predominantly binds to blood vessels of different calibers in a saturable fashion. In addition, binding to some epithelial basement membranes is seen. The localization pattern is similar to that reported for collagen XVIII, endostatin's parent molecule. In breast carcinomas, endostatin co-localizes largely with FGF-2. In a surprising contrast to FGF-2, endostatin binding is resistant to treatment with heparitinase, demonstrating that binding is not mediated by heparan sulfate proteoglycans. Furthermore, FGF-2 and heparin do not compete for endostatin binding, providing additional evidence for the discreteness of endostatin and FGF-binding sites.

Figure 1.

A to F: Endostatin binding in human tissues. Frozen sections of human tissues were incubated with recombinant HA-tagged endostatin (130 nmol/L), and bound endostatin was detected with anti-HA antibodies and Alexa-546-conjugated secondary antiserum (red channel). Double labeling was carried out on some sections (A, B, D, E, and F) with Alexa-488 conjugate (green channel) to determine location of other proteins as indicated. A: Capillaries in normal lung; HA-endostatin binding (red) and factor-VIII-related antigen (green). B: Capillary in breast cancer; co-localization of HA-endostatin binding (red) with perlecan (green). C: Normal renal cortex with glomerulus (G) and renal tubules (T) in the periphery. D: Normal breast tissue with branching duct (D) and stromal capillaries (V); HA-endostatin binding (red) and factor-VIII-related antigen (green). E and F: Infiltrating ductal carcinoma with angiogenic hot spots; HA-endostatin binding (red) and factor-VIII-related antigen (green). G: Ductal carcinoma in situ (DCIS) within ductal lumen (D) and with garland of newly formed capillaries (V). Strong signal within duct lumen is due to autofluorescence of necrotic or calcified material. H and I: Competition between endostatin and FGF-2. Frozen sections of human breast carcinomas were incubated with FGF-2, and bound growth factor was detected with anti-FGF-2 monoclonal antibody and Alexa-546-conjugated secondary antibody. H: FGF-2 (3 nmol/L) binding to blood vessels. I: FGF-2 (3 nmol/L) binding in the presence of 300-fold molar excess (1 μmol/L) of endostatin. The slide was preincubated with the competitor for 30 minutes before adding FGF-2. J to O: Comparison of endostatin and FGF-2 binding. Frozen sections of human skin (J and M) and breast carcinomas (K, L, N, and O) were incubated with HA-tagged endostatin (J, K, and L) or FGF-2 (M, N, and O). Bound protein was detected with anti-HA or anti-FGF-2 monoclonal primary antibodies and visualized by immunofluorescence. Treatment with heparitinase enzyme before the binding reaction was performed on L and O to determine whether the binding sites were heparan sulfates.