The antiangiogenic activity of cleaved high molecular weight kininogen is mediated through binding to endothelial cell tropomyosin

Abstract

Conformationally altered proteins and protein fragments derived from the extracellular matrix and hemostatic system may function as naturally occurring angiogenesis inhibitors. One example of such a protein is cleaved high molecular weight kininogen (HKa). HKa inhibits angiogenesis by inducing apoptosis of proliferating endothelial cells, effects mediated largely by HKa domain 5. However, the mechanisms underlying the antiangiogenic activity of HKa have not been characterized, and its binding site on proliferating endothelial cells has not been defined. Here, we report that the induction of endothelial cell apoptosis by HKa, as well as the antiangiogenic activity of HKa in the chick chorioallantoic membrane, was inhibited completely by antitropomyosin monoclonal antibody TM-311. TM-311 also blocked the high-affinity Zn2+-dependent binding of HKa to both purified tropomyosin and proliferating endothelial cells. Confocal microscopic analysis of endothelial cells stained with monoclonal antibody TM-311, as well as biotin labeling of cell surface proteins on intact endothelial cells, revealed that tropomyosin exposure was enhanced on the surface of proliferating cells. These studies demonstrate that the antiangiogenic effects of HKa depend on high-affinity binding to endothelial cell tropomyosin.

Fig 1.

(A) Inhibition of bFGF (10 ng/ml)-induced endothelial cell proliferation by 20 nM HKa (open bars) and 60 nM HKa D5 (black bars) is prevented by increasing concentrations of mAb TM-311. Proliferation was measured as described in Materials and Methods. (B) Effect of mAb TM-311 and antibodies against other endothelial cell HK or HKa-binding proteins on the inhibition of endothelial cell proliferation caused by 20 nM HKa. Proliferation was measured in response to 10 ng/ml bFGF. All antibodies were used at a concentration of 300 nM. The control antibody for polyclonal antiurokinase receptor (uPAR) and cytokeratin 1 (CK-1) antibodies was normal rabbit IgG (NRIgG), and that for mAb TM-311 and mAbs against the receptor for the globular heads of C1q (gC1qR) was MOPC-21.

Fig 2.

Effect of mAb TM-311 on HKa-induced endothelial cell apoptosis assessed by endonucleolytic cleavage of DNA. Endothelial cell DNA was isolated from cells cultured for 12 h in the presence of mAb TM-311 alone (lane 1), 20 nM HKa (lane 2), 20 nM HKa + 60 nM TM-311 (lane 3), 50 nM HKa D5 (lane 4), 50 nM HKa D5 + 150 nM mAb TM-311 (lane 5), 2 μM 2-methoxyestradiol (lane 6), or 2 μM 2-methoxyestradiol + 6 μM mAb TM-311 (lane 7) and analyzed as described in Materials and Methods. M, markers.

Fig 3.

Confocal laser scanning microscopic analysis of proliferating and confluent endothelial cells. (A) Proliferating endothelial cells stained with MOPC-21. (B) Proliferating endothelial cells stained with TM-311. (C) Confluent endothelial cells stained with TM-311. Cells were permeabilized by exposure to 0.1% Triton X-100 before staining.

Fig 4.

Immunoprecipitation of endothelial tropomyosin by mAb TM-311. Cell surface proteins on proliferating and confluent human umbilical vein endothelial cells were labeled with N-hydroxysuccinimide (sulfo)-LC-biotin. Equal amounts of protein from detergent extracts of each culture were immunoprecipitated by using TM-311 or MOPC-21. Immunoprecipitated proteins were separated by using 10% SDS/PAGE, transferred to poly(vinylidene difluoride), and detected by using streptavidin-peroxidase and chemiluminescence.

Fig 5.

(A) Specific binding of HKa to proliferating endothelial cells. Cells were cultured as in proliferation assays and incubated with increasing concentrations of biotin-HKa in the absence or presence of 10 μM Zn²⁺. Cell-bound biotin-HKa was detected by using streptavidin peroxidase and the peroxidase substrate turbo-TMB. The curve was fit by nonlinear regression, yielding a K_d of ≈2.5 nM. (B) Inhibition of the binding of biotin-HKa to proliferating endothelial cells by mAb TM-311. Biotin-HKa (20 nM) was incubated with endothelial cells in the presence of 10 μM Zn²⁺ and increasing concentrations of mAb TM-311. Cell-bound biotin-HKa was determined as described for A.

Fig 6.

(A) Specific binding of HKa to purified tropomyosin. Ninety-six-well plates were coated with tropomyosin and then incubated with increasing concentrations of biotin-HKa in the absence or presence of 10 μM Zn²⁺. Bound ligand was determined by using streptavidin-peroxidase and turbo-TMB, and the curve was fit by nonlinear regression, yielding a K_d of ≈1.6 nM. (B) Inhibition of the binding of 20 nM biotin-HKa to purified tropomyosin by mAb TM-311. (C) Inhibition of the binding of biotin-HKa (10 nM) to purified tropomyosin by HKa D5. The IC₅₀ for inhibition of HKa binding by HKa D5 was ≈8.1 nM.

Fig 7.

Cross-linking of HKa to endothelial cell surface proteins. Biotin-HKa was incubated with confluent (lanes 1 and 2) or proliferating (lanes 3 and 4) endothelial cells in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of a 20-fold molar excess of unlabeled HKa before cross-linking by using bis(sulfosuccinimidyl) suberate. Biotin-HKa was incubated also with MDA-MB-231 breast carcinoma cells under identical conditions (lane 5). Detergent extracts were separated by SDS/PAGE and then transferred to poly(vinylidene difluoride) and detected by chemiluminescence. The arrowhead denotes a prominent band of ≈140–150 kDa, the expected size of an HKa-tropomyosin complex. HUVEC, human umbilical vein endothelial cells.

Fig 8.

Effect of HKa and/or TM-311 on in vivo angiogenesis in the CAM. Angiogenesis is represented by the fine neovessels contacting and radiating from the filter disk in a spoke-like pattern. The effects of bFGF (A), bFGF + TM-311 (B), bFGF + HKa (C), and bFGF + HKa + TM-311 (D) are depicted. HKa inhibited bFGF-induced angiogenesis by ≈85%, and these effects were inhibited completely by mAb TM-311.