PMC

Fisp12 induces neovascularization in rat corneas. Hydron pellets containing test substances were formulated and implanted into corneas of rats as described in Materials and Methods and Table . The formation of new blood vessels was visualized by perfusion with colloidal carbon 7 days after implantation. Hydron pellets contained in Fisp12 (A), bFGF (B), Fisp12 storage buffer (C), and Fisp12 protein preincubated with anti-Fisp12 antibodies (D) are shown.

Fisp12 protects HMVECs from apoptosis. HMVECs were starved for 24 h prior to the addition to slides coated with 10 μg of laminin (Collaborative biosciences) per ml overnight at 4°C. Cells were then incubated in EBM for 20 h with or without the addition of Fisp12 protein, and apoptosis was monitored by using the TUNEL assay; the number of apoptotic cells was then counted. (A) Dose dependence of Fisp12-promoted cell survival. Values for HMVECs incubated in the absence of Fisp12 (control) or in the presence of indicated concentrations of Fisp12 are shown. Where indicated, Fisp12 was preincubated with anti-Fisp12 antibody prior to addition to the medium. Complete medium was added as a positive control. The percentages of apoptotic cells ± the SD from at least 500 cells counted are shown, and each experiment was done in triplicate. (B) Cell proliferation assay. HMVECs treated as described in panel A were labeled with BrdUrd for 24 h, and the percentages of cells incorporating label in the absence or presence of Fisp12 are shown. (C and D) Photomicrographs of HMVECs incubated in the absence or presence of 5 μg of Fisp12 per ml, respectively.

Fisp12 stimulates HMVEC migration through an α_vβ₃-dependent pathway. The migration of HMVECs was measured in a modified Boyden chamber assay. The cells placed in a lower chamber that migrated into the upper chamber were counted in three high-power fields for each condition ± the SD after a 4-h incubation at 37°C. As chemoattractants, bFGF (10 ng/ml), VEGF (1 ng/ml), vitronectin (5 μg/ml), and Fisp12 (1 μg/ml unless otherwise indicated) was placed in either the top or the bottom chamber, or both, as indicated. (A) Fisp12-stimulated cell migration is dose dependent. Cells that migrated into the upper well where the indicated amount of purified Fisp12 or the corresponding amount of the Fisp12 storage buffer was placed, were counted. The results are expressed as the percentage of bFGF-induced migration ± the SD. (B) Specific inhibition of Fisp12-induced cell migration by anti-Fisp12 antibodies. HMVEC migration was measured as described above by using either Fisp12 or bFGF as the chemoattractant. Where indicated, these proteins were preincubated with anti-Fisp12 antibodies (30 μg/ml) before addition to the upper wells. Neg., background migration in the absence of chemoattractant. (C) Fisp12 induces chemokinesis. The migration of HMVECs was measured in a checkerboard-type analysis. Fisp12 or bFGF were added to the upper chamber, the lower chamber, neither chamber, or both chambers as indicated. (D) Specific inhibition of Fisp12-induced HMVEC migration by anti-α_vβ₃ antibody. HMVEC migration was monitored by using Fisp12, vitronectin, bFGF, or VEGF as the chemoattractants. Where indicated cells were preincubated with 50 μg of LM609 per ml for 1 h before addition to the lower chamber. The results are expressed as the percentage of cells that migrated to VEGF. Neg., background migration in the absence of chemoattractant.

Fisp12 mediates HMVEC adhesion through integrin α_vβ₃. HMVECs were washed and harvested in PBS with 1 mM EDTA, resuspended in serum-free medium, and plated on microtiter wells coated with indicated substrates. After incubation at 37°C for 30 min, adherent cells were fixed and stained with methylene blue, followed by quantitation by measuring the absorbance at 620 nm. Data shown are the means of duplicate determinations, and similar results were obtained in at least three separate experiments. Error bars represent the standard deviation(s) (SD). (A) Dose dependence of adhesion to Fisp12. HMVECs were plated onto microtiter wells coated with the indicated concentrations of purified Fisp12. (B) Divalent cation dependence. HMVEC adhesion to either BSA- or Fisp12-coated plates was determined; EDTA (10 mM) or Ca²⁺ (20 mM) was added where indicated. (C) Inhibition by RGD peptides. HMVECs were incubated with either buffer (control) or 0.2 or 2.0 mM GRGDSP or GRGESP peptides, as indicated, prior to addition to the microtiter wells coated with Fisp12 (5 μg/ml), 2 μg of fibronectin per ml (FN), or 0.1 μg of vitronectin per ml (VN). (D) HMVEC adhesion to Fisp12 was dependent on integrin α_vβ₃. Microtiter wells were coated with BSA, Fisp12 (2.5 μg/ml), fibronectin (2 μg/ml), or vitronectin (0.1 μg/ml). HMVECs were incubated with either normal mouse immunoglobulin G or the anti-α_vβ₃ antibody LM609 (50 μg/ml) prior to plating, and adhesion was measured at 30 min thereafter.