Tissue inhibitors of metalloproteinases (TIMPs) suppress matrix metalloproteinase (MMP) activity critical for extracellular matrix turnover associated with both physiologic and pathologic tissue remodeling. We demonstrate here that TIMP-2 abrogates angiogenic factor-induced endothelial cell proliferation in vitro and angiogenesis in vivo independent of MMP inhibition. These effects require alpha 3 beta 1 integrin-mediated binding of TIMP-2 to endothelial cells. Further, TIMP-2 induces a decrease in total protein tyrosine phosphatase (PTP) activity associated with beta1 integrin subunits as well as dissociation of the phosphatase SHP-1 from beta1. TIMP-2 treatment also results in a concomitant increase in PTP activity associated with tyrosine kinase receptors FGFR-1 and KDR. Our findings establish an unexpected, MMP-independent mechanism for TIMP-2 inhibition of endothelial cell proliferation in vitro and reveal an important component of the antiangiogenic effect of TIMP2 in vivo.

One of the major problems in angiogenesis research remains the lack of suitable methods for quantifying the angiogenic response in vivo. We describe the development and application of the directed in vivo angiogenesis assay (DIVAA) and demonstrated that it is reproducible and quantitative. This assay consists of subcutaneous implantation of semiclosed silicone cylinders (angioreactors) into nude mice. Angioreactors are filled with only 18 micro l of extracellular matrix premixed with or without angiogenic factors. Vascularization within angioreactors is quantified by the intravenous injection of fluorescein isothiocyanate (FITC)-dextran before their recovery, followed by spectrofluorimetry. Angioreactors examined by immunofluorescence show cells and invading angiogenic vessels at different developmental stages. The minimally detectable angiogenic response requires 9 days after implantation and >/=50 ng/ml (P < 0.01) of either fibroblast growth factor-2 or vascular endothelial growth factor. Characterization of this assay system demonstrates that the FITC-labeled dextran quantitation is highly reproducible and that levels of FITC-dextran are not significantly influenced by vascular permeability. DIVAA allows accurate dose-response analysis and identification of effective doses of angiogenesis-modulating factors in vivo. TNP-470 potently inhibits angiogenesis (EC(50) = 88 pmol/L) induced by 500 ng/ml of fibroblast growth factor-2. This inhibition correlates with decreased endothelial cell invasion. DIVAA efficiently detects differences in anti-angiogenic potencies of thrombospondin-1 peptides (25 micro mol/L) and demonstrates a partial inhibition of angiogenesis ( approximately 40%) in a matrix metalloprotease (MMP)-2-deficient mouse compared with that in wild-type animals. Zymography of angioreactors from MMP-deficient and control animals reveals quantitative changes in MMP expression. These results support DIVAA as an assay to compare potencies of angiogenic factors or inhibitors, and for profiling molecular markers of angiogenesis in vivo.

CR-1 appears to have an important role in the multistep process of angiogenesis.

Purpose To determine the in vivo effect of doxycycline (doxy) on choroidal angiogenesis and pterygium growth by using a choroidal neovascular murine model (CNV), a directed in vivo angiogenesis assay (DIVAA) and a pterygium murine model. Design Experimental Study Participants 3 murine models were investigated with 4 mice minimum per group and 22 maximum per group. Methods Mice received water with or without doxycycline (Leiter's Pharmacy, San Jose, CA). For the CNV, the neovascular lesion volume was determined in choroid-retinal pigment epithelial (RPE) flat mounts using confocal microscopy seven days after laser induction. For DIVAA, silicone capsules containing 10,000 human pterygium epithelial cells were implanted in the flanks of mice subcutaneously. After eleven days, neovascularization (NV) was quantified using spectrofluorimetry after murine tail-vein injection of fluorescein isothiocyanate (FITC)-labeled dextran. A pterygium epithelial cell model was developed by injecting 10,000 human pterygium epithelial cells in the nasal subconjunctival space in athymic nude mice. Doxy was started on day six at 50 mg/kg/day; corneal lesions that resulted from the injections were compared at days six and fifteen. Main outcome measures Student's t-test was used to evaluate the data for the CNV and DIVAA models and histologic preparations were used to evaluate pterygia lesions. Results There was significantly less NV and lesion volume with doxy taken in drinking water versus plain water. With doxy treatment, the laser-induced CNV showed a maximal 66% decrease in choroidal blood vessel volume (p≤0.008) and the DIVAA showed a 30% reduction of blood vessel growth and migration (p<0.004). Histologic preparations demonstrated that pterygium cell lesions regressed when mice were administered doxy for 9 days. Conclusions Doxycycline significantly inhibited angiogenesis in three murine models. The most dramatic effect was found in the choroidal neovascularization model followed by the pterygia epithelial cell DIVAA model. The anterior segment pterygium model also showed regression histologically. This suggests that doxycycline may be successful as an adjunctive treatment for choroidal neovascularization and pterygia in humans; clinical trials would be necessary to determine if there is a benefit.