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  • Suppression of choroidal neovascularization and quantitative and qualitative inhibition of VEGF and CCL2 by heparin.

Suppression of choroidal neovascularization and quantitative and qualitative inhibition of VEGF and CCL2 by heparin.

Investigative ophthalmology & visual science (2011-02-08)
Daisuke Tomida, Koji M Nishiguchi, Keiko Kataoka, Tetsuhiro R Yasuma, Eiji Iwata, Ruka Uetani, Shu Kachi, Hiroko Terasaki
ABSTRACT

To study the effect of heparin on the development of laser-induced choroidal neovascularization (CNV) and to assess the underlying molecular mechanisms. Bone marrow transplantation (BMT) was conducted by intravenous injection of green fluorescence protein (GFP)-labeled bone marrow cells (1 × 10(7) cells) into irradiated (9 Gy) C57BL/6J mice. Laser photocoagulation was applied to induce CNV; subsequently, unfractionated heparin or phosphate-buffered saline was injected into mice that did or did not undergo BMT. The area of CNV, distribution of injected heparin, and quantities of infiltrating cells positive for Griffonia simplicifolia (GS) and GFP inside and outside the CNV were evaluated. Effects of heparin on the secretion of VEGF, CCL2, and TNF-α by ARPE19 cells and on the binding of VEGF, CCL2, TNF-α, and their receptors were analyzed in vitro. Intravitreal injection of heparin at higher doses reduced the size of the CNV. Heparin localized at the vascular structures and photoreceptor layers adjacent to the laser scar. Only GS-positive cells infiltrating outside the CNV were reduced significantly, but not those inside the CNV or those expressing GFP. Relative decreases in VEGF and CCL2 levels were observed in media of ARPE19 cells at higher heparin concentrations. In vitro binding assays revealed that heparin and porcine ocular fluid, respectively, suppressed the binding of VEGF to VEGFR2 and CCL2 to CCR2. Intravitreal heparin injection inhibited CNV development. Reduced VEGF and CCL2 secretion by RPE cells and suppression of VEGF-VEGFR2 and CCL2-CCR2 interactions at the laser site mediated by heparin may contribute to the pharmacologic effect.

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Gelatin blocking buffer, for Western blotting, powder blend