PAR–2 Mediated Angiogenesis in the OIR Model is Regulated by the Cytoplasmic Tail of Tissue Factor

Abstract

Abstract: : Purpose: The cytoplasmic tail of tissue factor has been shown to regulate PAR–2 mediated angiogenesis during development. To test if they have similar roles in pathological neo–vascularization, the role of tissue factor (TF) cytoplasmic tail and protease–activated receptor–2 (PAR–2) on retinal revascularization and neo–vascularization was tested in the mouse model for oxygen induced retinopathy (OIR). Methods: Neonatal TFΔCT mice, PAR–2–deficient mice, TFΔCT /PAR–2 deficient double mutants, and wild type (wt) littermates were exposed to 75 % hyperoxia at P7 for 5 days. Since TFΔCT have an accelerated rate of retinal vascularization, mice were placed in hyperoxia at P5 when retinal vascularization is comparable to a P7 wild type. At various timepoints after return to normoxia retinas were dissected, fixed in 4% PFA and methanol, and incubated with fluorescein conjugated isolectin Griffonia simplicifolia. Retinas were imaged using confocal microscopy and areas of obliteration and neo–vascular tufts were quantified from P17 retinas, 5 days after return to normoxia. Results: Immediately after hyperoxia exposure the extent of obliteration was similar in all mice. Five days later the area of obliteration was significantly smaller (66 %) in TFΔCT retinas compared to wt retinas, suggesting that loss of TF cytoplasmic tail results in enhanced revascularization of obliterated areas. Similar to developmental neovascularization, the enhanced revascularization observed in TFΔCT mice was dependent on PAR–2 signaling since TFΔCT/PAR–2 double knockouts reverted the extent of revascularization to the wt retinal vascular phenotype. No significant alteration in the extent of obliteration was observed in PAR–2 knockouts. The formation of neovascular tufts seemed to occur faster in TFΔCT mice than in the other mice. In TFΔCT/ PAR–2 double knockouts, tuft formation was reverted to wt level. Conclusions: Similar to that observed in developmental angiogenesis, regulation of PAR–2 signaling by the TF cytoplasmic tail is critical for regulation of pathological angiogenesis in the mouse OIR model.

Keywords: retinal neovascularization • transgenics/knock-outs • retina