Abstract
Purpose: :
Tissue factor (TF) dependent coagulation is activated when factor VII (VII) binds to TF on cells in the injured area, resulting in a proteolytically active TF/VIIa–complex that generates a fibrin clot. Recent data suggest that TF signaling can occur independent of downstream coagulation protease signaling. This TF dependent upstream signaling is mediated by protease activated receptors (PAR) 1 and 2. It is also known that phosphorylation of TF cytoplasmic tail is associated with PAR2 in neoangiogenesis in diabetic retinopathy. Here we investigate if deletion of TF cytoplasmic tail results in accelerated angiogenesis in the mouse model of oxygen induced retinopathy (OIR). Using specific inhibitors we further investigated whether TF–VIIa is involved in this effect and if it is mediated by PAR1 and/or PAR2.
Methods: :
Neonatal TFΔCT (lacking TF cytoplasmic tail), PAR1 and PAR2 knockout, TFΔCT PAR1 and TFΔCT PAR2 double mutants and wild type (WT) littermate mice were exposed to 75 % hyperoxia at P7 for 5 days. Eyes were harvested at P12 and P17, retinas dissected, fixed, and stained with fluorescein conjugated isolectin Griffonia simplicifolia. Retinas were imaged using confocal microscopy and areas of obliteration and neo–vascular tufts quantified. NAPc2, a TF–VIIa inhibitor, was administered IP daily after hyperoxia exposure.
Results: :
Deletion of TF cytoplasmic domain resulted in significantly accelerated revascularization of avascular retina. In TFΔCT/PAR2 double mutants, retinal revascularization rate was reverted back to the WT level, whereas in TFΔCT/PAR1 double mutants it remained comparable to TFΔCT mice. The revascularization rate in PAR1 and PAR2 deficient mice was comparable to WT mice. This demonstrates that accelerated angiogenesis in TFΔCT mice is PAR2, but not PAR1, mediated. Since PAR2 can be activated by ligands other than TF–VIIa complex, we tested if accelerated angiogenesis in the TFΔCT mice is mediated by proteolytic activation of PAR2 by the TF–VIIa complex. The specific inhibitor to TF–VIIa, NAPc2, delayed revascularization in TFΔCT mice and resulted in the wt vascular phenotype.
Conclusions: :
PAR2 activation by the TF–VIIa complex results in accelerated angiogenesis in TFΔCT mice in the OIR model, and TF dependent angiogenesis is dependent on PAR2 signaling. Since TF cytoplasmic tail phosphorylation is characteristic of pathological vessels in diabetic tissue, we suggest that inhibition of TF–VIIa–PAR2 signaling pathways may have therapeutic value for the treatment of ocular neovascular diseases.
Keywords: retinal neovascularization • retina