The Antiangiogenic Effect Of Vasostatin On Diabetic Retinopathy (dr) In Mice

Diabetic retinopathy is a frequent complication in the eye of individuals with long-term diabetes and remains the leading cause of blindness in working-age adults throughout the world. Nearly 80% of patients develop diabetic retinopathy after the first 2 decades of diabetes. The vascular changes present in diabetic retinas are well established as evidenced by the early breakdown of the blood-retinal barrier and neoangiogenesis and retinal detachment in the late phases. In this case, antiangiogenic therapy using drugs and food components is a recognized strategy for the prevention of diabetic retinopathy.Vasostatin, the N-terminal domain of calreticulin inclusive of amino acids 1-180, is a potent endogenous inhibitor of angiogenesis and tumour growth. The discovery of its inhibiting angiogenesis effect has made it possible to test the hypothesis that vasostatin may affect development of angiogenesis in diabetic retinas. To address this issue, the following studies were designed and performed.In the first study, streptozotocin-induced diabetic mice were used to evaluate whether locally application of recombinant human vasostatin affected angiogenesis and microvascular functions in diabetic retinas. Weight-matched 8-week-old mice received five intraperitoneal injections of streptozotocin (STZ) (100 mg/kg body wt). Control mice received injections with dissolved buffer alone. After 16 weeks of treatment by STZ, mouse eyes were implanted with 50μl of methylcellulose eye drops containing PBS or vasostatin (final concentration of 0.1mg/ml) were applied three times daily for 2 weeks. Flash electroretinography (FERG) was used to measure retinal microvascular function. For visualization of the retinal vascular pattern, whole retinas were stainied by FITC conjugated Griffonia simplicifolia isolectin B4. Compared with control mice, the amplitudes of Ops of FERG were reduced in diabetic mice. Wave latencies were also extended. Diabetic retinas showed remarkable neovascularization by increasing of blood capillary length and bud of new vessels. All of these abnormalities were significantly inhibited by application of vasostatin in diabetic mice.In the second study, human umbilical vein endothelial cells (HUVEC) were used to evaluate the antiangiogenic mechanism of vasostatin. HUVEC were treated with 0–3mg/ml under hypoxic conditions for 24h. After treatment, cell viability and apoptosis of HUVEC were detected using CCK-8 assay and Hoechst 33342 staining respectively. Cellular proteins were prepared for Western blot analysis to determin the expression of proteins in apoptosis signal pathway. Vasostatin significantly enhanced apoptosis of HUVEC under hypoxia in a dose dependent manner. Western blot analysis revealed that vasostatin suppressed the phosphorylation of Akt, and increased the phosphorylation of Bad in hypoxia-treated HUVEC.These results indicated that locally application of vasostatin may ameliorate functions of diabetic retinas, which was accompanied by an decrease of neovascularization. The antiangiogenic effects of vasostatin are associated with changes in growth factor-dependent Akt/Bad/caspase-3 signaling as well as induction of apoptosis in endothelial cells. Our findings suggest that vasostatin has potential as a therapeutic supplement for preventing diabetic retinopathy, and therefore future clinical study will be required to evaluate the efficacy and safety of vasostatin.