The Basis Of The Renin - Angiotensin System And Diabetic Retinopathy Study

AIMS Diabetic retinopathy, the most severe of the several ocular complications of diabetes, is a common cause of visual impairment and blindness. Although the pathogenesis of diabetic retinopathy is incompletely understood, recent evidence has suggested a pathogenic role for the renin-angiotensin system (RAS) and vascular endothelial growth factor (VEGF) in the eye in response to chronic hyperglycemia. Angiotensin â…¡ is a known stimulus for the expression of VEGF in diabetic retinas, angiotensin converting enzyme inhibitor treatment of diabetic rats reduced diabetes-associated changes in VEGF expression and vascular permeability. This action of angiotensin â…¡ is mediated by the angiotensin â…¡ type 1 (AT₁) receptor. However, the retinal status of angiotensin â…¡ type 2 (AT2) receptor in diabetic rat is still unclear, and the role of AT₂ receptor in inducing VEGF expression has been controversial. The aim of the present study was to evaluate the status of AT₂ receptor in adult diabetic rats, and to access the effects of AT₂ receptor blockade on retinal VEGF expression in experimental diabetic rats.METHODS Eight-week-old male Sprague-Dawley (SD) rats were injected intravenously with strepozotocin at a dose of 55mg/kg in citrate buffer to induce diabetes (n=24). Control rats received an intravenous injection of citrate buffer alone (n=8). Diabetic rats were further randomly allocated to no treatment or treatment with either an AT₁ or AT₂ receptor antagonist (n=8 per group). The AT₁ receptor antagonist,valsartan, was administered at a dose of 30mg/kg per day by once-daily oral gavage. The AT₂ receptor antagonist, PD123319, was administered at a dose of 830ng/min delivered by subcutaneous osmotic minipump implanted into the mid scapular region. The rats were sacrificed four weeks after the induction of diabetes. The retinal VEGF gene and protein expression were accessed by real time reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry respectively. The retinal expression of AT₁ and AT₂ receptors were evaluated by the same methods in control and 4 weeks diabetic rats.RESULTS VEGF gene expression was increased approximately 3-fold in the retina of diabetic rats when compared with control animals. Treatment with either valsartan or PD123319 attenuated VEGF gene expression to a similar level to that observed in the control rats. VEGF staining was predominantly in the inner limiting membrane, ganglion cell layer, and inner nuclear layer. Weak positive staining for VEGF was also found in the outer limiting membrane and retinal pigment epithelium. Retinal VEGF protein expression was increased significantly in diabetic rats compared with control animals and the increase was attenuated by treatment with either valsartan or PD123319. There was no significant difference in VEGF immunostaining in the retina of diabetic animals treated with either AT₁ or AT₂ receptor antagonist. Real-time RT-PCR revealed that AT₁ and AT₂ receptor mRNA were detected in the retinas of adult rats and increased in diabetic animals. Immunostaining for both AT₁ and AT₂ receptors was observed in blood vessels, inner limiting membrane, and the inner nuclear layer. There were no apparent changes in the intensity or distribution of immunostaining for the AT₁ and AT₂ receptors between the control and diabetic group.CONCLUSIONS These findings suggest that VEGF expression is modulated by AT₁ and AT₂ receptors, thereby implicating angiotensin â…¡ receptor subtypes in retinal diseases such as diabetic retinopathy. The blockade of the AT₂ receptor as well as AT) receptor may confer end organ protection in various retinal diseases.AIMS Angiotensin II , the major effector molecule of the renin-angiotensin system ( RAS ), is a known stimulus for the expression of vascular endothelial growth factor ( VEGF ). There is evidence that angiotensin II , VEGF and their cognate receptors participate in retinal angiogenesis. However, there remains controversy as to the role of the individual angiotensin II receptor subtypes in mediating this effect. The aims of the present study were, first, to explore the in vivo effects of angiotensin II on expression of VEGF in retina and second, to study the relative roles of the AT] receptor and AT2 receptor subtype on VEGF expression.METHODS Eight-week-old male Sprague-Dawley (SD) rats were infused with either angiotensin II ( tv=24 ) or vehicle ( n=8 ) for 14 days at a dose of 58.3ng/min delivered by subcutaneous osmotic minipump implanted in the mid scapular region. Angiotensin Il-infused rats received no treatment ( n=8 ), an ATi antagonist valsartan ( 30mg/kg/d ), or an AT2 receptor antagonist PD123319 (830ng/min). PD123319 was administered by a osmotic minipump implanted intraperitoneally. Gene expression of VEGF was assessed by real time reverse transcription polymerase chain reaction (RT-PCR). Protein expression was assessed by immunohistochemistry.RESULTS Angiotensin II infusion was associated with a significantreduction of body weight and elevation of systolic blood pressure ( SBP ). Treatment with valsartan prevented the decrease in body weight and increase in SBP. The AT2 receptor antagonist, PD123319, did not significantly influence the body weight and SBP in angiotensin II-infused rats. Gene and protein expression of retinal VEGF were increased by angiotensin II infusion. Treatment with either valsartan or PD123319 attenuated retinal VEGF gene and protein expression.CONCLUSIONS These findings extend the increasing evidence that the AT2 receptor , in addition to the AT] receptor subtype, plays an important role in mediating the Angiotenin II induced VEGF overexpression in retina. The blockade of the AT2 receptor as well as ATI receptor may confer end organ protection in various retinal diseases.AIMS Retinopathy is the most common chronic microvascualr complication of diabetes. In the normal retina, angiogenic homeostasis is controlled by the balance between angiogenic stimulators, such as vascular endothelial growth factor (VEGF), and angiogenic inhibitors, such as pigment epithelium-derived factor (PEDF). PEDF, initially identified as a potential neurotrophic factor, was shown to have potent anti-angiogenic activity as it specially inhibited the migration of endothelial cells, an essential step in angiogenesis. Increased intraocular expression of PEDF by gene therapy inhibits retinal and choroidal neovascularization. Intraocular level of PEDF was reduced in patients with angiogenic eye disease, including proliferative diabetic retinopathy (PDR). The content of PEDF in the aqueous humor of diabetic patients strongly predicts who among them will develop progression of retinopathy. It is previously demonstrated that the blockade of renin-angiotensin system (RAS) can reduce the expression of VEGF in diabetic retina. However, the effect of RAS on PEDF expression is still unknown. In this study, we analyzed the retinal expression PEDF in long term diabetic rats before PDR develops, and accessed the effect of angiotensin converting enzyme inhibitor (ACEI) on PEDF expression.METHODS Eight-week-old male Sprague-Dawley (SD) rats were injected intravenously with strepozotocin at a dose of 55mg/kg in citrate buffer to induce...