Localization of FN-SiRNA in rat retinal vascular cells after intravitreal injection
Retinal sections of the intravitreally injected Cy3-labeled FN-siRNA performed together with 4',6-diamidino-2-phenylindole (DAPI) were examined using confocal microscopy. FN-siRNA localization in the retinal vascular cells of the rat eyes was observed within 2 h post-injection. Based on the double stain from Cy3 and DAPI, fluorescence from the siRNA could be assigned to the nuclei of retinal vascular endothelial cells and pericytes (). The luminally located endothelial cell was distinguished from the abluminally located pericytes by its elongated nucleus.
Dose response study showing FN-SiRNA efficacy
The effectiveness of the FN-siRNA strategy was tested using increasing concentrations (0.5–6.0 µM) of siRNA to identify an optimal dose for ~30% downregulation of FN expression. A reduced FN level was observed in rat retinas after intravitreal injection at concentrations ≥1 μM at 1 week post-injection (). The 1 µM, 3 µM, and 6 µM dose significantly reduced FN expression (79±11% of control, p=0.03; 68±7% of control, p=0.01; 73±9% of control, p=0.02, respectively).
Time course study showing FN-SiRNA efficacy
A time course (0–6 weeks) study following intravitreal injection of FN-siRNA showed significant downregulation of FN expression in the retinas. After 1 week, retinal FN expression was reduced by approximately 35%, which persisted to the 6 week time point (). Scrambled siRNA showed no effect on the retinal FN protein level at any time point and showed no significant difference between the control (uninjected eyes) and the eyes injected with FN-siRNA.
Effect of FN-SiRNA on retinal FN protein level
Western blot analyses indicated a significant increase in FN expression in the retinas of diabetic rats compared to those of non-diabetic control rats (188±14.2% of control versus 100±7.4% of control, p<0.002), respectively. A significant downregulation in FN expression was observed in the retinas of diabetic rats that were intravitreally injected with FN-siRNA compared to those of uninjected diabetic control eyes or those of diabetic eyes injected with scrambled siRNA (145±19.9% of control versus 188±14.2% of control, p=0.03; 145±19.9% of control versus 183±15% of control, p=0.03, respectively; ).
Effect of reduced FN on retinal capillary basement membrane thickness
An electron microscopy analysis showed a significant increase (~40%) in retinal capillary BM thickness in the diabetic rats compared to those of non-diabetic control rats (72.5±5.0 nm versus 51.5±4.8 nm, p<0.001). Upon intravitreal injection with FN-siRNA, a significant reduction (~75%) in retinal capillary BM thickness was observed in the retinas of diabetic rats compared to those of uninjected control diabetic rats and diabetic rats intravitreally injected with scrambled siRNA (56.4±2.8 nm versus 72.5±5.0 nm, p=0.03; 56.4±2.8 nm versus 73.7±3.9 nm, p<0.01, respectively; ).
Effect of FN-SiRNA on the development of vascular lesions
In this study, we assessed two characteristic lesions of diabetic retinopathy—pericyte loss and acellular capillaries—both of which showed significant improvement in retinas treated with FN-siRNA. The increased pericyte loss (151±18%, p=0.02 of the control) and number of acellular capillaries (175±16%, p=0.01 of the control) observed in diabetic retinas was significantly reduced in retinas of diabetic rats treated with FN-siRNA (pericyte loss: 115±11%, p=0.02 of the control; acellular capillaries: 118±9%, p=0.01 of the control). The number of acellular capillaries () and the pericyte loss () in diabetic rats that received scrambled siRNA did not differ from those of the diabetic rats. There was a strong correlation between decreased pericyte loss and reduced BM thickening (r=0.71), as well as between a decreased number of acellular capillaries and reduced BM thickening (r=0.82).
Efficacy and safety of long-term application of FN-SiRNA
Retinas injected with siRNA and examined 1, 2, 4, and 6 weeks post-injection showed no physical evidence of redness or opacity in the eyes, and retinal capillaries and sections examined in parallel showed no infiltration of macrophages.
Blood glucose, bodyweight, and HbA1c levels
Fasting blood glucose levels were measured every alternate day and showed hyperglycemic conditions in diabetic animals (307±48 mg/dl) compared to normal animals (122±21 mg/dl). The HbA1c level measured at the three-month interval also confirmed a hyperglycemic state in diabetic animals (11.2±1.6%) versus normal animals (5.3±1.1%; ). A reduced bodyweight was observed in the diabetic animals, which indicates a diabetic condition in these animals.
Figure 8 HbA1c levels in experimental animals. HbA1c levels measured at the end of the study confirmed hyperglycemic status in diabetic animals. Diabetic animals injected with fibronectin (FN)-siRNA or scrambled siRNA also displayed a similar hyperglycemic condition. (more ...)