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Specific retinal diacylglycerol and protein kinase C beta isoform modulation mimics abnormal retinal hemodynamics in
S E Bursell1, C Takagi, A C Clermont
1Joslin Diabetes Center, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA.
Purpose:
Elevation of diacylglycerol (DAG) and protein kinase C (PKC) levels in diabetic vascular tissue is associated with abnormalities of retinal and renal hemodynamics. The object of this study was to determine whether direct elevation of retinal DAG levels, in the absence of diabetes or hyperglycemia, can mimic the hemodynamic abnormalities normally observed in diabetic rats. Retinal DAG levels were elevated using an inhibitor of DAG kinase that converts DAG to phosphatidic acid. The effectiveness of a specific PKC-beta isoform inhibitor introduced directly into the retinas of diabetic rats in reversing diabetes-related abnormal retinal hemodynamics was also investigated.
Methods:
For retinal blood flow studies, diacylglycerol kinase (DGK) inhibitor R59949, at various concentrations, was injected into the vitreous of nondiabetic Sprague-Dawley rats (n = 33), and a PKC-beta isoform-selective inhibitor LY333531 was injected into the vitreous of rats with streptozotocin (STZ)-induced diabetes of 2 weeks' duration (n = 21). Retinal hemodynamic changes were quantitated using video-based fluorescein angiography. Total DAG levels were assayed from five nondiabetic rat retinas after DGK inhibition and retinal PKC activities were assayed from six diabetic rat retinas after PKC-beta inhibition.
Results:
DGK inhibitor R59949 injected into the vitreous dose dependently increased the mean circulation time (MCT) and decreased retinal blood flow (EC50 = 10(-8) M). After 30 minutes, 10(-5) M R59949 induced a 1.7-fold increase in total retinal DAG levels, compared with the levels in vehicle-injected eyes, an increase in MCT from 0.87 +/- 0.05 seconds to 1.44 +/- 0.12 seconds (P < 0.01) and a decrease in retinal blood flow from 105.3 +/- 6.5 pixel2/second to 64.1 +/- 5 pixel2/second (P < 0.01). The effect of R59949 was sustained for 60 minutes after injection. These retinal hemodynamic parameters after DGK inhibition were comparable to those measured at baseline in rats with STZ-induced diabetes of 2 weeks' duration (MCT = 1.38 +/- 0.20 seconds; retinal blood flow = 68 +/- 11.2 pixel2/second). Intravitreal injection of the PKC-beta inhibitor (LY333531) at 10(-5) M in diabetic rats decreased by a factor of 1.6 the diabetes-related increased PKC activation, decreased the prolonged MCT (0.98 +/- 0.13 seconds; P < 0.01) and increased retinal blood flow (93.4 +/- 14.2 pixel2/second; P < 0.01). The measured retinal circulatory parameters after PKC inhibition in the retina were comparable to those measured at baseline in the nondiabetic rats.
Conclusions:
These results provide direct evidence that DAG elevation and subsequent PKC-beta isoform activation are the primary biochemical sequelae responsible for the development of the abnormal retinal hemodynamics observed in diabetic rats.