Abstract
Purpose:
To learn whether the increased TGF-β signaling induced by diabetes in retinal vessels contributes to microangiopathy and is a candidate target for interventions. We had detected an increase in TGF-β signaling in rat retinal vessels after 3 months of diabetes (Gerhardinger et al 2009) and the known roles of TGF-β in vascular homeostasis and pathologies make the cytokine a prime potential contributor to diabetic microangiopathy.
Methods:
TGF-β signaling and gene expression were evaluated in fresh retinal vessels isolated from rats with 3 months of streptozotocin-diabetes untreated or treated with SM16, a selective inhibitor of the type I TGF-β receptor ALK5. SM16 was given for 3 weeks mixed with the chow to deliver a dose of 6 mg/Kg bw/day (Anscher et al 2008). Diabetic rats received insulin to prevent catabolism. SM16-treated and untreated nondiabetic rats were used as controls. Smad2/3 phosphorylation and protein levels were measured by Western blot, gene expression by PCR Array (Rat Endothelial Cell Biology RT2 ProfilerTM) and RealTime PCR.
Results:
The retinal vessel preparations were similarly enriched in endothelial markers --at least 8-fold over the whole retina-- in all experimental groups. In rats with 3.5 months of diabetes and HbA1c double the control values, only three genes changed their expression level more than 2 fold with P < 0.05 when compared to control rats: endothelin 2; fibroblast growth factor 2; and placental growth factor (PGF), a VEGF family member that enhances vascular permeability. Treatment with SM16 prevented the diabetes-induced excess Smad2/3 phosphorylation without lowering the basal, and prevented PGF overexpression. In addition, SM16 changed the expression of several genes that had not been altered by diabetes alone, and in directions that would make the endothelium pro-inflammatory, thrombogenic, permeable, and prone to apoptosis (increased monocyte chemoattractant protein-1; and decreased tissue factor pathway inhibitor, thrombomodulin, occludin, VE-cadherin, Tie-2, and VEGF A). The above SM16 effects were not observed in normal rats treated with SM16.
Conclusions:
The effects of SM16 on the retinal vessels of diabetic rats were not mere drug toxicity. Rather, they suggest that the small increase in TGF-β signaling induced by diabetes has a protective role for the vascular endothelium.
Keywords: 499 diabetic retinopathy •
543 growth factors/growth factor receptors