Aldose reductase AR is an NADPH dependent aldo keto reductas

Aldose reductase (AR), is an NADPH-dependent aldo-keto reductase very well studied as a catalyst of glucose conversion to sorbitol in the polyol pathway [11], [12]. In the diabetic lens characterized by chronically high levels of glucose, AR is responsible for production of high levels of sorbitol and associated osmotic and oxidative stress [13], [14]. AR is also implicated in the pathogenesis of diabetic retinopathy, but most likely through its role in promoting inflammation in retinal PKI-402 such as retinal microglia and endothelial cells of the retinal vasculature [15], [16]. In our previous studies of AR-mediated pathogenesis of diabetic eye disease, we observed that a strain of transgenic mice designed for over-expression of human AR (AKR1B1) in the lens developed anterior subcapsular cataracts even in the absence of diabetes and hyperglycemia [17]. Histological analysis of cells associated with the subcapsular cataract revealed high levels of αSMA, a protein that is considered a key marker of cells that have undergone EMT. The aim of the present study was to further evaluate the impact of AR expression on markers of EMT in the lens. To do so we compared the expression of EMT marker genes in primary cultures of LEC from mutant mouse strains that either over-express human AR (AR-Tg) or are null for expression of AR (AR knock out, ARKO). Our studies revealed that elevated AR increases the basal expression levels of EMT marker genes such as αSMA, fibronectin, and Snail. In addition, elevated levels of activated ERK1/2 were observed in cells of AR-Tg mice, suggesting that the EMT-like phenotype of cells containing elevated levels of AR may be influenced by the activation of Smad-independent signaling pathways.
Materials and methods
Results
Discussion Cataract is one of the major causes of blindness among the world [29], [30]. Surgical removal of the opaque cataractous lens is a relatively safe procedure and results in a dramatic improvement in vision and quality of life. However, the wound response of the eye following removal of the degenerate lens can produce unwanted proliferation of lens epithelial cells left in the capsular bag following surgery. These cells undergo EMT as part of the wound response and cause PCO, a fibrotic wrinkling of the capsular bag and interruption of the light path needed to convey visual information to the retina. The end result of PCO is reduced visual acuity and significantly compromised quality of life to the patient and caregivers. Given the prevalence of cataract and the relatively high rate of PCO development following cataract surgery, especially among young patients [31], there is an urgent need for therapies to prevent cellular changes that lead to this blinding condition. Previous studies from our lab and those from Ramana and Srivastava have shown that AR inhibition attenuates cell proliferation, cell migration and EMT process on LECs [9], [32]. In this study, we investigated different level of AR expression on primary LECs. We observed the elevation of TGF-β2 gene in the LECs of AR-Tg mice in the comparison to WT mice (Fig. 1B). Elevation of AR increases flux of AR polyol pathway, which produces more oxidative stress due to NADPH/NADP+ and NAD+/NADH imbalance. Since oxidative stress is one of the mediators to induce TGF-β expression [33], AR-induced TGF-β2 elevation might be the result of oxidative stress (Fig. 1B). Foxe3 and Pax6 are markers expressed in lens and would be attenuated under TGF-β2-induced EMT process [25], [26]. We found both genes were dramatically downregulated in AR-Tg compared to WT mice (Fig. 1C), which suggests the EMT process is elevated by high levels of AR. We further investigated the EMT gene expression in WT and AR-Tg groups. We observed an increase in gene expression including α-SMA, fibronectin and Snail in LEC cultured from lenses of transgenic mice, and the elevated expression levels could be reduced by Sorbinil treatment (Fig. 2A and B). These data suggest that AR positively regulates EMT production and AR inhibition reduces it. Western blotting of fibronectin presented consistent results with gene transcript expression data (Fig. 2C). To further demonstrate the AR effect in vivo, we utilized immunohistochemical staining of α-SMA on the sections from WT and AR-Tg mice. Data showed that α-SMA expression is higher in transgenics compared to WT mice (Fig. 2D). In vitro and in vivo studies demonstrate the positive correlation of AR and EMT production. AR levels are elevated in diabetic patients [34]. Cataract occurs more frequently in diabetic patients and surgical removal of the opaque lens is the most common therapy. Since AR induces EMT marker expression, there would be an increased risk of PCO formation after diabetic cataract removal. In a potentially related area, it is interesting to note that fructose, the end product of AR polyol pathway, has been demonstrated to form advanced glycated end-products (AGEs) much faster than glucose [35]. Raghavan and colleagues recently reported that AGEs in lens capsule enhance EMT of LECs under TGF-β2 treatment [36], [37]. Accordingly, AR-induced AGEs production might be another stimulus for PCO development following cataract surgery.