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    • There two LXR isoforms LXR and LXR

    2018-11-01

    There two LXR isoforms, LXRα and LXRβ, that are closely related sharing 80% homology in their DNA and ligand binding domains (Zhao and Dahlman-Wright, 2010). Relative expression levels of LXRα and LXRβ vary among different tissues. In the retina, both isoforms are expressed with LXRα localized to the nerve fiber layer and RPE while LXRβ is expressed in all retinal layers and RPE as well (Dwyer et al., 2011). Our previous study using STZ-induced T1D model demonstrated that enhanced LXR activity is protective against diabetes-induced retinal vascular damage, whereas LXRα−/− and LXRα/β−/− caused retinal vascular damage and development of acellular amantadine hcl even in the absence of diabetes (Hazra et al., 2012). This study further expands these data demonstrating that LXR activation also prevented the development of acellular capillaries in T2D Leprdb/db model. Importantly, HRECs isolated from diabetic donors had decreased levels of LXRα and LXRβ as compared to control tissue. To further address the role of LXR in diabetes-induced retinal endothelial cell damage, we used BREC. Inflammatory cytokines have a well-accepted role in endothelial cell damage in DR, thus we used cytokine-stimulated BREC as an in vitro model of DR (Joussen et al., 2004). BREC treated with TNFα had reduced expression level of LXRα, that was accompanied by the reduction in RCT genes ABCA1 and ABCG1, as well as upregulation of IL-1β and IL-6 expression levels. Treatment with the LXR activating ligand, DMHCA, led to significant upregulation of ABCA1 and ABCG1, and downregulation of IL-1β and IL-6 expression both in the absence and presence of TNFα. These data are in agreement with a dual role of LXR through both activation of RCT by binding to the LXR response element on ABCA1 and ABCG1 gene promoters, and in stabilizing the NCoR complex to inhibit signaling through NFκB response element on inflammatory gene promoters in retinal endothelial cells. Although both LXRα and LXRβ isoforms are expressed in retinal endothelial cells, there was a more pronounced effect of diabetes and inflammation regulated through LXRα rather than LXRβ expression. In addition to direct effects of the diabetic metabolic milieu, activated macrophages can contribute to inflammatory conditions in the retina (Chakravarthy et al., 2016; Hazra et al., 2013). Both LXRα and LXRβ isoforms are expressed in macrophages. Activation of LXRs in macrophages suppresses the induction of inflammatory genes by LPS and leads to upregulation of RCT genes, ABCA1 and ABCG1, causing cholesterol efflux. Our data confirms that LXR activation using LXR agonist GW3965 inhibits LPS-induced cytokine production by macrophages. We further demonstrate that metabolic stimuli (palmitate, insulin, glucose) lead to reduced RCT genes (ABCA1) and increased cytokine production in the macrophages. Treatment with this LXR ligand reversed both ABCA1 inhibition and increase in IL-6 production in stimulated macrophages, strongly supporting a beneficial role of LXR activation in metabolically challenged macrophages in diabetes. Lastly, CAC dysfunction leading to inadequate retinal vascular repair is an important contributor to DR pathogenesis. We previously demonstrated the importance of LXR signaling in CAC function using “loss of function” studies where LXRα/β−/− animals had significantly decreased CAC migration when compared to wild-type animals. The data presented here show that activation of LXR prevents diabetes-induced loss of CAC migration and function. Taken together, the results of this study demonstrate that LXRs are downregulated in diabetes and activation of LXR signaling has the potential to restore normal cholesterol metabolism as well as to have anti-inflammatory effects in retina and retinal cells, as well as activated macrophages and CACs, thus affecting all multiple aspects of DR pathogenesis. It is important to note that although GW3965 activates LXR and prevents acellular capillary formation in mice, this compound has been shown to cause lipogenesis by activation of the SREBP-1c pathway leading to negative side (Quinet et al., 2004). Unlike GW3965, lipid LXR modulator DMHCA has less effect on SREBP-1, while inducing a robust activation of LXR leading to ABC upregulation (Fig. 4C). Additionally, the Quinet et al., group demonstrated that when DMHCA is administered in the presence of GW3965, DMHCA acts as a gene-selective functional antagonist and partially blocks GW3965-induced increased in SREBP-1c mRNA. Importantly, ABCA1 regulation is not affected and the ligand retains agonist properties for ABCA1 (Quinet et al., 2004). DMHCA also decreased atherosclerosis in the ApoE−/− mouse model without activating fatty acid synthesis in the liver, kidney or intestine (Kratzer et al., 2009). We tested DMHCA in BREC cell culture model in this study. DMHCA was highly effective at activating LXR-dependent RCT genes and at inhibiting expression of inflammatory genes in cytokine-stimulated BREC.