Cellular metabolic memory occurs in diabetic microvascular and macrovascular complications, but the underlying mechanisms remain unclear. Here, we investigate the role of sirtuin 1 (SIRT1) and metformin in this phenomenon. In bovine retinal capillary endothelial cells (BRECs) and retinas of diabetic rats, the inflammatory gene, nuclear factor-κB (NF-κB), and the proapoptotic gene, Bax, induced by hyperglycemia, remained elevated after returning to normoglycemia. BRECs with small interfering RNA–mediated SIRT1 knockdown had increased sensitivity to hyperglycemia stress, whereas SIRT1 overexpression or activation by metformin inhibited the increase of mitochondrial reactive oxygen species–mediated glyceraldehyde-3-phosphate dehydrogenase by poly (ADP-ribose) polymerase (PARP) activity through the upregulation of liver kinase B1/AMP-activated protein kinase (LKB1/AMPK), ultimately suppressing NF-κB and Bax expression. Furthermore, we showed that hyperglycemia led to PARP activation, which in turn may have downregulated SIRT1. Of importance, this study also demonstrated that metformin suppressed the “memory” of hyperglycemia stress in the diabetic retinas, which may be involved in the SIRT1/LKB1/AMPK pathway. Our data suggest that SIRT1 is a potential therapeutic target for the treatment of the cellular metabolic memory, and the use of metformin specifically for such therapy may be a new avenue of investigation in the diabetes field.

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BackgroundPancreatitis-associated protein (PAP) is a pancreatic secretory protein belongs to the group VII of C-type lectin family. Emerging evidence suggests that PAP plays a protective effect in inflammatory diseases. In the present study, we newly identified a 16-amino-acid peptide (named PAPep) derived from C-type lectin-like domain (CTLD) of human PAP with potent anti-inflammatory activity using both in vivo and in vitro assays.Methodology/Principal FindingsWe assessed the anti-inflammatory effect of PAPep on endotoxin-induced uveitis (EIU) in rats and demonstrated that intravitreal pretreatment of PAPep concentration-dependently attenuated clinical manifestation of EIU rats, reduced protein leakage and cell infiltration into the aqueous humor (AqH), suppressed tumor necrosis factor (TNF)-α, interleukin (IL)-6, intercellular adhesion molecule-1 (ICAM-1) and monocyte chemoattractant protein (MCP)-1 production in ocular tissues, and improved histopathologic manifestation of EIU. Furthermore, PAPep suppressed the LPS-induced mRNA expression of TNF-α and IL-6 in RAW 264.7 cells, inhibited protein expression of ICAM-1 in TNF-α-stimulated human umbilical vein endothelial cells (HUVECs) as well as U937 cells adhesion to HUVECs. Western blot analysis in ocular tissues and different cell lines revealed that the possible mechanism for this anti-inflammatory effect of PAPep may depend on its ability to inhibit the activation of NF-kB signaling pathway.Conclusions/SignificanceOur studies provide the first evidence that the sequence of PAPep is within the critically active region for the anti-inflammatory function of PAP and the peptide may be a promising candidate for the management of ocular inflammatory diseases.

a b s t r a c tEndotoxin-induced uveitis (EIU) is an animal model of acute ocular inflammation for the study of human endogenous anterior uveitis. The mechanisms accounting for the development of ocular inflammation remain hazy. MicroRNAs (mi-RNAs) have been proposed as novel regulators of inflammation. It remains unclear whether a microRNA-mediated regulatory mechanism is involved in LPS-induced EIU. In this study, we report that miR-93 expression in the eyes of EIU rats and LPSstimulated macrophages is significantly decreased. We also show that miR-93 inhibits NF-jB activation and pro-inflammatory cytokines by targeting IRAK4 expression. We further demonstrate that miR-93 inhibits IRAK4 expression by binding directly to the 3 0 -UTR of IRAK4. Our findings suggest that miR-93 is a negative regulator of the immune response in EIU.

BackgroundUveitis is a potentially sight-threatening form of ocular inflammation that affects the uvea in the wall of the eye. Currently available treatments for uveitis have exhibited profound adverse side effects. However, KS23 is a novel 23-amino-acid anti-inflammatory peptide derived from adiponectin that may have the capability to function as a safe alternative to these existing treatment options. We, therefore, evaluated the preventive effect of KS23 in experimental autoimmune uveitis (EAU).MethodsEAU was induced in mice via immunization with the peptide interphotoreceptor retinoid binding protein 161–180 (IRBP161–180). KS23 was then administered every 2 days via intraperitoneal injection to induce protection against EAU. Clinical and histopathological scores were employed to evaluate the disease progression. Inflammatory cytokines were also quantified using ELISA, and the expression levels of specific chemokines and chemokine receptors were assessed via qRT-PCR. In addition, the proportions of Th1 and Th17 cells were detected via flow cytometry, and the expression levels of specific proteins were quantified from the retina of mice using western blot analysis, to elucidate the specific mechanism of action employed by KS23 to suppress the inflammation associated with EAU.ResultsKS23 was found to significantly improve EAU-associated histopathological scores, while decreasing the expression of pro-inflammatory cytokines (IFN-γ, TNF-α, IL-6, and IL-17A), chemokines (LARC, RANTES, MIG, IP-10), and chemokine receptors (CCR6 and CXCR3). The proportions of Th1 and Th17 cells were also suppressed following intraperitoneal injection with KS23. The anti-inflammatory mechanism employed by KS23 was determined to be associated with the activation of AMPK and subsequent inhibition of NF-κB.ConclusionsKS23 decreased the proportions of Th1 and Th17 cells to effectively ameliorate the progression of EAU. It may, therefore, serve as a promising potential therapeutic agent for uveitis.

Plasma and vitreous LRG1 levels were elevated in patients with PDR. Leucine-rich-α2-glycoprotein (LRG1) might be a potential risk-warning marker for PDR.

Endothelial glycocalyx plays a significant role in the development and progression of diabetic complications. Endomucin (EMCN) is an anti-inflammatory membrane glycoprotein that is mainly expressed in venous and capillary endothelial cells. However, the function of EMCN in diabetic retinopathy (DR) progression is still completely unknown. We first investigated the change of EMCN expression in the retina and human retinal microvascular endothelial cells. We then overexpressed EMCN in the retina with adeno-associated virus and induced DR with streptozotocin (STZ). We analyzed EMCN's effect on the integrity of endothelial glycocalyx under conditions of DR. Furthermore, we investigated EMCN's protective effect against inflammation and blood-retinal barrier (BRB) destruction. We found that EMCN is specifically expressed in retinal endothelial cells and that its levels are decreased during hyperglycemia in vitro and in vivo. Overexpression of EMCN can restore the retinal endothelial glycocalyx of STZ-induced diabetic rats. Furthermore, EMCN overexpression can decrease leukocyte-endothelial adhesion to ameliorate inflammation and stabilize the BRB to inhibit vessel leakage in rats with DR. EMCN may protect patients with diabetes from retinal vascular degeneration by restoring the endothelial glycocalyx. EMCN may thus represent a novel therapeutic strategy for DR because it targets endothelial glycocalyx degradation associated with this disease.

a b s t r a c tThrombomodulin (TM) is a single-transmembrane glycoprotein receptor for thrombin, which is best known as a cofactor for thrombin-mediated activation of anticoagulant protein C. C-type lectin-like domain (CTLD) of TM has distinct coagulation/fibrinolysis-independent anti-inflammatory properties. Here we found anti-inflammatory effects of a novel peptide (GC31) from CTLD of TM in endotoxin-induced uveitis, which was characterized by a reduction of leukocyte counts, protein concentration, tumor necrosis factor (TNF)-a and monocyte chemoattractant protein (MCP)-1 levels in aqueous humor. Through in vitro experiments, we further found that GC31 suppressed TNF-a and interleukin (IL)-6 expressions in lipopolysaccharide (LPS)-stimulated macrophage-like RAW264.7 cells and interrupted LPS-induced nuclear factor-jB (NF-jB) activation. These data indicate a beneficial role of peptide GC31 in preventing intraocular inflammatory response, especially uveitis.