Abstract MP250: Reduction In Endothelial Aryl Hydrocarbon Receptor Nuclear Translocator (arnt) Mediates Vascular Dysfunction In Mice With Type 2 Diabetes Mellitus

Background: Endothelial dysfunction, especially at the microvasculature level, is one of the most deleterious events in diabetes. ARNT is a transcription factor that functions as a master regulator of glucose homeostasis, but its role in diabetic vascular complications is poorly understood. Results and method: We found a reduction in ARNT expression in microvascular endothelial cells (MVECs) derived from type 2 diabetic mice (db/db). Thus, we generated an inducible, EC-specific ARNT-knockout mutation ( Arnt ΔEC, ERT2) to address the hypothesis that aberrations in ARNT expression might contribute to the vascular deficiencies associated with diabetes. We show here that loss of ARNT in the endothelium mimics diabetic phenotypes, such as impairs blood flow recovery after hindlimb ischemia, delays wound healing, and exacerbates infiltration of pro-inflammatory neutrophils after myocardial infarction. Interestedly, the degree of these impairments in the KO mice was more remarkable in diabetic animals induced with high-fat chow. In addition, the siRNA-mediated knockdown of ARNT activity reduced tube formation and cell viability measurements in HUVECs cultured under high-glucose conditions. The Arnt ΔEC, ERT2 mutation also reduced measures of cell viability while increasing the production of reactive oxygen species (ROS) in MVECs isolated from mouse skeletal muscle, and the viability of Arnt ΔEC, ERT2 MVECs under high-glucose concentrations increased when the cells were treated with a ROS inhibitor. Conclusion: Collectively, these observations suggest that declines in endothelial ARNT expression contribute to the suppressed angiogenic phenotype in diabetic mice and that the cytoprotective effect of ARNT expression in ECs is at least partially mediated by declines in ROS production. Endothelial ARNT might be a critical mediator of endothelial function and could serve as a therapeutic target for diabetic complications.

Effects of multiple doses of simvastatin and artesunate monotherapy on SCHISTOSOMA MANSONI adult worms during infection in hyperlidemic mice

A single dose of simvastatin and of artesunate monotherapy cause damage to the reproductive system of schistosomes as well as severe tegumental damage in male worms recovered from mice fed high-fat chow. This study aims to investigate whether treatment with multipledose regimes may offer more antischistosomal activity advantages than single daily dosing in mice fed high-fat chow. For this purpose, nine weeks post-infection, Swiss Webster mice were gavaged with simvastatin (200 mg/kg) or artesunate (300 mg/kg) for five consecutive days and euthanized two weeks post-treatment. Adult worms were analyzed using brightfieldmicroscopy, confocal microscopy and scanning electron microscopy, presenting damages caused by simvastatin and artesunate to the reproductive system of males and females as well as tegument alterations, including peeling, sloughing areas, loss of tubercles, tegumental bubbles and tegument rupture exposing subtegumental tissue. The overall findings in this study revealed the potential antischistosomal activity of simvastatin and artesunate against Schistosoma mansoni adult worms, in addition to showing that multiple doses of either monotherapy caused severe damage to the tegument.KEY WORDS: Schistosoma mansoni; hyperlipidemia; simvastatin; artesunate; microscopy.

Repurposing anti-inflammasome NRTIs for improving insulin sensitivity and reducing type 2 diabetes development

Innate immune signaling through the NLRP3 inflammasome is activated by multiple diabetes-related stressors, but whether targeting the inflammasome is beneficial for diabetes is still unclear. Nucleoside reverse-transcriptase inhibitors (NRTI), drugs approved to treat HIV-1 and hepatitis B infections, also block inflammasome activation. Here, we show, by analyzing five health insurance databases, that the adjusted risk of incident diabetes is 33% lower in patients with NRTI exposure among 128,861 patients with HIV-1 or hepatitis B (adjusted hazard ratio for NRTI exposure, 0.673; 95% confidence interval, 0.638 to 0.710; P < 0.0001; 95% prediction interval, 0.618 to 0.734). Meanwhile, an NRTI, lamivudine, improves insulin sensitivity and reduces inflammasome activation in diabetic and insulin resistance-induced human cells, as well as in mice fed with high-fat chow; mechanistically, inflammasome-activating short interspersed nuclear element (SINE) transcripts are elevated, whereas SINE-catabolizing DICER1 is reduced, in diabetic cells and mice. These data suggest the possibility of repurposing an approved class of drugs for prevention of diabetes.

Resveratrol Inhibits MMP3 and MMP9 Expression and Secretion by Suppressing TLR4/NF-κB/STAT3 Activation in Ox-LDL-Treated HUVECs

Aim. Resveratrol is a natural plant polyphenol. The present study investigated the effects of resveratrol on the Toll-like receptor 4- (TLR4-) mediated expression and secretion of matrix metalloproteinases (MMPs) in oxidized low-density lipoprotein- (ox-LDL-) treated human umbilical vein endothelial cells (HUVECs). Methods. Protein expression was analyzed by immunoblotting. The secretion of MMPs was measured by an enzyme-linked immunosorbent assay. The animal experiments were performed with and without resveratrol treatment in high-fat chow-fed mice. Results. Resveratrol inhibited the expression of TLR4, MMP3, and MMP9 in ox-LDL- and lipopolysaccharide- (LPS-) treated HUVECs. Resveratrol reduced the secretion of MMP3 and MMP9 that was induced by ox-LDL and LPS. The TLR4 inhibitor CLI-095 similarly suppressed the expression and secretion of MMP3 and MMP9 in ox-LDL- and LPS-treated HUVECs. Resveratrol attenuated the phosphorylation of the transcription factors nuclear factor-κB (NF-κB) and signal transducer and activator of transcription 3 (STAT3) that was induced by ox-LDL and LPS. Resveratrol recovered Sirt1 expression. In the animal experiments, resveratrol decreased TLR4 expression in the aorta, MMP9 levels in plasma, and vascular structural changes in high-fat chow-fed mice, with no significant effect on plasma MMP3 levels. Conclusion. Resveratrol inhibited the TLR4-mediated expression and secretion of MMP3 and MMP9 in ox-LDL-treated HUVECs. The mechanism of action of resveratrol may be associated with the suppression of NF-κB and STAT3 phosphorylation and restoration of Sirt1 expression. Resveratrol exerts protective effects against vascular structural changes in high-fat chow-fed mice.

Supplementation with Nicotinamide Riboside Reduces Brain Inflammation and Improves Cognitive Function in Diabetic Mice

The purpose of this study is to investigate whether nicotinamide riboside (NR) can improve inflammation and cognitive function in diabetic mice. ICR male mice were fed for 14 weeks with either high-fat chow diet (HF, 60% kcal fat) or standard chow diet (CON, 10% kcal fat). HF, streptozotocin, and nicotinamide were used to induce hyperglycemia. NR or vehicle was delivered via stomach gavage for six weeks. Oral glucose tolerance test, Y-maze test, and nest construction test were conducted before and after the NR treatment period. NR treatment induced down-regulation of NLRP3, ASC, and caspase-1. NR reduced IL-1 expression significantly by 50% in whole brains of hyperglycemic mice. Other inflammatory markers including TNF-α and IL-6 were also attenuated by NR. Brain expression of amyloid-β precursor protein and presenilin 1 were reduced by NR. In addition, NR induced significant reduction of amyloid-β in whole brains of diabetic mice. NR treatment restored hyperglycemia-induced increases in brain karyopyknosis to the levels of controls. Nest construction test showed that NR improved hippocampus functions. Spatial recognition memory and locomotor activity were also improved by NR supplementation. These findings suggest that NR may be useful for treating cognitive impairment by inhibiting amyloidogenesis and neuroinflammation.

Abstract 392: Decreased ARNT Mediates Cardiovascular Endothelial Dysfunction in Diabetic Cardiomyopathy

Background: Endothelial dysfunction is thought to be one of the key risk factors leading to cardiac dysfunction. We have previously shown that ARNT is a critical regulator of cardiac metabolism and its deletion in the heart mimics diabetic cardiomyopathy. Here, we hypothesize that reduced ARNT expression in the endothelium may lead to endothelial dysfunction and contribute to diabetic cardiomyopathy. Methods and Results: Primary cardiac endothelial cells (mCVEC) were isolated from DB/DB mouse hearts and confirmed using vWF staining and flow cytometry. Isolated mCVEC from DB/DB mice showed more than a 50% reduction in ARNT protein levels, suggesting that endothelial ARNT may play a role in diabetic hearts. We generated a mouse with an endothelial specific ARNT deletion (ecARNT -/- ) by crossing ARNT flox/flox mice with Cre recombinase mice under the control of the VE-Cadherin promoter. Deletion of ARNT in the endothelium was achieved by the administration of oral tamoxifen chow. ecARNT -/- mice displayed cardiac hypertrophy and worsened cardiac function after high-fat chow feeding. In vitro studies were done using siRNA technology to knockdown ARNT in mCVEC. Knockdown of ARNT did not increase cell viability at base level, but led to impaired capillary-like endothelial tube formation and reduced cell migration in response to high glucose treatment. Nitric oxide (NO) production (a marker of endothelial dysfunction) and eNOS expression were also reduced after ARNT knockdown. To determine the underlying mechanisms by which ARNT may regulate endothelial metabolism we performed a DNA microarray and confirmed our results using RT-PCR. We discovered a significant induction of NF-kB and its target genes, including ELAM-1 and ICAM-1. These changes are similar to those we observed in mCVEC from DB/DB mouse hearts. Taken together, this data shows that a reduction in ARNT may regulate endothelial dysfunction in the diabetic heart through an inflammatory pathway. Conclusion: Endothelial ARNT may be a critical mediator of endothelial function and could serve as a therapeutic target for diabetic cardiomyopathy.

Abstract 626: Vascular Endothelial HSP60 and Von Willebrand Factor Induction Are Both Involved in Promoting Fatty-Streak Formation in ApoE Null Mice

Objectives: This study was designed to determine the murine atherosclerosis in ApoE null mice with inducible expression of human heat shock protein 60 (hHSP60) in vascular endothelium. Background: Autoimmunity to HSP60 may be involved in eliciting early atherosclerotic lesions. Most classical risk factors of atherosclerosis were previously shown in cell culture models to induce HSP60 expression in vascular ECs and in vivo particularly at regions predilected to lesions. However, it has not been demonstrated directly in an animal model whether HSP60 induction in ECs are capable of influencing lesion formation. Methods: We developed ApoE -/- ::Cdh5-CreER T2 ::G-Lox-HSP60 triple Tg mouse model with tamoxifen-induced hHSP60 expression in ECs. Eight week-old triple TG mice were fed by tamoxifen citrate-contained chow for 2 weeks, followed by high fat chow (HF) for additional 4 and 8 weeks before sacrificed for oil red staining for fatty streaks and IHC staining. Results: In tamoxifen-fed triple Tg mouse, vascular EC expression of hHSP60 increased fatty-streak formation and increased the severity of lesions in comparison with uninduced triple Tg or wildtype ApoE mouse. In triple Tg mouse, we found tamoxifen strongly induced HSP60 expression in ECs of the lesions and to a lesser degree in ECs of veins and other arterial regions not predilected to lesions. Concomitant VWF induction at ECs and subendothelial regions was observed at areas with increased HSP60 expression. Conclusions: hHSP60 induction at vascular ECs accelerates fatty-steak formation in ApoE null mice that may involve increasing local VWF expression.