The functional role of hemojuvelin in acute ischemic stroke.

Our study aimed to establish the role of hemojuvelin (HJV) in acute ischemic stroke (AIS). We performed immunohistochemistry for HJV expression in human brain tissues from 10 AIS and 2 non-stroke autopsy subjects. Plasma HJV was measured in 112 AIS patients within 48 h after stroke. The results showed significantly increased HJV expression in brain tissues from AIS patients compare to non-stroke subjects. After adjusting for clinical variables, plasma levels of HJV within 48 h after stroke were an independent predictor of poor functional outcome three months post-stroke (OR:1.78, 95% CI: 1.03-3.07; P = 0.038). In basic part, Western blotting showed that HJV expression in mice brains was apparent at 3 h after middle cerebral artery occlusion (MCAO), and increased significantly at 72 h. In cultured cortical neurons, expression of HJV protein increased remarkably 24 h after oxygen glucose deprivation (OGD), and small interfering RNAs (siHJV) transfected OGD neurons had a lower apoptotic rate. Importantly, 72 h post-MCAO, HJV knockout mice had significantly smaller infarcts and less expression of cleaved caspase-3 protein compared with wild-type mice. In summary, HJV participates in the mechanisms of post-stroke neuronal injury, and that plasma HJV levels can be a potential early outcome indicator for AIS patients.

Endoplasmic Reticulum Protein TXNDC5 Augments Myocardial Fibrosis by Facilitating Extracellular Matrix Protein Folding and Redox-Sensitive Cardiac Fibroblast Activation.

RATIONALE: Cardiac fibrosis plays a critical role in the pathogenesis of heart failure. Excessive accumulation of extracellular matrix (ECM) resulting from cardiac fibrosis impairs cardiac contractile function and increases arrhythmogenicity. Current treatment options for cardiac fibrosis, however, are limited, and there is a clear need to identify novel mediators of cardiac fibrosis to facilitate the development of better therapeutics. Exploiting coexpression gene network analysis on RNA sequencing data from failing human heart, we identified TXNDC5 (thioredoxin domain containing 5), a cardiac fibroblast (CF)-enriched endoplasmic reticulum protein, as a potential novel mediator of cardiac fibrosis, and we completed experiments to test this hypothesis directly. OBJECTIVE: The objective of this study was to determine the functional role of TXNDC5 in the pathogenesis of cardiac fibrosis. METHODS AND RESULTS: RNA sequencing and Western blot analyses revealed that TXNDC5 mRNA and protein were highly upregulated in failing human left ventricles and in hypertrophied/failing mouse left ventricle. In addition, cardiac TXNDC5 mRNA expression levels were positively correlated with those of transcripts encoding transforming growth factor ß1 and ECM proteins in vivo. TXNDC5 mRNA and protein were increased in human CF (hCF) under transforming growth factor ß1 stimulation in vitro. Knockdown of TXNDC5 attenuated transforming growth factor ß1-induced hCF activation and ECM protein upregulation independent of SMAD3 (SMAD family member 3), whereas increasing expression of TXNDC5 triggered hCF activation and proliferation and increased ECM protein production. Further experiments showed that TXNDC5, a protein disulfide isomerase, facilitated ECM protein folding and that depletion of TXNDC5 led to ECM protein misfolding and degradation in CF. In addition, TXNDC5 promotes hCF activation and proliferation by enhancing c-Jun N-terminal kinase activity via increased reactive oxygen species, derived from NAD(P)H oxidase 4. Transforming growth factor ß1-induced TXNDC5 upregulation in hCF was dependent on endoplasmic reticulum stress and activating transcription factor 6-mediated transcriptional control. Targeted disruption of Txndc5 in mice (Txndc5-/-) revealed protective effects against isoproterenol-induced cardiac hypertrophy, reduced fibrosis (by ≈70%), and markedly improved left ventricle function; post-isoproterenol left ventricular ejection fraction was 59.1±1.5 versus 40.1±2.5 (P<0.001) in Txndc5-/- versus wild-type mice, respectively. CONCLUSIONS: The endoplasmic reticulum protein TXNDC5 promotes cardiac fibrosis by facilitating ECM protein folding and CF activation via redox-sensitive c-Jun N-terminal kinase signaling. Loss of TXNDC5 protects against ß agonist-induced cardiac fibrosis and contractile dysfunction. Targeting TXNDC5, therefore, could be a powerful new therapeutic approach to mitigate excessive cardiac fibrosis, thereby improving cardiac function and outcomes in patients with heart failure.

Activation of AMP-Activated Protein Kinase by Adenine Alleviates TNF-Alpha-Induced Inflammation in Human Umbilical Vein Endothelial Cells.

The AMP-activated protein kinase (AMPK) signaling system plays a key role in cellular stress by repressing the inflammatory responses induced by the nuclear factor-kappa B (NF-κB) system. Previous studies suggest that the anti-inflammatory role of AMPK involves activation by adenine, but the mechanism that allows adenine to produce these effects has not yet been elucidated. In human umbilical vein endothelial cells (HUVECs), adenine was observed to induce the phosphorylation of AMPK in both a time- and dose-dependent manner as well as its downstream target acetyl Co-A carboxylase (ACC). Adenine also attenuated NF-κB targeting of gene expression in a dose-dependent manner and decreased monocyte adhesion to HUVECs following tumor necrosis factor (TNF-α) treatment. The short hairpin RNA (shRNA) against AMPK α1 in HUVECs attenuated the adenine-induced inhibition of NF-κB activation in response to TNF-α, thereby suggesting that the anti-inflammatory role of adenine is mediated by AMPK. Following the knockdown of adenosyl phosphoribosyl transferase (APRT) in HUVECs, adenine supplementation failed to induce the phosphorylation of AMPK and ACC. Similarly, the expression of a shRNA against APRT nullified the anti-inflammatory effects of adenine in HUVECs. These results suggested that the role of adenine as an AMPK activator is related to catabolism by APRT, which increases the cellular AMP levels to activate AMPK.

Identification of adenine modulating AMPK activation in NIH/3T3 cells by proteomic approach.

AMP-activated protein kinase (AMPK) is a metabolic master switch maintaining the energy homeostasis in cells and thought to modulate cellular response to stresses. Adenine as well as a pharmacological AMPK activator, 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), induced the phosphorylation of AMPK and acetyl-CoA carboxylase in NIH/3T3 cells. Administration of adenine or AICAR increased the expression and translocation of glucose transporter 4, enhanced the cellular glucose uptake, and elevated the intracellular ATP level. To better understand the proteomic changes in response to exogenous adenine treatment, we performed two-dimensional difference gel electrophoresis (2DE-DIGE) and grouped protein spots with similar intensities prior to MS analysis. These process allowed us to exclude these constant expressed proteins, reduce the coverage from abundant signals and increase the identification of middle/lower expressed proteins. Bioinformatics analysis on the proteomic alterations suggested that both of adenine and AICAR could induce up-regulation of a panel of proteins associated with glucose metabolism. We also found that adenine upregulated expression of the glycolytic enzyme, hexokinase 2, indicating a link between adenine and AMPK-mediated glycolysis. Taken together, by demonstrating the adenine-mediated proteome changes in NIH/3T3 cells, our study provides useful information for the characteristics of adenine-induced AMPK activation and development of efficient AMPK activator. BIOLOGICAL SIGNIFICANCE: AMPK is a fuel sensing enzyme that responds to a central role of energy homeostasis and contributes to the acceleration of insulin signaling. Recently, we have shown that exogenous adenine exerted anti-inflammatory effects through activation of AMPK, suggesting the treatment is a potent therapeutic strategy against hyperglycemia. Adenine had similar effects with 5-amino-4-imidazole-carboxamide riboside (AICAR, an AMPK activator) in modulating glucose uptake via AMPK-mediated signaling. In this study, we performed a 2DE-DIGE/MS-based approach to investigate the mechanism of exogenous adenine in NIH/3T3 cells. Our results provide evidence of a novel role for adenine in AMPK-mediated signaling and glucose metabolism and suggest potential therapeutic perspectives in insulin resistance and metabolic dysfunctions.

Aliskiren reduces portal pressure in portal hypertensive rats.

BACKGROUND: Aliskiren is a direct renin inhibitor used in the treatment for arterial hypertension. It can also augment nitric oxide (NO) production, which plays a crucial role in the pathogenesis of portal hypertension and modulation of porto-systemic collaterals. This study investigated the effects of aliskiren on portal pressure and porto-systemic collaterals of portal vein-ligated (PVL) rats. MATERIALS AND METHODS: Sham-operated and PVL rats received aliskiren (50 mg/kg per day) or distilled water (control) treatment for 10 days. The mean arterial pressure and portal pressure were measured by catheterization of the right femoral artery and mesenteric vein, while the superior mesenteric arterial blood flow was measured by Doppler technique. The left adrenal vein and superior mesentery artery were dissected for mRNA study. The PVL rats also underwent preincubation with (i) Krebs solution (control); (ii) 10(-4) M aliskiren; or (iii) 10(-4) M aliskiren plus nonselective NO inhibitor N(ω)-nitro-L-arginine (10(-4) M), followed by the addition of arginine vasopressin (AVP) to evaluate the collateral vascular responsiveness. RESULTS: Aliskiren had systemic arterial pressure- and portal pressure-lowering effects in PVL rats. Superior mesentery arterial resistance also decreased. The constitutive NO synthase was enhanced in the left adrenal vein and superior mesentery artery after aliskiren treatment. Aliskiren attenuated the collateral vasoconstrictive effects of AVP, but the vasodilatory effects were abolished after nonselective NO synthase inhibition. CONCLUSIONS: Chronic aliskiren use reduces portal pressure in portal hypertensive rats partly due to the modulation of splanchnic and collateral NO synthase.

Selective cyclooxygenase inhibition improves hepatic encephalopathy in fulminant hepatic failure of rat.

Prostaglandin plays an important role in the pathogenesis of hepatic encephalopathy. This study investigated the therapeutic effects of selective cyclooxygenase (COX) inhibitor on hepatic encephalopathy in thioacetamide-induced fulminant hepatic failure (FHF) rats. The selective COX-1 inhibitor (SC-560), COX-2 inhibitor (NS-398) or distilled water (control) was administered in the normal and FHF rats. The mortality rates were calculated and severity of hepatic encephalopathy was evaluated using Opto-Varimex activity sensors. Besides, the levels of blood ammonia, 6-keto-prostaglandin-F(1α) (PGF(1α), active metabolite of prostacyclin), tumor necrosis factor α (TNF-α) and liver biochemistry tests were measured. The hepatic mRNA expressions of nitric oxide synthase and COX were determined, and the liver histopathological changes were examined. The liver biochemistries and motor activities were similar among COX-1, COX-2 treated and control groups. SC-560 treatment improved the survival of FHF rats (mortality rates: SC-560 group 0%, control 33%; P=0.037). Besides, SC-560 treatment improved hepatic encephalopathy and decrease plasma levels of PGF(1α), but did not change TNF-α levels. There were no significant differences in liver biochemistry and ammonia levels except that the aspartate aminotransferase levels were lower in the NS-398 treated group. Both hepatic COX-1 and COX-2 mRNA expressions were attenuated after SC-560 treatment. The decreased COX-2 and increased constitutive nitric oxide synthase mRNA expressions were found after NS-398 treatment. Besides, the histopathology of liver got improved after selective COX inhibition. In conclusion, COX-1 inhibition by SC-560 decreases the mortalities and improves motor activities, suggesting COX-1, rather than COX-2, plays a major role in hepatic encephalopathy of FHF rats.