Chronic Restraint Stress Induces Gastric Mucosal Inflammation with Enhanced Oxidative Stress in a Murine Model.

BACKGROUND: Although the underlying mechanisms of chronic stress are still unknown, this condition has been related to the pathophysiology of gastric mucosal inflammation, whose development is accelerated by oxidative stress. The present study investigates how chronic stress influences gastric mucosal oxidative stress and inflammation. METHODS: Eight-week-old C57BL/6J male mice were subjected to two-week intermittent restraint stress. The expressions of CD11b (a specific for monocyte/macrophage), monocyte/macrophage cell surface markers (CD68 and F4/80), NADPH oxidase-4 (Nox-4) and 8-hydroxy-2'-deoxyguanosine (8-OHdG, a sensitive biomarker of oxidative stress) were determined using immunohistochemistry, RT-PCR, and enzyme-linked immunosorbent assay, respectively. The expressions of antioxidant enzymes, such as superoxide dismutase, catalase, and glutathione peroxidase, were examined by RT-PCR and Western blotting. The expressions of proinflammatory cytokines, including monocyte chemoattractant protein-1 (MCP-1), interleukin-1ß (IL-1ß), and tumor necrosis factor-α (TNF-α), were determined using immunohistochemistry and RT-PCR, respectively. RESULTS: Chronic stress increased the lymphocytic infiltration and inflammation within the gastric mucosa of mice. Stress remarkably increased the expression levels of CD11b and mRNA expression levels of CD68 and F4/80 in the mucosa of the stomach of stressed mice. Stress remarkably increased both mRNA and plasma concentrations of Nox-4 and 8-OHdG; and markedly reduced gastric mRNA and protein expression levels of antioxidant enzymes such as superoxide dismutase, catalase, and glutathione peroxidase. The expressions of proinflammatory cytokines (MCP-1, IL-1ß, and TNF-α) were predominantly observed in the gastric mucosal layers of the stressed mice. Furthermore, stress remarkably elevated the gastric mucosal mRNA expression levels of MCP-1, IL-1ß, and TNF-α. CONCLUSION: Two weeks of restraint stress induced gastric inflammation in the murine model with enhanced oxidative stress and reduced anti-oxidative system.

Chronic stress augments esophageal inflammation, and alters the expression of transient receptor potential vanilloid 1 and protease­activated receptor 2 in a murine model.

Stress is a pivotal factor for inflammation, reactive oxygen species (ROS) production and formation of visceral hypersensitivity (VH) in the process of gastroesophageal reflux disease (GERD). In the present study, the effects of stress on esophageal inflammation, oxidative stress and VH were investigated in a chronic restraint stress mouse model. C57BL/6J male mice were subjected to 2 weeks of intermittent restraint stress, and histopathological analysis revealed that stress induced esophageal inflammation and fibrosis, while no distinct changes were detected in non­stressed control mice. In addition, increased NADPH oxidase 4 expression was observed in the plasma and esophagus of stressed mice, indicating accumulation of ROS. The expression levels of antioxidants, including Mn­superoxide dismutase (MnSOD), Cu/Zn­SOD, catalase and glutathione peroxidase, were also analyzed using reverse transcription­quantitative polymerase chain reaction (RT­qPCR). In addition, transient receptor potential vanilloid 1 (TRPV­1) and protease­activated receptor 2 (PAR­2), which are crucial receptors for VH, were measured by immunohistochemistry and RT­qPCR. The results demonstrated that stress markedly reduced antioxidant expression, while it significantly upregulated TRPV­1 and PAR­2 expression levels in the mouse esophagus. Finally, 2 weeks of restraint stress significantly increased the esophageal and plasma levels of inflammatory cytokines, including interleukin (IL)­6, IL­8, interferon­Î³ and tumor necrosis factor­α. Taken together, the present study results indicated that stress­induced esophageal inflammation and ROS generation involves VH.

Anxiety and Depression among Hypertensive Outpatients in Afghanistan: A Cross-Sectional Study in Andkhoy City.

There is a relationship between mental and physical health. Depression and anxiety are linked with the development of several chronic diseases. The purpose of the present study was to determine the prevalence and factors associated with anxiety and depression among adult hypertensive outpatients in Afghanistan. Methods. Two hundred thirty-four consecutive hypertensive patients from December 2015 to August 2016 were recruited to complete the Hospital Anxiety and Depression Scale (HADS) questionnaire, which has scores for classifying the participants having anxiety and depression symptoms. Results. Of the total 234 patients, 81 (34.6%) were males and 153 (65.4%) were females. The mean age was 54.6 ± 12.7 for the hypertensive patients with anxiety and 63.8 ± 15.0 for the hypertensive patients with depression while this figure was 49.5 ± 10.2 for the adult participants in general population in Kabul city (Saeed, 2013). The prevalence of anxiety and depression (42.3% vs. 58.1%) among hypertensive persons is compared with the same mental disorders among Afghan refugees (39.3% vs. 22.1%) in Dalakee Refugee Camp (in Iran) (Hosseini Divkolaye and Burkle, 2017). Of the total participants, 99 had anxiety (42.3%), 136 had depression (58.1%), and 66 had (28.2%) comorbid anxiety-depression. Multivariate analysis was used. For anxiety age, female gender, smoking, diabetes mellitus, and 2 or more chronic diseases had a significant association. For depression, age and diabetes mellitus had a significant association, and for comorbid anxiety, depression, age, diabetes mellitus, and 2 or more chronic diseases had a significant association. Conclusion. This study shows that anxiety and depression are highly prevalent among hypertensive patients in an outpatient clinic in Afghanistan. There was an association between some sociodemographic and clinical characteristics and anxiety and depression. More studies are needed on a national level to inform the development of strategies for the prevention and control of psychological distress among patients with chronic diseases in Afghanistan.

Increased dipeptidyl peptidase-4 accelerates diet-related vascular aging and atherosclerosis in ApoE-deficient mice under chronic stress.

OBJECTIVES: Exposure to psychosocial stress is a risk factor for cardiovascular disease. Given that dipeptidyl peptidase-4 (DPP4) regulates several intracellular signaling pathways associated with glucagon-like peptide-1 (GLP-1) metabolism, we investigated the role of DPP4 in stress-related vascular senescence and atherosclerosis in apolipoprotein E-deficient (ApoE-/-) mice. METHODS AND RESULTS: ApoE-/- mice fed a high-fat (HF) diet were randomly assigned to one of non-stress and immobilized stress groups for 12weeks. Chronic stress accelerated vascular senescence and atherosclerotic plaque growth at the aortic roots. Stressed mice had increased levels of plasma DPP4 and decreased levels of plasma GLP-1 and adiponectin (APN) and adipose APN expression. Stress increased plaque macrophage infiltration, neovessel density, and elastin fragmentation, lessened the plaque collagen content, and increased the levels of toll-like receptor-2 (TLR2), TLR4, C-X-C chemokine receptor-4, cathepsins S and K, osteopontin, peroxisome proliferator-activated receptor-α, p16INK4A, p21, and gp91phox mRNAs and/or proteins. Stressed aortas had also increased matrix metalloproteinase-2 (MMP-2) and MMP-9 activities. DPP4 inhibition with anagliptin reversed stress-related atherosclerotic lesion formation, and this benefit was abrogated by APN blocking. In vitro, the GLP-1 receptor agonist exenatide stimulated APN expression in 3T3-L1 cells. CONCLUSIONS: These results indicate that the DPP4 inhibition-mediated benefits are likely attributable, at least in part, to attenuation of plaque inflammation, oxidative stress and proteolysis associated with GLP-1-mediated APN production in ApoE-/- mice under stress. Thus, DPP4 will be a novel therapeutic target for the treatment of stress-related cardiovascular disease.

Associations between proteinuria and cardiovascular risk factors among hypertensive patients in Andkhoy, Afghanistan.

Proteinuria in hypertension is an early marker of renal disease and a predictor for the progression of end stage renal disease, and cardiovascular diseases. This study was designed to determine the prevalence of proteinuria and its association with cardiovascular risk factors among adult hypertensive patients in Afghanistan. Five hundred fifty-five patients with a high blood pressure recorded in an outpatient clinic in Andkhoy, Afghanistan from December 2014 to May 2015, were included in this study. Data obtained from each patient, included demographic characteristics, body mass index, blood pressure patterns, cardiovascular history, cardiovascular risk factors, comorbidity, and current drug-therapy. Dipstick screening for proteinuria was performed with reagent test strips. The mean age of the patients was 57.9 ± 13.3 years, and a female predominance was observed (n = 333, 60%). The prevalence of proteinuria was 67.2%. The predictors of proteinuria were found to be age ≥65 years (odds ratio [OR] 1.02, 95% confidence interval [CI] 1.00-1.04), smoking (OR 1.88, 95% CI 1.17-3.02), heart failure (OR 2.23, 95% CI 1.13-4.41), and diabetes mellitus (OR 3.41, 95% CI 1.49-7.81). In conclusion, this study shows that proteinuria is highly prevalent among hypertensive outpatients in an outpatient clinic in Andkhoy, Afghanistan, especially in those with high cardiovascular risk.

Dipeptidyl peptidase- IV inhibitor alogliptin improves stress-induced insulin resistance and prothrombotic state in a murine model.

BACKGROUND: Stress evokes lipolytic release of free fatty acid (FFA) and low-grade inflammation in visceral adipose tissue, mediated by increased adipokine secretion, and contributes to glucose metabolism disorder and prothrombotic state. We tested the hypothesis that alogliptin, a dipeptidyl peptidase-4 inhibitor, can ameliorate the biological effects of chronic stress in mice. METHOD AND RESULTS: C57BL/6J mice were subjected to 2-week intermittent restraint stress and orally treated with vehicle or alogliptin (dose: 15 or 45mg/kg/day). Plasma levels of lipids, proinflammatory cytokines (monocyte chemoattractant protein-1, tumor necrosis factor-α, and interleukin-6), and 8-hydroxydeoxyguanosine were measured with enzyme-linked immunosorbent assay. Monocyte/macrophage accumulation in inguinal white adipose tissue (WAT) was examined by CD11b-positive cell count and mRNA expression of CD68 and F4/80 was examined by immunohistochemistry and RT-PCR, respectively. The mRNA levels of the above-mentioned proinflammatory cytokines, NADPH oxidase 4, adiponectin, and coagulation factors (plasminogen activation inhibitor-1 and tissue factor) in WAT were also assessed with RT-PCR. Glucose metabolism was assessed by glucose and insulin tolerance tests, plasma levels of DPP-4 activity, glucagon-like peptide-1, expression of DPP-4, insulin receptor substrate-1 and glucose transporter 4 in WAT and skeletal muscle. Alogliptin administration suppressed stress-induced FFA release, oxidative stress, adipose tissue inflammation, DPP-4 activation, and prothrombotic state in a dose-dependent manner, and improved insulin sensitivity in stressed mice. CONCLUSIONS: The results indicate that alogliptin improves stress-induced prothrombotic state and insulin resistance; suggesting that alogliptin could have beneficial therapeutic effects against cardiovascular complications in diabetic patients under stress.

Cathepsin S Activity Controls Injury-Related Vascular Repair in Mice via the TLR2-Mediated p38MAPK and PI3K-Akt/p-HDAC6 Signaling Pathway.

OBJECTIVE: Cathepsin S (CatS) participates in atherogenesis through several putative mechanisms. The ability of cathepsins to modify histone tail is likely to contribute to stem cell development. Histone deacetylase 6 (HDAC6) is required in modulating the proliferation and migration of various types of cancer cells. Here, we investigated the cross talk between CatS and HADC6 in injury-related vascular repair in mice. APPROACH AND RESULTS: Ligation injury to the carotid artery in mice increased the CatS expression, and CatS-deficient mice showed reduced neointimal formation in injured arteries. CatS deficiency decreased the phosphorylation levels of p38 mitogen-activated protein kinase, Akt, and HDAC6 and toll-like receptor 2 expression in ligated arteries. The genetic or pharmacological inhibition of CatS also alleviated the increased phosphorylation of p38 mitogen-activated protein kinase, Akt, and HDAC6 induced by platelet-derived growth factor BB in cultured vascular smooth muscle cells (VSMCs), and p38 mitogen-activated protein kinase inhibition and Akt inhibition decreased the phospho-HDAC6 levels. Moreover, CatS inhibition caused decrease in the levels of the HDAC6 activity in VSMCs in response to platelet-derived growth factor BB. The HDAC6 inhibitor tubastatin A downregulated platelet-derived growth factor-induced VSMC proliferation and migration, whereas HDAC6 overexpression exerted the opposite effect. Tubastatin A also decreased the intimal VSMC proliferation and neointimal hyperplasia in response to injury. Toll-like receptor 2 silencing decreased the phosphorylation levels of p38 mitogen-activated protein kinase, Akt, and HDAC6 and VSMC migration and proliferation. CONCLUSIONS: This is the first report detailing cross-interaction between toll-like receptor 2-mediated CatS and HDAC6 during injury-related vascular repair. These data suggest that CatS/HDAC6 could be a potential therapeutic target for the control of vascular diseases that are involved in neointimal lesion formation.

Indoxyl sulfate induces IL-6 expression in vascular endothelial and smooth muscle cells through OAT3-mediated uptake and activation of AhR/NF-κB pathway.

BACKGROUND/AIMS: Interleukin-6 (IL-6) is one of the inflammation biomarkers with highest predictive value for outcome in chronic kidney disease (CKD) patients. The present study aimed to determine the effects of indoxyl sulfate (IS) on IL-6 expression in vascular cells. METHODS: IS was administered to normo- and hypertensive rats. Human umbilical vein endothelial cells (HUVECs) and human aortic smooth muscle cells (HASMCs) were incubated with or without IS. RESULTS: Immunohistochemistry revealed that IS-administered rats showed increased expression of IL-6 in the aortic tissues. IS increased IL-6 expression in HUVECs and HASMCs in a time- and dose-dependent manner. Knockdown of organic anion transporter 3 (OAT3) using small interfering RNA (siRNA) inhibited IS-induced expression of IL-6 in HUVECs and HASMCs. IS induced activation of aryl hydrocarbon receptor (AhR) and nuclear factor-κB (NF-κB) subunit p65 in HUVECs and HASMCs. Both AhR siRNA and p65 siRNA inhibited IS-induced expression of IL-6. AhR siRNA inhibited IS-induced phosphorylation and nuclear translocation of p65 without change in total p65 level. However, p65 siRNA did not inhibit IS-induced nuclear translocation of AhR. Thus, AhR is responsible for IS-induced p65 signaling transduction. CONCLUSION: IS induces IL-6 expression in vascular endothelial and smooth muscle cells through OAT3/AhR/NF-κB pathway.

Indoxyl sulfate downregulates expression of Mas receptor via OAT3/AhR/Stat3 pathway in proximal tubular cells.

UNLABELLED: Renin-angiotensin system (RAS) plays a pivotal role in chronic kidney disease (CKD). Angiotensin converting enzyme-related carboxypeptidase 2 (ACE2)/angiotensin (Ang)-(1-7)/Mas receptor axis counteracts the deleterious actions of Ang II. ACE2 exerts its actions by cleaving Ang II into Ang-(1-7) which activates Mas receptor. This study aimed to determine if the expression of Mas receptor is altered in the kidneys of CKD rats, and if indoxyl sulfate (IS), a uremic toxin, affects the expression of Mas receptor in rat kidneys and cultured human proximal tubular cells (HK-2 cells). The expression of Mas receptor was examined in the kidneys of CKD and AST-120-treated CKD rats using immunohistochemistry. Further, the effects of IS on Mas receptor expression in the kidneys of normotensive and hypertensive rats were examined. The effects of IS on the expression of Mas receptor and phosphorylation of endothelial nitric oxide synthase (eNOS) in HK-2 cells were examined using immunoblotting. CKD rats showed reduced renal expression of Mas receptor, while AST-120 restored its expression. Administration of IS downregulated Mas receptor expression in the kidneys of normotensive and hypertensive rats. IS downregulated Mas receptor expression in HK-2 cells in a time- and dose-dependent manner. Knockdown of organic anion transporter 3 (OAT3), aryl hydrocarbon receptor (AhR), and signal transducer and activator of transcription 3 (Stat3) inhibited IS-induced downregulation of Mas receptor and phosphorylated eNOS. N-acetylcysteine, an antioxidant, also inhibited IS-induced downregulation of Mas receptor and phosphorylated eNOS. Ang-(1-7) attenuated IS-induced transforming growth factor-ß1 (TGF-ß1) expression. CONCLUSION: Mas receptor expression is reduced in the kidneys of CKD rats. IS downregulates renal expression of Mas receptor via OAT3/AhR/Stat3 pathway in proximal tubular cells. IS-induced downregulation of Mas receptor might be involved in upregulation of TGF-ß1 in proximal tubular cells.

Indoxyl sulfate enhances p53-TGF-ß1-Smad3 pathway in proximal tubular cells.

BACKGROUND/AIM: Indoxyl sulfate-induced activation of nuclear factor (NF)-ĸB promotes transforming growth factor (TGF)-ß1 in human proximal tubular cells (HK-2 cells). The present study aimed to elucidate the cross talk among indoxyl sulfate, p53 and TGF-ß1-Smad3 signaling in proximal tubular cells. METHODS: The effects of indoxyl sulfate on the expression of TGF-ß1, Smad3, and α-smooth muscle actin (α-SMA) were determined using HK-2 cells. As for in vivo experiments the following animals were used: Dahl salt-resistant normotensive rats (DN) and indoxyl sulfate-administered Dahl salt-resistant normotensive rats (DN+IS). RESULTS: Both indoxyl sulfate and nutlin-3, a specific p53 inducer, stimulated TGF-ß1 expression, which was suppressed by pifithrin-α, p-nitro, a p53 inhibitor. Further, indoxyl sulfate stimulated TGF-ß1-induced expression of α-SMA by enhancing Smad3 expression and TGF-ß1-induced Smad3 phosphorylation. Indoxyl sulfate induced phosphorylation of extracellular signal-regulated kinase (ERK). U0126, an inhibitor of ERK pathway, prevented indoxyl sulfate-induced upregulation of Smad3 expression. Immunohistochemistry demonstrated that TGF-ß1 and Smad3 were localized in renal tubular cells, and that indoxyl sulfate increased the TGF-ß1 and Smad3-positive area in the kidney. CONCLUSION: Indoxyl sulfate stimulates p53-induced TGF-ß1 expression and TGF-ß1-induced α-SMA expression in proximal tubular cells. Indoxyl sulfate-induced Smad3 accelerates TGF-ß1-induced α-SMA expression through ERK activation. Thus, indoxyl sulfate enhances p53-TGF-ß1-Smad3 pathway in proximal tubular cells.

Indoxyl sulfate upregulates renal expression of ICAM-1 via production of ROS and activation of NF-κB and p53 in proximal tubular cells.

AIMS: Intercellular adhesion molecule 1 (ICAM-1) plays an important role in adhesion of monocytes/macrophages to injured tubulointerstitial tissue. The present study aimed to determine if indoxyl sulfate, a uremic toxin, regulates renal expression of ICAM-1. MAIN METHODS: The effect of indoxyl sulfate on expression of ICAM-1 was determined using human proximal tubular cells (HK-2 cells) and the following animals: (1) Dahl salt-resistant normotensive rats (DN), (2) Dahl salt-resistant normotensive indoxyl sulfate-administered rats (DN+IS), (3) Dahl salt-sensitive hypertensive rats (DH), and (4) Dahl salt-sensitive hypertensive indoxyl sulfate-administered rats (DH+IS). KEY FINDINGS: DN+IS, DH, and DH+IS rats showed significantly increased mRNA expression of ICAM-1 in the kidneys compared with DN rats. DH+IS rats showed significantly increased mRNA expression of ICAM-1 in the kidneys compared with DH rats. Immunohistochemistry revealed that ICAM-1 was localized in the cytoplasm of renal tubular cells, and was most prominently expressed in DH+IS rats. Indoxyl sulfate upregulated mRNA and protein expression of ICAM-1 in HK-2 cells. Inhibitors of NADPH oxidase (diphenylene iodonium chloride), NF-κB (isohelenin) and p53 (pifithrin-α,p-nitro) suppressed indoxyl sulfate-induced expression of ICAM-1 mRNA and protein in HK-2 cells. SIGNIFICANCE: Indoxyl sulfate upregulated renal expression of ICAM-1 through production of reactive oxygen species (ROS) such as superoxide, and activation of NF-κB and p53 in proximal tubular cells. Further, administration of indoxyl sulfate promoted ICAM-1 expression in rat kidneys. Thus, accumulation of indoxyl sulfate in chronic kidney disease might be involved in the pathogenesis of tubulointerstitial injury through induction of ICAM-1 in the kidney.