Clinical Significance of Serum Kallistatin and ENOX1 Levels in Patients with Coronary Heart Disease.

BACKGROUND: Kallistatin and ENOX1 are regulators of inflammation and oxidative stress which are typical pathological reactions in atherosclerosis. However, there is limited information of kallistatin and ENOX1 in coronary heart disease (CHD). METHODS: Fifty healthy controls, 56 stable angina pectoris (SAP) patients, and 47 acute coronary syndrome (ACS) patients were included in this study. Levels of kallistatin and ENOX1 in serum were measured by ELISA. χ2 test was performed to analyze categorical data. ANOVA, Pearson correlation analysis, and multiple linear regression were performed to analyze the numerical data. Finally, receiver operating characteristic (ROC) curve was applied to assess the diagnostic value of kallistatin in CHD. RESULTS: Among the 153 participants, 59.5% were male and the average age was 63.8 ± 11.39 years. Compared with the control group, kallistatin expression was decreased in the SAP and ACS groups while expression of ENOX1 was increased in the ACS group (p < 0.05). Pearson correlation analysis showed that the kallistatin level was negatively correlated with the Gensini score (r = -0.210, p < 0.01), white blood cell (WBC) count (r = -0.283, p < 0.001), and triglyceride levels (r = -0.242, p < 0.01) and positively correlated with age (r = 0.353, p < 0.001) and high-density lipoprotein cholesterol (r = 0.310, p < 0.001). ENOX1 expression was positively correlated with WBC count (r = 0.244, p < 0.01), international normalized ratio (r = 0.177, p < 0.05), and Gensini score (r = 0.201, p < 0.05). Multiple linear regression showed that Cr, alanine transaminase, glucose, and kallistatin are independent predictors for Gensini score. The ROC curve showed that kallistatin had the highest diagnostic significance (p = 0.007) when the area under curve was 0.636, with a sensitivity of 0.735 and a specificity of 0.495. CONCLUSION: Expression of kallistatin was decreased in CHD patients and that of ENOX1 was increased in ACS patients. Kallistatin and ENOX1 were closely connected with the severity of CHD, and kallistatin may be helpful in the diagnosis of CHD.

Increased Expression of Ecto-NOX Disulfide-thiol Exchanger 1 (ENOX1) in Diabetic Mice Retina and its Involvement in Diabetic Retinopathy Development.

BACKGROUND/AIM: Diabetic retinopathy (DR) is a type of retinal damage caused by a complication of diabetes and is a major cause of blindness in working-age adults. Ecto-NOX disulfide-thiol exchanger 1 (ENOX1) is a member of the ecto-NOX family involved in the plasma membrane electron transport pathway. This study aimed to investigate the role of ENOX1 in the development of DR. MATERIALS AND METHODS: Human retinal endothelial cells (HRECs) and human retinal pigment epithelial cells (HREpiCs) exposed to a high concentration (25 mM) of D-glucose and type 2 diabetes (T2D) mice (+Leprdb/+Leprdb, db/db) with retinopathy were used as models to determine the ENOX1 expression levels there. RESULTS: Our results showed that ENOX1 expression levels did not significantly change in both HRECs and HREpiCs under hyperglycemic conditions for 48 h. Nevertheless, ENOX1 expression increased significantly in T2D mouse retinas, particularly in the photoreceptor layer, compared to the control mouse retinas. CONCLUSION: Different retinal ENOX1 expression in T2D mice and control mice suggested that ENOX1 may be involved in DR development.

Targeting Enox1 in tumor stroma increases the efficacy of fractionated radiotherapy.

The goal of this investigation was to clarify the question of whether targeting Enox1 in tumor stroma would synergistically enhance the survival of tumor-bearing mice treated with fractionated radiotherapy. Enox1, a NADH oxidase, is expressed in tumor vasculature and stroma. However, it is not expressed in many tumor types, including HT-29 colorectal carcinoma cells. Pharmacological inhibition of Enox1 in endothelial cells inhibited repair of DNA double strand breaks, as measured by γH2AX and 53BP1 foci formation, as well as neutral comet assays. For 4 consecutive days athymic mice bearing HT-29 hindlimb xenografts were injected with a small molecule inhibitor of Enox1 or solvent control. Tumors were then administered 2 Gy of x-rays. On day 5 tumors were administered a single 'top-up' fraction of 30 Gy, the purpose of which was to amplify intrinsic differences in the radiation fractionation regimen produced by Enox1 targeting. Pharmacological targeting of Enox1 resulted in 80% of the tumor-bearing mice surviving at 90 days compared to only 40% of tumor-bearing mice treated with solvent control. The increase in survival was not a consequence of reoxygenation, as measured by pimonidazole immunostaining. These results are interpreted to indicate that targeting of Enox1 in tumor stroma significantly enhances the effectiveness of 2 Gy fractionated radiotherapy and identifies Enox1 as a potential therapeutic target.

Age-related NADPH Oxidase (arNOX) Activity Correlated with Cartilage Degradation and Bony Changes in Age-related Osteoarthritis.

The purpose of this study was to investigate the age-related NADPH oxidase (arNOX) activity in patients with age-related knee osteoarthritis (OA). Serum and cartilage arNOX activities were determined using an oxidized ferricytochrome C reduction assay. Full-thickness knee joint cartilages obtained through total knee replacement surgery were graded according to the Outerbridge (OB) classification. Radiographic severity of OA was determined on Knee X-rays according to the Kellgren-Lawrence (K/L) grading system. Cartilage ß-galactosidase, HIF-1α, and GLUT-1 expression levels were evaluated as markers for tissue senescence, hypoxia, and glycolysis. Higher arNOX activities occurred with higher levels of cartilage ß-galactosidase, HIF-1α, and GLUT-1 (P = 0.002). arNOX activity in cartilages with surface defects (OB grade II, III) was higher than in those without the defects (OB grade 0, I) (P = 0.012). Cartilage arNOX activity showed a positive correlation with serum arNOX activity (r = -0.577, P = 0.023). Serum arNOX activity was significantly higher in the OA subgroup with bilateral ROA than in the OA with no or unilateral ROA (2.449 ± 0.81, 2.022 ± 0.251 nM/mL, respectively, P = 0.019). The results of this study demonstrate that OA itself is not a cause to increase arNOX activities, however, arNOX hyperactivity is related to a high degree of cartilage degradation, and a high grade and extent of ROA in age-related OA.

Novel genes FAM134C, C3orf10 and ENOX1 are regulated by NRF-1 and differentially regulate neurite outgrowth in neuroblastoma cells and hippocampal neurons.

Nuclear respiratory factor-1 (NRF-1) is a major transcription factor in the human genome and functions in neurite outgrowth in neuroblastoma cells. Whether genes downstream from NRF-1 differentially regulate axonal and dendritic outgrowth in neurons remains largely unknown. Three hypothetical genes, C3orf10, FAM134C, and ENOX1, were investigated because their NRF-1 response elements are 100% conserved in humans and mice. We found that NRF-1 positively regulates FAM134C and ENOX1, but negatively regulates C3orf10 in human neuroblastoma IMR-32 cells and primary rat cortical neurons. In IMR-32 cells, FAM134C positively regulates and C3orf10 negatively regulates neurite outgrowth, but ENOX1 plays no role in neurite outgrowth regulation. FAM134C but not C3orf10 mediates NRF-1-enhanced neurite outgrowth. In primary rat hippocampal neurons, Fam134c is predominantly expressed in the axon hillock and C3orf10 is ubiquitously expressed in all neurites and cell bodies at different developmental stages, suggesting their roles in axonal and dendritic outgrowth. Knockdown of Fam134c decreased both axonal length and the number of axonal collaterals and dendrites, however, knockdown of C3orf10 only increased the number of axonal collaterals and dendrites. Overall, we annotated FAM134C, C3orf10, and ENOX1 as NRF-1-regulated genes, which have differential effects on neurite outgrowth in neuroblastoma cells as well as neurons. This study provided an effective platform for annotating hypothetical genes in the human genome and increasing our knowledge in the molecular network underlying neuronal differentiation.