DNase 1 Protects From Increased Thrombin Generation and Venous Thrombosis During Aging: Cross‐Sectional Study in Mice and Humans

Background Human aging is associated with increased risk of thrombosis, but the mechanisms are poorly defined. We hypothesized that aging induces peroxide‐dependent release of neutrophil extracellular traps that contribute to thrombin generation and thrombosis. Methods and Results We studied C57BL6J mice and littermates of glutathione peroxidase‐1 transgenic and wild‐type mice at young (4 month) and old (20 month) ages and a healthy cohort of young (18–39 years) or middle‐aged/older (50–72 years) humans. In plasma, we measured thrombin generation potential and components of neutrophil extracellular traps (cell‐free DNA and citrullinated histone). Aged wild‐type mice displayed a significant increase in thrombin generation that was decreased in aged glutathione peroxidase‐1 transgenic mice. Both aged wild‐type and aged glutathione peroxidase‐1 transgenic mice demonstrated similar elevation of plasma cell‐free DNA compared with young mice. In contrast, plasma levels of citrullinated histone were not altered with age or genotype. Release of neutrophil extracellular traps from neutrophils in vitro was also similar between young and aged wild‐type or glutathione peroxidase‐1 transgenic mice. Treatment of plasma or mice with DNase 1 decreased age‐associated increases in thrombin generation, and DNase 1 treatment blocked the development of experimental venous thrombi in aged C57BL6J mice. Similarly, thrombin generation potential and plasma cell‐free DNA, but not citrullinated histone, were higher in middle‐aged/older humans, and treatment of plasma with DNase 1 reversed the increase in thrombin generation. Conclusions We conclude that DNase 1 limits thrombin generation and protects from venous thrombosis during aging, likely by hydrolyzing cell‐free DNA.

Assessment of Glutathione Peroxidase-1 (GPX1) Gene Expression as a Specific Diagnostic and Prognostic Biomarker in Malignant Pleural Mesothelioma

Malignant pleural mesothelioma (MPM) is a malignant tumor of the mesothelial lining of the thorax. It has been related to frequent exposure to asbestos. Diagnosis of malignant pleural mesothelioma is considered a criticizing problem for clinicians. Early diagnosis and sufficient surgical excision of MPM are considered the cornerstone success factors for the management of early MPM. Glutathione peroxidase-1 (GPX1) is an intracellular protein found to be extensively distributed in all cells, and it belongs to the GPX group. In the current study, we included ninety-eight patients with MPM that underwent surgery at the Zagazig University Hospital in Egypt. We assessed GPX1 gene expression level as it was thought to be related to pathogenicity of cancer in a variety of malignant tumors. We observed a significant elevation in GPX1-mRNA levels in MPM relative to the nearby normal pleural tissues. It was found to be of important diagnostic specificity in the differentiation of MPM from normal tissues. Moreover, we studied the survival of patients in correlation to the GPX1 expression levels and we reported that median overall survival was about 16 months in patients with high GPX1 expression levels, while it was found to be about 40 months in low GPX1 levels.

Aloe vera increases collagen fibres in extracellular matrix and mRNA expression of peroxiredoxin-6 in bovine ovarian cortical tissues cultured in vitro

Summary In vitro culture of ovarian tissue containing primordial follicles is an important tool to study the initiation of follicular populations and to develop efficient culture systems to support in vitro follicle growth. Considering that in vitro culture favours oxidative stress, it is very important to supplement culture medium with antioxidant substances such as Aloe vera extract. This study aims to evaluate the effects of different concentrations of Aloe vera on the distribution of collagen fibres in the extracellular matrix, follicular activation, development and survival in bovine ovarian cortical tissues cultured in vitro, as well as on expression of mRNAs for antioxidant enzymes [superoxide dismutase (SOD), catalase (CAT), peroxiredoxin 6 (PRDX6) and glutathione peroxidase 1 (GPX1)]. To this end, ovarian cortical tissues were cultured for 6 days in α-MEM alone or supplemented with different concentrations of Aloe vera extract (1.0, 5.0, 10.0 or 50.0%). After culture, fragments were fixed and processed histologically to evaluate follicular morphology and activation, as well as the extracellular matrix by staining with picrosirius red. The levels of mRNA for SOD, CAT, PRDX6 and GPX1 in cultured ovarian tissues were evaluated by real-time polymerase chain reaction (PCR). Ovarian tissues cultured with 10.0 or 50.0% Aloe vera had higher percentages of collagen fibres than tissues cultured in control medium. A significant increase in developing follicles was observed in ovarian tissues cultured in α-MEM alone or supplemented with 10% Aloe vera when compared with fresh control or tissues cultured with 1.0% Aloe vera. Presence of Aloe vera did not influence the percentage of morphologically normal follicles when compared with control medium. Ovarian tissues cultured with 50.0% Aloe vera had higher percentages of morphologically normal follicles than those cultured with 10.0% Aloe vera. Furthermore, 10% Aloe vera significantly increased mRNA levels for PRDX6. In conclusion, 10.0% Aloe vera improves extracellular matrix distribution in cultured tissues and increases the expression of mRNA for PRDX6 after 6 days in vitro.

Human Proximal Tubule Epithelial Cells (HK-2) as a Sensitive In Vitro System for Ochratoxin A Induced Oxidative Stress

Ochratoxin A (OTA) is a mycotoxin that is potentially carcinogenic to humans. Although its mechanism remains unclear, oxidative stress has been recognized as a plausible cause for the potent renal carcinogenicity observed in experimental animals. The effect of OTA on oxidative stress parameters in two cell lines of LLC-PK1 and HK-2 derived from the kidneys of pig and human, respectively, were investigated and compared. We found that the cytotoxicity of OTA on LLC-PK1 and HK-2 cells was dose- and time-dependent in both cell lines. Furthermore, increased intracellular reactive oxygen species (ROS) induced by OTA in both cell lines were observed in a time-dependent manner. Glutathione (GSH) was depleted by OTA at >48 h in HK-2 but not in LLC-PK1 cells. While the mRNA levels of glucose-6-phosphate dehydrogenase (G6PD) and glutathione peroxidase 1 (GPX1) in LLC-PK1 were down-regulated by 0.67- and 0.66-fold, respectively, those of catalase (CAT), glutathione reductase (GSR), and superoxide dismutase 1 (SOD) in HK-2 were up-regulated by 2.20-, 2.24-, and 2.75-fold, respectively, after 72 h exposure to OTA. Based on these results, we conclude that HK-2 cells are more sensitive to OTA-mediated toxicity than LLC-PK1, and OTA can cause a significant oxidative stress in HK-2 as indicated by changes in the parameter evaluated.

Physiological Functions of Thiol Peroxidases (Gpx1 and Prdx2) during Xenopus laevis Embryonic Development

Glutathione peroxidase 1 (Gpx1) and peroxiredoxin 2 (Prdx2) belong to the thiol peroxidase family of antioxidants, and have been studied for their antioxidant functions and roles in cancers. However, the physiological significance of Gpx1 and Prdx2 during vertebrate embryogenesis are lacking. Currently, we investigated the functional roles of Gpx1 and Prdx2 during vertebrate embryogenesis using Xenopus laevis as a vertebrate model. Our investigations revealed the zygotic nature of gpx1 having its localization in the eye region of developing embryos, whereas prdx2 exhibited a maternal nature and were localized in embryonic ventral blood islands. Furthermore, the gpx1-morphants exhibited malformed eyes with incompletely detached lenses. However, the depletion of prdx2 has not established its involvement with embryogenesis. A molecular analysis of gpx1-depleted embryos revealed the perturbed expression of a cryba1-lens-specific marker and also exhibited reactive oxygen species (ROS) accumulation in the eye regions of gpx1-morphants. Additionally, transcriptomics analysis of gpx1-knockout embryos demonstrated the involvement of Wnt, cadherin, and integrin signaling pathways in the development of malformed eyes. Conclusively, our findings indicate the association of gpx1 with a complex network of embryonic developmental pathways and ROS responses, but detailed investigation is a prerequisite in order to pinpoint the mechanistic details of these interactions.

Combination of Antioxidant Enzyme Overexpression and N‐Acetylcysteine Treatment Enhances the Survival of Bone Marrow Mesenchymal Stromal Cells in Ischemic Limb in Mice With Type 2 Diabetes

Background Therapy with mesenchymal stem cells remains a promising but challenging approach to critical limb ischemia in diabetes because of the dismal cell survival. Methods and Results Critical limb ischemia in type 2 diabetes mouse model was used to explore the impact of diabetic limb ischemia on the survival of bone marrow mesenchymal stromal cells (bMSCs). Inhibition of intracellular reactive oxygen species was achieved with concomitant overexpression of superoxide dismutase (SOD)‐1 and glutathione peroxidase‐1 in the transplanted bMSCs, and extracellular reactive oxygen species was attenuated using SOD‐3 overexpression and N‐acetylcysteine treatment. In vivo optical fluorescence imaging and laser Doppler perfusion imaging were used to track cell retention and determine blood flow in diabetic ischemic limb, respectively. Survival of the transplanted bMSCs was significantly decreased in diabetic ischemic limb compared with the control. In vitro study indicated that advanced glycation end products, not high glucose, significantly decreased the proliferation of bMSCs and increased their apoptosis associated with increased reactive oxygen species production and selective reduction of SOD‐1 and SOD‐3. In vivo study demonstrated that concomitant overexpression of SOD‐1, SOD‐3, and glutathione peroxidase‐1, or host treatment with N‐acetylcysteine, significantly enhanced in vivo survival of transplanted bMSCs, and improved critical limb ischemia in diabetic mice. Combination of triple antioxidant enzyme overexpression in bMSCs with host N‐acetylcysteine treatment further improved bMSC survival with enhanced circulatory and functional recovery from diabetic critical limb ischemia. Conclusions Simultaneous suppression of reactive oxygen species from transplanted bMSCs and host tissue could additively enhance bMSC survival in diabetic ischemic limb with increased therapeutic efficacy in diabetes.

Characterization and Expression Profiling of Glutathione Peroxidase 1 gene (GPX1) and Activity of GPX in Onychostoma macrolepis suffered from Thermal Stress

In this study, full-length cDNA of glutathione peroxidases 1 (GPX1) of Onychostoma macrolepis was cloned by RACE, and expression of GPX1 and activity of GPX in O. macrolepis suffered from heat stress were analyzed. Compared with the control group (24°C), the experimental fish were stressed for 0, 1, 3, 6, 12, 24, and 48 hours at the heated water (30°C). Liver had highest level and response speed in GPX1 expression among various tissues after heat stress, indicated that liver was the highest sensitive tissue to heat stress. When the water was raised to the heating temperature (30°C), the GPX activity decreased in fish serum, and the consumption of GPX eliminated the increase of ROS caused by heat stress within 3h. However, after 6h and 12h stress at 30°C, GPX activity was significantly higher than that at 0h (P<0.05), which is due to the rapid response of GPX to heat stress. In summary, fish showed a transient stress response and was acclimated to the new temperature after 24 h according to the overall expression of the GPX1 and the serum GPX activity, and both GPX1 and GPX play crucial roles in this process.