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• Research Paper Volume 13, Issue 8 pp 11061-11082

  microRNA-363-3p reduces endothelial cell inflammatory responses in coronary heart disease via inactivation of the NOX4-dependent p38 MAPK axis

  Relevance score: 11.851779

  Tao Zhou, Suining Li, Liehong Yang, Daokang Xiang

  Keywords: microRNA-363-3p, NADPH oxidase 4, p38 MAPK, coronary heart disease, endothelial cell

  Published in Aging on March 19, 2021

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  Coronary heart disease (CHD) is one of the leading causes of heart-associated deaths worldwide. This study aimed to investigate the mechanism by which microRNA-363-3p (miR-363-3p) regulates endothelial injury induced by inflammatory responses in CHD. The expression patterns of miR-363-3p, NADPH oxidase 4 (NOX4), and p38 MAPK/p-p38 MAPK were examined in an established atherosclerosis (AS) model in C57BL/6 mice and in isolated coronary arterial endothelial cells (CAECs) after gain- or loss-of-function experiments. We also measured the levels of inflammatory factors (IL-6, ICAM-1, IL-10 and IL-1β), hydrogen peroxide (H₂O₂), and catalase (CAT) activity, followed by detection of cell viability and apoptosis. In AS, miR-363-3p was downregulated and NOX4 was upregulated, while miR-363-3p was identified as targeting NOX4 and negatively regulating its expression. The AS progression was reduced in NOX4 knockout mice. Furthermore, miR-363-3p resulted in a decreased inflammatory response, oxidative stress, and cell apoptosis in CAECs while augmenting their viability via blockade of the p38 MAPK signaling pathway. Overall, miR-363-3p hampers the NOX4-dependent p38 MAPK axis to attenuate apoptosis, oxidative stress injury, and the inflammatory reaction in CAECs, thus protecting CAECs against CHD. This finding suggests the miR-363-3p-dependent NOX4 p38 MAPK axis as a promising therapeutic target for CHD.

• Research Paper Volume 13, Issue 1 pp 831-845

  Atorvastatin improves motor function, anxiety and depression by NOX2-mediated autophagy and oxidative stress in MPTP-lesioned mice

  Relevance score: 11.01424

  Junqiang Yan, Jiarui Huang, Anran Liu, Jiannan Wu, Hua Fan, Mengmeng Shen, Xiaoyi Lai, Hongxia Ma, Wenjie Sun, Jianxue Yang, Yunqi Xu

  Keywords: Parkinson’s disease, atorvastatin, oxidative stress, autophagy, NADPH oxidase 2

  Published in Aging on December 3, 2020

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  Parkinson’s disease (PD) is a neurodegenerative disease caused by the loss of dopaminergic neurons. It is characterized by static tremors, stiffness, slow movements, and gait disturbances, but it is also accompanied by anxiety and depression. Our previous study showed that atorvastatin could reduce the risk of PD, but the mechanism is still unclear. In this paper, Our findings showed that atorvastatin increased muscle capacity and the coordination of movement and improved anxiety and depression. Atorvastatin could decrease the expression of α-synuclein Ser129 and NADPH oxidase 2 (NOX2), increase the protein expression of LC3II/I, and promote autophagy flow. To further confirm that atorvastatin protection was achieved by inhibiting NOX2, we injected at midbrain with NOX2 shRNA (M) lentivirus and found that silent NOX2 produced the same effect as atorvastatin. Further research found that atorvastatin could reduce MPTP-induced oxidative stress damage, while inhibiting NOX2 decreased the antioxidative stress effect of atorvastatin. Our results suggest that atorvastatin can improve muscle capacity, anxiety and depression by inhibiting NOX2, which may be related to NOX2-mediated oxidative stress and autophagy. Atorvastatin may be identified as a drug that can effectively improve behavioral disorders. NOX2 may be a potential gene target for new drug development in PD.

• Research Paper Volume 12, Issue 10 pp 8968-8986

  Inhibition of circulating exosomal microRNA-15a-3p accelerates diabetic wound repair

  Relevance score: 12.262068

  Yuan Xiong, Lang Chen, Tao Yu, Chenchen Yan, Wu Zhou, Faqi Cao, Xiaomeng You, Yingqi Zhang, Yun Sun, Jing Liu, Hang Xue, Yiqiang Hu, Dong Chen, Bobin Mi, Guohui Liu

  Keywords: microRNA-15a-3p, exosome, diabetic foot ulcer, wound repair, NADPH oxidase 5

  Published in Aging on May 21, 2020

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  Diabetic foot ulcers are a common complication of diabetes, and are usually incurable in the clinic. Exosomes (carriers that transfer endogenous molecules) from diabetic patients’ blood have been demonstrated to suppress diabetic wound repair. In this study, we investigated the effects of circulating exosomal microRNA-15a-3p (miR-15a-3p) on diabetic wound repair. Exosomes were extracted from diabetic patients’ blood, and were found to inhibit diabetic wound repair in vitro and in vivo. miR-15a-3p was upregulated in diabetic exosomes, and impaired wound healing. When miR-15a-3p was knocked down in diabetic exosomes, their negative effects were partially reversed both in vitro and in vivo. NADPH oxidase 5 (NOX5) was identified as a potential target of miR-15a-3p, and the inhibition of NOX5 reduced the release of reactive oxygen species, thereby impairing the functionality of human umbilical vein endothelial cells. In summary, inhibition of circulating exosomal miR-15a-3p accelerated diabetic wound repair by activating NOX5, providing a novel therapeutic target for diabetic foot ulcer therapy.

  

  Features of exosomes derived from non-diabetic and diabetic patients. (A) DLS results, TEM images and WB results of Con-Exos. (B) DLS results, TEM images and WB results of Dia-Exos; scale bar: 100 nm. (C) PKH26-labeled Con-Exos were absorbed by HUVECs, as indicated by a red fluorescent signal. (D) PKH26-labeled Dia-Exos were taken up by HUVECs, as indicated by a red fluorescent signal.

  
  
  

  

  Dia-Exos hindered wound healing in vivo. (A) General view of wound closure after different treatments. Wounds are shown on days 0, 3, 5, 7, 10 and 14 post-wounding. (B) The wound closure rates of the three groups; n = 6 per group. (C, D) The MPU ratio at the wound area in each group was assessed through small animal doppler detection. The MPU ratio is the MPU of the wound area (region of interest 1) divided by the MPU of the area around the wound (region of interest 2). n = 6 per group. (E) Immunohistochemical analysis of CD31 in the wound site after different treatments. The number of CD31-positive cells was quantified in five random fields of view. Vessels with diameters of 2-10 μm were counted as individual vessels. n = 6 per group; scale bar: 100 μm. Data are the means ± SDs of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

  
  
  

  

  Dia-Exos impaired HUVEC angiogenesis and survival in vitro. (A) The effects of Dia-Exos on HUVEC proliferation were measured with a CCK-8 assay. (B, C) Flow cytometry was used to quantify the cell cycle distribution. (D) The effects of Dia-Exos on the proliferation-related genes Cyclin D1 and Cyclin D3 were assessed using qRT-PCR. (E) The effects of Dia-Exos on the apoptosis-related genes Bcl-2 and Bax were assessed using qRT-PCR. (F, G) A Transwell migration assay was used to assess the effects of Dia-Exos on HUVEC migration; scale bar: 100 μm. (H–J) A tube formation assay was used to assess the effects of Dia-Exos on HUVEC angiogenesis; scale bar: 200 μm. (K, L) The scratch assay results of the three groups; scale bar: 250 μm. Data are the means ± SDs of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

  
  
  

  

  Dia-Exos were enriched with miR-15a-3p, which altered HUVEC function. (A, B) An miRNA microarray dataset of non-diabetic foot wound patients and DFU patients retrieved from NCBI GEO (accession number: GSE80178) indicated that miR-15a-3p was upregulated in foot skin from diabetic patients. (C, D) MiR-15a-3p overexpression was found in serum and exosomes from the diabetic group; n = 10 per group. (E) Effects of the two kinds of exosomes on miR-15a-3p levels in the skin tissues of mice treated with Dia-Exos. (F) qRT-PCR indicated that antagomiR-15a-3p could partially counteract the overexpression of miR-15a-3p in HUVECs. (G) A CCK-8 assay was used to assess the effects of antagomiR-15a-3p on HUVEC proliferation. (H, I) Flow cytometry was used to quantify the cell cycle distribution. (J) qRT-PCR analysis indicated that antagomiR-15a-3p could restore the mRNA levels of Cyclin D1 and Cyclin D3. (K) The effects of antagomiR-15a-3p on the apoptosis-related genes Bcl-2 and Bax were measured using qRT-PCR. (L, M) A Transwell migration assay was used to measure the effects of miR-15a-3p on HUVEC migration; scale bar: 100 μm. (N–P) A tube formation assay was used to assess the effects of miR-15a-3p on HUVEC angiogenesis; scale bar: 200 μm. (Q, R) The scratch assay results of the three groups; scale bar: 250 μm. Data are the means ± SDs of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

  
  
  

  

  Inhibition of miR-15a-3p partially reversed the impaired functionality of HUVECs treated with Dia-Exos. (A) MiR-15a-3p levels in the three groups were measured using qRT-PCR. (B) CCK-8 assay results of the three groups. (C, D) Flow cytometry was used to quantify the cell cycle distribution in treated cells. (E) The qRT-PCR results of the proliferation-related genes Cyclin D1 and Cyclin D3. (F) The apoptosis-related genes Bcl-2 and Bax were assessed using qRT-PCR. (G, H) A Transwell migration assay was used to assess the effects of miR-15a-3p inhibition on HUVEC migration; scale bar: 100 μm. (I–K) A tube formation assay was used to assess the effects of miR-15a-3p inhibition on HUVEC angiogenesis; scale bar: 200 μm. Data are the means ± SDs of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

  
  
  

  

  Knockdown of miR-15a-3p partially reversed the negative effects of Dia-Exos on wound healing in vivo. (A) General view of the wounds among in the four groups on days 0, 3, 5, 7, 10 and 14 post-wounding; n = 6 per group. (B) The mice of wound closure in the four groups were quantified using digital images evaluated with ImageJ software; n = 6 per group. (C, D) The blood flow at the wounds in the four groups was evaluated using small animal doppler detection; n = 6 per group. (E) The expression of miR-15a-3p in the wound tissues of the four groups. (F, G) CD31 immunohistochemistry results of the four groups. The number of CD31-positive cells was quantified in five random fields of view. Vessels with diameters of 2-10 μm were counted as individual vessels. n = 6 per group; scale bar: 100 μm. Data are the means ± SDs of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

  
  
  

  

  MiR-15a-3p inhibits the NOX5/ROS signaling pathway. (A, B) The binding between miR-15a-3p and NOX5 was demonstrated with a luciferase reporter assay. (C) The effects of miR-15a-3p on NOX5 levels were assessed using qRT-PCR. (D) The intracellular release of ROS was measured in the three groups. (E, F) WB and qRT-PCR analyses were used to detect the efficacy of NOX5 siRNA. (G) The release of ROS decreased when NOX5 was silenced. (H) A CCK-8 assay was applied to assess cell proliferation after different treatments. (I, J) Flow cytometry was used to quantify the cell cycle distribution in treated cells. (K) The proliferation-related genes Cyclin D1 and Cyclin D3 were assessed using qRT-PCR. (L) The apoptosis-related genes Bcl-2 and Bax were assessed using qRT-PCR. (M, N) A Transwell migration assay was used to assess the effects of miR-15a-3p on HUVEC migration; scale bar: 100 μm. Data are the means ± SDs of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

  
  
  

  

  Schematic diagram of the proposed mechanisms by which inhibiting circulating exosomal miR-15a-3p enhanced the angiogenesis and survival of HUVECs.

  
  
  

• Research Paper Volume 12, Issue 6 pp 5362-5383

  Delphinidin attenuates pathological cardiac hypertrophy via the AMPK/NOX/MAPK signaling pathway

  Relevance score: 12.58227

  Youming Chen, Zhuowang Ge, Shixing Huang, Lei Zhou, Changlin Zhai, Yuhan Chen, Qiuyue Hu, Wei Cao, Yuteng Weng, Yanyan Li

  Keywords: delphinidin, cardiac hypertrophy, AMPK, NADPH oxidase, oxidative stress

  Published in Aging on March 25, 2020

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  Reactive oxygen species (ROS) play a pivotal role in the development of pathological cardiac hypertrophy. Delphinidin, a natural flavonoid, was reported to exert marked antioxidative effects. Therefore, we investigated whether delphinidin ameliorates pathological cardiac hypertrophy via inhibiting oxidative stress. In this study, male C57BL/6 mice were treated with DMSO or delphinidin after surgery. Neonatal rat cardiomyocytes (NRCMs) were treated with angiotensin II (Ang II) and delphinidin in vitro. Eighteen-month-old mice were administered delphinidin to investigate the effect of delphinidin on aging-related cardiac hypertrophy. Through analyses of hypertrophic cardiomyocyte growth, fibrosis and cardiac function, delphinidin was demonstrated to confer resistance to aging- and transverse aortic constriction (TAC)-induced cardiac hypertrophy in vivo and attenuate Ang II-induced cardiomyocyte hypertrophy in vitro by significantly suppressing hypertrophic growth and the deposition of fibrosis. Mechanistically, delphinidin reduced ROS accumulation upon Ang II stimulation through the direct activation of AMP-activated protein kinase (AMPK) and subsequent inhibition of the activity of Rac1 and expression of p47^phox. In addition, excessive levels of ERK1/2, P38 and JNK1/2 phosphorylation induced by oxidative stress were abrogated by delphinidin. Delphinidin was conclusively shown to repress pathological cardiac hypertrophy by modulating oxidative stress through the AMPK/NADPH oxidase (NOX)/mitogen-activated protein kinase (MAPK) signaling pathway.

  

  Chemical structure of delphinidin (Dp).

  
  
  

  

  Delphinidin attenuated cardiac hypertrophy and improved cardiac function induced by pressure overload in vivo. (A) Statistical differences in the heart weight/body weight (HW/BW) and heart weight/tibia length (HW/TL) ratios between sham and TAC mice treated with vehicle or delphinidin (n=8). (B) Echocardiographic parameters in sham and TAC mice treated with vehicle or delphinidin (n=8). (C) Left, Hematoxylin-eosin (H&E) staining was performed to assess hypertrophic growth of the hearts of sham and TAC mice treated with vehicle or delphinidin (n=8). Right, Statistical analysis of differences in cardiomyocyte size (n=8). (D) Quantitative dihydroethidium (DHE) staining (n=8). (E) Chemiluminescence lucigenin assay (n=8). (F) Quantitative real-time PCR (qRT-PCR) was performed to analyze the mRNA levels of hypertrophic genes (n=5). In A–E, **p<0.01 versus the sham group; ***p<0.001 versus the sham group; ns versus the TAC group; #p<0.05 versus the TAC group; ##p<0.01 versus the TAC group; ###p<0.001 versus the TAC group. qRT-PCR was performed to analyze the mRNA levels of hypertrophic genes (n=5). In A–E, **p<0.01 versus the sham group; ***p<0.001 versus the sham group; ns versus the TAC group; #p<0.05 versus the TAC group; ##p<0.01 versus the TAC group; ###p<0.001 versus the TAC group.

  
  
  

  

  Delphinidin attenuated pressure overload-induced myocardial fibrosis in vivo. (A) Left, Representative PSR staining of histological sections of the LV (n=8). Right, Statistical analysis of differences in cardiac fibrosis. (B) Quantitative real-time PCR (qRT-PCR) was performed to analyze the mRNA levels of fibrosis genes (n=5). In A–B, **p<0.01 versus the sham group; ***p<0.001 versus the sham group; ns versus the TAC group; ##p<0.01 versus the TAC group; ###p<0.001 versus the TAC group.

  
  
  

  

  Delphinidin inhibited Ang II-induced hypertrophy in NRCMs. (A) The Cell Counting Kit-8 assay was used to detect the cell viability of cardiomyocytes treated with different concentrations of delphinidin (n=4). (B) NRCMs were treated with Ang II (1 μM) for 24 hours in the presence of delphinidin (10 and 50 μM) or DMSO. α-Actinin staining was performed to determine cell size. Representative images (left) and quantified cell sizes (right) of each group are shown (scale bar=20 μm). Cell surface areas (μm²) were measured in 3 independent experiments with at least 100 cells counted for each condition. (C) qRT-PCR was performed to analyze the expression of hypertrophic genes. (D and E) Representative image and results of quantitative analysis of ROS generation measured by DCF-DA and DHE staining. (F) Statistical analysis of differences in nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity. A.U., arbitrary units. In A–F,**p<0.01 versus the control group; ***p<0.001 versus the control group; ns versus the Ang II group; #p<0.05 versus the Ang II group; ##p<0.01 versus the Ang II group; ###p<0.001 versus the Ang II group.

  
  
  

  

  Delphinidin downregulated NOX through activating AMPK. (A) Determination of rac1 activity. Cell lysates were affinity precipitated with GTP-PBD bound to glutathione-agarose beads. Precipitated GTP-Rac1 was detected by immunoblotting with anti-Rac1 antibody (n=5). ***p<0.001 versus the PBS group; #p<0.05 versus the Ang II group. (B) Expression of the NOX subunits p47^phox, p40^phox, p67^phox, gp91^phox, p22^phox (n=5). *p<0.05 versus the PBS group; ns versus the Ang II group; #p<0.05 versus the Ang II group. (C) Representative western blot analysis revealed AMPK phosphorylation levels (n=5). **p<0.01 versus the PBS group; ##p<0.01 versus the Ang II group. (D) Representative western blot analysis and GST pulldown analysis revealed the effect of delphinidin on the AMPK phosphorylation level and NADPH oxidase subunit p47^phox and Rac1 activity. **p<0.01 versus the PBS group; #p<0.05 versus the Ang II group; ##p<0.01 versus the Ang II group; $p<0.05 versus the Ang II+Dp group; $$p<0.01 versus the Ang II+Dp group. Bubbles and traces besides the main strips are parts of the blotting background in western blot.

  
  
  

  

  Compound C abrogated the effects of delphinidin on Ang II-induced cardiomyocyte hypertrophy and oxidative stress by blocking AMPK activity. (A) NRCMs were treated with Ang II (1 μM) for 24 hours in the presence of delphinidin (50 μM) or compound C. α-Actinin staining was performed to determine cell size. Representative images (A1) and quantified cell sizes (A2) of each group are shown (scale bar=20 μm). Cell surface areas (μm²) were measured in 3 independent experiments with at least 100 cells counted for each condition. (B) qRT-PCR was performed to analyze the expression of hypertrophic genes. (C, D), Representative image and quantitative analysis of ROS generation measured by DCF-DA and DHE staining. (E) Statistical analysis of differences in nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity. A.U., arbitrary units. In A–E, *p<0.05 versus the Ang II group; **p<0.01 versus the Ang II group; ***p<0.001 versus the Ang II group; #p<0.05 versus the Ang II+Dp group; ##p<0.01 versus the Ang II+Dp group; ###p<0.001 versus the Ang II+Dp group.

  
  
  

  

  Effect of delphinidin on the MAPK signaling pathway. (A1, B1) Representative western blots showing total and phosphorylated ERK, JNK, and P38. (A2, B2) Quantitative results of western blot analysis (n=4); *p<0.05; **p<0.01; ***p<0.001. Bubbles and traces besides the main strips are parts of the blotting background in western blot.

  
  
  

  

  Delphinidin reduced cardiac hypertrophy in aged mice. (A) Representative gross morphology of young and aged mice administered delphinidin and DMSO. (B) Statistical analysis of differences in the heart weight/tibia length (HW/TL) ratio (n=6). (C) Left ventricular ejection fraction and fractional shortening of young and aged mice administered delphinidin and DMSO (n=6). (D) Left, H&E staining was performed to assess hypertrophic growth of the hearts of young and aged mice administered with delphinidin and DMSO. Right, Statistical analysis of differences in cardiomyocyte size (n=6). (E) Left, Representative PSR staining of histological sections of the LV (n=6). Right, Statistical analysis of differences in cardiac fibrosis. (F) Quantitative real-time PCR (qRT-PCR) was performed to analyze the mRNA levels of hypertrophic genes and fibrosis genes (n=5). In B–F, *p<0.05, **p<0.01, ***p<0.001.

  
  
  

  

  Delphinidin reduced ROS production in the aged myocardium through inhibiting NOX by activating AMPK. (A) Quantitative dihydroethidium (DHE) staining (n=8). (B) Statistical analysis of differences in nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity. A.U., arbitrary units. (C) Representative western blot analysis revealed AMPK phosphorylation levels (n=4). (D) Determination of rac1 activity. The cell lysates were affinity precipitated with GTP-PBD bound to glutathione-agarose beads. Precipitated GTP-Rac1 was detected by immunoblotting with anti-Rac1 antibody (n=4). (E) Expression of the NOX subunits p47^phox, p40^phox, p67^phox, gp91^phox, and p22^phox (n=4). In A–D, *p<0.05, **p<0.01, ***p<0.001. In (E) *p<0.05 versus the young+DMSO group; **p<0.01 versus the young+DMSO group; ***p<0.001 versus the young+DMSO group; ns versus the aged+DMSO group; #p<0.05 versus the aged+DMSO group.

  
  
  

  

  Cartoon demonstrating that delphinidin attenuates pathological cardiac hypertrophy via the AMPK/NOX/MAPK signaling pathway.

  
  
  

• Research Paper Volume 5, Issue 7 pp 515-530

  Oxidative stress improves coronary endothelial function through activation of the pro-survival kinase AMPK

  Relevance score: 12.41467

  Ehtesham Shafique, Wing C. Choy, Yuhong Liu, Jun Feng, Brenda Cordeiro, Arthur Lyra, Mohammed Arafah, Abdulmounem Yassin-Kassab, Arthus V.D. Zanetti, Richard T. Clements, Cesario Bianchi, Laura E. Benjamin, Frank W. Sellke, Md. Ruhul Abid

  Keywords: Endothelial function, signal transduction, NADPH oxidase, reactive oxygen species, autophagy, aging

  Published in Aging on June 23, 2013

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  Age-associated decline in cardiovascular function is believed to occur from the deleterious effects of reactive oxygen species (ROS). However, failure of recent clinical trials using antioxidants in patients with cardiovascular disease, and the recent findings showing paradoxical role for NADPH oxidase-derived ROS in endothelial function challenge this long-held notion against ROS. Here, we examine the effects of endothelium-specific conditional increase in ROS on coronary endothelial function. We have generated a novel binary (Tet-ON/OFF) conditional transgenic mouse (Tet-Nox2:VE-Cad-tTA) that induces endothelial cell (EC)-specific overexpression of Nox2/gp91 (NADPH oxidase) and 1.8±0.42-fold increase in EC-ROS upon tetracycline withdrawal (Tet-OFF). We examined ROS effects on EC signaling and function. First, we demonstrate that endothelium-dependent coronary vasodilation was significantly improved in Tet-OFF Nox2 compared to Tet-ON (control) littermates. Using EC isolated from mouse heart, we show that endogenous ROS increased eNOS activation and nitric oxide (NO) synthesis through activation of the survival kinase AMPK. Coronary vasodilation in Tet-OFF Nox2 animals was CaMKKβ-AMPK-dependent. Finally, we demonstrate that AMPK activation induced autophagy and thus, protected ECs from oxidant-induced cell death. Together, these findings suggest that increased ROS levels, often associated with cardiovascular conditions in advanced age, play a protective role in endothelial homeostasis by inducing AMPK-eNOS axis.

  

  (A) Schematic used to make conditional binary transgenic mice (NVF). Tet-ON, tetracycline in the drinking water to suppress the transgene; Tet-OFF, withdrawal of tetracycline for four weeks to induce the transgene. (B) Frozen heart sections of 8 weeks old Tet-ON and Tet.OFF NVF (for 4 weeks) mice showing EC-specific (CD31, green) HA-tagged Nox2 (red) expression in coronary vessels of Tet-OFF animal. (C) Immunohistochemistry using anti-HA antibody on frozen heart sections. (D) Western blots using transgenic mouse heart EC (MHEC) lysates showing Nox2 overexpression in two independent lines of Tet-OFF animals compared to Tet-On and WT animals. (E) Q-PCR using MHEC RNAs from Tet-ON and Tet-OFF animals (n=6/group). (F) FACS analyses using DCF fluorescence of MHECs (n=6/group). All animals were 8 weeks old, Tet-OFF were without tetracycline for four weeks. Tet-ON MHECs were grown in medium containing tetracycline 2μg/mL. *p<0.05.

  
  
  

  

  (A) Endothelium-dependent dilation of coronary arterioles from Tet-ON (n=6) and Tet-OFF (n=6) NVF mice in response to VEGF. 22±3.72% increase in vasodilation in Tet-OFF vs. Tet-ON. (B) Endothelium-dependent dilation of coronary arterioles from Tet-ON (n=6) and Tet-OFF (n=6) NVF mice in response to acetylcholine (Ach). 25±2.43% increased dilation in Tet-OFF vs. Tet-ON. (C) Endothelium-independent dilation of coronary arterioles from Tet-ON (n=6) and Tet-OFF (n=6) mice in response to NO donor, SNP. (D) NO-cGMP signaling inhibitor ODQ (10 μmol/L) inhibited coronary vasorelaxation in both Tet-ON and Tet-OFF coronary vessels. n = 6/group. All coronary vessels were pre-constricted ex-vivo using U46619 prior to the addition of VEGF, Ach or SNP as indicated.

  
  
  

  

  (A) Western blots (WB) analyses of MHEC protein lysates from two independent lines of NVF Tet-ON and Tet-OFF mice as indicated. WB was carried out using anti-phospho-AMPK (p-AMPK), anti-p-Akt (ser473), anti-p-eNOS (ser1179) and anti-T-Akt (total) antibodies. T-Akt was used as loading control. Right panels, bar graphs show quantitative densitometric analysis of three independent experiments using NIH image J (-fold change expressed in mean ± S.E.M.). *p<0.05 was considered statistically significant. (B) Protein extracts from Tet-OFF MHEC transfected with control siRNA (Scram-si) or si-AMPK were subject to Western blots as described in the Methods. Membranes were sequentially blotted, stripped and re-probed with anti-AMPK, anti-p-eNOS and GAPDH antibodies as shown. Representative blots of two independent experiments are shown. (C) NO production, as measured using citruline assay as described in Methods, was 2.1±0.32-fold higher in Tet-OFF MHEC compared to Tet-ON. Si-AMPK significantly inhibited NO production in Tet-OFF MHEC. *p<0.05.

  
  
  

  

  (A) Isolated coronary vessels from Tet-ON and Tet-OFF transgenic mice (n=6/group) were subject to microvessel reactivity assay in the presence or absence of Compound C (80 μmol/L). Ach-mediated vasodilatation was inhibited by Compound C in the coronary vessels from Tet-OFF mice, whereas Compound C had no significant effect on the coronary vessels from Tet-ON mice. (B) Same as in (A), except pre-treatment was carried out using CaMKKβ-inhibitor STO-609 (50 nmol/L).

  
  
  

  

  (A) Western blot analyses of MHEC protein lysates from two independent lines of NVF Tet-ON and Tet-OFF mice as indicated. WB was carried out using anti-p-mTOR (p-mTOR), anti-p-70S 6k, and anti-4E-BP antibodies. GAPDH was used for loading control. Lower panels, bar graphs show quantitative densitometric analysis of three independent experiments of the p-mTOR and p-70S 6K bands (-fold change expressed in mean ± S.E.M.). *p<0.05 was considered statistically significant. (B) WB analyses of MHEC from two independent lines of NVF Tet-ON and Tet-OFF as in (A) except anti-LC3A (I and II) and ant-GAPDH antibodies were used. Arrow, induction of the autophagy marker LC3-II in Tet-OFF MHEC is indicated. Lower panel, bar graph showing quantitative analyses of LC3-II as indicated. *p<0.05.

  
  
  

  

  (A) Schematic presentation shows that GFP-LC3 (green) and mRFP-LC3 (red) signals are present on the autophagosome, whereas autolysosome contains only mRFP-LC3 (red) signals [40]. (B) Confocal microscopy of Adv-mRFP-GFP-LC3-transduced MHECs. (C) Quantification of red and green fluorophores using NIH ImageJ 1.47b demonstrate >1.5-fold increase in autophagy in Tet-OFF MHEC (n=50 cells). (D) Quantification of colocalization events (yellow) using spatial overlap of red and green was done in Fiji program (ImageJ 1.47h). 1.5-fold increase in overall autophagy in Tet-OFF MHEC (C), but no significant changes in the ratio of intracellular red vs. green in Tet-OFF MHEC (C), and no changes in the colocalization signals between Tet-ON vs. Tet-OFF (D) suggest an effective autophagic flux in Tet-OFF MHEC. *p<0.05.

  
  
  

  

  (A) Tet-ON and Tet-OFF MHEC were transduced with Adv-mRFP-GFP-LC3 and transfected with either si-scr or si-Nox2 as indicated. Quantification of red and green fluorophores using NIH ImageJ 1.47b demonstrate significant increase in autophagy in Tet-OFF MHEC (n=50 cells), which was abrogated by knockdown of Nox2. (B) Annexin V-FITC labeling was carried out to determine apoptotic MHEC as described in the Methods. Bar graph shows apoptotic cells as percentage of total viable cells population using three independent experiments. Autophagosome-lysosome fusion blocker chloroquine induced apoptosis in both Tet-ON and Tet-OFF MHEC. However, chloroquine-induced apoptosis was significantly higher in Tet-OFF MHEC.

  
  
  

  

  Nox2-induced ROS in vascular endothelium activates CaMKKβ-AMPK, which in turn, activates eNOS to induce NO-mediated vasodilatation and inhibits mTOR resulting in protective autophagy.

  
  
  

• Research Perspective Volume 2, Issue 12 pp 1012-1016

  The NADPH Oxidase Nox4 and Aging in the Heart

  Relevance score: 13.901667

  Tetsuro Ago, Shouji Matsushima, Junya Kuroda, Daniela Zablocki, Takanari Kitazono, Junichi Sadoshima

  Keywords: aging, ROS, mitochondria, NADPH oxidase, Nox4

  Published in Aging on December 27, 2010

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  Oxidative stress in mitochondria is believed to promote aging. Although passive leakage of electron from the mitochondrial electron transport chain has been considered as a major source of oxidative stress in the heart and the cardiomyocytes therein, enzymes actively producing reactive oxygen species may also exist in mitochondria. We have shown recently that Nox4, a member of the NADPH oxidase family, is localized on intracellular membranes, primarily at mitochondria, in cardiomyocytes. Mitochondrial expression of Nox4 is upregulated by cardiac stress and aging in the heart, where Nox4 could become a major source of oxidative stress. This raises an intriguing possibility that Nox4 may play an important role in mediating aging of the heart. Here we discuss the potential involvement of Nox4 in mitochondrial oxidative stress and aging in the heart.

• Research Paper pp undefined-undefined

  Comprehensive bioinformatics analysis of NOX4 as a biomarker for pan-cancer prognosis and immune infiltration

  Relevance score: 11.256583

  Yuying Liu, Hua Huang, Xijun Yang, Danhe Huang, Xiongwei Wang, Mingyu Yuan, Lianqing Hong

  Keywords: pan-cancer analysis, bioinformatics, NADPH oxidase 4, tumor immune microenvironment, immune checkpoint

  Published in Aging on Invalid Date

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  Background: NADPH oxidase 4 (NOX4) has been proven to be associated with the prognosis of tumors in multiple cancers and can serve as a potential immunotherapy target to provide new treatment options for various tumors. In this study, our aim is to conduct an in-depth investigation of NOX4 across a range of cancer types to determine the relationship between NOX4 and tumors.

  Methods: Utilizing large-scale transcriptomic and clinical data from public databases, a systematic examination of NOX4 expression patterns was performed in pan-cancer cohorts. Survival analysis, methylation analysis, and correlation studies were employed to assess the diagnostic and prognostic significance of NOX4 in diverse cancer types. Additionally, an exploration of the relationship between NOX4 expression and immune infiltration across various tumors was conducted.

  Results: The analyses unveiled a consistent upregulation of NOX4 expression in multiple cancer types relative to normal tissues, indicating its potential as a universal cancer biomarker. Elevated NOX4 expression significantly correlated with poor overall survival in several cancers. Furthermore, the study demonstrated a robust correlation between NOX4 expression and immune cell infiltration, signifying its involvement in the modulation of the tumor microenvironment.

  Conclusions: This study imparts valuable insights into the potential applications of NOX4 in cancer research, highlighting its significance as a multifaceted biomarker with diagnostic, prognostic, and immunomodulatory implications across diverse malignancies.