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Nox2 and Nox4 mediate tumour necrosis factor-α-induced ventricular remodelling in mice.

Moe KT, Yin NO, Naylynn TM, Khairunnisa K, Wutyi MA, Gu Y, Atan MS, Wong MC, Koh TH, Wong P - J. Cell. Mol. Med. (2011)

Bottom Line: ROS was significantly decreased by inhibitors of NADPH oxidase, but not by inhibitors of other ROS production systems.Nox2 and Nox4 siRNA significantly attenuated TNF-α-induced ROS and upregulation of IL-1β and IL-6 in cardiomyocytes.Our study highlights a novel TNF-α-induced chronic ventricular remodelling mechanism mediated by sequential regulation of Nox2 and Nox4 subunits.

View Article: PubMed Central - PubMed

Affiliation: Research and Development Unit, National Heart Centre Singapore, Singapore. moe.kyaw.thu@nhc.com.sg

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Related in: MedlinePlus

Increased ROS production in ventricles of TNF-α-injected mice. The ROS in the ventricular tissues were detected by incubating the cryostat sections with DHE (2 × 10−6 mol/L) for 30 min at 37°C. Representative of DHE staining of ventricular tissues were shown (A). Magnification = 40 × . Fluorescence intensities in the sections of TNFα-injected mice were normalized with controls. Data were expressed as arbitrary fluorescence units (AFU) ± S.D. (B). Mitochondrial ROS in the ventricular tissues were detected by incubating the sections with MitoSOX Red (5 μM) for 10 min at 37°C. Representative of MitoSOX Red staining of ventricular tissues were shown (C) and normalized fluorescence intensities were shown (D). ROS in the ventricular homogenates was determined with CM-H2DCFDA. The fluorescence intensities of ventricular homogenates of TNF-α-injected and control mice were shown (E). The source of ROS production in the ventricles of TNF-α-injected mice was determined using different inhibitors. NADPH oxidase inhibitors (DPI and apocynin) significantly decreased ROS (F). Data were expressed as relative light unit (RLU) ± S.D. SOD, superoxide dismutase; DPI, diphenyleneiodonium; L-NAME, NG-nitro-L-arginine methyl ester. Trolox equivalent antioxidant capacity (TEAC) assay was performed to detect capacity of antioxidants in the plasma and quantified as millimolar Trolox equivalent (G). Glutathione assay and superoxide dismutase assay were performed to determine the total GSH (H) and SOD levels (I) in the ventricles of TNF-α-injected and control mice respectively. *P < 0.05.
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fig01: Increased ROS production in ventricles of TNF-α-injected mice. The ROS in the ventricular tissues were detected by incubating the cryostat sections with DHE (2 × 10−6 mol/L) for 30 min at 37°C. Representative of DHE staining of ventricular tissues were shown (A). Magnification = 40 × . Fluorescence intensities in the sections of TNFα-injected mice were normalized with controls. Data were expressed as arbitrary fluorescence units (AFU) ± S.D. (B). Mitochondrial ROS in the ventricular tissues were detected by incubating the sections with MitoSOX Red (5 μM) for 10 min at 37°C. Representative of MitoSOX Red staining of ventricular tissues were shown (C) and normalized fluorescence intensities were shown (D). ROS in the ventricular homogenates was determined with CM-H2DCFDA. The fluorescence intensities of ventricular homogenates of TNF-α-injected and control mice were shown (E). The source of ROS production in the ventricles of TNF-α-injected mice was determined using different inhibitors. NADPH oxidase inhibitors (DPI and apocynin) significantly decreased ROS (F). Data were expressed as relative light unit (RLU) ± S.D. SOD, superoxide dismutase; DPI, diphenyleneiodonium; L-NAME, NG-nitro-L-arginine methyl ester. Trolox equivalent antioxidant capacity (TEAC) assay was performed to detect capacity of antioxidants in the plasma and quantified as millimolar Trolox equivalent (G). Glutathione assay and superoxide dismutase assay were performed to determine the total GSH (H) and SOD levels (I) in the ventricles of TNF-α-injected and control mice respectively. *P < 0.05.

Mentions: To detect ROS in the ex vivo ventricular tissues of TNF-α-injected and control mice, DHE staining was performed. No significant difference in fluorescent intensity was observed among day-1 TNF-α-injected and control mice (Fig. 1A and B). However, the fluorescent intensities of mice injected with TNF-α 7 and 28 days previously were significantly higher than control mice. Mitochondrial ROS in the ex vivo ventricular tissues of TNF-α-injected and control mice showed slight fluorescence intensity (Fig. 1C). However, there was no significant difference between TNF-α injected and control mice at all three time points (Fig. 1D). The ROS in the ventricular homogenates was assessed by CM-H2DCFDA fluorescence assay. Mice injected with TNF-α 7 and 28 days previously showed a significant increase in fluorescence compared to the controls, but not in the mice injected 1 day previously (Fig. 1E). The enzymatic sources of the ROS were examined using specific inhibitors in the fluorescence assay. ROS production was significantly reduced with SOD and abolished by NADPH oxidase inhibitors, DPI and apocynin. However, allopurinol, L-NAME and rotenone had no effect on TNF-α-induced ROS in the ventricles (Fig. 1F).


Nox2 and Nox4 mediate tumour necrosis factor-α-induced ventricular remodelling in mice.

Moe KT, Yin NO, Naylynn TM, Khairunnisa K, Wutyi MA, Gu Y, Atan MS, Wong MC, Koh TH, Wong P - J. Cell. Mol. Med. (2011)

Increased ROS production in ventricles of TNF-α-injected mice. The ROS in the ventricular tissues were detected by incubating the cryostat sections with DHE (2 × 10−6 mol/L) for 30 min at 37°C. Representative of DHE staining of ventricular tissues were shown (A). Magnification = 40 × . Fluorescence intensities in the sections of TNFα-injected mice were normalized with controls. Data were expressed as arbitrary fluorescence units (AFU) ± S.D. (B). Mitochondrial ROS in the ventricular tissues were detected by incubating the sections with MitoSOX Red (5 μM) for 10 min at 37°C. Representative of MitoSOX Red staining of ventricular tissues were shown (C) and normalized fluorescence intensities were shown (D). ROS in the ventricular homogenates was determined with CM-H2DCFDA. The fluorescence intensities of ventricular homogenates of TNF-α-injected and control mice were shown (E). The source of ROS production in the ventricles of TNF-α-injected mice was determined using different inhibitors. NADPH oxidase inhibitors (DPI and apocynin) significantly decreased ROS (F). Data were expressed as relative light unit (RLU) ± S.D. SOD, superoxide dismutase; DPI, diphenyleneiodonium; L-NAME, NG-nitro-L-arginine methyl ester. Trolox equivalent antioxidant capacity (TEAC) assay was performed to detect capacity of antioxidants in the plasma and quantified as millimolar Trolox equivalent (G). Glutathione assay and superoxide dismutase assay were performed to determine the total GSH (H) and SOD levels (I) in the ventricles of TNF-α-injected and control mice respectively. *P < 0.05.
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Related In: Results  -  Collection

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fig01: Increased ROS production in ventricles of TNF-α-injected mice. The ROS in the ventricular tissues were detected by incubating the cryostat sections with DHE (2 × 10−6 mol/L) for 30 min at 37°C. Representative of DHE staining of ventricular tissues were shown (A). Magnification = 40 × . Fluorescence intensities in the sections of TNFα-injected mice were normalized with controls. Data were expressed as arbitrary fluorescence units (AFU) ± S.D. (B). Mitochondrial ROS in the ventricular tissues were detected by incubating the sections with MitoSOX Red (5 μM) for 10 min at 37°C. Representative of MitoSOX Red staining of ventricular tissues were shown (C) and normalized fluorescence intensities were shown (D). ROS in the ventricular homogenates was determined with CM-H2DCFDA. The fluorescence intensities of ventricular homogenates of TNF-α-injected and control mice were shown (E). The source of ROS production in the ventricles of TNF-α-injected mice was determined using different inhibitors. NADPH oxidase inhibitors (DPI and apocynin) significantly decreased ROS (F). Data were expressed as relative light unit (RLU) ± S.D. SOD, superoxide dismutase; DPI, diphenyleneiodonium; L-NAME, NG-nitro-L-arginine methyl ester. Trolox equivalent antioxidant capacity (TEAC) assay was performed to detect capacity of antioxidants in the plasma and quantified as millimolar Trolox equivalent (G). Glutathione assay and superoxide dismutase assay were performed to determine the total GSH (H) and SOD levels (I) in the ventricles of TNF-α-injected and control mice respectively. *P < 0.05.
Mentions: To detect ROS in the ex vivo ventricular tissues of TNF-α-injected and control mice, DHE staining was performed. No significant difference in fluorescent intensity was observed among day-1 TNF-α-injected and control mice (Fig. 1A and B). However, the fluorescent intensities of mice injected with TNF-α 7 and 28 days previously were significantly higher than control mice. Mitochondrial ROS in the ex vivo ventricular tissues of TNF-α-injected and control mice showed slight fluorescence intensity (Fig. 1C). However, there was no significant difference between TNF-α injected and control mice at all three time points (Fig. 1D). The ROS in the ventricular homogenates was assessed by CM-H2DCFDA fluorescence assay. Mice injected with TNF-α 7 and 28 days previously showed a significant increase in fluorescence compared to the controls, but not in the mice injected 1 day previously (Fig. 1E). The enzymatic sources of the ROS were examined using specific inhibitors in the fluorescence assay. ROS production was significantly reduced with SOD and abolished by NADPH oxidase inhibitors, DPI and apocynin. However, allopurinol, L-NAME and rotenone had no effect on TNF-α-induced ROS in the ventricles (Fig. 1F).

Bottom Line: ROS was significantly decreased by inhibitors of NADPH oxidase, but not by inhibitors of other ROS production systems.Nox2 and Nox4 siRNA significantly attenuated TNF-α-induced ROS and upregulation of IL-1β and IL-6 in cardiomyocytes.Our study highlights a novel TNF-α-induced chronic ventricular remodelling mechanism mediated by sequential regulation of Nox2 and Nox4 subunits.

View Article: PubMed Central - PubMed

Affiliation: Research and Development Unit, National Heart Centre Singapore, Singapore. moe.kyaw.thu@nhc.com.sg

Show MeSH
Related in: MedlinePlus