Aldose reductase regulates hyperglycemia-induced HUVEC death via SIRT1/AMPK-α1/mTOR pathway

in Journal of Molecular Endocrinology
Correspondence should be addressed to K V Ramana: kvramana@utmb.edu

*(P B Pal and H Sonowal contributed equally to this work)

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Although hyperglycemia-mediated death and dysfunction of endothelial cells have been reported to be a major cause of diabetes associated vascular complications, the mechanisms through which hyperglycemia cause endothelial dysfunction is not well understood. We have recently demonstrated that aldose reductase (AR, AKR1B1) is an obligatory mediator of oxidative and inflammatory signals induced by growth factors, cytokines and hyperglycemia. However, the molecular mechanisms by which AR regulates hyperglycemia-induced endothelial dysfunction is not well known. In this study, we have investigated the mechanism(s) by which AR regulates hyperglycemia-induced endothelial dysfunction. Incubation of human umbilical vein endothelial cells (HUVECs) with high glucose (HG) decreased the cell viability and inhibition of AR prevented it. Further, AR inhibition prevented the HG-induced ROS generation and expression of BCL-2, BAX and activation of Caspase-3 in HUVECs. AR inhibition also prevented the adhesion of THP-1 monocytes on HUVECs, expression of iNOS and eNOS and adhesion molecules ICAM-1 and VCAM-1 in HG-treated HUVECs. Further, AR inhibition restored the HG-induced depletion of SIRT1 in HUVECs and increased the phosphorylation of AMPKα1 along-with a decrease in phosphorylation of mTOR in HG-treated HUVECs. Fidarestat decreased SIRT1 expression in HUVECs pre-treated with specific SIRT1 inhibitor but not with the AMPKα1 inhibitor. Similarly, knockdown of AR in HUVECs by siRNA prevented the HG-induced HUVECs cell death, THP-1 monocyte adhesion and SIRT1 depletion. Furthermore, fidarestat regulated the phosphorylation of AMPKα1 and mTOR, and expression of SIRT1 in STZ-induced diabetic mice heart and aorta tissues. Collectively, our data suggest that AR regulates hyperglycemia-induced endothelial death and dysfunction by altering the ROS/SIRT1/AMPKα1/mTOR pathway.

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    Inhibition AR protects HG-induced decrease in the HUVEC viability. (A) HUVECs were treated with HG (25 mM) without or with fidarestat (10 µM) in a medium containing 0.5% FBS for 24, 48 and 72 h. Cell viability was measured by MTT assay (represented as % change over control). (B) HUVECs were incubated with HG without or with fidarestat in 24-well plates for 72 h at 37°C in a CO2 incubator. After 72 h, the cells were stained with Calcein AM, washed twice in HBSS. Fluorescence images (20×) were obtained using a fluorescence microscope. (C) Dot plots showing Annexin V/PI-stained HUVECs analyzed by flow cytometry after 48-h treatment with HG (25 mM) without or with fidarestat in 0.5% FBS containing media. (D) Bars showing quantification of percentage Annexin V and PI-positive cells in different treatment condition shown in (C). Data acquisition was done using a BD LSRII Fortessa and analyzed by Flow Jo software. Representative data are shown. Bars represent the mean ± s.d. (n = 4). # P < 0.05 vs normal/untreated control (normal glucose; 5.5 mM); **P < 0.01 vs HG (high glucose; 19.5 mM glucose added to 5.5 mM normal glucose media). A full colour version of this figure is available at https://doi.org/10.1530/JME-19-0080.

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    AR inhibition suppresses HG-induced intracellular ROS production in HUVEC. Growth-arrested HUVECs were incubated with high glucose (25 mM) without or with fidarestat (10 µM) in tissue culture plates for 24 and 48 h at 37°C in a CO2 incubator. (A) Fluorescence microscopic images showing ROS in cells detected using CM-H2DCFDA dye. The cells were loaded with the fluorescent dye 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate acetyl ester (CM-H2DCFDA; 5 µM, 30 min), and washed with HBSS. Fluorescence images (20×) were obtained using a fluorescence microscope. Representative images are shown. (B) Bars showing the ratio of fluorescence intensities in 590 (red)/510 (green) after staining with 10 µM lipid peroxidation sensor dye provided with Image-iT Lipid peroxidation Kit for 30 min and recorded using a Synergy 2 microplate reader. Oxidative stress-induced oxidation of dye leads to a shift in fluorescence from red (590) to green (510) and the ratio of fluorescence intensity (590/510) provides a readout for lipid peroxidation. 590/510 ratio is inversely proportional to lipid peroxidation. Bars represent mean ± s.d. (n = 3). Representative data is shown. *P < 0.01 vs control. # P < 0.01 vs HG treated. A full colour version of this figure is available at https://doi.org/10.1530/JME-19-0080.

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    AR inhibition prevents HG-induced apoptotic cell death in HUVECs. HUVECs were incubated with HG (25 mM) without or with fidarestat (10 µM) and resveratrol (20 µM) for 48 h at 37°C in a CO2 incubator. (A) Equal amount of proteins from the isolated mitochondrial and cytosolic fractions were used for Western blot analyses for BCL-2, BAX, Cyt c, Cleaved PARP, Cleaved Caspase 3, COX-IV and GAPDH. COX-IV and GAPDH were used as a loading control for mitochondrial protein and cytosolic fraction, respectively. (B and C) Bars representing a densitometric analysis of Western blots shown in (A). Representative blots from the three independent analyses are shown.

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    Inhibition of AR prevents HG-induced monocyte adhesion and expression of adhesion molecules, eNOS, and iNOS. (A) Growth-arrested HUVECs were incubated with HG (25 mM) without or with fidarestat (10 µM) in chambered tissue culture slides for 48 h at 37°C in a CO2 incubator. Subsequently, Calcein AM-labeled THP-1 cells were added to the treated HUVECs for 12 h. The cells were then washed with HBSS. Fluorescence images (20×) were obtained using a fluorescence microscope. (B) HUVECs were incubated with HG without or with fidarestat (10 µM) and resveratrol (Res) (20 µM) in 96-well plates for 48 h at 37°C in a CO2 incubator. Subsequently, THP-1 cells were added to the treated HUVECs for 12 h and then washed with PBS. Cell viability (% over control) was measured by MTT assay. (C) HUVECs were incubated with HG without or with fidarestat (10 µM) and resveratrol (20 µM) for 48 h at 37°C in a CO2 incubator. Western blot analysis was performed by using specific ICAM-1, VCAM-1, iNOS, and eNOS antibodies. Representative blots are shown. (D) Bars representing a densitometric analysis of western blots shown in C. The bars represent the mean ± s.d. (n = 4). # P < 0.05 vs normal (normal glucose, 5.5 mM); **P < 0.01 vs HG (high glucose; 19.5 mM glucose added to 5.5 mM normal glucose media). A full colour version of this figure is available at https://doi.org/10.1530/JME-19-0080.

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    Aldose reductase inhibition activates SIRT 1 in HG-induced HUVECs. (A) Growth-arrested HUVECs were incubated with HG (25 mM) without or with fidarestat (10 µM) and resveratrol (20 µM) for 48 h at 37°C in a CO2 incubator. Western blot analysis was performed by using specific SIRT1 antibodies. (B) HUVEC were treated with HG in the presence of SIRT1 activator (Res: 20 µM) and SIRT1 inhibitor (SIRT1 inh. III: 5 µM) for 72 h. Cell viability (% over control) was measured by MTT assay. (C) Immunofluorescence staining was used to measure the expression for SIRT1 using antibodies against SIRT1. The images (40×) were taken in a fluorescence microscope. Scale bar = 100 µM. (D) Bars showing quantification of fluorescence intensity of SIRT1 immunofluorescence staining shown in Panel C. Fluorescent images were quantified using Image J software. Bars represent the mean ± s.d. (n = 6). # P < 0.001 vs normal (normal glucose; 5.5 mM); **P < 0.001 vs HG (high glucose; 19.5 mM glucose added to 5.5 mM normal glucose media). A full colour version of this figure is available at https://doi.org/10.1530/JME-19-0080.

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    AR inhibitor fidarestat regulates AMPKα1 and mTOR signaling in HUVECs. (A) Growth-arrested HUVECs were pre-treated with fidarestat (10 µM) for 4 h followed by incubation with HG (25 mM) for 0, 30, 60, 120 and 240 min. Western blot analyses were performed by using specific total and phospho-AMPKα1, total AMPKα1, phospho-mTOR and total mTOR antibodies. (B) HUVECs were treated with HG in the absence or presence of SIRT1 activator (Res: 20 µM) and SIRT1 inhibitor (SIRT1 inh. III: 5 µM) for 48 h. Western blot analyses were performed by using specific pAMPKα1, AMPKα1, phospho- mTOR and total mTOR antibodies. (C) HUVECs were treated with HG without or with fidarestat followed by treatment with SIRT1 (SIRT1 inh. III: 5 µM) and AMPK inhibitors (Compound C: 10 µM) for 48 h. Western blot analyses were performed by using specific SIRT1, pAMPKα1 and AMPKα1 antibodies. Representative blots from three independent analysis are shown.

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    Effect of AR knockdown on HUVEC viability, expression of SIRT1 and monocyte adhesion. (A) HUVECs were transfected with AR-siRNA or control-siRNA and cultured for 48 h at 37°C. Transfected and normal cells were treated with HG in the presence or absence of SIRT1 activator (Res: 20 µM) and SIRT1 inhibitor (SIRT1 inh. III: 5 µM) for 72 h. Cell viability (% over control) was measured by MTT assay. The inset shows the Western blot of AR expression in AR siRNA transfected cells. (B) The levels of SIRT1 protein expression was assayed by immunofluorescence staining. The images (40×) were taken in a fluorescence microscope. (C) AR-knockdown and normal cells were treated with HG in the presence or absence of SIRT1 activator (Res: 20 µM) and SIRT1 inhibitor (Sirt1 inh.III: 5 µM) for 48 h. Subsequently, THP-1 cells were added to the treated HUVECs for 12 h, washed with PBS and cell viability (% over control) was determined by MTT assay. (D) The AR-knockdown and normal cells were treated with HG for 48 h. Western blot analyses were performed by using specific SIRT1, pAMPKα1, AMPKα1 and GAPDH antibodies. (E). Bars showing densitometric analysis of western blots shown in D. The bars represent the mean ± s.d. (n = 4). # P < 0.001 vs normal (normal glucose; 5.5 mM); **P < 0.001 vs HG (high glucose; 19.5 mM glucose added to 5.5 mM normal glucose media). A full colour version of this figure is available at https://doi.org/10.1530/JME-19-0080.

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    AR inhibition increased the expression of SIRT1, pAMPKα1 and decreased pmTOR expression in STZ-induced diabetic mice hearts and aortae. Male C57BL/6 mice were made diabetic as described in the methods. The diabetic mice were treated with fidarestat (10 mg/kg/day i.p) for 6 days. The levels of (A) SIRT1, (B) pAMPKα1 and (C) pmTOR proteins were measured in heart tissues by Western blot analysis using specific antibodies. In another set of experiments, diabetic mice were treated with AR-inhibitor fidarestat for 21 days (10 mg/kg/day i.p). Thoracic aorta tissue sections were dissected and lysed and analyzed by western blotting using specific antibodies for (D) Sirt1, (E) p-AMPKα1 and (F) p-mTOR. A representative blot from each group is shown.

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