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Activation of protease calpain by oxidized and glycated LDL increases the degradation of endothelial nitric oxide synthase.

Dong Y, Wu Y, Wu M, Wang S, Zhang J, Xie Z, Xu J, Song P, Wilson K, Zhao Z, Lyons T, Zou MH - J. Cell. Mol. Med. (2008)

Bottom Line: Neither eNOS reduction nor any of these other effects were observed in cells exposed to native LDL.Reduction of intracellular Ca(2+) levels abolished eNOS reduction by HOG-LDL, as did pharmacological or genetic through calcium channel blockers or calcium chelator BAPTA or inhibition of NAD(P)H oxidase (with apocynin) or inhibition of calpain (calpain 1-specific siRNA).Consistent with these results, HOG-LDL impaired acetylcholine-induced endothelium-dependent vasorelaxation of isolated mouse aortas, and pharmacological inhibition of calpain prevented this effect.

View Article: PubMed Central - PubMed

Affiliation: Harold Hamm Oklahoma Diabetes Center, Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA.

ABSTRACT
Oxidation and glycation of low-density lipoprotein (LDL) promote vascular injury in diabetes; however, the mechanisms underlying this effect remain poorly defined. The present study was conducted to determine the effects of 'heavily oxidized' glycated LDL (HOG-LDL) on endothelial nitric oxide synthase (eNOS) function. Exposure of bovine aortic endothelial cells with HOG-LDL reduced eNOS protein levels in a concentration- and time-dependent manner, without altering eNOS mRNA levels. Reduced eNOS protein levels were accompanied by an increase in intracellular Ca(2+), augmented production of reactive oxygen species (ROS) and induction of Ca(2+)-dependent calpain activity. Neither eNOS reduction nor any of these other effects were observed in cells exposed to native LDL. Reduction of intracellular Ca(2+) levels abolished eNOS reduction by HOG-LDL, as did pharmacological or genetic through calcium channel blockers or calcium chelator BAPTA or inhibition of NAD(P)H oxidase (with apocynin) or inhibition of calpain (calpain 1-specific siRNA). Consistent with these results, HOG-LDL impaired acetylcholine-induced endothelium-dependent vasorelaxation of isolated mouse aortas, and pharmacological inhibition of calpain prevented this effect. HOG-LDL may impair endothelial function by inducing calpain-mediated eNOS degradation in a ROS- and Ca(2+)-dependent manner.

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HOG-LDL-induced increases in intracellular Ca2+ and decreases in eNOS are ROS-dependent. (A) Superoxide (O2−) production in BAECs exposed to HOG-LDL (100 μg/ml, time course). n= 4, *P < 0.01 Controls versus each time-point. (B) Hydrogen peroxide (H2O2) production in HOG-LDL-exposed BAECs. n= 4, **P < 0.01 versus control. (C) Effect of HOG-LDL on NADPH oxidase activation, as determined by membrane translocation of p47phox. BAECs exposed to agentension II (Ang II, 1 μM) served as a positive control. The Western blot shown is representative of four blots obtained from four separate experiments. (D) Effect of EGTA (2.5 mM, 1-hr pre-incubation) on HOG-LDL-induced O2− release in BAECs. n= 3, **P < 0.01 versus control, ##P < 0.01 versus HOG-LDL. (E) H2O2 production in BAECs treated with HOG-LDL ± EGTA. n= 3, *P < 0.05 versus control, #P < 0.05 versus HOG-LDL. (F) Effect of apocynin (100 μM) or DPI (10 μM) on intracellular Ca2+ levels in BAEC exposed to HOG-LDL. n= 3, **P < 0.01 versus control, ##P < 0.01 versus HOG-LDL. (G) Effect of apocynin or DPI on eNOS degradation by HOG-LDL (100 μg/ml, 6 hrs), n= 4, *P < 0.01 versus untreated controls or n-LDL, #P < 0.01 versus HOG-LDL.
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fig05: HOG-LDL-induced increases in intracellular Ca2+ and decreases in eNOS are ROS-dependent. (A) Superoxide (O2−) production in BAECs exposed to HOG-LDL (100 μg/ml, time course). n= 4, *P < 0.01 Controls versus each time-point. (B) Hydrogen peroxide (H2O2) production in HOG-LDL-exposed BAECs. n= 4, **P < 0.01 versus control. (C) Effect of HOG-LDL on NADPH oxidase activation, as determined by membrane translocation of p47phox. BAECs exposed to agentension II (Ang II, 1 μM) served as a positive control. The Western blot shown is representative of four blots obtained from four separate experiments. (D) Effect of EGTA (2.5 mM, 1-hr pre-incubation) on HOG-LDL-induced O2− release in BAECs. n= 3, **P < 0.01 versus control, ##P < 0.01 versus HOG-LDL. (E) H2O2 production in BAECs treated with HOG-LDL ± EGTA. n= 3, *P < 0.05 versus control, #P < 0.05 versus HOG-LDL. (F) Effect of apocynin (100 μM) or DPI (10 μM) on intracellular Ca2+ levels in BAEC exposed to HOG-LDL. n= 3, **P < 0.01 versus control, ##P < 0.01 versus HOG-LDL. (G) Effect of apocynin or DPI on eNOS degradation by HOG-LDL (100 μg/ml, 6 hrs), n= 4, *P < 0.01 versus untreated controls or n-LDL, #P < 0.01 versus HOG-LDL.

Mentions: [32]). Thus, we hypothesized that ROS mediate the elevation in intracellular Ca2+ and subsequent calpain activation by HOG-LDL. In accordance with this hypothesis, HOG-LDL increased O2− levels by ∼ 4-fold (P < 0.01), whereas N-LDL had no effect (Fig. 5A). In addition, H2O2 was increased approximately twofold following 6 hrs of HOG-LDL treatment (Fig. 5B).


Activation of protease calpain by oxidized and glycated LDL increases the degradation of endothelial nitric oxide synthase.

Dong Y, Wu Y, Wu M, Wang S, Zhang J, Xie Z, Xu J, Song P, Wilson K, Zhao Z, Lyons T, Zou MH - J. Cell. Mol. Med. (2008)

HOG-LDL-induced increases in intracellular Ca2+ and decreases in eNOS are ROS-dependent. (A) Superoxide (O2−) production in BAECs exposed to HOG-LDL (100 μg/ml, time course). n= 4, *P < 0.01 Controls versus each time-point. (B) Hydrogen peroxide (H2O2) production in HOG-LDL-exposed BAECs. n= 4, **P < 0.01 versus control. (C) Effect of HOG-LDL on NADPH oxidase activation, as determined by membrane translocation of p47phox. BAECs exposed to agentension II (Ang II, 1 μM) served as a positive control. The Western blot shown is representative of four blots obtained from four separate experiments. (D) Effect of EGTA (2.5 mM, 1-hr pre-incubation) on HOG-LDL-induced O2− release in BAECs. n= 3, **P < 0.01 versus control, ##P < 0.01 versus HOG-LDL. (E) H2O2 production in BAECs treated with HOG-LDL ± EGTA. n= 3, *P < 0.05 versus control, #P < 0.05 versus HOG-LDL. (F) Effect of apocynin (100 μM) or DPI (10 μM) on intracellular Ca2+ levels in BAEC exposed to HOG-LDL. n= 3, **P < 0.01 versus control, ##P < 0.01 versus HOG-LDL. (G) Effect of apocynin or DPI on eNOS degradation by HOG-LDL (100 μg/ml, 6 hrs), n= 4, *P < 0.01 versus untreated controls or n-LDL, #P < 0.01 versus HOG-LDL.
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fig05: HOG-LDL-induced increases in intracellular Ca2+ and decreases in eNOS are ROS-dependent. (A) Superoxide (O2−) production in BAECs exposed to HOG-LDL (100 μg/ml, time course). n= 4, *P < 0.01 Controls versus each time-point. (B) Hydrogen peroxide (H2O2) production in HOG-LDL-exposed BAECs. n= 4, **P < 0.01 versus control. (C) Effect of HOG-LDL on NADPH oxidase activation, as determined by membrane translocation of p47phox. BAECs exposed to agentension II (Ang II, 1 μM) served as a positive control. The Western blot shown is representative of four blots obtained from four separate experiments. (D) Effect of EGTA (2.5 mM, 1-hr pre-incubation) on HOG-LDL-induced O2− release in BAECs. n= 3, **P < 0.01 versus control, ##P < 0.01 versus HOG-LDL. (E) H2O2 production in BAECs treated with HOG-LDL ± EGTA. n= 3, *P < 0.05 versus control, #P < 0.05 versus HOG-LDL. (F) Effect of apocynin (100 μM) or DPI (10 μM) on intracellular Ca2+ levels in BAEC exposed to HOG-LDL. n= 3, **P < 0.01 versus control, ##P < 0.01 versus HOG-LDL. (G) Effect of apocynin or DPI on eNOS degradation by HOG-LDL (100 μg/ml, 6 hrs), n= 4, *P < 0.01 versus untreated controls or n-LDL, #P < 0.01 versus HOG-LDL.
Mentions: [32]). Thus, we hypothesized that ROS mediate the elevation in intracellular Ca2+ and subsequent calpain activation by HOG-LDL. In accordance with this hypothesis, HOG-LDL increased O2− levels by ∼ 4-fold (P < 0.01), whereas N-LDL had no effect (Fig. 5A). In addition, H2O2 was increased approximately twofold following 6 hrs of HOG-LDL treatment (Fig. 5B).

Bottom Line: Neither eNOS reduction nor any of these other effects were observed in cells exposed to native LDL.Reduction of intracellular Ca(2+) levels abolished eNOS reduction by HOG-LDL, as did pharmacological or genetic through calcium channel blockers or calcium chelator BAPTA or inhibition of NAD(P)H oxidase (with apocynin) or inhibition of calpain (calpain 1-specific siRNA).Consistent with these results, HOG-LDL impaired acetylcholine-induced endothelium-dependent vasorelaxation of isolated mouse aortas, and pharmacological inhibition of calpain prevented this effect.

View Article: PubMed Central - PubMed

Affiliation: Harold Hamm Oklahoma Diabetes Center, Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA.

ABSTRACT
Oxidation and glycation of low-density lipoprotein (LDL) promote vascular injury in diabetes; however, the mechanisms underlying this effect remain poorly defined. The present study was conducted to determine the effects of 'heavily oxidized' glycated LDL (HOG-LDL) on endothelial nitric oxide synthase (eNOS) function. Exposure of bovine aortic endothelial cells with HOG-LDL reduced eNOS protein levels in a concentration- and time-dependent manner, without altering eNOS mRNA levels. Reduced eNOS protein levels were accompanied by an increase in intracellular Ca(2+), augmented production of reactive oxygen species (ROS) and induction of Ca(2+)-dependent calpain activity. Neither eNOS reduction nor any of these other effects were observed in cells exposed to native LDL. Reduction of intracellular Ca(2+) levels abolished eNOS reduction by HOG-LDL, as did pharmacological or genetic through calcium channel blockers or calcium chelator BAPTA or inhibition of NAD(P)H oxidase (with apocynin) or inhibition of calpain (calpain 1-specific siRNA). Consistent with these results, HOG-LDL impaired acetylcholine-induced endothelium-dependent vasorelaxation of isolated mouse aortas, and pharmacological inhibition of calpain prevented this effect. HOG-LDL may impair endothelial function by inducing calpain-mediated eNOS degradation in a ROS- and Ca(2+)-dependent manner.

Show MeSH
Related in: MedlinePlus