Limits...
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.

Show MeSH

Related in: MedlinePlus

HOG-LDL-induced activation of calpain decreases eNOS levels and impairs endothelium-dependent relaxation in C57BL/6J mice aorta. (A) Western blot analysis of total eNOS levels in aortas incubated with HOG-LDL (100 μg/ml for 24 hrs) in the presence or absence of the calpain inhibitor III, MDL28170 (20 μM). n= 4 in each group, **P < 0.01 versus N-LDL, versus HOG-LDL. (B) Endothelium-dependent relaxation of HOG-LDL-exposed aortas treated with or without MDL28170. n= 5, *P < 0.05 for HOG-LDL versus untreated control or N-LDL, #P < 0.05 for HOG-LDL versus HOG-LDL + MDL28170. (C) Endothelium-independent relaxation in aortas treated with HOG-LDL ± MDL28170. Results (mean ± S.E.M.) are expressed as the rate of relaxation to the pre-contraction, n= 4.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2821526&req=5

fig06: HOG-LDL-induced activation of calpain decreases eNOS levels and impairs endothelium-dependent relaxation in C57BL/6J mice aorta. (A) Western blot analysis of total eNOS levels in aortas incubated with HOG-LDL (100 μg/ml for 24 hrs) in the presence or absence of the calpain inhibitor III, MDL28170 (20 μM). n= 4 in each group, **P < 0.01 versus N-LDL, versus HOG-LDL. (B) Endothelium-dependent relaxation of HOG-LDL-exposed aortas treated with or without MDL28170. n= 5, *P < 0.05 for HOG-LDL versus untreated control or N-LDL, #P < 0.05 for HOG-LDL versus HOG-LDL + MDL28170. (C) Endothelium-independent relaxation in aortas treated with HOG-LDL ± MDL28170. Results (mean ± S.E.M.) are expressed as the rate of relaxation to the pre-contraction, n= 4.

Mentions: Next, we determined whether HOG-LDL induces calpain-dependent eNOS degradation in intact aortas. Isolated mouse aortas were exposed to either N-LDL or HOG-LDL in the presence or absence of MDL28170, and eNOS protein levels were measured. Exposure of mouse aortas to MDL28170 alone (data not shown) or N-LDL (Fig. 6A) did not affect eNOS levels. In contrast, exposure of aortas to 100 μg/ml HOG-LDL for 24 hrs reduced eNOS protein levels by 75–80% (P < 0.05, Fig. 6A). Importantly, this reduction in aortic eNOS levels was almost completely blocked by co-administration of MDL28170 (Fig. 6A).


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 activation of calpain decreases eNOS levels and impairs endothelium-dependent relaxation in C57BL/6J mice aorta. (A) Western blot analysis of total eNOS levels in aortas incubated with HOG-LDL (100 μg/ml for 24 hrs) in the presence or absence of the calpain inhibitor III, MDL28170 (20 μM). n= 4 in each group, **P < 0.01 versus N-LDL, versus HOG-LDL. (B) Endothelium-dependent relaxation of HOG-LDL-exposed aortas treated with or without MDL28170. n= 5, *P < 0.05 for HOG-LDL versus untreated control or N-LDL, #P < 0.05 for HOG-LDL versus HOG-LDL + MDL28170. (C) Endothelium-independent relaxation in aortas treated with HOG-LDL ± MDL28170. Results (mean ± S.E.M.) are expressed as the rate of relaxation to the pre-contraction, n= 4.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2821526&req=5

fig06: HOG-LDL-induced activation of calpain decreases eNOS levels and impairs endothelium-dependent relaxation in C57BL/6J mice aorta. (A) Western blot analysis of total eNOS levels in aortas incubated with HOG-LDL (100 μg/ml for 24 hrs) in the presence or absence of the calpain inhibitor III, MDL28170 (20 μM). n= 4 in each group, **P < 0.01 versus N-LDL, versus HOG-LDL. (B) Endothelium-dependent relaxation of HOG-LDL-exposed aortas treated with or without MDL28170. n= 5, *P < 0.05 for HOG-LDL versus untreated control or N-LDL, #P < 0.05 for HOG-LDL versus HOG-LDL + MDL28170. (C) Endothelium-independent relaxation in aortas treated with HOG-LDL ± MDL28170. Results (mean ± S.E.M.) are expressed as the rate of relaxation to the pre-contraction, n= 4.
Mentions: Next, we determined whether HOG-LDL induces calpain-dependent eNOS degradation in intact aortas. Isolated mouse aortas were exposed to either N-LDL or HOG-LDL in the presence or absence of MDL28170, and eNOS protein levels were measured. Exposure of mouse aortas to MDL28170 alone (data not shown) or N-LDL (Fig. 6A) did not affect eNOS levels. In contrast, exposure of aortas to 100 μg/ml HOG-LDL for 24 hrs reduced eNOS protein levels by 75–80% (P < 0.05, Fig. 6A). Importantly, this reduction in aortic eNOS levels was almost completely blocked by co-administration of MDL28170 (Fig. 6A).

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