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Proteomic analysis of shear stress-mediated protection from TNF-alpha in endothelial cells.

ABSTRACT: Previous studies have shown that physiological levels of shear stress can protect endothelial cells (ECs) from apoptotic stimuli. Here, we differentiate between acute and chronic protection and demonstrate the use of proteomic technologies to uncover mechanisms associated with chronic protection of ECs. We hypothesized that changes in abundance of proteins associated with the TNF-alpha signaling cascade orchestrate shear stress-mediated protection from TNF-alpha when cells are preconditioned with shear prior to the exposure of apoptotic stimuli. Detection of cleaved caspase 3 through Western blot analysis confirmed chronic shear stress-mediated protection from TNF-alpha. In the presence of the nitric oxide synthase inhibitor, LNMA (N(omega)-monomethyl-l-arginine), chronic protection remained. Treatment with a de novo protein synthesis inhibitor, cycloheximide, eliminated this protective effect. Isotopic-labeling experiments, coupled with LC-MS/MS (liquid chromatography-tandem mass spectrometry) of isolated components of the TNF-alpha pathway revealed that CARD9, a known activator of the NF-kappaB pathway, was increased (60%) in sheared cells versus nonsheared cells. This result was confirmed through Western blot analysis. Our data suggest that de novo formation of proteins is required for protection from TNF-alpha in ECs chronically exposed to shear stress, and that CARD9 is a candidate protein in this response.

SUBMITTER: Freed JK

PROVIDER: S-EPMC3712086 | biostudies-literature | 2010 May

REPOSITORIES: biostudies-literature

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Proteomic analysis of shear stress-mediated protection from TNF-alpha in endothelial cells.

Freed Julie K JK Greene Andrew S AS

Microcirculation (New York, N.Y. : 1994) 20100501 4

Previous studies have shown that physiological levels of shear stress can protect endothelial cells (ECs) from apoptotic stimuli. Here, we differentiate between acute and chronic protection and demonstrate the use of proteomic technologies to uncover mechanisms associated with chronic protection of ECs. We hypothesized that changes in abundance of proteins associated with the TNF-alpha signaling cascade orchestrate shear stress-mediated protection from TNF-alpha when cells are preconditioned wit ...[more]

PMID: 20536739

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Project description:Oxidative stress plays an important role in type 2 diabetes-related endothelial dysfunction. We hypothesized that resveratrol protects against oxidative stress-induced endothelial dysfunction in aortas of diabetic mice by inhibiting tumor necrosis factor alpha (TNFalpha)-induced activation of NAD(P)H oxidase and preserving phosphorylation of endothelial nitric oxide synthase (eNOS).We examined endothelial-dependent vasorelaxation to acetylcholine (ACh) in diabetic mice (Lepr(db)) and normal controls (m Lepr(db)). Relaxation to ACh was blunted in Lepr(db) compared with m Lepr(db), whereas endothelial-independent vasorelaxation to sodium nitroprusside (SNP) was comparable. Resveratrol improved ACh-induced vasorelaxation in Lepr(db) without affecting dilator response to SNP. Impaired relaxation to ACh in Lepr(db) was partially reversed by incubating the vessels with NAD(P)H oxidase inhibitor apocynin and a membrane-permeable superoxide dismutase mimetic TEMPOL. Dihydroethidium (DHE) staining showed an elevated superoxide (O(2)(.-)) production in Lepr(db), whereas both resveratrol and apocynin significantly reduced O(2)(.-) signal. Resveratrol increased nitrite/nitrate levels and eNOS (Ser1177) phosphorylation, and attenuated H(2)O(2) production and nitrotyrosine (N-Tyr) content in Lepr(db) aortas. Furthermore, resveratrol attenuated the mRNA and protein expression of TNFalpha. Genetic deletion of TNFalpha in diabetic mice (db(TNF-)/db(TNF-)) was associated with a reduced NAD(P)H oxidase activity and vascular O(2)(.-) production and an increased eNOS (Ser1177) phosphorylation, suggesting that TNFalpha plays a pivotal role in aortic dysfunction in diabetes by inducing oxidative stress and reducing NO bioavailability.Resveratrol restored endothelial function in type 2 diabetes by inhibiting TNFalpha-induced activation of NAD(P)H oxidase and preserving eNOS phosphorylation, suggesting the potential for new treatment approaches to promote vascular health in metabolic diseases.

Dataset Information

Proteomic analysis of shear stress-mediated protection from TNF-alpha in endothelial cells.

Publications

Proteomic analysis of shear stress-mediated protection from TNF-alpha in endothelial cells.

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