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Activation of nuclear factor-kappa B accelerates vascular calcification by inhibiting ankylosis protein homolog expression.

Zhao G, Xu MJ, Zhao MM, Dai XY, Kong W, Wilson GM, Guan Y, Wang CY, Wang X - Kidney Int. (2012)

Bottom Line: Although chronic inflammation is one of the etiologic factors, the underlying mechanism is not fully understood.Furthermore, a rat chronic renal failure model, with increased serum TNF levels, activated NF-κB and decreased ANKH levels.Both human calcified atherosclerotic lesions and arteries from patients with chronic kidney disease had activated NF-κB and decreased ANKH expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Pathophysiology, Key Laboratory of Molecular Cardiovascular Science, School of Basic Medical Science, Peking University Health Science Center, Ministry of Education, Beijing, PR China.

ABSTRACT
Vascular calcification is a major risk factor of cardiovascular mortality, particularly for patients with end-stage renal disease and diabetes. Although chronic inflammation is one of the etiologic factors, the underlying mechanism is not fully understood. To clarify this, we studied how nuclear factor-kappa B (NF-κB) induction, a mediator of inflammation, might promote vascular calcification. Activation of NF-κB by tumor necrosis factor (TNF) promoted inorganic phosphate-induced calcification in human aortic smooth muscle cells. Pyrophosphate (an inhibitor of calcification) efflux to the extracellular matrix was suppressed along with the decreased expression of ankylosis protein homolog (ANKH), a transmembrane protein that controls pyrophosphate efflux of cells. The restoration of ANKH expression in these cells overcame the decreased pyrophosphate efflux and calcification. Tristetraprolin, a downstream product of NF-κB activation, may mediate destabilization of ANKH mRNA as its knockdown by shRNA increased ANKH expression and decreased calcification. Furthermore, a rat chronic renal failure model, with increased serum TNF levels, activated NF-κB and decreased ANKH levels. In contrast, the inhibition of NF-κB maintained ANKH expression and attenuated vascular calcification both in vivo and in vitro. Both human calcified atherosclerotic lesions and arteries from patients with chronic kidney disease had activated NF-κB and decreased ANKH expression. Thus, TNF-activated NF-κB promotes inflammation-accelerated vascular calcification by inhibiting ankylosis protein homolog expression and consequent pyrophosphate secretion.

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IκBα increased ANKH expression and decreased calcium deposition in in vivo and ex vivo models. (A) In the rat chronic renal failure (CRF) model, Ad-IκBα or Ad-GFP were mixed with matrix gel and smeared around the abdominal aorta. Calcium deposition was analyzed by von Kossa staining. Ade, adenine diet at 6 weeks. ANKH and p-p65 levels were detected by immunohistochemical staining; shows one representative image from 6 rats per group (x 400). (B) Western blot analysis of protein levels of ANKH and IκBα in abdominal aortas. (C) Serum TNF level in CRF rats (n=6 rats per group, *P<0.05). (D) Calcium content in human renal artery tissue cultured in DMEM with Pi (3.0 mM) and/or TNF (10 ng/ml) for 2 weeks (n=4 per group, *P<0.05).
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Figure 6: IκBα increased ANKH expression and decreased calcium deposition in in vivo and ex vivo models. (A) In the rat chronic renal failure (CRF) model, Ad-IκBα or Ad-GFP were mixed with matrix gel and smeared around the abdominal aorta. Calcium deposition was analyzed by von Kossa staining. Ade, adenine diet at 6 weeks. ANKH and p-p65 levels were detected by immunohistochemical staining; shows one representative image from 6 rats per group (x 400). (B) Western blot analysis of protein levels of ANKH and IκBα in abdominal aortas. (C) Serum TNF level in CRF rats (n=6 rats per group, *P<0.05). (D) Calcium content in human renal artery tissue cultured in DMEM with Pi (3.0 mM) and/or TNF (10 ng/ml) for 2 weeks (n=4 per group, *P<0.05).

Mentions: To further test whether NF-κB activation promoted vascular calcification in vivo, we used a rat CRF model of vascular calcification induced by an adenine diet. We blocked NF-κB in abdominal aortas using the super-repressor form of IκBα, with serine 32 and 36 replaced by alanines. Abdominal aortas after 3 weeks of the adenine diet were smeared with Ad-IκBα or Ad-GFP mixed with matrix gel. At 6 weeks after the diet, calcium deposition was significantly induced in rat aortas, as assessed by von Kossa staining (Figure 6A). Ad-GFP did not affect calcium deposition, but Ad-IκBα significantly inhibited calcium deposition. ANKH expression was significantly lower in CRF than control aortas with saline treatment (Figure 6A). In contrast, Ad-IκBα but not Ad-GFP treatment rescued the decreased ANKH levels (Figure 6A). To further confirm our results, we examined ANKH expression in aortas by western blot analysis and found that Ad-IκBα maintained the expression of ANKH (Figure 6B). Therefore, NF-κB promoted vascular calcification by inhibiting ANKH expression in vivo.


Activation of nuclear factor-kappa B accelerates vascular calcification by inhibiting ankylosis protein homolog expression.

Zhao G, Xu MJ, Zhao MM, Dai XY, Kong W, Wilson GM, Guan Y, Wang CY, Wang X - Kidney Int. (2012)

IκBα increased ANKH expression and decreased calcium deposition in in vivo and ex vivo models. (A) In the rat chronic renal failure (CRF) model, Ad-IκBα or Ad-GFP were mixed with matrix gel and smeared around the abdominal aorta. Calcium deposition was analyzed by von Kossa staining. Ade, adenine diet at 6 weeks. ANKH and p-p65 levels were detected by immunohistochemical staining; shows one representative image from 6 rats per group (x 400). (B) Western blot analysis of protein levels of ANKH and IκBα in abdominal aortas. (C) Serum TNF level in CRF rats (n=6 rats per group, *P<0.05). (D) Calcium content in human renal artery tissue cultured in DMEM with Pi (3.0 mM) and/or TNF (10 ng/ml) for 2 weeks (n=4 per group, *P<0.05).
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Figure 6: IκBα increased ANKH expression and decreased calcium deposition in in vivo and ex vivo models. (A) In the rat chronic renal failure (CRF) model, Ad-IκBα or Ad-GFP were mixed with matrix gel and smeared around the abdominal aorta. Calcium deposition was analyzed by von Kossa staining. Ade, adenine diet at 6 weeks. ANKH and p-p65 levels were detected by immunohistochemical staining; shows one representative image from 6 rats per group (x 400). (B) Western blot analysis of protein levels of ANKH and IκBα in abdominal aortas. (C) Serum TNF level in CRF rats (n=6 rats per group, *P<0.05). (D) Calcium content in human renal artery tissue cultured in DMEM with Pi (3.0 mM) and/or TNF (10 ng/ml) for 2 weeks (n=4 per group, *P<0.05).
Mentions: To further test whether NF-κB activation promoted vascular calcification in vivo, we used a rat CRF model of vascular calcification induced by an adenine diet. We blocked NF-κB in abdominal aortas using the super-repressor form of IκBα, with serine 32 and 36 replaced by alanines. Abdominal aortas after 3 weeks of the adenine diet were smeared with Ad-IκBα or Ad-GFP mixed with matrix gel. At 6 weeks after the diet, calcium deposition was significantly induced in rat aortas, as assessed by von Kossa staining (Figure 6A). Ad-GFP did not affect calcium deposition, but Ad-IκBα significantly inhibited calcium deposition. ANKH expression was significantly lower in CRF than control aortas with saline treatment (Figure 6A). In contrast, Ad-IκBα but not Ad-GFP treatment rescued the decreased ANKH levels (Figure 6A). To further confirm our results, we examined ANKH expression in aortas by western blot analysis and found that Ad-IκBα maintained the expression of ANKH (Figure 6B). Therefore, NF-κB promoted vascular calcification by inhibiting ANKH expression in vivo.

Bottom Line: Although chronic inflammation is one of the etiologic factors, the underlying mechanism is not fully understood.Furthermore, a rat chronic renal failure model, with increased serum TNF levels, activated NF-κB and decreased ANKH levels.Both human calcified atherosclerotic lesions and arteries from patients with chronic kidney disease had activated NF-κB and decreased ANKH expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Pathophysiology, Key Laboratory of Molecular Cardiovascular Science, School of Basic Medical Science, Peking University Health Science Center, Ministry of Education, Beijing, PR China.

ABSTRACT
Vascular calcification is a major risk factor of cardiovascular mortality, particularly for patients with end-stage renal disease and diabetes. Although chronic inflammation is one of the etiologic factors, the underlying mechanism is not fully understood. To clarify this, we studied how nuclear factor-kappa B (NF-κB) induction, a mediator of inflammation, might promote vascular calcification. Activation of NF-κB by tumor necrosis factor (TNF) promoted inorganic phosphate-induced calcification in human aortic smooth muscle cells. Pyrophosphate (an inhibitor of calcification) efflux to the extracellular matrix was suppressed along with the decreased expression of ankylosis protein homolog (ANKH), a transmembrane protein that controls pyrophosphate efflux of cells. The restoration of ANKH expression in these cells overcame the decreased pyrophosphate efflux and calcification. Tristetraprolin, a downstream product of NF-κB activation, may mediate destabilization of ANKH mRNA as its knockdown by shRNA increased ANKH expression and decreased calcification. Furthermore, a rat chronic renal failure model, with increased serum TNF levels, activated NF-κB and decreased ANKH levels. In contrast, the inhibition of NF-κB maintained ANKH expression and attenuated vascular calcification both in vivo and in vitro. Both human calcified atherosclerotic lesions and arteries from patients with chronic kidney disease had activated NF-κB and decreased ANKH expression. Thus, TNF-activated NF-κB promotes inflammation-accelerated vascular calcification by inhibiting ankylosis protein homolog expression and consequent pyrophosphate secretion.

Show MeSH
Related in: MedlinePlus