CR6 interacting factor 1 deficiency promotes endothelial inflammation by SIRT1 downregulation

<div><p>Aims</p><p>CR6 interacting factor 1 (CRIF1) deficiency impairs mitochondrial oxidative phosphorylation complexes, contributing to increased mitochondrial and cellular reactive oxygen species (ROS) production. CRIF1 downregulation has also been revealed to decrease sirtuin 1 (SIRT1) expression and impair vascular function. Inhibition of SIRT1 disturbs oxidative energy metabolism and stimulates nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)-induced inflammation. Therefore, we hypothesized that both CRIF1 deficiency-induced mitochondrial ROS production and SIRT1 reduction play stimulatory roles in vascular inflammation.</p><p>Methods and results</p><p>Plasma levels and mRNA expression of proinflammatory cytokines (tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6) were markedly elevated in endothelium-specific CRIF1-knockout mice and CRIF1-silenced endothelial cells, respectively. Moreover, CRIF1 deficiency-induced vascular adhesion molecule-1 (VCAM-1) expression was consistently attenuated by the antioxidant N-acetyl-cysteine and NF-κB inhibitor (BAY11). We next showed that siRNA-mediated CRIF1 downregulation markedly activated NF-κB. SIRT1 overexpression not only rescued CRIF1 deficiency-induced NF-κB activation but also decreased inflammatory cytokines (TNF-α, IL-1β, and IL-6) and VCAM-1 expression levels in endothelial cells.</p><p>Conclusions</p><p>These results strongly suggest that CRIF1 deficiency promotes endothelial cell inflammation by increasing VCAM-1 expression, elevating inflammatory cytokines levels, and activating the transcription factor NF-κB, all of which were inhibited by SIRT1 overexpression.</p></div

SIRT1-mediated CRIF1 deletion-induced inflammatory cytokine and VCAM-1 expression.

<p>(A) HUVECs were infected with β-Gal and SIRT1 adenovirus for 24 h, followed by transfection with CRIF1 (100 pmol) siRNA for 36 h. TNFα, IL-1β, and IL-6 mRNA levels were quantified using qPCR. (B) Whole cell lysates were collected and analyzed for VCAM-1 protein expression by western blotting. VCAM-1 protein levels were quantified by densitometric analysis (right panel). (C) The aorta from WT and CRIF1 EKO mice were infected with β-Gal (control) or SIRT1 adenovirus and incubated for 24 h. Representative immunohistochemical staining images of CD31 (green) and VCAM-1 (red). Scale bars indicate 50 um. Arrowhead indicates VCAM-1 expression. L indicates lumen. Quantified VCAM-1 expression was normalized to the β-Gal (WT) control group (right panel). n = 5 per group. All western blots are representative of three independent experiments (A, B). The data are presented as means ± SEM of three independent experiments. *p < 0.05 vs. control cells or WT mice. ^#p < 0.05 vs. CRIF1 cells or CRIF1 EKO mice.</p

SIRT1 mediated CRIF1 deficiency-induced NF-κB activation in HUVECs.

<p>(A) HUVECs were transfected with CRIF1 siRNA (50 and 100 pmol) and SIRT1 siRNA (50 and 100 pmol) for 48 h, and the cytosolic and nuclear fractions were isolated. The protein expression levels of cytosolic p65 and IκBα and nuclear p65 were detected by western blotting. p65 and IκBα protein levels were quantified by densitometric analysis (right panel). (B) HUVECs were infected with β-Gal and SIRT1 adenovirus for 24 h, followed by transfection with CRIF1 siRNA (100 pmol) for 36 h. Cytosolic p65 and IκBα and nuclear p65 protein expression levels were quantified by densitometric analysis (right panel). All western blots are representative of three independent experiments. The data are presented as means ± SEM of three independent experiments. ^#p < 0.05 vs. CRIF1 control cells. *p < 0.05 vs. SIRT1 control cells.</p

Schematic model summarizing the underlying mechanisms and function of CRIF1 on inflammation.

<p>Schematic model summarizing the underlying mechanisms and function of CRIF1 on inflammation.</p

NAC and NF-κB inhibitor decreased CRIF1 deletion-induced expression of VCAM-1 in HUVECs.

<p>Endothelial HUVECs were pretreated with NAC and NF-κB inhibitor (BAY11-7082) for 1 h and then transfected with CRIF1 siRNA (50, 100 pmol) for 48 h. (A) VCAM-1 protein expression was detected by western blotting and quantified by densitometric analysis. (B) The location and protein expression of VCAM-1 (red) and Hoechst (blue) in HUVECs were examined by immunofluorescence. Scale bars indicate 20 um. All western blots are representative of three independent experiments. The data are presented as means ± SEM of three independent experiments. *p < 0.05 vs. control cells. ^#p < 0.05 vs. CRIF1 siRNA cells.</p