The mitochondrial antioxidant enzyme manganese superoxide dismutase (MnSOD) and the zinc finger transcription factor Kruppel-like factor-4 (KLF4) are involved in the regulation of redox homeostasis, apoptosis and cell proliferation. We have shown that estrogen exerts antioxidative actions via induction of MnSOD in cultured rat aortic vascular smooth muscle cells (VSMC). The purpose of the present study was to investigate whether estrogen inhibits VSMC proliferation via alteration of KLF4 and MnSOD expression. In cultured rat aortic VSMC, estrogen binding to estrogen receptor-alpha led to rapid increase in KLF4 expression and reduction of cell proliferation by 50%. Protein separation revealed that KLF4 was shifted to the nucleus when VSMC were treated with estrogen. Estrogen-mediated induction of KLF4 and the antiproliferative effect involved activation of PI-3 kinase, Akt phosphorylation and induction of NO synthase activity. Experiments in freshly isolated denuded aortic segments revealed an increase in KLF4 abundance after estrogen treatment and demonstrated that eNOS is expressed in the media at low levels. Transfection experiments showed that estrogen-induced overexpression of MnSOD required KLF4 and that both KLF4 and MnSOD were indispensable for the observed antiproliferative effect of estrogen in VSMC. To confirm these data in vivo, we investigated neointima formation after carotid artery injury in wild-type (WT) and MnSOD+/- mice. Estrogen deficiency led to enhanced neointima formation and higher numbers of Ki67-positive proliferating cells in the neointima of ovariectomized WT and MnSOD+/- mice. Moreover, MnSOD+/- mice showed more extensive neointima formation and Ki67 immunostaining. Interestingly, estrogen replacement prevented neointima formation in WT mice but failed to completely inhibit neointima formation in MnSOD+/- mice. Cultured VSMC derived from MnSOD+/- mice showed enhanced proliferation as compared to WT VSMC, and estrogen treatment failed to inhibit proliferation in MnSOD+/- VSMC. In conclusion, these data demonstrate the importance of MnSOD and KLF4 for proliferation control in VSMC. Our results provide novel insights into how proliferation of VSMC is regulated by estrogen and may help to identify novel targets for the treatment of vascular diseases such as restenosis.

Background/Aims: Common genetic variations in vitamin D metabolism are associated with liver stiffness. Whether these genes are implicated in hepatic steatosis remains unclear. Here we aimed to analyse the association of common vitamin D pathway gene variants with liver steatosis. Methods: Liver steatosis was assessed non-invasively in 241 patients with chronic liver conditions by controlled attenuation parameter (CAP). The following polymorphisms were genotyped using TaqMan assays: group-specific component (GC) rs7041, 7-dehydrocholesterol reductase (DHCR7) rs12785878, cytochrome P450 2R1 (CYP2R1) rs10741657, ­vitamin D receptor (VDR) rs7974353. Chemiluminescence immunoassay determined serum 25-hydroxyvitamin D (25(OH) D) concentrations. Results: Vitamin D deficiency (defined by 25(OH)D concentrations <20 ng/mL) occurred in 66% of patients. Median CAP was 296 (100–400) dB/m. Patients with advanced steatosis (CAP ≥280 dB/m) had significantly (p = 0.033) lower 25(OH)D levels as compared to patients with CAP <280 dB/m. Moreover, the rare allele [T] in GC rs7041 was significantly (p = 0.018) associated with higher 25(OH)D levels in patients with CAP <280 dB/m. However, GC, DHCR7, CYP2R1, and VDR polymorphisms were not related to liver steatosis and obesity traits. Conclusions: Higher CAP values are associated with low serum 25(OH)D concentrations but not with common vitamin D pathway gene variants.

BackgroundThe development of hepatocellular carcinoma (HCC) is a common consequence of advanced liver fibrosis but the interactions between fibrogenesis and carcinogenesis are still poorly understood. Recently it has been shown that HCC promotion depends on Toll-like receptor (TLR) 4. Pre-cancerogenous events can be modelled in mice by the administration of a single dose of diethylnitrosamine (DEN), with HCC formation depending amongst others on interleukin (IL) 6 production. Mice lacking the hepatocanalicular phosphatidylcholine transporter ABCB4 develop liver fibrosis spontaneously, resemble patients with sclerosing cholangitis due to mutations of the orthologous human gene, and represent a valid model to study tumour formation in pre-injured cholestatic liver. The aim of this study was to investigate DEN-induced liver injury in TLR4-deficient mice with biliary fibrosis.MethodsABCB4-deficient mice on the FVB/NJ genetic background were crossed to two distinct genetic backgrounds (TLR4-sufficient C3H/HeN and TLR4-deficient C3H/HeJ) for more than 10 generations. The two congenic knockout and the two corresponding wild-type mouse lines were treated with a single dose of DEN for 48 hours. Phenotypic differences were assessed by measuring hepatic collagen contents, inflammatory markers (ALT, CRP, IL6) as well as hepatic apoptosis (TUNEL) and proliferation (Ki67) rates.ResultsHepatic collagen accumulation is significantly reduced in ABCB4-/-:TLR4-/-double-deficient mice. After DEN challenge, apoptosis, proliferation and inflammatory markers are decreased in TLR4-deficient in comparison to TLR4-sufficient mice. When combining ABCB4 and TLR4 deficiency with DEN treatment, hepatic IL6 expression and proliferation rates are lowest in fibrotic livers from the double-deficient line. Consistent with these effects, selective digestive tract decontamination in ABCB4-/- mice also led to reduced tumor size and number after DEN.ConclusionThis study demonstrates that liver injury upon DEN challenge depends on pre-existing fibrosis and genetic background. The generation of ABCB4-/: TLR4-/- double-deficient mice illustrates that TLR4-deficiency protects against hepatic injury in a preclinical mouse model of chronic liver disease.

Complement factor C5a has two known receptors, C5aR, which mediates proinflammatory effects, and C5L2, a potential C5a decoy receptor. We previously identified C5a/C5aR signaling as a potent profibrotic pathway in the kidney. Here we tested for the first time the role of C5L2 in renal fibrosis. In unilateral ureteral obstruction (UUO)-induced kidney fibrosis, the expression of C5aR and C5L2 increased similarly and gradually as fibrosis progressed and was particularly prominent in injured dilated tubules. Genetic deficiency of either C5aR or C5L2 significantly reduced UUO-induced tubular injury. Expression of key proinflammatory mediators, however, significantly increased in C5L2- compared with C5aR-deficient mice, but this had no effect on the number of renal infiltrating macrophages or T cells. Moreover, in C5L2 mice, the cytokine and matrix metalloproteinase-inhibitor tissue inhibitor of matrix metalloproteinase-1 was specifically enhanced. Consequently, in C5L2 mice the degree of renal fibrosis was similar to wild type (WT), albeit with reduced mRNA expression of some fibrosis-related genes. In contrast, C5aR mice had significantly reduced renal fibrosis compared with WT and C5L2 mice in UUO. In vitro experiments with primary tubular cells demonstrated that deficiency for either C5aR or C5L2 led to a significantly reduced expression of tubular injury and fibrosis markers. Vice versa, stimulation of WT tubular cells with C5a significantly induced the expression of these markers, whereas the absence of either receptor abolished this induction. In conclusion, in experimental renal fibrosis C5L2 and C5aR both contribute to tubular injury, and, while C5aR acts profibrotic, C5L2 does not play a role in extracellular matrix accumulation, arguing against C5L2 functioning simply as a decoy receptor.

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