Far-Infrared Therapy Induces the Nuclear Translocation of PLZF Which Inhibits VEGF-Induced Proliferation in Human Umbilical Vein Endothelial Cells

Many studies suggest that far-infrared (FIR) therapy can reduce the frequency of some vascular-related diseases. The non-thermal effect of FIR was recently found to play a role in the long-term protective effect on vascular function, but its molecular mechanism is still unknown. In the present study, we evaluated the biological effect of FIR on vascular endothelial growth factor (VEGF)-induced proliferation in human umbilical vein endothelial cells (HUVECs). We found that FIR ranging 3∼10 µm significantly inhibited VEGF-induced proliferation in HUVECs. According to intensity and time course analyses, the inhibitory effect of FIR peaked at an effective intensity of 0.13 mW/cm2 at 30 min. On the other hand, a thermal effect did not inhibit VEGF-induced proliferation in HUVECs. FIR exposure also inhibited the VEGF-induced phosphorylation of extracellular signal-regulated kinases in HUVECs. FIR exposure further induced the phosphorylation of endothelial nitric oxide (NO) synthase (eNOS) and NO generation in VEGF-treated HUVECs. Both VEGF-induced NO and reactive oxygen species generation was involved in the inhibitory effect of FIR. Nitrotyrosine formation significantly increased in HUVECs treated with VEGF and FIR together. Inhibition of phosphoinositide 3-kinase (PI3K) by wortmannin abolished the FIR-induced phosphorylation of eNOS and Akt in HUVECs. FIR exposure upregulated the expression of PI3K p85 at the transcriptional level. We further found that FIR exposure induced the nuclear translocation of promyelocytic leukemia zinc finger protein (PLZF) in HUVECs. This induction was independent of a thermal effect. The small interfering RNA transfection of PLZF blocked FIR-increased PI3K levels and the inhibitory effect of FIR. These data suggest that FIR induces the nuclear translocation of PLZF which inhibits VEGF-induced proliferation in HUVECs

Risk of Non-melanoma Skin Cancer in Patients with Chronic Kidney Disease and its Relationship to Uraemic Pruritus

This study investigated the risk of non-melanoma skin cancer (NMSC) in pre-dialysis patients with chronic kidney disease (CKD) and explored associated risk factors. A population-based cohort of 1,515,858 Taiwanese CKD patients was included. The standardized incidence ratio (SIR) for incident NMSC was determined. Compared with the general population, a 1.14-fold risk of NMSC was found in the CKD cohort. NMSC risk was significant in patients with pre-dialysis stage 5 CKD and anaemia (1.48-fold), and in those with uraemic pruritus after long-term antihistamine treatment (1.38-fold). A higher SIR for NMSC was found in younger patients with CKD (age < 70 years, 1.34-fold; age 20–39 years, 1.63-fold), stage 5 CKD with anaemia (age < 70 years, 2.09-fold), and uraemic pruritus (age <70 years, 2.22-fold). Pre-dialysis patients with CKD are at higher risk of NMSC, especially those with advanced-stage CKD, and those with uraemic pruritus

Oligosaccharides Ameliorate Acute Kidney Injury by Alleviating Cluster of Differentiation 44-Mediated Immune Responses in Renal Tubular Cells

Acute kidney injury (AKI) is a sudden episode of kidney damage that commonly occurs in patients admitted to hospitals. To date, no ideal treatment has been developed to reduce AKI severity. Oligo-fucoidan (FC) interferes with renal tubular cell surface protein cluster of differentiation 44 (CD44) to prevent renal interstitial fibrosis; however, the influence of oligosaccharides on AKI remains unknown. In this study, FC, galacto-oligosaccharide (GOS), and fructo-oligosaccharide (FOS) were selected to investigate the influence of oligosaccharides on AKI. All three oligosaccharides have been proven to be partially absorbed by the intestine. We found that the oligosaccharides dose-dependently reduced CD44 antigenicity and suppressed the hypoxia-induced expression of CD44, phospho-JNK, MCP-1, IL-1β, and TNF-α in NRK-52E renal tubular cells. Meanwhile, CD44 siRNA transfection and JNK inhibitor SP600125 reduced the hypoxia-induced expression of phospho-JNK and cytokines. The ligand of CD44, hyaluronan, counteracted the influence of oligosaccharides on CD44 and phospho-JNK. At 2 days post-surgery for ischemia–reperfusion injury, oligosaccharides reduced kidney inflammation, serum creatine, MCP-1, IL-1β, and TNF-α in AKI mice. At 7 days post-surgery, kidney recovery was promoted. These results indicate that FC, GOS, and FOS inhibit the hypoxia-induced CD44/JNK cascade and cytokines in renal tubular cells, thereby ameliorating AKI and kidney inflammation in AKI mice. Therefore, oligosaccharide supplementation is a potential healthcare strategy for patients with AKI

Author Correction: Simvastatin reduces the carcinogenic effect of 3-methylcholanthrene in renal epithelial cells through histone deacetylase 1 inhibition and RhoA reactivation

An amendment to this paper has been published and can be accessed via a link at the top of the paper

3-Methylcholanthrene, an AhR Agonist, Caused Cell-Cycle Arrest by Histone Deacetylation through a RhoA-Dependent Recruitment of HDAC1 and pRb2 to E2F1 Complex

<div><p>We previously showed that treating vascular endothelial cells with 3-methylcholanthrene (3MC) caused cell-cycle arrest in the Go/G1 phase; this resulted from the induction of p21 and p27 and a decreased level and activity of the cyclin-dependent kinase, Cdk2. We further investigated the molecular mechanisms that modulate cell-cycle regulatory proteins through the aryl-hydrocarbon receptor (AhR)/Ras homolog gene family, member A (RhoA) dependent epigenetic modification of histone. AhR/RhoA activation mediated by 3MC was essential for the upregulation of retinoblastoma 2 (pRb2) and histone deacetylase 1 (HDAC1), whereas their nuclear translocation was primarily modulated by RhoA activation. The combination of increased phosphatase and tensin homolog (PTEN) activity and decreased phosphatidylinositide 3-kinase (PI3K) activation by 3MC led to the inactivation of the Ras-cRaf pathway, which contributed to pRb2 hypophosphorylation. Increased HDAC1/pRb2 recruitment to the E2F1 complex decreased E2F1-transactivational activity and H3/H4 deacetylation, resulting in the downregulation of cell-cycle regulatory proteins (Cdk2/4 and Cyclin D3/E). Co-immunoprecipitation and electrophoretic mobility shift assay (EMSA) results showed that simvastatin prevented the 3MC-increased binding activities of E2F1 proteins in their promoter regions. Additionally, RhoA inhibitors (statins) reversed the effect of 3MC in inhibiting DNA synthesis by decreasing the nuclear translocation of pRb2/HDAC1, leading to a recovery of the levels of cell-cycle regulatory proteins. In summary, 3MC decreased cell proliferation by the epigenetic modification of histone through an AhR/RhoA-dependent mechanism that can be rescued by statins.</p></div

Effect of statins in preventing a 3MC-mediated decrease in cell-cycle regulatory proteins and in DNA incorporation induced by RhoA inactivation.

<p>Cells were pretreated with simvastatin or pravastatin for 1-cytosol and nuclear-cytosol of 3MC-treated MCVECs was analyzed to determine the action of statins in RhoA inactivation and their effect in preventing 3MC-mediated alterations in c-Raf/pRb2/HDAC1/histone deacetylation. (A) We analyzed fractionation after 1 h; thereafter, the levels of cell-cycle regulatory proteins were assessed by Western blot after 4 or 6 h of 3MC treatment. (B) Cells were transfected overnight with a plasmid containing DNRhoA, and 3MC treatment was administered for 6 h. A western blot analysis was conducted to examine the effect of DNRhoA on the cell-cycle regulatory proteins reduced by 3MC. GAPDH (or α-tubulin), Lamin A/C, and VE-cadherin were used as internal controls for the cytosol (or total), nuclear and membrane fractions, respectively, to verify equivalent loading. (C) Cells were pretreated with statins for 1 h; this was followed by the 3MC challenge, which was pulsed for 15 h with BrdU (Invitrogen; 0.75 μg/mL) incubation for the DNA incorporation assay. Fixed cells on coverslips were stained with a mouse anti-BrdU antibody conjugated with Texas Red. Red represents BrdU-positive staining. Identical fields were stained with DAPI (Invitrogen) to reveal the positions of cell nuclei. We recorded micrographs of the representative fields at 200× magnification (scale bar in white  = 250 μm). (D) Cell numbers were counted using a hemo-cytometer at the indicated time points in cells with various treatments. Data are presented as mean ± SEM of 3 independent experiments (*<i>P</i><0.05 vs. control group; ^#<i>P</i><0.05 vs. 3MC treatment alone).</p

Involvement of PTEN and PI3K in pRb2 hypophosphorylation induced by Ras/c-Raf inactivation.

<p>(A) Cells were pretreated with bpv (an inhibitor of PTEN) for 30 min or YS-49 (an activator of PI3K) for 1 h, followed by 1 h of 100 nM 3MC treatment. Total cell lysates, and extraction of nuclear and cytosolic proteins were analyzed using Western blot analysis for related signaling molecules involved in chromatin deaceylation. (B) Cells were transfected with a plasmid containing the DNRas gene to mimic the effect of 3MC. After 1 h of 3MC treatment, cells were harvested for Western blot analysis of the mentioned molecules in total and cytosolic-membrane extracts. GAPDH, Lamin A/C, and VE-cadherin were used as internal controls for the cytosol (or total), nuclear and membrane fractions, respectively, to verify equivalent loading. The lower panel shows the intensity of bands in the Western blots using densitometry. Data are presented as mean ± SEM of 3 independent experiments (*<i>P</i><0.05 vs. control group; ^#<i>P</i><0.05 vs. 3MC treatment alone).</p

Elimination of 3MC-mediated increases in E2F/HDAC1 binding to the E2F responsive element in the promoters of Cdk2/4 and CyclinD3/E1 by simvastatin treatment.

<p>(A) Cells were cultured and treated with 100 nM of 3MC for 1 h after 1 h of simvastatin pretreatment. Nuclear proteins were assayed for E2F binding activity by WT and mut probes in an EMSA assay, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092793#s2" target="_blank">Materials and Methods</a>. The term “100xcold” denotes a 100-fold molar excess of unlabeled oligonucleotides relative to the biotin-labeled probe; this was added to the binding assay to compete with the unlabeled oligonucleotides. The mobility of the E2F-E2F responsive element complex is indicated. Representative results of 3 experiments are shown. (B) A ChIP assay was performed in cells that received simvastatin pretreatment for 1 h, or followed by the 3MC challenge for 1 h, as indicated. The DNA associated with HDAC1 was immunoprecipitated with an anti-HDAC1 antibody; thereafter, PCR amplification was used to determine the extent of the association between HDAC1 and the functional E2F-binding sites in the promoters of Cdk2/4 and Cyclin D3/E1. An anti-GAPDH antibody was used as a negative control for the ChIP assays. Representative results of 3 experiments are shown, and data are presented as the mean ± SEM (*<i>P</i><0.05 vs. the control; ^#<i>P</i><0.05 vs. 3MC treatment alone).</p