Evaluating the risk of ischemic stroke at a young age in patients with autoimmune inflammatory rheumatic diseases: a population-based cohort study in Taiwan.

Background: Recent studies have demonstrated an increased incidence of ischemic stroke among patients with certain autoimmune inflammatory rheumatic diseases (AIIRDs). However, the associations between young stroke and AIIRDs have not been fully investigated. This study aimed to evaluate the risk of ischemic stroke among young patients with AIIRDs. Methods: The National Health Insurance Research Database in Taiwan was utilized to establish cohorts of patients with AIIRDs diagnosed between 2004 and 2015, who were compared with 1,000,000 control participants. Cox proportional hazards regression models were used to calculate the hazard ratio of ischemic stroke and young ischemic stroke for individual AIIRDs after adjustment for relative risk factors. Results: During the study period, a total of 64,120 patients with AIIRDss and 1,000,000 control patients were identified. The overall mean follow-up time was 5.33 years. There were 223 (0.8%) and 1,923 (0.3%) young ischemic stroke-related hospitalizations among patients with AIIRDs and controls, respectively. The incidence rate of young ischemic stroke was 0.08 in patients with rheumatoid arthritis, 0.08 in patients with Sjögren's syndrome, 0.26 in patients with systemic lupus erythematosus, 0.17 in patients with idiopathic inflammatory myositis, 0.24 in patients with systemic sclerosis, 0.05 in patients with Behçet's disease, and 0.44 in patients with systemic vasculitis, versus 0.05 per 100 person-years in the general population. The adjusted hazard ratios for young ischemic stroke were 1.07 (95% CI 0.70-1.43) for rheumatoid arthritis, 1.39 (95% CI 0.94-2.06) for Sjögren's syndrome, 5.79 (95% CI 4.68-7.17) for systemic lupus erythematosus, 2.07 for idiopathic inflammatory myositis (95% CI 0.98-4.38), 2.79 for systemic sclerosis (95% CI 1.38-5.63), 0.82 for Behçet's disease (95% CI 0.26-2.55), and 4.15 (95% CI 1.96-8.82) for systemic vasculitis. Conclusions: Patients younger than 50 years with systemic lupus erythematosus, systemic sclerosis, or systemic vasculitis have a significantly elevated risk of developing ischemic stroke. Further research is needed to elucidate the pathogenesis of accelerated atherosclerosis in these AIIRDs.

Premature coronary artery disease in patients with immune-mediated inflammatory disease: a population-based study.

BACKGROUND: The associations between premature atherosclerosis and immune-mediated inflammatory diseases (IMIDs) are not fully investigated. To determine whether IMIDs are associated with premature atherosclerosis, we examined the risk of incident coronary artery disease (CAD) in men less than 45 years old and women less than 50 years old with various forms of IMIDs compared with general population. METHODS: A population-based cohort was established and included patients with IMID, who were followed until the development of CAD, withdrawal from the insurance system, death, or 31 December 2016, whichever point came first. Patients with IMID included rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), primary Sjogren's syndrome (SjS), idiopathic inflammatory myositis, systemic sclerosis (SSc), Behcet's disease (BD), and systemic vasculitis (SV). The comparison group was 1 000 000 beneficiaries sampled at random from the whole population as matched control participants. The Kaplan-Meier method was used to compare the cumulative incidences of CAD in patients with and without IMID. RESULTS: Among 58 862 patients with IMID, 2139 (3.6%) developed CAD and 346 (1.3%) developed premature CAD. Relative to the comparison cohorts, the adjusted HRs for premature CAD were 1.43 (95% CI 1.09 to 1.86) for primary SjS, 2.85 (95% CI 2.63 to 3.43) for SLE, 3.18 (95% CI 1.99 to 5.09) for SSc and 2.27 (95% CI 1.01 to 5.07) for SV. CONCLUSIONS: Primary Sjogren's syndrome, SLE, SSc and SV are associated with an increased risk of premature CAD. Our findings will support essential efforts to improve awareness of IMID impacting young adults.

Association between 9-month isoniazid prophylaxis of latent tuberculosis and severe hepatitis in patients treated with TNF inhibitors.

To investigate associations between isoniazid for latent tuberculosis and risk of severe hepatitis, affecting patients with rheumatoid arthritis or ankylosing spondylitis whose treatment includes tumor necrosis factor inhibitors. Our self-controlled case series study analyzed Taiwan's National Health Insurance Database from 2003 to 2015 to identify RA or AS patients, aged ≥ 20 years, receiving TNF inhibitors and a 9-month single isoniazid treatment. The outcome of interest was hospitalization due to severe hepatitis. We defined risk periods by isoniazid exposure (days): 1-28, 29-56, 57-84, 85-168, 169-252, and 253-280. To compare risk of severe hepatitis in exposed and non-exposed periods, we performed conditional Poisson regressions to generate incidence rate ratios (IRR) and 95% confidence intervals, with adjustment of patients' baseline covariates including age, sex, HBV, HCV and related medication. Of 54,267 RA patients and 137,889 AS patients identified between 2000 and 2015, 11,221 (20.7%) RA and 4,208 (3.1%) AS patients underwent TNFi therapy, with 722 (5%) receiving isoniazid for latent tuberculosis. We identified 31 incident cases (4.3%) of hospitalization due to severe hepatitis. Of these hospitalization events, 5 occurred in the exposed periods, 25 occurred in the INH unexposed periods, and 1 occurred in the pre-exposure period. Compared with non-exposure, the risk of severe hepatitis was higher in exposed periods (incidence rate ratio [IRR]: 5.1, 95% CI: 1.57-16.55), especially 57-84 days (IRR: 17.29, 95% CI: 3.11-96.25) and 85-168 days (IRR:10.55, 95% CI: 1.90-58.51). The INH related fatal hepatotoxicity was not identified in our study. Our findings suggest an association between risk of severe hepatitis and exposure to isoniazid in patients with RA or AS under TNFi therapy, particularly within the exposed period 57-168 days. A close monitoring of liver function is mandatory to minimize the risk, especially within the first 6 months after initiation of 9 months isoniazid.

Aldehyde dehydrogenase induction in arsenic-exposed rat bladder epithelium.

Arsenic is widely distributed in the environment. Many human cancers, including urothelial carcinoma (UC), show a dose-dependent relationship with arsenic exposure in the south-west coast of Taiwan (also known as the blackfoot disease (BFD) areas). However, the molecular mechanisms of arsenic-mediated UC carcinogenesis has not yet been defined. In vivo study, the rat bladder epithelium were exposed with arsenic for 48 h. The proteins were extracted from untreated and arsenic-treated rat bladder cells and utilized two-dimensional gel electrophoresis and mass spectrometry. Selected peptides were extracted from the gel and identified by quadrupole-time of flight (Q-TOF) Ultima-Micromass spectra. The significantly difference expression of proteins in arsenic-treated groups as compared with untreated groups was confirmed by immunohistochemistry (IHC) and western blotting. We found that thirteen proteins were down-regulated and nine proteins were up-regulated in arsenic-treated rat bladder cells when compared with untreated groups. The IHC and western blotting results confirmed that aldehyde dehydrogenase (ALDH) protein was up-regulated in arsenic-treated rat bladder epithelium. Expression of ALDH protein was significantly higher in UC patients from BFD areas than those from non-BFD areas using IHC (p=0.018). In conclusion, the ALDH protein expression could be used as molecular markers for arsenic-induced transformation.

Roles of oxidative stress and the ERK1/2, PTEN and p70S6K signaling pathways in arsenite-induced autophagy.

Studies show that arsenite induces oxidative stress and modifies cellular function via phosphorylation of proteins and inhibition of DNA repair enzymes. Autophagy, which has multiple physiological and pathological roles in cellular function, is initiated by oxidative stress and is regulated by the signaling pathways of phosphatidylinositol 3-phosphate kinase (PI3K)/mammalian target of rapamycin (mTOR)/p70S6 kinase (p70S6K) and extracellular signaling-regulated protein kinase 1/2 (ERK1/2) that play important roles in oncogenesis. However, the effects of arsenite-induced oxidative stress on autophagy and on expression of related proteins are not fully understood. This study found that cells treated with sodium arsenite had reduced 8-oxoguanine DNA glycosylase 1 (OGG1) and increased 8-hydroxy-2'-deoxyguanosine (8-OHdG) and activating transcription factor (ATF) 3 in SV-40 immortalized human uroepithelial (SV-HUC-1) cells. Arsenite also increased the number of autophagosomes and increased levels of the autophagy markers Beclin-1 and microtubule-associated protein 1 light chain 3B. Reactive oxygen species scavenger decreased arsenite-induced autophagy in SV-HUC-1 cells. Our previous work showed that arsenite induced phosphorylation of the ERK1/2 signaling pathway. The current study further showed that arsenite decreased phosphatase and tensin homologue (PTEN) levels and increased phospho-p70S6 kinase (p-p70S6K) in SV-HUC-1 cells. However, both kinase inhibitor U0126 and the DNA (cytosine-5-)-methyltransferase 1 (DNMT1) inhibitor 5-aza-deoxycytidine abolished the effect of arsenite on expressions of PTEN and p-p70S6K. These results show that autophagy induced by arsenite exposure is mediated by oxidative stress, which regulates activation of the PTEN, p70S6K and ERK1/2 signaling pathways. Thus, this study clarifies the role of autophagy in arsenite-induced urothelial carcinogenesis.

Overexpression of autophagy-related 16-like 1 in patients with oral squamous cell carcinoma.

Dyregulation of autophagy has been reported in various human cancers including oral squamous cell carcinoma (OSCC). The objective of this study was to link expression of autophagy-related 16-like 1 (ATG16L1), a protein essential for autophagosome formation, to clinical outcome in a cohort of 90 OSCC patients. Expression level of ATG16L1 was assessed by immunohistochemistry and an immunoreactivity score (IRS), ranging from 0 to 9, was assigned to each case. The results were correlated with clinicopathological parameters and outcome of patients. Twenty-seven patients (30%) exhibited ATG16L1 overexpression as indicated by an IRS of 9. Overexpression of ATG16L1 was significantly associated with disease stage (p = 0.001), size (p = 0.031) of the tumor, lymph node metastasis (p = 0.004), and histological grade (p = 0.038). ATG16L1 overexpression significantly affected the overall survival (p = 0.020) and time to recurrence (p = 0.031) of OSCC patients in Kaplan-Meier analysis. The present study suggested that ATG16L1 may be used as a biomarker for selecting OSCC patients with a more aggressive phenotype.

The association between drinking habits and family relationships in 3 villages of indigenous Taiwanese.

Although the vast majority of indigenous peoples in Taiwan consume alcohol, little is known about the relationship between alcoholic behavior and family relationships. A total of 471 residents from 3 villages in Alishan township in Taiwan were interviewed using a questionnaire that asked for demographic information and included the Family Function Scale and questions regarding the individual's consumption of alcohol. It was found that 50% of the participants drink alcohol, and 71% of their family members consume alcohol; 47% of the respondents indicated excessive alcohol consumption (ie, were heavy drinkers). When individuals are knowledgeable about alcohol-related health issues, their families generally function better (odds ratio = 2.56; 95% confidence interval = 1.38-4.74; P < .01). Those who were moderate and heavy drinkers were 2.5 and 3.0 times, respectively, more likely to have poor family relationships than those who were light drinkers. It is necessary to promote the reduction of alcohol consumption among indigenous peoples.

Immunopositivity of Beclin-1 and ATG5 as indicators of survival and disease recurrence in oral squamous cell carcinoma.

AIM: To evaluate the expression and prognostic value of two autophagy-related (Atg) proteins, Beclin-1 and Atg5, in human oral squamous cell carcinoma (OSCC) and to correlate findings with clinical outcomes. PATIENTS AND METHODS: Immunohistochemistry for Beclin-1 and Atg5 was assessed in tumor specimens from 90 patients with OSCC. Immunopositivity was semi-quantitatively scored and receiver-operating characteristic curve analysis was used to determine the cut-off positivity score. RESULTS: 55 (61.1%) and 52 (57.8%) cases showed positive Beclin-1 and Atg5 staining, respectively. 40 tumors (44.4%) were positive for both Beclin-1 and Atg5 expression and 23 cases (25.6%) showed absence of both proteins. Beclin-1 expression significantly correlated with tumor grade (p=0.008) and lymph node metastasis (p=0.009). The expression of Atg5 was associated with tumor grade (p=0.016), advanced clinical stage (p<0.001), large tumor size (p=0.002), and lymph node metastasis (p<0.001). A significant difference in 3-year OS (p=0.050) and TTR (p=0.049) between the patients with Beclin-1 expression and those not showing Beclin-1 expression was found whereas the difference did not reach a statistical significance for Atg5 expression. 3-year OS and TTR differed significantly between patients with dual expression and those with double-negative expression (p=0.022 and p=0.026, respectively). CONCLUSION: Dual expression of tumor Beclin-1 and Atg5 expression may be an adverse prognostic indicator for OSCC.

ATG9A overexpression is associated with disease recurrence and poor survival in patients with oral squamous cell carcinoma.

ATG9A is an integral membrane protein required for autophagosome formation and a membrane carrier in the autophagy pathways. The present study was designed to investigate the expression of ATG9A in oral squamous cell carcinoma (OSCC). Clinically annotated tumor specimens from 90 patients with OSCC were subjected to immunohistochemistry using an antibody against ATG9A and immunoreactivity was scored using an immunoreactivity score (IRS). Scores were compared with clinical and pathologic data to assess association with outcome. Overexpression of ATG9A was defined as an IRS of ≥9 by receiver operating characteristics curve analysis and was identified in 25 (28 %) of 90 cases. ATG9A overexpression was associated with disease recurrence and overall survival (OS) in both univariate (p = 0.030 and 0.025, respectively) and multivariate (p = 0.026 and 0.038, respectively) Cox analyses. Kaplan-Meier plots also showed that patients with ATG9A overexpression had shorter 3-year OS (p = 0.017) and time to recurrence (p = 0.021) than those with low ATG9A expression. These results suggest that the presence of ATG9A in the cytoplasm of tumor cells may be an independent biomarker for disease recurrence and survival in patients with OSCC.

High LC3 expression correlates with poor survival in patients with oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is a destructive disease with very poor prognosis and no effective treatment. Autophagy is a dynamic cellular process involved in various physiological processes and diseases including cancer that degrades cytoplasmic proteins and organelles. The role of autophagy in the pathogenesis of OSCC is not yet understood. Microtubule-associated protein light chains 3 (LC3) is a reliable autophagosome markers for monitoring autophagy. In the present study, LC3 expression was determined in a cohort of 90 OSCC samples by immunohistochemistry. The results were correlated with clinical and pathological characteristics of patients. High LC3 expression (N = 57; 63.3%) correlated with stage (P < .0001), tumor size (P < .0001), and lymph node involvement (P = .0003) and with an increased risk of death (P < .0001; hazard ratio, 3.59) in a univariate analysis. In the multivariate analysis adjusted for grade, stage, and alcohol, betel, and tobacco consumption, high LC3 expression retained statistical significance with regard to survival (P = .0043; hazard ratio, 2.99). The Kaplan-Meier survival curve also showed that high LC3 expression was significantly associated with poor overall survival (P = .0001). Elevated LC3 expression, which corresponds to increased level of autophagy activity, is a frequent event and an indicator of poor prognosis in human OSCC.

Expression of WWOX and FHIT is downregulated by exposure to arsenite in human uroepithelial cells.

Ecological studies in Taiwan, Chile, Argentina, Bangladesh, and Mexico have confirmed significant dose-dependent associations between ingestion of arsenic-contaminated drinking water and the risk of various human malignancies. The FHIT and WWOX genes are active in common fragile sites FRA3B and FRA16D, respectively. Reduced expression of FHIT or WWOX is known to be an early indicator of carcinogen-induced cancers. However, the effect of arsenite on the expressions and molecular mechanisms of these markers is still unclear. The aims of this study were (i) to observe the expression of ATR, WWOX and FHIT proteins in urothelial carcinoma (UC) between endemic and non-endemic areas of blackfoot disease (BFD) by immunohistochemical analyses; (ii) to compare expression of these genes between arsenite-treated SV-HUC-1 human epithelial cells and rat uroepithelial cells; and (iii) to determine the role of DNMT and MEK inhibitors on expressions of WWOX and FHIT in response to arsenite in SV-HUC-1. The experiments revealed that expressions of ATR, WWOX and FHIT in UC significantly differed between BFD areas and non-BFD areas (p=0.003, 0.009 and 0.021, respectively). In fact, the results for the arsenite-treated groups showed that ATR, WWOX and FHIT are downregulated by arsenite in SV-HUC-1. However, the inhibitors suppressed the effects of arsenite on WWOX and FHIT proteins and mRNA expression. In conclusion, arsenite decreased expressions of ATR, WWOX and FHIT via ERK1/2 activation in SV-HUC-1 cells. These findings confirm that dysregulations of these markers may contribute to arsenite-induced carcinogenesis.

Aberrant deleted in liver cancer-1 expression is associated with tumor metastasis and poor prognosis in urothelial carcinoma.

This study explored the potential role of deleted in liver cancer-1 (DLC-1) as a prognostic indicator of cancer metastasis and survival in urothelial carcinoma (UC). Tissue microarrays were constructed from paraffin-embedded specimens from 88 UC patients, and immunohistochemical staining was performed to investigate the association of DLC-1 with clinicopathologic characteristics and clinical outcome. The DLC-1 expression showed a significant positive correlation with tumor location (p = 0.041) and a significant negative correlation with advanced histological grade (p = 0.013). In tumors with low DLC-1 expression, Bcl-2 positivity was observed in 24.4% of cases. The DLC-1 expression had significant negative associations with Bcl-2 expression (p = 0.032) and with highly metastatic UC (p = 0.032). Kaplan-Meier analysis showed that DLC-1 protein expression was negatively associated with both overall survival (OS) (p = 0.035) and with distant metastasis-free survival (DMFS) (p = 0.041), but not with disease-free survival. Multivariate analyses indicated that tumor size was the significant independent predictors of OS (p = 0.048); however, only DLC-1 expression was a significant independent predictor of DMFS (p = 0.019). In conclusion, reduced DLC-1 protein expression may be an important factor in tumor progression and a useful prognostic molecular marker in UC.

Melanin bleaching with dilute hydrogen peroxide: a simple and rapid method.

Melanins are naturally occurring pigments in both normal and pathologic tissues. Two common bleaching processes are potassium permanganate followed by oxalic acid treatment and dilute hydrogen peroxide (H2O2) process. The potassium permanganate/oxalic acid method is faster and more easily incorporated in conventional daily immunostaining protocols, whereas the dilute H2O2 method requires 24 hours. This study aimed to reduce melanin bleaching time by using a 10% H2O2 dilution. First, reaction time was reduced to 30 minutes by raising the temperature to 65°C. Second, containers with high thermal conductivity were used to improve bleaching effectiveness. Experimental comparisons of melanin treatments with H2O2 contained in an iron jar, a glass coplin jar, and a plastic steel jar obtained bleaching time of 20, 30, and 40 minutes, respectively. These modifications of the conventional bleaching method significantly improve the speed and efficiency of the procedure and are recommended when performing immunohistochemical studies.

The expression and significance of WWOX and ß-catenin in hepatocellular carcinoma.

WW domain-containing oxidoreductase (WWOX) is a novel tumor suppressor gene, and its expression is reduced in various cancers, including hepatocellular carcinoma (HCC). WWOX has been reported to be downregulated in HCC cell lines as well as in primary HCC tissues. It has been suggested that WWOX is implicated in Wnt/ß-catenin pathway, which is frequently affected in HCC. The aim of this study was to evaluate the expression of WWOX, ß-catenin and T-cell factor 4 (TCF4) in HCC. Our result showed that downregulation of WWOX in HCC was correlated with cytoplasmic accumulation of ß-catenin. In addition, strong nuclear TCF4 expression was associated with tumor grade and stage in HCC. In conclusion, our result implied that downregulation of WWOX might lead to accumulation of cytoplasmic ß-catenin and the subsequent activation of Wnt/ß-catenin signaling pathway in HCC.

Sodium arsenite-induced abnormalities in expressions of Caveolin-1, eNOS, IKKß, and COX-2 in SV-40 immortalized human uroepithelial cells and in urothelial carcinomas.

Arsenic, a known human carcinogen, is found throughout the crust of the earth. Prolonged arsenic exposure is a known cause of urothelial carcinoma (UC) and blackfoot disease (BFD). The aim of this study was to determine the effect of sodium arsenite on Caveolin-1 and downstream signaling molecules (eNOS, IKKß and COX-2) expression in human urothelial cells (SV-HUC-1). Immunohistochemical (IHC) staining of Caveolin-1, eNOS, IKKß, and COX-2 was also compared between UC patients from endemic and non-endemic areas of BFD in Taiwan. Immunocytochemical staining and Western blotting results revealed increased expression of Caveolin-1, IKKß, and COX-2 but decreased eNOS in SV-HUC-1 cells treated with low concentration of arsenite. Additionally, MEK inhibitor (U0126) significantly attenuated arsenite-induced expression of Caveolin-1, IKKß and COX-2 while reducing eNOS expression. The IHC staining of UCs revealed that expressions of Caveolin-1, IKKß, and COX-2 were significantly higher in patients from endemic areas of BFD compared to patients from non-endemic areas (p=0.011, p=0.002, p=0.0001) whereas eNOS was significantly lower (p=0.0001). The correlation observed between Caveolin-1 and downstream signaling molecule expression may be an important mechanism of arsenic-induced urothelial carcinogenesis.

Overexpression of annexin 1 in the development and differentiation of urothelial carcinoma.

This study investigates the expression of annexin 1 in urothelial carcinoma (UC) and its relation with clinicopathologic factors, and evaluates its potential clinical significance. Annexin 1 expression was analyzed by immunohistochemical staining with manual tissue microarrays and Western blot in UC. Immunohistochemical analysis of UC in tissue microarrays showed that annexin 1 protein was 76.5% (150/196) positive, which was markedly increased compared with that in the normal urothelium 20.8% (5/24) (p < 0.01). In addition, the positive expression rate of annexin 1 was higher in the high-grade UC (81.7%; 143/175) than in the low-grade UC (33.3%; 7/21). Western blot revealed that the expression of annexin 1 was low in low-grade UC, and markedly increased in high-grade UC. In conclusion, annexin 1 overexpression is observed in UC, which suggests it may be associated with tumorigenesis and its expression correlates with the differentiation of UC.

para-Phenylenediamine-induced autophagy in human uroepithelial cell line mediated mutant p53 and activation of ERK signaling pathway.

para-Phenylenediamine (p-PD) is a major aromatic amine that is a widely used commercial oxidative-type hair dye. Some epidemiologic studies have suggested that the use of p-PD-based hair dyes might be related to increased risk of human malignant tumors including bladder cancer. However, the effects of p-PD on autophagy in human uroepithelial cells (SV-HUC-1) is still unclear. In this study, we demonstrate that p-PD can activate the extracellular signaling-regulated protein kinase 1/2 (ERK1/2) signaling pathway in SV-HUC-1 cells. In addition, we observed that autophagosomes increased in p-PD-treated SV-HUC-1 cells as shown by electron microscopy. Our results showed incremental increase of the concentrations, Beclin-1 and microtubule-associated protein light chain 3B (LC3B), which are important regulators of autophagosomes. In contrast, the MEK inhibitor (U0126) was suppressed autophagy and the effect of p-PD on ERK1/2, Beclin-1 and LC3B proteins expression, except for mutant p53. In this study, we demonstrated that inactivation of p53 induces a potent autophagy response. Finally, our results suggest that p-PD can activate the ERK1/2 signaling pathway and mutant p53, leading to the stimulation of autophagy in SV-HUC-1 cells. These results provide us with new insights for the understanding of the mechanism of p-PD-induced cell death in urothelial cells.

Effects of DNMT and MEK inhibitors on the expression of RECK, MMP-9, -2, uPA and VEGF in response to arsenite stimulation in human uroepithelial cells.

It has been shown that the ingestion of arsenic-contaminated drinking water is closely correlated with risk of several cancers. The mechanism of arsenic-induced carcinogenesis is still unclear. The RECK, MMP-9, -2, uPA and VEGF are the most common dysregulation in human tumors and cancer cell lines. However, the effect of arsenite on these markers expression and the molecular mechanism are still unclear. The purpose of the study was to investigate the relationship between the expression of RECK, MMP-9, -2, uPA and VEGF in arsenite-treated human and rat uroepithelial cells. In addition, we also observed and compared the expression of these markers in urothelial carcinoma (UC) from Blackfoot disease (BFD) areas and non-Blackfoot disease (non-BFD) areas. We analyzed the arsenite causing cell proliferation, RECK, MMP-9, -2, uPA and VEGF expression by Western blotting, immunocytochemistry (ICC), RT-PCR, and gelatin zymography. We demonstrated the effect of arsenite on methylation status of RECK promoter as determined by using methylation-specific PCR (MSP). Our results show that arsenite downregulation of RECK is caused by epigenetic inactivation via promoter hypermethylation, and that levels of MMP-9, -2, uPA and VEGF were increased in human uroepithelial cells (SV-HUC-1). However, when the cells were pretreated with inhibitors (5-aza-CdR or U0126) for 24h, the effects of arsenite on RECK, MMP-9, -2, uPA and VEGF expression were suppressed. Indeed, we also found significant differences between the expression of RECK, MMP-9, -2, uPA and VEGF in UC from the BFD areas and non-BFD areas (p=0.006, 0.007, 0.003, <0.001 and 0.001 respectively), as detected by immunohistochemistry (IHC). In in vivo study, our results showed the RECK protein expression was reduced and the expression of MMP-9, -2, uPA and VEGF increased in arsenite treatment groups. In conclusion, our results support the notion that arsenite might cause the histologic changes, RECK, MMP-9, -2, uPA and VEGF dysregulation through epigenetic inactivation and ERK1/2 activation in SV-HUC-1 cells. These findings may provide a better understanding of the urothelial carcinogenesis of arsenite.

Pretreatment of vitamin D3 ameliorates lung and muscle injury induced by reperfusion of bilateral femoral vessels in a rat model.

BACKGROUND: Peripheral arterial occlusive disease (PAOD) is a challenge in peripheral vascular disease. Clinical observations show reperfusion of occluded vessels may cause compartment syndrome or remote organ injury. Less well known is the role of vitamin D3 in tissue injury; therefore, we attempted to determine whether vitamin D3 could alleviate local and remote organ injury induced by reperfusion of occluded vessels in animal models. METHODS: Twenty-four male Sprague-Dawley rats were randomized into four groups: saline + sham, saline + I/R, vitamin D3 + sham, and vitamin D3 + I/R group. After pretreatment for 5 d, the animals designed to I/R injury were subjected to 3 h of ischemia induced by bilateral femoral arteries clamp, followed by reperfusion of the vessels for 3 h on d 6. Left lung and left anterior tibial muscle tissue were harvested for wet/dry weight ratio and histopathologic analysis. Blood was collected for analysis of urea nitrogen (BUN), creatinine (Cr), aspartate aminotransferase (AST), alanine aminotransferase (ALT), tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), ionized calcium levels, and heme oxygenase-1 (HO-1). RESULTS: Compared with the saline + sham group, there was a significant increase in plasma IL-6 level in both saline + I/R and vitamin D3 + I/R groups and muscle, lung wet/dry weight ratio in the saline + I/R group (P < 0.05). Compared with the saline + I/R group, there was a significant decrease in plasma IL-6 level, muscle and lung wet/dry weight ratio in both vitamin D3 + sham and vitamin D3 + I/R groups, and leukocyte HO-1 expression in vitamin D3 + sham group (P < 0.05). Compared with the vitamin D3 + sham group, there was a significant increase in plasma IL-6 levels in the vitamin D3 + I/R group, and leukocyte HO-1 expression in vitamin D3 + sham group (P < 0.05). BUN, Cr, AST, ALT, TNF-α, ionized calcium levels did not differ significantly among the groups. CONCLUSIONS: Pretreatment of vitamin D3 ameliorates the systemic IL-6 levels, lung and muscle injury induced by ischemia followed by reperfusion of bilateral occluded vessels in a rat model.