miR-126-3p is essential for CXCL12-induced angiogenesis.

Atherosclerosis, in the ultimate stage of cardiovascular diseases, causes an obstruction of vessels leading to ischemia and finally to necrosis. To restore vascularization and tissue regeneration, stimulation of angiogenesis is necessary. Chemokines and microRNAs (miR) were studied as pro-angiogenic agents. We analysed the miR-126/CXCL12 axis and compared impacts of both miR-126-3p and miR-126-5p strands effects in CXCL12-induced angiogenesis. Indeed, the two strands of miR-126 were previously shown to be active but were never compared together in the same experimental conditions regarding their differential functions in angiogenesis. In this study, we analysed the 2D-angiogenesis and the migration assays in HUVEC in vitro and in rat's aortic rings ex vivo, both transfected with premiR-126-3p/-5p or antimiR-126-3p/-5p strands and stimulated with CXCL12. First, we showed that CXCL12 had pro-angiogenic effects in vitro and ex vivo associated with overexpression of miR-126-3p in HUVEC and rat's aortas. Second, we showed that 2D-angiogenesis and migration induced by CXCL12 was abolished in vitro and ex vivo after miR-126-3p inhibition. Finally, we observed that SPRED-1 (one of miR-126-3p targets) was inhibited after CXCL12 treatment in HUVEC leading to improvement of CXCL12 pro-angiogenic potential in vitro. Our results proved for the first time: 1-the role of CXCL12 in modulation of miR-126 expression; 2-the involvement of miR-126 in CXCL12 pro-angiogenic effects; 3-the involvement of SPRED-1 in angiogenesis induced by miR-126/CXCL12 axis.

The expanding roles of microRNAs in kidney pathophysiology.

MicroRNAs (miRNAs) are short single-stranded RNAs that control gene expression through base pairing with regions within the 3'-untranslated region of target mRNAs. These small non-coding RNAs are now increasingly known to be involved in kidney physiopathology. In this review we will describe how miRNAs were in recent years implicated in cellular and animal models of kidney disease but also in chronic kidney disease, haemodialysed and grafted patients, acute kidney injury patients and so on. At the moment miRNAs are considered as potential biomarkers in nephrology, but larger cohorts as well as the standardization of methods of measurement will be needed to confirm their usefulness. It will further be of the utmost importance to select specific tissues and biofluids to make miRNAs appropriate in day-to-day clinical practice. In addition, up- or down-regulating miRNAs that were described as deregulated in kidney diseases may represent innovative therapeutic methods to cure these disorders. We will enumerate in this review the most recent methods that can be used to deliver miRNAs in a specific and suitable way in kidney and other organs damaged by kidney failure, such as the cardiovascular system.

Effects of insulin analogs as an add-on to metformin on cutaneous microcirculation in type 2 diabetic patients.

BACKGROUND: A single insulin injection was shown to improve microcirculatory blood flow. Our aim was to examine the effects of 4weeks of insulin therapy by three randomly assigned insulin analog regimens (Detemir, Aspart, and their combination) on cutaneous blood flow (CBF) and microcirculatory endothelial function as an add-on to metformin in type 2 diabetic patients poorly controlled on oral antidiabetic treatment. METHODS: Fourty-two type 2 diabetic patients with no history of cardiovascular disease in secondary failure to oral antidiabetic agents had CBF measurements before and after acetylcholine (Ach) iontophoretic administration. CBF measurements were performed at fasting and after a standardized breakfast during the post-prandial period. Before randomization (Visit 1, V1) during the tests, participants took only metformin. The same tests were repeated after 4weeks of insulin treatment (Visit 2, V2). RESULTS: Thirty-four patients had good quality recordings for both visits. During V1, CBF and CBF response to Ach increased in the post-prandial period. After 4weeks of insulin treatment, metabolic parameters improved. Compared to V1, CBF at fasting did not increase at V2 but there was an improvement in endothelial function at fasting after Ach iontophoresis, without difference across insulin regimens. Oxidative stress markers were not modified, and E-selectin and vascular cell adhesion molecule 1 levels decreased after insulin treatment, without differences between insulin groups. CONCLUSIONS: A strategy of improving glycemic control for 4weeks with insulin analogs improves microcirculatory endothelial reactivity and reduces endothelial biomarkers at fasting, whatever the insulin regimen used. Insulin therapy associated to metformin is able to improve fasting microvascular endothelial function even before complete metabolic control.

Plasma vitamin D status and recurrent depressive symptoms in the French SU.VI.MAX cohort.

PURPOSE: Depression is a major public health issue because it is a common cause of disability worldwide. It has been suggested that an optimal vitamin D status may be related to fewer depressive symptoms, but findings are inconsistent. We aimed to investigate the association between plasma vitamin D at midlife and recurrent depressive symptoms and to test for a modulating effect by overall dietary quality. METHODS: The relationship between plasma 25-hydroxyvitamin D (25(OH)D) and recurrent depressive symptoms was evaluated among 1196 participants of the Supplémentation en Vitamines et Minéraux Antioxydants cohort with available data on the Center for Epidemiologic Studies-Depression Scale (CES-D) at baseline (1996-1997) and follow-up (2007-2009). Recurrent depressive symptoms were defined as a CES-D score ≥16 at baseline and follow-up. Prevalence ratios (PR) and 95 % confidence intervals (95 %-CI) were estimated using extensively adjusted Poisson regression models. Dietary quality was estimated using an index measuring adherence to the French national recommendations. RESULTS: Having 25(OH)D concentrations above 10 ng/mL was related to a lower probability of recurrent depressive symptoms: PR (95 %-CI) = 0.48 (0.33; 0.69); P < 0.0001). When comparing individuals with concentrations < versus ≥20 or < versus ≥30 ng/mL, no significant results were obtained. In contrast, among individuals with low dietary quality, a better vitamin D status was related to a lower probability of recurrent depressive symptoms independently of the applied cutoff. CONCLUSIONS: Plasma vitamin D might have a preventive role against recurrent depressive symptoms, notably among individuals with poor dietary quality. Our findings are relevant for the development of depression prevention programs.

Weight Status and Alcohol Intake Modify the Association between Vitamin D and Breast Cancer Risk.

BACKGROUND: Mechanistic hypotheses suggest that vitamin D may contribute to the prevention of breast cancer. However, epidemiologic evidence is inconsistent, suggesting a potential effect modification by individual factors. OBJECTIVE: Our objective was to perform exploratory analyses on the prospective associations between the plasma 25-hydroxyvitamin D [25(OH)D] concentration, polymorphisms of genes encoding for the vitamin D receptor (VDR) and vitamin D-binding protein (also known as gc-globulin or group-specific component, GC), and breast cancer risk, along with 2 potential modifiers: body mass index (BMI; in kg/m(2)) and alcohol intake. METHODS: A nested case-control study was set up in the SUpplémentation en VItamines et Minéraux Anti-oXydants (SU.VI.MAX) cohort (1994-2007), involving 233 women with breast cancer and 466 matched controls (mean ± SD age: 49 ± 6 y). The plasma total 25(OH)D concentration and gene polymorphisms were assessed on samples obtained at baseline. Conditional logistic regression models were computed. RESULTS: A higher plasma 25(OH)D concentration was associated with a decreased risk of breast cancer for women with a BMI < the median of 22.4 [OR quartile (Q)4 compared with Q1: 0.46; 95% CI: 0.23, 0.89; P-trend = 0.01, P-interaction = 0.002], whereas it was associated with an increased risk for women with a BMI ≥ the median (OR Q4 compared with Q1: 2.45; 95% CI: 1.13, 5.28; P-trend = 0.02, P-interaction = 0.002). A plasma 25(OH)D concentration ≥ 10 ng/mL was associated with a decreased risk of breast cancer for women with alcohol intakes ≥ the median of 7.1 g/d (OR ≥10 compared with <10 ng/mL: 0.50; 95% CI: 0.26, 0.95; P = 0.03, P-interaction = 0.03). The genetic analyses were consistent with the results observed with plasma 25(OH)D. CONCLUSION: In this prospective study, BMI and alcohol intake modified the association between vitamin D [plasma 25(OH)D and vitamin D-related gene polymorphisms] and breast cancer risk. These effect modifications suggest explanations for discrepancies in results of previous studies. This trial was registered at clinicaltrials.gov as NCT00272428.

A prospective study of plasma 25-hydroxyvitamin D concentration and prostate cancer risk.

Mechanistic hypotheses suggest that vitamin D and the closely related parathyroid hormone (PTH) may be involved in prostate carcinogenesis. However, epidemiological evidence is lacking for PTH and inconsistent for vitamin D. Our objectives were to prospectively investigate the association between vitamin D status, vitamin D-related gene polymorphisms, PTH and prostate cancer risk. A total of 129 cases diagnosed within the Supplémentation en Vitamines et Minéraux Antioxydants cohort were included in a nested case-control study and matched to 167 controls (13 years of follow-up). 25-Hydroxyvitamin D (25(OH)D) and PTH concentrations were assessed from baseline plasma samples. Conditional logistic regression models were computed. Higher 25(OH)D concentration was associated with decreased risk of prostate cancer (ORQ4 v. Q1 0·30; 95 % CI 0·12, 0·77; P trend=0·007). PTH concentration was not associated with prostate cancer risk (P trend=0·4) neither did the studied vitamin D-related gene polymorphisms. In this prospective study, prostate cancer risk was inversely associated with 25(OH)D concentration but not with PTH concentration. These results bring a new contribution to the understanding of the relationship between vitamin D and prostate cancer, which deserves further investigation.

Identification of a Pro-Angiogenic Potential and Cellular Uptake Mechanism of a LMW Highly Sulfated Fraction of Fucoidan from Ascophyllum nodosum.

Herein we investigate the structure/function relationships of fucoidans from Ascophyllum nodosum to analyze their pro-angiogenic effect and cellular uptake in native and glycosaminoglycan-free (GAG-free) human endothelial cells (HUVECs). Fucoidans are marine sulfated polysaccharides, which act as glycosaminoglycans mimetics. We hypothesized that the size and sulfation rate of fucoidans influence their ability to induce pro-angiogenic processes independently of GAGs. We collected two fractions of fucoidans, Low and Medium Molecular Weight Fucoidan (LMWF and MMWF, respectively) by size exclusion chromatography and characterized their composition (sulfate, fucose and uronic acid) by colorimetric measurement and Raman and FT-IR spectroscopy. The high affinities of fractionated fucoidans to heparin binding proteins were confirmed by Surface Plasmon Resonance. We evidenced that LMWF has a higher pro-angiogenic (2D-angiogenesis on Matrigel) and pro-migratory (Boyden chamber) potential on HUVECs, compared to MMWF. Interestingly, in a GAG-free HUVECs model, LMWF kept a pro-angiogenic potential. Finally, to evaluate the association of LMWF-induced biological effects and its cellular uptake, we analyzed by confocal microscopy the GAGs involvement in the internalization of a fluorescent LMWF. The fluorescent LMWF was mainly internalized through HUVEC clathrin-dependent endocytosis in which GAGs were partially involved. In conclusion, a better characterization of the relationships between the fucoidan structure and its pro-angiogenic potential in GAG-free endothelial cells was required to identify an adapted fucoidan to enhance vascular repair in ischemia.

Comparative toxicity evaluation of flower-shaped and spherical gold nanoparticles on human endothelial cells.

In this paper, we propose a multi-parametric in vitro study of the cytotoxicity of gold nanoparticles (GNPs) on human endothelial cell (HUVEC). The cytotoxicity is evaluated by incubating cells with six different GNP types which have two different morphologies: spherical and flower-shaped, two sizes (∼15 and ∼50 nm diameter) and two surface chemistries (as prepared form and PEGylated form). Our results showed that by increasing the concentration of GNPs the cell viability decreases with a toxic concentration threshold of 10 pM for spherical GNPs and of 1 pM for flower-shaped GNPs. Dark field images, flow cytometry and spreading test revealed that flower-shaped GNPs have more deleterious effects on the cell mechanisms than spherical GNPs. We demonstrated that the main parameter in the evaluation of the GNPs toxicity is the GNPs roughness and that this effect is independent on the surface chemistry. We assume that this behavior is highly related to the efficiency of the GNPs internalization within the cells and that this effect is enhanced due to the specific geometry of the flower-shaped GNPs.

Heparanase and Syndecan-4 Are Involved in Low Molecular Weight Fucoidan-Induced Angiogenesis.

Induction of angiogenesis is a potential treatment for chronic ischemia. Low molecular weight fucoidan (LMWF), the sulfated polysaccharide from brown seaweeds, has been shown to promote revascularization in a rat limb ischemia, increasing angiogenesis in vivo. We investigated the potential role of two heparan sulfate (HS) metabolism enzymes, exostosin-2 (EXT2) and heparanase (HPSE), and of two HS-membrane proteoglycans, syndecan-1 and -4 (SDC-1 and SDC-4), in LMWF induced angiogenesis. Our results showed that LMWF increases human vascular endothelial cell (HUVEC) migration and angiogenesis in vitro. We report that the expression and activity of the HS-degrading HPSE was increased after LMWF treatment. The phenotypic tests of LMWF-treated and EXT2- or HPSE-siRNA-transfected cells indicated that EXT2 or HPSE expression significantly affect the proangiogenic potential of LMWF. In addition, LMWF increased SDC-1, but decreased SDC-4 expressions. The effect of LMWF depends on SDC-4 expression. Silencing EXT2 or HPSE leads to an increased expression of SDC-4, providing the evidence that EXT2 and HPSE regulate the SDC-4 expression. Altogether, these data indicate that EXT2, HPSE, and SDC-4 are involved in the proangiogenic effects of LMWF, suggesting that the HS metabolism changes linked to LMWF-induced angiogenesis offer the opportunity for new therapeutic strategies of ischemic diseases.

Impact of cytokine gene variants on the prediction and prognosis of hepatocellular carcinoma in patients with cirrhosis.

BACKGROUND & AIMS: Genetic polymorphisms modulate the expression of proinflammatory cytokines. We prospectively assessed the influence of 6 single nucleotide polymorphisms (SNPs) in TNFα, IL6, and IL1ß genes on the risk of hepatocellular carcinoma (HCC) in patients with cirrhosis. METHODS: TNFα (G-238A, C-863A, G-308A), IL6 (C-174G), and IL1ß (C-31T, C-511T) SNPs were assessed in 232 alcoholics and 253 HCV-infected patients with biopsy-proven cirrhosis, prospectively followed-up and screened for HCC. Their influence on HCC development was assessed using the Kaplan-Meier method. RESULTS: These variants did not influence the risk of HCC in alcoholic patients. Conversely, two variants influenced the risk of HCC occurrence in patients with HCV-related cirrhosis, namely the TNFα-308 (A) allele (HR = 2.4 [1.6-3.7], Log-rank <0.0001) and the IL1ß-31 (T) allele (HR = 1.5 [1.1-2.1], Log-rank = 0.004). When stratifying HCV-infected patients into four genotypic associations expected to progressively increase TNFα and IL1ß production, we observed increasing risk of HCC occurrence (Log-rank <0.0001) from group 1 to 4. The TNFα-308 (A) allele was the only genetic trait independently associated with risk of HCC in these patients, along with older age, male gender, BMI, and platelet count. These variables led to construction of a predictive score able to separate patients with HCV-related cirrhosis into three subgroups with progressively increasing 5-year cumulative incidences of 4.7%, 14.1%, and 36.3%, respectively (Log-rank <0.0001). CONCLUSIONS: Genetic heterogeneity in the TNFα and IL1ß gene promoters influences the risk of HCC in patients with HCV-induced cirrhosis. These genetic data, when incorporated into clinical scores, are able to refine selection of risk classes of HCC.

Association between prediagnostic biomarkers of inflammation and endothelial function and cancer risk: a nested case-control study.

Experimental and prevalent case-control studies suggest an association between biomarkers of inflammation, endothelial function, and adiposity and cancer risk, but results from prospective studies have been limited. The authors' objective was to prospectively examine the relations between these biomarkers and cancer risk. A nested case-control study was designed within the Supplémentation en Vitamines et Minéraux Antioxydants (SU.VI.MAX) Study, a nationwide French cohort study, to include all first primary incident cancers diagnosed between 1994 and 2007 (n = 512). Cases were matched with randomly selected controls (n = 1,024) on sex, age (in 2-year strata), body mass index (weight (kg)/height (m)(2); <25 vs. ≥25), and SU.VI.MAX intervention group. Conditional logistic regression was used to study the associations between prediagnostic levels of high-sensitivity C-reactive protein (hs-CRP), adiponectin, leptin, soluble intercellular adhesion molecule 1 (sICAM-1), soluble vascular cell adhesion molecule 1, soluble E-selectin, and monocyte chemoattractant protein 1 and cancer risk. All statistical tests were 2-sided. Plasma sICAM-1 level was positively associated with breast cancer risk (for quartile 4 vs. quartile 1, multivariate odds ratio (OR) = 1.86, 95% confidence interval (CI): 1.06, 3.26; P(trend) = 0.048). Plasma hs-CRP level was positively associated with prostate cancer risk (for quartile 4 vs. quartile 1, multivariate OR = 3.04, 95% CI: 1.28, 7.23; P(trend) = 0.03). These results suggest that prediagnostic hs-CRP and sICAM-1 levels are associated with increased prostate and breast cancer risk, respectively.

PNPLA3 rs738409, hepatocellular carcinoma occurrence and risk model prediction in patients with cirrhosis.

BACKGROUND & AIMS: Several studies have reported an association between the genetic variant rs738409 (G) in the PNPLA3 gene and the risk of cirrhosis in various liver diseases. Our purpose was to assess the influence of this polymorphism on the risk of hepatocellular carcinoma (HCC) occurrence in two distinct longitudinal cohorts of patients with cirrhosis as well as its possible usefulness in HCC-risk model prediction. METHODS: PNPLA3 rs738409 genotypes were assessed in 279 patients with alcoholic- and 253 patients with HCV-related cirrhosis. These patients were followed-up and screened for the risk of HCC, and the influence of rs738409 on the occurrence of liver cancer was assessed using the Kaplan-Meier method, then according to the multivariate Cox model. RESULTS: In patients with HCV-related cirrhosis, rs738409 genotypes did not influence the risk of HCC development (log-rank = 0.7) or death (log-rank = 0.2). Conversely, in patients with alcoholic cirrhosis, the rs738409 (GG) genotype was an independent risk factor for HCC occurrence (HR = 1.72 [1.21-2.45], log-rank = 0.002) as well as older age, male gender, and higher BMI. Combining these features enabled HCC-risk stratification of this population into three groups with the 6-year cumulative incidence ranging from 3.4% (low risk, n = 58), 12.2% (intermediate risk, n = 163), and 51.7% (high risk, n = 58), respectively (HR = 4.3 [2.7-6.4]; log-rank <0.0001). CONCLUSIONS: This study provides key data that affirm the influence of the rs738409 (GG) genotype on the occurrence of HCC in patients with alcoholic cirrhosis. Its combination with clinical features refines the selection of patients at higher risk of liver cancer development.

ESM-1 expression in stromal cells is predictive of recurrence after radiofrequency ablation in early hepatocellular carcinoma.

BACKGROUND & AIMS: The prognosis of hepatocellular carcinoma (HCC) treated by radiofrequency ablation (RFA) is mainly linked to tumor recurrence. So far, no tissue biomarker of recurrence has been validated in biopsy samples. We aimed at investigating the prognostic value of tissue biomarkers in HCC biopsy samples of patients treated with RFA. METHODS: All consecutive naive patients from 3 university hospitals, with compensated cirrhosis, early-stage (BCLC 0/A) uninodular HCC treated with RFA, and available tumor biopsy, were included. Edmondson's grade, and the expression of cytokeratin 19, glutamine synthase, beta-catenin, epithelial cell adhesion molecule (EpCAM), and endothelial cell-specific molecule 1 (ESM-1) were assessed. Main clinical end points were overall and early recurrence. Statistical analyses were performed using Kaplan Meier, Log-rank test, and Cox models. RESULTS: 150 patients were included. Recurrence, death or liver transplantation occurred in 85, 51, and 12 patients, respectively. Median follow-up was 27months. ESM-1 expression by HCC stromal endothelial cells was observed in 58 patients (40%) and was associated with higher serum AFP levels, larger tumor, and more frequent expression of EpCAM and surrogate markers of activation of the Wnt-ß-catenin pathway. The 2 independent predictive factors of overall recurrence were serum AFP (HR 1.11 [1.002; 1.22], p=0.045) and ESM-1 expression (HR 1.56 [1.004; 2.43], p=0.048). ESM-1 expression was also an independent predictive factor of early recurrence (HR 1.81 [1.02; 3.21], p=0.042). CONCLUSIONS: ESM-1 expression by stromal endothelial cells, in tumor biopsy samples, has an independent predictive value of early recurrence after RFA.

Insulin analogs as an add-on to metformin after failure to oral treatment in type 2 diabetic patients increase diastole duration. The INSUlin Regimens and VASCular Functions (INSUVASC) study.

BACKGROUND: Fast acting insulin analogues are known to improve arterial stiffness. The combination of metformin with insulin represents a widely used therapeutic strategy in diabetes. We hypothesized that insulin treatment in patients with type 2 diabetes (T2D) with long-acting, fast-acting or basal bolus insulin as an add-on to metformin would provide additional improvement of arterial stiffness. METHODS: The INSUlin Regimens and VASCular Functions (INSUVASC) study is a pilot, randomized, open label three-arms study that included 42 patients with type 2 diabetes (T2D) in primary prevention, after a failure to oral antidiabetic agents. Arterial stiffness measurements were performed at fasting and after a standardized breakfast. During the first visit (V1) pre-randomization, participants took only metformin to perform the tests. The same tests were repeated after 4 weeks of insulin treatment during the second visit (V2). RESULTS: Data were available for final analysis in 40 patients, with a mean age of 53.6±9.7 years and a mean duration of diabetes of 10.6±5.6 years. Twenty-one were females (52.5%), hypertension and dyslipidemia were present in 18 (45%) and 17 patients (42.5%), respectively. After insulin treatment, the metabolic control was associated to a decrease in oxidative stress and improvement of endothelial functions, with a post prandial diastole duration increased and a decrease of the peripheral arterial stiffness, with a better post prandial pulse pressure ratio and ejection duration after insulin. In hypertensive patients, insulin treatment provided positive effects by decreasing the pulse wave velocity and improving reflection time. CONCLUSIONS: A short time treatment by insulin in addition to metformin improved myocardial perfusion. Moreover, insulin treatment in hypertensive patients provides a better hemodynamic profile in large arteries.

A variant in myeloperoxidase promoter hastens the emergence of hepatocellular carcinoma in patients with HCV-related cirrhosis.

BACKGROUND & AIMS: Genetic dimorphisms modulate the activities of several pro- or antioxidant enzymes, including myeloperoxidase (MPO), catalase (CAT), manganese superoxide dismutase (SOD2), and glutathione peroxidase 1 (GPx1). We assessed the role of the G(-463)A-MPO, T(-262)C-CAT, Ala16Val-SOD2, and Pro198Leu-GPx1 variants in modulating HCC development in patients with HCV-induced cirrhosis. METHODS: Two hundred and five patients with HCV-induced, biopsy-proven cirrhosis but without detectable HCC at inclusion were prospectively followed-up for HCC development. The influence of various genotypes on HCC occurrence was assessed with the Kaplan-Meier method. RESULTS: During follow-up (103.2±3.4 months), 84 patients (41%) developed HCC, and 66 died. Whereas the Ala16Val-SOD2 or Pro198Leu-GPx1 dimorphisms did not modulate the risk, HCC occurrence was increased in patients with either the homozygous GG-MPO genotype (HR=2.8 [1.7-4.4]; first quartile time to HCC occurrence: 45 vs. 96 months; LogRank <0.0001) or the homozygous CC-CAT genotype (HR=1.74 [1.06-2.82]; first quartile time to HCC occurrence: 55 vs. 96 months; LogRank=0.02). Compared to patients with neither of these two at risk factors, patients with only the CC-CAT genotype had a HR of 2.05 [0.9-4.6] (p=0.08) and patients with only the GG-MPO genotype had a HR of 3.8 [1.5-9.1] (p=0.002), while patients with both risk factors had an HR of 4.8 [2.2-10.4] (p<0.0001). However, only the GG-MPO genotype was independently associated with the HCC risk in multivariate Cox analysis. CONCLUSIONS: The high activity-associated GG-MPO genotype increases the rate of HCC occurrence in patients with HCV-induced cirrhosis.

Sulfatase 2 Along with Syndecan 1 and Glypican 3 Serum Levels are Associated with a Prognostic Value in Patients with Alcoholic Cirrhosis-Related Advanced Hepatocellular Carcinoma.

Purpose: Sulfatase 2 (SULF2) is an enzyme related to heparan sulfate modifications. Its expression, as for some heparan sulfate proteoglycans expression, has been linked to hepatocellular carcinoma (HCC) at mRNA level and immunohistochemistry staining on biopsy samples. This study aims to evaluate the prognostic value of serum levels of SULF2 in patients with alcoholic cirrhosis with or without HCC. Patients and Methods: Two hundred and eighty-seven patients with alcoholic cirrhosis were enrolled in this study: 164 without HCC, 57 with early HCC, and 66 with advanced HCC at inclusion. We analyzed the association between SULF2 serum levels and prognosis using Kaplan-Meier method and univariate and multivariate analysis using a Cox model. Results: Child-Pugh C Patients have higher serum levels of SULF2 than Child-Pugh A patients. Serum levels of SULF2 were also higher in patients with advanced HCC compared with the other groups. In patients with advanced HCC, high serum levels of SULF2 were associated with less favorable overall survival. Combination of SULF2 with Glypican 3 (GPC3) and Syndecan 1 (SDC1) serum levels enhanced the ability to discriminate worst prognostic in advanced HCC. Conclusion: SULF2 along with GPC3 and SDC1 serum levels have been shown to be associated with a prognostic value in advanced HCC.

Syndecan-1 Is Overexpressed in Human Thoracic Aneurysm but Is Dispensable for the Disease Progression in a Mouse Model.

Glycosaminoglycans (GAGs) pooling has long been considered as one of the histopathological characteristics defining thoracic aortic aneurysm (TAA) together with smooth muscle cells (SMCs) apoptosis and elastin fibers degradation. However, little information is known about GAGs composition or their potential implication in TAA pathology. Syndecan-1 (SDC-1) is a heparan sulfate proteoglycan that is implicated in extracellular matrix (ECM) interaction and assembly, regulation of SMCs phenotype, and various aspects of inflammation in the vascular wall. Therefore, the aim of this study was to determine whether SDC-1 expression was regulated in human TAA and to analyze its role in a mouse model of this disease. In the current work, the regulation of SDC-1 was examined in human biopsies by RT-qPCR, ELISA, and immunohistochemistry. In addition, the role of SDC-1 was evaluated in descending TAA in vivo using a mouse model combining both aortic wall weakening and hypertension. Our results showed that both SDC-1 mRNA and protein are overexpressed in the media layer of human TAA specimens. RT-qPCR experiments revealed a 3.6-fold overexpression of SDC-1 mRNA (p = 0.0024) and ELISA assays showed that SDC-1 protein was increased 2.3 times in TAA samples compared with healthy counterparts (221 ± 24 vs. 96 ± 33 pg/mg of tissue, respectively, p = 0.0012). Immunofluorescence imaging provided evidence that SMCs are the major cell type expressing SDC-1 in TAA media. Similarly, in the mouse model used, SDC-1 expression was increased in TAA specimens compared to healthy samples. Although its protective role against abdominal aneurysm has been reported, we observed that SDC-1 was dispensable for TAA prevalence or rupture. In addition, SDC-1 deficiency did not alter the extent of aortic wall dilatation, elastin degradation, collagen deposition, or leukocyte recruitment in our TAA model. These findings suggest that SDC-1 could be a biomarker revealing TAA pathology. Future investigations could uncover the underlying mechanisms leading to regulation of SDC-1 expression in TAA.

Angiogenic properties of the chemokine RANTES/CCL5.

Atherosclerosis is an inflammatory disease that is one of the leading causes of death in developed countries. This disease is defined by the formation of an atherosclerotic plaque, which is responsible for artery obstruction and affects the heart by causing myocardial infarction. The vascular wall is composed of three cell types and includes a monolayer of endothelial cells and is irrigated by a vasa vasorum. The formation of the vascular network from the vasa vasorum is a process involved in the destabilization of this plaque. Cellular and molecular approaches are studied by in vitro assay of activated endothelial cells and in in vivo models of neovascularization. Chemokines are a large family of small secreted proteins that have been shown to play a critical role in the regulation of angiogenesis during several pathophysiological processes such as ischaemia. Chemokines may exert their regulatory activity on angiogenesis directly by activating the vasa vasorum, or as a consequence of leucocyte infiltration through the endothelium, and/or by the induction of growth factor expression such as that of VEGF (vascular endothelial growth factor). The present review focuses on the angiogenic activity of the chemokines RANTES (regulated upon activation, normal T-cell expressed and secreted)/CCL5 (CC chemokine ligand 5). RANTES/CCL5 is released by many cell types such as platelets or smooth muscle cells. This chemokine interacts with GPCRs (G-protein-coupled receptors) and GAG (glycosaminoglycan) chains bound to HSPGs (heparan sulfate proteoglycans). Many studies have demonstrated, using RANTES/CCL5 mutated on their GAG or GPCR-binding sites, the involvement of these chemokines in angiogenic process. In the present review, we discuss two controversial roles of RANTES/CCL5 in the angiogenic process.

Glycosaminoglycans and syndecan-4 are involved in SDF-1/CXCL12-mediated invasion of human epitheloid carcinoma HeLa cells.

BACKGROUND: In addition to their physiologic effects in inflammation and angiogenesis, chemokines are involved in cancer pathology. The CXC-chemokine stromal cell-derived factor-1 (SDF-1)/CXCL12 mediates its biological activities through activation of G protein-coupled receptor CXCR4 and binds to glycosaminoglycans (GAGs). METHODS: Using Bio-coat cell migration chambers, specific antagonists, flow cytometry and RNA interference, we evaluate the involvement of heparan sulfate proteoglycans (HSPG) in the SDF-1/CXCL12-induced invasion of human cervix epitheloid carcinoma HeLa cells. RESULTS: The SDF-1/CXCL12-induced cell invasion is dependent on CXCR4. Furthermore, Protein Kinase C delta (PKC delta) and c-jun NH2-terminal kinase/stress-activated protein kinase (JNK/SAPK) are implicated in this event, but not extracellular signal-regulated kinase (ERK) 1/2. Moreover, the invasion of HeLa cells induced by SDF-1/CXCL12 was dependent on matrix metalloproteinase-9 (MMP-9). The pre-incubation of HeLa cells with heparin or with anti-heparan sulfate antibodies or with beta-d-xyloside inhibited SDF-1/CXCL12-mediated cell invasion. Furthermore, the down-regulation of syndecan-4, a heparan sulfate proteoglycan, decreased SDF-1/CXCL12-mediated HeLa cell invasion. GAGs, probably on syndecan-4, are involved in SDF-1/CXCL12-mediated cell chemotaxis. GENERAL SIGNIFICANCE: These data suggest that targeting the glycosaminoglycan/chemokine interaction could be a new therapeutic approach for carcinomas in which SDF-1/CXCL12 is involved.

Syndecan-1 and syndecan-4 are involved in RANTES/CCL5-induced migration and invasion of human hepatoma cells.

BACKGROUND: We previously demonstrated that the CC-chemokine Regulated upon Activation, Normal T cell Expressed and Secreted (RANTES)/CCL5 exerts pro-tumoral effects on human hepatoma Huh7 cells through its G protein-coupled receptor, CCR1. Glycosaminoglycans play major roles in these biological events. METHODS: In the present study, we explored 1/ the signalling pathways underlying RANTES/CCL5-mediated hepatoma cell migration or invasion by the use of specific pharmacological inhibitors, 2/ the role of RANTES/CCL5 oligomerization in these effects by using a dimeric RANTES/CCL5, 3/ the possible involvement of two membrane heparan sulfate proteoglycans, syndecan-1 (SDC-1) and syndecan-4 (SDC-4) in RANTES/CCL5-induced cell chemotaxis and spreading by pre-incubating cells with specific antibodies or by reducing SDC-1 or -4 expression by RNA interference. RESULTS AND CONCLUSION: The present data suggest that focal adhesion kinase phosphorylation, phosphoinositide 3-kinase-, mitogen-activated protein kinase- and Rho kinase activations are involved in RANTES/CCL5 pro-tumoral effects on Huh7 cells. Interference with oligomerization of the chemokine reduced RANTES/CCL5-mediated cell chemotaxis. This study also indicates that SDC-1 and -4 may be required for HepG2, Hep3B and Huh7 human hepatoma cell migration, invasion or spreading induced by the chemokine. These results also further demonstrate the involvement of glycosaminoglycans as the glycosaminoglycan-binding deficient RANTES/CCL5 variant, in which arginine 47 was replaced by lysine, was devoid of effect. GENERAL SIGNIFICANCE: The modulation of RANTES/CCL5-mediated cellular effects by targeting the chemokine-syndecan interaction could represent a new therapeutic approach for hepatocellular carcinoma.