Risk factors associated with brachial–ankle pulse wave velocity among peritoneal dialysis patients in Macao

BACKGROUND: Cardiovascular disease is the leading cause of mortality among peritoneal dialysis (PD) patients in Macao. Increased arterial stiffness determined by pulse wave velocity (PWV) has been established as an independent predictor of cardiovascular mortality in end-stage renal disease patients. The present study aims to investigate the relationship between arterial stiffness and its associated risk factors in chronic PD patients. METHODS: A total of 96 chronic PD patients (48 males/48 females) were included in the cross-sectional study. Arterial stiffness was assessed by brachial-ankle PWV (baPWV). Patients were divided into two subgroups according to mean baPWV value. On enrollment, clinical characteristics and biochemical parameters were collected. RESULTS: Compared with low baPWV group patients, high baPWV group patients were significant older (p<0.001) and more likely to have a high proportion of female gender (p=0.004) as well as previous CVD history (p=0.008). Serum albumin, pre-albumin levels and residual renal creatinine clearance (CCr) were significantly lower but the serum ferritin level was significantly higher in high baPWV group patients than in low baPWV group patients (all p<0.01). BaPWV was positively associated with age (r=0.534, p<0.001), Charlson comorbidity index (r=0.350, p<0.001) and serum ferritin level (r=0.340, p=0.001). Meanwhile, baPWV negatively correlated with serum albumin (r=−0.479, p<0.001), pre-albumin levels (r=−0.320, p=0.003) and residual renal CCr (r=−0.177, p=0.048). Age-adjusted partial correlation test found a significant correlation between baPWV and CRP (r=0.462, p<0.001). Multivariate regression analysis showed that baPWV was independently associated with age (p<0.001), serum albumin level (p=0.015), CRP (p=0.019) and residual renal CCr (p=0.045). CONCLUSION: Arterial stiffness, assessed by baPWV, had an independent correlation with age, serum albumin level, CRP level and residual renal CCr among PD patients in Macao

Sphingosine-1-Phosphate Enhances Satellite Cell Activation in Dystrophic Muscles through a S1PR2/STAT3 Signaling Pathway

Sphingosine-1-phosphate (S1P) activates a widely expressed family of G protein-coupled receptors, serves as a muscle trophic factor and activates muscle stem cells called satellite cells (SCs) through unknown mechanisms. Here we show that muscle injury induces dynamic changes in S1P signaling and metabolism in vivo. These changes include early and profound induction of the gene encoding the S1P biosynthetic enzyme SphK1, followed by induction of the catabolic enzyme sphingosine phosphate lyase (SPL) 3 days later. These changes correlate with a transient increase in circulating S1P levels after muscle injury. We show a specific requirement for SphK1 to support efficient muscle regeneration and SC proliferation and differentiation. Mdx mice, which serve as a model for muscular dystrophy (MD), were found to be S1P-deficient and exhibited muscle SPL upregulation, suggesting that S1P catabolism is enhanced in dystrophic muscle. Pharmacological SPL inhibition increased muscle S1P levels, improved mdx muscle regeneration and enhanced SC proliferation via S1P receptor 2 (S1PR2)-dependent inhibition of Rac1, thereby activating Signal Transducer and Activator of Transcription 3 (STAT3), a central player in inflammatory signaling. STAT3 activation resulted in p21 and p27 downregulation in a S1PR2-dependent fashion in myoblasts. Our findings suggest that S1P promotes SC progression through the cell cycle by repression of cell cycle inhibitors via S1PR2/STAT3-dependent signaling and that SPL inhibition may provide a therapeutic strategy for MD

Chemerin Suppresses Breast Cancer Growth by Recruiting Immune Effector Cells Into the Tumor Microenvironment

Infiltration of immune cells into the tumor microenvironment (TME) can regulate growth and survival of neoplastic cells, impacting tumorigenesis and tumor progression. Correlations between the number of effector immune cells present in a tumor and clinical outcomes in many human tumors, including breast, have been widely described. Current immunotherapies utilizing checkpoint inhibitors or co-stimulatory molecule agonists aim to activate effector immune cells. However, tumors often lack adequate effector cell numbers within the TME, resulting in suboptimal responses to these agents. Chemerin (RARRES2) is a leukocyte chemoattractant widely expressed in many tissues and is known to recruit innate leukocytes. CMKLR1 is a chemotactic cellular receptor for chemerin and is expressed on subsets of dendritic cells, NK cells, and macrophages. We have previously shown that chemerin acts as a tumor suppressive cytokine in mouse melanoma models by recruiting innate immune defenses into the TME. Chemerin/RARRES2 is down-regulated in many tumors, including breast, compared to normal tissue counterparts. Here, using a syngeneic orthotopic EMT6 breast carcinoma model, we show that forced overexpression of chemerin by tumor cells results in significant recruitment of NK cells and T cells within the TME. While chemerin secretion by EMT6 cells did not alter their phenotypic behavior in vitro, it did significantly suppress tumor growth in vivo. To define the cellular effectors required for this anti-tumor phenotype, we depleted NK cells or CD8+ T cells and found that either cell type is required for chemerin-dependent suppression of EMT6 tumor growth. Finally, we show significantly reduced levels of RARRES2 mRNA in human breast cancer samples compared to matched normal tissues. Thus, for the first time we have shown that increasing chemerin expression within the breast carcinoma TME can suppress growth by recruitment of NK and T cells, thereby supporting this approach as a promising immunotherapeutic strategy

Atrasentan and renal events in patients with type 2 diabetes and chronic kidney disease (SONAR): a double-blind, randomised, placebo-controlled trial

Background: Short-term treatment for people with type 2 diabetes using a low dose of the selective endothelin A receptor antagonist atrasentan reduces albuminuria without causing significant sodium retention. We report the long-term effects of treatment with atrasentan on major renal outcomes. Methods: We did this double-blind, randomised, placebo-controlled trial at 689 sites in 41 countries. We enrolled adults aged 18–85 years with type 2 diabetes, estimated glomerular filtration rate (eGFR)25–75 mL/min per 1·73 m 2 of body surface area, and a urine albumin-to-creatinine ratio (UACR)of 300–5000 mg/g who had received maximum labelled or tolerated renin–angiotensin system inhibition for at least 4 weeks. Participants were given atrasentan 0·75 mg orally daily during an enrichment period before random group assignment. Those with a UACR decrease of at least 30% with no substantial fluid retention during the enrichment period (responders)were included in the double-blind treatment period. Responders were randomly assigned to receive either atrasentan 0·75 mg orally daily or placebo. All patients and investigators were masked to treatment assignment. The primary endpoint was a composite of doubling of serum creatinine (sustained for ≥30 days)or end-stage kidney disease (eGFR <15 mL/min per 1·73 m 2 sustained for ≥90 days, chronic dialysis for ≥90 days, kidney transplantation, or death from kidney failure)in the intention-to-treat population of all responders. Safety was assessed in all patients who received at least one dose of their assigned study treatment. The study is registered with ClinicalTrials.gov, number NCT01858532. Findings: Between May 17, 2013, and July 13, 2017, 11 087 patients were screened; 5117 entered the enrichment period, and 4711 completed the enrichment period. Of these, 2648 patients were responders and were randomly assigned to the atrasentan group (n=1325)or placebo group (n=1323). Median follow-up was 2·2 years (IQR 1·4–2·9). 79 (6·0%)of 1325 patients in the atrasentan group and 105 (7·9%)of 1323 in the placebo group had a primary composite renal endpoint event (hazard ratio [HR]0·65 [95% CI 0·49–0·88]; p=0·0047). Fluid retention and anaemia adverse events, which have been previously attributed to endothelin receptor antagonists, were more frequent in the atrasentan group than in the placebo group. Hospital admission for heart failure occurred in 47 (3·5%)of 1325 patients in the atrasentan group and 34 (2·6%)of 1323 patients in the placebo group (HR 1·33 [95% CI 0·85–2·07]; p=0·208). 58 (4·4%)patients in the atrasentan group and 52 (3·9%)in the placebo group died (HR 1·09 [95% CI 0·75–1·59]; p=0·65). Interpretation: Atrasentan reduced the risk of renal events in patients with diabetes and chronic kidney disease who were selected to optimise efficacy and safety. These data support a potential role for selective endothelin receptor antagonists in protecting renal function in patients with type 2 diabetes at high risk of developing end-stage kidney disease. Funding: AbbVie

S1P activation of STAT3 is mediated via an S1PR2/RhoGTPase pathway.

<p>SC-derived myoblasts were treated with indicated dose of: A) Rac1 inhibitor, or B) RhoA inhibitor for 24 hours in complete media, and cell lysates were immunoblotted with indicated antibodies. Levels of total STAT3 (STAT3-T) and phosphorylated STAT3 (STAT3-P) were measured and compared to actin loading control. C) SC-derived myoblasts were treated with 10 µM S1PR2 antagonist JTE-013 (R2i) for 20 hours, cells were incubated in fresh media for 5 minutes with or without R2i, and activated Rac1 was pulled down with GST-PAK1 fusion protein and immunoblotted with Rac1 antibody. STAT3 activation (STAT3-P) also shown for this experiment. D) Myoblasts were transfected with GFP, dominant negative Rac1 (Rac1 DN) or wild type RhoA (RhoA WT) plasmid constructs. Forty-eight hours later, cell lysates were immunoblotted to detect total and phosphorylated STAT3, GFP, Rac1 and RhoA, with GAPDH used as a loading control. E) Representative micrographs (10×) of H&E stained 10 µm frozen sections taken from mdx gastrocnemius muscles 5 days post NTX-injury. Groups were treated with vehicle, THI, STAT3 inhibitor WP1066, or THI+WP1066. F) Quantification of regenerating myofibers/mm² (n = 3/group). Scale bar = 50 microns. G) Analysis of SCs by flow cytometry as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037218#pone-0037218-g005" target="_blank"><b>Figure 5F</b></a>. Data are means ± SD, n = 5/group, * indicates p≤0.05.</p

Function of SCs isolated from sphingolipid mutant mice.

<p>A) Proliferation of WT and SphK1 KO primary SCs isolated 3 days after NTX injury, measured by BrdU uptake of desmin-positive cells incubated with either SphK1 WT or KO serum. B) Representative immunofluorescence images for experiment in “A”. Desmin-stained cells, green; BrdU-stained nuclei, orange. C) Differentiation of SC-derived myoblasts as shown by myotube formation. D) Representative immunofluorescence images for experiment in “C”. Hoechst-stained nuclei, blue; eMHC-stained fibers, green. E) Proliferation of SPL heterozygous null (+/−) and littermate control (+/+) mouse primary SC isolated 3 days after injury. F) Representative images for experiment in “E”. Desmin-stained cells, green; BrdU-stained nuclei, orange. G) Differentiation of heterozygous null (+/−) and littermate control (+/+) mouse SC. H) Representative immunofluorescence images for experiment in “G”. Hoechst-stained nuclei, blue; eMHC-stained fibers, green. * indicates p≤0.05.</p

STAT3 and S1PR2 signaling interactions in muscle regeneration of WT and mdx mice.

<p>A) Mdx mice received NTX with or without THI treatment. Muscles were harvested on day 5 post-injury, and whole muscle extracts were examined for total STAT3 (STAT3-T) and phosphorylated STAT3 (STAT3-P) protein levels. Actin is used as a loading control. B) STAT3 activation in SC-derived myoblasts treated <i>in vitro</i> with 10 µM S1PR1 (R1i), S1PR2 (R2i), and S1PR3 (R3i) antagonists. S1PR1 is used as a loading control, as its expression is invariant under these conditions. C) WT SC-derived primary myoblasts were treated with S1PR2 antagonist (JTE-013) or vehicle, and whole cell extracts were examined for STAT3 phosphorylation, total STAT3 and p21 and p27 protein levels. Actin is used as a loading control. D) SC-derived primary myoblasts were treated with either 50 nM siRNA against S1PR2 or mock transfection. S1PR2 knockdown was confirmed by qRT-PCR. E) Whole cell extracts of control and S1PR2 knockdown cells were examined for phosphorylated STAT3 and actin protein levels by immunoblotting. p = 0.03 (siRNA vs. mock) for relative densitometric levels of STAT3-P/actin from two experiments performed in duplicates. F) WT mice were treated with NTX plus antagonist of S1PR2 (JTE-013) or vehicle by subcutaneous injection. Muscles were harvested 5 days post injury; regenerating fibers were imaged, and G) regenerating fibers were quantified. H) Flow cytometry analysis of SCs from WT mice treated with JTE-013 or vehicle at 5 days post injury; * indicates p≤0.05, data are means ± SD, n = 5/group.</p

SPL inhibition improves muscle regeneration and SC recruitment in MD.

<p>A) Treatment of WT mice with THI for 2 days (2D) or 5 days (5D) results in elevated muscle S1P levels compared to vehicle-treated controls at 5 days post injury (5DPI); * indicates p≤0.05. Data are means ± SD, n = 4–5/group. B) Mdx mice received NTX+THI or vehicle daily on days −1 to 5. On day 5, mice were euthanized and gastrocnemius muscles were harvested. Ten µm tissue cryosections were obtained every 200 µm in sets of 4. H&E stained sections were examined for point of maximal injury. Representative photographs are shown. Scale bar = 50 microns. Recovering WT control and resting mdx muscles are shown for comparison. C) Fiber quantification of experiment in “B”. Centrally nucleated myofibers were counted in a field containing only regenerating fibers and normalized to 1 mm² area. * p≤0.05. D) Quantification of SC <i>in situ</i> in mdx mouse muscles identified by M-cadherin or Pax-7 expression and localization beneath the basal lamina. * indicates p≤0.05. E) Flow cytometry analysis of freshly isolated muscle cells from mdx mice treated with THI or vehicle at 5 days post injury. SCs were identified as CD45-, CD31-, Sca1-, integrin alpha-7+ and CD34+ cells; * indicates p≤0.05, data are means ± SD, n  =  5/group.</p