Activation of MMP-9 and migration are inhibited in <i>Vnn1</i><i>^−/−</i><b>SMCs. </b>

<p><b><i>A</i></b><i>,</i> Serum-starved SMCs were treated with PDGF (10 ng/ml), cysteamine (500 ng/ml) or diamide (5 µM) for 48 h, and conditioned media analyzed for MMP-9 activity by gelatin zymography. <b><i>B</i></b><i>,</i> Densitometric analysis of enhanced MMP-9 activity in WT compared to <i>Vnn1^−/−</i> SMCs. Data pooled from 3 independent experiments for densitometry. <b><i>C</i></b><i>,</i> We assayed migration in SMCs treated with diamide (5 µM) or PDGF (10 ng/ml), as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039106#s4" target="_blank">Methods</a>. Total number of SMCs migrated/well after 48 h treatment with diamide or PDGF are shown. Data are mean ± SD of 3 independent experiments. *P<0.05 vs. control.</p

Relationships between PPARγ expression, GSH content, and vanin-1 in arteries and cultured SMCs.

<p><i>A–B</i><i>,</i> We treated cultured aortic SMCs with diamide (5 µM) and/or PDGF (10 ng/ml) for 48 h and PPARγ expression was analyzed by Western blot, and densitometry. P<0.05 for ^#<i>Vnn1^−/−</i> control vs. WT control SMCs; *<i>Vnn1^−/−</i> control vs. diamide treated SMCs; **WT SMCs vs. PDGF and diamide treated SMCs, respectively. <b><i>C–D</i></b><i>,</i> SMCs were transfected with PPARγ siRNA, and then PPARγ expression analyzed by Western blot. *P<0.05 for <i>Vnn1^−/−</i> vs. WT SMCs, # <i>Vnn1^−/−</i> control vs. PPARγ siRNA, **PPARγ siRNA WT SMCs. <b><i>E</i></b><i>,</i> WT and <i>Vnn1^−/−</i> SMCs were treated with cysteamine (500 ng/ml) or BSO (1 µM), and GSH content determined after deproteinization<b>.</b> Data pooled from 3 experiments done in triplicate. ^# P<0.05 <i>Vnn1^−/−</i> vs. WT SMCs, *control vs. PPARγ siRNA,**control vs. cysteamine in both <i>Vnn1^−/−</i> and WT SMCs, ^Λ control vs. BSO treatment in both <i>Vnn1^−/−</i> and WT SMCs. In Panel <b><i>F</i></b>, WT and <i>Vnn1^−/−</i> SMCs were treated with diamide (5 µM) or PDGF 10 ng/ml) for 24 h, and cell proliferation compared. *P<0.05 vs. control. <b><i>G</i></b><i>,</i> SMC proliferation using PPARγ siRNA knockdown. Data are mean ± SD of 3 independent experiments. *P<0.05 control vs. PPARγ siRNA, **PPARγ siRNA vs. PPARγ siRNA + PDGF in WT and <i>Vnn1^−/−</i> SMCs.</p

Constitutive vanin-1 expression in aorta and pantetheinase activity in cultured mouse aortic SMCs.

<p><i>A</i><i>,</i> Histologic sections of aortae from WT and <i>Vnn1^−/−</i> mice were immunohistochemically stained for vanin-1 (brown positive staining). <b><i>B</i></b>, Vanin-1 analyzed by aortic tissue Western blotting. <b><i>C</i></b><i>,</i> In isolated aortic SMCs, vanin-1 and vanin-3 isoenzyme mRNA expression levels were compared by real-time PCR, normalized to GAPDH mRNA in samples from WT and <i>Vnn1^−/−</i> mice. *P<0.05, WT vs. <i>Vnn1^−/−</i> control. <b><i>D</i></b><i>,</i> Aortic sections were incubated with the substrate pantothenate–AMC and constitutive vanin-1 immunofluorescence (green) is shown in carotid sections of WT and <i>Vnn1^−/−</i> mice. <b><i>E</i></b><i>,</i> Pantetheinase activity is shown from cultured SMC lysates of mice of indicated genotypes; results from SMCs of different animals are shown as distinct plots, with each result the mean ± SD of 3 independent experiments. *P<0.05 for WT vs. <i>Vnn1^−/−.</i></p

Immunohistochemical staining for PPARγ and the cell proliferation marker Ki-67 in uninjured and injured carotid arteries.

<p><i>A</i><i>,</i> Paraffin-embedded, uninjured and injured aortae of the indicated genotypes were cut into 6 µm sections for immunohistochemistry, with PPARγ positive media and neointimal cells staining dark brown (arrowheads) in methyl green counterstained sections. <b><i>B</i></b><i>,</i> Percentage of arterial media and neointimal PPARγ positive cells, determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039106#s4" target="_blank">Methods</a>. <b><i>C</i></b><i>,</i> Immunohistochemical detection of Ki-67 in representative WT and <i>Vnn1^−/−</i> carotid sections. <b><i>D</i></b><i>,</i> Percentage of arterial media and neointimal Ki-67 positive cells. Data are mean ± SD of 3 independent experiments. *P<0.05 WT vs. <i>Vnn1^−/−</i>.</p

Vanin-1 mediates SMC proliferation in response to PDGF.

<p>Vanin-1 was expressed in <i>Vnn1^−/−</i> SMCs by transfection using pCMV-VNN1 expression vector. <b><i>A</i></b><i>,</i> Pantetheinase activity was measured in <i>Vnn1^−/−</i> SMCs after transfection and <b><i>B</i></b><i>,</i> Western blot analysis confirmed vanin-1 expression after transfection. <b><i>C</i></b><i>,</i> Following pCMV-VNN1 or empty vector transfection, SMCs were treated with PDGF (10 ng/ml) for 24 h, and proliferation measured. Data are mean ± SD of 3 independent experiments. *P<0.05 <i>Vnn1^−/−</i> SMCs control vs. <i>Vnn1^−/−</i> + pCMV-VNN1+ PDGF.</p

Vanin-1 and cysteamine modulate SMC superoxide production and GSH content in response to diamide and PDGF.

<p><i>A</i>, SMCs isolated from <i>Vnn1^−/−</i> and WT mouse aortae were growth-arrested in 0.1% calf serum for 24 h, and exposed to dihydroethidium (DHE) (10 µM). Images were captured 30 min after stimulation with diamide (5 µM) and PDGF (10 ng/ml). <b><i>B</i></b><i>,</i> Superoxide production was quantified by flow cytometry (excitation and emission wavelengths 488 nm and 610 nm, respectively). <b><i>C</i></b><i>,</i> WT and <i>Vnn1^−/−</i> SMCs were treated with PDGF (10 ng/ml) for 24 h, and pantetheinase activity measured (*WT+PDGF vs. <i>Vnn1^−/−</i> +PDGFand WT control). <b><i>D</i></b><i>,</i> In SMCs treated with cysteamine (500 ng/ml) or BSO (1 µM) for 48 h, DHE fluorescence was measured. <b><i>E</i></b><i>,</i> WT and <i>Vnn1^−/−</i> SMCs were treated with PDGF (10 ng/ml) for 24 h, and GSH content measured via enzymatic recycling assay. Data are mean ± SD of 3 independent experiments. *P<0.05 vs. control; **control vs. PDGF in WT, # control vs. diamide in WT and <i>Vnn1^−/−</i>. <b><i>F</i></b><i>,</i> SMC superoxide production in response to cysteamine and BSO treatment was quantified by flow cytometry (excitation and emission wavelengths 488 nm and 610 nm, respectively). Data are mean ± SD of 3 independent experiments.</p

Effect of intensive urate lowering with combined verinurad and febuxostat on albuminuria in patients with type 2 diabetes: A randomized trial

Rationale & Objective: Hyperuricemia has been implicated in the development and progression of chronic kidney disease. Verinurad is a novel, potent, specific urate reabsorption inhibitor. We evaluated the effects on albuminuria of intensive urate-lowering therapy with verinurad combined with the xanthine oxidase inhibitor febuxostat in patients with hyperuricemia and type 2 diabetes mellitus (T2DM). Study Design: Phase 2, multicenter, prospective, randomized, double-blind, parallel-group, placebo-controlled trial. Setting & Participants: Patients 18 years or older with hyperuricemia, albuminuria, and T2DM. Intervention: Patients randomly assigned 1:1 to verinurad (9 mg) plus febuxostat (80 mg) or matched placebo once daily for 24 weeks. Outcomes: The primary end point was change in urinary albumin-creatinine ratio (UACR) from baseline after 12 weeks’ treatment. Secondary end points included safety and tolerability and effect on glomerular filtration

Synthesis, Detection, and Metabolism of Pyridone Ribosides, Products of NAD Overoxidation

Pyridone-containing adenine dinucleotides, ox-NAD, are formed by overoxidation of nicotinamide adenine dinucleotide (NAD+) and exist in three distinct isomeric forms. Like the canonical nucleosides, the corresponding pyridone-containing nucleosides (PYR) are chemically stable, biochemically versatile, and easily converted to nucleotides, di- and triphosphates, and dinucleotides. The 4-PYR isomer is often reported with its abundance increasing with the progression of metabolic diseases, age, cancer, and oxidative stress. Yet, the pyridone-derived nucleotides are largely under-represented in the literature. Here, we report the efficient synthesis of the series of ox-NAD and pyridone nucleotides and measure the abundance of ox-NAD in biological specimens using liquid chromatography coupled with mass spectrometry (LC-MS). Overall, we demonstrate that all three forms of PYR and ox-NAD are found in biospecimens at concentrations ranging from nanomolar to midmicromolar and that their presence affects the measurements of NAD(H) concentrations when standard biochemical redox-based assays are applied. Furthermore, we used liver extracts and 1H NMR spectrometry to demonstrate that each ox-NAD isomer can be metabolized to its respective PYR isomer. Together, these results suggest a need for a better understanding of ox-NAD in the context of human physiology since these species are endogenous mimics of NAD+, the key redox cofactor in metabolism and bioenergetics maintenance

Additional file 2: Table S2. of Sex differences in gout characteristics: tailoring care for women and men

Dosing of urate-lowering therapies among eligible patients. (DOCX 12 kb

Additional file 3: Figure S1. of Sex differences in gout characteristics: tailoring care for women and men

Comparison of the dietary servings among women and men with gout. The p values represent adjusted analyses adjusting for age, BMI, duration of gout, comorbidity burden [hypertension, diabetes, renal disease, hyperlipidemia], HCTZ use, other diuretic use and current use of a urate-lowering drug. (TIF 320 kb