Statins Inhibit Angiotensin II/Smad Pathway and Related Vascular Fibrosis, by a TGF-β-Independent Process

We have recently described that in an experimental model of atherosclerosis and in vascular smooth muscle cells (VSMCs) statins increased the activation of the Smad pathway by transforming growth factor-β (TGF-β), leading to an increase in TGF-β-dependent matrix accumulation and plaque stabilization. Angiotensin II (AngII) activates the Smad pathway and contributes to vascular fibrosis, although the in vivo contribution of TGF-β has not been completely elucidated. Our aim was to further investigate the mechanisms involved in AngII-induced Smad activation in the vasculature, and to clarify the beneficial effects of statins on AngII-induced vascular fibrosis. Infusion of AngII into rats for 3 days activates the Smad pathway and increases fibrotic-related factors, independently of TGF-β, in rat aorta. Treatment with atorvastatin or simvastatin inhibited AngII-induced Smad activation and related-fibrosis. In cultured rat VSMCs, direct AngII/Smad pathway activation was mediated by p38 MAPK and ROCK activation. Preincubation of VSMCs with statins inhibited AngII-induced Smad activation at all time points studied (from 20 minutes to 24 hours). All these data show that statins inhibited several AngII-activated intracellular signaling systems, including p38-MAPK and ROCK, which regulates the AngII/Smad pathway and related profibrotic factors and matrix proteins, independently of TGF-β responses. The inhibitory effect of statins on the AngII/Smad pathway could explain, at least in part, their beneficial effects on hypertension-induced vascular damage

Influence of dialysis membrane composition on plasma bisphenol A levels during online hemodiafiltration.

Bisphenol A (BPA) is an ubiquitous environmental toxin that is also found in dialyzers. Online hemodiafiltration (OL-HDF) more efficiently clears high molecular weight molecules, and this may improve BPA clearance. However, the BPA contents of dialysis membranes may be a source of BPA loading during OL-HDF.A prospective study assessed plasma BPA levels in OL-HDF patients using BPA-free (polynephron) or BPA-containing (polysulfone) dialyzers in a crossover design with two arms, after a run-in OL-HDF period of at least 6 months with the same membrane: 31 patients on polynephron at baseline were switched to polysulfone membranes for 3 months (polynephron-to-polysulfone) and 29 patients on polysulfone were switched to polynephron for 3 months (polysulfone-to-polynephron).After a run-in OL-HDF period of at least 6 months with the same membrane, baseline pre-dialysis BPA was lower in patients on polynephron (8.79±7.97 ng/ml) than in those on polysulfone (23.42±20.38 ng/mL, p<0.01), but still higher than in healthy controls (<2 ng/mL). After 3 months of polynephron-to-polysulfone switch, BPA was unchanged (8.98±7.88 to 11.14±15.98 ng/mL, ns) while it decreased on the polysulfone-to-polynephron group (23.42±20.38 to 11.41±12.38 ng/mL, p<0.01).OL-HDF for 3 months with BPA-free dialyzer membranes was associated to a significant decrease in predialysis BPA levels when compared to baseline BPA levels while on a BPA-containing membrane

Statins inhibit the AngII/Smad pathway in cultured vascular smooth muscle cells.

<p>Cells were pretreated with simvastatin (SV) for 48 hours before stimulation with AngII for 20 minutes (A, C) or 24 hours (B). Figures A and B show a representative Western blot and data as mean±SEM of 3–6 independent experiments. *p<0.05 vs. control, #p<0.05 vs. AngII. Figure C shows a representative confocal microscopy experiment of 3 independent observations.</p

TGF-β is not upregulated in the aorta of AngII-infused rats.

<p>Rats were infused with AngII (100 ng/kg per minute, subcutaneously) for 3 days. Some animals were also treated with simvastatin or atorvastatin (5 mg/Kg/day), starting 48 hours before AngII infusion. <b>A.</b> RNA was isolated from frozen samples of rat aorta and gene expression was evaluated by real time and expressed as mean±SEM of 10 animals per group. <b>B.</b> TGF-β protein levels were measured in aortic protein extracts by Elisa. Data are expressed as mean±SEM of 10 animals per group.</p

p38 MAPK and ROCK inhibition disminishes AngII mediated Smad2 phosphorylation in VSMCs.

<p>Cells were preincubated for 1 hour with the inhibitors before treatment with AngII (A) or TGF-β (B) for 20 minutes. Figure shows a representative Western blot and data as mean±SEM of 5–6 independent experiments. *p<0.05 vs control; # p<0.05 vs AngII.</p

Infusion of AngII for 3 days increases ECM-related proteins in rat aorta: Inhibitory effect of statins.

<p>Gene expression of profibrotic factors CTGF and PAI-1 (A) or ECM proteins, fibronectin and type I procollagen (B) was evaluated by real time PCR. Figure C and D show a representative Western blot and data of aortic fibronectin and type 1 collagen protein levels, respectively. Data are expressed as mean±SEM of 10 animals per group, *p<0.05 vs control; # p<0.05 vs AngII.</p

Plasma BPA concentration in patients on OL-HDF with polynephron (PN) or polysulfone (PS) membranes.

<p>Pre- and post-dialysis measurements are shown for the first (baseline) and the last (3-month) session after the switch. Prior to the baseline pre-dialysis sample, patients had been on OL-HDF with the opposite membrane for >6 months. Thus, baseline pre-dialysis values represent values corresponding to >6 months OL-HDF with the opposite membrane and were used as baseline values for the switch study, while baseline post-dialysis values were already obtained after the first session with the switch membrane. Data expressed as mean±SD. ** p<0.01. Pre- and Post- refers to pre-dialysis and post-dialysis (OL-HDF) session.</p

Plasma BPA concentration.

<p>Healthy subjects (n = 10); patients on online hemodiafiltration (HDF) (n = 58) and patients on conventional hemodialysis (HD) who were later enrolled in the present study (n = 18) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193288#pone.0193288.ref007" target="_blank">7</a>]. Data expressed as mean±SD. For HD and OL-HDF, values correspond to baseline predialysis values in the switch studies. At this point, patients had been on HD or OL-HDF for >6 months. * p<0.05; ** p = 0.017.</p

Study design: Timing of plasma sampling for BPA assessment.

<p>Baseline pre-dialysis plasma sampling took place after >6 months OL-HDF and prior to the first session with the switch dialyzer. Baseline post-dialysis sampling was obtained immediately after the first session with the switch dialyzer. Sampling was repeated pre- and post-dialysis after 3 months on the switch dialyzer.</p

Summary of clinical and biochemical variables.

<p>Summary of clinical and biochemical variables.</p