Synergy between Sphingosine 1-Phosphate and Lipopolysaccharide Signaling Promotes an Inflammatory, Angiogenic and Osteogenic Response in Human Aortic Valve Interstitial Cells

<div><p>Given that the bioactive lipid sphingosine 1-phosphate is involved in cardiovascular pathophysiology, and since lipid accumulation and inflammation are hallmarks of calcific aortic stenosis, the role of sphingosine 1-phosphate on the pro-inflammatory/pro-osteogenic pathways in human interstitial cells from aortic and pulmonary valves was investigated. Real-time PCR showed sphingosine 1-phosphate receptor expression in aortic valve interstitial cells. Exposure of cells to sphingosine 1-phosphate induced pro-inflammatory responses characterized by interleukin-6, interleukin-8, and cyclooxygenase-2 up-regulations, as observed by ELISA and Western blot. Strikingly, cell treatment with sphingosine 1-phosphate plus lipopolysaccharide resulted in the synergistic induction of cyclooxygenase-2, and intercellular adhesion molecule 1, as well as the secretion of prostaglandin E₂, the soluble form of the intercellular adhesion molecule 1, and the pro-angiogenic factor vascular endothelial growth factor-A. Remarkably, the synergistic effect was significantly higher in aortic valve interstitial cells from stenotic than control valves, and was drastically lower in cells from pulmonary valves, which rarely undergo stenosis. siRNA and pharmacological analysis revealed the involvement of sphingosine 1-phosphate receptors 1/3 and Toll-like receptor-4, and downstream signaling through p38/MAPK, protein kinase C, and NF-κB. As regards pro-osteogenic pathways, sphingosine 1-phosphate induced calcium deposition and the expression of the calcification markers bone morphogenetic protein-2 and alkaline phosphatase, and enhanced the effect of lipopolysaccharide, an effect that was partially blocked by inhibition of sphingosine 1-phosphate receptors 3/2 signaling. In conclusion, the interplay between sphingosine 1-phosphate receptors and Toll-like receptor 4 signaling leads to a cooperative up-regulation of inflammatory, angiogenic, and osteogenic pathways in aortic valve interstitial cells that seems relevant to the pathogenesis of aortic stenosis and may allow the inception of new therapeutic approaches.</p></div

S1P induces pro-inflammatory molecules in AVICs.

<p>A) S1P receptor expression in control and stenotic AVICs (mean ± SEM of the relative mRNA levels normalized to β-actin, n = 7–10), was assessed by quantitative RT-PCR. B–C) 1 µM S1P induced cytokine secretion, as observed in antibody arrays and ELISA assays (representative of n = 4, 12 h). Squares indicate positive controls; arrows, constitutive cytokines; ovals, S1P-induced cytokines. D–E) Comparison of S1P-mediated induction of COX-2 expression (mean ± SEM, n = 8–12, 1 µM S1P) and PGE₂ secretion (mean ± SEM, n = 4–5). ELISA data, expressed as pg, were normalized to the cell protein content (mg). *<i>p</i><0.05. White bars indicate control AVIC; black bars, stenotic AVIC. Gro indicates growth-regulated oncogene α, β, and γ; MCP-1, monocyte chemotactic protein-1; R, resting.</p

S1P cooperates with LPS to up-regulate pro-inflammatory molecules.

<p>A) Representative immunoblots show the kinetics of COX-2 and ICAM-1 induction upon activation in control AVIC (n = 8). B–C) Dose-dependent effect (n = 3, 12 h). D) Immunoblots with densitometry data normalized to β-tubulin levels (mean ± SEM, n = 10 pairs of control-stenotic AVICs activated for 12 h and processed in the same blot) revealed a stronger synergistic effect in stenotic than in control AVICs. E) Representative immunoblots of at least 5 experiments show no cooperative effect between S1P and Pam₃CSK4 in stenotic AVICs. F) Immunoblots with densitometry data (mean ± SEM, n = 8 pairs of AVIC-PVIC from the same heart processed in the same blot), demonstrate a higher effect in AVIC than in PVIC. White bars indicate control AVIC; black bars, stenotic AVIC; gray bars, PVIC. L indicates 1 µg/ml LPS; Pam/P, 100 ng/ml Pam₃CSK4; S, 1 µM S1P. #<i>p</i><0.05 for S1P+LPS vs. S1P and LPS;*<i>p</i><0.05 for the indicated pair.</p

Signaling routes implicated in the cooperative effect.

<p>A–B) AVIC were pre-treated with the indicated drugs, activated for 12 h, and analyzed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109081#pone-0109081-g004" target="_blank">Figure 4</a>. Representative immunoblots of AVIC lysates and densitometry data show inhibition of the cooperative effect on COX-2 and ICAM-1 (100% value) by NF-κB-SN50 and MAPK inhibitors (n = 8). C) Supernatants were analyzed for sICAM-1 as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109081#pone-0109081-g003" target="_blank">Figure 3</a> (n = 6). GF indicates 300 nM GF109203X; PD, 50 µM PD98059; SB, 10 µM SB203580; S+L, S1P+LPS; SN50; 50 µg/mL NF-κB SN50; SP, 10 µM SP600125. *<i>p</i><0.05 vs. S1P+LPS (100% value).</p

Several signaling cascades, mainly p38/MAPK, are involved in the cooperative effect.

<p>A–B) Cell lysates from activated stenotic AVIC were analyzed for the early phosphorylation of NF-κB and MAP kinases. Representative immunoblots and densitometry data show synergistic activation of p38 by S1P+LPS (n = 6). Sample at t = 0 was run in both gels for comparison purposes. C) Representative immunoblots of p38 phosphorylation in AVIC and PVIC from the same patient processed in parallel are shown. D) Densitometry data is expressed as the fold induction of p-p38 relative to resting values (t = 0) using data previously normalized to the reference gene, β-tubulin (mean ± SEM, n = 5–7). Color bars, as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109081#pone-0109081-g002" target="_blank">Figure 2</a>. *<i>p</i><0.05; #<i>p</i><0.05 for S1P+LPS vs. LPS and S1P (at the same time point).</p

Receptors involved in the cooperative effect.

<p>AVICs were pre-treated with the indicated drugs, activated for 12 h, and cell lysates and supernatants were analyzed by Western blot and ELISA, respectively. A) Representative immunoblots with densitometry data demonstrate inhibition of the cooperative effect on COX-2 and ICAM-1 expression (100% value) using S1P_1/3 antagonists (n = 6–12). B) Silencing S1P_1/3 attenuated the cooperative effect (n = 3 control AVIC). Scramble, siRNA control; vehicle, 0.1% DEPC. C) Immunoblots showed cooperation between FTY720 and LPS (n = 3). D) ELISA quantification of sICAM-1 levels show inhibition by S1P_1/3 antagonists (n = 6–10). E) Immunoblots demonstrate inhibition of the cooperative effect by TLR4 antagonists (n = 3). Cay indicates 5 µM CAY10614; CLI, 3 µM CLI-095; FTY, 1 µM FTY720; JTE, 10 µM JTE-013; S+L, S1P+LPS; PTX, 100 ng/ml <i>pertussis</i> toxin; R, resting; Sur, 10 µM suramin; W146, 10 µM W146. *<i>p</i><0.05 vs. S1P+LPS.</p

S1P cooperates with LPS to up-regulate pro-osteogenic markers and calcium deposition.

<p>A) AVIC and PVIC from the same patient were activated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109081#pone-0109081-g002" target="_blank">Figure 2</a> for 48 h and analyzed with a BMP-2 antibody. Immunoblots and densitometry data are representative of at least 3 pairs of AVIC-PVIC processed in the same blot. Data are expressed as fold induction of BMP2, normalized to β-tubulin, as compared to resting conditions (mean ± SEM, n = 6 pairs of AVIC-PVIC). B–G) AVIC and PVIC from the same patient were treated with conditioning media (CM) in the presence of 1 µg/ml LPS, 0.1 µM S1P or vehicle as indicated in Methods. B) Calcium deposition expressed as µg Ca²⁺/mg protein is shown (mean ± SEM, n = 3 pairs of AVIC and PVIC with a total of 11 replicates). C) ALP blue staining of control AVIC (n = 3). D) Representative ALP data is expressed as nmol/mg.h. E) Data from control AVIC and PVIC are expressed as fold increase of ALP activity (mean ± SEM, n = 12 AVIC, n = 8 PVIC) relative to data with growth media. F–G). ALP activity in control AVIC pre-treated with the indicated drugs before activation (n = 5–9) Drug concentrations were as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109081#pone-0109081-g004" target="_blank">Figures 4</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109081#pone-0109081-g005" target="_blank">5</a>. M indicates M199, growth media; white bars, AVIC; gray bars, PVIC. *<i>p</i><0.05; #<i>p</i><0.05 for S1P+LPS vs. LPS and S1P.</p

S1P cooperates with LPS to induce the secretion of pro-inflammatory and pro-angiogenic molecules.

<p>Supernatants from cells treated with the indicated ligands as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109081#pone-0109081-g002" target="_blank">Figure 2</a> were analyzed by ELISA. Data are expressed as pg/mg cell protein (mean ± SEM). A) Kinetics of PGE₂ secretion in control and stenotic AVIC, n = 4–5. B) PGE₂ secretion data from A at 12 h, mean ± SEM, n = 4–5. C) IL-6 secretion data at 12 h, representative of 4 independent experiments. D) VEGF secretion data at 12 h, mean ± SEM, n = 6. E) sICAM-1 secretion data, mean ± SEM, n = 5–10.) Abbreviations were as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109081#pone-0109081-g002" target="_blank">Figure 2</a>; color bars, as indicated in the corresponding panel. *<i>p</i><0.05; #<i>p</i><0.05 for S1P+LPS vs. LPS and S1P.</p

Effect of erythrodiol and uvaol on the fibrotic effect of angiotensin II in cardiac myofibroblasts.

<p>Time course of angiotensin II (Ang II; 1 µM)-stimulated collagen I protein production (A). Effect of erythrodiol (ERY; 5 µM) or uvaol (UVA; 5 µM) on CTGF (B), collagen I (C) and galectin 3 (D) protein expression in angiotensin II-treated cardiac myofibroblasts for 12 hours. Representative immunoblots of 4 experiments. Values are mean ± SEM of four assays. *p<0.05 <i>vs</i> vehicle. †p<0.05 vs angiotensin II. Quantification of band intensities was measured by densitometry and normalized to respective α-tubulin.</p

Representative immunocytochemistry images of cardiac myofibroblasts examined by fluorescence microscopy.

<p>Vimentin staining (A), α-smooth muscle actin (α-SMA) staining (B) and vimentin and α-SMA staining merged (C). Magnification 40X. Scale bar: 50 µm.</p