Sildenafil Counteracts the In Vitro Activation of CXCL-9, CXCL-10 and CXCL-11/CXCR3 Axis Induced by Reactive Oxygen Species in Scleroderma Fibroblasts

Abstract

Oxidative stress plays a key role in systemic sclerosis (SSc) pathogenesis, and an altered redox homeostasis might be responsible for abnormal inflammatory status, fibrosis and tissue damage extension. In this study, we explored the effect of the phosphodiesterase type 5 inhibitor sildenafil in modulating the activation of the CXCL-9, -10, -11/CXCR3 axis, which is fundamental in the perpetuation of inflammation in different autoimmune diseases, in the cell culture of SSc human dermal fibroblasts exposed to a pro-oxidant environment. We observed that sildenafil significantly reduced gene expression and release of CXCL-9, -10 and -11, inhibited the CXCR3 action and suppressed the activation of STAT1-, JNK- and p38MAPK pathways. This in vitro study on dermal fibroblasts supports clinical studies to consider the efficacy of sildenafil in preventing tissue damage and fibrosis in SSc by targeting central biomarkers of disease progression, vascular injuries and fibrosis and reducing the pro-inflammatory activation induced by oxidative stress.

Keywords: chemokines; fibrosis; reactive oxygen species; sildenafil; systemic sclerosis.

Figure 1

Supernatants from human healthy (black columns) and SSc (grey columns) fibroblasts cultures exposed to H₂O₂ (100 μM, 24 h) in the presence or absence of sildenafil (1 μM). Cells were analyzed for CXCL-9 (A), CXCL-10 (B) and CXCL-11 (C) content. Data are presented as the fold increase vs. control taken as 1 ± SEM (n = 3). Statistical significance was determined using ANOVA with Bonferroni’s post-hoc test. * p < 0.05 and ** p < 0.01 vs. relative control within group; ^# p < 0.05 and ^## p < 0.01 vs. H₂O_2; ^§ p < 0.05 vs. corresponding treatment between groups; c, control group; S, sildenafil. Experiments were repeated at least three times (n = 3) with essentially identical results.

Figure 2

CXCL-9 (A), CXCL-10 (B) and CXCL-11 (C) mRNA of SSc fibroblasts cultures exposed to H₂O₂ (100 μM, 24 h) in the presence or absence of sildenafil (1 μM). Data are shown as the fold increase vs. H₂O₂ taken as 100% ± SEM (n = 3). Statistical significance was determined using the Mann–Whitney t-test. ^§ p < 0.05 of H₂O₂ vs. H₂O₂ + S. S, sildenafil. Experiments were repeated at least three times (n = 3) with essentially identical results.

Figure 3

Representative Western blot images of proteins analyzed in SSc fibroblasts. Proteins extracted from SSc fibroblasts exposed to H₂O₂ (100 μM) with or without sildenafil (1 μM) were immunoblotted with antibodies against the total and phosphorylated form of STAT1 (A), JNK (B), P38MAPK (C) and CRYAB (D). Bars of the histograms show the ratio between the phosphorylated and total forms of the protein targets. Statistical significance was determined using ANOVA with Bonferroni’s post-hoc test. * p < 0.05 vs. relative control within group; ^§ p < 0.05. S, sildenafil. Experiments were repeated at least three times (n = 3) with essentially identical results.

Figure 4

(A) Healthy (H) (upper panels) and SSc (lower panels) fibroblasts were exposed to H₂O₂ (100 μM, 1 h) in the presence or absence of sildenafil (1 μM) pre-treatment. Fixed non-permeabilized cells were stained for CXCR3 antibody (red) and DAPI for nuclei (blue). (B) Histograms represent the fluorescence intensities of healthy (red columns) and SSc (pink columns) fibroblasts for CXCR3 in the plasma membrane. Statistical significance was determined using ANOVA with Bonferroni’s post-hoc test. * p < 0.05 and ** p < 0.01 vs. relative control within group; ^# p < 0.05 vs. H₂O_2; ^§ p < 0.05 and ^§§ p < 0.01 vs. corresponding treatment between groups; c, control group; S, sildenafil. Experiments were repeated at least three times (n = 3) with essentially identical results.

Figure 5

Suggested mechanism of action of sildenafil on SSc fibroblasts. After a pro-oxidant stimulus, the cell activates intracellular pathways that rapidly induce both the translocation of the CXCR3 receptor on the plasma membrane and the phosphorylation of downstream signaling molecules such as STAT1, JNK and p38MAPK. These proteins adapt the DNA response inducing the expression and release of the pro-inflammatory chemokines CXCL-9, CXCL-10 and CXCL-11. The binding of these chemokines to the specific receptor CXCR3 on the plasma membrane increases and amplifies the pro-inflammatory response towards autocrine/paracrine action and recruits CXCR3+ immune cells to tissue damage. The action of sildenafil results in an impairment of CXCR3 recruitment on the plasma membrane and in the counteraction of the transduction pathway responsible for chemokines expression and release. This can determine the reduction of the inflammatory activation loop at the site of damage.