Sildenafil, a Phosphodiesterase Type 5 Inhibitor, Downregulates Osteopontin in Human Peripheral Blood Mononuclear Cells

Abstract

The aim of this study was to investigate the ability of sildenafil to regulate osteopontin (OPN) gene and protein in peripheral blood mononuclear cells (PBMCs) from healthy blood donors. OPN is expressed by a wide variety of cell types, including immune cells. OPN functions are linked to various physiological and pathological conditions. Sildenafil is a selective inhibitor of type 5 phosphodiesterase. Sildenafil has recently been found to have immunomodulatory effects in animal models and in studies performed in humans. PMA-stimulated and unstimulated PBMCs from 16 healthy blood donors (men) were cultured with sildenafil (at concentrations of 400 ng/ml and 4 µg/ml). OPN level in culture supernatants was measured by enzyme-linked immunosorbent assay. The analysis of OPN gene expression was performed by real-time PCR. Cell viability was assessed by trypan blue staining. PMA plus ionomycin stimulation of PBMCs resulted in a significant increase of OPN production and gene expression (p < 0.001). Sildenafil significantly decreased OPN secretion (p < 0.05) and gene expression (p < 0.05) in stimulated PBMCs; however, had no effect on OPN in unstimulated PBMCs. Sildenafil did not affect PBMCs viability. Sildenafil downregulates OPN in PBMCs from healthy men. Despite accumulating evidence for the immunomodulatory effects of sildenafil on human immune system cells, further studies are needed to determine if this drug affects the level of cGMP and NF-κB in PBMCs. In addition, it is needed to evaluate sildenafil's activity in PBMCs from patients with elevated OPN levels.

Keywords: Immune response; Osteopontin; PDE5 inhibitor; Peripheral blood mononuclear cells; Sildenafil.

Fig. 1

Effects of sildenafil on osteopontin (OPN) production in peripheral blood mononuclear cells (PBMCs). Experiments were performed on phorbol myristate acetate (PMA) plus ionomycin-stimulated and unstimulated PBMCs after incubation with sildenafil (at concentrations of 400 ng/ml and 4 µg/ml). The level of OPN in supernatants was measured by enzyme-linked immunosorbent assay (ELISA, mean ± SD). Statistically significant differences were considered when p<0.05 for Student’s t test on 16 PBMCs samples. **p < 0.001 (unstimulated PBMCs vs. PMA + ionomycin-stimulated PBMCs with and without sildenafil incubation). *p < 0.05 (PBMCs + PMA + ionomycin vs. PBMCs + PMA + ionomycin + sildenafil 400 ng/ml, PBMCs + PMA + ionomycin vs. PBMCs + PMA + ionomycin + sildenafil 4 µg/ml and PBMCs + PMA + ionomycin + sildenafil 400 ng/ml vs. PBMCs + PMA + ionomycin + sildenafil 4 µg/ml)

Fig. 2

Relative osteopontin (OPN) expression in peripheral blood mononuclear cells (PBMCs) determined by RT-PCR (mean). Experiments were performed on phorbol myristate acetate (PMA) plus ionomycin-stimulated and unstimulated PBMCs after incubation with sildenafil (at a concentrations of 400 ng/ml and 4 µg/ml)