The phosphodiesterase 5 inhibitor sildenafil decreases the proinflammatory chemokine IL-8 in diabetic cardiomyopathy: in vivo and in vitro evidence

Figures

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  Fig. 1. Serum IL-8 levels in DCM patients before (baseline) and after 3 months sildenafil or placebo, vs. healthy subjects. DCM patients showed higher baseline levels of IL-8 vs. healthy subjects (149.14 ± 46.89 pg/ml vs. 16.17 ± 5.38 pg/ml); IL-8 was significantly reduced (23.7 ± 6 pg/ml) close to healthy levels in DCM patients after sildenafil. Conversely, serum IL-8 remained high after placebo (130.33 ± 89.74 pg/ml). *p < 0.05 vs. baseline; °°p < 0.01 vs. healthy. Results (mean ± SE) are expressed as serum IL-8 (pg/ml)

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  Fig. 2. Effect of sildenafil on circulating IL-8 in subjects with DCM. a IL-8 serum level categorized DCM patients as responders (r) and non-responders (nr) to sildenafil; positive 197.55 ± 101.8 pg/ml) or null/negative (− 16.05 ± 6.97 pg/ml) variation was observed after sildenafil intake; *p < 0.05 rvs. nr. Inset: Baseline BNP was significantly higher in r vs. nr, *p < 0.05. Data of IL-8 (mean ± SE) are expressed as serum variation vs. baseline level (pg/ml); BNP (mean ± SE) was expressed as pmol/l. b. Receiver operating characteristic (ROC) curve. The area under the ROC curve is 0.945 (95% confidence interval of 0.772 to 1.0, p < 0.01), with a sensitivity of 0.93 and a specificity of 0.90

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  Fig. 3. Effect of sildenafil on IL-8 release induced by inflammatory stimuli in Hfaec and PBMC. IL-8 protein release was significantly counteracted by sildenafil in Hfaec and PBMC (percent of inhibition 54.54% ± 1.09 and 32.80% ± 0.55); results (mean ± SE) are expressed as percent of inhibition, calculated on IFNγ + TNFα- or PHA-induced IL-8 maximal secretion, taken as 100%; ***p < 0.001, **p < 0.01. Inset: IL-8 mRNA expression was reduced only in PBMC treated with sildenafil. mRNA levels—relative to β-actin mRNA, used as endogenous control—are expressed as (mean ± SE) percent of inflammatory stimuli-induced IL-8 mRNA expression, taken as 1; **p < 0.01. Data are obtained from three to seven experiments in triplicate, using different cell preparations

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  Fig. 4. Effect of sildenafil on cGMP stabilization and PDE5 expression. a cGMP concentration was significantly higher in Hfaec treated with sildenafil (1 µM, 30 min); *p < 0.05 vs. control cells. Inset Western blot analysis showed no changes of PDE5 protein expression in Hfaec before and after sildenafil; β-actin was used as loading control. b cGMP amount in PBMC after 30 min of sildenafil (1 µM) showed a trend to increase though not statistically different vs. control. Inset Western blot analysis showed no changes in PDE5 protein expression before and after sildenafil; β-actin was is the loading control. Results in panel A (mean ± SE) are expressed as cGMP concentration (pmol/106 cells) and derived from two different preparations for each cell type; the blots in panel a and b are representative of data deriving from two/three different preparations of both cell types

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  Fig. 5. Effect of sildenafil on human cardiac, endothelial and immune cell viability. a Hfaec cell number did not change after sildenafil (1 µM) for 24 h or 48 h vs. control (without drug). b PBMC cell number was not modified by sildenafil (1 µM) after 24 h and 48 h vs. control cells. Results derived from at least four different cell preparations for each cell type and are expressed as (mean ± SE)