Phosphodiesterase-4 Inhibition Reduces Ischemic/Reperfusion Liver Injury in a Mouse Model for Sickle Cell Disease.

Ischemic/reperfusion (I/R) injury plays a critical role in the pathogenesis of several clinical manifestations of sickle cell disease (SCD) and it results from complex interactions between cells and plasma factors. Hepatic dysfunction has been described in SCD and the pathophysiology of liver I/R damage in SCD is only partially known. The liver is highly vascular organ with high metabolic rate, susceptible to stress-induced by hypoxia. Here, we studied the effects of acute and chronic hypoxia on liver function and pathology in transgenic sickle mice (SAD mice). We evaluated the following parameters at baseline, at 4, 48 and 168 hours (hrs) of exposure to hypoxia (8% oxygen) followed by 2 hrs reoxygenation (21% oxygen): complete blood counts with retic, red cell density, serum AST and ALT levels, liver pathology and expression by RT-PCR of the following genes: TNF-a, IL-1b as inflammatory response markers; TgfB1 (involved in control cell growth and extracellular matrix formation), mmp9 (marker of hepatic matrix remodeling), NfKb (transcription effector involved in liver cell death and regeneration), Icam1 (endothelial damage and vascular remodeling marker), in addition to heme oxygenase-1 (Homx1), eNOS (Nos3) and iNOS (Nos2). SAD mice at baseline exhibited

• liver infiltrates of inflammatory cells, no thrombi and a mild hepatic injury (pathological score: 1.4±0.02; n=5);

• up-regulation of TgfB1, Icam1, Nos3 and down-regulation of Nfkb compared to normoxic WT mice.

One SAD mice died at 48 hrs hypoxia and 2 SAD mice died at 96 hrs hypoxia. In SAD mice I/R induced:

• increased red cell density at 48 and 168 hrs of hypoxia;

• increased peripheral neutrophil count at 48 and 168 hrs;

• increased retic count at 168 hrs;

• increased serum levels of AST/ALT;

• increased liver cell inflammatory infiltrate at 48 hrs (n=5) and 168 hrs (n=6);

• increased pathological score at 48 hrs (2.3±0.02 n=5) and at 168 hrs (3.2±0.05 n=6; P<0.05).

In SAD mice I/R induced changes in the expression of the following genes: 4hrs: up-regulation of Nfkb, Ilb1, Nos2 and down-regulation of Tgfb1, Icam1, homx1 and Nos3; 48 hrs: up-regulation of Icam1, Nos2, Tnfa and down-regulation of TgfB1, Nos3, homx1, Nfkb, Ilb1; 168 hrs: up-regulation of TgfB1, Icam1, Nos 3, homx1, Nfkb, Ilb1, Nos2 and down-regulation of Nos2. In WT mice I/R induced

• increased neutrophils and retic count at 168 hrs;

• increased serum levels of AST/ALT at 48 and 168 hrs;

• increased liver cell inflammatory infiltrate at 168 hrs (n=7) and pathological score at 168 hrs and

• modulation of the following genes at 4hrs: up-regulation of Nfkb, Icam1, Ilb1, Nos2, mmp9 and down-regulation of TgfB1 and homx1; at 48 hrs: up-regulation of TgfB1, Icam1, homx1, Nos2, Tnfa, mmp9 and down-regulation of Ilb1; at 168 hrs: up-regulation of Icam1, Nfkb, Nos 3, Nos2, Tnfa, mmp9 and down-regulation of Il1b, homox1, TnfB1.

Then, we investigated the effects of a PDE-4 inhibitor (Rolipram) on hepatic I/R injury in sickle cell SAD mice, since cAMP cell content has been recently described to be crucial in I/R liver damage and PDE-4 is a key regulator of cAMP metabolism. In SAD mice, Rolipram reduced the hypoxia induced peripheral neutrophilia and serum AST/ALT increase, prevented the I/R liver injury as supported by reducing cell inflammatory infiltrate, pathological score (1.8±0.02, n= 6, P<0.05) and the modulation of the following genes: TgfB1, Icam1, Nos3, Ilb1, Tnfa and mmp9 These data suggest that the inhibition of PDE-4 protects from I/R related sickle cell liver injury, most likely by inducing over-expression of Nos3, reducing vascular activation, modulating matrix remodeling and immune-inflammatory response.