Combined Treatment with Hydroxyurea and a Phosphodiesterase 9 (PDE9) Inhibitor Decreases Hepatic Damage in an Animal Model of Sickle Cell Anemia

Sickle cell anemia (SCA) is associated with recurrent vaso-occlusive processes and chronic inflammation, which may result in severe liver damage, affecting patient morbidity and mortality. The phosphodiesterase 9 (PDE9) enzyme degrades intracellular cyclic guanylate monophosphate (cGMP) and is highly expressed in the hematopoietic cells of SCA patients (Almeida et al., 2008, Br J Haematol). The inhibition of this enzyme induces fetal hemoglobin production in erythroid cells and, in a mouse model of inflammatory SCA vaso-occlusion, prevents acute vaso-occlusive processes and improves animal survival when used together with hydroxyurea (HU) (Almeida et al., 2012 Blood). As PDE9 inhibitors (PDE9i) may represent a strategy to specifically amplify the activation of the nitric oxide/guanylyl cyclase pathway by HU in hematopoietic cells, we aimed to determine whether the administration of a PDE9i (BAY73-6691) and/or HU for 4 weeks can modulate chronic liver inflammation and intrahepatic fibrosis in an animal model of SCA. Chimeric SCA and control (CON) mice were generated from the bone marrow transplantation of Berkeley transgenic SCA mice and C57BL/6 mice, respectively, to irradiated C57BL/6 mice. Three months after transplantation, phenotyped SCA mice received a five-times weekly dose of vehicle (0.9% sodium chloride, n=5), PDE9i (1 mg/Kg BAY73-6691, n=3), HU (50 mg/Kg, n=3) or PDE9i plus HU (n=3) by gavage for 4 weeks. Fibrosis was visualized in liver sections by second harmonic generation (SHG) using confocal microscopy (LSM780 microscope, Carl Zeiss). NKT cell (CD16/32⁺CD3e⁺) quantification, as a percentage of liver granulocytes, was determined by flow cytometry. After surgical exposure of the liver, the vaso-occlusive processes were analyzed by intravital confocal microscopy. In contrast to vehicle-treated CON mice, vehicle-treated SCA animals presented extensive accumulation of thick fibers of collagen, characteristic of fibrosis, at 4 months post-transplantation. Collagen fibers were generally localized near blood vessels and were associated with a marked inflammatory infiltrate (Fig. 1). Vehicle-treated SCA mice also presented a significantly higher number of NKT cells in the liver, compared to CON mice (0.39% ± 0.10%, 0.17% ± 0.04%, respect., P<0.05). In association with these findings, intravital microscopy demonstrated increased neutrophil (neu) recruitment in liver sinusoids, compared to CON mice (12.7 ± 2.0 and 5.2 ± 1.5 neu/0.262 mm², P<0.001), usually in association with platelet aggregates, characteristic of vaso-occlusive processes (Fig. 1). Neither PDE9i nor HU, when administered alone, prevented the accumulation of thick fibers of collagen in the liver, or the formation of hepatic fibrosis and inflammatory infiltrates in SCA animals (Fig. 1). In contrast, the treatment of SCA mice with both PDE9i plus HU for 4 weeks markedly reduced fibrosis and the organization of liver collagen, as well as the inflammatory infiltrate, compared to SCA-vehicle animals (Fig. 1). All three treatments significantly diminished NKT cell numbers in SCA livers (0.13% ± 0.02, 0.14% ± 0.01, 0.14% ± 0.07 for PDE9i, HU and PDE9i+HU, respectively, P<0.05, compared to SCA-vehicle). In contrast, none of the treatments were capable of inhibiting neu recruitment in the liver sinusoids (20.1 ± 3.0, 15.0 ± 2.7, 12.5 ± 3.6 neu/0.262 mm², for PDE9i, HU and PDE9i+HU, respect., P>0.05, compared to SCA-vehicle), possibly due to the extremely small size of these vessels. Additionally, the formation of platelet-neutrophil heterocellular aggregates in sinusoids were not observed to be modulated by these agents (Fig. 1). In conclusion, while the oral treatment of SCA mice with PDE9i and/or HU for four weeks failed to significantly abrogate leukocyte recruitment in liver sinusoids, at the concentrations employed, the use of HU plus PDE9i in SCA mice effectively diminished the extensive hepatic damage, including fibrosis, that results from chronic liver inflammation, representing a potential pharmacological approach for the management of liver damage in SCA.

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