Increased Expression of the cGMP-Specific Phophodiesterase, PDE9A, in Sickle Cell Disease (SCD) Reticulocytes and Neutrophils, and Induction of Erythroid γ Globin Expression and Reduction of SCD Neutrophil Adhesion Following PDE9A Inhibition.
Authors: Almeida Camila B. , Saad Sara T.O. , Costa Fernando F. , Conran Nicola 
Published: October 2019

Abstract
Modulation of the levels of the NO second messenger, cyclic guanosine monophosphate (cGMP), has been identified as a possible therapeutic approach for SCD treatment. Up-regulation of cGMP-dependent signaling may induce γ-globin production in erythrocytic lineage cells and diminish the adhesive properties of leukocytes, the adhesion of which contributes to vaso-occlusion. Since the phosphodiesterase (PDE) enzymes, which degrade intracellular cyclic nucleotides, display differing cellular expressions, their inhibition can provide tissue-specific induction of cGMP. We studied the gene expressions of 3 types of cGMP-specific PDE (−1A, −5A, −9A) in the reticulocytes (ret) and neutrophils (neu) of healthy controls (AA), steady-state SCD patients (SS) and SCD patients on hydroxyurea therapy (SSHU; 20–30 mg/kg/day). Ret and neu were separated from whole blood before extracting mRNA, synthesizing cDNA and quantifying PDE gene expression by Real-Time PCR relative to β-actin and GAPDH expression. PDE1 was not found expressed in ret of any of the groups, but was found expressed in the neu of all three groups (≤14.2 rel expression). Intermediate PDE5 expression was observed in ret of all groups (≤16.8 rel express); PDE5 expression in neu was, however, almost undetectable in all groups (<0.3 rel. express). In contrast, high PDE9A expressions were found in ret of all groups with significantly higher expression (P<0.05, unpaired t test) in the ret of SS than SSHU and AA (68.3±10.4; 181.8±50.0; 54.8±22.2 rel express for AA, SS, SSHU, resp., n=6). Extremely high PDE9A expressions were also found in the neu of all groups, although PDE9A was significantly higher in SCD neu than in control neu (101.7±24.0; 722.8±102.2; 873.9±221.7 rel. express for AA, SS, SSHU, resp., n≥4, P<0.05 for SS/SSHU comp. AA). Comparison of PDE9A expression in diverse cell types that included T98G glial cells, liver, colon, skin, spleen, lymphnode, mammary, ovary, uterus, testicle and K562 erythroleukemic cells demonstrated very low level or virtually undetectable gene PDE9A expression in all of these tissue types (<6.7 rel express in all tissues), with the exception of K562 cells (96.8 rel. express). Data indicate that PDE9A may be expressed at higher levels in hematopoietic lineage cells. In keeping with our results, co-culture of K562 cells in the presence of the PDE1 and PDE5 inhibitors, vinpocetine (1–50μM) and sildenafil (50–200μg/ml), respectively, did not significantly increase intracellular cGMP, nor γ-globin expression (data not shown, P>0.05), suggesting that PDE1 and PDE5 are not suitable targets for pharmacological inhibition in erythroid lineage cells. In contrast, co-culture of K562 cells in the presence of the PDE9A-specific inhibitor, BAY 73-6691, for 48 h significantly increased intracellular γ-globin expression by 97.0±7.8% (1.0μM, n=3, P<0.05). Furthermore, in vitro static adhesion assays showed that co-incubation of SS neu with BAY 73-6691 reversed augmented SS neu adhesion to immobilized fibronectin (20μg/ml) to control neu adhesion levels (AA neu adhesion: 8.7±1.3%; SS neu adhesion: 12.6±1.4%, reduced to 7.2±1.3% with 1.0 μM BAY 73-6691, n=4, P<0.01 comp.basal SS adhesion). The PDE9A enzyme may constitute a relatively hematopoietic cell-specific target for pharmacological inhibition with a potential to induce γ-globin production and to inhibit cell adhesion mechanisms, with beneficial consequences for individuals with SCD.