Detection of the mRNA Transcription Level of Several Genes of the HIF and NO Metabolic Pathways in PBMCs of Sickle Cell Disease Patients Using Quantitative RT-PCR Assay.

Background. Pulmonary hypertension is a common complication of sickle cell disease but the pathophysiology is not completely understood.

Methods. Using quantitative RT-PCR we studied mRNA from PBMCs of seven patients with sickle cell disease having pulmonary hypertension, five patients without pulmonary hypertension and 10 controls with neither sickle cell disease nor pulmonary hypertension. We investigated transcripts of several HIF regulated genes including endothelin-1 (ET1), vascular endothelial growth factor (VEGF), and inducible nitric oxide synthase (iNOS), several genes of the NO metabolic pathway including cGMP-specific phosphodiesterase 5A (PDE5), soluble guanylate cyclase large subunit (sGC), and arginase 2 (Arg2), and a gene implicated in primary pulmonary hypertension, bone morphogenetic protein receptor type II (BMPR2). β-actin and diacylglycerol kinase alpha (DGKA) were used as housekeeping gene controls.

Results. HIF-regulated genes: Levels of ET1 transcripts were higher in patients with sickle cell disease and pulmonary hypertension than the other two groups while VEGF transcripts were higher in both sickle cell disease groups than controls and levels of iNOS transcripts were low and did not differ among the groups. Upregulation of ET1 is associated with the development of hypoxic pulmonary hypertension in both experimental animals and humans. NO pathway genes: PDE5, sGC, Arg2 transcripts were higher in patients with sickle cell disease and PH than the other two groups. Upregulation of PDE5 and Arg2 might be expected to contribute to reduced NO signaling. Primary pulmonary hypertension gene: BMPR2 transcripts were higher in patients with sickle cell disease and PH than the other two groups.

Discussion. These results are consistent with the possibility that complex molecular pathways, including dysregulation of HIF and NO pathways, may contribute to the development of pulmonary hypertension in sickle cell disease.