Molecular Phenotype of Sickle Chronic Lung Disease Revealed by Global Gene Expression Profiling
Authors: Tan Fang , Yu Tianwei , Li Yuhua , Ghosh Samit , Mosunjac Mario , Ofori-Acquah Solomon F 
Published: April 2020

Abstract
Emerging evidence indicate the concentration of circulating heme in patients who have sickle cell disease (SCD) is sufficient to contribute to vasculopathies such as pulmonary hypertension. Despite this significance, the identity of specific molecules and pathways responsible for heme-induced pulmonary complications in SCD remains poorly understood. This study was conceived with the idea that whole-genome expression profiling offered a rigorous approach to identify specific molecules involved in both pathological and vasculoprotective mechanisms of sickle chronic lung disease. Human pulmonary artery endothelial cells (PAECs) and pulmonary microvascular endothelial cells (PMVECs) were exposed to a concentration (0–25 mM) range of hemin for seven days and total RNA isolated and interrogated with Affymetrix U133 plus 2.0 Genechips. Microarray data from 24 independent experiments was analyzed using the Bioconductor in the R framework and GeneSpring to generate two unique lists of genes regulated by hemin in PAECs and PMVECs. Multiple genes widely known to be influenced by heme including heme oxygenase-1 (HO-1), ferritin, transferrin receptor, and delta-aminolevulinate synthase were altered as expected thus validating the experimental, statistical and bio-informatics approaches used in this study. The microarray expression data was validated for 26 transcripts in PAECs and 14 transcripts in PMVECs using low-density array multiplex quantitative RT-PCR. Our findings indicate that the cytoprotective response to hemin is markedly more enhanced in PMVECs than in PAECs as determined by the number and the magnitude of differential expression of genes in the oxidative stress response and glutathione metabolism pathways. This finding is supported by a higher basal expression of nuclear factor erythroid 2-related factor 2 (Nrf2) in PMVECs than in PAECs. Heterogeneity of these anti-oxidant phenotypes was confirmed at the protein level in a concentration-dependent manner for multiple enzymes regulated by Nrf2 including NAD(P)H:quinone oxidoreductase 1 (NQO1), which is critical for preventing participation of quinones in redox cycling and generation of reactive oxygen species. Moreover, while NQO1 expression increased 3-fold in PMVECs exposed to hemin for seven days no significant increase in NQO1 expression occurred following shorter periods of hemin treatment. The clinicopathological and pathophysiological relevance of these findings were investigated in post-mortem lung tissues of cases of sickle chronic lung disease and in transgenic mice with SCD. Compared to normal human lung tissues, NQO1 expression increased 3-to 5-fold in the endothelium of small caliber size vessels as well as in both large and small airway epithelium in severe cases of sickle chronic lung disease with extensive pulmonary vascular remodeling. On the contrary, no significant difference in NQO1 expression was detected in the lungs of wild-type mice and transgenic hemizygous or homozygous SCD mice lacking pulmonary vascular remodeling. We conclude that different pulmonary segments and specific anti-oxidant molecules respond uniquely to heme. Unraveling this complex heterogeneity is critical to improving understanding of the pathogenesis and treatment of lung complications in SCD. Induction of NQO1 or its upstream regulator Nrf2 offers a potentially attractive strategy to augment the anti-oxidant phenotype of PAECs to slow the progression of pulmonary vasculopathies in SCD.