Pulmonary hypertension (PH) is a severe complication and independent predictor of mortality in sickle cell disease (SCD). Hemolysis and nitric oxide scavenging contribute to endothelial dysfunction and vascular remodeling leading to PH in SCD, yet the precise pathogenesis remains incompletely understood. We hypothesize that products of intravascular hemolysis lead to inflammation, increased endothelial proliferation, endothelial to mesenchymal transition (EndoMT), and ultimately contribute to the development and progression of PH in SCD.
All experiments in the current study utilized human pulmonary artery endothelial cells (HPAEC). Cell proliferation was determined by WST assay (Millipore) and PCNA expression. Migration was determined by wound assay using electric cell-substrate impedance sensing (ECIS). Interleukin-6 (IL-6) and -8 (IL-8) secretion were evaluated by ELISA assay (BioLegend). Western blot analysis was used to quantify protein expression. Statistical analysis utilized t-test and ANOVA where appropriate.
HPAEC treated with 5 uM hemin showed increased proliferation (p<0.001) and migration (p<0.05) compared to control. HPAEC treated with hemin also showed increased IL-6 (p< 0.001) and IL-8 (p< 0.001) secretion. Increased protein expression of mesenchymal tissue markers, vimentin and alpha smooth muscle actin, were identified in HPAEC treated with hemin compared to control. Given the suggestion of increased EndoMT in HPAEC treated with hemin, we undertook experiments using an MLCK inhibitor, ML-7. Pretreatment with ML-7 almost fully prevented the increased proliferation seen in hemin treated HPAEC (p<0.01) and inhibited secretion of IL-6 and IL-8 secretion (p<0.001 for both).
Low dose (5 uM) hemin exposure leads to dysfunction of HPAEC characterized by increased proliferation and migration as well as cytokine release. Hemin treatment was associated with EndoMT as a potential mechanism for the development of PH in patients with chronic hemolysis. Targeting MLCK activity in HPAEC undergoing EndoMT may be a unique target in the early stages of PH development.