Vitamin D Repletion Mitigates Oxidative Stress Induced Pulmonary Artery Remodeling in Sickle Cell Disease

The lung is a primary target of end organ pathology in patients with sickle cell disease (SCD). Pulmonary artery remodeling, a common feature at autopsy in SCD, is characteristic of the pulmonary hypertension of SCD. In an autopsy cohort of 20 patients with SCD, we found that pulmonary artery remodeling was a pathology of the young patient. Patients with severe remodeling had less than a 15% probability of surviving to 25 years of age, whereas those with milder remodeling had an 80% probability of living beyond 50. Therefore, we wanted to understand the mechanisms underpinning pulmonary artery remodeling.

Using benchtop ion trap mass spectrometry on extracted pulmonary proteins from the autopsy study, we screened for oxidative "fingerprints" (protein modifications) that were specific for SCD. We found that the lungs from patients with SCD carried a profound oxidative burden. Oxidative modifications arising from the hydroxyl (∙OH), superoxide (∙O₂^-) peroxynitrite (ONOO-),and eosinophil peroxidase were all significantly elevated compared to samples prepared from identical lung blocks from unaffected controls at autopsy. However, of the oxidative modifications detected, only the signature from peroxynitrite, 3-nitro tyrosine, (3-NT) could differentiate those SCD patients with profound pulmonary artery remodeling from those with milder forms of this pathology. After adjusting for age, the 3-NT content was increased 16 fold in the severely remodeled pulmonary tissues. Using immunohistochemistry, we found that 3NT was exquisitely localized to remodeling vessels and areas of smooth muscle proliferation. We assayed plasma proteins from patients with SCD at steady state and found a consistently elevated 3-NT content when compared to control plasma. The 3-NT containing proteins in living patients correlated strongly with NT-proBNP, but inversely with plasma free hemoglobin at steady state.

Peroxynitrite, a strong smooth muscle mitogen, is formed via the reaction of NO with ∙O₂^-, therefore we reasoned that the presence of the superoxide radical was transforming NO into ONOO-. Extracellular (EC) superoxides are neutralized by EC-superoxide dismutases (SODs) to preserve the vaso-regulatory effects of NO. EC-SOD is tethered to the outer membrane of cells to allow for diffusion of NO without superoxide attack. Using immunohistochemistry, electron microscopy, and linear-ion trap mass spectrometry with comparative proteomics on paraffin-free lung tissue we noted, that pulmonary EC-SOD was 60-fold depleted in the remodeling pulmonary vessels and endothelium in patients with SCD at autopsy. The plasma of patients at steady state, compared to unaffected controls, however, contained increased, but inactive EC-SOD indicating an SCD-specific release of EC-SOD from the outer membrane of endothelial surfaces and into the plasma compartment.

Using an in vitro culture of human pulmonary endothelial cells, we quantified EC-SOD on the cell surface using ELISA. Plasma from patients with SCD significantly reduced the amount of EC-SOD detectable on the endothelial cells. Plasma from healthy control subjects was without effect.

We, and many other groups have demonstrated that patients with SCD have compromised flow mediated dilation (FMD) - an NO mediated process. We found that both plasma EC-SOD and 3-NT content of plasma proteins inversely correlated with FMD in patients with SCD. However, high dose vitamin D repletion (100,000 units ergocalciferol weekly) improved FMD within 8 weeks and reduced both plasma EC-SOD and 3NT content of plasma proteins, thus restoring NO function. After an 8 week washout period of the ergocalciferol, FMD values, plasma EC-SOD, and plasma 3-NT content returned to baseline, pathological steady state levels.

Furthermore, plasma from the 8 week ergocalciferol treated patients lost the ability to deplete EC-SOD from the endothelial cell surface in vitro, whereas after the washout, plasma from the same subject effectively removed EC-SOD from the endothelial cells.

Taken together these data suggest a new mechanism of pulmonary hypertension in patients with SCD, one in which NO in the presence of superoxide radical promotes pulmonary artery remodeling and predicts an early death. Vitamin D, through an as yet undetermined mechanism, appears to restore NO function, and re-localize EC-SOD to the appropriate vascular compartment.