Proteomic Identification of Dysregulated Apolipoprotein Expression in Pulmonary Hypertension Secondary to Sickle Cell Disease.
Authors: Yuditskaya Susan , Hoehn Gerard , Suffredini Anthony , Xu Xiuli , Munson Peter , Gladwin Mark , Kato Gregory J. 
Published: April 2020

Pulmonary hypertension (PHT) is emerging as a major complication and independent risk factor for sudden death among adults with sickle cell disease (SCD). The identification of novel plasma markers of PHT might allow for earlier diagnosis and more convenient screening than offered by the current use of Doppler echocardiography to identify patients with high tricuspid regurgitant jet velocity (TRV). We used surface enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF MS) to search for plasma biomarkers of PHT from clinical specimens. A cohort of 27 homozygous SCD patients with PHT (mean TRV=3.1±0.4 m/sec) was compared with a group of 28 homozygous SCD patients without PHT (mean TRV=1.4±0.5 m/sec), selected for similar age, gender and renal function. Plasma samples were fractionated on anion exchange columns by step pH elution (pH 3–9, organic) and applied to solid-phase protein chip surfaces (Ciphergen Biosystems ProteinChips). Univariate and multivariate analysis of SELDI-TOF data was accomplished by Random Forest classification/regression (RF), stepwise logistical regression, and Spearman correlations with clinically measured parameters. The identity of significant protein species was confirmed through MALDI-TOF and or LC-MS/MS. Results and conclusions: SELDI-TOF MS analysis resulted in 28 sets of 162 spectra each, resolving 955 peaks per patient. RF and stepwise logistical regression modeling of these peaks consistently showed lower abundance of a 28.1 kDa protein in PHT patients (p&lt;0.0001), identified by MALDI-TOF MS and LC-MS/MS as the oxidant scavenging protein apolipoprotein A-I (apo A-I). The 28.1 kDa intensities also correlated strongly with clinical assays of apo A-I (r=0.58, p&lt;0.0001) and HDL levels (r=0.50, p=0.0001). Comparison of PHT prevalence between patients whose apo A-I levels fell in the lowest quartile indicated a risk ratio (RR) of 2.0 compared to the upper quartile (p=0.06). Patients in the upper quartile of apo B levels had a higher prevalence of PHT than the lowest quartile (RR 2.3, p=0.05), with even higher risk indicated by a high ratio of apo B/apo A-I (RR 3.3, p=0.006), all known to be risk indicators in cardiovascular disease without SCD. In addition, a 13.4 kDa peak, identified as the inflammatory marker serum amyloid A-4 (SAA-4) by LC-MS/MS, was also important by RF and was abundant in the PHT positive group (p=0.0006). An 8.9 kDa peak, significant in combination with 28.1 kDa by stepwise logistical regression (ROC area=0.88; p=0.007) was identified by LC-MS/MS as apolipoprotein A-II. These results implicate the apolipoprotein pathway in the development of PHT vasculopathy in sickle cell disease. Their known function in regulating oxidative species supports the proposed role of oxidant stress in the sickle cell vasculopathy. Our findings are consistent with published data indicating that an apo A-I mimetic dramatically improves blood flow in the sickle cell mouse. Further studies of these apolipoproteins are warranted to validate their potential mechanistic involvement in sickle vasculopathy and as predictive markers of PHT.