Nontargeted Plasma Proteomic Analysis in Sickle Cell Disease Implicates Pathways Involving Cell Structure, Immunity and Heme and Oxygen Transport in the Occurrence of Leg Ulcers

Background: Leg ulcers are a recurrent and debilitating complication of sickle cell disease (SCD), significantly impacting quality of life (QOL) and decreasing survival. The pathogenesis of SCD leg ulcers is poorly understood, although previous studies have suggested that contributing factors may include systemic vasculopathy, anemia, and diminished endothelial function due to reduced nitric oxide (NO) bioavailability. Identification of biomarkers would advance our understanding of the pathophysiology of leg ulcers in SCD and could provide new therapeutic targets to improve leg ulcer treatment. Thus, we performed an untargeted plasma proteomic analysis in SCD patients with and without leg ulcers.

Methods: Plasma samples were obtained from SCD patients with leg ulcers enrolled in a prospective, double-blinded, randomized clinical trial of the safety and efficacy of 10 weeks' treatment with 2% topical sodium nitrite (NCT02863068). Baseline samples were available for 17 patients, and end-of-study samples were available for 15. Plasma samples from 32 SCD patients without leg ulcers were obtained from a larger study described elsewhere (Telen et al. Blood [abstr.] 2023). All patients had SS or Sb⁰ thalassemia hemoglobin genotypes. Mass spectrometry was utilized to quantify the relative abundance of 1,274 proteins; normalized protein values were log₂ transformed prior to differential analysis using limma. Four comparisons were considered: baseline with leg ulcers (n=17) vs no leg ulcers (n=32); nitrite (n=7) vs placebo (n=8) treated leg ulcers at end of study; ulcers that demonstrated a 25% reduction in size (n=8) vs those that did not (n=7) at end of study; and baseline (n=7) vs end of study (n=7) for patients treated with nitrite cream. Age and sex were included as covariates in all models. False discovery rate (FDR) q-values were generated to adjust for multiple testing; proteins with a q-value <0.05 and absolute value of log fold change (|logFC|) >2 were deemed significant. To identify clusters of proteins with highly correlated abundance, weighted correlation network analysis was done using WGCNA. Logistic regression models were then used to test for association between modules of co-abundant proteins and the four comparisons described above, using the same covariates. Bonferroni correction was applied to account for multiple testing (5 modules, adjusted p=0.01). Finally, pathway analysis was performed on proteins significantly associated with our comparisons of interest using functional annotation tools implemented in DAVID.

Results: Among analyzed patients (n=49; mean age 38 [range 22-63]), 53.1% were female and 61.2% were on hydroxyurea. These characteristics did not differ by cohort (p>0.05). Differential analysis identified 213 proteins that were differentially abundant in the baseline leg ulcer samples compared to non-ulcer bearing controls; all were down-regulated in the leg ulcer samples. The most significant protein was RPN1 (logFC=-2.09, q=1.35x10^-7), and the protein with the largest effect size was ESYT1 (logFC=-3.69, q=5.38x10^-6). Both proteins are associated with endoplasmic reticulum (ER). None of the other comparisons yielded significant results (q>0.05). Network analysis revealed five modules of co-abundant proteins, three of which were associated with the presence of leg ulcers (MEturquoise, p=7x10^-4; MEblue, p=0.02; and MEyellow, p=0.03). The association of leg ulcers with the MEturquoise module surpassed correction for multiple testing. Pathway analysis of proteins associated with leg ulcers, and those grouped into MEturquoise, returned many FDR-significant pathways, including cell structure pathways such as actin binding, focal adhesion, and cytoskeleton. Pathways of proteins in MEblue included immunoglobulins and those involved in adaptive immune response. Finally, proteins in MEyellow were within pathways involved in hereditary hemolytic anemia, hemoglobin complex, and oxygen transport.

Conclusions: The presence of leg ulcers in SCD patients is associated with protein signatures related to cell structure, immunity and hemoglobin-related perturbations, most of which appear to be downregulated in patients with ulcers compared to those without. Findings related to ER are interesting due to the role the ER plays in endothelial damage from inflammation and shear stress. Larger sample sizes will be needed to confirm these findings.

Kato:CSL Behring: Current Employment, Patents & Royalties: named on the patent held by the US DHHS for formulation of topical sodium nitrite, granted Apr 30, 2019. Minniti:BluebirdBio: Membership on an entity's Board of Directors or advisory committees; Emmaus Life Science: Consultancy; Fulcrum: Consultancy; Agios: Membership on an entity's Board of Directors or advisory committees; Guidepoint: Consultancy. Telen:CSL Behring: Research Funding; GBT/Pfizer: Research Funding; Novo Nordisk: Research Funding.