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Abstract
Objective Endothelial progenitor cells (EPCs) are essential for maintenance of vascular homeostasis and stability, which are key processes in the pathogenesis of systemic lupus erythematosus (SLE). The frequency and function of EPCs in SLE pathogenesis is controversial, since several works reported an increase in EPCs populations in patients with SLE compared to healthy controls (HC),1 while others showed no differences2 or even a decrease.3 Given the contradictory results, in this study we have used novel flow cytometry tools for the identification of EPCs specific subpopulations in patients with SLE and we have explored their association with the clinical characteristics.
The objective of this work was to identify EPCs specific subpopulations in patients with SLE using a novel flow cytometry tool.
Methods Peripheral blood mononuclear cells (PBMCs) were isolated from patients with SLE and healthy controls (HC). mRNA and surface protein expression were determined by quantitative PCR (qPCR) and flow cytometry. Clusters identification and characterization were performed using tSNE-CUDA dimensionality reduction algorithms.
Results tSNE-CUDA analysis identified eight different clusters in PBMCs from HC and patients with SLE. Three of these clusters had EPCs-like phenotype and the expression was elevated in patients with SLE. Among these clusters, only cluster 6 was significantly elevated in patients with SLE (figure 1A-C). Moreover, four SLE-associated subclusters were found mainly expressed in patients with SLE, being only present in patients in remission with SLE and significantly associated with the 2021 Definition of Remission in SLE (figure 1D-F). Importantly, we also identified specific clusters in SLE patients with organ damage, according to the Systemic Lupus International Collaborating Clinics (SLICC)/American College of Rheumatology damage index (SDI). These clusters showed an EPCs-like phenotype, but the expression of angiogenic markers was lower compared to HC or patients without organ damage, suggesting an impaired angiogenic function (figure 2).
Conclusion Our novel approach identified clusters of EPCs in patients with SLE that are associated with remission and damage. Therefore, these clusters might be useful biomarkers to predict disease progression and severity in SLE pathogenesis.
Patients with SLE present EPC clusters and subclusters that are involved in disease. (A) High dimensional characterization of EPC clusters in healthy controls (HC) and patients with systemic lupus erythematosus (SLE) using tSNE-CUDA dimensionality reduction algorithm. (B) Characterization of EPC clusters identified in (A) according to the expression of surface protein markers. (C) Percentage of cluster expression in HC and patients with SLE. (D) High dimensional characterization of EPC subclusters in patients with SLE (yellow) and HC (purple) using tSNE-CUDA dimensionality reduction algorithm. (E) Characterization of EPC clusters identified in (D) according to the expression of surface protein markers. (F) Percentage of subcluster expression for HC and patients with SLE patients stratified according to the 2021 Definition of Remission in SLE (DORIS) index. Data are shown as percentage of cells. Bars show the mean ± SEM. **** = p < 0.0001, using Mann-Whitney test.
Specific EPC populations in SLE patients with organ damage. (A) High dimensional characterization of EPC clusters in SLE patients positive (SDI+, green) or negative (SDI-, grey) for the Systemic Lupus International Collaborating Clinics Damage (SLICC) Index, using tSNE-CUDA dimensionality reduction algorithm. (B, C) Phenotypic characteristics (B) and percentage of expression (C) of specific clusters for SDI+ patients (5 and 8), SDI- patients (1 and 2) and shared clusters (3,4,6,7). (D) CD31, Tie2, CD105, CD309 protein expression cluster 6 in HC, SDI- and SDI+ patients. Data are shown as percentage of cells and Median Fluorescence Intensity (MFI). Bars show the mean ± SEM. * = p < 0.05; ** = p< 0.01; *** = p < 0.001, using Kruskal-Wallis test.
References
Rodríguez-Carrio J. et al. Circulating endothelial cells and their progenitors in systemic lupus erythematosus and early rheumatoid arthritis patients. Rheumatology (United Kingdom) 2012;51:1775–1784.
Grisar J. et al. Systemic lupus erythematosus patients exhibit functional deficiencies of endothelial progenitor cells. Rheumatology 2008;47:1476–1483.
Lee PY, et al. Type I interferon as a novel risk factor for endothelial progenitor cell depletion and endothelial dysfunction in systemic lupus erythematosus. Arthritis Rheum 2007;56:3759–3769.
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