Background/Purpose: The endothelium is a major target of pathogenic antiphospholipid antibodies (aPL) found in patients with antiphospholipid syndrome (APS). It is well established that aPL promote a thrombo-inflammatory state in endothelial cells and immune cells. More recently, altered immune cell metabolism and mitochondrial function were reported to contribute to pathology. It is not known if these mechanisms are affected in endothelium. Here, we examine how aPL impact endothelial metabolism and mitochondrial function using ex vivo blood-derived endothelial colony forming cells (ECFC) from patients with APS.

Methods: ECFC were isolated from peripheral blood mononuclear cells (PBMC) obtained from IgG aPL positive patients with thrombotic APS and healthy controls (HC). Experiments were performed at passage 4-5 (n=10-12 per group). Protein expression was assessed by immunoblot, respiratory and glycolytic measurements with Agilent extracellular flux assays (Seahorse), and mitochondrial network analysis by cytochrome c immunofluorescence using confocal microscopy. Non-parametric Mann-Whitney and Spearman’s data analysis was performed.

Results: Colony number per 10⁷ PBMC, days from PBMC seeding to first colony appearance and days to initial passage (from P0 to P1) were similar between patient and control ECFC. Diminished basal mitochondrial respiration and OXPHOS-linked ATP production (oxygen consumption rate, p< 0.05) in APS ECFC indicated mitochondrial dysfunction. Tendency towards a glycolytic phenotype was also evident. Reduced mitochondrial function and elevated glycolysis were more prominent in ECFC isolated from patients with a history of cardiovascular events (n=8 with stroke or myocardial infarction; n=4 without) and from patients not receiving hydroxychloroquine (HCQ, n=6 on HCQ and n=6 not on HCQ). OXPHOS immunoblot analysis correlated with basal respiration measurements (r=0.7, p=0.04). Concomitantly, we observed suppressed mitochondrial MnSOD expression along with reduced mitochondrial footprint and increased fragmentation of the endothelial mitochondrial network in APS versus HC ECFC consistent with mitochondrial stress.

Conclusion: Perturbed mitochondrial energy homeostasis and increased glycolysis are pronounced in APS patient derived ECFC, signifying pathological metabolic reprogramming. Given the importance of mitochondria in endothelial homeostasis, dysfunctional mitochondria may contribute to the pro-coagulant and inflammatory features of endotheliopathy in APS.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/evidence-for-mitochondrial-dysfunction-in-blood-derived-endothelial-colony-forming-cells-isolated-from-patients-with-antiphospholipid-syndrome/