Yalavarthi, Srilakshmi

BACKGROUND: Whereas antiphospholipid syndrome (APS) is best known for increasing the risk of macrovascular thrombosis, APS vasculopathy is characterized by the abnormal proliferation of endothelial and smooth muscle cells, leading to occlusion of small blood vessels in the skin, kidneys, and heart, among other organs. The underlying mechanisms remain unclear, and targeted treatment options for patients with APS are lacking. METHODS: To identify and analyze APS microvascular endothelial cells (MVECs), skin biopsies of patients with APS complicated by livedo racemosa were characterized using single-cell RNA sequencing. CCN1 (cellular communication network factor 1) and CCN2 were identified in skin and kidney biopsies by immunofluorescence microscopy and in plasma by ELISA. Healthy dermal MVECs were cultured with APS patient–derived serum or immunoglobulin G (IgG), and relevant signaling pathways were characterized using quantitative PCR, immunoblotting, and immunofluorescence microscopy. The proliferation and migration of vascular smooth muscle cells were determined after exposure to conditioned medium from APS IgG–stimulated MVECs. A mouse model of APS IgG–accelerated neointima formation was developed. Anti-CCN2 monoclonal antibodies were tested in vascular smooth muscle cell functional assays and the mouse model. RESULTS: Increased endothelial cell expression of CCN1 and CCN2 was identified by single-cell RNA sequencing and further confirmed in APS skin by microscopy as well as in APS plasma by ELISA. Exposure of healthy MVECs to patient IgG triggered the upregulation of CCN1 and CCN2 via Toll-like receptor 4– and YAP1 (yes-associated protein 1)–mediated signaling. CCN2, originating from APS IgG–stimulated MVECs led to the proliferation and migration of vascular smooth muscle cells, phenotypes that were inhibited by an anti-CCN2 antibody or depletion of EGFR (epidermal growth factor receptor). We further observed increased expression of CCN2, along with evidence of YAP1 nuclear translocation, in kidney vessels of APS nephropathy biopsies. Finally, CCN2 inhibition with an anti-CCN2 monoclonal antibody significantly reduced neointima thickening and cell proliferation in a mouse model of APS IgG–accelerated neointima formation. CONCLUSIONS: This study revealed activation of YAP1-mediated signaling in APS dermal microvessels and demonstrated that the YAP1 target CCN2 plays a role in facilitating pro-proliferative communication between MVECs and vascular smooth muscle cells. These findings offer insights into the cellular and molecular mechanisms underlying APS vasculopathy, providing potential therapeutic targets for patients.

ObjectiveWhile thrombosis and pregnancy loss are the best‐known clinical features of antiphospholipid syndrome (APS), many patients also exhibit “extra‐criteria” manifestations, such as thrombocytopenia. The mechanisms that drive APS thrombocytopenia are not completely understood, and no clinical biomarkers are available for predicting antiphospholipid antibody (aPL)–mediated thrombocytopenia. Calprotectin is a heterodimer of S100A8 and S100A9 that is abundant in the neutrophil cytoplasm and released upon proinflammatory neutrophil activation. Here, we sought to evaluate the presence, clinical associations, and potential mechanistic roles of circulating calprotectin in a cohort of primary APS and aPL‐positive patients.MethodsLevels of circulating calprotectin were determined in plasma by the QUANTA Flash chemiluminescent assay. A viability dye‐based platelet assay was used to assess the potential impact of calprotectin on aPL‐mediated thrombocytopenia.ResultsCirculating calprotectin was measured in 112 patients with primary APS and 30 aPL‐positive (without APS criteria manifestations or lupus) patients as compared to patients with lupus (without APS), patients with unprovoked venous thrombosis (without aPL), and healthy controls. Levels of calprotectin were higher in patients with primary APS and aPL‐positive patients compared to healthy controls. After adjustment for age and sex, calprotectin level correlated positively with absolute neutrophil count (r = 0.41, P < 0.001), positively with C‐reactive protein level (r = 0.34, P = 0.002), and negatively with platelet count (r = −0.24, P = 0.004). Mechanistically, we found that calprotectin provoked aPL‐mediated thrombocytopenia by engaging platelet surface toll‐like receptor 4 and activating the NLRP3‐inflammasome, thereby reducing platelet viability in a caspase‐1‐dependent manner.ConclusionThese data suggest that calprotectin has the potential to be a functional biomarker and a new therapeutic target for APS thrombocytopenia.image

Abstract Objective Leucocyte immunoglobulin-like receptor A3 (LILRA3) belongs to a family of leucocyte receptors. Our previous study reported LILRA3 transcripts were markedly upregulated in neutrophils from patients with APS. We undertook this study to investigate clinical implications of LILRA3 in APS and its potential role in APS-associated thrombosis. Methods Two independent cohorts were studied. The first consisted of 294 APS patients, 48 asymptomatic aPL carriers and 150 healthy controls (HCs) from Peking University People’s Hospital. The second included 99 APS patients, 25 aPL carriers and 40 HCs from United States APS centres. Serum or plasma concentrations of LILRA3 and MPO-DNA complexes were measured. Additionally, 35 patients with thrombotic APS (tAPS) were evaluated to determine potential effects of immunosuppressive therapy on serum concentrations of LILRA3 and MPO-DNA complexes. Results Both positivity and serum concentration of LILRA3 were significantly increased in APS patients, especially in those with tAPS. LILRA3-positive tAPS patients displayed more severe thrombotic manifestations. Serum LILRA3 was positively correlated with MPO-DNA complexes in LILRA3-positive tAPS. After immunosuppressive treatment, LILRA3 and MPO-DNA complexes were consistently decreased in tAPS patients. Key findings from the Peking cohort were confirmed in the United States cohort. Conclusion Our study provides first evidence that LILRA3 is aberrantly expressed in APS, especially in patients with tAPS. Serum LILRA3 correlated with MPO-DNA complexes, and the two indices were consistently decreased in tAPS patients after treatment. LILRA3 may play a role in thrombosis of APS and may serve as a biomarker and/or therapeutic target in tAPS.