Charis Pericleous

Summary: Inflammation contributes to primary antiphospholipid syndrome (PAPS) pathogenesis, but cytokine profiles and associated thrombotic phenotypes remain incompletely defined. A panel of 20 cytokines, chemokines and endothelial activation markers was analysed in 37 PAPS patients on stable anticoagulation and 12 healthy controls (HC). We compared concentrations between groups and performed unsupervised hierarchical clustering and principal component analysis (PCA) to identify inflammatory subgroups. Compared with HC, patients with PAPS exhibited significantly elevated levels of 14/20 analytes: granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), interferon (IFN)‐γ, interleukin (IL)‐1α, IL‐1β, IL‐6, IL‐10, IL‐12p70, IL‐13, IL‐17a, tumour necrosis factor‐α (TNF‐α), intercellular adhesion molecule‐1 (ICAM‐1), P‐selectin and macrophage inflammatory protein‐1β (MIP1β) (all p < 0.05). Unsupervised clustering identified three inflammatory antiphospholipid syndrome (APS) subgroups distinct from HC. A core cytokine signature comprising GM‐CSF, IL‐6, IL‐10, IL‐12p70, IL‐13 and TNF‐α was elevated across all APS clusters. One cluster, enriched for arterial thrombosis, demonstrated a more pronounced inflammatory phenotype, with increased IFN‐α. Comparison of phenotypes showed significantly higher concentrations of GM‐CSF, IFN‐α, IL‐1α, IL‐4, IL‐6, IL‐10, IL‐17a and TNF‐α in arterial versus venous thrombosis. Our findings demonstrate widespread cytokine dysregulation and distinct inflammatory subgroups in PAPS, with arterial thrombosis showing stronger inflammation than venous thrombosis, supporting divergent thrombo‐inflammatory mechanisms and potential precision immunomodulatory therapeutic targets.

Systemic lupus erythematosus (SLE) is an established, independent risk factor for cardiovascular disease (CVD), most notably premature atherosclerotic cardiovascular disease (ASCVD). This association is thought to relate to chronic immune mediated inflammation. Clonal expansion of haematopoietic stem and progenitor cells (HSPCs) with acquired mutations, but no evidence of a blood disorder, is known as clonal haematopoiesis of indeterminate potential (CHIP). CHIP is associated with a pro-inflammatory immune phenotype, driven predominantly by mutant myeloid cells, and is similarly strongly associated with ASCVD. When SLE and CHIP co-occur, there may be synergy of their canonical cellular and molecular inflammatory pathways or even convergence of pathways through shared mechanisms of immune dysregulation, which accelerate CVD. Furthermore, enrichment of HSPC clones carrying CHIP driver mutations has been observed in chronic inflammatory conditions, including SLE. In this review, we explore the emerging role of CHIP in the relationship between SLE and ASCVD, proposing it as a pathogenic nexus in a triangular inflammatory network. Defining these relationships will help early identification of high-risk individuals and facilitate therapeutic targeting of CHIP to mitigate ASCVD complications in SLE patients.

Background: Thrombus formation is regulated by the interplay between endothelial cells (EC), platelets and coagulation factors. However, most in vitro assays used to study thrombosis and develop anti-thrombotic therapies do not include the endothelium. Objective: To develop a thrombus-on-chip model that includes endothelium and whole blood and allows for manipulation of extracellular matrix (ECM), shear stress and the addition of multiple cell types. Methods: A cylindrical vessel was created in a collagen matrix using the needle-based fabrication technique in a microfluidic device. Human umbilical vein EC (HUVEC) or endothelial colony-forming cells (ECFC) were cultured in the channel, with continuous monodirectional media turnover. Immunostaining and permeability measurement validated confluence and integrity of the monolayer. To investigate thrombosis, whole blood from healthy donors was perfused through TNF-α-activated or untreated EC-lined vessels. Image analysis and time-lapse microscopy were used to quantify labelled platelet adhesion and fibrin deposition. Results: TNF-α treatment resulted in increased platelet adhesion and fibrin deposition compared to control vessels. TNF-induced endothelial activation was confirmed by upregulation of adhesion molecules ICAM-1, E-selectin and tissue factor (TF). Thrombus formation in TNF-α treated vessels was inhibited by an anti-TF antibody. In ECFC vessels, platelet adhesion and fibrin deposition were comparable to HUVEC, supporting feasibility of patient-based studies. Conclusions: We developed a perfused thrombus-on-chip model that combines key elements of thrombus formation including endothelium. The model is amenable to independent control of microenvironmental stimuli, crosstalk with tissue-specific cells, and the inclusion of patients’ own cells and blood for precision medicine studies. ESSENTIALS: - The endothelium is a key contributor to thrombosis - Standard in vitro methods to study thrombosis do not include the endothelium - We have developed a thrombus-on-a-chip model of thromboinflammation to measure live platelet adhesion and fibrin deposition on activated endothelium - Using endothelial colony forming cells (ECFC), we show that the method is suitable for patient studies with autologous endothelium and blood

BACKGROUND: Atherosclerotic plaques form unevenly due to disturbed blood flow, causing localized endothelial cell (EC) dysfunction. Obesity exacerbates this process, but the underlying molecular mechanisms are unclear. The transcription factor EPAS1 (HIF2A) has regulatory roles in endothelium, but its involvement in atherosclerosis remains unexplored. This study investigates the potential interplay between EPAS1, obesity, and atherosclerosis. METHODS: Responses to shear stress were analyzed using cultured porcine aortic EC exposed to flow in vitro coupled with metabolic and molecular analyses and by en face immunostaining of murine aortic EC exposed to disturbed flow in vivo. Obesity and dyslipidemia were induced in mice via exposure to a high-fat diet or through Leptin gene deletion. The role of Epas1 in atherosclerosis was evaluated by inducible endothelial Epas1 deletion, followed by hypercholesterolemia induction (adeno-associated virus-PCSK9 [proprotein convertase subtilisin/kexin type 9]; high-fat diet). RESULTS: En face staining revealed EPAS1 enrichment at sites of disturbed blood flow that are prone to atherosclerosis initiation. Obese mice exhibited substantial reduction in endothelial EPAS1 expression. Sulforaphane, a compound with known atheroprotective effects, restored EPAS1 expression and concurrently reduced plasma triglyceride levels in obese mice. Consistently, triglyceride derivatives (free fatty acids) suppressed EPAS1 in cultured EC by upregulating the negative regulator PHD2. Clinical observations revealed that reduced serum EPAS1 correlated with increased endothelial PHD2 and PHD3 in obese individuals. Functionally, endothelial EPAS1 deletion increased lesion formation in hypercholesterolemic mice, indicating an atheroprotective function. Mechanistic insights revealed that EPAS1 protects arteries by maintaining endothelial proliferation by positively regulating the expression of the fatty acid-handling molecules CD36 (cluster of differentiation 36) and LIPG (endothelial type lipase G) to increase fatty acid beta-oxidation. CONCLUSIONS: Endothelial EPAS1 attenuates atherosclerosis at sites of disturbed flow by maintaining EC proliferation via fatty acid uptake and metabolism. This endothelial repair pathway is inhibited in obesity, suggesting a novel triglyceride-PHD2 modulation pathway suppressing EPAS1 expression. These findings have implications for therapeutic strategies addressing vascular dysfunction in obesity.

Takayasu arteritis (TAK) and giant cell arteritis (GCA) are the primary forms of large vessel vasculitis (LVV) and can result in serious cardiovascular morbidity. Improved understanding of the molecular basis of these diseases is required to develop novel biomarkers and targeted treatments. Moreover, it is unclear whether shared or distinct pathogenic processes underpin the LVV spectrum. To address this, we performed plasma proteomic profiling, quantifying 184 plasma proteins using Olink immunoassays in two independent cohorts totalling 405 individuals. In Cohort 1, comparison of patients with TAK (N=96) and large vessel-GCA (LV-GCA) (N=35) versus healthy controls (HCs) (N=35) revealed 52 and 72 significant differentially abundant proteins, respectively. Correlation with disease activity status identified novel TAK and LV-GCA disease activity markers. Cohort 2 consisted of patients presenting acutely with possible cranial GCA (C-GCA); C-GCA was subsequently confirmed (n=150) or excluded (n=89). 31 proteins were associated with C-GCA. Analyses stratified by temporal artery biopsy results revealed enrichment of the proteomic signal in biopsy-proven GCA, suggesting the presence of distinct endotypes within C-GCA. Cross-disease comparison revealed that active TAK, LV-GCA and biopsy-proven C-GCA had highly similar plasma proteomic profiles. Twenty-six proteomic associations were shared across all three groups including IL6, monocyte/macrophage related proteins (CCL5, CCL7, CSF1), tissue remodelling proteins (VEGFA, TIMP1, TNC) and proteins not previously linked to LVV (TNFSF14, IL7R). We also observed disease-specific associations including increased CXCL9 in LV-GCA and C-GCA but not in TAK and decreases in the extracellular matrix protein COMP in TAK but not in LV-GCA or C-GCA. Evaluation of publicly available transcriptomic data from LV-GCA aortic tissue revealed that 47 of the 112 proteins significantly altered in ≥1 LVV type had significantly altered mRNA expression in LVV aortic tissue. Similarities in LVV proteomic profiles suggest shared pathobiology involving innate immunity, particularly monocyte/macrophages, lymphocyte homeostasis and tissue remodelling processes. Our results highlight a signature of immune-stromal cross talk in LVV and identify potential novel therapeutic targets in this axis (e.g. TNFSF14). The correspondence of plasma signatures to tissue phenotype highlights the potential for non-invasive monitoring of arterial inflammation and injury.

Beta-2-Glycoprotein I (β2GPI) is the main autoantigenic target of antiphospholipid syndrome (APS) with antibodies leading to clinical manifestations. There are two known structural isomers of β2GPI, a J shape and a circular shaped one. The transition between these structures is incompletely understood, with the functional implications unknown. β2GPI is a substrate of the protease plasmin, which cleaves within the fifth domain of β2GPI leading to altered cellular binding. Very little is currently known regarding the structure and function of this protein variant. We present the first comprehensive structural characterisation plasmin-clipped β2GPI and the associated implications for pathogenic antibody binding to this protein. Methods β2GPI was purified using a novel acid-free process from healthy control plasma and cleaved with plasmin. Cleavage was confirmed by SDS-PAGE. Structural characterisation was undertaken using dynamic light scattering (DLS), small angle X-ray scattering (SAXS), ion mobility mass spectrometry (IMMS) and molecular dynamics simulation (MD). Activity was tested using inhibition of β2GPI ELISAs with patient samples and cleaved β2GPI in the fluid phase and cellular binding by flow cytometry using HUVEC cells. Results DLS revealed a significantly smaller hydrodynamic radius for plasmin-clipped β2GPI (p=0.0043). SAXS and MD analysis indicated a novel S-like structure of β2GPI only present in the plasmin-clipped sample whilst IMMS showed a different structure distributions in plasmin clipped compared to non-clipped B2GPI. The increased binding of autoantibodies was shown for plasmin-clipped β2GPI (p=0.056), implying a greater exposure of pathogenic epitopes following cleavage. Conclusions Cleavage of β2GPI by plasmin results in the production of a unique S-shaped structural conformation and higher patient antibody binding. This novel structure may explain the loss of binding to phospholipids and increase in anti-angiogenic potential described previously for plasmin-clipped β2GPI.

Background: Prothrombotic antiphospholipid antibodies (aPL) found in patients with antiphospholipid syndrome (APS) are a recognised risk factor for ischemic stroke. However, it is unclear if aPL cause injury post thrombolysis leading to worse outcomes. We investigated whether aPL exacerbate reperfusion injury and sought to translate our findings in endothelial colony forming cells (ECFC) isolated from patients with APS. Methods: Transient ischemic stroke was induced in adult rats injected with serum-derived IgG from patients with APS (APS-IgG, containing aPL) or healthy controls (HC-IgG). Infarct size and intracellular signalling processes involved in ischemia-reperfusion injury were determined post reperfusion. In vitro , human umbilical vein endothelial cells (HUVEC) treated with IgG, as well as APS and HC ECFC, were exposed to hypoxia (0.1% O ₂ ). Cell death and relevant signalling mechanisms were assessed following reperfusion and compared to matched normoxic cultures. Results: In vivo , APS-IgG induced >2-fold larger infarcts and lower levels of active phosphorylated Akt, a key pro-survival kinase, compared to HC-IgG. In vitro , aPL-mediated cell death and suppression of Akt phosphorylation was confirmed in HUVEC exposed to IgG and hypoxia-reperfusion. Consistent with these findings, higher rates of cell death and reduced Akt phosphorylation following reperfusion were observed in ex vivo APS ECFC compared to HC ECFC. Treatment with the immunomodulating agent hydroxychloroquine ameliorated ECFC death and this effect was more pronounced in APS-derived cells. Conclusion: Patient-derived IgG aPL exacerbate cell death following reperfusion in a novel in vivo stroke model for APS, as well as in vitro HUVEC cultures. These observations are mimicked in ex vivo APS ECFC. Our findings describe a novel pathogenic role for aPL in mediating tissue injury in addition to their known thrombogenic properties and indicate potential for pharmacological intervention.