Background/Objectives: Hypertension is a multifactorial condition in which inflammation plays a pivotal role. Current preclinical models fail to fully capture the complexity of the underlying mechanisms, limiting therapeutic discovery, while compounds targeting endothelial inflammatory pathways may offer promising alternatives. Methods: An advanced in vitro model of hypertension, consisting of a bioreactor for endothelial cell culture coupled with a peristaltic pump to mimic blood circulation, and a pressure modulator as mechanical stimulus (Live-Pa System) to reproduce hemodynamic stimuli, was employed to investigate the effects of the imidazo-pyrazolyl urea (IPU) 3l, known for its chemotaxis inhibition properties. The effects of 3l were investigated on hypertension-related inflammatory/vasoconstrictor markers, alone or in combination with hypertensive stimuli (ANGII and/or Live-Pa). Results: IPU 3l effectively counteracted ANGII-induced inflammation by significantly reducing NF-κB activation across all experimental conditions (static, dynamic, and Live-Pa) and IL-8 secretion under static and dynamic conditions. Conclusions: IPU 3l exhibits a consistent anti-inflammatory profile, primarily through inhibition of ANGII-induced NF-κB activation across all experimental conditions, with additional context-dependent effects on IL-8 secretion. This study introduces a new strategy for drug discovery by distinguishing biochemical from mechanical stress, providing a clearer framework to interpret condition-specific pharmacological responses. Such insights, difficult to obtain with conventional in vitro or in vivo models, are essential for developing more effective cardiovascular therapies.

OBJECTIVE:Antiphospholipid antibodies (aPL) may directly affect placentation and trigger local complement-mediated inflammation/thrombosis. Obstetric complications in the antiphospholipid syndrome (APS) are still frequent despite therapy/close monitoring, and the identification of new risk biomarkers is an unmet need. We aimed to prospectively investigate the prognostic value of classification/nonclassification laboratory APS criteria and markers of complement activation for pregnancy complications, in particular preterm delivery (PTD). METHODS:We observed 60 women prospectively during pregnancy and postpartum: 33 aPL-positive (20 APS and 13 aPL-carriers) and 27 aPL-negative (5 systemic lupus erythematosus, 11 rheumatoid arthritis and 11 undifferentiated connective tissue diseases). aPL criteria tests, anti-phosphatidylserine/prothrombin (anti-PS/PT) IgG/IgM and anti-β2glycoprotein I-Domain I IgG, complement products (C3, C4, C3a, C5a, sC5b-9, MBL) and complement pathway activity (classical pathway [CP], alternative pathway [AP], and lectin pathway [LP]) were measured in 231 available specimens. RESULTS:PTD was experienced in 21% of aPL-positive and 15% of aPL-negative women. Anti-PS/PT IgG/IgM titers were significantly higher in PTD versus non-PTD women (P < 0.05) at almost all time points. At variance with CP and AP, LP activity in aPL-positive PTD was lower than in non-PTD, reaching a statistically significant difference compared with aPL-negative PTD patients (P < 0.05). Moreover, LP activity significantly correlated with anti-PS/PT IgG/IgM titers, both measured in the first trimester (P < 0.05), and all of them significantly correlated with the week of gestation at delivery (P < 0.05). CONCLUSION:The reduced LP activity and the higher anti-PS/PT IgG/IgM titers in PTD aPL-positive women during the first trimester suggest their use as early prognostic tools for PTD in aPL-positive pregnant women.

Solid‑phase assays for the detection of antiphospholipid antibodies (aPL) are central to the laboratory evaluation of antiphospholipid syndrome (APS). Growing insights into the molecular immunology underlying these assays challenge traditional interpretations. It is now evident that the antigenic drivers are phospholipids (PL)‑binding proteins - primarily β2-glycoprotein I (β2GPI) and prothrombin (PT). β2-glycoprotein I‑dependent aPL represent the dominant subset, recognizing discontinuous conformational epitopes spanning domains I-II (DI-DII). Solid matrices coated with anionic PL or partially oxidized polystyrene amplify DV anchoring through its lysine‑rich cationic loop, thereby increasing local antigen density and molecule orientation, ultimately enhancing functional avidity of intrinsically low‑affinity aPL. These antibodies contribute to reactivity across all three APS laboratory criteria assays: lupus anticoagulant (LA), anticardiolipin, and anti‑β2GPI. Beyond β2GPI, PT‑reactive aPL, detected by phosphatidylserine/prothrombin (aPS/PT) ELISAs, bind epitopes unveiled only when PT engages anionic PL in a Ca²⁺‑dependent conformational transition. Distinct antigenic specificities - type I (open‑conformation fragment‑1 epitope) and type II (C‑terminal Gla domain) - exhibit divergent affinities, matrix‑dependent binding behaviors, and heterogeneous LA activity. Antibodies detected through aPS/PT assays are associated with APS manifestations and often with LA (range 54.8-82.0%). Conversely, a smaller population of 'pure' lipid‑binding aPL, independent of β2GPI or PT, appears to target lysobisphosphatidic acid (LBPA) within the endothelial protein C receptor/LBPA complex, activating pro‑inflammatory/pro‑coagulant pathways. Although these antibodies may contribute to APS pathogenesis in a minority of patients, their detection remains technically challenging and is currently confined to research settings. Understanding the immunological characteristics of aPL subsets is essential for interpreting solid‑phase assays and improving APS management.

Hypertension stands as one of the most significant preventable risk factors for cardiovascular disease, which is the leading cause of mortality worldwide. There is a disturbing gap, in preclinical research, between simplified cell culture and complex in vivo models. To contribute to bridge this gap, we have developed a simplified but realistic in vitro dynamic model of hypertension allowing the discrimination between mechanical pressure effects and Angiotensin II's pharmacological action. We utilized an advanced bioreactor system capable of producing adjustable flow rates to culture human umbilical vein endothelial cells (HUVEC). This system allows for the investigation of the possible effects of Angiotensin II and/or an increase in intraluminal pressure (via the Live-Pa pressure-actuation device) exerted directly upon the HUVEC monolayer without simulating transmural pressure. Key hypertension-associated inflammatory markers, such as NF-kB, p38MAPK, Interleukins (IL)-6/8, and Endothelin-1 (ET-1), were subsequently assessed. Angiotensin II induced HUVEC NF-kB and p38MAPK phosphorylation, and elevated IL-6 and ET-1 secretion, with a trend in IL-8 increase. Live-Pa alone enhanced NF-kB and p38MAPK and influenced cytokine/chemokine secretion. Combined stimuli significantly augmented the inflammatory parameters as compared to unstimulated cells, suggesting a synergistic effect between chemical and mechanical stimuli. Overall, these in vitro results demonstrate both key consistencies (e.g., NF-kB and p38MAPK activation) and specific distinctions (e.g., no significant IL-6 increase in Live-Pa-exposed versus control HUVEC) when compared to published data from hypertensive versus normotensive animal models. The proposed advanced in vitro model may successfully reproduce some features of vascular function in hypertension and simulate hemodynamic conditions by controlled flow with adjustable pressure parameters. Crucially, this system allows discrimination between mechanical blood pressure effects and Angiotensin II's pharmacological action on the endothelium, paving the way for understanding pathophysiological mechanisms and developing new therapies. Established methods make it possible that studies on cultured endothelial cells will be better comparable to the results of in vivo studies, thus directly supporting the 3Rs framework-Replacement, Reduction, and Refinement-which is essential for high-standard and ethical research.

Anti-phospholipid syndrome (APS) is a systemic autoimmune disease characterized by thrombotic vascular occlusion and maternal morbidity. Anti-coagulants remain pivotal drugs for the management of APS, but a significant proportion of patients do not benefit from long-term anti-coagulation and may require an alternative therapy to prevent antibody deposition and vascular thrombosis. We have developed a therapeutic approach based on the use of safe polymeric nanoparticles that selectively target beta2-glycoprotein I (β2GPI) deposited on endothelial cells (tNPs). Their efficacy was tested in a rat model of APS developed by infusing patients' sera containing medium-high titer antibodies against domain I of β2GPI. The tNPs bearing a CH2-deleted anti-β2GPI recombinant antibody as a targeting agent recognize β2GPI deposited on endothelial cells but failed to induce blood clot formation. The tNPs infused into rats immediately before APS sera competed with patients' antibodies, preventing their binding to deposited β2GPI and, as a consequence, resulted in thrombus formations and occlusion of mesenteric vessels. Similar results were obtained by injecting tNPs 24 hours before the administration of patients' sera to induce blood clot formation. Our findings suggest that β2GPI-targeted polymeric nanoparticles represent a stable and safe approach to prevent thrombus formation and vessel occlusion in a rat model of APS and may be used to control thrombosis developing in APS patients as a result of acute triggering events.

Two-dimensional in vitro cultures have represented a milestone in biomedical and pharmacological research. However, they cannot replicate the architecture and interactions of in vivo tissues. Moreover, ethical issues regarding the use of animals have triggered strategies alternative to animal models. The development of three-dimensional (3D) models offers a relevant tool to investigate disease pathogenesis and treatment, modeling in vitro the in vivo environment. We aimed to develop a dynamic 3D in vitro model for culturing human endothelial cells (ECs) and skin fibroblasts, simulating the structure of the tissues mainly affected in systemic sclerosis (SSc), a prototypical autoimmune fibrotic vasculopathy. Dermal fibroblasts and umbilical vein ECs grown in scaffold or hydrogel, respectively, were housed in bioreactors under flow. Fibroblasts formed a tissue-like texture with the deposition of a new extracellular matrix (ECM) and ECs assembled tube-shaped structures with cell polarization. The fine-tuned dynamic modular system allowing 3D fibroblast/EC culture connection represents a valuable model of the in vivo interplay between the main players in fibrotic vasculopathy as SSc. This model can lead to a more accurate study of the disease’s pathogenesis, avoiding the use of animals, and to the development of novel therapies, possibly resulting in improved patient management.

The present review reports the history of our scientific collaboration with Professor Shoenfeld's group. The collaboration started at the end of the 80s and was mainly focused on studies on the pathogenetic mechanisms of the anti-phospholipid syndrome (APS). Following the initial collaborative studies on antibodies against endothelium in systemic autoimmune vasculitis, we were able to use a similar strategy in APS. This line of research has resulted in the characterization of beta 2 glycoprotein I (β2GPI)-dependent anti-phospholipid antibodies (aPL) as mechanisms capable of mediating an endothelial perturbation crucial for the pathogenesis of APS. Thanks to these studies, the collaboration has led to the characterization of the membrane receptors for β2GPI and the cellular signaling resulting from antibody binding. This mechanism has also been shown to mediate the aPL effect on other cell types involved in APS pathogenesis. Finally, the exchange of information made it possible to replicate and extend the setting of animal models of the syndrome, which proved to be valuable tools for understanding the pathogenesis of the syndrome. It has been a long story recently refueled by common studies on the similarity of pro-inflammatory and pro-coagulant endotheliopathy in APS and in COVID-19.

Antibodies against phospholipid (aPL)-binding proteins, in particular, beta 2 glycoprotein I (β2GPI), are diagnostic/classification and pathogenic antibodies in antiphospholipid syndrome (APS). β2GPI-aPL recognize their target on endothelium and trigger a pro-thrombotic phenotype which is amplified by circulating monocytes, platelets and neutrophils. Complement activation is required as supported by the lack of aPL-mediated effects in animal models when the complement cascade is blocked. The final result is a localized clot. A strong generalized inflammatory response is associated with catastrophic APS, the clinical variant characterized by systemic thrombotic microangiopathy. A two-hit hypothesis was suggested to explain why persistent aPL are associated with acute events only when a second hit allows antibody/complement binding by modulating β2GPI tissue presentation. β2GPI/β2GPI-aPL are also responsible for obstetric APS, being the molecule physiologically present in placental/decidual tissues. Additional mechanisms mediated by aPL with different characteristics have been reported, but their diagnostic/prognostic value is still a matter of research.

Introduction Antidepressant discontinuation may cause withdrawal syndrome in some cases. However, evidence on this syndrome related to individual antidepressants is limited, as well as about individual risk factors for severe reactions. Objectives To ascertain whether each individual antidepressant is associated with an increased reporting of withdrawal syndrome as compared with other medications, and to examine clinical risk factors for severe reactions. Methods We conducted a pharmacovigilance study, with a case/non-case design. We included reports of antidepressant-related withdrawal syndrome from the VigiBase, the WHO global database of individual case safety reports of suspected adverse drug reactions. We performed a disproportionality analysis (calculating reporting odds ratio (ROR) and the Bayesian information component (IC)) of reports of antidepressant-related withdrawal syndrome, comparing antidepressants to all other drugs and to buprenorphine (as a positive control). Antidepressants with significant disproportionate reporting were ranked in terms of clinical priority. We compared serious versus non-serious reactions to determine clinical risk factors for severe reactions. Results Based on 31,688 reports of antidepressant-related withdrawal syndrome, we detected a disproportionate reporting for 23 antidepressants. The ROR for antidepressants altogether, compared to all other drugs, was 14.26 (95%CI:14.08-14.45), 17.01 for other antidepressants (95%CI:16.73-17.29), 13.65 for SSRIs (95%CI:13.41-13.90) and 2.8 for tricyclics (95%CI:2.59-3.02). Based on clinical priority ranking, the strongest signals were found for paroxetine, duloxetine, venlafaxine and desvenlafaxine (figure 1), being comparable to buprenorphine. Severe reactions were more frequently reported in males, adolescents, persons with multiple medications, and with longer treatment duration. Image: Conclusions Antidepressants are associated with increased reporting of withdrawal syndrome compared with other medications, with differences between individual antidepressants. Clinicians should be aware of such differences, when prescribing and discontinuing these drugs, as well as of the risk to experience more severe withdrawal symptoms in specific cases. Disclosure of InterestC. Gastaldon: None Declared, G. Schoretsanitis Consultant of: Dr. Schoretsanitis has served as a consultant for HLS Therapeutics, E. Arzenton : None Declared, E. Raschi : None Declared, D. Papola: None Declared, G. Ostuzzi: None Declared, U. Moretti: None Declared, E. Seifritz Grant / Research support from: Dr. Seifritz has received educational grants, consulting fees and lecture honoraria from Janssen Cilag, Lundbeck, Angelini, Otsuka, Servier, Ricordati, Vifor, Sunovion, Schwabe and Mepha, Consultant of: Dr. Seifritz has received educational grants, consulting fees and lecture honoraria from Janssen Cilag, Lundbeck, Angelini, Otsuka, Servier, Ricordati, Vifor, Sunovion, Schwabe and Mepha, J. Kane Shareolder of: LB Pharmaceuticals and Vanguard Research Group, Consultant of: Dr. Kane has been a consultant and/or advisor for or has received honoraria from Alkermes, Allergan, LB Pharmaceuticals, H. Lundbeck, Intracellular Therapies, Janssen Pharmaceuticals, Johnson and Johnson, Merck, Minerva, Neurocrine, Newron, Otsuka, Pierre Fabre, Reviva, Roche, Sumitomo Dainippon, Sunovion, Takeda, Teva and UpToDate, G. Trifirò Grant / Research support from: he was the scientific director of a Master program on pharmacovigilance, pharmacoepidemiology and real-world evidence which has received non-conditional grant from various pharmaceutical companies; he coordinated a pharmacoepidemiology team at the University of Messina until Oct 2020, which has received funding for conducting observational studies from various pharmaceutical companies (Boehringer Ingelheim, Daichii Sankyo, PTC Pharmaceuticals). He is also scientific coordinator of the academic spin-off “INSPIRE srl” which has received funding for conducting observational studies from contract research organizations (RTI Health Solutions, Pharmo Institute N.V.)., Consultant of: Dr. Trifirò has served in the last three years on advisory boards/seminars funded by SANOFI, Eli Lilly, AstraZeneca, Abbvie, Servier, Mylan, Gilead, Amgen;, Speakers bureau of: Dr. Trifirò has served in the last three years on advisory boards/seminars funded by SANOFI, Eli Lilly, AstraZeneca, Abbvie, Servier, Mylan, Gilead, Amgen;, C. Barbui: None Declared

Introduction Evidence on neonatal withdrawal syndrome following antidepressant intrauterine exposure is limited, particularly for antidepressants other than selective serotonin reuptake inhibitors (SSRIs). Objectives To ascertain whether maternal antidepressant treatment may be associated with withdrawal syndrome in neonates, investigating the comparative reporting between individual antidepressants and classes. Methods We performed a case/non-case pharmacovigilance study, searching reports of withdrawal syndrome in newborns in the VigiBase, the WHO database of suspected adverse drug reactions. Disproportionality analysis was performed, estimating reporting odds ratio (ROR) and the Bayesian information component (IC). Antidepressants were compared to all other medications, to methadone, and within each class of antidepressants (SSRIs, tricyclics (TCA) and other antidepressants). Antidepressants were ranked in terms of clinical priority, based on a semiquantitative score. Results We retrieved 406 reports of neonatal withdrawal syndrome in 379 neonates related to 15 antidepressants. Compared to all other drugs, disproportionate reporting was detected for antidepressants altogether (ROR: 6.18, 95%CI:5.45-7.01), for TCAs (10.55, 95%CI:8.02-13.88), other antidepressants (ROR: 5.90, 95%CI:4.74-7.36) and SSRIs (ROR: 4.68, 95%CI:4.04-5.42). All antidepressants showed a significant disproportionality, apart from bupropion (figure 1). We did not find any disproportionate reporting for any antidepressant compared to methadone. The clinical priority ranking showed moderate clinical priority for all antidepressants, with the exception four, that had a weak one (figure 1). Most frequently reported symptoms were respiratory symptoms (n=106), irritability/agitation (n=75), tremor (n=52) and feeding problems (n=40). Image: Conclusions Exposure to antidepressants in utero is associated with moderate signals of disproportionate reporting for neonatal withdrawal syndrome for most antidepressants. Clinicians should pay extra attention to neonates with antidepressant-treated mothers. Disclosure of Interest C. Gastaldon: None Declared, E. Arzenton : None Declared, E. Raschi: None Declared, O. Spigset: None Declared, D. Papola: None Declared, G. Ostuzzi: None Declared, U. Moretti: None Declared, G. Trifirò Grant / Research support from: he was the scientific director of a Master program on pharmacovigilance, pharmacoepidemiology and real-world evidence which has received non-conditional grant from various pharmaceutical companies; he coordinated a pharmacoepidemiology team at the University of Messina until Oct 2020, which has received funding for conducting observational studies from various pharmaceutical companies (Boehringer Ingelheim, Daichii Sankyo, PTC Pharmaceuticals). He is also scientific coordinator of the academic spin-off “INSPIRE srl” which has received funding for conducting observational studies from contract research organizations (RTI Health Solutions, Pharmo Institute N.V.)., Consultant of: has served in the last three years on advisory boards/seminars funded by SANOFI, Eli Lilly, AstraZeneca, Abbvie, Servier, Mylan, Gilead, Amgen, Speakers bureau of: has served in the last three years on advisory boards/seminars funded by SANOFI, Eli Lilly, AstraZeneca, Abbvie, Servier, Mylan, Gilead, Amgen, C. Barbui: None Declared, G. Schoretsanitis Consultant of: Dr. Schoretsanitis has served as a consultant for HLS Therapeutics and Thermo Fisher Scientific.

The impact of JAK/STAT inhibitors, which are used in various inflammatory diseases, on cardiovascular risk is controversial and has recently raised safety concerns. Our study investigates the direct effects of tofacitinib on macrophage cholesterol metabolism, which is crucial for atherosclerosis plaque development and stability. Cultured human macrophages THP-1 were used to assess the impact of tofacitinib on cell cholesterol efflux and synthesis via radioisotopic methods, and on cholesterol uptake by measuring the cell cholesterol content with a fluorometric assay. The cholesterol acceptors and donors were either standard lipoproteins or sera from patients with juvenile idiopathic arthritis (JIA) and from control subjects. Tofacitinib significantly increased the macrophage cholesterol efflux to all acceptors; it reduced cholesterol uptake from both the normal and hypercholesterolemic sera; and it reduced cholesterol synthesis. The treatment of macrophages with tofacitinib was able to increase the cholesterol efflux and decrease cholesterol uptake when using sera from untreated JIA patients with active disease as cholesterol acceptors and donors, respectively. In conclusion, our in vitro data support the concept that tofacitinib has a favorable impact on macrophage cholesterol metabolism, even in the presence of sera from rheumatologic patients, and suggest that other mechanisms may be responsible for the cardiovascular risk associated with tofacitinib use in selected patient populations.