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Atherosclerotic plaque rupture with thrombosis is a major cause of cardiovascular events. The natural history of plaque progression is still unknown and the plaque destabilization unpredictable. We have recently suggested that P2X7 purinergic receptor expressed in carotid plaques (CP) might play a role a role in the pathogenesis of atherosclerosis. P2X7 activation by ATP leads to a broad spectrum of effects, including membrane blebbing, tissue factor (TF)-dependent thrombosis in mice, metalloproteases (MMPs) activation and release. MMPs deregulation may alter extracellular matrix network, affect vascular smooth muscle cell (VSMC) migration, contributing to plaque rupture. To study the P2X7 pathway role in MMPs regulation and in CP progression to thrombosis we developed functional models. We setup and characterized an ex-vivo tissue culture model, using human explants from either atherosclerotic (CP) or not (internal mammary artery, IMA) vessels of patients submitted to carotid endoarterectomy or to coronary artery bypass surgery. The preservation of vascular features in ex-vivo culture was verified at different time points (from 24 hours to 15 days) by evaluating the morphology (Hematoxylin/Eosin, Movat's), and the expression of wall cells (aSMA, sm22, Vimentin, CD68, FSP1) and extracellular matrix markers (Collagen type I), of P2X7, MMP9 and TF by confocal microscopy and/or biochemistry. ATP quantification and gel zymography in tissue protein extracts and culture supernatants evaluated the metabolic activity and the MMPs-related gelatinolysis of cultured vessels. A time frame of 4 days was identified as the maximum for structurally and functionally preserved vessel ex-vivo culture; functional loss was seen at day7, morphology alteration at days 10 and 15. P2X7 and MMP9 were previously detected in CP intima-media, thus to perform targeted functional studies we obtained VSMC from ateromasic areas of CP and from IMA. IMA fragments released cells for up to 3 months, CP for up to 2 months. VSMC were characterized similarly to tissue cultures; when freshly-explanted or at passage1, VSMC displayed P2X7, MMP9 molecules expression and gelatinolytic activity. At further passages P2X7 was undetected or unmounted on membrane, MMP9 expression and gelatinolysis absent. Treatment with ATP (50-200mM, 24hours), P2X7 antagonists (KN62, A74003) affected cell metabolism and gelatinolysis but not P2X7 expression. Our data showed that CP and IMA ex-vivo cultures and primary VSMC in vitro cultures represent suitable functional models to investigate the role of P2X7-MMP9 pathway in vascular pathology. [ABSTRACT FROM PUBLISHER]

Atherosclerosis is a dynamic progressive disease of the large arteries characterized by accumulation of lipids, inflammatory and smooth muscle cells (SMCs) and extracellular matrix. In situ growth or embolization of arterial thrombi following a plaque rupture may lead to acute coronary syndrome and stroke. Fibrous cap rupture may expose thrombogenic material, initiating platelet aggregation and coagulation pathways. These thrombotic changes result from activation of the clotting cascade by tissue factor (TF) and coagulation contact phase. Aim of the project is to study Carotid Plaque (CP) thrombogenicity and the vascular wall components responsible for thrombus formation. CP (n=12) from patients submitted to endoarterectomy for significant stenosis and internal mammary artery fragments (n=4, controls not atherosclerotic) from individuals undergoing coronary artery bypass surgery were snap-frozen and sectioned. Thrombogenesis induced by CP was evaluated ex-vivo under flow conditions using healthy volunteer blood drawn into citrate anticoagulant. Recalcified blood was perfused over CP cryosections at constant flow velocity and 37 °C, and the volume of platelet aggregates and fibrin deposited onto the surface was measured in real time by confocal videomicroscopy. The areas with same XY coordinates than those analyzed in blood flow experiments were relocalized in serial cryosections, to identify by histology (Movat's, Hematoxylin/Eosin) and confocal microscopy the vascular cell phenotypes and wall components involved in thrombus formation. Thrombi formed mainly over endothelium/intima layer (including fibrous cap); were abundant in the media rich in SMC and in foam cells, in the presence of FSP1, collagen type I, proteoglycans, fibrinogen and TF. The fibro-necro-calcific core was quite unreactive, except when TF was present. IMA wall layers were reactive with analogous pattern but showed a different amount of thrombus formation. Addition to blood before perfusion of anti-human TF monoclonal antibody, or antibody blocking factor XI activation, or a combination of them reduced the volume of platelet aggregates, and of fibrin to a different extent in the different vascular layers. These data suggest an in-situ role of vascular cells and matrix, as well as of intra-tissue TF in thrombus formation over CP, involving both TF and contact phase coagulation pathways. [ABSTRACT FROM PUBLISHER]

Microenvironmental signals strongly influence chronic lymphocytic leukemia (CLL) cells through the activation of distinct membrane receptors, such as B-cell receptors, and inflammatory receptors, such as Toll-like receptors (TLRs). Inflammatory pathways downstream of these receptors lead to NF-κB activation, thus protecting leukemic cells from apoptosis. Dimethyl fumarate (DMF) is an anti-inflammatory and immunoregulatory drug used to treat patients with multiple sclerosis and psoriasis in which it blocks aberrant NF-κB pathways and impacts the NRF2 antioxidant circuit. Our in vitro analysis demonstrated that increasing concentrations of DMF reduce ATP levels and lead to the apoptosis of CLL cells, including cell lines, splenocytes from Eµ-TCL1-transgenic mice, and primary leukemic cells isolated from the peripheral blood of patients. DMF showed a synergistic effect in association with BTK inhibitors in CLL cells. DMF reduced glutathione levels and activated the NRF2 pathway; gene expression analysis suggested that DMF downregulated pathways related to NFKB and inflammation. In primary leukemic cells, DMF disrupted the TLR signaling pathways induced by CpG by reducing the mRNA expression of NFKBIZ, IL6, IL10 and TNFα. Our data suggest that DMF targets a vulnerability of CLL cells linked to their inflammatory pathways, without impacting healthy donor peripheral blood mononuclear cells., (© 2024. The Author(s).)

Chronic lymphocytic leukemia (CLL) is a prototypic neoplasia in which malignant cells strongly depend on microenvironmental stimulations in the lymphoid tissues where they accumulate; leukemic cells are exposed to interaction with bystander and accessory cells, as well as inflammatory soluble mediators. Cell lines are frequently used to model the pathobiology of this disease; however, they do not always recapitulate leukemic cell growth and response to stimulation, and no data are available on Toll-like receptors (TLR) signaling in CLL cell lines. To address this gap, we analyzed HG3, MEC2, and PCL12 cell lines, before and after CpG stimulation, by RNA-sequencing followed by bioinformatic analyses and validation experiments. We identified NFKBIZ mRNA and the corresponding IkBz protein as robust markers of TLR9 activation in both MEC2 and PCL12, but not in HG3 cells. Next, we compared our current results with previous results obtained with primary CLL patient samples and were able to conclude that MEC2 is most similar to the patients' cells in terms of global responsiveness to TLR stimulation; in particular, MEC2 better resembles the samples of patients, as it is characterized by high expression levels of IkBz, but with a lower number of genes regulated., (© 2023 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Chronic lymphocytic leukemia (CLL) co-evolves with its own microenvironment where inflammatory stimuli including toll-like receptors (TLR) signaling can protect CLL cells from spontaneous and drug-induced apoptosis by upregulating IκBζ, an atypical co-transcription factor. To dissect IκBζ-centered signaling pathways, we performed a gene expression profile of primary leukemic cells expressing either high or low levels of IκBζ after stimulation, highlighting that IκBζ is not only an inflammatory gene but it may control metabolic rewiring of malignant cells thus pointing to a novel potential opportunity for therapy. We exploited the capacity of the dimethyl itaconate (DI), an anti-inflammatory electrophilic synthetic derivative of the metabolite Itaconate, to target IκBζ. CLL cells, murine leukemic splenocytes, and leukocytes from healthy donors were treated in vitro with DI that abolished metabolic activation and reduced cell viability of leukemic cells only, even in the presence of robust TLR prestimulation. RNA sequencing highlighted that in addition to the expected electrophilic stress signature observed after DI treatment, novel pathways emerged including the downregulation of distinct MHC class II complex genes. In conclusion, DI not only abrogated the proinflammatory effects of TLR stimulation but also targeted a specific metabolic vulnerability in CLL cells., (© 2023 The Authors. European Journal of Immunology published by Wiley-VCH GmbH.)

Renal cell carcinoma is highly inflamed, and tumor cells are embedded into a microenvironment enriched with IL1. While inflammatory pathways are well characterized in the immune system, less is known about these same pathways in epithelial cells; it is unclear if and how innate immune signals directly impact on cancer cells, and if we could we manipulate these for therapeutic purposes. To address these questions, we first focused on the inflammatory receptors belonging to the IL1- and Toll-like receptor family including negative regulators in a small cohort of 12 clear cell RCC (ccRCC) patients' samples as compared to their coupled adjacent normal tissues. Our data demonstrated that renal epithelial cancer cells showed a specific and distinctive pattern of inflammatory receptor expression marked by a consistent downregulation of the inhibitory receptor SIGIRR mRNA. This repression was confirmed at the protein level in both cancer cell lines and primary tissues. When we analyzed in silico data of different kidney cancer histotypes, we identified the clear cell subtype as the one where SIGIRR was mostly downregulated; nonetheless, papillary and chromophobe tumor types also showed low levels as compared to their normal counterpart. RNA-sequencing analysis demonstrated that IL1 stimulation of the ccRCC cell line A498 triggered an intrinsic signature of inflammatory pathway activation characterized by the induction of distinct "pro-tumor" genes including several chemokines, the autocrine growth factor IL6, the atypical co-transcription factor NFKBIZ, and the checkpoint inhibitor PD-L1. When we looked for the macroareas most represented among the differentially expressed genes, additional clusters emerged including pathways involved in cell differentiation, angiogenesis, and wound healing. To note, SIGIRR overexpression in A498 cells dampened IL1 signaling as assessed by a reduced induction of NFKBIZ. Our results suggest that SIGIRR downregulation unleashes IL1 signaling intrinsic to tumor cells and that manipulating this pathway may be beneficial in ccRCC., Competing Interests: Author MV is employed by Engitix Therapeutics Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Mantione, Sana, Vilia, Riba, Doglioni, Larcher, Capitanio and Muzio.)

Atherothrombosis exposes vascular components to blood. Currently, new antithrombotic therapies are emerging. Herein we investigated thrombogenesis of human arteries with/without atherosclerosis, and the interaction of coagulation and vascular components, we and explored the anti-thrombogenic efficacy of blockade of the P2X purinoceptor 7 (P2X7). A confocal blood flow videomicroscopy system was performed on cryosections of internal mammary artery (IMA) or carotid plaque (CPL) determining/localizing platelets and fibrin. Blood from healthy donors elicited thrombi over arterial layers. Confocal microscopy associated thrombus with tissue presence of collagen type I, laminin, fibrin(ogen) and tissue factor (TF). The addition of antibodies blocking TF (aTF) or factor XI (aFXI) to blood significantly reduced fibrin deposition, variable platelet aggregation and aTF + aFXI almost abolished thrombus formation, showing synergy between coagulation pathways. A scarce effect of aTF over sub-endothelial regions, more abundant in tissue TF and bundles of laminin and collagen type I than deep intima, may suggest tissue thrombogenicity as molecular structure-related. Consistently with TF-related vascular function and expression of P2X7, the sections from CPL but not IMA tissue cultures pre-treated with the P2X7 antagonist A740003 demonstrated poor thrombogenesis in flow experiments. These data hint to local targeting studies on P2X7 modulation for atherothrombosis prevention/therapy.

In recent years significant progress has been made in the clinical management of chronic lymphocytic leukemia (CLL) as well as other B-cell malignancies; targeting proximal B-cell receptor signaling molecules such as Bruton Tyrosine Kinase (BTK) and Phosphoinositide 3-kinase (PI3Kδ) has emerged as a successful treatment strategy. Unfortunately, a proportion of patients are still not cured with available therapeutic options, thus efforts devoted to studying and identifying new potential druggable targets are warranted. B-cell receptor stimulation triggers a complex cascade of signaling events that eventually drives the activation of downstream transcription factors including Nuclear Factor of Activated T cells (NFAT). In this review, we summarize the literature on the expression and function of NFAT family members in CLL where NFAT is not only overexpressed but also constitutively activated; NFAT controls B-cell anergy and targeting this molecule using specific inhibitors impacts on CLL cell viability. Next, we extend our analysis on other mature B-cell lymphomas where a distinct pattern of expression and activation of NFAT is reported. We discuss the therapeutic potential of strategies aimed at targeting NFAT in B-cell malignancies not overlooking the fact that NFAT may play additional roles regulating the inflammatory microenvironment., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Sana, Mantione, Angelillo and Muzio.)

Chronic lymphocytic leukaemia (CLL) cells are strongly influenced by microenvironmental signals through the activation of distinct membrane receptors including the B-cell receptor and toll-like receptors (TLR). Recapitulating TLR stimulation in vitro by treating CLL cells with the TLR9 ligand CpG can induce metabolic activation and protection from apoptosis. We hypothesized that interfering with TLR signalling may be beneficial for treating CLL, and we tested in preclinical studies the effect of a specific interleukin-1 receptor-associated kinase 4 (IRAK4) inhibitory small molecule on primary leukaemic cells isolated from the peripheral blood of patients. We observed that IRAK4, an upstream kinase of the TLR pathway, is expressed in patients with CLL, and lower IRAK4 mRNA levels associate with a better outcome. The specific IRAK4 inhibitor disrupted TLR signalling as assessed by reduction of the specific biomarkers NFKBIZ and interleukin-6 mRNAs, and restrained the protective effect of in vitro TLR stimulation on cell viability. To note, IRAK4 inhibitor induced p53 and triggered apoptosis. Co-treatment of CLL cells with increasing concentrations of IRAK4i and the Bruton tyrosine kinase inhibitor ibrutinib demonstrated a synergistic effect. Our results suggest that targetting IRAK4 may represent a novel approach in CLL and may be combined with other signalling inhibitors., (© 2020 British Society for Haematology and John Wiley & Sons Ltd.)

Background: Vascular smooth muscle cells exhibit phenotypic plasticity in response to microenvironmental stimuli and contribute to vascular remodelling through mechanisms only partially understood. In atherosclerosis, P2X-purinoceptor7 (P2X7) has been related to interleukin-1β (IL-1β) and metalloproteinase 9 (MMP9). The hypoxia-inducible factor-1alpha (HIF1α) was associated to remodelling. Here the activation of IL-1β and MMP9 was studied in relationship to P2X7 and HIF1α in cells exploited from human carotid plaque and internal mammary artery., Methods and Results: Migrating cells expressed HIF1α-regulated canopy FGF-signalling regulator 2 and CD117, and led to primary cells with SMC-like phenotype (VSMC), P2X7 + . We investigated in VSMC the effects of hypoxia, of treatment with tumour necrosis factor-α (TNFα) and/or with P2X7 antagonist, A740003. Quantitative RT-PCR showed that hypoxia unaffected IL-1β and down-regulated MMP9 mRNAs, without activating HIF1α. TNFα increased IL-1β mRNA via NLR Family Pyrin Domain-Containing 3, with production of proIL-1β but no rise of mature IL-1β. Zymography demonstrated that A740003 triggered MMP9 secretion from VSMC. Combination of A740003 with TNFα abrogated this effect. Combination was ineffective on IL-1β activation elicited by TNFα, but down-regulated HIF1α mRNA. A740003 induced the intracellular P2X7 aggregation and differently perturbed lysosome and mitochondria network compared to TNFα., Conclusions: Cells migration from human arteries leads to partially differentiated VSMC analogous to neointimal cells within atherosclerotic lesions. Down-regulated HIF1α in stimulated VSMC translates in resilience in atherosclerotic lesions. P2X7-independent partial activation of IL-1β elicited by TNFα underlines complexity of the cytokine secretion. Data also supported P2X7 as modulator of MMP9 secretion, important for atherosclerosis progression., (Copyright © 2018 Elsevier B.V. All rights reserved.)

In atherosclerosis, matrix metallopeptidases (MMPs) contribute to plaque rupture through weakening of the fibrous cap. Pleiotropic P2X purinoceptor 7 (P2X7), expressed in the carotid plaque (PL), is involved in interleukin 1 beta (IL-1β) release that may influence MMP9 generation, thus their possible modulation through acting on P2X7 was investigated. P2X7-related machinery was characterized and the effects of P2X7 antagonists (A740003, KN62) and MMPs inhibitors (Batimastat, Ro28-2653) were studied in ex-vivo tissue cultures of human PL's vs. non-atherosclerotic internal mammary artery (IMA) by using molecular biology, immune-biochemical and microscopy methodologies. We highlighted atherosclerosis-related differences between PLs and IMAs molecular patterns, and their responsivity to P2X7 antagonism. High IL-1β tissue content was associated with PLs morphology and instability/vulnerability. We demonstrated that A740003, but not KN62, decreased IL-1β and MMP9 independently from NLR family pyrin domain containing 3, but in relationship with patient's smoking status. Acting downstream P2X7 by MMPs inhibitors, diminished IL-1β mRNA without transcriptional effect at MMP9, possibly because the assumption of statin by patients. These data firstly demonstrated A740003 suitability as a specific tool to decrease inflammatory status in human vessels and might support the design of studies applying P2X7 antagonists for the local targeting and tailored therapy of atherosclerosis.