The calcium-sensing-receptor (CaSR) in adipocytes contributes to sex-differences in the susceptibility to high fat diet induced obesity and atherosclerosis.

BACKGROUND: Female mice are more resistant to obesogenic effects of a high-fat diet (HFD), compared to male mice. Although the underlying mechanisms are poorly understood, sex hormones seem to play an important role. Interestingly, the activity of the oestrogen receptor-α (ERα) is affected by the calcium-sensing-receptor (CaSR). Therefore, we investigated sex-differences upon diet-induced obesity and the role of adipocyte-specific CaSR herein. METHODS: Adipocyte-specific Casr deficient mice (AdipoqCre+Casrflox) and control mice (Casrflox) were injected with AAV8-PCSK9 to make them prone to develop atherosclerosis and fed an obesity-inducing diet for 12 weeks. FINDINGS: Female mice have lower visceral white adipose tissue (vWAT) mass compared to male mice, while this sex-difference is abolished upon adipocyte-specific Casr deficiency. Furthermore, while females showed elevated levels of inflammatory cytokines and CD3+CD8+ T cell accumulation in vWAT, compared to males, adipocyte-specific Casr deficiency abrogated this sex-phenotype and demonstrated an inhibition of inflammatory signalling pathways. The expression of Erα, as well as associated genes involved in adipocyte differentiation, was increased in female mice in a mostly adipocyte-specific Casr dependent manner. Interestingly, circulating lipid levels were reduced in female compared to male mice, which correlated with decreased atherosclerotic plaque formation. These systemic effects were abrogated upon adipocyte-specific Casr deficiency. INTERPRETATION: Our findings indicate that female mice show a more pronounced vWAT dysfunction compared to males upon obesity. This sex effect is abolished upon adipocyte-specific Casr deficiency. In contrast, females show diminished atherosclerotic plaque formation compared to males, an effect that was abrogated by adipocyte-specific Casr deficiency. FUNDING: This work was supported by a grant from the Interdisciplinary Center for Clinical Research within the faculty of Medicine at the RWTH Aachen University, by the Corona Foundation, by the Deutsche Forschungsgemeinschaft (DFG), the BMBF and Free State of Bavaria and the DZHK.

Navigating the Maze of Kinases: CaMK-like Family Protein Kinases and Their Role in Atherosclerosis.

Circulating low-density lipoprotein (LDL) levels are a major risk factor for cardiovascular diseases (CVD), and even though current treatment strategies focusing on lowering lipid levels are effective, CVD remains the primary cause of death worldwide. Atherosclerosis is the major cause of CVD and is a chronic inflammatory condition in which various cell types and protein kinases play a crucial role. However, the underlying mechanisms of atherosclerosis are not entirely understood yet. Notably, protein kinases are highly druggable targets and represent, therefore, a novel way to target atherosclerosis. In this review, the potential role of the calcium/calmodulin-dependent protein kinase-like (CaMKL) family and its role in atherosclerosis will be discussed. This family consists of 12 subfamilies, among which are the well-described and conserved liver kinase B1 (LKB1) and 5' adenosine monophosphate-activated protein kinase (AMPK) subfamilies. Interestingly, LKB1 plays a key role and is considered a master kinase within the CaMKL family. It has been shown that LKB1 signaling leads to atheroprotective effects, while, for example, members of the microtubule affinity-regulating kinase (MARK) subfamily have been described to aggravate atherosclerosis development. These observations highlight the importance of studying kinases and their signaling pathways in atherosclerosis, bringing us a step closer to unraveling the underlying mechanisms of atherosclerosis.

Myeloid cannabinoid CB1 receptor deletion confers atheroprotection in male mice by reducing macrophage proliferation in a sex-dependent manner.

AIMS: Although the cannabinoid CB1 receptor has been implicated in atherosclerosis, its cell-specific effects in this disease are not well understood. To address this, we generated a transgenic mouse model to study the role of myeloid CB1 signaling in atherosclerosis. METHODS AND RESULTS: Here, we report that male mice with myeloid-specific Cnr1 deficiency on atherogenic background developed smaller lesions and necrotic cores than controls, while only minor genotype differences were observed in females. Male Cnr1 deficient mice showed reduced arterial monocyte recruitment and macrophage proliferation with less inflammatory phenotype. The sex-specific differences in proliferation were dependent on estrogen receptor (ER)α-estradiol signaling. Kinase activity profiling identified a CB1-dependent regulation of p53 and cyclin-dependent kinases. Transcriptomic profiling further revealed chromatin modifications, mRNA processing and mitochondrial respiration among the key processes affected by CB1 signaling, which was supported by metabolic flux assays. Chronic administration of the peripherally-restricted CB1 antagonist JD5037 inhibited plaque progression and macrophage proliferation, but only in male mice. Finally, CNR1 expression was detectable in human carotid endarterectomy plaques and inversely correlated with proliferation, oxidative metabolism and inflammatory markers, suggesting a possible implication of CB1-dependent regulation in human pathophysiology. CONCLUSION: Impaired macrophage CB1 signaling is atheroprotective by limiting their arterial recruitment, proliferation and inflammatory reprogramming in male mice. The importance of macrophage CB1 signaling appears to be sex-dependent.

Identifying Differences in Molecular Characteristics Relevant for Remodeling of Periodontal Ligament Stem Cells from the Upper and Lower Jaw.

Periodontal defects' localization affects wound healing and bone remodeling, with faster healing in the upper jaw compared to the lower jaw. While differences in blood supply, innervation, and odontogenesis contribute, cell-intrinsic variances may exist. Few studies explored cell signaling in periodontal ligament stem cells (PDLSC), overlooking mandible-maxilla disparitiesUsing kinomics technology, we investigated molecular variances in PDLSC. Characterization involved stem cell surface markers, proliferation, and differentiation capacities. Kinase activity was analyzed via multiplex kinase profiling, mapping differential activity in known gene regulatory networks. Upstream kinase analysis identified stronger EphA receptor expression in the mandible, potentially inhibiting osteogenic differentiation. The PI3K-Akt pathway showed higher activity in lower-jaw PDLSC. PDLSC from the upper jaw exhibit superior proliferation and differentiation capabilities. Differential activation of gene regulatory pathways in upper vs. lower-jaw PDLSC suggests implications for regenerative therapies.

ITIH5 as a multifaceted player in pancreatic cancer suppression, impairing tyrosine kinase signaling, cell adhesion and migration.

Inter-alpha-trypsin inhibitor heavy chain 5 (ITIH5) has been identified as a metastasis suppressor gene in pancreatic cancer. Here, we analyzed ITIH5 promoter methylation and protein expression in The Cancer Genome Atlas (TCGA) dataset and three tissue microarray cohorts (n = 618), respectively. Cellular effects, including cell migration, focal adhesion formation and protein tyrosine kinase activity, induced by forced ITIH5 expression in pancreatic cancer cell lines were studied in stable transfectants. ITIH5 promoter hypermethylation was associated with unfavorable prognosis, while immunohistochemistry demonstrated loss of ITIH5 in the metastatic setting and worsened overall survival. Gain-of-function models showed a significant reduction in migration capacity, but no alteration in proliferation. Focal adhesions in cells re-expressing ITIH5 exhibited a smaller and more rounded phenotype, typical for slow-moving cells. An impressive increase of acetylated alpha-tubulin was observed in ITIH5-positive cells, indicating more stable microtubules. In addition, we found significantly decreased activities of kinases related to focal adhesion. Our results indicate that loss of ITIH5 in pancreatic cancer profoundly affects its molecular profile: ITIH5 potentially interferes with a variety of oncogenic signaling pathways, including the PI3K/AKT pathway. This may lead to altered cell migration and focal adhesion formation. These cellular alterations may contribute to the metastasis-inhibiting properties of ITIH5 in pancreatic cancer.

In situ lipid-loading activates peripheral dendritic cell subsets characterized by cellular ROS accumulation but compromises their capacity to prime naïve T cells.

BACKGROUND AND AIMS: Dendritic cells (DCs), professional antigen-presenting cells, play an important role in pathologies by controlling adaptive immune responses. However, their adaptation to and functionality in hypercholesterolemia, a driving factor in disease onset and progression of atherosclerosis remains to be established. METHODS: In this study, we addressed the immediate impact of high fat diet-induced hypercholesterolemia in low-density lipoprotein receptor deficient (Ldlr-/-) mice on separate DC subsets, their compartmentalization and functionality. RESULTS: While hypercholesterolemia induced a significant rise in bone marrow myeloid and dendritic cell progenitor (MDP) frequency and proliferation rate after high fat diet feeding, it did not affect DC subset numbers in lymphoid tissue. Hypercholesterolemia led to almost immediate and persistent augmentation in granularity of conventional DCs (cDCs), in particular cDC2, reflecting progressive lipid accumulation by these subsets. Plasmacytoid DCs were only marginally and transiently affected. Lipid loading increased co-stimulatory molecule expression and ROS accumulation by cDC2. Despite this hyperactivation, lipid-laden cDC2 displayed a profoundly reduced capacity to stimulate naïve CD4+ T cells. CONCLUSION: Our data provide evidence that in hypercholesterolemic conditions, peripheral cDC2 subsets engulf lipids in situ, leading to a more activated status characterized by cellular ROS accumulation while, paradoxically, compromising their T cell priming ability. These findings will have repercussions not only for lipid driven cardiometabolic disorders like atherosclerosis, but also for adaptive immune responses to pathogens and/or endogenous (neo) antigens under conditions of hyperlipidemia.

ADAM10 and ADAM17, Major Regulators of Chronic Kidney Disease Induced Atherosclerosis?

Chronic kidney disease (CKD) is a major health problem, affecting millions of people worldwide, in particular hypertensive and diabetic patients. CKD patients suffer from significantly increased cardiovascular disease (CVD) morbidity and mortality, mainly due to accelerated atherosclerosis development. Indeed, CKD not only affects the kidneys, in which injury and maladaptive repair processes lead to local inflammation and fibrosis, but also causes systemic inflammation and altered mineral bone metabolism leading to vascular dysfunction, calcification, and thus, accelerated atherosclerosis. Although CKD and CVD individually have been extensively studied, relatively little research has studied the link between both diseases. This narrative review focuses on the role of a disintegrin and metalloproteases (ADAM) 10 and ADAM17 in CKD and CVD and will for the first time shed light on their role in CKD-induced CVD. By cleaving cell surface molecules, these enzymes regulate not only cellular sensitivity to their micro-environment (in case of receptor cleavage), but also release soluble ectodomains that can exert agonistic or antagonistic functions, both locally and systemically. Although the cell-specific roles of ADAM10 and ADAM17 in CVD, and to a lesser extent in CKD, have been explored, their impact on CKD-induced CVD is likely, yet remains to be elucidated.

Endothelial ADAM10 controls cellular response to oxLDL and its deficiency exacerbates atherosclerosis with intraplaque hemorrhage and neovascularization in mice.

Introduction: The transmembrane protease A Disintegrin And Metalloproteinase 10 (ADAM10) displays a "pattern regulatory function," by cleaving a range of membrane-bound proteins. In endothelium, it regulates barrier function, leukocyte recruitment and angiogenesis. Previously, we showed that ADAM10 is expressed in human atherosclerotic plaques and associated with neovascularization. In this study, we aimed to determine the causal relevance of endothelial ADAM10 in murine atherosclerosis development in vivo. Methods and results: Endothelial Adam10 deficiency (Adam10 ecko ) in Western-type diet (WTD) fed mice rendered atherogenic by adeno-associated virus-mediated PCSK9 overexpression showed markedly increased atherosclerotic lesion formation. Additionally, Adam10 deficiency was associated with an increased necrotic core and concomitant reduction in plaque macrophage content. Strikingly, while intraplaque hemorrhage and neovascularization are rarely observed in aortic roots of atherosclerotic mice after 12 weeks of WTD feeding, a majority of plaques in both brachiocephalic artery and aortic root of Adam10ecko mice contained these features, suggestive of major plaque destabilization. In vitro, ADAM10 knockdown in human coronary artery endothelial cells (HCAECs) blunted the shedding of lectin-like oxidized LDL (oxLDL) receptor-1 (LOX-1) and increased endothelial inflammatory responses to oxLDL as witnessed by upregulated ICAM-1, VCAM-1, CCL5, and CXCL1 expression (which was diminished when LOX-1 was silenced) as well as activation of pro-inflammatory signaling pathways. LOX-1 shedding appeared also reduced in vivo, as soluble LOX-1 levels in plasma of Adam10ecko mice was significantly reduced compared to wildtypes. Discussion: Collectively, these results demonstrate that endothelial ADAM10 is atheroprotective, most likely by limiting oxLDL-induced inflammation besides its known role in pathological neovascularization. Our findings create novel opportunities to develop therapeutics targeting atherosclerotic plaque progression and stability, but at the same time warrant caution when considering to use ADAM10 inhibitors for therapy in other diseases.

Ex Vivo Perfusion System to Analyze Chemokine-Driven Leukocyte Adhesion.

At the moment, many researchers are using in vitro techniques to investigate chemokine-driven leukocyte adhesion/recruitment, for example, by using a transwell or flow chamber system. Here we describe a more physiologically relevant, sophisticated, and highly flexible method to study leukocyte adhesion ex vivo in fresh murine carotid arteries under arterial flow conditions. This model mimics an in vivo situation and allows the combination of leukocytes and arteries isolated from different donors in one experiment, generating information on both vascular and leukocyte adhesive properties of both donors. This method provides a versatile, highly physiologically relevant model to investigate leukocyte adhesion.

In Vitro (Trans)Migration Experiment Using Chemokines as Stimulatory Factor.

Transmigration assays, and the use of the Boyden chamber, became one of the most used tools to assess cell motility, invasion, and chemotaxis. The classical Boyden chamber consists of two compartments separated by a membrane representing a physical barrier, which cells have to overcome by active migration. A large variety of Boyden chambers are available and can be customized to fit the experiment by choosing pore size, density, and membrane type. The method described in this chapter intends to measure the migration of mouse T cells towards the chemoattractant CCL25, as a practical example of such (trans)migration experiment that can be further adopted to individual needs and requirements.

MicroRNA-26b Attenuates Platelet Adhesion and Aggregation in Mice.

Platelets are key regulators of haemostasis, making platelet dysfunction a major driver of thrombosis. Numerous processes that determine platelet function are influenced by microRNAs (miRs). MiR-26b is one of the highest-expressed miRs in healthy platelets, and its expression in platelets is changed in a diseased state. However, the exact effect of this miR on platelet function has not been studied yet. In this study, we made use of a whole-body knockout of miR-26b in ApoE-deficient mice in order to determine its impact on platelet function, thrombus formation and platelet signalling both ex vivo and in vivo. We show that a whole-body deficiency of miR-26b exacerbated platelet adhesion and aggregation ex vivo. Additionally, in vivo, platelets adhered faster, and larger thrombi were formed in mice lacking miR-26b. Moreover, isolated platelets from miR-26b-deficient mice showed a hyperactivated Src and EGFR signalling. Taken together, we show here for the first time that miR-26b attenuates platelet adhesion and aggregation, possibly through Src and EGFR signalling.

Endothelial Retargeting of AAV9 In Vivo.

Adeno-associated viruses (AAVs) are frequently used for gene transfer and gene editing in vivo, except for endothelial cells, which are remarkably resistant to unmodified AAV-transduction. AAVs are retargeted here toward endothelial cells by coating with second-generation polyamidoamine dendrimers (G2) linked to endothelial-affine peptides (CNN). G2CNN AAV9-Cre (encoding Cre recombinase) are injected into mTmG-mice or mTmG-pigs, cell-specifically converting red to green fluorescence upon Cre-activity. Three endothelial-specific functions are assessed: in vivo quantification of adherent leukocytes after systemic injection of - G2CNN AAV9 encoding 1) an artificial adhesion molecule (S1FG) in wildtype mice (day 10) or 2) anti-inflammatory Annexin A1 (Anxa1) in ApoE-/- mice (day 28). Moreover, 3) in Cas9-transgenic mice, blood pressure is monitored till day 56 after systemic application of G2CNN AAV9-gRNAs, targeting exons 6-10 of endothelial nitric oxide synthase (eNOS), a vasodilatory enzyme. G2CNN AAV9-Cre transduces microvascular endothelial cells in mTmG-mice or mTmG-pigs. Functionally, G2CNN AAV9-S1FG mediates S1FG-leukocyte adhesion, whereas G2CNN AAV9-Anxa1-application reduces long-term leukocyte recruitment. Moreover, blood pressure increases in Cas9-expressing mice subjected to G2CNN AAV9-gRNAeNOS . Therefore, G2CNN AAV9 may enable gene transfer in vascular and atherosclerosis models.

Loss of soluble guanylyl cyclase in platelets contributes to atherosclerotic plaque formation and vascular inflammation.

Variants in genes encoding the soluble guanylyl cyclase (sGC) in platelets are associated with coronary artery disease (CAD) risk. Here, by using histology, flow cytometry and intravital microscopy, we show that functional loss of sGC in platelets of atherosclerosis-prone Ldlr-/- mice contributes to atherosclerotic plaque formation, particularly via increasing in vivo leukocyte adhesion to atherosclerotic lesions. In vitro experiments revealed that supernatant from activated platelets lacking sGC promotes leukocyte adhesion to endothelial cells (ECs) by activating ECs. Profiling of platelet-released cytokines indicated that reduced platelet angiopoietin-1 release by sGC-depleted platelets, which was validated in isolated human platelets from carriers of GUCY1A1 risk alleles, enhances leukocyte adhesion to ECs. I mp or ta ntly, p ha rm ac ol ogical sGC stimulation increased platelet angiopoietin-1 release in vitro and reduced leukocyte recruitment and atherosclerotic plaque formation in atherosclerosis-prone Ldlr-/- mice. Therefore, pharmacological sGC stimulation might represent a potential therapeutic strategy to prevent and treat CAD.

Key Chemokine Pathways in Atherosclerosis and Their Therapeutic Potential.

The search to improve therapies to prevent or treat cardiovascular diseases (CVDs) rages on, as CVDs remain a leading cause of death worldwide. Here, the main cause of CVDs, atherosclerosis, and its prevention, take center stage. Chemokines and their receptors have long been known to play an important role in the pathophysiological development of atherosclerosis. Their role extends from the initiation to the progression, and even the potential regression of atherosclerotic lesions. These important regulators in atherosclerosis are therefore an obvious target in the development of therapeutic strategies. A plethora of preclinical studies have assessed various possibilities for targeting chemokine signaling via various approaches, including competitive ligands and microRNAs, which have shown promising results in ameliorating atherosclerosis. Developments in the field also include detailed imaging with tracers that target specific chemokine receptors. Lastly, clinical trials revealed the potential of various therapies but still require further investigation before commencing clinical use. Although there is still a lot to be learned and investigated, it is clear that chemokines and their receptors present attractive yet extremely complex therapeutic targets. Therefore, this review will serve to provide a general overview of the connection between various chemokines and their receptors with atherosclerosis. The different developments, including mouse models and clinical trials that tackle this complex interplay will also be explored.

Design, synthesis, and biological evaluation of novel pyrrolidinone small-molecule Formyl peptide receptor 2 agonists.

A series of Formyl peptide receptor 2 small molecule agonists with a pyrrolidinone scaffold, derived from a combination of pharmacophore modelling and docking studies, were designed and synthesized. The GLASS (GPCR-Ligand Association) database was screened using a pharmacophore model. The most promising novel ligand structures were chosen and then tested in cellular assays (calcium mobilization and ß-arrestin assays). Amongst the selected ligands, two pyrrolidinone compounds (7 and 8) turned out to be the most active. Moreover compound 7 was able to reduce the number of adherent neutrophils in a human neutrophil static adhesion assay which indicates its anti-inflammatory and proresolving properties. Further exploration and optimization of new ligands showed that heterocyclic rings, e.g. pyrazole directly connected to the pyrrolidinone scaffold, provide good stability and a boost in the agonistic activity. The compounds of most interest (7 and 30) were tested in an ERK phosphorylation assay, demonstrating selectivity towards FPR2 over FPR1. Compound 7 was examined in an in vivo mouse pharmacokinetic study. Compound 7 may be a valuable in vivo tool and help improve understanding of the role of the FPR2 receptor in the resolution of inflammation process.

SARS-CoV-2 seroprevalence survey among health care providers in a Belgian public multiple-site hospital.

Although the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic is lasting for more than 1 year, the exposition risks of health-care providers are still unclear. Available evidence is conflicting. We investigated the prevalence of antibodies against SARS-CoV-2 in the staff of a large public hospital with multiple sites in the Antwerp region of Belgium. Risk factors for infection were identified by means of a questionnaire and human resource data. We performed hospital-wide serology tests in the weeks following the first epidemic wave (16 March to the end of May 2020) and combined the results with the answers from an individual questionnaire. Overall seroprevalence was 7.6%. We found higher seroprevalences in nurses [10.0%; 95% confidence interval (CI) 8.9-11.2] than in physicians 6.4% (95% CI 4.6-8.7), paramedical 6.0% (95% CI 4.3-8.0) and administrative staff (2.9%; 95% CI 1.8-4.5). Staff who indicated contact with a confirmed coronavirus disease 2019 (COVID-19) colleague had a higher seroprevalence (12.0%; 95% CI 10.7-13.4) than staff who did not (4.2%; 95% CI 3.5-5.0). The same findings were present for contacts in the private setting. Working in general COVID-19 wards, but not in emergency departments or intensive care units, was also a significant risk factor. Since our analysis points in the direction of active SARS-CoV-2 transmission within hospitals, we argue for implementing a stringent hospital-wide testing and contact-tracing policy with special attention to the health care workers employed in general COVID-19 departments. Additional studies are needed to establish the transmission dynamics.

Synthesis and evaluation of novel cyclopentane urea FPR2 agonists and their potential application in the treatment of cardiovascular inflammation.

The discovery of natural specialized pro-resolving mediators and their corresponding receptors, such as formyl peptide receptor 2 (FPR2), indicated that resolution of inflammation (RoI) is an active process which could be harnessed for innovative approaches to tame pathologies with underlying chronic inflammation. In this work, homology modelling, molecular docking and pharmacophore studies were deployed to assist the rationalization of the structure-activity relationships of known FPR2 agonists. The developed pharmacophore hypothesis was then used in parallel with the homology model for the design of novel ligand structures and in virtual screening. In the first round of optimization compound 8, with a cyclopentane core, was chosen as the most promising agonist (ß-arrestin recruitment EC50 = 20 nM and calcium mobilization EC50 = 740 nM). In a human neutrophil static adhesion assay, compound 8 decreased the number of adherent neutrophils in a concentration dependent manner. Further investigation led to the more rigid cycloleucines (compound 22 and 24) with improved ADME profiles and maintaining FPR2 activity. Overall, we identified novel cyclopentane urea FPR2 agonists with promising ADMET profiles and the ability to suppress the inflammatory process by inhibiting the neutrophil adhesion cascade, which indicates their anti-inflammatory and pro-resolving properties.

Comparison of inhibitory effects of irreversible and reversible Btk inhibitors on platelet function.

All irreversible Bruton tyrosine kinase (Btk) inhibitors including ibrutinib and acalabrutinib induce platelet dysfunction and increased bleeding risk. New reversible Btk inhibitors were developed, like MK-1026. The mechanism underlying increased bleeding tendency with Btk inhibitors remains unclear. We investigated the effects of ibrutinib, acalabrutinib and MK-1026 on platelet function in healthy volunteers, patients and Btk-deficient mice, together with off-target effects on tyrosine kinase phosphorylation. All inhibitors suppressed GPVI- and CLEC-2-mediated platelet aggregation, activation and secretion in a dose-dependent manner. Only ibrutinib inhibited thrombus formation on vWF-co-coated surfaces, while on collagen this was not affected. In blood from Btk-deficient mice, collagen-induced thrombus formation under flow was reduced, but preincubation with either inhibitor was without additional effects. MK-1026 showed less off-target effects upon GPVI-induced TK phosphorylation as compared to ibrutinib and acalabrutinib. In ibrutinib-treated patients, GPVI-stimulated platelet activation, and adhesion on vWF-co-coated surfaces were inhibited, while CLEC-2 stimulation induced variable responses. The dual inhibition of GPVI and CLEC-2 signalling by Btk inhibitors might account for the increased bleeding tendency, with ibrutinib causing more high-grade bleedings due to additional inhibition of platelet-vWF interaction. As MK-1026 showed less off-target effects and only affected activation of isolated platelets, it might be promising for future treatment.