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.

Endothelial ACKR3 drives atherosclerosis by promoting immune cell adhesion to vascular endothelium.

Atherosclerosis is the foundation of potentially fatal cardiovascular diseases and it is characterized by plaque formation in large arteries. Current treatments aimed at reducing atherosclerotic risk factors still allow room for a large residual risk; therefore, novel therapeutic candidates targeting inflammation are needed. The endothelium is the starting point of vascular inflammation underlying atherosclerosis and we could previously demonstrate that the chemokine axis CXCL12-CXCR4 plays an important role in disease development. However, the role of ACKR3, the alternative and higher affinity receptor for CXCL12 remained to be elucidated. We studied the role of arterial ACKR3 in atherosclerosis using western diet-fed Apoe-/- mice lacking Ackr3 in arterial endothelial as well as smooth muscle cells. We show for the first time that arterial endothelial deficiency of ACKR3 attenuates atherosclerosis as a result of diminished arterial adhesion as well as invasion of immune cells. ACKR3 silencing in inflamed human coronary artery endothelial cells decreased adhesion molecule expression, establishing an initial human validation of ACKR3's role in endothelial adhesion. Concomitantly, ACKR3 silencing downregulated key mediators in the MAPK pathway, such as ERK1/2, as well as the phosphorylation of the NF-kB p65 subunit. Endothelial cells in atherosclerotic lesions also revealed decreased phospho-NF-kB p65 expression in ACKR3-deficient mice. Lack of smooth muscle cell-specific as well as hematopoietic ACKR3 did not impact atherosclerosis in mice. Collectively, our findings indicate that arterial endothelial ACKR3 fuels atherosclerosis by mediating endothelium-immune cell adhesion, most likely through inflammatory MAPK and NF-kB pathways.

Transcriptome signature of miRNA-26b KO mouse model suggests novel targets.

BACKGROUND: MicroRNAs (miRNAs) are short (20-24 nt) non-coding RNAs that are involved in post-transcriptional regulation of gene expression in multicellular organisms by affecting both the stability and translation of mRNAs. One of the miRNAs that has been shown to play a role in various pathologies like cancer, neurological disorders and cardiovascular diseases is miRNA-26b. However, these studies only demonstrated rather ambiguous associations without revealing a causal relationship. Therefore, the aim of this study is to establish and validate a mouse model which enables the elucidation of the exact role of miRNA-26b in various pathologies. RESULTS: A miRNA-26b-deficient mouse model was established using homologous recombination and validated using PCR. miRNA-26b-deficient mice did not show any physiological abnormalities and no effects on systemic lipid levels, blood parameters or tissue leukocytes. Using next generation sequencing, the gene expression patterns in miRNA-26b-deficient mice were analyzed and compared to wild type controls. This supported the already suggested role of miRNA-26b in cancer and neurological processes, but also revealed novel associations of miRNA-26b with thermogenesis and allergic reactions. In addition, detailed analysis identified several genes that seem to be highly regulated by miRNA-26b, which are linked to the same pathological conditions, further confirming the role of miRNA-26b in these pathologies and providing a strong validation of our mouse model. CONCLUSIONS: miRNA-26b plays an important role in various pathologies, although causal relationships still have to be established. The described mouse model of miRNA-26b deficiency is a crucial first step towards the identification of the exact role of miRNA-26b in various diseases that could identify miRNA-26b as a promising novel diagnostic or even therapeutic target in a broad range of pathologies.

Haematopoietic and cardiac GPR55 synchronize post-myocardial infarction remodelling.

While classical cannabinoid receptors are known to crucially impact on myocardial infarction (MI) repair, a function of the cannabinoid-sensitive receptor GPR55 herein is poorly understood. We investigated the role of GPR55 in cardiac physiology and post-MI inflammation and remodelling. Global GPR55-/- and wildtype (WT) mice were basally characterized or assigned to 1, 3 or 28 days permanent MI and subsequently analysed via pro-inflammatory and pro-hypertrophic parameters. GPR55-/- deficiency was basally associated with bradycardia, increased diastolic LV volume and sarcomere length and a subtle inflammatory phenotype. While infarct size and myeloid cell infiltration were unaffected by GPR55 depletion, acute cardiac chemokine production was prolonged post-MI. Concurrently, GPR55-/- hearts exhibited a premature expansion of pro-reparative and phagocytic macrophages paralleled by early up-regulation of extracellular matrix (ECM) factors 3 days post-MI, which could be mimicked by sole haematopoietic GPR55 depletion. Moreover, global GPR55 deficiency mitigated MI-induced foetal gene re-programming and cardiomyocyte hypertrophy, culminating in aggravated LV dilatation and infarct expansion. GPR55 regulates cardiac homeostasis and ischaemia responses by maintaining adequate LV filling and modulating three crucial processes post-MI: wound healing kinetics, cardiomyocyte hypertrophy and maladaptive remodelling.

The Microbiota Promotes Arterial Thrombosis in Low-Density Lipoprotein Receptor-Deficient Mice.

Atherosclerotic plaque development depends on chronic inflammation of the arterial wall. A dysbiotic gut microbiota can cause low-grade inflammation, and microbiota composition was linked to cardiovascular disease risk. However, the role of this environmental factor in atherothrombosis remains undefined. To analyze the impact of gut microbiota on atherothrombosis, we rederived low-density lipoprotein receptor-deficient (Ldlr-/- ) mice as germfree (GF) and kept these mice for 16 weeks on an atherogenic high-fat Western diet (HFD) under GF isolator conditions and under conventionally raised specific-pathogen-free conditions (CONV-R). In spite of reduced diversity of the cecal gut microbiome, caused by atherogenic HFD, GF Ldlr-/- mice and CONV-R Ldlr-/- mice exhibited atherosclerotic lesions of comparable sizes in the common carotid artery. In contrast to HFD-fed mice, showing no difference in total cholesterol levels, CONV-R Ldlr-/- mice fed control diet (CD) had significantly reduced total plasma cholesterol, very-low-density lipoprotein (VLDL), and LDL levels compared with GF Ldlr-/- mice. Myeloid cell counts in blood as well as leukocyte adhesion to the vessel wall at the common carotid artery of GF Ldlr-/- mice on HFD were diminished compared to CONV-R Ldlr-/- controls. Plasma cytokine profiling revealed reduced levels of the proinflammatory chemokines CCL7 and CXCL1 in GF Ldlr-/- mice, whereas the T-cell-related interleukin 9 (IL-9) and IL-27 were elevated. In the atherothrombosis model of ultrasound-induced rupture of the common carotid artery plaque, thrombus area was significantly reduced in GF Ldlr-/- mice relative to CONV-R Ldlr-/- mice. Ex vivo, this atherothrombotic phenotype was explained by decreased adhesion-dependent platelet activation and thrombus growth of HFD-fed GF Ldlr-/- mice on type III collagen.IMPORTANCE Our results demonstrate a functional role for the commensal microbiota in atherothrombosis. In a ferric chloride injury model of the carotid artery, GF C57BL/6J mice had increased occlusion times compared to colonized controls. Interestingly, in late atherosclerosis, HFD-fed GF Ldlr-/- mice had reduced plaque rupture-induced thrombus growth in the carotid artery and diminished ex vivo thrombus formation under arterial flow conditions.

Hematopoietic ChemR23 (Chemerin Receptor 23) Fuels Atherosclerosis by Sustaining an M1 Macrophage-Phenotype and Guidance of Plasmacytoid Dendritic Cells to Murine Lesions-Brief Report.

Objective- Expression of the chemokine-like receptor ChemR23 (chemerin receptor 23) has been specifically attributed to plasmacytoid dendritic cells (pDCs) and macrophages and ChemR23 has been suggested to mediate an inflammatory immune response in these cells. Because chemokine receptors are important in perpetuating chronic inflammation, we aimed to establish the role of ChemR23-deficiency on macrophages and pDCs in atherosclerosis. Approach and Results- ChemR23-knockout/knockin mice expressing eGFP (enhanced green fluorescent protein) were generated and after crossing with apolipoprotein E-deficient ( Apoe-/- ChemR23 e/e) animals were fed a western-type diet for 4 and 12 weeks. Apoe-/- ChemR23 e/e mice displayed reduced lesion formation and reduced leukocyte adhesion to the vessel wall after 4 weeks, as well as diminished plaque growth, a decreased number of lesional macrophages with an increased proportion of M2 cells and a less inflammatory lesion composition after 12 weeks of western-type diet feeding. Hematopoietic ChemR23-deficiency similarly reduced atherosclerosis. Additional experiments revealed that ChemR23-deficiency induces an alternatively activated macrophage phenotype, an increased cholesterol efflux and a systemic reduction in pDC frequencies. Consequently, expression of the pDC marker SiglecH in atherosclerotic plaques of Apoe-/- ChemR23 e/e mice was declined. ChemR23-knockout pDCs also exhibited a reduced migratory capacity and decreased CCR (CC-type chemokine receptor)7 expression. Finally, adoptive transfer of sorted wild-type and knockout pDCs into Apoe-/- recipient mice revealed reduced accumulation of ChemR23-deficient pDCs in atherosclerotic lesions. Conclusions- Hematopoietic ChemR23-deficiency increases the proportion of alternatively activated M2 macrophages in atherosclerotic lesions and attenuates pDC homing to lymphatic organs and recruitment to atherosclerotic lesions, which synergistically restricts atherosclerotic plaque formation and progression.

Whole body and hematopoietic ADAM8 deficiency does not influence advanced atherosclerotic lesion development, despite its association with human plaque progression.

Although A Disintegrin And Metalloproteinase 8 (ADAM8) is not crucial for tissue development and homeostasis, it has been implicated in various inflammatory diseases by regulating processes like immune cell recruitment and activation. ADAM8 expression has been associated with human atherosclerosis development and myocardial infarction, however a causal role of ADAM8 in atherosclerosis has not been investigated thus far. In this study, we examined the expression of ADAM8 in early and progressed human atherosclerotic lesions, in which ADAM8 was significantly upregulated in vulnerable lesions. In addition, ADAM8 expression was most prominent in the shoulder region of human atherosclerotic lesions, characterized by the abundance of foam cells. In mice, Adam8 was highly expressed in circulating neutrophils and in macrophages. Moreover, ADAM8 deficient mouse macrophages displayed reduced secretion of inflammatory mediators. Remarkably, however, neither hematopoietic nor whole-body ADAM8 deficiency in mice affected atherosclerotic lesion size. Additionally, except for an increase in granulocyte content in plaques of ADAM8 deficient mice, lesion morphology was unaffected. Taken together, whole body and hematopoietic ADAM8 does not contribute to advanced atherosclerotic plaque development, at least in female mice, although its expression might still be valuable as a diagnostic/prognostic biomarker to distinguish between stable and unstable lesions.

Evaluation of the BDCA2-DTR Transgenic Mouse Model in Chronic and Acute Inflammation.

BACKGROUND AND AIMS: Plasmacytoid dendritic cells (pDCs) are a small subset of dendritic cells and the main producers of type I interferons. Besides their contribution to tolerance, they are known to be involved in autoimmune diseases and have recently been implicated in atherosclerosis. However, their precise involvement, particularly in advanced lesion development, remains elusive. Hence, we investigated the role of pDCs in atherogenesis vs atheroprogression by specifically depleting this cell population using the BDCA2-DTR mouse model bred to Apolipoprotein E (Apoe-/-) deficient mice. METHODS AND RESULTS: Our results revealed that continuous diphtheria toxin-induced pDC depletion in Apoe-/- BDCA2-DTR mice receiving a high-fat diet (HFD) for 4 weeks did not alter lesion size or composition. Instead, these mice displayed increased B cell numbers and altered levels of inflammatory cytokines. Analysis of depletion efficiency showed that complete pDC depletion could only be sustained for one week and reoccurring pDCs sorted after 4 weeks did not express DTR anymore. Consequently, we analyzed lesion development in a model of partial carotid ligation, inducing established lesions after 5 weeks of HFD feeding, and only depleted pDCs during the last week of 5 weeks HFD feeding. Despite short-term, but efficient pDC depletion, we observed no differences in atherosclerotic lesion development, but changes in inflammatory cytokine titers. To assure the functionality of the BDCA2-DTR model in acute settings, we additionally examined the effect of pDC depletion in an indirect acute lung injury (iALI) model. This time, efficient pDC depletion resulted in a significantly reduced macrophage and neutrophil accumulation in the lung 12 hours after LPS challenge, underlining a pro-inflammatory role of pDCs in the innate immune response in iALI. CONCLUSION: Taken together, the BDCA2-DTR mouse model only allows efficient pDC depletion for one week, which subsequently restricts its usability to more acute but not chronic inflammatory disease models.

The role of screenings methods and risk profile assessments in prevention and health promotion programmes: an ethnographic analysis.

In prevention and health promotion interventions, screening methods and risk profile assessments are often used as tools for establishing the interventions' effectiveness, for the selection and determination of the health status of participants. The role these instruments fulfil in the creation of effectiveness and the effects these instruments have themselves remain unexplored. In this paper, we have analysed the role screening methods and risk profile assessments fulfil as part of prevention and health promotion programmes in the selection, enrolment and participation of participants. Our analysis showed, that screening methods and health risk assessments create effects as they objectify health risks and/or the health status of individuals, i.e., they select the individuals 'at risk' and indicate the lifestyle modifications these people are required to make in order to improve their health. Yet, these instruments also reduce the group of participants thereby decreasing the possible effect of interventions, as they provide the legitimisation for people to make choices to whether they enrol or not and what lifestyle changes they incorporate into their lives. In other words, they present a space of interaction, in which agency is distributed across the practice nurses, the participants and the instruments. Decisions were not just made upon the projection of the outcomes of these instruments; decisions that were made by both the patients and practice nurses were the resultant of their opinions on these outcomes that were formed in interaction with the instruments.