Biology subjects

Maegdefessel, L.

Publications and source records attributed to Maegdefessel, L..

12 recordsLinked to original sources

bioRxiv2023

Loss of myeloid cannabinoid CB1 receptor confers atheroprotection by reducing macrophage proliferation and immunometabolic reprogramming

Although the cannabinoid CB1 receptor has been implicated in atherosclerosis, its cell-specific effects in this disease are not well understood. 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 were reproducible in bone marrow derived macrophages and blunted by estradiol. Kinase activity profiling revealed a CB1-dependent regulation of p53 and cyclin-dependent kinases. Transcriptomic profiling further unveiled 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, hinting to a possible implication of CB1-dependent regulation in human pathophysiology. In conclusion, impaired CB1 signaling in macrophages is atheroprotective by limiting their arterial recruitment, proliferation and inflammatory reprogramming. The importance of macrophage CB1 signaling seems to be more pronounced in male mice. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/535832v2_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@1d0b2f4org.highwire.dtl.DTLVardef@94f1b4org.highwire.dtl.DTLVardef@8868edorg.highwire.dtl.DTLVardef@1a0b151_HPS_FORMAT_FIGEXP M_FIG C_FIG Graphical summary (created with BioRender.com)

immunology↗

bioRxiv2023

The novel coronary artery disease risk factor ADAMTS-7 modulates atherosclerotic plaque formation by degradation of TIMP-1

BackgroundThe ADAMTS7 locus was genome-wide significantly associated with coronary artery disease (CAD). Lack of the extracellular matrix (ECM) protease ADAMTS-7 was shown to reduce atherosclerotic plaque formation. ObjectiveTo identify molecular mechanisms and downstream targets of ADAMTS-7 mediating risk of atherosclerosis. MethodsTargets of ADAMTS-7 were identified by high-resolution mass spectrometry of atherosclerotic plaques from Apoe-/- and Apoe-/-Adamts7-/- mice. ECM proteins were identified using solubility profiling. Putative targets were validated using immunofluorescence, in vitro degradation assays, co-immunoprecipitation, and Forster resonance energy transfer (FRET)-based protein-protein interaction assays. ADAMTS7 expression was measured in fibrous caps of human carotid artery plaques. ResultsIn humans, ADAMTS7 expression was higher in caps of unstable as compared to stable carotid plaques. Compared to Apoe-/- mice, atherosclerotic aortas of Apoe-/- mice lacking Adamts-7 (Apoe-/-Adamts7-/-) contained higher protein levels of tissue inhibitor of metalloproteases 1 (Timp-1). In co-immunoprecipitation experiments, the catalytic domain of ADAMTS-7 bound to TIMP-1, which was degraded in the presence of ADAMTS-7 in vitro. ADAMTS-7 reduced the inhibitory capacity of TIMP-1 at its canonical target matrix metalloprotease 9 (MMP-9) As a downstream mechanism, we investigated collagen content in plaques of Apoe-/- and Apoe-/-Adamts7-/- mice after Western diet. Picrosirius red staining of the aortic root revealed less collagen as a readout of higher MMP-9 activity in Apoe-/- as compared to Apoe-/- Adamts7-/- mice. In order to facilitate high-throughput screening for ADAMTS-7 inhibitors with the aim to decrease TIMP-1 degradation, we designed a FRET-based assay targeting the ADAMTS-7 catalytic site. ConclusionADAMTS-7, which is induced in unstable atherosclerotic plaques, decreases TIMP-1 stability reducing its inhibitory effect on MMP-9, which is known to promote collagen degradation and is likewise genome-wide significantly associated with CAD. Disrupting the interaction of ADAMTS-7 and TIMP-1 might be a strategy to increase collagen content and plaque stability for reduction of atherosclerosis-related events.

molecular biology↗

bioRxiv2023

INKILN is a novel long noncoding RNA promoting vascular smooth muscle inflammation

BackgroundActivation of vascular smooth muscle cells (VSMCs) inflammation is vital to initiate vascular disease. However, the role of human-specific long noncoding RNAs (lncRNAs) in VSMC inflammation is poorly understood. MethodsBulk RNA-seq in differentiated human VSMCs revealed a novel human-specific lncRNA called INflammatory MKL1 Interacting Long Noncoding RNA (INKILN). INKILN expression was assessed in multiple in vitro and ex vivo models of VSMC phenotypic modulation and human atherosclerosis and abdominal aortic aneurysm (AAA) samples. The transcriptional regulation of INKILN was determined through luciferase reporter system and chromatin immunoprecipitation assay. Both loss- and gain-of-function approaches and multiple RNA-protein and protein-protein interaction assays were utilized to uncover the role of INKILN in VSMC proinflammatory gene program and underlying mechanisms. Bacterial Artificial Chromosome (BAC) transgenic (Tg) mice were utilized to study INKLIN expression and function in ligation injury-induced neointimal formation. ResultsINKILN expression is downregulated in contractile VSMCs and induced by human atherosclerosis and abdominal aortic aneurysm. INKILN is transcriptionally activated by the p65 pathway, partially through a predicted NF-{kappa}B site within its proximal promoter. INKILN activates the proinflammatory gene expression in cultured human VSMCs and ex vivo cultured vessels. Mechanistically, INKILN physically interacts with and stabilizes MKL1, a key activator of VSMC inflammation through the p65/NF-{kappa}B pathway. INKILN depletion blocks ILI{beta}-induced nuclear localization of both p65 and MKL1. Knockdown of INKILN abolishes the physical interaction between p65 and MKL1, and the luciferase activity of an NF-{kappa}B reporter. Further, INKILN knockdown enhances MKL1 ubiquitination, likely through the reduced physical interaction with the deubiquitinating enzyme, USP10. INKILN is induced in injured carotid arteries and exacerbates ligation injury-induced neointimal formation in BAC Tg mice. ConclusionsThese findings elucidate an important pathway of VSMC inflammation involving an INKILN/MKL1/USP10 regulatory axis. Human BAC Tg mice offer a novel and physiologically relevant approach for investigating human-specific lncRNAs under vascular disease conditions.

molecular biology↗

bioRxiv2022

The lncRNA landscape of cardiac resident macrophages and identification of Schlafenlnc as a regulator of macrophage migratory function

Cardiac resident macrophages (crMPs) were recently shown to exert pivotal functions in cardiac homeostasis and disease, but the underlying molecular mechanisms are largely unclear. Long non-coding RNAs (lncRNAs) are increasingly recognized as important regulatory molecules in a number of cell types, but neither the identity nor the molecular mechanisms of lncRNAs in crMPs are known. Here, we have employed deep RNA-seq and single cell RNA sequencing to resolve the crMP lncRNA landscape from healthy and diseased murine myocardium. CrMPs express previously unknown and highly cell type-specific lncRNAs, among which one lncRNA, termed Schlafenlnc, was particularly abundant and enriched in crMPs. We found Schlafenlnc to be necessary for migration-associated gene expression in macrophages in vitro and in vivo and essential for their adhesion and migration. Collectively, our data provide a basis to the systematic characterization of lncRNAs in crMPs and establish Schlafenlnc as a critical regulator of macrophage migratory functions.

cell biology↗

bioRxiv2022

PI 3-kinase isoform p110alpha controls smooth muscle cell functionality and protects against aortic aneurysm formation

BackgroundCatalytic class IA PI 3-kinase isoform p110 is a crucial regulator of cellular proliferation and survival in numerous cell types. While p110 is critically involved in pathogenic vascular remodeling, its physiological role for vascular integrity under stress conditions has not been studied. We report a protective function of smooth muscle p110 against abdominal aortic aneurysm (AAA) formation. Methods & ResultsIn mice lacking p110 in smooth muscle cells (sm-p110-/-), perfusion of the infrarenal aorta with porcine pancreatic elastase (PPE) yielded substantially enhanced AAA formation compared to wild type controls. This disease phenotype is partly attributable to a subtle preexisting vascular phenotype under basal conditions, as sm-p110-/- mice displayed a smaller media area, deranged aortic wall structure (detached smooth muscle cells, increased apoptotic cell death), and a diminished functional responsiveness of aortic rings to vasodilators. Furthermore, p110 is also implicated in regenerative processes during AAA development: Whereas wild type mice showed increased media hypertrophy, neointima formation and proliferation upon PPE intervention, these vascular remodeling processes were diminished in sm-p110-/- mice. Concomitantly, increased numbers of elastic fiber breaks and ECM degradation were detected in sm-p110-/- aorta. Mechanistically, we found that lack of p110 expression impaired smooth muscle cell proliferation, expression of contractile marker genes and production of elastin fibers. This phenotype largely depended on reduced phosphorylation and inactivation of FOXO1, as specific FOXO1 inhibition fully rescued proliferation of p110-/- smooth muscle cells, and knockdown of FOXO1 increased expression of calponin and elastin. ConclusionsSmooth muscle p110 protects against AAA disease by maintaining aortic wall homoeostasis and promoting SMC proliferation to compensate for cell loss during AAA development. Our findings have potential implications for current approaches aimed at p110 inhibition for cancer therapy and suggest new pharmacological strategies to activate p110 signaling in AAA disease.

cell biology↗

bioRxiv2022

Utilization of an Artery-on-a-chip to unravel novel regulators and therapeutic targets in vascular diseases

IntroductionOrgans-on-chips represent novel in vitro models that have the capacity to emulate aspects of human physiology and pathophysiology by incorporating features like tissue-multicellularity and exposure to organ-relevant physical environment. We developed an artery-on-a-chip with the objective to recapitulate the structure of the arterial wall composed of intimal and medial layers and the relevant hemodynamic forces that affect luminal cells. ResultsBy comparing arteries-on-chips exposed either to in vivo-like shear stress values or kept in static conditions, we identified a panel of novel genes modulated by shear stress. We next measured the expression pattern of shear stress-modulated genes in areas of the vascular tree affected by atherosclerotic plaques and aortic aneurysms, where disease development and progression are induced by alterations of shear stress. We obtained biopsies from patients affected by carotid artery disease (CAD), comprising the atherosclerotic plaque (diseased artery) and the adjacent region (non-diseased artery). From patients with abdominal aortic aneurysms (AAA), we obtained the aneurysmal portion (diseased aorta) and non-dilated adjacent segment (non-diseased aorta). Genes modulated by shear stress followed the same expression pattern in non-diseased segments of human vessels and were expressed by endothelial and smooth muscle cells as evidenced by immunofluorescence analysis and single cell RNA sequencing. Using mice and porcine models of vascular CAD and AAA, we confirmed that shear stress mediated targets are important in discriminating diseased and non-diseased vessel portions in vivo. Furthermore, we showed that our artery-on-a-chip can serve as a platform for drug-testing. We were able to reproduce the effects of a therapeutic agent previously used in AAA animal models in artery-on-a-chip systems and extend our understanding of its therapeutic effect through a multicellular structure. ConclusionsOur novel in vitro model is capable of mimicking important physiological aspects of human arteries, such as the response to shear stress, and can further shed light on the mechanism of action of potential therapeutics before they enter the clinical stage. TeaserThe artery-on-a-chip is a novel in vitro platform that enables the mimicry of human arteries and can be used to gain insights into the development and therapeutic targeting of vascular diseases.

cell biology↗

bioRxiv2022

Targeting long non-coding RNA NUDT6 enhances smooth muscle cell survival and limits vascular disease progression

Long non-coding RNAs (lncRNAs) orchestrate various biological processes and regulate the development of cardiovascular diseases. Their potential therapeutic benefit to tackle disease progression has recently been extensively explored. Our study investigates the role of lncRNA Nudix Hydrolase 6 (NUDT6) and its antisense target Fibroblast Growth Factor 2 (FGF2) in two vascular pathologies: abdominal aortic aneurysms (AAA) and carotid artery disease. Using tissue samples from both diseases, we detected a substantial increase of NUDT6, whereas FGF2 was downregulated. Targeting Nudt6 in vivo with antisense oligonucleotides in three murine and one porcine animal models of carotid artery disease and AAA limited disease progression. Restoration of FGF2 upon Nudt6 knockdown improved vessel wall morphology and fibrous cap stability. Overexpression of NUDT6 in vitro impaired smooth muscle cell (SMC) migration, while limiting their proliferation and augmenting apoptosis. By employing RNA pulldown followed by mass spectrometry as well as RNA immunoprecipitation, we identified Cysteine and Glycine Rich Protein 1 (CSRP1) as another direct NUDT6 interaction partner, regulating cell motility and SMC differentiation. Overall, the present study identifies NUDT6 as a well-conserved antisense transcript of FGF2. NUDT6 silencing triggers SMC survival and migration and could serve as a novel RNA-based therapeutic strategy in vascular diseases.

molecular biology↗

bioRxiv2022

The circular RNA Ataxia-telangiectasia mutated (cATM) regulates oxidative stress in smooth muscle cells in expanding abdominal aortic aneurysms

An abdominal aortic aneurysm (AAA) is a pathological widening of the aortic wall characterized by loss of smooth muscle cells (SMCs), extracellular matrix degradation, and local inflammation. This condition is often asymptomatic until rupture occurs, leading to high morbidity and mortality rates. Diagnosis is often accidental, and for now, the only available treatment option remains surgical intervention. Circular RNAs (circRNAs) are RNA loops that originated from backsplicing, which have received increasing attention as a novel class of functional non-coding RNAs contributing to cardiovascular physiology and disease. Their high structural stability, combined with a remarkable enrichment in body fluids, make circRNAs promising disease biomarkers. We aimed to investigate the contribution of circRNAs to AAA pathogenesis and their potential application as biomarkers for AAA diagnosis. Combined circRNA array and quantitative real-time PCR analysis revealed the presence of differentially expressed circular transcripts stemming from AAA-relevant gene loci. Among these, the circRNA to the Ataxia-telangiectasia mutated gene (cATM) was upregulated in human AAA tissue specimens, in AAA patient-derived SMCs, and serum samples collected from aneurysm patients. In control primary aortic SMCs, cATM increased upon angiotensin II stimulation, while its silencing triggered apoptosis. Furthermore, doxorubicin could induce cATM expression, supporting a link with acute stress response in SMCs. Constitutively higher cATM expressing AAA patient-derived SMCs were less vulnerable to oxidative stress-induced cell death and survival pathways enriched when compared to control SMCs. Taken together, this data supports the role of cATM in adapting SMCs to oxidative stress in the vascular AAA micromilieu. This molecular signature provides an additional parameter to be included in procedures for AAA screening in combination with already established practices.

molecular biology↗

bioRxiv2022

Aging-regulated TUG1 is dispensable for endothelial cell function

The evolutionary conserved Taurine Upregulated Gene 1 (TUG1) is a ubiquitously expressed gene that is one of the highest expressed genes in human and rodent endothelial cells (ECs). We here show that TUG1 expression decreases significantly in aging mouse carotid artery ECs and human ECs in vitro, indicating a potential role in the aging endothelial vasculature system. We therefore investigated if, and how, TUG1 might function in aging ECs, but despite extensive phenotyping found no alterations in basal EC proliferation, apoptosis, barrier function, migration, mitochondrial function, or monocyte adhesion upon TUG1 silencing in vitro. TUG1 knockdown did slightly and significantly decrease cumulative sprout length upon vascular endothelial growth factor A stimulation in human umbilical vein endothelial cells (HUVECs), though TUG1-silenced HUVECs displayed no transcriptome-wide mRNA expression changes explaining this effect. Further, ectopic expression of the highly conserved and recently discovered 153 amino acid protein translated from certain TUG1 transcript isoforms did not alter angiogenic sprouting in vitro. Our data show that, despite a high expression and strong evolutionary conservation of both the TUG1 locus and the protein sequence it encodes, TUG1 does not seem to play a major role in basic endothelial cell function.

cell biology↗

bioRxiv2021

MIF-2/D-DT is an atypical atherogenic chemokine that promotes advanced atherosclerosis and hepatic lipogenesis

Atherosclerosis is the underlying cause of cardiovascular diseases (CVDs) such as myocardial infarction and ischemic stroke. It is a lipid-triggered chronic inflammatory condition of the arterial vascular wall that is driven by various inflammatory pathways including atherogenic cytokines and chemokines. D-dopachrome tautomerase (D-DT), also known as macrophage migration inhibitory factor-2 (MIF-2), belongs to the MIF protein family, which is best known for its pathogenic role in a variety of inflammatory and immune conditions including CVDs. While MIF is well known as a promoter of atherogenic processes, MIF-2 has not been studied in atherosclerosis. Here, we investigated atherosclerosis in hyperlipidemic Mif-2-/-Apoe-/- mice and studied the role of MIF-2 in various atherogenic assays in vitro. We found that global Mif-2 deficiency as well as its pharmacological blockade by 4-CPPC protected against atherosclerotic lesion formation and vascular inflammation in models of early and advanced atherogenesis. On cellular level, MIF-2 promoted monocyte migration in 2D and 3D and monocyte arrest on aortic endothelial monolayers, promoted B-cell chemotaxis in vitro and B-cell homing in vivo, and increased macrophage foam cell formation. Dose curves and direct comparison in a 3D migration set-up suggest that MIF-2 may be a more potent chemokine than MIF for monocytes and B cells. We identify CXCR4 as a novel receptor for MIF-2. The evidence relies on a CXCR4 inhibitor, CXCR4 internalization experiments, MIF-2/CXCR4 binding studies by yeast-CXCR4 transformants, and fluorescence spectroscopic titrations with a soluble CXCR4 surrogate. Of note, Mif-2-/-Apoe-/- mice exhibited decreased plasma cholesterol and triglyceride levels, lower body weights, smaller livers, and profoundly reduced hepatic lipid accumulation compared to Apoe-/- mice. Mechanistic experiments in Huh-7 hepatocytes suggest that MIF-2 regulates the expression and activation of sterol-regulatory element binding protein-1 and -2 (SREBP-1, SREBP-2) to induce lipogenic downstream genes such as FASN and LDLR, while it attenuated the activation of the SREBP inhibiting AMPK pathway. Studies using receptor Inhibitors showed that SREBP activation and hepatic lipoprotein uptake by MIF-2 is mediated by both CXCR4 and CD74. Lastly and in line with a combined role of MIF-2 in vascular inflammation and hepatic lipid accumulation, MIF-2 was found to be profoundly upregulated in unstable human carotid plaques, underscoring a critical role for MIF-2 in advanced stages of atherosclerosis. Together, these data identify MIF-2 as a novel atherogenic chemokine and CXCR4 ligand that not only promotes lesion formation and vascular inflammation but also strongly affects hepatic lipogenesis in an SREBP-mediated manner, possibly linking atherosclerosis and hepatic steatosis.

immunology↗

bioRxiv2021

GPR55 in B cells limits atherosclerosis development and regulates plasma cell maturation

Identifying novel pathways regulating the adaptive immune response in chronic inflammatory diseases such as atherosclerosis is of particular interest in view of developing new therapeutic drugs. Here we report that the lipid receptor GPR55 is highly expressed by splenic B cells and inversely correlates with atheroma plaque size in mice. In human carotid endarterectomy specimen, GPR55 transcript levels were significantly lower in unstable compared to stable carotid plaques. To study the impact of GPR55 deficiency in atherosclerosis, we crossed Gpr55 knockout mice with apolipoprotein E (ApoE) knockout mice and subjected the mice to Western diet for 4 to 16 weeks. Compared to ApoE-/- controls, ApoE-/-Gpr55-/- mice developed larger plaques with increased necrotic core size, associated with elevated circulating and aortic leukocyte counts. Flow cytometry, immunofluorescence and RNA-sequencing analysis of splenic B cells in these mice revealed a hyperactivated B cell phenotype with disturbed plasma cell maturation and immunoglobulin (Ig)G antibody overproduction. The specific contribution of B cell GPR55 in atherosclerosis was further studied in mixed Gpr55-/-/{micro}MT bone marrow chimeras on low density receptor deficiency (Ldlr-/-) background, revealing that B-cell specific depletion of Gpr55 was sufficient to promote plaque development. Conversely, adoptive transfer of wildtype B cells into ApoE-/-Gpr55-/- mice blunted the proatherogenic phenotype. In vitro stimulation of splenocytes with the endogenous GPR55 ligand LPI promoted plasma cell proliferation and enhanced B cell activation marker expression, which was inhibited by the GPR55 antagonist CID16020046. Collectively, these discoveries provide new evidence for GPR55 as key modulator of the adaptive immune response in atherosclerosis. Targeting GPR55 could be useful to limit inflammation and plaque progression in patients suffering from atherosclerosis.

immunology↗

bioRxiv2021

Heterocomplexes between the Atypical Chemokine MIF and the CXC-Motif Chemokine CXCL4L1 Regulate Inflammation and Thrombus Formation

To fulfil their orchestrating function in immune cell trafficking in homeostasis and disease, a network of 49 chemokines and 23 receptors capitalizes on features of specificity, redundancy, and functional selectivity such as biased agonism. The discovery of the chemokine interactome, i.e. heteromeric chemokine-chemokine interactions, even across CC- and CXC-class borders, has further expanded the complexity within the network. Moreover, some inflammatory mediators, which are not structurally linked to classical CC-, CXC-, CX3C-, or C-chemokines, can bind to chemokine receptors and behave as atypical chemokines (ACKs). We identified the cytokine macrophage migration inhibitory factor (MIF) as an ACK that binds to the chemokine receptors CXCR2 and CXCR4 to promote atherogenic leukocyte recruitment. Here, we hypothesized that chemokine-chemokine interactions extend to ACKs and that MIF may form heterocomplexes with classical chemokines. We tested this hypothesis, applying an unbiased chemokine protein binding array. The platelet chemokine CXCL4L1, but not its variant CXCL4 or the CXCR2/CXCR4 ligands CXCL8 or CXCL12, was identified as a candidate interactor. MIF/CXCL4L1 complexation was verified by co-immunoprecipitation, surface plasmon-resonance analysis, and microscale thermophoresis, which also established high-affinity binding (KD{asymp}100-150 nM). The binding interface was predicted by peptide array-based mapping and molecular docking. We next determined whether heterocomplex formation modulates inflammatory and atherogenic activities of MIF. MIF-elicited T-cell chemotaxis as assessed in a 3D-matrix-based live cell-imaging set-up was abrogated, when cells were co-incubated with MIF and CXCL4L1. Heterocomplexation also blocked MIF-triggered migration of Egfp+ microglia in cortical cultures in situ. Of note, CXCL4L1 blocked the binding of Alexa-MIF to a soluble ectodomain mimic of CXCR4 and co-incubation with CXCL4L1 attenuated MIF-triggered dynamic mass redistribution in HEK293-CXCR4 transfectants, indicating that complex formation interferes with MIF/CXCR4 pathways. As MIF and CXCL4L1 are abundant platelet products, we finally tested their role in platelet activation. Multi-photon microscopy, FLIM- FRET, and proximity ligation assay visualized heterocomplexes in platelet aggregates and clinical human thrombus sections. Moreover, heterocomplex formation inhibited MIF- stimulated thrombus formation under flow and skewed the morphology of adhering platelets from a large to a small lamellipodia phenotype. Together, our study establishes a novel molecular interaction, adding to the complexity of the chemokine interactome and chemokine/receptor network. MIF/CXCL4L1, or more generally, ACK/CXC-motif chemokine heterocomplexes may be promising target structures to modulate inflammation and thrombosis.

immunology↗