Vascular CXCR4 Limits Atherosclerosis by Maintaining Arterial Integrity: Evidence From Mouse and Human Studies.

BACKGROUND: The CXCL12/CXCR4 chemokine ligand/receptor axis controls (progenitor) cell homeostasis and trafficking. So far, an atheroprotective role of CXCL12/CXCR4 has only been implied through pharmacological intervention, in particular, because the somatic deletion of the CXCR4 gene in mice is embryonically lethal. Moreover, cell-specific effects of CXCR4 in the arterial wall and underlying mechanisms remain elusive, prompting us to investigate the relevance of CXCR4 in vascular cell types for atheroprotection. METHODS: We examined the role of vascular CXCR4 in atherosclerosis and plaque composition by inducing an endothelial cell (BmxCreERT2-driven)-specific or smooth muscle cell (SMC, SmmhcCreERT2- or TaglnCre-driven)-specific deficiency of CXCR4 in an apolipoprotein E-deficient mouse model. To identify underlying mechanisms for effects of CXCR4, we studied endothelial permeability, intravital leukocyte adhesion, involvement of the Akt/WNT/ß-catenin signaling pathway and relevant phosphatases in VE-cadherin expression and function, vascular tone in aortic rings, cholesterol efflux from macrophages, and expression of SMC phenotypic markers. Finally, we analyzed associations of common genetic variants at the CXCR4 locus with the risk for coronary heart disease, along with CXCR4 transcript expression in human atherosclerotic plaques. RESULTS: The cell-specific deletion of CXCR4 in arterial endothelial cells (n=12-15) or SMCs (n=13-24) markedly increased atherosclerotic lesion formation in hyperlipidemic mice. Endothelial barrier function was promoted by CXCL12/CXCR4, which triggered Akt/WNT/ß-catenin signaling to drive VE-cadherin expression and stabilized junctional VE-cadherin complexes through associated phosphatases. Conversely, endothelial CXCR4 deficiency caused arterial leakage and inflammatory leukocyte recruitment during atherogenesis. In arterial SMCs, CXCR4 sustained normal vascular reactivity and contractile responses, whereas CXCR4 deficiency favored a synthetic phenotype, the occurrence of macrophage-like SMCs in the lesions, and impaired cholesterol efflux. Regression analyses in humans (n=259 796) identified the C-allele at rs2322864 within the CXCR4 locus to be associated with increased risk for coronary heart disease. In line, C/C risk genotype carriers showed reduced CXCR4 expression in carotid artery plaques (n=188), which was furthermore associated with symptomatic disease. CONCLUSIONS: Our data clearly establish that vascular CXCR4 limits atherosclerosis by maintaining arterial integrity, preserving endothelial barrier function, and a normal contractile SMC phenotype. Enhancing these beneficial functions of arterial CXCR4 by selective modulators might open novel therapeutic options in atherosclerosis.

Small but smart: MicroRNAs orchestrate atherosclerosis development and progression.

MicroRNAs (miRNAs) are short non-coding RNA able to bind specific sequences on target messenger RNAs (mRNAs) and thereby to post-transcriptionally modulate gene expression. Being expressed in all vertebrate cell types, miRNAs have emerged as key players in a wide array of biological processes, including cell proliferation, differentiation and apoptosis. Over the past decade, knowledge concerning the contribution of miRNAs to human pathology has grown with an astonishing pace. In particular, a major involvement of miRNAs in atherosclerosis as a leading cause of global mortality has been supported by ample evidence from in vitro, in vivo and clinical studies. This review aims to summarize and highlight current concepts of miRNA function in the continuum of atherogenesis ranging from risk factors (i.e. dyslipidemia, diabetes, hypertension), to endothelial dysfunction up to the events leading to plaque rupture. Areas in need for further research and potential perspectives for translational applications will be scrutinized. This article is part of a Special Issue entitled: MicroRNAs and lipid/energy metabolism and related diseases edited by Carlos Fernández-Hernando and Yajaira Suárez.

RECK (reversion-inducing cysteine-rich protein with Kazal motifs) regulates migration, differentiation and Wnt/ß-catenin signaling in human mesenchymal stem cells.

The membrane-anchored glycoprotein RECK (reversion-inducing cysteine-rich protein with Kazal motifs) inhibits expression and activity of certain matrix metalloproteinases (MMPs), thereby suppressing tumor cell metastasis. However, RECK's role in physiological cell function is largely unknown. Human mesenchymal stem cells (hMSCs) are able to differentiate into various cell types and represent promising tools in multiple clinical applications including the regeneration of injured tissues by endogenous or transplanted hMSCs. RNA interference of RECK in hMSCs revealed that endogenous RECK suppresses the transcription and biosynthesis of tissue inhibitor of metalloproteinases (TIMP)-2 but does not influence the expression of MMP-2, MMP-9, membrane type (MT)1-MMP and TIMP-1 in these cells. Knockdown of RECK in hMSCs promoted monolayer regeneration and chemotactic migration of hMSCs, as demonstrated by scratch wound and chemotaxis assay analyses. Moreover, expression of endogenous RECK was upregulated upon osteogenic differentiation and diminished after adipogenic differentiation of hMSCs. RECK depletion in hMSCs reduced their capacity to differentiate into the osteogenic lineage whereas adipogenesis was increased, demonstrating that RECK functions as a master switch between both pathways. Furthermore, knockdown of RECK in hMSCs attenuated the Wnt/ß-catenin signaling pathway as indicated by reduced stability and impaired transcriptional activity of ß-catenin. The latter was determined by analysis of the ß-catenin target genes Dickkopf1 (DKK1), axis inhibition protein 2 (AXIN2), runt-related transcription factor 2 (RUNX2) and a luciferase-based ß-catenin-activated reporter (BAR) assay. Our findings demonstrate that RECK is a regulator of hMSC functions suggesting that modulation of RECK may improve the development of hMSC-based therapeutical approaches in regenerative medicine.

Impairment of hypoxia-induced HIF-1α signaling in keratinocytes and fibroblasts by sulfur mustard is counteracted by a selective PHD-2 inhibitor.

Skin exposure to sulfur mustard (SM) provokes long-term complications in wound healing. Similar to chronic wounds, SM-induced skin lesions are associated with low levels of oxygen in the wound tissue. Normally, skin cells respond to hypoxia by stabilization of the transcription factor hypoxia-inducible factor 1 alpha (HIF-1α). HIF-1α modulates expression of genes including VEGFA, BNIP3, and MMP2 that control processes such as angiogenesis, growth, and extracellular proteolysis essential for proper wound healing. The results of our studies revealed that exposure of primary normal human epidermal keratinocytes (NHEK) and primary normal human dermal fibroblasts (NHDF) to SM significantly impaired hypoxia-induced HIF-1α stabilization and target gene expression in these cells. Addition of a selective inhibitor of the oxygen-sensitive prolyl hydroxylase domain-containing protein 2 (PHD-2), IOX2, fully recovered HIF-1α stability, nuclear translocation, and target gene expression in NHEK and NHDF. Moreover, functional studies using a scratch wound assay demonstrated that the application of IOX2 efficiently counteracted SM-mediated deficiencies in monolayer regeneration under hypoxic conditions in NHEK and NHDF. Our findings describe a pathomechanism by which SM negatively affects hypoxia-stimulated HIF-1α signaling in keratinocytes and fibroblasts and thus possibly contributes to delayed wound healing in SM-injured patients that could be treated with PHD-2 inhibitors.

MicroRNA-specific regulatory mechanisms in atherosclerosis.

During the past decade, the crucial role of microRNAs (miRs) controlling tissue homeostasis and disease in the cardiovascular system has become widely recognized. By controlling the expression levels of their targets, several miRs have been shown to modulate the function of endothelial cells, vascular smooth muscle cells, and macrophages, thereby regulating the development and progression of atherosclerosis. For instance, miR-155 can exacerbate early stages of atherosclerosis by increasing the inflammatory activation and disturbing efficient lipid handling in macrophages. Conversely, miRs can exert atheroprotective roles, as has been established for the complementary miR-126 strand pair, which forms a dual system sustaining the endothelial proliferative reserve and promoting endothelial regeneration to counteract atherogenic effects of disturbed flow and hyperlipidemia. Under some conditions, miRs are released from cells and are transported by microvesicles, ribonucleoprotein complexes, and lipoproteins, being remarkably stable in circulation. Conferred by such delivery modules, miRs can regulate target mRNAs in recipient cells, representing a new tool for cell-cell communication in the context of atherosclerotic disease. Here, we will discuss novel aspects of miR-mediated regulatory mechanisms, namely the regulation by competing RNA targets, miRNA tandems, or complementary miR strand pairs, as well as their potential diagnostic and therapeutic value in atherosclerosis. This article is part of a Special Issue entitled 'Non-coding RNAs'.

Tissue inhibitor of metalloproteinase-1 (TIMP-1) regulates mesenchymal stem cells through let-7f microRNA and Wnt/ß-catenin signaling.

Tissue inhibitor of metalloproteinases 1 (TIMP-1) is a matrix metalloproteinase (MMP)-independent regulator of growth and apoptosis in various cell types. The receptors and signaling pathways that are involved in the growth factor activities of TIMP-1, however, remain controversial. RNA interference of TIMP-1 has revealed that endogenous TIMP-1 suppresses the proliferation, metabolic activity, and osteogenic differentiation capacity of human mesenchymal stem cells (hMSCs). The knockdown of TIMP-1 in hMSCs activated the Wnt/ß-catenin signaling pathway as indicated by the increased stability and nuclear localization of ß-catenin in TIMP-1-deficient hMSCs. Moreover, TIMP-1 knockdown cells exhibited enhanced ß-catenin transcriptional activity, determined by Wnt/ß-catenin target gene expression analysis and a luciferase-based ß-catenin-activated reporter assay. An analysis of a mutant form of TIMP-1 that cannot inhibit MMP indicated that the effect of TIMP-1 on ß-catenin signaling is MMP independent. Furthermore, the binding of CD63 to TIMP-1 on the surface of hMSCs is essential for the TIMP-1-mediated effects on Wnt/ß-catenin signaling. An array analysis of microRNAs (miRNAs) and transfection studies with specific miRNA inhibitors and mimics showed that let-7f miRNA is crucial for the regulation of ß-catenin activity and osteogenic differentiation by TIMP-1. Let-7f was up-regulated in TIMP-1-depleted hMSCs and demonstrably reduced axin 2, an antagonist of ß-catenin stability. Our results demonstrate that TIMP-1 is a direct regulator of hMSC functions and reveal a regulatory network in which let-7f modulates Wnt/ß-catenin activity.

Caught in action: how MSCs modulate atherosclerotic plaque.

Atherosclerosis (AS) is a medical condition marked by the stiffening and constriction of the arteries. This is caused by the accumulation of plaque, a substance made up of fat, cholesterol, calcium, and other elements present in the blood. Over time, this plaque solidifies and constricts the arteries, restricting the circulation of oxygen-rich blood to the organs and other body parts. The onset and progression of AS involve a continuous inflammatory response, including the infiltration of inflammatory cells, foam cells derived from monocytes/macrophages, and inflammatory cytokines and chemokines. Mesenchymal stromal cells (MSCs), a type of multipotent stem cells originating from various body tissues, have recently been demonstrated to have a protective and regulatory role in diseases involving inflammation. Consequently, the transplantation of MSCs is being proposed as a novel therapeutic strategy for atherosclerosis treatment. This mini-review intends to provide a summary of the regulatory effects of MSCs at the plaque site to lay the groundwork for therapeutic interventions.

Targeting miR-497-5p rescues human keratinocyte dysfunction upon skin exposure to sulfur mustard.

Sulfur mustard (SM) is a highly toxic chemical warfare agent. Exposure to SM results in various pathologies including skin lesions with subsequent impaired wound healing. To date, there are no effective treatments available. Here we discover a SM-triggered pathomechanism involving miR-497-5p and its target survivin which contributes to keratinocyte dysfunction. Transcriptome analysis using RNA-seq in normal human epidermal keratinocytes (NHEK) revealed that SM evoked differential expression of 1896 mRNAs and 25 miRNAs with many of these RNAs known to be involved in keratinocyte function and wound healing. We demonstrated that keratinocyte differentiation and proliferation were efficiently regulated by miRNAs induced in skin cells after exposure to SM. The inhibition of miR-497-5p counteracted SM-induced premature differentiation and stimulated proliferation of NHEK. In addition, we showed that microneedle-mediated transdermal application of lipid-nanoparticles containing miR-497-5p inhibitor restored survivin biosynthesis and cellular functionality upon exposure to SM using human skin biopsies. Our findings expand the current understanding of SM-associated molecular toxicology in keratinocytes and highlight miR-497-5p as feasible clinical target for specific skin therapy in SM-exposed patients and beyond.

Properties and fate of human mesenchymal stem cells upon miRNA let-7f-promoted recruitment to atherosclerotic plaques.

AIMS: Atherosclerosis is a chronic inflammatory disease of the arteries leading to the formation of atheromatous plaques. Human mesenchymal stem cells (hMSCs) are recruited from the circulation into plaques where in response to their environment they adopt a phenotype with immunomodulatory properties. However, the mechanisms underlying hMSC function in these processes are unclear. Recently, we described that miRNA let-7f controls hMSC invasion guided by inflammatory cytokines and chemokines. Here, we investigated the role of let-7f in hMSC tropism to human atheromas and the effects of the plaque microenvironment on cell fate and release of soluble factors. METHODS AND RESULTS: Incubation of hMSCs with LL-37, an antimicrobial peptide abundantly found in plaques, increased biosynthesis of let-7f and N-formyl peptide receptor 2 (FPR2), enabling chemotactic invasion of the cells towards LL-37, as determined by qRT-PCR, flow cytometry, and cell invasion assay analysis. In an Apoe-/- mouse model of atherosclerosis, circulating hMSCs preferentially adhered to athero-prone endothelium. This property was facilitated by elevated levels of let-7f in the hMSCs, as assayed by ex vivo artery perfusion and two-photon laser scanning microscopy. Exposure of hMSCs to homogenized human atheromatous plaque material considerably induced the production of various cytokines, chemokines, matrix metalloproteinases, and tissue inhibitors of metalloproteinases, as studied by PCR array and western blot analysis. Moreover, exposure to human plaque extracts elicited differentiation of hMSCs into cells of the myogenic lineage, suggesting a potentially plaque-stabilizing effect. CONCLUSIONS: Our findings indicate that let-7f promotes hMSC tropism towards atheromas through the LL-37/FPR2 axis and demonstrate that hMSCs upon contact with human plaque environment develop a potentially athero-protective signature impacting the pathophysiology of atherosclerosis.

Tissue Inhibitor of Metalloproteinases-1 Interacts with CD74 to Promote AKT Signaling, Monocyte Recruitment Responses, and Vascular Smooth Muscle Cell Proliferation.

Tissue inhibitor of metalloproteinases-1 (TIMP-1), an important regulator of matrix metalloproteinases (MMPs), has recently been shown to interact with CD74, a receptor for macrophage migration inhibitory factor (MIF). However, the biological effects mediated by TIMP-1 through CD74 remain largely unexplored. Using sequence alignment and in silico protein-protein docking analysis, we demonstrated that TIMP-1 shares residues with both MIF and MIF-2, crucial for CD74 binding, but not for CXCR4. Subcellular colocalization, immunoprecipitation, and internalization experiments supported these findings, demonstrating that TIMP-1 interacts with surface-expressed CD74, resulting in its internalization in a dose-dependent manner, as well as with a soluble CD74 ectodomain fragment (sCD74). This prompted us to study the effects of the TIMP-1-CD74 axis on monocytes and vascular smooth muscle cells (VSCMs) to assess its impact on vascular inflammation. A phospho-kinase array revealed the activation of serine/threonine kinases by TIMP-1 in THP-1 pre-monocytes, in particular AKT. Similarly, TIMP-1 dose-dependently triggered the phosphorylation of AKT and ERK1/2 in primary human monocytes. Importantly, Transwell migration, 3D-based Chemotaxis, and flow adhesion assays demonstrated that TIMP-1 engagement of CD74 strongly promotes the recruitment response of primary human monocytes, while live cell imaging studies revealed a profound activating effect on VSMC proliferation. Finally, re-analysis of scRNA-seq data highlighted the expression patterns of TIMP-1 and CD74 in human atherosclerotic lesions, thus, together with our experimental data, indicating a role for the TIMP-1-CD74 axis in vascular inflammation and atherosclerosis.