G-Protein Coupled Receptor Targeting on Myeloid Cells in Atherosclerosis

Atherosclerosis, the underlying cause of the majority of cardiovascular diseases (CVDs), is a lipid-driven, inflammatory disease of the large arteries. Gold standard therapy with statins and the more recently developed proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors have improved health conditions among CVD patients by lowering low density lipoprotein (LDL) cholesterol. Nevertheless, a substantial part of these patients is still suffering and it seems that ‘just’ lipid lowering is insufficient. The results of the Canakinumab Anti-inflammatory Thrombosis Outcome Study (CANTOS) have now proven that inflammation is a key driver of atherosclerosis and that targeting inflammation improves CVD outcomes. Therefore, the identification of novel drug targets and development of novel therapeutics that block atherosclerosis-specific inflammatory pathways have to be promoted. The inflammatory processes in atherosclerosis are facilitated by a network of immune cells and their subsequent responses. Cell networking is orchestrated by various (inflammatory) mediators which interact, bind and induce signaling. Over the last years, G-protein coupled receptors (GPCRs) emerged as important players in recognizing these mediators, because of their diverse functions in steady state but also and specifically during chronic inflammatory processes – such as atherosclerosis. In this review, we will therefore highlight a selection of these receptors or receptor sub-families mainly expressed on myeloid cells and their role in atherosclerosis. More specifically, we will focus on chemokine receptors, both classical and atypical, formyl-peptide receptors, the chemerin receptor 23 and the calcium-sensing receptor. When information is available, we will also describe the consequences of their targeting which may hold promising options for future treatment of CVD

PCSK9: A Multi-Faceted Protein That Is Involved in Cardiovascular Biology.

Pro-protein convertase subtilisin/kexin type 9 (PCSK9) is secreted mostly by hepatocytes and to a lesser extent by the intestine, pancreas, kidney, adipose tissue, and vascular cells. PCSK9 has been known to interact with the low-density lipoprotein receptor (LDLR) and chaperones the receptor to its degradation. In this manner, targeting PCSK9 is a novel attractive approach to reduce hyperlipidaemia and the risk for cardiovascular diseases. Recently, it has been recognised that the effects of PCSK9 in relation to cardiovascular complications are not only LDLR related, but that various LDLR-independent pathways and processes are also influenced. In this review, the various LDLR dependent and especially independent effects of PCSK9 on the cardiovascular system are discussed, followed by an overview of related PCSK9-polymorphisms and currently available and future therapeutic approaches to manipulate PCSK9 expression

Small Things Matter: Relevance of MicroRNAs in Cardiovascular Disease

MicroRNAs (miRNAs) are short sequences of non-coding RNA that play an important role in the regulation of gene expression and thereby in many physiological and pathological processes. Furthermore, miRNAs are released in the extracellular space, for example in vesicles, and are detectable in various biological fluids, such as serum, plasma, and urine. Over the last years, it has been shown that miRNAs are crucial in the development of several cardiovascular diseases (CVDs). This review discusses the (patho)physiological implications of miRNAs in CVD, ranging from cardiovascular risk factors (i.e., hypertension, diabetes, dyslipidemia), to atherosclerosis, myocardial infarction, and cardiac remodeling. Moreover, the intriguing possibility of their use as disease-specific diagnostic and prognostic biomarkers for human CVDs will be discussed in detail. Finally, as several approaches have been developed to alter miRNA expression and function (i.e., mimics, antagomirs, and target-site blockers), we will highlight the miRNAs with the most promising therapeutic potential that may represent suitable candidates for therapeutic intervention in future translational studies and ultimately in clinical trials. All in all, this review gives a comprehensive overview of the most relevant miRNAs in CVD and discusses their potential use as biomarkers and even therapeutic targets

Inflammatory Mediators in Atherosclerotic Vascular Remodeling.

Atherosclerotic vascular disease remains the most common cause of ischemia, myocardial infarction, and stroke. Vascular function is determined by structural and functional properties of the arterial vessel wall, which consists of three layers, namely the adventitia, media, and intima. Key cells in shaping the vascular wall architecture and warranting proper vessel function are vascular smooth muscle cells in the arterial media and endothelial cells lining the intima. Pathological alterations of this vessel wall architecture called vascular remodeling can lead to insufficient vascular function and subsequent ischemia and organ damage. One major pathomechanism driving this detrimental vascular remodeling is atherosclerosis, which is initiated by endothelial dysfunction allowing the accumulation of intimal lipids and leukocytes. Inflammatory mediators such as cytokines, chemokines, and modified lipids further drive vascular remodeling ultimately leading to thrombus formation and/or vessel occlusion which can cause major cardiovascular events. Although it is clear that vascular wall remodeling is an elementary mechanism of atherosclerotic vascular disease, the diverse underlying pathomechanisms and its consequences are still insufficiently understood

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

IntroductionThe 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 resultsEndothelial Adam10 deficiency (Adam10ecko) 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.DiscussionCollectively, 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

Palmitoylethanolamide Promotes a Proresolving Macrophage Phenotype and Attenuates Atherosclerotic Plaque Formation

Objective Palmitoylethanolamide is an endogenous fatty acid mediator that is synthetized from membrane phospholipids by N-acyl phosphatidylethanolamine phospholipase D. Its biological actions are primarily mediated by PPAR- (peroxisome proliferator-activated receptors ) and the orphan receptor GPR55. Palmitoylethanolamide exerts potent anti-inflammatory actions but its physiological role and promise as a therapeutic agent in chronic arterial inflammation, such as atherosclerosis remain unexplored. Approach and Results First, the polarization of mouse primary macrophages towards a proinflammatory phenotype was found to reduce N-acyl phosphatidylethanolamine phospholipase D expression and palmitoylethanolamide bioavailability. N-acyl phosphatidylethanolamine phospholipase D expression was progressively downregulated in the aorta of apolipoprotein E deficient (ApoE(-/-)) mice during atherogenesis. N-acyl phosphatidylethanolamine phospholipase D mRNA levels were also downregulated in unstable human plaques and they positively associated with smooth muscle cell markers and negatively with macrophage markers. Second, ApoE(-/-) mice were fed a high-fat diet for 4 or 16 weeks and treated with either vehicle or palmitoylethanolamide (3 mg/kg per day, 4 weeks) to study the effects of palmitoylethanolamide on early established and pre-established atherosclerosis. Palmitoylethanolamide treatment reduced plaque size in early atherosclerosis, whereas in pre-established atherosclerosis, palmitoylethanolamide promoted signs of plaque stability as evidenced by reduced macrophage accumulation and necrotic core size, increased collagen deposition and downregulation of M1-type macrophage markers. Mechanistically, we found that palmitoylethanolamide, by activating GPR55, increases the expression of the phagocytosis receptor MerTK (proto-oncogene tyrosine-protein kinase MER) and enhances macrophage efferocytosis, indicative of proresolving properties. Conclusions The present study demonstrates that palmitoylethanolamide protects against atherosclerosis by promoting an anti-inflammatory and proresolving phenotype of lesional macrophages, representing a new therapeutic approach to resolve arterial inflammation

Corrigendum: Identification of Hypoxia Induced Metabolism Associated Genes in Pulmonary Hypertension.

[This corrects the article DOI: 10.3389/fphar.2021.753727.]

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

Adventitial lymphatic capillary expansion impacts on plaque T cell accumulation in atherosclerosis

During plaque progression, inflammatory cells progressively accumulate in the adventitia, paralleled by an increased presence of leaky vasa vasorum. We here show that next to vasa vasorum, also the adventitial lymphatic capillary bed is expanding during plaque development in humans and mouse models of atherosclerosis. Furthermore, we investigated the role of lymphatics in atherosclerosis progression. Dissection of plaque draining lymph node and lymphatic vessel in atherosclerotic ApoE(-/-)mice aggravated plaque formation, which was accompanied by increased intimal and adventitial CD3(+) T cell numbers. Likewise, inhibition of VEGF-C/D dependent lymphangiogenesis by AAV aided gene transfer of hVEGFR3-Ig fusion protein resulted in CD3(+) T cell enrichment in plaque intima and adventitia. hVEGFR3-Ig gene transfer did not compromise adventitial lymphatic density, pointing to VEGF-C/D independent lymphangiogenesis. We were able to identify the CXCL12/CXCR4 axis, which has previously been shown to indirectly activate VEGFR3, as a likely pathway, in that its focal silencing attenuated lymphangiogenesis and augmented T cell presence. Taken together, our study not only shows profound, partly CXCL12/CXCR4 mediated, expansion of lymph capillaries in the adventitia of atherosclerotic plaque in humans and mice, but also is the first to attribute an important role of lymphatics in plaque T cell accumulation and development.Peer reviewe

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/beta-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=1215) 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/beta-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