L-Wnk1 Deletion in Smooth Muscle Cells Causes Aortitis and Inflammatory Shift.

BACKGROUND: The long isoform of the Wnk1 (with-no-lysine [K] kinase 1) is a ubiquitous serine/threonine kinase, but its role in vascular smooth muscle cells (VSMCs) pathophysiology remains unknown. METHODS: AngII (angiotensin II) was infused in Apoe-/- to induce experimental aortic aneurysm. Mice carrying an Sm22-Cre allele were cross-bred with mice carrying a floxed Wnk1 allele to specifically investigate the functional role of Wnk1 in VSMCs. RESULTS: Single-cell RNA-sequencing of the aneurysmal abdominal aorta from AngII-infused Apoe-/- mice revealed that VSMCs that did not express Wnk1 showed lower expression of contractile phenotype markers and increased inflammatory activity. Interestingly, WNK1 gene expression in VSMCs was decreased in human abdominal aortic aneurysm. Wnk1-deficient VSMCs lost their contractile function and exhibited a proinflammatory phenotype, characterized by the production of matrix metalloproteases, as well as cytokines and chemokines, which contributed to local accumulation of inflammatory macrophages, Ly6Chi monocytes, and γδ T cells. Sm22Cre+Wnk1lox/lox mice spontaneously developed aortitis in the infrarenal abdominal aorta, which extended to the thoracic area over time without any negative effect on long-term survival. AngII infusion in Sm22Cre+Wnk1lox/lox mice aggravated the aortic disease, with the formation of lethal abdominal aortic aneurysms. Pharmacological blockade of γδ T-cell recruitment using neutralizing anti-CXCL9 antibody treatment, or of monocyte/macrophage using Ki20227, a selective inhibitor of CSF1 receptor, attenuated aortitis. Wnk1 deletion in VSMCs led to aortic wall remodeling with destruction of elastin layers, increased collagen content, and enhanced local TGF-ß (transforming growth factor-beta) 1 expression. Finally, in vivo TGF-ß blockade using neutralizing anti-TGF-ß antibody promoted saccular aneurysm formation and aorta rupture in Sm22 Cre+ Wnk1lox/lox mice but not in control animals. CONCLUSION: Wnk1 is a key regulator of VSMC function. Wnk1 deletion promotes VSMC phenotype switch toward a pathogenic proinflammatory phenotype, orchestrating deleterious vascular remodeling and spontaneous severe aortitis in mice.

Identification of Adipose Tissue as a Reservoir of Macrophages after Acute Myocardial Infarction.

Medullary and extra-medullary hematopoiesis has been shown to govern inflammatory cell infiltration and subsequently cardiac remodeling and function after acute myocardial infarction (MI). Emerging evidence positions adipose tissue (AT) as an alternative source of immune cell production. We, therefore, hypothesized that AT could act as a reservoir of inflammatory cells that participate in cardiac homeostasis after MI. To reveal the distinct role of inflammatory cells derived from AT or bone marrow (BM), chimeric mice were generated using standard repopulation assays. We showed that AMI increased the number of AT-derived macrophages in the cardiac tissue. These macrophages exhibit pro-inflammatory characteristics and their specific depletion improved cardiac function as well as decreased infarct size and interstitial fibrosis. We then reasoned that the alteration of AT-immune compartment in type 2 diabetes could, thus, contribute to defects in cardiac remodeling. However, in these conditions, myeloid cells recruited in the infarcted heart mainly originate from the BM, and AT was no longer used as a myeloid cell reservoir. Altogether, we showed here that a subpopulation of cardiac inflammatory macrophages emerges from myeloid cells of AT origin and plays a detrimental role in cardiac remodeling and function after MI. Diabetes abrogates the ability of AT-derived myeloid cells to populate the infarcted heart.

Emerging Roles of the Atypical Chemokine Receptor 3 (ACKR3) in Cardiovascular Diseases.

Chemokines, and their receptors play a crucial role in the pathophysiology of cardiovascular diseases (CVD). Chemokines classically mediate their effects by binding to G-protein-coupled receptors. The discovery that chemokines can also bind to atypical chemokine receptors (ACKRs) and initiate alternative signaling pathways has changed the paradigm regarding chemokine-related functions. Among these ACKRs, several studies have highlighted the exclusive role of ACKR3, previously known as C-X-C chemokine receptor type 7 (CXCR7), in CVD. Indeed, ACKR3 exert atheroprotective, cardioprotective and anti-thrombotic effects through a wide range of cells including endothelial cells, platelets, inflammatory cells, fibroblasts, vascular smooth muscle cells and cardiomyocytes. ACKR3 functions as a scavenger receptor notably for the pleiotropic chemokine CXCL12, but also as a activator of different pathways such as ß-arrestin-mediated signaling or modulator of CXCR4 signaling through the formation of ACKR3-CXCR4 heterodimers. Hence, a better understanding of the precise roles of ACKR3 may pave the way towards the development of novel and improved therapeutic strategies for CVD. Here, we summarize the structural determinant characteristic of ACKR3, the molecules targeting this receptor and signaling pathways modulated by ACKR3. Finally, we present and discuss recent findings regarding the role of ACKR3 in CVD.

Splenic Marginal Zone B Lymphocytes Regulate Cardiac Remodeling After Acute Myocardial Infarction in Mice.

BACKGROUND: Mature B lymphocytes alter the recovery of cardiac function after acute myocardial infarction (MI) in mice. Follicular B cells and marginal zone B (MZB) cells are spatially distinct mature B-cell populations in the spleen, and they exert specific functional properties. microRNA-21 (miR21)/hypoxia-inducible factor-α (HIF-α)-related pathways have been shown to govern B-cell functions. OBJECTIVES: The goal of this study was to unravel the distinct role of MZB cells and that of endogenous activation of miR21/HIF-α signaling in MZB cells during post-ischemic injury. METHODS: Acute MI was induced in mice by permanent ligation of the left anterior descending coronary artery. Cardiac function and remodeling were assessed by using echocardiography and immunohistochemistry. To determine the specific role of MZB cells, the study used mice with B-cell lineage-specific conditional deletion of Notch signaling, which leads to selection deficiency of MZB cells. To evaluate the role of the HIF-1α isoform, mice were generated with MZB-cell lineage-specific conditional deletion of Hif1a. RESULTS: Acute MI prompted an miR21-dependent increase in HIF-1α, particularly in splenic MZB cells. MZB cell deficiency and MZB cell-specific deletion of miR21 or Hif1a improved cardiac function after acute MI. miR21/HIF-1α signaling in MZB cells was required for Toll-like receptor dependent expression of the monocyte chemoattractant protein CCL7, leading to increased mobilization of inflammatory monocytes to the ischemic myocardium and to adverse post-ischemic cardiac remodeling. CONCLUSIONS: This work reveals a novel function for the miR21/HIF-1α pathway in splenic MZB cells with potential major implications for the modulation of cardiac function after acute MI.

Extracellular vesicles fail to trigger the generation of new cardiomyocytes in chronically infarcted hearts.

Background: Extracellular vesicles (EV) mediate the therapeutic effects of stem cells but it is unclear whether this involves cardiac regeneration mediated by endogenous cardiomyocyte proliferation. Methods: Bi-transgenic MerCreMer/ZEG (n = 15/group) and Mosaic Analysis With Double Markers (MADM; n = 6/group) mouse models underwent permanent coronary artery ligation and received, 3 weeks later, 10 billion EV (from human iPS-derived cardiovascular progenitor cells [CPC]), or saline, injected percutaneously under echo guidance in the peri-infarcted myocardium. Endogenous cardiomyocyte proliferation was tracked by EdU labeling and biphoton microscopy. Other end points, including cardiac function (echocardiography and MRI), histology and transcriptomics were blindly assessed 4-6 weeks after injections. Results: There was no proliferation of cardiomyocytes in either transgenic mouse strains. Nevertheless, EV improved cardiac function in both models. In MerCreMer/ZEG mice, LVEF increased by 18.3 ± 0.2% between baseline and the end-study time point in EV-treated hearts which contrasted with a decrease by 2.3 ± 0.2% in the PBS group; MADM mice featured a similar pattern as intra-myocardial administration of EV improved LVEF by 13.3 ± 0.16% from baseline whereas it decreased by 14.4 ± 0.16% in the control PBS-injected group. This functional improvement was confirmed by MRI and associated with a reduction in infarct size, the decreased expression of several pro-fibrotic genes and an overexpression of the anti-fibrotic miRNA 133-a1 compared to controls. Experiments with an anti-miR133-a demonstrated that the cardio-reparative effects of EV were partly abrogated. Conclusions: EV-CPC do not trigger cardiomyocyte proliferation but still improve cardiac function by other mechanisms which may include the regulation of fibrosis.

Extracellular vesicles from human cardiovascular progenitors trigger a reparative immune response in infarcted hearts.

AIMS: The cardioprotective effects of human induced pluripotent stem cell-derived cardiovascular progenitor cells (CPC) are largely mediated by the paracrine release of extracellular vesicles (EV). We aimed to assess the immunological behaviour of EV-CPC, which is a prerequisite for their clinical translation. METHODS AND RESULTS: Flow cytometry demonstrated that EV-CPC expressed very low levels of immune relevant molecules including HLA Class I, CD80, CD274 (PD-L1), and CD275 (ICOS-L); and moderate levels of ligands of the natural killer (NK) cell activating receptor, NKG2D. In mixed lymphocyte reactions, EV-CPC neither induced nor modulated adaptive allogeneic T cell immune responses. They also failed to induce NK cell degranulation, even at high concentrations. These in vitro effects were confirmed in vivo as repeated injections of EV-CPC did not stimulate production of immunoglobulins or affect the interferon (IFN)-γ responses from primed splenocytes. In a mouse model of chronic heart failure, intra-myocardial injections of EV-CPC, 3 weeks after myocardial infarction, decreased both the number of cardiac pro-inflammatory Ly6Chigh monocytes and circulating levels of pro-inflammatory cytokines (IL-1α, TNF-α, and IFN-γ). In a model of acute infarction, direct cardiac injection of EV-CPC 2 days after infarction reduced pro-inflammatory macrophages, Ly6Chigh monocytes, and neutrophils in heart tissue as compared to controls. EV-CPC also reduced levels of pro-inflammatory cytokines IL-1α, IL-2, and IL-6, and increased levels of the anti-inflammatory cytokine IL-10. These effects on human macrophages and monocytes were reproduced in vitro; EV-CPC reduced the number of pro-inflammatory monocytes and M1 macrophages, while increasing the number of anti-inflammatory M2 macrophages. CONCLUSIONS: EV-CPC do not trigger an immune response either in in vitro human allogeneic models or in immunocompetent animal models. The capacity for orienting the response of monocyte/macrophages towards resolution of inflammation strengthens the clinical attractiveness of EV-CPC as an acellular therapy for cardiac repair.

Dynamics of Cardiac Neutrophil Diversity in Murine Myocardial Infarction.

RATIONALE: After myocardial infarction, neutrophils rapidly and massively infiltrate the heart, where they promote both tissue healing and damage. OBJECTIVE: To characterize the dynamics of circulating and cardiac neutrophil diversity after infarction. METHODS AND RESULTS: We employed single-cell transcriptomics combined with cell surface epitope detection by sequencing to investigate temporal neutrophil diversity in the blood and heart after murine myocardial infarction. At day 1, 3, and 5 after infarction, cardiac Ly6G+ (lymphocyte antigen 6G) neutrophils could be delineated into 6 distinct clusters with specific time-dependent patterning and proportions. At day 1, neutrophils were characterized by a gene expression profile proximal to bone marrow neutrophils (Cd177, Lcn2, Fpr1), and putative activity of transcriptional regulators involved in hypoxic response (Hif1a) and emergency granulopoiesis (Cebpb). At 3 and 5 days, 2 major subsets of Siglecfhi (enriched for eg, Icam1 and Tnf) and Siglecflow (Slpi, Ifitm1) neutrophils were found. Cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) analysis in blood and heart revealed that while circulating neutrophils undergo a process of aging characterized by loss of surface CD62L and upregulation of Cxcr4, heart infiltrating neutrophils acquired a unique SiglecFhi signature. SiglecFhi neutrophils were absent from the bone marrow and spleen, indicating local acquisition of the SiglecFhi signature. Reducing the influx of blood neutrophils by anti-Ly6G treatment increased proportions of cardiac SiglecFhi neutrophils, suggesting accumulation of locally aged neutrophils. Computational analysis of ligand/receptor interactions revealed putative pathways mediating neutrophil to macrophage communication in the myocardium. Finally, SiglecFhi neutrophils were also found in atherosclerotic vessels, revealing that they arise across distinct contexts of cardiovascular inflammation. CONCLUSIONS: Altogether, our data provide a time-resolved census of neutrophil diversity and gene expression dynamics in the mouse blood and ischemic heart at the single-cell level, and reveal a process of local tissue specification of neutrophils in the ischemic heart characterized by the acquisition of a SiglecFhi signature.

nSMase2 (Type 2-Neutral Sphingomyelinase) Deficiency or Inhibition by GW4869 Reduces Inflammation and Atherosclerosis in Apoe-/- Mice.

OBJECTIVE: Atherosclerosis is a chronic multifactorial and inflammatory disease of large and medium arteries and the leading cause of cardiovascular diseases worldwide. The aim of this study was to investigate whether and how the nSMase2 (type 2-neutral sphingomyelinase), a key enzyme of sphingolipid metabolism, may contribute to the development of atherosclerotic lesions. APPROACH AND RESULTS: The role of nSMase2 in atherosclerosis was investigated in Apoe-/-;Smpd3fro/fro mice, mutant for nSMase2, and in Apoe-/-;Smpd3+/+ mice intraperitoneally injected with GW4869, a pharmacological nSMase2 inhibitor. The defect or inhibition of nSMase2 resulted in a reduction of atherosclerotic lesions and a decrease in macrophage infiltration and lipid deposition, although cholesterolemia remained unchanged. nSMase2 inhibition decreased the inflammatory response of murine endothelial cells to oxLDL (oxidized low-density lipoprotein), as assessed by the significant reduction of MCP-1 (monocyte chemoattractant protein 1), ICAM-1 (intercellular adhesion molecule-1), and VCAM-1 (vascular cell adhesion molecule-1) mRNA expressions and macrophage recruitment. Likewise, in RAW264.7 or in macrophages isolated from Apoe-/-/Smpd3fro/fro or Apoe-/-/Smpd3+/+ mice stimulated by lipopolysaccharides, nSMase2 inhibition resulted in a decrease in the expression of inflammatory molecules. Mechanistically, the anti-inflammatory response resulting from nSMase2 inhibition involves Nrf2 (nuclear factor [erythroid-derived 2]-like 2 or NF-E2-related factor-2) activation in both endothelial cells and macrophages, as assessed by the lack of protective effect of GW4869 in endothelial cells silenced for Nrf2 by small interfering RNAs, and in lipopolysaccharide-stimulated macrophages issued from Nrf2-KO mice. CONCLUSIONS: The genetic deficiency or inhibition of nSMase2 strongly decreases the development of atherosclerotic lesions in Apoe-/- mice, by reducing inflammatory responses through a mechanism involving the Nrf2 pathway. Inhibitors of nSMase2 may, therefore, constitute a novel approach to slow down atherosclerosis progression.