Lymphatic and Immune Cell Cross-Talk Regulates Cardiac Recovery After Experimental Myocardial Infarction

Objective: Lymphatics play an essential pathophysiological role in promoting fluid and immune cell tissue clearance. Conversely, immune cells may influence lymphatic function and remodeling. Recently, cardiac lymphangiogenesis has been proposed as a therapeutic target to prevent heart failure after myocardial infarction (MI). We investigated the effects of gene therapy to modulate cardiac lymphangiogenesis post-MI in rodents. Second, we determined the impact of cardiac-infiltrating T cells on lymphatic remodeling in the heart. Approach and Results: Comparing adenoviral versus adeno-associated viral gene delivery in mice, we found that only sustained VEGF (vascular endothelial growth factor)-C(C156S)therapy, achieved by adeno-associated viral vectors, increased cardiac lymphangiogenesis, and led to reduced cardiac inflammation and dysfunction by 3 weeks post-MI. Conversely, inhibition of VEGF-C/-D signaling, through adeno-associated viral delivery of soluble VEGFR3 (vascular endothelial growth factor receptor 3), limited infarct lymphangiogenesis. Unexpectedly, this treatment improved cardiac function post-MI in both mice and rats, linked to reduced infarct thinning due to acute suppression of T-cell infiltration. Finally, using pharmacological, genetic, and antibody-mediated prevention of cardiac T-cell recruitment in mice, we discovered that both CD4(+)and CD8(+)T cells potently suppress, in part through interferon-gamma, cardiac lymphangiogenesis post-MI. Conclusions: We show that resolution of cardiac inflammation after MI may be accelerated by therapeutic lymphangiogenesis based on adeno-associated viral gene delivery of VEGF-C-C156S. Conversely, our work uncovers a major negative role of cardiac-recruited T cells on lymphatic remodeling. Our results give new insight into the interconnection between immune cells and lymphatics in orchestration of cardiac repair after injury.Peer reviewe

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ISSN : 978-1-4244-0924-2International audienc

Dose-dependent activation of distinct hypertrophic pathways by serotonin in cardiac cells.

International audienceThere is substantial evidence supporting a hypertrophic action of serotonin [5-hydroxytryptamine (5-HT)] in cardiomyocytes. However, little is known about the mechanisms involved. We previously demonstrated that 5-HT-induced hypertrophy depends, in part, on the generation of reactive oxygen species by monoamine oxidase-A (MAO-A) (see Ref. 3). Cardiomyocytes express 5-HT(2) receptors, which may also participate in hypertrophy. Here, we analyzed the respective contribution of 5-HT(2) receptors and MAO-A in H9C2 cardiomyoblast hypertrophy. 5-HT induced a dose-dependent increase in [(3)H]leucine incorporation and stimulation of two markers of cardiac hypertrophy, ANF-luc and alphaSK-actin-luc reporter genes. Experiments using 1 microM 5-HT showed that hypertrophic response occurred independently from MAO-A. Using pharmacological inhibitors (M100907 and ketanserin), we identified a novel mechanism of action involving 5-HT(2A) receptors and requiring Ca(2+)/calcineurin/nuclear factor of activated T-cell activation. The activation of this hypertrophic pathway was fully prevented by 5-HT(2A) inhibitors and was unaffected by MAO inhibition. When 10 microM 5-HT was used, an additional hypertrophic response, prevented by the MAO inhibitors pargyline and RO 41-1049, was observed. Unlike the 5-HT(2A)-receptor-mediated H9C2 cell hypertrophy, MAO-A-dependent hypertrophic response required activation of extracellular-regulated kinases. In conclusion, our results show the existence of a dose-dependent shift of activation of distinct intracellular pathways involved in 5-HT-mediated hypertrophy of cardiac cells

Le système lymphatique cardiaque

International audience> Le système lymphatique est un réseau vas-culaire responsable du transport des graisses intestinales. Il participe à la surveillance immune et permet le maintien de l'homéostasie tissu-laire. Malgré un grand nombre d'évidences mon-trant l'importance des vaisseaux lymphatiques dans les maladies cardiovasculaires, le rôle des vaisseaux lymphatiques cardiaques n'a été que très peu étudié. En condition physiologique, le système lymphatique cardiaque régule dynami-quement le drainage des fluides interstitiels vers les ganglions médiastinaux afin de maintenir l'homéostasie du tissu cardiaque et de prévenir la formation d'un oedème. Après un infarctus du myocarde, les vaisseaux lymphatiques du coeur ischémique perdent leur fonction et contribuent au développement d'un oedème myocardique chronique qui aggrave la fibrose et la dysfonc-tion cardiaque. La stimulation de la lymphan-giogenèse cardiaque, fondée sur la délivrance de facteurs de croissance lymphangiogéniques comme le VEGF-C, pourrait représenter une nou-velle stratégie thérapeutique pour améliorer la fonction cardiaque. Cette revue met en évidence la chronologie des principales découvertes asso-ciées au développement et à la fonction lympha-tique cardiaque. < captés par des cellules présentatrices d'antigènes (CPA) afin d'initier des réponses immunitaires spécifiques contre des particules qui pour-raient être pathogènes (Figure 1) [3]. Les capillaires lymphatiques sont présents dans la peau et dans la plupart des organes internes, à l'exception de la moelle osseuse et des tissus avasculaires comme le cartilage, la cornée et l'épiderme. Dans l'intestin, les vaisseaux lymphatiques ont été initialement nom-més « veines blanches » en raison de leur coloration blanche observée en phase postprandiale dans la région mésentérique. Ils jouent un rôle primordial dans l'absorption des lipides intestinaux ; ils sont encore appelés « lactés » ou « lactéaux », en raison de cette particularité [4]. Majoritairement présents dans les villosités de l'intestin grêle, les vaisseaux lymphatiques sont à l'origine de l'absorption des lipides alimentaires, libérés sous la forme de chylomicrons par les entéro-cytes (Figure 1). Les vaisseaux lymphatiques jouent également un rôle important dans le transport réverse du cholestérol [5, 6]. La composition protéique de la lymphe est équivalente à celle du fluide inters-titiel qui est similaire, mais généralement moins concentrée, à celle du plasma sanguin, à l'exception de la lymphe intestinale qui contient une grande quantité de lipides intestinaux [7]. Cette lymphe est une émulsion trouble et laiteuse, souvent appelée « chyle ». Le drainage lymphatique peut être considérablement modifié à la suite d'une infection, d'un traumatisme, d'une chirurgie, d'une transplantation, d'un traitement, ou d'une maladie veineuse ou congénitale [8, 9]. La lymphangiogenèse, processus dirigeant la croissance de nou-veaux vaisseaux lymphatiques, apparaît au cours du développement embryonnaire. Elle se met en place à partir de bourgeonnements du système sanguin [1]. Elle est également impliquée dans de nombreux états pathologiques (Figure 1)

Platelet derived serotonin drives the activation of rat cardiac fibroblasts by 5-HT2A receptors.

International audiencePlatelet activation occurs in different acute and chronic heart diseases including myocardial infarction, obstructive hypertrophic cardiomyopathy and valve stenosis. Recent studies suggested that some factors secreted by activated platelets may participate in cardiac remodeling. In the present study, we investigated whether platelets and platelet-released serotonin (5-HT) are directly involved in the functional regulation of cardiac fibroblasts. Treatment of neonatal rat cardiac fibroblasts with platelet lysate, 5-HT and the 5-HT2A receptor agonist DOI increased the expression of alpha-SMA protein, a marker of fibroblast differentiation into myofibroblasts. Platelet lysate, 5-HT and DOI also induced a time-dependent stimulation of cardiac fibroblast migration that was inhibited by the 5-HT2A receptor antagonist ketanserin. Finally, incubation of cardiac fibroblasts with platelet lysate or 5-HT enhanced secretion of TGF-beta1 and expression of MMP-3 and MMP-13. As observed for fibroblast migration, these effects were prevented by ketanserin. These results demonstrated for the first time that factors released from platelet directly regulate cardiac fibroblasts by enhancing secretion of TGF-beta1 and MMPs and promoting their migration and differentiation. 5-HT released by platelets appears to be a major contributor of platelet effects which are mediated through 5-HT2A receptors

Extracellular Vesicles Produced by the Cardiac Microenvironment Carry Functional Enzymes to Produce Lipid Mediators In Situ

International audienceThe impact of the polyunsaturated fatty acids (PUFAs) at physiological concentrations on the composition of eicosanoids transported within the extracellular vesicles (EVs) of rat bone marrow mesenchymal stem cells and cardiomyoblasts was reported by our group in 2020. The aim of this article was to extend this observation to cells from the cardiac microenvironment involved in the processes of inflammation, namely mouse J774 macrophages and rat heart mesenchymal stem cells cMSCs. Moreover, to enhance our capacity to understand the paracrine exchange between these orchestrators of cardiac inflammation, we investigated some machinery involved in the eicosanoid’s synthesis transported by the EVs produced by these cells (including the two formerly described cells: bone marrow mesenchymal stem cells BM-MSC and cardiomyoblasts H9c2). We analyzed the oxylipin and the enzymatic content of the EVs collected from cell cultures supplemented (or not) with PUFAs. We prove that large eicosanoid profiles are exported in the EVs by the cardiac microenvironment cells, but also that these EVs carry some critical and functional biosynthetic enzymes, allowing them to synthesize inflammation bioactive compounds by sensing their environment. Moreover, we demonstrate that these are functional. This observation reinforces the hypothesis that EVs are key factors in paracrine signaling, even in the absence of the parent cell. We also reveal a macrophage-specific behavior, as we observed a radical change in the lipid mediator profile when small EVs derived from J774 cells were exposed to PUFAs. To summarize, we prove that the EVs, due to the carried functional enzymes, can alone produce bioactive compounds, in the absence of the parent cell, by sensing their environment. This makes them potential circulating monitoring entities

Extracellular vesicles of MSCs and cardiomyoblasts are vehicles for lipid mediators

International audienceRecent works reported the relevance of cellular exosomes in the evolution of different pathologies. However, most of these studies focused on the ability of exosomes to convey mi-RNA from cell to cell. The level of knowledge concerning the transport of lipid mediators by these nanovesicles is more than fragmented. The role of lipid mediators in the inflammatory signaling is fairly well described, in particular concerning the derivatives of the arachidonic acid (AA), called eicosanoïds or lipid mediators. The aim of the present work was to study the transport of these lipids within the extracellular vesicles of rat bone marrow mesenchymal stem cells (BM-MSC) and the cardiomyoblast cell line H9c2. We were able to characterize, for the first time, complete profiles of oxilipins within these nanovesicles. We studied also the impact on these profiles, of the polyunsaturated fatty acids (PUFAs) know to be precursors of the inflammatory signaling molecules (AA, eicosapentaenoic acid EPA and Docosahexaenoic acid DHA), at physiological concentrations. By growing the progenitor cells under PUFAs supplementation, we provide a comprehensive assessment of the beneficial effect of u-3 PUFA therapy. Actually, our results tend to support the resolving role of the inflammation that stromal cell-derived extracellular vesicles can have within the cardiac microenvironment

Vesicular monoamine transporter 1 mediates dopamine secretion in rat proximal tubular cells.

International audienceRenal dopamine, synthesized by proximal tubules, plays an important role in the regulation of renal sodium excretion. Although the renal dopaminergic system has been extensively investigated in both physiological and pathological situations, the mechanisms whereby dopamine is stored and secreted by proximal tubule cells remain obscure. In the present study we investigated whether vesicular monoamine transporters (VMAT)-1 and -2, which participate in amine storing and secretion, are expressed in rat renal proximal tubules, and we defined their involvement in dopamine secretion. By combining RT-PCR, Western blot, and immunocytochemistry we showed that VMAT-1 is the predominant isoform expressed in isolated proximal tubule cells. These results were confirmed by immunohistochemistry analysis of rat renal cortex showing that VMAT-1 was found in proximal tubules but not in glomeruli. Functional studies showed that, as previously reported for VMAT-dependent amine transporters, dopamine release by cultured proximal tubule cells was partially inhibited by disruption of intracellular H(+) gradient. In addition, dopamine secretion was prevented by the VMAT-1/VMAT-2 inhibitor reserpine but not by the VMAT-2 inhibitor tetrabenazine. Finally, we demonstrated that tubular VMAT-1 mRNA and protein expression were significantly upregulated during a high-sodium diet. In conclusion, our results show for the first time the expression of a VMAT in the renal proximal tubule and its involvement in regulation of dopamine secretion. These data represent the first step toward the comprehension of the role of this transporter in renal dopamine handling and its involvement in pathological situations

Role of endothelial AADC in cardiac synthesis of serotonin and nitrates accumulation.

Serotonin (5-HT) regulates different cardiac functions by acting directly on cardiomyocytes, fibroblasts and endothelial cells. Today, it is widely accepted that activated platelets represent a major source of 5-HT. In contrast, a supposed production of 5-HT in the heart is still controversial. To address this issue, we investigated the expression and localization of 5-HT synthesizing enzyme tryptophan hydroxylase (TPH) and L-aromatic amino acid decarboxylase (AADC) in the heart. We also evaluated their involvement in cardiac production of 5-HT. TPH1 was weakly expressed in mouse and rat heart and appeared restricted to mast cells. Degranulation of mast cells by compound 48/80 did not modify 5-HT cardiac content in mice. Western blots and immunolabelling experiments showed an abundant expression of AADC in the mouse and rat heart and its co-localization with endothelial cells. Incubation of cardiac homogenate with the AADC substrate (5-hydroxy-L-tryptophan) 5-HTP or intraperitoneal injection of 5-HTP in mice significantly increased cardiac 5-HT. These effects were prevented by the AADC inhibitor benserazide. Finally, 5-HTP administration in mice increased phosphorylation of aortic nitric oxide synthase 3 at Ser (1177) as well as accumulation of nitrates in cardiac tissue. This suggests that the increase in 5-HT production by AADC leads to activation of endothelial and cardiac nitric oxide pathway. These data show that endothelial AADC plays an important role in cardiac synthesis of 5-HT and possibly in 5-HT-dependent regulation of nitric oxide generation

Metformin Attenuates Postinfarction Myocardial Fibrosis and Inflammation in Mice

International audienceDiabetes is a major risk factor for the development of cardiovascular disease with a higher incidence of myocardial infarction. This study explores the role of metformin, a first-line antihyperglycemic agent, in postinfarction fibrotic and inflammatory remodeling in mice. Three-month-old C57BI/6J mice were submitted to 30 min cardiac ischemia followed by reperfusion for 14 days. Intraperitoneal treatment with metformin (5 mg/kg) was initiated 15 min after the onset of reperfusion and maintained for 14 days. Real-time PCR was used to determine the levels of COL3A1, αSMA, CD68, TNF-α and IL-6. Increased collagen deposition and infiltration of macrophages in heart tissues are associated with upregulation of the inflammation-associated genes in mice after 14 days of reperfusion. Metformin treatment markedly reduced postinfarction fibrotic remodeling and CD68-positive cell population in mice. Moreover, metformin resulted in reduced expression of COL3A1, αSMA and CD68 after 14 days of reperfusion. Taken together, these results open new perspectives for the use of metformin as a drug that counteracts adverse myocardial fibroticand inflammatory remodeling after MI