Kapka Miteva

Crosstalk between fibroblasts and inflammatory cells

Fibroblasts, which are traditionally recognized as a quiescent cell responsible for extracellular matrix production, are more and more appreciated as an active key player of the immune system. This review describes how fibroblasts and immune cells reciprocally influence the pathogenesis of fibrosis. An overview is given how fibroblasts are triggered by components of the innate and adaptive immunity on the one hand and how fibroblasts modulate immune cell behaviour via conditioning the cellular and cytokine microenvironment on the other hand. Finally, latest insights into the role of cardiac fibroblasts in the orchestration of inflammatory cell infiltration in the heart, and their impact on heart failure, are outlined.

Role of Heart Rate Reduction in the Prevention of Experimental Heart Failure: Comparison Between If-Channel Blockade and β-Receptor Blockade

To investigate whether heart rate reduction via If-channel blockade and &bgr;-receptor blockade prevents left ventricular (LV) dysfunction, we studied ivabradine and metoprolol in angiotensin II–induced heart failure. Cardiac dysfunction in C57BL/6J mice was induced by implantation of osmotic pumps for continuous subcutaneous dosing of angiotensin II (1.8 mg/kg per day SC) over a period of 3 weeks. Ivabradine (10 mg/kg per day) and metoprolol (90 mg/kg per day), which resulted in similar heart rate reduction, or placebo treatments were simultaneously started with infusion of angiotensin II. After 3 weeks, LV function was estimated by conductance catheter technique, cardiac remodeling assessed by estimation of cardiac hypertrophy, fibrosis, and inflammatory stress response by immunohistochemistry or PCR, respectively. Compared with controls, angiotensin II infusion resulted in hypertension in impaired systolic (LV contractility, stroke volume, end systolic elastance, afterload, index of arterial-ventricular coupling, and cardiac output; P<0.05) and diastolic (LV relaxation, LV end diastolic pressure, &tgr;, and stiffness constant &bgr;; P<0.05) LV function. This was associated with a significant increase in cardiac hypertrophy and fibrosis. Increased cardiac stress was also indicated by an increase in cardiac inflammation and apoptosis. Both ivabradine and metoprolol led to a similar reduction in heart rate. Metoprolol also reduced systolic blood pressure. Ivabradine led to a significant improvement in systolic and diastolic LV function (P<0.05). This was associated with less cardiac hypertrophy, fibrosis, inflammation, and cardiac apoptosis (P<0.05). Metoprolol treatment did not prevent the reduction in cardiac function and adverse remodeling, despite a reduction of the inflammatory stress response. Behind heart rate reduction, additional beneficial cardiac effects contribute to heart failure prevention with If-channel inhibition.

Mesenchymal stem cells improve murine acute coxsackievirus B3-induced myocarditis

Aims Coxsackievirus B3 (CVB3)-induced myocarditis, initially considered a sole immune-mediated disease, also results from a direct CVB3-mediated injury of the cardiomyocytes. Mesenchymal stem cells (MSCs) have, besides immunomodulatory, also anti-apoptotic features. In view of clinical translation, we first analysed whether MSCs can be infected by CVB3. Next, we explored whether and how MSCs could reduce the direct CVB3-mediated cardiomyocyte injury and viral progeny release, in vitro, in the absence of immune cells. Finally, we investigated whether MSC application could improve murine acute CVB3-induced myocarditis. Methods and results Phase contrast pictures and MTS viability assay demonstrated that MSCs did not suffer from CVB3 infection 4–12–24–48 h after CVB3 infection. Coxsackievirus B3 RNA copy number decreased in this time frame, suggesting that no CVB3 replication took place. Co-culture of MSCs with CVB3-infected HL-1 cardiomyocytes resulted in a reduction of CVB3-induced HL-1 apoptosis, oxidative stress, intracellular viral particle production, and viral progeny release in a nitric oxide (NO)-dependent manner. Moreover, MSCs required priming via interferon-γ (IFN-γ) to exert their protective effects. In vivo, MSC application improved the contractility and relaxation parameters in CVB3-induced myocarditis, which was paralleled with a reduction in cardiac apoptosis, cardiomyocyte damage, left ventricular tumour necrosis factor-α mRNA expression, and cardiac mononuclear cell activation. Mesenchymal stem cells reduced the CVB3-induced CD4− and CD8− T cell activation in an NO-dependent way and required IFN-γ priming. Conclusion We conclude that MSCs improve murine acute CVB3-induced myocarditis via their anti-apoptotic and immunomodulatory properties, which occur in an NO-dependent manner and require priming via IFN-γ.

Mesenchymal Stromal Cells but Not Cardiac Fibroblasts Exert Beneficial Systemic Immunomodulatory Effects in Experimental Myocarditis

Systemic application of mesenchymal stromal cells (MSCs) in inflammatory cardiomyopathy exerts cardiobeneficial effects. The mode of action is unclear since a sufficient and long-acting cardiac homing of MSCs is unlikely. We therefore investigated the regulation of the immune response in coxsackievirus B3 (CVB3)-induced acute myocarditis after intravenous application of MSCs. Wildtype mice were infected with CVB3 and treated with either PBS, human MSCs or human cardiac fibroblasts intravenously 1 day after infection. Seven days after infection, MSCs could be detected in the spleen, heart, pancreas, liver, lung and kidney, whereby the highest presence was observed in the lung. MSCs increased significantly the myocardial expression of HGF and decreased the expression of the proinflammatory cytokines TNFα, IL1β and IL6 as well as the severity of myocarditis and ameliorated the left ventricular dysfunction measured by conductance catheter. MSCs upregulated the production of IFNγ in CD4+ and CD8+ cells, the number of IL10-producing regulatory T cells and the apoptosis rate of T cells in the spleen. An increased number of CD4+CD25+FoxP3 could be found in the spleen as well as in the circulation. In contrast, application of human cardiac fibroblasts had no effect on the severity of myocarditis and the systemic immune response observed after MSCs-administration. In conclusion, modulation of the immune response in extracardiac organs is associated with cardiobeneficial effects in experimental inflammatory cardiomyopathy after systemic application of MSCs.

High-Density Lipoproteins Reduce Endothelial-to-Mesenchymal Transition

Objective—Endothelial-to-mesenchymal transition is an inflammation-induced process by which endothelial cells can transdifferentiate into fibroblasts. Based on the endothelial-protective and antifibrotic effects of high-density lipoproteins (HDL), we aimed to investigate whether HDL can reduce endothelial-to-mesenchymal transition. Approach and Results—Therefore, human aortic endothelial cells were stimulated with the profibrotic factor transforming growth factor (TGF)-&bgr;1 in the presence or absence of HDL. Their impact on the transition of endothelial cells to mesenchymal-like cells was analyzed. Phase contrast microscopy demonstrated that HDL abrogated the TGF-&bgr;1–induced spindle-shape morphology in human aortic endothelial cells. Furthermore, HDL decreased the TGF-&bgr;1–mediated induction of &agr;-smooth muscle actin expression and concomitant loss in endothelial cadherin expression, as shown by immunofluorescence staining and flow cytometry. In addition, HDL decreased the TGF-&bgr;1–induced collagen deposition in human aortic endothelial cells involving the scavenger receptor class B, type 1 and downstream phosphatidyl inositol-3-kinase following the findings that the HDL-mediated reduction was abrogated by scavenger receptor class B, type 1 siRNA knockdown and phosphatidyl inositol-3-kinase inhibition, respectively. The HDL-mediated reduction in endothelial-to-mesenchymal transition was associated with an induction of the inhibitory Smad, Smad 7. Conclusions—We provide the first in vitro evidence that the endothelial-protective and antifibrotic effects of HDL include the reduction in endothelial-to-mesenchymal transition.

Human Cardiac-Derived Adherent Proliferating Cells Reduce Murine Acute Coxsackievirus B3-Induced Myocarditis

Background Under conventional heart failure therapy, inflammatory cardiomyopathy typically has a progressive course, indicating a need for alternative therapeutic strategies to improve long-term outcomes. We recently isolated and identified novel cardiac-derived cells from human cardiac biopsies: cardiac-derived adherent proliferating cells (CAPs). They have similarities with mesenchymal stromal cells, which are known for their anti-apoptotic and immunomodulatory properties. We explored whether CAPs application could be a novel strategy to improve acute Coxsackievirus B3 (CVB3)-induced myocarditis. Methodology/Principal Findings To evaluate the safety of our approach, we first analyzed the expression of the coxsackie- and adenovirus receptor (CAR) and the co-receptor CD55 on CAPs, which are both required for effective CVB3 infectivity. We could demonstrate that CAPs only minimally express both receptors, which translates to minimal CVB3 copy numbers, and without viral particle release after CVB3 infection. Co-culture of CAPs with CVB3-infected HL-1 cardiomyocytes resulted in a reduction of CVB3-induced HL-1 apoptosis and viral progeny release. In addition, CAPs reduced CD4 and CD8 T cell proliferation. All CAPs-mediated protective effects were nitric oxide- and interleukin-10-dependent and required interferon-γ. In an acute murine model of CVB3-induced myocarditis, application of CAPs led to a decrease of cardiac apoptosis, cardiac CVB3 viral load and improved left ventricular contractility parameters. This was associated with a decline in cardiac mononuclear cell activity, an increase in T regulatory cells and T cell apoptosis, and an increase in left ventricular interleukin-10 and interferon-γ mRNA expression. Conclusions We conclude that CAPs are a unique type of cardiac-derived cells and promising tools to improve acute CVB3-induced myocarditis.

Mesenchymal stromal cells: a promising cell source for the treatment of inflammatory cardiomyopathy.

Inflammatory cardiomyopathy is associated with a diffuse inflammation in the heart accompanied with cardiac fibrosis, cardiomyocyte apoptosis, and reduced capillary density. On the other hand, mesenchymal stromal cells (MSCs) have immunomodulatory, anti-fibrotic, anti-apoptotic, and pro-angiogenic features, making them attractive candidates for the treatment of inflammatory cardiomyopathy. The potential of MSC application for the treatment of myocarditis is supported by their beneficial effects in experimental models of acute and chronic inflammatory cardiomopathy. This review summarizes the cardioprotective features of MSCs and describes how MSCs are primed by the inflammatory environment to exert their protective effects. In view of clinical translation, searching for the optimal source of MSC and delivery route, allowing efficient and non-invasive cell application, the differences between MSCs of distinct origin and between diverse routes of application are outlined.

Mesenchymal Stromal Cells Modulate Monocytes Trafficking in Coxsackievirus B3-Induced Myocarditis

Mesenchymal stromal cell (MSC) application in Coxsackievirus B3 (CVB3)‐induced myocarditis reduces myocardial inflammation and fibrosis, exerts prominent extra‐cardiac immunomodulation, and improves heart function. Although the abovementioned findings demonstrate the benefit of MSC application, the mechanism of the MSC immunomodulatory effects leading to a final cardioprotective outcome in viral myocarditis remains poorly understood. Monocytes are known to be a trigger of myocardial tissue inflammation. The present study aims at investigating the direct effect of MSC on the mobilization and trafficking of monocytes to the heart in CVB3‐induced myocarditis. One day post CVB3 infection, C57BL/6 mice were intravenously injected with 1 x 106 MSC and sacrificed 6 days later for molecular biology and flow cytometry analysis. MSC application reduced the severity of myocarditis, and heart and blood pro‐inflammatory Ly6Chigh and Ly6Cmiddle monocytes, while those were retained in the spleen. Anti‐inflammatory Ly6Clow monocytes increased in the blood, heart, and spleen of MSC‐treated CVB3 mice. CVB3 infection induced splenic myelopoiesis, while MSC application slightly diminished the spleen myelopoietic activity in CVB3 mice. Left ventricular (LV) mRNA expression of the chemokines monocyte chemotactic protein‐1 (MCP)−1, MCP‐3, CCL5, the adhesion molecules intercellular adhesion molecule‐1, vascular cell adhesion molecule‐1, the pro‐inflammatory cytokines interleukin‐6, interleukin‐12, tumor necrosis factor‐α, the pro‐fibrotic transforming growth factorβ1, and circulating MCP‐1 and MCP‐3 levels decreased in CVB3 MSC mice, while LV stromal cell‐derived factor‐1α RNA expression and systemic levels of fractalkine were increased in CVB3 MSC mice. MSC application in CVB3‐induced myocarditis modulates monocytes trafficking to the heart and could be a promising strategy for the resolution of cardiac inflammation and prevention of the disease progression. Stem Cells Translational Medicine 2017;6:1249–1261

Human Endomyocardial Biopsy Specimen-Derived Stromal Cells Modulate Angiotensin II-Induced Cardiac Remodeling

Cardiac‐derived adherent proliferating cells (CardAPs) are cells derived from human endomyocardial biopsy specimens; they share several properties with mesenchymal stromal cells. The aims of this study were to evaluate whether intramyocardial injection of CardAPs modulates cardiac fibrosis and hypertrophy in a mouse model of angiotensin II (Ang II)‐induced systolic heart failure and to analyze underlying mechanisms. Intramyocardial application of 200,000 CardAPs improved left ventricular function. This was paralleled by a decline in left ventricular remodeling, as indicated by a reduction in cardiac fibrosis and hypertrophy. CardAPs reduced the ratio of the left ventricle to body weight and cardiac myosin expression (heavy chain), and decreased the Ang II‐induced phosphorylation state of the cardiomyocyte hypertrophy mediators Akt, extracellular‐signal regulated kinase (ERK) 1, and ERK2. In accordance with the antifibrotic and antihypertrophic effects of CardAPs shown in vivo, CardAP supplementation with cardiac fibroblasts decreased the Ang II‐induced reactive oxygen species production, α‐SMA expression, fibroblast proliferation, and collagen production. Coculture of CardAPs with HL‐1 cardiomyocytes downregulated the Ang II‐induced expression of myosin in HL‐1. All antifibrotic and antihypertrophic features of CardAPs were mediated in a nitric oxide‐ and interleukin (IL)‐10‐dependent manner. Moreover, CardAPs induced a systemic immunomodulation, as indicated by a decrease in the activity of splenic mononuclear cells and an increase in splenic CD4CD25FoxP3, CD4‐IL‐10, and CD8‐IL‐10 T‐regulatory cells in Ang II mice. Concomitantly, splenocytes from Ang II CardAPs mice induced less collagen in fibroblasts compared with splenocytes from Ang II mice. We conclude that CardAPs improve Ang II‐induced cardiac remodeling involving antifibrotic and antihypertrophic effects via paracrine actions and immunomodulatory properties.

Immunomodulatory effects of mesenchymal stromal cells revisited in the context of inflammatory cardiomyopathy.

Myocarditis is a common inflammatory cardiomyopathy, associated with cardiomyocyte apoptosis, which can lead to chronic left ventricular dysfunction. Under conventional heart failure therapy, inflammatory cardiomyopathy typically has a progressive course, indicating a need for alternative therapeutic strategies to improve long-term outcomes. Experimental and clinical studies consistently support the application of cellular transplantation as a strategy to improve myocardial function. Mesenchymal stromal cells (MSCs) mediate distinct paracrine effects supporting endogenous regeneration, but most important are their remarkable immunoregulatory properties. In this review, an overview of current knowledge on immunopathology in myocarditis will be given. Furthermore, current research regarding the immunomodulatory properties of MSCs in the context of myocarditis will be discussed. Finally, the impact of MSC priming by the environment on their functionality and the advantages of systemic administration of MSCs under myocarditis are outlined.