Apolipoprotein A-I gene transfer exerts immunomodulatory effects and reduces vascular inflammation and fibrosis in ob/ob mice

Background Obesity is associated with vascular inflammation, fibrosis and reduced high-density lipoproteins (HDL)-cholesterol. We aimed to investigate whether adenoviral gene transfer with human apolipoprotein (apo) A-I (Ad.A-I), the main apo of HDL, could exert immunomodulatory effects and counteract vascular inflammation and fibrosis in ob/ob mice. Methods Ad.A-I transfer was performed in 8 weeks (w) old ob/ob mice, which were sacrificed 7 w later. The aorta was excised for mRNA analysis and the spleen for splenocyte isolation for subsequent flow cytometry and co-culture with murine fibroblasts. HDL was added to mononuclear cells (MNC) and fibroblasts to assess their impact on adhesion capacity and collagen deposition, respectively. Results Ad.A-I led to a 1.8-fold (p < 0.05) increase in HDL-cholesterol versus control ob/ob mice at the day of sacrifice, which was paralleled by a decrease in aortic TNF-α and VCAM-1 mRNA expression. Pre-culture of MNC with HDL decreased their adhesion to TNF-α-activated HAEC. Ad.A-I exerted immunomodulatory effects as evidenced by a downregulation of aortic NOD2 and NLRP3 mRNA expression and by a 12 %, 6.9 %, and 15 % decrease of the induced proliferation/activity of total splenic MNC, CD4+, and CD8+ cells in ob/ob Ad.A-I versus control ob/ob mice, respectively (p < 0.05). Ad.A-I further reduced aortic collagen I and III mRNA expression by 62 % and 66 %, respectively (p < 0.0005), and abrogated the potential of ob/ob splenocytes to induce the collagen content in murine fibroblasts upon co-culture. Finally, HDL decreased the TGF-ß1-induced collagen deposition of murine fibroblasts in vitro

Impact of Syndecan-2-Selected Mesenchymal Stromal Cells on the Early Onset of Diabetic Cardiomyopathy in Diabetic db/db Mice

Background: Mesenchymal stromal cells (MSCs) are an attractive cell type for cell therapy given their immunomodulatory, anti-fibrotic, and endothelial-protective features. The heparin sulfate proteoglycan, syndecan-2/CD362, has been identified as a functional marker for MSC isolation, allowing one to obtain a homogeneous cell product that meets regulatory requirements for clinical use. We previously assessed the impact of wild-type (WT), CD362(-), and CD362(+) MSCs on local changes in protein distribution in left ventricular (LV) tissue and on LV function in an experimental model of early-onset diabetic cardiomyopathy. The present study aimed to further explore their impact on mechanisms underlying diastolic dysfunction in this model. Materials: For this purpose, 1 x 10(6) WT, CD362(-), or CD362(+) MSCs were intravenously (i.v.) injected into 20-week-old diabetic BKS.Cg-m+/+Lepr(db)/BomTac, i.e., db/db mice. Control animals (db+/db) were injected with the equivalent volume of phosphate-buffered saline (PBS) alone. After 4 weeks, mice were sacrificed for further analysis. Results: Treatment with all three MSC populations had no impact on blood glucose levels in db/db mice. WT, CD362(-), and CD362(+) MSC application restored LV nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) levels in db/db mice, which correlated with a reduction in cardiomyocyte stiffness. Furthermore, all stromal cells were able to increase arteriole density in db/db mice. The effect of CD362(+) MSCs on NO and cGMP levels, cardiomyocyte stiffness, and arteriole density was less pronounced than in mice treated with WT or CD362(-) MSCs. Analysis of collagen I and III protein expression revealed that fibrosis had not yet developed at this stage of experimental diabetic cardiomyopathy. All MSCs reduced the number of cardiac CD3(+) and CD68(+) cells in db/db mice, whereas only splenocytes from CD362(-)- and CD362(+)-db/db mice exhibited a lower pro-fibrotic potential compared to splenocytes from db/db mice. Conclusion: CD362(+) MSC application decreased cardiomyocyte stiffness, increased myocardial NO and cGMP levels, and increased arteriole density, although to a lesser extent than WT and CD362(-) MSCs in an experimental model of early-onset diabetic cardiomyopathy without cardiac fibrosis. These findings suggest that the degree in improvement of cardiomyocyte stiffness following CD362(+) MSC application was insufficient to improve diastolic function

Sex- and age-related differences in the inflammatory properties of cardiac fibroblasts: impact on the cardiosplenic axis and cardiac fibrosis

Background: Age and sex are prominent risk factors for heart failure and determinants of structural and functional changes of the heart. Cardiac fibroblasts (cFB) are beyond their task as extracellular matrix-producing cells further recognized as inflammation-supporting cells. The present study aimed to evaluate the impact of sex and age on the inflammatory potential of cFB and its impact on the cardiosplenic axis and cardiac fibrosis. Materials: Left ventricles (LV) of 3- and 12-months old male and female C57BL/6J mice were harvested for immunohistochemistry, immunofluorescence and cFB outgrowth culture and the spleen for flow cytometry. LV-derived cFB and respective supernatants were characterized. Results: LV-derived cFB from 3-months old male mice exhibited a higher inflammatory capacity, as indicated by a higher gene expression of CC-chemokine ligand (CCL) 2, and CCL7 compared to cFB derived from 3-months old female mice. The resulting higher CCL2/chemokine C-X3-C motif ligand (Cx3CL1) and CCL7/Cx3CL1 protein ratio in cell culture supernatants of 3-months old male vs. female cFB was reflected by a higher migration of Ly6Chigh monocytes towards supernatant from 3-months old male vs. female cFB. In vivo a lower ratio of splenic pro-inflammatory Ly6Chigh to anti-inflammatory Ly6Clow monocytes was found in 3-months old male vs. female mice, suggesting a higher attraction of Ly6Chigh compared to Ly6Clow monocytes towards the heart in male vs. female mice. In agreement, the percentage of pro-inflammatory CD68+ CD206- macrophages was higher in the LV of male vs. female mice at this age, whereas the percentage of anti-inflammatory CD68+ CD206+ macrophages was higher in the LV of 3-months old female mice compared to age-matched male animals. In parallel, the percentage of splenic TGF-beta+ cells was higher in both 3- and 12-months old female vs. male mice, as further reflected by the higher pro-fibrotic potential of female vs. male splenocytes at both ages. In addition, female mice displayed a higher total LV collagen content compared to age-matched male mice, whereby collagen content of female cFB was higher compared to male cFB at the age of 12-months. Conclusion: Age- and sex-dependent differences in cardiac fibrosis and inflammation are related to age- and sex-dependent variations in the inflammatory properties of cardiac fibroblasts

Down-regulation of endothelial TLR4 signalling after apo A-I gene transfer contributes to improved survival in an experimental model of lipopolysaccharide-induced inflammation

The protective effects of high-density lipoprotein (HDL) under lipopolysaccharide (LPS) conditions have been well documented. Here, we investigated whether an effect of HDL on Toll-like receptor 4 (TLR4) expression and signalling may contribute to its endothelial-protective effects and to improved survival in a mouse model of LPS-induced inflammation and lethality. HDL cholesterol increased 1.7-fold (p < 0.005) and lung endothelial TLR4 expression decreased 8.4-fold (p < 0.005) 2 weeks after apolipoprotein (apo) A-I gene transfer. Following LPS administration in apo A-I gene transfer mice, lung TLR4 and lung MyD88 mRNA expression, reflecting TLR4 signalling, were 3.0-fold (p < 0.05) and 2.1-fold (p < 0.05) lower, respectively, than in LPS control mice. Concomitantly, LPS-induced lung neutrophil infiltration, lung oedema and mortality were significantly attenuated following apo A–I transfer. In vitro, supplementation of HDL or apo A–I to human microvascular endothelial cells-1 24 h before LPS administration reduced TLR4 expression, as assessed by fluorescent-activated cell sorting, and decreased the LPS-induced MyD88 mRNA expression and NF-κB activity, independently of LPS binding. In conclusion, HDL reduces TLR4 expression and signalling in endothelial cells, which may contribute significantly to the protective effects of HDL in LPS-induced inflammation and lethality

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

Inflammation als therapeutischer Ansatzpunkt bei Herzinsuffizienz

Inflammation plays a major role in the development of heart failure which prevalence is increasing. Despite the current effective pharmaceutical treatment, which targets the neurohumoral activation, the morbidity and mortality of heart failure is still substantial, urging for the search of novel therapeutical strategies. Given the importance of inflammation in the pathogenesis of heart failure, the aim of this cumulative study was to investigate the potential of different strategies to counteract inflammation in two severe models of inflammation: lipopolyssacharide (LPS)- and Coxsackievirus B3 (CVB3)-induced inflammation, in view of finding a novel anti-inflammatory strategy for the treatment of heart failure. The potential of high-density lipoproteins (HDL) and its main apolipoprotein (apo), apo A-I, which are known for their anti-inflammatory properties, to reduce LPS-induced inflammation and their impact on the innate immune system, on Toll-like receptor (TLR) 4 signalling, were elucidated in the first study. The second and third study were directed at evaluating the potential of mesenchymal stromal cells (MSCs) and the recently identified cardiac-derived adherent proliferating cells (CAPs) to reduce CVB3-induced inflammatory cardiomyopathy, respectively. The mode-of-action of MSCs and CAPs, particularly their immunomodulatory effects, and the impact of the inflammatory environment on their functionality were analyzed. Apo A-I gene transfer preceding LPS administration reduced TLR4 signalling in the lung, which was reflected by an attenuation in lung neutrophil infiltration, lung oedema, and mortality. In vitro, supplementation of HDL or apo A–I to endothelial cells prior to LPS reduced TLR4 expression and signalling, independently of the formation of LPS- HDL complexes. MSCs as well as CAPs were able to reduce the CVB3-induced cardiomyocyte apoptosis and activity of (splenic) mononuclear cells in vitro for which they required interferon-γ. These findings were in vivo translated by a reduction in cardiac apoptosis/damage and mononuclear cell activity and by an improvement in cardiac contractility. In conclusion, we demonstrated that the anti-inflammatory effects of HDL comprise the reduction in (endothelial) TLR4 expression and signalling. Furthermore, we showed that MSCs and the MSC-like CAPs improve CVB3-induced inflammatory cardiomyopathy and that MSCs and CAPs require the inflammatory environment to exert their cardioprotective/immunomodulatory effects. Further studies in (other) models of heart failure are still required to further assess the potential of those strategies for the treatment of heart failure.Inflammation spielt eine wesentliche Rolle bei der Entstehung einer Herzinsuffizienz, deren Prävalenz stetig ansteigt. Trotz der derzeit verfügbaren effektiven pharmazeutischen Behandlung, welche auf die neurohumorale Aktivierung abzielt, ist die Morbidität und Mortalität der Herzinsuffizienz noch immer erheblich, weshalb neue therapeutische Strategien benötigt werden. Aufgrund der Bedeutung der Inflammation für die Pathogenese der Herzinsuffizienz bestand das Ziel dieser gesamten Studie darin, das anti- inflammatorische Potenzial verschiedener Strategien in zwei akuten Modellen zu untersuchen: Lipopolysaccharid (LPS)- und Coxsackievirus B3 (CVB3)- induzierte Inflammation. Das Potenzial des Lipoproteins hoher Dichte (HDL) und seines wichtigsten Apolipoproteins (Apo), Apo A-I, welche für ihre anti- inflammatorischen Eigenschaften bekannt sind, die LPS-induzierte Inflammation zu reduzieren und deren Einfluss auf das angeborene Immunsystem, insbesondere auf den Toll-like Rezeptor (TLR) 4 Signalweg, wurden in der ersten Studie aufgeklärt. Die zweite und dritte Studie dienten der Evaluation des Potenzials mesenchymaler Stromazellen (MSCs) bzw. der erst kürzlich identifizierten und aus dem Herzen gewonnenen adhärenten proliferierenden Zellen (CAPs), die CVB3-induzierte inflammatorische Kardiomyopathie zu reduzieren. Die Wirkungsweise von MSCs und CAPs, speziell deren immunmodulatorische Effekte, und der Einfluss der inflammatorischen Umgebung auf deren Funktionalität wurden analysiert. Apo A-I Gentransfer vor der Anwendung von LPS reduzierte die TLR4 Antwort in der Lunge. Dies äußerte sich in einer verminderten Infiltration von Neutrophilen in die Lunge, Anzahl an Lungenödemen und verminderter Mortalität. Die in vitro Zugabe von HDL oder Apo A-I zu Endothelzellen vor dem Hinzufügen von LPS reduzierte die Expression und Antwort von TLR4 unabhängig von der Bildung von HDL-LPS Komplexen. Sowohl MSCs als auch CAPs benötigten Interferon-γ, um die CVB3-induzierte Apoptose der Kardiomyozyten sowie die Aktivität mononukleärer Zellen (aus der Milz) in vitro zu reduzieren. Gleiche Ergebnisse, wie die Reduktion der kardialen Apoptose/Verletzungen als auch der kardialen Aktivität mononukleärer Zellen wurden auch in vivo erzielt und führten zusätzlich zur Verbesserung der Herzkontraktilität. Abschließend ist zu sagen, dass die anti-inflammatorischen Effekte des HDLs eine Reduktion der (endothelialen) TLR4 Expression und Antwort beinhaltet. Weiterhin wurde gezeigt, dass MSCs und die den MSCs ähnlichen CAPs die CVB3-induzierte inflammatorische Kardiomyopathie verbessern. Zudem konnte nachgewiesen werden, dass sowohl MSCs als auch CAPs die inflammatorische Umgebung zur Aktivierung ihrer kardioprotektiven/immunmodulatorischen Effekte benötigen. Weitere Studien in (anderen) Modellen der Herzinsuffizienz sind notwendig, um das Potenzial dieser Strategien zur Behandlung dieser Krankheit abschließend beurteilen zu können

The E3 Ubiquitin Ligase Peli1 Deficiency Promotes Atherosclerosis Progression

Background: Atherosclerosis is a chronic inflammatory vascular disease and the main cause of death and morbidity. Emerging evidence suggests that ubiquitination plays an important role in the pathogenesis of atherosclerosis including control of vascular inflammation, vascular smooth muscle cell (VSMC) function and atherosclerotic plaque stability. Peli1 a type of E3 ubiquitin ligase has emerged as a critical regulator of innate and adaptive immunity, however, its role in atherosclerosis remains to be elucidated. Methods: Apoe&minus;/&minus; mice and Peli1-deficient Apoe&minus;/&minus; Peli1&minus;/&minus; mice were subject to high cholesterol diet. Post sacrifice, serum was collected, and atherosclerotic plaque size and parameters of atherosclerotic plaque stability were evaluated. Immunoprofiling and foam cell quantification were performed. Results: Peli1 deficiency does not affect atherosclerosis lesion burden and cholesterol levels, but promotes VSMCs foam cells formation, necrotic core expansion, collagen, and fibrous cap reduction. Apoe&minus;/&minus; Peli1&minus;/&minus; mice exhibit a storm of inflammatory cytokines, expansion of Th1, Th1, Th17, and Tfh cells, a decrease in regulatory T and B cells and induction of pro-atherogenic serum level of IgG2a and IgE. Conclusions: In the present study, we uncover a crucial role for Peli1 in atherosclerosis as an important regulator of inflammation and VSMCs phenotypic modulation and subsequently atherosclerotic plaque destabilization

Cardiac (myo)fibroblasts modulate the migration of monocyte subsets

Abstract Cardiac fibroblasts play an important role in the regulation of the extracellular matrix and are newly recognized as inflammatory supporter cells. Interferon (IFN)-γ is known to counteract transforming growth factor (TGF)-ß1-induced myofibroblast differentiation. This study aims at investigating in vitro how IFN-γ affects TGF-ß1-induced monocyte attraction. Therefore, C4 fibroblasts and fibroblasts obtained by outgrowth culture from the left ventricle (LV) of male C57BL6/j mice were stimulated with TGF-β1, IFN-γ and TGF-β1 + IFN-γ. Confirming previous studies, IFN-γ decreased the TGF-ß1-induced myofibroblast differentiation, as obviated by lower collagen I, III, α-smooth muscle actin (α-SMA), lysyl oxidase (Lox)-1 and lysyl oxidase-like (LoxL)-2 levels in TGF-β1 + IFN-γ- versus TGF-ß1-stimulated cardiac fibroblasts. TGF-β1 + IFN-γ-stimulated C4 and cardiac fibroblasts displayed a higher CC-chemokine ligand (CCL) 2, CCL7 and chemokine C-X3-C motif ligand (Cx3CL1) release versus sole TGF-ß1-stimulated fibroblasts. Analysis of migrated monocyte subsets towards the different conditioned media further revealed that sole TGF-β1- and IFN-γ-conditioned media particularly attracted Ly6Clow and Ly6Chigh monocytes, respectively, as compared to control media. In line with theses findings, TGF-β1 + IFN-γ-conditioned media led to a lower Ly6Clow/Ly6Chigh monocyte migration ratio compared to sole TGF-ß1 treatment. These differences in monocyte migration reflect the complex interplay of pro-inflammatory cytokines and pro-fibrotic factors in cardiac remodelling and inflammation