Basella alba L. (Malabar Spinach) as an Abundant Source of Betacyanins: Identification, Stability, and Bioactivity Studies on Natural and Processed Fruit Pigments.

The antioxidant and anti-inflammatory activities of acylated and decarboxylated gomphrenins, as well as Basella alba L. fruit extract, were investigated in relation to gomphrenin, known for its high biological potential. The most abundant natural acylated gomphrenins, namely, 6'-O-E-caffeoyl-gomphrenin (malabarin) and 6'-O-E-4-coumaroyl-gomphrenin (globosin), were isolated from B. alba extract for the studies. In addition, controlled thermal decarboxylation of gomphrenin in the purified B. alba extract at 65-75 °C resulted in the formation of the most prevalent decarboxylated products, including 17-decarboxy-gomphrenin and 2,17-bidecarboxy-gomphrenin, along with their isoforms. The structures of the decarboxylated pigments were confirmed by NMR analyses. Exploring the matrix effect on pigment reactivity revealed a tremendous increase in the stability of all betacyanins after the initial stage of extract purification using a cation exchanger under various conditions. This indicates the removal of a substantial portion of the unfavorable matrix from the extract, which presumably contains reactive species that could otherwise degrade the pigments. Furthermore, the high concentration of citrates played a significant role in favoring the formation of 2-decarboxy-gomphrenin to a considerable extent. In vitro screening experiments revealed that the tested compounds demonstrated strong anti-inflammatory properties in lipopolysaccharide (LPS)-activated human macrophages. This effect encompassed the selective inhibition of cytokine and chemokine release from activated macrophages, modulation of the chemotactic activity of immune cells, and the regulation of tissue remodeling mediators' release.

TNF-α protects from exacerbated myocarditis and cardiac death by suppressing expansion of activated heart-reactive CD4+ T cells.

AIMS: Tumour necrosis factor α (TNF-α) represents a classical pro-inflammatory cytokine, and its increased levels positively correlate with the severity of many cardiovascular diseases. Surprisingly, some heart failure patients receiving high doses of anti-TNF-α antibodies showed serious health worsening. This work aimed to examine the role of TNF-α signalling on the development and progression of myocarditis and heart-specific autoimmunity. METHODS AND RESULTS: Mice with genetic deletion of TNF-α (Tnf+/- and Tnf-/-) and littermate controls (Tnf+/+) were used to study myocarditis in the inducible and the transgenic T cell receptor (TCRM) models. Tnf+/- and Tnf-/- mice immunized with α-myosin heavy chain peptide (αMyHC) showed reduced myocarditis incidence, but the susceptible animals developed extensive inflammation in the heart. In the TCRM model, defective TNF-α production was associated with increased mortality at a young age due to cardiomyopathy and cardiac fibrosis. We could confirm that TNF-α as well as the secretome of antigen-activated heart-reactive effector CD4+ T (Teff) cells effectively activated the adhesive properties of cardiac microvascular endothelial cells (cMVECs). Our data suggested that TNF-α produced by endothelial in addition to Teff cells promoted leucocyte adhesion to activated cMVECs. Analysis of CD4+ T lymphocytes from both models of myocarditis showed a strongly increased fraction of Teff cells in hearts, spleens, and in the blood of Tnf+/- and Tnf-/- mice. Indeed, antigen-activated Tnf-/- Teff cells showed prolonged long-term survival and TNF-α cytokine-induced cell death of heart-reactive Teff. CONCLUSION: TNF-α signalling promotes myocarditis development by activating cardiac endothelial cells. However, in the case of established disease, TNF-α protects from exacerbating cardiac inflammation by inducing activation-induced cell death of heart-reactive Teff. These data might explain the lack of success of standard anti-TNF-α therapy in heart failure patients and open perspectives for T cell-targeted approaches.

Single-cell transcriptomics reveals subtype-specific molecular profiles in Nrf2-deficient macrophages from murine atherosclerotic aortas.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcriptional regulator of antioxidant and anti-inflammatory response in all cell types. It also activates the transcription of genes important for macrophage function. Nrf2 activity declines with age and has been closely linked to atherosclerosis, but its specific role in this vascular pathology is not clear. Atherosclerotic plaques contain several macrophage subsets with distinct, yet not completely understood, functions in the lesion development. The aim of this study was to analyze the transcriptome of diverse Nrf2-deficient macrophage subpopulations from murine atherosclerotic aortas. Mice with transcriptionally inactive Nrf2 in Cdh5-expressing cells (Nrf2 Cdh5tKO) were used in the experiments. These mice lack transcriptional Nrf2 activity in endothelial cells, but also in a proportion of leukocytes. We confirmed that the bone marrow-derived and tissue-resident macrophages isolated from Nrf2 Cdh5tKO mice exhibit a significant decline in Nrf2 activity. Atherosclerosis was induced in Nrf2 Cdh5tKO and appropriate control mice via adeno-associated viral vector (AAV)-mediated overexpression of murine proprotein convertase subtilisin/kexin type 9 (Pcsk9) in the liver and high-fat diet feeding. After 21 weeks, live aortic cells were sorted on FACS and single-cell RNA sequencing (scRNA-seq) was performed. Unsupervised clustering singled out 13 distinct aortic cell types. Among macrophages, 9 subclusters were identified. Differential gene expression analysis revealed cell subtype-specific expression patterns. A subset of inflammatory macrophages from atherosclerotic Nrf2 Cdh5tKO mice demonstrated downregulation of DNA replication genes (e.g. Mcm7, Lig1, Pola1) concomitant with upregulation of DNA damage sensor Atr gene. Atherosclerotic Nrf2 Cdh5tKO Lyve1+ resident macrophages showed strong upregulation of IFN-stimulated genes, as well as changes in the expression of death pathways-associated genes (Slc40a1, Bcl2a1). Furthermore, we observed subtype-specific expression of core ferroptosis genes (e.g. Cp, Hells, Slc40a1) in inflammatory versus tissue resident macrophages. This observation suggested a link between ferroptosis and inflammatory microenvironment appearing at a very early stage of atherogenesis. Our findings indicate that Nrf2 deficiency in aortic macrophages leads to subtype-specific transcriptomic changes associated with inflammation, iron homeostasis, cell injury or death pathways. This may help understanding the role of aging-associated decline of Nrf2 activity and the function of specific macrophage subtypes in atherosclerotic lesion development.

Autophagy in Heart Failure: Insights into Mechanisms and Therapeutic Implications.

Autophagy, a dynamic and complex process responsible for the clearance of damaged cellular components, plays a crucial role in maintaining myocardial homeostasis. In the context of heart failure, autophagy has been recognized as a response mechanism aimed at counteracting pathogenic processes and promoting cellular health. Its relevance has been underscored not only in various animal models, but also in the human heart. Extensive research efforts have been dedicated to understanding the significance of autophagy and unravelling its complex molecular mechanisms. This review aims to consolidate the current knowledge of the involvement of autophagy during the progression of heart failure. Specifically, we provide a comprehensive overview of published data on the impact of autophagy deregulation achieved by genetic modifications or by pharmacological interventions in ischemic and non-ischemic models of heart failure. Furthermore, we delve into the intricate molecular mechanisms through which autophagy regulates crucial cellular processes within the three predominant cell populations of the heart: cardiomyocytes, cardiac fibroblasts, and endothelial cells. Finally, we emphasize the need for future research to unravel the therapeutic potential associated with targeting autophagy in the management of heart failure.

The AP-1 transcription factor Fosl-2 drives cardiac fibrosis and arrhythmias under immunofibrotic conditions.

Fibrotic changes in the myocardium and cardiac arrhythmias represent fatal complications in systemic sclerosis (SSc), however the underlying mechanisms remain elusive. Mice overexpressing transcription factor Fosl-2 (Fosl-2tg) represent animal model of SSc. Fosl-2tg mice showed interstitial cardiac fibrosis, disorganized connexin-43/40 in intercalated discs and deregulated expression of genes controlling conduction system, and developed higher heart rate (HR), prolonged QT intervals, arrhythmias with prevalence of premature ventricular contractions, ventricular tachycardias, II-degree atrio-ventricular blocks and reduced HR variability. Following stimulation with isoproterenol Fosl-2tg mice showed impaired HR response. In contrast to Fosl-2tg, immunodeficient Rag2-/-Fosl-2tg mice were protected from enhanced myocardial fibrosis and ECG abnormalities. Transcriptomics analysis demonstrated that Fosl-2-overexpression was responsible for profibrotic signature of cardiac fibroblasts, whereas inflammatory component in Fosl-2tg mice activated their fibrotic and arrhythmogenic phenotype. In human cardiac fibroblasts FOSL-2-overexpression enhanced myofibroblast signature under proinflammatory or profibrotic stimuli. These results demonstrate that under immunofibrotic conditions transcription factor Fosl-2 exaggerates myocardial fibrosis, arrhythmias and aberrant response to stress.

Effects of faecal microbiota transplantation on the small intestinal mucosa in systemic sclerosis.

OBJECTIVES: In SSc, gastrointestinal tract (GIT) involvement is a major concern, with no disease-modifying and limited symptomatic therapies available. Faecal microbiota transplantation (FMT) represents a new therapeutic option for GIT-affliction in SSc, showing clinical promise in a recent controlled pilot trial. Here, we aim to investigate effects of FMT on duodenal biopsies collected from SSc patients by immunohistochemistry and transcriptome profiling. METHODS: We analysed duodenal biopsies obtained pre-intervention (week 0) and post-intervention (weeks 2 and 16) from nine SSc patients receiving an intestinal infusion of FMT (n = 5) or placebo (n = 4). The analysis included immunohistochemistry (IHC) with a selected immune function and fibrosis markers, and whole biopsy transcriptome profiling. RESULTS: In patients receiving FMT, the number of podoplanin- and CD64-expressing cells in the mucosa were lower at week 2 compared with baseline. This decline in podoplanin- (r = 0.94) and CD64-positive (r = 0.89) cells correlated with improved patient-reported lower GIT symptoms. Whole biopsy transcriptome profiling from week 2 showed significant enrichment of pathways critical for cellular and endoplasmic reticulum stress responses, microvillus and secretory vesicles, vascular and sodium-dependent transport, and circadian rhythm. At week 16, we found enrichment of pathways mandatory for binding activity of immunoglobulin receptors, T cell receptor complexes, and chemokine receptors, as well as response to zinc-ions. We found that 25 genes, including Matrix metalloproteinase-1 were upregulated at both week 2 and week 16. CONCLUSION: Combining selective IHC and unbiased gene expression analyses, this exploratory study highlights the potential for disease-relevant organ effects of FMT in SSc patients with GIT involvement. TRIAL REGISTRATION: ClinicalTrials.gov, http://clinicaltrials.gov, NCT03444220.

T Lymphocyte-Derived Exosomes Transport MEK1/2 and ERK1/2 and Induce NOX4-Dependent Oxidative Stress in Cardiac Microvascular Endothelial Cells.

Background: Activation of endothelial cells by inflammatory mediators secreted by CD4+ T lymphocytes plays a key role in the inflammatory response. Exosomes represent a specific class of signaling cues transporting a mixture of proteins, nucleic acids, and other biomolecules. So far, the impact of exosomes shed by T lymphocytes on cardiac endothelial cells remained unknown. Methods and Results: Supernatants of CD4+ T cells activated with anti-CD3/CD28 beads were used to isolate exosomes by differential centrifugation. Activation of CD4+ T cells enhanced exosome production, and these exosomes (CD4-exosomes) induced oxidative stress in cardiac microvascular endothelial cells (cMVECs) without affecting their adhesive properties. Furthermore, CD4-exosome treatment aggravated the generation of mitochondrial reactive oxygen species (ROS), reduced nitric oxide (NO) levels, and enhanced the proliferation of cMVECs. These effects were reversed by adding the antioxidant apocynin. On the molecular level, CD4-exosomes increased NOX2, NOX4, ERK1/2, and MEK1/2 in cMVECs, and ERK1/2 and MEK1/2 proteins were found in CD4-exosomes. Inhibition of either MEK/ERK with U0126 or ERK with FR180204 successfully protected cMVECs from increased ROS levels and reduced NO bioavailability. Treatment with NOX1/4 inhibitor GKT136901 effectively blocked excessive ROS and superoxide production, reversed impaired NO levels, and reversed enhanced cMVEC proliferation triggered by CD4-exosomes. The siRNA-mediated silencing of Nox4 in cMVECs confirmed the key role of NOX4 in CD4-exosome-induced oxidative stress. To address the properties of exosomes under inflammatory conditions, we used the mouse model of CD4+ T cell-dependent experimental autoimmune myocarditis. In contrast to exosomes obtained from control hearts, exosomes obtained from inflamed hearts upregulated NOX2, NOX4, ERK1/2, MEK1/2, increased ROS and superoxide levels, and reduced NO bioavailability in treated cMVECs, and these changes were reversed by apocynin. Conclusion: Our results point to exosomes as a novel class of bioactive factors secreted by CD4+ T cells in immune response and represent potential important triggers of NOX4-dependent endothelial dysfunction. Neutralization of the prooxidative aspect of CD4-exosomes could open perspectives for the development of new therapeutic strategies in inflammatory cardiovascular diseases.

Dersimelagon, a novel oral melanocortin 1 receptor agonist, demonstrates disease-modifying effects in preclinical models of systemic sclerosis.

BACKGROUND: Activation of melanocortin 1 receptor (MC1R) is known to exert broad anti-inflammatory and anti-fibrotic effects. The purpose of this study is to investigate the potential of dersimelagon, a novel oral MC1R agonist, as a therapeutic agent for systemic sclerosis (SSc). METHODS: The effects of dersimelagon phosphoric acid (MT-7117) on skin fibrosis and lung inflammation were evaluated in bleomycin (BLM)-induced SSc murine models that were optimized for prophylactic and therapeutic evaluation. Microarray-based gene expression analysis and serum protein profiling were performed in the BLM-induced SSc models. The effect of MT-7117 on transforming growth factor-ß (TGF-ß)-induced activation of human dermal fibroblasts was evaluated in vitro. Immunohistochemical analyses of MC1R expression in the skin of SSc patients were performed. RESULTS: Prophylactic treatment with MT-7117 (≥ 0.3 mg/kg/day p.o.) significantly inhibited skin fibrosis and lung inflammation, and therapeutic treatment with MT-7117 (≥ 3 mg/kg/day p.o.) significantly suppressed the development of skin fibrosis in the BLM-induced SSc models. Gene array analysis demonstrated that MT-7117 exerts an anti-inflammatory effect via suppression of the activation of inflammatory cells and inflammation-related signals; additionally, vascular dysfunction was extracted as the pathology targeted by MT-7117. Serum protein profiling revealed that multiple SSc-related biomarkers including P-selectin, osteoprotegerin, cystatin C, growth and differentiation factor-15, and S100A9 were suppressed by MT-7117. MT-7117 inhibited the activation of human dermal fibroblasts by suppressing TGF-ß-induced ACTA2 (encoding α-smooth muscle actin) mRNA elevation. MC1R was expressed by monocytes/macrophages, neutrophils, blood vessels (endothelial cells), fibroblasts, and epidermis (keratinocytes) in the skin of SSc patients, suggesting that these MC1R-positive cells could be targets for MT-7117. CONCLUSIONS: MT-7117 demonstrates disease-modifying effects in preclinical models of SSc. Investigations of its mechanism of action and target expression analyses indicate that MT-7117 exerts its positive effect by affecting inflammation, vascular dysfunction, and fibrosis, which are all key pathologies of SSc. The results of the present study suggest that MT-7117 is a potential therapeutic agent for SSc. A phase 2 clinical trial investigating the efficacy and tolerability of MT-7117 in patients with early, progressive diffuse cutaneous SSc is currently in progress.

Angiotensin II receptor 1 controls profibrotic Wnt/ß-catenin signalling in experimental autoimmune myocarditis.

AIMS: Angiotensin (Ang) II signalling has been suggested to promote cardiac fibrosis in inflammatory heart diseases; however, the underlying mechanisms remain obscure. Using Agtr1a-/- mice with genetic deletion of angiotensin receptor type 1 (ATR1) and the experimental autoimmune myocarditis (EAM) model, we aimed to elucidate the role of Ang II-ATR1 pathway in development of heart-specific autoimmunity and post-inflammatory fibrosis. METHODS AND RESULTS: EAM was induced in wild-type (WT) and Agtr1a-/- mice by subcutaneous injections with alpha myosin heavy chain peptide emulsified in complete Freund's adjuvant. Agtr1a-/- mice developed myocarditis to a similar extent as WT controls at day 21 but showed reduced fibrosis and better systolic function at day 40. Crisscross bone marrow chimaera experiments proved that ATR1 signalling in the bone marrow compartment was critical for cardiac fibrosis. Heart infiltrating, bone-marrow-derived cells produced Ang II, but lack of ATR1 in these cells reduced transforming growth factor beta (TGF-ß)-mediated fibrotic responses. At the molecular level, Agtr1a-/- heart-inflammatory cells showed impaired TGF-ß-mediated phosphorylation of Smad2 and TAK1. In WT cells, TGF-ß induced formation of RhoA-GTP and RhoA-A-kinase anchoring protein-Lbc (AKAP-Lbc) complex. In Agtr1a-/- cells, stabilization of RhoA-GTP and interaction of RhoA with AKAP-Lbc were largely impaired. Furthermore, in contrast to WT cells, Agtr1a-/- cells stimulated with TGF-ß failed to activate canonical Wnt pathway indicated by suppressed activity of glycogen synthase kinase-3 (GSK-3)ß and nuclear ß-catenin translocation and showed reduced expression of Wnts. In line with these in vitro findings, ß-catenin was detected in inflammatory regions of hearts of WT, but not Agtr1a-/- mice and expression of canonical Wnt1 and Wnt10b were lower in Agtr1a-/- hearts. CONCLUSION: Ang II-ATR1 signalling is critical for development of post-inflammatory fibrotic remodelling and dilated cardiomyopathy. Our data underpin the importance of Ang II-ATR1 in effective TGF-ß downstream signalling response including activation of profibrotic Wnt/ß-catenin pathway.

WNT/ß-Catenin Signaling Promotes TGF-ß-Mediated Activation of Human Cardiac Fibroblasts by Enhancing IL-11 Production.

Cardiac fibrosis is a pathological process associated with the development of heart failure. TGF-ß and WNT signaling have been implicated in pathogenesis of cardiac fibrosis, however, little is known about molecular cross-talk between these two pathways. The aim of this study was to examine the effect of exogenous canonical WNT3a and non-canonical WNT5a in TGF-ß-activated human cardiac fibroblasts. We found that WNT3a and TGF-ß induced a ß-catenin-dependent response, whereas WNT5a prompted AP-1 activity. TGF-ß triggered profibrotic signatures in cardiac fibroblasts, and co-stimulation with WNT3a or co-activation of the ß-catenin pathway with the GSK3ß inhibitor CHIR99021 enhanced collagen I and fibronectin production and development of active contractile stress fibers. In the absence of TGF-ß, neither WNT3a nor CHIR99021 exerted profibrotic responses. On a molecular level, in TGF-ß-activated fibroblasts, WNT3a enhanced phosphorylation of TAK1 and production and secretion of IL-11 but showed no effect on the Smad pathway. Neutralization of IL-11 activity with the blocking anti-IL-11 antibody effectively reduced the profibrotic response of cardiac fibroblasts activated with TGF-ß and WNT3a. In contrast to canonical WNT3a, co-activation with non-canonical WNT5a suppressed TGF-ß-induced production of collagen I. In conclusion, WNT/ß-catenin signaling promotes TGF-ß-mediated fibroblast-to-myofibroblast transition by enhancing IL-11 production. Thus, the uncovered mechanism broadens our knowledge on a molecular basis of cardiac fibrogenesis and defines novel therapeutic targets for fibrotic heart diseases.

Elevated Fibronectin Levels in Profibrotic CD14+ Monocytes and CD14+ Macrophages in Systemic Sclerosis.

Background: Systemic sclerosis (SSc) is an autoimmune disease characterized by overproduction of extracellular matrix (ECM) and multiorgan fibrosis. Animal studies pointed to bone marrow-derived cells as a potential source of pathological ECM-producing cells in immunofibrotic disorders. So far, involvement of monocytes and macrophages in the fibrogenesis of SSc remains poorly understood. Methods and Results: Immunohistochemistry analysis showed accumulation of CD14+ monocytes in the collagen-rich areas, as well as increased amount of alpha smooth muscle actin (αSMA)-positive fibroblasts, CD68+ and mannose-R+ macrophages in the heart and lungs of SSc patients. The full genome transcriptomics analyses of CD14+ blood monocytes revealed dysregulation in cytoskeleton rearrangement, ECM remodeling, including elevated FN1 (gene encoding fibronectin) expression and TGF-ß signalling pathway in SSc patients. In addition, single cell RNA sequencing analysis of tissue-resident CD14+ pulmonary macrophages demonstrated activated profibrotic signature with the elevated FN1 expression in SSc patients with interstitial lung disease. Peripheral blood CD14+ monocytes obtained from either healthy subjects or SSc patients exposed to profibrotic treatment with profibrotic cytokines TGF-ß, IL-4, IL-10, and IL-13 increased production of type I collagen, fibronectin, and αSMA. In addition, CD14+ monocytes co-cultured with dermal fibroblasts obtained from SSc patients or healthy individuals acquired a spindle shape and further enhanced production of profibrotic markers. Pharmacological blockade of the TGF-ß signalling pathway with SD208 (TGF-ß receptor type I inhibitor), SIS3 (Smad3 inhibitor) or (5Z)-7-oxozeaenol (TGF-ß-activated kinase 1 inhibitor) ameliorated fibronectin levels and type I collagen secretion. Conclusions: Our findings identified activated profibrotic signature with elevated production of profibrotic fibronectin in CD14+ monocytes and CD14+ pulmonary macrophages in SSc and highlighted the capability of CD14+ monocytes to acquire a profibrotic phenotype. Taking together, tissue-infiltrating CD14+ monocytes/macrophages can be considered as ECM producers in SSc pathogenesis.

Down-Regulation of Dkk-1 in Platelets of Patients With Axial Spondyloarthritis.

OBJECTIVE: Axial spondyloarthritis (SpA) is a chronic autoinflammatory disease with new bone formation, which is controlled by Wnt/ß-catenin signaling. Dkk-1 is an inhibitor of the Wnt pathway, and in humans, platelets represent a major source of Dkk-1. This study was undertaken to investigate whether levels of Dkk-1 in serum and platelet expression of DKK1 messenger RNA (mRNA) and Dkk-1 protein are affected in patients with axial SpA compared to healthy controls. METHODS: Forty-one patients with axial SpA and 35 healthy controls were enrolled in the study. Total serum Dkk-1 levels in all patients and healthy controls were measured by quantitative enzyme-linked immunosorbent assay. Platelet DKK1 mRNA was analyzed by quantitative reverse transcriptase-polymerase chain reaction in 20 patients with axial SpA and 20 controls, and Dkk-1 protein levels were measured by immunoblotting in 20 patients with axial SpA and 18 controls. RESULTS: We found a lower concentration of Dkk-1 in the serum from patients with axial SpA compared to the serum from healthy controls (P < 0.0001). Furthermore, the expression of Dkk-1 was significantly reduced both at the transcriptional level (P < 0.04) and at the protein level (P < 0.007) in platelets isolated from the blood of patients with axial SpA. CONCLUSION: Our preliminary observations suggest that dysfunction of the megakaryocyte/platelet axis might be responsible for reduced serum Dkk-1 levels in patients with axial SpA. Dkk-1 is down-regulated in the platelets of patients with axial SpA, a mechanism that might play a role in new bone formation.

The AP-1 Transcription Factor Fosl-2 Regulates Autophagy in Cardiac Fibroblasts during Myocardial Fibrogenesis.

BACKGROUND: Pathological activation of cardiac fibroblasts is a key step in development and progression of cardiac fibrosis and heart failure. This process has been associated with enhanced autophagocytosis, but molecular mechanisms remain largely unknown. METHODS AND RESULTS: Immunohistochemical analysis of endomyocardial biopsies showed increased activation of autophagy in fibrotic hearts of patients with inflammatory cardiomyopathy. In vitro experiments using mouse and human cardiac fibroblasts confirmed that blockade of autophagy with Bafilomycin A1 inhibited fibroblast-to-myofibroblast transition induced by transforming growth factor (TGF)-ß. Next, we observed that cardiac fibroblasts obtained from mice overexpressing transcription factor Fos-related antigen 2 (Fosl-2tg) expressed elevated protein levels of autophagy markers: the lipid modified form of microtubule-associated protein 1A/1B-light chain 3B (LC3BII), Beclin-1 and autophagy related 5 (Atg5). In complementary experiments, silencing of Fosl-2 with antisense GapmeR oligonucleotides suppressed production of type I collagen, myofibroblast marker alpha smooth muscle actin and autophagy marker Beclin-1 in cardiac fibroblasts. On the other hand, silencing of either LC3B or Beclin-1 reduced Fosl-2 levels in TGF-ß-activated, but not in unstimulated cells. Using a cardiac hypertrophy model induced by continuous infusion of angiotensin II with osmotic minipumps, we confirmed that mice lacking either Fosl-2 (Ccl19CreFosl2flox/flox) or Atg5 (Ccl19CreAtg5flox/flox) in stromal cells were protected from cardiac fibrosis. CONCLUSION: Our findings demonstrate that Fosl-2 regulates autophagocytosis and the TGF-ß-Fosl-2-autophagy axis controls differentiation of cardiac fibroblasts. These data provide a new insight for the development of pharmaceutical targets in cardiac fibrosis.

Regulation of Monocyte Adhesion and Type I Interferon Signaling by CD52 in Patients With Systemic Sclerosis.

OBJECTIVE: Systemic sclerosis (SSc) is characterized by dysregulation of type I interferon (IFN) signaling. CD52 is known for its immunosuppressive functions in T cells. This study was undertaken to investigate the role of CD52 in monocyte adhesion and type I IFN signaling in patients with SSc. METHODS: Transcriptome profiles of circulating CD14+ monocytes from patients with limited cutaneous SSc (lcSSc), patients with diffuse cutaneous SSc (dcSSs), and healthy controls were analyzed by RNA sequencing. Levels of CD52, CD11b/integrin αΜ, and CD18/integrin ß2 in whole blood were assessed by flow cytometry. CD52 expression was analyzed in relation to disease phenotype (early, lcSSc, dcSSc) and autoantibody profiles. The impact of overexpression, knockdown, and antibody blocking of CD52 was analyzed by gene and protein expression assays and functional assays. RESULTS: Pathway enrichment analysis indicated an increase in adhesion- and type I IFN-related genes in monocytes from SSc patients. These cells displayed up-regulated expression of CD11b/CD18, reduced expression of CD52, and enhanced adhesion to intercellular adhesion molecule 1 and endothelial cells. Changes in CD52 expression were consistent with the SSc subtypes, as well as with immunosuppressive treatments, autoantibody profiles, and monocyte adhesion properties in patients with SSc. Overexpression of CD52 led to decreased levels of CD18 and monocyte adhesion, while knockdown of CD52 increased monocyte adhesion. Experiments with the humanized anti-CD52 monoclonal antibody alemtuzumab in blood samples from healthy controls increased monocyte adhesion and CD11b/CD18 expression, and enhanced type I IFN responses. Monocytic CD52 expression was up-regulated by interleukin-4 (IL-4)/IL-13 via the STAT6 pathway, and was down-regulated by lipopolysaccharide and IFNs α, ß, and γ in a JAK1 and histone deacetylase IIa (HDAC IIa)-dependent manner. CONCLUSION: Down-regulation of the antiadhesion CD52 antigen in CD14+ monocytes represents a novel mechanism in the pathogenesis of SSc. Targeting of the IFN-HDAC-CD52 axis in monocytes might represent a new therapeutic option for patients with early SSc.

Complexity of TNF-α Signaling in Heart Disease.

Heart disease is a leading cause of death with unmet clinical needs for targeted treatment options. Tumor necrosis factor alpha (TNF-α) represents a master pro-inflammatory cytokine that plays an important role in many immunopathogenic processes. Anti-TNF-α therapy is widely used in treating autoimmune inflammatory disorders, but in case of patients with heart disease, this treatment was unsuccessful or even harmful. The underlying reasons remain elusive until today. This review summarizes the effects of anti-TNF-α treatment in patients with and without heart disease and describes the involvement of TNF-α signaling in a number of animal models of cardiovascular diseases. We specifically focused on the role of TNF-α in specific cardiovascular conditions and in defined cardiac cell types. Although some mechanisms, mainly in disease development, are quite well known, a comprehensive understanding of TNF-α signaling in the failing heart is still incomplete. Published data identify pathogenic and cardioprotective mechanisms of TNF-α in the affected heart and highlight the differential role of two TNF-α receptors pointing to the complexity of the TNF-α signaling. In the light of these findings, it seems that targeting the TNF-α pathway in heart disease may show therapeutic benefits, but this approach must be more specific and selectively block pathogenic mechanisms. To this aim, more research is needed to better understand the molecular mechanisms of TNF-α signaling in the failing heart.

The AP1 Transcription Factor Fosl2 Promotes Systemic Autoimmunity and Inflammation by Repressing Treg Development.

Regulatory T cells (Tregs) represent a major population in the control of immune homeostasis and autoimmunity. Here we show that Fos-like 2 (Fosl2), a TCR-induced AP1 transcription factor, represses Treg development and controls autoimmunity. Mice overexpressing Fosl2 (Fosl2tg) indeed show a systemic inflammatory phenotype, with immune infiltrates in multiple organs. This phenotype is absent in Fosl2tg × Rag2-/- mice lacking T and B cells, and Fosl2 induces T cell-intrinsic reduction of Treg development that is responsible for the inflammatory phenotype. Fosl2tg T cells can transfer inflammation, which is suppressed by the co-delivery of Tregs, while Fosl2 deficiency in T cells reduces the severity of autoimmunity in the EAE model. We find that Fosl2 could affect expression of FoxP3 and other Treg development genes. Our data highlight the importance of AP1 transcription factors, in particular Fosl2, during T cell development to determine Treg differentiation and control autoimmunity.

Long noncoding RNA H19X is a key mediator of TGF-ß-driven fibrosis.

TGF-ß is a master regulator of fibrosis, driving the differentiation of fibroblasts into apoptosis-resistant myofibroblasts and sustaining the production of extracellular matrix (ECM) components. Here, we identified the nuclear long noncoding RNA (lncRNA) H19X as a master regulator of TGF-ß-driven tissue fibrosis. H19X was consistently upregulated in a wide variety of human fibrotic tissues and diseases and was strongly induced by TGF-ß, particularly in fibroblasts and fibroblast-related cells. Functional experiments following H19X silencing revealed that H19X was an obligatory factor for TGF-ß-induced ECM synthesis as well as differentiation and survival of ECM-producing myofibroblasts. We showed that H19X regulates DDIT4L gene expression, specifically interacting with a region upstream of the DDIT4L gene and changing the chromatin accessibility of a DDIT4L enhancer. These events resulted in transcriptional repression of DDIT4L and, in turn, in increased collagen expression and fibrosis. Our results shed light on key effectors of TGF-ß-induced ECM remodeling and fibrosis.

Activated Cardiac Fibroblasts Control Contraction of Human Fibrotic Cardiac Microtissues by a ß-Adrenoreceptor-Dependent Mechanism.

Cardiac fibrosis represents a serious clinical problem. Development of novel treatment strategies is currently restricted by the lack of the relevant experimental models in a human genetic context. In this study, we fabricated self-aggregating, scaffold-free, 3D cardiac microtissues using human inducible pluripotent stem cell (iPSC)-derived cardiomyocytes and human cardiac fibroblasts. Fibrotic condition was obtained by treatment of cardiac microtissues with profibrotic cytokine transforming growth factor ß1 (TGF-ß1), preactivation of foetal cardiac fibroblasts with TGF-ß1, or by the use of cardiac fibroblasts obtained from heart failure patients. In our model, TGF-ß1 effectively induced profibrotic changes in cardiac fibroblasts and in cardiac microtissues. Fibrotic phenotype of cardiac microtissues was inhibited by treatment with TGF-ß-receptor type 1 inhibitor SD208 in a dose-dependent manner. We observed that fibrotic cardiac microtissues substantially increased the spontaneous beating rate by shortening the relaxation phase and showed a lower contraction amplitude. Instead, no changes in action potential profile were detected. Furthermore, we demonstrated that contraction of human cardiac microtissues could be modulated by direct electrical stimulation or treatment with the ß-adrenergic receptor agonist isoproterenol. However, in the absence of exogenous agonists, the ß-adrenoreceptor blocker nadolol decreased beating rate of fibrotic cardiac microtissues by prolonging relaxation time. Thus, our data suggest that in fibrosis, activated cardiac fibroblasts could promote cardiac contraction rate by a direct stimulation of ß-adrenoreceptor signalling. In conclusion, a model of fibrotic cardiac microtissues can be used as a high-throughput model for drug testing and to study cellular and molecular mechanisms of cardiac fibrosis.