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.

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. 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 (T_eff) cells effectively activated the adhesive properties of cardiac microvascular endothelial cells (cMVECs). Our data suggested that TNF-α produced by endothelial in addition to T_eff cells promoted leucocyte adhesion to activated cMVECs. Analysis of CD4⁺ T lymphocytes from both models of myocarditis showed a strongly increased fraction of T_eff cells in hearts, spleens, and in the blood of Tnf^+/− and Tnf^−/− mice. Indeed, antigen-activated Tnf^−/− T_eff cells showed prolonged long-term survival and TNF-α cytokine-induced cell death of heart-reactive T_eff. 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 T_eff. 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. A dual role of TNF-α in autoimmune myocarditis. The top panel illustrates that TNF-α can have a pro-inflammatory effect by activating cardiac endothelial cells and an anti-inflammatory effect by inducing activation-induced cell death of heart-reactive T cells. The bottom panel depicts two alternative scenarios in case of the absence of TNF-α.

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. 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-β)-mediated fibrotic responses. At the molecular level, Agtr1a^-/- heart-inflammatory cells showed impaired TGF-β-mediated phosphorylation of Smad2 and TAK1. In WT cells, TGF-β 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-β failed to activate canonical Wnt pathway indicated by suppressed activity of glycogen synthase kinase-3 (GSK-3)β and nuclear β-catenin translocation and showed reduced expression of Wnts. In line with these in vitro findings, β-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. 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-β downstream signalling response including activation of profibrotic Wnt/β-catenin pathway.

Exposure to environmental nanoparticles is related to the adverse impact on health, including cardiovascular system. Various forms of nanoparticles have been reported to interact with endothelium and induce inflammation. However, the potential role of nanoparticles in the pathogenesis of atherosclerosis and their mechanisms of action are still unclear. The aim of this study was to investigate the effect of two broadly used nanomaterials, which also occur in natural environment - silicon oxide (SiO₂) and ferric oxide (Fe₂O₃) in the form of nanoparticles (NPs) - on the development of atherosclerosis. We used apolipoprotein E-knockout mice exposed to silica and ferric oxide nanoparticles in a whole body inhalation chamber. Inhaled silica nanoparticles augmented the atherosclerotic lesions and increased the percentage of pro-inflammatory M1 macrophages in both the plaque and the peritoneum in apoE^-/- mice. Exposure to ferric oxide nanoparticles did not enhance atherogenesis process, however, it caused significant changes in the atherosclerotic plaque composition (elevated content of CD68-positive macrophages and enlarged necrotic core accompanied by the decreased level of M1 macrophages). Both silica and ferric oxide NPs altered the phenotype of T lymphocytes in the spleen by promoting polarization towards Th17 cells. Exposure to silica and ferric oxide nanoparticles exerts impact on atherosclerosis development and plaque composition. Pro-atherogenic abilities of silica nanoparticles are associated with activation of pro-inflammatory macrophages.

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.

Progressive cardiac fibrosis is a common cause of heart failure. Rho-associated, coiled-coil-containing protein kinases (ROCKs) have been shown to enhance fibrotic processes in the heart and in other organs. In this study, using wild-type, Rock1^+/− and Rock2^+/− haploinsufficient mice and mouse model of experimental autoimmune myocarditis (EAM) we addressed the role of ROCK1 and ROCK2 in development of myocarditis and postinflammatory fibrosis. We found that myocarditis severity was comparable in wild-type, Rock1^+/− and Rock2^+/− mice at day 21 of EAM. During the acute stage of the disease, hearts of Rock1^+/− mice showed unaffected numbers of CD11b⁺CD36⁺ macrophages, CD11b⁺CD36^–Ly6G^hiLy6c^hi neutrophils, CD11b⁺CD36^–Ly6G^–Ly6c^hi inflammatory monocytes, CD11b⁺CD36^–Ly6G^–Ly6c^– monocytes, CD11b⁺SiglecF⁺ eosinophils, CD11b⁺CD11c⁺ inflammatory dendritic cells and type I collagen-producing fibroblasts. Isolated Rock1^+/− cardiac fibroblasts treated with transforming growth factor-beta (TGF-β) showed attenuated Smad2 and extracellular signal-regulated kinase (Erk) phosphorylations that were associated with impaired upregulation of smooth muscle actin alpha (αSMA) protein. In contrast to cardiac fibroblasts, expanded Rock1^+/− heart inflammatory myeloid cells showed unaffected Smad2 activation but enhanced Erk phosphorylation following TGF-β treatment. Rock1^+/− inflammatory cells responded to TGF-β by a reduced transcriptional profibrotic response and failed to upregulate αSMA and fibronectin at the protein levels. Unexpectedly, in the EAM model wild-type, Rock1^+/− and Rock2^+/− mice developed a similar extent of cardiac fibrosis at day 40. In addition, hearts of the wild-type and Rock1^+/− mice showed comparable levels of cardiac vimentin, periostin and αSMA. In conclusion, despite the fact that ROCK1 regulates TGF-β-dependent profibrotic response, neither ROCK1 nor ROCK2 is critically involved in the development of postinflammatory fibrosis in the EAM model.