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

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

Integrins and their role in neoplastic transformation

Integryny należą do dużej rodziny heterodimerycznych transbłonowych receptorów pośredniczących w interakcjach pomiędzy komórkami oraz komórkami a macierzą zewnątrzkomórkową. Integryny wraz ze swoimi ligandami zaangażowane są w wiele procesów, takich jak adhezja, migracja i różnicowanie komórek. Co więcej, integryny modulują odpowiedź komórki na większość czynników wzrostu, cytokin i innych rozpuszczalnych czynników, odgrywając tym samym niezwykle istotną rolę w procesach zarówno fizjologicznych jak i patologicznych. Celem tej pracy jest przybliżenie podstawowych informacji na temat klasyfikacji integryn, struktury, procesu aktywacji na skutek związania z ligandem, funkcji w komórkach prawidłowych oraz pokazanie, że zaburzenia w ich ekspresji prowadzą do wielu schorzeń, w tym nowotworów. W oparciu o najnowszą literaturę przedstawione zostały także możliwe zastosowania terapii skierowanej przeciwko receptorom integrynowym w leczeniu nowotworów.Integrins form a large family of heterodimeric transmembrane receptors that mediate cell-cell and cell–extracellular matrix interactions. Integrins and their ligands are involved in multitude of processes such as cell adhesion, migration and differentiation. What is more, integrin mediated interactions modulate cellular response to most growth factors, cytokines and other soluble factors playing crucial role in both physiological and pathological processes. The aim of this review is to provide basic information on integrin classification, structure, process of activation by ligand binding, their functions in normal cells and to show that abnormalities in their expression lead to many diseases – focusing mostly on neoplasms. Subsequently, on the basis of current literature, possible ways of targeting integrin receptors in treatment of these diseases are presented

The influence of TGF-β1 on human gliobastoma multiforme invasiveness in vitro

Złośliwe nowotwory mózgu, a w szczególności glejak wielopostaciowy (GBM, ang. glioblastoma multiforme), stanowią jedno z największych wyzwań dzisiejszej onkologii. Pomimo dużego postępu w zakresie stosowanych metod neurochirurgii, chemio- i radioterapii mediana przeżyć pacjentów od momentu diagnozy GBM wynosi zaledwie 14 miesięcy. Problem w leczeniu nowotworów mózgu stanowi ich lokalizacja oraz obecność bariery krew-mózg. W przypadku GBM dodatkowy problem stanowi niezwykle inwazyjny charakter tego nowotworu sprawiający, że całkowita resekcja zmienionych tkanek jest praktycznie niemożliwa, oraz wysoki stopień heterogenności utrudniający opracowywanie terapii celowanych. Uważa się, że za nabywanie inwazyjnego potencjału oraz oporności na chemioterapię komórek nowotworowych odpowiada proces przejścia epitelialno-mezenchymalnego (EMT, ang. epitelial-mesenchymal transition). Proces ten może być inicjowany przez sygnały pochodzące ze środowiska komórek oraz mutacje w obrębie specyficznych genów. Jednym z głównych czynników napędzającym cały proces jest transformujący czynnik wzrostu β (TGF-β, ang. transforming growth factor β), który za pośrednictwem swoistych receptorów aktywuje ekspresję szeregu czynników transkrypcyjnych. Celem pracy było zbadanie wpływu długotrwałej ekspozycji komórek GBM na TGF-β1 na ich potencjał inwazyjny, zmiany fenotypowe oraz określenie, czy zmianom wywoływanym przez tą cytokinę towarzyszą zmiany w poziomach białek SNAIL1 oraz Cx43. Przeprowadzone badania wykazały, że długotrwała ekspozycja komórek linii GBM na TGF-β1 prowadzi do znacznych zmian w ich fenotypie oraz zwiększeniu ich potencjału do migracji oraz transmigracji. Ponadto, zmiany wywoływane przez TGF-β1 zdają się utrzymywać przez co najmniej 15 dni od zaprzestania eksponowania komórek na działanie tego czynnika. W czasie prowadzenia hodowli z TGF-β1 dochodziło również do akumulacji obu badanych białek, lecz ich poziom szybko malał po ponownym przeniesieniu komórek do zwykłej pożywki.Malignant brain tumors, most notably glioblastoma multiforme (GBM), present one of the greatest challenges in modern oncology. Despite numerous advances in neurosurgery, chemotherapy and radiotherapy median survival after diagnosis is only 14 months. The biggest issue in treating brain tumors lies in their localization and presence of the blood-brain barrier. In case of GBM successful treatment is also hindered due to its extremely invasive nature, rendering complete resection practically impossible and high heterogeneity, impeding the development of targeted therapies. It is believed, that epithelial-mesenchymal transition (EMT) is responsible for the acquisition of invasive potential and resistance to chemotherapy in cancer cells. EMT can be initiated by both extracellular cues and mutations in specific gene sequences, but transforming growth factor β (TGF-β) seems to be the main driving force of the process. TGF-β, by binding to its specific receptors, activates transcription of numerous transcription factors involved in switching cell phenotype from epithelial to mesenchymal. The aim of this study was to assess the long term effects of TGF-β1 exposure on GBM cell lines phenotypes, invasiveness, transmigration potential and changes in levels of SNAIL1 and Cx43 proteins. Conducted research has shown, that long term exposure to TGF-β1 leads to major changes in overall cell morphology, high increase in invasiveness and transmigration potential. Moreover, the changes caused by TGF-β1 seem to persist for at least 15 days after termination of supplementing cell medium with the cytokine. Exposition to TGF-β1 also led to the accumulation of both investigated proteins, but their level decreased rapidly after changing cell medium to one free of TGF-β1

Complexity of TNF-α\alpha 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

Haploinsufficient Rock1+/- and Rock2+/- Mice Are Not Protected from Cardiac Inflammation and Postinflammatory Fibrosis in Experimental Autoimmune Myocarditis

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-Ly6GhiLy6chi neutrophils, CD11b+CD36-Ly6G-Ly6chi 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

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