RESUMO
Endothelial-mesenchymal transition (EndMT) is a form of endothelial dysfunction wherein endothelial cells acquire a mesenchymal phenotype and lose endothelial functions, which contributes to the pathogenesis of intimal hyperplasia and atherosclerosis. The mitogen activated protein kinase 7 (MAPK7) inhibits EndMT and decreases the expression of the histone methyltransferase Enhancer-of-Zeste homologue 2 (EZH2), thereby maintaining endothelial quiescence. EZH2 is the catalytic subunit of the Polycomb Repressive Complex 2 that methylates lysine 27 on histone 3 (H3K27me3). It is elusive how the crosstalk between MAPK7 and EZH2 is regulated in the endothelium and if the balance between MAPK7 and EZH2 is disturbed in vascular disease. In human coronary artery disease, we assessed the expression levels of MAPK7 and EZH2 and found that with increasing intima/media thickness ratio, MAPK7 expression decreased, whereas EZH2 expression increased. In vitro, MAPK7 activation decreased EZH2 expression, whereas endothelial cells deficient of EZH2 had increased MAPK7 activity. MAPK7 activation results in increased expression of microRNA (miR)-101, a repressor of EZH2. This loss of EZH2 in turn results in the increased expression of the miR-200 family, culminating in decreased expression of the dual-specificity phosphatases 1 and 6 who may repress MAPK7 activity. Transfection of endothelial cells with miR-200 family members decreased the endothelial sensitivity to TGFß1-induced EndMT. In endothelial cells there is reciprocity between MAPK7 signaling and EZH2 expression and disturbances in this reciprocal signaling associate with the induction of EndMT and severity of human coronary artery disease.
Assuntos
Transdiferenciação Celular/fisiologia , Doença da Artéria Coronariana/patologia , Endotélio Vascular/patologia , Proteína Potenciadora do Homólogo 2 de Zeste/fisiologia , Mesoderma/patologia , Proteína Quinase 7 Ativada por Mitógeno/fisiologia , Transdução de Sinais/fisiologia , Túnica Íntima/patologia , Regiões 3' não Traduzidas/genética , Doença da Artéria Coronariana/enzimologia , Estenose Coronária/enzimologia , Estenose Coronária/patologia , Fosfatase 1 de Especificidade Dupla/biossíntese , Fosfatase 1 de Especificidade Dupla/genética , Fosfatase 6 de Especificidade Dupla/biossíntese , Fosfatase 6 de Especificidade Dupla/genética , Endotélio Vascular/enzimologia , Ativação Enzimática , Regulação da Expressão Gênica , Genes Reporter , Código das Histonas , Células Endoteliais da Veia Umbilical Humana , Humanos , Hiperplasia , Mesoderma/enzimologia , MicroRNAs/biossíntese , MicroRNAs/genética , Túnica Média/patologiaRESUMO
Understanding the response of endothelial cells to aligned myotubes is important to create an appropriate environment for tissue-engineered vascularized skeletal muscle. Part of the native tissue environment is the extracellular matrix (ECM). The ECM is a supportive scaffold for cells and allows cellular processes such as proliferation, differentiation, and migration. Interstitial matrix and basal membrane both comprise proteinaceous and polysaccharide components for strength, architecture, and volume retention. Virtually all cells are anchored to their basal lamina. One of the physical factors that affects cell behavior is topography, which plays an important role on cell alignment. We tested the hypothesis that topography-driven aligned human myotubes promote and support vascular network formation as a prelude to in vitro engineered vascularized skeletal muscle. Therefore, we used a PDMS-based topography substrate to investigate the influence of pre-aligned myotubes on the network formation of microvascular endothelial cells. The aligned myotubes produced a network of collagen fibers and laminin. This network supported early stages of endothelial network formation.
RESUMO
Understanding how endothelial cell phenotype is affected by topography could improve the design of new tools for tissue engineering as many tissue engineering approaches make use of topography-mediated cell stimulation. Therefore, we cultured human pulmonary microvascular endothelial cells (ECs) on a directional topographical gradient to screen the EC vascular-like network formation and alignment response to nano to microsized topographies. The cell response was evaluated by microscopy. We found that ECs formed unstable vascular-like networks that aggregated in the smaller topographies and flat parts whereas ECs themselves aligned on the larger topographies. Subsequently, we designed a mixed topography where we could explore the network formation and proliferative properties of these ECs by live imaging for three days. Vascular-like network formation continued to be unstable on the topography and were only produced on the flat areas and a fibronectin coating did not improve the network stability. However, an instructive adipose tissue-derived stromal cell (ASC) coating provided the correct environment to sustain the vascular-like networks, which were still affected by the topography underneath. It was concluded that large microsized topographies inhibit vascular endothelial network formation but not proliferation and flat and nano/microsized topographies allow formation of early networks that can be stabilized by using an ASC instructive layer.
RESUMO
Endothelial-mesenchymal transition occurs during intimal hyperplasia and neointima formation via mechanisms that are incompletely understood. Endothelial MAPK7 signaling is a key mechanosensitive factor that protects against endothelial-mesenchymal transition, but its signaling activity is lost in vessel areas that are undergoing pathological remodeling. At sites of vascular remodeling in mice and pigs, endothelial MAPK7 signaling was lost. The TGFß-induced microRNA-374b targets MAPK7 and its downstream effectors in endothelial cells, and its expression induces endothelial-mesenchymal transition. Gain-of-function experiments, where endothelial MAPK7 signaling was restored, precluded endothelial-mesenchymal transition. In human coronary artery disease, disease severity is associated with decreased MAPK7 expression levels and increased miR-374b expression levels. Endothelial-mesenchymal transition occurs in intimal hyperplasia and early lesion formation and is governed in part by microRNA-374b-induced silencing of MAPK7 signaling. Restoration of MAPK7 signaling abrogated these pathological effects in endothelial cells expressing miR-374b. Thus, our data suggest that the TGFß-miR-374b-MAPK7 axis plays a key role in the induction of endothelial-mesenchymal transition during intimal hyperplasia and early lesion formation and might pose an interesting target for antiatherosclerosis therapy. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.
Assuntos
Transição Epitelial-Mesenquimal/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , MicroRNAs/fisiologia , Proteína Quinase 7 Ativada por Mitógeno/antagonistas & inibidores , Animais , Doença da Artéria Coronariana/etiologia , Estenose Coronária/fisiopatologia , Vasos Coronários/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Camundongos Endogâmicos C57BL , Sus scrofa , Suínos , Túnica Íntima/metabolismo , Remodelação VascularRESUMO
Regenerative endodontics exploits the mineralization potential of stem cells from the apical papilla (SCAPs) in order to promote root maturation of permanent immature teeth. SCAPs may encounter post-disinfection residual bacteria either in planktonic or in biofilm growth mode. Bacterial components bind to Toll-like receptors (TLRs) and trigger pro-inflammatory responses. We hypothesized that biofilm-triggered TLR activation affects the mineralization potential of human SCAPs. SCAPs were challenged with conditioned media derived from standardized dual-species biofilms and planktonic bacterial cultures and their inflammatory status and mineralization capacity were studied. Bacterial products from both growth modes (planktonic vs. biofilm) compromised cell viability, proliferation and mineralization capacity of SCAPs, but in a species- and growth mode-dependent fashion. While TLR4 expression remained unaffected, TLR2 expression was upregulated coinciding with a pro-inflammatory activation of SCAPs. Moreover, TLR and its downstream TGF-ß-associated kinase (TAK1) appeared to be blocking mineralization, as inhibition of these factors restored it. In conclusion, bacterial products promoted the pro-inflammatory status and inhibited mineralization of human SCAPs in a TLR-, species-, and culture-dependent fashion. TLR2 emerged as the pivotal mediator of these responses and further research is warranted towards the judicious manipulation of SCAPs in order to modify the untoward events of TLR-priming and signaling.
Assuntos
Biofilmes/crescimento & desenvolvimento , Papila Dentária/citologia , Boca/microbiologia , Ápice Dentário/citologia , Adolescente , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Papila Dentária/imunologia , Regulação da Expressão Gênica , Humanos , MAP Quinase Quinase Quinases/metabolismo , Osteogênese , Células-Tronco/citologia , Células-Tronco/imunologia , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , Ápice Dentário/imunologia , Calcificação de Dente , Adulto JovemRESUMO
Endothelial-to-mesenchymal transition (EndMT) is characterized by the loss of endothelial cell markers and functions, and coincides with de novo expression of mesenchymal markers. EndMT is induced by TGFß1 and changes endothelial microRNA expression. We found that miR-20a is decreased during EndMT, and that ectopic expression of miR-20a inhibits EndMT induction. TGFß1 induces cellular hypertrophy in human umbilical vein endothelial cells and abrogates VE-cadherin expression, reduces endothelial sprouting capacity and induces the expression of the mesenchymal marker SM22α (also known as TAGLN). We identified ALK5 (also known as TGFBR1), TGFBR2 and SARA (also known as ZFYVE9) as direct miR-20a targets. Expression of miR-20a mimics abrogate the endothelial responsiveness to TGFß1, by decreasing ALK5, TGFBR2 and SARA, and inhibit EndMT, as indicated by the maintenance of VE-cadherin expression, the ability of the cells to sprout and the absence of SM22α expression. FGF2 increases miR-20a expression and inhibits EndMT in TGFß1-stimulated endothelial cells. In summary, FGF2 controls endothelial TGFß1 signaling by regulating ALK5, TGFBR2 and SARA expression through miR-20a. Loss of FGF2 signaling combined with a TGFß1 challenge reduces miR-20a levels and increases endothelial responsiveness to TGFß1 through elevated receptor complex levels and activation of Smad2 and Smad3, which culminates in EndMT.
Assuntos
Transdiferenciação Celular/fisiologia , Células Endoteliais/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Células-Tronco Mesenquimais/metabolismo , MicroRNAs/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Antígenos CD , Biomarcadores/metabolismo , Caderinas , Células Cultivadas , Células HEK293 , Células Endoteliais da Veia Umbilical Humana , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Serina Endopeptidases/metabolismo , Transdução de Sinais/fisiologiaRESUMO
BACKGROUND: Experimental clinical stem cell therapy has been used for more than a decade to alleviate the adverse aftermath of acute myocardial infarction (aMI). The post-infarcted myocardial microenvironment is characterized by cardiomyocyte death, caused by ischemia and inflammation. These conditions may negatively affect administered stem cells. As postnatal cardiomyocytes have a poor proliferation rate, while induction of proliferation seems even more rare. Thus stimulation of their proliferation rate is essential after aMI. In metaplastic disease, the pro-inflammatory cytokine interleukin-6 (IL-6) has been identified as potent mediators of the proliferation rate. We hypothesized that IL-6 could augment the proliferation rate of (slow-)dividing cardiomyocytes. METHODS: To mimic the behavior of therapeutic cells in the post-infarct cardiac microenvironment, human Adipose Derived Stromal Cells (ADSC) were cultured under hypoxic (2% O2) and pro-inflammatory conditions (IL-1ß) for 24h. Serum-free conditioned medium from ADSC primed with hypoxia and/or IL-1ß was added to rat neonatal cardiomyocytes and adult cardiomyocytes (HL-1) to assess paracrine-driven changes in cardiomyocyte proliferation rate and induction of myogenic signaling pathways. RESULTS: We demonstrate that ADSC enhance the proliferation rate of rat neonatal cardiomyocytes and adult HL-1 cardiomyocytes in a paracrine fashion. ADSC under hypoxia and inflammation in vitro had increased the interleukin-6 (IL-6) gene and protein expression. Similar to conditioned medium of ADSC, treatment of rat neonatal cardiomyocytes and HL-1 with recombinant IL-6 alone also stimulated their proliferation rate. This was corroborated by a strong decrease of cardiomyocyte proliferation after addition of IL-6 neutralizing antibody to conditioned medium of ADSC. The stimulatory effect of ADSC conditioned media or IL-6 was accomplished through activation of both Janus Kinase-Signal Transducer and Activator of Transcription (JAK/STAT) and Mitogen-Activated Protein (MAP) kinases (MAPK) mitogenic signaling pathways. CONCLUSION: ADSC are promising therapeutic cells for cardiac stem cell therapy. The inflammatory and hypoxic host post-MI microenvironment enhances the regenerative potential of ADSC to promote the proliferation rate of cardiomyocytes. This was achieved in paracrine manner, which warrants the development of ADSC conditioned medium as an "of-the-shelf" product for treatment of post-myocardial infarction complications.
Assuntos
Tecido Adiposo/metabolismo , Proliferação de Células , Sistema de Sinalização das MAP Quinases , Miócitos Cardíacos/citologia , Fator de Transcrição STAT3/metabolismo , Células Estromais/metabolismo , Tecido Adiposo/citologia , Tecido Adiposo/enzimologia , Animais , Sequência de Bases , Técnicas de Cocultura , Meios de Cultivo Condicionados , Primers do DNA , Ensaio de Imunoadsorção Enzimática , Humanos , Interleucina-6/metabolismo , Microscopia de Fluorescência , RNA Mensageiro/genética , Ratos , Células Estromais/citologia , Células Estromais/enzimologiaRESUMO
During heart development, cells from the proepicardial organ spread over the naked heart tube to form the epicardium. From here, epicardium-derived cells (EPDCs) migrate into the myocardium. EPDCs proved to be indispensable for the formation of the ventricular compact zone and myocardial maturation, by largely unknown mechanisms. In this study we investigated in vitro how EPDCs affect cardiomyocyte proliferation, cellular alignment and contraction, as well as the expression and cellular distribution of proteins involved in myocardial maturation. Embryonic quail EPDCs induced proliferation of neonatal mouse cardiomyocytes. This required cell-cell interactions, as proliferation was not observed in transwell cocultures. Western blot analysis showed elevated levels of electrical and mechanical junctions (connexin43, N-cadherin), sarcomeric proteins (Troponin-I, alpha-actinin), extracellular matrix (collagen I and periostin) in cocultures of EPDCs and cardiomyocytes. Immunohistochemistry indicated more membrane-bound expression of Cx43, N-cadherin, the mechanotransduction molecule focal adhesion kinase, and higher expression of the sarcoplasmic reticulum Ca(2+) ATPase (SERCA2a). Newly developed software for analysis of directionality in immunofluorescent stainings showed a quantitatively determined enhanced cellular alignment of cardiomyocytes. This was functionally related to increased contraction. The in vitro effects of EPDCs on cardiomyocytes were confirmed in three reciprocal in vivo models for EPDC-depletion (chicken and mice) in which downregulation of myocardial N-cadherin, Cx43, and FAK were observed. In conclusion, direct interaction of EPDCs with cardiomyocytes induced proliferation, correct mechanical and electrical coupling of cardiomyocytes, ECM-deposition and concurrent establishment of cellular array. These findings implicate that EPDCs are ideal candidates as adjuvant cells for cardiomyocyte integration during cardiac (stem) cell therapy.
Assuntos
Miócitos Cardíacos/citologia , Pericárdio/citologia , Pericárdio/metabolismo , Animais , Animais Recém-Nascidos , Western Blotting , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Meios de Cultivo Condicionados/farmacologia , CamundongosRESUMO
AIMS: Reciprocal plasticity exists between endothelial and mesenchymal lineages. For instance, mature endothelial cells adopt a smooth muscle-like phenotype through transforming growth factor beta-1 (TGFbeta1)-driven endothelial-to-mesenchymal transdifferentiation (EndMT). Peripheral blood contains circulating endothelial progenitor cells of which the endothelial colony-forming cells (ECFCs) harbour stem cell-like properties. Given the plasticity between endothelial and mesenchymal lineages and the stem cell-like properties of ECFCs, we hypothesized that ECFCs can give rise to smooth muscle-like progeny. METHODS AND RESULTS: ECFCs were stimulated with TGFbeta1, after which TGFbeta signalling cascades and their downstream effects were investigated. Indeed, EndMT of ECFCs resulted in smooth muscle-like progeniture. TGFbeta1-driven EndMT is mediated by ALK5 kinase activity, increased downstream Smad2 signalling, and reduced protein levels of inhibitor of DNA-binding protein 3. ECFCs lost expression of endothelial markers and endothelial anti-thrombogenic function. Simultaneously, mesenchymal marker expression was gained, cytoskeletal rearrangements occurred, and cells acquired a contractile phenotype. Transdifferentiated ECFCs were phenotypically stable and self-sustaining and, importantly, showed fibroblast growth factor-2 and angiopoietin-1-mediated pro-angiogenic paracrine properties. CONCLUSION: Our study is the first to demonstrate that ECFCs can give rise to smooth muscle-like progeny, with potential therapeutic benefits. These findings further illustrate that ECFCs are highly plastic, which by itself has implications for therapeutical use.
Assuntos
Linhagem da Célula , Transdiferenciação Celular , Células Endoteliais/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Neovascularização Fisiológica , Células-Tronco/metabolismo , Angiopoietina-1/metabolismo , Biomarcadores/metabolismo , Células Cultivadas , Citoesqueleto/metabolismo , Sangue Fetal/citologia , Fator 2 de Crescimento de Fibroblastos/metabolismo , Humanos , Proteínas Inibidoras de Diferenciação/metabolismo , Músculo Liso Vascular/citologia , Proteínas de Neoplasias/metabolismo , Comunicação Parácrina , Fenótipo , Proteínas Serina-Treonina Quinases/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Transdução de Sinais , Proteína Smad2/metabolismo , Fatores de Tempo , Fator de Crescimento Transformador beta1/metabolismo , VasoconstriçãoRESUMO
Targeting viral proteins early during infection may limit exacerbation of human cytomegalovirus infection. The viral chemokine-receptor homologue US28 interferes with leukocyte trafficking and, possibly, viral replication. Because US28 molecules are abundant on the surface of infected cells, this homologue is a potential target for antiviral therapy. To assess the relationship between US28 and disease activity, we measured, by quantitative reverse-transcription polymerase chain reaction, the levels of US28 and immediate-early (IE) 1 gene transcripts in the blood of lung-transplant recipients. We found that, during primary and secondary infection, the IE1 and US28 genes have early transcription kinetics and are expressed at similar levels. This may render US28 an attractive target for antiviral therapy.