RESUMO
During select pathological conditions, the heart can hypertrophy and remodel in either a dilated or concentric ventricular geometry, which is associated with lengthening or widening of cardiomyocytes, respectively. The mitogen-activated protein kinase kinase 1 (MEK1) and extracellular signal-related kinase 1 and 2 (ERK1/2) pathway has been implicated in these differential types of growth such that cardiac overexpression of activated MEK1 causes profound concentric hypertrophy and cardiomyocyte thickening, while genetic ablation of the genes encoding ERK1/2 in the mouse heart causes dilation and cardiomyocyte lengthening. However, the mechanisms by which this kinase signaling pathway controls cardiomyocyte directional growth as well as its downstream effectors are poorly understood. To investigate this, we conducted an unbiased phosphoproteomic screen in cultured neonatal rat ventricular myocytes treated with an activated MEK1 adenovirus, the MEK1 inhibitor U0126, or an eGFP adenovirus control. Bioinformatic analysis identified cytoskeletal-related proteins as the largest subset of differentially phosphorylated proteins. Phos-tag and traditional Western blotting were performed to confirm that many cytoskeletal proteins displayed changes in phosphorylation with manipulations in MEK1-ERK1/2 signaling. From this, we hypothesized that the actin cytoskeleton would be changed in vivo in the mouse heart. Indeed, we found that activated MEK1 transgenic mice and gene-deleted mice lacking ERK1/2 protein had enhanced non-sarcomeric actin expression in cardiomyocytes compared with wild-type control hearts. Consistent with these results, cytoplasmic ß- and γ-actin were increased at the subcortical intracellular regions of adult cardiomyocytes. Together, these data suggest that MEK1-ERK1/2 signaling influences the non-sarcomeric cytoskeletal actin network, which may be important for facilitating the growth of cardiomyocytes in length and/or width.NEW & NOTEWORTHY Here, we performed an unbiased analysis of the total phosphoproteome downstream of MEK1-ERK1/2 kinase signaling in cardiomyocytes. Pathway analysis suggested that proteins of the non-sarcomeric cytoskeleton were the most differentially affected. We showed that cytoplasmic ß-actin and γ-actin isoforms, regulated by MEK1-ERK1/2, are localized to the subcortical space at both lateral membranes and intercalated discs of adult cardiomyocytes suggesting how MEK1-ERK1/2 signaling might underlie directional growth of adult cardiomyocytes.
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Actinas , Miócitos Cardíacos , Camundongos , Ratos , Animais , Miócitos Cardíacos/metabolismo , Actinas/metabolismo , Sistema de Sinalização das MAP Quinases , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Transdução de Sinais , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Citoesqueleto/metabolismo , Camundongos Transgênicos , Hipertrofia/metabolismo , Hipertrofia/patologia , Proteínas do Citoesqueleto/metabolismo , Células CultivadasRESUMO
Introduction: The ribosomal protein L3-like (RPL3L) is a heart and skeletal muscle-specific ribosomal protein and paralogue of the more ubiquitously expressed RPL3 protein. Mutations in the human RPL3L gene are linked to childhood cardiomyopathy and age-related atrial fibrillation, yet the function of RPL3L in the mammalian heart remains unknown. Methods and Results: Here, we observed that mouse cardiac ventricles express RPL3 at birth, where it is gradually replaced by RPL3L in adulthood but re-expressed with induction of hypertrophy in adults. Rpl3l gene-deleted mice were generated to examine the role of this gene in the heart, although Rpl3l -/- mice showed no overt changes in cardiac structure or function at baseline or after pressure overload hypertrophy, likely because RPL3 expression was upregulated and maintained in adulthood. mRNA expression analysis and ribosome profiling failed to show differences between the hearts of Rpl3l null and wild type mice in adulthood. Moreover, ribosomes lacking RPL3L showed no differences in localization within cardiomyocytes compared to wild type controls, nor was there an alteration in cardiac tissue ultrastructure or mitochondrial function in adult Rpl3l -/- mice. Similarly, overexpression of either RPL3 or RPL3L with adeno-associated virus -9 in the hearts of mice did not cause discernable pathology. However, by 18 months of age Rpl3l -/- null mice had significantly smaller hearts compared to wild type littermates. Conclusion: Thus, deletion of Rpl3l forces maintenance of RPL3 expression within the heart that appears to fully compensate for the loss of RPL3L, although older Rpl3l -/- mice showed a mild but significant reduction in heart weight.
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OBJECTIVE: In this work, we have sought to define growth factor requirements and the signaling basis for different stages of human vascular morphogenesis and maturation. Approach and Results: Using a serum-free model of endothelial cell (EC) tube morphogenesis in 3-dimensional collagen matrices that depends on a 5 growth factor combination, SCF (stem cell factor), IL (interleukin)-3, SDF (stromal-derived factor)-1α, FGF (fibroblast growth factor)-2, and insulin (factors), we demonstrate that VEGF (vascular endothelial growth factor) pretreatment of ECs for 8 hours (ie, VEGF priming) leads to marked increases in the EC response to the factors which includes; EC tip cells, EC tubulogenesis, pericyte recruitment and proliferation, and basement membrane deposition. VEGF priming requires VEGFR2, and the effect of VEGFR2 is selective to the priming response and does not affect factor-dependent tubulogenesis in the absence of priming. Key molecule and signaling requirements for VEGF priming include RhoA, Rock1 (Rho-kinase), PKCα (protein kinase C α), and PKD2 (protein kinase D2). siRNA suppression or pharmacological blockade of these molecules and signaling pathways interfere with the ability of VEGF to act as an upstream primer of downstream factor-dependent EC tube formation as well as pericyte recruitment. VEGF priming was also associated with the formation of actin stress fibers, activation of focal adhesion components, upregulation of the EC factor receptors, c-Kit, IL-3Rα, and CXCR4 (C-X-C chemokine receptor type 4), and upregulation of EC-derived PDGF (platelet-derived growth factor)-BB, PDGF-DD, and HB-EGF (heparin-binding epidermal growth factor) which collectively affect pericyte recruitment and proliferation. CONCLUSIONS: Overall, this study defines a signaling signature for a separable upstream VEGF priming step, which can activate ECs to respond to downstream factors that are necessary to form branching tube networks with associated mural cells.
Assuntos
Indutores da Angiogênese/farmacologia , Comunicação Celular/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Neovascularização Fisiológica/efeitos dos fármacos , Pericitos/metabolismo , Fator A de Crescimento do Endotélio Vascular/farmacologia , Comunicação Celular/genética , Células Cultivadas , Técnicas de Cocultura , Regulação da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Neovascularização Fisiológica/genética , Fosforilação , Transdução de Sinais , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/agonistas , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Here we examine the question of how endothelial cells (ECs) develop their apical membrane surface domain during lumen and tube formation. We demonstrate marked apical membrane targeting of activated Src kinases to this apical domain during early and late stages of this process. Immunostaining for phosphotyrosine or phospho-Src reveals apical membrane staining in intracellular vacuoles initially. This is then followed by vacuole to vacuole fusion events to generate an apical luminal membrane, which is similarly decorated with activated phospho-Src kinases. Functional blockade of Src kinases completely blocks EC lumen and tube formation, whether this occurs during vasculogenic tube assembly or angiogenic sprouting events. Multiple Src kinases participate in this apical membrane formation process and siRNA suppression of Src, Fyn and Yes, but not Lyn, blocks EC lumen formation. We also demonstrate strong apical targeting of Src-GFP and Fyn-GFP fusion proteins and increasing their expression enhances lumen formation. Finally, we show that Src- and Fyn-associated vacuoles track and fuse along a subapically polarized microtubule cytoskeleton, which is highly acetylated. These vacuoles generate the apical luminal membrane in a stereotypically polarized, perinuclear position. Overall, our study identifies a critical role for Src kinases in creating and decorating the EC apical membrane surface during early and late stages of lumen and tube formation, a central event in the molecular control of vascular morphogenesis.
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Membrana Celular/metabolismo , Células Endoteliais/metabolismo , Endotélio Vascular/citologia , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Quinases da Família src/metabolismo , Animais , Células Endoteliais/citologia , Endotélio Vascular/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Neovascularização Fisiológica , Fosforilação , Transporte Proteico , Proteínas Proto-Oncogênicas c-fyn/genética , Transdução de Sinais , Vacúolos/metabolismo , Quinases da Família src/genéticaRESUMO
This review addresses fundamental mechanisms underlying how capillaries form in three-dimensional extracellular matrices and how endothelial cells (ECs) and pericytes co-assemble to form capillary networks. In addition to playing a critical role in supplying oxygen and nutrients to tissues, recent work suggests that blood vessels supply important signals to facilitate tissue development. Here, we hypothesize that another major function of capillaries is to supply signals to suppress major disease mechanisms including inflammation, infection, thrombosis, hemorrhage, edema, ischemic injury, fibrosis, autoimmune disease and tumor growth/progression. Capillary dysfunction plays a key pathogenic role in many human diseases, and thus, this suppressing function may be attenuated and central toward the initiation and progression of disease. We describe how capillaries form through creation of EC-lined tube networks and vascular guidance tunnels in 3D extracellular matrices. Pericytes recruit to the abluminal EC tube surface within these tunnel spaces, and work together to assemble the vascular basement membrane matrix. These processes occur under serum-free conditions in 3D collagen or fibrin matrices and in response to five key growth factors which are stem cell factor, interleukin-3, stromal-derived factor-1α, fibroblast growth factor-2 and insulin. In addition, we identified a key role for EC-derived platelet-derived growth factor-BB and heparin-binding epidermal growth factor in pericyte recruitment and proliferation to promote EC-pericyte tube co-assembly and vascular basement membrane matrix deposition. A molecular understanding of capillary morphogenesis and maturation should lead to novel therapeutic strategies to repair capillary dysfunction in major human disease contexts including cancer and diabetes.
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Células Endoteliais/citologia , Matriz Extracelular/metabolismo , Morfogênese/fisiologia , Neovascularização Fisiológica/fisiologia , Pericitos/citologia , Animais , Membrana Basal/metabolismo , HumanosRESUMO
Considerable progress has occurred toward our understanding of the molecular basis for vascular morphogenesis, maturation, and stabilization. A major reason for this progress has been the development of novel in vitro systems to investigate these processes in 3D extracellular matrices. In this chapter, we present models of human endothelial cell (EC) tube formation and EC-pericyte tube co-assembly using serum-free defined conditions in 3D collagen matrices. We utilize both human venous and arterial ECs and show that both cell types readily form tubes and induce pericyte recruitment and both ECs and pericytes work together to remodel the extracellular matrix environment by assembling the vascular basement membrane, a key step in capillary tube network maturation and stabilization. Importantly, we have shown that these events occur under serum-free defined conditions using the hematopoietic stem cell cytokines, SCF, IL-3, and SDF-1α and also including FGF-2. In contrast, the combination of VEGF and FGF-2 fails to support vascular tube morphogenesis or pericyte-induced tube maturation under the same serum-free defined conditions. Furthermore, we present novel assays whereby we have developed both human ECs and pericytes to induce specific genes using a doxycycline-regulated lentiviral system. In this manner, we can upregulate the expression of wild-type or mutant gene products at any stage of vascular morphogenesis or maturation in 3D matrices. These in vitro experimental approaches will continue to identify key molecular requirements and signaling pathways that control fundamental events in tissue vascularization under normal or pathologic conditions. Furthermore, these models will provide new insights into the development of novel disease therapeutic approaches where vascularization is an important pathogenic component and create new ways to assemble capillary tube networks with associated pericytes for tissue engineering applications.
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Técnicas de Cocultura/métodos , Doxiciclina/farmacologia , Células Endoteliais/citologia , Matriz Extracelular/genética , Morfogênese , Neovascularização Fisiológica , Pericitos/citologia , Técnicas de Cultura de Tecidos/métodos , Animais , Membrana Basal/metabolismo , Comunicação Celular/efeitos dos fármacos , Colágeno Tipo I/farmacologia , Citocinas/farmacologia , Células Endoteliais/efeitos dos fármacos , Matriz Extracelular/efeitos dos fármacos , Vetores Genéticos/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Lentivirus/metabolismo , Morfogênese/efeitos dos fármacos , Neovascularização Fisiológica/efeitos dos fármacos , Pericitos/efeitos dos fármacos , Ratos , Inibidor Tecidual de Metaloproteinase-3/metabolismoRESUMO
An important advance using in vitro EC tube morphogenesis and maturation models has been the development of systems using serum-free defined media. Using this approach, the growth factors and cytokines which are actually necessary for these events can be determined. The first model developed by our laboratory was such a system where we showed that phorbol ester was needed in order to promote survival and tube morphogenesis in 3D collagen matrices. Recently, we have developed a new system in which the hematopoietic stem cell cytokines, stem cell factor (SCF), interleukin-3 (IL-3), and stromal derived factor-1α (SDF-1α) were added in conjunction with FGF-2 to promote human EC tube morphogenesis in 3D collagen matrices under serum-free defined conditions. This new model using SCF, IL-3, SDF-1α, and FGF-2 also works well following the addition of pericytes where EC tube formation occurs, pericytes are recruited to the tubes, and vascular basement membrane matrix assembly occurs following EC-pericyte interactions. In this chapter, we describe several in vitro assay models that we routinely utilize to investigate the molecular requirements that are critical to EC tube formation and maturation events in 3D extracellular matrix environments.
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Células Endoteliais/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Neovascularização Fisiológica , Pericitos/metabolismo , Células Cultivadas , Quimiocina CXCL12/metabolismo , Matriz Extracelular/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Humanos , Interleucina-3/metabolismo , Morfogênese/fisiologia , Fator de Células-Tronco/metabolismoRESUMO
We describe a novel 3D fibrin matrix model using recombinant hematopoietic stem cell cytokines under serum-free defined conditions which promotes the assembly of human endothelial cell (EC) tubes with co-associated pericytes. Individual ECs and pericytes are randomly mixed together and EC tubes form that is accompanied by pericyte recruitment to the EC tube abluminal surface over a 3-5 day period. These morphogenic processes are stimulated by a combination of the hematopoietic stem cell cytokines, stem cell factor, interleukin-3, stromal derived factor-1α, and Flt-3 ligand which are added in conjunction with fibroblast growth factor (FGF)-2 into the fibrin matrix. In contrast, this tube morphogenic response does not occur under serum-free defined conditions when VEGF and FGF-2 are added together in the fibrin matrices. We recently demonstrated that VEGF and FGF-2 are able to prime EC tube morphogenic responses (i.e. added overnight prior to the morphogenic assay) to hematopoietic stem cell cytokines in collagen matrices and, interestingly, they also prime EC tube morphogenesis in 3D fibrin matrices. EC-pericyte interactions in 3D fibrin matrices leads to marked vascular basement membrane assembly as demonstrated using immunofluorescence and transmission electron microscopy. Furthermore, we show that hematopoietic stem cell cytokines and pericytes stimulate EC sprouting in fibrin matrices in a manner dependent on the α5ß1 integrin. This novel co-culture system, under serum-free defined conditions, allows for a molecular analysis of EC tube assembly, pericyte recruitment and maturation events in a critical ECM environment (i.e. fibrin matrices) that regulates angiogenic events in postnatal life.
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Citocinas/metabolismo , Células Endoteliais/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Pericitos/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Células Cultivadas , Meios de Cultura Livres de Soro , Fibrina/metabolismo , Imunofluorescência , Células-Tronco Hematopoéticas/imunologia , Humanos , Integrina alfa5beta1/metabolismo , Microscopia Eletrônica de TransmissãoRESUMO
OBJECTIVE: Although significant research has detailed angiogenesis during development and cancer, little is known about cardiac angiogenesis, yet it is critical for survival following pathological insult. The transcription factor c-Myc is a target of anticancer therapies because of its mitogenic and proangiogenic induction. In the current study, we investigate its role in cardiac angiogenesis in a cell-dependent and gene-specific context. METHODS AND RESULTS: Angiogenesis assays using c-Myc-deficient cardiac endothelial cells and fibroblasts demonstrate that c-Myc is essential to vessel formation, and fibroblast-mediated vessel formation is dependent on c-Myc expression in fibroblasts. Gene analyses revealed that c-Myc-mediated gene expression is unique in cardiac angiogenesis and varies in a cell-dependent manner. In vitro 3-dimensional cultures demonstrated c-Myc's role in the expression of secreted angiogenic factors, while also providing evidence for c-Myc-mediated cell-cell interactions. Additional in vivo vascular analyses support c-Myc's critical role in capillary formation and vessel patterning during development and also in response to a pathological stimulus where its expression in myocytes is required for angiogenic remodeling. CONCLUSIONS: These data demonstrate that proper c-Myc expression in cardiac fibroblasts and myocytes is essential to cardiac angiogenesis. These results have the potential for novel therapeutic applications involving the angiogenic response during cardiac remodeling.
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Vasos Coronários/citologia , Neovascularização Fisiológica/genética , Proteínas Proto-Oncogênicas c-myc/biossíntese , RNA/genética , Transdução de Sinais , Animais , Comunicação Celular , Células Cultivadas , Vasos Coronários/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Reação em Cadeia da Polimerase , Proteínas Proto-Oncogênicas c-myc/genéticaRESUMO
In the heart, electrical, mechanical, and chemical signals create an environment essential for normal cellular responses to developmental and pathologic cues. Communication between fibroblasts, myocytes, and endothelial cells, as well as the extracellular matrix, are critical to fluctuations in heart composition and function during normal development and pathology. Recent evidence suggests that cytokines play a role in cell-cell signaling in the heart. Indeed, we find that interactions between myocytes and cardiac fibroblasts results in increased interleukin-6 and tumor necrosis factor-alpha secretion. We also used confocal and transmission electron microscopy to observe close relationships and possible direct communication between these cells in vivo. Our results highlight the importance of direct cell-cell communication in the heart, and indicate that interactions between fibroblasts, myocytes, and capillary endothelium results in differential cytokine expression. Studying these cell-cell interactions has many implications for the process of cardiac remodeling and overall heart function during development and cardiopathology.
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Comunicação Celular , Espaço Extracelular/metabolismo , Fibroblastos/metabolismo , Miocárdio/metabolismo , Animais , Células Cultivadas , Técnicas de Cocultura , Endotélio/metabolismo , Matriz Extracelular/metabolismo , Fibroblastos/ultraestrutura , Interleucina-6/metabolismo , Camundongos , Microscopia Eletrônica de Transmissão , Miocárdio/ultraestrutura , Ratos , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND AND PURPOSE: Stress is an important risk factor for cardiovascular disease; however, most of the research on this topic has focused on incidence rather than outcome. The goal of this study was to determine the effects of prior exposure to chronic stress on ischemia-induced neuronal death, microglial activation, and anxiety-like behavior. METHODS: In Experiment 1, mice were exposed to 3 weeks of daily restraint (3 hours) and then subjected to either 8 minutes of cardiac arrest/cardiopulmonary resuscitation (CA/CPR) or sham surgery. Anxiety-like behavior, microglial activation, and neuronal damage were assessed on postischemic Day 4. In Experiment 2, mice were infused intracerebroventricularly with minocycline (10 microg/day) to determine the effect of inhibiting post-CA/CPR microglial activation on the development of anxiety-like behavior and neuronal death. RESULTS: CA/CPR precipitated anxiety-like behavior and increased microglial activation and neuronal damage within the hippocampus relative to sham surgery. Prior exposure to stress exacerbated these measures among CA/CPR mice, but had no significant effect on sham-operated mice. Treatment with minocycline reduced both neuronal damage and anxiety-like behavior among CA/CPR animals. Anxiety-like behavior was significantly correlated with measures of microglial activation but not neuronal damage. CONCLUSIONS: A history of stress exposure increases the pathophysiological response to ischemia and anxiety-like behavior, whereas inhibiting microglial activation reduces neuronal damage and mitigates the development of anxiety-like behavior after CA/CPR. Thus, modulating inflammatory signaling after cerebral ischemia may be beneficial in protecting the brain and preventing the development of affective disorders.
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Ansiedade/prevenção & controle , Reanimação Cardiopulmonar , Parada Cardíaca/tratamento farmacológico , Minociclina/uso terapêutico , Estresse Psicológico/complicações , Estresse Psicológico/tratamento farmacológico , Animais , Ansiedade/etiologia , Ansiedade/psicologia , Reanimação Cardiopulmonar/efeitos adversos , Parada Cardíaca/complicações , Parada Cardíaca/psicologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estresse Psicológico/psicologiaRESUMO
The formation and the patterning of the coronary vasculature are critical to the development and pathology of the heart. Alterations in cytokine signaling and biomechanical load can alter the vascular distribution of the vessels within the heart. Changes in the physical patterning of the vasculature can have significant impacts on the relationships of the pressure-flow network and distribution of critical growth and survival factors to the tissue. Interleukin-6 (IL-6) is a pleiotropic cytokine that regulates several biological processes, including vasculogenesis. Using both immunohistological and cardioangiographic analyses, we tested the hypothesis that IL-6-loss will result in decreased vessel density, along with changes in vascular distribution. Moreover, given the impact of vascular patterning on pressure-flow and distribution mechanics, we utilized non-Euclidean geometrical fractal analysis to quantify the changes in patterning resulting from IL-6-loss. Our analyses revealed that IL-6-loss results in a decreased capillary density and increase in intercapillary distances, but does not alter vessel size or diameter. We also observed that the IL-6-/- coronary vasculature had a marked increase in fractal dimension (D value), indicating that IL-6-loss alters vascular patterning. Characterization of IL-6-loss on coronary vasculature may lend insight into the role of IL-6 in the formation and patterning of the vascular bed.