RESUMO
Endothelial cell (EC) sensing of wall fluid shear stress (FSS) from blood flow governs vessel remodeling to maintain FSS at a specific magnitude or set point in healthy vessels. Low FSS triggers inward remodeling to restore normal FSS but the regulatory mechanisms are unknown. In this paper, we describe the signaling network that governs inward artery remodeling. FSS induces Smad2/3 phosphorylation through the type I transforming growth factor (TGF)-ß family receptor Alk5 and the transmembrane protein Neuropilin-1, which together increase sensitivity to circulating bone morphogenetic protein (BMP)-9. Smad2/3 nuclear translocation and target gene expression but not phosphorylation are maximal at low FSS and suppressed at physiological high shear. Reducing flow by carotid ligation in rodents increases Smad2/3 nuclear localization, while the resultant inward remodeling is blocked by the EC-specific deletion of Alk5. The flow-activated MEKK3/Klf2 pathway mediates the suppression of Smad2/3 nuclear translocation at high FSS, mainly through the cyclin-dependent kinase (CDK)-2-dependent phosphosphorylation of the Smad linker region. Thus, low FSS activates Smad2/3, while higher FSS blocks nuclear translocation to induce inward artery remodeling, specifically at low FSS. These results are likely relevant to inward remodeling in atherosclerotic vessels, in which Smad2/3 is activated through TGF-ß signaling.
Assuntos
Artérias Carótidas/fisiologia , Doenças das Artérias Carótidas/prevenção & controle , Células Endoteliais/fisiologia , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Estresse Mecânico , Remodelação Vascular , Animais , Artérias Carótidas/citologia , Doenças das Artérias Carótidas/metabolismo , Doenças das Artérias Carótidas/patologia , Células Endoteliais/citologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Transdução de Sinais , Proteína Smad2/genética , Proteína Smad3/genética , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismoRESUMO
Chemerin is a multifunctional protein initially characterized in our laboratory as a chemoattractant factor for leukocyte populations. Its main functional receptor is CMKLR1. We identified previously chemerin as an anti-tumoral factor inhibiting the vascularization of tumor grafts. We show here that overexpression of bioactive chemerin in mice results in a reduction of the density of the retinal vascular network during its development and in adults. Chemerin did not affect vascular sprouting during the post-natal development of the network, but rather promoted endothelial cell apoptosis and vessel pruning. This phenotype was reversed to normal in CMKLR1-deficient mice, demonstrating the role of this receptor. Chemerin inhibited also neoangiogenesis in a model of pathological proliferative retinopathy, and in response to hind-limb ischemia. Mechanistically, PTEN and FOXO1 antagonists could almost completely restore the density of the retinal vasculature, suggesting the involvement of the PI3-kinase/AKT pathway in the chemerin-induced vessel regression process.
Assuntos
Quimiocinas , Peptídeos e Proteínas de Sinalização Intercelular , Animais , Apoptose , Quimiocinas/metabolismo , Hipóxia , Peptídeos e Proteínas de Sinalização Intercelular/genética , CamundongosRESUMO
The role of fluid shear stress in vasculature development and remodeling is well appreciated. However, the mechanisms regulating these effects remain elusive. We show that abnormal flow sensing in lymphatic endothelial cells (LECs) caused by Sdc4 or Pecam1 deletion in mice results in impaired lymphatic vessel remodeling, including abnormal valve morphogenesis. Ablation of either gene leads to the formation of irregular, enlarged and excessively branched lymphatic vessels. In both cases, lymphatic valve-forming endothelial cells are randomly oriented, resulting in the formation of abnormal valves. These abnormalities are much more pronounced in Sdc4-/-; Pecam1-/- double-knockout mice, which develop severe edema. In vitro, SDC4 knockdown human LECs fail to align under flow and exhibit high expression of the planar cell polarity protein VANGL2. Reducing VANGL2 levels in SDC4 knockdown LECs restores their alignment under flow, while VANGL2 overexpression in wild-type LECs mimics the flow alignment abnormalities seen in SDC4 knockdown LECs. SDC4 thus controls flow-induced LEC polarization via regulation of VANGL2 expression.
Assuntos
Linfangiogênese/genética , Vasos Linfáticos/embriologia , Proteínas do Tecido Nervoso/metabolismo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/genética , Sindecana-4/genética , Animais , Linhagem Celular , Desenvolvimento Embrionário/genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Interferência de RNA , RNA Interferente Pequeno/genéticaRESUMO
Atherosclerotic plaque localization correlates with regions of disturbed flow in which endothelial cells (ECs) align poorly, whereas sustained laminar flow correlates with cell alignment in the direction of flow and resistance to atherosclerosis. We now report that in hypercholesterolemic mice, deletion of syndecan 4 (S4(-/-)) drastically increased atherosclerotic plaque burden with the appearance of plaque in normally resistant locations. Strikingly, ECs from the thoracic aortas of S4(-/-) mice were poorly aligned in the direction of the flow. Depletion of S4 in human umbilical vein endothelial cells (HUVECs) using shRNA also inhibited flow-induced alignment in vitro, which was rescued by re-expression of S4. This effect was highly specific, as flow activation of VEGF receptor 2 and NF-κB was normal. S4-depleted ECs aligned in cyclic stretch and even elongated under flow, although nondirectionally. EC alignment was previously found to have a causal role in modulating activation of inflammatory versus antiinflammatory pathways by flow. Consistent with these results, S4-depleted HUVECs in long-term laminar flow showed increased activation of proinflammatory NF-κB and decreased induction of antiinflammatory kruppel-like factor (KLF) 2 and KLF4. Thus, S4 plays a critical role in sensing flow direction to promote cell alignment and inhibit atherosclerosis.
Assuntos
Aterosclerose/metabolismo , Células Endoteliais/metabolismo , Transdução de Sinais , Sindecana-4/metabolismo , Animais , Aterosclerose/genética , Western Blotting , Células Cultivadas , Células Endoteliais/citologia , Feminino , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Confocal , NF-kappa B/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estresse Mecânico , Sindecana-4/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
An altered gut microbiota is associated with type 1 diabetes (T1D), affecting the production of short-chain fatty acids (SCFA) and glucose homeostasis. We previously demonstrated that enhancing serum acetate and butyrate using a dietary supplement (HAMSAB) improved glycemia in non-obese diabetic (NOD) mice and patients with established T1D. The effects of SCFA on immune-infiltrated islet cells remain to be clarified. Here, we performed single-cell RNA sequencing on islet cells from NOD mice fed an HAMSAB or control diet. HAMSAB induced a regulatory gene expression profile in pancreas-infiltrated immune cells. Moreover, HAMSAB maintained the expression of ß-cell functional genes and decreased cellular stress. HAMSAB-fed mice showed preserved pancreatic endocrine cell identity, evaluated by decreased numbers of poly-hormonal cells. Finally, SCFA increased insulin levels in human ß-like cells and improved transplantation outcome in NOD/SCID mice. Our findings support the use of metabolite-based diet as attractive approach to improve glucose control in T1D.
RESUMO
Ezrin, radixin, and moesin possess a very similar structure with a C-terminal actin-binding domain and a N-terminal FERM interacting domain. They are known to be involved in cytoskeleton organization in several cell types but their function in vascular smooth muscle cells (VSMC) is still unknown. The aim of this study was to investigate the role of ERM proteins in cell migration induced by PDGF, a growth factor involved in pathophysiological processes like angiogenesis or atherosclerosis. We used primary cultured VSMC obtained from rat aorta, which express the three ERM proteins. Simultaneous depletion of the three ERM proteins with specific siRNAs abolished the effects of PDGF on cell architecture and migration and markedly increased cell adhesion and focal adhesion size, while these parameters were only slightly affected by depletion of ezrin, radixin or moesin alone. Rac1 activation, cell proliferation, and Ca(2+) signal in response to PDGF were unaffected by ERM depletion. These results indicate that ERM proteins exert a redundant control on PDGF-induced VSMC migration by regulating focal adhesion turn-over and cell adhesion to substrate.
Assuntos
Movimento Celular/fisiologia , Proteínas do Citoesqueleto/fisiologia , Adesões Focais/fisiologia , Proteínas de Membrana/fisiologia , Proteínas dos Microfilamentos/fisiologia , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/fisiologia , Animais , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Proteínas do Citoesqueleto/genética , Adesões Focais/efeitos dos fármacos , Masculino , Proteínas de Membrana/genética , Proteínas dos Microfilamentos/genética , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Fator de Crescimento Derivado de Plaquetas/farmacologia , Pseudópodes/fisiologia , Ratos , Ratos WistarRESUMO
The endothelium is a mechanosensitive organ whose pleiotropic actions regulate vessel structure to adjust tissue perfusion. To do so, it possesses ion channels, receptor complexes, and signaling pathways responding to blood flow, whose activation will either maintain vascular integrity and quiescence or, on the contrary, remodel the vessel's structure in both health and disease. Recent studies have demonstrated the crucial role of endothelial inflammation, endothelial to mesenchymal transition (EndMT), and perturbed hemodynamics in the progression of pulmonary arterial hypertension and essential hypertension. These two distinct diseases share some common mechanistic cues, pointing towards new potential therapeutic approaches to treat them. In this review, we summarize these common mechanisms to map future drug development strategies targeting flow sensing mechanisms and vascular remodeling.
Assuntos
Hipertensão , Remodelação Vascular , Humanos , Remodelação Vascular/fisiologia , Transição Epitelial-Mesenquimal , Endotélio , Transdução de Sinais/fisiologia , Hipertensão/tratamento farmacológicoRESUMO
Ezrin, Radixin, Moesin binding phosphoprotein 50 (EBP50) is a scaffold protein that possesses two PDZ interacting domains. We have shown that, in isolated artery stimulated with noradrenaline, EBP50 interacts with several elements of the cytoskeleton. However, the contribution of EBP50 to the organization of the cytoskeleton is unknown. We have used primary cultured vascular smooth muscle cells to investigate the involvement of EBP50 in the regulation of cell architecture, motility and cell cycle, and to identify its target proteins and subsequent action mechanism. The results showed that depletion of EBP50 by siRNA transfection induced changes in cell architecture and increased cell migration. The same phenotype was induced by inhibition of myosin IIa and this effect was not additive in cells depleted for EBP50. Moreover, a larger proportion of binucleated cells was observed after EBP50 depletion, indicating a defect in cytokinesis. The identification, after co-immunoprecipitation, of a direct interaction of EBP50 with both tubulin and myosin IIa suggested that EBP50 could regulate cell migration and cytokinesis by linking myosin IIa fibers and microtubule network. Indeed, depletion of EBP50 also dismantled myosin IIa fibers and induced the formation of stable microtubules in lamellae expansions and Rac1 activation. This signaling cascade leads to the formation of lamellipodia, trailing tails and decrease of focal adhesion formation, triggering cell migration.
Assuntos
Proteínas de Transporte/metabolismo , Movimento Celular , Citocinese , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/fisiologia , Fosfoproteínas/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Aorta/citologia , Proteínas de Transporte/genética , Forma Celular , Células Cultivadas , Masculino , Microscopia de Fluorescência , Microtúbulos/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Cadeias Leves de Miosina/metabolismo , Miosina não Muscular Tipo IIA/genética , Miosina não Muscular Tipo IIA/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosfoproteínas/genética , Fosforilação , Cultura Primária de Células , Interferência de RNA , Ratos , Ratos Wistar , Trocadores de Sódio-HidrogênioRESUMO
Ezrin, radixin, and moesin (ERM) proteins are known to be substrates of Rho kinase (ROCK), a key player in vascular smooth muscle regulation. Their function in arteries remains to be elucidated. The objective of the present study was to investigate ERM phosphorylation and function in rat aorta and mesenteric artery and the influence of ERM-binding phosphoprotein 50 (EBP50), a scaffold partner of ERM proteins in several cell types. In isolated arteries, ERM proteins are phosphorylated by PKC and ROCK with different kinetics after either agonist stimulation or KCl-induced depolarization. Immunoprecipitation of EBP50 in noradrenaline-stimulated arteries allowed identification of its interaction with moesin and several other proteins involved in cytoskeleton regulation. This interaction was inhibited by Y27632, a ROCK inhibitor. Moesin or EBP50 depletion after small interfering RNA transfection by reverse permeabilization in intact mesenteric arteries both potentiated the contractility in response to agonist stimulation without any effect on contractile response induced by high KCl. This effect was preserved in ionomycin-permeabilized arteries. These results indicate that, in agonist-stimulated arteries, the activation of ROCK leads to the binding of moesin to EBP50, which interacts with several components of the cytoskeleton, resulting in a decrease in the contractile response.
Assuntos
Proteínas de Transporte/metabolismo , Artérias Mesentéricas/metabolismo , Proteínas dos Microfilamentos/metabolismo , Norepinefrina/metabolismo , Fosfoproteínas/metabolismo , Quinases Associadas a rho/metabolismo , Amidas/farmacologia , Animais , Anti-Hipertensivos/metabolismo , Aorta/efeitos dos fármacos , Aorta/metabolismo , Ionomicina/farmacologia , Masculino , Artérias Mesentéricas/efeitos dos fármacos , Contração Muscular/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Cloreto de Potássio/farmacologia , Ligação Proteica , Proteína Quinase C/análise , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Piridinas/farmacologia , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Wistar , Trocadores de Sódio-Hidrogênio , Quinases Associadas a rho/antagonistas & inibidoresRESUMO
Vertebrate tissues exhibit mechanical homeostasis, showing stable stiffness and tension over time and recovery after changes in mechanical stress. However, the regulatory pathways that mediate these effects are unknown. A comprehensive identification of Argonaute 2-associated microRNAs and mRNAs in endothelial cells identified a network of 122 microRNA families that target 73 mRNAs encoding cytoskeletal, contractile, adhesive and extracellular matrix (CAM) proteins. The level of these microRNAs increased in cells plated on stiff versus soft substrates, consistent with homeostasis, and suppressed targets via microRNA recognition elements within the 3' untranslated regions of CAM mRNAs. Inhibition of DROSHA or Argonaute 2, or disruption of microRNA recognition elements within individual target mRNAs, such as connective tissue growth factor, induced hyper-adhesive, hyper-contractile phenotypes in endothelial and fibroblast cells in vitro, and increased tissue stiffness, contractility and extracellular matrix deposition in the zebrafish fin fold in vivo. Thus, a network of microRNAs buffers CAM expression to mediate tissue mechanical homeostasis.
Assuntos
Células Endoteliais/metabolismo , Fibroblastos/metabolismo , Regulação da Expressão Gênica , MicroRNAs/genética , Regiões 3' não Traduzidas , Nadadeiras de Animais/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Homeostase/genética , Humanos , Camundongos Endogâmicos C57BL , MicroRNAs/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismoRESUMO
Blood circulation, facilitating gas exchange and nutrient transportation, is a quintessential feature of life in vertebrates. Any disruption to blood flow, may it be by blockade or traumatic rupture, irrevocably leads to tissue infarction or death. Therefore, it is not surprising that hemostasis and vascular adaptation measures have been evolutionarily selected to mitigate the adverse consequences of altered circulation. Blood vessels can be broadly categorized as arteries, veins, or capillaries, based on their structure, hemodynamics, and gas exchange. However, all of them share one property: they are lined with an epithelial sheet called the endothelium, which typically lies on a basement membrane. This endothelium is the primary interface between the flowing blood and the rest of the body, and it has highly specialized molecular mechanisms to detect and respond to changes in blood perfusion. The purpose of this commentary will be to highlight some of the recent developments in the research on blood flow sensing, vascular remodeling, and homeostasis and to discuss the development of innovative pharmaceutical approaches targeting mechanosensing mechanisms to prolong patient survival and improve quality of life.
Assuntos
Velocidade do Fluxo Sanguíneo/fisiologia , Homeostase/fisiologia , Resistência ao Cisalhamento/fisiologia , Doenças Vasculares/patologia , Remodelação Vascular/fisiologia , Animais , Velocidade do Fluxo Sanguíneo/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Homeostase/efeitos dos fármacos , Humanos , Resistência ao Cisalhamento/efeitos dos fármacos , Doenças Vasculares/tratamento farmacológico , Doenças Vasculares/metabolismo , Remodelação Vascular/efeitos dos fármacos , Vasodilatadores/farmacologia , Vasodilatadores/uso terapêuticoRESUMO
Flowing blood exerts a frictional force, fluid shear stress (FSS), on the endothelial cells that line the blood and lymphatic vessels. The magnitude, pulsatility, and directional characteristics of FSS are constantly sensed by the endothelium. Sustained increases or decreases in FSS induce vessel remodeling to maintain proper perfusion of tissue. In this review, we discuss these mechanisms and their relevance to physiology and disease, and propose a model for how information from different mechanosensors might be integrated to govern remodeling.
Assuntos
Remodelação Vascular/fisiologia , Animais , Fenômenos Biomecânicos , Células Endoteliais/fisiologia , Humanos , Mecanotransdução Celular/fisiologia , Modelos Biológicos , Estresse MecânicoRESUMO
Endothelial cells transduce the frictional force from blood flow (fluid shear stress) into biochemical signals that regulate gene expression and cell behavior via specialized mechanisms and pathways. These pathways shape the vascular system during development and during postnatal and adult life to optimize flow to tissues. The same pathways also contribute to atherosclerosis and vascular malformations. This Review covers recent advances in basic mechanisms of flow signaling and the involvement of these mechanisms in vascular physiology, remodeling, and these diseases. We propose that flow sensing pathways that govern normal morphogenesis can contribute to disease under pathological conditions or can be altered to induce disease. Viewing atherosclerosis and vascular malformations as instances of pathological morphogenesis provides a unifying perspective that may aid in developing new therapies.
Assuntos
Aterosclerose/fisiopatologia , Endotélio Vascular/fisiopatologia , Mecanotransdução Celular , Animais , Fenômenos Biomecânicos , Humanos , Placa Aterosclerótica/fisiopatologia , Remodelação VascularRESUMO
Morphogenesis of the vascular system is strongly modulated by mechanical forces from blood flow. Hereditary hemorrhagic telangiectasia (HHT) is an inherited autosomal-dominant disease in which arteriovenous malformations and telangiectasias accumulate with age. Most cases are linked to heterozygous mutations in Alk1 or Endoglin, receptors for bone morphogenetic proteins (BMPs) 9 and 10. Evidence suggests that a second hit results in clonal expansion of endothelial cells to form lesions with poor mural cell coverage that spontaneously rupture and bleed. We now report that fluid shear stress potentiates BMPs to activate Alk1 signaling, which correlates with enhanced association of Alk1 and endoglin. Alk1 is required for BMP9 and flow responses, whereas endoglin is only required for enhancement by flow. This pathway mediates both inhibition of endothelial proliferation and recruitment of mural cells; thus, its loss blocks flow-induced vascular stabilization. Identification of Alk1 signaling as a convergence point for flow and soluble ligands provides a molecular mechanism for development of HHT lesions.
Assuntos
Receptores de Activinas Tipo II/metabolismo , Mecanotransdução Celular , Estresse Mecânico , Telangiectasia Hemorrágica Hereditária/patologia , Malformações Arteriovenosas/patologia , Derivação Arteriovenosa Cirúrgica , Proteínas Morfogenéticas Ósseas/metabolismo , Proliferação de Células , Endoglina/metabolismo , Células Endoteliais/metabolismo , Deleção de Genes , Células HEK293 , Hemorreologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Pericitos/metabolismo , Fluxo Sanguíneo Regional , Retina/patologia , Transdução de Sinais , SolubilidadeRESUMO
Cerebral cavernous malformations (CCMs) are vascular malformations located within the central nervous system often resulting in cerebral hemorrhage. Pharmacological treatment is needed, since current therapy is limited to neurosurgery. Familial CCM is caused by loss-of-function mutations in any of Ccm1, Ccm2, and Ccm3 genes. CCM cavernomas are lined by endothelial cells (ECs) undergoing endothelial-to-mesenchymal transition (EndMT). This switch in phenotype is due to the activation of the transforming growth factor beta/bone morphogenetic protein (TGFß/BMP) signaling. However, the mechanism linking Ccm gene inactivation and TGFß/BMP-dependent EndMT remains undefined. Here, we report that Ccm1 ablation leads to the activation of a MEKK3-MEK5-ERK5-MEF2 signaling axis that induces a strong increase in Kruppel-like factor 4 (KLF4) in ECs in vivo. KLF4 transcriptional activity is responsible for the EndMT occurring in CCM1-null ECs. KLF4 promotes TGFß/BMP signaling through the production of BMP6. Importantly, in endothelial-specific Ccm1 and Klf4 double knockout mice, we observe a strong reduction in the development of CCM and mouse mortality. Our data unveil KLF4 as a therapeutic target for CCM.
Assuntos
Hemangioma Cavernoso do Sistema Nervoso Central/genética , Hemangioma Cavernoso do Sistema Nervoso Central/patologia , Fatores de Transcrição Kruppel-Like/metabolismo , Animais , Proteína Morfogenética Óssea 6/antagonistas & inibidores , Proteína Morfogenética Óssea 6/genética , Proteína Morfogenética Óssea 6/metabolismo , Proliferação de Células , Modelos Animais de Doenças , Progressão da Doença , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Células HEK293 , Hemangioma Cavernoso do Sistema Nervoso Central/metabolismo , Humanos , Proteína KRIT1 , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/antagonistas & inibidores , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/antagonistas & inibidores , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteína Quinase 7 Ativada por Mitógeno/metabolismo , Mutação , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Interferência de RNA , Transdução de Sinais , Proteína Smad1/metabolismo , Fator de Crescimento Transformador beta/metabolismoRESUMO
Endothelial responses to fluid shear stress are essential for vascular development and physiology, and determine the formation of atherosclerotic plaques at regions of disturbed flow. Previous work identified VE-cadherin as an essential component, along with PECAM-1 and VEGFR2, of a complex that mediates flow signaling. However, VE-cadherin's precise role is poorly understood. We now show that the transmembrane domain of VE-cadherin mediates an essential adapter function by binding directly to the transmembrane domain of VEGFR2, as well as VEGFR3, which we now identify as another component of the junctional mechanosensory complex. VEGFR2 and VEGFR3 signal redundantly downstream of VE-cadherin. Furthermore, VEGFR3 expression is observed in the aortic endothelium, where it contributes to flow responses in vivo. In summary, this study identifies a novel adapter function for VE-cadherin mediated by transmembrane domain association with VEGFRs.
Assuntos
Antígenos CD/metabolismo , Caderinas/metabolismo , Mecanotransdução Celular/fisiologia , Neovascularização Fisiológica/fisiologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Movimento Celular , Células Cultivadas , Endotélio Vascular/metabolismo , Células HEK293 , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Placa Aterosclerótica/patologia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Estrutura Terciária de Proteína , Interferência de RNA , RNA Interferente Pequeno , Estresse Mecânico , Estresse Fisiológico , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
The molecular mechanisms responsible for the development and progression of atherosclerotic lesions have not been fully established. Here, we investigated the role played by endothelial-to-mesenchymal transition (EndMT) and its key regulator FGF receptor 1 (FGFR1) in atherosclerosis. In cultured human endothelial cells, both inflammatory cytokines and oscillatory shear stress reduced endothelial FGFR1 expression and activated TGF-ß signaling. We further explored the link between disrupted FGF endothelial signaling and progression of atherosclerosis by introducing endothelial-specific deletion of FGF receptor substrate 2 α (Frs2a) in atherosclerotic (Apoe(-/-)) mice. When placed on a high-fat diet, these double-knockout mice developed atherosclerosis at a much earlier time point compared with that their Apoe(-/-) counterparts, eventually demonstrating an 84% increase in total plaque burden. Moreover, these animals exhibited extensive development of EndMT, deposition of fibronectin, and increased neointima formation. Additionally, we conducted a molecular and morphometric examination of left main coronary arteries from 43 patients with various levels of coronary disease to assess the clinical relevance of these findings. The extent of coronary atherosclerosis in this patient set strongly correlated with loss of endothelial FGFR1 expression, activation of endothelial TGF-ß signaling, and the extent of EndMT. These data demonstrate a link between loss of protective endothelial FGFR signaling, development of EndMT, and progression of atherosclerosis.
Assuntos
Doença da Artéria Coronariana/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Doença da Artéria Coronariana/genética , Doença da Artéria Coronariana/patologia , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismoRESUMO
Vascular remodeling under conditions of growth or exercise, or during recovery from arterial restriction or blockage is essential for health, but mechanisms are poorly understood. It has been proposed that endothelial cells have a preferred level of fluid shear stress, or 'set point', that determines remodeling. We show that human umbilical vein endothelial cells respond optimally within a range of fluid shear stress that approximate physiological shear. Lymphatic endothelial cells, which experience much lower flow in vivo, show similar effects but at lower value of shear stress. VEGFR3 levels, a component of a junctional mechanosensory complex, mediate these differences. Experiments in mice and zebrafish demonstrate that changing levels of VEGFR3/Flt4 modulates aortic lumen diameter consistent with flow-dependent remodeling. These data provide direct evidence for a fluid shear stress set point, identify a mechanism for varying the set point, and demonstrate its relevance to vessel remodeling in vivo.
Assuntos
Estresse Fisiológico , Veias Umbilicais/fisiologia , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/fisiologia , Remodelação Vascular , Animais , Endotélio Vascular/fisiologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Peixe-ZebraRESUMO
4-Aminopyridine is widely used as a Kv channel blocker. However, its mechanism of action is still a matter of debate. Extracellular calcium as well as 4-aminopyridine have been reported to interact with the activation kinetics of particular Kv channels. The objective of the present study was to investigate whether extracellular calcium could modulate the inhibition of Kv current by 4-aminopyridine in vascular myocytes. Kv current was recorded by using whole-cell patch-clamp in freshly isolated smooth muscle cells from rat mesenteric artery. Macroscopic properties of Kv current were not affected by change in extracellular calcium from 0 to 2mM. During a 10s depolarizing pulse, 4-aminopyridine inhibited the peak current without affecting the end-pulse current. The concentration-effect curve of 4-aminopyridine was shifted to the left in the presence of 2mM calcium compared to 0 calcium. After 4-aminopyridine washout, current recovery from block was slower in the presence than in the absence of calcium. Inhibition of Kv current by 4-aminopyridine (0.5mM) and the Kv2 blocker stromatoxin (50nM) was additive and stromatoxin did not alter the potentiation of 4-aminopyridine effect by extracellular calcium. These results showed that extracellular calcium modulated the inhibitory potency of 4-aminopyridine on Kv current in vascular myocytes. The component of Kv current that was inhibited by 4-aminopyridine in a calcium-sensitive manner was distinct from Kv2 current.
Assuntos
4-Aminopiridina/farmacologia , Cálcio/farmacologia , Miócitos de Músculo Liso/efeitos dos fármacos , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/fisiologia , Animais , Células Cultivadas , Masculino , Artérias Mesentéricas/citologia , Miócitos de Músculo Liso/fisiologia , Ratos , Ratos WistarRESUMO
Forces acting on cells govern many important regulatory events during development, normal physiology, and disease processes. Integrin-mediated adhesions, which transmit forces between the extracellular matrix and the actin cytoskeleton, play a central role in transducing effects of forces to regulate cell functions. Recent work has led to major insights into the molecular mechanisms by which these adhesions respond to forces to control cellular signaling pathways. We briefly summarize effects of forces on organs, tissues, and cells; and then discuss recent advances toward understanding molecular mechanisms.