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1.
J Int Med Res ; 48(7): 300060520939746, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32722979

RESUMO

The novel coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus-2 infection is a serious global concern. Increased morbidity and mortality is associated with older age, male gender, cardiovascular disease, diabetes, and smoking. As COVID-19 spreads from coastal borders, both state to state and country to country, our understanding of its pathophysiology has evolved. Age and type 2 diabetes mellitus (T2DM) play especially important roles in COVID-19 progression. T2DM is an age-related disease associated with metabolic syndrome, obesity, insulin resistance (hyperinsulinemia), hyperlipidemia, hypertension, hyperglycemia, and endothelial activation and dysfunction. This review evaluates the relationships and intersection between endothelial cell activation and dysfunction in T2DM and COVID-19. COVID-19 induces multiple injuries of the terminal bronchioles and alveolar blood-gas barrier and associated ultrastructural tissue remodeling. COVID-19 may unmask multiple vulnerabilities associated with T2DM including damage to the endothelial glycocalyx and multiple end-organ macro and microvascular diseases. Unmasking existing vulnerabilities in diabetic patients with COVID-19 is important. Globally, we must come together to better understand why T2DM is associated with increased COVID-19 morbidity and mortality.


Assuntos
Betacoronavirus , Infecções por Coronavirus/complicações , Infecções por Coronavirus/fisiopatologia , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/fisiopatologia , Células Endoteliais/fisiologia , Síndrome Metabólica/complicações , Síndrome Metabólica/fisiopatologia , Pneumonia Viral/complicações , Pneumonia Viral/fisiopatologia , Animais , Barreira Alveolocapilar/patologia , Barreira Alveolocapilar/fisiopatologia , Bronquíolos/patologia , Bronquíolos/fisiopatologia , Comorbidade , Infecções por Coronavirus/epidemiologia , Diabetes Mellitus Tipo 2/epidemiologia , Reposicionamento de Medicamentos , Células Endoteliais/patologia , Humanos , Síndrome Metabólica/epidemiologia , Modelos Biológicos , Pandemias , Pneumonia Viral/epidemiologia , Alvéolos Pulmonares/fisiologia , Alvéolos Pulmonares/fisiopatologia , Ratos , Cicatrização/fisiologia
2.
Nat Commun ; 11(1): 3653, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32694534

RESUMO

The vasculature represents a highly plastic compartment, capable of switching from a quiescent to an active proliferative state during angiogenesis. Metabolic reprogramming in endothelial cells (ECs) thereby is crucial to cover the increasing cellular energy demand under growth conditions. Here we assess the impact of mitochondrial bioenergetics on neovascularisation, by deleting cox10 gene encoding an assembly factor of cytochrome c oxidase (COX) specifically in mouse ECs, providing a model for vasculature-restricted respiratory deficiency. We show that EC-specific cox10 ablation results in deficient vascular development causing embryonic lethality. In adult mice induction of EC-specific cox10 gene deletion produces no overt phenotype. However, the angiogenic capacity of COX-deficient ECs is severely compromised under energetically demanding conditions, as revealed by significantly delayed wound-healing and impaired tumour growth. We provide genetic evidence for a requirement of mitochondrial respiration in vascular endothelial cells for neoangiogenesis during development, tissue repair and cancer.


Assuntos
Mitocôndrias/metabolismo , Neoplasias/patologia , Neovascularização Patológica/patologia , Neovascularização Fisiológica , Cicatrização/fisiologia , Trifosfato de Adenosina/metabolismo , Alquil e Aril Transferases/genética , Alquil e Aril Transferases/metabolismo , Animais , Linhagem Celular Tumoral/transplante , Respiração Celular , Modelos Animais de Doenças , Embrião de Mamíferos , Desenvolvimento Embrionário/fisiologia , Células Endoteliais/fisiologia , Endotélio Vascular/citologia , Endotélio Vascular/fisiologia , Feminino , Técnicas de Inativação de Genes , Humanos , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Mitocôndrias/genética , Neoplasias/irrigação sanguínea , Fosforilação Oxidativa
3.
Life Sci ; 257: 118013, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32603818

RESUMO

AIMS: Emerging literature illustrates critical roles of long noncoding RNAs (lncRNAs) in the progression of atherosclerosis. However, the biological functions and mechanism by which lncRNAs regulate the atherosclerosis remain unclear. MATERIALS AND METHODS: Human umbilical vein endothelial cells (HUVECs) were treated with oxidative low-density lipoprotein (ox-LDL). RNA and protein levels were respectively measured using RT-qPCR and western blot. Molecular interaction was detected using luciferase reporter assay and chromatin immunoprecipitation (ChIP). Proliferation and migration were measured using CCK-8 and wound healing assay. KEY FINDINGS: Here, results unveiled that lncRNA SNHG7 was remarkedly up-regulated in ox-LDL exposed HUVECs. Gain and loss of function experiments showed that the SNHG7 repressed the proliferation and migration of HUVECs. Mechanistically, transcription factor E2F1 was found to target the promoter region of lncRNA SNHG7 and accelerated its expression. Moreover, miR-186-5p was found to bind with the 3'-UTR of SNHG7, meanwhile miR-186-5p also bound with the MMP2 mRNA 3'-UTR. SIGNIFICANCE: In conclusion, these results show the essential roles of E2F1/SNHG7/miR-186-5p/MMP2 axis on the proliferation and migration of endothelial cells, providing a potential therapeutic target for atherosclerosis.


Assuntos
Aterosclerose/metabolismo , Células Endoteliais/metabolismo , RNA Longo não Codificante/metabolismo , Regiões 3' não Traduzidas , Apoptose/genética , Aterosclerose/fisiopatologia , Movimento Celular/genética , Proliferação de Células/genética , Progressão da Doença , Fator de Transcrição E2F1/metabolismo , Células Endoteliais/fisiologia , Técnicas de Silenciamento de Genes , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Lipoproteínas LDL , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/fisiologia
4.
Crit Care ; 24(1): 365, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32560665

RESUMO

In this viewpoint, we summarize the relevance of thromboinflammation in COVID-19 and discuss potential mechanisms of endothelial injury as a key point for the development of lung and distant organ dysfunction, with a focus on direct viral infection and cytokine-mediated injury. Entanglement between inflammation and coagulation and resistance to heparin provide a rationale to consider other therapeutic approaches in order to preserve endothelial function and limit microthrombosis, especially in severe forms. These strategies include nebulized heparin, N-acetylcysteine, plasma exchange and/or fresh frozen plasma, plasma derivatives to increase the level of endogenous anticoagulants (tissue factor pathway inhibitor, activated protein C, thrombomodulin, antithrombin), dipyridamole, complement blockers, different types of stem cells, and extracellular vesicles. An integrated therapy including these drugs has the potential to improve outcomes in COVID-19.


Assuntos
Infecções por Coronavirus/terapia , Células Endoteliais/fisiologia , Inflamação/prevenção & controle , Pneumonia Viral/terapia , Trombose/prevenção & controle , Infecções por Coronavirus/fisiopatologia , Humanos , Pandemias , Pneumonia Viral/fisiopatologia
5.
Life Sci ; 254: 117780, 2020 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-32407844

RESUMO

AIMS: In vivo studies suggest a positive influence of fresh frozen plasma (FFP) on endothelial properties and vascular barrier function, leading to improved outcomes in animal sepsis models as well as in major abdominal surgery. However, those effects are incompletely described. It was our aim to evaluate in vitro effects of FFP on endothelial key functions and to identify underlying mechanisms. MATERIALS AND METHODS: Human pulmonary microvascular endothelial cells (HPMECs) were prestimulated with LPS, followed by incubation with FFP. Permeability for FITC-dextran was assessed, and intercellular gap formation was visualized. NF-κB nuclear translocation and expression of pro-inflammatory, pro-adhesion, and leakage-related genes were evaluated, and monocyte adhesion to ECs was assessed. Intracellular cAMP levels as well as phosphorylation of functional proteins were analyzed. In patients undergoing major abdominal surgery, Syndecan-1 serum levels were assessed prior to and following FFP transfusion. KEY FINDINGS: Post-incubation of HPMVECs with FFP increased intracellular cAMP levels that had been decreased by preceding LPS stimulation. On one hand, this reduced endotoxin-mediated upregulation of IL-8, ICAM-1, VCAM-1, VEGF, and ANG-2. Impaired phosphorylation of functional proteins was restored, and intercellular cohesion and barrier function were rescued. On the other hand, NF-κB nuclear translocation as well as monocyte adhesion was markedly increased by the combination of LPS and FFP. Syndecan-1 serum levels were lower in surgery patients that were transfused with FFP compared to those that were not. SIGNIFICANCE: Our data provide evidence for a differential modulation of crucial endothelial properties by FFP, potentially mediated by elevation of intracellular cAMP levels.


Assuntos
Células Endoteliais/fisiologia , Endotélio Vascular/metabolismo , Plasma/fisiologia , Idoso , Adesão Celular/fisiologia , Permeabilidade da Membrana Celular/fisiologia , Células Cultivadas , AMP Cíclico/metabolismo , Dextranos/metabolismo , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/metabolismo , Junções Comunicantes/fisiologia , Humanos , Lipopolissacarídeos , Pessoa de Meia-Idade , Monócitos/fisiologia , NF-kappa B/metabolismo , Fosforilação , Sindecana-1/sangue
6.
PLoS One ; 15(5): e0232356, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32357159

RESUMO

Lymphatic systems play important roles in the maintenance of fluid homeostasis and undergo anatomical and physiological changes during inflammation and aging. While lymphatic endothelial cells (LECs) undergo mesenchymal transition in response to transforming growth factor-ß (TGF-ß), the molecular mechanisms underlying endothelial-to-mesenchymal transition (EndMT) of LECs remain largely unknown. In this study, we examined the effect of TGF-ß2 and tumor necrosis factor-α (TNF-α), an inflammatory cytokine, on EndMT using human skin-derived lymphatic endothelial cells (HDLECs). TGF-ß2-treated HDLECs showed increased expression of SM22α, a mesenchymal cell marker accompanied by increased cell motility and vascular permeability, suggesting HDLECs to undergo EndMT. Our data also revealed that TNF-α could enhance TGF-ß2-induced EndMT of HDLECs. Furthermore, both cytokines induced the production of Activin A while decreasing the expression of its inhibitory molecule Follistatin, and thus enhancing EndMT. Finally, we demonstrated that human dermal lymphatic vessels underwent EndMT during aging, characterized by double immunostaining for LYVE1 and SM22α. These results suggest that both TGF-ß and TNF-α signals play a central role in EndMT of LECs and could be potential targets for senile edema.


Assuntos
Ativinas/metabolismo , Células Endoteliais/fisiologia , Transição Epitelial-Mesenquimal/fisiologia , Receptores de Fatores de Crescimento Transformadores beta/fisiologia , Transdução de Sinais , Fator de Necrose Tumoral alfa/fisiologia , Células Endoteliais/metabolismo , Células HEK293 , Humanos , Vasos Linfáticos/citologia , Proteína Smad2/fisiologia , Transativadores/fisiologia , Quinases Associadas a rho/metabolismo
7.
Arterioscler Thromb Vasc Biol ; 40(6): 1510-1522, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32349535

RESUMO

OBJECTIVE: Endothelial Cav-1 (caveolin-1) expression plays a relevant role during atherogenesis by controlling NO production, vascular inflammation, LDL (low-density lipoprotein) transcytosis, and extracellular matrix remodeling. Additional studies have identified cholesterol-rich membrane domains as important regulators of autophagy by recruiting ATGs (autophagy-related proteins) to the plasma membrane. Here, we investigate how the expression of Cav-1 in the aortic endothelium influences autophagy and whether enhanced autophagy contributes to the atheroprotective phenotype observed in Cav-1-deficient mice. Approach and Results: To analyze the impact of Cav-1 deficiency on regulation of autophagy in the aortic endothelium during the progression of atherosclerosis, we fed Ldlr-/- and Cav-1-/-Ldlr-/- mice a Western diet and assessed autophagy in the vasculature. We observe that the absence of Cav-1 promotes autophagy activation in athero-prone areas of the aortic endothelium by enhancing autophagic flux. Mechanistically, we found that Cav-1 interacts with the ATG5-ATG12 complex and influences the cellular localization of autophagosome components in lipid rafts, which controls the autophagosome formation and autophagic flux. Pharmacological inhibition of autophagy attenuates the atheroprotection observed in Cav-1-/- mice by increasing endothelial inflammation and macrophage recruitment, identifying a novel molecular mechanism by which Cav-1 deficiency protects against the progression of atherosclerosis. CONCLUSIONS: These results identify Cav-1 as a relevant regulator of autophagy in the aortic endothelium and demonstrate that pharmacological suppression of autophagic flux in Cav-1-deficient mice attenuates the atheroprotection observed in Cav-1-/- mice. Additionally, these findings suggest that activation of endothelial autophagy by blocking Cav-1 might provide a potential therapeutic strategy for cardiovascular diseases including atherosclerosis.


Assuntos
Aterosclerose/prevenção & controle , Autofagia/fisiologia , Caveolina 1/deficiência , Endotélio Vascular/fisiopatologia , Vasculite/prevenção & controle , Adenina/análogos & derivados , Adenina/farmacologia , Animais , Aorta/patologia , Aorta/fisiopatologia , Aorta/ultraestrutura , Aterosclerose/etiologia , Autofagia/efeitos dos fármacos , Caveolina 1/análise , Caveolina 1/fisiologia , Dieta Ocidental , Células Endoteliais/química , Células Endoteliais/fisiologia , Células Endoteliais/ultraestrutura , Endotélio Vascular/química , Endotélio Vascular/ultraestrutura , Feminino , Humanos , Masculino , Microdomínios da Membrana/química , Microdomínios da Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células NIH 3T3 , Receptores de LDL/deficiência
8.
Kidney Int ; 98(2): 314-322, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32461141

RESUMO

The novel coronavirus disease COVID-19 originates in the lungs, but it may extend to other organs, causing, in severe cases, multiorgan damage, including cardiac injury and acute kidney injury. In severe cases, the presence of kidney injury is associated with increased risk of death, highlighting the relevance of this organ as a target of SARS-CoV-2 infection. COVID-19-associated tissue injury is not primarily mediated by viral infection, but rather is a result of the inflammatory host immune response, which drives hypercytokinemia and aggressive inflammation that affect lung parenchymal cells, diminishing oxygen uptake, but also endothelial cells, resulting in endotheliitis and thrombotic events and intravascular coagulation. The complement system represents the first response of the host immune system to SARS-CoV-2 infection, but there is growing evidence that unrestrained activation of complement induced by the virus in the lungs and other organs plays a major role in acute and chronic inflammation, endothelial cell dysfunction, thrombus formation, and intravascular coagulation, and ultimately contributes to multiple organ failure and death. In this review, we discuss the relative role of the different complement activation products in the pathogenesis of COVID-19-associated tissue inflammation and thrombosis and propose the hypothesis that blockade of the terminal complement pathway may represent a potential therapeutic option for the prevention and treatment of lung and multiorgan damage.


Assuntos
Betacoronavirus , Ativação do Complemento , Infecções por Coronavirus/imunologia , Pneumonia Viral/imunologia , Animais , Complemento C5a/fisiologia , Infecções por Coronavirus/complicações , Células Endoteliais/fisiologia , Humanos , Inflamação/etiologia , Lectina de Ligação a Manose/fisiologia , Camundongos , Pandemias , Pneumonia Viral/complicações , Trombose/etiologia , Doenças Vasculares/etiologia
9.
Neuron ; 107(2): 306-319.e9, 2020 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-32407670

RESUMO

Melanin-concentrating hormone (MCH)-expressing neurons are key regulators of energy and glucose homeostasis. Here, we demonstrate that they provide dense projections to the median eminence (ME) in close proximity to tanycytes and fenestrated vessels. Chemogenetic activation of MCH neurons as well as optogenetic stimulation of their projections in the ME enhance permeability of the ME by increasing fenestrated vascular loops and enhance leptin action in the arcuate nucleus of the hypothalamus (ARC). Unbiased phosphoRiboTrap-based assessment of cell activation upon chemogenetic MCH neuron activation reveals MCH-neuron-dependent regulation of endothelial cells. MCH neurons express the vascular endothelial growth factor A (VEGFA), and blocking VEGF-R signaling attenuates the leptin-sensitizing effect of MCH neuron activation. Our experiments reveal that MCH neurons directly regulate permeability of the ME barrier, linking the activity of energy state and sleep regulatory neurons to the regulation of hormone accessibility to the ARC.


Assuntos
Permeabilidade da Membrana Celular/fisiologia , Hormônios Hipotalâmicos/fisiologia , Eminência Mediana/fisiologia , Melaninas/fisiologia , Neurônios/fisiologia , Hormônios Hipofisários/fisiologia , Animais , Núcleo Arqueado do Hipotálamo/fisiologia , Vasos Sanguíneos/fisiologia , Capilares/fisiologia , Núcleo Celular/fisiologia , Núcleo Celular/ultraestrutura , Células Endoteliais/fisiologia , Leptina/fisiologia , Eminência Mediana/irrigação sanguínea , Camundongos , Cultura Primária de Células , Ratos , Ratos Sprague-Dawley , Receptores de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/biossíntese
10.
Yakugaku Zasshi ; 140(4): 513-519, 2020.
Artigo em Japonês | MEDLINE | ID: mdl-32238634

RESUMO

Repair of injured tissues requires angiogenesis, the growth of new blood vessels from pre-existing ones. Cutaneous wound healing is a complex and dynamic process by which skin tissue repairs itself after injury; however, how endothelial cells and pericytes form new blood vessels during cutaneous wound angiogenesis remains unclear. We recently developed a fluorescence-based live imaging system to analyze cutaneous wound angiogenesis in adult zebrafish. Employing this system, we found that endothelial cells and pericytes remain in a quiescent state in normal skin tissue, whereas cutaneous injury immediately activates both types of cells to induce angiogenesis. At 2 days post-injury (dpi), the injured vessels elongated, and some uninjured vessels became tortuous and began to sprout new branches. Then, vessel sprouting, elongation, bifurcation, and anastomosis progressively occurred to form the tortuous and disorganized vascular networks observed at 6 dpi. Thereafter, blood vessel tortuosity gradually decreased through the regression of excessive vessels, thereby leading to the formation of well-organized vessel networks at 42 dpi. Pericytes are thought to detach from the vessel wall to promote endothelial cell sprouting upon the induction of angiogenesis. However, not only endothelial cells but also pericytes proliferated to form pericyte-covered tortuous blood vessels in response to cutaneous injury, revealing an unexpected role of pericytes in cutaneous wound angiogenesis. Therefore, this live-imaging system for adult zebrafish is anticipated to make a valuable contribution to research advancements in understanding the angiogenesis that occurs during tissue repair.


Assuntos
Neovascularização Fisiológica/fisiologia , Imagem Óptica/métodos , Fenômenos Fisiológicos da Pele , Cicatrização/fisiologia , Animais , Células Endoteliais/fisiologia , Humanos , Modelos Animais , Pericitos/fisiologia , Peixe-Zebra
11.
Hum Cell ; 33(3): 479-489, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32277427

RESUMO

The paracrine secretion of angiogenic cytokines from adipose-derived stem cells (ADSCs) might promote endothelial cell angiogenesis, therefore promoting wound healing in injured tissues. Hypoxia is one of the common occurrence in injured tissues, during which angiogenesis is enhanced to improve the oxygen supply. In the present study, miR-590-3p, an anti-angiogenic miRNA, was predicted to target VEGFA, a key factor that can be transcriptionally upregulated by HIF1A during ADSC proliferation and tubule formation in response to hypoxic stimulation. Herein, we found that in response to hypoxic stimuli, HIF1A and VEGFA protein expressions were remarkably induced. In addition, ADSC viability was promoted. Incubation with conditioned medium from ADSCs stimulated by hypoxia significantly enhanced the angiogenic ability of human dermal microvascular endothelial cells (HDMECs), while the conditioned medium from VEGFA-silenced ADSCs significantly reversed the angiogenic ability of HDMECs. Regarding the molecular mechanism, it was verified that miR-590-3p binds to VEGFA; miR-590-3p inhibited VEGFA to affect the paracrine regulation by ADSCs, subsequently hindering the HDMEC angiogenesis. More importantly, the consequences of miR-590-3p-overexpressing conditioned medium on HDMEC angiogenesis were partially reversed by VEGFA-overexpressing conditioned medium. In conclusion, miR-590-3-5p/VEGFA axis modulates the paracrine secretion of VEGFA by ADSCs to affect the angiogenesis of HDMECs.


Assuntos
Derme/citologia , Células Endoteliais/fisiologia , Células-Tronco Mesenquimais/metabolismo , MicroRNAs/fisiologia , Neovascularização Fisiológica/genética , Comunicação Parácrina/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/fisiologia , Células Cultivadas , Expressão Gênica/genética , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , MicroRNAs/genética , MicroRNAs/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Cicatrização
12.
Int Heart J ; 61(2): 364-372, 2020 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-32132319

RESUMO

Atherosclerosis is a chronic inflammatory disease with multiple characteristic facets, including vascular inflammation, endothelial dysfunction, plaque development, impaired blood flow, and cholesterol deposition through dyslipidemia. Toll-like receptors (TLRs) of the innate immune system have been closely linked to the development of atherosclerotic lesions. TLR7 recognizes viral or endogenous single-stranded RNA, which is released during vascular apoptosis and necrosis. The role of TLR7 in vascular disease remains controversial, and therefore, we sought to investigate the effects of TLR7 stimulation in mice.Intravenous injection of a ligand for TLR7 (R848) induced a significant pro-inflammatory cytokine response in mice. This was associated with impaired reendothelialization upon acute denudation of the carotid artery, as measured by Evan's blue staining, and increased numbers of circulating endothelial microparticles (EMPs) and circulating Sca1/Flk1 positive cells as a marker for increased endothelial damage. Chronic subcutaneous stimulation of TLR7 in apolipoprotein E-deficient (ApoE-/-) mice increased aortic production of reactive oxygen species (ROS), the number of circulating EMPs, and most importantly, augmented the formation of atherosclerotic plaque when compared with vehicle-treated animals.Systemic stimulation of TLR7 leads to impaired reendothelialization upon acute vascular injury and is associated with the production of pro-inflammatory cytokines and increased levels of circulating EMPs and Sca1/Flk1 positive cells. Importantly, ApoE-/- mice chronically treated with R848 displayed increased atherosclerotic plaque development and elevated levels of ROS in the aortic tissue. In addition, TLR7-activation-induced apoptosis and impaired migration in human coronary artery endothelial cells and showed significant upregulation of the signaling cascade of IL-1 receptor-associated kinase (IRAK) 2 and IRAK4. Our data highlight the importance of fully understanding the pathomechanisms involved in atherogenesis, and further studies are necessary to identify the ligand-specific effects of TLR7 for possible therapeutic targeting.


Assuntos
Aterosclerose/etiologia , Glicoproteínas de Membrana/metabolismo , Receptor 7 Toll-Like/metabolismo , Animais , Apoptose , Aterosclerose/metabolismo , Movimento Celular , Micropartículas Derivadas de Células/metabolismo , Células Cultivadas , Citocinas/metabolismo , Células Endoteliais/fisiologia , Humanos , Imidazóis , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Camundongos Knockout para ApoE
13.
Am J Physiol Gastrointest Liver Physiol ; 318(4): G803-G815, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32116021

RESUMO

Liver sinusoidal endothelial cells (LSECs) are the first liver cells to encounter waste macromolecules, pathogens, and toxins in blood. LSECs are highly specialized to mediate the clearance of these substances via endocytic scavenger receptors and are equipped with fenestrae that mediate the passage of macromolecules toward hepatocytes. Although some transcription factors (TFs) are known to play a role in LSEC specialization, information about the specialized LSEC signature and its transcriptional determinants remains incomplete.Based on a comparison of liver, heart, and brain endothelial cells (ECs), we established a 30-gene LSEC signature comprising both established and newly identified markers, including 7 genes encoding TFs. To evaluate the LSEC TF regulatory network, we artificially increased the expression of the 7 LSEC-specific TFs in human umbilical vein ECs. Although Zinc finger E-box-binding protein 2, homeobox B5, Cut-like homolog 2, and transcription factor EC (TCFEC) had limited contributions, musculoaponeurotic fibrosarcoma (C-MAF), GATA binding protein 4 (GATA4), and MEIS homeobox 2 (MEIS2) emerged as stronger inducers of LSEC marker expression. Furthermore, a combination of C-MAF, GATA4, and MEIS2 showed a synergistic effect on the increase of LSEC signature genes, including liver/lymph node-specific ICAM-3 grabbing non-integrin (L-SIGN) (or C-type lectin domain family member M (CLEC4M)), mannose receptor C-Type 1 (MRC1), legumain (LGMN), G protein-coupled receptor 182 (GPR182), Plexin C1 (PLXNC1), and solute carrier organic anion transporter family member 2A1 (SLCO2A1). Accordingly, L-SIGN, MRC1, pro-LGMN, GPR182, PLXNC1, and SLCO2A1 protein levels were elevated by this combined overexpression. Although receptor-mediated endocytosis was not significantly induced by the triple TF combination, it enhanced binding to E2, the hepatitis C virus host-binding protein. We conclude that C-MAF, GATA4, and MEIS2 are important transcriptional regulators of the unique LSEC fingerprint and LSEC interaction with viruses. Additional factors are however required to fully recapitulate the molecular, morphological, and functional LSEC fingerprint.NEW & NOTEWORTHY Liver sinusoidal endothelial cells (LSECs) are the first liver cells to encounter waste macromolecules, pathogens, and toxins in the blood and are highly specialized. Although some transcription factors are known to play a role in LSEC specialization, information about the specialized LSEC signature and its transcriptional determinants remains incomplete. Here, we show that Musculoaponeurotic Fibrosarcoma (C-MAF), GATA binding protein 4 (GATA4), and Meis homeobox 2 (MEIS2) are important transcriptional regulators of the unique LSEC signature and that they affect the interaction of LSECs with viruses.


Assuntos
Células Endoteliais/fisiologia , Regulação da Expressão Gênica/fisiologia , Fígado/citologia , Animais , Marcadores Genéticos , Humanos , Fígado/metabolismo , Masculino , Especificidade de Órgãos , Ratos , Transcriptoma
14.
Am J Respir Cell Mol Biol ; 63(1): 104-117, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32160015

RESUMO

The extracellular matrix (ECM) increasingly emerges as an active driver in several diseases, including idiopathic pulmonary arterial hypertension (IPAH). The basement membrane (BM) is a specialized class of ECM proteins. In pulmonary arteries, the BM is in close contact and direct proximity to vascular cells, including endothelial cells. So far, the role of the BM has remained underinvestigated in IPAH. Here, we aimed to shed light on the involvement of the BM in IPAH, by addressing its structure, composition, and function. On an ultrastructural level, we observed a marked increase in BM thickness in IPAH pulmonary vessels. BM composition was distinct in small and large vessels and altered in IPAH. Proteoglycans were mostly responsible for distinction between smaller and larger vessels, whereas BM collagens and laminins were more abundantly expressed in IPAH. Type IV collagen and laminin both strengthened endothelial barrier integrity. However, only type IV collagen concentration dependently increased cell adhesion of both donor and IPAH-derived pulmonary arterial endothelial cells (PAECs) and induced nuclear translocation of mechanosensitive transcriptional coactivator of the hippo pathway YAP (Yes-activated protein). On the other hand, laminin caused cytoplasmic retention of YAP in IPAH PAECs. Accordingly, silencing of COL4A5 and LAMC1, respectively, differentially affected tight junction formation and barrier integrity in both donor and IPAH PAECs. Collectively, our results highlight the importance of a well-maintained BM homeostasis. By linking changes in BM structure and composition to altered endothelial cell function, we here suggest an active involvement of the BM in IPAH pathogenesis.


Assuntos
Membrana Basal/fisiopatologia , Células Endoteliais/fisiologia , Hipertensão Pulmonar Primária Familiar/fisiopatologia , Artéria Pulmonar/fisiopatologia , Adulto , Membrana Basal/metabolismo , Colágeno Tipo IV/metabolismo , Células Endoteliais/metabolismo , Matriz Extracelular/metabolismo , Matriz Extracelular/fisiologia , Proteínas da Matriz Extracelular/metabolismo , Hipertensão Pulmonar Primária Familiar/metabolismo , Feminino , Humanos , Laminina/metabolismo , Masculino , Proteoglicanas/metabolismo , Artéria Pulmonar/metabolismo
15.
Nat Protoc ; 15(3): 1066-1081, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32005982

RESUMO

Endothelial cells (ECs) are fundamental components of the blood vessels that comprise the vascular system; facilitate blood flow; and regulate permeability, angiogenesis, inflammatory responses and homeostatic tissue maintenance. Accumulating evidence suggests there is EC heterogeneity in vivo. However, isolation of fresh ECs from adult mice to investigate this further is challenging. Here, we describe an easy and reproducible protocol for isolation of different types of ECs and CD157+ vascular-resident endothelial stem cells (VESCs) by mechano-enzymatic tissue digestion followed by fluorescence-activated cell sorting. The procedure was established on liver tissue but can be used to isolate ECs from other organs with minimal modification. Preparation of single-cell suspensions can be completed in 2.5 h. We also describe assays for EC clonal and network formation, as well as transcriptomic analysis of isolated ECs. The protocol enables isolation of primary ECs and VESCs that can be used for a wide range of downstream analyses in vascular research.


Assuntos
Técnicas Citológicas/métodos , Células Endoteliais/fisiologia , Fígado/citologia , Células-Tronco/fisiologia , Animais , Camundongos
16.
Proc Natl Acad Sci U S A ; 117(10): 5463-5471, 2020 03 10.
Artigo em Inglês | MEDLINE | ID: mdl-32079726

RESUMO

Chronic pain is a major clinical problem of which the mechanisms are incompletely understood. Here, we describe the concept that PI16, a protein of unknown function mainly produced by fibroblasts, controls neuropathic pain. The spared nerve injury (SNI) model of neuropathic pain increases PI16 protein levels in fibroblasts in dorsal root ganglia (DRG) meninges and in the epi/perineurium of the sciatic nerve. We did not detect PI16 expression in neurons or glia in spinal cord, DRG, and nerve. Mice deficient in PI16 are protected against neuropathic pain. In vitro, PI16 promotes transendothelial leukocyte migration. In vivo, Pi16 -/- mice show reduced endothelial barrier permeability, lower leukocyte infiltration and reduced activation of the endothelial barrier regulator MLCK, and reduced phosphorylation of its substrate MLC2 in response to SNI. In summary, our findings support a model in which PI16 promotes neuropathic pain by mediating a cross-talk between fibroblasts and the endothelial barrier leading to barrier opening, cellular influx, and increased pain. Its key role in neuropathic pain and its limited cellular and tissue distribution makes PI16 an attractive target for pain management.


Assuntos
Fibroblastos/enzimologia , Neuralgia/genética , Proteínas Secretadas Inibidoras de Proteinases/genética , Animais , Movimento Celular , Dor Crônica , Modelos Animais de Doenças , Células Endoteliais/fisiologia , Gânglios Espinais , Leucócitos/fisiologia , Meninges/citologia , Camundongos Knockout , Traumatismos dos Nervos Periféricos/fisiopatologia , Nervo Isquiático/enzimologia
17.
Arterioscler Thromb Vasc Biol ; 40(4): 958-972, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32078339

RESUMO

OBJECTIVE: Angiocrine factors, mediating the endothelial-mural cell interaction in vascular wall construction as well as maintenance, are incompletely characterized. This study aims to investigate the role of endothelial cell-derived FSTL1 (follistatin-like protein 1) in vascular homeostasis. Approach and Results: Using conditional knockout mouse models, we show that loss of FSTL1 in endothelial cells (Fstl1ECKO) led to an increase of pulmonary vascular resistance, resulting in the heart regurgitation especially with tricuspid valves. However, this abnormality was not detected in mutant mice with Fstl1 knockout in smooth muscle cells or hematopoietic cells. We further showed that there was excessive αSMA (α-smooth muscle actin) associated with atrial endocardia, heart valves, veins, and microvessels after the endothelial FSTL1 deletion. There was also an increase in collagen deposition, as demonstrated in livers of Fstl1ECKO mutants. The SMAD3 (mothers against decapentaplegic homolog 3) phosphorylation (pSMAD3) was significantly enhanced, and pSMAD3 staining was colocalized with αSMA in vein walls, suggesting the activation of TGFß (transforming growth factor ß) signaling in vascular mural cells of Fstl1ECKO mice. Consistently, treatment with a TGFß pathway inhibitor reduced the abnormal association of αSMA with the atria and blood vessels in Fstl1ECKO mutant mice. CONCLUSIONS: The findings imply that endothelial FSTL1 is critical for the homeostasis of vascular walls, and its insufficiency may favor cardiovascular fibrosis leading to heart failure.


Assuntos
Endotélio Vascular/fisiopatologia , Fibrose/fisiopatologia , Proteínas Relacionadas à Folistatina/fisiologia , Proteína Smad3/fisiologia , Actinas/metabolismo , Animais , Modelos Animais de Doenças , Células Endoteliais/fisiologia , Proteínas Relacionadas à Folistatina/metabolismo , Homeostase , Humanos , Camundongos Knockout , Fosforilação , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta/fisiologia , Insuficiência da Valva Tricúspide/fisiopatologia , Resistência Vascular
18.
Oxid Med Cell Longev ; 2020: 4213541, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32051731

RESUMO

Angiotensin-converting enzyme 2 (ACE2) is an emerging cardiovascular protective target that mediates the metabolism of angiotensin (Ang) II into Ang (1-7). Our group has demonstrated that ACE2 overexpression enhances the function of endothelial progenitor cells (EPCs). Here, we investigated whether ACE2-primed EPCs (ACE2-EPCs) can protect cerebral microvascular endothelial cells (ECs) against injury and dysfunction in an in vitro model, with focusing on their exosomal and cytokine paracrine effects on endothelial mitochondria. Human EPCs were transfected with lentivirus containing null or human ACE2 cDNA (denoted as Null-EPCs and ACE2-EPCs, respectively). Their conditioned culture media, w/wo depletion of exosomes (ACE2-EPC-CMEX-, Null-EPC-CMEX-, ACE2-EPC-CM, and Null-EPC-CM), were used for coculture experiments. EC injury and dysfunction model was induced by Ang II before coculture. Apoptosis, angiogenic ability, mitochondrion functions (ROS production, membrane potential, fragmentation), and gene expressions (ACE2, Nox2, and Nox4) of ECs were analyzed. The supernatant was collected for measuring the levels of ACE2, Ang II/Ang-(1-7), and growth factors (VEGF and IGF). Our results showed that (1) ACE2-EPC-CM had higher levels of ACE2, Ang (1-7), VEGF, and IGF than that of Null-EPC-CM. (2) Ang II-injured ECs displayed an increase of apoptotic rate and reduction in tube formation and migration abilities, which were associated with ACE2 downregulation, Ang II/Ang (1-7) imbalance, Nox2/Nox4 upregulation, ROS overproduction, an increase of mitochondrion fragmentation, and a decrease of membrane potential. (3) ACE2-EPC-CM had better protective effects than Null-EPC-CM on Ang II-injured ECs, which were associated with the improvements on ACE2 expression, Ang II/Ang (1-7) balance, and mitochondrial functions. (4) ACE2-EPC-CMEX- and Null-EPC-CMEX- showed reduced effects as compared to ACE2-EPCs-CM and Null-EPCs-CM. In conclusion, our data demonstrate that ACE2 overexpression can enhance the protective effects of EPCs on ECs injury, majorly through the exosomal effects on mitochondrial function.


Assuntos
Células Endoteliais/fisiologia , Exossomos/metabolismo , Mitocôndrias/metabolismo , Peptidil Dipeptidase A/metabolismo , Sobrevivência Celular , Células Cultivadas , Técnicas de Cocultura , Meios de Cultivo Condicionados/metabolismo , Citocinas/metabolismo , Células Progenitoras Endoteliais , Humanos , Potenciais da Membrana , Comunicação Parácrina , Peptidil Dipeptidase A/genética , Espécies Reativas de Oxigênio/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
19.
Yakugaku Zasshi ; 140(1): 51-62, 2020.
Artigo em Japonês | MEDLINE | ID: mdl-31902886

RESUMO

The author has described two new functions of endothelial cells for efficient delivery of drugs to tissues. First, it was indicated that tight junction (TJ)-associated protein, claudin-1, exerts potent paracellular barrier function in cultured mouse lung microvascular endothelial cells (LMECs). This barrier was instantly and reversibly opened by reduction of TJ proteins expression via histamine H1 and H2 receptors. Histamine was biosynthesized by l-histidine decarboxylase from uptaken l-histidine, and biotransformed by type B of monoamine oxidase, suggesting that histamine concentration is controlled in rat brain MECs (BMECs) and LMECs. Moreover, uptake of l-histidine into BMECs and LMECs markedly increased with addition of ZnSO4. Second, it was suggested that drug-metabolizing enzymes such as CYP and flavin-containing monooxygenase exist in vascular endothelial cells exposed to blood and to aerobic conditions. These cells have the same ability to metabolize drugs as hepatocytes, demonstrating that vascular endothelial cells are a metabolic barrier against tissue transfer of drugs. From these results, it was suggested that reversible opening of TJ and selective inhibition of drug metabolism in vascular endothelial cells may be efficient delivery strategies of drugs to tissues. Finally, I hope that this research will lead to development of new drugs and possible re-evaluation of discontinued drugs.


Assuntos
Sistemas de Liberação de Medicamentos , Células Endoteliais/enzimologia , Células Endoteliais/fisiologia , Animais , Células Cultivadas , Claudina-1/genética , Claudina-1/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Células Endoteliais/metabolismo , Expressão Gênica , Histamina/biossíntese , Histidina/metabolismo , Camundongos , Oxigenases de Função Mista/metabolismo , Ratos , Receptores Histamínicos H1 , Receptores Histamínicos H2
20.
Plast Reconstr Surg ; 145(2): 420-431, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31985635

RESUMO

BACKGROUND: Secondary lymphedema is a refractory disease, for which adipose-derived stem cells have shown some therapeutic potential. However, the mechanism of this action remains poorly understood. METHODS: The authors identified podoplanin-expressing adipose-derived stem cells, which allowed them to divide adipose-derived stem cells into podoplanin-positive and podoplanin-negative groups that they characterized in vitro. The authors then used a mouse hindlimb model for lymphedema to trace the fate of podoplanin-positive, podoplanin-negative, and unsorted adipose-derived stem cells in vivo. RESULTS: When induced in culture, podoplanin-positive cells were noted to up-regulate the expression of lymphatic endothelial cell markers, including LYVE-1, and assumed a cobblestone morphology. In addition, a substantial increase in lymphangiogenic cytokines was detected in the podoplanin-positive supernatant. The above findings were largely absent from the podoplanin-negative and unsorted groups. In the mouse model, the implanted cells relieved the limb lymphedema by promoting lymphangiogenesis, with the podoplanin-positive group showing the most significant effect. Immunocolocalization further revealed that the podoplanin-positive cells incorporated into lymphatic vessels were positive for LYVE-1. CONCLUSIONS: These data demonstrated that actions by means of both paracrine and differentiation pathways were involved in the adipose-derived stem cell-mediated therapeutic effects. The podoplanin-positive cells possessed lymphatic paracrine and differentiation abilities and may represent lymphatic endothelial cell precursor cells. The podoplanin-negative cells, which constitute a considerable proportion of the adipose-derived stem cells, may play an important paracrine role by secreting mesenchymal stem cell-related factors.


Assuntos
Linfangiogênese/fisiologia , Vasos Linfáticos/fisiologia , Glicoproteínas de Membrana/metabolismo , Células-Tronco Mesenquimais/fisiologia , Animais , Biomarcadores/metabolismo , Células Cultivadas , Citocinas/metabolismo , Modelos Animais de Doenças , Células Endoteliais/fisiologia , Feminino , Proteínas de Fluorescência Verde , Linfedema/fisiopatologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neovascularização Fisiológica/fisiologia , Fenótipo
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