RESUMO
The increasing prevalence of diabetes has resulted in a global epidemic1. Diabetes is a major cause of blindness, kidney failure, heart attacks, stroke and amputation of lower limbs. These are often caused by changes in blood vessels, such as the expansion of the basement membrane and a loss of vascular cells2-4. Diabetes also impairs the functions of endothelial cells5 and disturbs the communication between endothelial cells and pericytes6. How dysfunction of endothelial cells and/or pericytes leads to diabetic vasculopathy remains largely unknown. Here we report the development of self-organizing three-dimensional human blood vessel organoids from pluripotent stem cells. These human blood vessel organoids contain endothelial cells and pericytes that self-assemble into capillary networks that are enveloped by a basement membrane. Human blood vessel organoids transplanted into mice form a stable, perfused vascular tree, including arteries, arterioles and venules. Exposure of blood vessel organoids to hyperglycaemia and inflammatory cytokines in vitro induces thickening of the vascular basement membrane. Human blood vessels, exposed in vivo to a diabetic milieu in mice, also mimic the microvascular changes found in patients with diabetes. DLL4 and NOTCH3 were identified as key drivers of diabetic vasculopathy in human blood vessels. Therefore, organoids derived from human stem cells faithfully recapitulate the structure and function of human blood vessels and are amenable systems for modelling and identifying the regulators of diabetic vasculopathy, a disease that affects hundreds of millions of patients worldwide.
Assuntos
Membrana Basal/patologia , Vasos Sanguíneos/patologia , Angiopatias Diabéticas/patologia , Modelos Biológicos , Organoides/patologia , Organoides/transplante , Proteínas Adaptadoras de Transdução de Sinal , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Artérias/citologia , Artérias/efeitos dos fármacos , Arteríolas/citologia , Arteríolas/efeitos dos fármacos , Membrana Basal/citologia , Membrana Basal/efeitos dos fármacos , Vasos Sanguíneos/citologia , Vasos Sanguíneos/efeitos dos fármacos , Vasos Sanguíneos/crescimento & desenvolvimento , Proteínas de Ligação ao Cálcio , Angiopatias Diabéticas/enzimologia , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Humanos , Hiperglicemia/complicações , Técnicas In Vitro , Mediadores da Inflamação/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Camundongos , Organoides/citologia , Organoides/efeitos dos fármacos , Pericitos/citologia , Pericitos/efeitos dos fármacos , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/efeitos dos fármacos , Receptor Notch3/metabolismo , Transdução de Sinais , Vênulas/citologia , Vênulas/efeitos dos fármacosRESUMO
The shuttling of leukocytes between the bloodstream and interstitial tissues involves different locomotion strategies that are governed by locally presented soluble and cell-bound signals. Recent studies have furthered our understanding of the rapidly advancing field of leukocyte migration, particularly regarding cellular and subcellular events at the level of the venular wall. Furthermore, emerging cellular models are now addressing the transition from an adherent mode to a non-adherent state, incorporating mechanisms that support an efficient migratory profile of leukocytes in the interstitial tissue beyond the venular wall.
Assuntos
Movimento Celular , Leucócitos/citologia , Vênulas/citologia , Animais , Membrana Basal/citologia , Membrana Basal/metabolismo , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Humanos , Leucócitos/metabolismo , Pericitos/citologia , Pericitos/metabolismo , Vênulas/metabolismoRESUMO
RATIONALE: Microvascular inflammation and endothelial dysfunction secondary to unchecked activation of endothelium play a critical role in the pathophysiology of sepsis and organ failure. The intrinsic signaling mechanisms responsible for dampening excessive activation of endothelial cells are not completely understood. OBJECTIVE: To determine the central role of YAP (Yes-associated protein), the major transcriptional coactivator of the Hippo pathway, in modulating the strength and magnitude of endothelial activation and vascular inflammation. METHODS AND RESULTS: Endothelial-specific YAP knockout mice showed increased basal expression of E-selectin and ICAM (intercellular adhesion molecule)-1 in endothelial cells, a greater number of adherent neutrophils in postcapillary venules and increased neutrophil counts in bronchoalveolar lavage fluid. Lipopolysaccharide challenge of these mice augmented NF-κB (nuclear factor-κB) activation, expression of endothelial adhesion proteins, neutrophil and monocyte adhesion to cremaster muscle venules, transendothelial neutrophil migration, and lung inflammatory injury. Deletion of YAP in endothelial cells also markedly augmented the inflammatory response and cardiovascular dysfunction in a polymicrobial sepsis model induced by cecal ligation and puncture. YAP functioned by interacting with the E3 ubiquitin-protein ligase TLR (Toll-like receptor) signaling adaptor TRAF6 (tumor necrosis factor receptor-associated factor 6) to ubiquitinate TRAF6, and thus promoted TRAF6 degradation and modification resulting in inhibition of NF-κB activation. TRAF6 depletion in endothelial cells rescued the augmented inflammatory phenotype in mice with endothelial cell-specific deletion of YAP. CONCLUSIONS: YAP modulates the activation of endothelial cells and suppresses vascular inflammation through preventing TRAF6-mediated NF-κB activation and is hence essential for limiting the severity of sepsis-induced inflammation and organ failure.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Células Endoteliais/fisiologia , Endotélio Vascular/fisiopatologia , Fosfoproteínas/fisiologia , Fator 6 Associado a Receptor de TNF/metabolismo , Vasculite/etiologia , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Permeabilidade Capilar , Adesão Celular , Proteínas de Ciclo Celular , Selectina E/metabolismo , Células Endoteliais/citologia , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Molécula 1 de Adesão Intercelular/metabolismo , Contagem de Leucócitos , Camundongos , Camundongos Knockout , Microvasos , Monócitos/fisiologia , NF-kappa B/metabolismo , Neutrófilos/citologia , Fosfoproteínas/deficiência , Fosfoproteínas/genética , Sepse/complicações , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Vênulas/citologia , Proteínas de Sinalização YAPRESUMO
UNLABELLED: Circulating factor VIII (FVIII) is derived from liver and from extrahepatic sources probably of endothelial origin, but the vascular sites of FVIII production remain unclear. Among organs profiled, only liver and lymph nodes (LNs) show abundant expression of F8 messenger RNA (mRNA). Transcriptomic profiling of subsets of stromal cells, including endothelial cells (ECs) from mouse LNs and other tissues, showed that F8 mRNA is expressed by lymphatic ECs (LECs) but not by capillary ECs (capECs), fibroblastic reticular cells, or hematopoietic cells. Among blood ECs profiled, F8 expression was seen only in fenestrated ECs (liver sinusoidal and renal glomerular ECs) and some high endothelial venules. In contrast, von Willebrand factor mRNA was expressed in capECs but not in LECs; it was coexpressed with F8 mRNA in postcapillary high endothelial venules. Purified LECs and liver sinusoidal ECs but not capECs from LNs secrete active FVIII in culture, and human and mouse lymph contained substantial FVIII: C activity. Our results revealed localized vascular expression of FVIII and von Willebrand factor and identified LECs as a major cellular source of FVIII in extrahepatic tissues.
Assuntos
Células Endoteliais/metabolismo , Endotélio Linfático/metabolismo , Endotélio Vascular/metabolismo , Fator VIII/biossíntese , Regulação da Expressão Gênica/fisiologia , Fator de von Willebrand/biossíntese , Animais , Capilares/citologia , Capilares/metabolismo , Células Endoteliais/citologia , Endotélio Linfático/citologia , Endotélio Vascular/citologia , Feminino , Humanos , Glomérulos Renais/irrigação sanguínea , Glomérulos Renais/citologia , Glomérulos Renais/metabolismo , Fígado/irrigação sanguínea , Fígado/citologia , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Especificidade de Órgãos , Vênulas/citologia , Vênulas/metabolismoRESUMO
A classical hallmark of acute inflammation is neutrophil infiltration of tissues, a multistep process that involves sequential cell-cell interactions of circulating leukocytes with IL-1- or TNF-activated microvascular endothelial cells (ECs) and pericytes (PCs) that form the wall of the postcapillary venules. The initial infiltrating cells accumulate perivascularly in close proximity to PCs. IL-17, a proinflammatory cytokine that acts on target cells via a heterodimeric receptor formed by IL-17RA and IL-17RC subunits, also promotes neutrophilic inflammation but its effects on vascular cells are less clear. We report that both cultured human ECs and PCs strongly express IL-17RC and, although neither cell type expresses much IL-17RA, PCs express significantly more than ECs. IL-17, alone or synergistically with TNF, significantly alters inflammatory gene expression in cultured human PCs but not ECs. RNA sequencing analysis identifies many IL-17-induced transcripts in PCs encoding proteins known to stimulate neutrophil-mediated immunity. Conditioned media from IL-17-activated PCs, but not ECs, induce pertussis toxin-sensitive neutrophil polarization, likely mediated by PC-secreted chemokines, and they also stimulate neutrophil production of proinflammatory molecules, including TNF, IL-1α, IL-1ß, and IL-8. Furthermore, IL-17-activated PCs, but not ECs, can prolong neutrophil survival by producing G-CSF and GM-CSF, delaying the mitochondrial outer membrane permeabilization and caspase-9 activation. Importantly, neutrophils exhibit enhanced phagocytic capacity after activation by conditioned media from IL-17-treated PCs. We conclude that PCs, not ECs, are the major target of IL-17 within the microvessel wall and that IL-17-activated PCs can modulate neutrophil functions within the perivascular tissue space.
Assuntos
Endotélio Vascular/fisiologia , Interleucina-17/imunologia , Neutrófilos/imunologia , Pericitos/fisiologia , Receptores de Interleucina-17/imunologia , Caspase 9/metabolismo , Células Cultivadas , Meios de Cultura , Citocinas/biossíntese , Citocinas/imunologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/imunologia , Fator Estimulador de Colônias de Granulócitos/biossíntese , Fator Estimulador de Colônias de Granulócitos/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/biossíntese , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Humanos , Interleucina-17/genética , Interleucina-17/farmacologia , Infiltração de Neutrófilos , Neutrófilos/fisiologia , Pericitos/efeitos dos fármacos , Pericitos/imunologia , Receptores de Interleucina-17/fisiologia , Análise de Sequência de RNA , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/farmacologia , Vênulas/citologia , Vênulas/imunologiaRESUMO
Microcirculation is the generic name for the finest level of the circulatory system and consists of arteriolar and venular networks located upstream and downstream of capillaries, respectively. Anatomically arterioles are surrounded by a monolayer of spindle-shaped smooth muscle cells (myocytes), while terminal branches of precapillary arterioles, capillaries and all sections of postcapillary venules are surrounded by a monolayer of morphologically different perivascular cells (pericytes). Pericytes are essential components of the microvascular vessel wall. Wrapped around endothelial cells, they occupy a strategic position at the interface between the circulating blood and the interstitial space. There are physiological differences in the responses of pericytes and myocytes to vasoactive molecules, which suggest that these two types of vascular cells could have different functional roles in the regulation of local blood flow within the same microvascular bed. Also, pericytes may play different roles in different microcirculatory beds to meet the characteristics of individual organs. Contractile activity of pericytes and myocytes is controlled by changes of cytosolic free Ca2+concentration. In this chapter, we attempt to summarize the results in the field of Ca2+ signalling in pericytes especially in light of their contractile roles in different tissues and organs. We investigate the literature and describe our results regarding sources of Ca2+, relative importance and mechanisms of Ca2+ release and Ca2+ entry in control of the spatio-temporal characteristics of the Ca2+ signals in pericytes, where possible Ca2+ signalling and contractile responses in pericytes are compared to those of myocytes.
Assuntos
Sinalização do Cálcio , Microcirculação , Pericitos/metabolismo , Arteríolas/citologia , Capilares/citologia , Humanos , Células Musculares/citologia , Vênulas/citologiaRESUMO
Although the homing of lymphocytes to GALT has been extensively studied, little is known about how high endothelial venules (HEVs) within Peyer's patches (PPs) are patterned to display dominantly mucosal addressin cell adhesion molecule 1 (MAdCAM-1). In this study, we report that Nkx2-3-deficient mice show gradual loss of MAdCAM-1 in PPs postnatally and increased levels of mRNA for peripheral lymph node addressin (PNAd) backbone proteins as well as enhanced expression of MECA79 sulfated glycoepitope at the luminal aspect of HEVs, thus replacing MAdCAM-1 with PNAd. Induction of PNAd in mutant PPs requires lymphotoxin ß receptor activity, and its upregulation needs the presence of mature T and B cells. Furthermore, treatment with MECA-79 anti-PNAd mAb in vivo effectively blocks lymphocyte homing to mutant PPs. Despite the replacement of MAdCAM-1 by PNAd in HEV endothelia, lymphocytes could efficiently home to PPs in mutant mice. We conclude that although Nkx2-3 activity controls the addressin balance of HEVs in GALT, the general HEV functionality is preserved independently from Nkx2-3, indicating a substantial plasticity in the specification of GALT HEV endothelium.
Assuntos
Linfócitos B/metabolismo , Proteínas de Homeodomínio/imunologia , Nódulos Linfáticos Agregados/metabolismo , Linfócitos T/metabolismo , Fatores de Transcrição/imunologia , Animais , Animais Recém-Nascidos , Anticorpos Monoclonais/farmacologia , Antígenos de Superfície/genética , Antígenos de Superfície/imunologia , Linfócitos B/citologia , Linfócitos B/imunologia , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/imunologia , Regulação da Expressão Gênica , Proteínas de Homeodomínio/genética , Mucosa Intestinal/metabolismo , Intestinos/citologia , Intestinos/imunologia , Linfonodos/citologia , Linfonodos/imunologia , Linfonodos/metabolismo , Receptor beta de Linfotoxina/genética , Receptor beta de Linfotoxina/imunologia , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Mucoproteínas , Nódulos Linfáticos Agregados/citologia , Nódulos Linfáticos Agregados/imunologia , Transdução de Sinais , Linfócitos T/citologia , Linfócitos T/imunologia , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Vênulas/citologia , Vênulas/imunologia , Vênulas/metabolismoRESUMO
Telocytes are cells with telopodes, which distinguish them from other interstitial cells. According to the study of lung, it was confirmed that telocytes were mainly distributed in the alveolar interstitial tissues connected tightly with alveolar epithelia cells and participated in the structure of air-blood barrier, in the small vein and bronchioles and in the interstitial space of smooth muscle participated in the frame structure of the blood and bronchioles. Telocytes are positive to CD34 and C-kit which expressed on the surface of hemopoietic stem cells, and are proposed to participate in the angiogenesis. In this chapter, we try to clarify the morphological characteristics of lung telocytes, both in tissues and culture, and introduce the experiences on the method of telocytes isolation and primary culture. The proteomics analysis of lung telocytes through iTRAQ (isobaric tags for relative and absolute quantification) was also discussed and it will provide new research directions in the future.
Assuntos
Células Endoteliais/citologia , Pulmão/citologia , Neovascularização Fisiológica , Telócitos/citologia , Animais , Antígenos CD34/genética , Antígenos CD34/metabolismo , Arteríolas/citologia , Arteríolas/metabolismo , Biomarcadores/metabolismo , Capilares/citologia , Capilares/metabolismo , Comunicação Celular , Células Endoteliais/metabolismo , Expressão Gênica , Humanos , Pulmão/irrigação sanguínea , Pulmão/metabolismo , Camundongos , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-kit/metabolismo , Telócitos/metabolismo , Vênulas/citologia , Vênulas/metabolismo , Vimentina/genética , Vimentina/metabolismoRESUMO
Neutrophil extravasation occurs across postcapillary venules, structures composed of endothelial cells (ECs), pericytes (PCs), and basement membrane (BM). We constructed composite models of the human postcapillary venule, combining ECs with PCs or PC-deposited BM, to better study this process. Quiescent and tumor necrosis factor α (TNF-α)-activated composites demonstrated in situ-like expression of cadherins, E-selectin, intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), platelet-endothelial cell adhesion molecule 1 (PECAM-1), CD99, and interleukin 8 (IL-8). After TNF-α activation, the ECs supported greater neutrophil adhesion (66.1 vs. 23.7% of input cells) and transmigration (35.1 vs. 7.20% of input cells) than did the PCs, but the composites behaved comparably (no significant difference) to ECs in both assays. TNF-α-activated EC-conditioned medium (CM) increased transmigration across the PCs, whereas TNF-α-activated PC-CM decreased transmigration across the ECs, and culturing on PC-derived BM decreased both adhesion to and transmigration across the ECs. Anti-very late antigen 4 (VLA-4; on neutrophils) inhibited adhesion to TNF-α-activated composites, but not to ECs alone. Anti-CD99 (expressed on all 3 cell types) inhibited transmigration across the composites (14.5% of control) more than across the ECs (39.0% of control), and venular shear stress reduced transmigration across the ECs (17.3% of static) more than across the composites (36.7% of static). These results provide proof of concept that our composite human EC/PC/BM venular construct can reveal new interactions in the inflammatory cascade.
Assuntos
Leucócitos/citologia , Modelos Biológicos , Vênulas/anatomia & histologia , Adesão Celular , Movimento Celular , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Humanos , Microscopia Eletrônica de Varredura , Vênulas/citologiaRESUMO
RATIONALE: Endothelial adherens junction proteins constitute an important element in the control of microvascular permeability. Platelet-activating factor (PAF) increases permeability to macromolecules via translocation of endothelial nitric oxide synthase (eNOS) to cytosol and stimulation of eNOS-derived nitric oxide signaling cascade. The mechanisms by which nitric oxide signaling regulates permeability at adherens junctions are still incompletely understood. OBJECTIVE: We explored the hypothesis that PAF stimulates hyperpermeability via S-nitrosation (SNO) of adherens junction proteins. METHODS AND RESULTS: We measured PAF-stimulated SNO of ß-catenin and p120-catenin (p120) in 3 cell lines: ECV-eNOSGFP, EAhy926 (derived from human umbilical vein), and postcapillary venular endothelial cells (derived from bovine heart endothelium) and in the mouse cremaster muscle in vivo. SNO correlated with diminished abundance of ß-catenin and p120 at the adherens junction and with hyperpermeability. Tumor necrosis factor-α increased nitric oxide production and caused similar increase in SNO as PAF. To ascertain the importance of eNOS subcellular location in this process, we used ECV-304 cells transfected with cytosolic eNOS (GFPeNOSG2A) and plasma membrane eNOS (GFPeNOSCAAX). PAF induced SNO of ß-catenin and p120 and significantly diminished association between these proteins in cells with cytosolic eNOS but not in cells wherein eNOS is anchored to the cell membrane. Inhibitors of nitric oxide production and of SNO blocked PAF-induced SNO and hyperpermeability, whereas inhibition of the cGMP pathway had no effect. Mass spectrometry analysis of purified p120 identified cysteine 579 as the main S-nitrosated residue in the region that putatively interacts with vascular endothelial-cadherin. CONCLUSIONS: Our results demonstrate that agonist-induced SNO contributes to junctional membrane protein changes that enhance endothelial permeability.
Assuntos
Junções Aderentes/metabolismo , Permeabilidade Capilar/fisiologia , Cateninas/metabolismo , Células Endoteliais/metabolismo , Transdução de Sinais/fisiologia , beta Catenina/metabolismo , Sequência de Aminoácidos , Animais , Permeabilidade Capilar/efeitos dos fármacos , Cateninas/genética , Bovinos , Proteínas de Fluorescência Verde/genética , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Dados de Sequência Molecular , Músculo Esquelético/irrigação sanguínea , Músculo Esquelético/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Nitrosação/fisiologia , Fator de Crescimento Derivado de Plaquetas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Vênulas/citologia , delta CateninaRESUMO
Protein kinase C (PKC)-θ is involved in T cell activation via regulating the avidity of the ß(2) integrin LFA-1 in the immunological synapse. LFA-1 also mediates leukocyte adhesion. To investigate the role of PKC-θ in neutrophil adhesion, we performed intravital microscopy in cremaster venules of mice reconstituted with bone marrow from LysM-GFP(+) (wild-type [WT]) and PKC-θ gene-deficient (Prkcq(-/-)) mice. Following stimulation with CXCL1, both WT and Prkcq(-/-) cells became adherent. Although most WT neutrophils remained adherent for at least 180 s, 50% of Prkcq(-/-) neutrophils were detached after 105 s and most by 180 s. Upon CXCL1 injection, rolling of all WT neutrophils stopped for 90 s, but rolling of Prkcq(-/-) neutrophils started 30 s after CXCL1 stimulation. A similar neutrophil adhesion defect was seen in vitro, and spreading of Prkcq(-/-) neutrophils was delayed. Prkcq(-/-) neutrophil recruitment was impaired in fMLP-induced transmigration into the cremaster muscle, thioglycollate-induced peritonitis, and LPS-induced lung injury. We conclude that PKC-θ mediates integrin-dependent neutrophil functions and is required to sustain neutrophil adhesion in postcapillary venules in vivo. These findings suggest that the role of PKC-θ in outside-in signaling following engagement of neutrophil integrins is relevant for inflammation in vivo.
Assuntos
Antígenos CD18/imunologia , Isoenzimas/imunologia , Antígeno-1 Associado à Função Linfocitária/imunologia , Neutrófilos/imunologia , Proteína Quinase C/imunologia , Vênulas/imunologia , Animais , Antígenos CD18/genética , Adesão Celular , Quimiocina CXCL1/farmacologia , Genes Reporter , Proteínas de Fluorescência Verde/genética , Sinapses Imunológicas , Isoenzimas/deficiência , Isoenzimas/genética , Ativação Linfocitária , Antígeno-1 Associado à Função Linfocitária/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia de Vídeo , Neutrófilos/citologia , Neutrófilos/fisiologia , Proteína Quinase C/deficiência , Proteína Quinase C/genética , Proteína Quinase C-theta , Vênulas/citologia , Vênulas/metabolismoRESUMO
OBJECTIVE: To elucidate shear-dependent effects of deformation of the endothelial glycocalyx on adhesion of circulating ligands in post-capillary venules, and delineate effect of MMPs. METHODS: Adhesion of WBCs and lectin-coated FLMs (0.1 µm diameter) to EC of post-capillary venules in mesentery was examined during acute reductions in shear rates (γ·, hemorrhagic hypotension). Adhesion was examined with or without superfusion with 0.5 µm doxycycline to inhibit MMPs. Thickness of the glycocalyx was measured by exclusion of fluorescent 70 kDa dextran from the EC surface. RESULTS: During superfusion with Ringers, rapid reductions in γ· resulted in a significant rise in WBC adhesion and a twofold rise in microsphere adhesion. With addition of doxycycline WBC and FLM adhesion increased twofold under high- and low-flow conditions. FLM adhesion was invariant with γ· throughout the network in the normal (high)-flow state. With reductions in γ·, thickness of the glycocalyx increased significantly, with or without doxycycline. CONCLUSIONS: The concurrent increase in WBC and FLM adhesion with increased thickness of the glycocalyx during reductions in shear suggests that glycocalyx core proteins recoil from their deformed steady-state configuration, which increases exposure of binding sites for circulating ligands.
Assuntos
Adesão Celular/fisiologia , Endotélio Vascular/metabolismo , Glicocálix/metabolismo , Lectinas/metabolismo , Leucócitos/metabolismo , Vênulas/metabolismo , Animais , Antibacterianos/farmacologia , Adesão Celular/efeitos dos fármacos , Doxiciclina/farmacologia , Elasticidade , Endotélio Vascular/citologia , Glicocálix/efeitos dos fármacos , Hipotensão/metabolismo , Hipotensão/fisiopatologia , Soluções Isotônicas/farmacologia , Leucócitos/citologia , Ligantes , Masculino , Metaloproteinases da Matriz/metabolismo , Veias Mesentéricas/citologia , Veias Mesentéricas/metabolismo , Microesferas , Ratos , Ratos Wistar , Fluxo Sanguíneo Regional/efeitos dos fármacos , Fluxo Sanguíneo Regional/fisiologia , Solução de Ringer , Circulação Esplâncnica/efeitos dos fármacos , Circulação Esplâncnica/fisiologia , Estresse Mecânico , Vênulas/citologiaRESUMO
The existence of a hydrodynamically relevant endothelial glycocalyx has been established in capillaries, venules, and arterioles in vivo. The glycocalyx is thought to consist primarily of membrane-bound proteoglycans with glycosaminoglycan side-chains, membrane-bound glypicans, and adsorbed plasma proteins. The proteoglycans found on the luminal surface of endothelial cells are syndecans-1, -2, and -4, and glypican-1. The extent to which any of these proteins might serve to anchor the glycocalyx to the endothelium has not yet been determined. To test whether syndecan-1, in particular, is an essential anchoring protein, we performed experiments to determine the hydrodynamically relevant glycocalyx thickness in syndecan-1 deficient (Sdc1(-/-)) mice. Micro-particle image velocimetry data were collected using a previously described method. Microviscometric analysis of these data consistently revealed the existence of a hydrodynamically relevant endothelial glycocalyx in Sdc1(-/-) mice in vivo. The mean glycocalyx thickness found in Sdc1(-/-) mice was 0.45±0.10 µm (N=15), as compared with 0.54±0.12 µm (N=11) in wild-type (WT) mice (p=0.03). The slightly thinner glycocalyx observed in Sdc1(-/-) mice relative to WT mice may be due to the absence of syndecan-1. These findings show that healthy Sdc1(-/-) mice are able to synthesize and maintain a hydrodynamically relevant glycocalyx, which indicates that syndecan-1 is not an essential anchoring protein for the glycocalyx in Sdc1(-/-) mice. This may also be the case for WT mice; however, Sdc1(-/-) mice might adapt to the lack of syndecan-1 by increasing the expression of other proteoglycans. In any case, syndecan-1 does not appear to be a prerequisite for the existence of an endothelial glycocalyx.
Assuntos
Células Endoteliais/metabolismo , Glicocálix/metabolismo , Sindecana-1/deficiência , Vênulas/metabolismo , Animais , Velocidade do Fluxo Sanguíneo , Adesão Celular , Hidrodinâmica , Análise dos Mínimos Quadrados , Leucócitos/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia de Fluorescência , Microscopia de Vídeo , Dinâmica não Linear , Fluxo Sanguíneo Regional , Sindecana-1/genética , Vênulas/citologiaRESUMO
ATP can differentially affect the micro- and macrovascular endothelial barrier. It has been shown that it can both increase and/or decrease macromolecule permeability of microvascular endothelial cells and microvessels, in vivo. We hypothesised that the barrier stabilising effect is mediated by ATP itself via P2 receptors, while barrier-disrupting effect is mediated by its metabolite adenosine via adenosine receptors. The effects of ATP, ADP, AMP and adenosine on barrier function were studied in cultured rat coronary microvascular endothelial monolayers (RCEC) in vitro, as well as in rat mesentery vessels, and in rat hearts in vivo. ATP and ADP showed a biphasic effect on permeability of RCEC monolayers with a reduction followed by a later increase in albumin permeability. The permeability decreasing effect of ATP was enhanced by ecto-nucleotidase inhibitor ARL67156 while permeability increasing effect was enhanced by apyrase, an extracellular ecto-nucleotidase. Moreover, the permeability increasing effect was abrogated by adenosine receptor antagonists, 8-phenyltheophylline (8-PT) and DMPX. Adenosine and adenosine receptor agonists 5'-(N-ethylcarboxamido)-adenosine (NECA), CGS21680, and R-PIA enhanced albumin permeability which was antagonised by 8-PT, A(1), and A(2) but not by A(3) receptor antagonists. Likewise, immunofluorescence microscopy of VE-cadherin and actin showed that NECA induces a disturbance of intercellular junctions. Pre-incubation of ATP antagonised the effects of NECA on permeability, actin cytoskeleton and intercellular junctions. Similar effects of the applied substances were observed in rat mesentery artery by determining the vascular leakage using intravital microscopy as well as in rat hearts by assessing myocardial water contents in vivo. In conclusion, the study demonstrates that in RCEC, ATP, ADP, and its metabolite adenosine play opposing roles on endothelial barrier function.
Assuntos
Trifosfato de Adenosina/farmacologia , Adenosina/farmacologia , Vasos Coronários/fisiologia , Agonistas do Receptor Purinérgico P1/farmacologia , Antagonistas de Receptores Purinérgicos P1/farmacologia , Vênulas/fisiologia , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Difosfato de Adenosina/farmacologia , Monofosfato de Adenosina/farmacologia , Adenosina-5'-(N-etilcarboxamida)/farmacologia , Animais , Caderinas/metabolismo , Células Cultivadas , Vasos Coronários/citologia , Vasos Coronários/efeitos dos fármacos , Vasos Coronários/metabolismo , Edema Cardíaco/patologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/fisiologia , Masculino , Miocárdio , Permeabilidade/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Antagonistas do Receptor Purinérgico P2/farmacologia , Ratos , Ratos Wistar , Receptores Purinérgicos P1/metabolismo , Teofilina/análogos & derivados , Teofilina/farmacologia , Vênulas/citologia , Vênulas/efeitos dos fármacos , Vênulas/metabolismoRESUMO
The barrier functions of myocardial precapillary arteriolar and postcapillary venular walls (PCA or PCV, respectively) are of considerable scientific and clinical interest (regulation of blood flow and recruitment of immune defense). Using enzyme histochemistry combined with confocal microscopy, we reexamined the cell architecture of human PCA and PVC and reconstructed appropriate in vitro models for studies of their barrier functions. Contrary to current opinion, the PCA endothelial tube is encompassed not by smooth muscle cells but rather by a concentric layer of pericytes cocooned in a thick, microparticle-containing extracellular matrix (ECM) that contributes substantially to the tightness of the arteriolar wall. This core tube extends upstream into the larger arterioles, there additionally enwrapped by smooth muscle. PCV consist of an inner layer of large, contractile endothelial cells encompassed by a fragile, wide-meshed pericyte network with a weakly developed ECM. Pure pericyte and endothelial cell preparations were isolated from PCA and PCV and grown in sandwich cultures. These in vitro models of the PCA and PCV walls exhibited typical histological and functional features. In both plasma-like (PLM) and serum-containing (SCM) media, the PCA model (including ECM) maintained its low hydraulic conductivity (L(P) = 3.24 ± 0.52·10(-8)cm·s(-1)·cmH(2)O(-1)) and a high selectivity index for transmural passage of albumin (SI(Alb) = 0.95 ± 0.02). In contrast, L(P) and SI(Alb) in the PCV model (almost no ECM) were 2.55 ± 0.32·10(-7)cm·s(-1)·cmH(2)O(-1) and 0.88 ± 0.03, respectively, in PLM, and 1.39 ± 0.10·10(-6)cm·s(-1)·cmH(2)O(-1) and 0.49 ± 0.04 in SCM. With the use of these models, systematic, detailed studies on the regulation of microvascular barrier properties now appear to be feasible.
Assuntos
Permeabilidade Capilar , Vasos Coronários/metabolismo , Células Endoteliais/metabolismo , Pericitos/metabolismo , Albuminas/metabolismo , Arteríolas/citologia , Arteríolas/metabolismo , Separação Celular , Células Cultivadas , Técnicas de Cocultura , Vasos Coronários/citologia , Meios de Cultura , Condutividade Elétrica , Matriz Extracelular/metabolismo , Humanos , Imuno-Histoquímica , Microscopia Confocal , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Fatores de Tempo , Vênulas/citologia , Vênulas/metabolismoRESUMO
The vascular pathobiology of sickle cell anemia involves inflammation, coagulation, vascular stasis, reperfusion injury, iron-based oxidative biochemistry, deficient nitric oxide (NO) bioavailability, and red cell sickling. These disparate pathobiologies intersect and overlap, so it is probable that multimodality therapy will be necessary for this disease. We have, therefore, tested a histone deacetylase (HDAC) inhibitor, trichostatin A (TSA), for efficacy in reducing endothelial activation. We found that pulmonary vascular endothelial VCAM-1 and tissue factor (TF) expression (both are indicators of endothelial activation) are powerfully and significantly inhibited by TSA. This is seen both with pretreatment before the inducing stress of hypoxia/reoxygenation (NY1DD sickle transgenic mouse), and upon longer-term therapy after endothelial activation has already occurred (hBERK1 sickle mouse at ambient air). In addition, TSA prevented vascular stasis in sickle mice, it exhibited activity as an iron chelator, and it induced expression of the antisickling hemoglobin, hemoglobin F. Notably, the TSA analog SAHA (suberoylanilide hydroxaminc acid) that is already approved for human clinical use exhibits the same spectrum of biologic effects as TSA. We suggest that SAHA possibly could provide true, multimodality, salubrious effects for prevention and treatment of the chronic vasculopathy of sickle cell anemia.
Assuntos
Anemia Falciforme/tratamento farmacológico , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Anemia Falciforme/genética , Anemia Falciforme/metabolismo , Animais , Células Cultivadas , Modelos Animais de Doenças , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Inibidores Enzimáticos/farmacologia , Hemoglobina Fetal/genética , Hemoglobina A/genética , Hemoglobina Falciforme/genética , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Quelantes de Ferro/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Veias Pulmonares/citologia , Fluxo Sanguíneo Regional/efeitos dos fármacos , Fluxo Sanguíneo Regional/fisiologia , Tromboplastina/metabolismo , Molécula 1 de Adesão de Célula Vascular/metabolismo , Vênulas/citologia , Vênulas/fisiologia , Vorinostat , Talassemia beta/tratamento farmacológico , Talassemia beta/genética , Talassemia beta/metabolismoRESUMO
Leukocyte transmigration occurs at specific locations (portals) on the endothelium, but the nature of these portals is not clear. Using intravital confocal microscopy of anesthetized mouse cremaster muscle in combination with immunofluorescence labeling, we showed that in microvessels transmigration is mainly junctional and preferentially occurs at tricellular endothelial junctional regions. Our data suggest that enrichment of ICAM-1 near approximately 43% of these junctions makes these locations preferred for transmigration by signaling the location of a nearby portal, as well as preparing the endothelial cell (EC) junctions, to accommodate leukocyte passage. Blockade of the extracellular domain of the ICAM-1 significantly reduced transmigration (by 68.8 + or - 4.5%) by reducing the ability of leukocytes to get to these portals. In contrast, blockade of the cytoplasmic tail of ICAM-1 reduced transmigration (by 71.1 + or - 7.0%) by disabling VE-cadherin rearrangement. Importantly, venular convergences are optimally equipped to support leukocyte transmigration. Differences in EC morphology result in a significantly higher number of tricellular junctions in convergences compared with straight venular regions (20.7 + or - 1.2 versus 12.43 + or - 1.1/6000 microm(2), respectively). Consequently, leukocyte adhesion and transmigration are significantly higher in convergences compared with straight regions (1.6- and 2.6-fold, respectively). Taken together, these data identify an important role for EC morphology and expression patterns of ICAM-1 in leukocyte transmigration.
Assuntos
Junções Aderentes/imunologia , Quimiotaxia de Leucócito/imunologia , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Migração e Rolagem de Leucócitos/imunologia , Transdução de Sinais/imunologia , Junções Aderentes/metabolismo , Sequência de Aminoácidos , Animais , Adesão Celular/imunologia , Endotélio Vascular/imunologia , Molécula 1 de Adesão Intercelular/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microvasos/citologia , Microvasos/imunologia , Microvasos/metabolismo , Dados de Sequência Molecular , Vênulas/citologia , Vênulas/imunologia , Vênulas/metabolismoRESUMO
Blood vessels are highly organized and complex structure, which are far more than simple tubes conducting the blood to almost any tissue of the body. The fine structure of the wall of blood vessels has been studied previously using the electron microscope, but the presence the telocytes associated with vasculature, a specific new cellular entity, has not been studied in depth. Interestingly, telocytes have been recently found in the epicardium, myocardium, endocardium, human term placenta, duodenal lamina propria and pleura. We show the presence of telocytes located on the extracellular matrix of blood vessels (arterioles, venules and capillaries) by immunohistochemistry and transmission electron microscopy. Also, we demonstrated the first evidence of a primary cilium in telocytes. Several functions have been proposed for these cells. Here, the telocyte-blood vessels cell proximity, the relationship between telocytes, exosomes and nervous trunks may have a special significance.
Assuntos
Arteríolas/ultraestrutura , Capilares/ultraestrutura , Células Intersticiais de Cajal/ultraestrutura , Vênulas/ultraestrutura , Animais , Arteríolas/citologia , Capilares/citologia , Células Cultivadas , Endocárdio/citologia , Endocárdio/ultraestrutura , Humanos , Células Intersticiais de Cajal/citologia , Microscopia Eletrônica de Transmissão , Miocárdio/citologia , Miocárdio/ultraestrutura , Pericárdio/citologia , Pericárdio/ultraestrutura , Pleura/citologia , Pleura/ultraestrutura , Ratos , Ratos Wistar , Vênulas/citologiaRESUMO
Compelling evidence continues to emerge suggesting that the glycocalyx surface layer on vascular endothelial cells plays a determining role in numerous physiological processes including inflammation, microvascular permeability, and endothelial mechanotransduction. Previous research has shown that enzymes degrade the glycocalyx, whereas inflammation causes shedding of the layer. To track the endogenous recovery of the glycocalyx in vivo, we used fluorescent microparticle image velocimetry (micro-PIV) in mouse cremaster muscle venules to estimate the hydrodynamically relevant glycocalyx thickness 1, 3, 5, and 7 days after enzymatic or cytokine-mediated degradation of the layer. Results indicate that after acute degradation of the glycocalyx, 5 to 7 days are required for the layer to endogenously restore itself to its native hydrodynamically relevant thickness in vivo. In light of these findings, and because demonstrable evidence has emerged that standard cell culture conditions are not conducive to providing the environment and/or cellular conditions necessary to produce and maintain a physiologically relevant cell surface glycocalyx in vitro, we sought to determine whether merely the passage of time would be sufficient to promote the production of a hydrodynamically relevant glycocalyx on a confluent monolayer of human umbilical vein endothelial cells (HUVECs). Using micro-PIV, we found that the hydrodynamically relevant glycocalyx was substantially absent 7 days postconfluence on HUVEC-lined cylindrical collagen microchannels maintained under standard culture conditions. Thus, it remains to be determined how a hydrodynamically relevant glycocalyx surface layer can be synthesized and maintained in culture before the endothelial cell culture model can be used to elucidate glycocalyx-mediated mechanisms of endothelial cell function.
Assuntos
Endotélio Vascular/citologia , Endotélio Vascular/fisiologia , Glicocálix/fisiologia , Glicocálix/ultraestrutura , Animais , Técnicas de Cultura de Células , Colágeno/fisiologia , Endotélio Vascular/patologia , Endotélio Vascular/fisiopatologia , Humanos , Inflamação/fisiopatologia , Camundongos , Microscopia/métodos , Músculo Esquelético/irrigação sanguínea , Permeabilidade , Reologia/métodos , Vênulas/citologia , Vênulas/fisiologiaRESUMO
Angioimmunoblastic T-cell lymphoma (AITL) is a T-cell lymphoma of follicular helper T-cell origin. Histologically, neoplastic T-cells proliferate to form clusters adjacent to or between arborizing high endothelial venules (HEVs). HEVs in normal lymph nodes express sulfated glycans called peripheral lymph node addressin (PNAd); however, it remains unclear whether PNAd is also expressed on HEVs in AITL. Furthermore, although it is widely accepted that HEVs are conspicuous in AITL due to their proliferation, quantitative histological support for this concept is lacking. To investigate these issues, we employed monoclonal antibodies recognizing PNAd, namely, MECA-79, HECA-452, and 297-11A, and performed quantitative immunohistochemical analysis of HEVs in 36 AITL-affected and 67 normal lymph nodes. Staining with all three antibodies confirmed that AITL HEVs express PNAd. Moreover, AITL HEVs were bound calcium-dependently by L-selectin-IgM fusion proteins, indicating that they function in the recruitment of L-selectin-expressing lymphocytes. Unexpectedly, HEV distribution density was not increased but rather decreased in AITL compared with normal lymph nodes, but HEV cross-sectional area in AITL was significantly greater than that seen in normal lymph nodes. Overall, these results indicate that the prominence of AITL HEVs is likely due to increased cross-sectional area rather than increased distribution density.