RESUMO
OBJECTIVE: The pathology of snake envenomation is closely tied to the severity of edema in the tissue surrounding the area of the bite. Elucidating the mechanisms that promote the development of such severe edema is critical to a better understanding of how to treat this life-threatening injury. We focused on one of the most abundant venom components in North American viper venom, crotamine, and the effects it has on the cells and function of the lymphatic system. METHODS: We used RT-PCR to identify the location and relative abundance of crotamine's cellular targets (Kvα channels) within the tissues and cells of the lymphatic system. We used calcium flux, nitrate production, and cell morphometry to determine the effects of crotamine on lymphatic endothelial cells. We used tracer transport, node morphometry, and node deposition to determine the effects of crotamine on lymph transport in vivo. RESULTS: We found that genes that encode targets of crotamine are highly present in lymphatic tissues and cells and that there is a differential distribution of those genes that correlates with phasic contractile activity. We found that crotamine potentiates calcium flux in human dermal lymphatic endothelial cells in response to stimulation with histamine and sheer stress (but not alone) and that it alters the production of nitric oxide in response to shear as well as changes the level of F-actin polymerization of those same cells. Crotamine alters lymphatic transport of large molecular weight tracers to local lymph nodes and is deposited within the node mostly in the immediate subcapsular region. CONCLUSION: This evidence suggests that snake venom components may have an impact on the function of the lymphatic system. This needs to be studied in greater detail as there are numerous venom components that may have effects on aspects of the lymphatic system. This would not only provide basic information on the pathobiology of snakebite but also provide targets for improved therapeutics to treat snakebite.
Assuntos
Mordeduras de Serpentes , Humanos , Células Endoteliais , Cálcio , Venenos de Serpentes/farmacologia , Venenos de Serpentes/químicaRESUMO
AIM: Fluid and macromolecule transport from the interstitium into and through lymphatic vessels is necessary for tissue homeostasis. While lymphatic capillary structure suggests that passive, paracellular transport would be the predominant route of macromolecule entry, active caveolae-mediated transcellular transport has been identified in lymphatic endothelial cells (LECs) in vitro. Caveolae also mediate a wide array of endothelial cell processes, including nitric oxide regulation. Thus, how does the lack of caveolae impact "lymphatic function"? METHODS: Various aspects of lymphatic transport were measured in mice constitutively lacking caveolin-1 ("CavKO"), the protein required for caveolae formation in endothelial cells, and in mice with a LEC-specific Cav1 gene deletion (Lyve1-Cre x Cav1flox/flox ; "LyCav") and ex vivo in their vessels and cells. RESULTS: In each model, lymphatic architecture was largely unchanged. The lymphatic conductance, or initial tissue uptake, was significantly higher in both CavKO mice and LyCav mice by quantitative microlymphangiography and the permeability to 70 kDa dextran was significantly increased in monolayers of LECs isolated from CavKO mice. Conversely, transport within the lymphatic system to the sentinel node was significantly reduced in anaesthetized CavKO and LyCav mice. Isolated, cannulated collecting vessel studies identified significantly reduced phasic contractility when lymphatic endothelium lacks caveolae. Inhibition of nitric oxide synthase was able to partially restore ex vivo vessel contractility. CONCLUSION: Macromolecule transport across lymphatics is increased with loss of caveolae, yet phasic contractility reduced, resulting in reduced overall lymphatic transport function. These studies identify lymphatic caveolar biology as a key regulator of active lymphatic transport functions.
Assuntos
Cavéolas , Vasos Linfáticos , Animais , Cavéolas/metabolismo , Caveolina 1 , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Camundongos , Óxido Nítrico Sintase/metabolismoRESUMO
Edema is common in preeclampsia (preE), a hypertensive disorder of pregnancy. Cardiotonic steroids (CTSs) such as marinobufagenin (MBG) are involved in the pathogenesis of preE. To assess whether CTSs are involved in the leakage of lymphatic endothelial cell (LEC), we evaluated their effect on monolayer permeability of LECs (MPLEC) in culture. A rat mesenteric LECs were treated with DMSO (vehicle), and CTSs (MBG, CINO, OUB) at concentrations of 1, 10, and 100 nM. Some LECs were pretreated with 1 µM L-NAME (N-Nitro-L-Arginine Methyl Ester) before adding 100 nM MBG or cinobufotalin (CINO). Expression of ß-catenin and vascular endothelial (VE)-cadherin in CTS-treated LECs was measured by immunofluorescence and MPLEC was quantified using a fluorescence plate reader. Western blot was performed to measure ß-catenin and VE-cadherin protein levels and myosin light chain 20 (MLC20) phosphorylation. MBG (≥ 1 nM) and CINO (≥ 10 nM) caused an increase (p < 0.05) in the MPLEC compared to DMSO while ouabain (OUB) had no effect. Pretreatment of LECs with 1 µM L-NAME attenuated (p < 0.05) the MPLEC. The ß-catenin expression in LECs was downregulated (p < 0.05) by MBG and CINO. However, there was no effect on the LECs tight junctions for the CINO group. VE-cadherin expression was downregulated (p < 0.05) by CINO, and MLC20 phosphorylation was upregulated (p < 0.05) by MBG. We demonstrated that MBG and CINO caused an increase in the MPLEC, which were attenuated by L-NAME pretreatment. The data suggest that CTSs exert their effect via nitric-oxide-dependent signaling pathway and may be involved in vascular leak syndrome of LEC lining in preE.
Assuntos
Bufanolídeos/farmacologia , Permeabilidade da Membrana Celular , Células Endoteliais/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Óxido Nítrico/metabolismo , Vasoconstritores/farmacologia , Animais , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Fosforilação , RatosRESUMO
A novel snake venom cysteine-rich secretory protein (svCRiSP), Hellerin, was purified from C. o. helleri venom using sequential reverse phase and cation-exchange chromatography. Gel electrophoresis, N-terminal sequencing, and LC-MS/MS sequencing identified a single protein with a molecular mass of approximately 24.8â¯kDa and confirmed its identity as a svCRiSP. Hellerin had cytotoxic effects on human umbilical vein endothelial cells (HUVECs) in a dose-dependent manner but not in human dermal lymphatic endothelial cells (HDLECs) and human dermal blood endothelial cells (HDBECs). Hellerin produced a dramatic increase in both blood vascular permeability in vivo, and in the trans-epithelial permeability of cultured HDLEC and HDBEC cells. This is the first study that describes the effect of a svCRiSP on vascular, blood and lymphatic permeability.
Assuntos
Permeabilidade Capilar/efeitos dos fármacos , Venenos de Crotalídeos/química , Proteínas de Répteis/farmacologia , Sequência de Aminoácidos , Animais , Linhagem Celular , Cromatografia Líquida , Venenos de Crotalídeos/isolamento & purificação , Crotalus , Cisteína , Células Endoteliais da Veia Umbilical Humana , Humanos , Proteínas de Répteis/química , Proteínas de Répteis/isolamento & purificação , Alinhamento de Sequência , Espectrometria de Massas em TandemRESUMO
Background and Aim: Lipedema is a common painful SAT disorder characterized by enlargement of fat primarily in the legs of women. Case reports of lipedema tissue samples demonstrate fluid and fibrosis in the interstitial matrix, increased macrophages, and adipocyte hypertrophy. The aims of this project are to investigate blood vasculature, immune cells, and structure of lipedema tissue in a cohort of women. Methods: Forty-nine participants, 19 controls and 30 with lipedema, were divided into groups based on body mass index (BMI): Non-Obese (BMI 20 to <30 kg/m2) and Obese (BMI 30 to <40 kg/m2). Histological sections from thigh skin and fat were stained with H&E. Adipocyte area and blood vessel size and number were quantified using ImageJ software. Markers for macrophages (CD68), mast cells (CD117), T cells (CD3), endothelial cells (CD31), blood (SMA), and lymphatic (D2-40 and Lyve-1) vessels were investigated by IHC and IF. Results: Non-Obese Lipedema adipocyte area was larger than Non-Obese Controls (p=0.005) and similar to Obese Lipedema and Obese Controls. Macrophage numbers were significantly increased in Non-Obese (p < 0.005) and Obese (p < 0.05) Lipedema skin and fat compared to Control groups. No differences in T lymphocytes or mast cells were observed when comparing Lipedema to Control in both groups. SMA staining revealed increased dermal vessels in Non-Obese Lipedema patients (p < 0.001) compared to Non-Obese Controls. Lyve-1 and D2-40 staining showed a significant increase in lymphatic vessel area but not in number or perimeter in Obese Lipedema participants (p < 0.05) compared to Controls (Obese and Non-Obese). Areas of angiogenesis were found in the fat in 30% of lipedema participants but not controls. Conclusion: Hypertrophic adipocytes, increased numbers of macrophages and blood vessels, and dilation of capillaries in thigh tissue of non-obese women with lipedema suggest inflammation, and angiogenesis occurs independent of obesity and demonstrates a role of altered vasculature in the manifestation of the disease.
Assuntos
Tecido Adiposo/patologia , Peso Corporal Ideal/fisiologia , Lipedema/fisiopatologia , Vasos Linfáticos/fisiopatologia , Obesidade/fisiopatologia , Coxa da Perna/patologia , Adipócitos/citologia , Adulto , Índice de Massa Corporal , Feminino , Humanos , Hipertrofia/patologia , Macrófagos/patologia , Obesidade/complicaçõesRESUMO
It is theorized that toxic agents are transported from the hyperpermeable gut of burn victims through the lymph, to the systemic circulation, causing global injury. We believe that immune cells respond to leakage of "toxic lymph" following trauma causing the attraction of these cells to the perilymphatic space. To test this, we utilized a model of burn on rats to examine changes in a single immune cell population associated with mesenteric lymphatic dysfunction. We examined the ability of serum from these animals to increase permeability in lymphatic endothelial monolayers and disrupt cellular junctions. We also treated burn animals with doxycycline, an inhibitor of microvascular permeability, and observed the effects on immune cell populations, morphometry, and lymphatic endothelial permeability. Burn injury increased the number of MHCII+ immune cells along the vessel (>50%). The size and shape of these cells also changed significantly following burn injury. Serum from burn animals increased lymphatic endothelial permeability (â¼1.5-fold) and induced breaks in VE-cadherin staining. Doxycycline treatment blocked the accumulation of immune cells along the vessel, whereas serum from doxycycline-treated animals failed to increase lymphatic endothelial permeability. The size of cells along the vessel in doxycycline-treated burn animals was not affected, suggesting that the cells already present on the lymphatic vessels still respond to substances in the lymph. These findings suggest that factors produced during burn can induce lymphatic endothelial barrier disruption and lymph produced during traumatic injury can influence the attraction and morphology of immune cell populations along the vessel.
Assuntos
Células Apresentadoras de Antígenos/efeitos dos fármacos , Queimaduras/tratamento farmacológico , Doxiciclina/farmacologia , Células Endoteliais/efeitos dos fármacos , Antígenos de Histocompatibilidade Classe II/imunologia , Vasos Linfáticos/efeitos dos fármacos , Animais , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/patologia , Antígenos CD/genética , Antígenos CD/imunologia , Biomarcadores/metabolismo , Queimaduras/genética , Queimaduras/imunologia , Queimaduras/patologia , Caderinas/genética , Caderinas/imunologia , Permeabilidade Capilar , Movimento Celular/efeitos dos fármacos , Tamanho Celular , Modelos Animais de Doenças , Células Endoteliais/imunologia , Células Endoteliais/patologia , Endotélio Linfático/efeitos dos fármacos , Endotélio Linfático/imunologia , Endotélio Linfático/patologia , Expressão Gênica , Antígenos de Histocompatibilidade Classe II/genética , Linfa/citologia , Linfa/efeitos dos fármacos , Linfa/imunologia , Vasos Linfáticos/imunologia , Vasos Linfáticos/patologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/patologia , Masculino , Mesentério/efeitos dos fármacos , Mesentério/imunologia , Mesentério/patologia , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Monócitos/patologia , Ratos , Ratos Sprague-DawleyRESUMO
Inflammatory bowel disease (IBD), which includes Crohn's disease and ulcerative colitis, is a chronic inflammatory condition of the gastrointestinal (GI) tract. Currently, it is treated with immunosuppressant or biologics that often induce severe adverse effects. Thus, there is an urgent clinical need for more specific treatments. To provide a valid therapeutic tool for IBD therapy, in this work we developed biomimetic nanovesicles by manipulating leukocyte membranes to exploit mechanisms of T-cell recruitment during inflammation. A subset of T-lymphocytes participates in homing to inflamed tissue in the gastrointestinal tract by overexpressing the α4ß7 integrin, which is responsible for binding to its receptor on the endothelial membrane, the mucosal addressin cell adhesion molecule 1. Based on this principle, we engineered biomimetic vesicles, referred to as specialized leukosomes (SLKs), which are leukocyte-like carriers 'doped' with the α4ß7 integrin over-induced in purified immune cells. We tested SLKs in an in vivo murine model of IBD induced by treatment with dextran sulfate sodium. Notably, treatment of IBD mice with SLKs allowed us to observe a reduction of inflammation (favorable modulation of both pro- and anti-inflammatory genes, as well as reduction of immune cells infiltration into the colon tissue), and a consequent enhanced intestinal repair (low epithelial damage). In this study, we demonstrate that biological-derived nanoparticles can be used not only as naturally targeted drug delivery systems, but also as nano-therapeutics endowed with intrinsic anti-inflammatory properties.
Assuntos
Anti-Inflamatórios/farmacologia , Materiais Biomiméticos , Doenças Inflamatórias Intestinais/tratamento farmacológico , Nanopartículas , Animais , Sulfato de Dextrana , Doenças Inflamatórias Intestinais/induzido quimicamente , Integrinas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Linfócitos T/imunologiaRESUMO
BACKGROUND: Lymphatic dysfunction has been linked to inflammation since the 1930s. Lymphatic function in the gut and mesentery is grossly underexplored in models of inflammatory bowel disease despite the use of lymphatic occlusion in early models of inflammatory bowel disease. Activation of the innate and adaptive immune system is a hallmark of TNBS-induced inflammation and is linked to disruption of the intrinsic lymph pump. Recent identification of crosstalk between lymphatic vessel resident immune cells and regulation of lymphatic vessel contractility underscore the importance of the timing of lymphatic dysfunction during tissue inflammation in response to TNBS. METHODS: To evaluate lymphatic function in TNBS induced inflammation, lymph was collected and flow measured from mesenteric lymphatics. Cellularity and cytokine profile of the lymph was also measured. Histopathology was performed to determine severity of injury and immunofluorescent staining of the mesentery was done to evaluate changes in the population of immune cells that reside near and on gastro-intestinal collecting lymphatics. RESULTS: Lymph transport fell 24 hours after TNBS administration and began recovering at 72 hours. Significant reduction of lymph flow preceded significant increase in histopathological score and occurred simultaneously with increased myeloperoxidase activity. These changes were preceded by increased MHCII cells surrounding mesenteric lymphatics leading to an altered lymphatic environment that would favor dysfunction. CONCLUSIONS: Alterations in environmental factors that effect lymphatic function occur before the development of gross GI inflammation. Reduced lymphatic function in TNBS-mediated inflammation is likely an early factor in the development of injury and that recovery of function is associated with resolution of inflammation.
Assuntos
Colo/irrigação sanguínea , Células Dendríticas/imunologia , Ileíte/etiologia , Íleo/patologia , Imunidade Celular , Isquemia/complicações , Vasos Linfáticos/patologia , Animais , Células Dendríticas/patologia , Modelos Animais de Doenças , Ileíte/imunologia , Ileíte/patologia , Íleo/irrigação sanguínea , Íleo/imunologia , Imuno-Histoquímica , Isquemia/imunologia , Isquemia/patologia , RatosRESUMO
BACKGROUND: Until now, there has been no tool available to provide lymphatic researchers the ability to perform experiments in tissue explants containing lymphatic vessels under tissue position- and lymphatic lumen-controlled conditions. METHODS AND RESULTS: In this article we provide technical details and description of the method of using the newly developed and implemented the position- and lymphatic lumen-controlled tissue chambers to study live lymphatic vessels and surrounding tissues ex vivo. In this study, we, for the first time, performed detailed comparative analysis of the contractile and pumping activity of rat mesenteric lymphatic vessels (MLVs) situated within tissue explants mounted in new tissue chambers and isolated, cannulated, and pressurized rat MLVs maintained in isolated vessel setups. We found no significant differences of the effects of both transmural pressure- and wall shear stress sensitivities of MLVs in tissue chambers and isolated MLVs. CONCLUSIONS: We conclude that this new experimental tool, a position- and lymphatic lumen-controlled tissue chamber, allows precise investigation of lymphatic function of MLVs interacting with elements of the tissue microenvironment. This method provides an important new set of experimental tools to investigate lymphatic function.
Assuntos
Vasos Linfáticos/anatomia & histologia , Animais , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
Proper lymphatic function is necessary for the transport of fluids, macromolecules, antigens and immune cells out of the interstitium. The lymphatic endothelium plays important roles in the modulation of lymphatic contractile activity and lymph transport, but it's role as a barrier between the lymph and interstitial compartments is less well understood. Alterations in lymphatic function have long been associated with edema and inflammation although the integrity of the lymphatic endothelial barrier during inflammation is not well-defined. In this paper we evaluated the integrity of the lymphatic barrier in response to inflammatory stimuli commonly associated with increased blood endothelial permeability. We utilized in vitro assays of lymphatic endothelial cell (LEC) monolayer barrier function after treatment with different inflammatory cytokines and signaling molecules including TNF-α, IL-6, IL-1ß, IFN-γ and LPS. Moderate increases in an index of monolayer barrier dysfunction were noted with all treatments (20-60 % increase) except IFN-γ which caused a greater than 2.5-fold increase. Cytokine-induced barrier dysfunction was blocked or reduced by the addition of LNAME, except for IL-1ß and LPS treatments, suggesting a regulatory role for nitric oxide. The decreased LEC barrier was associated with modulation of both intercellular adhesion and intracellular cytoskeletal activation. Cytokine treatments reduced the expression of VE-cadherin and increased scavenging of ß-catenin in the LECs and this was partially reversed by LNAME. Likewise the phosphorylation of myosin light chain 20 at the regulatory serine 19 site, which accompanied the elevated monolayer barrier dysfunction in response to cytokine treatment, was also blunted by LNAME application. This suggests that the lymphatic barrier is regulated during inflammation and that certain inflammatory signals may induce large increases in permeability.
Assuntos
Permeabilidade da Membrana Celular/efeitos dos fármacos , Citocinas/farmacologia , Células Endoteliais/citologia , Endotélio Vascular/citologia , Mediadores da Inflamação/farmacologia , Animais , Antígenos CD/metabolismo , Western Blotting , Caderinas/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Imunofluorescência , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ratos Sprague-Dawley , S-Nitroso-N-Acetilpenicilamina/farmacologia , Proteínas de Transporte Vesicular/metabolismoRESUMO
BACKGROUND: Ulcerative colitis (UC) is the most common form of inflammatory bowel disease in the USA. A key component of UC is the increase in inflammatory angiogenesis of the colon during active disease. This increase is driven to a great extent by the over expression of VEGF-A. Recently, VEGF165(b) (VEGF164(b) in mouse), an anti-angiogenic form of VEGF-A was described and its regulation was determined to be disturbed in many pathologies such as cancer and pre-eclampsia. RESULTS: The aims of this study were to examine the role of this inhibitory VEGF by expressing this molecule in a model of intestinal inflammation, and to evaluate its expression as a potential new therapeutic approach for treating UC. A modified model of TNBS colitis was used to determine the effects of rVEGF164(b) expression on colon inflammation. Expansion of the vascular system was assessed by immunhistochemical methods and macro- and microscopic measurements of inflammation in the colon were measured. Leukocyte invasion of the tissue was measured by myeloperoxidase assay and identification and counting of lymphoid follicles. Both angio- and lymphangiogenesis were reduced by expression of rVEGF164(b), which correlated with reduction in both gross and microscopic inflammatory scores. Leukocyte invasion of the tissue was also reduced by rVEGF164(b) expression. CONCLUSIONS: This is the first report using an endogenous inhibitory VEGF-A isoform for therapy in a model of experimental colitis. Inhibitory VEGF molecules play an important role in maintenance of gut homeostasis and may be dysregulated in UC. The results of this study suggest that restoration of rVEGF164(b) expression has anti-inflammatory activity in a TNBS model and warrants further examination as a possible therapeutic for UC.
Assuntos
Colite Ulcerativa/tratamento farmacológico , Substâncias Protetoras/uso terapêutico , Fator A de Crescimento do Endotélio Vascular/uso terapêutico , Animais , Vasos Sanguíneos/efeitos dos fármacos , Vasos Sanguíneos/patologia , Agregação Celular/efeitos dos fármacos , Colite Ulcerativa/sangue , Colite Ulcerativa/patologia , Colo/efeitos dos fármacos , Colo/imunologia , Colo/patologia , Sulfato de Dextrana , Modelos Animais de Doenças , Progressão da Doença , Células HEK293 , Humanos , Leucócitos/efeitos dos fármacos , Leucócitos/patologia , Vasos Linfáticos/efeitos dos fármacos , Vasos Linfáticos/patologia , Camundongos , Tamanho do Órgão/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Isoformas de Proteínas , Ácido Trinitrobenzenossulfônico , Fator A de Crescimento do Endotélio Vascular/sangue , Fator A de Crescimento do Endotélio Vascular/farmacologiaRESUMO
The principal function of the lymphatic system is to transport lymph from the interstitium to the nodes and then from the nodes to the blood. In doing so lymphatics play important roles in fluid homeostasis, macromolecular/antigen transport and immune cell trafficking. To better understand the genes that contribute to their unique physiology, we compared the transcriptional profile of muscular lymphatics (prenodal mesenteric microlymphatics and large, postnodal thoracic duct) to axillary and mesenteric arteries and veins isolated from rats. Clustering of the differentially expressed genes demonstrated that the lymph versus blood vessel differences were more profound than between blood vessels, particularly the microvessels. Gene ontology functional category analysis indicated that microlymphatics were enriched in antigen processing/presentation, IgE receptor signaling, catabolic processes, translation and ribosome; while they were diminished in oxygen transport, regulation of cell proliferation, glycolysis and inhibition of adenylate cyclase activity by G-proteins. We evaluated the differentially expressed microarray genes/products by qPCR and/or immunofluorescence. Immunofluorescence documented that multiple MHC class II antigen presentation proteins were highly expressed by an antigen-presenting cell (APC) type found resident within the lymphatic wall. These APCs also expressed CD86, a co-stimulatory protein necessary for T-cell activation. We evaluated the distribution and phenotype of APCs within the pre and postnodal lymphatic network. This study documents a novel population of APCs resident within the walls of muscular, prenodal lymphatics that indicates novel roles in antigen sampling and immune responses. In conclusion, these prenodal lymphatics exhibit a unique profile that distinguishes them from blood vessels and highlights the role of the lymphatic system as an immunovascular system linking the parenchymal interstitium, lymph nodes and the blood.
Assuntos
Artérias/metabolismo , Perfilação da Expressão Gênica/métodos , Imunidade/genética , Sistema Linfático/metabolismo , Veias/metabolismo , Animais , Apresentação de Antígeno/genética , Células Apresentadoras de Antígenos/metabolismo , Análise por Conglomerados , Imunofluorescência , Ontologia Genética , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/metabolismo , Masculino , Mesentério/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Vascular endothelial growth factors (VEGF) are a Janus-faced family of growth factors exerting both neuroprotective and maladaptive effects on the blood-brain barrier. For example, VEGFs are beneficial in promoting postischemic brain angiogenesis, but the newly formed vessels are leaky. We investigated the role of the naturally occurring murine inhibitory VEGF isoform VEGF165b in a mouse model of focal cerebral ischemia by middle cerebral artery occlusion and reperfusion (I/R) in male C57BL/6 mice. We investigated the roles of VEGF164/165 and VEGF165b in both brain and nonbrain endothelial barrier, angiogenesis, and neutrophil migration using oxygen glucose deprivation and reoxygenation as in vitro model. We investigated the role of VEGF165b in brain edema, neutrophil infiltration, ischemic brain damage, and neuronal death in vivo using an adenovirus encoding a recombinant VEGF164b isoform. Neither VEGF164/165 nor VEGF165b significantly altered brain endothelial barrier or angiogenesis in vitro. However, treatment of brain endothelial cells with VEGF165b increased neutrophil migration in vitro and exacerbated stroke injury by aggravating neutrophil infiltration and neurodegeneration in vivo. Our results indicate that alterations in the delicate balance in the relative levels of pro- and antiangiogenic VEGF isoforms can result in either adaptive or detrimental effects, depending on the VEGF isoform levels and on the duration and extent of injury.
Assuntos
Isquemia Encefálica/patologia , Encéfalo/patologia , Proteínas Recombinantes/efeitos adversos , Fator A de Crescimento do Endotélio Vascular/efeitos adversos , Adenoviridae/metabolismo , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Astrócitos/patologia , Comportamento Animal , Encéfalo/metabolismo , Edema Encefálico/complicações , Edema Encefálico/patologia , Isquemia Encefálica/sangue , Isquemia Encefálica/complicações , Linhagem Celular , Permeabilidade da Membrana Celular , Quimiotaxia , Modelos Animais de Doenças , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Humanos , Immunoblotting , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Infiltração de Neutrófilos/efeitos dos fármacos , Isoformas de Proteínas/efeitos adversos , Acidente Vascular Cerebral/sangue , Acidente Vascular Cerebral/complicações , Acidente Vascular Cerebral/patologia , Regulação para Cima/efeitos dos fármacosRESUMO
OBJECTIVE: Cytokines contribute to cerebro-vascular inflammatory and immune responses by inducing ECAMs' expression. Ischemic insults can be separated into aglycemic and hypoxic components. However, whether aglycemia, hypoxia or OGD plays a major role in dysregulating BBB or promotes immune cell infiltration via ECAMs' expression is not clear. We investigated how expression of ICAM-1, VCAM-1, MAdCAM-1, PECAM-1, E- and P-selectin in response to TNF-α, IL-1ß and IFN-γ was altered by aglycemia (A), hypoxia (H) or combined oxygen glucose deprivation (OGD). METHODS: A cell surface enzyme linked immunoabsorbent assay (cell surface ELISA) was used to analyze ECAM expression. RESULTS: We observed that ICAM-1 and PECAM-1 expressions were insensitive to hypoxia, aglycemia or OGD. Conversely, VCAM-1 and E-selectin were increased by hypoxia, but not by aglycemia. MAdCAM-1 and P-selectin were induced by hypoxia, and decreased by aglycemia. Patterns of cytokine-regulated ECAMs' expression were also modified by metabolic conditions. CONCLUSIONS: Our results indicate that patterns of inflammation-associated ECAMs represent cumulative influences from metabolic stressors, as well as cytokine activation. The expression of ECAMs following tissue injury reflects mechanistic interactions between metabolic disturbances, and alterations in tissue cytokines. Normalization of tissue metabolism, as well as cytokine profiles, may provide important targets for therapeutic treatment of inflammation.
Assuntos
Encéfalo/metabolismo , Moléculas de Adesão Celular/biossíntese , Citocinas/farmacologia , Células Endoteliais/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Animais , Encéfalo/patologia , Hipóxia Celular/efeitos dos fármacos , Linhagem Celular , Células Endoteliais/patologia , Glucose/metabolismo , Glucose/farmacologia , Camundongos , Edulcorantes/metabolismo , Edulcorantes/farmacologiaRESUMO
The glio-vascular unit (G-unit) plays a prominent role in maintaining homeostasis of the blood-brain barrier (BBB) and disturbances in cells forming this unit may seriously dysregulate BBB. The direct and indirect effects of cytokines on cellular components of the BBB are not yet unclear. The present study compares the effects of cytokines and cytokine-treated astrocytes on brain endothelial barrier. 3-dimensional transwell co-cultures of brain endothelium and related-barrier forming cells with astrocytes were used to investigate gliovascular barrier responses to cytokines during pathological stresses. Gliovascular barrier was measured using trans-endothelial electrical resistance (TEER), a sensitive index of in vitro barrier integrity. We found that neither TNF-α, IL-1ß or IFN-γ directly reduced barrier in human or mouse brain endothelial cells or ECV-304 barrier (independent of cell viability/metabolism), but found that astrocyte exposure to cytokines in co-culture significantly reduced endothelial (and ECV-304) barrier. These results indicate that the barrier established by human and mouse brain endothelial cells (and other cells) may respond positively to cytokines alone, but that during pathological conditions, cytokines dysregulate the barrier forming cells indirectly through astrocyte activation involving reorganization of junctions, matrix, focal adhesion or release of barrier modulating factors (e.g. oxidants, MMPs).
Assuntos
Astrócitos/efeitos dos fármacos , Astrócitos/fisiologia , Barreira Hematoencefálica/efeitos dos fármacos , Permeabilidade Capilar/efeitos dos fármacos , Citocinas/farmacologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/fisiologia , Animais , Astrócitos/citologia , Barreira Hematoencefálica/citologia , Barreira Hematoencefálica/fisiologia , Permeabilidade Capilar/fisiologia , Células Cultivadas , Técnicas de Cocultura , Impedância Elétrica , Células Endoteliais/citologia , Endotélio Vascular/citologia , Endotélio Vascular/fisiologia , Humanos , Interferon gama/farmacologia , Interleucina-1beta/farmacologia , Camundongos , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Inflammatory bowel diseases (IBD) are a complex group of diseases involving alterations in mucosal immunity and gastrointestinal physiology during both initiation and progressive phases of the disease. At the core of these alterations are endothelial cells, whose continual adjustments in structure and function coordinate vascular supply, immune cell emigration, and regulation of the tissue environment. Expansion of the endothelium in IBD (angiogenesis), mediated by inflammatory growth factors, cytokines and chemokines, is a hallmark of active gut disease and is closely related to disease severity. The endothelium in newly formed or inflamed vessels differs from that in normal vessels in the production of and response to inflammatory cytokines, growth factors, and adhesion molecules, altering coagulant capacity, barrier function and blood cell recruitment in injury. This review examines the roles of the endothelium in the initiation and propagation of IBD pathology and distinctive features of the intestinal endothelium contributing to these conditions.
Assuntos
Endotélio Vascular/fisiologia , Endotélio Vascular/fisiopatologia , Doenças Inflamatórias Intestinais/fisiopatologia , Intestinos/irrigação sanguínea , Animais , Coagulação Sanguínea/imunologia , Plaquetas/imunologia , Moléculas de Adesão Celular/metabolismo , Quimiocinas/imunologia , Neoplasias Colorretais/etiologia , Neoplasias Colorretais/patologia , Citocinas/imunologia , Células Endoteliais/citologia , Células Endoteliais/imunologia , Humanos , Doenças Inflamatórias Intestinais/patologia , Doenças Inflamatórias Intestinais/terapia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Intestinos/imunologia , Intestinos/patologia , Neovascularização Patológica/complicações , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Células-Tronco/fisiologia , Receptores Toll-Like/imunologiaRESUMO
OBJECTIVE: To investigate the effects of the murine inhibitory vascular endothelial growth factor (VEGF, rVEGF164b), we generated an adenoviral vector encoding rVEGF164b, and examined its effects on endothelial barrier, growth, and structure. METHOD: Mouse vascular endothelial cells (MVEC) proliferation was determined by an MTT assay. Barrier of MVEC monolayers was measured by trans-endothelial electrical resistance (TEER). Reorganization of actin and zonula occludens-1 (ZO-1) were determined by fluorescent microscopy. RESULTS: Mouse venous endothelial cells treated with murine VEGF-A (VEGF-A) (50 ng/mL) increased proliferation (60.7 ± 0.1%) within 24 hours (p < 0.05) and rVEGF164b inhibited VEGF-A-induced proliferation. TEER was significantly decreased by VEGF-A (81.7 ± 6.2% of control). Treatment with rVEGF164b at 50 ng/mL transiently reduced MVEC barrier (p < 0.05) at 30 minutes post-treatment (87.9 ± 1.7% of control TEER), and returned to control levels by 40 minutes post-treatment. Treatment with rVEGF164b prevented barrier changes by subsequent exposure to VEGF-A. Treatment of MVECS with VEGF-A reorganized F-actin and ZO-1, which was attenuated by rVEGF164b. CONCLUSIONS: VEGF-A may dysregulate endothelial barrier through junctional cytoskeleton processes, which can be attenuated by rVEGF164b. The VEGF-A stimulated MVEC proliferation, barrier dysregulation, and cytoskeletal rearrangement. However, rVEGF164b blocks these effects, therefore it may be useful for regulation studies of VEGF-A/VEGF-R signaling in many different models.
Assuntos
Células Endoteliais/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/farmacologia , Actinas/metabolismo , Adenoviridae/genética , Sequência de Aminoácidos , Animais , Permeabilidade Capilar/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura , Impedância Elétrica , Células Endoteliais/citologia , Células Endoteliais/fisiologia , Vetores Genéticos , Humanos , Proteínas de Membrana/metabolismo , Camundongos , Fosfoproteínas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/fisiologia , Proteína da Zônula de Oclusão-1RESUMO
BACKGROUND: Inflammatory Bowel Diseases (IBD) remain significant health problems in the US and worldwide. IBD is most often associated with eastern European ancestry, and is less frequently reported in other populations of African origin e.g. African Americans ('AAs'). Whether AAs represent an important population with IBD in the US remains unclear since few studies have investigated IBD in communities with a majority representation of AA patients. The Louisiana State University Health Sciences Center in Shreveport (LSUHSC-S) is a tertiary care medical center, with a patient base composed of 58% AA and 39% Caucasian (W), ideal for evaluating racial (AA vs. W) as well and gender (M vs. F) influences on IBD. METHODS: In this retrospective study, we evaluated 951 visits to LSUHSC-S for IBD (between 2000 to 2008) using non-identified patient information based on ICD-9 medical record coding (Crohn's disease 'CD'-555.0- 555.9 and ulcerative colitis 'UC'-556.0-556.9). RESULTS: Overall, there were more cases of CD seen than UC. UC and CD affected similar ratios of AA and Caucasian males (M) and females (F) with a rank order of WF > WM > AAF > AAM. Interestingly, in CD, we found that annual visits per person was the highest in AA M (10.7 ± 1.7); significantly higher (* -p < 0.05) than in WM (6.3 ± 1.0). Further, in CD, the female to male (F: M) ratio in AA was significantly higher (*- p < 0.05) (1.9 ± 0.2) than in Caucasians (F:M = 1.3 ± 0.1) suggesting a female dominance in AACD; no differences were seen in UC F: M ratios. CONCLUSION: Although Caucasians still represent the greatest fraction of IBD (~64%), AAs with IBD made up >1/3 (36.4%) of annual IBD cases from 2000-2008 at LSUHSC-S. Further studies on genetic and environments risks for IBD risk in AAs are needed to understand differences in presentation and progression in AAs and other 'non-traditional' populations.
Assuntos
Negro ou Afro-Americano , Centros Comunitários de Saúde , Doenças Inflamatórias Intestinais/etnologia , Visita a Consultório Médico/tendências , Adulto , Feminino , Humanos , Incidência , Masculino , Prevalência , Estudos Retrospectivos , Estados Unidos/epidemiologiaRESUMO
BACKGROUND: The pathophysiology of inflammatory bowel disease (IBD) includes leukocyte infiltration, blood and lymphatic remodeling, weight loss and protein enteropathy. The roles of angiopoietin-2 (Ang-2) in initiating gut inflammation, leukocyte infiltration and angiogenesis are not well understood. METHODS: Disease activity index, histopathological scoring, myeloperoxidase assay, immunohistochemistry and sodium dodecyl sulphate- polyacrylamide gel electrophoretic methods were employed in the present study to address the roles of Ang-2 in experimental colitis. RESULTS: Several important differences were seen in the development of experimental IBD in Ang-2(-/-) mice. Although weight change and disease activity differ only slightly in WT and Ang-2(-/-) + DSS treated mice, leukocyte infiltration, inflammation and blood and lymphatic vessel density is significantly attenuated compared to WT + DSS mice. Gut capillary fragility and water export (stool blood and form) appear significantly earlier in Ang-2(-/-) + DSS mice vs. WT. Colon lengths were also significantly reduced in Ang-2(-/-) and gut histopathology was less severe in Ang-2(-/-) compared to WT + DSS. Lastly, the decrease in serum protein content in WT + DSS was less severe in Ang-2(-/-) + DSS, thus protein losing enteropathy (PLE) a feature of IBD is relieved by Ang-2(-/-). CONCLUSION: These data demonstrate that in DSS colitis, Ang-2 mediates inflammatory hemangiogenesis, lymphangiogenesis and neutrophil infiltration to reduce some, but not all clinical features of IBD. The implications for Ang-2 manipulation in the development of IBD and other inflammatory diseases and treatments involving Ang-2 are discussed.