RESUMO
Coordinated navigation within tissues is essential for cells of the innate immune system to reach the sites of inflammatory processes, but the signals involved are incompletely understood. Here we demonstrate that NG2(+) pericytes controlled the pattern and efficacy of the interstitial migration of leukocytes in vivo. In response to inflammatory mediators, pericytes upregulated expression of the adhesion molecule ICAM-1 and released the chemoattractant MIF. Arteriolar and capillary pericytes attracted and interacted with myeloid leukocytes after extravasating from postcapillary venules, 'instructing' them with pattern-recognition and motility programs. Inhibition of MIF neutralized the migratory cues provided to myeloid leukocytes by NG2(+) pericytes. Hence, our results identify a previously unknown role for NG2(+) pericytes as an active component of innate immune responses, which supports the immunosurveillance and effector function of extravasated neutrophils and macrophages.
Assuntos
Molécula 1 de Adesão Intercelular/metabolismo , Oxirredutases Intramoleculares/metabolismo , Leucócitos/imunologia , Fatores Inibidores da Migração de Macrófagos/metabolismo , Pericitos/imunologia , Receptores de Reconhecimento de Padrão/imunologia , Anticorpos Bloqueadores/farmacologia , Arteríolas/imunologia , Capilares/imunologia , Comunicação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Movimento Celular/imunologia , Células Cultivadas , Humanos , Imunidade Inata , Mediadores da Inflamação/metabolismo , Molécula 1 de Adesão Intercelular/genética , Oxirredutases Intramoleculares/genética , Oxirredutases Intramoleculares/imunologia , Leucócitos/efeitos dos fármacos , Fatores Inibidores da Migração de Macrófagos/genética , Fatores Inibidores da Migração de Macrófagos/imunologia , Ativação de Neutrófilo/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Vênulas/imunologiaRESUMO
Trafficking of polymorphonuclear neutrophils (PMNs) during inflammation critically depends on the ß2 integrins lymphocyte function-associated antigen 1 (LFA-1) (CD11a/CD18) and macrophage-1 antigen (CD11b/CD18). Here, we identify coronin 1A (Coro1A) as a novel regulator of ß2 integrins that interacts with the cytoplasmic tail of CD18 and is crucial for induction of PMN adhesion and postadhesion events, including adhesion strengthening, spreading, and migration under flow conditions. Transition of PMN rolling to firm adhesion critically depends on Coro1A by regulating the accumulation of high-affinity LFA-1 in focal zones of adherent cells. Defective integrin affinity regulation in the genetic absence of Coro1A impairs leukocyte adhesion and extravasation in inflamed cremaster muscle venules in comparison with control animals. In a Helicobacter pylori mouse infection model, PMN infiltration into the gastric mucosa is dramatically reduced in Coro1A-/- mice, resulting in an attenuated gastric inflammation. Thus, Coro1A represents an important novel player in integrin biology, with key functions in PMN trafficking during innate immunity.
Assuntos
4-Butirolactona/análogos & derivados , Antígenos CD18/metabolismo , Movimento Celular , Imunidade Inata , Neutrófilos/citologia , Neutrófilos/metabolismo , 4-Butirolactona/metabolismo , Actinas/metabolismo , Animais , Sinalização do Cálcio , Adesão Celular , Gastrite/imunologia , Gastrite/microbiologia , Gastrite/patologia , Helicobacter pylori/fisiologia , Antígeno-1 Associado à Função Linfocitária/metabolismo , Antígeno de Macrófago 1/metabolismo , Camundongos Endogâmicos C57BL , Receptores Acoplados a Proteínas G/metabolismo , ReologiaRESUMO
OBJECTIVES: To determine predictors for long-term outcome in high-risk patients undergoing transcatheter edge-to-edge mitral valve repair (TMVR) for severe mitral regurgitation (MR). BACKGROUND: There is no data on predictors of long-term outcome in high-risk real-world patients. METHODS: From August 2009 to April 2011, 126 high-risk patients deemed inoperable were treated with TMVR in two high-volume university centers. RESULTS: MR could be successfully reduced to grade ≤2 in 92.1% of patients (116/126 patients). Long-term clinical follow-up up to 5 years (95.2% follow-up rate) revealed a mortality rate of 35.7% (45/126 patients). Repeat mitral valve treatment (surgery or intervention) was needed in 19 patients (15.1%). Long-term clinical improvement was demonstrated with 69% of patients being in NYHA class ≤II. In a multivariable Cox regression analysis, the post-procedural grade of MR (hazard ratio [HR] 1.55 per grade, P = 0.035), the left ventricular ejection fraction (HR 0.58 for difference between 75th and 25th percentile, P = 0.031) and the glomerular filtration rate (HR 0.33 for 75th vs 25th percentile, P < 0.001) were independent predictors for long-term mortality. Patients with primary MR and a post-procedural MR grade ≤1 had the most favorable long-term outcome. CONCLUSIONS: This study determines predictors of long-term clinical outcome after TMVR and demonstrates that the grade of residual MR determines long-term survival. Our data suggest that it might be of benefit reducing residual MR to the lowest possible MR grade using TMVR-especially in selected high-risk patients with primary MR who are not considered as candidates for surgical MVR.
Assuntos
Cateterismo Cardíaco , Implante de Prótese de Valva Cardíaca , Efeitos Adversos de Longa Duração , Insuficiência da Valva Mitral/cirurgia , Valva Mitral/cirurgia , Idoso , Cateterismo Cardíaco/métodos , Cateterismo Cardíaco/estatística & dados numéricos , Feminino , Alemanha/epidemiologia , Implante de Prótese de Valva Cardíaca/efeitos adversos , Implante de Prótese de Valva Cardíaca/métodos , Implante de Prótese de Valva Cardíaca/mortalidade , Humanos , Efeitos Adversos de Longa Duração/mortalidade , Efeitos Adversos de Longa Duração/cirurgia , Masculino , Pessoa de Meia-Idade , Insuficiência da Valva Mitral/diagnóstico , Insuficiência da Valva Mitral/fisiopatologia , Modelos de Riscos Proporcionais , Risco Ajustado/métodos , Fatores de Risco , Índice de Gravidade de Doença , Resultado do Tratamento , Função Ventricular EsquerdaRESUMO
To determine whether leukocytes need to open endothelial cell contacts during extravasation, we decided to generate mice with strongly stabilized endothelial junctions. To this end, we replaced VE-cadherin genetically by a VE-cadherin-α-catenin fusion construct. Such mice were completely resistant to the induction of vascular leaks by VEGF or histamine. Neutrophil or lymphocyte recruitment into inflamed cremaster, lung and skin were strongly inhibited in these mice, documenting the importance of the junctional route in vivo. Surprisingly, lymphocyte homing into lymph nodes was not inhibited. VE-cadherin-α-catenin associated more intensely with the actin cytoskeleton as demonstrated by its membrane mobility and detergent extractability. Our results establish the junctional route as the main pathway for extravasating leukocytes in several, although not in all tissues. Furthermore, in these tissues, plasticity of the VE-cadherin-catenin complex is central for the leukocyte diapedesis mechanism.
Assuntos
Antígenos CD/metabolismo , Caderinas/metabolismo , Permeabilidade Capilar , Leucócitos/fisiologia , Migração Transendotelial e Transepitelial , alfa Catenina/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Antígenos CD/genética , Caderinas/genética , Células Cultivadas , Endotélio Vascular/metabolismo , Feminino , Técnicas de Introdução de Genes , Inflamação/genética , Inflamação/metabolismo , Junções Intercelulares/metabolismo , Leucócitos/metabolismo , Pulmão/metabolismo , Linfonodos/metabolismo , Masculino , Camundongos , Músculo Esquelético/metabolismo , Pele/metabolismo , alfa Catenina/genéticaRESUMO
In vitro studies suggest that leukocytes locomote in an ameboid fashion independently of pericellular proteolysis. Whether this motility pattern applies for leukocyte migration in inflamed tissue is still unknown. In vivo microscopy on the inflamed mouse cremaster muscle revealed that blockade of serine proteases or of matrix metalloproteinases (MMPs) significantly reduces intravascular accumulation and transmigration of neutrophils. Using a novel in vivo chemotaxis assay, perivenular microinjection of inflammatory mediators induced directional interstitial migration of neutrophils. Blockade of actin polymerization, but not of actomyosin contraction abolished neutrophil interstitial locomotion. Multiphoton laser scanning in vivo microscopy showed that the density of the interstitial collagen network increases in inflamed tissue, thereby providing physical guidance to infiltrating neutrophils. Although neutrophils locomote through the interstitium without pericellular collagen degradation, inhibition of MMPs, but not of serine proteases, diminished their polarization and interstitial locomotion. In this context, blockade of MMPs was found to modulate expression of adhesion/signaling molecules on neutrophils. Collectively, our data indicate that serine proteases are critical for neutrophil extravasation, whereas these enzymes are dispensable for neutrophil extravascular locomotion. By contrast, neutrophil interstitial migration strictly relies on actin polymerization and does not require the pericellular degradation of collagen fibers but is modulated by MMPs.
Assuntos
Quimiotaxia de Leucócito/fisiologia , Inflamação/imunologia , Metaloproteinases da Matriz/fisiologia , Infiltração de Neutrófilos/fisiologia , Aminocaproatos/farmacologia , Animais , Aprotinina/farmacologia , Quimiotaxia de Leucócito/efeitos dos fármacos , Doenças do Sistema Imunitário/metabolismo , Doenças do Sistema Imunitário/patologia , Inflamação/metabolismo , Transtornos Leucocíticos/metabolismo , Transtornos Leucocíticos/patologia , Masculino , Metaloproteinases da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Infiltração de Neutrófilos/efeitos dos fármacos , Infiltração de Neutrófilos/imunologia , Peritonite/imunologia , Peritonite/patologia , Ácido Tranexâmico/farmacologia , Migração Transcelular de Célula/efeitos dos fármacos , Migração Transcelular de Célula/imunologiaRESUMO
Endothelial cell-selective adhesion molecule (ESAM) is specifically expressed at endothelial tight junctions and on platelets. To test whether ESAM is involved in leukocyte extravasation, we have generated mice carrying a disrupted ESAM gene and analyzed them in three different inflammation models. We found that recruitment of lymphocytes into inflamed skin was unaffected by the gene disruption. However, the migration of neutrophils into chemically inflamed peritoneum was inhibited by 70% at 2 h after stimulation, recovering at later time points. Analyzing neutrophil extravasation directly by intravital microscopy in the cremaster muscle revealed that leukocyte extravasation was reduced (50%) in ESAM(-/-) mice without affecting leukocyte rolling and adhesion. Depletion of >98% of circulating platelets did not abolish the ESAM deficiency-related inhibitory effect on neutrophil extravasation, indicating that it is only ESAM at endothelial tight junctions that is relevant for the extravasation process. Knocking down ESAM expression in endothelial cells resulted in reduced levels of activated Rho, a GTPase implicated in the destabilization of tight junctions. Indeed, vascular permeability stimulated by vascular endothelial growth factor was reduced in ESAM(-/-) mice. Collectively, ESAM at endothelial tight junctions participates in the migration of neutrophils through the vessel wall, possibly by influencing endothelial cell contacts.
Assuntos
Permeabilidade Capilar/imunologia , Moléculas de Adesão Celular/fisiologia , Movimento Celular/imunologia , Neutrófilos/patologia , Fator A de Crescimento do Endotélio Vascular/fisiologia , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Permeabilidade Capilar/genética , Moléculas de Adesão Celular/deficiência , Moléculas de Adesão Celular/genética , Comunicação Celular/genética , Comunicação Celular/imunologia , Movimento Celular/genética , Ativação Enzimática/genética , Ativação Enzimática/imunologia , Feminino , Masculino , Camundongos , Camundongos Knockout , Neutrófilos/imunologiaRESUMO
von Willebrand factor (VWF) is an important player in hemostasis but has also been suggested to promote inflammatory processes. Gene ablation of VWF causes a simultaneous defect in P-selectin expression making it difficult to identify VWF-specific functions. Therefore, we analyzed whether blocking antibodies against VWF would be able to interfere with neutrophil extravasation. We found that these antibodies inhibited neutrophil recruitment into thioglycollate-inflamed peritoneum and KC-stimulated cremaster by approximately 50%. Whereas platelet-VWF was not involved, the contribution of VWF to granulocyte recruitment was strictly dependent on the presence of platelets and the accessibility of their VWF-receptor glycoprotein Ib. Surprisingly, platelet P-selectin was largely dispensable for leukocyte extravasation, in agreement with our observation that anti-VWF antibodies did not affect leukocyte rolling and adhesion. Searching for possible effects downstream of leukocyte capture, we found that anti-VWF antibodies significantly inhibited thioglycollate-induced vascular permeability. The increase of permeability was independent of circulating granulocytes, showing that it was not a side effect of neutrophil diapedesis. Collectively, our results demonstrate that VWF-associated platelets strongly support neutrophil extravasation at a step downstream of leukocyte docking to the vessel wall. This step could be related to leukocyte diapedesis facilitated by destabilization of the endothelial barrier.
Assuntos
Movimento Celular , Leucócitos/citologia , Leucócitos/imunologia , Peritonite/imunologia , Fator de von Willebrand/imunologia , Animais , Anticorpos/imunologia , Plaquetas/imunologia , Permeabilidade Capilar , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/citologia , Neutrófilos/imunologia , Selectina-P/imunologia , Peritônio/imunologia , Complexo Glicoproteico GPIb-IX de Plaquetas/imunologiaRESUMO
Platelets play a key role in hemostasis and various diseases including arterial thrombosis. Glycoprotein VI (GPVI) mediates adhesion to collagen structures exposed at sites of vascular injury and subsequent platelet activation. We determined the effects of specific activation of GPVI on the human platelet proteome. Isolated human platelets were stimulated with an activating monoclonal antibody specific for GPVI. Platelet proteins were analyzed by 2-dimensional difference gel electrophoresis (2D-DIGE) and mass spectrometry. We identified 8 differentially abundant proteins associated with cell signaling, metabolism, organization and rearrangement of the cytoskeleton, and membrane trafficking. Differentially abundant proteins included aldose reductase (AR), beta-centractin, charged multivesicular body protein 3, Src substrate cortactin, ERp57, and pleckstrin. Importantly, GPVI-modulated protein abundance was functionally relevant. Correspondingly, AR enzyme activity significantly increased upon GPVI activation and inhibition of AR resulted in reduced platelet aggregation. Furthermore, ERp57 was released upon ligation of platelet GPVI and increased the activity of tissue factor, a major initiator of blood coagulation. In summary, GPVI activation results in differential changes in abundance of platelet proteins, including AR and ERp57, which support platelet aggregation and platelet-dependent coagulation. These results provide further insight into the mechanisms that underlie platelet activation through the GPVI receptor and may help to identify novel pharmacologic targets.
Assuntos
Biomarcadores/metabolismo , Plaquetas/metabolismo , Agregação Plaquetária , Glicoproteínas da Membrana de Plaquetas/metabolismo , Proteoma/análise , Trombose/metabolismo , Adulto , Anticorpos Monoclonais , Coagulação Sanguínea , Western Blotting , Eletroforese em Gel Bidimensional , Feminino , Citometria de Fluxo , Humanos , Masculino , Ativação Plaquetária , Transdução de Sinais , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Adulto JovemRESUMO
Leukocyte extravasation depends on various adhesion receptors at endothelial cell contacts. Here we have analyzed how mouse CD99 and CD99L2 cooperate with PECAM-1. We found that antibodies against mouse CD99 and PECAM-1 trap neutrophils between endothelial cells in in vitro transmigration assays. A sequential function, as has been suggested for human PECAM-1 and CD99, could not be demonstrated. In contrast to these in vitro results, blocking CD99 or CD99L2 or gene disruption of PECAM-1 trapped neutrophils in vivo between endothelial cells and the underlying basement membrane as revealed by electron microscopy and by 3-dimensional confocal fluorescence microscopy in the inflamed cremaster tissue. Leukocyte extravasation was inhibited in interleukin-1beta-inflamed peritoneum and in the cremaster by PECAM-1 gene disruption and was further attenuated by blocking antibodies against CD99 and CD99L2. In addition, CD99 and CD99L2 were required for leukocyte extravasation in the cremaster after stimulation with tumor necrosis factor-alpha, where the need for PECAM-1 is known to be bypassed. We conclude that CD99 and CD99L2 act independently of PECAM-1 in leukocyte extravasation and cooperate in an independent way to help neutrophils overcome the endothelial basement membrane.
Assuntos
Antígenos CD/fisiologia , Endotélio Vascular/metabolismo , Leucócitos/imunologia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/fisiologia , Antígeno 12E7 , Animais , Membrana Basal/imunologia , Membrana Basal/metabolismo , Adesão Celular , Movimento Celular , Células Cultivadas , Endotélio Vascular/citologia , Endotélio Vascular/imunologia , Imunofluorescência , Humanos , Inflamação , Leucócitos/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/imunologia , Neutrófilos/metabolismo , Peritônio/imunologiaRESUMO
OBJECTIVE: The cyclin-dependent kinase (CDK) inhibitor flavopiridol is currently being tested in clinical trials as anticancer drug. Beyond its cell death-inducing action, we hypothesized that flavopiridol affects inflammatory processes. Therefore, we elucidated the action of flavopiridol on leukocyte-endothelial cell interaction and endothelial activation in vivo and in vitro and studied the underlying molecular mechanisms. METHODS AND RESULTS: Flavopiridol suppressed concanavalin A-induced hepatitis and neutrophil infiltration into liver tissue. Flavopiridol also inhibited tumor necrosis factor-α-induced leukocyte-endothelial cell interaction in the mouse cremaster muscle. Endothelial cells were found to be the major target of flavopiridol, which blocked the expression of endothelial cell adhesion molecules (intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin), as well as NF-κB-dependent transcription. Flavopiridol did not affect inhibitor of κB (IκB) kinase, the degradation and phosphorylation of IκBα, nuclear translocation of p65, or nuclear factor-κB (NF-κB) DNA-binding activity. By performing a cellular kinome array and a kinase activity panel, we found LIM domain kinase-1 (LIMK1), casein kinase 2, c-Jun N-terminal kinase (JNK), protein kinase C (PKC), CDK4, CDK6, CDK8, and CDK9 to be influenced by flavopiridol. Using specific inhibitors, as well as RNA interference (RNAi), we revealed that only CDK9 is responsible for the action of flavopiridol. CONCLUSIONS: Our study highlights flavopiridol as a promising antiinflammatory compound and inhibition of CDK9 as a novel approach for the treatment of inflammation-associated diseases.
Assuntos
Comunicação Celular/fisiologia , Quinase 9 Dependente de Ciclina/antagonistas & inibidores , Endotélio Vascular/citologia , Flavonoides/uso terapêutico , Inflamação/prevenção & controle , Leucócitos/citologia , Piperidinas/uso terapêutico , Inibidores de Proteínas Quinases/uso terapêutico , Animais , Adesão Celular/efeitos dos fármacos , Comunicação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/fisiopatologia , Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Concanavalina A/efeitos adversos , Quinase 9 Dependente de Ciclina/metabolismo , Modelos Animais de Doenças , Selectina E/metabolismo , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Flavonoides/farmacologia , Humanos , Inflamação/metabolismo , Inflamação/fisiopatologia , Molécula 1 de Adesão Intercelular/metabolismo , Leucócitos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Piperidinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
Ischemia-reperfusion (IR) triggers tissue injury by activating innate immunity, for example, via TLR2 and TLR4. Surprisingly, TLR signaling in intrinsic renal cells predominates in comparison to intrarenal myeloid cells in the postischemic kidney. We hypothesized that immune cell activation is specifically suppressed in the postischemic kidney, for example, by single Ig IL-1-related receptor (SIGIRR). SIGIRR deficiency aggravated postischemic acute renal failure in association with increased renal CXCL2/MIP2, CCL2/MCP-1, and IL-6 mRNA expression 24 h after IR. Consistent with this finding interstitial neutrophil and macrophage counts were increased and tubular cell necrosis was aggravated in Sigirr-deficient vs wild-type IR kidneys. In vivo microscopy revealed increased leukocyte transmigration in the postischemic microvasculature of Sigirr-deficient mice. IL-6 and CXCL2/MIP2 release was much higher in Sigirr-deficient renal myeloid cells but not in Sigirr-deficient tubular epithelial cells after transient hypoxic culture conditions. Renal IR studies with chimeric mice confirmed this finding, as lack of SIGIRR in myeloid cells largely reproduced the phenotype of renal IR injury seen in Sigirr(-/-) mice. Additionally, clodronate depletion of dendritic cells prevented the aggravated renal failure in Sigirr(-/-) mice. Thus, loss of function mutations in the SIGIRR gene predispose to acute renal failure because SIGIRR prevents overshooting tissue injury by suppressing the postischemic activation of intrarenal myeloid cells.
Assuntos
Injúria Renal Aguda/etiologia , Células Dendríticas/fisiologia , Receptores de Interleucina-1/fisiologia , Traumatismo por Reperfusão/complicações , Animais , Contagem de Células , Quimiotaxia de Leucócito , Células Epiteliais , Túbulos Renais/patologia , Macrófagos/fisiologia , Camundongos , Camundongos Knockout , Microcirculação , Células Mieloides , Neutrófilos/fisiologia , Receptores de Interleucina-1/deficiência , Receptores de Interleucina-1/genética , Traumatismo por Reperfusão/imunologiaRESUMO
OBJECTIVE: Although the chemokines monocyte chemoattractant protein-1 (Ccl2/JE/MCP-1) and macrophage inflammatory protein-1alpha (Ccl3/MIP-1alpha) have recently been implicated in neutrophil migration, the underlying mechanisms remain largely unclear. METHODS AND RESULTS: Stimulation of the mouse cremaster muscle with Ccl2/JE/MCP-1 or Ccl3/MIP-1alpha induced a significant increase in numbers of firmly adherent and transmigrated leukocytes (>70% neutrophils) as observed by in vivo microscopy. This increase was significantly attenuated in mice receiving an inhibitor of RNA transcription (actinomycin D) or antagonists of platelet activating factor (PAF; BN 52021) and leukotrienes (MK-886; AA-861). In contrast, leukocyte responses elicited by PAF and leukotriene-B(4) (LTB(4)) themselves were not affected by actinomycin D, BN 52021, MK-886, or AA-861. Conversely, PAF and LTB(4), but not Ccl2/JE/MCP-1 and Ccl3/MIP-1alpha, directly activated neutrophils as indicated by shedding of CD62L and marked upregulation of CD11b. Moreover, Ccl2/JE/MCP-1- and Ccl3/MIP-1alpha-elicited leakage of fluorescein isothiocyanate dextran as well as collagen IV remodeling within the venular basement membrane were completely absent in neutrophil-depleted mice. CONCLUSIONS: Ccl2/JE/MCP-1 and Ccl3/MIP-1alpha mediate firm adherence and (subsequent) transmigration of neutrophils via protein synthesis and secondary generation of leukotrienes and PAF, which in turn directly activate neutrophils. Thereby, neutrophils facilitate basement membrane remodeling and promote microvascular leakage.
Assuntos
Quimiocina CCL2/metabolismo , Quimiocina CCL3/metabolismo , Inibidores de Lipoxigenase/farmacologia , Infiltração de Neutrófilos/fisiologia , Biossíntese de Proteínas/efeitos dos fármacos , 1-Alquil-2-acetilglicerofosfocolina Esterase/farmacologia , Animais , Benzoquinonas/farmacologia , Células Cultivadas , Quimiocina CCL2/farmacologia , Quimiocina CCL3/farmacologia , Quimiotaxia de Leucócito/efeitos dos fármacos , Quimiotaxia de Leucócito/fisiologia , Dactinomicina/farmacologia , Modelos Animais de Doenças , Indóis/farmacologia , Indometacina/farmacologia , Leucotrieno B4/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Infiltração de Neutrófilos/efeitos dos fármacos , Distribuição Aleatória , Sensibilidade e EspecificidadeRESUMO
BACKGROUND/AIMS: This study was designed to investigate the role of endothelial cell-selective adhesion molecule (ESAM), a recently discovered receptor expressed in endothelial tight junctions and platelets, for leukocyte migration in inflamed liver. METHODS: The role of ESAM for leukocyte migration in the liver was analyzed using ESAM-deficient mice in a model of warm hepatic ischemia-reperfusion (90min/30-360min). RESULTS: As shown by immunostaining, ESAM is expressed in sinusoids as well as in venules and is not upregulated upon I/R. Emigrated leukocytes were quantified in tissue sections. Postischemic neutrophil transmigration was significantly attenuated in ESAM-/- mice after 2h of reperfusion, whereas it was completely restored after 6h. In contrast, T-cell migration did not differ between ESAM+/+ and ESAM-/- mice. Using intravital microscopy, we demonstrate that ESAM deficiency attenuates I/R-induced vascular leakage after 30min of reperfusion. The I/R-induced elevation in AST/ALT activity, the sinusoidal perfusion failure, and the number of TUNEL-positive hepatocytes were comparable between ESAM+/+ and ESAM-/- mice. CONCLUSIONS: ESAM is expressed in the postischemic liver and mediates neutrophil but not T-cell transmigration during early reperfusion. ESAM deficiency attenuates I/R-induced vascular leakage and does not affect leukocyte adherence. Despite the effect on neutrophil migration, ESAM-deficiency does not protect from I/R-induced injury.
Assuntos
Moléculas de Adesão Celular/fisiologia , Endotélio Vascular/fisiopatologia , Inflamação/sangue , Contagem de Leucócitos , Circulação Hepática/fisiologia , Hepatopatias/sangue , Animais , Moléculas de Adesão Celular/deficiência , Moléculas de Adesão Celular/genética , Cruzamentos Genéticos , Feminino , Granulócitos/enzimologia , Inflamação/fisiopatologia , Antígenos Comuns de Leucócito/análise , Fígado/fisiopatologia , Hepatopatias/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microcirculação/fisiologia , Naftol AS D Esterase/sangueRESUMO
Endothelial barrier dysfunction is a hallmark of many severe pathologies, including sepsis or atherosclerosis. The cardiovascular hormone atrial natriuretic peptide (ANP) has increasingly been suggested to counteract endothelial leakage. Surprisingly, the precise in vivo relevance of these observations has never been evaluated. Thus, we aimed to clarify this issue and, moreover, to identify the permeability-controlling subcellular systems that are targeted by ANP. Histamine was used as important pro-inflammatory, permeability-increasing stimulus. Measurements of fluorescein isothiocyanate (FITC)-dextran extravasation from venules of the mouse cremaster muscle and rat hematocrit values were performed to judge changes of endothelial permeability in vivo. It is noteworthy that ANP strongly reduced the histamine-evoked endothelial barrier dysfunction in vivo. In vitro, ANP blocked the breakdown of transendothelial electrical resistance (TEER) induced by histamine. Moreover, as judged by immunocytochemistry and Western blot analysis, ANP inhibited changes of vascular endothelial (VE)-cadherin, beta-catenin, and p120(ctn) morphology; VE-cadherin and myosin light chain 2 (MLC2) phosphorylation; and F-actin stress fiber formation. These changes seem to be predominantly mediated by the natriuretic peptide receptor (NPR)-A, but not by NPR-C. In summary, we revealed ANP as a potent endothelial barrier protecting agent in vivo and identified adherens junctions and the contractile apparatus as subcellular systems targeted by ANP. Thus, our study highlights ANP as an interesting pharmacological compound opening new therapeutic options for preventing endothelial leakage.
Assuntos
Fator Natriurético Atrial/farmacologia , Permeabilidade Capilar/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Histamina/farmacologia , Animais , Células Cultivadas , Impedância Elétrica , Células Endoteliais/metabolismo , Endotélio Vascular/citologia , Fluoresceína-5-Isotiocianato/metabolismo , Corantes Fluorescentes/metabolismo , Hematócrito , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Ratos Sprague-Dawley , Veias Umbilicais/citologiaRESUMO
BACKGROUND: The aim of this study was to establish and validate a practical method to disperse nanoparticles in physiological solutions for biological in vitro and in vivo studies. RESULTS: TiO2 (rutile) dispersions were prepared in distilled water, PBS, or RPMI 1640 cell culture medium. Different ultrasound energies, various dispersion stabilizers (human, bovine, and mouse serum albumin, Tween 80, and mouse serum), various concentrations of stabilizers, and different sequences of preparation steps were applied. The size distribution of dispersed nanoparticles was analyzed by dynamic light scattering and zeta potential was measured using phase analysis light scattering. Nanoparticle size was also verified by transmission electron microscopy. A specific ultrasound energy of 4.2 x 105 kJ/m3 was sufficient to disaggregate TiO2 (rutile) nanoparticles, whereas higher energy input did not further improve size reduction. The optimal sequence was first to sonicate the nanoparticles in water, then to add dispersion stabilizers, and finally to add buffered salt solution to the dispersion. The formation of coarse TiO2 (rutile) agglomerates in PBS or RPMI was prevented by addition of 1.5 mg/ml of human, bovine or mouse serum albumin, or mouse serum. The required concentration of albumin to stabilize the nanoparticle dispersion depended on the concentration of the nanoparticles in the dispersion. TiO2 (rutile) particle dispersions at a concentration lower than 0.2 mg/ml could be stabilized by the addition of 1.5 mg/ml albumin. TiO2 (rutile) particle dispersions prepared by this method were stable for up to at least 1 week. This method was suitable for preparing dispersions without coarse agglomerates (average diameter < 290 nm) from nanosized TiO2 (rutile), ZnO, Ag, SiOx, SWNT, MWNT, and diesel SRM2975 particulate matter. CONCLUSION: The optimized dispersion method presented here appears to be effective and practicable for preparing dispersions of nanoparticles in physiological solutions without creating coarse agglomerates.
RESUMO
BACKGROUND: The Synergy between Percutaneous Coronary Intervention with TAXUS and Cardiac Surgery (SYNTAX)-score is a validated tool for risk stratification and revascularization strategy selection in patients with complex coronary artery disease. The aim of this study was to analyse its age-related prognostic value. METHODS: SYNTAX-score was calculated in 1331 all-comer patients undergoing percutaneous coronary intervention (PCI): 463 patients ≥ 75 years and 868 patients < 75 years. Outcomes of interest were all-cause mortality at one and two years. RESULTS: A significant interaction of age and SYNTAX-score for mortality was observed at two-year (P interaction = 0.019) but not at one-year follow-up (P interaction = 0.594). In multivariable analysis, SYNTAX-score independently predicted 1-year mortality in both age groups (< 75 years, hazard ratio (HR): 1.43, 95% confidence intervals (CI): 1.03-2.00, P = 0.034; and ≥ 75 years, HR: 1.37, 95% CI: 1.01-1.85, P = 0.042), but only two-year mortality among younger patients (< 75 years, HR: 1.33, 95% CI: 1.01-1.76, P = 0.041; and ≥ 75 years, HR: 1.11, 95% CI: 0.87-1.41, P = 0.394). SYNTAX-score tertiles were useful to stratify 1-year mortality in both, patients < 75 years (SYNTAX-score < 9, 3.8%; 9-20, 5.3%; ≥ 20, 10.3%; P = 0.004) and ≥ 75 years (SYNTAX-score < 11, 5.7%; 11-22.5, 16.1%; ≥ 22.5, 18.7%; P = 0.003), but two-year mortality only among patients < 75 years (SYNTAX-score < 9, 6.5%; 9-20, 7.6%; ≥ 20, 15%; P < 0.001) and not among ≥ 75 years old patients (SYNTAX-score < 11, 19.4%; 11-22.5, 26.3%; ≥ 22.5, 27.9%; P = 0.138). CONCLUSIONS: Age modifies the impact of the SYNTAX-score on longer-term mortality after PCI. Among patients < 75 years, the SYNTAX-score independently predicts the risk of death at one and two years after PCI, while among patients ≥ 75 years its predictive role is limited to the first year after PCI. Further studies are needed to evaluate the value of SYNTAX-score for selecting the most appropriate revascularization strategy among elderly patients.
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Matrix metalloproteinases-2 and -9 (MMP-2/9) are critically involved in degradation of extracellular matrix, and their inhibition is discussed as a promising strategy against hepatic ischemia-reperfusion (I/R) injury. Here, we analyzed the role of MMP-2 and -9 for leukocyte migration and tissue injury in sham-operated mice and in mice after I/R, treated with a MMP-2/9 inhibitor or vehicle. Using zymography, we show that the MMP-2/9 inhibitor abolished I/R-induced MMP-9 activation, whereas MMP-2 activity was not detectable in all groups. As demonstrated by intravital microscopy, MMP-9 inhibition attenuated postischemic rolling and adherence of total leukocytes in hepatic postsinusoidal venules, CD4+ T cell accumulation in sinusoids, and neutrophil transmigration. These effects were associated with reduction of plasma tumor necrosis factor alpha (TNF-alpha) levels and endothelial expression of CD62P. Motility of interstitially migrating leukocytes was assessed by near-infrared reflected light oblique transillumination microscopy in the postischemic cremaster muscle. Upon MMP-9 blockade, leukocyte migration velocity and curve-line and straight-line migration distances were reduced significantly as compared with the vehicle-treated I/R group. Postischemic sinusoidal perfusion failure, hepatocellular apoptosis, and alanine aminotransferase activity were only slightly reduced after MMP-9 inhibition, whereas aspartate aminotransferase activity and mortality were significantly lower. In conclusion, MMP-9 is involved in the early recruitment cascades of neutrophils and CD4+ T cells, promotes neutrophil and T cell transmigration during hepatic I/R, and is required for motility of interstitially migrating leukocytes. MMP-9 blockade is associated with an attenuation of TNF-alpha release and endothelial CD62P expression, weakly protects from early microvascular/hepatocellular I/R damage, but improves postischemic survival.
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Linfócitos T CD4-Positivos/fisiologia , Quimiotaxia de Leucócito/fisiologia , Isquemia/fisiopatologia , Fígado/irrigação sanguínea , Metaloproteinase 9 da Matriz/fisiologia , Neutrófilos/fisiologia , Traumatismo por Reperfusão/fisiopatologia , Alanina Transaminase/sangue , Animais , Antígenos Ly/análise , Apoptose , Aspartato Aminotransferases/sangue , Adesão Celular/efeitos dos fármacos , Quimiotaxia de Leucócito/efeitos dos fármacos , Dipeptídeos/farmacologia , Endotélio Vascular/patologia , Ativação Enzimática , Feminino , Fator de Crescimento de Hepatócito/biossíntese , Fator de Crescimento de Hepatócito/genética , Antígenos Comuns de Leucócito/análise , Fígado/metabolismo , Fígado/patologia , Metaloproteinase 2 da Matriz/fisiologia , Inibidores de Metaloproteinases de Matriz , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/patologia , Selectina-P/metabolismo , Peptídeos Cíclicos/farmacologia , Inibidores de Proteases/farmacologia , Transporte Proteico/efeitos dos fármacos , RNA Mensageiro/biossíntese , Fator de Necrose Tumoral alfa/análise , VênulasRESUMO
BACKGROUND: Clinical relevant cerebrovascular events (CVE) following transcatheter aortic valve implantation (TAVI) still remain a devastating complication associated with mortality and severe impairments. Therefore, identification of particularly modifiable predictors of this complication is clinically relevant and an important step for planning preventive strategies. METHODS: A total of 985 patients who underwent trans-femoral TAVI for aortic valve stenosis in our institution from February 2008 to January 2015 were considered. The influence of demographics, clinical and procedural data on the occurrence of CVE was assessed with a competing risk model with death as competing event. Clinical events were defined according to VARC-2 criteria. RESULTS: At a median follow-up of 838days, 95% CI 807-892, 59 patients experienced any CVE (5.9%) and the overall cumulative mortality rate was 46.1%. CVEs mainly occur later than 30days after TAVI (47.5%), 88.1% of them were of ischemic origin and 52.5% were disabling events. Independent predictors of CVEs were age (hazard ratio 1.05; 95% CI 1.01 to 1.09), history of CVE (hazard ratio 2.54; 95% CI 1.39 to 4.63) and use of balloon post-dilation (hazard ratio 1.85; 95% CI 1.08 to 3.18). CONCLUSION: In patients undergoing TAVI incidence of clinically relevant CVEs is frequent with half of the events occurring after the first 30days post-TAVI. Identification of balloon post-dilation as the only modifiable predictor of CVE risk at mid-term, urges its cautious performance after prosthesis implantation. CLINICALTRIALS. GOV IDENTIFIER: NCT02289339.
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Transtornos Cerebrovasculares/diagnóstico , Transtornos Cerebrovasculares/etiologia , Complicações Pós-Operatórias/diagnóstico , Complicações Pós-Operatórias/etiologia , Sistema de Registros , Substituição da Valva Aórtica Transcateter/efeitos adversos , Idoso , Idoso de 80 Anos ou mais , Estenose da Valva Aórtica/diagnóstico , Estenose da Valva Aórtica/cirurgia , Feminino , Seguimentos , Humanos , Masculino , Valor Preditivo dos Testes , Substituição da Valva Aórtica Transcateter/tendênciasRESUMO
Deep vein thrombosis (DVT) is a major cause of cardiovascular death. The sequence of events that promote DVT remains obscure, largely as a result of the lack of an appropriate rodent model. We describe a novel mouse model of DVT which reproduces a frequent trigger and resembles the time course, histological features, and clinical presentation of DVT in humans. We demonstrate by intravital two-photon and epifluorescence microscopy that blood monocytes and neutrophils crawling along and adhering to the venous endothelium provide the initiating stimulus for DVT development. Using conditional mutants and bone marrow chimeras, we show that intravascular activation of the extrinsic pathway of coagulation via tissue factor (TF) derived from myeloid leukocytes causes the extensive intraluminal fibrin formation characteristic of DVT. We demonstrate that thrombus-resident neutrophils are indispensable for subsequent DVT propagation by binding factor XII (FXII) and by supporting its activation through the release of neutrophil extracellular traps (NETs). Correspondingly, neutropenia, genetic ablation of FXII, or disintegration of NETs each confers protection against DVT amplification. Platelets associate with innate immune cells via glycoprotein Ibα and contribute to DVT progression by promoting leukocyte recruitment and stimulating neutrophil-dependent coagulation. Hence, we identified a cross talk between monocytes, neutrophils, and platelets responsible for the initiation and amplification of DVT and for inducing its unique clinical features.