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1.
Sci Rep ; 9(1): 15932, 2019 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-31685838

RESUMO

In advanced inflammatory disease, microvascular thrombosis leads to the interruption of blood supply and provokes ischemic tissue injury. Recently, intravascularly adherent leukocytes have been reported to shape the blood flow in their immediate vascular environment. Whether these rheological effects are relevant for microvascular thrombogenesis remains elusive. Employing multi-channel in vivo microscopy, analyses in microfluidic devices, and computational modeling, we identified a previously unanticipated role of leukocytes for microvascular clot formation in inflamed tissue. For this purpose, neutrophils adhere at distinct sites in the microvasculature where these immune cells effectively promote thrombosis by shaping the rheological environment for platelet aggregation. In contrast to larger (lower-shear) vessels, this process in high-shear microvessels does not require fibrin generation or extracellular trap formation, but involves GPIbα-vWF and CD40-CD40L-dependent platelet interactions. Conversely, interference with these cellular interactions substantially compromises microvascular clotting. Thus, leukocytes shape the rheological environment in the inflamed venular microvasculature for platelet aggregation thereby effectively promoting the formation of blood clots. Targeting this specific crosstalk between the immune system and the hemostatic system might be instrumental for the prevention and treatment of microvascular thromboembolic pathologies, which are inaccessible to invasive revascularization strategies.

3.
Sci Adv ; 5(6): eaav4275, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31223646

RESUMO

Carcinoma cells undergo epithelial-mesenchymal transition (EMT); however, contributions of EMT heterogeneity to disease progression remain a matter of debate. Here, we addressed the EMT status of ex vivo cultured circulating and disseminated tumor cells (CTCs/DTCs) in a syngeneic mouse model of metastatic breast cancer (MBC). Epithelial-type CTCs with a restricted mesenchymal transition had the strongest lung metastases formation ability, whereas mesenchymal-type CTCs showed limited metastatic ability. EpCAM expression served as a surrogate marker to evaluate the EMT heterogeneity of clinical samples from MBC, including metastases, CTCs, and DTCs. The proportion of epithelial-type CTCs, and especially DTCs, correlated with distant metastases and poorer outcome of patients with MBC. This study fosters our understanding of EMT in metastasis and underpins heterogeneous EMT phenotypes as important parameters for tumor prognosis and treatment. We further suggest that EpCAM-dependent CTC isolation systems will underestimate CTC numbers but will quantify clinically relevant metastatic cells.

4.
Acta Biomater ; 82: 24-33, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30296618

RESUMO

Rapid implant vascularization is a prerequisite for successful biomaterial engraftment. Vitronectin (VN) is a matricellular glycoprotein well known for its capability to interact with growth factors, proteases, and protease inhibitors/receptors. Since such proteins are highly relevant for angiogenic processes, we hypothesized that VN contributes to the tissue integration of biomaterials. Employing different in vivo and ex vivo microscopy techniques, engraftment of porous polyethylene (PPE) implants was analyzed in the dorsal skinfold chamber model in wild-type (WT) and VN-/- mice. Upon PPE implantation, vascularization of this biomaterial was severely compromised in animals lacking this matricellular protein. Proteome profiling revealed that VN deficiency does not cause major changes in angiogenic protein composition in the implants suggesting that VN promotes PPE vascularization via mechanisms modulating the activity of angiogenic factors rather than by directly enriching them in the implant. Consequently, surface coating with recombinant VN (embedded in Matrigel®) accelerated implant vascularization in WT mice by enhancing the maturation of a vascular network. Thus, VN contributes to the engraftment of PPE implants by promoting the vascularization of this biomaterial. Surface coating with VN might provide a promising strategy to improve the vascularization of PPE implants without affecting the host's integrity. STATEMENT OF SIGNIFICANCE: Porous polyethylene (PPE) is a biomaterial frequently used in reconstructive surgery. The proper vascularization of PPE implants is a fundamental prerequisite for its successful engraftment in host tissue. Although the overall biocompatibility of PPE is good, there are less favorable application sites for its use in tissue reconstruction mostly characterized by low blood supply. Employing advanced in vivo microscopy methods and proteomic analyses in genetically engineered mice, we here describe a previously unrecognized function of vitronectin (VN) that enables this abundantly present glycoprotein to particularly promote the vascularization of PPE biomaterial. These properties of VN specifically facilitate the formation of a dense vessel network within the implant which relies on modulating the activity of angiogenic mediators rather than on the enrichment of these factors in the implant. Consequently, surface coating with this matricellular protein effectively accelerated and intensified implant vascularization which might be beneficial for its implementation at unfavorable sites for implantation without affecting the host's integrity.


Assuntos
Materiais Revestidos Biocompatíveis , Implantes Experimentais , Neovascularização Fisiológica/efeitos dos fármacos , Polietileno , Vitronectina , Animais , Materiais Revestidos Biocompatíveis/química , Materiais Revestidos Biocompatíveis/farmacologia , Camundongos , Camundongos Knockout , Polietileno/química , Polietileno/farmacologia , Vitronectina/química , Vitronectina/farmacologia
5.
Arterioscler Thromb Vasc Biol ; 38(4): 829-842, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29371242

RESUMO

OBJECTIVE: Ischemia-reperfusion (I/R) injury significantly contributes to organ dysfunction and failure after myocardial infarction, stroke, and transplantation. In addition to its established role in the fibrinolytic system, plasminogen activator inhibitor-1 has recently been implicated in the pathogenesis of I/R injury. The underlying mechanisms remain largely obscure. APPROACH AND RESULTS: Using different in vivo microscopy techniques as well as ex vivo analyses and in vitro assays, we identified that plasminogen activator inhibitor-1 rapidly accumulates on microvascular endothelial cells on I/R enabling this protease inhibitor to exhibit previously unrecognized functional properties by inducing an increase in the affinity of ß2 integrins in intravascularly rolling neutrophils. These events are mediated through low-density lipoprotein receptor-related protein-1 and mitogen-activated protein kinase-dependent signaling pathways that initiate intravascular adherence of these immune cells to the microvascular endothelium. Subsequent to this process, extravasating neutrophils disrupt endothelial junctions and promote the postischemic microvascular leakage. Conversely, deficiency of plasminogen activator inhibitor-1 effectively reversed leukocyte infiltration, microvascular dysfunction, and tissue injury on experimental I/R without exhibiting side effects on microvascular hemostasis. CONCLUSIONS: Our experimental data provide novel insights into the nonfibrinolytic properties of the fibrinolytic system and emphasize plasminogen activator inhibitor-1 as a promising target for the prevention and treatment of I/R injury.


Assuntos
Músculos Abdominais/irrigação sanguínea , Fígado/irrigação sanguínea , Microvasos/metabolismo , Infiltração de Neutrófilos , Neutrófilos/metabolismo , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Traumatismo por Reperfusão/metabolismo , Músculos Abdominais/metabolismo , Músculos Abdominais/patologia , Animais , Antígenos CD18/metabolismo , Permeabilidade Capilar , Linhagem Celular , Modelos Animais de Doenças , Humanos , Cinética , Migração e Rolagem de Leucócitos , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microvasos/patologia , Ativação de Neutrófilo , Neutrófilos/transplante , Inibidor 1 de Ativador de Plasminogênio/deficiência , Inibidor 1 de Ativador de Plasminogênio/genética , Conformação Proteica , Receptores de LDL/metabolismo , Traumatismo por Reperfusão/patologia , Transdução de Sinais , Proteínas Supressoras de Tumor/metabolismo
6.
Nanomedicine (Lond) ; 13(6): 623-638, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29334311

RESUMO

AIM: To optimize the design of nanoparticles for diagnosis or therapy of vascular diseases, it is mandatory to characterize the determinants of nano-bio interactions in vascular lesions. MATERIALS & METHODS: Using ex vivo and in vivo microscopy, we analyzed the interactive behavior of quantum dots with different surface functionalizations in atherosclerotic lesions of ApoE-deficient mice. RESULTS: We demonstrate that quantum dots with different surface functionalizations exhibit specific interactive behaviors with distinct molecular and cellular components of the injured vessel wall. Moreover, we show a role for fibrinogen in the regulation of the spatio-temporal interaction dynamics in atherosclerotic lesions. CONCLUSION: Our findings emphasize the relevance of surface chemistry-driven nano-bio interactions on the differential in vivo behavior of nanoparticles in diseased tissue.


Assuntos
Apolipoproteínas E/genética , Aterosclerose/diagnóstico , Nanopartículas/química , Placa Aterosclerótica/diagnóstico , Animais , Aterosclerose/genética , Aterosclerose/fisiopatologia , Modelos Animais de Doenças , Humanos , Camundongos , Nanopartículas/administração & dosagem , Placa Aterosclerótica/genética , Placa Aterosclerótica/fisiopatologia , Pontos Quânticos
7.
FASEB J ; 31(11): 4796-4808, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28720647

RESUMO

CD4+ T cells recruited to the liver play a key role in the pathogenesis of ischemia/reperfusion (I/R) injury. The mechanism of their activation during alloantigen-independent I/R is not completely understood. We hypothesized that liver-resident dendritic cells (DCs) interact with CD4+ T cells in the postischemic liver and that modulation of DCs or T-cell-DC interactions attenuates liver inflammation. In mice, warm hepatic I/R (90/120-240 min) was induced. Tolerogenic DCs were generated in situ by pretreatment of animals with the vitamin D analog paricalcitol. A mAb-CD44 was used for blockade of CD4+ T-cell-DC interactions. As shown by 2-photon in vivo microscopy as well as confocal microscopy, CD4+ T cells were closely colocalized with DCs in the postischemic liver. Pretreatment with paricalcitol attenuated I/R-induced maturation of DCs (flow cytometry), CD4+ T-cell recruitment into the liver (intravital microscopy), and hepatocellular/microvascular damage (intravital microscopy, alanine aminotransferase/aspartate aminotransferase, histology). However, interruption of T-cell-DC interaction increased proinflammatory DC maturation and even enhanced tissue damage. Simultaneous treatment with an anti-CD44mAb completely abolished the beneficial effect of paricalcitol on T-cell migration and tissue injury. Our study demonstrates for the first time that hepatic DCs interact with CD4+ T cells in the postischemic liver in vivo; modulation of DCs and/or generation of tolerogenic DCs attenuates intrahepatic CD4+ T-cell recruitment and reduces I/R injury; and interruption of CD44-dependent CD4+ T-cell-DC interactions enhances tissue injury by preventing the modulatory effect of hepatic DCs on T cells, especially type 1 T helper effector cells. Thus, hepatic DCs are strongly involved in the promotion of CD4+ T-cell-dependent postischemic liver inflammation.-Funken, D., Ishikawa-Ankerhold, H., Uhl, B., Lerchenberger, M., Rentsch, M., Mayr, D., Massberg, S., Werner, J., Khandoga, A. In situ targeting of dendritic cells sets tolerogenic environment and ameliorates CD4+ T-cell response in the postischemic liver.


Assuntos
Comunicação Celular/imunologia , Células Dendríticas/imunologia , Fígado/imunologia , Traumatismo por Reperfusão/imunologia , Células Th1/imunologia , Animais , Células Dendríticas/patologia , Feminino , Fígado/patologia , Camundongos , Traumatismo por Reperfusão/patologia , Células Th1/patologia
8.
ACS Nano ; 11(2): 1498-1508, 2017 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-28135073

RESUMO

Advances in the engineering of nanoparticles (NPs), which represent particles of less than 100 nm in one external dimension, led to an increasing utilization of nanomaterials for biomedical purposes. A prerequisite for their use in diagnostic and therapeutic applications, however, is the targeted delivery to the site of injury. Interactions between blood-borne NPs and the vascular endothelium represent a critical step for nanoparticle delivery into diseased tissue. Here, we show that the endothelial glycocalyx, which constitutes a glycoprotein-polysaccharide meshwork coating the luminal surface of vessels, effectively controls interactions of carboxyl-functionalized quantum dots with the microvascular endothelium. Glycosaminoglycans of the endothelial glycocalyx were found to physically cover endothelial adhesion and signaling molecules, thereby preventing endothelial attachment, uptake, and translocation of these nanoparticles through different layers of the vessel wall. Conversely, degradation of the endothelial glycocalyx promoted interactions of these nanoparticles with microvascular endothelial cells under the pathologic condition of ischemia-reperfusion, thus identifying the injured endothelial glycocalyx as an essential element of the blood-tissue border facilitating the targeted delivery of nanomaterials to diseased tissue.


Assuntos
Barreira Hematoencefálica/metabolismo , Endotélio Vascular/metabolismo , Glicocálix/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Nanopartículas/metabolismo , Pontos Quânticos/metabolismo , Animais , Barreira Hematoencefálica/química , Células Cultivadas , Endotélio Vascular/química , Glicocálix/química , Células Endoteliais da Veia Umbilical Humana/química , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Nanopartículas/química , Pontos Quânticos/química
9.
Nanomedicine (Lond) ; 12(3): 207-217, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28078967

RESUMO

AIM: To assess the role of the endothelial glycocalyx (eGCX) for the uptake of nanoparticles by endothelial cells. METHODS: The expression of the eGCX on cultured human umbilical vein endothelial cells was determined by immunostaining of heparan sulfate. Enzymatic degradation of the eGCX was achieved by incubating the cells with eGCX-shedding enzymes. The uptake of 50-nm polystyrene nanospheres was quantified by confocal microscopy. RESULTS: Human umbilical vein endothelial cells expressed a robust eGCX when cultured for 10 days. The uptake of both carboxylated and aminated polystyrene nanospheres was significantly increased in cells in which the glycocalyx was enzymatically degraded, while it remained at a low level in cells with an intact glycocalyx. CONCLUSION: The eGCX constitutes a barrier against the internalization of blood-borne nanoparticles by endothelial cells.


Assuntos
Glicocálix/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Nanopartículas/metabolismo , Sobrevivência Celular , Células Cultivadas , Glicocálix/química , Heparitina Sulfato/química , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos , Hialuronoglucosaminidase/química , Nanopartículas/química , Neuraminidase/química , Polissacarídeo-Liase/química , Poliestirenos/química , Propriedades de Superfície
10.
Blood ; 128(19): 2327-2337, 2016 11 10.
Artigo em Inglês | MEDLINE | ID: mdl-27609642

RESUMO

Under steady-state conditions, aged neutrophils are removed from the circulation in bone marrow, liver, and spleen, thereby maintaining myeloid cell homeostasis. The fate of these aged immune cells under inflammatory conditions, however, remains largely obscure. Here, we demonstrate that in the acute inflammatory response during endotoxemia, aged neutrophils cease returning to the bone marrow and instead rapidly migrate to the site of inflammation. Having arrived in inflamed tissue, aged neutrophils were found to exhibit a higher phagocytic activity as compared with the subsequently recruited nonaged neutrophils. This distinct behavior of aged neutrophils under inflammatory conditions is dependent on specific age-related changes in their molecular repertoire that enable these "experienced" immune cells to instantly translate inflammatory signals into immune responses. In particular, aged neutrophils engage Toll-like receptor-4- and p38 MAPK-dependent pathways to induce conformational changes in ß2 integrins that allow these phagocytes to effectively accomplish their mission in the front line of the inflammatory response. Hence, ageing in the circulation might represent a critical process for neutrophils that enables these immune cells to properly unfold their functional properties for host defense.


Assuntos
Senescência Celular , Inflamação/imunologia , Inflamação/patologia , Neutrófilos/imunologia , Doença Aguda , Animais , Antígeno CD11b/metabolismo , Adesão Celular , Membrana Celular/metabolismo , Rastreamento de Células , Citocinas/metabolismo , Integrinas/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Modelos Biológicos , Fagocitose , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Receptor 4 Toll-Like/metabolismo
11.
Arterioscler Thromb Vasc Biol ; 36(9): 1891-9, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27386940

RESUMO

OBJECTIVE: Although the investigation on the importance of mitochondria-derived reactive oxygen species (ROS) in endothelial function has been gaining momentum, little is known on the precise role of the individual components involved in the maintenance of a delicate ROS balance. Here we studied the impact of an ongoing dysregulated redox homeostasis by examining the effects of endothelial cell-specific deletion of murine thioredoxin reductase 2 (Txnrd2), a key enzyme of mitochondrial redox control. APPROACH AND RESULTS: We analyzed the impact of an inducible, endothelial cell-specific deletion of Txnrd2 on vascular remodeling in the adult mouse after femoral artery ligation. Laser Doppler analysis and histology revealed impaired angiogenesis and arteriogenesis. In addition, endothelial loss of Txnrd2 resulted in a prothrombotic, proinflammatory vascular phenotype, manifested as intravascular cellular deposits, as well as microthrombi. This phenotype was confirmed by an increased leukocyte response toward interleukin-1 in the mouse cremaster model. In vitro, we could confirm the attenuated angiogenesis measured in vivo, which was accompanied by increased ROS and an impaired mitochondrial membrane potential. Ex vivo analysis of femoral arteries revealed reduced flow-dependent vasodilation in endothelial cell Txnrd2-deficient mice. This endothelial dysfunction could be, at least partly, ascribed to inadequate nitric oxide signaling. CONCLUSIONS: We conclude that the maintenance of mitochondrial ROS via Txnrd2 in endothelial cells is necessary for an intact vascular homeostasis and remodeling and that Txnrd2 plays a vitally important role in balancing mitochondrial ROS production in the endothelium.


Assuntos
Endotélio Vascular/enzimologia , Artéria Femoral/enzimologia , Inflamação/enzimologia , Isquemia/enzimologia , Mitocôndrias/enzimologia , Tiorredoxina Redutase 2/deficiência , Trombose/enzimologia , Remodelação Vascular , Vasodilatação , Animais , Células Cultivadas , Modelos Animais de Doenças , Células Progenitoras Endoteliais/enzimologia , Células Progenitoras Endoteliais/patologia , Endotélio Vascular/patologia , Endotélio Vascular/fisiopatologia , Artéria Femoral/patologia , Artéria Femoral/fisiopatologia , Artéria Femoral/cirurgia , Predisposição Genética para Doença , Inflamação/genética , Inflamação/patologia , Inflamação/fisiopatologia , Isquemia/genética , Isquemia/patologia , Isquemia/fisiopatologia , Ligadura , Potencial da Membrana Mitocondrial , Camundongos Knockout , Mitocôndrias/patologia , Neovascularização Fisiológica , Óxido Nítrico/metabolismo , Oxirredução , Fenótipo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Tiorredoxina Redutase 2/genética , Trombose/genética , Trombose/patologia , Trombose/fisiopatologia , Fatores de Tempo
12.
PLoS Biol ; 14(5): e1002459, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-27152726

RESUMO

Effective immune responses require the directed migration of leukocytes from the vasculature to the site of injury or infection. How immune cells "find" their site of extravasation remains largely obscure. Here, we identified a previously unrecognized role of platelets as pathfinders guiding leukocytes to their exit points in the microvasculature: upon onset of inflammation, circulating platelets were found to immediately adhere at distinct sites in venular microvessels enabling these cellular blood components to capture neutrophils and, in turn, inflammatory monocytes via CD40-CD40L-dependent interactions. In this cellular crosstalk, ligation of PSGL-1 by P-selectin leads to ERK1/2 MAPK-dependent conformational changes of leukocyte integrins, which promote the successive extravasation of neutrophils and monocytes to the perivascular tissue. Conversely, blockade of this cellular partnership resulted in misguided, inefficient leukocyte responses. Our experimental data uncover a platelet-directed, spatiotemporally organized, multicellular crosstalk that is essential for effective trafficking of leukocytes to the site of inflammation.


Assuntos
Plaquetas/fisiologia , Leucócitos/fisiologia , Vasculite/metabolismo , Animais , Antígenos CD40/metabolismo , Ligante de CD40/metabolismo , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Integrinas/metabolismo , Selectina L/metabolismo , Contagem de Leucócitos , Masculino , Glicoproteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Microvasos/metabolismo , Microvasos/patologia , Monócitos/metabolismo , Monócitos/patologia , Selectina-P/metabolismo , Vasculite/patologia
13.
Small ; 12(24): 3245-57, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-27120195

RESUMO

Nanotechnology holds great promise for a plethora of potential applications. The interaction of engineered nanomaterials with living cells, tissues, and organisms is, however, only partly understood. Microscopic investigations of nano-bio interactions are mostly performed with a few model nanoparticles (NPs) which are easy to visualize, such as fluorescent quantum dots. Here the possibility to visualize nonfluorescent NPs with multiphoton excitation is investigated. Signals from silver (Ag), titanium dioxide (TiO2 ), and silica (SiO2 ) NPs in nonbiological environments are characterized to determine signal dependency on excitation wavelength and intensity as well as their signal stability over time. Ag NPs generate plasmon-induced luminescence decaying over time. TiO2 NPs induce photoluminescent signals of variable intensities and in addition strong third harmonic generation (THG). Optimal settings for microscopic detection are determined and then applied for visualization of these two particle types in living cells, in murine muscle tissue, and in the murine blood stream. Silica NPs produce a THG signal, but in living cells it cannot be discriminated sufficiently from endogenous cellular structures. It is concluded that multiphoton excitation is a viable option for studies of nano-bio interactions not only for fluorescent but also for some types of nonfluorescent NPs.


Assuntos
Nanopartículas/química , Nanopartículas Metálicas/química , Pontos Quânticos , Dióxido de Silício/química , Titânio/química
14.
Arterioscler Thromb Vasc Biol ; 35(4): 899-910, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25722429

RESUMO

OBJECTIVE: Leukocyte recruitment to the site of inflammation is a key event in a variety of cardiovascular pathologies. Infiltrating neutrophils constitute the first line of defense that precedes a second wave of emigrating monocytes reinforcing the inflammatory reaction. The mechanisms initiating this sequential process remained largely obscure. APPROACH AND RESULTS: Using advanced in vivo microscopy and in vitro/ex vivo techniques, we identified individual spatiotemporal expression patterns of selectins and their principal interaction partners on neutrophils, resident/inflammatory monocytes, and endothelial cells. Coordinating the intraluminal trafficking of neutrophils and inflammatory monocytes to common sites of extravasation, selectins assign different sites to these immune cells for their initial interactions with the microvascular endothelium. Whereas constitutively expressed leukocyte L-selectin/CD62L and endothelial P-selectin/CD62P together with CD44 and P-selectin glycoprotein ligand-1/CD162 initiate the emigration of neutrophils, de novo synthesis of endothelial E-selectin/CD62E launches the delayed secondary recruitment of inflammatory monocytes. In this context, P-selectin/CD62P and L-selectin/CD62L together with P-selectin glycoprotein ligand-1/CD162 and CD44 were found to regulate the flux of rolling neutrophils and inflammatory monocytes, whereas E-selectin/CD62E selectively adjusts the rolling velocity of inflammatory monocytes. Moreover, selectins and their interaction partners P-selectin glycoprotein ligand-1/CD162 and CD44 differentially control the intraluminal crawling behavior of neutrophils and inflammatory monocytes collectively enabling the sequential extravasation of these immune cells to inflamed tissue. CONCLUSIONS: Our findings provide novel insights into the mechanisms initiating the sequential infiltration of the perivascular tissue by neutrophils and monocytes in the acute inflammatory response and might thereby contribute to the development of targeted therapeutic strategies for prevention and treatment of cardiovascular diseases.


Assuntos
Células Endoteliais/metabolismo , Selectina L/metabolismo , Migração e Rolagem de Leucócitos , Monócitos/metabolismo , Neutrófilos/metabolismo , Selectina-P/metabolismo , Peritonite/metabolismo , Migração Transendotelial e Transepitelial , Animais , Receptor 1 de Quimiocina CX3C , Citocinas/metabolismo , Modelos Animais de Doenças , Células Endoteliais/imunologia , Hemodinâmica , Receptores de Hialuronatos/metabolismo , Mediadores da Inflamação/metabolismo , Ligantes , Masculino , Glicoproteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microcirculação , Microvasos/imunologia , Microvasos/metabolismo , Microvasos/fisiopatologia , Monócitos/imunologia , Neutrófilos/imunologia , Peritonite/genética , Peritonite/imunologia , Peritonite/fisiopatologia , Receptores de Quimiocinas/genética , Receptores de Quimiocinas/metabolismo , Transdução de Sinais , Fatores de Tempo
15.
Arterioscler Thromb Vasc Biol ; 34(7): 1495-504, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24764453

RESUMO

OBJECTIVE: Neutrophil infiltration of the postischemic tissue considerably contributes to organ dysfunction on ischemia/reperfusion injury. Beyond its established role in fibrinolysis, tissue-type plasminogen activator (tPA) has recently been implicated in nonfibrinolytic processes. The role of this serine protease in the recruitment process of neutrophils remains largely obscure. APPROACH AND RESULTS: Using in vivo microscopy on the postischemic cremaster muscle, neutrophil recruitment and microvascular leakage, but not fibrinogen deposition at the vessel wall, were significantly diminished in tPA(-/-) mice. Using cell transfer techniques, leukocyte and nonleukocyte tPA were found to mediate ischemia/reperfusion-elicited neutrophil responses. Intrascrotal but not intra-arterial application of recombinant tPA induced a dose-dependent increase in the recruitment of neutrophils, which was significantly higher compared with stimulation with a tPA mutant lacking catalytic activity. Whereas tPA-dependent transmigration of neutrophils was selectively reduced on the inhibition of plasmin or gelatinases, neutrophil intravascular adherence was significantly diminished on the blockade of mast cell activation or lipid mediator synthesis. Moreover, stimulation with tPA caused a significant elevation in the leakage of fluorescein isothiocyanate dextran to the perivascular tissue, which was completely abolished on neutrophil depletion. In vitro, tPA-elicited macromolecular leakage of endothelial cell layers was abrogated on the inhibition of its proteolytic activity. CONCLUSIONS: Endogenously released tPA promotes neutrophil transmigration to reperfused tissue via proteolytic activation of plasmin and gelatinases. As a consequence, tPA on transmigrating neutrophils disrupts endothelial junctions allowing circulating tPA to extravasate to the perivascular tissue, which, in turn, amplifies neutrophil recruitment through the activation of mast cells and release of lipid mediators.


Assuntos
Quimiotaxia de Leucócito , Músculos/irrigação sanguínea , Infiltração de Neutrófilos , Neutrófilos/enzimologia , Traumatismo por Reperfusão/enzimologia , Ativador de Plasminogênio Tecidual/metabolismo , Animais , Permeabilidade Capilar , Células Cultivadas , Quimiotaxia de Leucócito/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Células Endoteliais/metabolismo , Fibrinogênio/metabolismo , Fibrinolisina/metabolismo , Gelatinases/metabolismo , Hemodinâmica , Humanos , Masculino , Mastócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microcirculação , Microvasos/metabolismo , Microvasos/fisiopatologia , Mutação , Infiltração de Neutrófilos/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Proteínas Recombinantes/administração & dosagem , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/imunologia , Traumatismo por Reperfusão/fisiopatologia , Fatores de Tempo , Ativador de Plasminogênio Tecidual/administração & dosagem , Ativador de Plasminogênio Tecidual/deficiência , Ativador de Plasminogênio Tecidual/genética , Migração Transendotelial e Transepitelial
16.
J Appl Toxicol ; 34(11): 1167-76, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24531921

RESUMO

Although carbon-based nanomaterials (CBNs) have been shown to exert prothrombotic effects in microvessels, it is poorly understood whether CBNs also have the potential to interfere with the process of leukocyte-endothelial cell interactions and whether the shape of CBNs plays a role in these processes. Thus, the aim of this study was to compare the acute effects of two differently shaped CBNs, fiber-shaped single-walled carbon nanotubes (SWCNT) and spherical ultrafine carbon black (CB), on thrombus formation as well as on leukocyte-endothelial cell interactions and leukocyte transmigration in the murine microcirculation upon systemic administration in vivo. Systemic administration of both SWCNT and CB accelerated arteriolar thrombus formation at a dose of 1 mg kg(-1) body weight, whereas SWCNT exerted a prothrombotic effect also at a lower dose (0.1 mg kg(-1) body weight). In vitro, both CBNs induced P-selectin expression on human platelets and formation of platelet-granulocyte complexes. In contrast, injection of fiber-shaped SWCNT or of spherical CB did not induce leukocyte-endothelial cell interactions or leukocyte transmigration. In vitro, both CBNs slightly increased the expression of activation markers on human monocytes and granulocytes. These findings suggest that systemic administration of CBNs accelerates arteriolar thrombus formation independently of the CBNs' shape, but does not induce leukocyte-endothelial cell interactions or leukocyte transmigration.


Assuntos
Microcirculação/efeitos dos fármacos , Nanotubos de Carbono/toxicidade , Fuligem/toxicidade , Animais , Arteríolas/efeitos dos fármacos , Arteríolas/patologia , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Humanos , Leucócitos/citologia , Leucócitos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Varredura , Selectina-P/genética , Selectina-P/metabolismo , Trombose/induzido quimicamente
17.
Blood ; 122(5): 770-80, 2013 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-23757732

RESUMO

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/imunologia
18.
Kidney Int ; 83(4): 647-61, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23325083

RESUMO

Ischemia-reperfusion activates innate immunity and sterile inflammation, resulting in acute kidney injury. Since pentraxin 3 (PTX3) regulates multiple aspects of innate immunity and tissue inflammation, we tested whether PTX3 would be involved in renal ischemia-reperfusion injury. Renal pedicle clamping increased PTX3 serum levels, as well as PTX3 expression, inside the kidney but predominantly in CD45/CD11c(+) cells, a subpopulation of intrarenal mononuclear phagocytes. Lack of PTX3 aggravated postischemic acute kidney injury as evidenced by massive tubular necrosis, and TNF and IL-6 release, as well as massively increased neutrophil and macrophage infiltrates at 24 h. This was followed by tubular atrophy, interstitial fibrosis, and kidney shrinking 10 weeks later. In vivo microscopy uncovered increased leukocyte adhesion and transmigration in postischemic microvessels of Ptx3-deficient mice. Furthermore, injection of recombinant PTX3 up to 6 h after reperfusion prevented renal leukocyte recruitment and postischemic kidney injury. Thus, local PTX3 release from a subpopulation of intrarenal mononuclear phagocytes or delayed PTX3 treatment limits postischemic renal inflammation. Conversely, Ptx3 loss-of-function mutations predispose to postischemic acute kidney injury and subsequent chronic kidney disease.


Assuntos
Lesão Renal Aguda/prevenção & controle , Proteína C-Reativa/metabolismo , Rim/irrigação sanguínea , Rim/imunologia , Proteínas do Tecido Nervoso/metabolismo , Insuficiência Renal Crônica/prevenção & controle , Traumatismo por Reperfusão/prevenção & controle , Lesão Renal Aguda/sangue , Lesão Renal Aguda/genética , Lesão Renal Aguda/imunologia , Lesão Renal Aguda/patologia , Animais , Atrofia , Proteína C-Reativa/administração & dosagem , Proteína C-Reativa/deficiência , Proteína C-Reativa/genética , Células Cultivadas , Modelos Animais de Doenças , Feminino , Fibrose , Mediadores da Inflamação/metabolismo , Injeções , Interleucina-6/metabolismo , Rim/patologia , Necrose Tubular Aguda/imunologia , Necrose Tubular Aguda/patologia , Necrose Tubular Aguda/prevenção & controle , Leucócitos/imunologia , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/administração & dosagem , Proteínas do Tecido Nervoso/sangue , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Infiltração de Neutrófilos , Selectina-P/metabolismo , Proteínas Recombinantes/administração & dosagem , Insuficiência Renal Crônica/sangue , Insuficiência Renal Crônica/genética , Insuficiência Renal Crônica/imunologia , Insuficiência Renal Crônica/patologia , Traumatismo por Reperfusão/sangue , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/imunologia , Traumatismo por Reperfusão/patologia , Fatores de Tempo , Migração Transendotelial e Transepitelial , Fator de Necrose Tumoral alfa/metabolismo
19.
Blood ; 120(4): 880-90, 2012 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-22674804

RESUMO

Initial observations suggested that C-C motif chemokines exclusively mediate chemotaxis of mononuclear cells. In addition, recent studies also implicated these chemotactic cytokines in the recruitment of neutrophils. The underlying mechanisms remained largely unknown. Using in vivo microscopy on the mouse cremaster muscle, intravascular adherence and subsequent paracellular transmigration of neutrophils elicited by the chemokine (C-C motif) ligand 3 (CCL3, synonym MIP-1α) were significantly diminished in mice with a deficiency of the chemokine (C-C motif) receptor 1 (Ccr1(-/-)) or 5 (Ccr5(-/-)). Using cell-transfer techniques, neutrophil responses required leukocyte CCR1 and nonleukocyte CCR5. Furthermore, neutrophil extravasation elicited by CCL3 was almost completely abolished on inhibition of G protein-receptor coupling and PI3Kγ-dependent signaling, while neutrophil recruitment induced by the canonical neutrophil attractants chemokine (C-X-C motif) ligand 1 (CXCL1, synonym KC) or the lipid mediator platetelet-activating factor (PAF) was only partially reduced. Moreover, Ab blockade of ß(2) integrins, of α(4) integrins, or of their putative counter receptors ICAM-1 and VCAM-1 significantly attenuated CCL3-, CXCL1-, or PAF-elicited intravascular adherence and paracellular transmigration of neutrophils. These data indicate that the C-C motif chemokine CCL3 and canonical neutrophil attractants exhibit both common and distinct mechanisms for the regulation of intravascular adherence and transmigration of neutrophils.


Assuntos
Movimento Celular , Quimiocina CCL3/fisiologia , Quimiotaxia de Leucócito/fisiologia , Neutrófilos/metabolismo , Animais , Proteínas de Transporte/metabolismo , Células Cultivadas , Quimiocina CCL2/fisiologia , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Citometria de Fluxo , Integrinas/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Neutrófilos/citologia , Receptores CCR1/metabolismo , Receptores CCR5/metabolismo , Molécula 1 de Adesão de Célula Vascular/metabolismo
20.
J Am Soc Nephrol ; 23(8): 1375-88, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22677551

RESUMO

In AKI, dying renal cells release intracellular molecules that stimulate immune cells to secrete proinflammatory cytokines, which trigger leukocyte recruitment and renal inflammation. Whether the release of histones, specifically, from dying cells contributes to the inflammation of AKI is unknown. In this study, we found that dying tubular epithelial cells released histones into the extracellular space, which directly interacted with Toll-like receptor (TLR)-2 (TLR2) and TLR4 to induce MyD88, NF-κB, and mitogen activated protein kinase signaling. Extracellular histones also had directly toxic effects on renal endothelial cells and tubular epithelial cells in vitro. In addition, direct injection of histones into the renal arteries of mice demonstrated that histones induce leukocyte recruitment, microvascular vascular leakage, renal inflammation, and structural features of AKI in a TLR2/TLR4-dependent manner. Antihistone IgG, which neutralizes the immunostimulatory effects of histones, suppressed intrarenal inflammation, neutrophil infiltration, and tubular cell necrosis and improved excretory renal function. In summary, the release of histones from dying cells aggravates AKI via both its direct toxicity to renal cells and its proinflammatory effects. Because the induction of proinflammatory cytokines in dendritic cells requires TLR2 and TLR4, these results support the concept that renal damage triggers an innate immune response, which contributes to the pathogenesis of AKI.


Assuntos
Lesão Renal Aguda/metabolismo , Histonas/metabolismo , Fator 88 de Diferenciação Mieloide/metabolismo , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , Lesão Renal Aguda/imunologia , Animais , Permeabilidade Capilar , Citocinas/metabolismo , Células Endoteliais/fisiologia , Células Epiteliais/metabolismo , Injeções Intra-Arteriais , Rim/patologia , Túbulos Renais/metabolismo , Leucócitos/fisiologia , Lipopolissacarídeos , Camundongos , Camundongos Endogâmicos C57BL , Necrose , Artéria Renal , Traumatismo por Reperfusão/prevenção & controle
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