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Following a myocardial infarction (MI), the prognosis of patients is highly dependent upon the re-establishment of perfusion not only in the occluded coronary artery, but also within the coronary microcirculation. However, our fundamental understanding of the pathophysiology of the tiniest blood vessels of the heart is limited primarily because no current clinical imaging tools can directly visualise them. Moreover, in vivo experimental studies of the beating heart using intravital imaging have also been hampered due to obvious difficulties related to significant inherent contractile motion, movement of the heart brought about by nearby lungs and its location in an anatomically challenging position for microscopy. However, recent advances in microscopy techniques, and the development of fluorescent reporter mice and fluorescently conjugated antibodies allowing visualisation of vascular structures, thromboinflammatory cells and blood flow, have allowed us to overcome some of these challenges and increase our basic understanding of cardiac microvascular pathophysiology. In this review, the elegant attempts of the pioneers in intravital imaging of the beating heart will be discussed, which focussed on providing new insights into the anatomy and physiology of the healthy heart microvessels. The reviews end with the more recent studies that focussed on disease pathology and increasing our understanding of myocardial thromboinflammatory cell recruitment and flow disturbances, particularly in the setting of diseases such as MI.
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Circulação Coronária , Vasos Coronários , Animais , Camundongos , Microcirculação , Circulação Coronária/fisiologia , Vasos Coronários/diagnóstico por imagem , Coração/diagnóstico por imagem , MicrovasosRESUMO
Concern regarding the quality of cold perfusion (QOP) during macroscopic assessment of procured kidneys is a common reason for discard. In the UK, QOP is routinely graded by both retrieving and implanting teams during back-bench surgery as: 1 (good), 2 (fair), 3 (poor) or 4 (patchy). We evaluated the association of this grading with organ utilization, graft outcomes, and agreement between teams. Data on all deceased-donor kidneys procured between January 2000 and December 2016 were analyzed for discard rates, while association with graft outcomes was studied in single adult transplants. Of 31,167 kidneys procured, 90.6%, 5.7%, 1.7%, and 2.1% were assigned grades 1, 2, 3, and 4, respectively, at retrieval. QOP was an independent risk factor of discard, with the highest rates observed in grade 3 kidneys (41.8%), compared to 6.5% in grade 1 (aOR 7.67, 95% CI 5.44-10.82, p < .001). Grading at retrieval was an independent predictor of delayed graft function (p = .019) and primary non-function (p = .001), but not long-term graft survival (p = .111). Implanting grade was an independent predictor of all three outcomes (p < .001, p < .001, and p = .002, respectively). Consistency of grading between teams was poor (Kappa = 0.179). QOP influences utilization and predicts outcomes, but a standardized and validated scoring system is required.
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Transplante de Rim , Obtenção de Tecidos e Órgãos , Adulto , Estudos de Coortes , Sobrevivência de Enxerto , Humanos , Rim , Perfusão , Doadores de Tecidos , Reino UnidoRESUMO
Interactions between platelets, leukocytes and the vessel wall provide alternative pathological routes of thrombo-inflammatory leukocyte recruitment. We found that when platelets were activated by a range of agonists in whole blood, they shed platelet-derived extracellular vesicles which rapidly and preferentially bound to blood monocytes compared to other leukocytes. Platelet-derived extracellular vesicle binding to monocytes was initiated by P-selectin-dependent adhesion and was stabilised by binding of phosphatidylserine. These interactions resulted in the progressive transfer of the platelet adhesion receptor GPIbα to monocytes. GPIbα+-monocytes tethered and rolled on immobilised von Willebrand Factor or were recruited and activated on endothelial cells treated with TGF-ß1 to induce the expression of von Willebrand Factor. In both models monocyte adhesion was ablated by a function-blocking antibody against GPIbα. Monocytes could also bind platelet-derived extracellular vesicle in mouse blood in vitro and in vivo Intratracheal instillations of diesel nanoparticles, to model chronic pulmonary inflammation, induced accumulation of GPIbα on circulating monocytes. In intravital experiments, GPIbα+-monocytes adhered to the microcirculation of the TGF-ß1-stimulated cremaster muscle, while in the ApoE-/- model of atherosclerosis, GPIbα+-monocytes adhered to the carotid arteries. In trauma patients, monocytes bore platelet markers within 1 hour of injury, the levels of which correlated with severity of trauma and resulted in monocyte clearance from the circulation. Thus, we have defined a novel thrombo-inflammatory pathway in which platelet-derived extracellular vesicles transfer a platelet adhesion receptor to monocytes, allowing their recruitment in large and small blood vessels, and which is likely to be pathogenic.
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Plaquetas , Vesículas Extracelulares , Animais , Células Endoteliais , Humanos , Inflamação , Camundongos , Monócitos , Complexo Glicoproteico GPIb-IX de PlaquetasRESUMO
We investigated the adhesive behavior of mesenchymal stem cells (MSC) in blood, which might influence their fate when infused as therapy. Isolated human bone marrow MSC (BMMSC) or umbilical cord MSC (UCMSC) adhered efficiently from flow to the matrix proteins, collagen, or fibronectin, but did not adhere to endothelial selectins. However, when suspended in blood, BMMSC no longer adhered to collagen, while UCMSC adhered along with many aggregated platelets. Neither MSC adhered to fibronectin from flowing blood, although the fibronectin surface did become coated with a platelet monolayer. UCMSC induced platelet aggregation in platelet rich plasma, and caused a marked drop in platelet count when mixed with whole human or mouse blood in vitro, or when infused into mice. In contrast, BMMSC did not activate platelets or induce changes in platelet count. Interestingly, isolated UCMSC and BMMSC both adhered to predeposited platelets. The differences in behavior in blood were attributable to expression of podoplanin (an activating ligand for the platelet receptor CLEC-2), which was detected on UCMSC, but not BMMSC. Thus, platelets were activated when bound to UCMSC, but not BMMSC. Platelet aggregation by UCMSC was inhibited by recombinant soluble CLEC-2, and UCMSC did not cause a reduction in platelet count when mixed with blood from mice deficient in CLEC-2. We predict that both MSC would carry platelets in the blood, but their interaction with vascular endothelium would depend on podoplanin-induced activation of the bound platelets. Such interactions with platelets might target MSC to damaged tissue, but could also be thrombotic. Stem Cells 2018;36:1062-1074.
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Plaquetas/metabolismo , Adesão Celular/genética , Células-Tronco Mesenquimais/metabolismo , Animais , Humanos , CamundongosRESUMO
Ca2+ entry via Orai1 store-operated Ca2+ channels in the plasma membrane is critical to cell function, and Orai1 loss causes severe immunodeficiency and developmental defects. The tetraspanins are a superfamily of transmembrane proteins that interact with specific 'partner proteins' and regulate their trafficking and clustering. The aim of this study was to functionally characterize tetraspanin Tspan18. We show that Tspan18 is expressed by endothelial cells at several-fold higher levels than most other cell types analyzed. Tspan18-knockdown primary human umbilical vein endothelial cells have 55-70% decreased Ca2+ mobilization upon stimulation with the inflammatory mediators thrombin or histamine, similar to Orai1-knockdown. Tspan18 interacts with Orai1, and Orai1 cell surface localization is reduced by 70% in Tspan18-knockdown endothelial cells. Tspan18 overexpression in lymphocyte model cell lines induces 20-fold activation of Ca2+ -responsive nuclear factor of activated T cell (NFAT) signaling, in an Orai1-dependent manner. Tspan18-knockout mice are viable. They lose on average 6-fold more blood in a tail-bleed assay. This is due to Tspan18 deficiency in non-hematopoietic cells, as assessed using chimeric mice. Tspan18-knockout mice have 60% reduced thrombus size in a deep vein thrombosis model, and 50% reduced platelet deposition in the microcirculation following myocardial ischemia-reperfusion injury. Histamine- or thrombin-induced von Willebrand factor release from endothelial cells is reduced by 90% following Tspan18-knockdown, and histamine-induced increase of plasma von Willebrand factor is reduced by 45% in Tspan18-knockout mice. These findings identify Tspan18 as a novel regulator of endothelial cell Orai1/Ca2+ signaling and von Willebrand factor release in response to inflammatory stimuli.
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Cálcio/metabolismo , Traumatismo por Reperfusão Miocárdica/genética , Proteína ORAI1/genética , Tetraspaninas/genética , Trombose Venosa/genética , Fator de von Willebrand/genética , Animais , Linfócitos B/citologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Galinhas , Modelos Animais de Doenças , Regulação da Expressão Gênica , Células HEK293 , Células HeLa , Histamina/farmacologia , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Transporte de Íons/efeitos dos fármacos , Células Jurkat , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Fatores de Transcrição NFATC/genética , Fatores de Transcrição NFATC/metabolismo , Proteína ORAI1/metabolismo , Transdução de Sinais , Tetraspaninas/metabolismo , Trombina/farmacologia , Trombose Venosa/metabolismo , Trombose Venosa/patologia , Fator de von Willebrand/metabolismoRESUMO
BACKGROUND & AIMS: There is growing interest in the use of bone marrow cells to treat liver fibrosis, however, little is known about their antifibrotic efficacy or the identity of their effector cell(s). Sphingosine-1-phosphate (S1P) mediates egress of immune cells from the lymphoid organs into the lymphatic vessels; we investigated its role in the response of hematopoietic stem cells (HSCs) to liver fibrosis in mice. METHODS: Purified (c-kit+/sca1+/lin-) HSCs were infused repeatedly into mice undergoing fibrotic liver injury. Chronic liver injury was induced in BoyJ mice by injection of carbon tetrachloride (CCl4) or placement on a methionine-choline-deficient diet. Some mice were irradiated and given transplants of bone marrow cells from C57BL6 mice, with or without the S1P antagonist FTY720; we then studied HSC mobilization and localization. Migration of HSC lines was quantified in Transwell assays. Levels of S1P in liver, bone marrow, and lymph fluid were measured using an enzyme-linked immunosorbent assay. Liver tissues were collected and analyzed by immunohistochemical quantitative polymerase chain reaction and sphingosine kinase activity assays. We performed quantitative polymerase chain reaction analyses of the expression of sphingosine kinase 1 and 2, sphingosine-1-phosphate lyase 1, and sphingosine-1-phosphate phosphatase 1 in normal human liver and cirrhotic liver from patients with alcohol-related liver disease (n = 6). RESULTS: Infusions of HSCs into mice with liver injury reduced liver scarring based on picrosirius red staining (49.7% reduction in mice given HSCs vs control mice; P < .001), and hepatic hydroxyproline content (328 mg/g in mice given HSCs vs 428 mg/g in control mice; P < .01). HSC infusion also reduced hepatic expression of α-smooth muscle actin (0.19 ± 0.007-fold compared with controls; P < .0001) and collagen type I α 1 chain (0.29 ± 0.17-fold compared with controls; P < .0001). These antifibrotic effects were maintained with infusion of lymphoid progenitors that lack myeloid potential and were associated with increased numbers of recipient neutrophils and macrophages in liver. In studies of HSC cell lines, we found HSCs to recruit monocytes, and this process to require C-C motif chemokine receptor 2. In fibrotic liver tissue from mice and patients, hepatic S1P levels increased owing to increased hepatic sphingosine kinase-1 expression, which contributed to a reduced liver:lymph S1P gradient and limited HSC egress from the liver. Mice given the S1P antagonist (FTY720) with HSCs had increased hepatic retention of HSCs (1697 ± 247 cells in mice given FTY720 vs 982 ± 110 cells in controls; P < .05), and further reductions in fibrosis. CONCLUSIONS: In studies of mice with chronic liver injury, we showed the antifibrotic effects of repeated infusions of purified HSCs. We found that HSCs promote recruitment of endogenous macrophages and neutrophils. Strategies to reduce SIP signaling and increase retention of HSCs in the liver could increase their antifibrotic activities and be developed for treatment of patients with liver fibrosis.
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Movimento Celular/efeitos dos fármacos , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/fisiologia , Cirrose Hepática/prevenção & controle , Lisofosfolipídeos/antagonistas & inibidores , Lisofosfolipídeos/metabolismo , Esfingosina/análogos & derivados , Actinas/metabolismo , Aldeído Liases/genética , Animais , Linhagem Celular , Doença Hepática Crônica Induzida por Substâncias e Drogas/complicações , Colágeno Tipo I/metabolismo , Cadeia alfa 1 do Colágeno Tipo I , Feminino , Cloridrato de Fingolimode/uso terapêutico , Expressão Gênica , Humanos , Imunossupressores/uso terapêutico , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática/etiologia , Cirrose Hepática/genética , Cirrose Hepática/patologia , Linfa/metabolismo , Macrófagos , Masculino , Proteínas de Membrana/genética , Camundongos , Monócitos , Neutrófilos , Monoéster Fosfórico Hidrolases/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Esfingosina/antagonistas & inibidores , Esfingosina/metabolismoRESUMO
To commemorate the auspicious occasion of the 30th anniversary of IPC, leading pioneers in the field of cardioprotection gathered in Barcelona in May 2016 to review and discuss the history of IPC, its evolution to IPost and RIC, myocardial reperfusion injury as a therapeutic target, and future targets and strategies for cardioprotection. This article provides an overview of the major topics discussed at this special meeting and underscores the huge importance and impact, the discovery of IPC has made in the field of cardiovascular research.
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Precondicionamento Isquêmico Miocárdico , Traumatismo por Reperfusão Miocárdica , Animais , HumanosRESUMO
Mesenchymal stem cells (MSCs) have shown therapeutic promise in many experimental and clinical models of inflammation. However, a commonly reported feature of MSC transplantation is poor homing to injured tissues. Previously, we have shown that pretreatment with cytokines/chemical factors enhances hematopoietic SC adhesion within intestinal microvasculature following ischemia-reperfusion (IR) injury. Using intravital microscopy, the ability of similar pretreatment strategies to enhance the recruitment of murine MSCs to murine intestinal microvasculature following IR injury was investigated. Primary MSCs were isolated from bone marrow and selected on the basis of platelet-derived growth factor receptor-α and SC antigen-1 positivity (PDGFRα(+) /Sca-1(+) ). MSC recruitment was similar in IR injured gut mucosa when compared with sham operated controls, with limited cell adhesion observed. MSCs appeared contorted in microvessels, suggesting physical entrapment. Although not recruited specifically by injury, MSC administration significantly reduced neutrophil recruitment and improved tissue perfusion in the severely injured jejunum. Vasculoprotective effects were not demonstrated in the lesser injured ileum. Pretreatment of MSCs with tumor necrosis factor (TNF)-α, CXCL12, interferon (IFN)-γ, or hydrogen peroxide did not enhance their intestinal recruitment. In fact, TNFα and IFNγ removed the previous therapeutic ability of transplanted MSCs to reduce neutrophil infiltration and improve perfusion in the jejunum. We provide direct evidence that MSCs can rapidly limit leukocyte recruitment and improve tissue perfusion following intestinal IR injury. However, this study also highlights complexities associated with strategies to improve MSC therapeutic efficacy. Future studies using cytokine/chemical pretreatments to enhance MSC recruitment/function require careful consideration and validation to ensure therapeutic function is not impeded.
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Movimento Celular/fisiologia , Íleo/irrigação sanguínea , Íleo/lesões , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/fisiologia , Traumatismo por Reperfusão/prevenção & controle , Animais , Movimento Celular/efeitos dos fármacos , Citocinas/metabolismo , Citocinas/farmacologia , Íleo/metabolismo , Masculino , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Traumatismo por Reperfusão/metabolismoRESUMO
Introduction: Investigating coronary microvascular perfusion responses after myocardial infarction (MI) would aid in the development of flow preserving therapies. Laser speckle contrast imaging (LSCI) is a powerful tool used for real-time, non-contact, full-field imaging of blood flow in various tissues/organs. However, its use in the beating heart has been limited due to motion artifacts. Methods: In this paper, we report the novel use of LSCI, combined with custom speckle analysis software (SpAn), to visualise and quantitate changes in ventricular perfusion in adult and aged mice undergoing ischaemia-reperfusion (IR) injury. The therapeutic benefit of inhibiting the actions of the pro-inflammatory cytokine interleukin-36 (IL-36) was also investigated using an IL-36 receptor antagonist (IL-36Ra). Results: Imaging from uncovered and covered regions of the left ventricle demonstrated that whilst part of the LSCI flux signal was derived from beating motion, a significant contributor to the flux signal came from ventricular microcirculatory blood flow. We show that a biphasic flux profile corresponding to diastolic and systolic phases of the cardiac cycle can be detected without mathematically processing the total flux data to denoise motion artifacts. Furthermore, perfusion responses to ischaemia and postischaemia were strong, reproducible and could easily be detected without the need to subtract motion-related flux signals. LSCI also identified significantly poorer ventricular perfusion in injured aged mice following IR injury which markedly improved with IL-36Ra. Discussion: We therefore propose that LSCI of the heart is possible despite motion artifacts and may facilitate future investigations into the role of the coronary microcirculation in cardiovascular diseases and development of novel therapies.
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BACKGROUND: Free labile hemin acts as a damage-associated molecular pattern during acute and chronic hemolysis and muscle injury, supporting platelet activation and thrombosis. OBJECTIVES: To investigate the anti-thrombotic potential of hydroxychloroquine on hemolysis-induced platelet activation and arterial thrombosis. METHODS: The effect of hydroxychloroquine on hemin-induced platelet activation and hemolysis-induced platelet recruitment and aggregation was measured in washed platelets and hemolyzed blood, respectively. Its effect on ferric-chloride (FeCl3)-induced arterial thrombosis and lung perfusion following hemin injection was assessed in wild-type mice. RESULTS: Erythrocyte lysis and endothelial cell activation cooperatively supported platelet aggregation and thrombosis at arterial shear stress. This thrombotic effect was reversed by hydroxychloroquine. In a purified system, hydroxychloroquine inhibited platelet build-up on immobilized von Willebrand factor in hemolyzed blood without altering initial platelet recruitment. Hydroxychloroquine inhibited hemin-induced platelet activation and phosphatidylserine exposure independently of reactive oxygen species generation. In the presence of hemin, hydroxychloroquine did not alter glycoprotein VI shedding but reduced C-type-lectin-like-2 expression on platelets. In vivo, hydroxychloroquine reversed pulmonary perfusion decline induced by exogenous administration of hemin. In arterial thrombosis models, hydroxychloroquine inhibited ferric-chloride-induced thrombosis in the carotid artery and reduced von Willebrand factor accumulation in the thrombi. CONCLUSION: Hydroxychloroquine inhibited hemolysis-induced arterial thrombosis ex vivo and improved pulmonary perfusion in hemin-treated mice, supporting a potential benefit of its use as an adjuvant therapy in hemolytic diseases to limit arterial thrombosis and to improve organ perfusion.
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Hemina , Hemólise , Hidroxicloroquina , Pulmão , Ativação Plaquetária , Trombose , Animais , Hidroxicloroquina/farmacologia , Hemólise/efeitos dos fármacos , Hemina/farmacologia , Trombose/tratamento farmacológico , Trombose/sangue , Pulmão/efeitos dos fármacos , Pulmão/irrigação sanguínea , Ativação Plaquetária/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Agregação Plaquetária/efeitos dos fármacos , Compostos Férricos , Humanos , Masculino , Cloretos , Modelos Animais de Doenças , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Fator de von Willebrand/metabolismoRESUMO
UNLABELLED: Human bone marrow mesenchymal stem cells (hMSCs) have shown benefit in clinical trials of patients with liver disease. Efficient delivery of cells to target organs is critical to improving their effectiveness. This requires an understanding of the mechanisms governing cellular engraftment into the liver. Binding of hMSCs to normal/injured liver tissue, purified extracellular matrices, and human hepatic sinusoidal endothelial cells (HSECs) were quantified in static and flow conditions. To define the mechanisms underpinning hMSC interactions, neutralizing adhesion molecule antibodies were used. Fluorescently labelled hMSCs were infused intraportally into CCl(4) -injured mice with and without neutralizing antibodies. hMSCs expressed high levels of CD29/ß1-integrin and CD44. Using liver tissue binding assays, hMSC adhesion was greatest in diseased human liver versus normal liver (32.2 cells/field versus 20.5 cells/field [P = 0.048]). Neutralizing antibodies against CD29 and CD44 reduced hMSC binding to diseased liver by 34% and 35%, respectively (P = 0.05). hMSCs rolled at 528 µm/second on HSECs in flow assays. This rolling was abolished by CD29 blockade on hMSCs and vascular cell adhesion molecule-1 (VCAM-1) blockade on HSECs. Firm adhesion to HSECs was reduced by CD29 (55% [P = 0.002]) and CD44 (51% [P = 0.04]) blockade. Neutralizing antibodies to CD29 and CD44 reduced hepatic engraftment of hMSCs in murine liver from 4.45 cells/field to 2.88 cells/field (P = 0.025) and 2.35 cells/field (P = 0.03), respectively. hMSCs expressed modest levels of chemokine receptors including CCR4, CCR5, and CXCR3, but these made little contribution to hMSC adhesion in this setting. CONCLUSION: hMSCs bind preferentially to injured liver. Rolling of hMSCs is regulated by CD29/VCAM-1, whereas CD29/CD44 interactions with VCAM-1, fibronectin, and hyaluronan on HSECs determine firm adhesion both in vitro and in vivo as demonstrated using a murine model of liver injury.
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Movimento Celular , Receptores de Hialuronatos/fisiologia , Integrina beta1/fisiologia , Fígado/lesões , Fígado/patologia , Células-Tronco Mesenquimais/fisiologia , Animais , Humanos , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Aims: Risks and outcomes of myocardial infarction (MI) are different between men and women and some studies have demonstrated that the latter have a higher risk of mortality. Whilst there are many reasons for this, it may also partially be linked to stronger innate and adaptive immune responses mounted by females compared to males. However, little is known about how sex impacts the coronary microvessels, the site where inflammatory processes take place, after an MI. Intravital and laser speckle microscopy was used to image coronary microvessels and ventricular perfusion in vivo in response to myocardial ischaemia-reperfusion (IR) injury in male and female mice. Interleukin-36 (IL-36) is the latest addition to the IL-1 superfamily of pro-inflammatory cytokines and has recently been shown to mediate inflammation in a number of non-cardiovascular diseases. Its role in mediating potential sex-related microcirculatiory pertubations in the heart are unknown. Therefore, the vasculoprotective efficacy of an IL-36 receptor antagonist (IL-36Ra) was also investigated. Methods and results: Immunostaining and flow cytometry demonstrated higher expression of IL-36 and its receptor in female hearts, an observation confirmed in human samples. Intravital imaging of the anaesthetised mouse beating heart identified significantly greater neutrophil recruitment in female hearts, but a greater burden of thrombotic disease in male hearts. Male mice had reduced functional capillary density and were unable to restore perfusion to baseline values as effectively as females. However, female mice had significantly larger infarcts. Interestingly, IL-36Ra decreased inflammation, improved perfusion, and reduced infarct size in both sexes despite increasing platelet presence in male hearts. Mechanistically, this was explained by IL-36Ra attenuating endothelial oxidative damage and VCAM-1 expression. Importantly, IL-36Ra administration during ischaemia was critical for vasculoprotection to be realised. Conclusion: This novel study identified notable sex-related differences in the coronary microcirculatory response to myocardial IR injury which may explain why some studies have noted poorer outcomes in women after MI. Whilst contemporary MI treatment focuses on anti-platelet strategies, the heightened presence of neutrophils in female IR injured coronary microvessels necessitates the development of an effective anti-inflammatory approach for treating female patients. We also emphasise the importance of early intervention during the ischaemic period in order to maximise therapeutic effectiveness.
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Introduction: Opening occluded coronary arteries in patients with myocardial infarction (MI) damages the delicate coronary microvessels through a process called myocardial ischaemia-reperfusion injury. Although mesenchymal stromal cells (MSCs) have the potential to limit this injury, clinical success remains limited. This may be due to (i) poor MSC homing to the heart (ii) infused MSCs, even if derived from the same site, being a heterogeneous population with varying therapeutic efficacy and (iii) conventional 2D culture of MSCs decreasing their homing and beneficial properties. This study investigated whether 3D culture of two distinctly different bone marrow (BM)-derived MSC sub-populations could improve their homing and coronary vasculoprotective efficacy. Methods: Intravital imaging of the anaesthetised mouse beating heart was used to investigate the trafficking and microvascular protective effects of two clonally-derived BM-derived MSC lines, namely CD317neg MSCs-Y201 and CD317pos MSCs-Y202, cultured using conventional monolayer and 3D hanging drop methods. Results: 3D culture consistently improved the adhesive behaviour of MSCs-Y201 to various substrates in vitro. However, it was their differential ability to reduce neutrophil events within the coronary capillaries and improve ventricular perfusion in vivo that was most remarkable. Moreover, dual therapy combined with heparin further improved the vasculoprotection afforded by 3D cultured MSCs-Y201 by also modifying platelet as well as neutrophil recruitment, which subsequently led to the greatest salvage of viable myocardium. Therapeutic benefit could mechanistically be explained by reductions in coronary endothelial oxidative stress and intercellular adhesion molecule-1 (ICAM-1)/vascular cell adhesion molecule-1 (VCAM-1) expression. However, since this was noted by both 2D and 3D cultured MSCs-Y201, therapeutic benefit is likely explained by the fact that 3D cultured MSCs-Y201 were the most potent sub-population at reducing serum levels of several pro-inflammatory cytokines. Conclusion: This novel study highlights the importance of not only 3D culture, but also of a specific CD317neg MSC sub-population, as being critical to realising their full coronary vasculoprotective potential in the injured heart. Since the smallest coronary blood vessels are increasingly recognised as a primary target of reperfusion injury, therapeutic interventions must be able to protect these delicate structures from inflammatory cells and maintain perfusion in the heart. We propose that relatively feasible technical modifications in a specific BM-derived MSC sub-population could achieve this.
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Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais , Traumatismo por Reperfusão Miocárdica , Camundongos , Animais , Humanos , Heparina/farmacologia , Heparina/metabolismo , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/metabolismo , Traumatismo por Reperfusão Miocárdica/terapia , Traumatismo por Reperfusão Miocárdica/metabolismo , MicrovasosRESUMO
BACKGROUND: Aspirin and platelet P2Y12 inhibitors, such as ticagrelor, suboptimally inhibit microvascular thrombosis during ST-elevation myocardial infarction. Glycoprotein (GP) IIb/IIIa inhibitors may further inhibit this but cause excessive bleeding. OBJECTIVES: We investigated whether combination of glenzocimab, a GPVI inhibitor, with aspirin and ticagrelor provides additional antithrombotic effects, as GPVI has a critical role in atherothrombosis but minimal involvement in hemostasis. METHODS: We investigated the effects of glenzocimab (monoclonal antibody Fab fragment) using blood from healthy donors and patients with acute coronary syndrome treated with aspirin and ticagrelor. Platelets were stimulated with multiple agonists, including atherosclerotic plaque, from patients undergoing carotid endarterectomy. RESULTS: Aspirin and ticagrelor partially inhibited atherosclerotic plaque-induced platelet aggregation by 48% compared with control (34 ± 3 vs 65 ± 4 U; P < .001). Plaque-induced platelet aggregation, adhesion, secretion, and activation were critically dependent on GPVI activation. Glenzocimab alone reduced plaque-induced aggregation by 75% compared with control (16 ± 4 vs 65 ± 4 U; P < .001) and by >95% when combined with aspirin and ticagrelor (3 ± 1 vs 65 ± 4 U; P < .001). Glenzocimab reduced platelet aggregation, adhesion, and thrombin generation when added to blood of aspirin- and ticagrelor-treated patients with acute coronary syndrome. Glenzocimab shared several antithrombotic effects with the GPIIb/IIIa inhibitor eptifibatide with less effect on general hemostasis assessed by rotational thromboelastometry. In a murine intravital model of ST-elevation myocardial infarction, genetic depletion of GPVI reduced microvascular thrombosis. CONCLUSION: Addition of glenzocimab to aspirin and ticagrelor enhances platelet inhibition via multiple mechanisms of atherothrombosis. Compared with a GPIIb/IIIa inhibitor, glenzocimab shares multiple antithrombotic effects, with less inhibition of mechanisms involved in general hemostasis.
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Síndrome Coronariana Aguda , Placa Aterosclerótica , Infarto do Miocárdio com Supradesnível do Segmento ST , Trombose , Humanos , Animais , Camundongos , Inibidores da Agregação Plaquetária/farmacologia , Ticagrelor/farmacologia , Fibrinolíticos/efeitos adversos , Síndrome Coronariana Aguda/tratamento farmacológico , Ativação Plaquetária , Aspirina/farmacologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas , Trombose/tratamento farmacológico , Trombose/prevenção & controleRESUMO
BACKGROUND & AIMS: IL-17 secreting CD4 (Th17) and CD8 (Tc17) T cells have been implicated in immune-mediated liver diseases, but the molecular basis for their recruitment and positioning within the liver is unknown. METHODS: The phenotype and migratory behaviour of human liver-derived Th17 and Tc17 cells were investigated by flow cytometry and chemotaxis and flow-based adhesion assays. The recruitment of murine Th17 cells to the liver was studied in vivo using intra-vital microscopy. RESULTS: IL-17(+) T cells comprised 1-3% of the T cell infiltrate in inflammatory liver diseases and included both CD4 (Th17) and CD8 (Tc17) cells. They expressed RORC and the IL-23 receptor and included subsets that secreted IL-22 and interferon-γ. Th17 and Tc17 cells expressed high levels of CXCR3 and CCR6, Tc17 cells also expressed CXCR6. Binding to human sinusoidal endothelium from flow was dependent on ß1 and ß2 integrins, CXCR3, and, in the case of Th17 cells, VAP-1. Th17 recruitment via sinusoids in mice with liver inflammation was reduced by treatment with antibodies against CXCR3 ligands, confirming the role of CXCR3 in Th17 recruitment in vivo. In human liver, IL-17(+) cells were detected in portal infiltrates close to inflamed bile ducts expressing the CCR6 ligand CCL20. Cytokine-treated human cholangiocytes secreted CCL20 and induced CCR6-dependent migration of Th17 cells suggesting that local cholangiocyte chemokine secretion localises Th17 cells to bile ducts. CONCLUSIONS: CXCR3 promotes recruitment of Th17 cells from the blood into the liver in both human and murine liver injury. Their subsequent positioning near bile ducts is dependent on cholangiocyte-secreted CCL20.
Assuntos
Hepatite/patologia , Hepatopatias/patologia , Receptores CCR6/metabolismo , Receptores CXCR3/metabolismo , Células Th17/patologia , Animais , Sistema Biliar/metabolismo , Sistema Biliar/patologia , Movimento Celular/fisiologia , Células Cultivadas , Quimiocina CCL20/metabolismo , Modelos Animais de Doenças , Hepatite/metabolismo , Humanos , Hepatopatias/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , Células Th17/metabolismoRESUMO
Following myocardial infarction (MI), elderly patients have a poorer prognosis than younger patients, which may be linked to increased coronary microvessel susceptibility to injury. Interleukin-36 (IL-36), a newly discovered proinflammatory member of the IL-1 superfamily, may mediate this injury, but its role in the injured heart is currently not known. We first demonstrated the presence of IL-36(α/ß) and its receptor (IL-36R) in ischemia/reperfusion-injured (IR-injured) mouse hearts and, interestingly, noted that expression of both increased with aging. An intravital model for imaging the adult and aged IR-injured beating heart in real time in vivo was used to demonstrate heightened basal and injury-induced neutrophil recruitment, and poorer blood flow, in the aged coronary microcirculation when compared with adult hearts. An IL-36R antagonist (IL-36Ra) decreased neutrophil recruitment, improved blood flow, and reduced infarct size in both adult and aged mice. This may be mechanistically explained by attenuated endothelial oxidative damage and VCAM-1 expression in IL-36Ra-treated mice. Our findings of an enhanced age-related coronary microcirculatory dysfunction in reperfused hearts may explain the poorer outcomes in elderly patients following MI. Since targeting the IL-36/IL-36R pathway was vasculoprotective in aged hearts, it may potentially be a therapy for treating MI in the elderly population.
Assuntos
Infarto do Miocárdio , Traumatismo por Reperfusão Miocárdica , Idoso , Animais , Humanos , Interleucinas , Camundongos , Microcirculação , Infarto do Miocárdio/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Infiltração de NeutrófilosRESUMO
Platelets play a fundamental role in hemostasis and thrombosis. They are also involved in pathologic conditions resulting from blocked blood vessels, including myocardial infarction and ischemic stroke. Platelet adhesion, activation, and aggregation at sites of vascular injury are regulated by a diverse repertoire of tyrosine kinase-linked and G protein-coupled receptors. Src family kinases (SFKs) play a central role in initiating and propagating signaling from several platelet surface receptors; however, the underlying mechanism of how SFK activity is regulated in platelets remains unclear. CD148 is the only receptor-like protein tyrosine phosphatase identified in platelets to date. In the present study, we show that mutant mice lacking CD148 exhibited a bleeding tendency and defective arterial thrombosis. Basal SFK activity was found to be markedly reduced in CD148-deficient platelets, resulting in a global hyporesponsiveness to agonists that signal through SFKs, including collagen and fibrinogen. G protein-coupled receptor responses to thrombin and other agonists were also marginally reduced. These results highlight CD148 as a global regulator of platelet activation and a novel antithrombotic drug target.
Assuntos
Ativação Plaquetária/genética , Trombose/genética , Animais , Antígenos de Superfície/metabolismo , Plaquetas/metabolismo , Adesão Celular/efeitos dos fármacos , Células Cultivadas , Fibrinogênio/farmacologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Agregação Plaquetária/efeitos dos fármacos , Glicoproteínas da Membrana de Plaquetas/metabolismo , Glicoproteínas da Membrana de Plaquetas/fisiologia , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Fosfatases/fisiologia , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/genética , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/metabolismo , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/fisiologia , Receptores de IgG/genética , Transdução de Sinais/imunologiaRESUMO
Anti-myeloperoxidase (anti-MPO) antibodies have been implicated in the pathogenesis of small-vessel vasculitis, but the molecular mechanisms by which these antibodies contribute to disease are unknown. For determination of how anti-MPO antibodies affect inflammatory cell recruitment in small-vessel vasculitis, intravital microscopy was used to monitor leukocyte behavior in the accessible cremasteric microvessels under various experimental conditions. After local pretreatment of the cremaster muscle with cytokines (TNF-alpha, IL-1beta, or keratinocyte-derived chemokine), administration of anti-MPO IgG to wild-type mice reduced leukocyte rolling in favor of augmented adhesion to and transmigration across the endothelium. This led to a decrease in the number of systemic circulating leukocytes and, similar to the early events in the development of vasculitic lesions, an increase in leukocyte recruitment to renal and pulmonary tissue. TNF-alpha led to the greatest recruitment of inflammatory cells, and IL-1beta led to the least. When anti-CD18 was co-administered, anti-MPO IgG did not affect leukocyte rolling, adhesion, or transmigration; similarly, anti-MPO IgG did not produce these effects in Fc receptor gamma chain-/- mice. This study provides direct in vivo evidence of enhanced leukocyte-endothelial cell interactions in the presence of anti-MPO IgG and highlights the critical roles of Fcgamma receptors and beta2 integrins in mediating these interactions. In addition, it suggests that neutrophils primed by cytokines in the presence of anti-MPO IgG can have systemic effects and target specific vascular beds.
Assuntos
Anticorpos Anticitoplasma de Neutrófilos/imunologia , Comunicação Celular/imunologia , Células Endoteliais/imunologia , Leucócitos/imunologia , Circulação Renal/imunologia , Vasculite/imunologia , Animais , Anticorpos Anticitoplasma de Neutrófilos/metabolismo , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Antígenos CD18/imunologia , Antígenos CD18/metabolismo , Comunicação Celular/efeitos dos fármacos , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Hemorragia/imunologia , Hemorragia/metabolismo , Hemorragia/patologia , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Interleucina-1beta/farmacologia , Glomérulos Renais/imunologia , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Migração e Rolagem de Leucócitos/efeitos dos fármacos , Migração e Rolagem de Leucócitos/imunologia , Leucócitos/citologia , Leucócitos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microcirculação/imunologia , Microcirculação/metabolismo , Peroxidase/imunologia , Peroxidase/metabolismo , Receptores de IgG/genética , Receptores de IgG/imunologia , Fator de Necrose Tumoral alfa/farmacologia , Vasculite/metabolismo , Vasculite/patologiaRESUMO
Although mortality rates from cardiovascular disease in the developed world are falling, the prevalence of cardiovascular disease (CVD) is not. Each year, the number of people either being diagnosed as suffering with CVD or undergoing a surgical procedure related to it, such as percutaneous coronary intervention, continues to increase. In order to ensure that we can effectively manage these diseases in the future, it is critical that we fully understand their basic physiology and their underlying causative factors. Over recent years, the important role of the cardiac microcirculation in both acute and chronic disorders of the heart has become clear. The recruitment of inflammatory cells into the cardiac microcirculation and their subsequent activation may contribute significantly to tissue damage, adverse remodeling, and poor outcomes during recovery. However, our basic understanding of the cardiac microcirculation is hampered by an historic inability to image the microvessels of the beating heart-something we have been able to achieve in other organs for over 100 years. This stems from a couple of clear and obvious difficulties related to imaging the heart-firstly, it has significant inherent contractile motion and is affected considerably by the movement of lungs. Secondly, it is located in an anatomically challenging position for microscopy. However, recent microscopic and technological developments have allowed us to overcome some of these challenges and to begin to answer some of the basic outstanding questions in cardiac microvascular physiology, particularly in relation to inflammatory cell recruitment. In this review, we will discuss some of the historic work that took place in the latter part of last century toward cardiac intravital, before moving onto the advanced work that has been performed since. This work, which has utilized technology such as spinning-disk confocal and multiphoton microscopy, has-along with some significant advancements in algorithms and software-unlocked our ability to image the "business end" of the cardiac vascular tree. This review will provide an overview of these techniques, as well as some practical pointers toward software and other tools that may be useful for other researchers who are considering utilizing this technique themselves.
Assuntos
Doenças Cardiovasculares/patologia , Vasos Coronários/patologia , Inflamação/imunologia , Microscopia Intravital/métodos , Algoritmos , Animais , Doenças Cardiovasculares/diagnóstico , Movimento Celular , História do Século XX , História do Século XXI , Humanos , Microscopia Intravital/história , Microcirculação , Contração MiocárdicaRESUMO
AIM: Few haematopoietic stem cells (HSCs) injected systemically for therapeutic purposes actually reach sites of injury as the vast majority become entrapped within pulmonary capillaries. One promising approach to maintain circulating HSC numbers would be to separate subpopulations with smaller size and/or greater deformability from a heterogeneous population. This study tested whether this could be achieved using label-free microfluidic devices. METHODS: 2 straight (A-B) and 3 spiral (C-E) devices were fabricated with different dimensions. Cell sorting was performed at different flow rates after which cell diameter and stiffness were determined using micromanipulation. Cells isolated using the most efficient device were tested intravitally for their ability to home to the mouse injured gut. RESULTS: Only straight Device B at a high flow rate separated HSCs with different mechanical properties. Side outlets collected mostly deformable cells (nominal rupture stress/σ R = 6.81 kPa; coefficient of variation/CV = 0.31) at a throughput of 2.3 × 105 cells/min. All spiral devices at high flow rates separated HSCs with different stiffness and size. Inner outlets collected mostly deformable cells in Devices C (σ R = 25.06 kPa; CV = 0.26), D (σ R = 22.21 kPa; CV = 0.41), and E (σ R = 29.26 kPa; CV = 0.27) at throughputs of 2.3 × 105 cells/min, 1.5 × 105 cells/min, and 1.6 × 105 cells/min, respectively. Since Device C separated cells with higher efficiency and throughput, it was utilized to test the homing ability of separated cells in vivo. Significantly more deformable cells were observed trafficking through the injured gut-interestingly, increased retention was not observed. CONCLUSION: This study applied microfluidics to separate subpopulations from one stem cell type based on their intrinsic mechanical heterogeneity. Fluid dynamics within curved devices most effectively separated HSCs. Such devices may benefit cellular therapy.