RESUMEN
Mesothelin (MSLN) is an attractive immuno-oncology target, but the development of MSLN-targeting therapies has been impeded by tumor shedding of soluble MSLN (sMSLN), on-target off-tumor activity, and an immunosuppressive tumor microenvironment. We sought to engineer an antibody-based, MSLN-targeted T-cell engager (αMSLN/αCD3) with enhanced ability to discriminate high MSLN-expressing tumors from normal tissue, and activity in the presence of sMSLN. We also studied the in vivo antitumor efficacy of this molecule (NM28-2746) alone and in combination with the multifunctional checkpoint inhibitor/T-cell co-activator NM21-1480 (αPD-L1/α4-1BB). Cytotoxicity and T-cell activation induced by NM28-2746 were studied in co-cultures of peripheral blood mononuclear cells and cell lines exhibiting different levels of MSLN expression, including in the presence of soluble MSLN. Xenotransplant models of human pancreatic cancer were used to study the inhibition of tumor growth and stimulation of T-cell infiltration into tumors induced by NM28-2746 alone and in combination with NM21-1480. The bivalent αMSLN T-cell engager NM28-2746 potently induced T-cell activation and T-cell mediated cytotoxicity of high MSLN-expressing cells but had much lower potency against low MSLN-expressing cells. A monovalent counterpart of NM28-2746 had much lower ability to discriminate high MSLN-expressing from low MSLN-expressing cells. The bivalent molecule retained this discriminant ability in the presence of high concentrations of sMSLN. In xenograft models, NM28-2746 exhibited significant tumor suppressing activity, which was significantly enhanced by combination therapy with NM21-1480. NM28-2746, alone or in combination with NM21-1480, may overcome shortcomings of previous MSLN-targeted immuno-oncology drugs, exhibiting enhanced discrimination of high MSLN-expressing cell activity in the presence of sMSLN.
Asunto(s)
Antineoplásicos , Mesotelina , Humanos , Proteínas Ligadas a GPI/genética , Linfocitos T , Leucocitos Mononucleares/metabolismo , Antineoplásicos/farmacologíaRESUMEN
P2Y12 blockade improves patient outcomes after myocardial infarction. As well as antithrombotic effects, anti-inflammatory effects may contribute to this beneficial clinical outcome. Here we aimed to identify potential anti-inflammatory effects of P2Y12 receptor blockers on monocytes and macrophages. Using flow cytometry, migration assays, flow chambers and RNA microarrays, we investigated the effects of adenosine diphosphate (ADP) and P2Y12 receptor blockers on blood monocytes, THP-1 monocytes and THP-1 monocytes after differentiation to macrophages. P2Y12 -expressing platelets can form aggregates with monocytes in circulating blood. Mediated by platelets, ADP results in activation of the integrin receptor Mac-1 on blood monocytes, as detected by the conformation-specific single-chain antibody MAN-1. Via the same association with platelets, THP-1 monocyte adhesion to the endothelial intercellular adhesion molecule 1 (ICAM-1) is induced by ADP. P2Y12 receptor blockers prevent these ADP effects on monocytes. Interestingly, in contrast to THP-1 monocytes, THP-1 monocytes, after differentiation to macrophages, directly expressed the P2Y12 receptor and consequently ADP was found to be a potent chemoattractant. Again, P2Y12 receptor blockers antagonised this effect. Accordingly, stimulation of THP-1 macrophages with ADP caused a substantial change in gene expression pattern and upregulation of several genes associated with inflammation and atherogenesis. These data establish novel anti-inflammatory effects of P2Y12 receptor blockers on monocytes and macrophages, which are expected to contribute to cardiovascular risk reduction.
Asunto(s)
Síndrome Coronario Agudo/patología , Antiinflamatorios/farmacología , Enfermedad de la Arteria Coronaria/patología , Inflamación/tratamiento farmacológico , Macrófagos/efectos de los fármacos , Monocitos/efectos de los fármacos , Antagonistas del Receptor Purinérgico P2Y/farmacología , Síndrome Coronario Agudo/sangre , Adenosina Difosfato/metabolismo , Estudios de Casos y Controles , Enfermedad de la Arteria Coronaria/sangre , Humanos , Inflamación/metabolismo , Inflamación/patología , Macrófagos/metabolismo , Monocitos/metabolismo , Fosforilación , Receptores Purinérgicos P2Y12 , Células THP-1RESUMEN
The monocyte ß2-integrin Mac-1 is crucial for leukocyte-endothelium interaction, rendering it an attractive therapeutic target for acute and chronic inflammation. Using phage display, a Designed-Ankyrin-Repeat-Protein (DARPin) was selected as a novel binding protein targeting and blocking the αM I-domain, an activation-specific epitope of Mac-1. This DARPin, named F7, specifically binds to activated Mac-1 on mouse and human monocytes as determined by flow cytometry. Homology modelling and docking studies defined distinct interaction sites which were verified by mutagenesis. Intravital microscopy showed reduced leukocyte-endothelium adhesion in mice treated with this DARPin. Using mouse models of sepsis, myocarditis and ischaemia/reperfusion injury, we demonstrate therapeutic anti-inflammatory effects. Finally, the activated Mac-1-specific DARPin is established as a tool to detect monocyte activation in patients receiving extra-corporeal membrane oxygenation, as well as suffering from sepsis and ST-elevation myocardial infarction. The activated Mac-1-specific DARPin F7 binds preferentially to activated monocytes, detects inflammation in critically ill patients, and inhibits monocyte and neutrophil function as an efficient new anti-inflammatory agent.
Asunto(s)
Antiinflamatorios/farmacología , Proteínas de Repetición de Anquirina Diseñadas/farmacología , Antígeno de Macrófago-1/metabolismo , Monocitos/efectos de los fármacos , Infarto del Miocardio/tratamiento farmacológico , Miocarditis/tratamiento farmacológico , Miocardio/metabolismo , Sepsis/tratamiento farmacológico , Animales , Técnicas de Visualización de Superficie Celular , Células Cultivadas , Proteínas de Repetición de Anquirina Diseñadas/genética , Modelos Animales de Enfermedad , Epítopos , Oxigenación por Membrana Extracorpórea , Humanos , Antígeno de Macrófago-1/genética , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/metabolismo , Simulación del Acoplamiento Molecular , Monocitos/inmunología , Monocitos/metabolismo , Infarto del Miocardio/inmunología , Infarto del Miocardio/metabolismo , Infarto del Miocardio/fisiopatología , Miocarditis/inmunología , Miocarditis/metabolismo , Miocarditis/fisiopatología , Miocardio/inmunología , Miocardio/patología , Prueba de Estudio Conceptual , Unión Proteica , Infarto del Miocardio con Elevación del ST/inmunología , Infarto del Miocardio con Elevación del ST/metabolismo , Sepsis/inmunología , Sepsis/metabolismo , Sepsis/fisiopatología , Función Ventricular Izquierda/efectos de los fármacosRESUMEN
Actin filaments are highly dynamic structures involved in many cellular processes including cell-to-cell/substrate association and cell motility. The actin cytoskeleton is tightly regulated by actin-binding proteins, which include the members of the ADF (actin-depolymerizing factor)/cofilin family. The members of the LIM kinase family of proteins (LIMK1 and 2) regulate actin dynamics by controlling the binding affinity of ADF/cofilin towards actin. LIMK2 has two major splice variants, LMK2a and LIMK2b. We have generated mice lacking LIMK2a expression (LIMK2a KO), to study its specific role in the regulation of the actin cytoskeleton. The LIMK2a KO mice showed a significant prolonged bleeding complication upon injuries compared to wild type mice. This prolonged bleeding prompted us to check the expression of the LIMK2 protein in platelets as it was previously suggested that it is not expressed in platelets. We showed that human and mouse express LIMK2 in platelets and using our LIMK2a KO mice we have identified a potential key role for LIMK2 in platelet functions including platelet spreading, aggregation and thrombus formation.
Asunto(s)
Plaquetas/metabolismo , Quinasas Lim/metabolismo , Agregación Plaquetaria , Citoesqueleto de Actina/metabolismo , Animales , Plaquetas/fisiología , Células Cultivadas , Humanos , Quinasas Lim/genética , Ratones , Ratones Endogámicos C57BLRESUMEN
BACKGROUND: Microvesicles (MVs) are small cell-derived vesicles, which are mainly released by activated cells. They are part of a communication network delivering biomolecules, for example, inflammatory molecules, via the blood circulation to remote cells in the body. Platelet-derived MVs are known to induce vascular inflammation. Research on the mediators and mechanisms of their inflammatory effects has attracted major interest. We hypothesize that specific lipids are the mediators of vascular inflammation caused by platelet-derived MVs. METHODS AND RESULTS: Liquid chromatography electrospray ionization-tandem mass spectrometry was used for lipid profiling of platelet-derived MVs. Lysophosphatidylcholine (LPC) was found to be a major component of platelet-derived MVs. Investigating the direct effects of LPC, we found that it induces platelet activation, spreading, migration and aggregation as well as formation of inflammatory platelet-monocyte aggregates. We show for the first time that platelets express the LPC receptor G2AR, which mediates LPC-induced platelet activation. In a mouse model of atherosclerotic plaque instability/rupture, circulating LPC was detected as a surrogate marker of plaque instability. These findings were confirmed by matrix-assisted laser desorption ionization imaging, which showed that the LPC concentration of human plaques was highest in vulnerable plaque regions. CONCLUSION: LPC is a major component of platelet-derived MVs and via its interaction with G2AR on platelets contributes to platelet activation, spreading, migration and aggregation and ultimately to vascular inflammation. Circulating LPC reports on atherosclerotic plaque instability in mice and is significantly increased in unstable areas of atherosclerotic plaques in both mice and humans, linking LPC to plaque instability.
Asunto(s)
Aterosclerosis/metabolismo , Plaquetas/metabolismo , Micropartículas Derivadas de Células/metabolismo , Lisofosfatidilcolinas/análisis , Animales , Movimiento Celular , Células Cultivadas , Regulación de la Expresión Génica , Humanos , Inflamación , Lípidos/química , Espectrometría de Masas , Ratones , Microscopía Fluorescente , Monocitos/citología , Permeabilidad , Placa Aterosclerótica/metabolismo , Activación Plaquetaria , Agregación PlaquetariaRESUMEN
Actin is highly abundant in platelets, and its function is dependent on its structure. Actin filaments (F-actin) are dynamic structures involved in many cellular processes including platelet shape changes and adhesion. The actin cytoskeleton is tightly regulated by actin-binding proteins, which include members of the actin depolymerising factor (ADF)/cofilin family. LIM kinase (LIMK) and its phosphatase slingshot (SSH-1L) regulate actin dynamics by controlling the binding affinity of ADF/cofilin towards actin. We hypothesised that the inhibition of LIMK activity may prevent the changes in platelet shape and their function during activation by controlling the dynamics of F-actin. Our results demonstrate that in platelet, inhibition of LIMK by small LIMK inhibitors controls the level of filamentous actin leading to decreased platelet adhesion and aggregation. These findings encourage further studies on controlling platelet function via the cytoskeleton.
Asunto(s)
Plaquetas/metabolismo , Quinasas Lim/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Transducción de Señal , Terapia Trombolítica , Citoesqueleto de Actina/efectos de los fármacos , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animales , Plaquetas/efectos de los fármacos , Cofilina 1/metabolismo , Citoesqueleto/efectos de los fármacos , Citoesqueleto/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Hemorragia/tratamiento farmacológico , Humanos , Quinasas Lim/metabolismo , Ratones , Fosfoproteínas Fosfatasas/metabolismo , Fosforilación/efectos de los fármacos , Adhesividad Plaquetaria/efectos de los fármacos , Inhibidores de Proteínas Quinasas/uso terapéutico , Cola (estructura animal) , Trombosis/tratamiento farmacológico , Quinasas Asociadas a rho/antagonistas & inhibidores , Quinasas Asociadas a rho/metabolismoRESUMEN
High mobility group box 1 (HMGB1) acts as both a nuclear protein that regulates gene expression, as well as a pro-inflammatory alarmin that is released from necrotic or activated cells. Recently, HMGB1-expression in human atherosclerotic plaques was identified. Therapeutic blockade of HMGB1 reduced the development of diet-induced atherosclerosis in ApoE knockout mice. Thus, we hypothesised an interaction between HMGB1 and activated platelets. Binding of recombinant HMGB1 to platelets was assessed by flow cytometry. HMGB1 bound to thrombin-activated human platelets (MFI 2.49 vs 25.01, p=0.0079). Blood from wild-type, TLR4 and RAGE knockout mice was used to determine potential HMGB1 receptors on platelets. HMGB1 bound to platelets from wild type C57Bl6 (MFI 2.64 vs 20.3, p< 0.05), and TLR4-/- mice (MFI 2.11 vs 25.65, p< 0.05) but failed to show binding to platelets from RAGE-/- mice (p > 0.05). RAGE expression on human platelets was detected by RT-PCR with mRNA extracted from highly purified platelets and confirmed by Western blot and immunofluorescence microscopy. Platelet activation increased RAGE surface expression (MFI 4.85 vs 6.74, p< 0.05). Expression of HMGB1 in human coronary artery thrombi was demonstrated by immunohistochemistry and revealed high expression levels. Platelets bind HMGB1 upon thrombin-induced activation. Platelet specific expression of RAGE could be detected at the mRNA and protein level and is involved in the binding of HMGB1. Furthermore, platelet activation up-regulates platelet surface expression of RAGE. HMGB1 is highly expressed in platelet-rich human coronary artery thrombi pointing towards a central role for HMGB1 in atherothrombosis, thereby suggesting the possibility of platelet targeted anti-inflammatory therapies for atherothrombosis.
Asunto(s)
Plaquetas/fisiología , Trombosis Coronaria/metabolismo , Vasos Coronarios/metabolismo , Proteína HMGB1/metabolismo , Receptor para Productos Finales de Glicación Avanzada/metabolismo , Animales , Células Cultivadas , Vasos Coronarios/patología , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Activación Plaquetaria , Unión Proteica , Receptor para Productos Finales de Glicación Avanzada/genética , Trombina/metabolismo , Receptor Toll-Like 4/genéticaRESUMEN
BACKGROUND: Costimulatory cascades such as the CD40L-CD40 dyad enhance immune cell activation and inflammation during atherosclerosis. Here, we tested the hypothesis that CD40 directly modulates traits of the metabolic syndrome in diet-induced obesity in mice. METHODS AND RESULTS: To induce the metabolic syndrome, wild-type or CD40(-/-) mice consumed a high-fat diet for 20 weeks. Unexpectedly, CD40(-/-) mice exhibited increased weight gain, impaired insulin secretion, augmented accumulation of inflammatory cells in adipose tissue, and enhanced proinflammatory gene expression. This proinflammatory and adverse metabolic phenotype could be transplanted into wild-type mice by reconstitution with CD40-deficient lymphocytes, indicating a major role for CD40 in T or B cells in this context. Conversely, therapeutic activation of CD40 signaling by the stimulating antibody FGK45 abolished further weight gain during the study, lowered glucose levels, improved insulin sensitivity, and suppressed adipose tissue inflammation. Mechanistically, CD40 activation decreased the expression of proinflammatory cytokines in T cells but not in B cells or macrophages. Finally, repopulation of lymphocyte-free Rag1(-/-) mice with CD40(-/-) T cells provoked dysmetabolism and inflammation, corroborating a protective role of CD40 on T cells in the metabolic syndrome. Finally, levels of soluble CD40 showed a positive association with obesity in humans, suggesting clinical relevance of our findings. CONCLUSIONS: We present the surprising finding that CD40 deficiency on T cells aggravates whereas activation of CD40 signaling improves adipose tissue inflammation and its metabolic complications. Therefore, positive modulation of the CD40 pathway might describe a novel therapeutic concept against cardiometabolic disease.
Asunto(s)
Tejido Adiposo/inmunología , Aterosclerosis/inmunología , Antígenos CD40/genética , Antígenos CD40/inmunología , Síndrome Metabólico/inmunología , Obesidad/inmunología , Adipocitos/inmunología , Adipocitos/metabolismo , Traslado Adoptivo , Animales , Aterosclerosis/genética , Aterosclerosis/metabolismo , Ligando de CD40/inmunología , Ligando de CD40/metabolismo , Humanos , Inflamación/genética , Inflamación/inmunología , Inflamación/metabolismo , Resistencia a la Insulina/genética , Resistencia a la Insulina/inmunología , Activación de Linfocitos/inmunología , Masculino , Síndrome Metabólico/genética , Síndrome Metabólico/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Obesidad/genética , Obesidad/metabolismo , Transducción de Señal/inmunología , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
Previous studies have reported that C-reactive protein (CRP) interacting with low-density lipoproteins (LDL) affects macrophage activation and LDL uptake. However, the physiological relevance of CRP-LDL interaction with circulating monocytes remains elusive. Moreover, recent studies have shown that CRP exists in two isoforms with partly opposing characteristics pentameric (pCRP) and monomeric CRP (mCRP). Here we investigated the effects of CRP interacting with minimally modified low-density lipoprotein (mmLDL) interaction in regard to events involved in formation of atherosclerotic plaque. We analyzed the effect of mmLDL on human monocytes and found a substantial increase in monocyte activation as evaluated by CD11b/CD18 expression and increased monocyte adhesion under static and under shear flow conditions to human endothelial cells. Monocyte adhesion and activation was attenuated by pCRP via the prevention of mmLDL binding to monocytes. These anti-inflammatory properties of pCRP were lost when it dissociates to the monomeric form. Our results elucidate the physiological relevance of the CRP-mmLDL interaction and furthermore confirm the importance of the previously described pCRP dissociation to mCRP as a localized inflammatory "activation" mechanism.
Asunto(s)
Aterosclerosis/metabolismo , Proteína C-Reactiva/metabolismo , Mediadores de Inflamación/metabolismo , Inflamación/prevención & control , Lipoproteínas LDL/metabolismo , Monocitos/metabolismo , Aterosclerosis/inmunología , Proteína C-Reactiva/química , Antígeno CD11b/metabolismo , Antígenos CD18/metabolismo , Adhesión Celular , Células Cultivadas , Técnicas de Cocultivo , Células Endoteliales/inmunología , Células Endoteliales/metabolismo , Humanos , Inflamación/inmunología , Inflamación/metabolismo , Mediadores de Inflamación/química , Monocitos/inmunología , Unión Proteica , Conformación Proteica , Factores de TiempoRESUMEN
AIMS: Elevated serum C-reactive protein (CRP) following myocardial infarction (MI) is associated with poor outcomes. Although animal studies have indicated a direct pathogenic role of CRP, the mechanism underlying this remains elusive. Dissociation of pentameric CRP (pCRP) into pro-inflammatory monomers (mCRP) may directly link CRP to inflammation. We investigated whether cellular microparticles (MPs) can convert pCRP to mCRP and transport mCRP following MI. METHODS AND RESULTS: MPs enriched in lysophosphatidylcholine were obtained from cell cultures and patient whole-blood samples collected following acute MI and control groups. Samples were analysed by native western blotting and flow cytometry. MPs were loaded with mCRP in vitro and incubated with endothelial cells prior to staining with monoclonal antibodies. In vitro experiments demonstrated that MPs were capable of converting pCRP to mCRP which could be inhibited by the anti-CRP compound 1,6 bis-phosphocholine. Significantly more mCRP was detected on MPs from patients following MI compared with control groups by western blotting and flow cytometry (P = 0.0005 for association). MPs containing mCRP were able to bind to the surface of endothelial cells and generate pro-inflammatory signals in vitro, suggesting a possible role of MPs in transport and delivery of pro-inflammatory mCRP in vascular disease. CONCLUSION: Circulating MPs can convert pCRP to pro-inflammatory mCRP in patients following MI, demonstrating for the first time mCRP generation in vivo and its detection in circulating blood. MPs can bind to cell membranes and transfer mCRP to the cell surface, suggesting a possible mCRP transport/delivery role of MPs in the circulation.
Asunto(s)
Proteína C-Reactiva/metabolismo , Micropartículas Derivadas de Células/metabolismo , Lisofosfolípidos/metabolismo , Infarto del Miocardio/sangre , Anciano , Anciano de 80 o más Años , Línea Celular , Células Endoteliales/metabolismo , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
BACKGROUND: Adipose tissue inflammation fuels the metabolic syndrome. We recently reported that CD40L--an established marker and mediator of cardiovascular disease--induces inflammatory cytokine production in adipose cells in vitro. Here, we tested the hypothesis that CD40L deficiency modulates adipose tissue inflammation in vivo. METHODOLOGY/PRINCIPAL FINDINGS: WT or CD40L(-/-) mice consumed a high fat diet (HFD) for 20 weeks. Inflammatory cell recruitment was impaired in mice lacking CD40L as shown by a decrease of adipose tissue macrophages, B-cells, and an increase in protective T-regulatory cells. Mechanistically, CD40L-deficient mice expressed significantly lower levels of the pro-inflammatory chemokine MCP-1 both, locally in adipose tissue and systemically in plasma. Moreover, levels of pro-inflammatory IgG-antibodies against oxidized lipids were reduced in CD40L(-/-) mice. Also, circulating low-density lipoproteins and insulin levels were lower in CD40L(-/-) mice. However, CD40L(-/-) mice consuming HFD were not protected from the onset of diet-induced obesity (DIO), insulin resistance, and hepatic steatosis, suggesting that CD40L selectively limits the inflammatory features of diet-induced obesity rather than its metabolic phenotype. Interestingly, CD40L(-/-) mice consuming a low fat diet (LFD) showed both, a favorable inflammatory and metabolic phenotype characterized by diminished weight gain, improved insulin tolerance, and attenuated plasma adipokine levels. CONCLUSION: We present the novel finding that CD40L deficiency limits adipose tissue inflammation in vivo. These findings identify CD40L as a potential mediator at the interface of cardiovascular and metabolic disease.
Asunto(s)
Autoanticuerpos/inmunología , Subgrupos de Linfocitos B/inmunología , Ligando de CD40/deficiencia , Ligando de CD40/inmunología , Dieta/efectos adversos , Inmunoglobulina G/inmunología , Paniculitis/etiología , Animales , Autoanticuerpos/sangre , Subgrupos de Linfocitos B/metabolismo , Quimiocinas/genética , Metabolismo Energético/genética , Hígado Graso/genética , Regulación de la Expresión Génica , Inmunoglobulina G/sangre , Resistencia a la Insulina/genética , Metabolismo de los Lípidos , Lípidos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Obesidad/etiología , Oxidación-Reducción , Paniculitis/genética , Paniculitis/inmunologíaRESUMEN
AIMS: Circulating microRNAs (miRNAs) have attracted major interest as biomarkers for cardiovascular diseases. Since RNases are abundant in circulating blood, there needs to be a mechanism protecting miRNAs from degradation. We hypothesized that microparticles (MP) represent protective transport vehicles for miRNAs and that these are specifically packaged by their maternal cells. METHODS AND RESULTS: Conventional plasma preparations, such as the ones used for biomarker detection, are shown to contain substantial numbers of platelet-, leucocyte-, and endothelial cell-derived MP. To analyse the widest spectrum of miRNAs, Next Generation Sequencing was used to assess miRNA profiles of MP and their corresponding stimulated and non-stimulated cells of origin. THP-1 (monocytic origin) and human umbilical vein endothelial cell (HUVEC) MP were used for representing circulating MP at a high purity. miRNA profiles of MP differed significantly from those of stimulated and non-stimulated maternal THP-1 cells and HUVECs, respectively. Quantitative reverse transcription-polymerase chain reaction of miRNAs which have been associated with cardiovascular diseases also demonstrated significant differences in miRNA profiles between platelets and their MP. Notably, the main fraction of miRNA in plasma was localized in MP. Furthermore, miRNA profiles of MP differed significantly between patients with stable and unstable coronary artery disease. CONCLUSION: Circulating MP represent transport vehicles for large numbers of specific miRNAs, which have been associated with cardiovascular diseases. miRNA profiles of MP are significantly different from their maternal cells, indicating an active mechanism of selective 'packaging' from cells into MP. These findings describe an interesting mechanism for transferring gene-regulatory function from MP-releasing cells to target cells via MP circulating in blood.
Asunto(s)
Micropartículas Derivadas de Células/fisiología , Enfermedad de la Arteria Coronaria/sangre , Enfermedad de la Arteria Coronaria/fisiopatología , MicroARNs/sangre , Índice de Severidad de la Enfermedad , Anciano , Transporte Biológico/fisiología , Biomarcadores/sangre , Plaquetas/citología , Plaquetas/efectos de los fármacos , Plaquetas/patología , Células Cultivadas , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Femenino , Perfilación de la Expresión Génica , Humanos , Lipopolisacáridos/farmacología , Masculino , Persona de Mediana Edad , Monocitos/citología , Monocitos/efectos de los fármacos , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Beta-amyloid (Aß) plaques and local inflammation are central to the pathogenesis of Alzheimer's disease. Although an association between circulating pentameric C-reactive protein (pCRP) and Alzheimer's disease has been reported no pathomechanistic link has been established. We hypothesized that Aß plaques induce the dissociation of pCRP to individual monomers (mCRP), which possess strong pro-inflammatory properties not shared with pCRP and localizing inflammation to Alzheimer's plaques. pCRP was incubated with Aß plaques generated in vitro and with non-aggregated Aß(42) peptide. pCRP dissociation to mCRP was found only when co-incubated with Aß plaques. Furthermore, sections of frontal cortex from brains of patients with and without Alzheimer's disease were stained with antibodies specific for mCRP and pCRP. There was significantly more mCRP in the cortex of Alzheimer's disease patients (P ≤ 0.01). In contrast, there was no significant difference in pCRP staining. These findings establish that Aß plaques possess a previously unrecognized potential to dissociate pentameric CRP to monomeric CRP. The existence of mCRP but not pCRP in the brains of Alzheimer's disease patients strongly indicates that this newly described biological effect of Aß plaques is relevant in Alzheimer pathobiology; potentially localizing and amplifying inflammation via the strong pro-inflammatory effects of locally generated mCRP.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Encéfalo/metabolismo , Proteína C-Reactiva/metabolismo , Placa Amiloide/metabolismo , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/patología , Encéfalo/patología , Proteína C-Reactiva/química , Femenino , Humanos , Inflamación , Masculino , Placa Amiloide/patologíaRESUMEN
RATIONALE: CD40L figures prominently in chronic inflammatory diseases such as atherosclerosis. However, since CD40L potently regulates immune function and hemostasis by interaction with CD40 receptor and the platelet integrin GPIIb/IIIa, its global inhibition compromises host defense and generated thromboembolic complications in clinical trials. We recently reported that CD40L mediates atherogenesis independently of CD40 and proposed Mac-1 as an alternate receptor. OBJECTIVE: Here, we molecularly characterized the CD40L-Mac-1 interaction and tested whether its selective inhibition by a small peptide modulates inflammation and atherogenesis in vivo. METHODS AND RESULTS: CD40L concentration-dependently bound to Mac-1 I-domain in solid phase binding assays, and a high-affinity interaction was revealed by surface-plasmon-resonance analysis. We identified the motif EQLKKSKTL, an exposed loop between the α1 helix and the ß-sheet B, on Mac-1 as binding site for CD40L. A linear peptide mimicking this sequence, M7, specifically inhibited the interaction of CD40L and Mac-1. A cyclisized version optimized for in vivo use, cM7, decreased peritoneal inflammation and inflammatory cell recruitment in vivo. Finally, LDLr(-/-) mice treated with intraperitoneal injections of cM7 developed smaller, less inflamed atherosclerotic lesions featuring characteristics of stability. However, cM7 did not interfere with CD40L-CD40 binding in vitro and CD40L-GPIIb/IIIa-mediated thrombus formation in vivo. CONCLUSIONS: We present the novel finding that CD40L binds to the EQLKKSKTL motif on Mac-1 mediating leukocyte recruitment and atherogenesis. Specific inhibition of CD40L-Mac-1 binding may represent an attractive anti-inflammatory treatment strategy for atherosclerosis and other inflammatory conditions, potentially avoiding the unwanted immunologic and thrombotic effects of global inhibition of CD40L.
Asunto(s)
Aterosclerosis/metabolismo , Ligando de CD40/metabolismo , Quimiotaxis de Leucocito/fisiología , Antígeno de Macrófago-1/metabolismo , Trombosis/etiología , Secuencias de Aminoácidos , Animales , Aterosclerosis/genética , Aterosclerosis/prevención & control , Tiempo de Sangría , Coagulación Sanguínea/efectos de los fármacos , Coagulación Sanguínea/fisiología , Células CHO , Células Cultivadas , Cricetinae , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Moleculares , Fragmentos de Péptidos/farmacología , Fragmentos de Péptidos/uso terapéutico , Péptidos Cíclicos/farmacología , Peritonitis/sangre , Peritonitis/prevención & control , Conformación Proteica , Mapeo de Interacción de Proteínas , Estructura Terciaria de Proteína , Receptores de LDL/deficiencia , Proteínas Recombinantes de Fusión/fisiología , Resonancia por Plasmón de SuperficieRESUMEN
BACKGROUND: Staphylococcus aureus (S. aureus) is a common pathogen capable of causing life-threatening infections. Staphylococcal superantigen-like protein 5 (SSL5) has recently been shown to bind to platelet glycoproteins and induce platelet activation. This study investigates further the interaction between SSL5 and platelet glycoproteins. Moreover, using a glycan discovery approach, we aim to identify potential glycans to therapeutically target this interaction and prevent SSL5-induced effects. METHODOLOGY/PRINCIPAL FINDINGS: In addition to platelet activation experiments, flow cytometry, immunoprecipitation, surface plasmon resonance and a glycan binding array, were used to identify specific SSL5 binding regions and mediators. We independently confirm SSL5 to interact with platelets via GPIbα and identify the sulphated-tyrosine residues as an important region for SSL5 binding. We also identify the novel direct interaction between SSL5 and the platelet collagen receptor GPVI. Together, these receptors offer one mechanistic explanation for the unique functional influences SSL5 exerts on platelets. A role for specific families of platelet glycans in mediating SSL5-platelet interactions was also discovered and used to identify and demonstrate effectiveness of potential glycan based inhibitors in vitro. CONCLUSIONS/SIGNIFICANCE: These findings further elucidate the functional interactions between SSL5 and platelets, including the novel finding of a role for the GPVI receptor. We demonstrate efficacy of possible glycan-based approaches to inhibit the SSL5-induced platelet activation. Our data warrant further work to prove SSL5-platelet effects in vivo.
Asunto(s)
Plaquetas/efectos de los fármacos , Glicoproteínas de Membrana/metabolismo , Activación Plaquetaria/efectos de los fármacos , Glicoproteínas de Membrana Plaquetaria/metabolismo , Polisacáridos/farmacología , Staphylococcus aureus/metabolismo , Superantígenos/farmacología , Plaquetas/metabolismo , Secuencia de Carbohidratos , Relación Dosis-Respuesta a Droga , Epítopos/metabolismo , Células HL-60 , Humanos , Glicoproteínas de Membrana/química , Datos de Secuencia Molecular , Complejo GPIb-IX de Glicoproteína Plaquetaria , Polisacáridos/química , Polisacáridos/metabolismo , Especificidad por Sustrato , Sulfatos/metabolismo , Superantígenos/química , Superantígenos/metabolismo , Tirosina/metabolismoRESUMEN
OBJECTIVE: Hypothermia is used in various clinical settings to inhibit ischemia-related organ damage. However, prothrombotic effects have been described as potential side effects. This study aimed to elucidate the mechanism of hypothermia-induced platelet activation and subsequent prothrombotic events and to develop preventative pharmacological strategies applicable during clinically used hypothermia. METHODS AND RESULTS: Platelet function was investigated ex vivo and in vivo at clinically used hypothermia (28°C/18°C). Hypothermic mice demonstrated increased expression of platelet activation marker P-selectin, platelet-leukocyte aggregate formation, and thrombocytopenia. Intravital microscopy of FeCl(3)-injured murine mesenteric arteries revealed increased platelet thrombus formation with hypothermia. Ex vivo flow chamber experiments indicated increased platelet-fibrinogen adhesion under hypothermia. We show that hypothermia results in reduced ADP hydrolysis via reduction of CD39 (E-NTPDase1) activity, resulting in increased levels of ADP and subsequent augmented primary and secondary platelet activation. In vivo administration of ADP receptor P(2)Y(12) antagonists and recombinant soluble CD39 prevented hypothermia-induced thrombus formation and thrombocytopenia, respectively. CONCLUSIONS: The platelet agonist ADP plays a key role in hypothermia-induced platelet activation. Inhibition of receptor binding or hydrolysis of ADP has the potential to protect platelets against hypothermia-induced activation. Our findings provide a rational basis for further evaluation of novel antithrombotic strategies in clinically applied hypothermia.
Asunto(s)
Adenosina Difosfato/sangre , Plaquetas/efectos de los fármacos , Fibrinolíticos/farmacología , Hipotermia Inducida , Activación Plaquetaria/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/farmacología , Trombosis/prevención & control , Análisis de Varianza , Animales , Antígenos CD/sangre , Antígenos CD/farmacología , Apirasa/sangre , Apirasa/farmacología , Plaquetas/metabolismo , Fibrinógeno/metabolismo , Humanos , Hidrólisis , Hipotermia Inducida/efectos adversos , Leucopenia/sangre , Leucopenia/etiología , Glicoproteínas de Membrana/sangre , Ratones , Ratones Endogámicos C57BL , Selectina-P/sangre , Adhesividad Plaquetaria/efectos de los fármacos , Complejo GPIb-IX de Glicoproteína Plaquetaria , Antagonistas del Receptor Purinérgico P2Y/farmacología , Receptores Purinérgicos P2Y1/sangre , Receptores Purinérgicos P2Y1/efectos de los fármacos , Receptores Purinérgicos P2Y12/sangre , Receptores Purinérgicos P2Y12/efectos de los fármacos , Proteínas Recombinantes/farmacología , Trombocitopenia/sangre , Trombocitopenia/etiología , Trombosis/sangre , Trombosis/etiología , Factor de von Willebrand/metabolismoRESUMEN
The adhesion of leukocytes to endothelium plays a central role in the development of atherosclerosis and thus represents an attractive therapeutic target for anti-atherosclerotic therapies. Vascular cell adhesion molecule-1 (VCAM-1) mediates both the initial tethering and the firm adhesion of leukocytes to endothelial cells. Our work evaluates the feasibility of using the cytoskeletal anchorage of VCAM-1 as a target for gene therapy. As a proof of concept, integrin alphaIIbbeta3-mediated cell adhesion with clearly defined cytoskeletal anchorage was tested. We constructed fusion proteins containing the intracellular domain of beta3 placed at various distances to the cell membrane. Using cell adhesion assays and immunofluorescence, we established fusion constructs with competitive and dominant negative inhibition of cell adhesion. With the goal being the transfer of the dominant negative mechanism towards VCAM-1 inhibition, we constructed a fusion molecule containing the cytoplasmic domain of VCAM-1. Indeed, VCAM-1 mediated leukocyte adhesion can be inhibited via transfection of DNA encoding the designed VCAM-1 fusion protein. This is demonstrated in adhesion assays under static and flow conditions using CHO cells expressing recombinant VCAM-1 as well as activated endothelial cells. Thus, we are able to describe a novel approach for dominant negative inhibition of leukocyte adhesion to endothelial cells. This approach warrants further development as a novel gene therapeutic strategy that aims for a locally restricted effect at atherosclerotic areas of the vasculature.
Asunto(s)
Adhesión Celular/fisiología , Movimiento Celular/fisiología , Citoesqueleto/metabolismo , Técnicas de Transferencia de Gen , Proteínas Recombinantes/metabolismo , Molécula 1 de Adhesión Celular Vascular/metabolismo , Animales , Antígenos CD7/genética , Antígenos CD7/metabolismo , Células CHO , Cricetinae , Cricetulus , Selectina E/genética , Selectina E/metabolismo , Humanos , Molécula 1 de Adhesión Intercelular/genética , Molécula 1 de Adhesión Intercelular/metabolismo , Monocitos/citología , Monocitos/metabolismo , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/genética , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Proteínas Recombinantes/genética , Molécula 1 de Adhesión Celular Vascular/genéticaRESUMEN
Celastrol is an active ingredient of the traditional Chinese medicinal plant, Tripterygium wilfordii Hook F, which is known especially for its anti-inflammatory effects. However, on the cellular and molecular levels, celastrol's mechanism of action is only poorly understood. Because platelets contribute to inflammatory events, this study investigates the effects of celastrol on platelet function using flow cytometry, aggregometry, and adhesion assays. In in vitro experiments with human platelets, celastrol inhibits adenosine-5-diphosphate (ADP)-induced expression of the platelet activation marker P-selectin and glycoprotein IIb/IIIa activation with 50% inhibition values of 1.62 and 1.86 microM, respectively. Celastrol also inhibits thrombin-stimulated and phorbol 12-myristate 13-acetate-stimulated P-selectin expression on platelets. Furthermore, ADP-stimulated platelet adhesion on fibrinogen is partially prevented by 5 microM celastrol. In platelet aggregometry, celastrol (0.05-0.5 mM) inhibits ADP-induced aggregation of platelet-rich plasma. Moreover, 12 male C57BL/6J mice were randomly grouped to receive intraperitoneal treatment with either celastrol (2 mg x kg x day) or vehicle. After 4 weeks of the respective treatment, celastrol inhibited 2 and 20 microM ADP-stimulated platelet fibrinogen binding by 34.5% (P < 0.01) and 28.9% (P < 0.05), respectively, compared with controls. In conclusion, these results indicate that celastrol exerts inhibitory effects on platelets. This new finding contributes to the understanding of antithrombotic and also anti-inflammatory effects of celastrol.
Asunto(s)
Activación Plaquetaria/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/farmacología , Tripterygium/química , Triterpenos/farmacología , Adulto , Animales , Antiinflamatorios no Esteroideos/farmacología , Adhesión Celular/efectos de los fármacos , Medicamentos Herbarios Chinos/farmacología , Femenino , Citometría de Flujo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Concentración Osmolar , Selectina-P/metabolismo , Triterpenos Pentacíclicos , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Distribución AleatoriaRESUMEN
C-reactive protein (CRP) is a predictor of cardiovascular risk. It circulates as a pentamer (pentameric CRP) in plasma. The in vivo existence of monomeric (m)CRP has been postulated, but its function and source are not clear. We show that mCRP is deposited in human aortic and carotid atherosclerotic plaques but not in healthy vessels. pCRP is found neither in healthy nor in diseased vessels. As source of mCRP, we identify a mechanism of dissociation of pCRP to mCRP. We report that activated platelets, which play a central role in cardiovascular events, mediate this dissociation via lysophosphatidylcholine, which is present on activated but not resting platelets. Furthermore, the dissociation of pCRP to mCRP can also be mediated by apoptotic monocytic THP-1 and Jurkat T cells. The functional consequence is the unmasking of proinflammatory effects of CRP as demonstrated in experimental settings that are pathophysiologically relevant for atherogenesis: compared to pCRP, mCRP induces enhanced monocyte chemotaxis; monocyte activation, as determined by conformational change of integrin Mac-1; generation of reactive oxygen species; and monocyte adhesion under static and physiological flow conditions. In conclusion, we demonstrate mCRP generation via pCRP dissociation on activated platelets and H(2)O(2)-treated apoptotic THP-1 and Jurkat T cells, thereby identifying a mechanism of localized unmasking of the proinflammatory properties of CRP. This novel mechanism provides a potential link between the established cardiovascular risk marker, circulating pCRP, and localized platelet-mediated inflammatory and proatherogenic effects.
Asunto(s)
Enfermedades de la Aorta/sangre , Aterosclerosis/sangre , Plaquetas/metabolismo , Proteína C-Reactiva/metabolismo , Enfermedades de las Arterias Carótidas/sangre , Mediadores de Inflamación/sangre , Inflamación/sangre , Activación Plaquetaria , Enfermedades de la Aorta/inmunología , Enfermedades de la Aorta/patología , Apoptosis , Aterosclerosis/inmunología , Aterosclerosis/patología , Autopsia , Plaquetas/inmunología , Proteína C-Reactiva/química , Enfermedades de las Arterias Carótidas/inmunología , Enfermedades de las Arterias Carótidas/patología , Adhesión Celular , Quimiotaxis de Leucocito , Células Endoteliales/metabolismo , Humanos , Inflamación/inmunología , Inflamación/patología , Mediadores de Inflamación/química , Células Jurkat , Lisofosfatidilcolinas/sangre , Antígeno de Macrófago-1/metabolismo , Lípidos de la Membrana/sangre , Microdominios de Membrana/metabolismo , Monocitos/metabolismo , Conformación Proteica , Especies Reactivas de Oxígeno/metabolismo , Receptores de IgG/metabolismo , Proteínas Recombinantes/metabolismo , Relación Estructura-Actividad , Factores de TiempoRESUMEN
OBJECTIVE: The Jarvik 2000 (Jarvik Heart, Inc, New York, NY) is a thumb-sized high-speed impeller pump that is used as a ventricular assist device in patients with terminal heart failure. Because the Jarvik 2000 is designed for long-term use, it is a central question whether the mechanical forces inside the pump affect blood components. This study evaluated the potential association of the high rotational speed of the Jarvik 2000 with platelet activation, which may result in thromboembolic events. METHODS: The study group comprised patients with terminal heart failure who were supported with the Jarvik 2000. All were men and received 100 mg aspirin daily. In 8 patients, soluble platelet activation markers (P-selectin and sCD40L), platelet counts, and hemolysis markers (haptoglobin and lactate dehydrogenase levels) were determined. In 5 patients, P-selectin expression and platelet receptor glycoprotein IIb/IIIa activation were determined with flow cytometry and compared with a control group of 5 healthy men. Platelet activation was measured at various rotational device speeds. RESULTS: After Jarvik 2000 implantation, increased hemolysis was observed, but platelet activation markers and platelet counts were not affected. Increased rotational speed (8000 to 12,000 rpm) of the device also did not result in increased platelet activation. CONCLUSION: The Jarvik 2000 was not associated with detectable platelet activation, despite high rotational impeller speeds.