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COVID-19 , Monocitos , SARS-CoV-2 , Tromboplastina , Trombosis , Humanos , COVID-19/inmunología , COVID-19/sangre , COVID-19/complicaciones , Monocitos/inmunología , Monocitos/metabolismo , Tromboplastina/metabolismo , Trombosis/sangre , Trombosis/inmunología , Trombosis/etiología , SARS-CoV-2/inmunologíaRESUMEN
Neutrophils can contribute to inflammatory disease propagation via innate mechanisms intended for inflammation resolution. For example, neutrophil extracellular traps (NETs) are necessary for trapping pathogens but can contribute to clot formation and blood flow restriction, that is, ischemia. Currently, no therapeutics in the clinic directly target NETs despite the known involvement of NETs contributing to mortality and increased disease severity. Vascular-deployed particle-based therapeutics are a novel and robust alternative to traditional small-molecule drugs by enhancing drug delivery to cells of interest. This work designs a high-throughput assay to investigate the immunomodulatory behavior and functionality of salicylic acid-based polymer-based particle therapeutics against NETosis in human neutrophils. Briefly, this work finds that polymeric composition plays a role, and particle size can also influence rates of NETosis. Salicylate-based polymeric (Poly-SA) particles are found to functionally inhibit NETosis depending on the particle size and concentration exposed to neutrophils. This work demonstrates the high throughput method can help fast-track particle-based therapeutic optimization and design, more efficiently preparing this innovative therapeutics for the clinic.
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BACKGROUND: SARS-CoV-2 infections cause COVID-19 and are associated with inflammation, coagulopathy, and high incidence of thrombosis. Myeloid cells help coordinate the initial immune response in COVID-19. Although we appreciate that myeloid cells lie at the nexus of inflammation and thrombosis, the mechanisms that unite the two in COVID-19 remain largely unknown. METHODS: In this study, we used systems biology approaches including proteomics, transcriptomics, and mass cytometry to define the circulating proteome and circulating immune cell phenotypes in subjects with COVID-19. RESULTS: In a cohort of subjects with COVID-19 (n=35), circulating markers of inflammation (CCL23 [C-C motif chemokine ligand 23] and IL [interleukin]-6) and vascular dysfunction (ACE2 [angiotensin-converting enzyme 2] and TF [tissue factor]) were elevated in subjects with severe compared with mild COVID-19. Additionally, although the total white blood cell counts were similar between COVID-19 groups, CD14+ (cluster of differentiation) monocytes from subjects with severe COVID-19 expressed more TF. At baseline, transcriptomics demonstrated increased IL-6, CCL3, ACOD1 (aconitate decarboxylase 1), C5AR1 (complement component 5a receptor), C5AR2, and TF in subjects with severe COVID-19 compared with controls. Using stress transcriptomics, we found that circulating immune cells from subjects with severe COVID-19 had evidence of profound immune paralysis with greatly reduced transcriptional activation and release of inflammatory markers in response to TLR (Toll-like receptor) activation. Finally, sera from subjects with severe (but not mild) COVID-19 activated human monocytes and induced TF expression. CONCLUSIONS: Taken together, these observations further elucidate the pathological mechanisms that underlie immune dysfunction and coagulation abnormalities in COVID-19, contributing to our growing understanding of SARS-CoV-2 infections that could also be leveraged to develop novel diagnostic and therapeutic strategies.
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COVID-19 , Monocitos , Tromboplastina , Trombosis , Adulto , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Biomarcadores/sangre , COVID-19/inmunología , COVID-19/sangre , COVID-19/complicaciones , Monocitos/inmunología , Monocitos/metabolismo , Proteómica/métodos , SARS-CoV-2/fisiología , Tromboplastina/metabolismo , Tromboplastina/genética , Trombosis/inmunología , Trombosis/sangre , Trombosis/etiologíaRESUMEN
Background Ectonucleotidases maintain vascular homeostasis by metabolizing extracellular nucleotides, modulating inflammation and thrombosis, and potentially, myocardial flow through adenosine generation. Evidence implicates dysfunction or deficiency of ectonucleotidases CD39 or CD73 in human disease; the utility of measuring levels of circulating ectonucleotidases as plasma biomarkers of coronary artery dysfunction or disease has not been previously reported. Methods and Results A total of 529 individuals undergoing clinically indicated positron emission tomography stress testing between 2015 and 2019 were enrolled in this single-center retrospective analysis. Baseline demographics, clinical data, nuclear stress test, and coronary artery calcium score variables were collected, as well as a blood sample. CD39 and CD73 levels were assessed as binary (detectable, undetectable) or continuous variables using ELISAs. Plasma CD39 was detectable in 24% of White and 8% of Black study participants (P=0.02). Of the clinical history variables examined, ectonucleotidase levels were most strongly associated with underlying liver disease and not other traditional coronary artery disease risk factors. Intriguingly, detection of circulating ectonucleotidase was inversely associated with stress myocardial blood flow (2.3±0.8 mL/min per g versus 2.7 mL/min per g±1.1 for detectable versus undetectable CD39 levels, P<0.001) and global myocardial flow reserve (Pearson correlation between myocardial flow reserve and log(CD73) -0.19, P<0.001). A subanalysis showed these differences held true independent of liver disease. Conclusions Vasodilatory adenosine is the expected product of local ectonucleotidase activity, yet these data support an inverse relationship between plasma ectonucleotidases, stress myocardial blood flow (CD39), and myocardial flow reserve (CD73). These findings support the conclusion that plasma levels of ectonucleotidases, which may be shed from the endothelial surface, contribute to reduced stress myocardial blood flow and myocardial flow reserve.
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Enfermedad de la Arteria Coronaria , Imagen de Perfusión Miocárdica , Humanos , Estudios Retrospectivos , Tomografía Computarizada por Rayos X , Adenosina , Vasos Coronarios/diagnóstico por imagen , Perfusión , Imagen de Perfusión Miocárdica/métodos , Enfermedad de la Arteria Coronaria/diagnóstico , Circulación CoronariaRESUMEN
The combination of inflammation and thrombosis is a hallmark of many cardiovascular diseases. Under such conditions, platelets are recruited to an area of inflammation by forming platelet-leukocyte aggregates via interaction of PSGL-1 on leukocytes and P-selectin on activated platelets, which can bind to the endothelium. While particulate drug carriers have been utilized to passively redirect leukocytes from areas of inflammation, the downstream impact of these carriers on platelet accumulation in thromboinflammatory conditions has yet to be studied. Here, we explore the ability of polymeric particles to divert platelets away from inflamed blood vessels both in vitro and in vivo. We find that untargeted and targeted micron-sized polymeric particles can successfully reduce platelet adhesion to an inflamed endothelial monolayer in vitro in blood flow systems and in vivo in a lipopolysaccharide-induced, systemic inflammation murine model. Our data represent initial work in developing cargo-free, anti-platelet therapeutics specifically for conditions of thromboinflammation.
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Neutrófilos , Trombosis , Humanos , Animales , Ratones , Neutrófilos/metabolismo , Inflamación/metabolismo , Tromboinflamación , Trombosis/metabolismo , Plaquetas/metabolismo , Leucocitos/metabolismo , Selectina-P/metabolismoRESUMEN
BACKGROUND: Small airway inflammation and fibrosis or bronchiolitis obliterans (BO) is the predominant presentation of chronic lung allograft dysfunction (CLAD) post-lung transplantation. Carbon monoxide (CO) is a critical endogenous signaling transducer with known anti-inflammatory and anti-fibrotic effects but its therapeutic potential in CLAD remains to be fully elucidated. METHODS: Here we investigate the effect of inhaled CO in modulating chronic lung allograft rejection pathology in a murine orthotopic lung transplant model of BO (B6D2F1/JâDBA/2J). Additionally, the effects of CO on the activated phenotype of mesenchymal cells isolated from human lung transplant recipients with CLAD were studied. RESULTS: Murine lung allografts treated with CO (250 ppm × 30 minutes twice daily from days 7 to 40 post-transplantation) demonstrated decreased immune cell infiltration, fibrosis, and airway obliteration by flow cytometry, trichrome staining, and morphometric analysis, respectively. Decreased total collagen, with levels comparable to isografts, was noted in CO-treated allografts by quantitative hydroxyproline assay. In vitro, CO (250 ppm × 16h) was effective in reversing the fibrotic phenotype of human CLAD mesenchymal cells with decreased collagen I and ß-catenin expression as well as an inhibitory effect on ERK1/2 MAPK, and mTORC1/2 signaling. Sildenafil, a phosphodiesterase 5 inhibitor, partially mimicked the effects of CO on CLAD mesenchymal cells and was partially effective in decreasing collagen deposition in murine allografts, suggesting the contribution of cGMP-dependent and -independent mechanisms in mediating the effect of CO. CONCLUSION: These results suggest a potential role for CO in alleviating allograft fibrosis and mitigating chronic rejection pathology post-lung transplant.
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Bronquiolitis Obliterante , Trasplante de Pulmón , Humanos , Animales , Ratones , Monóxido de Carbono , Aloinjertos/patología , Trasplante de Pulmón/efectos adversos , Fibrosis , Pulmón/patología , Bronquiolitis Obliterante/etiología , Bronquiolitis Obliterante/prevención & control , Colágeno , Rechazo de InjertoRESUMEN
PURPOSE: Acute heart failure (AHF) syndromes manifest increased inflammation and vascular dysfunction; however, mechanisms that integrate the two in AHF remain largely unknown. The glycocalyx (GAC) is a sugar-based shell that envelops all mammalian cells. Much GAC research has focused on its role in vascular responses, with comparatively little known about how the GAC regulates immune cell function. METHODS: In this study, we sought to determine if GAC degradation products are elevated in AHF patients, how these degradation products relate to circulating inflammatory mediators, and whether the monocyte GAC (mGAC) itself modulates monocyte activation. Inflammatory markers and GAC degradation products were profiled using ELISAs. Flow cytometry was used to assess the mGAC and RNA-seq was employed to understand the role of the mGAC in regulating inflammatory activation programs. RESULTS: In a cohort of hospitalized AHF patients (n = 17), we found that (1) the GAC degradation product heparan sulfate (HS) was elevated compared with age-matched controls (4396 and 2903 ng/mL; p = 0.01) and that (2) HS and soluble CD14 (a marker of monocyte activation) levels were closely related (Pearson's r = 0.65; p = 0.002). Mechanistically, Toll-like receptor (TLR) activation of human monocytes results in GAC remodeling and a decrease in the mGAC (71% compared with no treatment; p = 0.0007). Additionally, we found that ex vivo enzymatic removal of HS and disruption of the mGAC triggers human monocyte activation and amplifies monocyte inflammatory responses. Specifically, using RNA-seq, we found that enzymatic degradation of the mGAC increases transcription of inflammatory (IL6, CCL3) and vascular (tissue factor/F3) mediators. CONCLUSION: These studies indicate that the mGAC is dynamically remodeled during monocyte activation and that mGAC remodeling itself may contribute to the heightened inflammation associated with AHF.
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People with kidney disease are disproportionately affected by atherosclerosis for unclear reasons. Soluble urokinase plasminogen activator receptor (suPAR) is an immune-derived mediator of kidney disease, levels of which are strongly associated with cardiovascular outcomes. We assessed suPAR's pathogenic involvement in atherosclerosis using epidemiologic, genetic, and experimental approaches. We found serum suPAR levels to be predictive of coronary artery calcification and cardiovascular events in 5,406 participants without known coronary disease. In a genome-wide association meta-analysis including over 25,000 individuals, we identified a missense variant in the plasminogen activator, urokinase receptor (PLAUR) gene (rs4760), confirmed experimentally to lead to higher suPAR levels. Mendelian randomization analysis in the UK Biobank using rs4760 indicated a causal association between genetically predicted suPAR levels and atherosclerotic phenotypes. In an experimental model of atherosclerosis, proprotein convertase subtilisin/kexin-9 (Pcsk9) transfection in mice overexpressing suPAR (suPARTg) led to substantially increased atherosclerotic plaques with necrotic cores and macrophage infiltration compared with those in WT mice, despite similar cholesterol levels. Prior to induction of atherosclerosis, aortas of suPARTg mice excreted higher levels of CCL2 and had higher monocyte counts compared with WT aortas. Aortic and circulating suPARTg monocytes exhibited a proinflammatory profile and enhanced chemotaxis. These findings characterize suPAR as a pathogenic factor for atherosclerosis acting at least partially through modulation of monocyte function.
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Aterosclerosis , Receptores del Activador de Plasminógeno Tipo Uroquinasa , Animales , Ratones , Aterosclerosis/genética , Biomarcadores , Estudio de Asociación del Genoma Completo , Monocitos , Proproteína Convertasa 9 , Receptores del Activador de Plasminógeno Tipo Uroquinasa/genética , Factores de Riesgo , Activador de Plasminógeno de Tipo Uroquinasa , HumanosRESUMEN
The relationship between regional variabilities in airflow (ventilation) and blood flow (perfusion) is a critical determinant of gas exchange efficiency in the lungs. Hypoxic pulmonary vasoconstriction is understood to be the primary active regulator of ventilation-perfusion matching, where upstream arterioles constrict to direct blood flow away from areas that have low oxygen supply. However, it is not understood how the integrated action of hypoxic pulmonary vasoconstriction affects oxygen transport at the system level. In this study we develop, and make functional predictions with a multi-scale multi-physics model of ventilation-perfusion matching governed by the mechanism of hypoxic pulmonary vasoconstriction. Our model consists of (a) morphometrically realistic 2D pulmonary vascular networks to the level of large arterioles and venules; (b) a tileable lumped-parameter model of vascular fluid and wall mechanics that accounts for the influence of alveolar pressure; (c) oxygen transport accounting for oxygen bound to hemoglobin and dissolved in plasma; and (d) a novel empirical model of hypoxic pulmonary vasoconstriction. Our model simulations predict that under the artificial test condition of a uniform ventilation distribution (1) hypoxic pulmonary vasoconstriction matches perfusion to ventilation; (2) hypoxic pulmonary vasoconstriction homogenizes regional alveolar-capillary oxygen flux; and (3) hypoxic pulmonary vasoconstriction increases whole-lobe oxygen uptake by improving ventilation-perfusion matching.
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Hipoxia/fisiopatología , Modelos Biológicos , Circulación Pulmonar/fisiología , Relación Ventilacion-Perfusión/fisiología , Algoritmos , Animales , Arteriolas/fisiopatología , Fenómenos Biofísicos , Biología Computacional , Simulación por Computador , Humanos , Pulmón/irrigación sanguínea , Pulmón/fisiopatología , Oxígeno/fisiología , Intercambio Gaseoso Pulmonar/fisiología , Ratas , Vasoconstricción/fisiología , Vénulas/fisiopatologíaRESUMEN
Purinergic signaling is discussed as a potential therapeutic target to reduced COVID-19 severity.
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Infecciones por Coronavirus/inmunología , Síndrome de Liberación de Citoquinas/inmunología , Dipiridamol/uso terapéutico , Neumonía Viral/inmunología , Purinérgicos/uso terapéutico , Trombosis/inmunología , Adenosina Difosfato/inmunología , Adenosina Trifosfato/inmunología , Betacoronavirus , COVID-19 , Infecciones por Coronavirus/tratamiento farmacológico , Síndrome de Liberación de Citoquinas/tratamiento farmacológico , Reposicionamiento de Medicamentos , Trampas Extracelulares/inmunología , Humanos , Pandemias , Inhibidores de Fosfodiesterasa/uso terapéutico , Neumonía Viral/tratamiento farmacológico , SARS-CoV-2 , Trombosis/tratamiento farmacológico , Tratamiento Farmacológico de COVID-19RESUMEN
Triggering events for acute aortic dissections are incompletely understood. We sought to investigate whether there is an association between admission for acute type A aortic dissection (ATAAD) to the University of Michigan Medical Center and the reported annual influenza activity by the Michigan Department of Health and Human Services. From 1996-2019 we had 758 patients admitted for ATAAD with 3.1 admissions per month during November-March and 2.5 admissions per month during April-October (p = 0.01). Influenza reporting data by the Michigan Department of Health and Human Services became available in 2009. ATAAD admissions for the period 2009-2019 (n = 455) were 4.8 cases/month during peak influenza months compared to 3.5 cases/month during non-peak influenza months (p = 0.001). ATAAD patients admitted during influenza season had increased in-hospital mortality (11.0% vs. 5.8%, p = 0.024) and increased 30-day mortality (9.7 vs. 5.4%, p = 0.048). The results point to higher admission rates for ATAAD during months with above average influenza rates. Future studies need to investigate whether influenza virus infection affects susceptibility for aortic dissection, and whether this risk can be attenuated with the annual influenza vaccine in this patient population.
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Aneurisma de la Aorta/mortalidad , Disección Aórtica/mortalidad , Brotes de Enfermedades , Mortalidad Hospitalaria , Gripe Humana/epidemiología , Admisión del Paciente/estadística & datos numéricos , Enfermedad Aguda , Anciano , Disección Aórtica/etiología , Aneurisma de la Aorta/etiología , Susceptibilidad a Enfermedades/etiología , Femenino , Humanos , Gripe Humana/complicaciones , Masculino , Michigan/epidemiología , Persona de Mediana Edad , Riesgo , Estaciones del Año , Factores de TiempoRESUMEN
OBJECTIVE: CD73 is an ectonucleotidase which catalyzes the conversion of AMP (adenosine monophosphate) to adenosine. Adenosine has been shown to be anti-inflammatory and vasorelaxant. The impact of ectonucleotidases on age-dependent atherosclerosis remains unclear. Our aim was to investigate the role of CD73 in age-dependent accumulation of atherosclerosis. Approach and results: Mice doubly deficient in CD73 and ApoE (apolipoprotein E; (cd73-/-/apoE-/-) were generated, and the extent of aortic atherosclerotic plaque was compared with apoE-/- controls at 12, 20, 32, and 52 weeks. By 12 weeks of age, cd73-/-/apoE-/- mice exhibited a significant increase in plaque (1.4±0.5% of the total vessel surface versus 0.4±0.1% in apoE-/- controls, P<0.005). By 20 weeks of age, this difference disappeared (2.9±0.4% versus 3.3±0.7%). A significant reversal in phenotype emerged at 32 weeks (9.8±1.2% versus 18.3±1.4%; P<0.0001) and persisted at the 52 week timepoint (22.4±2.1% versus 37.0±2.1%; P<0.0001). The inflammatory response to aging was found to be comparable between cd73-/-/apoE-/- mice and apoE-/- controls. A reduction in lipolysis in CD73 competent mice was observed, even with similar plasma lipid levels (cd73-/-/apoE-/- versus apoE-/- at 12 weeks [16.2±0.7 versus 9.5±1.4 nmol glycerol/well], 32 weeks [24.1±1.5 versus 7.4±0.4 nmol/well], and 52 weeks [13.8±0.62 versus 12.7±2.0 nmol/well], P<0.001). CONCLUSIONS: At early time points, CD73 exerts a subtle antiatherosclerotic influence, but with age, the pattern reverses, and the presence of CD73 promoted suppression of lipid catabolism.
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5'-Nucleotidasa/genética , Aterosclerosis/genética , Regulación del Desarrollo de la Expresión Génica , ARN/genética , 5'-Nucleotidasa/biosíntesis , Animales , Aorta Torácica/metabolismo , Aorta Torácica/patología , Aterosclerosis/metabolismo , Aterosclerosis/patología , Cromatografía Líquida de Alta Presión , Modelos Animales de Enfermedad , Femenino , Citometría de Flujo , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , FenotipoRESUMEN
Deep vein thrombosis (DVT), caused by alterations in venous homeostasis is the third most common cause of cardiovascular mortality; however, key molecular determinants in venous thrombosis have not been fully elucidated. Several lines of evidence indicate that DVT occurs at the intersection of dysregulated inflammation and coagulation. The enzyme ectonucleoside tri(di)phosphohydrolase (ENTPD1, also known as CD39) is a vascular ecto-apyrase on the surface of leukocytes and the endothelium that inhibits intravascular inflammation and thrombosis by hydrolysis of phosphodiester bonds from nucleotides released by activated cells. Here, we evaluated the contribution of CD39 to venous thrombosis in a restricted-flow model of murine inferior vena cava stenosis. CD39-deficiency conferred a >2-fold increase in venous thrombogenesis, characterized by increased leukocyte engagement, neutrophil extracellular trap formation, fibrin, and local activation of tissue factor in the thrombotic milieu. This was orchestrated by increased phosphorylation of the p65 subunit of NFκB, activation of the NLRP3 inflammasome, and interleukin-1ß (IL-1ß) release in CD39-deficient mice. Substantiating these findings, an IL-1ß-neutralizing antibody attenuated the thrombosis risk in CD39-deficient mice. These data demonstrate that IL-1ß is a key accelerant of venous thrombo-inflammation, which can be suppressed by CD39. CD39 inhibits in vivo crosstalk between inflammation and coagulation pathways, and is a critical vascular checkpoint in venous thrombosis.
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Antígenos CD/metabolismo , Apirasa/metabolismo , Inflamasomas/metabolismo , Interleucina-1beta/metabolismo , Trombosis de la Vena/metabolismo , Animales , Antígenos CD/genética , Apirasa/genética , Modelos Animales de Enfermedad , Trampas Extracelulares/genética , Trampas Extracelulares/metabolismo , Humanos , Inflamasomas/genética , Proteína Antagonista del Receptor de Interleucina 1/farmacología , Interleucina-1beta/genética , Ratones , Ratones Noqueados , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Neutrófilos/metabolismo , Factor de Transcripción ReIA/genética , Factor de Transcripción ReIA/metabolismo , Trombosis de la Vena/genética , Trombosis de la Vena/patologíaRESUMEN
Potentiation of neutrophil extracellular trap (NET) release is one mechanism by which antiphospholipid antibodies (aPL Abs) effect thrombotic events in patients with antiphospholipid syndrome (APS). Surface adenosine receptors trigger cyclic AMP (cAMP) formation in neutrophils, and this mechanism has been proposed to regulate NETosis in some contexts. Here we report that selective agonism of the adenosine A2A receptor (CGS21680) suppresses aPL Ab-mediated NETosis in protein kinase A-dependent fashion. CGS21680 also reduces thrombosis in the inferior vena cavae of both control mice and mice administered aPL Abs. The antithrombotic medication dipyridamole is known to potentiate adenosine signaling by increasing extracellular concentrations of adenosine and interfering with the breakdown of cAMP. Like CGS21680, dipyridamole suppresses aPL Ab-mediated NETosis via the adenosine A2A receptor and mitigates venous thrombosis in mice. In summary, these data suggest an anti-inflammatory therapeutic paradigm in APS, which may extend to thrombotic disease in the general population.
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Agonistas del Receptor de Adenosina A2/farmacología , Adenosina/análogos & derivados , Síndrome Antifosfolípido/tratamiento farmacológico , Trampas Extracelulares/efectos de los fármacos , Neutrófilos/efectos de los fármacos , Fenetilaminas/farmacología , Trombosis de la Vena/tratamiento farmacológico , Adenosina/inmunología , Adenosina/metabolismo , Adenosina/farmacología , Animales , Anticuerpos Antifosfolípidos/sangre , Síndrome Antifosfolípido/genética , Síndrome Antifosfolípido/inmunología , Síndrome Antifosfolípido/patología , AMP Cíclico/inmunología , AMP Cíclico/metabolismo , Dipiridamol/farmacología , Modelos Animales de Enfermedad , Trampas Extracelulares/inmunología , Trampas Extracelulares/metabolismo , Fibrinolíticos/farmacología , Regulación de la Expresión Génica , Humanos , Inmunoglobulina G/sangre , Masculino , Ratones , Ratones Endogámicos C57BL , Neutrófilos/inmunología , Receptor de Adenosina A2A/genética , Receptor de Adenosina A2A/inmunología , Transducción de Señal , Vena Cava Inferior/efectos de los fármacos , Vena Cava Inferior/inmunología , Vena Cava Inferior/metabolismo , Trombosis de la Vena/genética , Trombosis de la Vena/inmunología , Trombosis de la Vena/patologíaRESUMEN
Objective- Leukocyte flux contributes to thrombus formation in deep veins under pathological conditions, but mechanisms that inhibit venous thrombosis are incompletely understood. Ectonucleotide di(tri)phosphohydrolase 1 ( ENTPD1 or Cd39), an ectoenzyme that catabolizes extracellular adenine nucleotides, is embedded on the surface of endothelial cells and leukocytes. We hypothesized that under venous stasis conditions, CD39 regulates inflammation at the vein:blood interface in a murine model of deep vein thrombosis. Approach and Results- CD39-null mice developed significantly larger venous thrombi under venous stasis, with more leukocyte recruitment compared with wild-type mice. Gene expression profiling of wild-type and Cd39-null mice revealed 76 differentially expressed inflammatory genes that were significantly upregulated in Cd39-deleted mice after venous thrombosis, and validation experiments confirmed high expression of several key inflammatory mediators. P-selectin, known to have proximal involvement in venous inflammatory and thrombotic events, was upregulated in Cd39-null mice. Inferior vena caval ligation resulted in thrombosis and a corresponding increase in both P-selectin and VWF (von Willebrand Factor) levels which were strikingly higher in mice lacking the Cd39 gene. These mice also manifest an increase in circulating platelet-leukocyte heteroaggregates suggesting heterotypic crosstalk between coagulation and inflammatory systems, which is amplified in the absence of CD39. Conclusions- These data suggest that CD39 mitigates the venous thromboinflammatory response to flow interruption.
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Antígenos CD/fisiología , Apirasa/fisiología , Quimiotaxis de Leucocito/fisiología , Hemorreología , Vasculitis/enzimología , Trombosis de la Vena/enzimología , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Antígenos CD/genética , Apirasa/deficiencia , Apirasa/genética , Plaquetas/fisiología , Adhesión Celular , Regulación de la Expresión Génica , Redes Reguladoras de Genes , Ligadura , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Selectina-P/biosíntesis , Selectina-P/genética , Receptores Purinérgicos P2Y1/metabolismo , Vasculitis/fisiopatología , Vena Cava Inferior , Trombosis de la Vena/fisiopatología , Factor de von Willebrand/biosíntesis , Factor de von Willebrand/genéticaRESUMEN
A common thread underlying vascular or tissue injury is the loss of plasmalemmal integrity and the passive (or even active) spillage of intracellular contents into the circulation. Purinergic nucleotides, which serve as energy shuttling moieties within cells, are among the contents released into the bloodstream, where they signal danger and trigger thrombosis and inflammation. To regain vascular homeostasis, vascular cells have evolved highly conserved mechanisms to transact the catalytic degradation of extracellular nucleotides such as adenosine triphosphate (ATP) and adenosine diphosphate (ADP). CD39, the main endothelial ectonucleotidase which cleaves ATP and ADP, plays an essential role in ridding the bloodstream of these danger signals, thereby sustaining vascular homeostasis. Studies herein describe the upregulation of endothelial CD39 gene by steady laminar shear forces, and conversely, its downregulation under turbulent flow conditions. CD39 appears to be a critical ectonucleotidase which suppresses atherogenesis under experimental hyperlipidemic conditions in mice, and which also significantly mitigates pathologic vascular remodeling and development of pulmonary arterial hypertension in mice placed under chronic hypoxic conditions. Together, these data reveal that CD39 opposes pathologic vascular remodeling under hyperlipidemic or hypoxic conditions. CD39 can therefore be viewed as a critical vascular homeostatic regulator to sustain vascular quiescence and to protect against pathological vascular remodeling in diseases as diverse as atherosclerosis and pulmonary arterial hypertension.
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Apirasa/metabolismo , Aterosclerosis/enzimología , Hipertensión Pulmonar/enzimología , Arteria Pulmonar/enzimología , Remodelación Vascular , Animales , Apirasa/genética , Aterosclerosis/genética , Aterosclerosis/patología , Aterosclerosis/fisiopatología , Proliferación Celular , Células Cultivadas , Modelos Animales de Enfermedad , Regulación Enzimológica de la Expresión Génica , Humanos , Hipertensión Pulmonar/genética , Hipertensión Pulmonar/patología , Hipertensión Pulmonar/fisiopatología , Mecanotransducción Celular , Placa Aterosclerótica , Arteria Pulmonar/patología , Arteria Pulmonar/fisiopatología , Flujo Sanguíneo Regional , Transducción de SeñalRESUMEN
OBJECTIVES: CD39 and CD73 are surface enzymes that jut into the extracellular space where they mediate the step-wise phosphohydrolysis of the autocrine and paracrine danger signals ATP and ADP into anti-inflammatory adenosine. Given the role of vascular and immune cells' "purinergic halo" in maintaining homeostasis, we hypothesized that the ectonucleotidases CD39 and CD73 might play a protective role in lupus. METHODS: Lupus was modeled by intraperitoneal administration of pristane to three groups of mice: wild-type (WT), CD39-/-, and CD73-/-. After 36 weeks, autoantibodies, endothelial function, kidney disease, splenocyte activation/polarization, and neutrophil activation were characterized. RESULTS: As compared with WT mice, CD39-/- mice developed exaggerated splenomegaly in response to pristane, while both groups of ectonucleotidase-deficient mice demonstrated heightened anti-ribonucleoprotein production. The administration of pristane to WT mice triggered only subtle dysfunction of the arterial endothelium; however, both CD39-/- and CD73-/- mice demonstrated striking endothelial dysfunction following induction of lupus, which could be reversed by superoxide dismutase. Activated B cells and plasma cells were expanded in CD73-/- mice, while deficiency of either ectonucleotidase led to expansion of TH17 cells. CD39-/- and CD73-/- mice demonstrated exaggerated neutrophil extracellular trap release, while CD73-/- mice additionally had higher levels of plasma cell-free DNA. CONCLUSION: These data are the first to link ectonucleotidases with lupus autoimmunity and vascular disease. New therapeutic strategies may harness purinergic nucleotide dissipation or signaling to limit the damage inflicted upon organs and blood vessels by lupus.
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The surface properties of nanoparticles (NPs) are a major factor that influences how these nanomaterials interact with biological systems. Interactions between NPs and macrophages of the reticuloendothelial system (RES) can reduce the efficacy of NP diagnostics and therapeutics. Traditionally, to limit NP clearance by the RES system, the NP surface is neutralized with molecules like poly(ethylene glycol) (PEG) which are known to resist protein adsorption and RES clearance. Unfortunately, PEG modification is not without drawbacks including difficulties with the synthesis and associations with immune reactions. To overcome some of these obstacles, we neutralized the NP surface by acetylation and compared this modification to PEGylation for RES clearance and tumor-specific targeting. We found that acetylation was comparable to PEGylation in reducing RES clearance. Additionally, we found that dendrimer acetylation did not impact folic acid (FA)-mediated targeting of tumor cells whereas PEG surface modification reduced the targeting ability of the NP. These results clarify the impact of different NP surface modifications on RES clearance and cell-specific targeting and provide insights into the design of more effective NPs.
Asunto(s)
Ácido Fólico/farmacología , Macrófagos/química , Nanopartículas/química , Animales , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Ácido Fólico/química , Humanos , Células KB , Ratones , Estructura Molecular , Polietilenglicoles/síntesis química , Polietilenglicoles/química , Células RAW 264.7 , Relación Estructura-Actividad , Propiedades de SuperficieRESUMEN
BACKGROUND: Cerebral tissue damage after an ischemic event can be exacerbated by inflammation and thrombosis. Elevated extracellular ATP and ADP levels are associated with cellular injury, inflammation, and thrombosis. Ectonucleoside triphosphate diphosphohydrolase-1 (CD39), an enzyme expressed on the plasmalemma of leukocytes and endothelial cells, suppresses platelet activation and leukocyte infiltration by phosphohydrolyzing ATP/ADP. To investigate the effects of increased CD39 in an in vivo cerebral ischemia model, we developed a transgenic mouse expressing human CD39 (hCD39). METHODS: A floxed-stop sequence was inserted between the promoter and the hCD39 transcriptional start site, generating a mouse in which the expression of hCD39 can be controlled tissue-specifically using Cre recombinase mice. We generated mice that express hCD39 globally or in myeloid-lineage cells only. Cerebral ischemia was induced by middle cerebral artery occlusion. Infarct volumes were quantified by MRI after 48 hours. RESULTS: Both global and transgenic hCD39- and myeloid lineage CD39-overexpressing mice (transgenic, n=9; myeloid lineage, n=6) demonstrated significantly smaller cerebral infarct volumes compared with wild-type mice. Leukocytes from ischemic and contralateral hemispheres were analyzed by flow cytometry. Although contralateral hemispheres had equal numbers of macrophages and neutrophils, ischemic hemispheres from transgenic mice had less infiltration (n=4). Transgenic mice showed less neurological deficit compared with wild-type mice (n=6). CONCLUSIONS: This is the first report of transgenic overexpression of CD39 in mice imparting a protective phenotype after stroke, with reduced leukocyte infiltration, smaller infarct volumes, and decreased neurological deficit. CD39 overexpression, either globally or in myeloid lineage cells, quenches postischemic leukosequestration and reduces stroke-induced neurological injury.
Asunto(s)
Antígenos CD/biosíntesis , Antígenos CD/genética , Apirasa/biosíntesis , Apirasa/genética , Isquemia Encefálica/genética , Isquemia Encefálica/metabolismo , Linaje de la Célula/fisiología , Transgenes/fisiología , Animales , Isquemia Encefálica/prevención & control , Expresión Génica , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Células Mieloides/fisiologíaRESUMEN
Mechanical complications of myocardial infarction (MI) are often fatal. Little is known about endogenous factors that predispose to myocardial rupture after MI. Ectonucleoside triphosphate diphosphohydrolase (CD39) could be a critical mediator of propensity to myocardial rupture after MI due to its role in modulating inflammation and thrombosis. Using a model of permanent coronary artery ligation, rupture was virtually abrogated in cd39-/- mice versus cd39+/+ controls, with elevated fibrin and collagen deposition and marked neutrophil and macrophage influx. Macrophages were found to display increased surface expression of CD301 and CD206, marking a reparative phenotype, driven by increased extracellular ATP and IL-4 in the infarcted myocardium of cd39-/- mice. A myeloid-specific CD39-knockout mouse also demonstrated protection from rupture, with an attenuated rupture phenotype, suggesting that complete ablation of CD39 provides the greatest degree of protection in this model. Absence of CD39, either globally or in a myeloid lineage-restricted fashion, skews the phenotype toward alternatively activated (reparative) macrophage infiltration following MI. These studies reveal a previously unrecognized and unexpected role of endogenous CD39 to skew macrophage phenotype and promote a propensity to myocardial rupture after MI.