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The rising incidences of atherosclerosis have necessitated efforts to identify novel targets for therapeutic interventions. In the present study, we observed increased expression of the mechanosensitive calcium channel Piezo1 transcript in mouse and human atherosclerotic plaques, correlating with infiltration of PIEZO1-expressing macrophages. In vitro administration of Yoda1, a specific agonist for PIEZO1, led to increased foam cell apoptosis and enhanced phagocytosis by macrophages. Mechanistically, PIEZO1 activation resulted in intracellular F-actin rearrangement, elevated mitochondrial ROS levels and induction of mitochondrial fragmentation upon PIEZO1 activation, as well as increased expression of anti-inflammatory genes. In vivo, ApoE-/- mice treated with Yoda1 exhibited regression of atherosclerosis, enhanced stability of advanced lesions, reduced plaque size and necrotic core, increased collagen content, and reduced expression levels of inflammatory markers. Our findings propose PIEZO1 as a novel and potential therapeutic target in atherosclerosis.
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Apoptosis , Aterosclerosis , Células Espumosas , Canales Iónicos , Macrófagos , Fagocitosis , Animales , Canales Iónicos/metabolismo , Canales Iónicos/genética , Aterosclerosis/metabolismo , Aterosclerosis/patología , Aterosclerosis/genética , Ratones , Células Espumosas/metabolismo , Células Espumosas/patología , Humanos , Macrófagos/metabolismo , Ratones Endogámicos C57BL , Tiofenos/farmacología , Masculino , Especies Reactivas de Oxígeno/metabolismo , Placa Aterosclerótica/patología , Placa Aterosclerótica/metabolismo , Placa Aterosclerótica/genética , Mitocondrias/metabolismo , Pirazinas , TiadiazolesRESUMEN
OBJECTIVE: The aim of this study was to evaluate the effect of standardized care guidelines featuring next-day discharge on hospital length of stay (LOS), outcomes, patient experience, and healthcare consumption in patients receiving ventriculoperitoneal shunts (VPSs) for idiopathic normal pressure hydrocephalus (iNPH). METHODS: This single-center population-based observational cohort study included 271 adult iNPH patients who underwent VPS surgery at Karolinska University Hospital. Groups were compared before and after the implementation of new standardized discharge guidelines. The primary outcome was the hospital LOS. Secondary outcomes included postoperative complications, patient experience, and frequency of unscheduled outpatient follow-up appointments. RESULTS: A total of 121 patients were treated with variable hospitalization periods. One hundred fifty patients were managed under the new guidelines stipulating next-day discharge. There was no significant difference in positive surgical outcome (66% vs 71%, p = 0.372) or rates of complication (p ≥ 0.066) between groups. The standardized guidelines group had a significantly shorter period of inpatient hospitalization (1 vs 2 days, p < 0.001) and demonstrated lower levels of unscheduled outpatient healthcare consumption (p < 0.001) while maintaining levels of positive patient experience (p = 0.828). CONCLUSIONS: Standardized discharge guidelines featuring next-day discharge are safe for iNPH patients undergoing VPS surgery, maintain the quality of the patient experience, and are an effective method for mitigating healthcare consumption in an expanding patient group.
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Central nervous system (CNS) tumors account for almost a third of pediatric cancers and are the largest contributor to cancer-related death in children. Cranial radiation therapy (CRT) is, often in combination with chemotherapy and surgery, effective in the treatment of high-grade childhood brain cancers, but it has been associated with late complications in 50-90% of survivors, such as decline in cognition and mood, decreased social competence, and fatigue. A leading hypothesis to explain the decline in cognition, at least partially, is injury to the neural stem and progenitor cells (NSPCs), which leads to apoptosis and altered fate choice, favoring gliogenesis over neurogenesis. Hence, treatments harnessing neurogenesis are of great relevance in this context. Lithium, a well-known mood stabilizer, has neuroprotective and antitumor effects and has been found to reverse irradiation-induced damage in rodents, at least in part by regulating the expression of the glutamate decarboxylase 2 gene (Gad2) via promoter demethylation in rat NSPCs. Additionally, lithium was shown to rescue irradiation-induced cognitive defects in mice. Here, we show that irradiation (IR) alone or in combination with lithium chloride (LiCl) caused major changes in gene expression and global DNA methylation in iPSC-derived human NSPCs (hNSPCs) compared to untreated cells, as well as LiCl-only-treated cells. The pattern of DNA methylation changes after IR-treatment alone was stochastic and observed across many different gene groups, whereas differences in DNA methylation after LiCl-treatment of irradiated cells were more directed to specific promoters of genes, including genes associated with neurogenesis, for example GAD2. Interestingly, IR and IR + LiCl treatment affected the promoter methylation and expression of several genes encoding factors involved in BMP signaling, including the BMP antagonist gremlin1. We propose that lithium in addition to promoting neuronal differentiation, also represses glial differentiation in hNSPCs with DNA methylation regulation being a key mechanism of action.
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Metilación de ADN , Litio , Niño , Humanos , Ratas , Ratones , Animales , Litio/farmacología , Neurogénesis , Expresión Génica , Compuestos de Litio/farmacologíaRESUMEN
Long non-coding RNAs (lncRNAs) orchestrate various biological processes and regulate the development of cardiovascular diseases. Their potential therapeutic benefit to tackle disease progression has recently been extensively explored. Our study investigates the role of lncRNA Nudix Hydrolase 6 (NUDT6) and its antisense target fibroblast growth factor 2 (FGF2) in two vascular pathologies: abdominal aortic aneurysms (AAA) and carotid artery disease. Using tissue samples from both diseases, we detected a substantial increase of NUDT6, whereas FGF2 was downregulated. Targeting Nudt6 in vivo with antisense oligonucleotides in three murine and one porcine animal model of carotid artery disease and AAA limited disease progression. Restoration of FGF2 upon Nudt6 knockdown improved vessel wall morphology and fibrous cap stability. Overexpression of NUDT6 in vitro impaired smooth muscle cell (SMC) migration, while limiting their proliferation and augmenting apoptosis. By employing RNA pulldown followed by mass spectrometry as well as RNA immunoprecipitation, we identified Cysteine and Glycine Rich Protein 1 (CSRP1) as another direct NUDT6 interaction partner, regulating cell motility and SMC differentiation. Overall, the present study identifies NUDT6 as a well-conserved antisense transcript of FGF2. NUDT6 silencing triggers SMC survival and migration and could serve as a novel RNA-based therapeutic strategy in vascular diseases.
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Aneurisma de la Aorta Abdominal , Enfermedades de las Arterias Carótidas , ARN Largo no Codificante , Animales , Ratones , Aneurisma de la Aorta Abdominal/genética , Aneurisma de la Aorta Abdominal/terapia , Aneurisma de la Aorta Abdominal/metabolismo , Apoptosis/genética , Proliferación Celular/genética , Progresión de la Enfermedad , Factor 2 de Crecimiento de Fibroblastos/genética , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Porcinos , Oligonucleótidos AntisentidoRESUMEN
Extracellular vesicles (EVs) can be functionalized to display specific protein receptors on their surface. However, surface-display technology typically labels only a small fraction of the EV population. Here, we show that the joint display of two different therapeutically relevant protein receptors on EVs can be optimized by systematically screening EV-loading protein moieties. We used cytokine-binding domains derived from tumour necrosis factor receptor 1 (TNFR1) and interleukin-6 signal transducer (IL-6ST), which can act as decoy receptors for the pro-inflammatory cytokines tumour necrosis factor alpha (TNF-α) and IL-6, respectively. We found that the genetic engineering of EV-producing cells to express oligomerized exosomal sorting domains and the N-terminal fragment of syntenin (a cytosolic adaptor of the single transmembrane domain protein syndecan) increased the display efficiency and inhibitory activity of TNFR1 and IL-6ST and facilitated their joint display on EVs. In mouse models of systemic inflammation, neuroinflammation and intestinal inflammation, EVs displaying the cytokine decoys ameliorated the disease phenotypes with higher efficacy as compared with clinically approved biopharmaceutical agents targeting the TNF-α and IL-6 pathways.
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Vesículas Extracelulares , Enfermedades Neuroinflamatorias , Animales , Citocinas , Inflamación , Ratones , Factor de Necrosis Tumoral alfaRESUMEN
BACKGROUND: Long noncoding RNAs (lncRNAs) are important regulators of biological processes involved in vascular tissue homeostasis and disease development. The present study assessed the functional contribution of the lncRNA myocardial infarction-associated transcript (MIAT) to atherosclerosis and carotid artery disease. METHODS: We profiled differences in RNA transcript expression in patients with advanced carotid artery atherosclerotic lesions from the Biobank of Karolinska Endarterectomies. The lncRNA MIAT was identified as the most upregulated noncoding RNA transcript in carotid plaques compared with nonatherosclerotic control arteries, which was confirmed by quantitative real-time polymerase chain reaction and in situ hybridization. RESULTS: Experimental knockdown of MIAT, using site-specific antisense oligonucleotides (LNA-GapmeRs) not only markedly decreased proliferation and migration rates of cultured human carotid artery smooth muscle cells (SMCs) but also increased their apoptosis. MIAT mechanistically regulated SMC proliferation through the EGR1 (Early Growth Response 1)-ELK1 (ETS Transcription Factor ELK1)-ERK (Extracellular Signal-Regulated Kinase) pathway. MIAT is further involved in SMC phenotypic transition to proinflammatory macrophage-like cells through binding to the promoter region of KLF4 and enhancing its transcription. Studies using Miat-/- and Miat-/-ApoE-/- mice, and Yucatan LDLR-/- mini-pigs, as well, confirmed the regulatory role of this lncRNA in SMC de- and transdifferentiation and advanced atherosclerotic lesion formation. CONCLUSIONS: The lncRNA MIAT is a novel regulator of cellular processes in advanced atherosclerosis that controls proliferation, apoptosis, and phenotypic transition of SMCs, and the proinflammatory properties of macrophages, as well.
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Aterosclerosis/genética , Placa Aterosclerótica/genética , ARN Largo no Codificante/metabolismo , Animales , Humanos , RatonesRESUMEN
As a consequence of the success of present-day cancer treatment, radiotherapy-induced vascular disease is emerging. This disease is caused by chronic inflammatory activation and is likely orchestrated in part by microRNAs. In irradiated versus nonirradiated conduit arteries from patients receiving microvascular free tissue transfer reconstructions, irradiation resulted in down-regulation of miR-29b and up-regulation of miR-146b. miR-29b affected inflammation and adverse wound healing through its targets pentraxin-3 and dipeptidyl-peptidase 4. In vitro and in vivo, we showed that miR-29b overexpression therapy, through inhibition of pentraxin-3 and dipeptidyl-peptidase 4, could dampen the vascular inflammatory response.
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BACKGROUND: Long noncoding RNAs have emerged as critical molecular regulators in various biological processes and diseases. Here we sought to identify and functionally characterize long noncoding RNAs as potential mediators in abdominal aortic aneurysm development. METHODS: We profiled RNA transcript expression in 2 murine abdominal aortic aneurysm models, Angiotensin II (ANGII) infusion in apolipoprotein E-deficient ( ApoE-/-) mice (n=8) and porcine pancreatic elastase instillation in C57BL/6 wild-type mice (n=12). The long noncoding RNA H19 was identified as 1 of the most highly upregulated transcripts in both mouse aneurysm models compared with sham-operated controls. This was confirmed by quantitative reverse transcription-polymerase chain reaction and in situ hybridization. RESULTS: Experimental knock-down of H19, utilizing site-specific antisense oligonucleotides (LNA-GapmeRs) in vivo, significantly limited aneurysm growth in both models. Upregulated H19 correlated with smooth muscle cell (SMC) content and SMC apoptosis in progressing aneurysms. Importantly, a similar pattern could be observed in human abdominal aortic aneurysm tissue samples, and in a novel preclinical LDLR-/- (low-density lipoprotein receptor) Yucatan mini-pig aneurysm model. In vitro knock-down of H19 markedly decreased apoptotic rates of cultured human aortic SMCs, whereas overexpression of H19 had the opposite effect. Notably, H19-dependent apoptosis mechanisms in SMCs appeared to be independent of miR-675, which is embedded in the first exon of the H19 gene. A customized transcription factor array identified hypoxia-inducible factor 1α as the main downstream effector. Increased SMC apoptosis was associated with cytoplasmic interaction between H19 and hypoxia-inducible factor 1α and sequential p53 stabilization. Additionally, H19 induced transcription of hypoxia-inducible factor 1α via recruiting the transcription factor specificity protein 1 to the promoter region. CONCLUSIONS: The long noncoding RNA H19 is a novel regulator of SMC survival in abdominal aortic aneurysm development and progression. Inhibition of H19 expression might serve as a novel molecular therapeutic target for aortic aneurysm disease.
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Aneurisma de la Aorta Abdominal/genética , Músculo Liso Vascular/metabolismo , ARN Largo no Codificante/genética , Angiotensina II , Animales , Aorta Abdominal/metabolismo , Aorta Abdominal/patología , Aneurisma de la Aorta Abdominal/inducido químicamente , Aneurisma de la Aorta Abdominal/metabolismo , Aneurisma de la Aorta Abdominal/patología , Apoptosis , Estudios de Casos y Controles , Células Cultivadas , Dilatación Patológica , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados para ApoE , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Elastasa Pancreática , ARN Largo no Codificante/metabolismo , Receptores de LDL/genética , Receptores de LDL/metabolismo , Porcinos , Porcinos Enanos , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Regulación hacia ArribaRESUMEN
miRNAs are potential regulators of carotid artery stenosis and concordant vulnerable atherosclerotic plaques. Hence, we analyzed miRNA expression in laser captured micro-dissected fibrous caps of either ruptured or stable plaques (n = 10 each), discovering that miR-21 was significantly downregulated in unstable lesions. To functionally evaluate miR-21 in plaque vulnerability, miR-21 and miR-21/apolipoprotein-E double-deficient mice (Apoe-/-miR-21-/-) were assessed. miR-21-/- mice lacked sufficient smooth muscle cell proliferation in response to carotid ligation injury. When exposing Apoe-/-miR-21-/- mice to an inducible plaque rupture model, they presented with more atherothrombotic events (93%) compared with miR-21+/+Apoe-/- mice (57%). We discovered that smooth muscle cell fate in experimentally induced advanced lesions is steered via a REST-miR-21-REST feedback signaling pathway. Furthermore, Apoe-/-miR-21-/- mice presented with more pronounced atherosclerotic lesions, greater foam cell formation, and substantially higher levels of arterial macrophage infiltration. Local delivery of a miR-21 mimic using ultrasound-targeted microbubbles into carotid plaques rescued the vulnerable plaque rupture phenotype. In the present study, we identify miR-21 as a key modulator of pathologic processes in advanced atherosclerosis. Targeted, lesion site-specific overexpression of miR-21 can stabilize vulnerable plaques.
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Aterosclerosis/genética , Aterosclerosis/patología , MicroARNs/genética , Animales , Apoptosis/genética , Enfermedades de las Arterias Carótidas/genética , Enfermedades de las Arterias Carótidas/patología , Modelos Animales de Enfermedad , Fibrosis , Perfilación de la Expresión Génica , Técnicas de Transferencia de Gen , Genotipo , Humanos , Inmunohistoquímica , Lipoproteínas LDL/metabolismo , Macrófagos/metabolismo , Macrófagos/patología , Masculino , Ratones , Ratones Noqueados , MicroARNs/administración & dosificación , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Placa Aterosclerótica/genética , Placa Aterosclerótica/patologíaRESUMEN
BACKGROUND AND AIMS: Increased proinsulin relative to insulin levels have been associated with subclinical atherosclerosis (measured by carotid intima-media thickness (cIMT)) and are predictive of future cardiovascular disease (CVD), independently of established risk factors. The mechanisms linking proinsulin to atherosclerosis and CVD are unclear. A genome-wide meta-analysis has identified nine loci associated with circulating proinsulin levels. Using proinsulin-associated SNPs, we set out to use a Mendelian randomisation approach to test the hypothesis that proinsulin plays a causal role in subclinical vascular remodelling. METHODS: We studied the high CVD-risk IMPROVE cohort (n = 3345), which has detailed biochemical phenotyping and repeated, state-of-the-art, high-resolution carotid ultrasound examinations. Genotyping was performed using Illumina Cardio-Metabo and Immuno arrays, which include reported proinsulin-associated loci. Participants with type 2 diabetes (n = 904) were omitted from the analysis. Linear regression was used to identify proinsulin-associated genetic variants. RESULTS: We identified a proinsulin locus on chromosome 15 (rs8029765) and replicated it in data from 20,003 additional individuals. An 11-SNP score, including the previously identified and the chromosome 15 proinsulin-associated loci, was significantly and negatively associated with baseline IMTmean and IMTmax (the primary cIMT phenotypes) but not with progression measures. However, MR-Eggers refuted any significant effect of the proinsulin-associated 11-SNP score, and a non-pleiotropic SNP score of three variants (including rs8029765) demonstrated no effect on baseline or progression cIMT measures. CONCLUSIONS: We identified a novel proinsulin-associated locus and demonstrated that whilst proinsulin levels are associated with cIMT measures, proinsulin per se is unlikely to have a causative effect on cIMT.
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Enfermedades de las Arterias Carótidas/genética , Análisis de la Aleatorización Mendeliana , Polimorfismo de Nucleótido Simple , Proinsulina/genética , Remodelación Vascular/genética , Enfermedades Asintomáticas , Enfermedades de las Arterias Carótidas/sangre , Enfermedades de las Arterias Carótidas/diagnóstico por imagen , Enfermedades de las Arterias Carótidas/fisiopatología , Grosor Intima-Media Carotídeo , Cromosomas Humanos Par 15 , Europa (Continente) , Femenino , Frecuencia de los Genes , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Humanos , Modelos Lineales , Masculino , Fenotipo , Proinsulina/sangre , Sitios de Carácter Cuantitativo , Factores de RiesgoRESUMEN
RATIONALE: In the search for markers and modulators of vascular disease, microRNAs (miRNAs) have emerged as potent therapeutic targets. OBJECTIVE: To investigate miRNAs of clinical interest in patients with unstable carotid stenosis at risk of stroke. METHODS AND RESULTS: Using patient material from the BiKE (Biobank of Karolinska Endarterectomies), we profiled miRNA expression in patients with stable versus unstable carotid plaque. A polymerase chain reaction-based miRNA array of plasma, sampled at the carotid lesion site, identified 8 deregulated miRNAs (miR-15b, miR-29c, miR-30c/d, miR-150, miR-191, miR-210, and miR-500). miR-210 was the most significantly downregulated miRNA in local plasma material. Laser capture microdissection and in situ hybridization revealed a distinct localization of miR-210 in fibrous caps. We confirmed that miR-210 directly targets the tumor suppressor gene APC (adenomatous polyposis coli), thereby affecting Wnt (Wingless-related integration site) signaling and regulating smooth muscle cell survival, as well as differentiation in advanced atherosclerotic lesions. Substantial changes in arterial miR-210 were detectable in 2 rodent models of vascular remodeling and plaque rupture. Modulating miR-210 in vitro and in vivo improved fibrous cap stability with implications for vascular disease. CONCLUSIONS: An unstable carotid plaque at risk of stroke is characterized by low expression of miR-210. miR-210 contributes to stabilizing carotid plaques through inhibition of APC, ensuring smooth muscle cell survival. We present local delivery of miR-210 as a therapeutic approach for prevention of atherothrombotic vascular events.
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MicroARNs/administración & dosificación , MicroARNs/biosíntesis , Placa Aterosclerótica/metabolismo , Placa Aterosclerótica/terapia , Animales , Aterosclerosis/metabolismo , Aterosclerosis/patología , Aterosclerosis/terapia , Estenosis Carotídea/metabolismo , Estenosis Carotídea/patología , Estenosis Carotídea/terapia , Células Cultivadas , Estudios de Cohortes , Células Endoteliales/metabolismo , Células Endoteliales/patología , Humanos , Captura por Microdisección con Láser/métodos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , MicroARNs/análisis , Placa Aterosclerótica/patología , Ratas , Ratas Sprague-Dawley , Accidente Cerebrovascular/metabolismo , Accidente Cerebrovascular/patología , Accidente Cerebrovascular/prevención & controlRESUMEN
Type 2 diabetes and cardiovascular disease are complex disorders involving metabolic and inflammatory mechanisms. Here we investigated whether sCD93, a group XIV c-type lectin of the endosialin family, plays a role in metabolic dysregulation or carotid intima-media thickness (IMT). Although no association was observed between sCD93 and IMT, sCD93 levels were significantly lower in subjects with type 2 diabetes (n = 901, mean ± SD 156.6 ± 40.0 ng/mL) compared with subjects without diabetes (n = 2,470, 164.1 ± 44.8 ng/mL, P < 0.0001). Genetic variants associated with diabetes risk (DIAGRAM Consortium) did not influence sCD93 levels (individually or combined in a single nucleotide polymorphism score). In a prospective cohort, lower sCD93 levels preceded the development of diabetes. Consistent with this, a cd93-deficient mouse model (in addition to apoe deficiency) demonstrated no difference in atherosclerotic lesion development compared with apoe(-/-) cd93-sufficient littermates. However, cd93-deficient mice showed impaired glucose clearance and insulin sensitivity (compared with littermate controls) after eating a high-fat diet. The expression of cd93 was observed in pancreatic islets, and leaky vessels were apparent in cd93-deficient pancreases. We further demonstrated that stress-induced release of sCD93 is impaired by hyperglycemia. Therefore, we propose CD93 as an important component in glucometabolic regulation.
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Grosor Intima-Media Carotídeo , Glicoproteínas de Membrana/metabolismo , Receptores de Complemento/metabolismo , Animales , Apolipoproteínas E , Aterosclerosis/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Células Endoteliales/metabolismo , Femenino , Genotipo , Humanos , Masculino , Glicoproteínas de Membrana/deficiencia , Glicoproteínas de Membrana/genética , Ratones , Ratones Noqueados , Páncreas/metabolismo , Receptores de Complemento/deficiencia , Receptores de Complemento/genéticaRESUMEN
IL-5 is a Th2 cytokine which activates eosinophils and is suggested to have an atheroprotective role. Genetic variants in the IL5 locus have been associated with increased risk of CAD and ischemic stroke. In this study we aimed to identify genetic variants associated with IL-5 concentrations and apply a Mendelian randomisation approach to assess IL-5 levels for causal effect on intima-media thickness in a European population at high risk of coronary artery disease. We analysed SNPs within robustly associated candidate loci for immune, inflammatory, metabolic and cardiovascular traits. We identified 2 genetic loci for IL-5 levels (chromosome 5, rs56183820, BETA=0.11, P=6.73E(-5) and chromosome 14, rs4902762, BETA=0.12, P=5.76E(-6)) and one for eosinophil count (rs72797327, BETA=-0.10, P=1.41E(-6)). Both chromosome 5 loci were in the vicinity of the IL5 gene, however the association with IL-5 levels failed to replicate in a meta-analysis of 2 independent cohorts (rs56183820, BETA=0.04, P=0.2763, I(2)=24, I(2)-P=0.2516). No significant associations were observed between SNPs associated with IL-5 levels or eosinophil count and IMT measures. Expression quantitative trait analyses indicate effects of the IL-5 and eosinophil-associated SNPs on RAD50 mRNA expression levels (rs12652920 (r2=0.93 with rs56183820) BETA=-0.10, P=8.64E(-6) and rs11739623 (r2=0.96 with rs72797327) BETA=-0.23, P=1.74E(-29), respectively). Our data do not support a role for IL-5 levels and eosinophil count in intima-media thickness, however SNPs associated with IL-5 and eosinophils might influence stability of the atherosclerotic plaque via modulation of RAD50 levels.
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Cromosomas Humanos Par 5/genética , Enfermedad de la Arteria Coronaria/genética , Eosinófilos/metabolismo , Sitios Genéticos/genética , Interleucina-5/genética , Ácido Anhídrido Hidrolasas , Anciano , Grosor Intima-Media Carotídeo , Cromosomas Humanos Par 14/genética , Enfermedad de la Arteria Coronaria/sangre , Enzimas Reparadoras del ADN/genética , Proteínas de Unión al ADN/genética , Europa (Continente) , Femenino , Expresión Génica , Predisposición Genética a la Enfermedad/genética , Genética de Población , Estudio de Asociación del Genoma Completo/métodos , Humanos , Interleucina-5/sangre , Recuento de Leucocitos , Modelos Lineales , Masculino , Persona de Mediana Edad , Placa Aterosclerótica/sangre , Placa Aterosclerótica/genética , Polimorfismo de Nucleótido Simple , Factores de RiesgoRESUMEN
OBJECTIVE: Atherosclerosis is an inflammatory condition, and rupture of atherosclerotic plaques is a major cause of cardiovascular disease (CVD). Lysophosphatidylcholine (LPC) is generated in low-density lipoprotein (LDL) during oxidation and/or enzymatic modification and has been implicated in atherosclerosis. Annexin A5 (ANXA5) is an antithrombotic and atheroprotective plasma protein. Here, we demonstrate novel pro-inflammatory and atherogenic properties of LPC, and inhibitory effects of ANXA5. METHODS: Endothelial cells and macrophages (differentiated from, THP-1 a monocytic cell line) were co-cultured. Expression of MMP-9 and OxLDL uptake by macrophages were studied by flow cytometry. The effect of LPC on leukotriene B4 (LTB4) synthesis in macrophages was studied by enzyme immunoassay (EIA). Chemotactic properties of LPC were investigated using a mouse intra-peritoneal recruitment model. RESULTS: Co-culture of macrophages and endothelial cells enhanced MMP-9 expression in both cell types. This effect was increased by LPC and diminished by ANXA5. Likewise, LPC induced LTB4 production by macrophages, whereas native LDL or phosphatidylcholine (PTC) had no effect. ANXA5 inhibited uptake of OxLDL in macrophages. LPC induced cell infiltration in vivo, as determined by increased cell count in mouse peritoneal exudates, and this effect was inhibited by ANXA5. CONCLUSIONS: ANXA5 could potentially play an important protective role in both atherogenesis and atherosclerotic plaque rupture by reducing pro-inflammatory effects of OxLDL and LPC as well as inhibiting OxLDL binding and uptake by macrophages. The possibility that ANXA5 could be developed into a novel therapy against CVD deserves further study.
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Anexina A5/farmacología , Lisofosfatidilcolinas/farmacología , Placa Aterosclerótica/inducido químicamente , Placa Aterosclerótica/prevención & control , Animales , Anexina A5/metabolismo , Transporte Biológico/efectos de los fármacos , Línea Celular , Movimiento Celular/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/citología , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Inflamación/metabolismo , Leucotrieno B4/biosíntesis , Lipoproteínas LDL/metabolismo , Lisofosfatidilcolinas/metabolismo , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Placa Aterosclerótica/metabolismo , Placa Aterosclerótica/patologíaRESUMEN
Remodeling of extracellular matrix (ECM) plays an important role in both atherosclerosis and aneurysm disease. Serine protease inhibitor A3 (serpinA3) is an inhibitor of several proteases such as elastase, cathepsin G and chymase derived from mast cells and neutrophils. In this study, we investigated the putative role of serpinA3 in atherosclerosis and aneurysm formation. SerpinA3 was expressed in endothelial cells and medial smooth muscle cells in human atherosclerotic lesions and a 14-fold increased expression of serpinA3n mRNA was found in lesions from Apoe-/- mice compared to lesion-free vessels. In contrast, decreased mRNA expression (-80%) of serpinA3 was found in biopsies of human abdominal aortic aneurysm (AAA) compared to non-dilated aortas. Overexpression of serpinA3n in transgenic mice did not influence the development of atherosclerosis or CaCl2-induced aneurysm formation. In situ zymography analysis showed that the transgenic mice had lower cathepsin G and elastase activity, and more elastin in the aortas compared to wild-type mice, which could indicate a more stable aortic phenotype. Differential vascular expression of serpinA3 is clearly associated with human atherosclerosis and AAA but serpinA3 had no major effect on experimentally induced atherosclerosis or AAA development in mouse. However, serpinA3 may be involved in a phenotypic stabilization of the aorta.
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Aneurisma/metabolismo , Aterosclerosis/metabolismo , Inhibidores de Serina Proteinasa/metabolismo , alfa 1-Antiquimotripsina/metabolismo , Aneurisma/genética , Animales , Aterosclerosis/genética , Cloruro de Calcio/farmacología , Catepsina G/metabolismo , Línea Celular , Citocinas/farmacología , Células Endoteliales/metabolismo , Activación Enzimática/efectos de los fármacos , Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Mediadores de Inflamación/farmacología , Mastocitos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Elastasa Pancreática/metabolismo , ARN Mensajero/metabolismo , alfa 1-Antiquimotripsina/genéticaRESUMEN
INTRODUCTION: Collagen-induced arthritis (CIA) is a mouse model for rheumatoid arthritis (RA) and is induced after immunization with type II collagen (CII). CIA, like RA, is an autoimmune disease leading to destruction of cartilage and joints, and both the priming and inflammatory phases have been suggested to be dependent on proteases. In particular, the cysteine proteases have been proposed to be detrimental to the arthritic process and even immunomodulatory. A natural inhibitor of cysteine proteases is cystatin C. METHODS: Cystatin C-deficient, sufficient and heterozygous mice were tested for onset, incidence and severity of CIA. The effect of cystatin C-deficiency was further dissected by testing the inflammatory effector phase of CIA; that is, collagen antibody-induced arthritis model and priming phase, that is, T cell response both in vivo and in vitro. In addition, in order to determine the importance of T cells and antigen-presenting cells (APCs), these cell populations were separated and in vitro T cell responses determined in a mixed co-culture system. Finally, flow cytometry was used in order to further characterize cell populations in cystatin C-deficient mice. RESULTS: Here, we show that mice lacking cystatin C, develop arthritis at a higher incidence and an earlier onset than wild-type controls. Interestingly, when the inflammatory phase of CIA was examined independently from immune priming then cystatin C-deficiency did not enhance the arthritis profile. However, in line with the enhanced CIA, there was an increased T cell and B cell response as delayed-type hypersensitivity reaction and anti-CII antibody titers were elevated in the cystatin C-deficient mice after immunization. In addition, the ex vivo naïve APCs from cystatin C-deficient mice had a greater capacity to stimulate T cells. Interestingly, dendritic cells had a more activated phenotype in naïve cystatin C-deficient mice. CONCLUSIONS: The lack of cystatin C enhances CIA and primarily affects in vivo priming of the immune system. Although the mechanism of this is still unknown, we show evidence for a more activated APC compartment, which would elevate the autoimmune response towards CII, thus resulting in an enhanced development of chronic arthritis.
Asunto(s)
Artritis Experimental/inmunología , Artritis Reumatoide/inmunología , Autoinmunidad/inmunología , Cistatina C/inmunología , Inflamación/inmunología , Animales , Presentación de Antígeno/inmunología , Artritis Experimental/genética , Artritis Reumatoide/genética , Autoanticuerpos/inmunología , Autoantígenos/inmunología , Autoinmunidad/genética , Separación Celular , Colágeno Tipo II/inmunología , Colágeno Tipo II/toxicidad , Cistatina C/genética , Citometría de Flujo , Hipersensibilidad Tardía/genética , Hipersensibilidad Tardía/inmunología , Inflamación/genética , Activación de Linfocitos/inmunología , Ratones , Ratones Noqueados , Linfocitos T/inmunologíaRESUMEN
OBJECTIVE: To establish a new animal model in DRB1*0401 (DR4)-transgenic mice in which T cell tolerance to self type II collagen (CII) can be broken and allow for the development of autoimmune arthritis, to investigate the role of posttranslational modifications of the CII(259-273) epitope in the induction and breaking of tolerance of DR4-restricted T cells, and to characterize DR4-restricted T cell recognition of the immunodominant CII(259-273) epitope. METHODS: DR4-transgenic mice expressing either the entire human CII protein (HuCII) or only the immunodominant T cell epitope of heterologous CII (MMC) in joint cartilage were established on different genetic backgrounds, and susceptibility to collagen-induced arthritis (CIA) was tested. RESULTS: HuCII mice displayed stronger T cell tolerance to heterologous CII than did MMC mice. On the B10 background, arthritis developed only in MMC mice with a defective oxidative burst. However, MMC mice on the C3H background were susceptible to arthritis also with a functional oxidative burst. Significant recall responses in tolerized mice were detected only against the nonglycosylated CII(259-273) epitope. Recognition of the CII(259-273) epitope was heterogeneous, but the majority of T cells in DR4 mice specifically recognized the nonglycosylated side chain of lysine at position 264. CONCLUSION: It is possible to break tolerance to self CII and induce arthritis in DR4 mice. However, arthritis susceptibility is tightly controlled by the genetic background and by the source of the transgenic element for expressing the heterologous CII peptide as a self CII protein in the joint. In contrast to CIA in A(q)-expressing mice, the nonglycosylated CII(259-273) epitope is clearly immunodominant in both tolerized and nontolerized DR4 mice.
Asunto(s)
Artritis Experimental/inmunología , Colágeno Tipo II/inmunología , Antígeno HLA-DR4/inmunología , Autotolerancia/inmunología , Linfocitos T/inmunología , Animales , Artritis Experimental/genética , Femenino , Predisposición Genética a la Enfermedad , Antígeno HLA-DR4/genética , Humanos , Masculino , Ratones , Ratones Transgénicos , Ratas , Autotolerancia/genéticaRESUMEN
alpha2beta1 integrins are normally confined to the proliferating basal layers of the epidermis. However, during wound healing and in psoriasis, these integrins are expressed on keratinocytes in suprabasal layers correlating with a less differentiated phenotype. Transgenic mice expressing alpha2beta1 integrins under the involucrine promoter have previously been demonstrated, to various degrees, spontaneously develop a skin disorder resembling psoriasis. Herein, we show that a mild epidermal wounding induces a uniform acanthosis together with an influx of immune cells. The disease initiates as a normal wound healing process and is completely restored in wildtype mice by day 14. However, in the integrin transgenic mice a chronic inflammation develops, a process that can be compared to the Koebner phenomenon in psoriatic patients. In this study, we have followed the integrin transgenic mice for five weeks, where substantial keratinocyte hyper-proliferation, inflammatory infiltration and high cytokine levels within the skin can still be observed. In addition, draining lymph nodes were dramatically increased in size and contained highly activated T cells, as well as APCs secreting large amounts of pro-inflammatory cytokines. Furthermore, the systemic immune response was affected with increased spleen size, elevated cytokine levels in the serum and altered lymphocyte trafficking patterns, very much resembling what is seen in psoriasis patients. Finally, CD4(+) T cell depletion was not able to affect the onset or progression of skin inflammation. This suggests that altered keratinocyte differentiation and proliferation can drive a skin inflammation and cause chronic immune cell activation both at a local and systemic level.