RESUMO
Coronavirus disease 2019 (COVID-19) is a mild to moderate respiratory tract infection, however, a subset of patients progress to severe disease and respiratory failure. The mechanism of protective immunity in mild forms and the pathogenesis of severe COVID-19 associated with increased neutrophil counts and dysregulated immune responses remain unclear. In a dual-center, two-cohort study, we combined single-cell RNA-sequencing and single-cell proteomics of whole-blood and peripheral-blood mononuclear cells to determine changes in immune cell composition and activation in mild versus severe COVID-19 (242 samples from 109 individuals) over time. HLA-DRhiCD11chi inflammatory monocytes with an interferon-stimulated gene signature were elevated in mild COVID-19. Severe COVID-19 was marked by occurrence of neutrophil precursors, as evidence of emergency myelopoiesis, dysfunctional mature neutrophils, and HLA-DRlo monocytes. Our study provides detailed insights into the systemic immune response to SARS-CoV-2 infection and reveals profound alterations in the myeloid cell compartment associated with severe COVID-19.
Assuntos
Infecções por Coronavirus/imunologia , Células Mieloides/imunologia , Mielopoese , Pneumonia Viral/imunologia , Adulto , Idoso , Antígenos CD11/genética , Antígenos CD11/metabolismo , COVID-19 , Células Cultivadas , Infecções por Coronavirus/sangue , Infecções por Coronavirus/patologia , Feminino , Antígenos HLA-DR/genética , Antígenos HLA-DR/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Células Mieloides/citologia , Pandemias , Pneumonia Viral/sangue , Pneumonia Viral/patologia , Proteoma/genética , Proteoma/metabolismo , Proteômica , Análise de Célula ÚnicaRESUMO
BACKGROUND: Early in the host-response to infection, neutrophils release calprotectin, triggering several immune signalling cascades. In acute infection management, identifying infected patients and stratifying these by risk of deterioration into sepsis, are crucial tasks. Recruiting a heterogenous population of patients with suspected infections from the emergency department, early in the care-path, the CASCADE trial aimed to evaluate the accuracy of blood calprotectin for detecting bacterial infections, estimating disease severity, and predicting clinical deterioration. METHODS: In a prospective, observational trial from February 2021 to August 2022, 395 patients (n = 194 clinically suspected infection; n = 201 controls) were enrolled. Blood samples were collected at enrolment. The accuracy of calprotectin to identify bacterial infections, and to predict and identify sepsis and mortality was analysed. These endpoints were determined by a panel of experts. RESULTS: The Area Under the Receiver Operating Characteristic (AUROC) of calprotectin for detecting bacterial infections was 0.90. For sepsis within 72 h, calprotectin's AUROC was 0.83. For 30-day mortality it was 0.78. In patients with diabetes, calprotectin had an AUROC of 0.94 for identifying bacterial infection. CONCLUSIONS: Calprotectin showed notable accuracy for all endpoints. Using calprotectin in the emergency department could improve diagnosis and management of severe infections, in combination with current biomarkers. CLINICAL TRIAL REGISTRATION NUMBER: DRKS00020521.
Assuntos
Biomarcadores , Complexo Antígeno L1 Leucocitário , Sepse , Humanos , Complexo Antígeno L1 Leucocitário/sangue , Sepse/sangue , Sepse/diagnóstico , Sepse/mortalidade , Biomarcadores/sangue , Estudos Prospectivos , Masculino , Feminino , Pessoa de Meia-Idade , Idoso , Infecções Bacterianas/sangue , Infecções Bacterianas/diagnóstico , Infecções Bacterianas/mortalidade , Curva ROC , Adulto , Idoso de 80 Anos ou mais , Serviço Hospitalar de EmergênciaRESUMO
INTRODUCTION: Chronic activation of the mineralocorticoid receptor (MR) leads to pathological processes like inflammation and fibrosis during cardiorenal disease. Modulation of immunological processes in the heart or kidney may serve as a mechanistic and therapeutic interface in cardiorenal pathologies. In this study, we investigated anti-inflammatory/-fibrotic and immunological effects of the selective nonsteroidal MR antagonists finerenone (FIN) in the deoxycorticosterone acetate (DOCA)-salt model. METHODS: Male C57BL6/J mice were uninephrectomized and received a DOCA pellet implantation (2.4 mg/day) plus 0.9% NaCl in drinking water (DOCA-salt) or received a sham operation and were orally treated with FIN (10 mg/kg/day) or vehicle in a preventive study design. Five weeks after the procedure, blood pressure (BP), urinary albumin/creatinine ratio (UACR), glomerular and tubulointerstitial damage, echocardiographic cardiac function, as well as cardiac/renal inflammatory cell content by FACS analysis were assessed. RESULTS: BP was significantly reduced by FIN. FACS analysis revealed a notable immune response due to DOCA-salt exposure. Especially, infiltrating renal RORγt γδ-positive T cells were upregulated, which was significantly ameliorated by FIN treatment. This was accompanied by a significant reduction of UACR in FIN-treated mice. In the heart, FIN reduced DOCA-salt-induced cardiac hypertrophy, cardiac fibrosis and led to an improvement of the global longitudinal strain. Cardiac actions of FIN were not associated with a regulation of cardiac RORγt γδ-positive T cells. DISCUSSION/CONCLUSION: The present study shows cardiac and renal protective effects of FIN in a DOCA-salt model. The cardiorenal protection was accompanied by a reduction of renal RORγt γδ T cells. The observed actions of FIN may provide a potential mechanism of its efficacy recently observed in clinical trials.
Assuntos
Hipertensão Renal , Hipertensão , Naftiridinas , Linfócitos T , Animais , Pressão Sanguínea , Acetato de Desoxicorticosterona , Fibrose , Hipertensão/tratamento farmacológico , Hipertensão Renal/patologia , Rim/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Naftiridinas/farmacologia , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/uso terapêuticoRESUMO
We detected delayed and reduced antibody and T-cell responses after BNT162b2 vaccination in 71 elderly persons (median age 81 years) compared with 123 healthcare workers (median age 34 years) in Germany. These data emphasize that nonpharmaceutical interventions for coronavirus disease remain crucial and that additional immunizations for the elderly might become necessary.
Assuntos
COVID-19 , Adulto , Idoso , Idoso de 80 Anos ou mais , Vacina BNT162 , Vacinas contra COVID-19 , Alemanha/epidemiologia , Humanos , SARS-CoV-2 , Linfócitos T , VacinaçãoRESUMO
OBJECTIVES: The rapid diagnosis of acute infections and sepsis remains a serious challenge. As a result of limitations in current diagnostics, guidelines recommend early antimicrobials for suspected sepsis patients to improve outcomes at a cost to antimicrobial stewardship. We aimed to develop and prospectively validate a new, 29-messenger RNA blood-based host-response classifier Inflammatix Bacterial Viral Non-Infected version 2 (IMX-BVN-2) to determine the likelihood of bacterial and viral infections. DESIGN: Prospective observational study. SETTING: Emergency Department, Campus Benjamin Franklin, Charité-Universitätsmedizin Berlin, Germany. PATIENTS: Three hundred twelve adult patients presenting to the emergency department with suspected acute infections or sepsis with at least one vital sign change. INTERVENTIONS: None (observational study only). MEASUREMENTS AND MAIN RESULTS: Gene expression levels from extracted whole blood RNA was quantified on a NanoString nCounter SPRINT (NanoString Technologies, Seattle, WA). Two predicted probability scores for the presence of bacterial and viral infection were calculated using the IMX-BVN-2 neural network classifier, which was trained on an independent development set. The IMX-BVN-2 bacterial score showed an area under the receiver operating curve for adjudicated bacterial versus ruled out bacterial infection of 0.90 (95% CI, 0.85-0.95) compared with 0.89 (95% CI, 0.84-0.94) for procalcitonin with procalcitonin being used in the adjudication. The IMX-BVN-2 viral score area under the receiver operating curve for adjudicated versus ruled out viral infection was 0.83 (95% CI, 0.77-0.89). CONCLUSIONS: IMX-BVN-2 demonstrated accuracy for detecting both viral infections and bacterial infections. This shows the potential of host-response tests as a novel and practical approach for determining the causes of infections, which could improve patient outcomes while upholding antimicrobial stewardship.
Assuntos
Infecções Bacterianas/diagnóstico , RNA Mensageiro/análise , Viroses/diagnóstico , Idoso , Idoso de 80 Anos ou mais , Área Sob a Curva , Infecções Bacterianas/sangue , Infecções Bacterianas/fisiopatologia , Berlim , Biomarcadores/análise , Biomarcadores/sangue , Serviço Hospitalar de Emergência/organização & administração , Serviço Hospitalar de Emergência/estatística & dados numéricos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , RNA Mensageiro/sangue , Curva ROC , Viroses/sangue , Viroses/fisiopatologiaRESUMO
Periodontitis is a chronic inflammatory disease leading to progressive connective tissue degradation and loss of the tooth-supporting bone. Clinical and experimental studies suggest that hepatocyte growth factor (HGF) is involved in the dysregulated fibroblast-epithelial cell interactions in periodontitis. The aim of this study was to explore effects of HGF to impact fibroblast-induced collagen degradation. A patient-derived experimental cell culture model of periodontitis was applied. Primary human epithelial cells and fibroblasts isolated from periodontitis-affected gingiva were co-cultured in a three-dimensional collagen gel. The effects of HGF neutralizing antibody on collagen gel degradation were tested and transcriptome analyses were performed. HGF neutralizing antibody attenuated collagen degradation and elicited expression changes of genes related to extracellular matrix (ECM) and cell adhesion, indicating that HGF signaling inhibition leads to extensive impact on cell-cell and cell-ECM interactions. Our study highlights a potential role of HGF in periodontitis. Antagonizing HGF signaling by a neutralizing antibody may represent a novel approach for periodontitis treatment.
Assuntos
Fator de Crescimento de Hepatócito , Periodontite , Fibroblastos , Gengiva , Humanos , Modelos TeóricosRESUMO
There is prevailing evidence to suggest a decisive role for platelet-derived growth factors (PDGF) and their receptors in primary myelofibrosis. While PDGF receptor ß (PDGFRß) expression is increased in bone marrow stromal cells of patients correlating with the grade of myelofibrosis, knowledge on the precise role of PDGFRß signaling in myelofibrosis is sparse. Using the Gata-1low mouse model for myelofibrosis, we applied RNA sequencing, protein expression analyses, multispectral imaging and, as a novel approach in bone marrow tissue, an in situ proximity ligation assay to provide a detailed characterization of PDGFRß signaling and regulation during development of myelofibrosis. We observed an increase in PDGFRß and PDGF-B protein expression in overt fibrotic bone marrow, along with an increase in PDGFRß-PDGF-B interaction, analyzed by proximity ligation assay. However, PDGFRß tyrosine phosphorylation levels were not increased. We therefore focused on regulation of PDGFRß by protein tyrosine phosphatases as endogenous PDGFRß antagonists. Gene expression analyses showed distinct expression dynamics among PDGFRß-targeting phosphatases. In particular, we observed enhanced T-cell protein tyrosine phosphatase protein expression and PDGFRß-T-cell protein tyrosine phosphatase interaction in early and overt fibrotic bone marrow of Gata-1low mice. In vitro, T-cell protein tyrosine phosphatase (Ptpn2) knockdown increased PDGFRß phosphorylation at Y751 and Y1021, leading to enhanced downstream signaling in fibroblasts. Furthermore, Ptpn2 knockdown cells showed increased growth rates when exposed to low-serum growth medium. Taken together, PDGF signaling is differentially regulated during myelofibrosis. Protein tyrosine phosphatases, which have so far not been examined during disease progression, are novel and hitherto unrecognized components in myelofibrosis.
Assuntos
Mielofibrose Primária , Animais , Camundongos , Fosforilação , Fator de Crescimento Derivado de Plaquetas/genética , Fator de Crescimento Derivado de Plaquetas/metabolismo , Mielofibrose Primária/genética , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Transdução de SinaisRESUMO
BACKGROUND: The 2019 coronavirus disease (COVID-19) caused by the SARS-CoV-2 virus has an impact on all aspects of patient care. Serum ferritin generally represents a biomarker of choice when iron deficiency is suspected. However, ferritin is also an acute-phase-protein exhibiting elevated serum concentration in various inflammatory diseases. Here we focus on the role of serum ferritin for diagnostic and clinical management of patients with COVID-19 in comparison with other infectious and non-infectious diseases. METHODS: We examined scientific articles listed in PubMed reporting on ferritin in various infectious and non-infectious diseases. We then compared these results with nine current COVID-19 ferritin reports published in 2020. RESULTS: Several non-infectious, as well as non-COVID-19 infectious diseases, are characterised by a partly dramatic elevation of serum ferritin levels. All COVID-19 studies published between February and May 2020, which documented laboratory serum ferritin, indicate ferritin as a biomarker of COVID-19 severity in hospitalised patients. CONCLUSIONS: Serum ferritin may be considered both a prognostic and stratifying biomarker that can also contribute to therapeutic decision-making concerning patients with COVID-19. It should be emphasised, however, that most scientific reports refer to cohorts in the Asian region. Further validation in other cohorts is urgently required.
Assuntos
Biomarcadores/sangue , COVID-19/sangue , Doenças Transmissíveis/sangue , Ferritinas/sangue , Inflamação/sangue , COVID-19/epidemiologia , COVID-19/virologia , Doenças Transmissíveis/diagnóstico , Feminino , Humanos , Inflamação/diagnóstico , Masculino , Pandemias , Prognóstico , SARS-CoV-2/fisiologia , Sensibilidade e EspecificidadeRESUMO
Background Lactate dehydrogenase (LD) activity is routinely monitored for therapeutic risk stratification of malignant diseases, but is also prone to preanalytical influences. Methods We systematically analyzed the impact of defined preanalytical conditions on the hemolysis-susceptible parameters LD, potassium (K) and hemolysis index in vacuum blood collection tubes (serum [SE], heparin plasma [HP]). Blood was collected by venipuncture from healthy volunteers. Tubes were either filled or underfilled to approximately 50%, then processed directly or stored at room temperature for 4 h. Potassium (K), sodium (Na), chloride (Cl), LD, creatine kinase (CK), total cholesterol, and indices for hemolysis, icterus, and lipemia were analyzed. Filling velocity was determined in a subset of tubes. Findings in healthy volunteers were reconfirmed in an in-patient cohort (n = 74,751) that was analyzed for plasma yield and LD data distribution. Results LD activity was higher in HP compared to SE. Underfilling led to higher LD values (SE: +21.6%; HP: +28.3%), K (SE: +4.2%; HP: +5.3%), and hemolysis index (SE: +260.8%; HP: +210.0%), while other analytes remained largely unchanged. Filling velocity of tubes was approximately 3-fold higher in the first half compared to the second half in both HP and SE collection tubes. Importantly, plasma yield also inversely correlated with LD in routine patients. By calculating reference limits, the lowest plasma yield quartile of the patient cohort displayed LD values clearly exceeding current reference recommendations. Conclusions Underfilling of tubes leads to a higher proportion of blood aspirated with high velocity and relevant elevations in LD. This finding should be considered in cases of clinically implausible elevated LD activities.
Assuntos
Heparina/química , L-Lactato Desidrogenase/sangue , Flebotomia/métodos , Adulto , Feminino , Hemólise , Humanos , L-Lactato Desidrogenase/normas , Masculino , Pessoa de Meia-Idade , Flebotomia/instrumentação , Flebotomia/normas , Potássio/sangue , Fase Pré-Analítica , Sódio/sangueRESUMO
BACKGROUND: Obesity is associated with cardiovascular complications, including pulmonary hypertension (PH). Reports suggest that peroxisome proliferator-activated receptor-γ (PPARγ) has direct action in preventing vascular remodelling in PH. Here we dissected the specific role of high-fat-diet (HFD)-induced obesity and vascular smooth muscle cell (VSMC)-PPARγ for remodelling of small pulmonary arteries. METHODS: Wild-type (WT) and VSMC-specific PPARγ-knockout (SmPparγ-/-) mice were fed a low-fat-diet (LFD, 10% kcal from fat) or HFD (60% kcal from fat) for 24 weeks. Mice were metabolically phenotyped (e.g. weight development, insulin/glucose tolerance) at the beginning, and after 12 and 24 weeks, respectively. At 24 weeks additionally pulmonary pressure, heart structure, pulmonary vascular muscularization together with gene and protein expression in heart and lung tissues were determined. RESULTS: HFD increased right ventricular systolic pressure (RVSP) to a similar extent in WT and SmPparγ-/- mice. HFD decreased glucose tolerance and insulin sensitivity in both WT and SmPparγ-/- mice. Importantly, the increase in RVSP correlated with the degree of insulin resistance. However, VSMC-PPARγ deficiency increased pulmonary vascular muscularization independently of the diet-induced rise in RVSP. This increase was associated with elevated expression of early growth response protein 1 in heart and osteopontin in lung tissue. CONCLUSIONS: Here we demonstrate a correlation of insulin resistance and pulmonary pressure. Further, deficiency of PPARγ in VSMCs diet-independently leads to increased pulmonary vascular muscularization.
Assuntos
Hipertensão Pulmonar/metabolismo , Músculo Liso Vascular/metabolismo , Obesidade/metabolismo , PPAR gama/deficiência , Artéria Pulmonar/metabolismo , Remodelação Vascular/fisiologia , Animais , Células Cultivadas , Dieta Hiperlipídica/efeitos adversos , Hipertensão Pulmonar/patologia , Resistência à Insulina/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso Vascular/patologia , Obesidade/patologia , Artéria Pulmonar/patologia , Distribuição AleatóriaRESUMO
BACKGROUND: The overall prognosis of non-small cell lung cancer (NSCLC) is poor, and currently only patients with localized disease are potentially curable. Therefore, preferably non-invasively determined biomarkers that detect NSCLC patients at early stages of the disease are of high clinical relevance. The aim of this study was to identify and validate novel protein markers in plasma using the highly sensitive DNA-assisted multiplex proximity extension assay (PEA) to discriminate NSCLC from other lung diseases. METHODS: Plasma samples were collected from a total of 343 patients who underwent surgical resection for different lung diseases, including 144 patients with lung adenocarcinoma (LAC), 68 patients with non-malignant lung disease, 83 patients with lung metastasis of colorectal cancers and 48 patients with typical carcinoid. One microliter of plasma was analyzed using PEA, allowing detection and quantification of 92 established cancer related proteins. The concentrations of the plasma proteins were compared between disease groups. RESULTS: The comparison between LAC and benign samples revealed significantly different plasma levels for four proteins; CXCL17, CEACAM5, VEGFR2 and ERBB3 (adjusted p-value < 0.05). A multi-parameter classifier was developed to discriminate between samples from LAC patients and from patients with non-malignant lung conditions. With a bootstrap aggregated decision tree algorithm (TreeBagger), a sensitivity of 93% and specificity of 64% was achieved to detect LAC in this risk population. CONCLUSIONS: By applying the highly sensitive PEA, reliable protein profiles could be determined in microliter amounts of plasma. We further identified proteins that demonstrated different plasma concentration in defined disease groups and developed a signature that holds potential to be included in a screening assay for early lung cancer detection.
Assuntos
Adenocarcinoma de Pulmão/sangue , Proteínas Sanguíneas/análise , Carcinoma Pulmonar de Células não Pequenas/sangue , Detecção Precoce de Câncer/métodos , Neoplasias Pulmonares/sangue , Programas de Rastreamento/métodos , Idoso , Antígeno Carcinoembrionário/sangue , Quimiocinas CXC/sangue , Estudos de Coortes , Confiabilidade dos Dados , Feminino , Proteínas Ligadas por GPI/sangue , Humanos , Imunoensaio/métodos , Masculino , Modelos Biológicos , Curva ROC , Receptor ErbB-3/sangue , Sensibilidade e Especificidade , Estatísticas não Paramétricas , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/sangueRESUMO
Monocyte migration is a key element in atherosclerosis. LDL-C facilitates monocyte migration via induction of CCR2. PCSK9 regulates cell surface expression of the LDL-R and is expressed in vascular smooth muscle cells (VSMCs). The present study was done to investigate the regulation of PCSK9 in VSMCs and its impact on monocyte function. METHODS AND RESULTS: PCSK9 mRNA and protein levels were upregulated in VSMCs by the TLR-4 ligand LPS, whereas TGF-ß or angiotensin II had no effect. Induction of PCSK9 was selectively inhibited by TLR-4 blockade and further downstream by the SAPK/JNK-inhibitor SP600125, whereas inhibitors of ERK1/2, p38 or PI3-kinase pathways had no effect. Incubation of monocytes in conditioned media from LPS-stimulated VSMCs resulted in a significant reduction of LDL-R levels on monocytes, comparable to the effects of recombinant PCSK9. LDL-C increased monocyte CCR2 expression, which augmented monocyte migration towards MCP-1. This LDL-C dependent monocyte chemotaxis was inhibited by supernatants from LPS-stimulated VSMCs, similar to recombinant PCSK9 and a specific LDL-R blocking antibody. CONCLUSION: PCSK9 is regulated in VSMCs by TLR-4 - SAPK/JNK signaling, a pathway important in inflammation and metabolism. VSMC-derived PCSK9 reduces monocyte LDL-R expression, affecting LDL-C/LDL-R-mediated CCR2-expression on monocytes, which is crucial to cell motility and atherogenesis.
Assuntos
Monócitos/imunologia , Pró-Proteína Convertase 9/imunologia , Receptores CCR2/imunologia , Animais , Aterosclerose/imunologia , Linhagem Celular , Células Cultivadas , Quimiotaxia de Leucócito , Humanos , Inflamação/imunologia , Lipopolissacarídeos/imunologia , Masculino , Monócitos/citologia , Músculo Liso Vascular/citologia , Músculo Liso Vascular/imunologia , Ratos Sprague-Dawley , Receptores CCR2/análise , Receptor 4 Toll-Like/imunologiaRESUMO
The renin-angiotensin system (RAS) and obesity have been implicated in vascular outward remodeling, including aneurysms, but the precise mechanisms are not yet understood. We investigated the effect of the angiotensin receptor type 1 (AT1-receptor) antagonist telmisartan on aortic outward remodeling in a diet-induced obesity model in mice. C57/Black6J mice were fed either a low-fat diet (LFD) or a high-fat diet (HFD) for 14 weeks. One group of HFD mice was additionally exposed to telmisartan (3 mg/kg per day) for the last 4 weeks. HFD led to aortic outward remodeling, characterized by increased proteolysis, along with structural changes, such as fragmentation of elastic fibers and decreased elastin content. Vascular damage was associated with up-regulation of matrix metalloproteinase (MMP)-2 (MMP-2), MMP-3, MMP-12, cathepsin D, and cathepsin B. HFD aortae exhibited an enhanced inflammatory status, characterized by tumor necrosis factor α (TNF-α) and interleukin-1ß (IL-1ß) colocalized with adipocytes in the adventitia. HFD resulted in a significant increase in aortic dimensions, evident by ultrasound measurements. Telmisartan abolished aortic dilatation and preserved elastin content. HFD induced enhanced expression of aortic MMP-2, MMP-9, and TNF-α was abrogated by telmisartan. Adventitial proteolytic and inflammatory factors were also examined in samples from human abdominal aneurysms. The expression of TNF-α, IL-1ß, and MMP-9 was higher in the adventitial fat of diseased vessels compared with healthy tissues. Finally, adipocytes treated with TNF-α showed enhanced MMP-2, MMP-3, and cathepsin D, which was prevented by telmisartan. Taken together, HFD in mice induced aortic dilatation with up-regulation of matrix degrading and inflammatory pathways similar to those seen in human aortic aneurysmatic tissue. The HFD-induced vascular pathology was reduced by AT1-receptor antagonist telmisartan.
Assuntos
Bloqueadores do Receptor Tipo 1 de Angiotensina II/administração & dosagem , Aorta/metabolismo , Obesidade/metabolismo , Receptor Tipo 1 de Angiotensina/metabolismo , Doenças Vasculares/fisiopatologia , Animais , Aorta/efeitos dos fármacos , Dieta Hiperlipídica/efeitos adversos , Humanos , Metaloproteinase 12 da Matriz/genética , Metaloproteinase 12 da Matriz/metabolismo , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 3 da Matriz/genética , Metaloproteinase 3 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/complicações , Obesidade/etiologia , Obesidade/genética , Receptor Tipo 1 de Angiotensina/genética , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Doenças Vasculares/tratamento farmacológico , Doenças Vasculares/etiologia , Remodelação VascularRESUMO
OBJECTIVE: Drug-eluting coronary stents reduce restenosis rate and late lumen loss compared with bare-metal stents; however, drug-eluting coronary stents may delay vascular healing and increase late stent thrombosis. The peroxisome proliferator-activated receptor-delta (PPARδ) exhibits actions that could favorably influence outcomes after drug-eluting coronary stents placement. APPROACH AND RESULTS: Here, we report that PPARδ ligand-coated stents strongly reduce the development of neointima and luminal narrowing in a rabbit model of experimental atherosclerosis. Inhibition of inflammatory gene expression and vascular smooth muscle cell (VSMC) proliferation and migration, prevention of thrombocyte activation and aggregation, and proproliferative effects on endothelial cells were identified as key mechanisms for the prevention of restenosis. Using normal and PPARδ-depleted VSMCs, we show that the observed effects of PPARδ ligand GW0742 on VSMCs and thrombocytes are PPARδ receptor dependent. PPARδ ligand treatment induces expression of pyruvate dehydrogenase kinase isozyme 4 and downregulates the glucose transporter 1 in VSMCs, thus impairing the ability of VSMCs to provide the increased energy demands required for growth factor-stimulated proliferation and migration. CONCLUSIONS: In contrast to commonly used drugs for stent coating, PPARδ ligands not only inhibit inflammatory response and proliferation of VSMCs but also prevent thrombocyte activation and support vessel re-endothelialization. Thus, pharmacological PPARδ activation could be a promising novel strategy to improve drug-eluting coronary stents outcomes.
Assuntos
Angioplastia com Balão/instrumentação , Aorta/efeitos dos fármacos , Doenças da Aorta/prevenção & controle , Aterosclerose/prevenção & controle , Fármacos Cardiovasculares/administração & dosagem , Stents Farmacológicos , PPAR delta/agonistas , Esteroides/administração & dosagem , Trombose/prevenção & controle , Angioplastia com Balão/efeitos adversos , Animais , Aorta/metabolismo , Aorta/patologia , Doenças da Aorta/metabolismo , Doenças da Aorta/patologia , Aterosclerose/metabolismo , Aterosclerose/patologia , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Lesões das Artérias Carótidas/metabolismo , Lesões das Artérias Carótidas/patologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Doença da Artéria Coronariana/metabolismo , Doença da Artéria Coronariana/patologia , Doença da Artéria Coronariana/terapia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Metabolismo Energético/efeitos dos fármacos , Transportador de Glucose Tipo 1/genética , Transportador de Glucose Tipo 1/metabolismo , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Camundongos Knockout , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Neointima , PPAR delta/deficiência , PPAR delta/genética , PPAR delta/metabolismo , Ativação Plaquetária/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Piruvato Desidrogenase Quinase de Transferência de Acetil , Ratos , Ratos Sprague-Dawley , Reepitelização/efeitos dos fármacos , Recidiva , Transdução de Sinais/efeitos dos fármacos , Trombose/etiologia , Trombose/metabolismo , Trombose/patologia , Fatores de TempoAssuntos
COVID-19 , Moléculas de Adesão Celular , Serviço Hospitalar de Emergência , Humanos , Prognóstico , SARS-CoV-2RESUMO
AIM: Degradation of extracellular matrices is an integral part in periodontitis. For antagonizing this pathophysiological mechanism, we aimed at identifying gene expression profiles in disease progression contributing periodontitis-associated fibroblasts (PAFs) versus normal gingival fibroblasts to determine their molecular repertoire, and exploit it for therapeutic intervention. MATERIALS AND METHODS: Applying an exploratory analysis using a small number of microarrays in combination with a three dimensional (3D) in vitro culture model that incorporates some aspects of periodontitis, PAFs were initially characterized by gene-expression analyses, followed by targeted gene down-regulation and pharmacological intervention in vitro. Further, immunohistochemistry was applied for phosphorylation analyses in tissue specimens. RESULTS: PAFs were characterized by 42 genes being commonly up-regulated >1.5-fold, and by five genes that were concordantly down-regulated (<0.7-fold). Expression of vascular endothelial growth factor (VEGF)-receptor 1 (Flt-1) was highly enhanced, and was thus further explored in in vitro culture models of periodontal fibroblasts without accounting for the microbiome. Phosphorylation of the VEGF-receptor 1 was enhanced in PAFs. Receptor inhibition by a specific VEGF-receptor inhibitor or intrinsic down-regulation by RNAi of the VEGF-receptor kinase in 3D gel cultures resulted in significant reduction in collagen degradation associated with increased tissue inhibitor of metalloproteinase expression, suggesting that Flt-1 may contribute to periodontitis. CONCLUSION: Based on the finding that VEGF-receptor kinase inhibition impaired collagen degradation pathways, Flt-1 may represent a candidate for therapeutic approaches in periodontitis.
Assuntos
Periodontite , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Regulação para Baixo , Fibroblastos , Gengiva , HumanosRESUMO
RATIONALE: The nuclear receptor peroxisome proliferator-activated receptor-γ (PPARγ) is an important regulator of gene transcription in vascular cells and mediates the vascular protection observed with antidiabetic glitazones. OBJECTIVE: To determine the molecular mechanism of ligand-dependent transrepression in vascular smooth muscle cells and their impact on the vascular protective actions of PPARγ. METHODS AND RESULTS: Here, we report a molecular pathway in vascular smooth muscle cells by which ligand-activated PPARγ represses transcriptional activation of the matrix-degrading matrix metalloproteinase-9 (MMP-9) gene, a crucial mediator of vascular injury. PPARγ-mediated transrepression of the MMP-9 gene was dependent on the presence of the high-mobility group A1 (HMGA1) protein, a gene highly expressed in vascular smooth muscle cells, newly identified by oligonucleotide array expression analysis. Transrepression of MMP-9 by PPARγ and regulation by HMGA1 required PPARγ SUMOylation at K367. This process was associated with formation of a complex between PPARγ, HMGA1, and the SUMO E2 ligase Ubc9 (ubiquitin-like protein SUMO-1 conjugating enzyme). After PPARγ ligand stimulation, HMGA1 and PPARγ were recruited to the MMP-9 promoter, which facilitated binding of SMRT (silencing mediator of retinoic acid and thyroid hormone receptor), a nuclear corepressor involved in transrepression. The relevance of HMGA1 for vascular PPARγ signaling was underlined by the complete absence of vascular protection through a PPARγ ligand in HMGA1(-/-) mice after arterial wire injury. CONCLUSIONS: The present data suggest that ligand-dependent formation of HMGA1-Ubc9-PPARγ complexes facilitates PPARγ SUMOylation, which results in the prevention of SMRT corepressor clearance and induction of MMP-9 transrepression. These data provide new information on PPARγ-dependent vascular transcriptional regulation and help us to understand the molecular consequences of therapeutic interventions with PPARγ ligands in the vasculature.
Assuntos
Proteína HMGA1a/metabolismo , Músculo Liso Vascular/metabolismo , PPAR gama/metabolismo , Transdução de Sinais/fisiologia , Transcrição Gênica/fisiologia , Animais , Endotelina-1/metabolismo , Artéria Femoral/efeitos dos fármacos , Artéria Femoral/lesões , Artéria Femoral/metabolismo , Proteína HMGA1a/deficiência , Proteína HMGA1a/genética , Masculino , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Knockout , Modelos Animais , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/lesões , NF-kappa B/metabolismo , Tiazolidinedionas/farmacologia , Enzimas de Conjugação de Ubiquitina/metabolismoRESUMO
Protein tyrosine phosphatases (PTPs) regulate multiple signaling pathways. Disruption of tyrosine phosphorylation through imbalanced action between protein tyrosine kinases (RTKs) and PTPs is a hallmark of metabolic disorders, including insulin resistance. A representative member of the receptor-type PTP family, PTPRJ (DEP-1), was previously identified as a negative regulator of insulin signaling and possesses post-translational glycosylation sites. In this regard, it seems of great importance to decipher the structure of PTPRJ's glycosylation, particularly in the context of metabolic disturbances, but this has not been done in detail. Thus, here we aimed at characterizing the glycosylation pattern of PTPRJ in liver. We show that N-glycosylation accounts for up to half of PTPRJ's molecular weight. Applying mass spectrometry, we detected increased levels of high-mannose structures in PTPRJ in liver tissue of obese mice compared to lean littermates. In addition, complex neutral structures without fucose were also elevated in PTPRJ of high-fat diet (HFD) mice. Conversely, complex fucosylated N-glycans as well as sialylated bi- and triantennary N-glycans, were significantly reduced in PTPRJ of HFD-derived liver tissue compared to LFD by â¼two fold (P≤.01, P≤.0001 and P≤.001, respectively). In congruence with these findings, the mannosidase MAN2A1, responsible for the conversion of high-mannose to complex N-glycans, was significantly downregulated under HFD conditions. Here we present for the first time that HFD-induced obesity impacts on the glycosylation pattern of the insulin signaling component PTPRJ in liver. These findings may inspire new research on the glycosylation of PTPs in metabolic diseases and may open up new therapeutic approaches.
Assuntos
Dieta Hiperlipídica , Glicosilação , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores , Animais , Camundongos , Dieta Hiperlipídica/efeitos adversos , Insulina/metabolismo , Fígado/metabolismo , Manose/metabolismo , Polissacarídeos , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/metabolismoRESUMO
BACKGROUND: Epidemiological data suggest that sex independently contributes to cardiovascular risk. Clinical trials are often hampered by the enrollment of few female patients. METHODS AND RESULTS: The Ongoing Telmisartan Alone and in Combination With Ramipril Global End Point Trial (ONTARGET) and the parallel Telmisartan Randomized Assessment Study in ACE Intolerant Subjects With Cardiovascular Disease (TRANSCEND) included a large proportion of female patients (9378 female versus 22 168 male patients). Differences in male and female patients enrolled in ONTARGET/TRANSCEND were analyzed for the primary 4-fold end point (composite of cardiovascular death, myocardial infarction, stroke, or admission to hospital for heart failure), a secondary 3-fold end point (cardiovascular death, myocardial infarction, stroke), and individual components of the primary composite. Baseline characteristics included age, ethnicity, body mass index, physical activity, tobacco use, alcohol consumption, formal education, clinical diagnosis for study entry, patient history, and concomitant medication. Patients were followed up until death or the end of the study (median, 56 months). Compared with male patients, female patients had a 19% significantly lower risk for the 4-fold end point and 21% for the 3-fold end point (after adjustment for study, treatment, and the above baseline values). Similarly, the adjusted risk for cardiovascular death (17%) and myocardial infarction (22%), but not for stroke and hospitalization for heart failure, was also significantly lower in women. Diabetic female patients were characterized by a higher risk for acute myocardial infarction compared with diabetic male patients, whereas alcohol consumption resulted in significantly lower risk in women. CONCLUSIONS: In our analysis made up of 70.3% male and 29.7% female patients, an ≈20% lower risk for the combined cardiovascular end points in female patients was observed despite treatment with cardioprotective agents. This difference was driven primarily by a significantly lower incidence of myocardial infarction. Thus, we demonstrate in a large interventional trial that sex greatly affects the occurrence of cardiovascular events in patients with vascular disease or high-risk diabetes mellitus. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT00153101.