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Sepsis-associated encephalopathy (SAE) is associated with increased risk of long-term cognitive impairment. SAE is driven, at least in part, by brain endothelial dysfunction in response to systemic cytokine signaling. However, the mechanisms driving SAE and its consequences remain largely unknown. Here, we performed translating ribosome affinity purification and RNA-sequencing (TRAP-seq) from the brain endothelium to determine the transcriptional changes after an acute endotoxemic (LPS) challenge. LPS induced a strong acute transcriptional response in the brain endothelium that partially correlates with the whole brain transcriptional response and suggested an endothelial-specific hypoxia response. Consistent with a crucial role for IL-6, loss of the main regulator of this pathway, SOCS3, leads to a broadening of the population of genes responsive to LPS, suggesting that an overactivation of the IL-6/JAK/STAT3 pathway leads to an increased transcriptional response that could explain our prior findings of severe brain injury in these mice. To identify any potential sequelae of this acute response, we performed brain TRAP-seq following a battery of behavioral tests in mice after apparent recovery. We found that the transcriptional response returns to baseline within days post-challenge, but reductions in gene expression regulating protein translation and respiratory electron transport remained. We observed that mice that recovered from the endotoxemic shock showed mild, sex-dependent cognitive impairment, suggesting that the acute brain injury led to sustained effects. A better understanding of the transcriptional and non-transcriptional changes in response to shock is needed in order to prevent and/or revert the devastating consequences of septic shock.
Assuntos
Encéfalo , Disfunção Cognitiva , Lipopolissacarídeos , Camundongos Endogâmicos C57BL , Animais , Masculino , Encéfalo/metabolismo , Encéfalo/efeitos dos fármacos , Disfunção Cognitiva/metabolismo , Feminino , Lipopolissacarídeos/farmacologia , Camundongos , Encefalopatia Associada a Sepse/metabolismo , Endotélio/metabolismo , Endotélio/efeitos dos fármacos , Endotoxinas/farmacologia , Endotoxinas/toxicidadeRESUMO
PURPOSE: Newer treatment options offer the promise of improved outcomes for metastatic and unresectable melanoma. This investigation was performed to review these modalities for cutaneous eyelid and orbital disease. METHODS: A search for articles that were related to this subject was performed in the PubMed database, and the bibliographies of these manuscripts were reviewed to ensure capture of the appropriate literature. Data was abstracted and analyzed. RESULTS: Historically, patients who suffer from melanoma of the ocular adnexa have fared poorly. Approaches that employ BRAF and mitogen-associated protein kinase inhibitors, immunotherapy, and novel cellular therapies improve outcomes and survival rates, although the side effect profiles of these agents are problematic. Most of the existing strategies have not explored ocular adnexal disease specifically, and treatment plans are generally adapted from the general cutaneous oncology literature. CONCLUSIONS: Thanks to advances in our comprehension of the cellular biology of the disease, the management of unresectable and metastatic melanoma has evolved considerably over the past several years. Newer modalities will likely continue to improve survival and reduce adverse events.
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Purpose: Overactivation of the mitogen activated kinase pathway has been associated with rosacea. We hypothesised that inhibitors of this pathway can be repurposed to alleviate rosacea symptoms. Methods: In order to test this hypothesis, we designed a double-blind, randomised, placebo-controlled phase I clinical trial to assess the safety and tolerability of a first-in-kind topical formulation of a MEK kinase inhibitor, trametinib. Subjects applied daily trametinib-containing cream (0.05 mg in 0.5 mL) to one cheek and cream without inhibitor to the other for consecutive 21 days. Skin irritation scores and blood samples were obtained during visits on days 8, 15 and 22. Results: On analysis of high-performance liquid chromatography, no systemic trametinib absorption was detected during this treatment period. Subjects demonstrated a slight but significant improvement in both cheeks, regardless of drug contents. No adverse effects were reported during this time. Conclusions: Topical trametinib was well tolerated at a dose of 0.05 mg per day without meaningful systemic absorption or local adverse events. A dose escalation trial is warranted to determine optimal dosing to treat rosacea while avoiding the adverse effects of systemic treatment.
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Sepsis-associated encephalopathy (SAE) is a common manifestation in septic patients that is associated with increased risk of long-term cognitive impairment. SAE is driven, at least in part, by brain endothelial dysfunction in response to systemic cytokine signaling. However, the mechanisms driving SAE and its consequences remain largely unknown. Here, we performed translating ribosome affinity purification and RNA-sequencing (TRAP-seq) from the brain endothelium to determine the transcriptional changes after an acute endotoxemic (LPS) challenge. LPS induced a strong acute transcriptional response in the brain endothelium that partially correlates with the whole brain transcriptional response and suggested an endothelial-specific hypoxia response. Consistent with a crucial role for IL-6, loss of the main regulator of this pathway, SOCS3, leads to a broadening of the population of genes responsive to LPS, suggesting that an overactivation of the IL-6/JAK/STAT3 pathway leads to an increased transcriptional response that could explain our prior findings of severe brain injury in these mice. To identify any potential sequelae of this acute response, we performed brain TRAP-seq following a battery of behavioral tests in mice after apparent recovery. We found that the transcriptional response returns to baseline within days post-challenge. Despite the transient nature of the response, we observed that mice that recovered from the endotoxemic shock showed mild, sex-dependent cognitive impairment, suggesting that the acute brain injury led to sustained, non-transcriptional effects. A better understanding of the transcriptional and non-transcriptional changes in response to shock is needed in order to prevent and/or revert the devastating consequences of septic shock.
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Transient early endosome (EE)-mitochondria interactions can mediate mitochondrial iron translocation, but the associated mechanisms are still elusive. We showed that Divalent Metal Transporter 1 (DMT1) sustains mitochondrial iron translocation via EE-mitochondria interactions in triple-negative MDA-MB-231, but not in luminal A T47D breast cancer cells. DMT1 silencing increases labile iron pool (LIP) levels and activates PINK1/Parkin-dependent mitophagy in MDA-MB-231 cells. Mitochondrial bioenergetics and the iron-associated protein profile were altered by DMT1 silencing and rescued by DMT1 re-expression. Transcriptomic profiles upon DMT1 silencing are strikingly different between 2D and 3D culture conditions, suggesting that the environment context is crucial for the DMT1 knockout phenotype observed in MDA-MB-231 cells. Lastly, in vivo lung metastasis assay revealed that DMT1 silencing promoted the outgrowth of lung metastatic nodules in both human and murine models of triple-negative breast cancer cells. These findings reveal a DMT1-dependent pathway connecting EE-mitochondria interactions to mitochondrial iron translocation and metastatic fitness of breast cancer cells.
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Neoplasias da Mama , Ferro , Animais , Feminino , Humanos , Camundongos , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Endossomos/metabolismo , Ferro/metabolismo , Mitocôndrias/metabolismo , MitofagiaRESUMO
PURPOSE: The neutrophil-to-lymphocyte ratio (NLR) is a relatively novel biomarker to distinguish between acute stresses. This study was performed to determine whether the NLR may discern infectious orbital maladies from idiopathic orbital inflammation (IOI). METHODS: The NLR was calculated by a review of the initial blood draws of adult patients who presented to the emergency department at a single academic medical center. Statistical comparisons were performed to identify the significance of these results. RESULTS: Ten patients with IOI, 12 patients with necrotizing fasciitis (NF), and 12 patients with orbital cellulitis (OC) presented to the emergency department. The groups were not statistically significantly different in terms of age or gender. The mean NLRs were 3.48 (standard deviation = 1.80), 13.5 (standard deviation = 14.5), and 8.15 (standard deviation = 6.56) for IOI, NF, and OC, respectively. Patients with IOI had statistically significantly lower NLRs than patients with NF ( p = 0.037) and OC ( p = 0.034). However, the NLRs of patients with OC were not statistically significantly different from those of patients with NF ( p = 0.27). CONCLUSIONS: The NLR appears to distinguish IOI from infectious etiologies, but does not discern between variants of infection. These results should be juxtaposed against appropriate imaging and clinical evaluations, but elevated NLR values may heighten clinicians' concerns for an infectious process and encourage them to initiate appropriate management steps.
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Doenças Transmissíveis , Celulite Orbitária , Adulto , Humanos , Neutrófilos , Linfócitos , Celulite Orbitária/diagnóstico , Inflamação , Estudos RetrospectivosRESUMO
Endothelial dysfunction is a crucial factor in promoting organ failure during septic shock. However, the underlying mechanisms are unknown. Here, we show that kidney injury after lipopolysaccharide (LPS) insult leads to strong endothelial transcriptional and epigenetic responses. Furthermore, SOCS3 loss leads to an aggravation of the responses, demonstrating a causal role for the STAT3-SOCS3 signaling axis in the acute endothelial response to LPS. Experiments in cultured endothelial cells demonstrate that IL-6 mediates this response. Furthermore, bioinformatics analysis of in vivo and in vitro transcriptomics and epigenetics suggests a role for STAT, AP1 and interferon regulatory family (IRF) transcription factors. Knockdown of STAT3 or the AP1 member JunB partially prevents the changes in gene expression, demonstrating a role for these transcription factors. In conclusion, endothelial cells respond with a coordinated response that depends on overactivated IL-6 signaling via STAT3, JunB and possibly other transcription factors. Our findings provide evidence for a critical role of IL-6 signaling in regulating shock-induced epigenetic changes and sustained endothelial activation, offering a new therapeutic target to limit vascular dysfunction.
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Metilação de DNA , Células Endoteliais , Metilação de DNA/genética , Interleucina-6/genética , Lipopolissacarídeos , EndotélioRESUMO
Aging is associated with nonresolving inflammation and tissue dysfunction. Resolvin D2 (RvD2) is a proresolving ligand that acts through the G-protein-coupled receptor called GPR18. Unbiased RNA sequencing revealed increased Gpr18 expression in macrophages from old mice, and in livers from elderly humans, which was associated with increased steatosis and fibrosis in middle-aged (MA) and old mice. MA mice that lacked GPR18 on myeloid cells had exacerbated steatosis and hepatic fibrosis, which was associated with a decline in Mac2+ macrophages. Treatment of MA mice with RvD2 reduced steatosis and decreased hepatic fibrosis, correlating with increased Mac2+ macrophages, increased monocyte-derived macrophages, and elevated numbers of monocytes in the liver, blood, and bone marrow. RvD2 acted directly on the bone marrow to increase monocyte-macrophage progenitors. A transplantation assay further demonstrated that bone marrow from old mice facilitated hepatic collagen accumulation in young mice. Transient RvD2 treatment to mice transplanted with bone marrow from old mice prevented hepatic collagen accumulation. Together, this study demonstrates that RvD2-GPR18 signaling controls steatosis and fibrosis and provides a mechanistic-based therapy for promoting liver repair in aging.
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Medula Óssea , Fígado Gorduroso , Pessoa de Meia-Idade , Humanos , Camundongos , Animais , Idoso , Medula Óssea/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Envelhecimento , Cirrose Hepática , Fibrose , Colágeno/genética , Camundongos Endogâmicos C57BLRESUMO
PURPOSE: Recently, several new therapies have emerged to address locally advanced cutaneous basal cell and squamous cell carcinomas. Given the constraints of the ocular adnexa and orbit, this review was designed to discuss the role of these modalities in this region. METHODS: A PubMed search was carried out to analyze the utility of United States Food and Drug Administration-approved therapies to address these malignancies. The data presented in the identified investigations were analyzed and abstracted. RESULTS: Multiple novel interventions may be useful in the management of periocular cutaneous basal cell and squamous cell carcinomas, including imiquimod, hedgehog inhibitors, and immunotherapy. While many of these treatments have not been specifically explored in the orbit and ocular adnexa, the literature generally shows favorable response rates. However, adverse events were common in these studies. CONCLUSIONS: Several novel treatments may address periorbital cutaneous malignancies, and these therapies may be particularly useful in patients with unresectable disease and those who are poor surgical candidates.
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Carcinoma Basocelular , Carcinoma de Células Escamosas , Neoplasias Cutâneas , Estados Unidos , Humanos , Carcinoma Basocelular/cirurgia , Proteínas Hedgehog/uso terapêutico , Neoplasias Cutâneas/patologia , Carcinoma de Células Escamosas/terapiaRESUMO
Increased circulating levels of soluble interleukin (IL)-6 receptor α (sIL-6Rα) are commonly observed during inflammatory responses, allowing for IL-6 signaling in cells that express the ubiquitous receptor subunit gp130 but not IL-6Rα, such as endothelial cells. Activation of Toll-like receptor (TLR)-4 or the tumor necrosis factor (TNF) receptor leads to NF-κB-dependent increases in endothelial IL-6 expression. Thus, we hypothesize that danger signals may induce autocrine IL-6 signaling within the endothelium via sIL-6Rα-mediated trans-signaling. In support of this hypothesis, we recently demonstrated that conditional deletion in the endothelium of the IL-6 signaling inhibitor SOCS3 leads to rapid mortality in mice challenged with the TLR-4 agonist endotoxin through increases in vascular leakage, thrombosis, leukocyte adhesion, and a type I-like interferon response. Here, we sought to directly test a role for sIL-6Rα in LPS-treated human umbilical vein and dermal blood microvascular endothelial cells. We show that cotreatment with sIL-6Rα dramatically increases the loss of barrier function and the expression of COX2 and tissue factor mRNA levels induced by LPS. This cotreatment led to strong activation of STAT1 and STAT3 while not affecting LPS-induced activation of p38 and NF-κB signaling. Similar results were obtained when sIL-6Rα was added to a TNF challenge. JAK inhibition by pretreatment with ruxolitinib or by SOCS3 overexpression blunted LPS and sIL-6R synergistic effects, whereas SOCS3 knockdown further increased the response. Together, these findings demonstrate that IL-6 signaling downstream of NF-κB activation leads to a strong endothelial activation and may explain the acute endotheliopathy observed during critical illness.
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Células Endoteliais , Interleucina-6 , Animais , Células Endoteliais/metabolismo , Endotélio/metabolismo , Endotoxinas/metabolismo , Endotoxinas/toxicidade , Humanos , Interleucina-6/metabolismo , Lipopolissacarídeos/toxicidade , Camundongos , NF-kappa B/metabolismo , Proteína 3 Supressora da Sinalização de Citocinas/genética , Proteína 3 Supressora da Sinalização de Citocinas/metabolismo , Proteínas Supressoras da Sinalização de Citocina/metabolismoRESUMO
PURPOSE: Rosacea is a common cause of ocular surface disease. Specific immunologic features have been implicated in its pathogenesis, including toll-like receptors, mitogen-associated kinase, and nuclear factor kappa-B. Myeolid differentiation factor 88 (MYD88) has been associated with these elements, suggesting a role for this protein in rosacea. This study was designed to compare the expression of MYD88 in the eyelids of patients with and without this disease. METHODS: Western blotting for MYD88 was performed in 14 control patients and 15 patients with rosacea. Bands were quantified and normalized to actin. Immunohistochemical staining for MYD88 was performed in a different cohort of 12 patients with rosacea and 12 controls, and positively-staining cells were counted across five consecutive 40x fields. Statistical analyses compared the differences between the two groups via a dedicated software package. RESULTS: On western blotting, the mean ratios of MYD88 to actin were 13.8 (standard deviation = 14.1) and 44.3 (standard deviation = 39.6) in control and rosacea patients, respectively (p = .002). On immunohistochemistry, the mean numbers of positively-staining cells were 12.1/40x field (standard deviation = 9.61/40x field) and 27.4/40x (standard deviation = 18.7/40x field) in control and rosacea patients, respectively (p = .0438). CONCLUSIONS: MYD88 is enriched in eyelid specimens of rosacea. This finding further implicates the innate immune system in the pathogenesis of rosacea, and is consistent with previous reports regarding the role of this protein in ocular surface disease and the previously-implicated cellular features of the disease. Inhibition of MYD88 may be a successful treatment strategy to manage rosacea.
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Fator 88 de Diferenciação Mieloide/metabolismo , Rosácea , Actinas/metabolismo , Pálpebras/patologia , Humanos , NF-kappa B/metabolismo , Rosácea/patologiaRESUMO
Rosacea is a common chronic skin disease affecting mostly people aged 40 and above, with currently no cure. When it affects the eyelids and periocular skin, it leads to dry eye and potentially corneal damage. Research performed over the last decade shed light into the potential mechanisms leading to skin hypersensitivity and provided promising avenues for development of novel, rational therapeutics aimed at reducing the skin inflammatory state. In this review, we discuss the current knowledge on the mechanisms of rosacea in general and of periocular skin-affecting disease in particular, identify key questions that remain to be answered in future research, and offer a disease model that can explain the key characteristics of this disease, with particular emphasis on a potential positive feedback loop that could explain both the acute and chronic features of rosacea.
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Rosácea , Pálpebras , Humanos , PeleRESUMO
SOCS3 is the main inhibitor of the JAK/STAT3 pathway. This pathway is activated by interleukin 6 (IL-6), a major mediator of the cytokine storm during shock. To determine its role in the vascular response to shock, we challenged mice lacking SOCS3 in the adult endothelium (SOCS3iEKO) with a nonlethal dose of lipopolysaccharide (LPS). SOCS3iEKO mice died 16-24 hours postinjection after severe kidney failure. Loss of SOCS3 led to an LPS-induced type I IFN-like program and high expression of prothrombotic and proadhesive genes. Consistently, we observed intraluminal leukocyte adhesion and neutrophil extracellular trap-osis (NETosis), as well as retinal venular leukoembolization. Notably, heterozygous mice displayed an intermediate phenotype, suggesting a gene dose effect. In vitro studies were performed to study the role of SOCS3 protein levels in the regulation of the inflammatory response. In human umbilical vein endothelial cells, pulse-chase experiments showed that SOCS3 protein had a half-life less than 20 minutes. Inhibition of SOCS3 ubiquitination and proteasomal degradation led to protein accumulation and a stronger inhibition of IL-6 signaling and barrier function loss. Together, our data demonstrate that the regulation of SOCS3 protein levels is critical to inhibit IL-6-mediated endotheliopathy during shock and provide a promising therapeutic avenue to prevent multiorgan dysfunction through stabilization of endothelial SOCS3.
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Endotélio Vascular/patologia , Endotoxemia/imunologia , Proteína 3 Supressora da Sinalização de Citocinas/metabolismo , Animais , Modelos Animais de Doenças , Endotoxemia/diagnóstico , Endotoxemia/mortalidade , Endotoxemia/patologia , Heterozigoto , Células Endoteliais da Veia Umbilical Humana , Humanos , Interleucina-6/metabolismo , Lipopolissacarídeos/administração & dosagem , Lipopolissacarídeos/imunologia , Camundongos , Camundongos Knockout , Proteólise , Índice de Gravidade de Doença , Proteína 3 Supressora da Sinalização de Citocinas/análise , Proteína 3 Supressora da Sinalização de Citocinas/genética , UbiquitinaçãoRESUMO
Ca2+ /calmodulin-dependent protein kinase II (CaMKII) is a ubiquitous serine threonine kinase with established roles in physiological and pathophysiological vascular remodeling. Based on our previous study demonstrating that CaMKIIδ promotes thrombin-induced endothelial permeability and recent reports that CaMKII may contribute to inflammatory remodeling in the heart, we investigated CaMKIIδ-dependent regulation of endothelial function downstream of an interleukin-6 (IL-6)/JAK/STAT3 signaling axis. Upon treatment with IL-6 and its soluble receptor (sIL-6r), CaMKIIδ expression is significantly induced in HUVEC. Using pharmacological inhibitors of JAK and siRNA targeting STAT3, we demonstrated that activation of STAT3 is sufficient to induce CaMKIIδ expression. Under these conditions, rather than promoting IL-6-induced permeability, we found that CaMKIIδ promotes endothelial cell migration as measured by live cell imaging of scratch wound closure and single-cell motility analysis. In a similar manner, endothelial cell proliferation was attenuated upon knockdown of CaMKIIδ as determined by growth curves, cell cycle analysis, and capacitance of cell-covered electrodes as measured by ECIS. Using inducible endothelial-specific STAT3 knockout mice, we demonstrate that STAT3 signaling promotes developmental angiogenesis in the neonatal mouse retina assessed at postnatal day 6. CaMKIIδ expression in retinal endothelium was attenuated in these animals as measured by qPCR. STAT3's effects on angiogenesis were phenocopied by the endothelial-specific knockout of CaMKIIδ, with significantly reduced vascular outgrowth and number of junctions in the developing P6 retina. For the first time, we demonstrate that transcriptional regulation of CaMKIIδ by STAT3 promotes endothelial motility, proliferation, and in vivo angiogenesis.
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Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Interleucina-6/metabolismo , Janus Quinases/metabolismo , Vasos Retinianos/fisiologia , Fator de Transcrição STAT3/metabolismo , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Movimento Celular , Regulação da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana , Humanos , Interleucina-6/genética , Janus Quinases/genética , Camundongos , Neovascularização Fisiológica , Isoformas de Proteínas , Interferência de RNA , Retina , Fator de Transcrição STAT3/genética , Regulação para CimaRESUMO
The COVID19 pandemic has caused more than a million of deaths worldwide, primarily due to complications from COVID19-associated acute respiratory distress syndrome (ARDS). Controversy surrounds the circulating cytokine/chemokine profile of COVID19-associated ARDS, with some groups suggesting that it is similar to patients without COVID19 ARDS and others observing substantial differences. Moreover, although a hyperinflammatory phenotype associates with higher mortality in non-COVID19 ARDS, there is little information on the inflammatory landscape's association with mortality in patients with COVID19 ARDS. Even though the circulating leukocytes' transcriptomic signature has been associated with distinct phenotypes and outcomes in critical illness including ARDS, it is unclear whether the mortality-associated inflammatory mediators from patients with COVID19 are transcriptionally regulated in the leukocyte compartment. Here, we conducted a prospective cohort study of 41 mechanically ventilated patients with COVID19 infection using highly calibrated methods to define the levels of plasma cytokines/chemokines and their gene expressions in circulating leukocytes. Plasma IL1RA and IL8 were found positively associated with mortality, whereas RANTES and EGF negatively associated with that outcome. However, the leukocyte gene expression of these proteins had no statistically significant correlation with mortality. These data suggest a unique inflammatory signature associated with severe COVID19.
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COVID-19/metabolismo , COVID-19/patologia , Inflamação/metabolismo , Síndrome do Desconforto Respiratório/mortalidade , SARS-CoV-2 , Idoso , COVID-19/mortalidade , Estudos de Coortes , Citocinas/genética , Citocinas/metabolismo , Feminino , Regulação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
OBJECTIVE: Atherosclerosis predominantly forms in regions of oscillatory shear stress while regions of laminar shear stress are protected. This protection is partly through the endothelium in laminar flow regions expressing an anti-inflammatory and antithrombotic gene expression program. Several molecular pathways transmitting these distinct flow patterns to the endothelium have been defined. Our objective is to define the role of the MEF2 (myocyte enhancer factor 2) family of transcription factors in promoting an atheroprotective endothelium. Approach and Results: Here, we show through endothelial-specific deletion of the 3 MEF2 factors in the endothelium, Mef2a, -c, and -d, that MEF2 is a critical regulator of vascular homeostasis. MEF2 deficiency results in systemic inflammation, hemorrhage, thrombocytopenia, leukocytosis, and rapid lethality. Transcriptome analysis reveals that MEF2 is required for normal regulation of 3 pathways implicated in determining the flow responsiveness of the endothelium. Specifically, MEF2 is required for expression of Klf2 and Klf4, 2 partially redundant factors essential for promoting an anti-inflammatory and antithrombotic endothelium. This critical requirement results in phenotypic similarities between endothelial-specific deletions of Mef2a/c/d and Klf2/4. In addition, MEF2 regulates the expression of Notch family genes, Notch1, Dll1, and Jag1, which also promote an atheroprotective endothelium. In contrast to these atheroprotective pathways, MEF2 deficiency upregulates an atherosclerosis promoting pathway through increasing the amount of TAZ (transcriptional coactivator with PDZ-binding motif). CONCLUSIONS: Our results implicate MEF2 as a critical upstream regulator of several transcription factors responsible for gene expression programs that affect development of atherosclerosis and promote an anti-inflammatory and antithrombotic endothelium. Graphic Abstract: A graphic abstract is available for this article.
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Aterosclerose/metabolismo , Endotélio Vascular/metabolismo , Fatores de Transcrição MEF2/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Aterosclerose/genética , Aterosclerose/patologia , Endotélio Vascular/patologia , Feminino , Regulação da Expressão Gênica , Homeostase , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/deficiência , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Fatores de Transcrição MEF2/deficiência , Fatores de Transcrição MEF2/genética , Masculino , Camundongos , Camundongos Knockout , Receptores Notch/genética , Transdução de Sinais , Transativadores/metabolismoRESUMO
OBJECTIVE: Plaque necrosis is a key feature of defective resolution in atherosclerosis. Recent evidence suggests that necroptosis promotes plaque necrosis; therefore, we sought to determine how necroptotic cells (NCs) impact resolution programs in plaques. Approach and Results: To investigate the role(s) of necroptosis in advanced atherosclerosis, we used mice deficient of Mlkl, an effector of necroptosis. Mlkl-/- mice that were injected with a gain-of-function mutant PCSK9 (AAV8-gof-PCSK9) and fed a Western diet for 16 weeks, showed significantly less plaque necrosis, increased fibrous caps and improved efferocytosis compared with AAV8-gof-PCSK9 injected wt controls. Additionally, hypercholesterolemic Mlkl-/- mice had a significant increase in proresolving mediators including resolvin D1 (RvD1) and a decrease in prostanoids including thromboxane in plaques and in vitro. We found that exuberant thromboxane released by NCs impaired the clearance of both apoptotic cells and NCs through disruption of oxidative phosphorylation in macrophages. Moreover, we found that NCs did not readily synthesize RvD1 and that exogenous administration of RvD1 to macrophages rescued NC-induced defective efferocytosis. RvD1 also enhanced the uptake of NCs via the activation of p-AMPK (AMP-activated protein kinase), increased fatty acid oxidation, and enhanced oxidative phosphorylation in macrophages. CONCLUSIONS: These results suggest that NCs derange resolution by limiting key SPMs and impairing the efferocytic repertoire of macrophages. Moreover, these findings provide a molecular mechanism for RvD1 in directing proresolving metabolic programs in macrophages and further suggests RvD1 as a potential therapeutic strategy to limit NCs in tissues. Graphic Abstract: A graphic abstract is available for this article.
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Ácidos Docosa-Hexaenoicos/metabolismo , Ácidos Graxos/metabolismo , Macrófagos/metabolismo , Necroptose/fisiologia , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacologia , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Apoptose , Feminino , Macrófagos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Necrose , Fosforilação Oxidativa , Fagocitose , Placa Aterosclerótica/metabolismo , Placa Aterosclerótica/patologia , Prostaglandinas/metabolismo , Proteínas Quinases/deficiência , Proteínas Quinases/genéticaRESUMO
Temperature measurement at the nanoscale has brought insight to a wide array of research interests in modern chemistry, physics, and biology. These measurements have been enabled by the advent of nanothermometers, which relay nanoscale temperature information through the analysis of their intrinsic photophysical behavior. In the past decade, several nanothermometers have been developed including dyes, nanodiamonds, fluorescent proteins, nucleotides, and nanoparticles. However, temperature measurement using intact DNA has not yet been achieved. Here, we present a method to study the temperature sensitivity of the DNA molecule within a physiologic temperature range when complexed with fluorescent dye. We theoretically and experimentally report the temperature sensitivity of the DNA-Hoechst 33342 complex in different sizes of double-stranded oligonucleotides and plasmids, showing its potential use as a nanothermometer. These findings allow for extending the thermal study of DNA to several research fields including DNA nanotechnology, optical tweezers, and DNA nanoparticles.
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Nanodiamantes , Nanotecnologia , DNA , Corantes Fluorescentes , TemperaturaRESUMO
We performed RNA-seq and high-resolution mass spectrometry on 128 blood samples from COVID-19-positive and COVID-19-negative patients with diverse disease severities and outcomes. Quantified transcripts, proteins, metabolites, and lipids were associated with clinical outcomes in a curated relational database, uniquely enabling systems analysis and cross-ome correlations to molecules and patient prognoses. We mapped 219 molecular features with high significance to COVID-19 status and severity, many of which were involved in complement activation, dysregulated lipid transport, and neutrophil activation. We identified sets of covarying molecules, e.g., protein gelsolin and metabolite citrate or plasmalogens and apolipoproteins, offering pathophysiological insights and therapeutic suggestions. The observed dysregulation of platelet function, blood coagulation, acute phase response, and endotheliopathy further illuminated the unique COVID-19 phenotype. We present a web-based tool (covid-omics.app) enabling interactive exploration of our compendium and illustrate its utility through a machine learning approach for prediction of COVID-19 severity.