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Sepsis is a dysregulated systemic inflammatory response to an infection, which can lead to multiple organ dysfunction syndrome that includes the kidney. Leukocyte recruitment is an important process of the host immune defense in response to sepsis. Endothelial cells (EC) actively regulate leukocyte recruitment by expressing adhesion molecules following the activation of dedicated intracellular signal transduction pathways. Previous studies reported that the expression of adhesion molecules was associated with the activation of endothelial NF-κB p65 and MAPK c-Jun pathways in vitro in response to conditions that mimic processes that occur in inflammation. This study aimed to investigate the spatiotemporal patterns of leukocyte recruitment, expression of adhesion molecules, and endothelial nuclear p65 and c-Jun localization in renal microvascular beds of septic mice. Here, we used a cecal ligation and puncture (CLP) sepsis mouse model and RT-qPCR and immunohistochemical staining. We showed that neutrophils, macrophages, and T lymphocytes were all present in the kidney, yet only neutrophils accumulated in a spatiotemporally discernible pattern, mainly in glomeruli at 4 hours after CLP-sepsis initiation. E-selectin, not VCAM-1, was expressed in glomeruli at the same time point. In a subset of mice at 72 hours after CLP-sepsis started, VCAM-1 expression was prominent in glomerular EC, which was not related to changes in mmu-microRNA(miR)-126a-3p levels, a short noncoding microRNA previously shown to inhibit the translation of VCAM-1 mRNA into protein. Nuclear localization of p65 and c-Jun occurred in EC of all microvascular segments at 4 and 7 hours after CLP-sepsis initiation. In summary, sepsis-induced recruitment of neutrophils, E-selectin expression, and NF-κB p65 and MAPK c-Jun pathway activation coincided in glomeruli at the early stage of the disease. In the other microvascular beds, sepsis led to NF-κB p65 and MAPK c-Jun pathway activation with limited expression of E-selectin and no association with VCAM-1 expression or leukocyte recruitment.
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BACKGROUND: Acute kidney injury (AKI) in sepsis patients increases patient mortality. Endothelial cells are important players in the pathophysiology of sepsis-associated AKI (SA-AKI), yet knowledge regarding their spatiotemporal involvement in coagulation disbalance and leukocyte recruitment is lacking. This study investigated the identity and kinetics of responses of different microvascular compartments in kidney cortex in response to SA-AKI. METHODS: Laser microdissected arterioles, glomeruli, peritubular capillaries, and postcapillary venules from kidneys of mice subjected to cecal ligation and puncture (CLP) were analyzed using RNA sequencing. Differential expression and pathway enrichment analyses identified genes involved in coagulation and inflammation. A selection of these genes was evaluated by RT-qPCR in microvascular compartments of renal biopsies from patients with SA-AKI. The role of two identified genes in lipopolysaccharide-induced endothelial coagulation and inflammatory activation were determined in vitro in HUVEC using siRNA-based gene silencing. RESULTS: CLP-sepsis in mice induced altered expression of approximately 400 genes in the renal microvasculature, with microvascular compartments exhibiting unique spatiotemporal responses. In mice, changes in gene expression related to coagulation and inflammation were most extensive in glomeruli at early and intermediate time points, with high induction of Plat, Serpine1, Thbd, Icam1, Stat3, and Ifitm3. In human SA-AKI, PROCR and STAT3 were induced in postcapillary venules, while SERPINE1 expression was diminished. IFITM3 was increased in arterioles and glomeruli. In vitro studies revealed that STAT3 and IFITM3 partly control endothelial coagulation and inflammatory activation. CONCLUSION: Renal microvascular compartments in mice and humans exhibited heterogeneous changes in coagulation- and inflammation-related gene expression in response to SA-AKI. Additional research should aim at understanding the functional consequences of the here described heterogeneous microvascular responses to establish the usefulness of identified genes as therapeutic targets in SA-AKI.
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Coagulação Sanguínea , Inflamação , Microvasos , Sepse , Animais , Sepse/complicações , Sepse/genética , Camundongos , Humanos , Inflamação/genética , Inflamação/patologia , Microvasos/patologia , Microvasos/metabolismo , Masculino , Rim/metabolismo , Rim/patologia , Rim/irrigação sanguínea , Camundongos Endogâmicos C57BL , Injúria Renal Aguda/genética , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/etiologia , Injúria Renal Aguda/patologiaRESUMO
Endothelial cells in blood vessels in the kidney exert different functions depending on the (micro)vascular bed they are located in. The present study aimed to investigate microRNA and mRNA transcription patterns that underlie these differences. We zoomed in on microvascular compartments in the mouse renal cortex by laser microdissecting the microvessels prior to small RNA- and RNA-sequencing analyses. By these means, we characterized microRNA and mRNA transcription profiles of arterioles, glomeruli, peritubular capillaries, and postcapillary venules. Quantitative RT-PCR, in situ hybridization, and immunohistochemistry were used to validate sequencing results. Unique microRNA and mRNA transcription profiles were found in all microvascular compartments, with dedicated marker microRNAs and mRNAs showing enriched transcription in a single microvascular compartment. In situ hybridization validated the localization of microRNAs mmu-miR-140-3p in arterioles, mmu-miR-322-3p in glomeruli, and mmu-miR-451a in postcapillary venules. Immunohistochemical staining showed that von Willebrand factor protein was mainly expressed in arterioles and postcapillary venules, whereas GABRB1 expression was enriched in glomeruli, and IGF1 was enriched in postcapillary venules. More than 550 compartment-specific microRNA-mRNA interaction pairs were identified that carry functional implications for microvascular behavior. In conclusion, our study identified unique microRNA and mRNA transcription patterns in microvascular compartments of the mouse kidney cortex that underlie microvascular heterogeneity. These patterns provide important molecular information for future studies into differential microvascular engagement in health and disease.NEW & NOTEWORTHY Renal endothelial cells display a high level of heterogeneity depending on the (micro)vascular bed they reside in. The molecular basis contributing to these differences is poorly understood yet of high importance to increase understanding of microvascular engagement in the kidney in health and disease. This report describes m(icro)RNA expression profiles of microvascular beds in the mouse renal cortex and uncovers microvascular compartment-specific m(icro)RNAs and miRNA-mRNA pairs, thereby revealing important molecular mechanisms underlying renal microvascular heterogeneity.
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MicroRNAs , Transcriptoma , Camundongos , Animais , Células Endoteliais/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Rim/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Endothelial cells (ECs) in the microvasculature in organs are active participants in the pathophysiology of sepsis. Tyrosine protein kinase receptor Tie2 (Tek; Tunica interna Endothelial cell Kinase) is thought to play a role in their inflammatory response, yet data are inconclusive. We investigated acute endotoxemia-induced changes in the expression of Tie2 and inflammation-associated endothelial adhesion molecules E-selectin and VCAM-1 (vascular cell adhesion molecule-1) in kidneys and lungs in inducible, EC-specific Tie2 knockout mice. The extent of Tie2 knockout in healthy mice differed between microvascular beds, with low to absent expression in arterioles in kidneys and in capillaries in lungs. In kidneys, Tie2 mRNA dropped more than 70% upon challenge with lipopolysaccharide (LPS) in both genotypes, with no change in protein. In renal arterioles, tamoxifen-induced Tie2 knockout was associated with higher VCAM-1 protein expression in healthy conditions. This did not increase further upon challenge of mice with LPS, in contrast to the increased expression occurring in control mice. Also, in lungs, Tie2 mRNA levels dropped within 4 h after LPS challenge in both genotypes, while Tie2 protein levels did not change. In alveolar capillaries, where tamoxifen-induced Tie2 knockout did not affect the basal expression of either adhesion molecule, a 4-fold higher E-selectin protein expression was observed after exposure to LPS compared to controls. The here-revealed heterogeneous effects of absence of Tie2 in ECs in kidney and lung microvasculature in health and in response to acute inflammatory activation calls for further in vivo investigations into the role of Tie2 in EC behavior.
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Endotoxemia , Molécula 1 de Adesão de Célula Vascular , Camundongos , Animais , Molécula 1 de Adesão de Célula Vascular/genética , Molécula 1 de Adesão de Célula Vascular/metabolismo , Endotoxemia/metabolismo , Selectina E/genética , Selectina E/metabolismo , Células Endoteliais/metabolismo , Lipopolissacarídeos/farmacologia , Lipopolissacarídeos/metabolismo , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , RNA Mensageiro/metabolismoRESUMO
Tyrosine-protein kinase receptor Tie2, also known as Tunica interna Endothelial cell Kinase or TEK plays a prominent role in endothelial responses to angiogenic and inflammatory stimuli. Here we generated a novel inducible Tie2 knockout mouse model, which targets mature (micro)vascular endothelium, enabling the study of the organ-specific contribution of Tie2 to these responses. Mice with floxed Tie2 exon 9 alleles (Tie2floxed/floxed) were crossed with end-SCL-Cre-ERT transgenic mice, generating offspring in which Tie2 exon 9 is deleted in the endothelial compartment upon tamoxifen-induced activation of Cre-recombinase (Tie2ΔE9). Successful deletion of Tie2 exon 9 in kidney, lung, heart, aorta, and liver, was accompanied by a heterogeneous, organ-dependent reduction in Tie2 mRNA and protein expression. Microvascular compartment-specific reduction in Tie2 mRNA and protein occurred in arterioles of all studied organs, in renal glomeruli, and in lung capillaries. In kidney, lung, and heart, reduced Tie2 expression was accompanied by a reduction in Tie1 mRNA expression. The heterogeneous, organ- and microvascular compartment-dependent knockout pattern of Tie2 in the Tie2floxed/floxed;end-SCL-Cre-ERT mouse model suggests that future studies using similar knockout strategies should include a meticulous analysis of the knockout extent of the gene of interest, prior to studying its role in pathological conditions, so that proper conclusions can be drawn.
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Células Endoteliais , Tamoxifeno , Animais , Células Endoteliais/metabolismo , Integrases , Camundongos , Camundongos Knockout , Camundongos Transgênicos , RNA Mensageiro/metabolismo , Receptor TIE-2/genética , Receptor TIE-2/metabolismo , Tamoxifeno/metabolismo , Tamoxifeno/farmacologiaRESUMO
BACKGROUND: The mechanisms driving acute kidney injury (AKI) in critically ill COVID-19 patients are unclear. We collected kidney biopsies from COVID-19 AKI patients within 30 min after death in order to examine the histopathology and perform mRNA expression analysis of genes associated with renal injury. METHODS: This study involved histopathology and mRNA analyses of postmortem kidney biopsies collected from patients with COVID-19 (n = 6) and bacterial sepsis (n = 27). Normal control renal tissue was obtained from patients undergoing total nephrectomy (n = 12). The mean length of ICU admission-to-biopsy was 30 days for COVID-19 and 3-4 days for bacterial sepsis patients. RESULTS: We did not detect SARS-CoV-2 RNA in kidney biopsies from COVID-19-AKI patients yet lung tissue from the same patients was PCR positive. Extensive acute tubular necrosis (ATN) and peritubular thrombi were distinct histopathology features of COVID-19-AKI compared to bacterial sepsis-AKI. ACE2 mRNA levels in both COVID-19 (fold change 0.42, p = 0.0002) and bacterial sepsis patients (fold change 0.24, p < 0.0001) were low compared to control. The mRNA levels of injury markers NGAL and KIM-1 were unaltered compared to control tissue but increased in sepsis-AKI patients. Markers for inflammation and endothelial activation were unaltered in COVID-19 suggesting a lack of renal inflammation. Renal mRNA levels of endothelial integrity markers CD31, PV-1 and VE-Cadherin did not differ from control individuals yet were increased in bacterial sepsis patients (CD31 fold change 2.3, p = 0.0006, PV-1 fold change 1.5, p = 0.008). Angiopoietin-1 mRNA levels were downregulated in renal tissue from both COVID-19 (fold change 0.27, p < 0.0001) and bacterial sepsis patients (fold change 0.67, p < 0.0001) compared to controls. Moreover, low Tie2 mRNA expression (fold change 0.33, p = 0.037) and a disturbed VEGFR2/VEGFR3 ratio (fold change 0.09, p < 0.0001) suggest decreased microvascular flow in COVID-19. CONCLUSIONS: In a small cohort of postmortem kidney biopsies from COVID-19 patients, we observed distinct histopathological and gene expression profiles between COVID-19-AKI and bacterial sepsis-AKI. COVID-19 was associated with more severe ATN and microvascular thrombosis coupled with decreased microvascular flow, yet minimal inflammation. Further studies are required to determine whether these observations are a result of true pathophysiological differences or related to the timing of biopsy after disease onset.
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COVID-19/patologia , Expressão Gênica/genética , Rim/patologia , Rim/fisiopatologia , Sepse/patologia , Injúria Renal Aguda/etiologia , Injúria Renal Aguda/fisiopatologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Análise de Variância , COVID-19/genética , COVID-19/fisiopatologia , Estado Terminal/terapia , Feminino , Humanos , Unidades de Terapia Intensiva/organização & administração , Unidades de Terapia Intensiva/estatística & dados numéricos , Masculino , Pessoa de Meia-Idade , Sepse/genética , Sepse/fisiopatologia , Escore Fisiológico Agudo SimplificadoRESUMO
Neglecting tissue heterogeneity during the analysis of microRNA (miRNA) levels results in average signals from an unknown mixture of different cell types that are difficult to interpret. Here we demonstrate the technical requirements needed to obtain high-quality, quantitative miRNA expression information from tumor tissue compartments obtained by laser microdissection (LMD). Furthermore, we show the significance of disentangling tumor tissue heterogeneity by applying the newly developed protocols for combining LMD of tumor tissue compartments with RT-qPCR analysis to reveal compartment-specific miRNA expression signatures. An important advantage of this strategy is that the miRNA signature can be directly linked to histopathology. In summary, combining LMD and RT-qPCR is a powerful approach for spatial miRNA expression analysis in complex tissues, enabling discovery of disease mechanisms, biomarkers and drug candidates.
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Perfilação da Expressão Gênica/métodos , Microdissecção e Captura a Laser/métodos , MicroRNAs/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Animais , Biomarcadores/metabolismo , HumanosRESUMO
Tyrosine kinase receptor (Tie2) is mainly expressed by endothelial cells. In animal models mimicking critical illness, Tie2 levels in organs are temporarily reduced. Functional consequences of these reduced Tie2 levels on microvascular endothelial behavior are unknown. We investigated the effect of partial deletion of Tie2 on the inflammatory status of endothelial cells in different organs. Newly generated heterozygous Tie2 knockout mice (exon 9 deletion, ΔE9/Tie2) exhibiting 50% reduction in Tie2 mRNA and protein, and wild-type littermate controls (Tie2), were subjected to hemorrhagic shock and resuscitation (HS + R), or challenged with i.p. lipopolysaccharide (LPS). Kidney, liver, lung, heart, brain, and intestine were analyzed for mRNA levels of adhesion molecules E-selectin, vascular cell adhesion molecule 1 (VCAM-1), and intercellular cell adhesion molecule 1 (ICAM-1), and CD45. Exposure to HS + R did not result in different expression responses of these molecules between organs from Tie2 or Tie2 mice and sham-operated mice. In contrast, the LPS-induced mRNA expression levels of E-selectin, VCAM-1, and ICAM-1, and CD45 in organs were attenuated in Tie2 mice when compared with Tie2 mice in kidney and liver, but not in the other organs studied. Furthermore, reduced expression of E-selectin and VCAM-1 protein, and reduced influx of CD45 cells upon LPS exposure, was visible in a microvascular bed-specific pattern in kidney and liver of Tie2 mice compared with controls. In contrast to the hypothesis that a disbalance in the Ang/Tie2 system leads to increased microvascular inflammation, heterozygous deletion of Tie2 is associated with an organ-restricted, microvascular bed-specific attenuation of endothelial inflammatory response to LPS.
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Células Endoteliais/metabolismo , Microvasos/metabolismo , Receptor TIE-2/metabolismo , Animais , Selectina E/genética , Selectina E/metabolismo , Células Endoteliais/patologia , Inflamação/induzido quimicamente , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Antígenos Comuns de Leucócito/genética , Antígenos Comuns de Leucócito/metabolismo , Lipopolissacarídeos/toxicidade , Camundongos , Camundongos Knockout , Microvasos/patologia , Especificidade de Órgãos , Receptor TIE-2/genética , Molécula 1 de Adesão de Célula Vascular/genética , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
Hemorrhagic shock (HS) is associated with low blood pressure due to excessive loss of circulating blood and causes both macrocirculatory and microcirculatory dysfunction. Fluid resuscitation after HS is used in the clinic to restore tissue perfusion. The persistent microcirculatory damage caused by HS and/or resuscitation can result in multiple organ damage, with the kidney being one of the involved organs. The kidney microvasculature consists of different segments that possess a remarkable heterogeneity in functional properties. The aim of this study was to investigate the inflammatory responses of these different renal microvascular segments, i.e., arterioles, glomeruli, and postcapillary venules, to HS and resuscitation (HS/R) in mice and to explore the effects of intervention with a nuclear factor-kappa B (NF-κB) inhibitor on these responses. We found that HS/R disturbed the balance of the angiopoietin-Tie2 ligand-receptor system, especially in the glomeruli. Furthermore, endothelial adhesion molecules, proinflammatory cytokines, and chemokines were markedly upregulated by HS/R, with the strongest responses occurring in the glomerular and postcapillary venous segments. Blockade of NF-κB signaling during the resuscitation period only slightly inhibited HS/R-induced inflammatory activation, possibly because NF-κB p65 nuclear translocation already occurred during the HS period. In summary, although all three renal microvascular segments were activated upon HS/R, responses of endothelial cells in glomeruli and postcapillary venules to HS/R, as well as to NF-κB inhibition were stronger than those in arterioles. NF-κB inhibition during the resuscitation phase does not effectively counteract NF-κB p65 nuclear translocation initiating inflammatory gene transcription.
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Rim , Microcirculação , Microvasos , NF-kappa B/metabolismo , Ressuscitação , Choque Hemorrágico , Transdução de Sinais , Animais , Citocinas/biossíntese , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Rim/irrigação sanguínea , Rim/metabolismo , Rim/patologia , Masculino , Camundongos , Microvasos/metabolismo , Microvasos/patologia , Choque Hemorrágico/metabolismo , Choque Hemorrágico/patologia , Choque Hemorrágico/terapia , Regulação para CimaRESUMO
To identify mechanisms associated with sepsis-acute kidney injury based on the expression levels of renal injury biomarkers, neutrophil gelatinase-associated lipocalin, and kidney injury molecule-1 in renal biopsies which may allow the identification of sepsis-acute kidney injury patient subtypes. DESIGN: Prospective, clinical laboratory study using "warm" human postmortem sepsis-acute kidney injury kidney biopsies. SETTING: Research laboratory at university teaching hospital. SUBJECTS: Adult patients who died of sepsis in the ICU and control patients undergoing tumor nephrectomy. MEASUREMENTS AND MAIN RESULTS: Reverse transcription quantitative polymerase chain reaction and immunohistochemical staining were used to quantify messenger RNA and protein expression levels of neutrophil gelatinase-associated lipocalin and kidney injury molecule-1 in the kidney of sepsis-acute kidney injury patients and control subjects. Morphometric analysis was used to quantify renal and glomerular neutrophil gelatinase-associated lipocalin and kidney injury molecule-1 protein levels. Neutrophil gelatinase-associated lipocalin and kidney injury molecule-1 messenger RNA and protein levels were increased in kidneys of sepsis-acute kidney injury patients compared with control kidney tissue. Neutrophil gelatinase-associated lipocalin was localized in the distal tubules, collecting ducts, the adventitia of the renal arterioles, and in the glomerular tufts of renal biopsies from sepsis-acute kidney injury patients. In contrast, kidney injury molecule-1 was localized at the brush border of the proximal tubules. There was no correlation between neutrophil gelatinase-associated lipocalin and kidney injury molecule-1 levels. Furthermore, renal neutrophil gelatinase-associated lipocalin and kidney injury molecule-1 levels were not associated with the extent of renal injury, the severity of critical illness, or serum creatinine levels at either ICU admission or day of expiration. By laser microdissecting glomeruli, followed by reverse transcription quantitative polymerase chain reaction, we identified heterogenous glomerular neutrophil gelatinase-associated lipocalin production in the kidney of sepsis-acute kidney injury patients. CONCLUSION: We found differences in the expression of neutrophil gelatinase-associated lipocalin and kidney injury molecule-1 in patients with the same syndrome "sepsis-acute kidney injury" meaning there is no single pathway leading to sepsis-acute kidney injury. This underscores the beliefs that there are many/different pathophysiological pathways that can cause sepsis-acute kidney injury. Hence, patients with criteria that meet the definitions of both acute kidney injury and sepsis can be divided into subtypes based on pathophysiological features.
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Sepsis is a severe systemic inflammatory response to infection. Endothelial activation and dysfunction play a critical role in the pathophysiology of sepsis and represent an important therapeutic target to reduce sepsis mortality. Interferon regulatory factor 1 (IRF-1) was recently identified as a downstream target of TNF-α-mediated signal transduction in endothelial cells. The aim of this study was to explore the importance of IRF-1 as a regulator of lipopolysaccharide (LPS)-induced endothelial proinflammatory activation. We found that renal IRF-1 was upregulated by LPS in vivo as well as in LPS-stimulated endothelial cells in vitro. Furthermore, we identified intracellular retinoic acid inducible gene-I (RIG-I) as a regulator of LPS-mediated IRF-1 induction. IRF-1 depletion specifically resulted in diminished induction of VCAM-1 in response to LPS, but not of E-selectin or ICAM-1, which was independent of NFκB signaling. When both IRF-1 and the RIG-I adapter protein mitochondrial antiviral signaling (MAVS) were absent, VCAM-1 induction was not additionally inhibited, suggesting that MAVS and IRF-1 reside in the same signaling pathway. Surprisingly, E-selectin and IL-6 induction were no longer inhibited by MAVS knockdown when IRF-1 was also absent, revealing a redundant endothelial activation pathway. In summary, we report an IRF-1-mediated proinflammatory signaling pathway that specifically regulates LPS-mediated VCAM-1 expression, independent of NFκB.
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Endotélio/metabolismo , Fator Regulador 1 de Interferon/metabolismo , Rim/fisiologia , Sepse/imunologia , Molécula 1 de Adesão de Célula Vascular/metabolismo , Animais , Modelos Animais de Doenças , Endotélio/patologia , Regulação da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana , Humanos , Fator Regulador 1 de Interferon/genética , Lipopolissacarídeos/imunologia , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , RNA Interferente Pequeno/genética , Receptores de Superfície Celular , Transdução de Sinais , Fator de Necrose Tumoral alfa/metabolismo , Molécula 1 de Adesão de Célula Vascular/genéticaRESUMO
In patients with sepsis-induced multi-organ dysfunction syndrome, diverging patterns of oedema formation and loss of function in organs such as lung and kidney suggest that endothelial permeability-regulating molecular responses are differentially regulated. This potential differential regulation has been insufficiently studied at the level of components of adherens and tight junctions. We hypothesized that such a regulation by endothelial cells in sepsis takes place in an organ-specific manner. We addressed our hypothesis by studying by quantitative real time polymerase chain reaction the expression of a predefined subset of EC permeability-related molecules (occludin, claudin-5, PV-1, CD-31, endomucin, Angiopoietin-1, Angiopoietin-2, Tie2, VEGFA, VEGFR1, VEGFR2, and VE-cadherin) in kidney and lung after systemic lipopolysacharide injection in mice, and in kidneys of patients who died of sepsis. We showed that baseline endothelial expression of permeability-related molecules differs in mouse kidney and lung. Moreover, we showed differential regulation of these molecules after lipopolysacharide injection in the two mouse organs. In lung we found a decrease in expression levels of molecules of the adherence and tight junctions complex and related signaling systems, compatible with increased permeability. In contrast, in kidney we found expression patterns of these molecules compatible with decreased permeability. Finally, we partially corroborated our findings in mouse kidney in human kidneys from septic patients. These findings may help to understand the clinical difference in the extent of oedema formation in kidney and lung in sepsis-associated organ failure.
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Rim/metabolismo , Pulmão/metabolismo , Angiopoietina-1/genética , Angiopoietina-1/metabolismo , Animais , Western Blotting , Claudina-5/genética , Claudina-5/metabolismo , Humanos , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Ocludina/genética , Ocludina/metabolismo , Receptor TIE-2/genética , Receptor TIE-2/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sepse , Sialomucinas/genética , Sialomucinas/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Sepsis is a systemic inflammatory response to infections associated with organ failure that is the most frequent cause of death in hospitalized patients. Exaggerated endothelial activation, altered blood flow, vascular leakage, and other disturbances synergistically contribute to sepsis-induced organ failure. The underlying signaling events associated with endothelial proinflammatory activation are not well understood, yet they likely consist of molecular pathways that act in an endothelium-specific manner. We found that LPS, a critical factor in the pathogenesis of sepsis, is internalized by endothelial cells, leading to intracellular signaling without the need for priming as found recently in immune cells. By identifying a novel role for retinoic acid-inducible gene-I (RIG-I) as a central regulator of endothelial activation functioning independent of TLR4, we provide evidence that the current paradigm of TLR4 solely being responsible for LPS-mediated endothelial responses is incomplete. RIG-I, as well as the adaptor protein mitochondrial antiviral signaling protein, regulates NF-κB-mediated induction of adhesion molecules and proinflammatory cytokine expression in response to LPS. Our findings provide essential new insights into the proinflammatory signaling pathways in endothelial cells and suggest that combined endothelial-specific inhibition of RIG-I and TLR4 will provide protection from aberrant endothelial responses associated with sepsis.
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Proteína DEAD-box 58/metabolismo , Células Endoteliais/imunologia , Inflamação/imunologia , Lipopolissacarídeos/imunologia , Transdução de Sinais , Receptor 4 Toll-Like , Animais , Células Endoteliais/patologia , Inflamação/patologia , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Receptor 4 Toll-Like/imunologiaRESUMO
OBJECTIVE: To investigate the consequences of histone deacetylase inhibition by histone deacetylase inhibitor valproic acid and IκB kinase/nuclear factor-κB signaling blockade by IκB kinase inhibitor BAY11-7082 on (microvascular) endothelial cell behavior in vitro as well as in mice subjected to hemorrhagic shock/resuscitation in vivo. DESIGN: Prospective, randomized laboratory investigation using an established mouse model of hemorrhagic shock. SETTING: Research laboratory at university teaching hospital. SUBJECTS: Endothelial cells and C57BL/6 male mice. INTERVENTIONS: Endothelial cells were incubated with tumor necrosis factor-α in the absence or presence of valproic acid or BAY11-7082 in vitro. Mice were subjected to hemorrhagic shock by blood withdrawn until the mean arterial pressure of 30 mm Hg and maintained at this pressure for 90 minutes. At 90 minutes, subgroups of mice were resuscitated with 4% human albumin in the absence or presence of vehicle, valproic acid (300 µg/g body weight) or BAY11-7082 (400 µg per mouse). Mice were killed 1 hour and 4 hours after resuscitation. MEASUREMENTS AND MAIN RESULTS: Valproic acid and BAY11-7082 selectively diminished tumor necrosis factor-α-induced endothelial proinflammatory activation in vitro. In vivo, both systemic and local inflammatory responses were significantly induced by hemorrhagic shock/resuscitation. The decreased histone acetylation in kidneys after hemorrhagic shock/resuscitation was restored by valproic acid treatment. In glomerular endothelial cells, the nuclear translocation of nuclear factor-κB, which was induced by hemorrhagic shock/resuscitation, was eliminated by BAY11-7082 treatment while enhanced in the presence of valproic acid. Both valproic acid and BAY11-7082 significantly attenuated the hemorrhagic shock/resuscitation-induced protein expression of endothelial cell adhesion molecules E-selectin and vascular cell adhesion molecule-1 in the microvasculature of kidneys and liver, although messenger RNA expression levels of these molecules analyzed in whole-organ lysates of kidneys, lungs, and liver were not extensively affected. The reduced protein expression of adhesion molecules was paralleled by diminishing the adhesion/transmigration of neutrophils in kidneys and liver after hemorrhagic shock/resuscitation. CONCLUSION: Suppression of histone deacetylase activity and blockade of IκB kinase/nuclear factor-κB signaling during resuscitation ameliorate microvascular endothelial proinflammatory responses in organs in mice after hemorrhagic shock.
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Células Endoteliais/metabolismo , Histona Desacetilases/metabolismo , Quinase I-kappa B/antagonistas & inibidores , NF-kappa B/antagonistas & inibidores , Choque Hemorrágico/fisiopatologia , Animais , Modelos Animais de Doenças , Inibidores de Histona Desacetilases/farmacologia , Mediadores da Inflamação/imunologia , Rim/patologia , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Nitrilas/farmacologia , Estudos Prospectivos , RNA Mensageiro/metabolismo , Ressuscitação , Transdução de Sinais , Sulfonas/farmacologia , Ácido Valproico/farmacologiaRESUMO
In recent years much research in RNA nanotechnology has been directed to develop an efficient and clinically suitable delivery system for short interfering RNA (siRNA). The current study describes the in vivo siRNA delivery using PEGylated antibody-targeted SAINT-based-lipoplexes (referred to as antibody-SAINTPEGarg/PEG2%), which showed superior siRNA delivery capacity and effective down-regulation of VE-cadherin gene expression in vitro in inflammation-activated primary endothelial cells of different vascular origins. PEGylation of antibody-SAINTPEGarg resulted in more desirable pharmacokinetic behavior than that of non-PEGylated antibody-SAINTPEGarg. To create specificity for inflammation-activated endothelial cells, antibodies against vascular cell adhesion molecule-1 (VCAM-1) were employed. In TNFα-challenged mice, these intravenously administered anti-VCAM-1-SAINTPEGarg/PEG2% homed to VCAM-1 protein expressing vasculature. Confocal laser scanning microscopy revealed that anti-VCAM-1-SAINTPEGarg/PEG2% co-localized with endothelial cells in lung postcapillary venules. Furthermore, they did not exert any liver and kidney toxicity. Yet, lack of in vivo gene silencing as assessed in whole lung and in laser microdissected lung microvascular segments indicates that in vivo internalization and/or intracellular trafficking of the delivery system and its cargo in the target cells are not sufficient, and needs further attention, emphasizing the essence of evaluating siRNA delivery systems in an appropriate in vivo animal model at an early stage in their development.
Assuntos
Anticorpos/metabolismo , Antígenos CD/metabolismo , Caderinas/metabolismo , Endotélio Vascular/metabolismo , Inflamação/metabolismo , Lipídeos/química , Pulmão/irrigação sanguínea , Polietilenoglicóis/química , Compostos de Piridínio/química , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transfecção/métodos , Molécula 1 de Adesão de Célula Vascular/metabolismo , Animais , Anticorpos/química , Antígenos CD/genética , Caderinas/genética , Modelos Animais de Doenças , Endotélio Vascular/imunologia , Regulação da Expressão Gênica , Inflamação/induzido quimicamente , Inflamação/genética , Inflamação/prevenção & controle , Masculino , Camundongos Endogâmicos C57BL , Microscopia Confocal , RNA Interferente Pequeno/química , RNA Interferente Pequeno/genética , Fatores de Tempo , Distribuição Tecidual , Fator de Necrose Tumoral alfa , Molécula 1 de Adesão de Célula Vascular/imunologia , Vênulas/metabolismoRESUMO
The pivotal role of endothelial cells in the pathology of inflammatory diseases raised interest in the development of short interfering RNA (siRNA) delivery devices for selective pharmacological intervention in the inflamed endothelium. The current study demonstrates endothelial specific delivery of siRNAs and downregulation of inflammatory genes in activated endothelium in vivo by applying a novel type of targeted liposomes based on the cationic amphiphile SAINT-C18 (1-methyl-4-(cis-9-dioleyl)methyl-pyridinium-chloride). To create specificity for inflamed endothelial cells, these so-called SAINT-O-Somes were harnessed with antibodies against vascular cell adhesion protein 1 (VCAM-1). In TNFα challenged mice, intravenously administered anti-VCAM-1 SAINT-O-Somes exerted long circulation times and homed to VCAM-1 expressing endothelial cells in inflamed organs. The formulations were devoid of liver and kidney toxicity. Using anti-VCAM-1 SAINT-O-Somes we successfully delivered siRNA to knock down VE-cadherin mRNA in inflamed renal microvasculature, as demonstrated by using laser microdissection of different microvascular beds prior to analysis of gene expression. Using the same strategy, we demonstrated local attenuation of endothelial inflammatory response towards lipopolysaccharide in kidneys of mice treated with anti-VCAM-1 SAINT-O-Somes containing NFκB p65 specific siRNA. This study is the first demonstration of a novel, endothelial specific carrier that is suitable for selective in vivo delivery of siRNAs into inflamed microvascular segments and interference with disease associated endothelial activation.
Assuntos
Anticorpos/administração & dosagem , Antígenos CD/genética , Caderinas/genética , Compostos de Piridínio/administração & dosagem , RNA Interferente Pequeno/administração & dosagem , Fator de Transcrição RelA/genética , Molécula 1 de Adesão de Célula Vascular/imunologia , Animais , Encéfalo/metabolismo , Células Cultivadas , Regulação para Baixo , Endotélio Vascular/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Inflamação/metabolismo , Rim/efeitos dos fármacos , Rim/metabolismo , Lipossomos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Pulmão/metabolismo , Masculino , Camundongos , Miocárdio/metabolismo , Compostos de Piridínio/farmacocinética , RNA Interferente Pequeno/farmacocinética , Distribuição Tecidual , Fator de Necrose Tumoral alfa/farmacologia , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
BACKGROUND: In sepsis and various other inflammatory conditions, elevated circulating levels of angiopoietin-2 (Ang2) are detected, but the precise functional role of Ang2 in these conditions is not well understood. Here, we investigated the contribution of Ang2 to the inflammatory response and renal function impairment in a mouse model of endotoxaemia. METHODS: Ang2-deficient mice and wild-type littermates were challenged with lipopolysaccharide [LPS; 1500 EU/g, intraperitoneal (i.p.)]. In additional experiments, wild-type C57Bl/6 mice were depleted of circulating neutrophils by antibody treatment (NIMPR14) prior to LPS challenge to study the role of neutrophils in regulating LPS-induced cytokine release. After 8 or 24 h of LPS challenge, the mice were sacrificed and organs were harvested. Quantitative reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay were performed for endothelial adhesion molecules (P-selectin, E-selectin, VCAM-1 and ICAM-1) and plasma cytokines (TNF-α, IL-6, KC, MIP-2), respectively. To assess renal function, blood urea nitrogen levels in plasma and albumin-to-creatinine ratio in urine were measured. RESULTS Upon LPS challenge, expression levels of various endothelial adhesion molecules in Ang2-deficient mice were reduced in an organ-specific manner. In contrast, in these mice, plasma levels of TNF-α and IL-6 were significantly increased compared with their wild-type littermates, possibly due to decreased neutrophil glomerular influx. Importantly, the absence of Ang2 did not protect the mice from acute kidney injury (AKI) upon LPS challenge. CONCLUSIONS The absence of Ang2 release upon LPS challenge induces pleotropic effects with regard to endothelial activation and systemic inflammation, but does not protect mice from LPS-induced AKI.
Assuntos
Injúria Renal Aguda/patologia , Angiopoietina-2/fisiologia , Citocinas/metabolismo , Endotoxinas/toxicidade , Inflamação/etiologia , Lipopolissacarídeos/toxicidade , Injúria Renal Aguda/induzido quimicamente , Injúria Renal Aguda/metabolismo , Animais , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Ensaio de Imunoadsorção Enzimática , Inflamação/metabolismo , Inflamação/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismoRESUMO
The precise molecular effects that antiangiogenic drugs exert on tumor vasculature remain to be poorly understood. We therefore set out to investigate the molecular and architectural changes that occur in the vasculature of two different tumor types that both respond to vascular endothelial growth factor receptor 2 (VEGFR2) inhibitor therapy. Mice bearing Lewis lung carcinoma (LLC) or B16.F10 melanoma were treated with vandetanib (ZD6474), a VEGFR2/epidermal growth factor receptor (EGFR)/REarranged during Transfection (RET) kinase inhibitor, resulting in a significant 80% reduction in tumor outgrowth. Although in LLC the vascular density was not affected by vandetanib treatment, it was significantly decreased in B16.F10. In LLC, vandetanib treatment induced a shift in vascular gene expression toward stabilization, as demonstrated by upregulation of Tie2 and N-cadherin and downregulation of Ang2 and integrin ß3. In contrast, only eNOS and P-selectin responded to vandetanib treatment in B16.F10 vasculature. Strikingly, vandetanib reduced protein expression of VEGFR2 in both models, whereas mRNA remained unaffected. Analysis of miR-296 expression allowed us to exclude a role for the recently proposed microRNA-296 in VEGFR2 posttranslational control in LLC and B16.F10 in vivo. Our data demonstrate that VEGFR2/EGFR inhibition through vandetanib slows down both LLC and B16.F10 tumor growth. Yet, the underlying molecular changes in the vasculature that orchestrate the antitumor effect differ between tumor types. Importantly, in both models, vandetanib treatment induced loss of its pharmacological target, which was not directly related to miR-296 expression. Validation of our observations in tumor biopsies from VEGFR2 inhibitor-treated patients will be essential to unravel the effects of VEGFR2 inhibitor therapy on tumor vasculature in relation to therapeutic efficacy.
Assuntos
Inibidores da Angiogênese/uso terapêutico , Carcinoma Pulmonar de Lewis/tratamento farmacológico , Melanoma Experimental/tratamento farmacológico , MicroRNAs/fisiologia , Neovascularização Patológica/tratamento farmacológico , Piperidinas/uso terapêutico , Quinazolinas/uso terapêutico , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Inibidores da Angiogênese/administração & dosagem , Inibidores da Angiogênese/farmacologia , Animais , Carcinoma Pulmonar de Lewis/irrigação sanguínea , Carcinoma Pulmonar de Lewis/genética , Carcinoma Pulmonar de Lewis/patologia , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Imuno-Histoquímica , Injeções Intraperitoneais , Masculino , Melanoma Experimental/irrigação sanguínea , Melanoma Experimental/genética , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Piperidinas/administração & dosagem , Piperidinas/farmacologia , Quinazolinas/administração & dosagem , Quinazolinas/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/biossíntese , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
ERG is a member of the ETS transcription factor family that is highly enriched in endothelial cells (ECs). To further define the role of ERG in regulating EC function, we evaluated the effect of ERG knock-down on EC lumen formation in 3D collagen matrices. Blockade of ERG using siRNA completely interferes with EC lumen formation. Quantitative PCR (QPCR) was used to identify potential downstream gene targets of ERG. In particular, we identified RhoJ as the Rho GTPase family member that is closely related to Cdc42 as a target of ERG. Knockdown of ERG expression in ECs led to a 75% reduction in the expression of RhoJ. Chromatin immunoprecipitation and transactivation studies demonstrated that ERG could bind to functional sites in the proximal promoter of the RhoJ gene. Knock-down of RhoJ similarly resulted in a marked reduction in the ability of ECs to form lumens. Suppression of either ERG or RhoJ during EC lumen formation was associated with a marked increase in RhoA activation and a decrease in Rac1 and Cdc42 activation and their downstream effectors. Finally, in contrast to other Rho GTPases, RhoJ exhibits a highly EC-restricted expression pattern in several different tissues, including the brain, heart, lung, and liver.
Assuntos
Vasos Sanguíneos/crescimento & desenvolvimento , Células Endoteliais/metabolismo , Transativadores/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Western Blotting , Técnicas de Silenciamento de Genes , Humanos , Imunoprecipitação , Lasers , Camundongos , Camundongos Nus , Microdissecção , Morfogênese , RNA Interferente Pequeno , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/fisiologia , Regulador Transcricional ERGRESUMO
Previous research suggests that in hypertrophic scars (HSs), an excess of microvessels is present compared with normotrophic scars (NSs). The aim of our study was to quantify vascular densities in HSs and normotrophic scars and to provide an insight into the kinetics of changes in the expression of angiogenic factors in time during wound healing and HS formation. Human presternal wound healing after cardiothoracic surgery through a sternotomy incision was investigated in a standardized manner. Skin biopsies were collected at consecutive time points, i.e., during surgery and 2, 4, 6, 12, and 52 weeks postoperatively. The expression levels of angiopoietin-1, angiopoietin-2, Tie-2, vascular endothelial growth factor, and urokinase-type plasminogen activator were measured by real-time reverse transcription-polymerase chain reaction. Quantification of angiogenesis and cellular localization of the proteins of interest were based on immunohistochemical analysis. Microvessel densities were higher in the HSs compared with the normotrophic scars 12 weeks (p=0.017) and 52 weeks (p=0.030) postoperatively. Angiopoietin-1 expression was lower in the hypertrophic group (p<0.001), which, together with a nonsignificant increase of angiopoietin-2 expression, represented a considerable decrease in the angiopoietin-1/angiopoietin-2 ratio in the hypertrophic group 4 weeks (p=0.053), 12 weeks (p<0.001), and 52 weeks (p<0.001) postoperatively. The expression of urokinase-type plasminogen activator was up-regulated during HS formation (p=0.008). Vascular endothelial growth factor expression was not significantly different when comparing both groups. In summary, the differential expression of angiopoietin-1, angiopoietin-2, and urokinase-type plasminogen activator in time is associated with an increased vascular density in HSs compared with normotrophic scars.