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Objective: Endothelial-to-mesenchymal transition (EndMT) is a transdifferentiation process in which endothelial cells (ECs) adopt a mesenchymal-like phenotype. Over the past few years, it became clear that EndMT can contribute to several cardiovascular pathologies. However, the molecular pathways underlying the development of EndMT remain incompletely understood. Since the epigenetic enzyme Enhancer of Zeste Homolog 2 (EZH2) and its concomitant mark H3K27Me3 have been shown to be elevated in many cardiovascular diseases that associate with EndMT, we hypothesized that H3K27Me3 is a determinant for the susceptibility of EndMT. Methods: To study the association between H3K27Me3 and EndMT, a knockdown model of EZH2 in human endothelial cells (HUVEC) was utilized to reduce H3K27Me3 abundance, followed by induction of EndMT using TGFß1. The expression of molecular markers of EndMT and fibrogenesis were analysed. Results: In cultured HUVECs, a reduction of H3K27Me3 abundance facilitates EndMT but mitigates fibrogenesis as shown by a decreased expression of collagen I and III. In HUVEC, H3K27Me3 abundance directly affects the expression of miR29c, a collagen-targeting miRNA. Additionally, knockdown of miR-29c in HUVEC with low H3K27Me3 abundance partly restored the expression of collagen I and III. Expectedly, in rats with perivascular fibrosis an increased abundance of H3K27Me3 associated with a decreased expression of miR-29c. Conclusion: our data shows that endothelial fibrogenesis underlies an epigenetic regulatory pathway and we demonstrate that a decreased abundance of H3K27Me3 in ECs blunts fibrogenesis in part in a miR-29c dependent manner. Therefore, a reduction of H3K27Me3 could serve as a novel therapeutical strategy to mitigate fibrogenesis and may prove to be beneficial in fibrogenic diseases including atherosclerosis, cardiac fibrosis, and PAH.
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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
Major surgery induces systemic inflammation leading to pro-inflammatory activation of endothelial cells. Endothelial inflammation is one of the drivers of postoperative organ damage, including acute kidney injury Tumour Necrosis Factor alpha (TNF-α) is an important component of surgery-induced pro-inflammatory activation of endothelial cells. Kinases, the backbone of signalling cascades, can be targeted by pharmacological inhibition. This is a promising treatment option to interfere with excessive endothelial inflammation. In this study, we identified activated kinases as potential therapeutic targets. These targets were pharmacologically inhibited to reduce TNF-α-induced pro-inflammatory signalling in endothelial cells. Kinome profiling using PamChip arrays identified 64 protein tyrosine kinases and 88 serine-threonine kinases, the activity of which was determined at various timepoints (5-240 min) following stimulation with 10 ng/ml TNF-α in Human umbilical vein endothelial cells in vitro. The PTKs Axl and Fyn were selected based on high kinase activity profiles. Co-localisation experiments with the endothelial-specific protein CD31 showed Axl expression in endothelial cells of glomeruli and Fyn in arterioles and glomeruli of both control and TNF-α-exposed mice. Pharmacological inhibition with Axl inhibitor BMS-777607 and Fyn inhibitor PP2 significantly reduced TNF-α-induced pro-inflammatory activation of E-selectin, VCAM-1, ICAM-1, IL-6 and IL-8 at mRNA and VCAM-1, ICAM-1, and IL-6 at protein level in HUVEC in vitro. Upon pharmacological inhibition with each inhibitor, leukocyte adhesion to HUVEC was also significantly reduced, however to a minor extent. In conclusion, pre-treatment of endothelial cells with kinase inhibitors BMS-777607 and PP2 reduces TNF-α-induced endothelial inflammation in vitro.
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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
Sepsis is a life-threatening condition often leading to multiple organ failure for which currently no pharmacological treatment is available. Endothelial cells (EC) are among the first cells to respond to pathogens and inflammatory mediators in sepsis and might be a sentinel target to prevent the occurrence of multiple organ failure. Lipopolysaccharide (LPS) is a Gram-negative bacterial component that induces endothelial expression of inflammatory adhesion molecules, cytokines, and chemokines. This expression is regulated by a network of kinases, the result of which in vivo enables leukocytes to transmigrate from the blood into the underlying tissue, causing organ damage. We hypothesised that besides the known kinase pathways, other kinases are involved in the regulation of EC in response to LPS, and that these can be pharmacologically targeted to inhibit cell activation. Using kinome profiling, we identified 58 tyrosine kinases (TKs) that were active in human umbilical vein endothelial cells (HUVEC) at various timepoints after stimulation with LPS. These included AXL tyrosine kinase (Axl), focal adhesion kinase 1 (FAK1), and anaplastic lymphoma kinase (ALK). Using siRNA-based gene knock down, we confirmed that these three TKs mediate LPS-induced endothelial inflammatory activation. Pharmacological inhibition with FAK1 inhibitor FAK14 attenuated LPS-induced endothelial inflammatory activation and leukocyte adhesion partly via blockade of NF-κB activity. Administration of FAK14 after EC exposure to LPS also resulted in inhibition of inflammatory molecule expression. In contrast, inhibition of ALK with FDA-approved inhibitor Ceritinib attenuated LPS-induced endothelial inflammatory activation via a pathway that was independent of NF-κB signalling while it did not affect leukocyte adhesion. Furthermore, Ceritinib administration after start of EC exposure to LPS did not inhibit inflammatory activation. Combined FAK1 and ALK inhibition attenuated LPS-induced endothelial activation in an additive manner, without affecting leukocyte adhesion. Summarising, our findings suggest the involvement of FAK1 and ALK in mediating LPS-induced inflammatory activation of EC. Since pharmacological inhibition of FAK1 attenuated endothelial inflammatory activation after the cells were exposed to LPS, FAK1 represents a promising target for follow up studies.
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Quinase do Linfoma Anaplásico/antagonistas & inibidores , Anti-Inflamatórios/farmacologia , Quinase 1 de Adesão Focal/antagonistas & inibidores , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Inflamação/prevenção & controle , Lipopolissacarídeos/toxicidade , Inibidores de Proteínas Quinases/farmacologia , Aminopiridinas/farmacologia , Quinase do Linfoma Anaplásico/genética , Quinase do Linfoma Anaplásico/metabolismo , Quinase 1 de Adesão Focal/genética , Quinase 1 de Adesão Focal/metabolismo , Perfilação da Expressão Gênica , Células HL-60 , Células Endoteliais da Veia Umbilical Humana/enzimologia , Humanos , Inflamação/enzimologia , Inflamação/genética , Análise Serial de Proteínas , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Piridonas/farmacologia , Pirimidinas/farmacologia , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais , Sulfonas/farmacologia , Fatores de Tempo , Transcriptoma , Receptor Tirosina Quinase AxlRESUMO
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
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
OBJECTIVE: Human aging is associated with remodeling of the immune system. While most studies on immunosenescence have focused on adaptive immunity, the effects of aging on innate immunity are not well understood. Here, we investigated whether aging affects cytokine responses to a wide range of well-defined pattern recognition receptor (PRR) ligands, such as ligands for Toll-like receptors (TLRs), C-type lectin receptors (CLRs), NOD-like receptors (NLRs), retinoic-acid-inducible gene-I like receptors (RLRs) and the cytosolic DNA sensor absent in melanoma 2 (AIM2). METHOD: Blood was collected from 16 young (20-39 years) and 18 elderly (60-84 years) healthy participants. Pro-inflammatory cytokine (TNF-α, IL-1ß, IL-6, and IL-8) production in a whole blood assay (WBA) after stimulation with TLR ligands (Pam3csk4, poly(I:C), LPS, CpG), CLR ligand (ß-glucan), NLR ligand (MDP), RLR ligands (5'ppp-dsDNA and poly(I:C)/lyovec) and the AIM2 ligand (poly(dA:dT) was assessed by ELISA. TLR2 and TLR4 expression by leukocytes and monocytes was determined by flow-cytometry. Expression of AIM2 by peripheral blood mononuclear cells (PBMC) was assessed by qRT-PCR and Western blot. RESULT: Cytokine responses to Pam3csk4, poly(I:C) and CpG, ß-glucan, MDP, 5'ppp-dsDNA and poly(I:C)/lyovec were comparable between young and old participants. We observed a higher IL-8 response following stimulation of elderly blood samples with the TLR4 ligand LPS, which was associated with higher proportions of TLR4 expressing monocytes. Interestingly, stimulation of whole blood cells with the AIM2 ligand poly(dA:dT) resulted in significantly lower cytokine responses in old participants. Moreover, these lower cytokine responses were associated with lower AIM2 protein expression and activation in PBMC of old participants. CONCLUSION: Our findings reveal an age-dependent reduction of AIM2 expression and activation which may explain reduced cytokine responses to the cytosolic DNA mimic poly(dA:dT) in healthy elderly individuals. Reduced AIM2-mediated sensing with age may contribute to increased vulnerability to bacterial or viral infections in the elderly.