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Fibrosis is a hallmark of chronic disease. Although fibroblasts are involved, it is unclear to what extent endothelial cells also might contribute. We detected increased expression of the transcription factor Sox9 in endothelial cells in several different mouse fibrosis models. These models included systolic heart failure induced by pressure overload, diastolic heart failure induced by high-fat diet and nitric oxide synthase inhibition, pulmonary fibrosis induced by bleomycin treatment, and liver fibrosis due to a choline-deficient diet. We also observed up-regulation of endothelial SOX9 in cardiac tissue from patients with heart failure. To test whether SOX9 induction was sufficient to cause disease, we generated mice with endothelial cell-specific overexpression of Sox9, which promoted fibrosis in multiple organs and resulted in signs of heart failure. Endothelial Sox9 deletion prevented fibrosis and organ dysfunction in the two mouse models of heart failure as well as in the lung and liver fibrosis mouse models. Bulk and single-cell RNA sequencing of mouse endothelial cells across multiple vascular beds revealed that SOX9 induced extracellular matrix, growth factor, and inflammatory gene expression, leading to matrix deposition by endothelial cells. Moreover, mouse endothelial cells activated neighboring fibroblasts that then migrated and deposited matrix in response to SOX9, a process partly mediated by the secreted growth factor CCN2, a direct SOX9 target; endothelial cell-specific Sox9 deletion reversed these changes. These findings suggest a role for endothelial SOX9 as a fibrosis-promoting factor in different mouse organs during disease and imply that endothelial cells are an important regulator of fibrosis.
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Insuficiência Cardíaca , Fatores de Transcrição , Animais , Humanos , Camundongos , Modelos Animais de Doenças , Células Endoteliais , Fibrose , Peptídeos e Proteínas de Sinalização Intercelular , Cirrose Hepática/complicações , Fatores de Transcrição SOX9/genéticaRESUMO
We addressed if hyperfiltration can be assessed transcutaneously in male diabetic obese mice (BTBRob/ob) at 12 and 24 wk and how this relates to glomerular parameters indicative for hyperfiltration. Transcutaneous assessment of FITC-Sinistrin clearance [transcutaneous assessment of glomerular filtration rate (tGFR)] was compared against classical plasma clearance. Kidney from SV620C-01-PEI perfused mice were harvested at 24 wk and processed for tissue clearing and classical histology. Perfusion patterns of glomerular capillaries, glomerular size, and vasodilation of the afferent arterioles were assessed. Although at 12 wk FITC-Sinistrin half-life (t1/2) for both tGFR and plasma clearance suggested hyperfiltration, this was not significant anymore at 24 wk. In kidneys of diabetic mice the diameter of the afferent arteriole was significantly larger and positively correlated with glomerular size. Glomerular perfusion pattern in these mice was heterogeneous ranging from non- to well-perfused glomeruli. Nonperfused glomerular areas displayed a strong periodic acid-Schiff's (PAS) positive staining. Collectively our data demonstrate that tGFR is a valid method to detect hyperfiltration. Hyperfiltration occurs early in BTBRob/ob mice and disappears with disease progression as a consequence of a reduced filtration surface. It remains to be assessed if tGFR is also a valid method in diabetic mice with severely compromised renal function.NEW & NOTEWORTHY tGFR measurement is a relatively new method to assess kidney function in conscious rodents, which can be repeated multiple times in the same animal to track the course of the disease and/or the effect of potential treatments. Since the literature was inconclusive on the suitability of this technique in obese mice, we validated it for the first time against classical plasma clearance in the commonly used BTBRob/ob mouse model.
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Diabetes Mellitus Experimental , Nefropatias Diabéticas , Nefropatias , Masculino , Camundongos , Animais , Taxa de Filtração Glomerular , Camundongos Obesos , FluoresceínasRESUMO
In diabetic nephropathy (DN), glomerular endothelial cells (GECs) and podocytes undergo pathological alterations, which are influenced by metabolic changes characteristic of diabetes, including hyperglycaemia (HG) and elevated methylglyoxal (MGO) levels. However, it remains insufficiently understood what effects these metabolic factors have on GEC and podocytes and to what extent the interactions between the two cell types can modulate these effects. To address these questions, we established a co-culture system in which GECs and podocytes were grown together in close proximity, and assessed transcriptional changes in each cell type after exposure to HG and MGO. We found that HG and MGO had distinct effects on gene expression and that the effect of each treatment was markedly different between GECs and podocytes. HG treatment led to upregulation of "immediate early response" genes, particularly those of the EGR family, as well as genes involved in inflammatory responses (in GECs) or DNA replication/cell cycle (in podocytes). Interestingly, both HG and MGO led to downregulation of genes related to extracellular matrix organisation in podocytes. Crucially, the transcriptional responses of GECs and podocytes were dependent on their interaction with each other, as many of the prominently regulated genes in co-culture of the two cell types were not significantly changed when monocultures of the cells were exposed to the same stimuli. Finally, the changes in the expression of selected genes were validated in BTBR ob/ob mice, an established model of DN. This work highlights the molecular alterations in GECs and podocytes in response to the key diabetic metabolic triggers HG and MGO, as well as the central role of GEC-podocyte crosstalk in governing these responses.
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Diabetes Mellitus Experimental , Nefropatias Diabéticas , Podócitos , Camundongos , Animais , Podócitos/metabolismo , Nefropatias Diabéticas/patologia , Glomérulos Renais/patologia , Células Endoteliais/metabolismo , Óxido de Magnésio/farmacologia , Diabetes Mellitus Experimental/metabolismo , Transdução de Sinais , Camundongos Endogâmicos , Glucose/metabolismo , ApoptoseRESUMO
In vivo tissue engineering (TE) techniques like the AV loop model provide an isolated and well-defined microenvironment to study angiogenesis-related cell interactions. Functional visualization of the microvascular network within these artificial tissue constructs is crucial for the fundamental understanding of vessel network formation and to identify the underlying key regulatory mechanisms. To facilitate microvascular tracking advanced fluorescence imaging techniques are required. We studied the suitability of microporous polylactic acid (PLA) scaffolds with known low autofluorescence to form axial vascularized tissue constructs in the AV loop model and to validate these scaffolds for fluorescence-based perfusion imaging. Compared to commonly used collagen elastin (CE) scaffolds, the total number of vessels and cells in PLA scaffolds was lower. In detail, CE-based constructs exhibited significantly higher vessel numbers on day 14 and 28 (d14: 316 ± 53; d28: 610 ± 74) compared to the respective time points in PLA-based constructs (d14: 144 ± 18; d28: 327 ± 34; each p < 0.05). Analogously, cell counts in CE scaffolds were higher compared to corresponding PLA constructs (d14: 7661.25 ± 505.93 and 5804.04 ± 716.59; d28: 11211.75 + 1278.97 and 6045.71 ± 572.72, p < 0.05). CE scaffolds showed significantly higher vessel densities in proximity to the main vessel axis compared to PLA scaffolds (200-400 µm and 600-800 µm on day 14; 400-1000 µm and 1400-1600 µm on day 28). CE scaffolds had significantly higher cell counts on day 14 at distances from 800 to 2000 µm and at distances from 400 to 1600 µm on day 28. While the total number of vessels and cells in PLA scaffolds were lower, both scaffold types were ideally suited for axial vascularization techniques. The intravascular perfusion of PLA-based constructs with fluorescence dye MHI148-PEI demonstrated dye specificity against vascular walls of low- and high-order branches as well as capillaries and facilitated the fluorescence-based visualization of microcirculatory networks. Fluorophore tracking may contribute to the development of automated quantification methods after 3D reconstruction and image segmentation. These technologies may facilitate the characterization of key regulators within specific subdomains and add to the current understanding of vessel formation in axially vascularized tissue constructs.
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Neovascularização Fisiológica , Alicerces Teciduais , Humanos , Microcirculação , Engenharia Tecidual/métodos , Neovascularização Patológica , Poliésteres , Colágeno , PerfusãoRESUMO
Bone morphogenetic protein (BMP)-9, a member of the TGFß-family of cytokines, is believed to be mainly produced in the liver. The serum levels of BMP-9 were reported to be reduced in newly diagnosed diabetic patients and BMP-9 overexpression ameliorated steatosis in the high fat diet-induced obesity mouse model. Furthermore, injection of BMP-9 in mice enhanced expression of fibroblast growth factor (FGF)21. However, whether BMP-9 also regulates the expression of the related FGF19 is not clear. Because both FGF21 and 19 were described to protect the liver from steatosis, we have further investigated the role of BMP-9 in this context. We first analyzed BMP-9 levels in the serum of streptozotocin (STZ)-induced diabetic rats (a model of type I diabetes) and confirmed that BMP-9 serum levels decrease during diabetes. Microarray analyses of RNA samples from hepatic and intestinal tissue from BMP-9 KO- and wild-type mice (C57/Bl6 background) pointed to basal expression of BMP-9 in both organs and revealed a down-regulation of hepatic Fgf21 and intestinal Fgf19 in the KO mice. Next, we analyzed BMP-9 levels in a cohort of obese patients with or without diabetes. Serum BMP-9 levels did not correlate with diabetes, but hepatic BMP-9 mRNA expression negatively correlated with steatosis in those patients that did not yet develop diabetes. Likewise, hepatic BMP-9 expression also negatively correlated with serum LPS levels. In situ hybridization analyses confirmed intestinal BMP-9 expression. Intestinal (but not hepatic) BMP-9 mRNA levels were decreased with diabetes and positively correlated with intestinal E-Cadherin expression. In vitro studies using organoids demonstrated that BMP-9 directly induces FGF19 in gut but not hepatocyte organoids, whereas no evidence of a direct induction of hepatic FGF21 by BMP-9 was found. Consistent with the in vitro data, a correlation between intestinal BMP-9 and FGF19 mRNA expression was seen in the patients' samples. In summary, our data confirm that BMP-9 is involved in diabetes development in humans and in the control of the FGF-axis. More importantly, our data imply that not only hepatic but also intestinal BMP-9 associates with diabetes and steatosis development and controls FGF19 expression. The data support the conclusion that increased levels of BMP-9 would most likely be beneficial under pre-steatotic conditions, making supplementation of BMP-9 an interesting new approach for future therapies aiming at prevention of the development of a metabolic syndrome and liver steatosis.
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Diabetes Mellitus Experimental , Fígado Gorduroso , Humanos , Ratos , Camundongos , Animais , Fator 2 de Diferenciação de Crescimento/metabolismo , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/metabolismo , Obesidade/complicações , Obesidade/metabolismo , Fígado/metabolismo , Fígado Gorduroso/metabolismo , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/metabolismo , RNA Mensageiro/metabolismoRESUMO
Diabetic retinopathy, also defined as microvascular complication of diabetes mellitus, affects the entire neurovascular unit with specific aberrations in every compartment. Neurodegeneration, glial activation and vasoregression are observed consistently in models of diabetic retinopathy. However, the order and the severity of these aberrations varies in different models, which is also true in patients. In this study, we analysed rat models of diabetic retinopathy with similar phenotypes to identify key differences in the pathogenesis. For this, we focussed on intercellular junction-associated gene expression, which are important for the communication and homeostasis within the neurovascular unit. Streptozotocin-injected diabetic Wistar rats, methylglyoxal supplemented Wistar rats and polycystin-2 transgenic (PKD) rats were analysed for neuroretinal function, vasoregression and retinal expression of junction-associated proteins. In all three models, neuroretinal impairment and vasoregression were observed, but gene expression profiling of junction-associated proteins demonstrated nearly no overlap between the three models. However, the differently expressed genes were from the main classes of claudins, connexins and integrins in all models. Changes in Rcor1 expression in diabetic rats and Egr1 expression in PKD rats confirmed the differences in upstream transcription factor level between the models. In PKD rats, a possible role for miRNA regulation was observed, indicated by an upregulation of miR-26b-5p, miR-122-5p and miR-300-3p, which was not observed in the other models. In silico allocation of connexins revealed not only differences in regulated subtypes, but also in affected retinal cell types, as well as connexin specific upstream regulators Sox7 and miR-92a-3p. In this study, we demonstrate that, despite their similar phenotype, models for diabetic retinopathy exhibit significant differences in their pathogenic pathways and primarily affected cell types. These results underline the importance for more sensitive diagnostic tools to identify pathogenic clusters in patients as the next step towards a desperately needed personalized therapy.
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Diabetes Mellitus Experimental , Retinopatia Diabética , MicroRNAs , Ratos , Animais , Retinopatia Diabética/genética , Retinopatia Diabética/metabolismo , Ratos Wistar , Diabetes Mellitus Experimental/metabolismo , Retina/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Fenótipo , Expressão GênicaRESUMO
Acute kidney injury (AKI) is characterized by a rapid reduction in renal function and glomerular filtration rate (GFR). The broadly used anti-cancer chemotherapeutic agent cisplatin often induces AKI as an adverse drug side effect. Therapies targeted at the reversal of AKI and its potential progression to chronic kidney disease or end-stage renal disease are currently insufficiently effective. Mesenchymal stromal cells (MSCs) possess diverse immunomodulatory properties that confer upon them significant therapeutic potential for the treatment of diverse inflammatory disorders. Human dermal MSCs expressing ATP-Binding Cassette member B5 (ABCB5) have shown therapeutic efficacy in clinical trials in chronic skin wounds or recessive dystrophic epidermolysis bullosa. In preclinical studies, ABCB5+ MSCs have also shown to reverse metabolic reprogramming in polycystic kidney cells, suggesting a capacity for this cell subset to improve also organ function in kidney diseases. Here, we aimed to explore the therapeutic capacity of ABCB5+ MSCs to improve renal function in a preclinical rat model of cisplatin-induced AKI. First, the anti-apoptotic and immunomodulatory capacity was compared against research-grade adipose stromal cells (ASCs). Then, cross-species immunomodulatory capacity was checked, testing first inhibition of mitogen-driven peripheral blood mononuclear cells and then modulation of macrophage function. Finally, therapeutic efficacy was evaluated in a cisplatin AKI model. First, ABCB5+ MSCs suppressed cisplatin-induced apoptosis of human conditionally-immortalized proximal tubular epithelial cells in vitro, most likely by reducing oxidative stress. Second, ABCB5+ MSCs inhibited the proliferation of either human or rat peripheral blood mononuclear cells, in the human system via the Indoleamine/kynurenine axis and in the murine context via nitric oxide/nitrite. Third, ABCB5+ MSCs decreased TNF-α secretion after lipopolysaccharide stimulation and modulated phagocytosis and in both human and rat macrophages, involving prostaglandin E2 and TGF-ß1, respectively. Fourth, clinical-grade ABCB5+ MSCs grafted intravenously and intraperitoneally to a cisplatin-induced AKI murine model exerted modulatory effects on mRNA expression patterns toward an anti-inflammatory and pro-regenerative state despite an apparent lack of amelioration of renal damage at physiologic, metabolic, and histologic levels. Our results demonstrate anti-inflammatory and pro-regenerative effects of clinical grade ABCB5+ MSCs in vitro and in vivo and suggest potential therapeutic utility of this cell population for treatment or prevention of cisplatin chemotherapy-induced tissue toxicity.
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Injúria Renal Aguda , Células-Tronco Mesenquimais , Humanos , Ratos , Camundongos , Animais , Cisplatino/efeitos adversos , Modelos Animais de Doenças , Leucócitos Mononucleares/metabolismo , Rim/patologia , Células-Tronco Mesenquimais/metabolismo , Injúria Renal Aguda/induzido quimicamente , Injúria Renal Aguda/terapia , Injúria Renal Aguda/patologia , RNA Mensageiro/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATPRESUMO
[This corrects the article DOI: 10.3389/fimmu.2020.589818.].
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Dysregulation in glomerular hemodynamics favors hyperfiltration in diabetic kidney disease (DKD). Although carnosine supplementation ameliorates features of DKD, its effect on glomerular vasoregulation is not known. We assessed the influence of carnosine and carnosinase-1 (CN1) on afferent glomerular arteriole vasodilation and its association with glomerular size, hypertrophy, and nephrin expression in diabetic BTBRob/ob mice. Two cohorts of mice including appropriate controls were studied: i.e., diabetic mice that received oral carnosine supplementation (cohort 1) and human (h)CN1 transgenic (TG) diabetic mice (cohort 2). The lumen area ratio (LAR) of the afferent arterioles and glomerular parameters were measured by conventional histology. Three-dimensional analysis using a tissue clearing strategy was also used. In both cohorts, LAR was significantly larger in diabetic BTBRob/ob versus nondiabetic BTBRwt/ob mice (0.41 ± 0.05 vs. 0.26 ± 0.07, P < 0.0001 and 0.42 ± 0.06 vs. 0.29 ± 0.04, P < 0.0001) and associated with glomerular size (cohort 1: r = 0.55, P = 0.001 and cohort 2: r = 0.89, P < 0.0001). LAR was partially normalized by oral carnosine supplementation (0.34 ± 0.05 vs. 0.41 ± 0.05, P = 0.004) but did not differ between hCN1 TG and wild-type BTBRob/ob mice. In hCN1 TG mice, serum CN1 concentrations correlated with LAR (r = 0.90, P = 0.006). Diabetic mice displayed decreased nephrin expression and increased glomerular hypertrophy. This was not significantly different in hCN1 TG BTBRob/ob mice (P = 0.06 and P = 0.08, respectively). In conclusion, carnosine and CN1 may affect intraglomerular pressure in an opposing manner through the regulation of afferent arteriolar tone. This study corroborates previous findings on the role of carnosine in the progression of DKD.NEW & NOTEWORTHY Dysregulation in glomerular hemodynamics favors hyperfiltration in diabetic kidney disease (DKD). Although carnosine supplementation ameliorates features of DKD, its effect on glomerular vasoregulation is not known. We assessed the influence of carnosine and carnosinase-1 (CN1) on afferent glomerular arteriole vasodilation and its association with glomerular size, hypertrophy, and nephrin expression in diabetic BTBRob/ob mice. Our results provide evidence that carnosine feeding and CN1 overexpression likely affect intraglomerular pressure through vasoregulation of the afferent arteriole.
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Carnosina , Diabetes Mellitus Experimental , Nefropatias Diabéticas , Animais , Arteríolas/metabolismo , Carnosina/metabolismo , Carnosina/farmacologia , Nefropatias Diabéticas/tratamento farmacológico , Nefropatias Diabéticas/patologia , Dipeptidases , Humanos , Hipertrofia , Camundongos , Camundongos Endogâmicos , Camundongos Transgênicos , VasodilataçãoRESUMO
Cystic kidney disease (CKD) is a heterogeneous group of genetic disorders and one of the most common causes of end-stage renal disease. Here, we investigate the potential effects of long-term human stem cell treatment on kidney function and the gene expression profile of PKD/Mhm (Cy/+) rats. Human adipose-derived stromal cells (ASC) and human skin-derived ABCB5+ stromal cells (2 × 106) were infused intravenously or intraperitoneally monthly, over 6 months. Additionally, ASC and ABCB5+-derived conditioned media were administrated intraperitoneally. The gene expression profile results showed a significant reprogramming of metabolism-related pathways along with downregulation of the cAMP, NF-kB and apoptosis pathways. During the experimental period, we measured the principal renal parameters as well as renal function using an innovative non-invasive transcutaneous device. All together, these analyses show a moderate amelioration of renal function in the ABCB5+ and ASC-treated groups. Additionally, ABCB5+ and ASC-derived conditioned media treatments lead to milder but still promising improvements. Even though further analyses have to be performed, the preliminary results obtained in this study can lay the foundations for a novel therapeutic approach with the application of cell-based therapy in CKD.
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Previous evidence suggests that resistance training in combination with specific collagen peptides (CP) improves adaptive responses of the muscular apparatus. Although beneficial effects have been repeatedly demonstrated, the underlying mechanisms are not well understood. Therefore, the primary objective of the present randomized trial was to elucidate differences in gene expression pathways related to skeletal muscle signal transduction following acute high-load resistance exercise with and without CP intake. Recreationally active male participants were equally randomized to high-load leg extension exercise in combination with 15 g CP or placebo (PLA) supplementation. Muscle biopsies from the vastus lateralis muscle were obtained at baseline as well as 1, 4 and 24 h post exercise to investigate gene expression using next generation sequencing analysis. Several important anabolic pathways including PI3K-Akt and MAPK pathways were significantly upregulated at 1 and 4 h post-exercise. Significant between-group differences for both pathways were identified at the 4 h time point demonstrating a more pronounced effect after CP intake. Gene expression related to the mTOR pathway demonstrated a higher visual increase in the CP group compared to PLA by trend, but failed to achieve statistically significant group differences. The current findings revealed a significantly higher upregulation of key anabolic pathways (PI3K-Akt, MAPK) in human skeletal muscle 4 h following an acute resistance training combined with intake of 15 g of specific collagen peptides compared to placebo. Further investigations should examine potential relationships between upregulated gene expression and changes in myofibrillar protein synthesis as well as potential long-term effects on anabolic pathways on the protein level.
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Colorectal cancer (CRC) is a high-incidence malignancy worldwide which still needs better therapy options. Therefore, the aim of the present study was to investigate the responses of normal or malignant human intestinal epithelium to bone morphogenetic protein (BMP)-9 and to find out whether the application of BMP-9 to patients with CRC or the enhancement of its synthesis in the liver could be useful strategies for new therapy approaches. In silico analyses of CRC patient cohorts (TCGA database) revealed that high expression of the BMP-target gene ID1, especially in combination with low expression of the BMP-inhibitor noggin, is significantly associated with better patient survival. Organoid lines were generated from human biopsies of colon cancer (T-Orgs) and corresponding non-malignant areas (N-Orgs) of three patients. The N-Orgs represented tumours belonging to three different consensus molecular subtypes (CMS) of CRC. Overall, BMP-9 stimulation of organoids promoted an enrichment of tumour-suppressive gene expression signatures, whereas the stimulation with noggin had the opposite effects. Furthermore, treatment of organoids with BMP-9 induced ID1 expression (independently of high noggin levels), while treatment with noggin reduced ID1. In summary, our data identify the ratio between ID1 and noggin as a new prognostic value for CRC patient outcome. We further show that by inducing ID1, BMP-9 enhances this ratio, even in the presence of noggin. Thus, BMP-9 is identified as a novel target for the development of improved anti-cancer therapies of patients with CRC.
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Neoplasias do Colo , Neoplasias Colorretais , Fator 2 de Diferenciação de Crescimento , Proteína Morfogenética Óssea 2 , Proteína Morfogenética Óssea 4/farmacologia , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Neoplasias Colorretais/genética , Fator 2 de Diferenciação de Crescimento/genética , Humanos , Proteína 1 Inibidora de Diferenciação , Fígado/metabolismo , Transdução de SinaisRESUMO
Optical tissue clearing (OTC) methods render tissue transparent by matching the refractive index within a sample to enable three-dimensional (3D) imaging with advanced microscopes. The application of OTC method in mediastinal organs in mice remains poorly understand. Our aim was to establish a simple protocol pipeline for 3D imaging of the mediastinal organs in mice. Trachea, oesophagus, thymus and heart were harvested from mice after retrograde perfusion via the abdominal aorta. We combined and optimized antibody labelling of thick tissue samples, OTC with cheap and non-toxic solvent ethyl cinnamate (ECi), and light-sheet fluorescence microscopy (LSFM) or laser confocal fluorescence microscopy (LCFM) to visualize the vasculature of those tissues. A high degree of optical transparency of trachea, oesophagus, thymus and heart was achieved after ECi-based OTC. With anti-CD31 antibody immunofluorescence labelling before ECi-based OTC, the vasculature of these tissues with their natural morphology, location and organizational network was imaged using LSFM or LCFM. This simple protocol pipeline provides an easy-to-setup and comprehensive way to study the vasculature of mediastinal organs in 3D without any special equipment. We anticipate that it will facilitate diverse applications in biomedical research of thoracic diseases and even other organs.
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Sistema Cardiovascular/anatomia & histologia , Imageamento Tridimensional/métodos , Animais , Imuno-Histoquímica/métodos , Camundongos , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Tórax/anatomia & histologiaRESUMO
Expression of the immediate-early response gene IER2 has been associated with the progression of several types of cancer, but its functional role is poorly understood. We found that increased IER2 expression in human melanoma is associated with shorter overall survival, and subsequently investigated the mechanisms through which IER2 exerts this effect. In experimental melanoma models, sustained expression of IER2 induced senescence in a subset of melanoma cells in a p53/MAPK/AKT-dependent manner. The senescent cells produced a characteristic secretome that included high levels of the extracellular phosphoglycoprotein osteopontin. Nuclear localization of the IER2 protein was critical for both the induction of senescence and osteopontin secretion. Osteopontin secreted by IER2-expressing senescent cells strongly stimulated the migration and invasion of non-senescent melanoma cells. Consistently, we observed coordinate expression of IER2, p53/p21, and osteopontin in primary human melanomas and metastases, highlighting the pathophysiological relevance of IER2-mediated senescence in melanoma progression. Together, our study reveals that sustained IER2 expression drives melanoma invasion and progression through stimulating osteopontin secretion via the stochastic induction of senescence.
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Biomarcadores Tumorais/metabolismo , Senescência Celular , Regulação Neoplásica da Expressão Gênica , Proteínas Imediatamente Precoces/metabolismo , Melanoma/patologia , Osteopontina/metabolismo , Transativadores/metabolismo , Animais , Apoptose , Biomarcadores Tumorais/genética , Proliferação de Células , Humanos , Proteínas Imediatamente Precoces/genética , Melanoma/genética , Melanoma/metabolismo , Camundongos , Invasividade Neoplásica , Osteopontina/genética , Prognóstico , Transativadores/genética , Células Tumorais CultivadasRESUMO
Blue light regulates biological function in various cells, such as proliferation, oxidative stress, and cell death. We employed blue light illumination on human umbilical vein endothelial cells utilizing a LED device at 453 nm wavelength and revealed a novel biphasic response on human umbilical vein endothelial cells (HUVECs). The results showed that low fluence blue light irradiation promoted the fundamental cell activities, including cell viability, migration and angiogenesis by activating the angiogenic pathways such as the VEGF signaling pathway. In contrast, high fluence illumination caused the opposite effect on those activities by upregulating pro-apoptotic signaling cascades like ferroptosis, necroptosis and the p53 signaling pathways. Our results provide an underlying insight into photobiomodulation by blue light and may help to implement potential treatment strategies for treating angiogenesis-dependent diseases.
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Adipose tissue (AT) is a key metabolic organ which functions are rhythmically regulated by an endogenous circadian clock. Feeding is a "zeitgeber" aligning the clock in AT with the external time, but mechanisms of this regulation remain largely unclear. We tested the hypothesis that postprandial changes of the hormone insulin directly entrain circadian clocks in AT and investigated a transcriptional-dependent mechanism of this regulation. We analyzed gene expression in subcutaneous AT (SAT) of obese subjects collected before and after the hyperinsulinemic-euglycemic clamp or control saline infusion (SC). The expressions of core clock genes PER2, PER3, and NR1D1 in SAT were differentially changed upon insulin and saline infusion, suggesting insulin-dependent clock regulation. In human stem cell-derived adipocytes, mouse 3T3-L1 cells, and AT explants from mPer2Luc knockin mice, insulin induced a transient increase of the Per2 mRNA and protein expression, leading to the phase shift of circadian oscillations, with similar effects for Per1 Insulin effects were dependent on the region between -64 and -43 in the Per2 promoter but not on CRE and E-box elements. Our results demonstrate that insulin directly regulates circadian clocks in AT and isolated adipocytes, thus representing a primary mechanism of feeding-induced AT clock entrainment.
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Tecido Adiposo/efeitos dos fármacos , Relógios Circadianos/efeitos dos fármacos , Ritmo Circadiano/efeitos dos fármacos , Insulina/farmacologia , Células 3T3-L1 , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Tecido Adiposo/metabolismo , Animais , Humanos , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/genética , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/metabolismo , Proteínas Circadianas Period/genética , Proteínas Circadianas Period/metabolismo , Regiões Promotoras Genéticas/efeitos dos fármacosRESUMO
In the last few years, the focus of phototherapy has shifted toward the visible (400-700 nm) part of the electromagnetic spectrum of light. Lately, it has been demonstrated that visible light (VL) can have both beneficial and detrimental effects, especially on the skin. Previously and until now, the most harmful effects on the skin are associated with ultraviolet radiation (UVR). After exposure to natural light, the most evident and immediate change is observed on skin pigmentation. Various wavelengths within the visible spectrum have been reported to alter skin pigmentation. However, the underlying mechanisms are incompletely understood so far. The article aims to shed light on the progress made in the photobiology field (photobiomodulation, PBM) to study the role of visible light on skin melanocytes.
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Melanócitos , Pigmentação da Pele , Pele , Raios Ultravioleta , Melanócitos/efeitos da radiação , Fotobiologia , Pele/efeitos da radiação , Raios Ultravioleta/efeitos adversosRESUMO
Vascular dysfunction and vasoregression are hallmarks of a variety of inflammatory central nervous system disorders and inflammation-related retinal diseases like diabetic retinopathy. Activation of microglia and the humoral innate immune system are contributing factors. Anti-inflammatory approaches have been proposed as therapies for neurovascular diseases, which include the modulation of microglial activation. The present study aimed at investigating the effects of microglial activation by clodronate-coated liposomes on vasoregression in a model of retinal degeneration. Clodronate treatment over 5 weeks led to an increase in activated CD74+ microglia and completely prevented acellular capillaries and pericyte loss. Gene expression analyses indicated that vasoprotection was due to the induction of vasoprotective factors such as Egr1, Stat3, and Ahr while expression of pro-inflammatory genes remained unchanged. We concluded that activated microglia led to a shift toward induction of pleiotropic protective pathways supporting vasoprotection in neurovascular retinal diseases.
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Hirschsprung disease (HSCR, OMIM 142623) involves congenital intestinal obstruction caused by dysfunction of neural crest cells and their progeny during enteric nervous system (ENS) development. HSCR is a multifactorial disorder; pathogenetic variants accounting for disease phenotype are identified only in a minority of cases, and the identification of novel disease-relevant genes remains challenging. In order to identify and to validate a potential disease-causing relevance of novel HSCR candidate genes, we established a complementary study approach, combining whole exome sequencing (WES) with transcriptome analysis of murine embryonic ENS-related tissues, literature and database searches, in silico network analyses, and functional readouts using candidate gene-specific genome-edited cell clones. WES datasets of two patients with HSCR and their non-affected parents were analysed, and four novel HSCR candidate genes could be identified: ATP7A, SREBF1, ABCD1 and PIAS2. Further rare variants in these genes were identified in additional HSCR patients, suggesting disease relevance. Transcriptomics revealed that these genes are expressed in embryonic and fetal gastrointestinal tissues. Knockout of these genes in neuronal cells demonstrated impaired cell differentiation, proliferation and/or survival. Our approach identified and validated candidate HSCR genes and provided further insight into the underlying pathomechanisms of HSCR.
Assuntos
Doença de Hirschsprung/genética , Membro 1 da Subfamília D de Transportadores de Cassetes de Ligação de ATP/genética , Animais , Diferenciação Celular/genética , Linhagem Celular , Proliferação de Células/genética , Sobrevivência Celular/genética , Simulação por Computador , ATPases Transportadoras de Cobre/genética , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Técnicas de Inativação de Genes , Humanos , Lactente , Masculino , Camundongos , Proteínas Inibidoras de STAT Ativados/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Sequenciamento do ExomaRESUMO
Articular cartilage (AC) is an avascular tissue composed of scattered chondrocytes embedded in a dense extracellular matrix, in which nourishment takes place via the synovial fluid at the surface. AC has a limited intrinsic healing capacity, and thus mainly surgical techniques have been used to relieve pain and improve function. Approaches to promote regeneration remain challenging. The microfracture (MF) approach targets the bone marrow (BM) as a source of factors and progenitor cells to heal chondral defects in situ by opening small holes in the subchondral bone. However, the original function of AC is not obtained yet. We hypothesize that mechanical stimulation can mobilize mesenchymal stromal cells (MSCs) from BM reservoirs upon MF of the subchondral bone. Thus, the aim of this study was to compare the counts of mobilized human BM-MSCs (hBM-MSCs) in alginate-laminin (alginate-Ln) or collagen-I (col-I) scaffolds upon intermittent mechanical loading. The mechanical set up within an established bioreactor consisted of 10% strain, 0.3 Hz, breaks of 10 s every 180 cycles for 24 h. Contrary to previous findings using porcine MSCs, no significant cell count was found for hBM-MSCs into alginate-Ln scaffolds upon mechanical stimulation (8 ± 5 viable cells/mm3 for loaded and 4 ± 2 viable cells/mm3 for unloaded alginate-Ln scaffolds). However, intermittent mechanical stimulation induced the mobilization of hBM-MSCs into col-I scaffolds 10-fold compared to the unloaded col-I controls (245 ± 42 viable cells/mm3 vs. 22 ± 6 viable cells/mm3, respectively; p-value < 0.0001). Cells that mobilized into the scaffolds by mechanical loading did not show morphological changes. This study confirmed that hBM-MSCs can be mobilized in vitro from a reservoir toward col-I but not alginate-Ln scaffolds upon intermittent mechanical loading, against gravity.