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Single-cell RNA sequencing (scRNA-seq) remains state-of-the-art for transcriptomic cell-mapping. Here, we provide a protocol to generate high-resolution scRNA-seq of rare cardiomyocyte populations (e.g., regenerating/dividing, etc.) from mouse and zebrafish hearts as well as induced pluripotent stem cells, collected in time to achieve detailed transcriptomic insight. We describe the serial steps of viability staining, methanol fixation, storage, and cell sorting to preserve RNA integrity suited for scRNA-seq as well as the quality assessment of the data as shown by examples. For complete details on the use and execution of this protocol, please refer to Bak et al.1.
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Rationale: Extracellular vesicles (EVs) are thought to mediate intercellular communication during development and disease. Yet, biological insight to intercellular EV transfer remains elusive, also in the heart, and is technically challenging to demonstrate. Here, we aimed to investigate biological transfer of cardiomyocyte-derived EVs in the neonatal heart. Methods: We exploited CD9 as a marker of EVs, and generated two lines of cardiomyocyte specific EV reporter mice: Tnnt2-Cre; double-floxed inverted CD9/EGFP and αMHC-MerCreMer; double-floxed inverted CD9/EGFP. The two mouse lines were utilized to determine whether developing cardiomyocytes transfer EVs to other cardiac cells (non-myocytes and cardiomyocytes) in vitro and in vivo and investigate the intercellular transport pathway of cardiomyocyte-derived EVs. Results: Genetic tagging of cardiomyocytes was confirmed in both reporter mouse lines and proof of concept in the postnatal heart showed that, a fraction of EGFP+/MYH1- non-myocytes exist firmly demonstrating in vivo cardiomyocyte-derived EV transfer. However, two sets of direct and indirect EGFP +/- cardiac cell co-cultures showed that cardiomyocyte-derived EGFP+ EV transfer requires cell-cell contact and that uptake of EGFP+ EVs from the medium is limited. The same was observed when co-cultiring with mouse macrophages. Further mechanistic insight showed that cardiomyocyte EV transfer occurs through type I tunneling nanotubes. Conclusion: While the current notion assumes that EVs are transferred through secretion to the surroundings, our data show that cardiomyocyte-derived EV transfer in the developing heart occurs through nanotubes between neighboring cells. Whether these data are fundamental and relate to adult hearts and other organs remains to be determined, but they imply that the normal developmental process of EV transfer goes through cell-cell contact rather than through the extracellular compartment.
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Comunicação Celular , Técnicas de Cocultura , Vesículas Extracelulares , Miócitos Cardíacos , Animais , Vesículas Extracelulares/metabolismo , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/citologia , Camundongos , Comunicação Celular/fisiologia , Nanotubos , Coração/fisiologia , Tetraspanina 29/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Fluorescência Verde/genética , Animais Recém-Nascidos , Camundongos TransgênicosRESUMO
BACKGROUND: Breast cancer-related lymphedema (BCRL) is a debilitating sequela affecting up to one in three breast cancer survivors. Current treatments are palliative and does not address the underlying lymphatic injury. Recently, preclinical and non-randomized studies have shown promising results using Adipose-Derived Regenerative Cells (ADRCs) and lipotransfer in alleviating BCRL through regeneration of lymphatic tissue. However no randomized controlled trial has been performed in an attempt to eliminate a placebo effect. METHODS: This randomized, double-blinded, placebo-controlled trial included patients with no-option, persistent disabling unilateral BCRL. Patients were randomly assigned in a 1:1 ratio to receive either autologous ADRCs (4.20x10 7±1.75x10 7 cells) and 30cc lipotransfer or placebo (saline) to the axilla. The primary outcome was a change in BCRL volume one year after treatment. Secondary outcomes included changes in the quality of life, indocyanine green lymphangiography stage, bioimpedance, and safety. RESULTS: Eighty patients were included, of which 39 were allocated to ADRCs and lipotransfer treatment and 41 to placebo treatment. Baseline characteristics were similar in both groups. One year after treatment, no objective improvements were observed in the treatment or placebo groups. In contrast, significant subjective improvements were noted for both the treatment and placebo groups. CONCLUSION: This trial failed to confirm a benefit of ADRCs and lipotransfer in the treatment of BCRL. These non-confirmatory results suggest that ADRC and lipotransfer should not be recommended for alleviating BCRL at this time. However, we cannot exclude that repeated treatments or higher doses of ADRCs or lipotransfer could yield a clinical effect.
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BACKGROUND: Heart failure due to myocardial infarction (MI) involves fibrosis driven by epicardium-derived cells (EPDCs) and cardiac fibroblasts, but strategies to inhibit and provide cardio-protection remains poor. The imprinted gene, non-canonical NOTCH ligand 1 (Dlk1), has previously been shown to mediate fibrosis in the skin, lung and liver, but very little is known on its effect in the heart. METHODS: Herein, human pericardial fluid/plasma and tissue biopsies were assessed for DLK1, whereas the spatiotemporal expression of Dlk1 was determined in mouse hearts. The Dlk1 heart phenotype in normal and MI hearts was assessed in transgenic mice either lacking or overexpressing Dlk1. Finally, in/ex vivo cell studies provided knowledge on the molecular mechanism. RESULTS: Dlk1 was demonstrated in non-myocytes of the developing human myocardium but exhibited a restricted pericardial expression in adulthood. Soluble DLK1 was twofold higher in pericardial fluid (median 45.7 [34.7 (IQR)) µg/L] from cardiovascular patients (n = 127) than in plasma (median 26.1 µg/L [11.1 (IQR)]. The spatial and temporal expression pattern of Dlk1 was recapitulated in mouse and rat hearts. Similar to humans lacking Dlk1, adult Dlk1-/- mice exhibited a relatively mild developmental, although consistent cardiac phenotype with some abnormalities in heart size, shape, thorax orientation and non-myocyte number, but were functionally normal. However, after MI, scar size was substantially reduced in Dlk1-/- hearts as compared with Dlk1+/+ littermates. In line, high levels of Dlk1 in transgenic mice Dlk1fl/fl xWT1GFPCre and Dlk1fl/fl xαMHCCre/+Tam increased scar size following MI. Further mechanistic and cellular insight demonstrated that pericardial Dlk1 mediates cardiac fibrosis through epithelial to mesenchymal transition (EMT) of the EPDC lineage by maintaining Integrin ß8 (Itgb8), a major activator of transforming growth factor ß and EMT. CONCLUSIONS: Our results suggest that pericardial Dlk1 embraces a, so far, unnoticed role in the heart augmenting cardiac fibrosis through EMT. Monitoring DLK1 levels as well as targeting pericardial DLK1 may thus offer new venues for cardio-protection.
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Transição Epitelial-Mesenquimal , Infarto do Miocárdio , Adulto , Animais , Humanos , Camundongos , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Cicatriz/metabolismo , Cicatriz/patologia , Transição Epitelial-Mesenquimal/genética , Fibrose , Ligantes , Camundongos Transgênicos , Infarto do Miocárdio/genética , Pericárdio/metabolismo , Tórax/patologiaRESUMO
Cellular heterogeneity represents a major challenge for regenerative treatment using freshly isolated Adipose Derived Regenerative Cells (ADRCs). Emerging data suggest superior efficacy of ADRCs as compared to the ex vivo expanded and more homogeneous ADRCs (= ASCs) for indications involving (micro)vascular deficiency, however, it remains unknown which ADRC cell subtypes account for the improvement. Surprisingly, we found regarding erectile dysfunction (ED) that the number of injected CD31+ ADRCs correlated positively with erectile function 12 months after one bolus of autologous ADRCs. Comprehensive in vitro and ex vivo analyses confirmed superior pro-angiogenic and paracrine effects of human CD31+ enriched ADRCs compared to the corresponding CD31- and parent ADRCs. When CD31+, CD31- and ADRCs were co-cultured in aortic ring- and corpus cavernous tube formation assays, the CD31+ ADRCs induced significantly higher tube development. This effect was corroborated using conditioned medium (CM), while quantitative mass spectrometric analysis suggested that this is likely explained by secretory pro-angiogenic proteins including DKK3, ANGPT2, ANAX2 and VIM, all enriched in CD31+ ADRC CM. Single-cell RNA sequencing showed that transcripts of the upregulated and secreted proteins were present in 9 endothelial ADRC subsets including endothelial progenitor cells in the heterogenous non-cultured ADRCs. Our data suggest that the vascular benefit of using ADRCs in regenerative medicine is dictated by CD31+ ADRCs.
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Máculas Acústicas , Líquidos Corporais , Humanos , Masculino , Proteínas Angiogênicas , Bioensaio , Transporte Biológico , Meios de Cultivo CondicionadosRESUMO
Whereas cardiomyocytes (CMs) in the fetal heart divide, postnatal CMs fail to undergo karyokinesis and/or cytokinesis and therefore become polyploid or binucleated, a key process in terminal CM differentiation. This switch from a diploid proliferative CM to a terminally differentiated polyploid CM remains an enigma and seems an obstacle for heart regeneration. Here, we set out to identify the transcriptional landscape of CMs around birth using single cell RNA sequencing (scRNA-seq) to predict transcription factors (TFs) involved in CM proliferation and terminal differentiation. To this end, we established an approach combining fluorescence activated cell sorting (FACS) with scRNA-seq of fixed CMs from developing (E16.5, P1, and P5) mouse hearts, and generated high-resolution single-cell transcriptomic maps of in vivo diploid and tetraploid CMs, increasing the CM resolution. We identified TF-networks regulating the G2/M phases of developing CMs around birth. ZEB1 (Zinc Finger E-Box Binding Homeobox 1), a hereto unknown TF in CM cell cycling, was found to regulate the highest number of cell cycle genes in cycling CMs at E16.5 but was downregulated around birth. CM ZEB1-knockdown reduced proliferation of E16.5 CMs, while ZEB1 overexpression at P0 after birth resulted in CM endoreplication. These data thus provide a ploidy stratified transcriptomic map of developing CMs and bring new insight to CM proliferation and endoreplication identifying ZEB1 as a key player in these processes.
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Miócitos Cardíacos , Transcriptoma , Animais , Camundongos , Proliferação de Células , Genes Homeobox , Ploidias , Poliploidia , Homeobox 1 de Ligação a E-box em Dedo de Zinco , Dedos de ZincoRESUMO
Spinal cord injury (SCI) initiates detrimental cellular and molecular events that lead to acute and delayed neuroinflammation. Understanding the role of the inflammatory response in SCI requires insight into the temporal and cellular synthesis of inflammatory mediators. We subjected C57BL/6J mice to SCI and investigated inflammatory reactions. We examined activation, recruitment, and polarization of microglia and infiltrating immune cells, focusing specifically on tumor necrosis factor (TNF) and its receptors TNFR1 and TNFR2. In the acute phase, TNF expression increased in glial cells and neuron-like cells, followed by infiltrating immune cells. TNFR1 and TNFR2 levels increased in the delayed phase and were found preferentially on neurons and glial cells, respectively. The acute phase was dominated by the infiltration of granulocytes and macrophages. Microglial/macrophage expression of Arg1 increased from 1-7 days after SCI, followed by an increase in Itgam, Cx3cr1, and P2ry12, which remained elevated throughout the study. By 21 and 28 days after SCI, the lesion core was populated by galectin-3+, CD68+, and CD11b+ microglia/macrophages, surrounded by a glial scar consisting of GFAP+ astrocytes. Findings were verified in postmortem tissue from individuals with SCI. Our findings support the consensus that future neuroprotective immunotherapies should aim to selectively neutralize detrimental immune signaling while sustaining pro-regenerative processes.
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Erectile dysfunction is a common complication associated with type 2 diabetes mellitus (T2DM) and after prostatectomy in relation to cancer. The regenerative effect of cultured adipose-derived stem cells (ASCs) for ED therapy has been documented in multiple preclinical trials as well as in recent Pase 1 trials in humans. However, some studies indicate that diabetes negatively affects the mesenchymal stem cell pool, implying that ASCs from T2DM patients could have impaired regenerative capacity. Here, we directly compared ASCs from age-matched diabetic Goto-Kakizaki (ASCGK) and non-diabetic wild type rats (ASCWT) with regard to their phenotypes, proteomes and ability to rescue ED in normal rats. Despite ASCGK exhibiting a slightly lower proliferation rate, ASCGK and ASCWT proteomes were more or less identical, and after injections to corpus cavernosum they were equally efficient in restoring erectile function in a rat ED model entailing bilateral nerve crush injury. Moreover, molecular analysis of the corpus cavernosum tissue revealed that both ASCGK and ASCWT treated rats had increased induction of genes involved in recovering endothelial function. Thus, our finding argues that T2DM does not appear to be a limiting factor for autologous adipose stem cell therapy when correcting for ED.
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Diabetes Mellitus Tipo 2/complicações , Disfunção Erétil/terapia , Transplante de Células-Tronco , Tecido Adiposo/citologia , Animais , Células Cultivadas , Disfunção Erétil/etiologia , Masculino , Ratos , Células-TroncoRESUMO
BACKGROUND: Rotator cuff (RC) tendon tear leads to impaired shoulder function and pain. The supraspinatus (SS) tendon is most often affected, but the biological response of the SS muscle to SS tendon tear is largely unknown. This study aimed to investigate time-dependent muscle inflammation, degeneration, fatty infiltration, and regeneration in experimental SS tear conditions. METHODS: Forty-five C57BL/6 mice were subjected to SS tendon tear and allowed to recover for 1, 3, 5, 7, 14, or 28 days. The extent of muscle damage was examined using histologic, flow cytometric, proteomic, and chemiluminescence analyses. RESULTS: We found that muscle inflammation peaked around day 5 with increased monocyte infiltration and increased cytokine levels in the ipsilateral compared to the contralateral SS muscle. Bioinformatics analysis of proteomics on mice that survived 5 days after RC tendon tear revealed upregulated proteins involved in "neutrophil activation involved in immune response" and "extracellular matrix organization," whereas "skeletal muscle tissue development and contraction" and "respiratory electron transport chain" were among the most downregulated. Histologic analysis of collagen showed increased collagen accumulation and fatty infiltration of the ipsilateral SS over time. Finally, we observed time- and lesion-dependent changes in satellite cell and fibro-adipogenic progenitor populations. CONCLUSION: Altogether, we demonstrate that the SS muscle shows severe signs of acute inflammation, early degeneration, and fatty infiltration, as well as reduced regenerative potential following SS tendon tear.
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Lesões do Manguito Rotador , Manguito Rotador , Tecido Adiposo/patologia , Animais , Humanos , Inflamação , Camundongos , Camundongos Endogâmicos C57BL , Atrofia Muscular/patologia , Proteômica , Manguito Rotador/patologiaRESUMO
Small diameter (<6 mm) vessel grafts still pose a challenge for scientists worldwide. Decellularised umbilical artery (dUA) remains promising as small diameter tissue engineered vascular graft (TEVG), yet their immunogenicity remains unknown. Herein, we evaluated the host immune responses, with a focus on the innate part, towards human dUA implantation in mice, and confirmed our findings in an ex vivo allogeneic human setup. Overall, we did not observe any differences in the number of circulating white blood cells nor the number of monocytes among three groups of mice (1) dUA patch; (2) Sham; and (3) Mock throughout the study (day -7 to 28). Likewise, we found no difference in systemic inflammatory and anti-inflammatory cytokine levels between groups. However, a massive local remodelling response with M2 macrophages were observed in the dUA at day 28, whereas M1 macrophages were less frequent. Moreover, human monocytes from allogeneic individuals were differentiated into macrophages and exposed to lyophilised dUA to maximize an eventual M1 response. Yet, dUA did not elicit any immediate M1 response as determined by the absence of CCR7 and CXCL10. Together this suggests that human dUA elicits a minimal pro-inflammatory response further supporting its use as a TEVG in an allogeneic setup.
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Prótese Vascular , Quimiocina CXCL10/imunologia , Macrófagos/imunologia , Monócitos/imunologia , Receptores CCR7/imunologia , Artérias Umbilicais , Animais , Feminino , Humanos , CamundongosRESUMO
Ischemic heart disease is one of the leading causes of deaths worldwide. A major hindrance to resolving this challenge lies in the mammalian hearts inability to regenerate after injury. In contrast, zebrafish retain a regenerative capacity of the heart throughout their lifetimes. Apex resection (AR) is a popular zebrafish model for studying heart regeneration, and entails resecting 10-20% of the heart in the apex region, whereafter the regeneration process is monitored until the heart is fully regenerated within 60 days. Despite this popularity, video tutorials describing this technique in detail are lacking. In this paper we visualize and describe the entire AR procedure including anaesthesia, surgery, and recovery. In addition, we show that the concentration and duration of anaesthesia are important parameters to consider, to balance sufficient levels of sedation and minimizing mortality. Moreover, we provide examples of how zebrafish heart regeneration can be assessed both in 2D (immunohistochemistry of heart sections) and 3D (analyses of whole, tissue cleared hearts using multiphoton imaging). In summary, this paper aims to aid beginners in establishing and conducting the AR model in their laboratory, but also to spur further interest in improving the model and its evaluation.
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Anestesia em Procedimentos Cardíacos/métodos , Recursos Audiovisuais , Procedimentos Cirúrgicos Cardíacos/métodos , Coração/diagnóstico por imagem , Regeneração/fisiologia , Aminobenzoatos , Anestésicos , Animais , Técnicas de Imagem Cardíaca , Proliferação de Células , Humanos , Cinetocardiografia/métodos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/fisiologia , Peixe-ZebraRESUMO
To date, a wide range of materials, from synthetic to natural or a mixture of these, has been explored, modified, and examined as small-diameter tissue-engineered vascular grafts (SD-TEVGs) for tissue regeneration either in vitro or in vivo. However, very limited success has been achieved due to mechanical failure, thrombogenicity or intimal hyperplasia, and improvements of the SD-TEVG design are thus required. Here, in vivo studies investigating novel and relative long (10 times of the inner diameter) SD-TEVGs in large animal models and humans are identified and discussed, with emphasis on graft outcome based on model- and graft-related conditions. Only a few types of synthetic polymer-based SD-TEVGs have been evaluated in large-animal models and reflect limited success. However, some polymers, such as polycaprolactone (PCL), show favorable biocompatibility and potential to be further modified and improved in the form of hybrid grafts. Natural polymer- and cell-secreted extracellular matrix (ECM)-based SD-TEVGs tested in large animals still fail due to a weak strength or thrombogenicity. Similarly, native ECM-based SD-TEVGs and in-vitro-developed hybrid SD-TEVGs that contain xenogeneic molecules or matrix seem related to a harmful graft outcome. In contrast, allogeneic native ECM-based SD-TEVGs, in-vitro-developed hybrid SD-TEVGs with allogeneic banked human cells or isolated autologous stem cells, and in-body tissue architecture (IBTA)-based SD-TEVGs seem to be promising for the future, since they are suitable in dimension, mechanical strength, biocompatibility, and availability.
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Prótese Vascular , Engenharia Tecidual , Animais , Implante de Prótese Vascular , Matriz Extracelular/metabolismo , Rejeição de Enxerto/etiologia , Humanos , Modelos AnimaisRESUMO
Background: Polycaprolactone (PCL) scaffolds exhibit high biocompatibility and are attractive as vascular conduits. Materials & methods: PCL tubes were cultivated in bioreactor with human adipose regenerative cells to assess ex vivo cytocompatibility, whereas in vivo PCL tube patency was evaluated in sheep carotid bypass with and without antithrombotic treatment. Results:Ex vivo results revealed increasing adipose regenerative cells on PCL using dynamic bioreactor culturing. In vivo data showed that 67% (2/3) of grafts in the antithrombotic group were patent at day 28, while 100% (3/3) of control grafts were occluded already during the first week due to thrombosis. Histology showed that patent PCL grafts were recellularized by host cells. Conclusion: PCL tubes may work as small diameter vascular scaffolds under antithrombotic treatment.
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Fibrinolíticos , Enxerto Vascular , Animais , Prótese Vascular , Fibrinolíticos/farmacologia , Poliésteres , OvinosRESUMO
Patients with breast cancer-related lymphedema (BCRL) have reduced quality of life and arm function. Current treatments are palliative, and treatments improving lymphedema are lacking. Preclinical studies have suggested that adipose-derived regenerative cells (ADRCs) can alleviate lymphedema. We, therefore, aimed to assess whether ADRCs can alleviate lymphedema in clinical reality with long-term follow-up. We treated 10 patients with BCRL using ADRCs and a scar-releasing lipotransfer to the axillary region, and all patients were followed 1, 3, 6, 12, and 48 months after treatment. The primary endpoint was change in arm volume. Secondary endpoints were safety, change in lymphedema symptoms, quality of life, lymphedema-associated cellulitis, and conservative treatment use. There was no significant decrease in BCRL volume after treatment. However, self-reported upper extremity disability and arm heaviness and tension improved. Six patients reduced their use of conservative BCRL treatment. Five patients felt that their BCRL had improved substantially, and four of these would redo the treatment. We did not observe any cases of locoregional breast cancer recurrence. In this phase I study with 4 years of follow-up, axillary delivered ADRCs and lipotransfer were safe and feasible and improved BCRL symptoms and upper extremity function. Randomized controlled trials are needed to confirm the results of this study.
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Tecido Adiposo , Linfedema Relacionado a Câncer de Mama , Neoplasias da Mama , Transplante de Células-Tronco , Tecido Adiposo/citologia , Tecido Adiposo/transplante , Linfedema Relacionado a Câncer de Mama/terapia , Neoplasias da Mama/complicações , Neoplasias da Mama/terapia , Feminino , Seguimentos , Humanos , Recidiva Local de Neoplasia , Qualidade de VidaRESUMO
Muscle stem cells, also called muscle satellite cells (MuSCs), are responsible for skeletal muscle regeneration and are sustained in an undifferentiated and quiescent state under steady conditions. The calcitonin receptor (CalcR)-protein kinase A (PKA)-Yes-associated protein 1 (Yap1) axis is one pathway that maintains quiescence in MuSCs. Although CalcR signaling in MuSCs has been identified, the critical CalcR signaling targets are incompletely understood. Here, we show the relevance between the ectopic expression of delta-like non-canonical Notch ligand 1 (Dlk1) and the impaired quiescent state in CalcR-conditional knockout (cKO) MuSCs. Dlk1 expression was rarely detected in both quiescent and proliferating MuSCs in control mice, whereas Dlk1 expression was remarkably increased in CalcR-cKO MuSCs at both the mRNA and protein levels. It is noteworthy that all Ki67+ non-quiescent CalcR-cKO MuSCs express Dlk1, and non-quiescent CalcR-cKO MuSCs are enriched in the Dlk1+ fraction by cell sorting. Using mutant mice, we demonstrated that PKA-activation or Yap1-depletion suppressed Dlk1 expression in CalcR-cKO MuSCs, which suggests that the CalcR-PKA-Yap1 axis inhibits the expression of Dlk1 in quiescent MuSCs. Moreover, the loss of Dlk1 rescued the quiescent state in CalcR-cKO MuSCs, which indicates that the ectopic expression of Dlk1 disturbs quiescence in CalcR-cKO. Collectively, our results suggest that ectopically expressed Dlk1 is responsible for the impaired quiescence in CalcR-cKO MuSCs.
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Proteínas de Ligação ao Cálcio/metabolismo , Músculo Esquelético/metabolismo , Receptores da Calcitonina/metabolismo , Células Satélites de Músculo Esquelético/metabolismo , Animais , Diferenciação Celular/fisiologia , Divisão Celular/fisiologia , Proliferação de Células/fisiologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células-Tronco/metabolismoRESUMO
Spinal cord injury (SCI) is a devastating condition consisting of an instant primary mechanical injury followed by a secondary injury that progresses for weeks to months. The cytokine tumor necrosis factor (TNF) plays an important role in the pathophysiology of SCI. We investigated the effect of myeloid TNF ablation (peripheral myeloid cells (macrophages and neutrophils) and microglia) versus central myeloid TNF ablation (microglia) in a SCI contusion model. We show that TNF ablation in macrophages and neutrophils leads to reduced lesion volume and improved functional outcome after SCI. In contrast, TNF ablation in microglia only or TNF deficiency in all cells had no effect. TNF levels tended to be decreased 3 h post-SCI in mice with peripheral myeloid TNF ablation and was significantly decreased 3 days after SCI. Leukocyte and microglia populations and all other cytokines (IL-1ß, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, and IFNγ) and chemokines (CCL2, CCL5, and CXCL1) investigated, in addition to TNFR1 and TNFR2, were comparable between genotypes. Analysis of post-SCI signaling cascades demonstrated that the MAPK kinase SAPK/JNK decreased and neuronal Bcl-XL levels increased post-SCI in mice with ablation of TNF in peripheral myeloid cells. These findings demonstrate that peripheral myeloid cell-derived TNF is pathogenic in SCI.
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Deleção de Genes , Células Mieloides/metabolismo , Traumatismos da Medula Espinal/patologia , Traumatismos da Medula Espinal/fisiopatologia , Fator de Necrose Tumoral alfa/metabolismo , Animais , Receptor 1 de Quimiocina CX3C/metabolismo , Inflamação/patologia , Sistema de Sinalização das MAP Quinases , Macrófagos/metabolismo , Camundongos , Microglia/metabolismo , Microglia/patologia , Atividade Motora , Neutrófilos/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Recuperação de Função Fisiológica , Fatores de Transcrição STAT/metabolismo , Medula Espinal/patologia , Proteína bcl-X/metabolismoAssuntos
Alicerces Teciduais , Artérias Umbilicais , Animais , Prótese Vascular , Artérias Carótidas , Humanos , Ovinos , Engenharia TecidualRESUMO
Myocardial infarction (MI) is a worldwide condition that affects millions of people. This is mainly caused by the adult human heart lacking the ability to regenerate upon injury, whereas zebrafish have the capacity through cardiomyocyte proliferation to fully regenerate the heart following injury such as apex resection (AR). But a systematic overview of the methods used to evidence heart regrowth and regeneration in the zebrafish is lacking. Herein, we conducted a systematical search in Embase and Pubmed for studies on heart regeneration in the zebrafish following injury and identified 47 AR studies meeting the inclusion criteria. Overall, three different methods were used to assess heart regeneration in zebrafish AR hearts. 45 out of 47 studies performed qualitative (37) and quantitative (8) histology, whereas immunohistochemistry for various cell cycle markers combined with cardiomyocyte specific proteins was used in 34 out of 47 studies to determine cardiomyocyte proliferation qualitatively (6 studies) or quantitatively (28 studies). For both methods, analysis was based on selected heart sections and not the whole heart, which may bias interpretations. Likewise, interstudy comparison of reported cardiomyocyte proliferation indexes seems complicated by distinct study designs and reporting manners. Finally, six studies performed functional analysis to determine heart function, a hallmark of human heart injury after MI. In conclusion, our data implies that future studies should consider more quantitative methods eventually taking the 3D of the zebrafish heart into consideration when evidencing myocardial regrowth after AR. Furthermore, standardized guidelines for reporting cardiomyocyte proliferation and sham surgery details may be considered to enable inter study comparisons and robustly determine the effect of given genes on the process of heart regeneration.
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Coração/fisiologia , Regeneração/fisiologia , Medicina Regenerativa/métodos , Peixe-Zebra/fisiologia , Animais , Testes de Função Cardíaca , Miócitos Cardíacos/citologiaRESUMO
BACKGROUND: The imprinted gene Delta like non-canonical Notch ligand 1 (Dlk1) is considered an inhibitor of adipogenesis, but its in vivo impact on fat mass indeed remains elusive and controversial. METHODS: Fat deposits were assessed by MRI and DXA scanning in two cohorts of non-diabetic men, whereas glucose disposal rate (GDR) was determined during euglycemic hyperinsulinemic clamp. Blood analyte measurements were used for correlation and mediation analysis to investigate how age, BMI, and fat percentage affect the relation between DLK1 and GDR. Confirmatory animal studies performed in normal (NC) and high fat diet (HFD) fed Dlk1+/+ and Dlk1-/- mice included DXA scanning, glucose tolerance tests (GTTs), blood measurements, and skeletal muscle glucose uptake studies by positron emission tomography (PET), histology, qRT-PCR, and in vitro cell studies. FINDINGS: Overall, DLK1 is positively correlated with fat amounts, which is consistent with a negative linear relationship between DLK1 and GDR. This relationship is not mediated by age, BMI, or fat percentage. In support, DLK1 also correlates positively with HOMA-IR and ADIPO-IR in these humans, but has no linear relationship with the early diabetic inflammation marker MCP-1. In Dlk1-/- mice, the increase in fat percentage and adipocyte size induced by HFD is attenuated, and these animals are protected against insulin resistance. These Dlk1 effects seem independent of gluconeogenesis, but at least partly relies on increased in vivo glucose uptake in skeletal muscles by Dlk1 regulating the major glucose transporter Glut4 in vivo as well as in two independent cell lines. INTERPRETATION: Thus, instead of an adipogenic inhibitor, Dlk1 should be regarded as a factor causally linked to obesity and insulin resistance, and may be used to predict development of type 2 diabetes. FUND: The Danish Diabetes Academy supported by the Novo Nordisk Foundation, The Danish National Research Council (#09-073648), The Lundbeck Foundation, University of Southern Denmark, and Dep. Of Clinical Biochemistry and Pharmacology/Odense University Hospital, the Swedish Research Council, the Swedish Diabetes Foundation, the Strategic Research Program in Diabetes at Karolinska Institute and an EFSD/Lilly grant.