RESUMO
Macrophages regulate cutaneous wound healing by immune surveillance, tissue repair and remodelling. The depletion of dermal macrophages during the early and middle stages of wound healing has a detrimental impact on wound closure, characterised by reduced vessel density, fibroblast and myofibroblast proliferation, delayed re-epithelization and abated post-healing fibrosis and scar formation. However, in some animal species, oral mucosa and foetal life, cutaneous wounds can heal normally and remain scarless without any involvement of macrophages. These paradoxical observations have created much controversy on macrophages' indispensable role in skin wound healing. Advanced knowledge gained by characterising macrophage subsets, their plasticity in switching phenotypes and molecular drivers provides new insights into their functional importance during cutaneous wound healing. In this review, we highlight the recent findings on skin macrophage subsets, their functional role in adult cutaneous wound healing and the potential benefits of targeting them for therapeutic use.
Assuntos
Cicatriz , Cicatrização , Animais , Cicatrização/fisiologia , Cicatriz/patologia , Pele/patologia , Macrófagos , Fibroblastos/patologiaRESUMO
Wnt signaling controls blood vessel growth, regression and patterning during embryonic and postnatal life. Macrophages are major producers of Wnt ligands and angiogenic growth factors. It regulates vascular development and specification during embryogenesis and wound healing. Macrophage dysregulation in wound healing impairs vessel regeneration and delay wound closure. During cutaneous wound healing, the endovascular progenitors (EVPs) proliferate and differentiate into mature endothelial (D) cells in response to signals produced by perivascular cells, including macrophages, governing blood vessels regeneration. However, the role of macrophage's Wnt production on endothelial cells, especially the EVPs during wound healing is currently unknown. Here we used a cutaneous excisional wound model in mice with conditional deletion of Wnt secretion by myeloid cells (Wlsfl/flLysM-Cre+ ) to assess the kinetics of endothelial subpopulations (including EVP), myeloid infiltration, collagen deposition and wound closure. Deletion of Wls expression by myeloid cells did not affect wound closure and collagen deposition, indicating that myeloid Wls expression does not promote wound healing and regeneration. Myeloid-specific Wls deletion elevated the EVP population during the peak of angiogenesis, yet without affecting blood vessel density. Wounds in Wlsfl/flLysM-Cre+ animals showed unperturbed myeloid infiltration and differentiation. Overall, our data indicate that macrophage Wnt production shapes EVP kinetics without major relevance to wound healing. These findings extend the knowledge of macrophage and endothelial molecular crosstalk and position myeloid-derived Wnt production as a regulator of endovascular progenitor.
Assuntos
Células Endoteliais , Via de Sinalização Wnt , Animais , Diferenciação Celular/genética , Células Endoteliais/metabolismo , Macrófagos , Camundongos , Cicatrização/genéticaRESUMO
A need to identify a stem cell source for human endothelial colony forming cells (ECFCs) and mesenchymal stem cells (MSCs) that is high yield is crucial for their implementation in ischemia. Our lab has developed an isolation protocol to do this using full-term human villous placental tissue. This protocol describes enzymatic tissue digestion followed by MACS and FACS, achieving an 8 times greater yield versus traditional isolation techniques and delivering pure fetal stem cell colonies within 21-28 days cell culture. For complete details on the use and execution of this protocol, please refer to Patel et al. (2013) and Patel et al. (2014).
Assuntos
Células-Tronco Mesenquimais , Placenta , Técnicas de Cultura de Células/métodos , Células Endoteliais , Feminino , Feto , Humanos , GravidezRESUMO
Little is known regarding the molecular differences between basal cell carcinoma (BCC) subtypes, despite clearly distinct phenotypes and clinical outcomes. In particular, infiltrative BCCs have poorer clinical outcomes in terms of response to therapy and propensity for dissemination. In this project, we aimed to use exome sequencing and RNA sequencing to identify somatic mutations and molecular pathways leading to infiltrative BCCs. Using whole-exome sequencing of 36 BCC samples (eight infiltrative) combined with previously reported exome data (58 samples), we determine that infiltrative BCCs do not contain a distinct somatic variant profile and carry classical UV-induced mutational signatures. RNA sequencing on both datasets revealed key differentially expressed genes, such as POSTN and WISP1, suggesting increased integrin and Wnt signaling. Immunostaining for periostin and WISP1 clearly distinguished infiltrative BCCs, and nuclear ß-catenin staining patterns further validated the resulting increase in Wnt signaling in infiltrative BCCs. Of significant interest, in BCCs with mixed morphology, infiltrative areas expressed WISP1, whereas nodular areas did not, supporting a continuum between subtypes. In conclusion, infiltrative BCCs do not differ in their genomic alteration in terms of initiating mutations. They display a specific type of interaction with the extracellular matrix environment regulating Wnt signaling.
Assuntos
Carcinoma Basocelular/genética , Neoplasias Cutâneas/genética , Idoso , Proteínas de Sinalização Intercelular CCN/análise , Carcinoma Basocelular/classificação , Carcinoma Basocelular/patologia , Moléculas de Adesão Celular/análise , Feminino , Humanos , Masculino , Mutação , Proteínas Proto-Oncogênicas/análise , Neoplasias Cutâneas/classificação , Neoplasias Cutâneas/patologiaRESUMO
To better understand the influence of ultraviolet (UV) irradiation on the initial steps of skin carcinogenesis, we examine patches of labeled keratinocytes as a proxy for clones in the interfollicular epidermis (IFE) and measure their size variation upon UVB irradiation. Multicolor lineage tracing reveals that in chronically irradiated skin, patches near hair follicles (HFs) increase in size, whereas those far from follicles do not change. This is explained by proliferation of basal epidermal cells within 60 µm of HF openings. Upon interruption of UVB, patch size near HFs regresses significantly. These anatomical differences in proliferative behavior have significant consequences for the cell of origin of basal cell carcinomas (BCCs). Indeed, a UV-inducible murine BCC model shows that BCC patches are more frequent, larger, and more invasive near HFs. These findings have major implications for the prevention of field cancerization in the epidermis.
Assuntos
Epiderme/metabolismo , Neoplasias Induzidas por Radiação/patologia , Raios Ultravioleta , Animais , Carcinoma Basocelular/metabolismo , Carcinoma Basocelular/patologia , Proliferação de Células , Ciclina D1/metabolismo , Modelos Animais de Doenças , Epiderme/efeitos da radiação , Folículo Piloso/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neoplasias Induzidas por Radiação/metabolismo , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Células-Tronco/citologia , Células-Tronco/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
BACKGROUND: Basal Cell Carcinoma is the most common tumour and yet much remains to be determined regarding the molecular mechanisms that leads to its development. Hedgehog signal activation is sufficient for BCC induction, but the molecular mediators of BCC growth are not well understood. SoxF transcription factor Sox18 has been identified in human BCC, but its role in growth of the tumour is as yet unknown. OBJECTIVE: To determine if Sox18 is involved in the regulation of Basal Cell Carcinoma growth. METHODS: We analysed the function of Sox18 by combining a dominant negative Sox18 mouse model, Sox18+/OP with murine BCC RESULTS: We determine that Sox18 is ectopically expressed in the epidermal cells of a murine model of Basal Cell Carcinoma. We then show that dominant negative mutation of Sox18 increases the severity of murine Basal Cell Carcinoma. Finally, decreased Hey1 in Sox18+/OP BCC suggests Sox18 may negatively regulate BCC progression via Notch signaling. CONCLUSIONS: These data suggest that Sox18 is a hedgehog regulated mediator of tumour suppression within Basal Cell Carcinoma epidermis.
Assuntos
Carcinoma Basocelular/genética , Epiderme/patologia , Regulação Neoplásica da Expressão Gênica , Fatores de Transcrição SOXF/metabolismo , Neoplasias Cutâneas/genética , Animais , Carcinoma Basocelular/diagnóstico , Carcinoma Basocelular/patologia , Modelos Animais de Doenças , Expressão Ectópica do Gene , Proteínas Hedgehog/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Mutação , Receptor Patched-1/genética , Receptores Notch/metabolismo , Fatores de Transcrição SOXF/genética , Índice de Gravidade de Doença , Transdução de Sinais/genética , Neoplasias Cutâneas/diagnóstico , Neoplasias Cutâneas/patologiaRESUMO
The cellular and molecular profiles that govern the endothelial heterogeneity of the circulatory system have yet to be elucidated. Using a data-driven approach to study the endothelial compartment via single-cell RNA sequencing, we characterized cell subpopulations within and assigned them to a defined endothelial hierarchy. We show that two transcriptionally distinct endothelial populations exist within the aorta and, using two independent trajectory analysis methods, confirm that they represent transitioning cells rather than discrete cell types. Gene co-expression analysis revealed crucial regulatory networks underlying each population, including significant metabolic gene networks in progenitor cells. Using mitochondrial activity assays and phenotyping, we confirm that endovascular progenitors display higher mitochondrial content compared to differentiated endothelial cells. The identities of these populations were further validated against bulk RNA sequencing (RNA-seq) data obtained from normal and tumor-derived vasculature. Our findings validate the heterogeneity of the aortic endothelium and previously suggested hierarchy between progenitor and differentiated cells.
Assuntos
Aorta/metabolismo , Biomarcadores/metabolismo , Diferenciação Celular , Linhagem da Célula , Endotélio Vascular/metabolismo , Análise de Célula Única/métodos , Transcriptoma , Animais , Aorta/citologia , Endotélio Vascular/citologia , Feminino , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco/citologia , Células-Tronco/metabolismoRESUMO
Tumor vascularization is a hallmark of cancer central to disease progression and metastasis. Current anti-angiogenic therapies have limited success prompting the need to better understand the cellular origin of tumor vessels. Using fate-mapping analysis of endothelial cell populations in melanoma, we report the very early infiltration of endovascular progenitors (EVP) in growing tumors. These cells harbored self-renewal and reactivated the expression of SOX18 transcription factor, initiating a vasculogenic process as single cells, progressing towards a transit amplifying stage and ultimately differentiating into more mature endothelial phenotypes that comprised arterial, venous and lymphatic subtypes within the core of the tumor. Molecular profiling by RNA sequencing of purified endothelial fractions characterized EVPs as quiescent progenitors remodeling the extracellular matrix with significant paracrine activity promoting growth. Functionally, EVPs did not rely on VEGF-A signaling whereas endothelial-specific loss of Rbpj depleted the population and strongly inhibited metastasis. The understanding of endothelial heterogeneity opens new avenues for more effective anti-vascular therapies in cancer.
Assuntos
Transformação Celular Neoplásica/patologia , Células Progenitoras Endoteliais/patologia , Melanoma Experimental/patologia , Neovascularização Patológica/patologia , Fatores de Transcrição SOXF/metabolismo , Animais , Diferenciação Celular , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Endotélio Vascular/citologia , Endotélio Vascular/patologia , Matriz Extracelular/patologia , Feminino , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/genética , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , Vasos Linfáticos/citologia , Vasos Linfáticos/patologia , Masculino , Melanoma Experimental/irrigação sanguínea , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Invasividade Neoplásica/patologia , Fatores de Transcrição SOXF/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Endothelial colony forming cells (ECFC) and mesenchymal stem cells (MSC) combined have great potential to be used for cell therapy of ischemic vascular diseases. However, to improve allogeneic stem cell engraftment the use of immunosuppression, such as cyclosporine has been suggested. Our aim was to assess the impact of cyclosporine on hind limb revascularisation upon MSC and ECFC combination therapy. Balb/c immunocompetent mice subjected to hind limb ischemia (right femoral artery ligation) were given both human ECFC and MSC (weekly intramuscular injections) with or without cyclosporine (daily injection). Surprisingly, mice receiving cyclosporine had a significant decrease in reperfusion based on laser Doppler imaging compared to vehicle controls and had poorer limb survival. In vitro, the downstream calcineurin target NFATC4 was highly expressed in the self-renewing fraction of ECFCs. ECFCs cultured with cyclosporine had reduced colony formation capacity and tube formation in Matrigel. Lastly, ECFC displayed increased proliferation and loss of capacity for long term culture when in the presence of cyclosporine clearly showing a loss of quiescence and progenitor function. Our findings demonstrate the deleterious impact of cyclosporine on ECFC function, with significant impact on ECFC-based allogeneic cellular therapy. Stem Cells Translational Medicine 2019;8:162&7.