RESUMO
Regenerative capabilities of the endothelium rely on vessel-resident progenitors termed endothelial colony forming cells (ECFCs). This study aimed to investigate if these progenitors are impacted by conditions (i.e., obesity or atherosclerosis) characterized by increased serum levels of oxidized low-density lipoprotein (oxLDL), a known inducer of Endothelial-to-Mesenchymal Transition (EndMT). Our investigation focused on understanding the effects of EndMT on the self-renewal capabilities of progenitors and the associated molecular alterations. In the presence of oxLDL, ECFCs displayed classical features of EndMT, through reduced endothelial gene and protein expression, function as well as increased mesenchymal genes, contractility, and motility. Additionally, ECFCs displayed a dramatic loss in self-renewal capacity in the presence of oxLDL. RNA-sequencing analysis of ECFCs exposed to oxLDL validated gene expression changes suggesting EndMT and identified SOX9 as one of the highly differentially expressed genes. ATAC sequencing analysis identified SOX9 binding sites associated with regions of dynamic chromosome accessibility resulting from oxLDL exposure, further pointing to its importance. EndMT phenotype and gene expression changes induced by oxLDL in vitro or high fat diet (HFD) in vivo were reversed by the silencing of SOX9 in ECFCs or the endothelial-specific conditional knockout of Sox9 in murine models. Overall, our findings support that EndMT affects vessel-resident endothelial progenitor's self-renewal. SOX9 activation is an early transcriptional event that drives the mesenchymal transition of endothelial progenitor cells. The identification of the molecular network driving EndMT in vessel-resident endothelial progenitors presents a new avenue in understanding and preventing a range of condition where this process is involved.
Assuntos
Lipoproteínas LDL , Fatores de Transcrição SOX9 , Lipoproteínas LDL/metabolismo , Lipoproteínas LDL/farmacologia , Animais , Fatores de Transcrição SOX9/metabolismo , Fatores de Transcrição SOX9/genética , Camundongos , Humanos , Aterosclerose/metabolismo , Aterosclerose/patologia , Aterosclerose/genética , Transição Epitelial-Mesenquimal , Camundongos Endogâmicos C57BL , Masculino , Células Progenitoras Endoteliais/metabolismo , Células Progenitoras Endoteliais/citologia , Autorrenovação Celular , Células Endoteliais/metabolismoRESUMO
INTRODUCTION: Targeted routine antenatal prophylaxis with anti-D immunoglobulin (Ig) only to RhD-negative pregnant women who carry RhD-positive fetuses (determined by fetal RHD genotyping) has reduced D-alloimmunization significantly when administered in addition to postnatal prophylaxis. Achieving high analysis sensitivity and few false-negative fetal RHD results will make RhD typing of the newborn redundant. Postnatal prophylaxis can then be given based on the result of fetal RHD genotyping. Terminating routine RhD typing of the newborns in cord blood will streamline maternity care. Accordingly, we compared the results of fetal RHD genotyping with RhD typing of the newborns. METHODS: Fetal RHD genotyping was performed, and antenatal anti-D Ig was administered at gestational week 24 and 28, respectively. Data for 2017-2020 are reported. RESULTS: Ten laboratories reported 18,536 fetal RHD genotypings, and 16,378 RhD typing results of newborns. We found 46 false-positive (0.28%) and seven false-negative (0.04%) results. Sensitivity of the assays was 99.93%, while specificity was 99.24%. CONCLUSION: Few false-negative results support the good analysis quality of fetal RHD genotyping. Routine cord blood RhD typing will therefore be discontinued nationwide and postnatal anti-D Ig will now be given based on the result of fetal RHD genotyping.
Assuntos
Serviços de Saúde Materna , Isoimunização Rh , Gravidez , Feminino , Humanos , Recém-Nascido , Diagnóstico Pré-Natal/métodos , Sangue Fetal , Genótipo , Isoimunização Rh/genética , Isoimunização Rh/prevenção & controle , Sistema do Grupo Sanguíneo Rh-Hr/genética , Feto , Imunoglobulina rho(D)/uso terapêutico , Imunoglobulina rho(D)/genéticaRESUMO
Interfollicular epidermal (IFE) homeostasis is a major physiological process allowing maintenance of the skin barrier function. Despite progress in our understanding of stem cell populations in different hair follicle compartments, cellular mechanisms of IFE maintenance, in particular, whether a hierarchy of progenitors exists within this compartment, have remained controversial. We here used multicolour lineage tracing with Brainbow transgenic labels activated in the epidermis to track individual keratinocyte clones. Two modes of clonal progression could be observed in the adult murine dorsal skin. Clones attached to hair follicles showed rapid increase in size during the growth phase of the hair cycle. On the other hand, clones distant from hair follicles were slow cycling, but could be mobilized by a proliferative stimulus. Reinforced by mathematical modelling, these data support a model where progenitor cycling characteristics are differentially regulated in areas surrounding or away from growing hair follicles. Thus, while IFE progenitors follow a non-hierarchical mode of development, spatiotemporal control by their environment can change their potentialities, with far-reaching implications for epidermal homeostasis, wound repair and cancer development.
Assuntos
Proliferação de Células , Células Epidérmicas , Folículo Piloso/citologia , Queratinócitos/fisiologia , Células-Tronco/fisiologia , Animais , Diferenciação Celular , Técnicas Citológicas , Camundongos , Modelos Teóricos , Pele/citologia , Análise Espaço-TemporalRESUMO
SOX family proteins SOX2 and SOX18 have been reported as being essential in determining hair follicle type; however, the role they play during development remains unclear. Here, we demonstrate that Sox18 regulates the normal differentiation of the dermal papilla of all hair types. In guard (primary) hair dermal condensate (DC) cells, we identified transient Sox18 in addition to SOX2 expression at E14.5, which allowed fate tracing of primary DC cells until birth. Similarly, expression of Sox18 was detected in the DC cells of secondary hairs at E16.5 and in tertiary hair at E18.5. Dominant-negative Sox18 mutation (opposum) did not prevent DC formation in any hair type. However, it affected dermal papilla differentiation, restricting hair formation especially in secondary and tertiary hairs. This Sox18 mutation also prevented neonatal dermal cells or dermal papilla spheres from inducing hair in regeneration assays. Microarray expression studies identified WNT5A and TNC as potential downstream effectors of SOX18 that are important for epidermal WNT signalling. In conclusion, SOX18 acts as a mesenchymal molecular switch necessary for the formation and function of the dermal papilla in all hair types.
Assuntos
Diferenciação Celular/genética , Folículo Piloso/embriologia , Cabelo/embriologia , Fatores de Transcrição SOXF/fisiologia , Animais , Derme/embriologia , Derme/metabolismo , Embrião de Mamíferos , Células Epidérmicas , Epiderme/embriologia , Feminino , Genes Dominantes , Genes de Troca/fisiologia , Cabelo/metabolismo , Folículo Piloso/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Fatores de Transcrição SOXF/genéticaRESUMO
BACKGROUND: During adult life, blood vessel formation is thought to occur via angiogenic processes involving branching from existing vessels. An alternate proposal suggests that neovessels form from endothelial progenitors able to assemble the intimal layers. We here aimed to define vessel-resident endothelial progenitors in vivo in a variety of tissues in physiological and pathological situations such as normal aorta, lungs, and wound healing, tumors, and placenta, as well. METHODS: Based on protein expression levels of common endothelial markers using flow cytometry, 3 subpopulations of endothelial cells could be identified among VE-Cadherin+ and CD45- cells. RESULTS: Lineage tracing by using Cdh5creERt2/Rosa-YFP reporter strategy demonstrated that the CD31-/loVEGFR2lo/intracellular endothelial population was indeed an endovascular progenitor (EVP) of an intermediate CD31intVEGFR2lo/intracellular transit amplifying (TA) and a definitive differentiated (D) CD31hiVEGFR2hi/extracellular population. EVP cells arose from vascular-resident beds that could not be transferred by bone marrow transplantation. Furthermore, EVP displayed progenitor-like status with a high proportion of cells in a quiescent cell cycle phase as assessed in wounds, tumors, and aorta. Only EVP cells and not TA and D cells had self-renewal capacity as demonstrated by colony-forming capacity in limiting dilution and by transplantation in Matrigel plugs in recipient mice. RNA sequencing revealed prominent gene expression differences between EVP and D cells. In particular, EVP cells highly expressed genes related to progenitor function including Sox9, Il33, Egfr, and Pdfgrα. Conversely, D cells highly expressed genes related to differentiated endothelium including Ets1&2, Gata2, Cd31, Vwf, and Notch. The RNA sequencing also pointed to an essential role of the Sox18 transcription factor. The role of SOX18 in the differentiation process was validated by using lineage-tracing experiments based on Sox18CreERt2/Rosa-YFP mice. Besides, in the absence of functional SOX18/SOXF, EVP progenitors were still present, but TA and D populations were significantly reduced. CONCLUSIONS: Our findings support an entirely novel endothelial hierarchy, from EVP to TA to D, as defined by self-renewal, differentiation, and molecular profiling of an endothelial progenitor. This paradigm shift in our understanding of vascular-resident endothelial progenitors in tissue regeneration opens new avenues for better understanding of cardiovascular biology.
Assuntos
Células Endoteliais/metabolismo , Células-Tronco/metabolismo , Animais , Antígenos CD/metabolismo , Aorta/metabolismo , Aorta/patologia , Transplante de Medula Óssea , Caderinas/metabolismo , Diferenciação Celular , Células Endoteliais/citologia , Endotélio Vascular/citologia , Feminino , Antígenos Comuns de Leucócito/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Fisiológica , Placenta/metabolismo , Placenta/patologia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Gravidez , Fatores de Transcrição SOXF/metabolismo , Células-Tronco/citologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Ferimentos e Lesões/patologia , Ferimentos e Lesões/terapiaRESUMO
The prospect of using endothelial progenitors is currently hampered by their low engraftment upon transplantation. We report that mesenchymal stem/stromal cells (MSCs), independent of source and age, improve the engraftment of endothelial colony forming cells (ECFCs). MSC coculture altered ECFC appearance to an elongated mesenchymal morphology with reduced proliferation. ECFC primed via MSC contact had reduced self-renewal potential, but improved capacity to form tube structures in vitro and engraftment in vivo Primed ECFCs displayed major differences in transcriptome compared to ECFCs never exposed to MSCs, affecting genes involved in the cell cycle, up-regulating of genes influencing mesenchymal transition, adhesion, extracellular matrix. Inhibition of NOTCH signaling, a potential upstream regulator of mesenchymal transition, in large part modulated this gene expression pattern and functionally reversed the mesenchymal morphology of ECFCs. The collective results showed that primed ECFCs survive better and undergo a mesenchymal transition that is dependent on NOTCH signaling, resulting in significantly increased vasculogenic potential.-Shafiee, A., Patel, J., Wong, H. Y., Donovan, P., Hutmacher, D. W., Fisk, N. M., Khosrotehrani, K. Priming of endothelial colony-forming cells in a mesenchymal niche improves engraftment and vasculogenic potential by initiating mesenchymal transition orchestrated by NOTCH signaling.
Assuntos
Células Endoteliais/metabolismo , Células-Tronco Mesenquimais/fisiologia , Receptores Notch/metabolismo , Transdução de Sinais/fisiologia , Animais , Diferenciação Celular , Proliferação de Células , Técnicas de Cocultura , Feminino , Regulação da Expressão Gênica/fisiologia , Humanos , Camundongos , Placenta , Gravidez , Receptores Notch/genéticaRESUMO
Since the discovery of endothelial colony forming cells (ECFC), there has been significant interest in their therapeutic potential to treat vascular injuries. ECFC cultures display significant heterogeneity and a hierarchy among cells able to give rise to high proliferative versus low proliferative colonies. Here we aimed to define molecularly this in vitro hierarchy. Based on flow cytometry, CD34 expression levels distinguished two populations. Only CD34 + ECFC had the capacity to reproduce high proliferative potential (HPP) colonies on replating, whereas CD34- ECFCs formed only small clusters. CD34 + ECFCs were the only ones to self-renew in stringent single-cell cultures and gave rise to both CD34 + and CD34- cells. Upon replating, CD34 + ECFCs were always found at the centre of HPP colonies and were more likely in G0/1 phase of cell cycling. Functionally, CD34 + ECFC were superior at restoring perfusion and better engrafted when injected into ischemic hind limbs. Transcriptomic analysis identified cyclin-dependent kinase (CDK) cell cycle inhibiting genes (p16, p21, and p57), the Notch signaling pathway (dll1, dll4, hes1, and hey1), and the endothelial cytokine il33 as highly expressed in CD34 + ECFC. Blocking the Notch pathway using a γ-secretase inhibitor (DAPT) led to reduced expression of cell cycle inhibitors, increased cell proliferation followed by a loss of self-renewal, and HPP colony formation capacity reflecting progenitor exhaustion. Similarly shRNA knockdown of p57 strongly affected self-renewal of ECFC colonies. ECFC hierarchy is defined by Notch signalling driving cell cycle regulators, progenitor quiescence and self-renewal potential.
Assuntos
Antígenos CD34/metabolismo , Células Progenitoras Endoteliais/transplante , Neovascularização Fisiológica/genética , Receptores Notch/genética , Lesões do Sistema Vascular/terapia , Animais , Linhagem da Célula/genética , Proliferação de Células/genética , Autorrenovação Celular/genética , Inibidor de Quinase Dependente de Ciclina p27/genética , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Inibidor de Quinase Dependente de Ciclina p57/antagonistas & inibidores , Inibidor de Quinase Dependente de Ciclina p57/genética , Dipeptídeos/administração & dosagem , Células Progenitoras Endoteliais/metabolismo , Citometria de Fluxo , Membro Posterior/patologia , Membro Posterior/transplante , Humanos , Neovascularização Fisiológica/efeitos dos fármacos , Receptores Notch/antagonistas & inibidores , Receptores Notch/biossíntese , Medicina Regenerativa , Transdução de Sinais/efeitos dos fármacos , Lesões do Sistema Vascular/metabolismo , Lesões do Sistema Vascular/patologiaAssuntos
Carcinoma de Células Escamosas , Transplante de Órgãos , Neoplasias Cutâneas , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/prevenção & controle , Quimioprevenção , Método Duplo-Cego , Humanos , Transplante de Órgãos/efeitos adversos , Projetos Piloto , Sirolimo/efeitos adversos , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/prevenção & controle , TransplantadosRESUMO
The main carcinogen for keratinocyte skin cancers (KCs) such as basal and squamous cell carcinomas is ultraviolet (UV) radiation. There is growing evidence that accumulation of mutations and clonal expansion play a key role in KC development. The relationship between UV exposure, epidermal mutation load, and KCs remains unclear. Here, we examined the mutation load in both murine (n = 23) and human (n = 37) epidermal samples. Epidermal mutations accumulated in a UV dose-dependent manner, and this mutation load correlated with the KC burden. Epidermal ablation (either mechanical or laser induced), followed by spontaneous healing from underlying epithelial adnexae reduced the mutation load markedly in both mouse (n = 8) and human (n = 6) clinical trials. In a model of UV-induced basal cell carcinoma, epidermal ablation reduced incident lesions by >80% (n = 5). Overall, our findings suggest that mutation burden is strongly associated with KC burden and represents a target to prevent subsequent KCs.
Assuntos
Carcinoma Basocelular , Neoplasias Cutâneas , Humanos , Camundongos , Animais , Acúmulo de Mutações , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Pele/patologia , Epiderme/patologia , Carcinoma Basocelular/patologia , Raios Ultravioleta/efeitos adversos , MutaçãoRESUMO
The origin of basal cell carcinomas (BCC) remains elusive. This protocol combines the use of an ultraviolet (UV)-inducible BCC murine model (K14CrexPtchwt/lox) and an optimized protocol for florescent whole-mount cytokeratin 17 immunostaining to address the spatiotemporal dynamics of BCC formation. The high-resolution three-dimensional confocal images are an excellent tool to visualize and quantify the distribution of skin cancers at a given time point. For complete details on the use and execution of this protocol, please refer to Roy et al. (2020).
Assuntos
Carcinoma Basocelular , Neoplasias Experimentais , Neoplasias Cutâneas , Raios Ultravioleta/efeitos adversos , Animais , Carcinoma Basocelular/metabolismo , Carcinoma Basocelular/patologia , Camundongos , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Coloração e RotulagemRESUMO
Endothelial to mesenchymal transition (EndMT) is a leading cause of fibrosis and disease, however its mechanism has yet to be elucidated. The endothelium possesses a profound regenerative capacity to adapt and reorganize that is attributed to a population of vessel-resident endovascular progenitors (EVP) governing an endothelial hierarchy. Here, using fate analysis, we show that two transcription factors SOX9 and RBPJ specifically affect the murine EVP numbers and regulate lineage specification. Conditional knock-out of Sox9 from the vasculature (Sox9fl/fl/Cdh5-CreER RosaYFP) depletes EVP while enhancing Rbpj expression and canonical Notch signalling. Additionally, skin wound analysis from Sox9 conditional knock-out mice demonstrates a significant reduction in pathological EndMT resulting in reduced scar area. The converse is observed with Rbpj conditionally knocked-out from the murine vasculature (Rbpjfl/fl/Cdh5-CreER RosaYFP) or inhibition of Notch signaling in human endothelial colony forming cells, resulting in enhanced Sox9 and EndMT related gene (Snail, Slug, Twist1, Twist2, TGF-ß) expression. Similarly, increased endothelial hedgehog signaling (Ptch1fl/fl/Cdh5-CreER RosaYFP), that upregulates the expression of Sox9 in cells undergoing pathological EndMT, also results in excess fibrosis. Endothelial cells transitioning to a mesenchymal fate express increased Sox9, reduced Rbpj and enhanced EndMT. Importantly, using topical administration of siRNA against Sox9 on skin wounds can substantially reduce scar area by blocking pathological EndMT. Overall, here we report distinct fates of EVPs according to the relative expression of Rbpj or Notch signalling and Sox9, highlighting their potential plasticity and opening exciting avenues for more effective therapies in fibrotic diseases.
Assuntos
Células Endoteliais/metabolismo , Endotélio/metabolismo , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , Fatores de Transcrição SOX9/metabolismo , Transdução de Sinais/genética , Animais , Diferenciação Celular/genética , Linhagem da Célula , Endotélio/citologia , Feminino , Técnicas de Inativação de Genes , Proteínas Hedgehog/metabolismo , Humanos , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Interferente Pequeno , Receptores Notch/metabolismo , Fatores de Transcrição SOX9/genética , Fator de Crescimento Transformador beta/metabolismo , Cicatrização/genéticaRESUMO
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
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
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
Skin wound healing in adults is characterized by a peak of angiogenesis followed by regression of the excessive vasculature in parallel with collagen deposition and fibrosis in the wound. We hypothesized that regressing vessels in healing wounds were in fact entering an endothelial to mesenchymal transition contributing to scarring. Using vascular-specific fate tracking (Cdh5-creERt2/ROSA-YFP mice), full-thickness excisional wounds were analyzed to reveal a time-dependent transition from endothelial phenotype characterized by vascular endothelial-cadherin, CD31, and CD34 toward a mesenchymal phenotype characterized by alpha-smooth muscle actin and fibroblast-specific protein 1 expression. We next conditionally ablated RBPJ in the vasculature (Rbpjfl/fl/Cdh5-creERt2ROSA-YFP) to evaluate the role of canonical Notch signaling in this process. Endothelial to mesenchymal transition was clearly accelerated after the loss of Notch signaling within the vasculature. The acceleration of endothelial to mesenchymal transition resulted in delayed wound healing, increased fibrosis, and extensive scar tissue formation, with the rapid loss of key endothelial genes and proteins and upregulation of mesenchymal protein expression (alpha-smooth muscle actin and fibroblast-specific protein 1) in vessels. Our findings here uncover a cellular contributor to skin wound scarring through the process of endothelial to mesenchymal transition in skin wounds and demonstrate the importance of Notch signaling in regulating this critical process during healing.
Assuntos
Células Endoteliais/patologia , Mesoderma/patologia , Receptores Notch/fisiologia , Transdução de Sinais/fisiologia , Cicatrização , Ferimentos e Lesões/patologia , Animais , Antígenos CD34/análise , Fibrose , Humanos , Camundongos , Neovascularização Patológica , Molécula-1 de Adesão Celular Endotelial a Plaquetas/análise , Fator de Crescimento Transformador beta1/fisiologia , Ferimentos e Lesões/fisiopatologiaRESUMO
In this study, direct ionization mass spectrometry (DI-MS) has been developed for rapid differentiation of Ganoderma (known as Lingzhi in Chinese), a very popular and valuable herbal medicine. Characteristic mass spectra can be generated by DI-MS directly from the raw herbal medicines with the application of a high voltage and solvents. Rapid differentiation of the Ganoderma species that are officially stated in the Chinese pharmacopoeia from easily confused Ganoderma species could be achieved based on this method, as the acquired DI-MS spectra showed that ganoderic acids, the major active components of Ganoderma, could be found only in the official Ganoderma species but not in the confused Ganoderma species. In addition, classification of wild and cultivated Ganoderma and potential differentiation of Ganoderma from different geographical locations could be accomplished based on principal component analysis (PCA) or hierarchical clustering analysis (HCA). The method is rapid, simple and reproducible, and can be further extended to analysis of other herbal medicines.
Assuntos
Medicamentos de Ervas Chinesas/química , Ganoderma/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Triterpenos/análise , Análise por Conglomerados , Medicamentos de Ervas Chinesas/classificação , Desenho de Equipamento , Ganoderma/classificação , Análise de Componente Principal , Espectrometria de Massas por Ionização por Electrospray/economia , Espectrometria de Massas por Ionização por Electrospray/instrumentação , Fatores de TempoRESUMO
In this study, direct ionization mass spectrometry (DI-MS) for rapid authentication of Gastrodiae rhizoma (known as Tianma in Chinese), a popular herbal medicine, has been developed. This method is rapid, simple and allows direct generation of characteristic mass spectra from the raw herbal medicines with the application of some solvents and a high voltage. The acquired DI-MS spectra showed that gastrodin, parishin B/parishin C and parishin, the major active components of Gastrodiae rhizoma, could be found only in genuine Gastrodiae rhizoma samples, but not in counterfeit samples, thus allowing rapid authentication of Gastrodiae rhizoma. Moreover, wild and cultivated Gastrodiae rhizoma could be classified and Gastrodiae rhizoma from different geographical locations could be differentiated based on their different intensity ratios of characteristic ions or principal component analysis (PCA). This method is simple, rapid, reproducible, and can be extended to analyze other herbal medicines.