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1.
Angiogenesis ; 2024 May 11.
Article in English | MEDLINE | ID: mdl-38733496

ABSTRACT

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.

2.
Fetal Diagn Ther ; 50(4): 276-281, 2023.
Article in English | MEDLINE | ID: mdl-37379821

ABSTRACT

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.


Subject(s)
Maternal Health Services , Rh Isoimmunization , Pregnancy , Female , Humans , Infant, Newborn , Prenatal Diagnosis/methods , Fetal Blood , Genotype , Rh Isoimmunization/genetics , Rh Isoimmunization/prevention & control , Rh-Hr Blood-Group System/genetics , Fetus , Rho(D) Immune Globulin/therapeutic use , Rho(D) Immune Globulin/genetics
3.
EMBO J ; 35(24): 2658-2670, 2016 12 15.
Article in English | MEDLINE | ID: mdl-27797819

ABSTRACT

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.


Subject(s)
Cell Proliferation , Epidermal Cells , Hair Follicle/cytology , Keratinocytes/physiology , Stem Cells/physiology , Animals , Cell Differentiation , Cytological Techniques , Mice , Models, Theoretical , Skin/cytology , Spatio-Temporal Analysis
4.
Development ; 144(10): 1887-1895, 2017 05 15.
Article in English | MEDLINE | ID: mdl-28512199

ABSTRACT

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.


Subject(s)
Cell Differentiation/genetics , Hair Follicle/embryology , Hair/embryology , SOXF Transcription Factors/physiology , Animals , Dermis/embryology , Dermis/metabolism , Embryo, Mammalian , Epidermal Cells , Epidermis/embryology , Female , Genes, Dominant , Genes, Switch/physiology , Hair/metabolism , Hair Follicle/metabolism , Male , Mice , Mice, Transgenic , SOXF Transcription Factors/genetics
5.
Circulation ; 135(8): 786-805, 2017 02 21.
Article in English | MEDLINE | ID: mdl-27899395

ABSTRACT

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.


Subject(s)
Endothelial Cells/metabolism , Stem Cells/metabolism , Animals , Antigens, CD/metabolism , Aorta/metabolism , Aorta/pathology , Bone Marrow Transplantation , Cadherins/metabolism , Cell Differentiation , Endothelial Cells/cytology , Endothelium, Vascular/cytology , Female , Leukocyte Common Antigens/metabolism , Mice , Mice, Inbred C57BL , Neovascularization, Physiologic , Placenta/metabolism , Placenta/pathology , Platelet Endothelial Cell Adhesion Molecule-1/metabolism , Pregnancy , SOXF Transcription Factors/metabolism , Stem Cells/cytology , Vascular Endothelial Growth Factor Receptor-2/metabolism , Wounds and Injuries/pathology , Wounds and Injuries/therapy
6.
FASEB J ; 31(2): 610-624, 2017 02.
Article in English | MEDLINE | ID: mdl-28045376

ABSTRACT

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.


Subject(s)
Endothelial Cells/metabolism , Mesenchymal Stem Cells/physiology , Receptors, Notch/metabolism , Signal Transduction/physiology , Animals , Cell Differentiation , Cell Proliferation , Coculture Techniques , Female , Gene Expression Regulation/physiology , Humans , Mice , Placenta , Pregnancy , Receptors, Notch/genetics
7.
Stem Cells ; 34(4): 902-12, 2016 Apr.
Article in English | MEDLINE | ID: mdl-26732848

ABSTRACT

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.


Subject(s)
Antigens, CD34/metabolism , Endothelial Progenitor Cells/transplantation , Neovascularization, Physiologic/genetics , Receptors, Notch/genetics , Vascular System Injuries/therapy , Animals , Cell Lineage/genetics , Cell Proliferation/genetics , Cell Self Renewal/genetics , Cyclin-Dependent Kinase Inhibitor p27/genetics , Cyclin-Dependent Kinase Inhibitor p27/metabolism , Cyclin-Dependent Kinase Inhibitor p57/antagonists & inhibitors , Cyclin-Dependent Kinase Inhibitor p57/genetics , Dipeptides/administration & dosage , Endothelial Progenitor Cells/metabolism , Flow Cytometry , Hindlimb/pathology , Hindlimb/transplantation , Humans , Neovascularization, Physiologic/drug effects , Receptors, Notch/antagonists & inhibitors , Receptors, Notch/biosynthesis , Regenerative Medicine , Signal Transduction/drug effects , Vascular System Injuries/metabolism , Vascular System Injuries/pathology
10.
Sci Adv ; 9(19): eadf2384, 2023 05 10.
Article in English | MEDLINE | ID: mdl-37163607

ABSTRACT

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.


Subject(s)
Carcinoma, Basal Cell , Skin Neoplasms , Humans , Mice , Animals , Mutation Accumulation , Skin Neoplasms/genetics , Skin Neoplasms/pathology , Skin/pathology , Epidermis/pathology , Carcinoma, Basal Cell/pathology , Ultraviolet Rays/adverse effects , Mutation
11.
STAR Protoc ; 2(1): 100329, 2021 03 19.
Article in English | MEDLINE | ID: mdl-33644769

ABSTRACT

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).


Subject(s)
Carcinoma, Basal Cell , Neoplasms, Experimental , Skin Neoplasms , Ultraviolet Rays/adverse effects , Animals , Carcinoma, Basal Cell/metabolism , Carcinoma, Basal Cell/pathology , Mice , Neoplasms, Experimental/metabolism , Neoplasms, Experimental/pathology , Skin Neoplasms/metabolism , Skin Neoplasms/pathology , Staining and Labeling
12.
Nat Commun ; 12(1): 2564, 2021 05 07.
Article in English | MEDLINE | ID: mdl-33963183

ABSTRACT

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.


Subject(s)
Endothelial Cells/metabolism , Endothelium/metabolism , Immunoglobulin J Recombination Signal Sequence-Binding Protein/metabolism , SOX9 Transcription Factor/metabolism , Signal Transduction/genetics , Animals , Cell Differentiation/genetics , Cell Lineage , Endothelium/cytology , Female , Gene Knockout Techniques , Hedgehog Proteins/metabolism , Humans , Immunoglobulin J Recombination Signal Sequence-Binding Protein/genetics , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , RNA, Small Interfering , Receptors, Notch/metabolism , SOX9 Transcription Factor/genetics , Transforming Growth Factor beta/metabolism , Wound Healing/genetics
13.
Cell Rep ; 31(9): 107702, 2020 06 02.
Article in English | MEDLINE | ID: mdl-32492418

ABSTRACT

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.


Subject(s)
Epidermis/metabolism , Neoplasms, Radiation-Induced/pathology , Ultraviolet Rays , Animals , Carcinoma, Basal Cell/metabolism , Carcinoma, Basal Cell/pathology , Cell Proliferation , Cyclin D1/metabolism , Disease Models, Animal , Epidermis/radiation effects , Hair Follicle/pathology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Neoplasms, Radiation-Induced/metabolism , Skin Neoplasms/metabolism , Skin Neoplasms/pathology , Stem Cells/cytology , Stem Cells/metabolism , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism
14.
Nat Commun ; 10(1): 18, 2019 01 03.
Article in English | MEDLINE | ID: mdl-30604758

ABSTRACT

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.


Subject(s)
Cell Transformation, Neoplastic/pathology , Endothelial Progenitor Cells/pathology , Melanoma, Experimental/pathology , Neovascularization, Pathologic/pathology , SOXF Transcription Factors/metabolism , Animals , Cell Differentiation , Cell Line, Tumor , Cell Transformation, Neoplastic/genetics , Endothelium, Vascular/cytology , Endothelium, Vascular/pathology , Extracellular Matrix/pathology , Female , Immunoglobulin J Recombination Signal Sequence-Binding Protein/genetics , Immunoglobulin J Recombination Signal Sequence-Binding Protein/metabolism , Lymphatic Vessels/cytology , Lymphatic Vessels/pathology , Male , Melanoma, Experimental/blood supply , Mice , Mice, Inbred C57BL , Mice, Transgenic , Neoplasm Invasiveness/pathology , SOXF Transcription Factors/genetics , Vascular Endothelial Growth Factor A/metabolism
15.
Cell Rep ; 27(9): 2748-2758.e3, 2019 05 28.
Article in English | MEDLINE | ID: mdl-31141696

ABSTRACT

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.


Subject(s)
Aorta/metabolism , Biomarkers/metabolism , Cell Differentiation , Cell Lineage , Endothelium, Vascular/metabolism , Single-Cell Analysis/methods , Transcriptome , Animals , Aorta/cytology , Endothelium, Vascular/cytology , Female , Gene Expression Profiling , Gene Regulatory Networks , Mice , Mice, Inbred C57BL , Stem Cells/cytology , Stem Cells/metabolism
16.
J Invest Dermatol ; 138(5): 1166-1175, 2018 05.
Article in English | MEDLINE | ID: mdl-29248546

ABSTRACT

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.


Subject(s)
Endothelial Cells/pathology , Mesoderm/pathology , Receptors, Notch/physiology , Signal Transduction/physiology , Wound Healing , Wounds and Injuries/pathology , Animals , Antigens, CD34/analysis , Fibrosis , Humans , Mice , Neovascularization, Pathologic , Platelet Endothelial Cell Adhesion Molecule-1/analysis , Transforming Growth Factor beta1/physiology , Wounds and Injuries/physiopathology
17.
Anal Chim Acta ; 999: 99-106, 2018 Jan 25.
Article in English | MEDLINE | ID: mdl-29254579

ABSTRACT

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.


Subject(s)
Drugs, Chinese Herbal/chemistry , Ganoderma/chemistry , Spectrometry, Mass, Electrospray Ionization/methods , Triterpenes/analysis , Cluster Analysis , Drugs, Chinese Herbal/classification , Equipment Design , Ganoderma/classification , Principal Component Analysis , Spectrometry, Mass, Electrospray Ionization/economics , Spectrometry, Mass, Electrospray Ionization/instrumentation , Time Factors
18.
Anal Chim Acta ; 938: 90-7, 2016 Sep 28.
Article in English | MEDLINE | ID: mdl-27619090

ABSTRACT

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.


Subject(s)
Mass Spectrometry/methods , Rhizome/chemistry , Principal Component Analysis
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