RESUMEN
The importance of lymphatic vessels in a myriad of human diseases is rapidly gaining recognition; lymphatic vessel dysfunction is a feature of disorders including congenital lymphatic anomalies, primary lymphoedema and obesity, while improved lymphatic vessel function increases the efficacy of immunotherapy for cancer and neurological disease and promotes cardiac repair following myocardial infarction. Understanding how the growth and function of lymphatic vessels is precisely regulated therefore stands to inform the development of novel therapeutics applicable to a wide range of human diseases. Lymphatic vascular development is initiated during embryogenesis following establishment of the major blood vessels and the onset of blood flow. Lymphatic endothelial progenitor cells arise from a combination of venous and non-venous sources to generate the initial lymphatic vascular structures in the vertebrate embryo, which are then further ramified and remodelled to elaborate an extensive lymphatic vascular network. Signalling mediated via vascular endothelial growth factor (VEGF) family members and vascular endothelial growth factor receptor (VEGFR) tyrosine kinases is crucial for development of both the blood and lymphatic vascular networks, though distinct components are utilised to different degrees in each vascular compartment. Although much is known about the regulation of VEGFA/VEGFR2 signalling in the blood vasculature, less is understood regarding the mechanisms by which VEGFC/VEGFD/VEGFR3 signalling is regulated during lymphatic vascular development. This review will focus on recent advances in our understanding of the cellular and molecular mechanisms regulating VEGFA-, VEGFC- and VEGFD-mediated signalling via VEGFRs which are important for driving the construction of lymphatic vessels during development and disease.
Asunto(s)
Vasos Linfáticos/metabolismo , Receptores de Factores de Crecimiento Endotelial Vascular/metabolismo , Transducción de Señal/fisiología , Animales , Células Endoteliales/metabolismo , Humanos , Linfangiogénesis/fisiologíaRESUMEN
Lymphatic vessels are an integral component of the cardiovascular system, serving important roles in fluid homeostasis, lipid absorption, and immune cell trafficking. Defining the mechanisms by which the lymphatic vasculature is constructed and remodeled into a functional vascular network not only provides answers to fascinating biological questions, but is fundamental to understanding how lymphatic vessel growth and development goes awry in human pathologies. While long recognized as dysfunctional in lymphedema and exploited as a route of tumor metastasis, recent work has highlighted important roles for lymphatic vessels in modulating immune responses, regulating salt-sensitive hypertension and important for lung inflation at birth. Substantial progress in our understanding of the signaling pathways important for development and morphogenesis of the lymphatic vasculature has been made in recent years. Here, we review advances in our knowledge of the best characterized of these signaling pathways, that involving the vascular endothelial growth factor (VEGF) family members VEGF-C and VEGF-D, together with their receptors VEGFR2 and VEGFR3. Recent work has defined multiple levels at which signal transduction by means of this key axis is regulated; these include control of ligand processing and bioavailability, modulation of receptor activation by interacting proteins, and regulation of receptor endocytosis and trafficking.
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Linfangiogénesis/fisiología , Vasos Linfáticos/embriología , Transducción de Señal/fisiología , Células Madre/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Receptor 3 de Factores de Crecimiento Endotelial Vascular/metabolismo , Animales , Humanos , Vasos Linfáticos/citología , Células Madre/citología , Factor C de Crecimiento Endotelial Vascular/metabolismo , Factor D de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Recent work has established that heterozygous germline GATA2 mutations predispose carriers to familial myelodysplastic syndrome (MDS)/acute myeloid leukemia (AML), "MonoMAC" syndrome, and DCML deficiency. Here, we describe a previously unreported MDS family carrying a missense GATA2 mutation (p.Thr354Met), one patient with MDS/AML carrying a frameshift GATA2 mutation (p.Leu332Thrfs*53), another with MDS harboring a GATA2 splice site mutation, and 3 patients exhibiting MDS or MDS/AML who have large deletions encompassing the GATA2 locus. Intriguingly, 2 MDS/AML or "MonoMAC" syndrome patients with GATA2 deletions and one with a frameshift mutation also have primary lymphedema. Primary lymphedema occurs as a result of aberrations in the development and/or function of lymphatic vessels, spurring us to investigate whether GATA2 plays a role in the lymphatic vasculature. We demonstrate here that GATA2 protein is present at high levels in lymphatic vessel valves and that GATA2 controls the expression of genes important for programming lymphatic valve development. Our data expand the phenotypes associated with germline GATA2 mutations to include predisposition to primary lymphedema and suggest that complete haploinsufficiency or loss of function of GATA2, rather than missense mutations, is the key predisposing factor for lymphedema onset. Moreover, we reveal a crucial role for GATA2 in lymphatic vascular development.
Asunto(s)
Factor de Transcripción GATA2/genética , Mutación de Línea Germinal , Leucemia Mieloide Aguda/genética , Vasos Linfáticos/metabolismo , Linfedema/congénito , Síndromes Mielodisplásicos/genética , Adolescente , Adulto , Animales , Células Cultivadas , Niño , Femenino , Factor de Transcripción GATA2/metabolismo , Factor de Transcripción GATA2/fisiología , Mutación de Línea Germinal/fisiología , Humanos , Recién Nacido , Linfangiogénesis/genética , Linfedema/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Monocitos/patología , Síndrome , Adulto JovenRESUMEN
During lymphangiogenesis in the mammalian embryo, a subset of vascular endothelial cells in the cardinal veins is reprogrammed to adopt a lymphatic endothelial fate. The prevailing model of lymphangiogenesis contends that these lymphatic precursor cells migrate away from the cardinal veins and reassemble peripherally as lymph sacs from which a lymphatic vasculature is generated. However, this model fails to account for a number of observations that, as a result, have remained anecdotal. Here, we use optical projection tomography, confocal microscopy and in vivo live imaging to uncover three key stages of lymphatic vascular morphogenesis in the mouse embryo at high resolution. First, we define territories or "pre-lymphatic clusters" of Prox1-positive lymphatic endothelial progenitor cells along the antero-posterior axis of the cardinal veins. Second, these pre-lymphatic clusters undergo progressive extrusion ("ballooning") to generate primitive lymph sacs. Third, lymphatic vessels emerge by a combination of mechanisms including sprouting from the lymph sacs and direct delamination of streams of cells from the cardinal veins. Our data support a new model for lymphatic vascular patterning and morphogenesis, as a basis for identifying the molecular cues governing these processes.
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Linfangiogénesis , Vasos Linfáticos/embriología , Venas/embriología , Animales , Proteínas de Homeodominio/análisis , Ratones , Proteínas Supresoras de Tumor/análisisRESUMEN
Piezo channels are critical cellular sensors of mechanical forces. Despite their large size, ubiquitous expression, and irreplaceable roles in an ever-growing list of physiological processes, few Piezo channel-binding proteins have emerged. In this work, we found that MyoD (myoblast determination)-family inhibitor proteins (MDFIC and MDFI) are PIEZO1/2 interacting partners. These transcriptional regulators bind to PIEZO1/2 channels, regulating channel inactivation. Using single-particle cryogenic electron microscopy, we mapped the interaction site in MDFIC to a lipidated, C-terminal helix that inserts laterally into the PIEZO1 pore module. These Piezo-interacting proteins fit all the criteria for auxiliary subunits, contribute to explaining the vastly different gating kinetics of endogenous Piezo channels observed in many cell types, and elucidate mechanisms potentially involved in human lymphatic vascular disease.
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Canales Iónicos , Factores Reguladores Miogénicos , Humanos , Microscopía por Crioelectrón , Células HEK293 , Activación del Canal Iónico , Canales Iónicos/química , Canales Iónicos/genética , Canales Iónicos/metabolismo , Cinética , Enfermedades Linfáticas/genética , Mutación , Factores Reguladores Miogénicos/química , Factores Reguladores Miogénicos/genética , Factores Reguladores Miogénicos/metabolismo , Dominios Proteicos , Mioblastos/metabolismo , Animales , RatonesRESUMEN
Central conducting lymphatic anomaly (CCLA), characterized by the dysfunction of core collecting lymphatic vessels including the thoracic duct and cisterna chyli, and presenting as chylothorax, pleural effusions, chylous ascites, and lymphedema, is a severe disorder often resulting in fetal or perinatal demise. Although pathogenic variants in RAS/mitogen activated protein kinase (MAPK) signaling pathway components have been documented in some patients with CCLA, the genetic etiology of the disorder remains uncharacterized in most cases. Here, we identified biallelic pathogenic variants in MDFIC, encoding the MyoD family inhibitor domain containing protein, in seven individuals with CCLA from six independent families. Clinical manifestations of affected fetuses and children included nonimmune hydrops fetalis (NIHF), pleural and pericardial effusions, and lymphedema. Generation of a mouse model of human MDFIC truncation variants revealed that homozygous mutant mice died perinatally exhibiting chylothorax. The lymphatic vasculature of homozygous Mdfic mutant mice was profoundly mispatterned and exhibited major defects in lymphatic vessel valve development. Mechanistically, we determined that MDFIC controls collective cell migration, an important early event during the formation of lymphatic vessel valves, by regulating integrin ß1 activation and the interaction between lymphatic endothelial cells and their surrounding extracellular matrix. Our work identifies MDFIC variants underlying human lymphatic disease and reveals a crucial, previously unrecognized role for MDFIC in the lymphatic vasculature. Ultimately, understanding the genetic and mechanistic basis of CCLA will facilitate the development and implementation of new therapeutic approaches to effectively treat this complex disease.
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Quilotórax , Vasos Linfáticos , Linfedema , Factores Reguladores Miogénicos , Animales , Quilotórax/genética , Quilotórax/metabolismo , Células Endoteliales , Femenino , Humanos , Hidropesía Fetal/genética , Hidropesía Fetal/metabolismo , Vasos Linfáticos/patología , Linfedema/genética , Linfedema/metabolismo , Ratones , Factores Reguladores Miogénicos/genética , EmbarazoRESUMEN
The atypical cadherin FAT4 has established roles in the regulation of planar cell polarity and Hippo pathway signaling that are cell context dependent. The recent identification of FAT4 mutations in Hennekam syndrome, features of which include lymphedema, lymphangiectasia, and mental retardation, uncovered an important role for FAT4 in the lymphatic vasculature. Hennekam syndrome is also caused by mutations in collagen and calcium binding EGF domains 1 (CCBE1) and ADAM metallopeptidase with thrombospondin type 1 motif 3 (ADAMTS3), encoding a matrix protein and protease, respectively, that regulate activity of the key prolymphangiogenic VEGF-C/VEGFR3 signaling axis by facilitating the proteolytic cleavage and activation of VEGF-C. The fact that FAT4, CCBE1, and ADAMTS3 mutations underlie Hennekam syndrome suggested that all 3 genes might function in a common pathway. We identified FAT4 as a target gene of GATA-binding protein 2 (GATA2), a key transcriptional regulator of lymphatic vascular development and, in particular, lymphatic vessel valve development. Here, we demonstrate that FAT4 functions in a lymphatic endothelial cell-autonomous manner to control cell polarity in response to flow and is required for lymphatic vessel morphogenesis throughout development. Our data reveal a crucial role for FAT4 in lymphangiogenesis and shed light on the mechanistic basis by which FAT4 mutations underlie a human lymphedema syndrome.
Asunto(s)
Cadherinas/metabolismo , Polaridad Celular , Células Endoteliales/metabolismo , Linfangiogénesis , Vasos Linfáticos/metabolismo , Animales , Cadherinas/genética , Células Endoteliales/patología , Femenino , Factor de Transcripción GATA2/genética , Factor de Transcripción GATA2/metabolismo , Humanos , Vasos Linfáticos/patología , Linfedema/genética , Linfedema/metabolismo , Linfedema/patología , Ratones , Ratones Transgénicos , SíndromeRESUMEN
OBJECTIVE: To determine, using objective measures, the outcome of ex vivo cultured limbal epithelial stem cell (LESC) transplantation performed in compliance with good manufacturing practice using a novel culture system without 3T3 feeder cells. DESIGN: Prospective, noncomparative, interventional case series. PARTICIPANTS: Ten eyes of 10 patients with profound LESC deficiency arising from chemical injury (4 eyes), aniridia (3 eyes), ectodermal dysplasia (1 eye), Reiger's anomaly with Pax6 haploinsufficiency (1 eye), and unknown cause (1 eye). METHODS: Allogeneic (7 eyes) or autologous (3 eyes) corneal LESCs were cultured on human amniotic membrane. Tissue was transplanted to the recipient eye after superficial keratectomy. Impression cytology and confocal microscopy were performed 6 months after surgery with clinical follow-up to 13 months. Success was defined as an improvement in the defined clinical parameters of LESC deficiency, an improvement in visual acuity, the restoration of a more normal corneal phenotype on impression cytology, and the appearance of a regular hexagonal basal layer of cells on corneal confocal microscopy. MAIN OUTCOME MEASURES: Clinical parameters of LESC deficiency (loss of epithelial transparency, superficial corneal vascularization, epithelial irregularity, and epithelial breakdown), visual acuity, impression cytology and cytokeratin expression profiles, and in vivo confocal corneal confocal microscopy. RESULTS: The success rate using this technique was 60% (autografts 33%, allografts 71%). All patients with a successful outcome experienced an improvement in visual acuity of >/=2 lines Snellen acuity. Preoperatively, CK3+ and CK19+ cells accounted for 12+/-2.4% (mean +/- standard error of the mean) and 80+/-2.15% of cells, respectively, whereas postoperatively these accounted for 69+/-6.43% (P<0.0001) and 30+/-6.34% (P<0.0001) of cells, respectively. Goblet cells accounted for 8+/-1.19% of cells preoperatively and 1+/-0.35% of cells postoperatively (P<0.0001). CONCLUSIONS: These data demonstrate that it is possible to culture LESCs ex vivo in compliance with good manufacturing practice regulations. A set of objective outcome measures that confirm the efficiency of this technique in treating LESC deficiency is described. The widespread use of such standardized and objective outcome measures would facilitate a comparison between the different culture methods in use.
Asunto(s)
Enfermedades de la Córnea/cirugía , Epitelio Corneal/citología , Limbo de la Córnea/citología , Trasplante de Células Madre , Células Madre/citología , Adulto , Anciano , Recuento de Células , Trasplante de Células/métodos , Células Cultivadas , Enfermedades de la Córnea/patología , Femenino , Humanos , Inmunosupresores/administración & dosificación , Masculino , Microscopía Confocal , Persona de Mediana Edad , Estudios Prospectivos , Trasplante Autólogo , Trasplante Homólogo , Resultado del Tratamiento , Agudeza VisualRESUMEN
Ex vivo cultured limbal epithelial stem cells have been used successfully to treat corneal limbal stem cell deficiency. We identified 17 reports of the application of this novel cell-based therapy in humans. In addition we identified four reports of the use of culture oral mucosal epithelial cells to treat limbal stem cell deficiency. We examined these reports to discern the success rate, complication rate, visual outcome, whether there is an optimal technique and which patients are the most likely to benefit. We also discuss the different culture methods employed and the regulations governing cell banks that are providing this service. We found that the techniques used to cultivate and transplant cells varied, but that no individual method was clearly superior. The reported success rate is similar across all studies for both allografts and autografts. The clinical indications for this treatment are not clearly defined as indicated by the variety of disorders treated. Follow-up is limited and the long-term success rate is yet to be established. Nonetheless, we conclude that there is sufficient evidence to support the continued use and refinement of this procedure as a treatment for corneal stem cell deficiency.
Asunto(s)
Enfermedades de la Córnea/cirugía , Epitelio Corneal/citología , Trasplante de Células Madre/métodos , Células Cultivadas , Células Epiteliales/trasplante , HumanosRESUMEN
Numerous cytogenetic and molecular studies of breast cancer have identified frequent loss of heterozygosity (LOH) of the long arm of human chromosome 16. On the basis of these data, the likely locations of breast cancer tumor suppressor genes are bands 16q22.1 and 16q24.3. We have mapped the CBFA2T3 (MTG16) gene, previously cloned as a fusion partner of the AML1 protein from a rare (16;21) leukemia translocation, to the 16q24.3 breast cancer LOH region. The expression of CBFA2T3 was significantly reduced in a number of breast cancer cell lines and in primary breast tumors, including early ductal carcinomas in situ, when compared with nontransformed breast epithelial cell lines and normal breast tissue. Reintroduction of CBFA2T3 into different breast tumor derived cell lines with decreased expression of this gene reduced colony growth on plastic and in soft agar. CBFA2T3 was shown to function as a transcriptional repressor when tethered to the GAL4 DNA-binding domain in a reporter gene assay and, therefore, has the potential to be a transcriptional repressor in normal breast epithelial cells. Taken together, these findings suggest that CBFA2T3 is a likely candidate for the breast cancer tumor suppressor gene that is the target for the frequent 16q24 LOH in breast neoplasms.
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Neoplasias de la Mama/genética , Cromosomas Humanos Par 16/genética , Genes Supresores de Tumor , Pérdida de Heterocigocidad , Fosfoproteínas , Proteínas/genética , Proteínas Supresoras de Tumor , División Celular/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Biosíntesis de Proteínas , Proteínas/fisiología , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Neoplásico/biosíntesis , ARN Neoplásico/genética , Proteínas Represoras , Células Tumorales CultivadasRESUMEN
Heterozygous germline mutations in the zinc finger transcription factor GATA2 have recently been shown to underlie a range of clinical phenotypes, including Emberger syndrome, a disorder characterized by lymphedema and predisposition to myelodysplastic syndrome/acute myeloid leukemia (MDS/AML). Despite well-defined roles in hematopoiesis, the functions of GATA2 in the lymphatic vasculature and the mechanisms by which GATA2 mutations result in lymphedema have not been characterized. Here, we have provided a molecular explanation for lymphedema predisposition in a subset of patients with germline GATA2 mutations. Specifically, we demonstrated that Emberger-associated GATA2 missense mutations result in complete loss of GATA2 function, with respect to the capacity to regulate the transcription of genes that are important for lymphatic vessel valve development. We identified a putative enhancer element upstream of the key lymphatic transcriptional regulator PROX1 that is bound by GATA2, and the transcription factors FOXC2 and NFATC1. Emberger GATA2 missense mutants had a profoundly reduced capacity to bind this element. Conditional Gata2 deletion in mice revealed that GATA2 is required for both development and maintenance of lymphovenous and lymphatic vessel valves. Together, our data unveil essential roles for GATA2 in the lymphatic vasculature and explain why a select catalogue of human GATA2 mutations results in lymphedema.
Asunto(s)
Factor de Transcripción GATA2/metabolismo , Vasos Linfáticos/embriología , Linfedema/embriología , Mutación , Animales , Elementos de Facilitación Genéticos , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Factor de Transcripción GATA2/genética , Eliminación de Gen , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Células K562 , Vasos Linfáticos/patología , Linfedema/genética , Linfedema/patología , Ratones , Factores de Transcripción NFATC/genética , Factores de Transcripción NFATC/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Limbal stem cell deficiency (LSCD) is an eye disorder in which the stem cells responsible for forming the surface skin of the cornea are destroyed by disease. This results in pain, loss of vision, and a cosmetically unpleasant appearance. Many new treatments, including stem cell therapies, are emerging for the treatment of this condition, but assessment of these new technologies is severely hampered by the lack of biomarkers for this disease or validated tools for assessing its severity. The aims of this study were to design and test the reliability of a tool for grading LSCD, to define a set of core outcome measures for use in evaluating treatments for this condition, and to demonstrate their utility. This was achieved by using our defined outcome set (which included the Clinical Outcome Assessment in Surgical Trials of Limbal stem cell deficiency [COASTL] tool) to evaluate the 3-year outcomes for allogeneic ex vivo cultivated limbal epithelial transplantation (allo-CLET) in patients who had bilateral total LSCD secondary to aniridia or Stevens-Johnson syndrome. The results demonstrate that our new grading tool for LSCD, the COASTL tool, is reliable and repeatable, and that improvements in the biomarkers used in this tool correlate positively with improvements in visual acuity. The COASTL tool showed that following allo-CLET there was a decrease in LSCD severity and an increase in visual acuity up to 12 months post-treatment, but thereafter LSCD severity and visual acuity progressively deteriorated.
Asunto(s)
Aniridia/cirugía , Epitelio Corneal/patología , Limbo de la Córnea/patología , Complicaciones Posoperatorias/patología , Índice de Severidad de la Enfermedad , Síndrome de Stevens-Johnson/patología , Aloinjertos , Aniridia/patología , Biomarcadores/metabolismo , Células Cultivadas , Opacidad de la Córnea/patología , Opacidad de la Córnea/cirugía , Trasplante de Córnea/métodos , Bases de Datos Factuales , Epitelio Corneal/cirugía , Estudios de Seguimiento , Humanos , Limbo de la Córnea/cirugía , Reproducibilidad de los Resultados , Trasplante de Células Madre/métodos , Síndrome de Stevens-Johnson/cirugía , Resultado del TratamientoRESUMEN
Despite the importance of blood vessels and lymphatic vessels during development and disease, the signalling pathways underpinning vessel construction remain poorly characterised. Primary mouse endothelial cells have traditionally proven difficult to culture and as a consequence, few assays have been developed to dissect gene function and signal transduction pathways in these cells ex vivo. Having established methodology for the purification, short-term culture and transfection of primary blood (BEC) and lymphatic (LEC) vascular endothelial cells isolated from embryonic mouse skin, we sought to optimise robust assays able to measure embryonic LEC proliferation, migration and three-dimensional tube forming ability in vitro. In the course of developing these assays using the pro-lymphangiogenic growth factors FGF2 and VEGF-C, we identified previously unrecognised roles for FGFR1 signalling in lymphangiogenesis. The small molecule FGF receptor tyrosine kinase inhibitor SU5402, but not inhibitors of VEGFR-2 (SU5416) or VEGFR-3 (MAZ51), inhibited FGF2 mediated LEC proliferation, demonstrating that FGF2 promotes proliferation directly via FGF receptors and independently of VEGF receptors in primary embryonic LEC. Further investigation revealed that FGFR1 was by far the predominant FGF receptor expressed by primary embryonic LEC and correspondingly, siRNA-mediated FGFR1 knockdown abrogated FGF2 mediated LEC proliferation. While FGF2 potently promoted LEC proliferation and migration, three dimensional tube formation assays revealed that VEGF-C primarily promoted LEC sprouting and elongation, illustrating that FGF2 and VEGF-C play distinct, cooperative roles in lymphatic vascular morphogenesis. These assays therefore provide useful tools able to dissect gene function in cellular events important for lymphangiogenesis and implicate FGFR1 as a key player in developmental lymphangiogenesis in vivo.
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Células Endoteliales/metabolismo , Linfangiogénesis/fisiología , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/metabolismo , Transducción de Señal , Animales , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Separación Celular , Células Endoteliales/citología , Femenino , Factor 2 de Crecimiento de Fibroblastos/farmacología , Linfangiogénesis/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Cultivo Primario de Células , Factor C de Crecimiento Endotelial Vascular/farmacologíaRESUMEN
Human amniotic membrane (HAM) is employed as a substrate for the ex-vivo expansion of limbal epithelial cells (LECs) used to treat corneal epithelial stem cell deficiency in humans. The optimal method of HAM preparation for this purpose is unknown. This study evaluated the ability of different preparations of stored HAM to serve as substrates for LEC expansion ex-vivo. The effect of removing the amniotic epithelial cells (decellularisation) from HAM prior to seeding of LECs, the effect of glycerol cryopreservation and the effect of peracetic acid (PAA) sterilization and antibiotic disinfection were evaluated using different HAM test groups. Human LECs were cultured on each preparation and the following outcomes were assessed: confluence of growth, cell density, cell morphology and expression of the putative LESC markers deltaN-p63alpha and ABCG2. Removing amniotic epithelial cells prior to seeding of LECs resulted in a higher percentage of confluence but a lower cell density than intact HAM suggesting that decellularisation does not increase proliferation, but rather that it facilitates migration of LECs resulting in larger cells. Decellularisation did not affect the percentage of cells expressing the putative LESC markers deltaN-p63alpha (< or =4% in both intact and acellular groups) and ABCG2 (< or =3% in both intact and acellular groups). Glycerol cryopreservation of HAM resulted in poor morphology and a low proportion of cells expressing deltaN-p63alpha (< or =6%) and ABCG2 (< or =8%). HAM frozen at -80 degrees C in Hank's Balanced Salt Solution (HBSS) was superior, demonstrating excellent morphology of cultured LECs and high levels of deltaN-p63alpha (< or =68%) and ABCG2 (< or =62%) expression (p<0.001). The use of PAA or antibiotics to decontaminate HAM does not appear to affect this function. The variables affecting the ability of HAM to serve as a substrate for LEC expansion ex-vivo are poorly understood. The use of glycerol as a cryoprotectant impairs this ability whereas simple frozen HAM appears to work extremely well for this purpose.
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Amnios/metabolismo , Células Epiteliales/citología , Limbo de la Córnea/citología , Ensayo de Materiales/métodos , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Transportadoras de Casetes de Unión a ATP/metabolismo , Antibacterianos/farmacología , Recuento de Células , Proliferación Celular/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Criopreservación , Células Epiteliales/efectos de los fármacos , Glicerol , Humanos , Proteínas de Neoplasias/metabolismo , Factores de Tiempo , Transactivadores/metabolismo , Factores de Transcripción , Proteínas Supresoras de Tumor/metabolismoRESUMEN
The corneal epithelium is continuously renewed by a population of stem cells that reside in the corneoscleral junction, otherwise known as the limbus. These limbal epithelial stem cells (LESC) are imperative for corneal maintenance with deficiencies leading to in-growth of conjunctival cells, neovascularisation of the corneal stroma and eventual corneal opacity and visual loss. One such disease that has traditionally been thought to be due to LESC deficiency is aniridia, a pan-ocular congenital eye disease due to mutations in the PAX6 gene. Corneal changes or aniridia related keratopathy (ARK) seen in aniridia are typical of LESC deficiency. However, the pathophysiology behind ARK is still ill defined, with current theories suggesting it may be caused by a deficiency in the stem cell niche and adjacent corneal stroma, with altered wound healing responses also playing a role (Ramaesh et al, International Journal of Biochemistry & Cell Biology 37:547-557, 2005) or abnormal epidermal differentiation of LESC (Li et al., The Journal of Pathology 214:9, 2008). PAX6 is considered the master control gene for the eye and is required for normal eye development with expression continuing in the adult cornea, thus inferring a role for corneal repair and regeneration (Sivak et al., Developments in Biologicals 222:41-54, 2000). Studies of models of Pax6 deficiency, such as the small eyed (sey) mouse, should help to reveal the intrinsic and extrinsic mechanisms involved in normal LESC function.
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Epitelio Corneal/citología , Limbo de la Córnea/citología , Células Madre/citología , Animales , Epitelio Corneal/metabolismo , Proteínas del Ojo/genética , Proteínas de Homeodominio/genética , Humanos , Limbo de la Córnea/metabolismo , Ratones , Modelos Biológicos , Mutación , Factor de Transcripción PAX6 , Factores de Transcripción Paired Box/genética , Proteínas Represoras/genética , Células Madre/metabolismoRESUMEN
PURPOSE: To investigate the effect of human serum, an ocular therapy, on human corneal fibroblast (HCF) wound-healing activities. METHODS: The water soluble tetrazolium reagent-1, chemotactic chambers, fibroblast-populated type-I collagen gels, zymography, and Western blotting were used to assess HCF proliferation, migration, contraction, and matrix metalloproteinase (MMP) activity and levels, respectively. Fibroblasts were obtained from human donor corneas. Human serum, fibroblast culture medium (FCM; Dulbeccos Minimal Essential Medium/10% newborn calf serum) with and without calf serum supplementation, and 0.3% hypromellose were compared. RESULTS: Proliferation and migration were maximal in 1% human serum. Relaxed gel contraction was maximal for fibroblasts cultured in 10%, 50%, and 100% serum and FCM. Whereas in stressed gels, maximal contraction was induced when fibroblasts were cultured in 50% and 100% serum. In low serum concentrations, greater MMP-2 activity was detected than MMP-1 and MMP-9. CONCLUSIONS: Low concentrations of human serum stimulated HCF migration, proliferation, and MMP activity. High concentrations produced greater matrix contraction. This may have implications for the therapeutic use of autologous serum.
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Sustancia Propia/citología , Fibroblastos/fisiología , Suero/fisiología , Cicatrización de Heridas/fisiología , Western Blotting , Técnicas de Cultivo de Célula , Movimiento Celular/fisiología , Proliferación Celular , Dipéptidos/farmacología , Humanos , Queratina-12/metabolismo , Queratina-3/metabolismo , Inhibidores de la Metaloproteinasa de la Matriz , Metaloproteinasas de la Matriz/metabolismo , Microscopía Confocal , Soluciones Oftálmicas , Inhibidores de Proteasas/farmacología , Vimentina/metabolismoRESUMEN
The complex mechanisms by which transforming growth factor beta (TGFbeta) regulate re-epithelialisation following injury of stratified epithelia are not fully understood. TGFbeta signals via binding to distinct receptors activating downstream effectors, including Smads which initiate transcription of target genes. However, studies have shown that TGFbeta can also signal independently of Smads through MAPK pathways, demonstrating the diversity of TGFbeta signalling. Connective tissue growth factor (CTGF) is strongly induced by and acts downstream of TGFbeta causing pathophysiology in tissues by inducing matrix deposition, conversion of fibroblasts into contractile myofibroblasts (e.g. dermis and corneal stroma) and stimulation of epithelial-to-mesenchymal transition (e.g. kidney and lung) all of which are known to cause fibrosis. However, a role for CTGF in epithelial cell function which does not involve direct contribution to fibrosis has not been demonstrated. We show for the first time that synthesis of CTGF in cultures of human corneal epithelial cells is induced by TGFbeta through the Ras/MEK/ERK MAPK signalling pathway and that this is required for re-epithelialisation to occur through cell migration. These data reveal a novel function for CTGF in the regulation of epithelial tissue repair beyond its established role in fibrosis, and further highlight the complexity of TGFbeta regulation of epithelial cell function.
Asunto(s)
Córnea/metabolismo , Células Epiteliales/metabolismo , Proteínas Inmediatas-Precoces/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Sistema de Señalización de MAP Quinasas/fisiología , Factor de Crecimiento Transformador beta/metabolismo , Cicatrización de Heridas/fisiología , Línea Celular , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Células Cultivadas , Factor de Crecimiento del Tejido Conjuntivo , Córnea/citología , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Humanos , Proteínas Inmediatas-Precoces/efectos de los fármacos , MAP Quinasa Quinasa 1/efectos de los fármacos , MAP Quinasa Quinasa 1/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteína Quinasa 3 Activada por Mitógenos/efectos de los fármacos , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Regeneración/efectos de los fármacos , Regeneración/fisiología , Factor de Crecimiento Transformador beta/farmacología , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiología , Cicatrización de Heridas/efectos de los fármacos , Proteínas ras/efectos de los fármacos , Proteínas ras/metabolismoRESUMEN
It is anticipated that stem cell (SC) therapy will enable the regeneration of diseased tissues and organs. Understanding SC niches is an essential step toward realizing this goal. By virtue of its optical transparency and physical separation of SC and transient amplifying cell compartments, the human cornea provides a unique opportunity to visualize and observe a population of adult stem cells, limbal epithelial stem cells (LESCs), in their niche environment. To date, the characteristics of the LESC niche have remained unclear. State-of-the-art imaging techniques were used to construct a three-dimensional (3D) view of the entire human corneal limbus and identify the structural characteristics of the LESC niche. Two distinct candidate LESC niche structures were identified. Cells within these structures express high levels of the putative limbal stem cell markers p63alpha and ABCG2; however, current methods cannot identify for certain which exact cells within this cell population are truly LESCs. These structures could be located and observed in vivo in normal human subjects, but not in patients with clinically diagnosed corneal LESC deficiency. The distribution of these structures around the corneal circumference is not uniform. Biopsies targeted to limbal regions rich in LESC niche structures yielded significantly higher numbers of LESCs in culture. Our findings demonstrate how adult stem cell niches can be identified and observed in vivo in humans and provide new biological insight into the importance of LESC niche structures in maintaining normal LESC function. Finally, the concept of targeted biopsy of adult SC niches improves stem cell yield and may prove to be essential for the successful development of novel adult stem cell therapies. Disclosure of potential conflicts of interest is found at the end of this article.
Asunto(s)
Células Madre Adultas/citología , Biopsia/métodos , Diagnóstico por Imagen , Epitelio Corneal/citología , Limbo de la Córnea/citología , Adolescente , Adulto , Células Madre Adultas/ultraestructura , Anciano , Anciano de 80 o más Años , Tamaño de la Célula , Células Cultivadas , Niño , Preescolar , Epitelio Corneal/patología , Femenino , Humanos , Limbo de la Córnea/patología , Limbo de la Córnea/ultraestructura , Masculino , Persona de Mediana Edad , Células del Estroma/citologíaRESUMEN
The concept of stem cell therapy has engaged the attention of the public and scientists alike. Intensive research effort is focused upon understanding the biology and therapeutic potential of embryonic and adult stem cells, with the eventual goal of treating such pathologies as Parkinson's disease, diabetes, neurological injury and degenerations and cancer. Ex vivo expansion and transplantation of limbal epithelial stem cells to the corneas to treat blinding ocular surface disease was one of the first stem cell therapies to successfully reach the clinic. However, limbal epithelial stem cell research and therapy delivery has remained largely within the noncommercial academic clinician-scientist environment from which it was originally pioneered. In our experience, gaining regulatory approval has been as great a hurdle as surmounting the scientific challenges of stem cell therapy. Based upon our model of delivering 'accredited' limbal epithelial stem cell therapy to patients in compliance with Good Manufacturing Practice and the new European Union Tissues and Cells Directive, we address the key regulatory questions. This may help colleagues who are developing innovative academic research-driven stem cell therapies regarding donor consent, raw materials, quality assurance, laboratory specification, indemnity and funding.