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1.
Biology (Basel) ; 13(3)2024 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-38534415

RESUMO

The corneal epithelium (CE) is spread between two domains, the outer vascularized limbus and the avascular cornea proper. Epithelial cells undergo constant migration from the limbus to the vision-critical central cornea. Coordinated with this migration, the cells undergo differentiation changes where a pool of unique stem/precursor cells at the limbus yields the mature cells that reach the corneal center. Differentiation is heralded by the expression of the corneal-specific Krt12. Processing data acquired by scRNA-Seq showed that the increase in Krt12 expression occurs in four distinct steps within the limbus, plus a single continuous increase in the cornea. Differential gene analysis demonstrated that these domains reflect discreet stages of CE differentiation and yielded extensive information of the genes undergoing down- or upregulation in the sequential transition from less to more differentiate conditions. The approach allowed the identification of multiple gene cohorts, including (a) the genes which have maximal expression in the most primitive, Krt12-negative cell cohort, which is likely to include the stem/precursor cells; (b) the sets of genes that undergo continuous increase or decrease along the whole differentiation path; and (c) the genes showing maximal positive or negative correlation with the changes in Krt12.

2.
Cell Signal ; 113: 110963, 2024 01.
Artigo em Inglês | MEDLINE | ID: mdl-37931692

RESUMO

Following wounding, endogenously secreted TGFßs drive resident and bone marrow-derived cells to convert into α-smooth actin (SMA)-rich, contractile myofibroblasts. The TGFß effect is initiated by the phosphorylation of SMADs 2 and 3 (SMAD2/3). This event has been referred to as the canonical response to TGFß. TGFß also elicits other responses viewed as parallel events not directly connected to the SMAD activation, and thus referred to as noncanonical. A recognized response is the phosphorylation of the -activated kinase (TAK1/MAP3K), an upstream component of the mitogen-activated protein kinase (MAPK) cascade. We have now examined the relationship between these two effects of TGFß1 at their earliest stages. The bulk of the studies were carried out with primary fibroblasts derived from the human cornea. The results' widespread relevance was confirmed in critical experiments with dermal-, and Tenon's capsule-derived fibroblasts. Cells were treated with kinase inhibitors or targeting siRNAs followed by induction by 2 ng/ml TGFß1, and/or 10 ng/ml TNF-α. Cells were collected after 1 to 30 min for Western blot analysis and assayed for the accumulation of phosphorylated TAK1, ASK1, JNK1/2, p38, HPS27, MELK, SMAD2/3, and GAPDH. The effect of the kinase inhibitors on α-SMA expression and α-SMA stress fiber organization was also tested. For the immediate response to TGFß1 we found that a) activation of the MAPK pathway was completed within 1 min after the addition of TGFß1; b) phosphorylation of JNK1/2 was fully dependent on TAK1 and ASK1 activity, c) phosphorylation of MELK was fully dependent on JNK1/2 activity; d) phosphorylation of ASK1 depends on MELK activity, indicating the existence of an ASK1-MELK positive activation feedback loop; e) phosphorylation of SMAD2/3 started only after a 5 min period and reached a nadir after 10-15 min, f) the latter phosphorylation was fully blocked by inhibition of TAK1, ASK1, JNK1/2, and MELK, and siRNA-driven MELK downregulation; g) the inhibitors equally blocked the α-SMA protein expression, stress fiber development, and cell morphology changes at 72 h. These results demonstrate that the activation of the canonical pathway is fully subordinate to the activity of the MAPK pathway, challenging the concept of canonical and noncanonical TGFß pathways and that SMAD2/3 activation is mediated by MELK, a kinase not previously associated with rapid pharmacological responses.


Assuntos
Zíper de Leucina , Miofibroblastos , Humanos , Fosforilação , Miofibroblastos/metabolismo , Fator de Crescimento Transformador beta1/farmacologia , Fator de Crescimento Transformador beta1/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Actinas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteína Smad2/metabolismo
3.
Invest Ophthalmol Vis Sci ; 63(2): 3, 2022 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-35103750

RESUMO

Purpose: The transcription factor c-Myc (Myc) plays central regulatory roles in both self-renewal and differentiation of progenitors of multiple cell lineages. Here, we address its function in corneal epithelium (CE) maintenance and repair. Methods: Myc ablation in the limbal-corneal epithelium was achieved by crossing a floxed Myc mouse allele (Mycfl/fl) with a mouse line expressing the Cre recombinase gene under the keratin (Krt) 14 promoter. CE stratification and protein localization were assessed by histology of paraffin and plastic sections and by immunohistochemistry of frozen sections, respectively. Protein levels and gene expression were determined by western blot and real-time quantitative PCR, respectively. CE wound closure was tracked by fluorescein staining. Results: At birth, mutant mice appeared indistinguishable from control littermates; however, their rates of postnatal weight gain were 67% lower than those of controls. After weaning, mutants also exhibited spontaneous skin ulcerations, predominantly in the tail and lower lip, and died 45 to 60 days after birth. The mutant CE displayed an increase in stratal thickness, increased levels of Krt12 in superficial cells, and decreased exfoliation rates. Accordingly, the absence of Myc perturbed protein and mRNA levels of genes modulating differentiation and proliferation processes, including ΔNp63ß, Ets1, and two Notch target genes, Hey1 and Maml1. Furthermore, Myc promoted CE wound closure and wound-induced hyperproliferation. Conclusions: Myc regulates the balance among CE stratification, differentiation, and surface exfoliation and promotes the transition to the hyperproliferative state during wound healing. Its effect on this balance may be exerted through the control of multiple regulators of cell fate, including isoforms of tumor protein p63.


Assuntos
Lesões da Córnea/genética , Epitélio Corneano/patologia , Regulação da Expressão Gênica , Genes myc/genética , Homeostase/fisiologia , Transativadores/genética , Animais , Proliferação de Células , Células Cultivadas , Lesões da Córnea/metabolismo , Lesões da Córnea/patologia , Modelos Animais de Doenças , Epitélio Corneano/metabolismo , Genes Supressores de Tumor , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Confocal , RNA/genética , Transativadores/biossíntese
4.
BMC Ophthalmol ; 20(1): 325, 2020 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-32762738

RESUMO

BACKGROUND: The sparsity of established tools for the grading of limbal stem cell deficiency hinder objective assessments of the clinical outcome of cultivated limbal epithelial cell transplantation. To advance towards the development of standards for the comparison of the outcomes of these bio-surgical protocols we have now applied a battery of recognized objective and patient-declared subjective outcome criteria to the autologous modality of cultivated limbal epithelial cell transplantation. METHODS: The prospective study involved ten patients (M/F = 9/1; mean age = 42.1 years) displaying overt unilateral limbal stem cell deficiency complying with the inclusion criteria described in Methods. Limbal biopsies were obtained from the contralateral eye and their outgrowths after 2-week cultures were transplanted on the affected eye after pannus resection. Outcomes were followed up for 12 months. The objective tests were scores for best-corrected visual acuity (BCVA); using the LogMAR scale, a multiparametric ocular surface score (OSS), and the Schirmer's test. Subjective scores were based on patient answers to a) perception of visual improvement/pain; b) the 25-item National Eye Institute Visual Function Questionnaire (NEI-VFQ 25); and c) the 12-item Ocular Surface Disease Index Questionnaire (OSDI). All procedures were performed under good manufacture practices using solely xeno-free reagents. In all cases, a single biopsy was divided into two pieces and they were expanded in order to prevent outgrowth failure. In 5 patients, both biopsies generated healthy culture sheet. In those cases the lesser outgrowth were used for immune-histological characterization. RESULTS: The experimental parallel outgrowth samples showed a similar percent of p63α+ cells. PreOp and 12-month PostOp BCVAs and OSSs were, respectively, 1.15 ± 0.70; 0.21 ± 0.13 and 7.40 ± 2.01; 2,30 ± 1.30, (p < 0.05). Patient's responses to all three question sets except ocular pain were consistent with significant improvement (p < 0.05). CONCLUSION: Objective clinical metrics demonstrate that in patients with limbal stem cell deficiency, cultivated limbal epithelial cell transplantation improves vision and ocular surface health and subjective visual perceptions.


Assuntos
Queimaduras Químicas , Doenças da Córnea , Epitélio Corneano , Queimaduras Oculares , Limbo da Córnea , Adulto , Queimaduras Químicas/cirurgia , Transplante de Células , Queimaduras Oculares/induzido quimicamente , Humanos , Estudos Prospectivos , Células-Tronco , Transplante Autólogo , Resultado do Tratamento , Acuidade Visual
5.
Adv Protein Chem Struct Biol ; 118: 273-288, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31928728

RESUMO

Exfoliation syndrome (XFS) is an age-related disease defined by the deposition of aggregated fibrous material (XFM) in the peri-cellular space. Principal morbidity occurs in the eye, where XFM accumulates on the anterior ocular tissues. GWAS have found that certain genetic variants of lysyl oxidase-like 1 (LOXL1), a matrix cross-linking enzyme that is required for elastic fiber formation confer risk for the development of XFS, but are not a single causative factor as many genetically affected individuals do not develop XFS or subsequent glaucoma (XFG). We have found that XFG cells display defects in lysosomes, microtubules, autophagy, and mitochondria resembling defects found in cells from age-related syndromes, such as the main neurodegenerative diseases. In the majority of these diseases, the determining cellular factor is a protein containing intrinsically disordered regions (IDRs) and displaying a high propensity for aggregation. We have found that in XFG patient-derived cells, LOXL1 protein is actively subjected to autophagic clearance, suggesting that LOXL1 is undergoing aggregation. In silico analysis demonstrates that LOXL1's first 369 aa constitute an IDR with the highest disorder probability peak centering around the known risk positions. Experimentally, we have found over-expression of either unmodified LOXL1 or fluorescent chimeras preserving the well-structured N-terminus cause copious intracellular aggregation and that aggregation wanes when the high IDR peaks are deleted. Overall, our work suggests that XFS/G results from the aggregation of the LOXL1 protein coupled with a reduction of cellular proteostasis capabilities in aging, resulting in a chronic build-up of LOXL1-containing protein aggregates.


Assuntos
Aminoácido Oxirredutases/metabolismo , Síndrome de Exfoliação/metabolismo , Dobramento de Proteína , Autofagia , Síndrome de Exfoliação/genética , Humanos , Proteínas Intrinsicamente Desordenadas/metabolismo , Mutação , Ligação Proteica
6.
Sci Rep ; 8(1): 4055, 2018 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-29497083

RESUMO

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

7.
J Glaucoma ; 27(3): 197-201, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-27977481

RESUMO

In this short report we review previous work toward the identification of the protein and cellular sources of exfoliation glaucoma and described our recent finding on dysfunction of autophagy in Tenon capsule fibroblasts obtained from exfoliation syndrome glaucoma patients at the time of surgery and discuss the potential implications of these findings for understanding the cellular sources of the disease.


Assuntos
Autofagia/fisiologia , Síndrome de Exfoliação/etiologia , Glaucoma/etiologia , Síndrome de Exfoliação/metabolismo , Síndrome de Exfoliação/patologia , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibroblastos/fisiologia , Glaucoma/metabolismo , Glaucoma/patologia , Humanos , Pressão Intraocular/fisiologia , Masculino , Cápsula de Tenon/metabolismo , Cápsula de Tenon/patologia
8.
Sci Rep ; 7(1): 15241, 2017 11 10.
Artigo em Inglês | MEDLINE | ID: mdl-29127331

RESUMO

Wnt signaling plays an important role in the regulation of self-renewal in stem cells. Here we investigated the effect of CHIR99021, the primary transducer of the Wnt signaling canonical pathway, and IWP2, a wide action Wnt signal blocker, on the growth and differentiation of the limbal epithelial progenitor cells when these cells are cultured in two different, common culture approaches, outgrowth from limbal biopsy explants and isolated cell seeded in low calcium medium. Consistent with their expected effects, irrespective of the culture system, IWP2 decreased total ß-catenin while CHIR99021 increased it in nuclear localization. However, IWP2 increased stem/progenitor cell marker (p63α and ABCG2) content and clonogenic capacity in the explants but had opposite effects on isolated cells. CHIR99021 reduced the growth rate, stem/progenitor cell marker content and clonogenic capacity in the explants but also had the opposite effect on the isolated cells. These results show that the outcome of Wnt/ß-catenin signaling modification is dependent on the culture systems. Transplantation of limbal epithelial sheets from explant cultures is one of the standard treatments of limbal stem cell deficiency. Our study shows that Wnt-associated activity has a strong negative impact on stem/progenitor cell preservation in limbal explant cultures.


Assuntos
Epitélio Corneano/metabolismo , Limbo da Córnea/metabolismo , Piridinas/farmacologia , Pirimidinas/farmacologia , Células-Tronco/metabolismo , Via de Sinalização Wnt/efeitos dos fármacos , beta Catenina/metabolismo , Antígenos de Diferenciação/metabolismo , Técnicas de Cultura de Células , Epitélio Corneano/citologia , Feminino , Humanos , Limbo da Córnea/citologia , Masculino , Células-Tronco/citologia
9.
PLoS One ; 12(4): e0174905, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28380010

RESUMO

The DNA intercalating dye Hoechst 33342 or its close analog DCV are actively removed from cells by the multidrug resistance transporter ABCG2, a protein overexpressed in metastatic cells and somatic stem cells. In bivariate blue-red flow cytometry fluorescent plots active Hoechst or DCV efflux combined with a concentration dependent bathochromic shifts of these nuclear dyes leads to the segregation of the transporter-rich cells into a distinct cell cohort tilted towards the shorter wavelength axis of the plot, the cohort is generically known as the side population (SP). This feature has facilitated the surface marker-independent isolation of live stem cells. A drawback, though, is the known toxicity of Hoechst dyes. In this study we show that JC1, a bathochromic mitochondrial membrane potential-sensitive dye applied at proper concentration, can yield flow cytometry fluorescent emission bivariate plots containing a low JC1 accumulation (JC1low) cohort. Using a combination of multiple cell lines, ABC-transporter inhibitors and viral vector-driven insertion of the ABCG2 gene or ABCG2 and ABCB1 shRNAs we demonstrate that JC1low can be generated by either of the two aforementioned multidrug resistance transporters. Complete wash out of mitochondrial bound JC1 required more than 24 h. In spite of this tight binding, the dye did not affect either the mitochondrial membrane potentials or the proliferation rate. In contrast, contemporaneous with its nuclear accumulation, Hoechst 33342 or DVC, caused changes in the fluorescent emission of mitochondrial membrane potential sensitive dyes resembling the effects caused by the mitochondrial uncoupler FCCP. In a number of cell lines exposure to Hoechst resulted in marked slow-down of proliferation and abolition of ABCG2 transport activity during the subsequent 2 days but in K562 cells the exposure induced cell extended death. Overall, its lack of toxicity vis. a vis. the toxicity and genotoxicity of the DNA intercalating dyes makes JC1 an ideal tool for isolating live cells expressing high multidrug resistance transport activity.


Assuntos
Benzimidazóis/uso terapêutico , Carbocianinas/uso terapêutico , Citometria de Fluxo/métodos , Corantes Fluorescentes/uso terapêutico , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Células CACO-2 , Linhagem Celular , Epitélio Corneano/citologia , Células HEK293 , Humanos , Células K562 , Potencial da Membrana Mitocondrial/efeitos dos fármacos , RNA Interferente Pequeno/metabolismo
10.
PLoS One ; 11(7): e0157404, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27391778

RESUMO

PURPOSE: To test the hypothesis that autophagy dysfunction is involved in exfoliation syndrome (XFS), a systemic disorder of extracellular elastic matrices that causes a distinct form of human glaucoma. METHODS: Fibroblasts derived from tenon tissue discards (TFs) from filtration surgery to relieve intraocular pressure in XFS patients were compared against age-matched TFs derived from surgery in primary open-angle glaucoma (POAG) patients or from strabismus surgery. Differential interference contrast light, and electron microscopy were used to examine structural cell features. Immunocytochemistry was used to visualize LOXL1 and Fibulin-5, lysosomes, endosomes, Golgi, and microtubules. Light scatter, Cyto-IDTM and JC1 flow cytometry were used to measure relative cell size, autophagic flux rate and mitochondrial membrane potential (MMPT), respectively. Enhanced autophagy was induced by serum withdrawal. RESULTS: In culture, XFS-TFs were 1.38-fold larger (by light scatter ratio, p = 0.05), proliferated 42% slower (p = 0.026), and were morphologically distinct in 2D and 3D culture compared to their POAG counterparts. In extended 3D cultures, XFS-TFs accumulated 8-10 times more Fibulin-5 than the POAG-TFs, and upon serum withdrawal, there were marked deficiencies in relocation of endosomes and lysosomes to the perinuclear area. Correspondingly, the XFS-TFs displayed significant accumulation of the autophagasome marker LC3 II (3.9 fold increase compared to POAG levels, p = 0.0001) and autophagic flux rate as measured by Cyto-ID dye was 53% lower in XFS-TFs than in POAG-TFs (p = 0.01), indicating reduced clearance of autophagasomes. Finally the percent of cells with diminished MMPT was 3-8 times larger in the XFS-TFs than in POAG-TFs (p = 0.02). CONCLUSIONS: Our results provide for the first time a link between XFS pathology to autophagy dysfunction, a major contributor to multiple age related diseases systemically throughout the body, in the brain and in the retina. A diminished capacity for degradation of denatured protein and aging cellular organelles may underpin the development of extracellular protein aggregates in XFS.


Assuntos
Autofagia , Síndrome de Exfoliação/cirurgia , Fibroblastos/metabolismo , Glaucoma de Ângulo Aberto/cirurgia , Idoso , Idoso de 80 Anos ou mais , Aminoácido Oxirredutases/genética , Pré-Escolar , Síndrome de Exfoliação/metabolismo , Feminino , Glaucoma de Ângulo Aberto/metabolismo , Humanos , Pressão Intraocular , Luz , Masculino , Potenciais da Membrana , Pessoa de Meia-Idade , Membranas Mitocondriais/metabolismo , Espalhamento de Radiação , Estrabismo/cirurgia
11.
Mol Vis ; 22: 138-49, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26937166

RESUMO

PURPOSE: To determine the corneal regenerative capacity of sequentially generated primary, secondary, and tertiary limbal explant outgrowths in a limbal stem cell deficiency (LSCD) surgical model. METHODS: Two-millimeter-long limbal shallow biopsies were surgically excised from the upper quadrant of the right eye of rabbits and set on preserved amniotic membrane for explant culture. After the generation of primary outgrowth, the biopsies were sequentially transferred to new amniotic membrane to generate secondary and then tertiary outgrowths. Eighteen rabbits were subjected to a 360° limbal peritomy extending into the scleral zone and combined with superficial keratectomy of the corneal periphery and thorough mechanical debridement of the central cornea in their left eye. Right eye outgrowths, six of each generation, were engrafted on the ocular surface. Clinical outcomes (neovascularization, corneal clarity, and corneal fluorescein staining) were graded after 6 months. Post-mortem corneas were compared with histology, immunochemistry for p63 and Krt3, ABCG2-dependent dye exclusion, and capacity for outgrowths in explant culture. RESULTS: Immunohistology and western blot of the outgrowths for p63 and Krt3 indicated no differences in expression between the primary and tertiary outgrowths for these two markers of growth and differentiation. Clinically, all rabbits treated with amniotic membrane alone developed severe LSCD. Most rabbits grafted with cell outgrowths from all three outgrowth generations achieved stable (>6 months) recovery of the ocular surface. There were partial failures of grafts performed with two secondary and tertiary outgrowths. However, Kruskal-Wallis statistical analysis of the clinical scores yielded no significant difference between the three groups (p=0.524). Histology showed full anatomic recovery of grafts made with primary and tertiary outgrowths. Krt3 and p63 expression throughout the whole limbal corneal epithelium with primary or tertiary outgrowths was not distinguishable from each other. The percentage of dye-excluding cells present within this zone and the capacity of the explant epithelial outgrowth of the regenerated peripheral corneal zone were also on par with those of the donor corneas. The Krt3-negative cells that characterize the basal epithelial layer of the normal limbus could not be found in any regenerated cornea from the primary to tertiary outgrowths. CONCLUSIONS: Our results demonstrate that in rabbits post-primary explant outgrowths retain the capacity for LSCD recovery found in primary explants.


Assuntos
Córnea/fisiologia , Doenças da Córnea/terapia , Modelos Animais de Doenças , Epitélio Corneano/citologia , Limbo da Córnea/patologia , Transplante de Células-Tronco , Células-Tronco/patologia , Âmnio , Animais , Biópsia , Western Blotting , Técnicas de Cultura de Células , Doenças da Córnea/fisiopatologia , Epitélio Corneano/metabolismo , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Queratina-3/metabolismo , Coelhos , Recuperação de Função Fisiológica/fisiologia , Regeneração/fisiologia , Alicerces Teciduais , Fatores de Transcrição/metabolismo , Transplante Autólogo
12.
Exp Eye Res ; 145: 48-57, 2016 04.
Artigo em Inglês | MEDLINE | ID: mdl-26554938

RESUMO

Outgrowths of limbal epithelium by explant culture are used to treat limbal stem cell deficiency (LSCD). The explant culture medium is always complemented with serum, a complex solution which includes TGFß. Since TGFß is a cytostatic effector for epithelial proliferation we examined its effect on these cultures. Limbal biopsies were set on explant culture in DMEM/F12 with 5 ng/ml EGF and cholera toxin (ChT), ITS, and 5% FBS, henceforth SHEM or a) SHEMSB=SHEM plus SB431542 an inhibitor of TGFß signaling; b) sfSHEM = SHEM with FBS replaced by 0.05% Albumax II; and c) sfSHEMSB and sfSHEMA83 = sfSHEM plus, respectively, SB431542 or A-83-01, another TGFß inhibitor. After the initial outgrowths reached 3 cm in diameter, the limbal biopsies were serially transferred up to six times onto new inserts. Biopsy explant outgrowths were trypsinized and cell yield, morphology and stem-cell related JC-1 exclusion (IOVS, 52:4330) were determined by flow cytometry. Cells we plated at low density seeding to compare relative clonal proliferative activity. The expression of three proteins whose levels are associated with growth and differentiation states, Krt3, connexin 43 and p63 were determined by immunohistology and/or Western blot. Cell yield in rabbit, relative to SHEM (in %) were, SHEMSB, 104 ± 13 (p > 0.95); sfSHEM: 5 ± 3; and sfSHEMSB, 94 ± 18 (p > 0.95). Cell size and morphology, JC1 dye exclusion, Krt3, p63 and connexin 43 content, proliferation efficiency and the preservation of extended proliferative potential of the serially cultured biopsies were similar for SHEM, SHEMSB and sfSHEMSB. The only differences observed where reduced expression of Krt3 and increased preservation of p63 in the FBS-free medium. Removal of EGF from sfSHEMSB reduced yield by 92 ± 6% (p < 0.05). Removal of Albumax and ChT to establish a xeno-free medium caused a small, non-statistical decrease in growth rates. Equivalent results were observed in a preliminary experiment in human. These results suggest that in the absence serum endogenously generated TGFß act as an autocrine cytostatic agent and that TGFß inhibitors allow explant culture in xeno-free, chemically defined medium. Furthermore, the pro-growth effect of serum in limbal explant cultures may result exclusively from neutralization of the TGFß cytostatic effect.


Assuntos
Meios de Cultura Livres de Soro/farmacologia , Limbo da Córnea/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Western Blotting , Diferenciação Celular , Células Cultivadas , Imuno-Histoquímica , Limbo da Córnea/citologia , Modelos Animais , RNA/genética , Coelhos , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais , Fator de Crescimento Transformador beta/genética
13.
Mol Vis ; 19: 2542-50, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24357922

RESUMO

PURPOSE: An alternative autologous tissue for ocular surface reconstruction is a potential treatment for the patients with bilateral limbal stem cell deficiency. For the purpose of regenerative procedures in patients, it is desirable to eliminate the involvement of xenogeneic components, such as nonhuman sera and feeder cells. In the present study, we examined the behavior and phenotypic features of cultured conjunctival epithelial sheets generated in serum- and 3T3-free culture conditions when transplanted into the de-epithelialized limbal corneal surface. METHODS: Epithelial cells from normal conjunctiva obtained by neutral protease digestion were expanded by culture in a serum-free low-calcium medium and set in an air-liquid interface culture for 14 days. The resulting multilayered epithelial sheets were grafted onto rabbit ocular surfaces made epithelial-free by alkali treatment. Pre-grafted and post-grafted epithelia were analyzed by electron microscopy and immunohistochemistry. RESULTS: At graft time the cultured epithelial sheet consisted of 6-8 layers of properly stratified epithelium that displayed a CK19(+)/MUC5AC(+)/ CK3 (-)/CK12(-) phenotype, consistent with the conjunctival epithelial lineage. Two weeks after xeno-grafting the in vivo epithelium consisted of 5-6 well compacted layers expressing the precursor cell-related protein p63, the proliferation marker Ki67, desmosomes, hemidesmosomes and its integrin (ß4), and the corneal specific cytokeratins CK3, and CK12. Conjunctival goblet cell mucin (MUC5AC) was not visible. The engrafted epithelium stained positively for the anti-human nuclei antibody, confirming that the epithelial cells on the rabbit corneas were of human origin. CONCLUSIONS: Our results suggest that conjunctival epithelial sheets generated in serum- and 3T3-free culture conditions can acquire the corneal epithelial phenotype when transferred to the in vivo corneal stromal environment.


Assuntos
Transdiferenciação Celular/genética , Túnica Conjuntiva/citologia , Células Epiteliais/citologia , Epitélio Corneano/imunologia , Sobrevivência de Enxerto/imunologia , Regeneração/fisiologia , Adulto , Idoso , Animais , Biomarcadores/metabolismo , Transdiferenciação Celular/imunologia , Túnica Conjuntiva/imunologia , Túnica Conjuntiva/transplante , Meios de Cultura Livres de Soro , Células Epiteliais/imunologia , Células Epiteliais/transplante , Epitélio Corneano/lesões , Epitélio Corneano/patologia , Feminino , Expressão Gênica , Humanos , Queratinas/genética , Queratinas/imunologia , Antígeno Ki-67/genética , Antígeno Ki-67/imunologia , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Pessoa de Meia-Idade , Mucina-5AC/genética , Mucina-5AC/imunologia , Coelhos , Técnicas de Cultura de Tecidos , Transplante Heterólogo
14.
PLoS One ; 8(10): e77300, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24098582

RESUMO

Injuring mouse corneas with alkali causes myofibroblast expression leading to tissue opacification. However, in transient receptor potential vanilloid 1 channel (TRPV1-/-) knockout mice healing results in transparency restoration. Since TGFß is the primary inducer of the myofibroblast phenotype, we examined the mechanism by which TRPV1 affects TGFß-induced myofibroblast development. Experiments were performed in pig corneas and human corneal fibroblasts (HCFs). Immunohistochemical staining of α-smooth muscle actin (α-SMA) stress fibers was used to visualize myofibroblasts. Protein and phosphoprotein were determined by Western blotting. siRNA transfection silenced TRPV1 gene expression. Flow cytometry with a reactive oxygen species (ROS) reporting dye analyzed intracellular ROS. [Ca2+]I was measured by loading HCF with fura2. In organ cultured corneas, the TRPV1 antagonist capsazepine drastically reduced by 75% wound-induced myofibroblast development. In HCF cell culture, TGF-ß1 elicited rapid increases in Ca2+ influx, phosphorylation of SMAD2 and MAPKs (ERK1/2, JNK1/2 and p38), ROS generation and, after 72 hrs myofibroblast development. SMAD2 and p38 activation continued for more than 16 h, whereas p-ERK1/2 and p-JNK1/2 waned within 90 min. The long-lived SMAD2 activation was dependent on activated p38 and vice versa, and it was essential to generate a > 13-fold increase in α-SMA protein and a fully developed myofibroblast phenotype. These later changes were markedly reduced by inhibition of TRPV1 or reduction of the ROS generation rate. Taken together our results indicate that in corneal derived fibroblasts, TGFß- induced myofibroblast development is highly dependent on a positive feedback loop where p-SMAD2-induced ROS activates TRPV1, TRPV1 causes activation of p38, the latter in turn further enhances the activation of SMAD2 to establish a recurrent loop that greatly extends the residency of the activated state of SMAD2 that drives myofibroblast development.


Assuntos
Córnea/metabolismo , Opacidade da Córnea/genética , Miofibroblastos/metabolismo , Proteína Smad2/genética , Canais de Cátion TRPV/genética , Fator de Crescimento Transformador beta/genética , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Actinas/genética , Actinas/metabolismo , Álcalis , Animais , Cálcio/metabolismo , Lesões da Córnea , Opacidade da Córnea/induzido quimicamente , Opacidade da Córnea/metabolismo , Retroalimentação Fisiológica , Regulação da Expressão Gênica , Humanos , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteína Quinase 8 Ativada por Mitógeno/genética , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Proteína Quinase 9 Ativada por Mitógeno/genética , Proteína Quinase 9 Ativada por Mitógeno/metabolismo , Miofibroblastos/patologia , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Proteína Smad2/metabolismo , Suínos , Canais de Cátion TRPV/antagonistas & inibidores , Canais de Cátion TRPV/metabolismo , Técnicas de Cultura de Tecidos , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
15.
Exp Eye Res ; 107: 121-9, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23232207

RESUMO

Corneal wound healing in mice subsequent to an alkali burn results in dysregulated inflammation and opacification. Transient receptor potential vanilloid subtype 1 (TRPV1) channel activation in all tissue layers by endogenous ligands contributes to this sight compromising outcome since in TRPV1 knockout mice wound healing results instead in tissue transparency restoration. However, it is not known if primary human stromal fibroblasts exhibit such expression even though functional TRPV1 expression is evident in an immortalized human corneal epithelial cell line. In primary human corneal fibroblasts (HCF), TRPV1 gene expression and localization were identified based on the results of quantitative RT-PCR and immunocytochemistry, respectively. Western blot analysis identified a 100 kD protein corresponding to TRPV1 protein expression in a positive control. Single-cell fluorescence imaging detected in fura2-AM loaded cells Ca(2+) transients that rose 1.8-fold above the baseline induced by a selective TRPV1 agonist, capsaicin (CAP), which were blocked by a TRPV1 antagonist, capsazepine (CPZ) or exposure to a Ca(2+) free medium. The whole-cell mode of the planar patch-clamp technique identified TRPV1-induced currents that rose 1.76-fold between -60 and +130 mV. CAP-induced time dependent changes in the phosphorylation status of mitogen activated protein kinase (MAPK) signaling mediators that led to a 2.5-fold increase in IL-6 release after 24 h. This rise did not occur either in TRPV1 siRNA gene silenced cells or during exposure to SB203580 (10 µM), a selective p38 MAPK inhibitor. Taken together, identification of functional TRPV1 expression in HCF suggests that in vivo its activation by injury contributes to corneal opacification and inflammation during wound healing. These undesirable effects may result in part from increases in IL-6 expression mediated by p-p38 MAPK signaling.


Assuntos
Ceratócitos da Córnea/metabolismo , Regulação da Expressão Gênica/fisiologia , Canais de Cátion TRPV/genética , Western Blotting , Capsaicina/farmacologia , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Humanos , Imuno-Histoquímica , Interleucina-6/metabolismo , Técnicas de Patch-Clamp , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Canais de Cátion TRPV/metabolismo , Transfecção , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
16.
Invest Ophthalmol Vis Sci ; 52(7): 4330-7, 2011 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-21421882

RESUMO

PURPOSE: To determine changes in ABCG2-transport-dependent dye exclusion in outgrowths from limbal explants. METHODS: Human or rabbit limbal strips were deposited onto inserts. Over a month, the segments were twice transferred to new inserts. Fresh tissue (FT) cells, obtained by sequential dispase-trypsin digestion and the cells growing from the explant cultures, were characterized for ABCG2-dependent efflux by flow cytometry using a newly identified substratum, JC1. Rabbit cells were sorted into JC1-excluding (JC1(low)) and main (JC1(main)) cohorts and seeded with feeder 3T3 cells to determine colony formation efficiency (CFE). RESULTS: The JC1(low) cells were all Hoechst 33342-excluding cells and vice versa, establishing the physical equivalence between JC1(low) and the side population (SP). JC1(low) cell content was reduced by three ABCG2-specific inhibitors: FTC, Ko143, and glafenine. JC1(low) percentiles for the fresh human and rabbit cells were 1.4% and 4.1% and CFEs for rabbit JC1(low) and JC1(main) were 1.2% and 5.3%. In contrast, the respective JC1(low) percentiles in the first and second outgrowths were 19.5% and 27.4% and 25.8% and 32.5%, and the rabbit JC1(low) and JC1(main) CFEs were 12.3% and 0.9%. Thus, although in FT the contribution of the JC1(low) cohort to the CFE is minimal, in the explant culture the phenotype incorporates >80% of the CFE. CONCLUSIONS: ABCG2-dependent dye exclusion undergoes a large expansion in explant culture and becomes associated with a high CFE. The transport increase is more pronounced at late outgrowth times, suggesting permanence of stem cells within the explant.


Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Benzimidazóis/farmacocinética , Carbocianinas/farmacocinética , Proliferação de Células , Corantes Fluorescentes/farmacocinética , Limbo da Córnea/citologia , Limbo da Córnea/metabolismo , Proteínas de Neoplasias/metabolismo , Células 3T3 , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Adulto , Idoso , Animais , Benzimidazóis/toxicidade , Transporte Biológico , Cadáver , Carbocianinas/toxicidade , Separação Celular , Ensaio de Unidades Formadoras de Colônias , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Corantes Fluorescentes/toxicidade , Humanos , Técnicas In Vitro , Membranas Artificiais , Camundongos , Pessoa de Meia-Idade , Polietilenotereftalatos , Coelhos , Coloração e Rotulagem , Fatores de Tempo
17.
Mol Vis ; 16: 2109-20, 2010 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-21139686

RESUMO

PURPOSE: To compare the global gene expression profile of stratified epithelia generated in vitro using simian virus 40 (SV40) immortalized human corneal epithelial cells with the previously reported gene expression of normal human corneal epithelia. METHODS: Immortalized cells expanded in submerged culture were grown in an air-liquid interface of liquid permeable collagen-coated filters to foster stratification and differentiation. Stratified epithelia displaying resistances exceeding 300 Ω·cm2 were dissolved in an RNA purification lysis buffer. Purified RNA was used to globally determine gene expression levels using high-density single-channel oligonucleotide microarrays. Raw hybridization readings were converted into relative gene expression levels using Robust Multi-array Average (RMA) algorithm. Expression levels for selected genes were validated by real-time RT-qPCR. The biologic significance of the gene expression profiles was interpreted with the help of several microarray software analysis tools and ad hoc thematical analysis. RESULTS: The stratified cell culture to native epithelial comparison identified over- and under-expression in 22% and 14% of the probed genes, respectively. The larger expression decreases occurred in genes intimately associated with both the stratified epithelial lineage at large such as keratin 14 and the corneal phenotype, such as keratin 12, connexin 43, aldehyde dehydrogenases (ALDHs), and paired box gene 6 (PAX6) and its whole downstream transcriptome. Overexpression related to genes associated with cell cycling stimulation. CONCLUSIONS: The results indicate that the stratified corneal epithelial cell model generated using SV40 immortalized cells may be useful only in certain research applications. Extrapolations of studies with these cells to actual tissue cells should be done with a great deal of caution.


Assuntos
Técnicas de Cultura de Células/métodos , Células Epiteliais/metabolismo , Células Epiteliais/virologia , Epitélio Corneano/citologia , Perfilação da Expressão Gênica , Vírus 40 dos Símios/fisiologia , Linhagem Celular Transformada , Linhagem da Célula/genética , Células Epiteliais/citologia , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Especificidade de Órgãos/genética , RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para Cima/genética
18.
Mol Vis ; 16: 1696-704, 2010 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-20806045

RESUMO

PURPOSE: Dual specificity phosphatases (DUSPs) modulate the duration and magnitude of phospho-activation of Erk1/2, p38 and JNK1/2, the terminal kinases (TKs) of the mitogen activated protein kinase (MAPK) cascades. Three DUSPs, DUSP1, DUSP5, and DUSP6, are overexpressed in ocular surface side population stem cells (SPSCs). Our objective was to identify the impact of these enzymes on TK phosphorylation and proliferation of corneal epithelial cells. METHODS: SV40 immortalized (sv) and expanded fresh human corneal epithelial cells (efHCECs) were transduced with lentivectors to elicit expression of shRNAmir against DUSP1, DUSP5, and JNK1 to thereby create the DUSP1i, DUSP5i and JNKi cell sublines, or overexpress DUSP6 (henceforth DUSP6(+)), respectively. TK phosphorylation status and proliferation rates were determined by immunoblotting and (3)H thymidine uptake. RESULTS: In both ef and svHCECs, EGF supplementation after a 24 h serum starvation caused a rapid 5-15 min spike in the phosphorylation of all three TK types. This was followed by gradual decreases to low phosphorylation levels within one h. These declines coincided with dramatic increases in DUSP1 and DUSP5 protein expression. In DUSP1i, the DUSP1 increase was abolished. All 3 TKs maintained high phosphorylation levels for at least 90 min and proliferation rates were unchanged from non-transduced cells. In DUSP5i, the DUSP5 protein increase was prevented, the post peak phosphorylation decrease occurred only on Erk1/2 and the proliferation rate increased by 50%-60%. In JNK1i, JNK1 was essentially knocked out and proliferation rates were also markedly elevated. At steady-state, DUSP1i maintained high levels of pJNK1/2 expression. In DUSP6(+) Erk1/2 phosphorylation was prevented and proliferation rates decreased to less than 50%. CONCLUSIONS: DUSP5 and DUSP6 selectively control ERK pathway activity and proliferation. The lack of an effect of DUSP1 knockdown on proliferation can be attributed to its pan-MAPK effect. The expected augmented proliferative response due to enhanced and prolonged phosphorylation of Erk1/2 following DUSP1 knockdown does not occur because a pJNK1/2 antiproliferative effect is simultaneously unleashed.


Assuntos
Fosfatase 6 de Especificidade Dupla/metabolismo , Fosfatases de Especificidade Dupla/metabolismo , Células Epiteliais/citologia , Células Epiteliais/enzimologia , Epitélio Corneano/citologia , Linhagem Celular Transformada , Proliferação de Células , Densitometria , Fosfatase 1 de Especificidade Dupla , Fosfatase 6 de Especificidade Dupla/genética , Fosfatases de Especificidade Dupla/genética , Regulação Enzimológica da Expressão Gênica , Vetores Genéticos/genética , Humanos , Lentivirus/genética , Fosforilação , Timidina/metabolismo , Transdução Genética
19.
Invest Ophthalmol Vis Sci ; 51(11): 5601-9, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20538990

RESUMO

PURPOSE: To determine whether resolvin E1 (RvE1), an endogenous oxygenation product of eicosapentaenoic acid (EPA), induces increases in migration in human corneal epithelial cells (HCECs) and to identify signal pathways mediating this response. METHODS: Migration was measured with the scratch wound assay. Western blot analysis identified changes in the phosphorylation status of prospective intracellular signal transduction mediators. Immunocytochemistry probed for intracellular paxillin localization and actin reorganization. RESULTS: RvE1 enhanced HCEC migratory rates to levels comparable to those induced by epidermal growth factor (EGF). These increases were accompanied by increases in the phosphorylation status of epidermal growth factor receptor (EGFR), Akt, p38 MAPK, GSK-3α/ß, and paxillin, which essentially persisted for up to 60 minutes. The EGFR inhibitor AG1478 blocked the subsequent effects of RvE1 to induce increases in phosphorylation status and cell migration. The PI3-K inhibitor LY294002 or wortmannin or the p38 inhibitor BIRB796 blocked resolvin-induced increases in cell migration. Either the matrix metalloproteinase (MMP) inhibitor GM6001 or the specific heparin-bound EGF-like growth factor inhibitor CRM197 suppressed RvE1-induced stimulation of EGFR/PI3-K/Akt phosphorylation and cell migration. CONCLUSIONS: RvE1 enhances HCEC migration through MMP and sheddase-mediated EGFR transactivation. This response is dependent on PI3-K and p38-linked signaling eliciting paxillin (Tyr118) phosphorylation.


Assuntos
Movimento Celular/efeitos dos fármacos , Ácido Eicosapentaenoico/análogos & derivados , Epitélio Corneano/citologia , Receptores ErbB/genética , Ativação Transcricional , Actinas/metabolismo , Western Blotting , Movimento Celular/fisiologia , Relação Dose-Resposta a Droga , Ácido Eicosapentaenoico/antagonistas & inibidores , Ácido Eicosapentaenoico/farmacologia , Inibidores Enzimáticos/farmacologia , Epitélio Corneano/metabolismo , Receptores ErbB/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Humanos , Paxilina/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/fisiologia , Cicatrização/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
20.
J Pharm Sci ; 99(2): 1087-98, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19623615

RESUMO

ATP-binding cassette (ABC) transporters are able to efflux their substrate drugs from the cells. We compared expression of efflux proteins in normal human corneal epithelial tissue, primary human corneal epithelial cells (HCEpiC), and corneal epithelial cell culture model (HCE model) based on human immortal cell line. Expression of multidrug resistance protein 1 (MDR1), multidrug resistance-associated protein 1-6 (MRP1-6) and breast cancer resistance protein (BCRP) was studied using quantitative RT-PCR, Western blot, and immunohistochemistry. Only MRP1, MRP5, and BCRP were expressed in the freshly excised human corneal epithelial tissue. Expression of MRP1 and MRP5 was localized predominantly in the basal cells of the central cornea and limbus. Functional efflux activity was shown in the cell models, but they showed over-expression of most efflux transporters compared to that of normal corneal epithelium. In conclusion, MRP1, MRP5, and BCRP are expressed in the corneal epithelium, but MDR1, MRP2, MRP3, MRP4, and MRP6 are not significantly expressed. HCE cell model and commercially available primary cells deviate from this expression profile.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Epitélio Corneano/metabolismo , Transportadores de Cassetes de Ligação de ATP/biossíntese , Western Blotting , Cálcio/metabolismo , Células Cultivadas , Células Epiteliais/metabolismo , Fluoresceínas/metabolismo , Humanos , Imuno-Histoquímica , Técnicas In Vitro , RNA Mensageiro/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa
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