RESUMEN
Heterozygous mutation of PAX6 in humans leads to congenital aniridia (OMIM 106210) which is typified by congenital iris and foveal defects, and later onset glaucoma, aniridic keratopathy, and cataract. Mice heterozygous for Pax6 mutations phenocopy many aspects of aniridia including the iris defects, keratopathy and cataract, although Pax6 mutant mice have small lenses, a phenotype which is not typically reported in human aniridia, perhaps due to difficulties in measuring lens diameter during typical ophthalmic examinations as the lens periphery is shielded by the iris. In order to overcome this, records of patients diagnosed with congenital aniridia between April 2015 and May 2021 at the Necker-Enfants Malades Hospital, and genetically confirmed with a disease-causing PAX6 variant, were retrospectively reviewed for those with normal axial length whose iris defects allowed visualization of the lens margins and corneal diameter to allow calculation of a lens/corneal diameter ratio. This value was compared with values obtained from a cohort of patients with Sjödell grade IV oculocutaneous albinism type 1 (OCA1; OMIM 203100) which allowed visualization of the lens periphery via iris transillumination. This analysis revealed that patients with congenital aniridia had a significantly lower lens/corneal ratio when compared to those with albinism, suggesting that humans haploinsufficient for PAX6, like mice, rats, frogs, and zebrafish, exhibit reductions in lens size.
Asunto(s)
Aniridia , Catarata , Enfermedades de la Córnea , Humanos , Ratones , Ratas , Animales , Factor de Transcripción PAX6/genética , Factores de Transcripción Paired Box/genética , Estudios Retrospectivos , Pez Cebra , Aniridia/genética , Aniridia/diagnóstico , Mutación , Catarata/genética , Catarata/congénito , Proteínas de Homeodominio/genética , Proteínas del Ojo/genéticaRESUMEN
Age is a major risk factor for cataract (ARC). However, the influence of aging on the lens transcriptome is under studied. Lens epithelial (LEC) and fiber cells (LFC) were isolated from young (3 month old) and aged (24 month old) C57BL/6J mice, and the transcriptome elucidated via RNAseq. EdgeR estimated differential gene expression in pairwise contrasts, and Advaita's Ipathway guide and custom R scripts were used to evaluate the potential biological significance of differentially expressed genes (DEGs). This analysis revealed age-dependent decreases in lens differentiation marker expression in both LECs and LFCs, with gamma crystallin transcripts downregulating nearly 50 fold in aged LFCs. The expression of the transcription factors Hsf4 and Maf, which are known to activate lens fiber cell preferred genes, are downregulated, while FoxE3, which represses gamma crystallin expression, is upregulated in aged fibers. Aged LECs upregulate genes controlling the immune response, complement pathways, and cellular stress responses, including glutathione peroxidase 3 (Gpx3). Aged LFCs exhibit broad changes in the expression of genes regulating cell communication, and upregulate genes involved in antigen processing/presentation and cholesterol metabolism, while changes in the expression of mitochondrial respiratory chain genes are consistent with mitochondrial stress, including upregulation of NDufa4l2, which encodes an alternate electron transport chain protein. However, age did not profoundly affect the response of LECs to injury as both young and aged LECs upregulate inflammatory gene signatures at 24 h post injury to similar extents. These RNAseq profiles provide a rich data set that can be mined to understand the genetic regulation of lens aging and how this impinges on the pathophysiology of age related cataract.
Asunto(s)
Envejecimiento/genética , Catarata/genética , Factores de Transcripción Forkhead/genética , Regulación de la Expresión Génica , Factores de Transcripción del Choque Térmico/genética , Proteínas Proto-Oncogénicas c-maf/genética , Transcriptoma/genética , Animales , Catarata/metabolismo , Modelos Animales de Enfermedad , Femenino , Factores de Transcripción Forkhead/biosíntesis , Factores de Transcripción del Choque Térmico/biosíntesis , Proteínas de Choque Térmico , Cristalino/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Proto-Oncogénicas c-maf/biosíntesis , ARN/genética , gamma-Cristalinas/biosíntesis , gamma-Cristalinas/genéticaRESUMEN
The transcriptome of mammalian tissues differs between males and females, and these differences can change across the lifespan, likely regulating known sexual dimorphisms in disease prevalence and severity. Cataract, the most prevalent disease of the ocular lens, occurs at similar rates in young individuals, but its incidence is elevated in older women compared to men of the same age. However, the influence of sex on the lens transcriptome was unknown. RNAseq based transcriptomic profiling of young adult C57BL/6J mouse lens epithelial and fiber cells revealed that few genes are differentially expressed between the sexes. In contrast, lens cells from aged (24 month old) male and female C57BL/6J mice differentially expressed many genes, including several whose expression is lens preferred. Like cataracts, posterior capsular opacification (PCO), a major sequela of cataract surgery, may also be more prevalent in women. Lens epithelial cells isolated from mouse eyes 24 h after lens fiber cell removal exhibited numerous transcriptomic differences between the sexes, including genes implicated in complement cascades and extracellular matrix regulation, and these differences are much more pronounced in aged mice than in young mice. These results provide an unbiased basis for future studies on how sex affects the lens response to aging, cataract development, and cataract surgery.
Asunto(s)
Opacificación Capsular/genética , Extracción de Catarata/efectos adversos , Matriz Extracelular/genética , Regulación de la Expresión Génica , Cristalino/metabolismo , Complicaciones Posoperatorias/epidemiología , Transcriptoma/genética , Animales , Opacificación Capsular/metabolismo , Opacificación Capsular/cirugía , Modelos Animales de Enfermedad , Células Epiteliales/metabolismo , Células Epiteliales/patología , Matriz Extracelular/metabolismo , Femenino , Perfilación de la Expresión Génica/métodos , Cápsula del Cristalino/metabolismo , Cápsula del Cristalino/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Complicaciones Posoperatorias/genética , Complicaciones Posoperatorias/metabolismo , Factores Sexuales , Cicatrización de Heridas/genéticaRESUMEN
Congenital aniridia is caused by heterozygous mutations in the PAX6 gene. In this disease, congenital iris and foveal hypoplasia is associated with juvenile onset cataract, glaucoma, and corneal keratopathy. In rodents, Pax6 mutations result in a congenital reduction in ocular size that is not typically described in human aniridia. Here, the ocular morphometry of aniridia patients is compared with the lens phenotype of Pax6+/tm1/Pgr mice to reveal whether there are species differences in Pax6 regulation of lens development and homeostasis. Ultrasound biometry (UBM) revealed that eleven percent of aniridia patients exhibited mild microphthalmia while the anterior chamber depth of aniridic eyes was significantly reduced from 6 months of age onward. Although aniridic lens thickness was normal from birth, it was significantly decreased in aniridic lenses older than 30. Notably, 86% of aniridic lenses exhibited cataractous changes in this cohort. In addition, a significant proportion of aniridia patients develop lens subluxation as they age associated with reduced lens diameter as measured by anterior segment optical coherence tomography (AS-OCT). Analysis of young adult Pax6+/tm1/Pgr mouse lenses by micro-computed tomography (microCT), bright field and dark field imaging revealed that they are reduced in size but did not exhibit overt cataracts at this age. Overall, this study reveals that congenital microphthalmia as assessed by axial length, or microphakia, as assessed by lens thickness, are not typical in human aniridia, although these are primary manifestations of Pax6 mutations in mice, suggesting that PAX6 regulates some aspects of lens development differently between these species.
Asunto(s)
Aniridia/patología , Catarata/patología , Cristalino/patología , Microftalmía/patología , Adolescente , Adulto , Anciano , Animales , Aniridia/genética , Cámara Anterior/patología , Longitud Axial del Ojo/patología , Catarata/genética , Niño , Preescolar , Modelos Animales de Enfermedad , Femenino , Humanos , Lactante , Masculino , Ratones , Ratones Mutantes , Microftalmía/genética , Microscopía Acústica , Persona de Mediana Edad , Factor de Transcripción PAX6/genética , Fenotipo , Microscopía con Lámpara de Hendidura , Tomografía de Coherencia Óptica , Adulto JovenRESUMEN
Lens epithelial cells differentiate into lens fibers (LFs) in response to a fibroblast growth factor (FGF) gradient. This cell fate decision requires the transcription factor Prox1, which has been hypothesized to promote cell cycle exit in differentiating LF cells. However, we find that conditional deletion of Prox1 from mouse lenses results in a failure in LF differentiation despite maintenance of normal cell cycle exit. Instead, RNA-seq demonstrated that Prox1 functions as a global regulator of LF cell gene expression. Intriguingly, Prox1 also controls the expression of fibroblast growth factor receptors (FGFRs) and can bind to their promoters, correlating with decreased downstream signaling through MAPK and AKT in Prox1 mutant lenses. Further, culturing rat lens explants in FGF increased their expression of Prox1, and this was attenuated by the addition of inhibitors of MAPK. Together, these results describe a novel feedback loop required for lens differentiation and morphogenesis, whereby Prox1 and FGFR signaling interact to mediate LF differentiation in response to FGF.
Asunto(s)
Proteínas de Homeodominio/metabolismo , Cristalino/citología , Cristalino/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Factores de Crecimiento de Fibroblastos/farmacología , Proteínas de Homeodominio/genética , Ratones , Ratones Endogámicos C57BL , Receptores de Factores de Crecimiento de Fibroblastos/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Proteínas Supresoras de Tumor/genéticaRESUMEN
The appropriate spatial and temporal regulation of canonical Wnt signaling is vital for eye development. However, the literature often conflicts on the distribution of canonical Wnt signaling in the eye. Here, using a sensitive mouse transgenic reporter line, we report a detailed re-evaluation of the spatiotemporal dynamics of canonical Wnt signaling in the developing eye. Canonical Wnt activity was dynamic in the optic vesicle and later in the retina, while it was absent from the ectodermal precursors of the lens and corneal epithelium. However, later in corneal development, canonical Wnt reporter activity was detected in corneal stroma and endothelium precursors as they form from the neural crest, although this was lost around birth. Interestingly, while no canonical Wnt signaling was detected in the corneal limbus or basal cells at any developmental stage, it was robust in adult corneal wing and squamous epithelial cells. While canonical Wnt reporter activity was also absent from the postnatal lens, upon lens injury intended to model cataract surgery, it upregulated within 12â¯h in remnant lens epithelial cells, and co-localized with alpha smooth muscle actin in fibrotic lens epithelial cells from 48â¯h post-surgery onward. This pattern correlated with downregulation of the inhibitor of canonical Wnt signaling, Dkk3. These data demonstrate that canonical Wnt signaling is dynamic within the developing eye and upregulates in lens epithelial cells in response to lens injury. As canonical Wnt signaling can collaborate with TGFß to drive fibrosis in other systems, these data offer the first evidence in a lens-injury model that canonical Wnt may synergize with TGFß signaling to drive fibrotic posterior capsular opacification (PCO).
Asunto(s)
Opacificación Capsular/metabolismo , Desarrollo Embrionario/fisiología , Cristalino/embriología , Cápsula Posterior del Cristalino/metabolismo , Vía de Señalización Wnt/fisiología , Animales , Opacificación Capsular/patología , Modelos Animales de Enfermedad , Ojo/embriología , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Proteínas Fluorescentes Verdes/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal , Cápsula Posterior del Cristalino/patología , Análisis Espacio-TemporalRESUMEN
Lens fiber cells are highly elongated cells with complex membrane morphologies that are critical for the transparency of the ocular lens. Investigations into the molecular mechanisms underlying lens fiber cell elongation were first reported in the 1960s, however, our understanding of the process is still poor nearly 50 years later. This review summarizes what is currently hypothesized about the regulation of lens fiber cell elongation along with the available experimental evidence, and how this information relates to what is known about the regulation of cell shape/elongation in other cell types, particularly neurons.
Asunto(s)
Diferenciación Celular/fisiología , Forma de la Célula/fisiología , Cristalino/citología , Actinas/metabolismo , Animales , Citoesqueleto/fisiología , Humanos , Cristalino/embriología , Morfogénesis , Tubulina (Proteína)/metabolismoRESUMEN
Failure of lens fiber cell denucleation (LFCD) is associated with congenital cataracts, but the pathobiology awaits elucidation. Recent work has suggested that mechanisms that direct the unidirectional process of LFCD are analogous to the cyclic processes associated with mitosis. We found that lens-specific mutations that elicit an unfolded-protein response (UPR) in vivo accumulate p27(Cdkn1b), show cyclin-dependent kinase (Cdk)-1 inhibition, retain their LFC nuclei, and are cataractous. Although a UPR was not detected in lenses expressing K6W-Ub, they also accumulated p27 and showed failed LFCD. Induction of a UPR in human lens epithelial cells (HLECs) also induced accumulation of p27 associated with decreased levels of S-phase kinase-associated protein (Skp)-2, a ubiquitin ligase that regulates mitosis. These cells also showed decreased lamin A/C phosphorylation and metaphase arrest. The suppression of lamin A/C phosphorylation and metaphase transition induced by the UPR was rescued by knockdown of p27. Taken together, these data indicate that accumulation of p27, whether related to the UPR or not, prevents the phosphorylation of lamin A/C and LFCD in maturing LFCs in vivo, as well as in dividing HLECs. The former leads to cataract and the latter to metaphase arrest. These results suggest that accumulation of p27 is a common mechanism underlying retention of LFC nuclei.
Asunto(s)
Catarata/metabolismo , Catarata/patología , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Cristalino/metabolismo , Respuesta de Proteína Desplegada/fisiología , Animales , Línea Celular , Núcleo Celular/metabolismo , Células Epiteliales/metabolismo , Femenino , Humanos , Lamina Tipo A/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mitosis/fisiología , Fosforilación/fisiología , Proteínas Quinasas Asociadas a Fase-S/metabolismoRESUMEN
Integrins are heterodimeric cell surface molecules that mediate cell-extracellular matrix (ECM) adhesion, ECM assembly, and regulation of both ECM and growth factor induced signaling. However, the developmental context of these diverse functions is not clear. Loss of ß1-integrin from the lens vesicle (mouse E10.5) results in abnormal exit of anterior lens epithelial cells (LECs) from the cell cycle and their aberrant elongation toward the presumptive cornea by E12.5. These cells lose expression of LEC markers and initiate expression of the Maf (also known as c-Maf) and Prox1 transcription factors as well as other lens fiber cell markers. ß1-integrin null LECs also upregulate the ERK, AKT and Smad1/5/8 phosphorylation indicative of BMP and FGF signaling. By E14.5, ß1-integrin null lenses have undergone a complete conversion of all lens epithelial cells into fiber cells. These data suggest that shortly after lens vesicle closure, ß1-integrin blocks inappropriate differentiation of the lens epithelium into fibers, potentially by inhibiting BMP and/or FGF receptor activation. Thus, ß1-integrin has an important role in fine-tuning the response of the early lens to the gradient of growth factors that regulate lens fiber cell differentiation.
Asunto(s)
Diferenciación Celular/genética , Proteínas de Homeodominio/biosíntesis , Integrina beta1/genética , Cristalino/metabolismo , Organogénesis/genética , Proteínas Supresoras de Tumor/biosíntesis , Animales , Adhesión Celular/genética , Epitelio/crecimiento & desarrollo , Epitelio/metabolismo , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Integrina beta1/metabolismo , Cristalino/crecimiento & desarrollo , Ratones , Proteínas Proto-Oncogénicas c-maf/biosíntesis , Proteínas Proto-Oncogénicas c-maf/genética , Transducción de Señal , Proteínas Supresoras de Tumor/genéticaRESUMEN
Posterior capsular opacification (PCO) is the major complication arising after cataract treatment. PCO occurs when the lens epithelial cells remaining following surgery (LCs) undergo a wound healing response producing a mixture of α-smooth muscle actin (α-SMA)-expressing myofibroblasts and lens fibre cells, which impair vision. Prior investigations have proposed that integrins play a central role in PCO and we found that, in a mouse fibre cell removal model of cataract surgery, expression of αV integrin and its interacting ß-subunits ß1, ß5, ß6, ß8 are up-regulated concomitant with α-SMA in LCs following surgery. To test the hypothesis that αV integrins are functionally important in PCO pathogenesis, we created mice lacking the αV integrin subunit in all lens cells. Adult lenses lacking αV integrins are transparent and show no apparent morphological abnormalities when compared with control lenses. However, following surgical fibre cell removal, the LCs in control eyes increased cell proliferation, and up-regulated the expression of α-SMA, ß1-integrin, fibronectin, tenascin-C and transforming growth factor beta (TGF-ß)-induced protein within 48 hrs, while LCs lacking αV integrins exhibited much less cell proliferation and little to no up-regulation of any of the fibrotic markers tested. This effect appears to result from the known roles of αV integrins in latent TGF-ß activation as αV integrin null lenses do not exhibit detectable SMAD-3 phosphorylation after surgery, while this occurs robustly in control lenses, consistent with the known roles for TGF-ß in fibrotic PCO. These data suggest that therapeutics antagonizing αV integrin function could be used to prevent fibrotic PCO following cataract surgery.
Asunto(s)
Opacificación Capsular/metabolismo , Opacificación Capsular/patología , Extracción de Catarata/efectos adversos , Integrina alfaV/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Actinas/biosíntesis , Animales , Opacificación Capsular/etiología , Proliferación Celular , Células Epiteliales/metabolismo , Transición Epitelial-Mesenquimal , Cápsula del Cristalino/metabolismo , Cápsula del Cristalino/patología , Ratones , Miofibroblastos/metabolismo , Miofibroblastos/patología , Proteína smad3/biosíntesis , Cicatrización de HeridasRESUMEN
We report analysis of the ocular lens phenotype of the recessive, larval lethal zebrafish mutant, lama1 (a69/a69). Previous work revealed that this mutant has a shortened body axis and eye defects including a defective hyaloid vasculature, focal corneal dysplasia, and loss of the crystalline lens. While these studies highlight the importance of laminin α1 in lens development, a detailed analysis of the lens defects seen in these mutants was not reported. In the present study, we analyze the lenticular anomalies seen in the lama1 (a69/a69) mutants and show that the lens defects result from the anterior extrusion of lens material from the eye secondary to structural defects in the lens capsule and developing corneal epithelium associated with basement membrane loss. Our analysis provides further insights into the role of the lens capsule and corneal basement membrane in the structural integrity of the developing eye.
Asunto(s)
Laminina/genética , Cristalino/metabolismo , Cristalino/patología , Mutación , Proteínas de Pez Cebra/genética , Pez Cebra/genética , Animales , Membrana Basal/metabolismo , Colágeno Tipo IV/metabolismo , Córnea/metabolismo , Córnea/patología , Laminina/metabolismo , Cápsula del Cristalino/metabolismo , FenotipoRESUMEN
Cataract surgery (CS) is an effective treatment for cataracts, a major cause of visual disability worldwide. However, CS leads to ocular inflammation, and in the long term, it can result in posterior capsular opacification (PCO) and/or lens dislocation driven by the post-surgical overgrowth of lens epithelial cells (LECs) and their conversion to myofibroblasts and/or aberrant fiber cells. However, the molecular mechanisms by which CS results in inflammation and PCO are still obscure because most in vitro models do not recapitulate the wound healing response of LECs seen in vivo, while traditional animal models of cataract surgery, such as rabbits, do not allow the genetic manipulation of gene expression to test mechanisms. Recently, our laboratory and others have successfully used genetically modified mice to study the molecular mechanisms that drive the induction of proinflammatory signaling and LEC epithelial to mesenchymal transition, leading to new insight into PCO pathogenesis. Here, we report the established protocol for modeling cataract surgery in mice, which allows for robust transcriptional profiling of the response of LECs to lens fiber cell removal via RNAseq, the evaluation of protein expression by semi-quantitative immunofluorescence, and the use of modern mouse genetics tools to test the function of genes that are hypothesized to participate in the pathogenesis of acute sequelae like inflammation as well as the later conversion of LECs to myofibroblasts and/or aberrant lens fiber cells.
Asunto(s)
Extracción de Catarata , Catarata , Cristalino , Animales , Ratones , Conejos , Transición Epitelial-Mesenquimal , Cristalino/cirugía , Extracción de Catarata/efectos adversos , Catarata/etiología , InflamaciónRESUMEN
The late embryonic mouse lens requires the transcription factor ATF4 for its survival although the underlying mechanisms were unknown. Here, RNAseq analysis revealed that E16.5 Atf4 null mouse lenses downregulate the mRNA levels of lens epithelial markers as well as known markers of late lens fiber cell differentiation. However, a comparison of this list of differentially expressed genes (DEGs) with other known transcriptional regulators of lens development indicated that ATF4 expression is not directly controlled by the previously described lens gene regulatory network. Pathway analysis revealed that the Atf4 DEG list was enriched in numerous genes involved in nutrient transport, amino acid biosynthesis, and tRNA charging. These changes in gene expression likely result in the observed reductions in lens free amino acid and glutathione levels, which would result in the observed low levels of extractable lens protein, finally leading to perinatal lens disintegration. These data demonstrate that ATF4, via its function in the integrated stress response, is likely to play a crucial role in mediating the adaption of the lens to the avascularity needed to maintain lens transparency.
Asunto(s)
Cristalino , Animales , Ratones , Cristalino/metabolismo , Regulación de la Expresión Génica , Diferenciación Celular , Factores de Transcripción/metabolismo , Ratones Noqueados , Aminoácidos/metabolismoRESUMEN
Transforming growth factor-ß (TGF-ß) has roles in embryonic development, the prevention of inappropriate inflammation and tumour suppression. However, TGF-ß signalling also regulates pathological epithelial-to-mesenchymal transition (EMT), inducing or progressing a number of diseases ranging from inflammatory disorders, to fibrosis and cancer. However, TGF-ß signalling does not proceed linearly but rather induces a complex network of cascades that mutually influence each other and cross-talk with other pathways to successfully induce EMT. Particularly, there is substantial evidence for cross-talk between αV integrins and TGF-ß during EMT, and anti-integrin therapeutics are under development as treatments for TGF-ß-related disorders. However, TGF-ß's complex signalling network makes the development of therapeutics to block TGF-ß-mediated pathology challenging. Moreover, despite our current understanding of integrins and TGF-ß function during EMT, the precise mechanism of their role during physiological versus pathological EMT is not fully understood. This review focuses on the circle of regulation between αV integrin and TGF-ß signalling during TGF-ß induced EMT, which pose as a significant driver to many known TGF-ß-mediated disorders.
Asunto(s)
Transición Epitelial-Mesenquimal/genética , Integrina alfaV/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Animales , Adhesión Celular , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Fibrosis , Regulación de la Expresión Génica , Genes Reguladores , Humanos , Inflamación/genética , Inflamación/metabolismo , Inflamación/patología , Integrina alfaV/genética , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transducción de Señal , Factor de Crecimiento Transformador beta/genéticaRESUMEN
The unfolded protein response is a set of cell signaling pathways recently recognized to be activated in the lens during both normal development and endoplasmic reticulum stress induced by either unfolded proteins or oxidative damage. While mutations in the gene for connexin 50 are known to cause autosomal dominant cataracts, it has not been previously reported whether mutant connexins can activate the unfolded protein response in the lens. Mice homozygous for the S50P or G22R mutation of connexin 50 have reduced amounts of connexin 50 protein at the cell membrane, with some intracellular staining consistent with retention in the endoplasmic reticulum. Connexin 50 mutants have elevated levels of BiP expression in both lens epithelial and fiber cells from E15.5 with the most robust elevation detected in newborns. While this elevation decreases in magnitude postnatally, BiP expression is still abnormally high in adults, particularly in the perinuclear endoplasmic reticulum of cell nuclei that are inappropriately retained in adult homozygous mutant lenses. Xbp1 splicing was elevated in lenses from both connexin mutants studied, while Atf4 and Atf6 levels were not majorly affected. Overall, these data suggest that UPR may be a contributing factor to the phenotype of connexin 50 mutant lenses even though the relatively modest extent of the response suggests that it is unlikely to be a major driver of the pathology.
Asunto(s)
Catarata/metabolismo , Conexinas/genética , Proteínas del Ojo/genética , Cristalino/metabolismo , Mutación , Respuesta de Proteína Desplegada/fisiología , Factor de Transcripción Activador 4/genética , Factor de Transcripción Activador 6/genética , Animales , Animales Recién Nacidos , Western Blotting , Proteínas de Unión al ADN/genética , Chaperón BiP del Retículo Endoplásmico , Proteínas de Choque Térmico/metabolismo , Ratones , Ratones Endogámicos C57BL , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Factores de Transcripción del Factor Regulador X , Factores de Transcripción/genética , Proteína 1 de Unión a la X-BoxRESUMEN
Cataracts are treated by lens fiber cell removal followed by intraocular lens (IOL) implantation into the lens capsule. While effective, this procedure leaves behind numerous lens epithelial cells (LECs) which undergo a wound healing response that frequently leads to posterior capsular opacification (PCO). In order to elucidate the acute response of LECs to lens fiber cell removal which models cataract surgery (post cataract surgery, PCS), RNA-seq was conducted on LECs derived from wild type mice at 0 and 6 h PCS. This analysis found that LECs upregulate the expression of numerous proinflammatory cytokines and profibrotic regulators by 6 h PCS suggesting rapid priming of pathways leading to inflammation and fibrosis PCS. LECs also highly upregulate the expression of numerous immediate early transcription factors (IETFs) by 6 h PCS and immunolocalization found elevated levels of these proteins by 3 h PCS, and this was preceded by the phosphorylation of ERK1/2 in injured LECs. Egr1 and FosB were among the highest expressed of these factors and qRT-PCR revealed that they also upregulate in explanted mouse lens epithelia suggesting potential roles in the LEC injury response. Analysis of lenses lacking either Egr1 or FosB revealed that both genes may regulate a portion of the acute LEC injury response, although neither gene was essential for expression of either proinflammatory or fibrotic markers at later times PCS suggesting that IETFs may work in concert to mediate the LEC injury response following cataract surgery.
Asunto(s)
Opacificación Capsular , Extracción de Catarata , Lesiones Oculares , Cápsula del Cristalino , Cristalino , Ratones , Animales , Cápsula del Cristalino/metabolismo , Cápsula del Cristalino/patología , Cristalino/metabolismo , Células Epiteliales/metabolismo , Opacificación Capsular/metabolismo , Lesiones Oculares/metabolismo , Factores de Transcripción/metabolismo , FibrosisRESUMEN
Hyaluronan is an oligosaccharide found in the pericellular matrix of numerous cell types and hyaluronan-induced signaling is known to facilitate fibrosis and cancer progression in some tissues. Hyaluronan is also commonly instilled into the eye during cataract surgery to protect the corneal endothelium from damage. Despite this, little is known about the distribution of hyaluronan or its receptors in the normal ocular lens. In this study, hyaluronan was found throughout the mouse lens, with apparently higher concentrations in the lens epithelium. CD44, a major cellular receptor for hyaluronan, is expressed predominately in mouse secondary lens fiber cells born from late embryogenesis into adulthood. Surgical removal of lens fiber cells from adult mice resulted in a robust upregulation of CD44 protein, which preceded the upregulation of alpha-smooth muscle actin expression typically used as a marker of epithelial-mesenchyma transition in this model of lens epithelial cell fibrosis. Mice lacking the CD44 gene had morphologically normal lenses with a response to lens fiber cell removal similar to wildtype, although they exhibited an increase in cell-associated hyaluronan. Overall, these data suggest that lens cells have a hyaluronan-containing pericellular matrix whose structure is partially regulated by CD44. Further, these data indicate that CD44 upregulation in the lens epithelium may be an earlier marker of lens injury responses in the mouse lens than the upregulation of alpha-smooth muscle actin.
Asunto(s)
Receptores de Hialuranos/genética , Cristalino/lesiones , Actinas/genética , Actinas/metabolismo , Animales , Embrión de Mamíferos/metabolismo , Epitelio/lesiones , Femenino , Receptores de Hialuranos/metabolismo , Ácido Hialurónico/metabolismo , Cristalino/embriología , Ratones , Ratones Endogámicos C57BL , Regulación hacia ArribaRESUMEN
Western blotting (WB), enzyme-linked immunosorbent assay (ELISA) and flow cytometry (FC) have long been used to assess and quantitate relative protein expression in cultured cells and tissue samples. However, WB and ELISA have limited ability to meaningfully quantitate relative protein levels in tissues with complex cell composition, while tissue dissociation followed by FC is not feasible when tissue is limiting and/or cells difficult to isolate. While protein detection in tissue using immunofluorescent (IF) probes has traditionally been considered a qualitative technique, advances in probe stability and confocal imaging allow IF data to be easily quantitated, although reproducible quantitation of relative protein expression requires careful attention to appropriate controls, experiment design, and data collection. Here we describe the methods used to quantify the data presented in Shihan et al. Matrix Biology, 2020 which lays out a workflow where IF data collected on a confocal microscope can be used to quantitate the relative levels of a molecule of interest by measuring mean fluorescent intensity across a region of interest, cell number, and the percentage of cells in a sample "positive" for staining with the fluorescent probe of interest. Overall, this manuscript discusses considerations for collecting quantifiable fluorescent images on a confocal microscope and provides explicit methods for quantitating IF data using FIJI-ImageJ.
RESUMEN
Fibrotic posterior capsular opacification (PCO), a major complication of cataract surgery, is driven by transforming growth factor-ß (TGF-ß). Previously, αV integrins were found to be critical for the onset of TGF-ß-mediated PCO in vivo; however, the functional heterodimer was unknown. Here, ß8 integrin-conditional knockout (ß8ITG-cKO) lens epithelial cells (LCs) attenuated their fibrotic responses, while both ß5 and ß6 integrin-null LCs underwent fibrotic changes similar to WT at 5 days post cataract surgery (PCS). RNA-Seq revealed that ß8ITG-cKO LCs attenuated their upregulation of integrins and their ligands, as well as known targets of TGF-ß-induced signaling, at 24 hours PCS. Treatment of ß8ITG-cKO eyes with active TGF-ß1 at the time of surgery rescued the fibrotic response. Treatment of WT mice with an anti-αVß8 integrin function blocking antibody at the time of surgery ameliorated both canonical TGF-ß signaling and LC fibrotic response PCS, and treatment at 5 days PCS, after surgically induced fibrotic responses were established, largely reversed this fibrotic response. These data suggest that αVß8 integrin is a major regulator of TGF-ß activation by LCs PCS and that therapeutics targeting αVß8 integrin could be effective for fibrotic PCO prevention and treatment.
Asunto(s)
Opacificación Capsular/prevención & control , Catarata/prevención & control , Integrinas/uso terapéutico , Animales , Humanos , RatonesRESUMEN
Human diseases caused by mutations in extracellular matrix genes are often associated with an increased risk of cataract and lens capsular rupture. However, the underlying mechanisms of cataract pathogenesis in these conditions are still unknown. Using two different mouse models, we show that the accumulation of collagen chains in the secretory pathway activates the stress signaling pathway termed unfolded protein response (UPR). Transgenic mice expressing ectopic Col4a3 and Col4a4 genes in the lens exhibited activation of IRE1, ATF6, and PERK associated with expansion of the endoplasmic reticulum and attenuation of general protein translation. The expression of the transgenes had adverse effects on lens fiber cell differentiation and eventually induced cell death in a group of transgenic fiber cells. In Col4a1(+/Deltaex40) mutant mice, the accumulation of mutant chains also caused low levels of UPR activation. However, cell death was not induced in mutant lenses, suggesting that low levels of UPR activation are not proapoptotic. Collectively, the results provide in vivo evidence for a role of UPR in cataract formation in response to accumulation of terminally unfolded proteins in the endoplasmic reticulum.