Response of lens epithelial cells to injury: role of lumican in epithelial-mesenchymal transition

PURPOSE: Lens epithelial cells (LECs) undergo epithelial-mesenchcymal transition (EMT) after injury and transform into myofibroblasts positive for alpha-smooth muscle actin (alphaSMA), an established marker of this process. Lumican is a keratan sulfate proteoglycan core protein. This study was conducted to examine whether human and mouse LECs express lumican after injury. To determine whether lumican may modulate EMT of LECs in response to injury or to exposure to transforming growth factor-beta2 (TGFbeta2), alphaSMA expression by the LECs was examined in lumican (Lum)-knockout mice in vivo and in organ culture. METHODS: Human postoperative capsular specimens and healing, injured mouse lenses at various intervals were immunostained for lumican or alphaSMA. alphaSMA was also immunolocalized in healing, injured lenses of Lum-knockout mice. Finally, expression of lumican and alphaSMA was examined in lenses of Lum-knockout mice incubated with TGFbeta2. RESULTS: Lumican was immunolocalized in matrix in human postoperative capsular opacification. Lumican and alphaSMA were upregulated in mouse LECs from 8 hours and day 5 after an injury, respectively. LECs accumulated adjacent to the capsular break were of epithelial shape in Lum(-/-) mice and fibroblast-like in Lum(+/-) mice during healing. alphaSMA expression by LECs was significantly delayed in Lum(-/-) mice, indicating that lumican may modulate injury-induced EMT in LECs. TGFbeta2-induced EMT appeared to be suppressed in organ-cultured lenses of Lum(-/-) mice compared with those of Lum(+/+) mice. CONCLUSIONS: Human capsular opacification contains lumican, and mouse LECs upregulate lumican and alphaSMA in response to injury. Loss of lumican perturbs EMT of mouse LECs.     

Intravitreal injection of TGFbeta induces cataract in rats

PURPOSE: In a previous study, it was determined that TGFbeta induces cataractous changes in the rat lens in vitro. The purpose of the present study was to determine whether the introduction of biologically active TGFbeta into the vitreous stimulates cataractous changes in the rat lens in situ. METHODS: TGFbeta was injected into the vitreous of the left eye of anesthetized adult male Wistar rats. The right eye received sterile vehicle as a control. Three to four months after injection, animals were killed, and lenses were enucleated and examined for cataractous changes. RESULTS: All lenses from control eyes remained transparent and maintained normal cellular architecture throughout. In contrast, lenses from TGFbeta-injected eyes displayed cloudiness in the cortex. In some lenses, distinct opacities were also apparent at the equator and extending some distance toward the anterior and posterior poles. Histologically, the opacities corresponded to subcapsular plaques containing aberrant cells and accumulations of extracellular matrix. In addition, cortical fibers in the anterior and posterior of all lenses displayed variable degrees of swelling, and many retained their nuclei. In some regions, the fiber cells appeared to have degenerated to form large homogeneous areas. The cellular architecture of the equator of these lenses was also disrupted and, in the most severe case, no bow zone was apparent with nucleated cells extending to the posterior pole. CONCLUSION: The introduction of active TGFbeta into the vitreous induced lenses to undergo cataractous changes. In addition to the TGFbeta-induced changes in the epithelium that were reported previously, cataractous changes observed in this study also involved the lens fiber cells and resembled changes observed in human posterior subcapsular and cortical cataracts.     

TGFbeta-Smad signalling in postoperative human lens epithelial cells

AIMS: To localise Smads3/4 proteins in lens epithelial cells (LECs) of fresh and postoperative human specimens. Smads3/4 are involved in signal transduction between transforming growth factor beta (TGFbeta) cell surface receptors and gene promoters. Nuclear localisation of Smads indicates achievement of endogenous TGFbeta signalling in cells. METHODS: Three circular sections of the anterior capsule, one lens, and 17 capsules undergoing postoperative healing were studied. Immunohistochemistry was performed for Smads3/4 in paraffin sections of the specimens. The effect of exogenous TGFbeta2 on Smad3 subcellular localisation was examined in explant cultures of extracted human anterior lens epithelium. RESULTS: The cytoplasm, but not the nuclei, of LECs of uninjured lenses was immunoreactive for Smads3/4. In contrast, nuclear immunoreactivity for Smads3/4 was detected in LECs during capsular healing. Nuclei positive for Smads3/4 were observed in monolayered LECs adjacent to the regenerated lens fibres of Sommerring's ring. Interestingly, the nuclei of LECs that were somewhat elongated, and appeared to be differentiating into fibre-like cells, were negative for Smads3/4. Fibroblast-like, spindle-shaped lens cells with nuclear immunoreactivity for nuclear Smads3/4 were occasionally observed in the extracellular matrix accumulated in capsular opacification. Exogenous TGFbeta induced nuclear translocation of Smad3 in LECs of anterior capsule specimens in explant culture. CONCLUSIONS: This is consistent with TGFbeta induced Smad signalling being involved in regulating the behaviour of LECs during wound healing after cataract surgery.     

Epithelial transdifferentiation and cataract in the human lens

Anterior subcapsular cataracts cause a serious loss of vision and are normally associated with ocular trauma, inflammation or clinical skin conditions. They appear to be accompanied by epithelial cell growth and transdifferentiation where unscheduled production of a number of proteins, including alpha smooth muscle actin (alpha-sma), occurs. Clinical studies have also revealed an up-regulation of the TGFbeta signalling pathway in such cataracts. The present study, using phase contrast and immunofluorescent techniques, was undertaken to investigate the extent of alpha-sma expression in traumatic cataracts, in capsulorhexis specimens obtained during cataract surgery and in aged human lenses from donor eyes. The donor lenses were also exposed to trauma or TGFbeta in culture to observe their relative contribution to alpha-sma production. Dense anterior subcapsular cataracts were relatively rare (<1%), but all showed a pronounced up-regulation of alpha-sma, which was located both in anterior cells of normal appearance and in nucleated fibroblastic cells lying beneath the anterior epithelium. Surprisingly, more than 50% of capsulorhexis specimens from mature cataracts showed expression of alpha-sma, although to a limited extent. Alpha-sma was not expressed in any of the clear donor lenses and culture for 8 days in EMEM did not induce expression. Interestingly, unlike their young animal counterparts, human lenses failed to show the presence of alpha-sma when exposed to 10 ng ml(-1) TGFbeta. However, after culture, lenses with pre-existing cortical opacities did express alpha-sma, as did clear lenses subjected to injury or trauma. It appears that the greater the stress, the greater is the expression of alpha-sma. Cataract, and especially cortical cataract, should therefore be seen as associated with stress-induced signalling pathways in the lens that lead to the transdifferentiation of the anterior epithelial cells.     

Aberrant lens fiber differentiation in anterior subcapsular cataract formation: a process dependent on reduced levels of Pax6

PURPOSE: TGFbeta can induce development in lenses of opaque subcapsular fibrotic plaques that have many features of human subcapsular cataracts. To understand further the events associated with the onset and progression of TGFbeta-induced cataract, several different models for anterior subcapsular cataract (ASC) were used and characterized. METHODS: Anterior subcapsular plaques were induced in rat lenses cultured with TGFbeta and in transgenic mice overexpressing TGFbeta in the lens. ASC was also examined in lenses of mice haploinsufficient for Pax6, as well as in human biopsy specimens. Immunofluorescence and in situ hybridization labeling were used to examine changes in patterns of gene expression associated with cataract formation in these models. RESULTS: Examination of TGFbeta-induced cataract in transgenic mice established that the subcapsular plaques are composed of a heterogenous cell population: a population of myofibroblastic cells as well as a population of lens-fiber-like cells. Further support for phenotypic change comes from the observation that the cells in these plaques no longer expressed lens epithelial markers, such as Pax6 and Connexin43. Subsequent examination of human biopsy specimens of ASC, as well as lenses from Pax6-deficient mice, showed that the anterior subcapsular plaques in both cases were also composed of a heterogenous population of cells. In contrast, anterior subcapsular plaques that developed in vitro in response to TGFbeta did not have this same cellular heterogeneity, as no fiber-like cells were present. CONCLUSIONS: These findings suggest that in vivo, during TGFbeta-induced cataract formation, some lens epithelial cells transform into myofibroblastic cells, whereas others differentiate into fiber cells. As this pathologic change is accompanied by altered expression of genes characteristic of the normal lens epithelial cell phenotype and as lenses from Pax6-deficient mice exhibit development of anterior subcapsular plaques closely resembling those induced by TGFbeta in transgenic mice, the authors propose that a reduction in Pax6 levels may be essential for this pathologic process to progress. Furthermore, it is clear from these in vitro studies that TGFbeta alone cannot reproduce the same morphologic and molecular changes associated with ASC formation in vivo, indicating that additional molecule(s) in the eye are important in this process.     

Downregulated expression of ADAM9 in anterior polar cataracts

PURPOSE: To determine whether ADAM (a disintegrin and metalloproteinase) is regulated in lens epithelial cells (LECs) of patients with anterior polar cataracts and by transforming growth factor (TGF)-beta 1 in cultured LECs. SETTING: Department of Ophthalmology and Visual Science, College of Medicine, The Catholic University of Korea, Seoul, Korea. METHODS: Lens epithelial cells attached to the anterior capsules of human cataractous lenses with nuclear and anterior subcapsular cataracts and noncataractous lenses were analyzed by reverse transcribed-polymerase chain reaction for the expression of ADAMs. The effect of TGF-beta 1 on ADAM gene expression was also tested in mouse lens epithelial explants and cultured LEC lines (alpha TN-4 and HLE B-3). RESULTS: Significantly reduced expression of mRNA for ADAM9 was observed in LECs from patients with anterior polar cataracts. The expression of mRNA for ADAM9 was downregulated by TGF-beta 1 in cultured human LECs. Treatment of cultured mouse LECs with TGF-beta 1 led to a reduction in ADAM1 mRNA. CONCLUSIONS: ADAMs are expressed and regulated in LECs. The downregulated expression of ADAM9 may serve as a marker for anterior polar cataracts in addition to previously known proteins, fibronectin, alpha-SMA, and beta ig-h3. The functions of this protein in lens pathology require further investigation.     

Temporal changes in MMP mRNA expression in the lens epithelium during anterior subcapsular cataract formation

Transforming growth factor beta (TGFβ) has been known to play a role in anterior subcapsular cataract (ASC) formation and posterior capsule opacification (PCO), both of which are fibrotic pathologies of the lens. Several models have been utilized to study ASC formation, including the TGFβ1 transgenic mouse model and the ex-vivo rat lens model. A distinct characteristic of ASC development within these models includes the formation of isolated fibrotic plaques or opacities which form beneath the lens capsule. A hallmark feature of ASC formation is the epithelial to mesenchymal transition (EMT) of lens epithelial cells (LECs) into myofibroblasts. Recently, the matrix metalloproteinases (MMPs) have been implicated in the formation of these cataracts through their involvement in EMT. In the present study, we sought to further investigate the role of MMPs in subcapsular cataract formation in a time course manner, through the examination of gene expression and morphological changes which occur during this process. RT-QPCR and immunohistochemical analysis was carried out on lenses treated with TGFβ for a period of 2, 4 and 6 days. Laser capture microdissection (LCM) was utilized to specifically isolate cells within the plaque region and cells from the adjacent epithelium in lenses treated for a 6 day period. Multilayering of LECs was observed as early as day 2, which preceded the presence of alpha smooth muscle actin (α-SMA) immunoreactivity that was evident following 4 days of treatment with TGFβ. A slight reduction in E-cadherin mRNA was detected at day 2, although this was not significant until the day 4 time point. Importantly, our results also indicate an early induction of MMP-9 mRNA following 2 days of TGFβ treatment, whereas MMP-2 was found to be upregulated at the later 4 day time point. Further experiments using FHL 124 cells show an induction in MMP-2 protein levels following treatment with recombinant MMP-9. Together these findings suggest an upstream role for MMP-9 in ASC formation.     

The human anterior lens capsule: cell density, morphology and mitotic index in normal and cataractous lenses

The morphology, cell density and mitotic index of human lens epithelial cells were determined according to the region of the opacity as follows: (1) nuclear; (2) posterior subcapsular; (3) cortical; (4) nuclear and posterior subcapsular; (5) cortical and posterior subcapsular. Epithelial abnormalities, consisting of enlarged, polymorphous cells and intercellular clefts, were found in cataractous lenses involving posterior subcapsular and/or cortical opacities. In contrast to the normal pattern of cell density, the cell density of cataractous epithelia in these categories decreased from the central to the peripheral epithelium. This decrease in cell density was due to the presence of swollen cells and intercellular clefts. The central and pre-equatorial mitotic index of cataractous lens epithelia was not significantly different (range 0.002-0.008%) and no correlation was found between cataract type and mitotic index (no information was available for the equatorial region). The low mitotic index observed reflects the mature age range of cataractous lenses and in one normal lens epithelium from a 23-yr-old donor, a higher value of 0.015% was found. The relevance of these epithelial abnormalities to the normal function of the epithelium is discussed.     

TGFbeta induces morphological and molecular changes similar to human anterior subcapsular cataract

BACKGROUND: Transforming growth factor beta (TGFbeta) has been shown to induce subcapsular plaques in cultured rat lenses as well as in lenses of transgenic mice. In the present study the authors have extended their analysis of these cataract models to determine how closely they mimic human cataract. In particular, they studied the maturation of cataract in the transgenic model to determine if it develops similar features as previously described for anterior subcapsular cataract (ASC) in humans. Furthermore, they investigated whether both of these animal models express the range of molecular markers that have now been shown to be present in human ASC. METHODS: Histology and periodic acid Schiff staining were used to study the development and maturation of subcapsular plaques in transgenic mice overexpressing TGFbeta1 in the lens. Immunolabelling methods were used to identify the molecular markers for ASC in both the transgenic mouse model and in rat lenses cultured with TGFbeta2. RESULTS: Histological analysis showed that the subcapsular plaques that develop in adult transgenic mouse lenses bear a striking similarity to mature human ASC, including the formation of a new epithelial-like layer extending between the subcapsular plaque and the underlying fibre mass. All known molecular markers for human ASC were induced in both rodent models, including collagen types I and III, tenascin, and fibronectin. They also identified the presence of desmin in these plaques, a putative novel marker for human cataract. CONCLUSIONS: In both transgenic mouse and rat lens culture models TGFbeta induces markers similar to those found in human ASC. Atypical expression of these cataract markers is also characteristic of posterior capsular opacification (PCO). The molecular markers expressed are typical of a myofibroblastic/fibroblastic phenotype and suggest that a common feature of ASC and PCO may be induction of an epithelial-mesenchymal transition by TGFbeta.     

Effect of corticosteroids on electrolyte transport of the isolated human and rabbit lens

In the isolated human lens, short circuit current was inhibited by pharmacological concentrations of 6-methylprednisolone and opacities occurred in the posterior subcapsular region in some lenses. The effect was seen only when the anterior (epithelial) surface of the lens was exposed. There was an increase of the short circuit current in the rabbit lens by 6-methyl-prednisolone and the lenses remained clear. Methylprednisolone effects were seen in spite of Na-K-ATPase inhibition by ouabain. Aldosterone had no effect on the translenticular potential difference, short circuit current and transparency. The data are discussed with respect to corticosteroid receptors in the lens epithelium and to the pathogenesis of steroid-associated cataract in man.     

Growth spurt in a lens with a posterior subcapsular cataract

Over a period of 5 years, the lenses of a patient with a unilateral posterior subcapsular cataract were documented using Scheimpflug slit-image photography. For the first time ever, a growth spurt was observed of the cataractous lens. It is postulated that this could be a reflection of either a mitotic spurt of the lens epithelium or a failure of compaction of the the deeper lens fibres.     

Lens epithelial cell proliferation in human posterior capsule opacification specimens

In previous in vitro studies on capsular bags it was shown that, after a sham extracapsular cataract extraction (ECCE) on human donor eyes, lens epithelial cells (LECs) show, in the short term, a dramatically elevated mitotic activity as compared to that in the intact lens. The long term in vivo proliferation of LECs in human lenses after ECCE and intraocular lens (IOL) implantation has not been studied until now. In the present study, the mitotic activity of LECs in human post-mortem eyes with posterior capsule opacification (PCO) was investigated. Human lenses with signs of PCO were dissected from donor eyes and incubated in MEM, supplemented with fetal calf serum, for 1 day (n = 10) or 7 days (n = 9). Six additional specimens were cultured for 7 days after removal of the IOL and lens fibres. After the incubation period, mitotic activity was estimated using the BrdU procedure and the Ki67 proliferating cell marker.The mean number of BrdU-positive nuclei in the intact PCO specimens was at a level of 7.5 (day 1) and 6.5 (day 7). Removal of the IOL and the lens fibres leads to a ten-fold increase in BrdU positive cells (mean = 84.5). No correlation with donor age was found. The Ki67 observations corroborate the BrdU results.The results demonstrate that after an initial rise in proliferative activity, as shown in the capsular bag model, the mitotic activity of LECs returns to a rate comparable to that in intact cultured non-cataractous lenses. As in control lenses, removal of lens fibres significantly elevated the proliferative activity of the remaining LECs. Suppression by newly formed differentiated lens fibres in the in vivo capsular bag may be responsible for this return to control levels of mitotic activity of LECs in the PCO specimens.     

Growth factor deposition in anterior subcapsular cataract

PURPOSE: To characterize immunohistochemically the distribution of growth factors and extracellular matrix (ECM) components in an anterior subcapsular cataract (ASC) and to determine the role of growth factors in the development of ASC. SETTING: Department of Ophthalmology, Wakayama Medical University, Wakayama, Japan. METHODS: During cataract surgery in 22 patients, anterior capsules with an ASC were obtained. Sections of each specimen were immunostained with a panel of antibodies against ECM components, growth factors, cytoskeletal components, and signal transduction-related molecules. RESULTS: Collagen types I, V, and VI; fibronectin; fibrillin-1; and latent transforming growth factor beta binding protein-1 (LTBP-1) were localized to the ECM in ASC tissues. Collagen IV was localized to the ECM and the capsule. Lens epithelial cells (LECs) were positive for alpha-smooth muscle actin (alphaSMA). Lens epithelial cells and ECM stained for transforming growth factor beta2 (TGFbeta2) and TGFbeta3 in all samples, but TGFbeta1 latency-associated peptide (TGFbeta1-LAP) were detected in some samples. Fibroblast growth factor-2 (FGF-2) and hepatocyte growth factor-alpha (HGF-alpha) were localized to the ECM. Lens epithelial cells with nuclear staining for Erk-1, the mitogen-activated protein kinase (MAP kinase) cascade-related molecule, and Smad3, 1 of the Smad family members involving TGFbeta signaling, were detected. CONCLUSIONS: Matrix components (ie, collagen types, fibronectin, fibrillin-1), as well as growth factors such as TGFbeta1-LAP, TGFbeta2, TGFbeta3, FGF-2, and HGF-alpha, were detected in ASC. Fibrillin-1 might serve as a repository for TGFbetas. These growth factors may modulate the phenotypic alteration and behavior of LECs. The MAP kinase cascade and TGFbeta signaling are both activated in LECs in ASC.     

A new method for documenting lens opacities

We tested an anterior segment camera and digital analyzer on 32 eyes of 22 patients to determine whether its measurement of lens opacities correlated with measurements obtained by a standardized clinical grading system. The lenses were graded clinically for nuclear opacity, nuclear color, cortical opacity, and posterior subcapsular opacity. The lenses were then photographed and analyzed with this new device, and the results were compared. The camera system showed good reproducibility. Its results correlated well with the clinical gradings for nuclear capacity (P = .001) and cortical opacity (P = .001) but less well with posterior subcapsular opacity (P = .3), although there were only seven eyes with posterior subcapsular opacities. This camera system could help document and follow up lens opacity with more accuracy and reproducibility than has been previously possible.     

Study of experimental cataract produced by sugar alcohols in the organ-cultured mammalian ocular lens

Freshly isolated rabbit lenses were cultured in isosmolar TC-199 medium or hyperosmolar medium containing 180 mM sorbitol or mannitol. These experiments were performed to investigate the probable effects of hyperosmolar media on lens clarity and the ability of lens fiber cells to synthesize membrane intrinsic protein, MP-26. The data from these experiments show that incubation in hyperosmolar medium causes depressed MP-26 synthesis, whereas the presence of sugar alcohols in the culture medium induced anterior and posterior subcapsular opacities. The cation levels of lenses incubated in iso- and hyperosmolar medium were also measured. Data from these experiments revealed that although the experimental lenses display prominent opacities, their cation levels are generally similar to those of control lenses. It is proposed that the observed lens opacities are due to the presence of sugar alcohols in the culture medium and not to hyperosmolar shock.     

Cortical and subcapsular cataracts: significance of physical forces

Cortical cataracts usually begin with either sharp limited clear fluid clefts, resulting in opaque spokes, or clear lamellar separations, resulting in cuneiform opacities. They do not commence prior to 45 years of age. From this age on an increase in lens nuclei hardening can be detected. Therefore, during disaccommodation in older lenses, mechanical shear stresses must develop between the soft remaining cortices and the harder nuclei. These shear stresses are significant regarding the different cortical ruptures in predisposed lenses: in a privileged radial direction (according to zonular traction) of the sharp limited cortical spokes, or in parallel microridges at the commencement of lamellar separations, as observed when a rubber surface slides against a harder object. In pure cortical cataracts the ion pump (K+ > Na+) and investigated metabolic parameters remain largely intact. Therefore, it is not surprising that, in contrast to subcapsular cataracts, subcapsular opacities do not occur. Subcapsular cataracts are known to be caused by a variety of factors: aging, diabetes, corticosteroids, iridocyclitis, or X-ray, among many others. In contrast to cortical cataracts, subcapsular cataracts were found to be closely associated with ion pump damage (Na+ > K+) and a variety of metabolic activity alterations. In subcapsular cataracts passive fluids (from the vitreous and camera anterior) enter externally through the lens capsule. This initially forms numerous free clear, secondary grey, subcapsular fluid vacuoles. If the ion pump (metabolic barrier) is more severely damaged fluids may also enter the lens nucleus (secondary grey nuclear cataract), which rarely results in intumescent cataract. In cortical and subcapsular cataracts and lens perforations the main cause of grey opalescence appears to be the result of lens proteins (water-soluble crystalline) coming into direct contact with free fluids (water). In cortical cataracts this happens in the area of sharp limited mechanical cortical ruptures (fluid clefts), and in subcapsular cataracts during passive, external fluid entry, resulting in subcapsular fluid vacuoles and opacities, and also later grey-white nuclear opacities. The importance of water contact with water-soluble lens crystallines in behalf of light scattering and turbidness also has been investigated experimentally.     

细胞浆信息传递介质Smad 4在兔晶状体上皮细胞增殖中的作用

目的：探讨细胞浆信息传递介质Smad4在兔晶状体上皮细胞增殖及转化中的作用。方法：将健康白色家兔63只随机分成实验组（49只）及对照组（14只）,实验组兔双眼行透明晶状体皮质吸除术,对照组兔不做处理。在术后1及3d,1周、1、2、3及5个月分别处死7只实验组兔及2只对照组兔,分别应用原位杂交及免疫组化技术检测晶状体上皮细胞中Smad 4mRNA及增殖细胞核抗原（proliferating cell nuclear antigen,PCNA）的表达。结果：正常兔晶状体上皮细胞胞浆中Smad 4mRNA表达呈弱阳性。术后1d晶状体赤道部及前囊下部Smad 4mRNA无表达,吸光度（A）值与术前相比,差异有显著意义（P＜0．01）;术后3d Smad 4mRNA呈阳性表达,且随时间延长表达逐渐增强,晶状体赤道部细胞在术后1周时Smad 4mRNA表达最强;晶状体前囊下部细胞在术中1个月时Smad 4mRNA表达最强。PCNA的表达在术后1d最强,之后逐渐减弱,术后1个月时恢复至术前水平。术后1－2个月,晶状体赤道部出现转化的成纤维细胞,Smad 4mRNA呈阳性表达。结论：Smad 4mRNA在正常晶状体上皮细胞胞浆中有表达,Smad 4参与晶状体上皮细胞的增殖代谢过程,可能促使晶状体上皮细胞转化为成纤维细胞。     

Tgfbeta receptor expression in lens: implications for differentiation and cataractogenesis

TGFbeta induces changes characteristic of some forms of cataract. However, the responsiveness of lens epithelial cells to TGFbeta is age-dependent; weanling and adult, but not neonatal, lens epithelial cells respond. This study investigated TGFbeta receptor (TbetaRI and TbetaRII) expression during rat lens development and the effects of FGF-2 on TGFbeta responsiveness and TbetaR expression. Immunofluorescence, immunoblotting, RT-PCR and in situ hybridization were used to examine the spatio-temporal expression patterns of TbetaR. Lens explants were used to investigate the effects of FGF-2 on TGFbeta responsiveness and TbetaR expression. In the lens epithelium, little or no immunoreactivity was detected at P3 but at P21 there was distinct reactivity for TbetaRI and TbetaRII. Reactivity for both receptors was also found in the differentiating fibers in the transitional zone and cortex at both ages. Western blotting of lens membrane extracts identified multiple molecular weight forms of TbetaRI (30, 50, 90 kDa) and TbetaRII (70-120 kDa). In situ hybridization with a rat probe for Alk5 (TbetaRI) showed that the lens expresses Alk5 mRNA in epithelium and fibers throughout development. A rat TbetaRII probe revealed distinct expression of a TbetaRII mRNA in lens fibers throughout development and in the lens epithelium at P21 but not at P3. In vitro studies showed that lens epithelial explants from P9 rats did not undergo cataractous changes in response to TGFbeta but P13 explants did. Addition of FGF-2 to P9 explants induced increased TbetaR immunoreactivity and enhanced the competency of lens epithelial cells to respond to TGFbeta. These data indicate that the overall increased expression of TGFbeta receptors in lens epithelium during postnatal development (P3-P21) underlies an age-related change in TGFbeta responsiveness. The results also suggest that lens cells may express multiple forms of TbetaR. Expression of TbetaR in lens fibers throughout lens development and the induction of enhanced TbetaR expression by FGF suggest a role for TGFbeta signaling during FGF-induced responses and fiber differentiation.     

The comparability of estimates of retroilluminated lens opacities as judged from film-based and digital imaging

PURPOSE: Neitz film-based retroillumination cameras, the standard for documenting retroilluminated lens opacities for epidemiologic studies, are no longer produced. A digital imaging system is now available for imaging these opacities. We sought to compare gradings of images from both systems. DESIGN: Comparison of technique. METHODS: One hundred fourteen lenses were imaged with both systems and graded according to protocols. Concordance between the methods was compared using kappa statistics. RESULTS: There was moderate concordance for cortical opacities (kappa = 0.63) and good concordance for posterior subcapsular opacities (kappa = 0.83). Grades from digital images slightly underestimated the frequency and severity of cortical cataract. CONCLUSION: Digital imaging of retroilluminated lens opacities results in similar classification of the severity of opacities. It will be useful for epidemiologic studies of cortical (CC) and posterior subcapsular cataracts (PSC).