Cannabinoid receptor 1 suppresses transient receptor potential vanilloid 1-induced inflammatory responses to corneal injury.

Cannabinoid receptor type 1 (CB1)-induced suppression of transient receptor potential vanilloid type 1 (TRPV1) activation provides a therapeutic option to reduce inflammation and pain in different animal disease models through mechanisms involving dampening of TRPV1 activation and signaling events. As we found in both mouse corneal epithelium and human corneal epithelial cells (HCEC) that there is CB1 and TRPV1 expression colocalization based on overlap of coimmunostaining, we determined in mouse corneal wound healing models and in human corneal epithelial cells (HCEC) if they interact with one another to reduce TRPV1-induced inflammatory and scarring responses. Corneal epithelial debridement elicited in vivo a more rapid wound healing response in wildtype (WT) than in CB1(-/-) mice suggesting functional interaction between CB1 and TRPV1. CB1 activation by injury is tenable based on the identification in mouse corneas of 2-arachidonylglycerol (2-AG) with tandem LC-MS/MS, a selective endocannabinoid CB1 ligand. Suppression of corneal TRPV1 activation by CB1 is indicated since following alkali burning, CB1 activation with WIN55,212-2 (WIN) reduced immune cell stromal infiltration and scarring. Western blot analysis of coimmunoprecipitates identified protein-protein interaction between CB1 and TRPV1. Other immunocomplexes were also identified containing transforming growth factor kinase 1 (TAK1), TRPV1 and CB1. CB1 siRNA gene silencing prevented suppression by WIN of TRPV1-induced TAK1-JNK1 signaling. WIN reduced TRPV1-induced Ca(2+) transients in fura2-loaded HCEC whereas pertussis toxin (PTX) preincubation obviated suppression by WIN of such rises caused by capsaicin (CAP). Whole cell patch clamp analysis of HCEC showed that WIN blocked subsequent CAP-induced increases in nonselective outward currents. Taken together, CB1 activation by injury-induced release of endocannabinoids such as 2-AG downregulates TRPV1 mediated inflammation and corneal opacification. Such suppression occurs through protein-protein interaction between TRPV1 and CB1 leading to declines in TRPV1 phosphorylation status. CB1 activation of the GTP binding protein, G(i/o) contributes to CB1 mediated TRPV1 dephosphorylation leading to TRPV1 desensitization, declines in TRPV1-induced increases in currents and pro-inflammatory signaling events.

Characterization of S-glutathionyl hemoglobin in homozygous sickle cell disease.

S-glutathionyl hemoglobin is a proposed biomarker of oxidative stress but has not been measured in sickle cell disease patients. Unlike the S-glutathionyl adduct of normal adult hemoglobin, S-glutathionyl sickle hemoglobin (HbSSG) cannot be directly measured by capillary isoelectric focusing, because it coelutes with fetal hemoglobin (HbF). This suggests that HbF, measured in sickle cell patients with or without hydroxyurea therapy, might contain endogenous HbSSG. As S-glutathionyl hemoglobin can form during sample storage, HbSSG could falsely elevate HbF levels in stored samples. We measured HbSSG based on the quantitative difference in the heterogeneous HbF/HbSSG peak before and after hemolysates were treated with dithiothreitol. Paired t tests showed that dithiothreitol reduced HbF/HbSSG in blood from pediatric sickle cell patients (n=25, mean decrease+/-SD=1.0%+/-0.6, P<0.05) but not in normal infants (n=25). Higher HbF levels in hydroxyurea-treated patients (n=5) were not attributable to HbSSG. HbSSG increased significantly within 1 day in samples stored at -20 degrees C but was unchanged in samples stored 60 days at-70 degrees C. We conclude that blood from sickle cell patients contained up to 2.2% HbSSG, and that endogenous HbSSG is a minor interferent in the measurement of HbF in fresh blood but a major interferent in improperly stored samples.

A case of novel de novo paired box gene 6 (PAX6) mutation with early-onset diabetes mellitus and aniridia.

BACKGROUND: Paired box gene 6 (PAX6) is a transcription factor involved in eye development. Mutations of PAX6 cause congenital eye anomalies, such as aniridia. PAX6 is also involved in the development of the endocrine pancreas, and reported to be a genetic factor common to aniridia and glucose intolerance, although the latter is usually mild. Here, we describe a case of PAX6 mutation with early-onset diabetes mellitus. CASE REPORT: A 27-year-old woman was referred to our clinic. She was diagnosed having diabetes at the age of 15 with negative glutamic acid decarboxylase (GAD) antibody. Insulin treatment was started at age 24. Because she had aniridia, PAX6 gene mutation was investigated and a heterozygous 2-bp deletion (c.402del2) was identified. Her parents did not have aniridia and PAX6 mutations. Heterozygous PAX6 mutation may cause glucose intolerance. However, cases of early-onset diabetes mellitus have not been reported. Her parents did not have diabetes, but their insulinogenic indices were low (0.25 and 0.3, respectively). We thought her early-onset diabetes was partly as a result of PAX6 mutation and partly because of an unknown insulin secretory defect inherited from her parents. We could not find any mutations in HNF-1alpha, -1beta, -4alpha, IPF-1, ISL-1, BEAT2/NeuroD1, PAX4, and amylin genes. CONCLUSIONS: We report a case of PAX6 gene mutation with early-onset diabetes mellitus and aniridia. Low insulin secretory capacity in her parents suggested that her insulin secretory defect is as a result of not only PAX6 mutation but other genetic factors inherited from her parents.

Pediatric Hodgkin's and non-Hodgkin's lymphomas: a retrospective analysis at the Children's Hospital of New Orleans.

Lymphomas constitute 10-12% of childhood cancers and are the third most common childhood malignancy. A retrospective analysis of thirty-six patients from the tumor registry of Children's Hospital of New Orleans was conducted during the period from 1995-2000. Patients were divided based on patient and tumor characteristics with recurrence and survival data compared to the Surveillance, Epidemiology and End Results (SEER) data of the National Cancer Institute. There were 18 patients (51%) with Non-Hodgkin's Lymphoma (NHL) and 17 (49%) with Hodgkin's disease (HD). Our sample had a similar distribution compared to the national population cohort except for different gender distribution in our HD patients. Also, most of our patients (63%) presented with advanced disease (Stages III and IV). Seventeen percent had recurrence of disease and 80% achieved remission, of which two patients developed secondary leukemia. Overall mortality was 17%. The survival in patients with HD was 94% which is comparable if not slightly superior to the national data. In patients with NHL, survival was 72% which was marginally lower than the national results (80%), most likely due to more advanced disease. Increased awareness in the pediatric community of the signs and symptoms of childhood lymphoma should result in earlier diagnosis and improved survival.

Advanced glycation end-products in sickle cell anaemia.

Tissue accumulation of advanced glycation end-products (AGEs) has been implicated in the oxidant-induced vascular pathology of diabetes and other diseases. Because homozygous sickle cell anaemia (SCA) is a state of oxidative stress, we tested the hypothesis that circulating AGE levels are elevated in SCA. Blood was obtained from age- and race-matched children classified as either non-sickle cell controls, SCA without vaso-occlusive crisis (SCA - VOC), or SCA with vaso-occlusive crisis (SCA + VOC). Plasma and red blood cell (RBC) AGE levels were measured by immunoassay. RBC levels of reduced (GSH) and oxidized (GSSG) glutathione were measured by capillary electrophoresis as an indicator of endogenous antioxidant status. The results showed that plasma AGE levels and the rate of RBC AGE accumulation were significantly higher in patients with SCA compared with controls. GSH was not different between groups but was significantly inversely correlated with plasma AGEs in both controls and patients with SCA. GSSG was significantly lower and GSH/GSSG higher in SCA + VOC patients, suggesting that GSH/GSSG might be an objective indicator of acute VOC or a risk factor for VOC. We conclude that circulating AGE levels are strongly influenced by endogenous antioxidant status and may play a role in the vascular pathology of SCA.

Deposition of silicone oil droplets in the residual anterior lens capsule after vitrectomy and lensectomy in rabbits.

AIM: To examine the histology of preserved anterior lens capsule in vitrectomised and lensectomised rabbit eyes with and without silicone oil tamponade. METHODS: Forty adult Japanese albino rabbits received two port vitrectomy and lensectomy with or without silicone oil tamponade in one eye under both general and topical anaesthesia. Anterior lens capsule was preserved during operation. After healing intervals residual anterior capsule was histologically observed under light or electron microscopy. RESULTS: Immediately after operation, cuboidal lens epithelial cells were observed on the posterior surface of the preserved anterior capsule. During healing intervals in eyes with or without silicone oil tamponade, regenerated lens structure of Sommerring's ring and fibrous tissue formed in the peripheral and central areas of the residual capsule, respectively. Ultrastructural observation revealed the presence of many vacuoles amid matrix accumulation on the posterior capsular surface, suggesting the deposition of emulsified silicone oil droplets. CONCLUSION: Lens epithelial cells produce regenerated lenticular structure and fibrous tissue on the residual capsule following vitrectomy and lensectomy in rabbits. Silicone oil droplets formed by its emulsification deposit in extracellular matrix accumulated on the posterior surface of the anterior capsule. Emulsified silicone may potentially enhance opacification of residual anterior capsule following pars plana vitrectomy by silicone oil deposition and subsequent activation of lens epithelial cells.

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.

Tubedown-1 in remodeling of the developing vitreal vasculature in vivo and regulation of capillary outgrowth in vitro.

Tubedown-1 (tbdn-1) is a mammalian homologue of the N-terminal acetyltransferase subunit NAT1 of Saccharomyces cerevisiae and copurifies with an acetyltransferase activity. Tbdn-1 expression in endothelial cells becomes downregulated during the formation of capillary-like structures in vitro and is regulated in vivo in a manner which suggests a functional role in dampening blood vessel development. Here we show that tbdn-1 is expressed highly in the vitreal vascular network (tunica vasculosa lentis and vasa hyaloidea propria) during the pruning and remodeling phases of this transient structure. The vitreal blood vessels of mice harboring a targeted inactivation of TGF-beta2 fail to remodel and abnormally accumulate, a phenomenon reminiscent of the ocular pathology resembling persistent fetal vasculature (PFV) in humans. Since suppression of normal tbdn-1 expression has been previously observed in retinal vessel proliferation, we analyzed vitreal vascular changes and tbdn-1 expression in TGF-beta2(-/-) eyes. The nuclei of vitreal vessel endothelial cells in TGF-beta2(-/-) eyes express proliferating cell nuclear antigen (PCNA) and exhibit increased levels of active (P42/44)mitogen-activated protein kinase (phospho-(P42/44)MAPK), characteristics consistent with proliferative endothelial cells. In contrast to normal vitreal vessels, collagen IV expression exhibited a disorganized pattern in the TGF-beta2(-/-) vitreal vessels, suggesting vessel disorganization and possibly a breakdown of vessel basal laminae. Moreover, vitreal vessels of TGF-beta2(-/-) mice lack expression of pericyte markers (CD13, alpha smooth muscle actin) and show ultrastructural changes consistent with pericyte degeneration. The accumulating vitreal blood vessels of TGF-beta2(-/-) mice, while maintaining expression of the endothelial marker von Willebrand Factor, show a significant decrease in the expression of tbdn-1. We addressed the functional role of tbdn-1 in the regulation of vitreal blood vessels using an in vitro model of choroid-retina capillary outgrowth. Clones of the RF/6A fetal choroid-retina endothelial cell line showing suppression of tbdn-1 levels after overexpression of an antisense TBDN-1 cDNA display a significant increase in the formation of capillary-like structures in vitro compared with controls. These findings suggest that tbdn-1 inhibits capillary-like formation in vitro and may serve to dampen vitreal blood vessel formation preceding the regression of the vitreal vasculature during development. Our results also suggest that tbdn-1 may participate with TGF-beta2 in regulating normal development of the vitreal vasculature.

Latent TGFbeta binding protein-1 and fibrillin-1 in human capsular opacification and in cultured lens epithelial cells.

BACKGROUND/AIM: It was previously reported that collagenous extracellular matrix (ECM) in human capsular opacification contained isoforms of transforming growth factor beta (TGFbeta). In the present study, the authors performed immunohistochemistry to examine whether ECM in human capsular opacification and in cultures of bovine lens epithelial cells (LECs) contained latent TGFbeta binding protein-1 (LTBP-1), TGFbeta1 latency associated peptide (beta1-LAP), and fibrillin-1, a suspected ligand of LTBP-1 as well as a component of the extracellular microfibrillar apparatus. The aim of the study was to further clarify the mechanism of TGFbeta1 deposition in ECM of capsular opacification. METHODS: Human capsular opacification specimens and uninjured lens capsules, as well as cultured bovine LECs, were processed for immunohistochemistry using antibodies against LTBP-1, beta1-LAP, fibrillin-1, and collagen type I. RESULTS: LTBP-1, beta1-LAP, and fibrillin-1 all were localised to the ECM in human capsular opacification. Uninjured lens epithelium stained for beta1-LAP, but not for LTBP-1 and fibrillin-1. ECM deposited in confluent LEC cultures stained for LTBP-1, beta1-LAP, and fibrillin-1, while cultures with only sparse cellularity were unstained for LTBP-1 or fibrillin-1. CONCLUSIONS: LECs upregulate LTBP-1 and fibrillin-1 during postoperative healing. LTBP-1, beta1-LAP, and fibrillin-1 colocalised to the ECM in capsular opacification and in confluent LEC cultures. TGFbeta1 is considered to deposit in ECM in the large latent form. ECM secreted by LEC may function as a scavenger or repository of TGFbeta.

Effects of antiinflammatory drugs on migration of the rabbit corneal epithelium.

PURPOSE: To evaluate and compare the effects of diclofenac sodium 0.1% and betamethasone phosphate 0.1% on corneal wound healing. SETTING: Department of Ophthalmology, Wakayama Medical College, Wakayama, Japan. METHODS: Using the method described by Nishida et al., corneal epithelial spreading was measured in vitro in a rabbit corneal block in the presence or absence of 2 antiinflammatory agents at various doses. RESULTS: At clinical doses (1 microg/mL and 10 microg/mL), the drugs did not suppress migration of the corneal epithelium. At high doses (20 microg/mL, 50 microg/mL, and 100 microg/mL), they did inhibit the migration. There was no between-group difference in corneal epithelial migration at clinical doses. At high doses, corneal epithelial migration was inhibited in the diclofenac sodium group compared with the betamethasone group. CONCLUSIONS: At clinical doses, diclofenac sodium and betamethasone did not inhibit corneal epithelial migration. However, these drugs should be prescribed cautiously in patient with high-risk diseases such as diabetes mellitus and glaucoma.

Lens epithelial cell regeneration of a capsule-like structure during postoperative healing in rabbits.

PURPOSE: To determine whether lens epithelial cells (LECs) can regenerate the lens capsule during healing after lens extraction and intraocular lens (IOL) implantation. SETTING: Department of Ophthalmology, Wakayama Medical College, Japan. METHODS: Extracapsular lens extraction and IOL implantation were performed in 5 adult albino rabbits. Lens capsules were examined histologically and immunohistochemically 3 and 5 months later. RESULTS: Lens epithelial cells proliferated and regenerated lens fibers within the capsular bag. A multilayered homogenous capsule-like structure was present in the equatorial region. The structures contained type IV collagen but not type I collagen. CONCLUSION: Lens epithelial cells can regenerate lens capsule-like structures during healing after lens extraction. Postoperative LECs without phenotypic conversion to a fibroblastic type may produce this structure.

Comparison of Scheimpflug images of posterior capsule opacification and histological findings in rabbits and humans.

PURPOSE: To compare the posterior capsule opacification in Scheimpflug photographic images produced by an electronic anterior eye segment analysis system with the histopathological findings in rabbits and humans. SETTING: Department of Ophthalmology, Wakayama Medical College, Japan. METHODS: Opacified posterior capsules were photographed using the EAS-1000 system (Nidek) and were then extracted during vitreous surgery for proliferative diabetic retinopathy or proliferative vitreoretinopathy in 2 patients. In rabbits, phacoemulsification and aspiration (PEA) with intraocular lens (IOL) implantation was performed. The IOL was implanted in the bag or in the sulcus. After intervals of healing, the posterior capsule was photographed with the EAS-1000 and the animals were then killed. In both clinical and experimental specimens, the posterior capsule was processed for light microscopic histology and immunohistochemistry. RESULTS: Opacified human capsules were well imaged by the EAS-1000. Histology showed that lens epithelial cells proliferated with and without an accumulation of extracellular matrix. Details such as rolling of the capsulotomy edge were seen well. Regenerated lens fibers of Soemmering's ring were seen as a mass within the capsule. In the rabbit model, Scheimpflug images accurately represented the capsules as they appeared histologically. CONCLUSION: The EAS-1000 system provided faithful, relatively high-resolution images that corresponded to the histologic findings in the posterior capsules after PEA-IOL surgery in humans and rabbits.

Smad translocation and growth suppression in lens epithelial cells by endogenous TGFbeta2 during wound repair.

To determine whether endogenous TGFbeta affects lens epithelial cells during repair after an anterior capsule injury in mice, we studied translocation of Smad proteins, which carry the TGFbeta signal from cell surface receptors to promoters in nuclei. We immunolocalized Smads in murine lenses at intervals up to 8 weeks following capsular injury. Effects of injecting TGFbeta neutralizing antibodies on Smad4 location and cell proliferation were examined at 24 hr after injury. Finally, we examined whether exogenous TGFbeta2 induced Smad nuclear translocation in murine lenses in organ culture. Cell proliferation was quantitated by 5-bromo-2'-deoxyuridine (BrdU) labelling. In uninjured lenses, Smads were located in the cytoplasm. In injured lenses, nuclear localization of Smads was observed in cells next to the capsular break from 8 to 24 hr after the injury, and was observed peripheral to the break at 48 hr. Nuclear Smads then continued to be observed occasionally in a minority of cells. Injection of antibodies neutralizing TGFbeta2, but not TGFbeta1 or TGFbeta3, inhibited Smad4 nuclear translocation and resulted in the appearance of BrdU-positive anterior epithelial cells. With the lenses in culture, transient nuclear localization of Smads occurred between 3 and 24 hr in response to continuous exposure to TGFbeta2. No nuclear translocation was seen at 48 hr. Endogenous TGFbeta2 affects lens cells during wound repair after anterior capsule injury, inhibiting lens cell proliferation during the early phase. Nuclear translocation of Smads in lens epithelial cells is transient even with continuous exposure to TGFbeta2.

Accumulation of latent transforming growth factor-beta binding protein-1 and TGF beta 1 in extracellular matrix of filtering bleb and of cultured human subconjunctival fibroblasts.

PURPOSE: To examine immunohistochemically whether extracellular matrix (ECM) of the filtering bleb and of cultured human subconjunctival fibroblasts contains latent TGF beta binding protein-1 (LTBP-1) and TGF beta. METHODS: An enucleated human eye that had undergone trabeculectomy and cultured human subconjunctival fibroblasts were processed for light microscopic immunohistochemistry. Antibodies against LTBP-1, collagen types, fibrillin-1 and TGF beta s were used. TGF beta 1 was located by detecting beta 1-latency associated peptide (LAP). RESULTS: LTBP-1, beta 1-LAP and fibrillin-1 were all located in the subepithelial ECM as well as in the basal epithelial cells of the conjunctiva over the filtering bleb. TGF beta 2 and beta 3 were immunolocated to epithelium and/or fibroblasts/keratocytes. ECM deposited in confluent fibroblast cultures was positive for beta 1-LAP, LTBP-1 and fibrillin-1, whereas sparse cells were negative. CONCLUSIONS: LTBP-1, beta 1-LAP and fibrillin-1 are co-localized to the ECM of the filtering bleb and of cultured conjunctival fibroblasts. Both conjunctival epithelium and fibroblasts are considered to be the source of TGF beta in healing bleb. ECM secreted by in vivo and in vitro subconjunctival fibroblasts may works as a scavenger or repository of TGF beta.

Characterization of Corn1 mice: Alteration of epithelial and stromal cell gene expression.

PURPOSE: Corn1 is an autosomal recessive mutation characterized by corneal epithelial hyperplasia and stromal neovascularization. The aim of the present study is to examine the expression patterns of specific epithelial and stromal proteins in corn/corn1 mutant mice. METHODS: Immunohistochemistry with antibodies directed against keratins 1, 4, 5, 12, and 14 as well as loricrin, filaggrin, and involucrin were performed in corn1/corn1 and wild type, A.By/SnJ strain, mice at 4 weeks of age. Western blot hybridization was performed to confirm the presence of involucrin in corneas. In situ and northern blot hybridization were used to evaluate the expression of keratin 12, lumican, and keratocan in these mice. RESULTS: In corn1/corn1 mice, focal areas of corneal epithelial hyperplasia alternate with epithelium with normal appearance. Both regions of normal and hyperplastic corneal epithelium were labeled by anti-keratin 12 antibodies through all corneal epithelial layers. The anti-keratin 14 antibody only labeled the basal cell layer in normal epithelial areas, whereas it labeled both basal and suprabasal cell layers in hyperplastic areas. In wild type mice, anti-keratin 12 antibodies labeled all corneal epithelial layers, whereas anti-keratin 14 labeled the basal corneal epithelial cells only. Positive staining by anti-involucrin antibody was demonstrated in the basal corneal epithelial layer of wild type mice and normal areas of corn1/corn1 mice. Similarly, as observed with anti-keratin 14 antibody, the anti-involucrin antibody labeled both basal and suprabasal cell layers of hyperplastic corneal epithelium of corn1/corn1 mice. Antibodies against keratin 1, keratin 4, loricrin, and fillagrin did not label the corneas of wild type mice or corn1/corn1 mice. Northern hybridization indicated that the expressions of keratocan and lumican mRNA levels were up regulated in corn1/corn1 mice, but keratin 12 mRNA remained similar to that of the wild type mice. In situ hybridization revealed that the lumican mRNA was detected in epithelial and stromal cells of corn1/corn1 mice, whereas keratocan mRNA was only detected in stromal cells. CONCLUSIONS: Hyperproliferative epithelial cells of corn1/corn1 mice have increased levels of expression of keratin 14 and involucrin, but do not exhibit the phenotypical characteristics of cornification. These observations indicate that factors associated with the phenotypes of corn1/corn1 mice do not alter the cornea-type epithelial differentiation of keratin 12 expression, but cause aberrant expression of lumican by corneal epithelial cells.

Extracellular matrix components in a case of retrocorneal membrane associated with syphilitic interstitial keratitis.

PURPOSE: A web-like retrocorneal membrane (RCM) is an uncommon complication of chronic syphilitic interstitial keratitis. Extracellular matrix components have not yet been defined in this structure, although previous histologic examinations have suggested the presence of collagen. We examined the presence and distribution of extracellular matrix components in a patient with an RCM. METHODS: A specimen of the opaque cornea affected by syphilitic interstitial keratitis with RCM formation was obtained during penetrating keratoplasty in a 62-year-old woman and was evaluated by histology, immunohistochemistry, and scanning electron microscopy (SEM). Antibodies against collagen types I, III, and IV; fibronectin; vimentin; alpha-smooth muscle actin (alpha-SMA); heat shock protein 47 (Hsp 47); proliferating cell nuclear antigen (PCNA); and Ki67 were used. RESULTS: Histologic analysis detected multiple concentric, acellular layers positive for collagen types I, III, and IV. The corneal endothelial cells (CECs) were positive for vimentin, collagen I, fibronectin, and Hsp 47 but not for alpha-SMA. Furthermore, the CECs were negative for PCNA and Ki67, indicating that they were not proliferating. SEM revealed the RCM was covered by CECs with a fibroblastic appearance. CONCLUSION: RCM associated with syphilitic interstitial keratitis contained collagen types I, III, and IV and fibroblast-like CECs. These CECs may secrete the extracellular matrix components found in the RCM. Hsp 47 up-regulation in the CECs may play an important role in RCM formation. These findings provide further insights into the phenotypic modulation of CECs.

Disappearance of desmosomal components in rat corneal epithelium during wound healing.

PURPOSE: Epithelial migration is essential for healing of the ablated corneal epithelium. To reveal the mechanism which enables the corneal epithelial cells to dissociate during migration, we investigated the immunolocalization of the components of the desmosome, which is the main constituent of the intercellular junction attaching the intermediate filaments to the cell surface, desmoplakin 1, desmoglein and plakoglobin, in the corneal epithelium during wound healing in rats. METHODS: Under general anesthesia with ether inhalation, Wistar rats (n = 48) underwent removal of the central corneal epithelium of one eye with a small trephine and a scalpel. After various intervals of healing (5 min; 1, 3, 6, 9, 12, 24, 48 h; 1 and 2 weeks), the animals were killed and the affected eye was excised. Cryosections of each anterior segment of the eye were fixed with cold acetone and treated with primary antibodies against desmoplakin 1, desmoglein and plakoglobin. Immunolocalization of these substances was visualized by the peroxidase-diaminobenzidine reaction. RESULTS: A marked immunoreactivity for these desmosome components was detected at the intercellular junction in the normal corneal and conjunctival epithelium. At 6-24 h after epithelial ablation, a weak immunoreactivity for desmoplakin 1 and plakoglobin was observed in the migrating epithelium. At 48 h after epithelial ablation, a marked immunoreactivity for these desmosome components was seen again. At 6-48 h after epithelial ablation, a weak immunoreactivity for desmoglein was observed in the migrating epithelium. At 1 week after epithelial ablation, a marked immunoreactivity for this desmosome component reappeared. The regenerated epithelium gradually exhibited normal immunolocalization of the proteins. CONCLUSIONS: The desmosome components were considered to be degraded or destroyed prior to epithelial migration and reconstructed during healing of the squamous epithelium.

Collagens XII and XIV (FACITs) in capsular opacification and in cultured lens epithelial cells.

PURPOSE: We previously reported that extracellular matrix (ECM) accumulation in human capsular opacification included collagen types I, III, IV, V, and VI. To further characterize the ECM in capsular opacification we performed immunohistochemistry to localize collagen types XII and XIV (fibril-associated collagens with interrupted triple helices, or FACITs) in specimens of human capsular opacification and in cultures of bovine lens epithelial cells (LECs). METHODS: Cryosections and paraffin sections of human capsular opacification specimens or uninjured lens capsules, as well as cultured bovine LECs, were processed for immunohistochemistry using antibodies against collagen types I to VI, XII, and XIV. A rat crystalline lens was punctured through the central cornea and the eye was processed for immunohistochemistry for FACITs after healing intervals. RESULTS: In the absence of injury human LECs were unstained for FACITs, but as early as 10 days after operation, LECs in healing capsules were immunoreactive. Collagen types I, III, IV, V, and VI were also detected. ECM deposited in confluent LEC cultures stained for FACITs. Normal rat LECs were not stained for FACITs, but ECM accumulated in injured lens stained for them. CONCLUSIONS: LECs up-regulate FACITs during post-opera-tive healing. FACITs, as well as other collagen types, are deposited in ECM in healing injured rat lens, in human capsular opacification and in LEC cultures. ECM components may regulate LEC behavior during postoperative healing.

TGFbeta2 in corneal morphogenesis during mouse embryonic development.

To examine the roles of TGFbeta isoforms on corneal morphogenesis, the eyes of mice that lack TGFbetas were analyzed at different developmental stages for cell proliferation, migration and apoptosis, and for expression patterns of keratin 12, lumican, keratocan and collagen I. Among the three Tgfb(-/-) mice, only Tgfb2(-/-) mice have abnormal ocular morphogenesis characterized by thin corneal stroma, absence of corneal endothelium, fusion of cornea to lens (a Peters'-like anomaly phenotype), and accumulation of hyaline cells in vitreous. In Tgfb2(-/-) mice, fewer keratocytes were found in stroma that has a decreased accumulation of ECM; for example, lumican, keratocan and collagen I were greatly diminished. The absence of TGFbeta2 did not compromise cell proliferation, nor enhance apoptosis. The thinner stroma resulting from decreased ECM synthesis may account for the decreased cell number in the stroma of Tgfb2 null mice. Keratin 12 expression was not altered in Tgfb2(-/-) mice, implicating normal corneal type epithelial differentiation. Delayed appearance of macrophages in ocular tissues was observed in Tgfb2(-/-) mice. Malfunctioning macrophages may account for accumulation of cell mass in vitreous of Tgfb2 null mice.

Expression of transcription factor AP-1 in rat lens epithelial cells during wound repair.

We examined the spatial and temporal expression patterns of proteins and mRNAs of the components of transcription factor activator protein 1 (AP-1) to examine the activation pattern of lens epithelial cells during lens wound repair following an anterior capsular injury. One eye of adult Wistar rats (n = 106) were used. After making a lens anterior capsule incision with a hypodermic needle, the affected eye was enucleated 0 and 30 min, 1, 3, 5, 8, 10, 15, 20, 24 hr after injury. Forty six globes were processed for in situ hybridization with oligonucleotide probes for c-fos, fosB, c-jun, junB and junD mRNAs, and 60 globes were immunohistochemically analysed using anti-c-Fos and anti-c-Jun antibodies. Normal lens epithelial cells expressed mRNA signals for junD, but not for c-fos, fosB, c-jun, and junB. mRNAs for c-fos, fosB, c-jun, and junB were detected in the whole lens epithelium from the vicinity to the wound to the equator from 30 min to 8 hr post-injury with their peaks after 30 min to 1 hr, but were no longer detected at 10 hr or later. Expression of c-fos mRNA in the equatorial lens cells was more marked than that of c-jun mRNA. Immunohistochemistry showed that c-Fos protein was expressed in the lens epithelial cells in both the anterior and equatorial regions of the injured lens from 1 to 10 hr after injury, and was no longer detected at 12 hr. C-Jun protein was detected only in the equatorial lens cells from 1 to 5 hr after injury, and was no longer detected at 8 hr. Lens epithelial cells except those in the equatorial region did not express c-Jun protein. These findings indicate that transcriptional activation of lens epithelial cells is initiated in the very early phase after the lens injury, i.e. 30 min post-injury, suggesting that AP-1 may play important roles in regulating lens cell behavior during lens wound repair in rats.