Changes of extracellular matrix of the cornea in diabetes mellitus

Purpose

Differences in corneal viscoelasticity due to diabetes have been reported to have a protective effect on the progression of glaucoma and the development and progression of keratoconus. Due to longterm changes of tissue in diabetes mellitus, biomechanical changes of the cornea because of glycation and modified extracellular matrix may be detectable. The purpose of the study was to determine whether there is a difference in corneal biomechanical properties, characterized by corneal hysteresis (CH) and central corneal thickness (CCT), between diabetic and normal subjects, and relate these to the duration of diabetes.

Method

In a cross sectional study, a group of 484 eyes including 99 eyes of diabetic individuals was evaluated. CH as measured with the Ocular Response Analyzer, CCT (Orbscan II), Goldmann applanation tonometry (GAT) and slit-lamp examination were obtained from each patient. Linear mixed models were applied for statistical evaluation.

Results

CH showed a significant decrease with age (-0.036 mmHg/year, p?<?0.01) while CCT increased significantly (+0.7 µm/year, p?<?0.001). CH was significantly higher in diabetic eyes with an average difference of +0.55 mmHg (after correcting for age, IOP and CCT). This was not related to the duration of diabetes (mean 12.6?±?9.0y, p?=?0.522). CCT did not differ with regard to diabetes. Intraclass correlation coefficients were 81% and 50% for CCT and CH respectively.

Conclusion

CH is assumed to be an indicator for acquired changes of tissue such as diabetes-mediated. CCT is a more characteristic parameter for the individual patient. CH may provide more information about changes of the extracellular matrix in diabetes, and therefore offer a new monitoring parameter.     

Changes in extracellular matrix components after excimer laser photoablation in rat cornea

PURPOSE: To learn more about corneal wound healing after excimer laser photoablation. METHODS: Observations were made on the chronological changes in type I collagen, fibronectin, laminin, and type IV collagen after excimer ablation of rat cornea. Immunohistochemical techniques were used. RESULTS: There was no obvious change in the localization of type I collagen in the ablated area, but the localization of fibronectin, laminin, and type IV collagen changed remarkably. One day after ablation, immunofluorescent staining for fibronectin increased on the ablated surface. Subsequently, the fluorescence of fibronectin, laminin, and type IV collagen increased remarkably; in particular, the localization of these extracellular matrix proteins was sustained in the shallow layer of the stroma until about 24 weeks after ablation. Hematoxylin-eosin staining indicated that keratocytes temporarily disappeared 1 day after ablation, and activated keratocytes then migrated to the ablated areas. CONCLUSIONS: These results suggest that activated keratocytes might be synthesizing the extracellular matrix components. Therefore sustained responses of keratocytes may be induced by excimer laser photoablation.     

Immunohistochemical characterization of extracellular matrix in the developing human cornea

Collagen, fibronectin and laminin are important components of the extracellular matrix of the human cornea. We used the immunofluorescence technique with polyclonal antibodies directed against these proteins and to bullous pemphigoid antigen (BPA), in order to study their distribution in human corneas from 8 weeks of gestation to term and in adult corneas. Immunoreactivity was observed with antibodies to type I collagen in the limbus and the corneal stroma at 8 weeks of gestation. At 11 weeks of gestation it was found in epithelial basement membrane (EBM) and Descemet's membrane (DM) and continued thus throughout fetal and adult life. Type II collagen was not detected in fetal or adult cornea. Type III collagen was detected during 8-20th weeks of gestation in the EBM, DM and stroma. After 27th weeks of gestation, type III collagen could no longer be detected in the central cornea. Type IV collagen was detected in the EBM as early as 8 weeks of gestation and remained positive throughout fetal and adult life. Descemet's membrane was negative for type IV collagen at 8 weeks of gestation and became positive thereafter. Immunostaining for fibronectin in DM was negative at 8 weeks of gestation, followed by patchy staining of corneal stroma and EBM up to the age of 37 weeks of gestation. Staining in the EBM was negative or variable up to 70 years of age, and then became positive again in a 77 year old individual. Staining for LN was positive in the EBM after 8 weeks of gestation. Staining was negative in DM at that age, but became positive after 9 weeks of gestation. Staining for BPA was negative at 8-9 weeks of gestation, then gradually became positive.     

Immunogold fine structural localization of extracellular matrix components in aged human cornea

Using the immunogold technique combined with cryoultramicrotomy and London Resin white (LR white) embedding, we studied the fine structural distribution of types I–IV collagen and laminin in corneal tissue from seven enucleated human eyes (age range, 63–78 years). Type II collagen was not identified in any corneal layer. Type I and type III collagen were distributed in a similar fashion in striated collagen fibrils in Bowman's layer and in the stroma. Type IV collagen was located only in the posterior non-banded region of Descemet's membrane. Laminin was identified in subepithelial anchoring plaques and the sub-endothelial region of Descemet's membrane in accordance with its recognized adhesive function.Supported by the W.H. Ross Foundation for the Prevention of Blindness Offprint requests to: G.E. Marshall     

A review of manifestations of diabetes mellitus in the anterior eye and cornea

Diabetes mellitus can lead to blindness through its effects on the retina and lens, yet diabetes is a systemic disease influencing the entire eye. The current interest in extended wear contact lenses emphasizes the need for the practitioners to have a thorough knowledge of the pathophysiology of the anterior eye and cornea of the diabetic. The current literature is reviewed, and its relevance to contact lens wear is discussed.     

Localization of extracellular matrix proteins after holmium YAG laser radiation in rat cornea

Purpose: To understand corneal responses to holmium YAG (Ho:YAG) laser radiation, we used immunofluorescent microscopy to examine changes in the localization of extracellular matrix proteins. Methods: Rats were radiated with an Ho:YAG laser. On days 1, 3, and 7 after radiation, the eyes were enucleated and frozen. The cryosections were stained by immunofluorescent microscopy using antibodies against type I collagen, fibronectin, type IV collagen, and laminin.Results: One day after Ho:YAG laser radiation, contraction of the stromal collagen fibrils was observed. Keratocytes could not be observed at the radiated stromal region on day 1 after radiation. One week after radiation, keratocytes returned to the radiated area. Although the stromal collagen fibrils were contracted, they were stained by an antibody against type I collagen. Dense fluorescence for fibronectin was observed at the margin of the stromal acellular zone. Both laminin and type IV collagen were observed at the basement membrane under the corneal epithelium regardless of whether the corneas were radiated or not. Conclusions: These results suggest that Ho:YAG laser radiation might be useful for collagen contraction of the stroma, without causing serious damage to the corneal epithelium or the basement membrane.     

Localization of extracellular matrix proteins after holmium YAG laser radiation in rat cornea]

PURPOSE: To understand corneal responses to holmium YAG (Ho: YAG) laser radiation, we used immunofluorescent microscopy to examine changes in the localization of extracellular matrix proteins. METHODS: Rats were radiated with an Ho: YAG laser. On days 1, 3, and 7 after radiation, the eyes were enucleated and frozen. The cryosections were stained by immunofluorescent microscopy using antibodies against type I collagen, fibronectin, type IV collagen, and laminin. RESULTS: One day after Ho: YAG laser radiation, contraction of the stromal collagen fibrils was observed. Keratocytes could not be observed at the radiated stromal region on day 1 after radiation. One week after radiation, keratocytes returned to the radiated area. Although the stromal collagen fibrils were contracted, they were stained by an antibody against type I collagen. Dense fluorescence for fibronectin was observed at the margin of the stromal acellular zone. Both laminin and type IV collagen were observed at the basement membrane under the corneal epithelium regardless of whether the corneas were radiated or not. CONCLUSIONS: These results suggest that Ho: YAG laser radiation might be useful for collagen contraction of the stroma, without causing serious damage to the corneal epithelium or the basement membrane.     

Immunohistochemical study of localization of extracellular matrix after holmium YAG laser irradiation in rat cornea

PURPOSE: To better understand the corneal responses to holmium YAG (Ho:YAG) laser irradiation, we used immunofluorescent microscopy to examine changes in the localization of the extracellular matrix components, which play important roles in the maintenance of corneal morphology and functions. METHODS: Rats were irradiated with a Ho:YAG laser. On days 1, 3, and 7 after irradiation, the eyes were enucleated and frozen. Cryosections were made with a cryostat and were stained with antibodies against type I collagen, fibronectin, type IV collagen, or laminin for immunohistochemical study. RESULTS: One day after Ho:YAG laser irradiation, contraction of the stromal collagen fibrils was observed. Keratocytes could not be observed at the irradiated stromal region on day 1 after irradiation. One week later, however, keratocytes returned to the irradiated area. Although the stromal collagen fibrils had contracted, they were stained by an antibody against type I collagen. Dense fluorescence for fibronectin was observed at the margin of the stromal acellular zone. Both laminin and type IV collagen were observed at the basement membrane under the corneal epithelium, regardless of whether or not the corneas had been irradiated. CONCLUSION: These results suggest that Ho:YAG laser irradiation might be useful for the collagen contraction of stroma, without causing serious damage to the corneal epithelium and the basement membrane.     

Changes in the vascular wall in the conjunctiva of patients with diabetes mellitus

Venous capillary wall in type II diabetic patients' conjunctiva was examined. The study was performed on conjunctiva fragments removed from diabetic patients during the operation of senile cataract. The fragments were fixed in Lillie's solution then studied by using optical microscopy with usual but histochemical stainings, too. Conjunctiva fragments removed from patients of the same age, being operated by senile cataract and one conjunctiva from ten years old patient were examined by using the same techniques as they could be compared. Computer determinations of the external and internal diameter of the same capillary in all three groups were performed. Venous capillary wall thickening by type IV collagen hyperproduction in all the aged patients was noted. This kind of thickening is more revealed in aged diabetic person. Type I-III collagen presence in the capillary wall of the diabetic patients was noted, too. The average value of the differential between the external and internal diameter was increased in the diabetic patients with diabetic retinopathy. Venous capillary wall thickening in the diabetic patients is due to the basal membrana both by means of the type IV collagen hyperproduction and presence of the I-III collagen, too.     

Changes in extracellular matrix proteins and actin during corneal endothelial growth

Basement membranes influence growth, shape and differentiation of cells and tissues. However, the role and influence of Descemet's membrane during corneal development is not understood. To address this question, the relationships between cell growth and fibronectin, laminin and actin distribution in the developing rat corneal endothelium in vivo has been examined. During fetal development, rat corneal endothelial cells undergo DNA synthesis and mitosis. However, at day 14 of gestation both processes begin to decline and neither can be detected in endothelium of 1-month-old animals. By this time cell number has increased to approximately 100,000 and tissue area has increased 25-fold. However, as the tissue area increased, cell density decreased, indicating that cell spreading occurred in order to maintain tissue integrity. Changes in endothelial growth were accompanied by changes in the distribution of laminin, fibronectin and actin. Laminin and fibronectin were diffusely localized within endothelial cells in newborn animals. By 4 weeks of age, no proliferation was demonstrated and both extracellular matrix proteins were localized in pericellular patterns. Actin, on the other hand, which appeared diffuse at 16 days in utero, was distributed at or near the cell membrane by 19 days in utero. Thus, the reorganization of extracellular matrix glycoproteins and actin may indicate important roles for these components in regulating the growth and formation of the corneal endothelium in vivo.     

Changes of tear film and ocular surface in diabetes mellitus

This study was performed to investigate the changes of tear film and ocular surface in diabetic patients, as well as the ocular and systemic factors related to these changes. We assessed the scoring of keratoepitheliopathy, corneal sensitivity test, tear film break-up time (BUT), Schirmer test, and conjunctival impression cytology in 94 eyes of 47 patients with noninsulin-dependent diabetes mellitus and in 60 eyes of 30 normal subjects. The degree of keratoepitheliopathy was severe, and the corneal sensitivity, BUT, and tear secretion were significantly reduced in the diabetic patients. Conjunctival impression cytology showed a higher grade of conjunctival squamous metaplasia and lower goblet cell density in the diabetic patients. All parameters were related to the status of metabolic control, diabetic neuropathy, and stage of diabetic retinopathy. We think that diabetic patients with poor metabolic control, neuropathy, and advanced stage of retinopathy should be examined for tear film and ocular surface changes.     

Changes in the extracellular matrix of the human optic nerve head in primary open-angle glaucoma

Using immunofluorescent staining, we were able to characterize the changes in composition and distribution of the macromolecules making up the extracellular matrix of the lamina cribrosa of the glaucomatous human optic nerve head. In tissue adjacent to the glaucomatous cups, there was marked disorganization and loss of fibers of elastin within the cores of the cribriform plates. Collagen type VI, normally sparse, increased in quantity considerably throughout the lamina cribrosa in glaucomatous eyes with all degrees of damage. Collagen type IV and other basement membrane macromolecules appeared to extend into nerve bundles, presumably filling in spaces previously occupied by nerves. There was no appreciable change in the postlaminar region, which indicates the specificity of the extracellular matrix changes in the lamina cribrosa. Our results indicate that changes in the extracellular matrix play an important role in the progression of the glaucomatous process and may be a causative agent of the disease.     

Changes of meibomian glands in patients with type 2 diabetes mellitus

AIM: To investigate the morphological changes of meibomian glands in patients with type 2 diabetes mellitus (DM). METHODS: Of 118 eyes (118 patients) with type 2 DM (DM group) and 100 eyes of 100 control subjects (control group) were enrolled. After completing an ocular surface disease index (OSDI) questionnaire, the non-invasive tear film break-up time (NI-BUT) and the structure of the meibomian glands (MGs, meibography) were assessed by the Keratograph 5M system. Partial or complete loss of MG was scored for each eyelid from grade 0 (no loss) to grade 3 (lost area was >2/3 of the total MG area), which were also examined by laser scanning confocal microscopy (LSCM). The primary outcomes were meibomian gland acinar unit density (MGAUD), meibomian gland acinar longest diameter (MGALD) and meibomian gland acinar shortest diameter (MGASD). RESULTS: Compared with control group, the OSDI was significantly higher in DM group (Z=-5.916; P<0.001), while the NI-BUT was significantly lower (Z=-7.765; P<0.001). Keratograph showed that there were more MGs dropout in DM group than that in control group. The meiboscore was significantly higher in DM group compared with control group (Z=-3.937; P<0.001). LSCM revealed that there were cytological alterations of MGs in DM group compared with control group, which included enlargement of MG acinar units and decreased in density of MG acinar units. Specifically, there were lower MGAUD, larger MGALD and MGASD in DM group than control group (Z=-10.120, -9.4442, -7.771; P<0.001). CONCLUSION: Compared with the normal control participants, the patients with type 2 DM had more unstable tear films and severe symptoms of dry eye. Using Keratograph 5M system and LSCM, we found that the patients with type 2 DM had more significant morphological and cytological changes and dysfunction in MGs.     

Maspin: synthesis by human cornea and regulation of in vitro stromal cell adhesion to extracellular matrix.

PURPOSE: Maspin, a tumor-suppressor protein that regulates cell migration, invasion, and adhesion, is synthesized by many normal epithelial cells, but downregulated in invasive epithelial tumor cells. The purpose of this study was to determine whether cells in the normal human cornea express maspin and whether maspin affects corneal stromal cell adhesion to extracellular matrix molecules. METHODS: Maspin expression was analyzed by immunodot blot, Western blot, and RT-PCR analyses in cells obtained directly from human corneas in situ. Maspin protein and mRNA were also studied in primary and passaged cultures of corneal stromal cells using Western blot analysis, RT-PCR, and immunofluorescence microscopy. Maspin cDNA was cloned and sequenced from human corneal epithelial cells and expressed in a yeast system. The recombinant maspin was used to study attachment of cultured human corneal stromal cells to extracellular matrices. RESULTS: Maspin mRNA and micromolar amounts of the protein were found in all three layers of the human cornea in situ, including the stroma. Maspin was also detected in primary and first-passage corneal stromal cells, but its expression was downregulated in subsequent passages. Late-passage stromal cells, which did not produce maspin, responded to exogenous recombinant maspin as measured by increased cell adhesion not only to fibronectin, similar to mammary gland tumor epithelial cells, but also to type I collagen, type IV collagen, and laminin. CONCLUSIONS: The corneal stromal cell is the first nonepithelial cell type shown to synthesize maspin. Loss of maspin expression in late-passage corneal stromal cells in culture and their biological response to exogenous maspin suggests a role for maspin on the stromal cells in the cornea. Maspin may function within the cornea to regulate cell adhesion to extracellular matrix molecules and perhaps to regulate the migration of activated fibroblasts during corneal stromal wound healing.     

Immunogold fine structural localization of extracellular matrix components in aged human cornea II. Collagen types V and VI

Using immunogold immunocytochemical techniques we studied the distribution of collagen types V and VI in corneal tissue from seven enucleated human eyes (age range, 63–78 years). Results obtained by cryoultramicrotomy were marginally more intense than those obtained using London Resin white (LR white) embedding. Type V collagen was present in the striated collagen fibrils in Bowman's layer, in the stroma and in a thin, non-banded anterior zone of Descemet's membrane. Our results suggest that types I, IIl and V collagen co-distribute in striated collagen fibrils. By contrast, type VI collagen was located in fine filaments in the interfibrillar matrix of the stroma, in Bowman's layer and in the anchoring plaques of the sub-epithelial basement-membrane complex. This implies an importance in epithelial adhesion which was previously unsuspected. Keratocyte bodies were electron-dense, amorphous extracellular deposits of matrix-like material, and these were labelled with types III, V and VI collagen antibodies. Long-spacing collagen was observed in the corneal stroma, and this deposit did not contain any of the collagen types studied.Supported by the W.H. Ross Foundation for the Prevention of Blindness Offprint requests to: G.E. Marshall