In vitro studies in VCP-associated multisystem proteinopathy suggest altered mitochondrial bioenergetics.

Mitochondrial dysfunction has recently been implicated as an underlying factor to several common neurodegenerative diseases, including Parkinson's disease, Alzheimer's and amyotrophic lateral sclerosis (ALS). Valosin containing protein (VCP)-associated multisystem proteinopathy is a new hereditary disorder associated with inclusion body myopathy, Paget disease of bone (PDB), frontotemporal dementia (FTD) and ALS. VCP has been implicated in several transduction pathways including autophagy, apoptosis and the PINK1/Parkin cascade of mitophagy. In this report, we characterized VCP patient and mouse fibroblasts/myoblasts to examine their mitochondrial dynamics and bioenergetics. Using the Seahorse XF-24 technology, we discovered decreased spare respiratory capacity (measurement of extra ATP that can be produced by oxidative phosphorylation in stressful conditions) and increased ECAR levels (measurement of glycolysis), and proton leak in VCP human fibroblasts compared with age- and sex-matched unaffected first degree relatives. We found decreased levels of ATP and membrane potential, but higher mitochondrial enzyme complexes II+III and complex IV activities in the patient VCP myoblasts when compared to the values of the control cell lines. These results suggest that mutations in VCP affect the mitochondria's ability to produce ATP, thereby resulting in a compensatory increase in the cells' mitochondrial complex activity levels. Thus, this novel in vitro model may be useful in understanding the pathophysiology and discovering new drug targets of mitochondrial dynamics and physiology to modify the clinical phenotype in VCP and related multisystem proteinopathies (MSP).

2-ethylpyridine, a cigarette smoke component, causes mitochondrial damage in human retinal pigment epithelial cells in vitro.

PURPOSE: Our goal was to identify the cellular and molecular effects of 2-ethylpyridine (2-EP, a component of cigarette smoke) on human retinal pigment epithelial cells (ARPE-19) in vitro. MATERIALS AND METHODS: ARPE-19 cells were exposed to varying concentrations of 2-EP. Cell viability (CV) was measured by a trypan blue dye exclusion assay. Caspase-3/7 and caspase-9 activities were measured by fluorochrome assays. The production of reactive oxygen/nitrogen species (ROS/RNS) was detected with a 2',7'-dichlorodihydrofluorescein diacetate dye assay. The JC-1 assay was used to measure mitochondrial membrane potential (ΔΨm). Mitochondrial redox potential was measured using a RedoxSensor Red kit and mitochondria were evaluated with Mitotracker dye. RESULTS: After 2-EP exposure, ARPE-19 cells showed significantly decreased CV, increased caspase-3/7 and caspase-9 activities, elevated ROS/RNS levels, decreased ΔΨm value and decreased redox fluorescence when compared with control samples. CONCLUSIONS: These results show that 2-EP treatment induced cell death by caspase-dependent apoptosis associated with an oxidative stress and mitochondrial dysfunction. These data represent a possible mechanism by which smoking contributes to age-related macular degeneration and other retinal diseases and identify mitochondria as a target for future therapeutic interventions.

Increased expression of tenascin-C-binding epithelial integrins in human bullous keratopathy corneas.

We previously found an abnormal deposition of an extracellular matrix glycoprotein, tenascin-C (TN-C), in human corneas with pseudophakic/aphakic bullous keratopathy (PBK/ABK). In this work, we studied cellular TN-C receptors in normal and PBK/ABK corneas. Cryostat sections of normal and PBK/ABK corneas were stained by immuno-fluorescence for TN-C receptors: alpha2, alpha8, alpha9, alphaVbeta3, beta1, and beta6 integrins, and annexin II. Beta6 integrin mRNA levels were assessed by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) using beta2-microglobulin gene to normalize the samples. In PBK/ABK compared to normal corneas, relatively minor changes were observed for alpha2 and beta1 integrins, and for annexin II. Alpha8, alpha9, and beta6 subunits of TN-C receptors, alpha8beta1 alpha9beta1, and alphaVbeta6, respectively, were absent from normal central corneas but were found in the central epithelium of PBK/ABK corneas. Beta6 integrin showed the most significant accumulation. It correlated best with the expression of TN-C rather than with the expression of other alphaVbeta6 ligands, fibronectin, and vitronectin. RT-PCR analysis also showed elevated levels of beta6 mRNA in PBK/ABK compared to normal corneas. Therefore, accumulation of TN-C in PBK/ABK corneas was accompanied by an increased expression of its three binding integrins, especially alphaVbeta6 in the corneal epithelium. The interaction of tenascin-C with these integrins may contribute to the fibrotic process that occurs in PBK/ABK corneas.

Altered expression of growth factors and cytokines in keratoconus, bullous keratopathy and diabetic human corneas.

The purpose of this study was to identify the growth factors and cytokines present in normal and diseased corneas. Total RNA was isolated from normal and diseased corneas. cDNA was synthesized from individual corneas and semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) was performed with primers to IL-1alpha, 1IL-8, PDGF-B, BMP-2, BMP-4, IGF-I, TGF-beta2, FGF-2, and VEGF. After normalization to beta2-microglobulin, several factors were identified that were significantly different from normal. Antibodies to IGF-I, BMP-2, VEGF and TGF-beta2 were used for immunohistochemistry. A total of 93 corneas were used for this study including 31 normal, 20 keratoconus, 19 bullous keratopathy (pseudophakic and aphakic, PBK/ABK), and 23 diabetic corneas. The VEGF RNA levels were significantly decreased in the keratoconus and PBK/ABK corneas but increased in the diabetic corneas. BMP-2 gene expression was lower than normal in the PBK/ABK and diabetic corneas. IGF-I and BMP-4 RNA levels were increased in PBK/ABK. In the immunohistochemical studies, the protein patterns paralleled those found at the mRNA level. The only exception was IGF-I in diabetic corneas that showed increased staining in the epithelium and its basement membrane without a significant increase in mRNA levels. TGF-beta2 mRNA and protein levels were similar to normal in all diseased corneas. Thus, no alterations in the tested growth factors/cytokines were unique to keratoconus corneas. In contrast, PBK/ABK corneas had specific significant elevations of BMP-4 and IGF-I. Diabetic corneas were unique in their increased VEGF mRNA levels. These data suggest that while some growth factor/cytokine alterations are non-specific and can be found in multiple corneal diseases, there are others that are unique to that disease.

Effects of cholesterol and apolipoprotein E on retinal abnormalities in ApoE-deficient mice.

PURPOSE: To examine the pathologic changes in the retina of apolipoprotein E (apoE)-deficient mice fed a high-cholesterol diet. METHODS: ApoE-deficient mice (ApoE) were maintained on either regular mouse chow (ApoE-R) or a high-cholesterol diet (ApoE-C) for 25 weeks. Age-matched control C57BL/6J mice (C57) were also maintained on either regular mouse chow (C57-R) or a cholesterol-containing diet (C57-C). Retinal function was assessed by dark-adapted electroretinography (ERG). The eyes were embedded, sectioned, and analyzed by histologic and immunohistochemical methods, as well as by light and transmission electron microscopy. RESULTS: After the 25-week feeding period, ERG tracings of ApoE-C mice revealed significant increases of a- and b-wave implicit times when compared with the C57-R group of mice. In addition, there were reductions in oscillatory potential (OP) amplitudes in the ApoE-C group. However, a- and b-wave amplitudes appeared to be unchanged among the four groups of mice. Light microscopic examination of the retinas showed that compared with control C57-R mice, ApoE-C mice had significantly lower cell numbers in the inner and outer nuclear layers (85.1% +/- 4.6%, P < 0.05 and 81.4% +/- 3.7%, P < 0.01 of C57-R controls, respectively). Transmission electron microscopy of apoE-deficient mice revealed cells of the inner nuclear layer with condensation of nuclear chromatin and perinuclear vacuolization in focal areas. Bruch's membrane was also found to be thicker, and its elastic lamina appeared disorganized and discontinuous. Immunohistochemistry demonstrated diminished or no immunoreactivity for carbonic anhydrase II and calretinin in the retinal layers of apoE-deficient mice. CONCLUSIONS: Overall, there were increasing abnormalities of retinal function and cellular morphology among the four groups of mice in the order of C57-R < C57-C < ApoE-R < ApoE-C. These findings suggest that apoE and/or cholesterol play an important role in retinal function.

Identification of cell types in human diseased corneas.

PURPOSE: Activated myofibroblasts and macrophages are often found in corneal wound models. The current study was performed to determine whether human diseased corneas that had active tissue remodeling and enzyme activities also possessed myofibroblasts, macrophages, major histocompatibility complex class II cells, and/or CD-68-positive cells. METHODS: Normal, keratoconus, keratoconus with hydrops, bullous keratopathy, map-dot-fingerprint dystrophy, failed grafts, and acid burn/neovascularized corneas were collected, frozen in OCT, sectioned, and stained with antibodies to alpha smooth muscle actin (myofibroblast marker), CD14 (macrophage marker), CD68 (lysosomal membrane marker), and HLA-DR (major histocompatibility complex class II cells). Selective histochemical stains identified lysosomal enzymes. RESULTS: Normal and map-dot-fingerprint dystrophy corneas lacked antibody and enzyme staining. Keratoconus corneas were positive for CD68, HLA-DR, and lysosomal enzymes but were negative for CD14 and smooth muscle actin. Bullous keratopathy corneas had CD68-, CD14-, and HLA-DR-positive cells, relatively normal enzyme levels, and were smooth muscle actin-negative. Failed graft corneas had significant numbers of CD68-, CD14-, and HLA-DR-positive cells and increased acid phosphatase, but these corneas were smooth muscle actin-negative. Ulcerated and vascularized corneas had positive staining with all antibodies that were examined. Cultured stromal cells from normal corneas were CD68-positive, CD14-negative, and alpha smooth muscle actin-negative, and they produced lysosomal enzymes. CONCLUSIONS: The current study demonstrates that increased presence of lysosomal enzymes, corneal remodeling, and fibrosis can occur in the absence of myofibroblasts and/or macrophages.

Overexpression of matrix metalloproteinase-10 and matrix metalloproteinase-3 in human diabetic corneas: a possible mechanism of basement membrane and integrin alterations.

We have previously described decreased immunostaining of nidogen-1/entactin; laminin chains alpha1, alpha5, beta1,gamma1; and epithelial integrin alpha3beta1 in human diabetic retinopathy (DR) corneas. Here, using 142 human corneas, we tested whether these alterations might be caused by decreased gene expression levels or increased degradation. By semiquantitative reverse transcription-polymerase chain reaction, gene expression levels of the alpha1, alpha5, and beta1 laminin chains; nidogen-1/entactin; integrin alpha3 and beta1 chains in diabetic and DR corneal epithelium were similar to normal. Thus, the observed basement membrane and integrin changes were unlikely to occur because of a decreased synthesis. mRNA levels of matrix metalloproteinase-10 (MMP-10/stromelysin-2) were significantly elevated in DR corneal epithelium and stroma, and of MMP-3/stromelysin-1, in DR corneal stroma. No such elevation was seen in keratoconus corneas. These data were confirmed by immunostaining, zymography, and Western blotting. mRNA levels of five other proteinases and of three tissue inhibitors of MMPs were similar to normal in diabetic and DR corneal epithelium and stroma. The data suggest that alterations of laminins, nidogen-1/entactin, and epithelial integrin in DR corneas may occur because of an increased proteolytic degradation. MMP-10 overexpressed in the diabetic corneal epithelium seems to be the major contributor to the observed changes in DR corneas. Such alterations may bring about epithelial adhesive abnormalities clinically seen in diabetic corneas.

Hyaluronidase treatment, collagen fibril packing, and normal transparency in rabbit corneas.

PURPOSE: Hyaluronidase treatment is the initial step of corneaplasty, a treatment under development that induces stromal softening and involves the application of a custom designed forming lens to achieve modification of refractive error. The purpose of this investigation was to examine changes in the arrangement of stromal collagen fibrils after hyaluronidase treatment. METHODS: Rabbit corneas were evaluated by slit-lamp microscopy at 0, 2 and 7 days after treatment and haze was assessed by subjective observation. Molecular and interfibrillar Bragg spacing of corneal collagen were measured from synchrotron x-ray scattering patterns. Transmission electron microscopy and digital image analysis were used to calculate radial distribution functions from the positions of collagen fibrils. The calculated fibril sizes and positions were also used to predict the transmission of visible light through these corneas. RESULTS: Hyaluronidase-treated corneas were shown to have a decreased interfibrillar Bragg spacing of 15% to 21%. Fibril hydration did not change. Transparency of these corneas remained unaltered. CONCLUSIONS: Hyaluronidase reduced the hydration of the corneal stroma, which led to a more compacted collagen fibril arrangement. This compression was predicted to cause a small reduction in the transmission of visible light through the cornea but not to a point likely to cause visual impairment.

Everett Kinsey lecture. The elusive causes of keratoconus: a working hypothesis.

PURPOSE: Keratoconus is a diseasethat has been recognized clinically for many years. However, it is only more recently that a better understanding has been achieved in the area of keratoconus pathogenesis. The purpose of this paper is to summarize the completed research, review ongoing studies, and present a hypothesis for keratoconus pathology. METHODS: We used immunochemistry and molecular techniques to characterize keratoconus corneas. RESULTS AND CONCLUSIONS: Our hypothesis attempts to incorporate many of the recognized biochemical and molecular abnormalities found in the keratoconus corneas. Our hypothesis states: 1) there is abnormal processing of the free radicals and superoxides within the keratoconus corneas; 2) there is a build-up of destructive aldehydes or peroxynitrites within the corneas; 3) the cells that are damaged irreversibly undergo the process of apoptosis; and 4) the cells that are damaged reversibly undergo wound healing or repair. As part of the wound healing process, various degradative enzymes and wound healing factors are upregulated, which leads to focal areas of corneal thinning and fibrosis. Future studies will be directed to testthis working hypothesis and determine if these theories are valid.

Analysis of gene expression in human bullous keratopathy corneas containing limiting amounts of RNA.

PURPOSE: To validate the use of polymerase chain reaction (PCR)-amplified full-length cDNA as a substitute for mRNA in nucleic acid array and gene expression analysis. METHODS: Total RNA was isolated from age-matched normal autopsy corneas and pseudophakic bullous keratopathy (PBK) corneas. Full-length cDNA was generated and PCR amplified using the Smart cDNA synthesis technology. Southern blot analysis of this cDNA was compared with Northern blot analysis of the RNA. Amplified cDNA was used to probe a commercial gene array. By immunohistochemistry, the expression pattern of several adhesion molecules represented on the array was assessed. RESULTS: The cDNA produced by the Smart cDNA system gave results very similar to those of northern blot analysis when examined for beta2-microglobulin, Rab geranylgeranyl transferase, and tenascin-C. This cDNA obtained from normal or PBK corneas was labeled and used to probe a 588 gene array (Clontech). Among other differences, beta6 integrin was detected only with the PBK probe, beta-catenin was markedly elevated in PBK, and beta4 integrin appeared to be reduced in PBK. Immunohistochemical patterns of these proteins were consistent with the hybridization signals on the gene array. CONCLUSIONS: Smart cDNA synthesis and nucleic acid arrays were combined and validated for the first time to identify differential gene expression in normal and diseased corneas. These techniques require very little RNA such as that equivalent to a half of a single cornea, which is useful when the amount of tissue is limiting. Altered expression of adhesive proteins beta6 integrin and beta-catenin may be related to the formation of epithelial bullae and microcystic changes in PBK patients.

Basement membrane and growth factor gene expression in normal and diabetic human retinas.

PURPOSE: Recently, we found abnormal accumulation of several extracellular matrix components in retinal basement membranes in human diabetic retinopathy (DR). Others have described increased levels of various growth factors within the vitreous of DR patients. This study examined mRNA levels of these extracellular matrix components and growth factors within human retinal tissues. METHODS: Total retinal RNA was analyzed by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). RT-PCR products were identified by Southern blotting. Samples were normalized with respect to beta2-microglobulin cDNA. Twenty-one retinas were analyzed: 6 normal, 7 diabetic without DR and 8 diabetic with DR. RESULTS: In diabetic retinas without DR, the expression levels of most genes were similar to normal. In DR retinas, tenascin-C mRNA expression increased compared to both normal and diabetics without DR. By RT-PCR and Northern blotting, mainly small tenascin-C mRNA isoforms were expressed, and some of them were elevated in DR retinas. Fibronectin mRNA was elevated in DR compared to normal retinas, possibly due to the overexpression of extradomain A-containing isoform (ED-A+, or cellular fibronectin). In DR retinas, gene expression of vascular endothelial growth factor and placenta growth factor was elevated compared to normal, although mRNA for these growth factors receptors (VEGFR-1/Flt-1 and VEGFR-2/KDR) did not change significantly. Transforming growth factor-beta1 mRNA also increased in DR retinas. CONCLUSIONS: The data suggest that proliferative DR development may be associated with increased retinal expression of vascular endothelial growth factor, placenta growth factor and transforming growth factor-beta1 that possibly triggers the deposition of small tenascin-C isoforms in the blood vessel walls. Angiogenesis-stimulating tenascin-C may further promote diabetic retinal neovascularization.

Enhanced expression of a transmembrane phosphotyrosine phosphatase (LAR) in keratoconus cultures and corneas.

The purpose of the study was to identify genes that are differentially expressed in normal versus keratoconus corneas. Total RNA isolated from corneal stromal cell cultures was reverse-transcribed and then amplified by the polymerase chain reaction (PCR) using defined, arbitrary primers. The products were displayed on polyacrylamide gels and bands that were differentially expressed were excised, re-amplified and subcloned. The resulting clones were sequenced and utilized as probes for Northern blots with cultured cell RNA or Southern blots of corneal cDNA. One of the products that appeared to be more highly expressed in keratoconus cultures and corneas displayed 100% homology with leukocyte common antigen related protein (LAR), a transmembrane phosphotyrosine phosphatase. Western analyses and immunohistochemistry with monoclonal and/or polyclonal antibodies to LAR were used to examine keratocyte cultures and fresh frozen normal, keratoconus and pseudophakic bullous corneas. We identified a gene product with 100% homology to LAR that is expressed at the RNA level in keratoconus corneas and cell cultures but is found only at low or undetectable levels in normal cultures and normal and pseudophakic bullous keratopathy (PBK) corneas. By Western blotting and immunofluorescence with specific LAR antibodies, the protein was identified in keratoconus stromal cell cultures but not in normal cultures. When fresh frozen tissue was examined, LAR protein was localized to numerous stromal cells throughout central keratoconus corneas, while no central staining was seen in normal or bullous keratopathy corneas. LAR, a transmembrane phosphotyrosine phosphatase, is more highly expressed in keratoconus corneas and stromal cell cultures as demonstrated by differential display, Northern analyses, immunohistochemistry and Western blotting.

Extracellular matrix changes in human corneas after radial keratotomy.

Extracellular matrix and basement membrane alterations were identified in human corneas after radial keratotomy. Ten normal and five radial keratotomy autopsy corneas (two at 6 months post surgery, and three at 3 years post surgery) were studied by immunofluorescence with antibodies to 28 extracellular matrix and basement membrane components. Outside of radial keratotomy scars, all studied components had a normal distribution. Of stromal extracellular matrix, only type III collagen accumulated around the scars. The basement membrane around epithelial plugs had a normal composition except for type IV collagen. Its alpha1-alpha2 chains, normally present only in the limbal basement membrane, appeared around all plugs. alpha3 and alpha4 chains were very weak or absent in these areas, contrary to nonscarred areas. This basement membrane pattern was similar to the normal limbal but not to the central corneal pattern. Keratin 3 also had a limbal-like, suprabasal expression in the plug epithelium. The stroma around the scars accumulated tenascin-C, fibrillin-1, types VIII and XIV collagen, all of which were absent from normal corneal basement membrane and extracellular matrix. Only tenascin-C showed less staining in anterior scars 3 years post surgery than 6 months post surgery, but still persisted in posterior scars. Incomplete scar healing was evident even 3 years post radial keratotomy. It was manifested by the accumulation of abnormal extracellular matrix in the anterior and posterior scars and by the limbal-like pattern of type IV collagen isoforms in the basement membrane around epithelial plugs.

Human corneal epithelial basement membrane and integrin alterations in diabetes and diabetic retinopathy.

Corneas of diabetic patients have abnormal healing and epithelial adhesion, which may be due to alterations of the corneal extracellular matrix (ECM) and basement membrane (BM). To identify such alterations, various ECM and BM components and integrin receptors were studied by immunofluorescence on sections of normal and diabetic human corneas. Age-matched corneas from 15 normal subjects, 10 diabetics without diabetic retinopathy (DR), and 12 diabetics with DR were used. In DR corneas, the composition of the central epithelial BM was markedly altered, compared to normal or non-DR diabetic corneas. In most cases the staining for entactin/nidogen and for chains of laminin-1 (alpha1beta1gamma1) and laminin-10 (alpha5beta1gamma1 was very weak, discontinuous, or absent over large areas. Other BM components displayed less frequent changes. The staining for alpha3beta1 (VLA-3) laminin binding integrin was also weak and discontinuous in DR corneal epithelium. Components of stromal ECM remained unchanged even in DR corneas. Therefore, distinct changes were identified in the composition of the epithelial BM in DR corneas. They may be due to increased degradation or decreased synthesis of BM components and related integrins. These alterations may directly contribute to the epithelial adhesion and wound healing abnormalities found in diabetic corneas.

Expression of tenascin-C splice variants in normal and bullous keratopathy human corneas.

PURPOSE: To characterize the expression patterns of tenascin-C (TN-C) splice variants in normal corneas and in those affected by pseudophakic-aphakic bullous keratopathy (PBK-ABK). METHODS: Alternatively spliced variants of TN-C mRNA from normal and age-matched human corneas with PBK-ABK were analyzed by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) and Southern blot hybridization, using beta2-microglobulin as a housekeeping gene to normalize the samples. Normal and PBK-ABK corneas were studied by immunofluorescence and western blot analysis with antibodies to specific fibronectin type III-like (FN-III) repeats of TN-C. RESULTS: Tenascin-C mRNA expression was detected in epithelial, stromal, and endothelial cells of normal and PBK-ABK central corneas, although the protein was seen only in diseased corneas. Assessed by RT-PCR, PBK-ABK corneas expressed approximately three times more total TN-C mRNA than did normal corneas. Four major TN-C mRNA variants (with no FN-III insertional repeats or with retained insertional repeats D, A1, or A1+D) and three minor variants (with retained repeats A1+A2, A1+A2+D, or A1+A2+B+D) were much more abundant in PBK-ABK than in normal corneas. Repeat A1 was more abundant in PBK-ABK TN-C protein than repeats A2, A3, B, or D. Major TN-C variants in PBK-ABK corneas were in the range of 190 kDa to 240 kDa. CONCLUSIONS: Expression of TN-C mRNA and protein is higher in PBK-ABK corneas than in normal corneas. This increase mainly concerns relatively small TN-C splice variants that may affect corneal cell adhesion and migration and contribute to the exacerbation of PBK-ABK.

Increased expression of fibrillin-1 in human corneas with bullous keratopathy.

PURPOSE: To characterize the expression of fibrillins, microfibril components, in human corneas with pseudophakic/aphakic (PBK/ABK) bullous keratopathy. METHODS: Normal and PBK/ABK corneas were stained by immunofluorescence for fibrillin-1 and -2. The expression of fibrillin-1 messenger RNA (mRNA) was studied by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) and Southern analysis. RESULTS: Only fibrillin-1 was detected in normal and diseased corneas. As described previously, in normal corneas, it was found in the limbal stroma and basement membrane (BM) and in the peripheral corneal epithelial BM for a short distance near the limbus. Central corneal BM, stroma, and Descemet's membrane were negative. All PBK/ABK corneas were positive for fibrillin-1, which was detected in fibrillar deposits at the endothelial face of Descemet's membrane, in the epithelial BM, subepithelial fibrosis areas, and posterior collagenous layer. By RT-PCR, low levels of fibrillin-1 mRNA were detected in normal corneas, and they increased significantly in PBK/ABK corneas. CONCLUSION: The deposition of fibrillin-1, together with tenascin-C, in PBK/ABK corneas may be part of an abnormal fibrotic/wound-healing process that occurs during the development of postsurgical corneal edema with the formation of bullae and posterior collagenous layer.

Novel splice variants of human tenascin-C mRNA identified in normal and bullous keratopathy corneas.

PURPOSE: Pseudophakic/aphakic bullous keratopathy (PBK/ABK) human corneas accumulate an extracellular matrix glycoprotein tenascin-C (TN-C), an important modulator of cell adhesion and migration. Here, the purpose was to identify specific TN-C mRNA splice variants in normal and PBK/ABK human corneas. METHODS: Conventional and semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) with primers to alternatively spliced (insertional) and constitutive fibronectin type II-like repeats of TN-C was used. Splice variants were identified by cloning and sequencing of RT-PCR products or by Southern blot analysis. RESULTS: The majority of corneal TN-C mRNA species corresponded to relatively small forms of the protein. Four previously unidentified TN-C mRNA splice variants were found in normal and PBK/ABK corneas that contained insertional repeats A1+A2+B+D, A1+A2+D, A1+B+D, or A1+D. Variants with insertional repeats A1+A2 or A1, previously described in mouse and rat, were also identified in human corneas. Semiquantitative RT-PCR showed that novel TN-C mRNA variants were dramatically elevated in PBK/ABK compared to normal corneas. CONCLUSION: TN-C protein was found in PBK/ABK but not in normal corneas; however, both normal and diseased corneas contained mRNA for 15 different TN-C isoforms. PBK/ABK corneas had elevated levels of six relatively small TN-C mRNA variants including five novel ones. These specific isoforms may adversely affect adhesion and migration of corneal cells thus contributing to the exacerbation of PBK/ABK.

Localization of TIMP-1, TIMP-2, TIMP-3, gelatinase A and gelatinase B in pathological human corneas.

PURPOSE: Determine the tissue distribution patterns for tissue inhibitors of metalloproteinases (TIMP-1, TIMP-2, TIMP-3), gelatinase A and gelatinase B in normal and pathologic corneas. METHODS: Corneas were examined by immunohistochemistry, using antibodies to TIMP-1, TIMP-2, TIMP-3, gelatinase A or gelatinase B. RESULTS: In normal corneas, TIMP-1 antibody stained the epithelium and endothelium. TIMP-2 and TIMP-3 stained the epithelium, keratocytes and endothelium. Gelatinase A staining was weak and restricted to the epithelial cells. Radial keratotomy scars showed increased staining for TIMP-1 and TIMP-2 around the epithelial cell plug and along the incision. Bullous keratopathy corneas showed TIMP staining patterns similar to normal corneas and increased gelatinase A staining in regions of subepithelial fibrosis. Stromal scars of keratoconus corneas also had increased staining with TIMP-1 and TIMP-2 antibodies. In many keratoconus corneas, the TIMP-3 staining pattern was similar to normal corneas. However, in some keratoconus corneas, when Bowman's layer was missing, the stroma beneath was completely devoid of TIMP-3 antibody staining. No gelatinase B was seen in either the normal or diseased corneas. CONCLUSION: These data suggest that TIMP-1 and TIMP-2 are important for scar formation and corneal remodeling, since they were found in increased amounts at radial keratotomy incision sites and keratoconus scars. The significance of the focal stromal defects in TIMP-3 staining, associated with absence of Bowman's layer on keratoconus corneas, needs to be elucidated. At the stages of disease examined in this study, gelatinase B may not play a significant role in these pathological processes, since it was not seen in any of the corneas examined.

Alterations of corneal extracellular matrix after multiple refractive procedures: a clinical and immunohistochemical study.

PURPOSE: To describe the clinical course and alterations of the corneal extracellular matrix (ECM) and basement membrane (BM) in a cornea after hexagonal keratotomy, transverse keratotomies, and keratomileusis. METHODS: Frozen sections of this cornea and of 12 normal corneas were studied by immunofluorescence with specific antibodies. The patient history was analyzed to allow a clinical correlation. RESULTS: In the treated cornea, keratotomy scars and subepithelial fibrosis with neovascularization were seen. Around and beneath the epithelial plugs and along the keratotomy scars, deposits of types III, VI, VIII, and XIV collagen; fibrillin-1; fibronectin; and tenascin-C were found, together with short streaks of types IV (alpha 1-alpha 2) and VII collagen, laminin-1 and -5, entactin, and perlecan. alpha 3-alpha 4 Type IV collagen chains were abnormally absent from the BM around the epithelial plugs. At the edges of the keratomileusis flap, subepithelial fibrosis areas were found, with abnormal deposits of eight different collagen types, perlecan, fibronectin, fibrillin-1, and tenascin-C. The major part of the flap interface did not show ECM abnormalities. ECM alterations outside the scarred areas included the appearance of tenascin-C in the stroma and of alpha 1-alpha 2 type IV collagen in the epithelial BM, and the disappearance of fibronectin from Descemet's membrane. CONCLUSION: Five years after surgery, the treated cornea still presented BM abnormalities at sites of keratotomy scars and epithelial plugs. Several ECM components were abnormally expressed outside the scarred areas, consistent with an ongoing fibrosis in the treated cornea.

Abnormalities of the extracellular matrix in keratoconus corneas.

PURPOSE: To study alterations of the extracellular matrix (ECM) and basement membrane (BM) components in human keratoconus corneas. METHODS: Fifteen normal and 13 keratoconus corneas were characterized by immunofluorescence with antibodies to 23 ECM and BM components. RESULTS: Keratoconus staining patterns for posterior nonscarred regions and Descemet's membrane were normal. We focused on three areas of keratoconus corneas: (a) nonscarred anterior corneal regions, (b) scarred anterior and posterior corneal regions, and (c) gaps in Bowman's layer. In each of these areas, consistent ECM and BM changes could be found. Nonscarred regions showed decreased staining of the epithelial BM for entactin/nidogen, fibronectin, alpha 3-alpha 5 chains of type IV collagen, and chains of laminin-1. In contrast, scarred regions had greater than normal staining of the epithelial BM for these same components and also for laminin-5, perlecan, and type VII collagen. In the Bowman's layer gaps/breaks, focal fibrotic deposits containing type VIII collagen, fibrillin-1, tenascin-C, alpha 1-alpha 2 type IV collagen, and normal stromal ECM and epithelial BM components were seen. Fibrotic regions were largely restricted to areas where, because of the lack of Bowman's layer, the epithelium was in contact with the stroma. CONCLUSIONS: In a single keratoconus cornea, abnormalities in the ECM/BM patterns were not uniform. This may reflect locally increased protease activity (where few if any BM components are found) and ongoing wound healing (where more BM or ECM components or both are found).