M. C. Kenney

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 non-scarred regions and Descemets 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 Bowmans 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, α3-α5 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 Bowmans layer gaps/breaks, focal fibrotic deposits containing type VIII collagen, fibrillin-1, tenascin-C, α1-α2 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 Bowmans 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).

Basement membrane abnormalities in human eyes with diabetic retinopathy.

Vascular and parenchymal basement membranes (BMs) are thickened in diabetes, but alterations in individual BM components in diabetic eyes, especially in diabetic retinopathy (DR), are obscure. To identify abnormalities in the distribution of specific constituents, we analyzed cryostat sections of human eyes obtained at autopsy (seven normal, five diabetic without DR, and 13 diabetic with DR) by immunofluorescence with antibodies to 30 BM and extracellular matrix components. In non-DR eyes, no qualitative changes of ocular BM components were seen. In some DR corneas, epithelial BM was stained discontinuously for laminin-1, entactin/nidogen, and alpha3-alpha4 Type IV collagen, in contrast to non-DR corneas. Major BM alterations were found in DR retinas compared to normals and non-DR diabetics. The inner limiting membrane (retinal BM) of DR eyes had accumulations of fibronectin (including cellular) and Types I, III, IV (alpha1-alpha2), and V collagen. The BM zone of new retinal blood vessels in neovascularized areas accumulated tenascin and Type XII collagen, whereas normal, diabetic, and adjacent DR retinas showed only weak and irregular staining. In preretinal membranes, perlecan, bamacan, and Types VI, VIII, XII, and XIV collagen were newly identified. Diabetic BM thickening appears to involve qualitative alterations of specific BM markers at an advanced disease stage, with the appearance of DR.

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

PURPOSEnTo 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.nnnMETHODSnFrozen 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.nnnRESULTSnIn 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 Descemets membrane.nnnCONCLUSIONnFive 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.

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 Descemets membrane were negative. All PBK/ABK corneas were positive for fibrillin-1, which was detected in fibrillar deposits at the endothelial face of Descemets 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.

CLEAVAGE OF HUMAN CORNEAL TYPE VI COLLAGEN ALPHA 3 CHAIN BY MATRIX METALLOPROTEINASE-2

The human cornea contains significant amounts of type VI collagen and matrix metalloproteinase-2 (MMP-2), but there has been no established relation between these two components. The objective of this study was to determine whether corneal type VI collagen was susceptible to digestion by MMP-2. Human corneas were frozen and then pulverized in liquid nitrogen. The type VI collagen was isolated by sequential extractions with sodium chloride buffer, guanidinium chloride solution, and guanidinium chloride/dithiothreitol solution. Visualization of type VI collagen alpha 3(VI) chain was made by using Western blots with specific monoclonal antibodies. The extracts were incubated up to 24 h with isolated, activated MMP-2. Within 4 h of incubation, two lower molecular weight bands (approximately 190 and 170 kDa) appeared. These bands increased in intensity with time but were not further digested into smaller fragments. This cleavage activity was inhibited by ethylenediaminetetraacetic acid (EDTA). Because type VI collagen represents approximately 25% of the corneal dry weight, its degradation properties may be important for the integrity of the stroma in scarring episodes and corneal diseases, such as keratoconus.

The Effects of Commercially Available Preservative-Free FDA-Approved Triamcinolone (Triesence®) on Retinal Cells in Culture

PURPOSEnTo evaluate the effects of Triesence® (TRI), a new preservative-free triamcinolone approved by the U.S. Food and Drug Administration (FDA) for intraocular use, on human retina pigment epithelial (ARPE-19) and rat neurosensory (R28) cells in culture.nnnMETHODSnARPE-19 and R28 cell cultures were treated 24 h with 1,000, 500, 200, or 100 μg/mL of crystalline (cTRI) or 1,000, 500, or 200 μg/mL of solubilized (sTRI). TRI was solubilized by centrifuging the drug, discarding the supernatant containing the vehicle and then resuspending the drug pellet in an equivalent amount of Dimethyl sulfoxide to achieve the same concentration as the commercial preparation. Percentage of cell viability (CV) was evaluated by a trypan blue dye-exclusion assay. The mitochondrial membrane potential (ΔΨm) was analyzed with the JC-1 assay. The caspase-3/7 activity was measured by a fluorochrome assay.nnnRESULTSnIn the ARPE-19 cultures, the cTRI caused a decrease in CV at 1,000 μg/mL (13.03±6.51; P<0.001), 500 μg/mL (28.87±9.3; P<0.001), 200 μg/mL (54.93±5.61; P<0.001), and 100 μg/mL (82.53±0.65; P<0.005) compared with the untreated controls (96.98±0.16). In R28 cultures, the cTRI treatment also reduced CV values significantly (P<0.001) for the 1,000 μg/mL (22.73±2.44), 500 μg/mL (34.63±1.91), 200 μg/mL (58.70±1.39), and 100 μg/m (75.33±2.47) compared with the untreated controls (86.08±3.54). Once the TRI was solubilized (sTRI), the CV and ΔΨm remained similar to the untreated controls for both ARPE-19 and R28 cells. The sTRI treatment with 1,000, 500, and 200 μg/mL increased in caspase-3/7 activity in ARPE-19 cells (P<0.01) and in R28 cells (P<0.05) compared with dimethyl sulfoxide equivalent controls.nnnCONCLUSIONnThe crystalline form of TRI (cTRI) can cause a significant decrease in CV to cultured retinal cells. Once the TRI is solubilized (sTRI), at the same concentrations, the cells remain viable with no decrease in CV or ΔΨm. The sTRI can, however, increase caspase-3/7 activity, thus suggesting some degree of apoptosis.

Effects of dexamethasone on human trabecular meshwork cells in vitro

PurposeTo study the effects of dexamethasone sodium phosphate (Dex) on human trabecular meshwork (HTM) cells in vitro.MethodsHTM cells were treated with Dex 2xa0mg/ml, 1xa0mg/ml, 0.5xa0mg/ml, 0.25xa0mg/ml, 0.1xa0mg/ml, or 0.05xa0mg/ml for 24xa0h. Cell viability was measured by a trypan blue exclusion test. Caspase-3/7, -8, -9 and -12 activities were measured by fluorochrome assays as mean signal intensity (msi) to assess apoptosis. Mitochondrial dehydrogenase activity was determined by a WST assay to quantify mitochondrial damage.ResultsMean cell viabilities of HTM cells exposed to Dex at the higher doses of 2xa0mg/ml, 1xa0mg/ml, and 0.5xa0mg/ml were reduced: 11.9xa0%u2009±u20093.5 (Pu2009<u20090.001), 31.2xa0%u2009±u20093.2 (Pu2009<u20090.001), and 76.6xa0%u2009±u20094.4 (Pu2009<u20090.01). At the lower doses of 0.25xa0mg/ml, 0.1xa0mg/ml or 0.05xa0mg/ml, no significant cell viability reductions were seen: 96.3xa0%u2009±u20090.7 (Pu2009>u20090.05), 95.3xa0%u2009±u20092.5 (Pu2009>u20090.05) and 93.8xa0%u2009±u20092.3 (Pu2009>u20090.05), respectively compared to untreated HTM cells (97.0xa0%u2009±u20091.9). Caspase-3/7 activity (msi) of HTM cells exposed to Dex 2, 1 or 0.5xa0mg/ml was 21068u2009±u20092498 (Pu2009<u20090.001), 26994u2009±u20093104 (Pu2009<u20090.001) and 20416u2009±u20091150 (Pu2009<u20090.001) compared to untreated HTM cells 1148u2009±u2009803. Caspase-9 activity (msi) of HTM cells after exposure to Dex 2, 1 or 0.5xa0mg/ml was 14188u2009±u20091203 (Pu2009<u20090.001), 13256u2009±u20091564 (Pu2009<u20090.001) and 15041u2009±u20091584 (Pu2009<u20090.001) compared to untreated HTM cells 1748u2009±u2009524. The lower doses of Dex did not significantly increase caspase-3/7 or -9 activities. There were no increases for caspase-8 or -12 activities at any of the tested Dex doses. The WST assay showed mitochondrial dehydrogenase activities of 14.3u2009±u20090.7 (Pu2009<u20090.001), 9.6u2009±u20090.3 (Pu2009<u20090.001) and 56.0u2009±u20097.6 (Pu2009<u20090.001) at 2xa0mg/ml, 1xa0mg/ml and 0.5xa0mg/ml Dex compared to untreated HTM cells (186.1u2009±u200915.0).ConclusionsDex at 0.25, 0.1 and 0.05xa0mg/ml clinical dose did not cause significant reduction in cell viability, increased apoptosis, or mitochondrial dysfunction of HTM cells in vitro. At high doses (2, 1 or 0.5xa0mg/ml) Dex caused apoptosis via mitochondrial pathways.

In vitro evidence for mycophenolic acid dose-related cytotoxicity in human retinal cells.

Purpose: Mycophenolic acid (MPA) is an immunosuppressive agent that controls noninfectious uveitis. Intravitreal MPA delivery may be a potential adjuvant therapy in patients who have to discontinue steroid or immunosuppressive systemic therapy because of side effects. The aims of this study are to evaluate the in vitro effects of MPA over human retinal pigment epithelium (ARPE-19) and human Muller cells (MIO M-1). Methods: ARPE-19 cells and MIO M-1 cells were exposed to 25, 50, and 100 µg/mL of MPA (Roche Bioscience, Palo Alto, CA) for 24 hours. Toxicity was evaluated by trypan blue dye-exclusion cell viability assay, caspase-3/7 apoptosis-related assay, and JC-1 mitochondrial membrane potential assay. Results: The MPA (25 µg/mL and 50 µg/mL) did not cause reduction in cell viability or significant change in caspase-3/7 activity in both cell lines tested. Mycophenolic acid (100 µg/mL) caused a significant decrease in cell viability (P < 0.01) and higher caspase-3/7 activity (P < 0.05) in both cell lines compared with untreated cells. The JC-1 mitochondrial membrane potential did not show statistically significant differences for both cell lines and all concentration tested when compared with untreated controls (P > 0.05). Conclusion: Intraocular delivery may be a potential alternative for the treatment of noninfectious uveitis, either by intravitreal injection or sustained-release drug-delivery systems, in doses of 50 µg/mL or lower.