Topical retinoic acid induces corneal strengthening by upregulating transglutaminase 2 in murine cornea.

Transglutaminase 2 (TG2) is the most abundant crosslinking enzyme in murine and human cornea, while retinoids are well-known inducers of TG2 expression. This study aims to determine if the retinoic acid supplementation can increase corneal stiffness by crosslinking through upregulating the corneal TG2 expression. The right eyes of C57BL/6 mice were treated with 2 × 10-2M retinol palmitate (VApal) eyedrops or control eyedrops and hold for 30 min, once a day for 28 consecutive days. The WB and qPCR results showed increased expression of TG2 in murine cornea with the prolongation of VApal eyedrop application. After 28 days of VApal eyedrop treatment, the increased TG2 were found catalytically active and distributed in corneal epithelium and stroma as detected by 5-(biotinamido) pentylamine (5-BP) incorporation method and immunofluorescence staining. The transmission electron microscope image revealed that VApal treated cornea manifested with increased collagen density in anterior and middle layer of stroma. The higher elastic module was found among VApal treated cornea by nano-indentation test. In cultured corneal epithelial cells and keratocytes, all-trans retinoid acid (ATRA) treatment increased the content of TG2 in cell lysis and in culture medium. These results indicate that retinoic acid induce the reinforcement of the cornea by TG2 mediated crosslinking via increasing the TG2 expression in corneal epithelium and keratocyte. As TG2 was found to be less in the cornea of keratoconus patients in several RNA-sequencing studies, retinoic acid could serve as a non-invasive prevention method for keratoconus progression.

Exacerbated corneal inflammation and neovascularization in the HO-2 null mice is ameliorated by biliverdin.

Heme oxygenase (HO-1 and HO-2) represents an intrinsic cytoprotective and anti-inflammatory system based on its ability to modulate leukocyte migration and to inhibit expression of inflammatory cytokines and proteins. HO-2 deletion leads to unresolved corneal inflammation and chronic inflammatory complications including ulceration, perforation and neovascularization. We examined the consequences of HO-2 deletion on hemangiogenesis and lymphangiogenesis in the model of suture-induced inflammatory neovascularization. An 8.0 silk suture was placed at the corneal apex of wild type and HO-2 null mice. Neovascularization was assessed by vital microscopy and quantified by image analysis. Hemangiogenesis and lymphangiogenesis were determined by immunofluorescence staining using anti-CD31 and anti-LYVE-1 antibodies, respectively. Inflammation was quantified by histology and myeloperoxidase activity. The levels of HO-1 expression and inflammatory cytokines were determined by real time PCR and ELISA, respectively. Corneal sutures produced a consistent inflammatory response and a time-dependent neovascularization. The response in HO-2 null mice was associated with a greater increase compared to the wild type in the number of leukocytes (827,600+/-129,000 vs. 294,500+/-57,510; p<0.05), neovessels measured by vital microscopy (21.91+/-1.05 vs. 12.77+/-1.55 mm; p<0.001) 4 days after suture placement. Hemangiogenesis but not lymphangiogenesis was more pronounced in HO-2 null mice compared to wild type mice. Induction of HO-1 in sutured corneas was greatly attenuated in HO-2 null corneas and treatment with biliverdin diminished the exaggerated inflammatory and neovascular response in HO-2 null mice. The demonstration that the inflammatory responses, including expression of proinflammatory proteins, inflammatory cell influx and hemangiogenesis are exaggerated in HO-2 knockout mice strongly supports the notion that the HO system is critical for controlling the inflammatory and neovascular response in the cornea. Hence, pharmacological amplification of this system may constitute a novel therapeutic strategy for the treatment of corneal disorders associated with excessive inflammation and neovascularization.

Dose-dependent in vitro inhibition of rabbit corneal matrix metalloproteinases by an extract of Pothomorphe umbellata after alkali injury.

The in vitro ability of Pothomorphe umbellata ethanolic crude extract to inhibit matrix metalloproteinase (MMP) in normal cornea and in cornea after alkali injury was demonstrated. Corneas of albino rabbits were injured with 1 N NaOH for 20 s. After 48 h the corneas were excised, homogenized and analyzed for MMP-9 (92 kDa), pro-MMP-2 (72 kDa) and MMP-2 (67 kDa) activity by gelatin zymography. The activity was also measured in untreated corneas. After electrophoresis of 20 microg protein, gels were incubated with 50, 100, or 250 microg/mL lyophilized hydroethanolic (1:1) root crude extract of P. umbellata standardized for 4-nerolidylcatechol (7.09%). The activity of the enzymes was compared with that of untreated gel. At 48 h after injury, the activity of all MMPs was increased compared with untreated eyes. When the gels were incubated with P. umbellata extract the activity of MMP-2, pro-MMP-2 and MMP-9 decreased in a dose-dependent manner. MMP-9 activity decreased by approximately 50% after incubation with 50 microg/mL and was completely abolished at 100 and 250 microg/mL of the extract. After incubation with 50 microg/mL the activity of pro-MMP-2 and MMP-2 also decreased by 50%. The activity of pro-MMP-2 was almost completely abolished after incubation with 250 microg/mL of the extract. For MMP-2 the incubation with 100 or 250 microg/mL of the extract of P. umbellata promoted a 10-fold decrease in activity. In conclusion, P. umbellata root crude extract can be useful as an alternative therapy to control MMP activity after corneal injury.

Dose-dependent in vitro inhibition of rabbit corneal matrix metalloproteinases by an extract of Pothomorphe umbellata after alkali injury

The in vitro ability of Pothomorphe umbellata ethanolic crude extract to inhibit matrix metalloproteinase (MMP) in normal cornea and in cornea after alkali injury was demonstrated. Corneas of albino rabbits were injured with 1 N NaOH for 20 s. After 48 h the corneas were excised, homogenized and analyzed for MMP-9 (92 kDa), pro-MMP-2 (72 kDa) and MMP-2 (67 kDa) activity by gelatin zymography. The activity was also measured in untreated corneas. After electrophoresis of 20 æg protein, gels were incubated with 50, 100, or 250 µg/mL lyophilized hydroethanolic (1:1) root crude extract of P. umbellata standardized for 4-nerolidylcatechol (7.09 percent). The activity of the enzymes was compared with that of untreated gel. At 48 h after injury, the activity of all MMPs was increased compared with untreated eyes. When the gels were incubated with P. umbellata extract the activity of MMP-2, pro-MMP-2 and MMP-9 decreased in a dose-dependent manner. MMP-9 activity decreased by approximately 50 percent after incubation with 50 µg/mL and was completely abolished at 100 and 250 µg/mL of the extract. After incubation with 50 µg/mL the activity of pro-MMP-2 and MMP-2 also decreased by 50 percent. The activity of pro-MMP-2 was almost completely abolished after incubation with 250 µg/mL of the extract. For MMP-2 the incubation with 100 or 250 µg/mL of the extract of P. umbellata promoted a 10-fold decrease in activity. In conclusion, P. umbellata root crude extract can be useful as an alternative therapy to control MMP activity after corneal injury.

Chromium-picolinate induced ocular changes: Protective role of ascorbic acid.

Chromium-picolinate (Cr-picolinate) is a popular nutritional supplement; however its safety has been questioned with regard to its ability to act as a clastogen. The aim of the present work was to evaluate the biochemical, histological and morphological changes in the cornea and lens following oral administration of Cr-picolinate and the possible protective effect of Vitamin C. Ninety male Sprague-Dawley rats were divided into five groups included the control group, the groups treated with Cr-picolinate (0.8 and 1.5 mg/100 g b.w.) alone or in combination with Vitamin C (0.5 mg/100 g b.w.) for 8 weeks. The results indicated that the high dose of Cr-picolinate induced a significant decrease in SOD, GSH, Na(+)-, K(+)-ATPase levels, and a significant increase in MDA level. Severe morphological and histological changes in the cornea and lens accompanied with a decrease in the total soluble protein of the lens homogenate and changes in the crystalline fractions in lens. Vitamin C supplementation succeeded to restore these changes to great extent. It could be concluded that consumption of Cr-picolinate for a long time induced several hazards to cornea and lens. Supplementation with extra amounts of Vitamin C may be useful to restrain the Cr-picolinate induced ocular changes.

Inhibitors of cytochrome P450 4A suppress angiogenic responses.

Cytochrome P450 enzymes of the 4A family (CYP4A) convert arachidonic acid to 20-hydroxyeicosatetraenoic acid (20-HETE) in blood vessels of several vascular beds. The present study examined the effects of inhibiting the formation of 20-HETE with N-hydroxy-N'-(4-butyl-2-methylphenol) formamidine (HET0016) on the mitogenic response of vascular endothelial growth factor (VEGF) in human umbilical vein endothelial cells (HUVECs) in vitro, and on growth factor-induced angiogenesis in the cornea of rats in vivo. HET0016 (10 micromol/L and 20 microg, respectively) abolished the mitogenic response to VEGF in HUVECs and the angiogenic response to VEGF, basic fibroblast growth factor, and epidermal growth factor in vivo by 80 to 90% (P < 0.001). Dibromododecenyl methylsulfonimide (DDMS), a structurally and mechanistically different inhibitor of 20-HETE synthesis, also abolished angiogenic responses when tested with VEGF. Additionally, administration of the stable 20-HETE agonist, 20-hydroxyeicosa-6(Z) 15(Z)-dienoic acid (WIT003) induced mitogenesis in HUVECs and angiogenesis in the rat cornea in vivo. We studied the ability of HET0016 to alter the angiogenic response in the rat cornea to human glioblastoma cancer cells (U251). When administered locally into the cornea, HET0016 (20 microg) reduced the angiogenic response to U251 cancer cells by 70%. These results suggest that a product of CYP4A product, possibly 20-HETE, plays a critical role in the regulation of angiogenesis and may provide a useful target for reduction of pathological angiogenesis.

The protective effect of alpha-lipoic acid against oxidative damage in rabbit conjunctiva and cornea exposed to ultraviolet radiation.

PURPOSE: The purpose of this study was to determine the protective effect of alpha-lipoic acid against oxidative damage in rabbit conjunctiva and cornea exposed to ultraviolet radiation. METHODS: 20 rabbits weighing 2,500- 3,000 g were used, and we divided them into 4 groups with 5 randomly selected rabbits. The rabbits were exposed to 2 J/cm(2)/h of ultraviolet A radiation (UVA) in the range of 320-405 nm for 12 h per day within 90 days. The control group did not undergo any procedure, the UVA group was only exposed to UVA radiation. The PUVA group was treated with 8-methoxypsoralen and UVA. The alpha-lipoic acid group was administered 8-methoxypsoralen + UVA + alpha-lipoic acid. At the end of 90 days, the rabbits were killed by decapitation, and the eyes were enucleated. Both eyes of each rabbit were used for biochemical evaluation. Conjunctival and corneal free malondialdehyde (MDA), glutathione peroxidase (GSH-PX) and superoxide dismutase (SOD) levels were compared among the groups. RESULTS: Conjunctival free MDA levels were lower in the alpha-lipoic acid group compared with the UVA and PUVA groups (p < 0.05, p < 0.001, respectively). Both conjunctival SOD levels (p < 0.05, p < 0.01, respectively) and conjunctival GSH-PX levels (p < 0.01, p < 0.001, respectively) were higher in the alpha-lipoic acid group compared with other groups. Corneal free MDA levels were lower in the alpha-lipoic acid group compared with the UVA and PUVA groups (p < 0.01, p < 0.001, respectively). Both corneal SOD levels (p < 0.01, p < 0.01, respectively) and corneal GSH-PX levels (p < 0.01, p < 0.01, respectively) were higher in the alpha-lipoic acid group compared with the other groups. CONCLUSION: alpha-Lipoic acid which is considered as potent antioxidant protects the eye from the damaging effect of ultraviolet exposure.

The effect of excimer laser keratectomy on corneal glutathione peroxidase activities and aqueous humor selenium levels in rabbits.

BACKGROUND: The formation of free oxygen radicals has been demonstrated in the corneal tissue after 193 nm laser irradiation. Cornea has several defense mechanisms that protect against oxidative damage. One of them, glutathione peroxidase (GPx), catalyzes the destruction of hydrogen peroxide and lipid hydroperoxide. Selenium is a trace element which is incorporated into the selenoenzyme GPx. In the present study, the effect of excimer laser keratectomy on corneal GPx activities and aqueous humor selenium concentrations in rabbits was evaluated. METHODS: Animals were divided into five groups, and all groups were compared: controls (group 1), after epithelial scraping (group 2), transepithelial photorefractive keratectomy(PRK; group 3), superficial traditional PRK (50 microm; group 4) and deep traditional PRK (100 microm; group 5). Corneal GPx activities were measured by a modification of the coupled assay procedure. Aqueous humor selenium concentrations were determined using hydride generation atomic absorption spectrometry. RESULTS: Corneal GPx activities were significantly lower only in group 5 ( P<0.05), and the selenium concentration in the aqueous humor did not change in any group. CONCLUSION: Deep corneal photoablation inhibits GPx enzyme activities in the cornea. Therefore, antioxidants may be useful in reducing free radical-mediated complications after excimer laser corneal photoablation.

Corneal stimulation of MMP-1, -9 and uPA by platelet-activating factor is mediated by cyclooxygenase-2 metabolites.

PURPOSE: This study was undertaken to evaluate the significance of cyclooxygenase-2 (COX-2) activity on urokinase plasminogen activator (uPA) and matrix metalloproteinases (MMPs)-1 and -9 induction in cornea following platelet-activating factor (PAF) treatment. METHODS: Corneal organ cultures were pre-treated with increasing concentrations of COX-2-specific inhibitors NS398 or nimesulide prior to PAF stimulation. To determine the effect of exogenous prostaglandins (PGs) on uPA, MMP-1 and MMP-9 levels, corneas were pre-treated with COX-2 inhibitors followed by the addition of 2.5 microM PGD2, PGE2 or PGF2alpha. The levels of uPA and MMP-9 were assayed by casein and gelatin zymography, respectively. MMP-1 levels were determined by Western Blot analysis. RESULTS: The increase in uPA, MMP-9 and MMP-1 levels detected in corneal organ cultures treated with 100 nM cPAF was blocked by 5 microM NS398 and 10 microM nimesulide, concentrations at which these inhibitors selectively inhibit COX-2 activity. Furthermore, pre-incubation with COX-2 inhibitors, followed by supplementation with PGD2, PGE2 or PGF 2alpha, increases uPA, MMP-9 and MMP-1 levels in corneas similar to and in some cases greater than that produced by cPAF treatment alone. CONCLUSIONS: During corneal injury and inflamation, PAF is an important factor in the activation of proteolytic cascades, which could lead to corneal epithelial defects and ultimately ulceration. One important goal in treating these defects is to modulate the activity of enzymes that destroy the extracellular matrix. Our results suggest that COX-2 induction following PAF stimulation and subsequent eicosanoid release may play a crucial role in the induction of uPA, MMP-1 and MMP-9 enzymes. Specific COX-2 inhibition could therefore block the actions of PAF when inflammation is sustained.

cDNA cloning of 15-lipoxygenase type 2 and 12-lipoxygenases of bovine corneal epithelium.

Bovine corneal epithelium contains arachidonate 12- and 15-lipoxygenase activity, while human corneal epithelium contains only 15-lipoxygenase activity. Our purpose was to identify the corneal 12- and 15-lipoxygenase isozymes. We used cDNA cloning to isolate the amino acid coding nucleotide sequences of two bovine lipoxygenases. The translated sequence of one lipoxygenase was 82% identical with human 15-lipoxygenase type 2 and 75% identical with mouse 8-lipoxygenase, whereas the other translated nucleotide sequence was 87% identical with human 12-lipoxygenase of the platelet type. Expression of 15-lipoxygenase type 2 and platelet type 12-lipoxygenase mRNAs were detected by Northern analysis. In addition to these two lipoxygenases, 12-lipoxygenase of leukocyte (tracheal) type was detected by polymerase chain reaction (PCR), sequencing, and Northern analysis. Finally, PCR and sequencing suggested that human corneal epithelium contains 15-lipoxygenase types 1 and 2.

Discovery of a second 15S-lipoxygenase in humans.

The lipoxygenase metabolism of arachidonic acid occurs in specific blood cell types and epithelial tissues and is activated in inflammation and tissue injury. In the course of studying lipoxygenase expression in human skin, we detected and characterized a previously unrecognized enzyme that at least partly accounts for the 15S-lipoxygenase metabolism of arachidonic acid in certain epithelial tissues. The cDNA was cloned from human hair roots, and expression of the mRNA was detected also in prostate, lung, and cornea; an additional 16 human tissues, including peripheral blood leukocytes, were negative for the mRNA. The cDNA encodes a protein of 676 amino acids with a calculated molecular mass of 76 kDa. The amino acid sequence has approximately 40% identity to the known human 5S-, 12S-, and 15S-lipoxygenases. When expressed in HEK 293 cells, the newly discovered enzyme converts arachidonic acid exclusively to 15S-hydroperoxyeicosatetraenoic acid, while linoleic acid is less well metabolized. These features contrast with the previously reported 15S-lipoxygenase, which oxygenates arachidonic acid mainly at C-15, but also partly at C-12, and for which linoleic acid is an excellent substrate. The different catalytic activities and tissue distribution suggest a distinct function for the new enzyme compared with the previously reported human 15S-lipoxygenase.

Changes in rat corneal matrix metalloproteinases and serine proteinases under vitamin A deficiency.

PURPOSE: Vitamin A deficiency alters the transparency of the cornea due to epithelial cell keratinization and increases the susceptibility of the cornea to ulceration. The purpose of this study was to determine the effect of vitamin A deficiency on rat corneal matrix metalloproteinases and serine proteinases. METHODS: Four dietary groups of male WAG/RijMCW rats were prepared: (1) Vitamin A deficient rats were raised on a casein-based retinoid deficient diet; (2) Retinol repleted rats were raised on the retinoid deficient diet. On the eighty-sixth day on this diet, the rats were fed retinyl palmitate and then given free access to the retinyl palmitate-supplemented control diet; (3) The weight-matched, pair-fed rats were restricted in their intake of the retinyl palmitate-supplemented diet so that their weight gain matched that of the A-rats; (4) The non-restricted rats were given free access to the retinyl palmitate-supplemented diet. The animals were killed at the late plateau stage for weight of the deficiency (102-106 days). Zymography was used to study proteinases in the corneal extracts. RESULTS: Vitamin A deficient and control rat corneas contain multiple matrix metalloproteinases and serine proteinases. The matrix metalloproteinases at 90/92 kDa (gelatinase B) and 66/63/57 kDa (gelatinase A) were significantly decreased in the corneas of the vitamin A deficient rats relative to the control corneas. Corneas from the four groups of rats contained 76, 45, 38, 28 and 22 kDa proteinases that cleaved casein. Only the vitamin A deficient corneas contained a 50 kDa casein cleaving enzyme. The 76, 45, 38 and 28 kDa serine proteinases were significantly lower in the vitamin A deficient corneas. The major 22 kDa enzyme was not altered by the deficiency. All casein cleaving proteinases were inhibited by phenylmethylsulfonyl fluoride and chymostatin except for a minor 76 kDa band. The activity of this band was not altered by inhibitors for the other classes of proteinases, ethylenediaminetetraacetic acid, E-64 or pepstatin. The concentrations of the 61, 52 and 40 kDa plasminogen activators were not altered by the deficiency. CONCLUSIONS: Alterations in corneal proteinases under vitamin A deficiency conditions may be involved in the characteristic changes observed in the cornea under vitamin A deficiency conditions: decreased exfoliation of epithelial cells, increased levels of keratofibrils in the corneal keratocytes, increased stromal keratocyte degradation and increased susceptibility towards ulceration.

Effect of vitamin A deficiency on the early response to experimental Pseudomonas keratitis.

PURPOSE: Vitamin A-deficient humans and animals are more susceptible to infections than are healthy humans and animals. This study compares the early corneal response (within 24 hours) to an experimental Pseudomonas aeruginosa infection between vitamin A deficient and control rats. METHODS: Male WAG/Rij/MCW rats were fed either a vitamin A- deficient diet (A-) or the same diet with retinyl palmitate added back in a nonrestricted manner (N) or under pair-fed conditions (A+) to yield weight-matched rats. Some A-rats were repleted wih retinyl palmitate 16 days before being killed and then given free access to the retinyl palmitate-supplemented diet (R). Twenty-four hours before being killed, the corneas of anesthetized rats were scratched and P. aeruginosa organisms were applied to the corneal surface. The rats were killed using an overdose of sodium pentobarbital. Corneas were either processed for light and electron microscopic examination or extracted for proteinase and myeloperoxidase determination. Corneal myeloperoxidase concentrations relative to neutrophil myeloperoxidase concentrations were used to determine the number of neutrophils in the cornea. Zymography was used to study caseinases, gelatinases, and plasminogen activators. Reverse zymography was used to detect proteinase inhibitors. Similar results were noted at early, mid, and late weight plateau stages of vitamin A deficiency. RESULTS: Ulceration occurred within 24 hours when low numbers of P. aeruginosa (10(4) cpu) were applied topically onto scratched A- corneas, whereas no ulceration was observed in the A+, R, and N corneas. When higher numbers of P. aeruginosa (10(7)-10(8)) were applied to the scratched corneas, all corneas became ulcerated within 24 hours. The extent of ulceration in the control corneas was greater than that in A- corneas by a factor of two. Only the A- corneas contained inflammatory cells with unusual striated deposits in phagolysosomes. The total number of neutrophils in the cornea and the concentrations of caseinases, plasminogen activators, and gelatinases in the infected corneal extracts were similar; however, the concentrations of cysteine proteinase inhibitors were elevated under A- conditions. CONCLUSIONS: Vitamin A deficiency alters the response of the cornea to a P. aeruginosa infection during the first 24 hours. The alterations observed are probably due to multiple factors: an insufficient tear film for bacterial clearance and migration of neutrophils, epithelial keratinization, alterations in corneal wound healing, and changes in polymorphonuclear function.

Localization of urokinase-type plasminogen activator in human eyes: an immunocytochemical study.

We investigated the presence of urokinase-type plasminogen activator (u-PA) in various structures of the human eye by using a biotin-strepavidin complex immunoperoxidase technique with a monoclonal antibody specific for human u-PA. A moderate to intense reaction product was seen in the corneal endothelium, episclera, sclera, iris muscles, lens equator and posterior capsule, peripheral vitreous, epithelium of pars plana, retinal pigment epithelium, axons, uveal fibroblasts, optic nerve fibers, and extraocular muscles. A weak reaction product was seen in the conjunctival and corneal epithelium, the trabecular meshwork, melanocytes and stroma of the iris and choroid, and the posterior layers of the retina. u-PA was not localized in the corneal stroma, conjunctival goblet cells, and certain parts of the lens. Comparison of the present results with those of our previous study (Tripathi, Geanon and Tripathi, 1987, Ophthalmology, 94, 1434-8) on the localization of tissue plasminogen activator (t-PA) in the eye indicate that the distribution of u-PA and t-PA overlaps in many tissues, but differs significantly in others, and that this finding may be related to their complementary and specific roles. As in other body systems, u-PA in ocular tissues is probably involved in processes such as fibrinolysis, wound healing, turnover of intra- and extracellular macromolecules, muscles and neuromuscular regeneration, synapse formation, and maintenance of general neuronal function.

[The use of lysosomotropic preparations in treating severe experimental chemical eye burns]. / Primenenie lizosomotropnykh preparatov v lechenii tiazhelykh khimicheskikh ozhogov v éksperimente.

The action of membranotropic preparations--mildronat and phosphaden on the course of a severe burn process of the cornea with changes in lysosomal membranes revealed in pathogenesis has been studied in 80 rabbits. As a marking lysosomal enzyme, acid phosphatase was used. Besides this, peculiarities of the clinical course of the burn process have been studied, when treated by common methods and in a complex with the mentioned preparations. The results of the study have shown expressed stabilizing action of the preparations on lysosomal membranes of the cornea in early terms of the treatment, correlative relationship between results of biochemical investigations and clinical manifestations of the action of membranotropic preparations, high effectiveness of therapeutic action of mildronat as compared with phosphaden. The results obtained can serve as a foundation for the usage of the preparations in complex treatment of patients with severe chemical burns of the eye.