Up-Regulation of Matrix Metalloproteinase-2 by Scleral Monocyte-Derived Macrophages Contributes to Myopia Development.

Myopia is a leading cause of visual impairment worldwide. This sight-compromising condition is associated with scleral thinning, extracellular matrix remodeling, and inappropriate optical axial length elongation. Although macrophages are present in the sclera, their involvement in this condition is unknown. By using a form-deprivation myopia (FDM) mouse model, we found that both the scleral macrophage density and their matrix metalloproteinase-2 (MMP-2) expression levels increased in myopic eyes. Partial scleral macrophage depletion by clodronate shifted the refraction toward hyperopia in both the form-deprived and the untreated fellow eyes compared with their respective counterparts in the vehicle-injected control mice. However, this procedure did not alter susceptibility to FDM. FDM development was 59% less in the macrophage-specific Mmp2 deletion (LysMCreMmp-2fl/fl) mice than in their Cre-negative littermates (Mmp2fl/fl mice). Moreover, the expression of scleral C-C motif chemokine ligand-2 (CCL2), which is a potent monocyte chemoattractant recruiting monocytes to tissue sites, was increased during myopia progression. However, the increase in the density of scleral macrophages and myopia development were suppressed in fibroblast-specific Ccl2 deletion mice. These declines suggested that the increase in scleral macrophage density in myopic eyes stems from the up-regulation of scleral Ccl2 expression in fibroblasts, which, in turn, promotes monocytes recruitment. In summary, scleral monocyte-derived macrophages contribute to myopia development through enhancing MMP-2 expression in mice.

Cause and Effect Relationship between Changes in Scleral Matrix Metallopeptidase-2 Expression and Myopia Development in Mice.

Myopia is a serious sight-compromising condition in which decreases in scleral biomechanical strength are associated with protease up-regulation resulting in thinning of its collagenous framework and changes in the extracellular matrix composition. Matrix metallopeptidase (MMP)-2 is one of the known proteases mediating these alterations. To determine whether MMP-2 up-regulation precedes myopia development, the direct effects of gain and loss in Mmp2 gene function were evaluated on refractive development and form deprivation myopia in mice. Four weeks after injecting an adeno-associated virus serotype 8 packaged Mmp2 overexpression vector (AAV8-Mmp2), scleral MMP-2 up-regulation was accompanied by significant myopia in a normal visual environment. In contrast, AAV8 packaging with shRNA targeting Mmp2 inhibited rises in MMP-2 expression induced by form deprivation by 54% and reduced myopia development by 23% compared with eyes injected with an irrelevant scrambled sequence. Because opposing changes in MMP-2 protein expression levels had corresponding effects on myopia progression, up-regulation of this protease contributes to inducing this condition. This notion of a cause-and-effect relationship between MMP-2 up-regulation and myopia development is supported by showing that form-deprived myopia development was attenuated by 27% in fibroblast-specific Mmp2 deletion (S100a4creMmp2fl/fl) mice relative to Cre-negative littermates (Mmp2fl/fl). Therefore, MMP-2 is a potential drug target for inhibiting myopia progression.

LOXL3 Function Beyond Amino Oxidase and Role in Pathologies, Including Cancer.

Lysyl oxidase like 3 (LOXL3) is a copper-dependent amine oxidase responsible for the crosslinking of collagen and elastin in the extracellular matrix. LOXL3 belongs to a family including other members: LOX, LOXL1, LOXL2, and LOXL4. Autosomal recessive mutations are rare and described in patients with Stickler syndrome, early-onset myopia and non-syndromic cleft palate. Along with an essential function in embryonic development, multiple biological functions have been attributed to LOXL3 in various pathologies related to amino oxidase activity. Additionally, various novel roles have been described for LOXL3, such as the oxidation of fibronectin in myotendinous junction formation, and of deacetylation and deacetylimination activities of STAT3 to control of inflammatory response. In tumors, three distinct roles were described: (1) LOXL3 interacts with SNAIL and contributes to proliferation and metastasis by inducing epithelial-mesenchymal transition in pancreatic ductal adenocarcinoma cells; (2) LOXL3 is localized predominantly in the nucleus associated with invasion and poor gastric cancer prognosis; (3) LOXL3 interacts with proteins involved in DNA stability and mitosis completion, contributing to melanoma progression and sustained proliferation. Here we review the structure, function and activity of LOXL3 in normal and pathological conditions and discuss the potential of LOXL3 as a therapeutic target in various diseases.

Evaluation of MMP2 as a candidate gene for high myopia.

PURPOSE: Matrix metalloproteinase 2 (MMP2) has been shown to be expressed in the human sclera, and is increased in the sclera of the eye with myopia induced by form deprivation in chicks when compared with the control eye. The purpose of this study was to examine the relationship between high myopia and MMP2 in a mainland Han Chinese population. METHODS: Four hundred unrelated patients with high myopia and 400 normal controls in a mainland Han Chinese population were studied. All the subjects were genotyped for 20 tag single nucleotide polymorphisms (SNPs) in MMP2 with the dye terminator-based SNaPshot method. The distribution of the genotypes in the cases and controls was compared with a χ(2) test. Screening for mutations in the coding regions and the adjacent intronic regions of MMP2 was performed in 200 patients with high myopia and 200 normal controls by direct sequencing. RESULTS: None of the 20 tested SNPs showed significant association with high myopia in this study. Seven variations were detected upon sequencing of the coding regions and the adjacent intronic regions of MMP2 in 200 subjects with high myopia and 200 normal controls. One novel variation, c.1287G>A (p.K429K), was detected in 79 of the 200 patients with high myopia (65 heterozygous and 14 homozygous) and in 84 of the 200 controls (67 heterozygous and 17 homozygous). The c.1810G>A mutation (p. Arg500His) was detected in three of the 200 patients with high myopia but not in the controls. The five other variations, known as polymorphisms, were detected in the case and control groups. CONCLUSIONS: We found no evidence that MMP2 is responsible for high myopia in these Han Chinese subjects and hence is unlikely to be important in the genetic predisposition to high myopia. Our results imply that MMP2 may not play a major role in high myopia in the Han Chinese population.

Matrix metalloproteinases and educational attainment in refractive error: evidence of gene-environment interactions in the Age-Related Eye Disease Study.

PURPOSE: A previous study of Old Order Amish families showed an association of ocular refraction with markers proximal to matrix metalloproteinase (MMP) genes MMP1 and MMP10 and intragenic to MMP2. A candidate gene replication study of association between refraction and single nucleotide polymorphisms (SNPs) within these genomic regions was conducted. DESIGN: Candidate gene genetic association study. PARTICIPANTS: Two thousand participants drawn from the Age-Related Eye Disease Study (AREDS) were chosen for genotyping. After quality-control filtering, 1912 individuals were available for analysis. METHODS: Microarray genotyping was performed using the HumanOmni 2.5 bead array (Illumina, Inc., San Diego, CA). Single nucleotide polymorphisms originally typed in the previous Amish association study were extracted for analysis. In addition, haplotype tagging SNPs were genotyped using TaqMan assays. Quantitative trait association analyses of mean spherical equivalent refraction were performed on 30 markers using linear regression models and an additive genetic risk model while adjusting for age, sex, education, and population substructure. Post hoc analyses were performed after stratifying on a dichotomous education variable. Pointwise (P(emp)) and multiple-test study-wise (P(multi)) significance levels were calculated empirically through permutation. MAIN OUTCOME MEASURES: Mean spherical equivalent refraction was used as a quantitative measure of ocular refraction. RESULTS: The mean age and ocular refraction were 68 years (standard deviation [SD], 4.7 years) and +0.55 diopters (D; SD, 2.14 D), respectively. Pointwise statistical significance was obtained for rs1939008 (P(emp) = 0.0326). No SNP attained statistical significance after correcting for multiple testing. In stratified analyses, multiple SNPs reached pointwise significance in the lower-education group: 2 of these were statistically significant after multiple testing correction. The 2 highest-ranking SNPs in Amish families (rs1939008 and rs9928731) showed pointwise P(emp)<0.01 in the lower-education stratum of AREDS participants. CONCLUSIONS: This study showed suggestive evidence of replication of an association signal for ocular refraction to a marker between MMP1 and MMP10. Evidence of a gene-environment interaction between previously reported markers and education on refractive error also was shown. Variants in MMP1 through MMP10 and MMP2 regions seem to affect population variation in ocular refraction in environmental conditions less favorable for myopia development.

Effects of intravitreal insulin and insulin signaling cascade inhibitors on emmetropization in the chick.

PURPOSE: Intravitreal insulin has been shown to be a powerful stimulator of myopia in chickens, in particular if the retinal image is degraded or defocused. In most tissues, the insulin receptor activates two main signaling pathways: a) the mitogen-activated protein kinase (MAPK) cascade (e.g., mitogen-activated protein kinasem kinase [MEK] and extracellular regulated kinase [ERK]) and b) the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway. In the current study, insulin was injected, and these pathways were separately inhibited to determine which is activated when the retinal image is defocused by spectacle lenses. METHODS: Chicks were treated with either +7 D, -7 D, or no lenses. They were intravitreally injected with insulin, the MEK inhibitor U0126, the PI3K inhibitor Ly294002, or a combination of insulin and one of the inhibitors. Refractions and ocular dimension were measured at the beginning and after four days of treatment. The retinal proteins of the chicks were measured with western blots after 2 h and four days of treatment. Incubation occurred with anti-Akt1, anti-Erk1/2, anti-phospho-Akt(Thr308), and anti-phospho-Erk1/2((Thr202/Tyr204)) antibodies, and the ratio between the relative intensity of the phospho-form and the total-form was calculated. RESULTS: Chicks wearing positive lenses and injected with saline and with PI3K inhibitor compensated for the imposed defocus and became hyperopic. Insulin injections and insulin plus PI3K inhibitor injections prevented lens-induced hyperopia, whereas the MEK inhibitor alone and insulin plus MEK inhibitor had no effect. Obviously, the MEK inhibitor suppressed the effect of insulin on eye growth in the plus lens-treated animals. Chicks treated with negative lenses and injected with insulin, or with insulin plus MEK inhibitor, overcompensated for the imposed defocus. This effect of insulin was not detected in eyes injected with PI3K inhibitor plus insulin, suggesting that the PI3K inhibitor suppressed the effects of insulin in minus lens-treated animals. Insulin increased the ratio of phospho-Akt/total-Akt in animals with normal visual exposure but even more so in chicks wearing plus or minus lenses. The increase was blocked by simultaneous PI3K inhibitor injections in control eyes but not in lens-treated eyes. Insulin also increased the ratio of phospho-ERK/total-ERK in animals with normal visual exposure and in animals wearing positive lenses, compared to U0126- and Ly294002-injected eyes. In contrast, no significant activation of the MEK/ERK pathway was observed in the negative lens-treated animals. CONCLUSIONS: Intravitreal insulin promoted axial eye growth and stimulated both signaling pathways. The PI3K/Akt pathway was activated in control and plus and minus lens-treated eyes, but the MEK/ERK pathway was activated only with positive lenses or no lenses. With negative lenses, insulin did not stimulate the MEK/ERK signaling cascade. Independent of the pathway stimulated after insulin binding, the effect on insulin was always the same: an increase in eye growth.

Inhibiting the neuronal isoform of nitric oxide synthase has similar effects on the compensatory choroidal and axial responses to myopic defocus in chicks as does the non-specific inhibitor L-NAME.

In birds, the choroid plays a role in the visual regulation of eye growth, thickening in response to myopic defocus, and thinning in response to hyperopic defocus, in both cases moving the retina towards the image plane. This response is rapid, occurring within hours of the defocus stimulus. These changes are consistently associated with slower changes in the sclera, that result in the appropriate changes in axial elongation, decreasing growth in response to myopic defocus and increasing it in response to hyperopic defocus. The molecular mechanisms underlying the scleral response involve changes in the synthesis of extracellular matrix molecules, however, those underlying the changes in choroidal thickness are not known. However, evidence suggests that it may involve the gaseous signal molecule nitric oxide, as nitric oxide is a potent smooth muscle relaxant, and injections of the non-specific nitric oxide synthase inhibitor L-NAME transiently inhibits the thickening response. Interestingly, it also dis-inhibits ocular growth, in accordance with a mechanistic link between the two responses. If nitric oxide is part of the signal cascade underlying the visual regulation of eye growth, it would be important to ascertain the source of the molecule. As a first step towards doing so, we used various more specific NOS inhibitors and studied their effects on the choroidal and growth responses. Birds (7-12 days old) were fitted with +10 D lenses on one eye. On that day, single intravitreal injections (30 microl) of the following inhibitors were used: nNOS inhibitor N(omega)-propyl-L-arginine (n=12), iNOS inhibitor L-NIL (n=16), eNOS/iNOS inhibitor L-NIO (n=15), non-specific inhibitor L-NMMA (n=30) or physiological saline (n=18). Ocular dimensions were measured using high-frequency A-scan ultrasonography at the start of the experiment, and at 7, 24 and 48 h after. We found that the nNOS inhibitor N(omega)-propyl-L-arginine had the same inhibitory effects on the choroidal response, and dis-inhibition of the growth response, as did L-NAME; neither of the other inhibitors had any effect except L-NMMA. We conclude that the choroidal compensatory response is influenced by nNOS, possibly from the intrinsic choroidal neurons, or the parasympathetic innervation from the ciliary and/or pterygopalatine ganglia.

Correlation of axial length and myopic macular degeneration to levels of molecular factors in the aqueous.

To elucidate the molecular processes associated with the development of myopic macular degeneration (MMD), we measured the intraocular concentrations of molecular factors in emmetropic and myopic eyes. This is a retrospective clinic-based case-control study that included eyes undergoing routine cataract surgery whereby aqueous humour samples were obtained. We measured the concentrations of pigment epithelium derived factor(PEDF), matrix metalloproteinase 2(MMP-2), tissue inhibitor of metalloproteinase(TIMP-2), vascular endothelial growth factor isoform A(VEGF-A), interleukin 8(IL-8), interleukin 6(IL-6), C-reactive protein(CRP), angiopoietin 2(Ang2), and amphiregulin. 38 eyes (axial length (AL): 22.4-32.4 mm), including 12 highly myopic (HM) eyes (AL ≥ 26.5 mm) without MMD and 12 HM eyes with MMD but without neovascularization were included. Eyes with MMD were found to have significantly lower VEGF-A levels (p = 0.007) and higher MMP-2 levels (p = 0.02) than control eyes after adjusting for age and gender. MMP-2 levels correlated positively (r = 0.58, p = 0.002), while VEGF-A levels correlated negatively with longer axial length (r = -0.75, p < 0.001). Both the concentrations of VEGF-A (P = 0.25) and MMP-2 (P = 0.69) were not significantly associated with MMD after adjusting for AL. These findings suggest that the predominant mechanism underlying the development of non-neovascular MMD may be axial elongation, driven in part by MMP-2 related mechanisms.

MMP-2 participates in the sclera of guinea pig with form-deprivation myopia via IGF-1/STAT3 pathway.

OBJECTIVE: To investigate the expression changes of MMP-2 (matrix metalloproteinases-2) mediated by IGF-1 (insulin-like growth factors-1) STAT3 (signal transducer and activator of transcription 3) pathway in the sclera of the form-deprivation myopia guinea pigs. MATERIALS AND METHODS: Twenty-four three-week-old guinea pigs were randomly divided into 4 groups: group A (Control), B, C and D. Guinea pigs in group A were sacrificed after 21 days without any special treatment. Guinea pigs in group B were sacrificed 7 days after receiving stitch in the right eye. Guinea pigs in group C were sacrificed 14 days after receiving stitch in the right eye. Guinea pigs in group D were sacrificed 21 days after receiving stitch in the right eye. Eyeball refraction and axial length of guinea pigs were measured before sacrifice. Eyeballs of guinea pigs were enucleated after sacrifice. The expressions of IGF-1, STAT3 and MMP-2 in scleral tissue were detected by Western blot. RESULTS: Axial length extension and myopia appeared in the right eye of guinea pigs in group B. The expressions of IGF-1, STAT3 and MMP-2 in the sclera significantly increased after 7 days of occlusion compared with that in control group A (p<0.05). In the right eye of group C, the axial prolongation and myopia formation appeared after 14-day occlusion. The expressions of IGF-1, STAT3 and MMP-2 in sclera significantly increased compared with that in group A (p<0.05). In the right eye of group D, the axial extension and myopia formation occurred. IGF-1, STAT3 and MMP-2 in scleral significantly upregulated 21 days after occlusion (p<0.05). Furthermore, at different stages of deprivation, protein expressions of MMP-2 and IGF-1 in sclera were positively correlated (r = 0.962, p<0.01). CONCLUSIONS: Form-deprivation of guinea pigs lead to increased expressions of IGF-1, STAT3 and MMP-2 in the sclera and myopia of guinea pigs. The expressions of IGF-1, STAT3 and MMP-2 increased progressively over the time of deprivation. Additionally, overexpression of MMP-2 mediated by IGF-1/STAT3 pathway in sclera might promote the formation of myopia.

Crosslinking Enzyme Lysyl Oxidase Modulates Scleral Remodeling in Form-Deprivation Myopia.

PURPOSE: Scleral remodeling causes the excessive ocular elongation that underlies myopia. Lysyl oxidase (LOX), a copper-containing amine oxidase, can catalyze collagen and elastin crosslinking. The purpose of this study was to investigate the role of LOX in scleral remodeling in form-deprivation myopia (FDM). METHODS: Seventy-five guinea pigs were randomly divided into five groups as follows: a normal control group, an FDM group, an FDM plus ß-aminopropionitrile (BAPN) group, an FDM plus TGF-ß1 (TGF-ß1) group, and an FDM plus vehicle group. A translucent diffuser was used to induce FDM, and intravitreal injection was used to administer BAPN, TGF-ß1 or vehicle. The scleral LOX and collagen gene and protein levels and the posterior scleral ultrastructure and biomechanics were measured. RESULTS: In the FDM group, both the scleral LOX and collagen gene and protein levels were significantly lower than those in the control eyes. The collagen fibril diameters were significantly decreased in the FDM group compared with the diameters in the control group. A significant decrease in LOX gene and protein expression was observed after BAPN injection, and an increase was observed after TGF-ß1 treatment compared with the levels in the FDM group. Additionally, the scleral collagen fibrils were significantly decreased in the BAPN-treated eyes but increased in the TGF-ß1-treated eyes compared with the FDM eyes. The ultimate stress and Young's modulus of the sclera were lowest in the BAPN group, followed by the FDM group and the TGF-ß1 group. The ultimate strain (%) of the sclera was lowest in the TGF-ß1 group, followed by the FDM group and the BAPN group. CONCLUSION: LOX expression was significantly lowered in myopic sclera. Modulating LOX expression induced a change in both the scleral collagen fibril diameter and the scleral biomechanics. Therefore, LOX may play a key role in the myopia scleral remodeling procedure.

Time-course of changes to nitric oxide signaling pathways in form-deprivation myopia in guinea pigs.

The aim of this study was to investigate the time-course change of nitric oxide synthase (NOS) activity and cyclic GMP (cGMP) concentration in the posterior retina, choroid and sclera after differing periods of form-deprivation in guinea pigs. Three groups of guinea pigs were subjected to monocular FD for 7, 14 or 21 days. NOS activity and cGMP concentrations in ocular tissues of FD eyes and control eyes were analyzed by radioimmunoassay. The presence of NOS isoforms was detected by immunohistochemistry. Guinea pigs presented with considerable myopia after 14 days of FD. Retinal NOS activity in the FD group was lower than in the control group after 7 days of FD and was higher than in the control group after 14 and 21 days of FD. The choroidal and scleral NOS activities in the FD groups were higher than in the control groups after 21 days. The cGMP concentrations in the FD groups were higher than in the control groups at 21 days of the retinal, choroidal, and scleral tissues. Furthermore, the retinal cGMP concentration in the FD group was also significantly elevated at 14 days relative to the control group. We detected expression of three NOS isoforms in guinea pig ocular tissues. Our main observations were a change in NOS activity and an up-regulation in cGMP concentrations in posterior ocular tissues during the development of myopia. The function of elevated NOS activity may be mediated by cGMP.

[Effect of pirenzepine on form deprivation myopia in chicks and its possible mechanism].

OBJECTIVE: To observe the effect of M1-selective muscarinic antagonist, pirenzepine, on form deprivation myopia and investigate the expression of MMP-2 and its inhibitor TIMP-2 in the fibrous sclera in order to better understand the mechanism by which pirenzepine inhibits myopia. METHODS: 40 chicks after birth one day were divided into 4 groups randomly: I. Control group; II. Form deprivation group; III. Vehicle application group; IV. Pirenzepine injected group. Form deprivation myopia was established in right eyes of group II, III, IV by placement of a translucent occluder. The deprived eyes of group III and IV received daily subconjunctival administration of vehicle PBS and pirenzepine respectively. Optical measures such as refraction, axial length, equatorial diameter were made at the end of the experiment. Total RNA and protein were extracted from the posterior fibrous sclera chicks. The expression of MMP-2 and TIMP-2 mRNA and protein were investigated with RT-PCR and Western blot analysis respectively. RESULTS: Refraction status, axial length, equatorial diameter of the eyes in pirenzepine injected group were significantly lower when compared with form deprivation group (P < 0.01), but the parameters were higher when compared with normal control group therefore relatively myopic changes were detected. There were no significant difference between drug control and pirenzepine injected group when optical measures and the expression of MMP-2, TIMP-2 were concerned (P > 0.05). The expressions (mRNA and protein) of both MMP-2 and TIMP-2 were significantly different in form deprivation group when compared with normal control group (MMP-2 mRNA increased by 143.51%, P < 0.01; protein increased by 114.60%, P < 0.01; TIMP-2 mRNA decreased by 55.05%, P < 0.01; protein decreased by 53.73%, P < 0.01). In pirenzepine injected group the relative expression of MMP-2 mRNA and protein were decreased obviously by 41.95% (P < 0.01) and by 36.16% (P < 0.01), while TIMP-2 mRNA and protein expression was increased significantly by 72.46% (P < 0.01) and by 53.05% (P < 0.01) respectively compared with the form deprived group. CONCLUSION: Subconjunctivally administration of the M1 selective muscarinic antagonist, pirenzepine, partly prevents or restrains form deprivation induced myopia. It may exert its inhibitory effect by modulating the expression of MMP-2 and TIMP-2 in fibrous sclera.

Selective regulation of MMP and TIMP mRNA levels in tree shrew sclera during minus lens compensation and recovery.

PURPOSE: In juvenile tree shrews, a minus-power lens placed in front of the eye produces increased axial elongation and a myopic shift in refractive state that compensates for the power of the lens. Scleral tissue remodeling and modulation of the mechanical properties of the sclera occur during lens compensation. In this study, the time course of changes in scleral mRNA levels of three MMPs and three TIMPs during compensation for a minus lens and during recovery was investigated, to determine which, if any, are temporally associated with changes in the mechanical properties of the sclera and the axial elongation rate. METHODS: Competitive RT-PCR was used to measure the levels of mRNA for MT1-MMP, MMP-2, MMP-3, TIMP-1, TIMP-2, and TIMP-3 in the scleras of tree shrews that had received either 1, 2, 4, or 11 days of monocular -5-D lens treatment, or 11 days of -5-D lens treatment followed by 2 or 4 days of recovery. RESULTS: Relative to their control eyes, treated eye MT1-MMP and MMP-2 mRNA levels were significantly higher, and TIMP-3 levels were lower by 1 to 4 days of minus lens treatment. These differential effects were absent by 11 days of treatment when the treated eyes had compensated for the lens. The levels of all three TIMPs spiked upward in both eyes after 2 days of recovery. The differential changes in MT1-MMP, MMP-2, and TIMP-3 mRNA levels were all restricted to the treated eye and were temporally associated with the differential changes in axial elongation, refractive state, and the previously measured changes in creep rate. CONCLUSIONS: The observed changes in MT1-MMP, MMP-2, TIMP-2, and TIMP-3 mRNA are consistent with visually modulated MT1-MMP activation of MMP-2 and with MT1-MMP degradation of scleral extracellular matrix components. These data constitute further evidence that visual signals modulate gene expression of selected MMPs and TIMPs to control scleral remodeling, the mechanical properties of the sclera, axial elongation, and refractive state.

[Recent findings on scleral extracellular matrix and matrix metalloproteinases in the development of myopia].

Myopia is the most common eye disorder worldwide. So far there is no effective treatment for the disease. Recent findings indicated that the remodeling of scleral extracellular matrix (ECM) plays an important role in the development of myopia. The dynamic changes of matrix metalloproteinases in myopia (MMPs) activity, one of the most important enzymes that regulates the ECM, has a significant correlation with myopia. This review gives the latest findings about ECM and MMPs in the development of myopia.

[Dynamic changes of MMP-2 activity in the posterior sclera of chicks with form-deprivation myopia].

OBJECTIVE: To investigate the effect of form-deprivation on level of gelatinase in the posterior sclera in chicks. METHODS: Fifty 1-day-old chicks were monocularly deprived to establish the animal model of form-deprivation myopia (FDM). According to the duration of form-deprivation the experimental chicks were divided randomly and equivalently into 5 groups, which were deprived for 3, 7, 14, 21 and 30 days respectively. Meanwhile the other eyes of the deprived chicks were used as self-control groups and chicks of the same days were chosen randomly as the normal control groups for each FDM group. At each form-deprivation point the changes of degree of diopters and axial length of chicks in each group were recorded. The levels of gelatinase in posterior sclera of the experimental eyes were measured by gelatin enzymography. RESULTS: Compared with the normal and self-control groups, the levels of MMP-2 activity in FDM groups were much higher (P <0.01). With the increase of the time of monocular deprivation these changes became more significant and reached the top after 14 days' deprivation with an inter-group statistical difference (P <0.01). The dynamic changes of MMP-2 activity were the same as those of axial length and degree of diopters in each experimental groups. There was positive correlation between the MMP-2 activity and axial length (r = 0.989, P < 0.01). But there was a negative correlation between the MMP-2 activity and refractive degree. CONCLUSION: Increase of MMP-2 activity in the posterior sclera of chicks would be a direct key factor to trigger sclera ECM remodeling process in chick FDM.

Plasminogen activator activity in tears after excimer laser photorefractive keratectomy.

PURPOSE: To quantify changes of plasminogen activator activity in tear fluid during corneal re-epithelialization after excimer laser photorefractive keratectomy (PRK). METHODS: Tear samples were collected with glass capillaries from 77 eyes of 42 patients immediately before and immediately after PRK treatment and on postoperative days 3 and 5. In 20 patients, the contralateral eye was similarly sampled to serve as control. Plasminogen activator activity in the tear samples was measured by a spectrophotometric method using human plasminogen and chromogenic peptide substrate, D-valyl-L-leucyl-L-lysine-p-nitroanilide (S-2251). RESULTS: In tears of all eyes that underwent PRK, the plasminogen activator activities were lower immediately after PRK than were the preoperative values. For patient eyes with normal wound healing, tear plasminogen activator activities were significantly elevated above the preoperative level on the third postoperative day and then returned to the preoperative level by the fifth postoperative day. In contrast, tear plasminogen activator activities remained low through the third postoperative day in all (six) eyes in which haze developed after 3 to 6 months. The contralateral control eyes showed no appreciable change in plasminogen activator activity over the 5-day period. CONCLUSIONS: Plasminogen activator activity levels measured in tears of excimer laser PRK-treated eyes may serve as a predictor of wound healing. Extended low levels of plasminogen activator activity through the third postoperative day correlate with the development of corneal healing abnormalities (haze). The low plasminogen activator activity could be not only an accompanying sign but also a cause of defective corneal wound healing.

Increased latent gelatinase activity in the sclera of visually deprived chicks.

PURPOSE: Gelatinase activity was measured in the normal chick sclera and in sclera of form-deprived (myopic) eyes to assess the role of this metalloproteinase in ocular elongation associated with experimental myopia. METHODS: Gelatinases were extracted from anterior and posterior regions of normal chick sclera and sclera from eyes that had been form-vision deprived for 11 days. Gelatinase activity in the extracts was determined by measuring the digestion of 3H-gelatin after incubation with the extracts in the absence or presence of 1 mM aminophenylmercuric acetate (APMA) to activate latent gelatinases. Scleral gelatinases were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis gelatin zymography and immunoprecipitation analyses. RESULTS: No significant differences were detected in gelatinase activity between normal and deprived posterior sclera in the absence of APMA. However, when scleral extracts were incubated with APMA, extracts from the posterior sclera of deprived eyes contained significantly more gelatinase activity than paired controls (+127%, P = 0.0105). In contrast, no differences in active or latent gelatinase activity were detected in extracts from the anterior sclera. Removal of tissue inhibitors of metalloproteinases (TIMP) from control scleral extracts by reduction and alkylation resulted in a 222% increase in gelatinolytic activity after APMA-activation (P < or = 0.001), whereas similar treatment of deprived scleral extracts resulted in only a 76% increase in gelatinolytic activity (P < or = 0.001). A 65/58-kd doublet was the major gelatinolytic species from control and deprived posterior sclera that represent the proenzyme and active forms of the 72-kd gelatinase (MMP-2). CONCLUSIONS: These data indicate that visual deprivation is associated with an increased amount of the 72-kd progelatinase and a decreased amount of TIMP within the posterior sclera. Therefore, an imbalance between the levels of 72-kd progelatinase and its inhibitor may play a role in the remodeling processes of the posterior sclera during the development of form-deprivation myopia.

Form-deprivation myopia induces activation of scleral matrix metalloproteinase-2 in tree shrew.

PURPOSE: To investigate whether structural changes to the sclera during form-deprivation myopia are caused by active tissue remodeling, the gelatinase activity of tree shrew scleras was studied in normal animals, form-vision deprived animals, and animals recovering from myopia. METHODS: Infant tree shrews were monocularly deprived (MD) of form vision with translucent occluders for 5 days. Recovery animals were allowed 3 days of binocularly unoccluded vision after the period of form deprivation. Eyes were removed and dissected to provide scleral samples corresponding to equatorial and posterior regions. Gelatinase activity was assessed by quantitative sodium dodecyl sulfate-polyacrylamide gel electrophoresis or SDS-PAGE, gelatin, zymography of scleral matrix metalloproteinase (MMP) extracts. RESULTS: The major gelatinolytic species present in tree shrew sclera was found to be MMP-2 (gelatinase A). In normal (nondeprived) animals, most of the MMP-2 was found to be in the latent form (the ratio of active-to-latent MMP-2 was 0.23 +/- 0.05 and 0.34 +/- 0.06 in equatorial and posterior samples, respectively; n = 10 eyes from five animals). After 5 days of MD, there was a threefold increase in the amount of active scleral MMP-2 in myopic eyes compared to contralateral control eyes, whereas latent MMP-2 activity levels were not altered significantly. This increase in active MMP-2 was seen in both the equatorial and posterior sclera of myopic eyes (active-to-latent MMP-2 ratios were 0.53 +/- 0.10 and 0.81 +/- 0.09 in equatorial and posterior regions, respectively; n = 6 animals). Contralateral control eyes had levels of both active and latent MMP-2 not significantly different from normal eyes. After only 3 days of unoccluded vision, previously deprived eyes that were now recovering from myopia had a fivefold lower level of active MMP-2 than that seen in deprived eyes after 5 days of MD. In fact, active (and latent) MMP-2 levels were reduced in recovering eyes even below the levels found in their contralateral control eyes. Active-to-latent ratios for recovering eyes were 0.11 +/- 0.03 and 0.15 +/- 0.03 in equatorial and posterior sclera, respectively (n = 5 animals). CONCLUSIONS: These results demonstrate that form-deprivation myopia and recovery from myopia alter scleral catabolism and provide further support for the theory that changes in eye size during mammalian refractive development are the result of active tissue remodeling rather than passive scleral stretching alone.

The organophosphate pesticide chlorpyrifos affects form deprivation myopia.

PURPOSE: The effects of the anti-cholinesterase organophosphate pesticide chlorpyrifos (CPF) on the refractive development of the eye were examined. Form deprivation was used to induce eye growth to address the previously reported relationship between organophosphate pesticide use and the incidence of myopia. METHODS: Chickens, a well-established animal model for experimental myopia and organophosphate neurotoxicity, were dosed with chlorpyrifos (3 mg/kg per day, orally, from day 2 to day 9 after hatching) or corn oil vehicle (VEH) with or without monocular form deprivation (MFD) over the same period. The set of dependent measures included the refractive state of each eye measured using retinoscopy, axial dimensions determined with A-scan ultrasound, and intraocular pressure. RESULTS: Dosing with CPF yielded an inhibition of 35% butyrylcholinesterase in plasma and 45% acetylcholinesterase in brain. MFD resulted in a significant degree of myopia in form-deprived eyes resulting from significant lengthening of the vitreal chamber of the eye. CPF significantly reduced the effect of MFD, resulting in less myopic eyes (mean refraction: VEH-MFD = -16.2 +/- 2.3 diopters; CPF-MFD = -11.1 +/- 1.8 diopters) with significantly shorter vitreal chambers. Nonoccluded eyes were, on average, slightly hyperopic. Treatment with CPF for 1 week in the absence of MFD led to no significant change in ocular dimensions or refraction relative to controls. CONCLUSIONS: The use of form deprivation as a challenge suggests that CPF treatment interferes with the visual regulation of eye growth.

Prolidase deficiency associated with pathologic myopia.

Deficiency of prolidase, a key enzyme in proline metabolism, is extremely rare and is usually associated with skin lesions, recurrent infections, characteristic facies, mental retardation, and splenomegaly. These clinical features are largely due to inhibition of normal recycling of proline, which causes an alteration in the metabolism of collagen and other proline-rich proteins. The case of a 25-year-old with all the recognized characteristics of prolidase deficiency is reported. Pathologic myopia, which has not been hitherto described in association with prolidase deficiency, is added to the clinical spectrum of this rare disorder.