BMP-2 Is Involved in Scleral Remodeling in Myopia Development

The development of myopia is associated with scleral remodeling, but it is unclear which factors regulate this process. This study investigated bone morphogenetic protein-2 (BMP-2) expression in the sclera of guinea pigs with lens-induced myopia (LIM) and after recovery from myopia and evaluated the effect of BMP-2 on extracellular matrix (ECM) synthesis in human scleral fibroblasts (HSFs) cultured in vitro. Lens-induced myopia was brought about in two groups of guinea pigs (the lens-induced myopia and myopia recovery groups) by placing -4.00 D lenses on the right eye for three weeks. The left eye served as a contralateral control. In the recovery group, the lenses were removed after one week. The refractive power and axial length of the eyes were measured, and the BMP-2 expression levels in the sclera were measured. After three weeks, the lens-induced eyes acquired relative myopia in both groups of guinea pigs. Immunostaining of the eyeballs revealed significantly decreased BMP-2 expression in the posterior sclera of the myopic eyes compared to the contralateral eyes. One week after lens removal, BMP-2 expression recovered, and no differences were observed between the experimental and contralateral eyes in the recovery group. HSFs were cultured with BMP-2 or transforming growth factor-β1 (TGF-β1). Type I and type III collagen synthesis was significantly up-regulated following BMP-2 treatment in culture after one and two weeks, but the ratio of type III to type I collagen mRNA was not increased. Biosynthesis of glycosaminoglycan (GAG) and aggrecan was increased in HSFs treated with BMP-2. Some chondrogenesis-associated genes expression increased in HSFs treated with BMP-2. From this study, we concluded that BMP-2 is involved in scleral remodeling in the development and recovery of lens-induced myopia.     

Increased aggrecan (cartilage proteoglycan) production in the sclera of myopic chicks

A previously characterized chick model of myopia was used to evaluate biochemical changes in the sclera which are associated with ocular enlargement and myopia. Chicks were monocularly occluded for 10 days and the DNA, hydroxyproline, and glycosaminoglycan contents of the sclera were compared between the normal and the myopic eyes. No significant differences could be detected in total DNA or hydroxyproline content. There was, however, a 34% increase in glycosaminoglycans and a 20.7% decrease in cell density within the posterior sclera of myopic eyes. The biosynthesis of scleral proteoglycans was determined by measuring 35SO4 incorporation in the sclera of chicks visually occluded for 5, 10, and 15 days. No differences could be detected in 35SO4 incorporation into the cornea or the anterior sclera. However, 35SO4 incorporation was significantly increased in the posterior sclera of myopic eyes by 64% at Day 5, 39% at Day 10, and 49% at Day 15. When fractionated on Sepharose CL-4B, scleral proteoglycans were resolved into two peaks which were identified by Western blot analysis as aggrecan (cartilage proteoglycan) and decorin. Furthermore, Western blot and dot blot analyses indicated that significantly more aggrecan core protein was present in the sclera of myopic eyes compared with equivalent amounts of sclera from control eyes. These results indicate that increased synthesis and accumulation of aggrecan, which increases the volume of extracellular matrix in the posterior sclera, are responsible for the ocular enlargement observed in this model of myopia.     

Retinoscleral control of scleral remodelling in refractive development: a role for endogenous FGF-2?

AIMS: Studies in avian models of myopia have shown that refractive error development can be influenced by exogenously delivered fibroblast growth factor (FGF)-2. The present study sought to determine whether endogenous FGF-2 was associated with retinoscleral signalling or scleral remodelling during changes in refractive error in a mammalian model of myopia. METHODS: Myopia was induced in tree shrews over a 5-day period. One group of animals was then allowed 3 days of recovery from the induced myopia. Endogenous levels of FGF-2 were measured in scleral and retinal homogenates using ELISA. Real-time PCR was used to investigate scleral FGF-2 and FGF receptor (FGFR)-1 mRNA expression. RESULTS: No difference in FGF-2 content was found in posterior scleral or retinal extracts of myopic eyes (scleral -4+/-9%, retinal +23+/-17%) or recovering eyes (scleral -10+/-18%, retinal +1+/-13%), when compared with contralateral control eyes. In addition, no significant changes were found in scleral FGF-2 mRNA expression in myopic or recovering eyes (+106+/-56% and +14+/-12% respectively, P=0.21). However, FGF-2 concentration was significantly higher in anterior, relative to posterior, scleral regions in all animals (1602+/-105 vs 1030+/-50pg/mg respectively P<0.001). Expression of scleral FGFR-1 mRNA was upregulated in myopic eyes (+186+/-32%, P=0.01) but returned to control eye levels during recovery (+63+/-20%). CONCLUSIONS: The findings indicate that alterations in endogenous retinal or scleral FGF-2 levels are not associated with changes in scleral remodelling in this mammalian model of myopia. However, the reversible changes found in FGFR-1 expression in the sclera of myopic eyes mean that an indirect role for FGF-2 in the control of scleral remodelling is implicated. The anteroposterior difference found in scleral FGF-2 concentration indicates a role for this cytokine in the control of normal scleral growth and development and, presumably, eye size.     

cAMP Level Modulates Scleral Collagen Remodeling,a Critical Step in the Development of Myopia

The development of myopia is associated with decreased ocular scleral collagen synthesis in humans and animal models. Collagen synthesis is, in part, under the influence of cyclic adenosine monophosphate (cAMP). We investigated the associations between cAMP, myopia development in guinea pigs, and collagen synthesis by human scleral fibroblasts (HSFs). Form-deprived myopia (FDM) was induced by unilateral masking of guinea pig eyes. Scleral cAMP levels increased selectively in the FDM eyes and returned to normal levels after unmasking and recovery. Unilateral subconjunctival treatment with the adenylyl cyclase (AC) activator forskolin resulted in a myopic shift accompanied by reduced collagen mRNA levels, but it did not affect retinal electroretinograms. The AC inhibitor SQ22536 attenuated the progression of FDM. Moreover, forskolin inhibited collagen mRNA levels and collagen secretion by HSFs. The inhibition was reversed by SQ22536. These results demonstrate a critical role of cAMP in control of myopia development. Selective regulation of cAMP to control scleral collagen synthesis may be a novel therapeutic strategy for preventing and treating myopia.     

Isoform-specific changes in scleral transforming growth factor-beta expression and the regulation of collagen synthesis during myopia progression

The development of high myopia is associated with altered scleral extracellular matrix biochemistry. Previous studies highlight the importance of collagen turnover in this process, yet it is unclear which factors control scleral remodeling. This study used a mammalian model of myopia to investigate the capacity of TGF (transforming growth factor)-beta1, -beta2, and -beta3 to influence scleral remodeling in myopia. RT-PCR confirmed the presence of all mammalian TGF-beta isoforms in scleral tissue and scleral fibroblasts. Myopia was experimentally induced via monocular deprivation of pattern vision, and animals were allocated to two groups depending on the duration of treatment (1 or 5 days). Down-regulation of each isoform was apparent after only 1 day of myopia development (TGF-beta1, -32%; TGF-beta2, -27%; TGF-beta3, -42%). Whereas the decrease in TGF-beta1 and -beta3 expression was relatively constant between the two time points, differential down-regulation of TGF-beta2 was found between days 1 (-27%) and 5 (-50%). In vitro experiments, using primary scleral fibroblasts, demonstrated the capacity of all isoforms to increase collagen production in a dose-dependent manner. Changes in TGF-beta levels, which mimicked those during myopia induction, caused an approximately 15% reduction in collagen synthesis, which is qualitatively similar to those previously reported in vivo. These data represent the first demonstration of TGF-beta3 expression in the sclera and implicate all three TGF-beta isoforms in the control of scleral remodeling during myopia development. In addition, the early alterations in TGF-beta expression levels may reflect a role for these cytokines in mediating the retinoscleral signal that controls myopic eye growth.     

Measurement of the synthesis rates of collagens and total protein in rabbit muscle.

1. Collagen- and total-protein-synthesis rates were determined in rabbit muscle by continuous infusion of radioactive proline. 2. The precursor pool of free proline used for collagen synthesis was defined by measuring the specific radioactivity of hydroxy-proline in isolated type I procollagen. The specific radioactivities of type I procollagen were about 40% of those for free proline in the homogenate. 3. The mean ratio (+/- S.E.M.) between the fractional synthesis rates of muscle collagen and total protein was 0.99 +/- 0.10, where the total protein values were based on specific radioactivities of the homogenate free proline pools. 4. Types I, III and V collagen were solubilized by pepsin and isolated by fractional precipitation with NaCl. The fractional synthesis rates of types I and III collagens were very similar. Type V collagen samples had higher specific radioactivities than the other collagens, but this was not necessarily indicative of a higher rate of synthesis because of uncertainty about the cellular origin of this collagen and, hence, the specific radioactivity of its precursor proline pool.     

Post-translationally Abnormal Collagens of Prolyl 3-Hydroxylase-2 Null Mice Offer a Pathobiological Mechanism for the High Myopia Linked to Human LEPREL1 Mutations

Myopia, the leading cause of visual impairment worldwide, results from an increase in the axial length of the eyeball. Mutations in LEPREL1, the gene encoding prolyl 3-hydroxylase-2 (P3H2), have recently been identified in individuals with recessively inherited nonsyndromic severe myopia. P3H2 is a member of a family of genes that includes three isoenzymes of prolyl 3-hydroxylase (P3H), P3H1, P3H2, and P3H3. Fundamentally, it is understood that P3H1 is responsible for converting proline to 3-hydroxyproline. This limited additional knowledge also suggests that each isoenzyme has evolved different collagen sequence-preferred substrate specificities. In this study, differences in prolyl 3-hydroxylation were screened in eye tissues from P3h2-null (P3h2^n/n) and wild-type mice to seek tissue-specific effects due the lack of P3H2 activity on post-translational collagen chemistry that could explain myopia. The mice were viable and had no gross musculoskeletal phenotypes. Tissues from sclera and cornea (type I collagen) and lens capsule (type IV collagen) were dissected from mouse eyes, and multiple sites of prolyl 3-hydroxylation were identified by mass spectrometry. The level of prolyl 3-hydroxylation at multiple substrate sites from type I collagen chains was high in sclera, similar to tendon. Almost every known site of prolyl 3-hydroxylation in types I and IV collagen from P3h2^n/n mouse eye tissues was significantly under-hydroxylated compared with their wild-type littermates. We conclude that altered collagen prolyl 3-hydroxylation is caused by loss of P3H2. We hypothesize that this leads to structural abnormalities in multiple eye tissues, but particularly sclera, causing progressive myopia.     

Activation of collagen synthesis in primary culture of rat liver parenchymal cells (hepatocytes)

An increase in collagen synthesis by hepatic parenchymal cells (hepatocytes) was observed during 8 days in primary culture by the quantification of total [3H]hydroxyproline as a marker of total collagen synthesis and the ratio of [3H]hydroxyproline in the high-molecular-weight fraction to total [3H]hydroxyproline as a marker of collagen degradation after incubation of the cells with [3H]proline for 24 h. Type analysis of the collagen produced by the cells after 8 days in culture showed the presence of type I and type III collagens in addition to the components corresponding to type IV and type V (alpha A and alpha B) collagens. Only the latter two types were found in the collagens produced by the cells after 2 days in primary culture. The purity of the hepatocytes inoculated was 97%, and the majority of the contaminating small cells were erythrocytes. The rate of serum albumin synthesis, which is a typical function of the hepatocytes, was constant or increased during the culture period. Immuno-electron microscopic observation indicated the production of type I collagen by the hepatocytes after 8 days in primary culture. These results are explained only by the activation of collagen synthesis in the day-8 hepatocytes in primary culture.     

Inhibition of hypoxia-induced proliferation and collagen synthesis by vasonatrin peptide in cultured rat pulmonary artery smooth muscle cells

The aim of the present research is to investigate the effects of vasonatrin peptide (VNP) on hypoxia-induced proliferation and collagen synthesis in pulmonary artery smooth muscle cells (PASMCs). Smooth muscle cells isolated from rat pulmonary artery were cultured and used at passages 3-5. Cell proliferation and collagen synthesis were evaluated by cell counts, [(3)H] thymidine and [(3)H] proline incorporation. The results showed that cells exposed to hypoxia for 24 h exhibited a significant increase in [(3)H] thymidine (93%) and [(3)H] proline (52%) incorporation followed by a significant increase in cell number (47%) at 48 h in comparison with the respective normoxic controls. VNP reduced hypoxia-stimulated increase in cell proliferation in a concentration-dependent manner from 10(-8) to 10(-6) mol/L and attenuated hypoxia-induced collagen synthesis ranging from 10(-6) to 10(-5) mol/L, which is similar to but more potent than both ANP and CNP. The action of VNP on PASMCs was mimicked by 8-bromo-cGMP (10(-4) mol/L, the membrane-permeable cGMP analog), and blocked by HS-142-1 (2 x 10(-5) mol/L), the particulate guanylyl cyclase-coupled natriuretic peptide receptor antagonist, or KT-5823 (10(-6) mol/L), the cGMP-dependent protein kinase (PKG) inhibitor. The results suggest that VNP inhibits hypoxia-stimulated proliferation and collagen synthesis in cultured rat PASMCs via particulate guanylyl cyclase-coupled receptors through cGMP/PKG dependent mechanisms.     

Metabolism of rabbit skin collagen. Differences in the apparent turnover rates of type-I- and type-III-collagen precursors determined by constant intravenous infusion of labelled amino acids.

Growing rabbits were infused for up to 10 h with labelled proline, tyrosine and leucine to achieve plateau conditions within body free pools, for [3H]proline infusion, blood free-proline specific radioactivity remained constant after about 1 h. For individual animals, type-I- and type-III-collagen precursors were isolated by precipitation with (NH4)2SO4 and DEAE-cellulose chromatography. Experiments where 3H- and 14C-labelled proline and tyrosine were infused concurrently for different periods of time showed that type I procollagen reached plateau specific radioactivity within 3 h and 90% of the plateau value after 2 h infusion, corresponding to a calculated apparent t 1/2 of less than 26 min. Plateau values for type I procollagen were taken as precursor amino acid pool specific radioactivities. The type-III-collagen-precursor fractions consistently showed lower rates of label incorporation and, by assuming that both type I and type III collagens are synthesized from the same amino acid pools, kinetic analysis revealed an apparent t 1/2 for the isolated type-III-collagen precursors of 3.9 h. For proline, there were large variations between animals in the ratio between the precursor pool for collagen synthesis and the skin homogenate free pool (0.31 +/- 0.13, mean +/- S.D.), so that collagen-synthesis rates based solely on total tissue free-pool values for proline are subject to large and inconsistent errors.     

紫外光-核黄素交联法对豚鼠巩膜生物力学特性的影响

目的探索紫外光-核黄素交联法对巩膜织张力和强度的影响。方法交联组和对照组皆选右眼为实验眼,交联组采用波长为（370±5）nm、辐射强度定为3.0 mW/cm2的紫外线和0.1%核黄素为光敏剂对豚鼠赤道部巩膜面进行胶原交联,对照组不进行交联处理。术后一个月取交联组交联区巩膜条带和对照组相应区域的巩膜条带,进行生物力学测试,并对眼球各组织进行HE染色光镜和透射电镜检测。结果交联组巩膜的生物力学特性增强,赤道部交联组巩膜试件断裂时的极限应力增加了147%,弹性模量显著增加了193%,极限应变降低了21.9%;后极部交联组巩膜试件断裂时的极限应力增加了108%,弹性模量显著增加了191%,极限应变降低了40.42%。HE染色光镜检查结果显示形态学无病理改变,透射电镜结果显示交联组交联区的巩膜成纤维细胞增生活跃。结论紫外光—核黄素交联法可以有效地提高巩膜的生物力学特性,增强巩膜组织的张力和强度,有望作为治疗高度病理性近视的一种方法。     

Effects of prostaglandin E1 on collagen production and degradation in human fetal lung fibroblasts

We examined the effects of prostaglandin E1 on the production and degradation of collagen in human fetal lung fibroblasts. Percentage collagen production was determined by incubating confluent cultures for 6 h with [3H]proline and either [14C]glycine or [14C]leucine and measuring the relative amounts of radioactivity incorporated into collagenase-sensitive and collagenase-insensitive material. Percentage collagen degradation was determined by measuring hydroxy[14C]proline in a low-molecular-weight fraction relative to total hydroxy[14C]proline. Prostaglandin E1, when present at a concentration as low as 0.25 micrograms/ml, reduced net collagen production by a factor of one-half, from 8 +/- 2 to 4 +/- 1% (P less than 0.05). In contrast, the change in percentage degradation was relatively gradual, rising steadily from the control value of 15 +/- 2 to 33 +/- 2% at 4 micrograms/ml (P less than 0.05). The increase in degradation, while significant, could not account for the total decrease in collagen production. We conclude that prostaglandin E1 exerts its inhibitory effect on collagen production in two essentially independent ways: lowering the rate of synthesis and increasing intracellular degradation. However, the decrease in synthesis is greater than the increase in degradation.     

Bleomycin-induced synthesis of type I procollagen by human lung and skin fibroblasts in culture

Bleomycin is a chemotherapeutic agent sometimes associated with pulmonary fibrosis and skin lesions in patients undergoing treatment. We examined the mechanisms of increased collagen deposition on bleomycin-induced fibrosis by incubating human lung and skin fibroblast cultures with [14C]proline; the synthesis of [14C]hydroxyproline relative to DNA or cell protein was taken as an index of procollagen formation. Procollagen synthesis by lung cells in the presence of 0.1 and 1.0 microgram/ml bleomycin was significantly increased and similar results were obtained with skin fibroblasts. The relative synthesis of genetically distinct types of collagen was measured by isolating the newly synthesized type I and type III procollagens by DEAE-cellulose chromatography. The proportion of type III procollagen of total newly synthesized procollagen in control lung fibroblast cultures was 17.4 +/0 0.6% (mean +/- S.E.) while the corresponding value in cells incubated in 1 microgram/ml bleomycin was 12.5 +/- 0.6% (n = 6, P < 0.01). Similar results were obtained when the ratios of newly synthesized type I and type III collagens were estimated by interrupted polyacrylamide disc gel electrophoresis in sodium dodecyl sulfate after a limited proteolytic digestion with pepsin. The results indicate that the increased procollagen synthesis induced by bleomycin in fibroblast cultures is predominantly directed towards the synthesis of type I procollagen.     

Influence of ascorbic acid on ribosomal patterns and collagen biosynthesis in healing wounds of scorbutic guinea pigs

Scorbutic guinea pigs were wounded and the influence of administering ascorbic acid 6 days later was studied with respect to cellular morphology, ribosomal distribution and protein synthesis. Electron-microscopic studies revealed that the dilated endoplasmic reticulum observed in the fibroblasts of scorbutic wound tissue had reverted to a normal configuration 24h after intraperitoneal injection of 100mg of ascorbate. Quantitative determination of the distribution of free and membrane-bound ribosomes indicated a significant increase in membrane-bound ribosomes in wound tissue from ascorbate-supplemented (recovery) animals. Sucrose-density-gradient centrifugation indicated a significant increase in the proportion of large membrane-bound polyribosomes in the range 300-350S and a concomitant decrease in 80S monoribosomes in the ribosome sedimentation profile of recovery tissue. Determination of the synthesis of non-diffusible [(3)H]hydroxyproline in scorbutic and recovery wounds showed a 3-4-fold stimulation in peptidyl-proline hydroxylation in recovery tissues. Studies carried out in which scorbutic and recovery tissues were incubated with [(14)C]leucine indicated that general protein synthesis, as measured by (14)C incorporated into non-diffusible material/mug of DNA, was unaltered by ascorbate supplementation. Similar studies of [(3)H]proline incorporation suggested that in recovery tissues there was a small but significant increase in [(3)H]proline incorporated/mug of DNA, which probably represents an increase in protocollagen synthesis. This observation correlates well with the increase seen in recovery tissues of large polyribosomes on which collagen precursor polypeptides are known to be synthesized. Preliminary characterization of the repair collagen synthesized by recovery animals showed it to be a typical Type I collagen having the chain composition (alpha(1))(2)alpha(2). The extent of glycosylation of the hydroxylysine of the newly synthesized collagen was greater than that reported for either normal guinea-pig dermal collagen or dermal scar collagen.     

Scleral Cross-Linking Using Riboflavin UVA Irradiation for the Prevention of Myopia Progression in a Guinea Pig Model: Blocked Axial Extension and Altered Scleral Microstructure

PurposeTo develop methods of collagen cross-linking (CXL) in the sclera for the treatment of progressive myopia and to investigate the biomechanical and histological changes that occur in as a result.MethodsTwenty 14-day-old guinea pigs were divided into 3 groups: the cross-linking group (CL, n = 8), non cross-linking group (NCL, n = 8), and control group (n = 4). The scleras of the right eyes of the guinea pigs in the CL group were surgically exposed and riboflavin was dropped onto the irradiation zone for 20 seconds prior to ultraviolet-A (UVA) irradiation. The same procedure was conducted on the NCL group but without UVA irradiation. No procedure was conducted on the control group. The right eyes of the guinea pigs in the CL and NCL groups were then fitted with -10.00DS optics for six weeks. Retinoscopy and the axial lengths (AXL) were measured at baseline, and at the second, fourth and sixth weeks post-treatment in all three groups. All animal subjects were euthanized after the sixth week and then biomechanical and histopathological examinations of the scleras were conducted.ResultsThe mean AXL of the NCL group was longer than both the control and CL groups at six weeks (P = 0.001). The mean refractive error in the NCL group was statistically significantly more negative than both the control and the CL groups at six weeks (P = 0.001). The scleral collagen fiber arrangements of the CL and control groups were denser and more regularly distributed than the NCL group. Ultimate stress of the sclera was lowest in the NCL group, followed by the CL then the control group (P<0.05). Ultimate strain (%) of the sclera was lowest in the CL group followed by the NCL and then the control group (P<0.05).ConclusionOur study demonstrates that scleral CXL using riboflavin UVA irradiation effectively prevents the progression of myopia by increasing scleral biomechanical strength in a guinea pig model.     

Abnormal collagen deposition in synovia after collagen type V immunization in rabbits

Sinovitis in Scleroderma (SSc) is rare, usually aggressive and fully resembles rheumatoid arthritis. Experimental models of SSc have been used in an attempt to understand its pathogenesis. Previous studies done in our laboratory had already revealed the presence of a synovial remodeling process in rabbits immunized with collagen V. To validate the importance of collagen type V and to explore the quantitative relationship between this factor and synovia remodeling as well as the relationship between collagen type V and other collagens, we studied the synovial tissue in immunized rabbits. Rabbits (N=10) were immunized with collagen V plus Freund's adjuvant and compared with animals inoculated with adjuvant only (N=10). Synovial tissues were submitted to histological analysis, immunolocalization to collagen I, III and V and biochemical analysis by eletrophoresis, immunoblot and densitometric method. The synovial tissue presented an intense remodeling process with deposits of collagen types I, III and V after 75 and 120 days of immunization, mainly distributed around the vessels and interstitium of synovial extracellular matrix. Densitometric analysis confirmed the increased synthesis of collagen I, III and V chains (407.69+/-80.31; 24.46+/-2.58; 70.51+/-7.66, respectively) in immunized rabbits when compared with animals from control group (164.91+/-15.67; 12.89+/-1.05; 32+/-3.57) (p<0.0001). We conclude that synovial remodeling observed in the experimental model can reflect the articular compromise present in patients with scleroderma. Certainly, this experimental model induced by collagen V immunization will bring new insights in to pathogenic mechanisms and allow the testing of new therapeutic strategies to ameliorate the prognosis for scleroderma patients.     

Biosynthesis of skin collagens in normal and diabetic mice.

Synthesis of collagens in vitro was studied on minced mouse skins incubated with [3H]-proline in organ-culture conditions. A comparative study was carried out on genetically diabetic mice (KK strain) and control mice (Swiss strain). After incubation, neutral-salt-soluble and acid-soluble collagens were extracted. The insoluble dermis was digested by pepsin and type I and type III collagens separated by differential precipitation in neutral salt solutions. Type I and Type III collagens were characterized by ion-exchange and molecular-sieve chromatography, amino acid analysis and by the characterization of CNBr peptides. In diabetic-mouse skin, the relative proportion of type III collagen was significantly higher than in control-mouse skin. The incorporation of radioactively labelled proline into hydroxyproline of type III collagen was significantly faster in diabetic-mouse skin than in control-mouse skin.No significant modifications in the total collagen content of the skin or of their rates of synthesis were observed between the two strains. Alteration in the ratio of type III to type I collagen in the diabetic-mouse skin can be interpreted as a sign of alteration of the regulation of collagen biosynthesis and may be related to the structural alterations observed in the diabetic intercellular matrix.