Recombinant α2(IV)NC1 domain of type IV collagen is an effective regulator of retinal capillary endothelial cell proliferation and inhibits pre-retinal neovascularisation

Background A recombinant form of the α2(IV)NC1 domain of type IV collagen has been shown to have potent anti-angiogenic activity although this peptide has not been studied in the context of proliferative retinopathies. In the current investigation we examined the potential for α2(IV)NC1 to regulate retinal microvascular endothelial cell function using a range of in vitro and in vivo assay systems. Materials and methods α2(IV)NC1 at concentrations between 0.1 and 1 μg/ml was added to retinal microvascular endothelial cells (RMECs) followed by assessment of cell attachment, proliferation and survival. This agent was also tested within a novel in vitro three-dimensional retinal angiogenesis assay and the number of angiogenic sprouts quantified. α2(IV)NC1 was also delivered intra-vitreally to mice with oxygen-induced proliferative retinopathy (OIR) and neovascularisation evaluated in comparison with vehicle-treated controls. Results RMECs treated with α2(IV)NC1 (0.1, 0.5 and 1 μg/ml) showed delayed attachment at 3 h post-seeding, although this deficit had been restored at the 6-h time point. BrdU assay of DNA replication revealed that confluent RMECs treated with α2(IV)NC1 showed no measurable response in comparison with vehicle-treated controls. By contrast, proliferation of sub-confluent RMECs was significantly reduced by α2(IV)NC1 at 0.5 μg/ml (P<0.01). α2(IV)NC1 also induced apoptosis in RMECs and inhibited angiogenesis of pre-existing retinal vascular networks in vitro (P<0.001). Intra-vitreal injection of α2(IV)NC1 in the OIR model significantly inhibited pre-retinal neovascularisation compared with vehicle-treated controls (P<0.001). Conclusion α2(IV)NC1 inhibits angiogenesis in the retinal microvasculature. This recombinant protein has potential for the treatment of neovascularisation in proliferative retinopathies. BioStratum Inc. did not sponsor this research in any way. None of the authors are paid consultants with this company.     

Localization of collagen XVIII and the endostatin portion of collagen XVIII in aged human control eyes and eyes with age-related macular degeneration

PURPOSE: Endostatin, a C-terminal fragment of collagen XVIII (coll XVIII) formed by proteolysis, specifically inhibits endothelial cell migration and proliferation in vitro and potently inhibits angiogenesis and tumor growth in vivo. The purpose of this study was to examine the immunolocalization of endostatin and coll XVIII in the retina and choroid of human donor tissue sections from aged control donor eyes and to determine whether the localization or relative levels are changed in age-related macular degeneration (AMD). METHODS: Ocular tissues were obtained from six aged control donors (age range, 75-86 years; mean age, 80.5 years) without evidence or history of chorioretinal disease and from nine donors with AMD (age range, 74-105 years; mean age, 88.6 years). Tissues were cryopreserved, and streptavidin alkaline phosphatase immunohistochemistry was performed with goat anti-human and mouse anti-human endostatin antibodies and rabbit anti-mouse coll XVIII. Blood vessels were identified with mouse anti-human CD-34 antibody in adjacent sections. Pigment in RPE and choroidal melanocytes was bleached. Three independent observers scored the immunohistochemical reaction product. RESULTS: In aged control eyes, coll XVIII and endostatin (the endostatin portion of coll XVIII) immunoreactivity was observed in large retinal blood vessels and in capillaries in some individuals, but the internal limiting membrane (ILM) had the most intense retinal immunostaining. There was no significant difference in immunoreactivity to both antibodies in retinal blood vessels in aged control eyes. In the choroid, endostatin and coll XVIII were localized to blood vessels, Bruch's membrane, and RPE basal lamina. AMD retina and choroid had a similar pattern and intensity of coll XVIII immunostaining, as observed in control eyes but reaction product was more diffuse in the choroid. Endostatin immunoreactivity was significantly higher in ILM (P = 0.037) in AMD retina and significantly lower in the choriocapillaris, Bruch's membrane, and RPE basal lamina of AMD choroids (P < 0.05) and completely negative in some areas of AMD choroids. CONCLUSIONS: These data suggest that reduced levels of the endostatin portion of coll XVIII in Bruch's membrane, RPE basal lamina, intercapillary septa, and choriocapillaris in eyes with AMD may be permissive for choroidal neovascularization.     

Modulation of type III collagen synthesis in bovine corneal endothelial cells

Bovine corneal endothelial cells in culture synthesize predominantly type III collagen, unlike rabbit corneal endothelial cultures which synthesize type IV collagen. In an attempt to document whether this type III collagen synthesis by bovine cells is a tissue culture-specific phenomenon, collagens synthesized by organ culture of bovine Descemet's membrane/corneal endothelium complex were compared with those of subsequent tissue culture cells, up to the eighth passage. The biosynthetically labeled collagens were analyzed on SDS electrophoresis. The soluble fractions of tissues extracted with neutral salt followed by pepsin digestion contained only type I collagen; no other radiolabeled collagens were detected in organ culture. When pepsin treatment was eliminated, type IV collagen was identified in the tissue extract by immunoblot analysis using monoclonal antibody; type III collagen failed to show a positive band by immunoblot analysis. The pepsin-treated medium fraction of the primary culture contained types I, III and V collagen; type IV collagen was identified by either the characteristic electrophoretic mobility or by immunoblot analysis only prior to the proteolysis step. The subsequent subcultures continued to synthesize types I, III and V collagen, but type IV collagen was no longer detectable from the third passage on. No substantial quantitative changes in the expression of individual collagens were observed during subculture. From the primary culture, type I collagen accounted for 30%, type III for 60% and type V for 10%. Enhanced expression of type III collagen was observed in the eighth passage and in primary cultures grown on type I collagen matrix.(ABSTRACT TRUNCATED AT 250 WORDS)     

Localization of collagen XVIII and endostatin in the human eye

PURPOSE: To investigate the localization of endostatin, a potent angiogenesis inhibitor, and its progenitor collagen XVIII in the human eye. METHODS: Twelve normal human eyes were investigated. Immunohistochemistry of the anterior and posterior eye segment was performed using a polyclonal endostatin and collagen XVIII antibody and a monoclonal collagen XVIII antibody. Specificity of the antibodies was confirmed by Western blot analysis. RESULTS: The antibody against collagen XVIII stained Bowman's membrane, the lens capsule, the trabecular meshwork, and all epithelial and endothelial basal membranes in the anterior and posterior eye segment. In contrast, the antibody against endostatin showed a more distinct staining pattern. Intense intracellular staining for endostatin was present in the lens epithelium and in the non-pigmented epithelium of the ciliary body. Extracellular presence of endostatin could be detected in the lens capsule and all border membranes lining the aqueous humor including the anterior surface of the iris. The choroid was unstained. In the retina, staining was restricted to the inner limiting membrane and to endothelial cells of larger vessels. CONCLUSIONS: Our results show that there is a ring of specifically endostatin expressing structures forming a "barrier" around the anterior chamber and the vitreous. This might physiologically prevent vessels from sprouting into these avascular compartments.     

Histopathologic and electron-microscopic futures of corneal and scleral collagen fibers in osteogenesis imperfecta type III

Background: This report describes the histopathologic and electron-microscopie features of an eye from a patient with osteogenesis imperfecta type III. In particular, the diameters of corneal stromal and scleral collagen fibers were determined. Methods: The eyes of an 18-year-old white male with osteogenesis imperfecta type III were examined by light arid electron microscopy arid the pathological features were compared with an age-matched control eye. Results: The cornea was clear. The sclera had a blue color and was moderately thinned, especially at the equator. Light microscopy revealed absence of Bowman's layer. Transmission electron microscopy confirmed complete absence of Bowman's layer without evidence of scarring or inflammation. The collagen fibers of the corneal stromal lamellae were about 25% narrower than in the control, but the cornea was otherwise unremarkable ultrastructurally. The collagen fibers of the sclera were approximately 50% narrower than in the control and were much more uniform in size. Prominent portions of clastic fibers, which are usually only present in a small number in the inner portion of the sclera, were prescrit throughout the sclera. Conclusion: We propose that it is the uniformity of the scleral collagen fibers which gives the sclera translucence, producing the blue color often observed clinically in osteogenesis imperfecta. Absence of Bowman's layer of the cornea did not interfere with the stability of the cornea in this case. This appears to be the first published pathological examination of the eye in ostcogenesis imperfecta type III.     

Type IV collagen and corneal epithelial adhesion and migration. Effects of type IV collagen fragments and synthetic peptides on rabbit corneal epithelial cell adhesion and migration in vitro

Type IV collagen, a 500-kilodalton (alpha 1)2(alpha 2)1 heterotrimer with noncollagenous domains (NC1) is the major molecule in most basement membranes in the body. In addition to its structural role as scaffolding, type IV collagen is involved in promoting adhesion and migration of various cell types in vitro, including rabbit corneal epithelial cells. This study assessed the effect of purified proteolytic fragments of type IV collagen and selected synthetic peptides derived from the alpha 1 and alpha 2 chains that are related to the adhesion and directed migration of dissociated primary cultured rabbit epithelial cells. Two homologous peptides (HEP-1 and HEP-2) derived from alpha 1 and alpha 2 NC1 regions were found to promote epithelial cell adhesion. A peptide (HEP-3) derived from an interruption of the triple helix of type IV collagen was effective in promoting corneal epithelial cell migration in both chemotaxis and haptotaxis assays. The helical fragment of type IV collagen promoted both directed migration and ample adhesion, indicating that there may be at least another moiety in the helical region responsible for cell adhesion. The results with these peptides revealed to some extent how corneal epithelial cells react at the molecular level with type IV collagen. They could serve as the basis for therapeutic agents to modify corneal epithelial behavior in situations of perturbed wound healing.     

Avian corneal nerves: co-distribution with collagen type IV and acquisition of substance P immunoreactivity

Within the avian cornea collagen type IV is preferentially and characteristically localized to the epithelial and endothelial basement membranes. In the present paper, we demonstrate that collagen type IV also is present within the corneal stroma coincident with the development and distribution of corneal nerves indicating that intra-stromal fibers are associated with Schwann cells or an equivalent cell type. We also demonstrate intra-stromal fibers of collagen type IV orthogonal to the epithelial basement membrane. These novel structures are most prominent on the tenth day of development and become progressively less distinct until they are no longer detectable on the eighteenth day of development. Substance P immunoreactivity is prominently expressed by nerves innervating the epithelium. The first substance P immunoreactive nerves are detected on the twelfth day of development, concomitant with the initiation of epithelial innervation and not the extension of nerves through the stroma. Such nerve fibers become more numerous with progressive development and demonstrate extensive association with both basal and superficial epithelial cells. Thus, the avian cornea is richly supplied with substance P primary afferents. The expression of substance P immunoreactivity correlates directly with the initiation of innervation of the corneal epithelium.     

Posterior corneal elevation changes and characteristic analysis 1 year after corneal collagen cross-linking for keratoconus

International Ophthalmology - To investigate the changes in posterior corneal elevations (PCEs) in the circular areas and local points after corneal collagen cross-linking (CXL) for the treatment...     

准分子激光角膜切削术后转化生长因子β及Ⅰ、Ⅲ型胶原的免疫组化观察

目的观察准分子激光角膜切削术（ｐｈｏｔｏｒｅｆｒａｃｔｉｖｅｋｅｒａｔｅｃｔｏｍｙ,ＰＲＫ）术后,猴角膜愈合过程中角膜转化生长因子β（ｔｒａｎｓｆｏｒｍｉｎｇｇｒｏｗｔｈｆａｃｔｏｒｂｅｔａ,ＴＧＦβ）的表达,以及Ⅰ、Ⅲ型胶原的表达情况,探讨ＴＧＦβ与ＰＲＫ术后角膜愈合过程中Ⅰ、Ⅲ型胶原合成的关系。方法对３只（６只眼）恒河猴行ＰＲＫ治疗。输入－１０００Ｄ治疗程序,切削深度为１０３μｍ。分别于术后１、３及６个月行光镜、电镜及免疫组化检查。结果术后１个月,ＴＧＦβ在角膜上皮层染色阳性,上皮下也可见少量成纤维细胞染色阳性;３个月,角膜上皮染色减弱;６个月时染色呈阴性;对照组为阴性。Ⅰ、Ⅲ型胶原于术后１、３及６个月时均有较强的阳性染色,对照组Ⅰ型胶原染色阳性,Ⅲ型胶原染色阴性。结论ＴＧＦβ参与ＰＲＫ术后角膜的愈合过程,并且可能与术后角膜上皮下新生胶原中Ⅰ、Ⅲ型胶原的合成有关。     

乙醇对角膜上皮瓣活性及基底膜分离定位研究

目的 探讨不同乙醇作用时间对角膜上皮瓣活性的影响,并确定角膜上皮瓣的解剖分离层面.方法 按标准准分子激光角膜上皮瓣下磨镶术(LASEK)方法制备7只尸体眼角膜上皮瓣,其中对照组1只眼,其余6只分为A、B、C 3个组,每组2只眼,乙醇浸润时间分别为20、30和40 S.对7只眼进行HE、增殖细胞核抗原(PCNA)、层黏连蛋白免疫组化及Ⅶ型胶原免疫荧光染色,评价角膜上皮瓣细胞活性,确定LASEK手术中角膜上皮瓣的解剖分离层面,采用X2检验分析.结果 角膜上皮瓣为复层上皮细胞组织,没有Bowman's层和角膜基质成分,PCNA阳性细胞率为A组B组C组,其laminin染色在角膜床和上皮瓣基底膜一侧均有呈片段样棕色条纹状,其collagenⅦ染色阳性反应呈一条绿色线形,而且只存在于角膜床,上皮瓣基底膜侧不着色.结论 角膜上皮瓣活性随乙醇浸润时间延长而降低;乙醇浸润法制备角膜上皮瓣的组织分离层面位于角膜上皮基底膜内,并且定位于基底膜的透明层和致密层之间.     

Role of corneal collagen fibrils in corneal disorders and related pathological conditions

The cornea is a soft tissue located at the front of the eye with the principal function of transmitting and refracting light rays to precisely sense visual information. Corneal shape, refraction, and stromal stiffness are to a large part determined by corneal fibrils, the arrangements of which define the corneal cells and their functional behaviour. However, the modality and alignment of native corneal collagen lamellae are altered in various corneal pathological states such as infection, injury, keratoconus, corneal scar formation, and keratoprosthesis. Furthermore, corneal recuperation after corneal pathological change is dependent on the balance of corneal collagen degradation and contraction. A thorough understanding of the characteristics of corneal collagen is thus necessary to develop viable therapies using the outcome of strategies using engineered corneas. In this review, we discuss the composition and distribution of corneal collagens as well as their degradation and contraction, and address the current status of corneal tissue engineering and the progress of corneal cross-linking.     

Recurrence of lattice corneal dystrophy type 1 in the corneal grafts of two siblings

Amyloid was identified by light and electron microscopy within corneal grafts of two male siblings from a family with lattice corneal dystrophy type 1. These deposits indicate a recurrent disease within the donor tissue, and we believe this reflects an infiltration of the grafts by genetically abnormal host corneal fibroblasts. One of the patients developed bilateral recurrent disease in the grafts eight and 13 years after penetrating keratoplasty. His brother required a regraft 16 years after a penetrating keratoplasty. Although both of these patients required regrafts because of impaired visual acuity, the decrease in visual acuity in one case was not solely the result of reaccumulation of amyloid, but was at least in part caused by a plaque of fibrous tissue behind the cornea.     

In vitro effect of corneal collagen cross-linking on corneal hydration properties and stiffness

Background

The purpose of this study is to evaluate in-vitro the immediate effect of corneal collagen cross-linking (CXL) on corneal hydration and stiffness.

Methods

Forty-two corneal buttons from freshly enucleated porcine eyes were immersed in riboflavin 0.1% in dextran 20% dilution for 3 h in order for their hydration to reach equilibrium. Corneal buttons where divided into two groups; the first group was stored in dark conditions while the other group was irradiated with UV radiation (370 nm) for 30 min to simulate CXL according to the clinically applied protocol. After irradiation, all corneas were immersed in dextran 20% solution for 3 additional hours. Subsequently, each button underwent weighing, thickness measurement, and was mounted in a special device for the measurement of force versus deformation by compression. Finally, all corneal buttons were dehydrated for 48 h in a desiccating oven set at 62 °C and weighed again to obtain their dry mass. Hydration (%) of each button was calculated.

Results

Mean corneal hydration in the irradiated and the non-irradiated group of corneas was 69.8 and 72.2%, respectively (p?<?0.001). Differences in thickness and compressibility were not statistically significant. Thickness and hydration were positively correlated (Pearson’s r?=?0.714, p?<?0.001).

Conclusions

CXL causes corneal dehydration that can be detected immediately after the procedure. This phenomenon may contribute to increased mechanical stiffness of the cornea. A change in stiffness by means of compressibility could not be detected in porcine corneas.     

Extracellular matrices in the developing avian eye: type V collagen in corneal and noncorneal tissues

We have used immunofluorescence histochemistry to examine the temporal and spatial deposition of type V collagen in the extracellular matrices of the developing chick cornea and other selected ocular structures. Tissue sections from animals ranging in age from 4-day-old embryos to 1-day post-hatching were examined by indirect immunofluorescence employing monoclonal antibodies specific for conformational dependent sites in this molecule. In eyes from embryos younger than 6 days of development, no type V staining could be detected. Thus, the epithelially derived primary corneal stroma, which is already well formed at this time, contains little if any of this molecule. Its appearance was concomitant with the physical swelling of the primary stroma and invasion of pericorneal mesenchymal cells. Staining was initially localized in the anterior cornea; subsequently, all corneal matrices showed intense reactions, including the stroma proper and Bowman's and Descemet's membranes. In adjacent noncorneal tissues, the appearance of type V collagen occurred later in development. In some of these, such as in the ciliary body, the pattern of acquisition involved initial deposition at an epithelial-mesenchymal interface with subsequent progression of fibrous strands out into the surrounding mesenchymal tissue. Eventually, all ocular structures with a dense connective tissue component showed staining, but the intensity was appreciably less than that within the cornea. We have previously reported that in all mature tissues except Bowman's membrane, type V collagen is present in an "antigenically masked" form, and that unmasking can be achieved by pretreatment of the tissue sections with dilute acetic acid.(ABSTRACT TRUNCATED AT 250 WORDS)