Marita Uusitalo

Structure, development and function of cytoskeletal elements in non-neuronal cells of the Human Eye

The cytoskeleton, of which the main components in the human eye are actin microfilaments, intermediate filaments and microtubules with their associated proteins, is essential for the normal growth, maturation, differentiation, integrity and function of its cells. These components interact with intra- and extracellular environment and each other, and their profile frequently changes during development, according to physiologic demands, and in various diseases. The ocular cytoskeleton is unique in many ways. A special pair of cytokeratins, CK 3 and 12, has apparently evolved only for the purposes of the corneal epithelium. However, other cytokeratins such as CK 4, 5, 14, and 19 are also important for the normal ocular surface epithelia, and other types may be acquired in keratinizing diseases. The intraocular tissues, which have a relatively simple cytoskeleton consisting mainly of vimentin and simple epithelial CK 8 and 18, differ in many details from extraocular ones. The iris and lens epithelium characteristically lack cytokeratins in adults, and the intraocular muscles all have a cytoskeletal profile of their own. The dilator of the iris contains vimentin, desmin and cytokeratins, being an example of triple intermediate filament expression, but the ciliary muscle lacks cytokeratin and the sphincter of the iris is devoid even of vimentin. Conversion from extraocular-type cytoskeletal profile occurs during fetal life. It seems that posttranslational modification of cytokeratins in the eye may also differ from that of extraocular tissues. So far, it has not been possible to reconcile the cytoskeletal profile of intraocular tissues with their specific functional demands, but many theories have been put forward. Systematic search for cytoskeletal elements has also revealed novel cell populations in the human eye. These include transitional cells of the cornea that may represent stem cells on migration, myofibroblasts of the scleral spur and juxtacanalicular tissue that may modulate aqueous outflow, and subepithelial matrix cells of the ciliary body and myofibroblasts of the choroid that may both participate in accommodation. In contrast to the structure and development of the ocular cytoskeleton, changes that take place in ocular disease have not been analysed systematically. Nevertheless, potentially meaningful changes have already been observed in corneal dystrophies (Meesmanns dystrophy, posterior polymorphous dystrophy and iridocorneal endothelial syndrome), degenerations (pterygium) and inflammatory diseases (Pseudomonas keratitis), in opacification of the lens (anterior subcapsular and secondary cataract), in diseases characterized by proliferation of the retinal pigment epithelium (macular degeneration and proliferative vitreoretinopathy), and in intraocular tumours (uveal melanoma). In particular, upregulation of alpha-smooth muscle actin seems to be a relatively general response typical of spreading and migrating corneal stromal and lens epithelial cells, trabecular cells and retinal pigment epithelial cells.

Cell types of secondary cataract: an immunohistochemical analysis with antibodies to cytoskeletal elements and macrophages

Abstract• Background: The study was carried out to identify cell types of secondary cataract after extracapsular cataract extraction and implantation of an intraocular lens. • Methods: Twenty-five formalin-fixed, paraffin-embedded pseudophakic human eyes with secondary cataract, obtained at autopsy, were studied and compared to a specimen from an anterior subcapsular cataract with a panel of six monoclonal antibodies (MAbs, to vimentin, cytokeratin (CK) 8 and 18, desmin, α-smooth muscle actin, and the CD68 epitope of macrophages by the avidin-biotinylated peroxidase complex (ABC) method. • Results: MAb Vim 3B4 to vimentin immunolabeled spindle-shaped cells in 16 of 17 central plaques of secondary cataract as well as cells in all 16 Soemmerings ring cataracts. Spindle-shaped cells reacted with MAb CAM 5.2 to CK 8 in 13 of 18 eyes, but only one specimen was labeled with MAb CY-90 to CK 18. No immunoreaction was seen with MAb D33 to desmin, whereas MAb 1A4 to α-smooth muscle actin immunolabeled spindle-shaped cells in 15 of 18 plaques of secondary cataract. Macrophages were seen with MAb PG-M1 in 13 of 19 secondary cataracts. In the anterior subcapsular cataract, spindle-shaped cells under a wrinkled but otherwise intact capsule reacted with MAb Vim 3B4 to vimentin, MAb CAM 5.2 to CK 8, and MAb 1A4 to α-smooth muscle actin. •Conclusion: Spindle-shaped cells in secondary and anterior subcapsular cataracts react with antibodies to vimentin, CK 8 and α-smooth muscle actin, suggesting them to be metaplastic epithelial cells that derive from the lens epithelium. α-Smooth muscle actin persists in them at least 10 years postoperatively, but CK 8 starts to disappear after 3 years. Macrophages are one possible modulator of this transdifferentiation.

Collagen XVIII/endostatin shows a ubiquitous distribution in human ocular tissues and endostatin-containing fragments accumulate in ocular fluid samples.

BackgroundThe endostatin domain of type XVIII collagen (ColXVIII) inhibits neovascularization and regulates cell migration and matrix turnover. This study was designed to demonstrate the protein and gene expression patterns of ColXVIII/endostatin in the human eye and to ascertain whether endostatin is detectable in ocular fluid samples.MethodsTwenty human eyes enucleated on account of choroidal melanoma were used for immunohistochemical stainings with antibodies against ColXVIII and endostatin. In situ hybridization was used to localize cells responsible for the production of mRNA for ColXVIII. Tear fluid, aqueous humor, and vitreous gel samples were used for Western immunoblotting to detect endostatin fragments in these samples.ResultsColXVIII was immunolocalized to almost all ocular structures, namely the basement membranes (BMs) of the corneal and conjunctival epithelia, Descement’s membrane, the anterior border layer and posterior pigmented epithelium of the iris, the BMs of the pigmented and non-pigmented ciliary epithelia, the internal wall of Schlemm’s canal and trabeculae, the ciliary and iris muscle cells, the BMs of the pigment epithelium of the retina, and the internal limiting membrane. Universal expression was seen in the BMs of vascular endothelial cells, and in fibroblasts located in the conjunctiva, the iris, and the ciliary body. Endostatin showed a corresponding pattern, but additional immunostaining was present in the corneal and conjunctival epithelial cells. Most epithelial and mesenchymal cells expressed the mRNA for ColXVIII. Endostatin-containing fragments varying in size were detected in tear fluid, aqueous humor and vitreous gel samples.ConclusionsPractically all structures of the human eye contain ColXVIII/endostatin, emphasizing its possible important structural and functional role in the human eye. Furthermore, ocular fluid samples contain endostatin fragments, which may contribute to the antiangiogenic properties of the eye.

Immunohistochemical localization of chondroitin sulfate proteoglycan and tenascin in the human eye compared with the HNK-1 epitope

Abstract•Background: A previous study revealed the HNK-1 epitope in the human ciliary body beneath the ciliary epithelium. The molecules bearing this 3-sulphoglucuronic acid-containing oligosaccharide epitope in the eye remain unknown. As chondroitin sulphate proteoglycan (CSPG) and tenascin are potential candidates as bearers of the HNK-1 epitope, their distribution in the human eye was compared with that of the HNK-1 epitope. • Methods: Fifty-five formalin-fixed, paraffin-embedded human eyes, including 20 normal eyes and 35 eyes with exfoliation syndrome or glaucoma, were studied immunohistochemically with monoclonal antibody (MAb) CS-56 to CSPG, MAb TN2 to tenascin, and MAbs HNK-I and VC1.1 to the HNK-1 epitope. Additionally, four frozen lens capsules with exfoliation material were studied by indirect immunofluoresence. • Results: A population of dendritic cells in the inner connective tissue layer of the ciliary body and exfoliation material were immunoreactive with antibodies to the HNK-1 epitope, but no labelling for CSPG and tenascin was seen in them, including frozen sections. The inner surface of the nonpigmented ciliary epithelium was reactive for the HNK-1 epitope, and at the ora serrata also for CSPG. In some eyes with glaucoma, immunoreaction for CSPG and tenascin was seen beneath the epithelium and endothelium of the cornea. The nerve fibre layer of the retina was labelled for tenascin. In the sclera, all antibodies labelled the ground substance, and in some large blood vessels immunoreaction for CSPG and tenascin was seen subendothelially. • Conclusion: Apart from the sclera, the distribution of CSPG and tenascin was different from that of the HNK-1 epitope, suggesting that this carbohydrate epitope may not be borne by these molecules in the human ciliary body.

The HNK-1 Carbohydrate Epitope in the Eye: Basic Science and Functional Implications

The HNK-1 carbohydrate epitope is part of many cell membrane and extracellular matrix molecules. It has been implicated in cell to cell and cell to extracellular matrix adhesion, and antibodies to the HNK-1 epitope are emerging as a versatile tool in eye research. They have been used to identify a novel cell type in the human eye, the subepithelial matrix cells that reside in the inner connective tissue layer (ICTL) of the ciliary body. Although these cells resemble fibroblasts in ultrastructure, they form a distinct cell population that differs in its antigenic profile from fibroblasts of other tissues. These cells are associated with the elastic fiber system of the ICTL. Other structures in the human eye that harbor the HNK-1 epitope in a nonrandom pattern are the ciliary and iris epithelia, the zonular lamella, the lens capsule, the retina, glial cells of the optic and ciliary nerves, and scleral fibroblasts. The HNK-1 epitope in the eye appears early during embryonic development and is phylogenetically conserved, but many interspecies differences exist in its distribution. The role of the HNK-1 epitope may be to structurally stabilize the ciliary body and the retina, and to participate in zonular attachments. The HNK-1 epitope has been linked with many common eye diseases. The subepithelial matrix cells seem to be susceptible to undergo irreversible damage as a result of glaucoma, thermal injury, and tissue compression. This epitope has proved to be useful in identifying intraocular deposits of exfoliation syndrome. It can explain the adhesiveness of exfoliation material. Intraocular exfoliation material differs in HNK-1 immunoreactivity from the extraocular fibrillopathy of exfoliation syndrome and its presence in fellow eyes also argues against the concept of unilateral exfoliation syndrome. The HNK-1 epitope is found in the extracellular matrix of secondary cataract and anterior subcapsular cataract, and it may contribute to their pathogenesis. Finally, the HNK-1 epitope can be used to trace neuroepithelial derivatives of the optic vesicle in developmental anomalies and in tumors of the eye. Eventual identification of molecules that bear the HNK-1 epitope in the eye will likely shed light on many aspects of ocular physiology and pathobiology

The HNK-1 epitope in the inner connective tissue layer of the human ciliary body in exfoliation syndrome and various types of glaucoma

Possible changes in the expression of the HNK-1 carbohydrate epitope in the inner connective tissue layer of the human ciliary body, located between the ciliary epithelium and muscle, was studied using 2 formalin-fixed, paraffin-embedded eyes with exfoliation syndrome, 33 eyes with different types of glaucoma, and 21 morphologically normal control eyes. A strong immunoreaction delineating cell processes was observed in this layer with monoclonal antibodies HNK-1 and VC1.1 recognizing the HNK-1 epitope in control specimens, whereas partly granular immunoreaction was present in eyes with exfoliation syndrome. Exfoliation material was also immunoreactive. In all types of advanced glaucoma, the immunoreaction was mostly granular in nature and greatly diminished. No difference in HNK-1 immunoreactivity between control and glaucoma eyes was seen in the retina and ciliary epithelium. Elevated intraocular pressure, either directly or by decreasing blood flow to the ciliary body, may cause degenerative or metabolic changes in the inner connective tissue layer cells that bear or secrete molecules sharing the HNK-1 epitope. The partly granular immunoreactivity in eyes with exfoliation syndrome only indicates changes in this epitope even without an increase in intraocular pressure.

Differential distribution of the HNK-1 carbohydrate epitope in the vertebrate retina

The expression of the cell adhesion-related HNK-1 carbohydrate epitope in the retina and ciliary body was studied in different vertebrates and in man. A series of eyes from 4 fish, 5 bird, and 9 mammalian species was analyzed by immunohistochemistry with monoclonal antibodies (MAb) HNK-1 and VC1.1 to the HNK-1 epitope, and with MAb SY38 to synaptophysin. Additionally, 7 morphologically normal human eyes were studied. In all fishes, as well as in baboons and man, the radial glia and all retinal layers except the photoreceptor cell layer were immunoreactive for the HNK-1 epitope. In all birds, the nerve fiber layer and both plexiform layers were labelled. In nonprimate mammals only the plexiform layers were immunoreactive. Fine differences in this general immunoreaction pattern were seen in different species. Mab SY38 labeled both plexiform layers of mammals only. In the ciliary body, immunoreaction for the HNK-1 epitope was seen in the inner connective tissue layer only in man, but the ciliary nerves were labelled in all species except the mouse and rat. The HNK-1 epitope seems to be phylogenetically conserved in the retina, where the HNK-1 immunoreactive plexiform layers possibly are overlapped with HNK-1 reactive radial glial cells in fishes and primates. Instead in the inner connective tissue layer of the ciliary body, the HNK-1 epitope is not phylogenetically conserved.

The HNK-1 Carbohydrate Epitope and the Human Eye in Health and Disease.

The HNK-1 carbohydrate epitope is part of many cell membrane and extracellular matrix molecules, several of which have been implicated in cell adhesion. It is a versatile tool in eye research. In the human eye this epitope is present in the retina, the optic and ciliary nerves, the ciliary and iris epithelia, the zonular lamella, and the sciera. It is phylogenetically conserved, but the positive cell types vary from species to species. In addition to revealing interspecies differences in the vertebrate retina, the HNK-1 epitope has been used to identify a novel cell type in the eye: the subepithelial matrix cells that reside in the inner connective tissue layer (ICTL) of the ciliary body. Although these cells resemble fibroblasts in ultrastructure, they form a distinct cell population that differs in antigenic profile from fibroblasts in other tissues. The HNK-1 epitope is also associated with the elastic fiber system of the ICTL, which may be produced by the subepithelial matrix cells. It may help to structurally stabilize the ciliary body and the retina. The HNK-1 epitope is also involved in many important eye diseases. The subepithelial matrix cells seem to be susceptible to irrreversible atrophy as a result of glaucoma, thermal injury, and tissue compression. On the other hand, the HNK-1 epitope is found in the extracellular matrix of secondary cataracts and may contribute to its pathogenesis. Finally, this epitope has proved to be useful in identifying deposits of exfoliation material, and in tracing neuroepithelial derivatives in developmental anomalies and tumors of the eye.