Ocular Basement Membranes Research Articles

Neurological involvement in monogenic podocytopathies.

Genetic studies of hereditary nephrotic syndrome (NS) have identified more than 50 genes that, if mutated, are responsible for monogenic forms of steroid-resistant NS (SRNS), either isolated or syndromic. Most of these genes encode proteins expressed in the podocyte with various functions such as transcription factors, mitochondrial proteins, or enzymes, but mainly structural proteins of the slit diaphragm (SD) as well as cytoskeletal binding and regulator proteins. Syndromic NS is sometimes associated with neurological features. Over recent decades, various studies have established links between the physiology of podocytes and neurons, both morphologically (slit diaphragm and synapse) and functionally (signaling platforms). Variants in genes expressed in different compartments of the podocyte and neurons are responsible for phenotypes associating kidney lesions with proteinuria (mainly Focal and Segmental Glomerulosclerosis (FSGS) or Diffuse Mesangial Sclerosis (DMS)) and central and/or peripheral neurological disorders. The Galloway-Mowat syndrome (GAMOS, OMIM#251300) associates neurological defects, microcephaly, and proteinuria and is caused by variants in genes encoding proteins of various functions (microtubule cytoskeleton regulation (WDR73), regulation of protein synthesis via transfer RNAs (KEOPS and WDR4 complexes)). Pierson syndrome (OMIM#609049) associating congenital nephrotic syndrome and central neurological and ophthalmological anomalies is secondary to variants in LAMB2, involved in glomerular and ocular basement membranes. Finally, Charcot-Marie-Tooth-FSGS (OMIM#614455) combines peripheral sensory-motor neuropathy and proteinuria and arises from INF2 variants, resulting in cytoskeletal polymerization defects. This review focuses on genetic syndromes associating nephrotic range proteinuria and neurological involvement and provides the latest advances in the description of these neuro-renal disorders.

The human Descemet's membrane and lens capsule: Protein composition and biomechanical properties

The Descemet's membrane (DM) and the lens capsule (LC) are two ocular basement membranes (BMs) that are essential in maintaining stability and structure of the cornea and lens. In this study, we investigated the proteomes and biomechanical properties of these two materials to uncover common and unique properties. We also screened for possible protein changes during diabetes. LC-MS/MS was used to determine the proteomes of both BMs. Biomechanical measurements were conducted by atomic force microscopy (AFM) in force spectroscopy mode, and complemented with immunofluorescence microscopy. Proteome analysis showed that all six existing collagen IV chains represent 70% of all LC-protein, and are thus the dominant components of the LC. The DM on the other hand is predominantly composed of a single protein, TGF-induced protein, which accounted for around 50% of all DM-protein. Four collagen IV-family members in DM accounted for only 10% of the DM protein. Unlike the retinal vascular BMs, the LC and DM do not undergo significant changes in their protein compositions during diabetes. Nanomechanical measurements showed that the endothelial/epithelial sides of both BMs are stiffer than their respective stromal/anterior-chamber sides, and both endothelial and stromal sides of the DM were stiffer than the epithelial and anterior-chamber sides of the LC. Long-term diabetes did not change the stiffness of the DM and LC. In summary, our analyses show that the protein composition and biomechanical properties of the DM and LC are different, i.e., the LC is softer than DM despite a significantly higher concentration of collagen IV family members. This finding is unexpected, as collagen IV members are presumed to be responsible for BM stiffness. Diabetes had no significant effect on the protein composition and the biomechanical properties of both the DM and LC.

Remodeling of epithelial cells and basement membranes in a corneal deficiency model with long-term follow-up

The ocular surface consists of the cornea, conjunctiva, and the limbus that is located in the transitional zone between the cornea and conjunctiva. The corneal epithelial cells are generated through the mitosis of corneal epithelial stem cells in the limbus. This study investigated a rabbit corneal deficiency model prepared by the surgical removal of the corneal and limbal epithelia, which express cytokeratin 12 (K12). After the surgery, K13-expressing conjunctival epithelium migrated onto the corneal surface and completely covered the surface, leading to neovascularization and corneal opacification. However, at 24 and 48 weeks after the surgery, K12-expressing cornea-like cells reappeared on the model ocular surface. These cells formed an island surrounded by invaded conjunctiva and were isolated from the limbus. Interestingly, in the 24-week model surface, α1(IV) and α2(IV) collagen chains, which are normally found in the basement membrane of the native limbus and conjunctiva, and not in the cornea, were continuously deposited throughout the entire basement membrane, including the basement membrane under cornea-like cells. By contrast, in the 48-week model surface, α1(IV) and α2(IV) collagen chains were absent from the basement membrane beneath the central part of cornea-like cells and were localized below the invaded conjunctiva and the transitional zone between cornea-like cells and the invaded conjunctiva, which had similar distribution to the native ocular basement membrane. Moreover, K12, K14, p63, vimentin, and α1(IV) and α2(IV) collagen chains, which are colocalized in the native limbus, were all present at the transitional zone of the 48-week model surface. Therefore, a limbus-like structure appeared to be reconstructed on the surface of the 48-week model as a stem cell niche. This study should aid in the understanding of human corneal deficiency, the correlation between the epithelial cell phenotype and the composition of the basement membrane, and the epithelial stem cell niche.

Diabetes-induced morphological, biomechanical, and compositional changes in ocular basement membranes

The current study investigates the structural and compositional changes of ocular basement membranes (BMs) during long-term diabetes. By comparing retinal vascular BMs and the inner limiting membrane (ILM) from diabetic and non-diabetic human eyes by light and transmission electron microscopy (TEM), a massive, diabetes-related increase in the thickness of these BMs was detected. The increase in ILM thickness was confirmed by atomic force microscopy (AFM) on native ILM flat-mount preparations. AFM also detected a diabetes-induced increase in ILM stiffness. The changes in BM morphology and biophysical properties were accompanied by partial changes in the biochemical composition as shown by immunocytochemistry and western blots: agrin, fibronectin and tenascin underwent relative increases in concentration in diabetic BMs as compared to non-diabetic BMs. Fibronectin and tenascin were particularly high in the BMs of outlining microvascular aneurisms. The present data showed that retinal vascular BMs and the ILM undergo morphological, biomechanical and compositional changes during long-term diabetes. The increase in BM thickness not only resulted from an up-regulation of the standard BM proteins, but also from the expression of diabetes-specific extracellular matrix proteins that are not normally found in retinal BMs.

Laminin β1 and γ1 Containing Laminins Are Essential for Basement Membrane Integrity in the Zebrafish Eye

In this study recessive zebrafish mutations in the genes encoding laminin beta1 (lamb1) and laminin gamma1 (lamc1) were used to determine the functions of these laminin proteins during ocular basement membrane formation and during zebrafish eye development. Ocular defects in lamb1 and lamc1 mutants were characterized by using a combination of histology, immunohistochemistry, in situ hybridization, and transmission electron microscopy. The results demonstrated that zebrafish lamb1 and lamc1 mutants possess defects in two ocular basement membranes--the lens capsule and the inner limiting membrane--whereas Bruch's membrane is largely unaffected. lamb1 and lamc1 mutants possess severe lens dysplasias that result from a compromise in lens capsule integrity. Inner limiting membrane continuity is irregular in these mutants, and these irregularities result in small retinal ectopias that extend from the retina into the interstitial space between the retina and the lens. At late embryonic stages (e.g., 5-7 days after fertilization), retinal lamination defects are also observed in a subset of laminin mutants. The results demonstrate that laminin beta1 and -gamma1 containing laminins are essential for the integrity of the lens capsule basement membrane and inner limiting membrane in the zebrafish eye.

Clusterin Deficiency in Eyes with Pseudoexfoliation Syndrome May Be Implicated in the Aggregation and Deposition of Pseudoexfoliative Material

To determine the role of the extracellular chaperone clusterin in the pathophysiology of pseudoexfoliation (PEX) syndrome/glaucoma, which is characterized by the stable deposition of abnormal extracellular fibrillar material in anterior segment tissues. Real-time PCR, in situ hybridization, and immunohistochemistry were applied to analyze the mRNA and protein expression of clusterin in PEX eyes of patients without and with glaucoma and to compare them with eyes of patients with primary open-angle glaucoma and angle-closure glaucoma and with normal control eyes. Aqueous levels of clusterin were determined by Western blot analysis. Real-time PCR and Western blot analysis were used to study the effect of TGF-beta1, which is significantly increased in the aqueous humor of PEX eyes, on clusterin expression by nonpigmented ciliary epithelial cells in vitro. Clusterin mRNA was ubiquitously expressed in most ocular cells and tissues, particularly in the epithelium of ciliary processes, whereas the protein was mostly located to extracellular structures, such as ocular basement membranes and stromal fibers. Real-time PCR and in situ hybridization displayed significant downregulation of clusterin mRNA in all anterior segment tissues of PEX eyes, irrespective of the presence or type of glaucoma, compared with normal and glaucomatous control eyes, whereas posterior segment tissues did not show any differential expression. A generally decreased immunoreactivity, but a prominent binding of clusterin to all PEX deposits, could be observed in ocular tissues of PEX eyes. Clusterin levels in aqueous humor were significantly reduced in eyes of patients with PEX syndrome compared with normal and glaucomatous control eyes. The expression of clusterin mRNA and protein in nonpigmented ciliary epithelial cells was significantly downregulated by TGF-beta1 in vitro. Considering the known role of clusterin as a highly efficient extracellular chaperone, its deficiency in the anterior segment of PEX eyes may promote the stress-induced aggregation and stable deposition of the pathologic extracellular matrix product characteristic of PEX syndrome.

Tensile mechanical and creep properties of Descemet's membrane and lens capsule

Descemet's membrane (DM) and the lens capsule (LC) are two ocular basement membrane structures which in comparison with other basement membranes have exceptional thicknesses which increase with age. Both membranes are supposed to contain networks of type IV collagen and laminin linked together with nidogen/entactin and containing other glycoproteins and proteoglycans. DM is a unique basement membrane which in addition contains fine filaments of type VIII collagen arranged in a hexagonal lattice. The mechanical functions of the LC are in lens suspension and accommodation, and its mechanical properties, previously investigated, are of great interest from a surgical point of view. DM serves as an endothelial basement membrane. Otherwise, its physiological function is unknown but may be one of mechanical support, filtration, or fluid barrier. Data on the mechanical properties of DM or the supramolecular assembly of type VIII collagen are very scarce or absent. The aim of this study was to determine and compare the mechanical properties of the two ocular membranes in order to elucidate the properties of DM in the light of those of LC. The human eyes were from testamentary donors and rat, cow, and sow eyes were obtained from normal animals. The tensile mechanical properties were determined by a volume–strain procedure and creep properties by subjecting the membranes from the latter three species to a constant axial stress. In rat, cow, and sow, DM was less strained to obtain a fixed moderate stress value (0·5 MPa) and showed to be 3·4- to 5·2-fold stiffer and to attain 2·7- to 4·6-fold higher stress at a strain value of 0·10 when compared with LC. The maximal strain, stiffness and stress were found to be less than those of the LC. In humans, DM and LC showed very similar mechanical properties. The instantaneous creep of DM was found to be less than that of LC indicating a higher stiffness of DM in the axial direction. In conclusion, depending on the species, DM showed to possess from one-fourth to similar mechanical strength as that of LC, and, in rat, cow, and sow, DM appeared to be stiffer in both lateral and axial directions at moderate strain values when compared with LC.

Age-Dependent Iris Abnormalities in Collagen XVIII/Endostatin Deficient Mice with Similarities to Human Pigment Dispersion Syndrome

Collagen XVIII is expressed in ocular basement membranes (BMs) and inactivating mutations cause Knobloch syndrome, with several ocular abnormalities. In this study we investigated ocular structures in collagen XVIII/endostatin (Col18a1(-/-))-deficient mice to elucidate the role of this extracellular matrix component in the eye. Eyes of Col18a1(-/-) and control mice were examined by light and transmission electron microscopy, laser scanning ophthalmoscopy, and fluorescence angiography. Immunohistochemical analysis of neuronal, epithelial, and immune cells in the eye was performed with antibodies against established cell markers. Col18a1(-/-) mice showed a disruption of the posterior iris pigment epithelial (IPE) cell layer with release of melanin granules. The BM of the posterior IPE was attached to the lens and the nonpigmented epithelium of the ciliary body, which was flattened in mutant mice. In aged mutant mice a severe thickening of the stromal iris BM zone was found, and pigmented cells migrated out of the iris and covered the retina along the inner limiting membrane (ILM), sometimes penetrating into the retina. These cells resembled iris clump cells, and immunohistochemistry demonstrated that they were macrophage-like cells. Furthermore, morphologically abnormal retinal vasculature was seen by fluorescence angiography. The abnormalities in the iris and ciliary body of Col18a1(-/-) mice demonstrate an important role of collagen XVIII for the function of ocular BMs. The absence of this collagen alters the properties of BMs and leads to severe defects in the iris, showing striking similarities to human pigment dispersion syndrome. In addition, loss of collagen XVIII creates changes that allow clump cells to migrate out of the iris. These cells have not been well characterized previously. In the current study we showed that they are macrophage-like cells and are able to penetrate the ILM in mutant mice. The disease mechanism of human pigment dispersion syndrome is not well understood, but Col18a1(-/-) mice may serve as a model and demonstrate the potential importance of alterations in extracellular matrix components in this disease.

The anterior lens capsule used as support material in RPE cell-transplantation.

To investigate the use of an ocular basement membrane as support material for transplanted porcine RPE cells. Porcine RPE cells were grown on bovine corneal extracellular matrix (ECM), isolated bovine- and porcine lens capsules, and tissue culture plastic. Cell density, and cell morphology were studied by phase contrast microscopy and transmission electron microscopy. RPE cells grown on porcine anterior lens capsule and on ECM obtained better morphology and higher final cell density than cells grown on plastic and on bovine anterior lens capsule. It was possible to transplant the porcine anterior lens capsule to the subretinal space in pigs. Within two weeks of observation, the lens capsule was well tolerated in the subretinal space. The anterior lens capsule seems to be promising as support material for use in RPE cell-transplantation.

Absence of ocular manifestations in autosomal dominant Alport syndrome associated with haematological abnormalties

Most patients with Alport syndrome have X-linked or autosomal recessive disease that is characterised by renal failure, hearing loss, and, in nearly 75% of the cases, a dot-and-fleck retinopathy and anterior lenticonus. There are only case reports of individuals with the rare autosomal dominant form, who can have haematuria or renal failure, deafness, and, in addition, low platelet counts and neutrophil inclusions. The ocular features of autosomal dominant inheritance have not been described. We have examined the eyes in the members of two families where Alport syndrome was diagnosed on the basis of the clinical features and family history, and where autosomal dominant inheritance was confirmed by father-to-son disease transmission, the associated haematological abnormalities, and haplotypes that segregated with the recently described locus at chromosome 22q. In Family A, the eyes of two individuals with haematuria, hearing loss, and haematological abnormalities and of nine unaffected family members were examined. In Family B, the eyes of two individuals with renal failure, normal hearing, and haematological abnormalities were examined. None of the affected or unaffected members in either family had a dot-and-fleck retinopathy, anterior lenticonus, a history suggesting recurrent corneal erosions, or corneal dystrophy. These results indicate that the protein abnormality in autosomal dominant Alport syndrome does not produce the retinopathy and lenticonus typical of X-linked and autosomal recessive disease. This may be because the abnormal protein is not present or is less important in the ocular basement membranes than elsewhere, or because the presence of a normal allele in autosomal dominant disease compensates for the defective allele.

An immunoelectron microscope study of the aged human lens capsule

The distribution of types I–IV collagen and laminin was studied in seven aged human lens capsules using the immunogold EM technique on LR White embedded tissue. Samples were taken from the anterior, equatorial and posterior regions. Labelling for type II collagen was not observed. Type IV collagen was evenly distributed throughout the thickness of the capsule but was absent from the zonules. However, an unexpected finding was strong labelling for types I and III collagen, again evenly distributed throughout the capsule. The presence of type III collagen makes the lens capsule unique among ocular basement membranes. Laminin was present in linear densities, zonular lamellae and zonular fibres, suggesting that linear densities are an integral part of the zonular apparatus.

Distribution of Acidic and Basic Fibroblast Growth Factors (FGF) in the Foetal Rat Eye: Implications for Lens Development

Previously we reported that, in vitro, lens cells proliferate, migrate or differentiate in response to low, medium and high concentrations of FGF respectively. To examine further the role of FGF in lens development we used immunohistochemistry to study the distribution of aFGF and bFGF in the eye of the 20 day rat foetus. Strong aFGF-like reactivity was localised in a band of cells near the lens equator which included the germinative zone where most cell proliferation occurs and the transitional zone where epithelial cells differentiate into fibres. The closely apposed inner epithelial layer of the ciliary and iridial retina also reacted strongly. Reactivity for aFGF was also found in the epidermis and in the corneal and conjunctival epithelia. In the neural retina, reactivity was found in the nerve fibre layer and in isolated cells of the inner plexiform layer. bFGF-like reactivity was found in the retinal ganglion cell layer, extra-ocular muscles and associated with endothelial cells of the hyaloid, lenticular and choroid vasculatures. Pre-digestion of sections with hyaluronidase caused loss of cell-associated reactivity but revealed strong bFGF-like reactivity in ocular basement membranes, in particular, the lens capsule. The sensitivity of this capsular bFGF localisation to heparinase indicates that bFGF in the extracellular matrix is complexed with heparan sulphate proteoglycans. The results of this study are consistent with the hypothesis that FGF plays an important role in lens development via both autocrine and paracrine mechanisms.

Distribution of Acidic and Basic Fibroblast Growth Factors (FGF) in the Foetal Rat Eye: Implications for Lens Development

Previously we reported that, in vitro, lens cells proliferate, migrate or differentiate in response to low, medium and high concentrations of FGF respectively. To examine further the role of FGF in lens development we used immunohistochemistry to study the distribution of aFGF and bFGF in the eye of the 20 day rat foetus. Strong aFGF-like reactivity was localised in a band of cells near the lens equator which included the germinative zone where most cell proliferation occurs and the transitional zone where epithelial cells differentiate into fibres. The closely apposed inner epithelial layer of the ciliary and iridial retina also reacted strongly. Reactivity for aFGF was also found in the epidermis and in the corneal and conjunctival epithelia. In the neural retina, reactivity was found in the nerve fibre layer and in isolated cells of the inner plexiform layer. bFGF-like reactivity was found in the retinal ganglion cell layer, extra-ocular muscles and associated with endothelial cells of the hyaloid, lenticular and choroid vasculatures. Pre-digestion of sections with hyaluronidase caused loss of cell-associated reactivity but revealed strong bFGF-like reactivity in ocular basement membranes, in particular, the lens capsule. The sensitivity of this capsular bFGF localisation to heparinase indicates that bFGF in the extracellular matrix is complexed with heparan sulphate proteoglycans. The results of this study are consistent with the hypothesis that FGF plays an important role in lens development via both autocrine and paracrine mechanisms.

Monoclonal antibody analysis of ocular basement membranes during development

As a first step in a study of the role(s) of basement membranes in ocular morphogenesis, we have produced a variety of monoclonal antibodies against native lens capsule from adult chicks, and have used these reagents to stain histologica sections of ocular tissues from 4 1 2 - to 18-day-old chicken embryos. Four different patterns of immunofluorescence were observed in sections of corneas of 18-day-old chicken embryos stained with these antibodies. The antibodies in group 1 stained the basement membranes of both the corneal epithelium and the endothelium (as well as Descemet's membrane). Those in groups 2 and 3 stained only the epithelial or endothelial basement membranes, respectively. The group 4 antibody stained the corneal stroma as well as Bowman's membrane and Descemet's membrane. The antibodies in group 1 could be further subdivided into groups 1a and 1b on the basis of temporal differences in the onset of staining in corneas from 4 1 2 - to 7-day-old embryos. Thus, this series of monoclonal antibodies appears to recognize at least five different antigenic determinants. When these antibodies were used to stain sections of eyes at different stages of development, we found that the characteristic differential staining of some basement membranes was maintained throughout development, while the staining properties of others changed. This indicates that many of the ocular basement membranes may differ from one another in composition or conformation, and that at least some of them may undergo developmental changes. We also noticed a similarity in the pattern of fluorescence associated with the basement membranes of the limbal blood vessels and the corneal endothelium that is consistent with the hypothesis that the corneal endothelium is derived from the early periocular vascular endothelium. Our observations of developing corneas also revealed that the antigen recognized by the group 4 antibody may be produced by both the corneal epithelium and the stromal fibroblasts. The suitability of monoclonal antibodies for probing basement membrane heterogeneity is discussed.