Loss of Notch1 Disrupts the Barrier Repair in the Corneal Epithelium

The corneal epithelium is the outermost layer of the cornea that directly faces the outside environment, hence it plays a critical barrier function. Previously, conditional loss of Notch1 on the ocular surface was found to cause inflammation and keratinization of the corneal epithelium. This was in part attributed to impaired vitamin A metabolism, loss of the meibomian glands and recurrent eyelid trauma. We hypothesized that Notch1 plays an essential role in the corneal epithelial barrier function and is a contributing factor in the pathologic changes in these mice. Notch1 was conditionally deleted in adult Notch1^flox/flox, K14-Cre-ERT^+/- mice using hydroxy-tamoxifen. The results indicated that conditional deletion of Notch1 on the ocular surface leads to progressive impairment of the epithelial barrier function before the onset of corneal opacification and keratinization. Loss of the barrier was demonstrated both by an increase in in vivo corneal fluorescein staining and by enhanced penetration of a small molecule through the epithelium. Corneal epithelial wounding resulted in significant delay in recovery of the barrier function in conditional Notch1^-/- mice compared to wild type. Mice with conditional deletion of Notch1 did not demonstrate any evidence of dry eyes based on aqueous tear production and had normal conjunctival goblet cells. In a calcium switch experiment in vitro, Notch1^-/- cells demonstrated delayed membrane localization of the tight junction protein ZO-1 consistent with a defect in the epithelial tight junction formation. These findings highlight the role of Notch1 in epithelial differentiation and suggest that intrinsic defects in the corneal epithelial barrier recovery after wounding is an important contributing factor to the development of inflammatory keratinization in Notch1^-/- mice.     

Expression of specific keratin markers by rabbit corneal, conjunctival, and esophageal epithelia during vitamin A deficiency

Using an in vivo rabbit model system, we have studied the morphological and biochemical changes in corneal, conjunctival, and esophageal epithelia during vitamin A deficiency. Light and electron microscopy showed that the three epithelia undergo different degrees of morphological keratinization. Corneal and conjunctival epithelia became heavily keratinized, forming multiple layers of superficial, anucleated cornified cells. In contrast, esophageal epithelium underwent only minor morphological changes. To correlate morphological alterations with the expression of specific keratin molecules, we have analyzed the keratins from these epithelia by the immunoblot technique using the subfamily-specific AE1 and AE3 monoclonal antikeratin antibodies. The results indicate that during vitamin A deficiency, all three epithelia express an AE1-reactive, acidic 56.5-kd keratin and an AE3-reactive, basic 65-67-kd keratin. Furthermore, the expression of these two keratins correlated roughly with the degree of morphological keratinization. AE2 antibody (specific for the 56.5- and 65-67-kd keratins) stained keratinized corneal epithelial sections suprabasally, as in the epidermis, suggesting that these two keratins are expressed mainly during advanced stages of keratinization. These two keratins have previously been suggested to represent markers for epidermal keratinization. Our present data indicate that they can also be expressed by other stratified epithelia during vitamin A deficiency-induced keratinization, and suggest the possibility that they may play a role in the formation of the densely packed tonofilament bundles in cornified cells of keratinized tissues.     

Thoughts on the pulmonary blood-gas barrier

The pulmonary blood-gas barrier is an extraordinary structure because of its extreme thinness, immense strength, and enormous area. The essential components of the barrier were determined early in evolution and have been highly conserved. For example, the barriers of the African, Australian, and South American lungfish that date from as much as 400 million years ago have essentially the same structure as in the modern mammal or bird. In the evolution of vertebrates from bony fishes through amphibia, reptiles, and ultimately mammals and birds, changes in the pulmonary circulation occurred to limit the stresses in the blood-gas barrier. Only in mammals and birds is there a complete separation of the pulmonary and systemic circulations, which is essential to protect the extremely thin barrier from the necessary high-vascular pressures. To provide the blood-gas barrier with its required strength, evolution has exploited the high ultimate tensile strength of type IV collagen in basement membrane. Nevertheless, stress failure of the barrier occurs under physiological conditions in galloping Thoroughbred racehorses and also apparently in elite human athletes at maximal exercise. The human blood-gas barrier maintains its integrity during all but the most extreme physiological conditions. However, many pathological conditions cause stress failure. The structure of the blood-gas barrier is apparently continually regulated in response to wall stress, and this regulation is essential to maintain the extreme thinness but adequate strength. The mechanisms of this regulation remain to be elucidated and constitute one of the fundamental problems in lung biology.     

EPIDERMAL METAPLASIA OF THE CORNEAL ANTERIOR EPITHELIUM IN THE CHICK EMBRYO

The corneal anterior epithelium of younger chick embryos can be changed into a keratinized epidermis, when it is cultured in vitro combined with 6 1/2-day dorsal dermis. Even if a Millipore filter is inserted between the corneal anterior epithelium and underlying dorsal dermis, the epithelium undergoes similar metaplastic changes. In older embryos, however, the epithelium gradually loses the competence for the keratinization. Cultivation of cornea (anterior epithelium, stroma and endothelium) of 6 1/2- or 10-day embryos results in maintenance of its original pattern, and the epithelium fails to differentiate into a keratinized epidermis. The dermis isolated from 8 1/2-day dorsal or 12 1/2-day tarsometatarsal skin is not so effective in inducing the epidermal metaplasia. The mesenchyme of 5 1/2-day proventriculus or 5 1/2-day gizzard fails to bring about any endodermal metaplasia of the corneal epithelium. The corneal stroma, on the other hand, has no inhibitory action on the keratinization of the epidermis obtained from 6 1/2-day dorsal skin.     

PACAP through EGFR transactivation preserves human corneal endothelial integrity

The corneal endothelium is composed of a single hexagonal-shaped cells layer adherent to the Descemet's membrane. The primary function of these cells is maintaining of tissue clarity by regulating its hydration. Trauma, aging or other pathologies cause their loss, counterbalanced by enlargement of survived cells unable to guarantee an efficient fluid pumping to and from the stroma. Regenerative medicine using human corneal endothelial cells (HCECs) isolated from peripheral corneal-scleral tissue of a donor could be an attractive solution, overcoming transplantation problems. In a previous study, we have demonstrated that HCECs treatment with pituitary adenylate cyclase–activating polypeptide (PACAP) following growth factors deprivation prevents their degeneration. However, the molecular mechanism mediating this effect has not been clarified, yet. Here, we have shown for the first time the expression of PACAP and its receptor (PAC1R) in human corneal endothelium and demonstrated that this peptide, selectively binding to PAC1R, induces epidermal growth factor receptor (EGFR) phosphorylation and the MAPK/ERK1/2 signaling pathway activation. In conclusion, our data have suggested that PACAP could represent an important trophic factor in maintaining human corneal endothelial integrity through EGFR transactivation. Therefore, PACAP, as well as epidermal growth factor and fibroblast growth factor, could co-operate to guarantee tissue physiological functioning by supporting corneal endothelial barrier integrity.     

Structural changes in trichocyte keratin intermediate filaments during keratinization

The so-called hard alpha-keratins, such as quill and hair, have a composite structure in which intermediate filaments (IF) are embedded in a sulfur-rich matrix. Recent studies of these trichocyte keratin IF have revealed that substantial changes in the molecular architecture take place when oxidation of the cysteine residues occurs as part of the terminal differentiation/keratinization process. Recent cryoelectron microscope studies suggest that the IF has a tubular structure prior to keratinization, but transmission electron micrographs of thin sections of fully keratinized fibers exhibit a "ring-core" structure. In the present contribution we develop a generic model for the IF in the reduced state based on cross-linking studies and discuss two possibilities for the way in which this structure may be modified during the keratinization process.     

Loricrin-like immunoreactivity during keratinization in lizard epidermis

Two modalities of keratinization are present in lizard epidermis: alpha (soft-pliable corneous layers) and beta (hard and inflexible corneous layers). While beta-keratinization is probably due to the synthesis of a new (beta)-keratin gene product, alpha keratinization resembles in part that of mammalian epidermis. The goal of this study was to test whether a sulfur-rich molecule similar to the mammalian corneous cell envelope protein loricrin is also present in lizard epidermis. This was done using X-ray microanalysis and immunocytochemical and ultrastructural methods. In the epidermis of the lizard Podarcis muralis small (0.1-0.3 microm) to large (1-5 microm) keratohyalin-like granules (KHLGs) are produced in alpha-keratinizing cells, especially in the clear layer. Small KHLGs contain sulfur and show weak filaggrin-like and stronger loricrin-like immunoreactivities. The latter is also present in keratinizing alpha-layers but is absent in the beta layers. Large KHLGs in the clear layer derive from the aggregation of the small granules with other components, including lipid material. These large granules show some loricrin-like immunoreactivity and contain sulfur and phosphorous, histidine, but not filaggrin-like immunoreactivity. It is suggested here that phosphorous derives from their phospholipid component. The present study shows that the modality of alpha-keratinization of lizard epidermis resembles that of mammals and suggests that the basic molecular mechanisms of keratin aggregation and formation of the corneous cell envelope were already present in the therapsid line of reptiles from which mammals evolved.     

Fine structure of the horny teeth of the lamprey,Entosphenus japonicus

The fine structure of the horny teeth of the lamprey, Entosphenus japonicus, was examined by light- and electron-microscopy. Most of the horny teeth consisted of two horny and two nonhorny layers. The primary horny layer was well keratinized, and the cells were closely packed and intensely interdigitated, being joined together by many modified desmosomes. The plasma membrane of the horny cell, unlike the membranes of other vertebrates, was not thickened. The intercellular spaces were filled with electron-dense material. Microridges were seen on the free surface. Structures resembling microridges were found on the underside of the primary horny layer. The secondary horny layer displayed various stages of keratinization. The keratinization started at the apex and developed toward the base. In the early stage of keratinization, the superficial cells became cylindrical and were arranged in a row forming a dome-shaped line. Their nuclei were situated in the basal part of the cells. The appearance of the nonhorny layers varied according to the degree of keratinization of the horny layers beneath them. The nonhorny cells were joined together by many desmosomes and possessed many tonofilament bundles. The replacement and keratinization of the horny teeth are discussed in the light of these results.     

Quantitation of corneal fibril diameters from bony fish using low temperature preparative methods

Low-temperature dehydration and embedding techniques have been used to preserve the transverse structure of corneal collagen fibrils from nine bony fish. The diameters measured all lie close to a value of 25.5 nm, in contrast to the smaller (and more diverse) diameters measured from “conventionally” prepared controls. The results are consistent with our earlier studies on the corneas from mammals, amphibians, birds, reptiles, and cartilaginous and bony fish which showed that the collagen fibrils from the bony fish were significantly smaller than those from animals of the other vertebrate classes. Thus, on the basis of the enhanced ability of the low-temperature preparative techniques to preserve collagen fibril structure the “in vivo” corneal collagen fibril diameters have been revised from 17 to 25.5 nm for bony fish (this work) and from 25 to 36 nm for all other classes of vertebrates (our previous work).     

Sealing the live part of the skin: the integrated meshwork of desmosomes, tight junctions and curvilinear ridge structures in the cells of the uppermost granular layer of the human epidermis

In the literature the question of whether a system structurally and functionally related to the barrier function of the tight junctions (TJs) of polarized epithelia exists in the epidermis has been and still is controversially discussed. We have systematically addressed this question in a study of the granular layer of fetal and adult human epidermis, combining different light and electron microscopic methods. We show that the lateral membranes of the cells of the stratum granulosum are connected by an extended subapical complex system integrating desmosomes and TJ structures identified as sites of close membrane-membrane contact and as regions of membrane-to-membrane apposition that in immunoelectron microscopy are positive for TJ marker proteins, notably occludin, indicative of an extended, probably continuous TJ barrier. In addition, we have noted in freeze-fractures of the apical membrane attaching this layer to the basalmost membrane of the stratum corneum an extended system integrating desmosomes with intramembraneous ridge configurations that appear as strands, circles, lariats or complex meshworks showing numerous continuities with the desmosomes. In some regions this system interconnecting desmosomes with curvilinear ridge structures occupies the major part of the plasma membrane. The molecular organizations and possible functional contributions of both structural systems positioned at the border between the living portion of the epidermis and the corneal layer are discussed, in particular in relation to the formation of a stable association between the two layers and of a barrier to the paracellular flow of molecules and particles. It is also discussed whether similar structures occur in other keratinizing stratified squamous epithelia, in squamous metaplasias and in tumors derived from such tissues.     

Age-related changes in the ultrastructural organization of the human gingival epithelium

By means of transmissive and scanning electron microscopy 103 gingival bioptates in practically healthy persons at the age of 18-80 years have been studied. At ageing essential changes take place in all structural elements of the epithelium. The basal membrane is intermittent and loose. In cytoplasm of the cells of the basal layer epithelium the amount of microfilaments increases essentially, and as a result it becomes electron opaque. Tonofibrillar fasciculi of the spinous layer cells are fragmented, their contours are indistinct. In cytoplasm of the granular layer cells amount of keratohyalin granules increases, their size becomes large and their typical form is lost. In cytoplasm of the basal, spinous and granular layer cells the amount of organells decreases. Mitochondria acquire the appearance of electron translucent cavities with discomplexic, and sometimes, destroyed cristae. Rather great changes occur in intercellular interrelations. In all the layers some intercellular spaces are widen, in the spaces formed isolated desmosomes and other debries of cellular structures are formed. Sharp changes of microrelief of the granular layer epitheliocytes are observed. The ultrastructural rearrangements of epitheliocytes, revealed in the human gingiva, demonstrate certain disturbances in keratinization processes, in mechanical firmness, as well as in barrier function of the epithelial layer.     

Corneal cell proteins and ocular surface pathology

The cornea is a transparent and avascular tissue that functions as the major refractive structure for the eye. A wide variety of growth factors, chemokines, cytokines and their receptors are synthesized by corneal epithelial and stromal cells, and are found in tears. These molecules function in corneal wound healing and in inflammatory responses. Proteoglycans and glycoproteins are essential for normal corneal function, both at the air-epithelial interface and within the extracellular matrix. The ocular MUC mucins may play roles in forming the mucus layer of the tear film, in regulating tear film spread, and in inhibiting the adhesion of pathogens to the ocular surface. Lumican, keratocan and mimecan are the major keratan sulfate proteoglycans of the corneal stroma. They are essential, along with other proteoglycans and interfibrillar proteins, including collagens type VI and XII, for the maintenance of corneal transparency. Corneal epithelial cells interact with a specialized extracellular matrix structure, the basement membrane, composed of a specific subset of collagen type IV and laminin isoforms in addition to ubiquitous extracellular matrix molecules. Matrix metalloprotein-ases have been identified in normal corneal tissue and cells and may play a role in the development of ulcerative corneal diseases. Changes in extracellular matrix molecule localization and synthesis have been noted in other types of corneal diseases as well, including bullous keratopathy and keratoconus.     

Comparative observations on corneas,with special reference to Bowman's layer and Descemet's membrane in mammals and amphibians

Corneas of tadpole, mouse, rat, guinea pig, rabbit, cat, cattle, and human were examined by TEM and SEM in a comparative study. The differences between species were noted mainly by using TEM. Bowman's layer showed a tendency to be well developed in higher mammals. Tadpoles lack a Bowman's layer, lower mammals have a thin Bowman's layer, and higher mammals have a thick Bowman's layer. The boundary between the substantia propria and Descemet's membrane was distinct in higher mammals. On the other hand, there are no differences in thickness of the collagen fibrils that constitute Bowman's layer and those of the substantia propria. NaOH digestion was utilized for SEM preparation. SEM imaging revealed a textured appearance of the epithelial side of Bowman's layer. In Descemet's membrane, fibrous long spacing (FLS) fiber-like structures, which are arranged in parallel to the endothelium, were observed by both TEM and SEM. To our knowledge, this is the first report of SEM observations of FLS fiber-like structures on the endothelial surface of Descemet's membrane. SEM at a plane normal to the plane of the cornea showed that Descemet's membrane has a piled laminar structure. Descemet's membrane is closely associated with the collagen layer of the substantia propria. Collagen fibrils invading from the substantia propria into Descemet's membrane were observed with both TEM and SEM.     

Physiological adaptation in relation to hyperosmotic stress in the epidermis of a fresh-water teleost Barbus sophor (Cypriniformes, Cyprinidae): a histochemical study.

The changes in distribution of mucopolysaccharides, glycogen and protein bound sulphydryl groups of cysteine in the various cellular components of the epidermis of Barbus sophor along with its structural alterations as a result of hyperosmotic stress, have been described using histochemical techniques. The hyperosmotic saline induces a cyclic secretion of acid mucopolysaccharides by the mucous cells. Simultaneously the polygonal cells also show a marked disturbance in the processes of mucogenesis and keratinization, indicating an inverse relationship between the degree of keratinization and the amount of mucus secreted by epidermis. The role of glycogen in the polygonal cells has been discussed in relation to disturbed mucogenesis. The appearance of intercellular spaces in basal layer and middle layer has been correlated with the passage for movement of increased amounts of nutrients through the skin.     

Size-selective junctional barrier and Ca(2+)-independent cell adhesion in the testis of Cynops pyrrhogaster: expression and function of occludin

In urodeles which has testicular structure different from that in mammals, blood-testis barrier was reported to exist like in mammals. However, molecular and functional analyses of the components of the blood-testis barrier in urodeles have not been reported yet. Toward elucidation of the barrier functions and their molecular components in newt testis, we aimed to isolate occludin cDNAs and obtained two kinds of occludin partial cDNAs (occludin 1 and 2) encoding the putative second extracellular loop. Immunoblot and immunofluorescence studies using antibodies against peptides each corresponding to a part of the second extracellular loop of occludin 1 and 2, and those against beta-catenin and zonula occludens-1 (ZO-1) showed that occludin, as well as beta-catenin and ZO-1, was expressed not only in Sertoli cells but also in germ cells throughout all the stages from spermatogonia to elongate spermatids. Tracer experiments revealed a size-selective barrier which allows small molecules ( approximately 500 Da) to get into cysts through Sertoli cells' barrier, but not larger ones (>1.9 kDa) in the stages from spermatogonia to almost mature sperm. No occludin peptides corresponding to a part of the second extracellular loop destroyed the junctional barrier, while both the peptides and antibodies significantly inhibited reaggregation of the dissociated testicular cells which was to a large extent Ca(2+)-independent. These results indicate that the second extracellular loop of occludin is involved in cell adhesion rather than in size-selective barrier in newt testis, though the possibility cannot be excluded that the peptides were not long enough to inhibit the barrier function.     

Proteomic analysis of rabbit tear fluid: Defensin levels after an experimental corneal wound are correlated to wound closure

The cornea is the major refracting optical element of the eye and therefore critical for forming a retinal image. The exposed surface of the eye is protected from pathogens by the innate immune system whose components include defensins, naturally occurring peptides with antimicrobial properties, and the physical barrier formed by the outer epithelial layer of the cornea. The proteomic approach has revealed that tear levels of defensins are correlated with the course of healing of an experimental corneal wound. Tears were collected from New Zealand White rabbits prior to (day 0) and daily for 5 days (days 1-5) following a standard unilateral 6 mm diameter corneal epithelial abrasion. Tear protein profiles obtained from wounded and contra-lateral control eyes were compared using SELDI ProteinChip technology. Peptides and proteins of interest were purified by RP-HPLC and characterized by nanoESI-MS/MS. Mass spectra of tears on post-wound day 1, revealed 13 peaks whose level decreased and five that increased. During wound healing the tear protein profile correlated with wound closure. An important finding was that the levels of rabbit defensins (NP-1 and NP-2), which were elevated after wounding returned to normal levels by the time the corneal abrasion healed. Relative quantification of NP-2 in tear fluid prior to (day 0) and after corneal wounding (days 1- 3) was determined using iTRAQ technology. A corneal wound eliminates the barrier function of innate immunity and puts the cornea at risk from microbial attack until the epithelial cells restore the surface barrier. The increased availability of defensins in the tears during healing suggests that these peptides could protect the cornea from microbial attack during a period of increased vulnerability.     

Avian scale development. X. Dermal induction of tissue-specific keratins in extraembryonic ectoderm

Epidermal-dermal tissue interactions regulate morphogenesis and tissue-specific keratinization of avian skin appendages. The morphogenesis of scutate scales differs from that of reticulate scales, and the keratin polypeptides of their epidermal surfaces are also different. Do the inductive cues which initiate morphogenesis of these scales also establish the tissue-specific keratin patterns of the epidermis, or does the control of tissue-specific keratinization occur at later stages of development? Unlike feathers, scutate and reticulate scales can be easily separated into their epidermal and dermal components late in development when the major events of morphogenesis have been completed and keratinization will begin. Using a common responding tissue (chorionic epithelium) in combination with scutate and reticulate scale dermises, we find that these embryonic dermises, which have completed morphogenesis, can direct tissue-specific statification and keratinization. In other words, once a scale dermis has acquired its form, through normal morphogenesis, it is no longer able to initiate morphogenesis of that scale, but it can direct tissue-specific stratification and keratinization of a foreign ectodermal epithelium, which itself has not undergone scale morphogenesis.     

Immunocytochemical and autoradiographic studies on the process of keratinization in avian epidermis suggests absence of keratohyalin

The process of keratinization in apteric avian epidermis and in scutate scales of some avian species has been studied by autoradiography for histidine and immunohistochemistry for keratins and other epidermal proteins. Acidic or basic alpha-keratins are present in basal, spinosus, and transitional layers, but are not seen in the corneous layer. Keratinization-specific alpha-keratins (AE2-positive) are observed in the corneous layer of apteric epidermis but not in that of scutate scales, which contain mainly beta-keratin. Alpha-keratin bundles accumulate along the plasma membrane of transitional cells of apteric epidermis. In contrast to the situation in scutate scales, in the transitional layer and in the lowermost part of the corneous layer of apteric epidermis, filaggrin-like, loricrin-like, and transglutaminase immunoreactivities are present. The lack of isopeptide bond immunoreactivity suggests that undetectable isopeptide bonds are present in avian keratinocytes. Using immunogold ultrastructural immunocytochemistry a low but localized loricrin-like and, less, filaggrin-like labeling is seen over round-oval granules or vesicles among keratin bundles of upper spinosus and transitional keratinocytes of apteric epidermis. Filaggrin-and loricrin-labeling are absent in alpha-keratin bundles localized along the plasma membrane and in the corneous layer, formerly considered keratohyalin. Using ultrastructural autoradiography for tritiated histidine, occasional trace grains are seen among these alpha-keratin bundles. A different mechanism of redistribution of matrix and corneous cell envelope proteins probably operates in avian keratinocytes as compared to that of mammals. Keratin bundles are compacted around the lipid-core of apteric epidermis keratinocytes, which do not form complex chemico/mechanical-resistant corneous cell envelopes as in mammalian keratinocytes. These observations suggest that low amounts of matrix proteins are present among keratin bundles of avian keratinocytes and that keratohyalin granules are absent.     

The permeability barrier in the epidermis of the grass snake during the resting stage of the sloughing cycle

Summary Tracer and freeze-fracture replication techniques show that there are two morphologically and topographically distinct permeability barriers in the epidermis of the grass snake. Tight junctions interconnect the apico-lateral plasma membranes of the uppermost living cells, establishing an ionic or osmotic gradient between the stratum germinativum and alpha layer. The second barrier is formed by intercellular lipid sheets in the overlying mesos layer, which are very similar to the barrier found in the stratum corneum of mammals.