Interactions of extracellular collagen and corneal fibroblasts: Morphologic and biochemical changes of rabbit corneal cells cultured in a collagen matrix

Summary Corneal fibroblasts, also known as keratocytes are surrounded by an extracellular matrix of collagen in vivo. To understand the physiology and pathology of these corneal fibroblasts, it is important to study their interactions with this extracellular matrix. We cultured rabbit corneal fibroblasts on tissue culture plastic dishes or in a hydrated collagen gel and compared the changes in morphology and mitotic activity. Corneal fibroblasts on plastic dishes were flattened and widely spread, whereas those in collagen gel became spindle-shaped with long processes. Examination with an electron microscope revealed that the corneal fibroblasts in collagen gel formed gap junctions with neighboring cells. Gap junctions were hardly ever observed between corneal fibroblasts cultured on plastic dishes. Corneal fibroblasts cultured in a collagen matrix showed much less incorporation of [³H]thymidine than did corneal fibroblasts cultured on plastic, and this incorporation decreased with increasing concentration of collagen. Our present results suggest that the morphologic and biochemical characteristics of corneal fibroblasts cultured in collagen gel are different from those cultured on plastic. This research was supported in part by grants from the Ministry of Education, Science and Culture of Japan, by a grant from Osaka Eye Bank, Osaka, Japan, and by an intramural research fund of Kinki University. Part of this research was presented at the annual meeting of the Japanese Ophthalmological Society (May 1985) at Kyoto, Japan, and at the annual meeting of the Association for Research in Vision and Ophthalmology (May 1987) at Sarasota, FL.     

Extracellular compartments in matrix morphogenesis: collagen fibril, bundle, and lamellar formation by corneal fibroblasts

The regulation of collagen fibril, bundle, and lamella formation by the corneal fibroblasts, as well as the organization of these elements into an orthogonal stroma, was studied by transmission electron microscopy and high voltage electron microscopy. Transmission and high voltage electron microscopy of chick embryo corneas each demonstrated a series of unique extracellular compartments. Collagen fibrillogenesis occurred within small surface recesses. These small recesses usually contained between 5 and 12 collagen fibrils with typically mature diameters and constant intrafibrillar spacing. The lateral fusion of the recesses resulted in larger recesses and consequent formation of prominent cell surface foldings. Within these surface foldings, bundles that contained 50-100 collagen fibrils were formed. The surface foldings continued to fuse and the cell surface retracted, forming large surface-associated compartments in which bundles coalesced to form lamellae. High voltage electron microscopy of 0.5 micron sections cut parallel to the corneal surface revealed that the corneal fibroblasts and their processes had two major axes at approximately right angles to one another. The surface compartments involved in the production of the corneal stroma were aligned along the fibroblast axes and the orthogonality of the cell was in register with that of the extracellular matrix. In this manner, corneal fibroblasts formed collagen fibrils, bundles, and lamellae within a controlled environment and thereby determined the architecture of the corneal stroma by the configuration of the cell and its associated compartments.     

Uptake of India ink particles and latex beads by corneal fibroblasts

Summary The fate of India ink particles and polystyrene latex beads injected into the corneal stroma of rabbits was studied by the naked eye, light microscopy, and electron microscopy. All the injected ink particles or latex beads were unchanged in shape, size, and number for at least 6 months. India ink particles and latex beads were endocytosed by the corneal fibroblasts within 3–4 days after injection. Numerous ink particles were packed into vacuoles, 0.5–10 m in diameter, which occupy a large volume of the cytoplasm of the cell body and processes of fibroblasts in and near the injected area. Each latex bead, 0.72 m in diameter, is usually enclosed in one vesicle, and a large number of vesicles are distributed throughout the cytoplasm. In corneal tissue removed 10 min after injection of India ink and cultured for 3 or 7 days, uptake of many ink particles by the fibroblasts was seen. By this experiment, the contribution of the blood-derived cells was completely excluded, and it is more distinctly shown that the corneal fibroblast has a strong endocytotic activity.The uptake and long-term storage of ink particles and latex beads by the corneal fibroblast are reactions that protect the organ without inflammation from the injury and harm by non-toxic foreign materials.A part of this study was published in Kinki Daigaku Igaku Zasshi in Japanese as a Ph. D. thesis by Atsuko Ueda. This study was supported by grants from the Ministry of Education, Science and Culture, Japan, the Osaka Eye Bank, Osaka, Japan, and an intramural Research Fund of Kinki University, Japan     

Interpretation of the meridional X-ray diffraction pattern from collagen fibrils in corneal stroma

The low angle X-ray diffraction pattern from corneal stroma can be interpreted as arising from the equivalent of sharp meridional reflections due to the packing of molecules along the collagen fibrils and an equatorial pattern due to the packing of these fibrils within lamellae.Axial electron density profiles for corneal collagen fibrils have been produced by combining intensity data from the meridional pattern with two independent sets of phases. The first set was obtained using an electron microscopical technique, whereas the second set consisted of calculated tendon collagen phases given in the literature. Substantial agreement between the two electron density profiles was found.A quantitative analysis of the difference between the electron density profiles of rat tail tendon and corneal collagen showed that the step between the gap and overlap regions is smaller in cornea than in tendon. This is probably due to the binding of non-collagenous material in the gap region as occurs in bone and other tissue. Two peaks corresponding to regions where electron density is greater in the cornea are situated at the gap/overlap junctions. A third region where the corneal collagen is more electron dense is located near the centre of the gap region. The proximity of these peaks to the positions of hydroxylysine residues along the fibril axis suggests that they may be the major sites at which sugars are bound to corneal collagen.     

Proteomic analysis of the soluble fraction from human corneal fibroblasts with reference to ocular transparency

The transparent corneal stroma contains a population of corneal fibroblasts termed keratocytes, which are interspersed between the collagen lamellae. Under normal conditions, the keratocytes are quiescent and transparent. However, after corneal injury the keratocytes become activated and transform into backscattering wound-healing fibroblasts resulting in corneal opacification. At present, the most popular hypothesis suggests that particular abundant water-soluble proteins called enzyme-crystallins are involved in maintaining corneal cellular transparency. Specifically, corneal haze development is thought to be related to low levels of cytoplasmic enzyme-crystallins in reflective corneal fibroblasts. To further investigate this hypothesis, we have used a proteomic approach to identify the most abundant water-soluble proteins in serum-cultured human corneal fibroblasts that represent an in vitro model of the reflective wound-healing keratocyte phenotype. Densitometry of one-dimensional gels revealed that no single protein isoform exceeded 5% of the total water-soluble protein fraction, which is the qualifying property of a corneal enzyme-crystallin according to the current definition. This result indicates that wound-healing corneal fibroblasts do not contain enzyme-crystallins. A total of 254 protein identifications from two-dimensional gels were performed representing 118 distinct proteins. Proteins protecting against oxidative stress and protein misfolding were prominent, suggesting that these processes may participate in the generation of cytoplasmic light-scattering from corneal fibroblasts.     

Cell junctions in the rapidly moving edge of chick and quail blastoderms during gastrulation

Summary The intercellular contacts of the migrating edge of chick and quail blastoderms during gastrulation were studied by transmission electron microscopy of thin sections and of freeze-fracture replicas. Tight junctions and gap junctions as well as desmosomes were found. Tight junctions were organized as single junctional strands or as a complex of numerous junctional strands interposed between the lamellae and the bodies of the cells building up the margin of overgrowth. The function of these intercellular junctions is considered in relation to the locomotion of the margin of overgrowth cells.     

Freeze-fracture studies of the developing cell surface. I. The plasmalemma of the corneal fibroblast

The freeze-fracture technique was used to study changes in the corneal fibroblast cell membrane during morphogenesis in chick embryos. Fibroblasts migrate into the acellular primary corneal stroma on day 6 of embryogenesis, moving between the orthogonal layers of collagen fibrils which serve as their substratum. Morphometric analysis of the intramembrane particles (IMP) reveals their concentration on the P face to decrease from 756 to 534/mum2 from day 6 to day 14. After day 14, fibroblast migration and cell division cease and the stroma condenses due to dehydration, so that by day 18 all of the layers of fibroblasts are extremely flattened and the cornea has taken on its mature, transparent form. The cell membranes of the terminally differentiated, highly compacted fibroblasts are rich in IMP (1,300/MUM2, P face). In seeking to relate the particle increase to cell differentiation, we analyzed synthetic events taking place at this time, but no correlation, we analyzed synthetic events taking place at this time, but no correlation with 25SO4 or proline-3H incorporation was found. The event which seems best correlated with the doubling of P face particles between days 15 and 18 is the dehydration and condensation of the stroma, an event which is associated with cessation of both cell division and migration. Thyroxine stimulates premature condensation of the stroma, whereas thiouracil delays condensation, but neither of these treatments affects IMP concentration. Interestingly, IMP concentration on the filopodia of migrating fibroblasts is similar to that on the cell bodies, suggesting that the new membrane has the same composition as the pre-existing membrane. Observations are also presented on tight and gap junctions between fibroblasts and on the relation of extracellular matrix to the outer etched surface of the fibroblast plasmalemma.     

A descriptive and quantitative study of the keratocytes of the corneal stroma of albino rabbits using transmission electron microscopy

The present morphometric study was designed to assess the dimensions and shape of keratocytes and their nuclei by transmission electron microscopy, and to assess these features in relation to the stromal lamellae. Corneas from 10 albino rabbits were fixed in 2% glutaraldehyde in cacodylate buffer (pH 7.4, 300 mOsm/kg) and embedded in Spurr's epoxy resin. Both transverse and coronal thin sections through the corneal stroma were prepared. The stromal lamellae had an average thickness of 2.45+/-1.15 microm. The average cell thickness of the keratocytes was 1.34+/-0.46 microm (range 0.49-4.76 microm), with the apparent cell thickness being related to the average anterior-posterior thickness of the adjacent lamellae (r = 0.424, P = 0.001)). The relative length and thickness of the cell nucleus, in transverse section, was measured to be 0.65+/-0.13 and 0.76+/-0.10 of the cell body section respectively. As assessed by planimetry, the area of the keratocyte cell body viewed in coronal section was 292+/-118 microm2, with a nucleus-to-cytoplasm ratio of 0.437+/-0.295. The electron micrographs confirmed the presence of gap junctions between keratocyte cell processes, and the occasional presence of centrioles in the cells. Some keratocyte processes were observed to extend from one face of the lamellae to the other, suggesting anterior-to-posterior cell communication. These studies indicate that the keratocyte cell thickness is influenced by the physical pressure exerted by adjacent stromal lamellae. The cell nucleus, while a dominant feature in transverse section, has a normal size in relation to the cell cytoplasm when viewed in coronal section.     

The fine structure of the hypostome and mouth of hydra

Summary The normal morphology of the hypostome and mouth of hydra were examined by transmission electron microscopy with conventional thin sections and freeze-fracture replicas. Myonemes of the hypostome are small in diameter, have gap and intermediate-type cell junctions within each epithelial layer and are associated with the opposite epithelial layer by transmesogleal processes and gap junctions. Nematocysts and sensory cells are aggregated in the circumoral region. The fine structure of adherent flagella arising from gastrodermal gland cells, and the transition region at the mouth between epidermis and gastrodermis are described in detail for the first time. The possible functional significance of the findings is discussed.     

The cell junctions of the notochord of Xenopus laevis tadpoles

The cell junctions of the notochord of Xenopus laevis tadpoles were examined with the electron microscope using thin sections, lanthanum tracer experiments, and freeze-fracture replicas. Both the peripheral and vacuolated cells of the notochord are connected by numerous spot desmosomes characterized by an intercellular desmogloea and intermediate filaments on the cytoplasmic sides. The peripheral cells also display numerous hemidesmosomes facing the underlying basal lamina. Staining with rhodamine-phalloidin for F-actin yielded negative results and suggested that adhaerens-type junctions are absent. Tracer experiments with lanthanum and freeze-fracture replicas clearly revealed the presence of gap junctions between both cell types but no indications of tight junctions were found and no intercellular barrier existed for tracer infiltration of the notochord.     

Collagen fibril assembly by corneal fibroblasts in three-dimensional collagen gel cultures: small-diameter heterotypic fibrils are deposited in the absence of keratan sulfate proteoglycan.

Extracellular matrix assembly is a multistep process and the various steps in collagen fibrillogenesis are thought to be influenced by a number of factors, including other noncollagenous matrix molecules. The synthesis and deposition of extracellular matrix by corneal fibroblasts grown within three-dimensional collagen gel cultures were examined to elucidate the factors important in the establishment of tissue-specific matrix architecture. Corneal fibroblasts in collagen gel cultures form layers and deposit small-diameter collagen fibrils (approximately 25 nm) typical of the mature corneal stroma. The matrix synthesized contains type VI collagen in a filamentous network and type I and type V collagen assembled as heterotypic fibrils. The amount of type V collagen synthesized is relatively high and comparable to that seen in the corneal stroma. This matrix is deposited between cell layers in a manner reminiscent of the secondary corneal stroma, but is not deposited as densely or as organized as would be found in situ. No keratan sulfate proteoglycan, a proteoglycan found only in the corneal stroma, was synthesized by the fibroblasts in the collagen gel cultures. The assembly and deposition of small-diameter fibrils with a collagen composition and structure identical to that seen in the corneal stroma in the absence of proteoglycans typical of the secondary corneal stroma imply that although proteoglycan-collagen interactions may function in the establishment of interfibrillar spacing and lamellar organization, collagen-collagen interactions are the major parameter in the regulation of fibril diameter.     

Ultrastructure, histological distribution, and freeze-fracture immunocytochemistry of gap junctions in rat brain and spinal cord

The historical development of concepts of gap junctions as sites for electrical, ionic, and metabolic coupling is reviewed, from the initial discovery of gap junctions linking heart cells, to the current concepts that gap junctions represent 'electrotonic synapses' between neurons. The ultrastructure and immunocytochemistry of gap junctions in heart, brain, and spinal cord of adult rats is examined using conventional thin sections, negative staining, grid-mapped freeze-fracture replicas, and immunogold-labeled freeze-fracture replicas. We review evidence for neuronal gap junctions at 'mixed' (combined electrical and chemical) synapses throughout adult rat spinal cord. We also show immunogold labeling of connexin43 in astrocyte and ependymocyte gap junctions and of connexin32 in oligodendrocyte gap junctions. Ultrastructural and freeze-fracture immunocytochemical methods have provided for definitive determination of the number, size, histological distribution, and connexin composition of gap junctions between neurons in all regions of the central nervous systems of vertebrate species.     

Corneal cell-matrix interactions: type VI collagen promotes adhesion and spreading of corneal fibroblasts.

Type VI collagen is a nonfibrillar collagen present as a network throughout the chick secondary stroma. Immunolocalization of type VI collagen both in the chick corneal stroma and in other systems demonstrates that type VI collagen is present associated with cells and between striated fibrils. We hypothesize that type VI collagen may function in cell-matrix interactions important in corneal development. To examine this possibility, we have isolated and characterized bovine corneal type VI collagen and determined that the chain composition and morphology of type VI collagen isolated from cornea is similar to that isolated from other sources. The tissue form of type VI collagen was localized to filaments forming a network around fibrils and close to corneal fibroblasts. We then analyzed relative attachment and spreading on type VI collagen as compared to the other collagens present in the secondary stroma, and found that although corneal fibroblasts attach equally well to type VI and type I collagen, cells spread to a much greater extent on type VI collagen. Although corneal fibroblasts do have an RGD-dependent receptor which functions during adhesion to fibronectin, attachment to type VI collagen is RGD-independent unless the molecule is denatured. Blocking of the RGD-dependent receptor with soluble RGD peptides results in no change in attachment or spreading. These data imply a role for type VI collagen in cell-matrix interactions during corneal stroma development.     

A freeze-fracture study of exocytosis and reflexive gap junctions in human ovarian decidual cells

Fine-structural features of ovarian decidual cells and their mode of secretion were examined by means of freeze-fracture microscopy. Unique cortical peduncular processes contained secretory vesicles within the expanded peduncle tip, the membrane-leaflets of which exhibited a particle-poor E face adjacent to the vesicle lumen and a P face containing a greater particle number. Exocytosis from attached peduncles involved release of vesicular profiles 40-55 nm in diameter; small particles 8.5-11.5 nm in diameter were also observed at degranulation sites. In fractures revealing the E face of the plasmalemma, cytoplasmic portals at the bases of peduncular stalks were distinguishable from endocytic vesicles. The frequent occurrence of reflexive gap junctions associated with peduncles was shown by freeze-fracture. However, there appeared to be no consistent spatial relationship between gap junctions, secretory peduncles, or sites of exocytosis. Freeze-fracture analysis of the topography of reflexive gap junctional profiles revealed that such gap junctions share basic similarities with intercellular gap jum particle-free aisles. The finding in the present study of reflexive gap junctions occurring between peduncles and the cell soma, as well as between peduncles, suggests that the original definitiof the same cell should be broadened to include any gap junctional specialization formed between portions of the plasma membrane of one cell.     

The preimplantation mammalian embryo: characterization of intercellular junctions and their appearance during development.

Junctions in developing mammalian embryos were investigated with lanthanum tracer and freeze-fracture methods. The outermost blastomeres of mouse morulae possess focal tight junctions which become zonular and exclude lanthanum, thereby separating the “inner” cells from the maternal environment. This compartmentalization, creating a microenvironment inside the embryo, may be required for cell determination and for the accumulation of fluid during blastocoel expansion. Desmosomes appear for the first time at the blastocyst stage in the trophoblast junctional complex which also is characterized by gap junctions and a zonula occludens with underlying microfilament-like material and microtubules. Both gap and tight junctions have been visualized in freeze-fracture replicas of rabbit blastocysts. The zonula occludens forms a permeability barrier which is consistent with the high transtrophoblast electrical resistance. Mouse presumptive and mature inner cell mass (ICM) cells were associated by frequent gap junctions whereas junctional complexes were absent. Trophoblast gap and adhering junctions and cytoplasmic processes appeared to fix the ICM to one pole of the embryo and partially isolate it from the blastocoel. These findings support the idea that the ICM and trophoblast communicate upon implantation and require that the intercellular junctions between them be dissembled if the ICM is to migrate to a mesometrial position.     

Gap junctions in the cardiac muscle cells of the lamprey

Summary Electron microscopy of both thin sections and freeze-fracture replicas has demonstrated the occurrence of gap junctions (nexuses) in the cardiac muscle cells of the lamprey. These gap junctions are identical in basic structure with those found in the mammalian heart. However, they are much smaller (less than 0.5 in diameter), and more irregularly distributed than the typical gap junction in the mammalian heart. These small gap junctions seem to provide a structural basis for the electrical coupling between cardiac muscle cells in the lower vertebrates.In addition, the well developed sarcoplasmic reticulum and subsurface cisternae, which contain an electron dense spheroidal cast, are frequently observed in the cardiac muscles of the lamprey.This work is supported by a research grant from the Ministry of Education, Japan     

Gap junctions between microvilli of an oocyte and follicle cells in the teleost (Plecoglossus altivelis)

In the teleost, Plecoglossus altivelis, intercellular junctions between microvilli of an oocyte and follicle cells were studied by electron microscopy. Microvilli, which were radiated from an oocyte and arrived at the surface of follicle cells, established contact with follicle cells. These contact areas appeared to be a seven-layered membrane with an overall thickness of about 18 microns by standard fixation. In freeze-fracture replicas, many small aggregates of intramembraneous particles were revealed on the cleavage faces of cytoplasmic membranes of follicle cells. These morphological evidences suggest that in the teleost gap junctions exist between the oocyte and follicle cells, especially on the surface of follicle cells.     

THE APPEARANCE AND STRUCTURE OF INTERCELLULAR CONNECTIONS DURING THE ONTOGENY OF THE RABBIT OVARIAN FOLLICLE WITH PARTICULAR REFERENCE TO GAP JUNCTIONS

Lanthanum tracer and freeze-fracture electron microscope techniques were used to study junctional complexes between granulosa cells during the differentiation of the rabbit ovarian follicle. For convenience we refer to cells encompassing the oocyte, before antrum and gap junction formation, as follicle cells. After the appearance of an antrum and gap junctions we call the cells granulosa cells. Maculae adherentes are found at the interfaces of oocyte-follicle-granulosa cells throughout folliculogenesis. Gap junctions are first detected in follicles when the antrum appears. In early antral follicles typical large gap junctions are randomly distributed between granulosa cells. In freeze-fracture replicas, they are characterized by polygonally packed 90-Å particles arranged in rows separated by nonparticulate A-face membrane. A particle-sparse zone surrounds gap junctions and is frequently occupied by small particle aggregates of closely packed intramembranous particles. The gap junctions of granulosa cells appear to increase in size with further differentiation of the follicle. The granulosa cells of large Graafian follicles are adjoined by small and large gap junctions; annular gap junctions are also present. The large gap junctions are rarely surrounded by a particle-free zone on their A-faces, but are further distinguished by particle rows displaying a higher degree of organization.     

Characterization of intercellular junctions in the preimplantation mouse embryo by freeze-fracture and thin-section electron microscopy

Intercellular junction formation in preimplantation mouse embryos was investigated with thin-section and freeze-fracture electron microscopy. At the four-cell stage, regions of close membrane apposition with focal points of membrane contact and occasional underlying cytoplasmic densities were observed between blastomeres of thin-sectioned embryos. Corresponding intramembrane specializations were not, however, observed in freeze-fractured embryos. At the 8- to 16-cell stage, small gap and macula occludens junctions and complexes of these junctions were observed at all levels between blastomeres of freeze-fractured embryos. As development progressed from the early to mid 8- to 16-cell stage, the size of the occludens/gap junction complexes increased, forming fascia occludens/gap junction complexes. At the morula stage, gap junctions and occludens/gap junction complexes were observed on both presumptive trophoblast and inner cell-mass cells. Zonula occludens junctions were first observed at the morula stage on presumptive trophoblast cells of freeze-fractured embryos. The number of embryos possessing zonula occludens junctions increased at the mid compared to the early morula stage. At the blastocyst stage, junctional complexes consisting of zonula occludens, macula adherens, and gap junctions were observed between trophoblast cells of freeze-fractured and thin-sectioned embryos. Isolated gap and occludens junctions, adherens junctions, and occludens/gap junction complexes were observed on trophoblast and inner cell-mass cells.     

Size-Dependent Diffusion of Dextrans in Excised Porcine Corneal Stroma

Delivery of therapeutic agents to the eye requires efficient transport through cellular and extracellular barriers. We evaluated the rate of diffusive transport in excised porcine corneal stroma using fluorescently labeled dextran molecules with hydrodynamic radii ranging from 1.3 to 34 nm. Fluorescence correlation spectroscopy (FCS) was used to measure diffusion coefficients of dextran molecules in the excised porcine corneal stroma. The preferential sensitivity of FCS to diffusion along two dimensions was used to differentially probe diffusion along the directions parallel to and perpendicular to the collagen lamellae of the corneal stroma. In order to develop an understanding of how size affects diffusion in cornea, diffusion coefficients in cornea were compared to diffusion coefficients measured in a simple buffer solution. Dextran molecules diffuse more slowly in cornea as compared to buffer solution. The reduction in diffusion coefficient is modest however (67% smaller), and is uniform over the range of sizes that we measured. This indicates that, for dextrans in the 1.3 to 34 nm range, the diffusion landscape of corneal stroma can be represented as a simple liquid with a viscosity approximately 1.5 times that of water. Diffusion coefficients measured parallel vs. perpendicular to the collagen lamellae were indistinguishable. This indicates that diffusion in the corneal stroma is not highly anisotropic. Our results support the notion that the corneal stroma is highly permeable and isotropic to transport of hydrophilic molecules and particles with hydrodynamic radii up to at least 34 nm.