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.     

Immunofluorescent localization of collagen types I, II, and III in the embryonic chick eye.

The appearance and distribution of type I, II, and III collagens in the developing chick eye were studied by specific antibodies and indirect immunofluorescence. At stage 19, only type I collagen was detected in the primary corneal stroma, in the vitreous body, and along the lens surface. At later stages, type I collagen was located in the primary and secondary corneal stroma and in the fibrous sclera, but not around the lens. Type II collagen was first observed at stage 20 in the primary corneal stroma, neural retina, and vitreous body. It was particularly prominent at the interface of the neural retina and vitreous body and, from stage 30 on, in the cartilaginous sclera. The primary corneal stroma consisted of a mixture of type I and II collagens between stages 20 and 27. Invasion of the primary corneal stroma by mesenchyme and subsequent deposition of fibroblast-derived collagen corresponded with a pronounced increase of type I collagen, throughout the entire stroma, and of type II collagen, in the subepithelial region. Type II collagen was also found in Bowman's and Descemet's membranes. A transient appearance of type III collagen was observed in the corneal epithelial cells, but not in the stroma (stages 20–30). The fully developed cornea contained both type I and II collagens, but no type III collagen. Type III collagen was prominent in the fibrous sclera, iris, nictitating membrane, and eyelids.     

Fibroblast-collagen interactions in the formation of the secondary stroma of the chick cornea

Fibroblasts invade the primary corneal stroma of the 6-day-old chick embryo eye. The way in which these cells build the secondary stroma has been studied by microscope examination of the stroma during the subsequent 8 Days. Eyes were embedded in low viscosity nitrocellulose, and 30-micrometer tangential sections of cornea were cut and stained with azan (giving blue collagen and red cells). These sections were sufficiently thick to include enough cells and collagen for stromal organization to be visible under Nomarski optics. Three days after invasion, the fibroblasts extend along collagen bundles in the posterior region of the stroma; surprisingly, fibroblasts near the epithelium are more rounded. The collagen itself is organized in orthogonal bundles rather than in sheets. Measurements show that posterior bundles increase in size with time while anterior stroma si similar in diameter to primary stroma. These observations confirm that the epithelium continues to deposit primary stroma up to at least the 14th day. They show, moreover, that fibroblasts deposit collagen fibrils on extant stroma and that the farther a bundle is from the epithelium, and hence the longer the period since it was first laid down, the wider it is likely to be. Analysis of the results and existing data on hyaluronic acid levels in the stroma suggests that Bowman's membrane, the region of anterior stroma that remains uncolonized by cells, is, during this period at least, primary stroma laid down but as yet unswollen.     

Acquisition of type IX collagen by the developing avian primary corneal stroma and vitreous

Previous investigations from our laboratory and others have demonstrated that type II collagen, once thought to be a cartilage-specific molecule, is also a component of both the primary corneal stroma and the vitreous of embryonic chickens. In the present immunohistochemical study we have examined the expression in these embryonic matrices of another "cartilage-specific" collagen, type IX, along with type II. In the cornea, type IX collagen is in the primary stroma, but is not detectable in the mature, secondary stroma. Even within the primary stroma this collagen has a brief, transitory existence. It first appears in the peripheral stroma at the time the endothelial cells begin to migrate along its posterior surface, and spreads throughout the stroma during the following 24-36 hr. The epitopes on type IX collagen then suddenly become undetectable just before this matrix swells and becomes populated by the periocular mesenchymal cells (future keratocytes). In comparison, collagen type II (along with type I) is present in the stroma before and long after these events. Deposition of immunodetectable type IX collagen in the developing corneal stroma thus seems to be independent of type II. In the vitreous, we observed type IX collagen along with type II as soon as authentic vitreous could be identified and at all subsequent stages of development. In this tissue, therefore, the expression of collagen types IX and II appears to be coordinate.     

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.     

Changes in distribution of the long form of type XII collagen during chicken corneal development.

The expression and distribution of the long form of Type XII collagen were investigated histochemically during chicken corneal development using a monoclonal antibody (P3D11) raised against the N-terminal domain of chicken Type XII collagen. Specificity of the antibody was confirmed by immunoprecipitation before and after bacterial collagenase digestion. Immunofluorescent microscopic studies showed that during chicken cornea formation, the long form of Type XII collagen is initially detected on Day 3 embryo (stage 19) in the sub-epithelial matrix of the corneal periphery and in the matrix around the optic cup. On Day 5 embryo (stage 27) the long form was expressed in the primary stroma. Thereafter, as the secondary stroma was formed, the long form localized in the sub-epithelial and sub-endothelial matrices and in the anterior region of the limbus (corneoscleral junction) before the formation of Descemet's and Bowman's membranes. After hatching, the immunoreactivity decreased predominantly in the sub-epithelial and sub-endothelial matrices but remained at the anterior region of the limbus. Immunoelectron microscopic examination demonstrated that the long form localizes in the Descemet's and Bowman's membranes and along the collagen fibrils in the stroma with a periodic repeat. Based on the distribution of the long form of Type XII collagen in the sub-epithelial and sub-endothelial matrices and limbus, it was suggested that the long form of Type XII collagen is involved in formation of the Descemet's and Bowman's membranes and in stabilization of the limbus.     

Control of corneal differentiation by extracellular materials. Collagen as a promoter and stabilizer of epithelial stroma production

The primary stroma of the cornea of the chick embryo consists of orthogonally arranged collagen fibrils embedded in glycosaminoglycan (GAG) produced by the epithelium under the early inductive influence of the lens. The experiments reported here were designed to test whether or not the collagen of the lens basement lamina is capable of stimulating corneal epithelium to produce primary stroma. Enzymatically isolated 5-day-old corneal epithelia were grown for 24 hr in vitro in the presence of ³⁵SO₄ or proline-³H on various substrata. Epithelia cultured on lens capsule synthesized 2.5 times as much GAG (as measured by incorporation of label into CPC precipitable material) and almost 3 times as much collagen (assayed by hot TCA extraction or collagenase sensitivity) as when cultured on Millipore filter or other noncollagenous substrata. A similar stimulatory response was observed when epithelium was combined with chemically pure chondrosarcoma collagen, NaOH-extracted lens capsule, vitreous humor, frozen-killed corneal stroma or cartilage, or tendon collagen gels; in the latter case, the magnitude of the effect can be shown to be related to concentration of the collagen in the gel. All of the collagenous substrata stimulate not only extracellular matrix production, but also polymerization of corneal-type matrix, as judged by ultrastructural criteria and by the association of more radioactivity with the tissue than the medium. Since purified chondrosarcoma collagen is as effective as lens capsule, the stimulatory effect on collagen and GAG synthesis by corneal epithelium is not specific for basal lamina (lens capsule) collagen.     

Type VI collagen: immunohistochemical identification as a filamentous component of the extracellular matrix of the developing avian corneal stroma

Selected stages of the developing chicken cornea have been examined for type VI collagen, employing monoclonal antibodies specific for this molecule. By immunofluorescence, the molecule is not detectable in 5 1/2 day corneas, a time at which the epithelial-derived, acellular primary stroma is the only corneal matrix present. One day later, the presumptive stromal fibroblasts have invaded this stroma and have initiated synthesis of the secondary (mature) stroma. By that time, a strong fluorescent signal for the type VI collagen molecule is detectable throughout the stroma. It is present in all subsequent ages examined. The molecule is not restricted to the cornea, and is present in most stromal matrices examined, including those of the sclera, eyelid, and nictitating membrane. Immunoelectron microscopy was also performed, utilizing a colloidal gold-labeled secondary antibody. These data show that the type VI collagen is not a component of the striated collagen fibrils, but instead is assembled in the form of thin filaments. The monoclonal antibody bound to the filaments at periodic intervals of about 100 nm.     

Immunofluorescence study of the extracellular matrix of the human placenta

Distribution of collagen types I, III, IV, V and fibronectin in human placental villi has been studied by indirect immunofluorescence. During 9-12 weeks of pregnancy the extracellular matrix of villi represents a network of filaments organized in bundles and aggregates that contain collagen types I and III and finer filaments of collagen types IV and V. Collagen type IV is regularly detected in basal membrane of capillaries and particularly in villous epithelium, collagen type V and fibronectin are occasionally detected there. Marked immunofluorescent reaction on collagen types IV and V and fibronectin, and weak reaction on collagen type III is observed in cellular islets around cytotrophoblasts. In the fetus born in term placental villi have uniform immunofluorescence in thick basal membranes of fetal capillaries and of chorionic epithelium. The immunofluorescent reaction specific for all collagen types is uniform in villous stroma. Distribution of different collagen types and fibronectin, including the unusual localization of membrane collagen type IV, in villous stroma and cellular islets of early and mature placenta is discussed.     

Differential expression of type XIV collagen/undulin by human mammary gland intralobular and interlobular fibroblasts

Immunolocalisation of type XIV collagen/undulin in the human mammary gland revealed greater deposition in the interlobular stroma than in the intralobular stroma. The interlobular stroma is located between the breast lobules and their associated intralobular stroma. Fibroblasts isolated from the interlobular stroma synthesised 3- to 5-fold more type XIV collagen/undulin than intralobular fibroblasts, but synthesised type I and type IV collagens in similar amounts. The differential expression of type XIV collagen/undulin was maintained with passage in culture. The results suggest a role for type XIV collagen/undulin in stabilising dense collagen fibrils. The maintenance of two types of structurally distinct stromas may be important during developmental processes in the mammary gland.     

Collagen type I and type V are present in the same fibril in the avian corneal stroma

The distribution, supramolecular form, and arrangement of collagen types I and V in the chicken embryo corneal stroma were studied using electron microscopy, collagen type-specific monoclonal antibodies, and a preembedding immunogold method. Double-label immunoelectron microscopy with colloidal gold-tagged monoclonal antibodies was used to simultaneously localize collagen type I and type V within the chick corneal stroma. The results definitively demonstrate, for the first time, that both collagens are codistributed within the same fibril. Type I collagen was localized to striated fibrils throughout the corneal stroma homogeneously. Type V collagen could be localized only after pretreatment of the tissue to partially disrupt collagen fibril structure. After such pretreatments the type V collagen was found in regions where fibrils were partially dissociated and not in regions where fibril structure was intact. When pretreated tissues were double labeled with antibodies against types I and V collagen coupled to different size gold particles, the two collagens colocalized in areas where fibril structure was partially disrupted. Antibodies against type IV collagen were used as a control and were nonreactive with fibrils. These results indicate that collagen types I and V are assembled together within single fibrils in the corneal stroma such that the interaction of these collagen types within heterotypic fibrils masks the epitopes on the type V collagen molecule. One consequence of the formation of such heterotypic fibrils may be the regulation of corneal fibril diameter, a condition essential for corneal transparency.     

The influence of cell shape on the induction of functional differentiation in mouse mammary cells in vitro

Summary To define more clearly the in vitro conditions permissive for hormonal induction of functional differentiation, we cultured dissociated normal mammary cells from prelactating mice in or on a variety of substrates. Cultivation of an enriched epithelial cell population in association with living adult mammary stroma in the presence of lactogenic hormones resulted in both morphological and biochemical differentiation. This differentiation, however, was not enhanced over that seen when the cells were associated with killed stroma, provided that the killed stroma had a flexibility similar to that of the living stroma. Cells cultured in inflexible killed stroma usually did not differentiate. Cells cultured within the flexible environment of a collagen gel, but removed from the gas-medium interface, differentiated in a manner similar to those cultured in flexible stroma. Cells cultured on the surface of an attached collagen gel were squamous, and their basolateral surfaces were sequestered from the medium; they did not differentiate. Cells cultured on floating collagen gels were cuboidal-columnar, with basolateral surfaces exposed to the medium, and showed good functional differentiation. Cells cultured on inflexible floating collagen gels were extremely flattened and had exposed basolateral surfaces, and showed no evidence of functional differentiation. We infer that assumption of cuboidal to columnar shapes similar to those of mammary cells in vivo may be important to the induction of functional differentiation in vitro. The additional requirement of basolateral cell surface exposure also is important. This work was supported by U.S. Public Health Service Grants CA-05045 and CA-09041 from the National Cancer Institute, Bethesda, MD.     

Changes in the refractive index of the stroma and its extrafibrillar matrix when the cornea swells

The transparency of the corneal stroma is critically dependent on the hydration of the tissue; if the cornea swells, light scattering increases. Although this scattering has been ascribed to the disruption caused to the arrangement of the collagen fibrils, theory predicts that light scattering could increase if there is an increased mismatch in the refractive indices of the collagen fibrils and the material between them. The purpose of this article is to use Gladstone and Dale's law of mixtures to calculate volume fractions for a number of different constituents in the stroma, and use these to show how the refractive indices of the stroma and its constituent extrafibrillar material would be expected to change as more solvent enters the tissue. Our calculations predict that solvent entering the extrafibrillar space causes a reduction in its refractive index, and hence a reduction in the overall refractive index of the bovine stroma according to the equation n'(s) = 1.335 + 0.04/(0.22 + 0.24 H'), where n'(s) is the refractive index and H' is the hydration of the swollen stroma. This expression is in reasonable agreement with our experimental measurements of refractive index versus hydration in bovine corneas. When the hydration of the stroma increases from H = 3.2 to H = 8.0, we predict that the ratio of the refractive index of the collagen fibrils to that of the material between them increases from 1.041 to 1.052. This change would be expected to make only a small contribution to the large increase in light scattering observed when the cornea swells to H = 8.     

The roles of types XII and XIV collagen in fibrillogenesis and matrix assembly in the developing cornea

Corneal transparency depends on the architecture of the stromal extracellular matrix, including fibril diameter, packing, and lamellar organization. The roles of collagen types XII and XIV in regulation of corneal fibrillogenesis and development were examined. The temporal and spatial expression patterns were analyzed using semi-quantitative RT-PCR, in situ hybridization, Western analysis, and immunohistochemistry. Expression of types XII and XIV collagens in cornea development demonstrated that type XII collagen mRNA levels are constant throughout development (10D-adult) while type XIV mRNA is highest in early embryonic stages (10D-14D), decreasing significantly by hatching. The spatial expression patterns of types XII and XIV collagens demonstrated a homogeneous signal in the stroma for type XIV collagen, while type XII collagen shows segregation to the sub-epithelial and sub-endothelial stroma during embryonic stages. The type XII collagen in the anterior stroma was an epithelial product during development while fibroblasts contributed in the adult. Type XIV collagen expression was highest early in development and was absent by hatching. Both types XII and type XIV collagen have different isoforms generated by alternative splicing that may alter specific interactions important in fibrillogenesis, fibril-fibril interactions, and higher order matrix assembly. Analysis of these splice variants demonstrated that the long XII mRNA levels were constant throughout development, while the short XII NC3 mRNA levels peaked early (12D) followed by a decrease. Both type XIV collagen NC1 splice variants are highest during early stages (12D-14D) decreasing by 17D of development. These data suggest type XII collagen may have a role in development of stromal architecture and maintenance of fibril organization, while type XIV collagen may have a role in regulation of fibrillogenesis.     

Differential roles for two gelatinolytic enzymes of the matrix metalloproteinase family in the remodelling cornea.

We have documented changes in collagenolytic/gelatinolytic enzymes of the matrix metalloproteinase family (MMP) in remodelling rabbit cornea. MMP-2 (65 kDa gelatinase) in the proenzyme form is synthesized by the cells of the normal corneal stroma. After keratectomy the level of MMP-2 is increased in the stroma and enzyme appears in both pro- and activated forms. In addition, corneal cells synthesize MMP-9 (92 kDa gelatinase) in the proenzyme form after keratectomy; expression occurs in both the epithelial as well as stromal corneal layers. Changes in expression of both enzymes are precisely localized to the repairing portion of cornea, but demonstrate important differences in timing that correlate with the timing of specific events of matrix remodelling. Our data suggest that each of the gelatinases plays a different role in tissue remodelling after injury. We hypothesize that MMP-2 performs a surveillance function in normal cornea, catalyzing degradation of collagen molecules that occasionally become damaged. After wounding, this enzyme appears to participate in the prolonged process of collagen remodelling in the corneal stroma that eventually results in functional regeneration of the tissue. MMP-9 expression does not correlate with stromal remodelling, but we suggest that the enzyme might play a part in controlling resynthesis of the epithelial basement membrane.     

Bovine corneal stroma contains a structural glycoprotein located in the gap region of the collagen fibrils

Treatment of bovine corneal stroma using SDS-containing extracting solutions removes a 135,000 MW glycoprotein from the main collagen framework of the tissue. Low-angle synchrotron X-ray diffraction patterns obtained from corneas extracted in this way indicate that the glycoprotein has been removed from the gap regions of the collagen fibrils and is thus an important structural component of the corneal stroma. The glycoprotein (GP 135) shares a number of properties with one of the subunits of type VI collagen, but tests have so far failed to establish their identity.     

Ageing of the human corneal stroma: structural and biochemical changes.

High and low angle X-ray diffraction patterns from the corneal stroma give information about the mean intermolecular spacing of the collagen molecules and the mean interfibrillar spacing of the collagen fibrils, respectively. X-ray data were collected, using a high intensity synchrotron source, from human corneas and sclera at approximately physiological hydration. The spacings were measured as a function of tissue age. Between birth and 90 years there is an increase in the cross-sectional area associated with each molecule in corneal collagen from approx. 3.04 nm2 to 3.46 nm2, and an increase in scleral collagen from approx. 2.65 nm2 to 3.19 nm2. These changes may be due to an increase in the extent of non-enzymic cross-linking between collagen molecules over the age range. We have investigated this possibility by measuring collagen glycation using the thiobarbituric acid assay and the subsequent advanced glycation end-products (AGEs) using fluorescence emission. The results obtained have shown an age-related increase in glycation and AGEs in both tissues. We have also demonstrated a decrease in the interfibrillar spacing of corneal collagen with increasing age which may be related to changes in the proteoglycan composition of the interfibrillar matrix.     

X-ray diffraction studies of the corneal stroma.

A low-angle diffraction pattern has been obtained from corneal stroma. This pattern arises both from the arrangement of the collagen fibrils and from the packing of the tropocollagen molecules along the axes of the fibrils. The spacing arising from the packing of the fibrils increases homogeneously on swelling although the tissue as a whole swells only radially referred to the intact eye. The necessary rearrangement of the fibrils for this type of swelling to occur might result in the formation of regions devoid of collagen fibrils and the water not in the lattice of collagen fibrils could be synonymous with the lakes postulated by Benedek (1971) to explain the loss of transparency on swelling.The spacings due to the packing of the tropocollagen molecules are unusual in that, although they index as the third and fifth orders of the well-known 66 nm repeat, the first order of this spacing is absent. Calculation of the Patterson function for corneal collagen leads to peaks in electron density separated by distances of 0.38 and 0.24 of the repeat distance.     

Basement membrane collagen requirements for attachment and growth of mammary epithelium.

In vivo mammary epithelial cells rest upon a basement membrane composed in part of type IV collagen which is synthesized by these cells. In this study, basement membrane collagen is shown to be selectively recognized by normal mammary ducts and alveoli for attachment and growth when compared to the types of collagen derived from stroma (types I or III) or cartilage (type II). Cell attachment and growth on type I collagen is inhibited by the proline analogue, cis-hydroxyproline, which blocks normal collagen production. These effects of cis-hydroxyproline are not apparent when a basement membrane collagen substratum is provided. Unlike normal mammary epithelium, mammary fibroblasts show little preference for the collagen to which they will attach. A requirement of type IV collagen synthesis for normal mammary epithelial cell attachment and growth on stromal collagen in vitro may have significance in vivo where a basement membrane scaffold may be necessary for normal mammary morphogenesis and growth.     

The spatial organization of Descemet''s membrane-associated type IV collagen in the avian cornea

The organization of type IV collagen in the unconventional basement membrane of the corneal endothelium (Descemet's membrane) was investigated in developing chicken embryos using anti-collagen mAbs. Both immunofluorescence histochemistry and immunoelectron microscopy were performed. In mature embryos (greater than 15 d of development), the type IV collagen of Descemet's membrane was present as an array of discrete aggregates of amorphous material at the interface between Descemet's membrane and the posterior corneal stroma. Immunoreactivity for type IV collagen was also observed in the posterior corneal stroma as irregular plaques of material with a morphology similar to that of the Descemet's membrane-associated aggregates. This arrangement of Descemet's membrane-associated type IV collagen developed from a subendothelial mat of type IV collagen-containing material. This mat, in which type IV collagen-specific immunoreactivity was always discontinuous, first appeared at the time a confluent endothelium was established, well before the onset of Descemet's membrane formation. Immunoelectron microscopy of mature corneas revealed that the characteristic nodal matrix of Descemet's membrane itself was unreactive for type IV collagen, but was penetrated at intervals by projections of type IV collagen-containing material. These projections frequently appeared to contact cell processes from the underlying corneal endothelium. This spatial arrangement of type IV collagen suggests that it serves to suture the corneal endothelium/Descemet's membrane to the dense interfacial matrix of the posterior stroma.