Molecular Vision 2014; 20:638-648
Received 17 October 2013 | Accepted 11 May 2014 | Published 13 May 2014
1Structural Biophysics Group, School of Optometry and Vision Sciences, Cardiff University, Wales, UK; 2Department of Ophthalmology, Kyoto Prefectural University of Medicine, Hirokoji Kawaramachi, Kyoto, Japan
Correspondence to: Andrew Quantock, Structural Biophysics Group, School of Optometry and Vision Sciences, Cardiff University, Maindy Road, Cardiff CF24 4HQ, Wales, UK; Phone: +44 (0)29 20875064; FAX: +44 (0)29 20874859; email: address: QuantockAJ@cf.ac.uk
Purpose: Type VI collagen is a primary component of the extracellular matrix of many connective tissues. It can form distinct aggregates depending on tissue structure, chemical environment, and physiology. In the current study we examine the ultrastructure and mode of aggregation of type VI collagen molecules in the human trabecular meshwork.
Methods: Trabecular meshwork was dissected from donor human eyes, and three-dimensional transmission electron microscopy of type VI collagen aggregates was performed.
Results: Electron-dense collagen structures were detected in the human trabecular meshwork and identified as collagen type VI assemblies based on the three-dimensional spatial arrangement of the type VI collagen molecules, the 105-nm axial periodicity of the assemblies themselves, and their characteristic double bands, which arose from the globular domains of the type VI collagen molecules. Sulfated proteoglycans were also seen to associate with the assemblies either with the globular domain or the inner rod-like segments of the tetramers.
Conclusions: No extended structural regularity in the organization of type VI collagen assemblies within the trabecular meshwork was evident, and the lateral separation of the tetramers forming the assemblies varied, as did the angle formed by the main axes of adjacent tetramers. This is potentially reflective of the specific nature of the trabecular meshwork environment, which facilitates aqueous outflow from the eye, and we speculate that extracellular matrix ions and proteins might prevent a more tight packing of type VI collagen tetramers that form the assemblies.
Type VI collagen is a primary component of the extracellular matrix of many connective tissues [1,2], notably skin  and cornea. It was originally identified in pepsin fragments of human aortic intima  and designated as “intima collagen.” After further electron microscopy and biochemical studies [3,5], intima collagen was renamed type VI collagen  and a detailed structural model for this type of collagen was proposed [7-13].
Type VI collagen is a heterotrimer and was long considered to be composed of three  genetically distinct alpha 1 (VI), alpha 2 (VI), and alpha 3 (VI) chains . However, recently three novel collagen VI alpha chains have been discovered in humans: alpha 4 (VI), alpha 5 (VI), and alpha 6 (VI) chains [16,17]. After secretion into the extracellular matrix, the alpha chains assemble as type VI collagen molecules  that possess a central collagenous triple-helical segment, about 105 nm long, delimited at both ends by a globular domain (Figure 1A). Pairs of collagen VI molecules coil together in an antiparallel fashion  with a 30-nm axial shift to form dimers (Figure 1B) . Two dimers associate laterally with no axial shift to give rise to a tetramer (Figure 1C) , which can be considered as the basic structural unit of collagen VI microfibrils (Figure 1D) [7,18]. Cysteine residues form disulfide bridges between monomers in dimers and between dimers in tetramers , and they stabilize the main collagen VI structural unit. Tetramers can be described as possessing an inner rod-like region and two outer rod-like regions separated by and ending with the collagen VI globular domains of the four molecules forming them (Figure 1C).
Unlike the more abundant type I collagen , type VI collagen gives rise to cross-banded aggregates that are characterized by a pattern of pairs of transverse bands approximately 30 nm apart with a periodicity of about 105 nm [6,9,18]. In particular, noncovalent bonds between the C- and N- terminal globular domains at each end  of the outer rod-like regions of two adjacent tetramers produce end-to-end, cross-banded, pseudofibrillar aggregates (Figure 1D) with a repeated ca. 30–75-nm spacing [5,20]. The same interactions between globular domains of the tetramers also account for the formation of more extended collagen VI aggregates  resembling open networks. The basic way in which collagen type VI tetramers can assemble explains the organization of morphologically distinct aggregates [22-24], which also depends on the structure and physiology of the tissue [25-28] in which they are found.
Type VI collagen interacts with extracellular matrix constituents [29,30] in several connective tissues [31-33]. Collagen type VI is associated with several human disorders. For example, individuals having Bethlem myopathy exhibit mutations of the collagen type VI genes (COL6A1, COL6A2, COL6A3) that affect the N- terminal domains [34-36]. Collagen type VI assemblies are also involved with pathogenesis of ocular disorders, including age-related macular degeneration  and Sorsby's fundus dystrophy . Here, we examine the three-dimensional organization of collagen type VI aggregates in the human trabecular meshwork (Figure 2), the extracellular matrix of which provides flow resistance to aqueous humor leaving the anterior chamber of the eye  and which thus plays a role in the regulation of intraocular pressure.
Ocular tissues containing trabecular meshwork were dissected out by one of us (MU, an ocular surgeon) from both eyes of a 69-year-old female donor. Tissue, which was obtained from the Bristol Eye Bank, Bristol, UK, with full informed consent, was immediately fixed in 2.5% glutaraldehyde in 25 mM sodium acetate buffer (pH 5.7) containing 0.1 M MgCl2 and 0.05% cuprolinic blue dye (the dye (Polysciences Inc., Warrington, PA) was included to stain sulfated proteoglycans). Samples were then washed in sodium acetate buffer to remove the glutaraldehyde fixative and cuprolinic blue dye. Immersion in 0.5% sodium tungstate was performed to add contrast to the stained proteoglycans, and dehydration was accomplished by exposing the tissue to a graded series of ethanol (50%–100%). Following conventional processing with epoxy resin embedding (Agar Scientific, Stansted, UK), ultrathin sections approximately 90 nm thick were cut on an Ultracut E ultramicrotome (Leica Microsystems (UK) Ltd, Milton Keynes, UK) and contrasted for transmission electron microscopy by staining with saturated aqueous uranyl acetate for 12 min followed by four washes in filtered distilled water (1 min per wash). Sections were examined with a JEOL 1010 transmission electron microscope (Jeol UK, Welwyn Garden City, UK) operating at 80 kV. Experiments were conducted in accordance with the ethical principles that have their origin in the Declaration of Helsinki and in line with the requirements of the UK Human Tissue Act.
For electron tomography, 120-nm-thick sections were cut and collected on naked 200 mesh copper grids. After uranyl acetate staining as indicated above, 10 nm colloidal gold particles (BBI, Cardiff, UK) were deposited on both sides of the grid to serve as fiducial markers. A 92 single-axis tilt series of electron micrographs of collagen type VI assemblies was obtained from +60° to −60° in either 1° and 2° increments. Images were acquired using a Gatan ORIUS SC1000 charge coupled device camera (Gatan, Pleasanton, CA) at 20,000X magnification. Micrographs were aligned at a pixel size of 0.4 nm based on the position of the colloidal gold particles on each image. Tomographic reconstructions of longitudinal sections of trabecular meshwork type VI collagen aggregates and stained proteoglycan filaments were generated with the IMOD software package (Boulder, CO) . Segmentation of the tomograms, three-dimensional analysis, and visualization were accomplished via the use of EM3D software (College Station, TX) . Visualization of the reconstructed image stacks and the generation of animated stereo views were achieved with the 3DViewer plug-in of the ImageJ software package (Bethesda, MD) [42,43].
Transverse, banded, collagenous assemblies were found to be widespread throughout the extracellular matrix of the human trabecular meshwork beams and the juxtacanalicular region. These assemblies, displaying an axial periodicity of 109±13 nm (mean±standard deviation, n=47), formed clusters of different sizes, which often lined the intratrabecular spaces and which were surrounded by interstitial collagen fibrils displaying the typical 67-nm axial periodicity (Figure 2 and Figure 3). These assemblies, from their general appearance and periodicity, closely resembled the assemblies of collagen VI molecules found in other tissues  and matched the 100–120-nm periodicity of presumed type VI collagen long-spacing collagen documented in the human trabecular meshwork . One main difference was that in the trabecular meshwork the transverse bands were single instead of double bands. In typical collagen VI assemblies, the transverse bands arise from the electron dense material of the globular N- and C-terminal domains of the collagen type VI molecules and are split into pairs (Figure 4). These double bands are characteristic and indicative of collagen type VI assemblies and come about as a consequence of the axial alignment of the N- and C-globular domains of the outer rod-like regions of collagen type VI tetramers, which are about 30 nm apart axially (Figure 1C,D).
In the aggregates studied here, as well as in those of collagen VI described previously in other tissues [3,5-13], electron dense filaments were seen running perpendicular, or nearly perpendicular, to the transverse bands and crossing them. In the case of the collagen VI assemblies, these filaments were interpreted as arising from the inner and outer rod-like segments of the type VI collagen tetramers that form the assemblies (Figure 5) [37,38].
The many structural similarities between the assemblies in this study of trabecular meshwork and the collagen VI assemblies examined elsewhere [12,37,38,45] suggested that they were all made of collagen VI, with the tetramers arranged in essentially the same way. An exhaustive search at the microscope, which included tilting the specimens at different angles, produced clear evidence of single bands splitting into double bands in a small number of assemblies (Figure 4). These double bands were revealed only for a limited tilt angle range, appearing as single transverse bands at a 0° tilt angle, splitting into double bands at higher angles (about 20° in the cases shown in Figure 4), and reverting to single but much narrower bands at higher tilt angles. The axial distance between double bands in these assemblies was not regular, ranging between 112 nm and 85 nm. This would be expected if the tetramers in the assemblies were forming transient links and had not yet settled into a stable configuration. The ratio between the smaller and the larger gaps between the double bands in the most regular assemblies was about 0.2, which is consistent with that of other collagen VI assemblies. The existence of the double bands confirmed the presence of collagen VI in the assemblies.
To achieve a better understanding of the way in which collagen type VI molecules interact with each other in the trabecular meshwork, we generated three-dimensional tomographic reconstructions of the assemblies from single-axis tilt series of electron micrographs. This type of analysis can reveal details of the molecular organization of collagen type VI aggregates that are not accessible from conventional two-dimensional transmission electron microscopy (Figure 5) and can offer new insights into the way collagen type VI molecules are assembled in the matrix of the human trabecular meshwork. In Figure 6 we present one typical three-dimensional reconstruction that we produced of a type VI collagen assembly. Figure 6A shows an 8.57-nm thick slice through the reconstruction, while Figure 6B shows a surface rendering of the same reconstruction. Figure 6C represents a manual segmentation of the same view (see also Appendix 1, Appendix 2, and Appendix 3). In the surface rendering in Figure 6B, the presence of globular domains (highlighted with red arrows) was evident within a transverse band. Globular domains such as these, when not interacting with other material of the extracellular matrix, are likely to be responsible for the double bands seen in typical collagen VI assemblies (and in the assemblies seen in Figure 4). In the trabecular meshwork, the inner rod-like segments of the collagen type VI tetramers assemble with an unsystematic irregular lateral arrangement (Figure 6B; yellow arrowheads), with no evidence of a regular lateral repeat.
Our three-dimensional reconstructions of the trabecular meshwork showed the presence of sulfated proteoglycan filaments, which as well as populating the fibrillar matrix were also seen to co-associate with collagen type VI assemblies (Figure 5, Figure 6, and Appendix 4). The stained filaments were identical in nature to those seen in two-dimensional electron micrographs of trabecular meshwork published by other investigators [46,47], who confirmed they were proteoglycans by specific enzyme digestion protocols. Proteoglycans were found to associate close to the N- and C- globular domains of type VI collagen as well as to the rod-like segments (Figure 6; see Appendix 4, Appendix 5, and Appendix 6). Moreover, proteoglycans seemed to vary in morphology and size, which is consistent with the fact that different types of proteoglycans can interact with collagen type VI aggregates [47-49]. There was also some evidence of interaction between separate assemblies leading to the formation of larger collagen VI complexes (e.g., Figure 6). In general, it can be seen that the alignment between distinct assemblies within a larger complex occurred by keeping the transverse bands in the axial register (Figure 5 and Figure 6; see Appendix 4, Appendix 5, and Appendix 6).
Type VI collagen can form morphologically distinct aggregates with molecular organization that appears to be related to tissue morphology and physiology [22-28]. Various human pathologies have been associated with collagen type VI [34,35], for example, Bethlem myopathy, an autosomal-dominant, inherited, muscular disorder . There are also indications that type VI collagen assemblies are associated with eye disorders, such as age-related macular degeneration, the leading cause of blindness in the Western world, and Sorsby's fundus dystrophy [37,38]. Previous workers have demonstrated that collagen type VI is a key extracellular matrix component of the trabecular meshwork [51-53]. The current investigation is the first to study the architecture and association with proteoglycans of collagen type VI assemblies in the human trabecular meshwork by three-dimensional electron tomography.
Our three-dimensional reconstructions showed that collagen type VI tetramers-identified as such from their morphology, periodicity, and the characteristic double-banded structure arising from the arrangement of the globular domains-were aggregated in a more disordered way in the trabecular meshwork compared to other collagen type VI-rich tissues in which the collagen VI assemblies show an extended structural regularity [37,38,45]. This suggests that the molecular or physiologic environment of the trabecular meshwork matrix could interfere with the typical organization of collagen VI assemblies. Our proposed structural model for the collagen VI assemblies in the trabecular meshwork (Figure 7) was based on the interaction of collagen type VI tetramers through their globular domains. These interactions provide stability to the network of type VI collagen. Collagen VI tetramers are the building blocks in this model and they assemble together via noncovalent bonds between the N-terminal globular domains of one tetramer and the C-terminal globular domains of an adjacent tetramer. The electron dense mass of the globular domains would produce, in a different environment, two transverse bands at the position of the C- and N- terminal domains. The fact that single-banded assemblies were largely prevalent suggests that extra material accumulates in the space between the globular domains. It is possible that one or more of the proteins present in the trabecular meshwork interact with the N- and C-terminal globular domains or with the outer rod-like segments of the tetramers, explaining why double-banded aggregates are not always visible in the human trabecular meshwork. It is apparent from our data that there was no extended lateral regularity in the arrangement of the collagen type VI tetramers since not only was their separation variable but so were the relative angles that the inner and outer rod-like segments of the tetramers make with the transverse bands of the assemblies. This might be an effect of the ionic micro-environment of the trabecular matrix itself. In fact, trabecular meshwork cells have the potential to manipulate the aqueous humor outflow by altering the extracellular ionic micro-environment of their matrix, modulating extracellular matrix composition , including, for example, the size and degree of extension of meshwork proteoglycans. Following the principle of ionic modulation of flow resistance by the trabecular meshwork cells, a simple supposition would be that changes in the physicochemical properties of the trabecular meshwork would also affect the interactions between the globular domains of the type VI collagen molecules. On this basis, interactions between the globular domains that hold together adjacent tetramers would be disturbed, and this would have an effect on the structural stability of collagen type VI aggregates.
A second possibility is that the structural irregularity of the collagen type VI assemblies in the human trabecular meshwork is the consequence of interactions between the N- and C-terminal domains, which are somehow modulated by other factors or molecules. We should also consider that trabecular meshwork cells sense changes in mechanical stress or intraocular pressure that might trigger remodeling of the matrix to adjust the outflow resistance and maintain intraocular pressure homeostasis. Several matrix proteins are often involved in reorganization of the extracellular matrix and changes in cell-matrix interactions, and some of these proteins may interact with the collagen VI assemblies. Sulfated proteoglycans have been documented previously in the human trabecular meshwork where they interact with fibrillar collagen  and are likely to contribute to aqueous outflow resistance. Here, we demonstrated that these negatively charged macromolecules co-associate with collagen type VI assemblies. In a three-dimensional view, it is clear that while the binding site for a proportion of proteoglycans was located on rod-like segments of the collagen VI tetramers, there were also some proteoglycans associated with the transverse bands where the globular domains of collagen type VI are found. Our findings align with other studies that have shown that collagen type VI interacts with membrane-associated chondroitin sulfate proteoglycan , and also with biglycan and decorin [47,49], members of the small leucine-rich repeat proteoglycan family. The interaction of NG2 and collagen type VI is thought to be important in extracellular matrix organization as well as in cell-matrix interactions that determine cell morphology with respect to the matrix. The possibility of multiple proteoglycan binding sites is also in line with previous investigations, indicating that biglycan and decorin are localized near the N-terminal region of the triple helical domain  and the following globular domain of type VI collagen tetramers. Previous studies of human trabecular meshwork have indicated a decrease in the numbers of stained cuprolinic blue-proteoglycan complexes with age  but an increase in the amount of collagen type VI . These age-related alterations in matrix composition might influence the molecular ultrastructure and interactions in the human trabecular meshwork. It is hoped that new insights into the structure of collagen type VI and associated matrix structures of the trabecular meshwork in the three-dimensional view will shed more light on the function of the tissue and its role in the outflow pathway. As was previously mentioned, the human trabecular meshwork provides flow resistance to aqueous humor leaving the anterior chamber of the eye and thus likely plays a role in the regulation of intraocular pressure . If collagen type VI plays a role in the maintenance of the aqueous humor outflow pathway in the trabecular meshwork, one might hypothesize that alterations in its ultrastructure and interactions with other extracellular matrix components might impact upon pathological changes in the trabecular meshwork.
Appendix 1. 3D surface-rendered reconstruction of type VI collagen aggregates in the human trabecular meshwork.
Appendix 2. A surface representation of a 3D reconstruction of the tilted type VI collagen aggregates.
Appendix 3. 3D surface reconstruction of multiple type VI collagen aggregates in the human trabecular meshwork.
Appendix 4. Tomographic reconstruction and segmentation of type VI collagen molecules in the human trabecular meshwork.
Appendix 5. Manually-segmented reconstructed volume of the collagen VI molecules and proteoglycans in the aggregates.
Appendix 6. Manually-segmented reconstructed volume of the aggregates in Appendix 3.
This work was supported by a Cardiff University Professor Sir Martin Evans’ President's Studentship to EK, and by collaborative research grants from the Japan Society for the Promotion of Sciences and the Japan Eye Bank.