Type VII Collagen Expression in the Human Vitreoretinal Interface, Corpora Amylacea and Inner Retinal Layers

Type VII collagen, as a major component of anchoring fibrils found at basement membrane zones, is crucial in anchoring epithelial tissue layers to their underlying stroma. Recently, type VII collagen was discovered in the inner human retina by means of immunohistochemistry, while proteomic investigations demonstrated type VII collagen at the vitreoretinal interface of chicken. Because of its potential anchoring function at the vitreoretinal interface, we further assessed the presence of type VII collagen at this site. We evaluated the vitreoretinal interface of human donor eyes by means of immunohistochemistry, confocal microscopy, immunoelectron microscopy, and Western blotting. Firstly, type VII collagen was detected alongside vitreous fibers6 at the vitreoretinal interface. Because of its known anchoring function, it is likely that type VII collagen is involved in vitreoretinal attachment. Secondly, type VII collagen was found within cytoplasmic vesicles of inner retinal cells. These cells resided most frequently in the ganglion cell layer and inner plexiform layer. Thirdly, type VII collagen was found in astrocytic cytoplasmic inclusions, known as corpora amylacea. The intraretinal presence of type VII collagen was confirmed by Western blotting of homogenized retinal preparations. These data add to the understanding of vitreoretinal attachment, which is important for a better comprehension of common vitreoretinal attachment pathologies

Collageen type VII in het humane oog: structurele verbindingen : Onderzoek naar de distributie en locatie van intra-oculair collageen type VII, met name in het corpus ciliare.

Purpose: To investigate the distribution and localisation of type VII collagen (Col VII) in the human eye, with special interest in the ciliary body. Methods: Human donor eyes were obtained from the Corneabank, Beverwijk. The postmortem time did not exceed 48 hours. These eyes were embedded in paraffin and Epon or dissected for Western blotting. Sections were cut and stained immunohistochemically (IHC) using mouse monoclonal and rabbit polyclonal primary antibodies against Col VII, according to appropriate protocols for corresponding analyzing techniques. These techniques were light- and transmission electron microscopy (LM and TEM). Secondary antibodies were chosen accordingly. For TEM technique, gold labeled secondary antibodies were used. Western blots were performed on pooled homogenized ocular substructures of ciliary body, choroid, lens capsule with zonules and retina with ILM. These substructures were derived from the donor eyes by postmortem dissection. Setting: laboratory Background: Recently, type VII collagen has been demonstrated in the human retina. The function of this type of collagen - at least in skin - is to mechanically secure tissue layers to each other. The fashion in which ocular tissue layers are fastened has not yet been elucidated.

Type VII collagen in the intraocular environment

Het proefschrift probeert de eigenschappen van Collageen type VII (Col VII), zoals het in het intraoculaire milieu voorkomt, verder in kaart te brengen met behulp van immunohistochemie, -fluorescentie, -blotting en -electronenmicroscopie. Col VII is een ankereiwit, dat normaliter de basaalmembranen van dermale weefsels vasthecht aan de stromale onderlaag. Deze verankering komt tot stand door ankerfibrillen. Dit zijn structuren die ontstaan door laterale aggregatie van de Col VII moleculen, waarbij beide uiteinden van de aggregaten vastgrijpen in de bovenliggende basaalmembraan. De lussen die zo ontstaan ‘vangen’ stromale vezels, waardoor het epitheliale basaalmembraan aan het stroma wordt vastgehecht. Recent onderzoek wees uit dat de retina Col VII tot expressie brengt. Het is alleen onduidelijk of Col VII ankerfibrillen vormt in het intraoculaire milieu, of anderszins een hechtende functie heeft. Het voornaamste doel is om meer eigenschappen van intraoculair Col VII te achterhalen, en -indien mogelijk- een uitspraak te kunnen doen over de functie van Col VII in het intraoculaire milieu. Het ankereiwit kon worden aangetoond op de grenslaag van retina naar glasvocht, het accommodatie systeem (lens, zonulen en corpus ciliare), en in verscheidene intraoculaire bloedvaten. Hoewel het onduidelijk blijft of Col VII in deze structuren ook een hechtende functie heeft, blijkt uit recente literatuur dat Col VII mogelijk ook kan fungeren als een biologische signaalmolecuul in angiogenese of tumorgenese, of een bijkomende factor is in sclerose. Verder onderzoek is nodig om de exacte rol van Col VII in het intraoculaire milieu te ontrafelen

Type VII Collagen in the Human Accommodation System: Expression in Ciliary Body, Zonules, and Lens Capsule

To investigate intraocular expression of COL7A1 and its protein product type VII collagen, particularly at the accommodation system. Eyes from 26 human adult donors were used. COL7A1 expression was analyzed in ex vivo ciliary epithelium by microarray. Type VII collagen distribution was examined by Western blot analysis, immunohistochemistry. and immuno-electron microscopy. COL7A1 is expressed by pigmented and nonpigmented ciliary epithelia. Type VII collagen is distributed particularly at the strained parts of the accommodation system. Type VII collagen was associated with various basement membranes and with ciliary zonules. Anchoring fibrils were not visualized. Type VII collagen distribution at strained areas suggests a supporting role in tissue integrit

Western blot confirms type VII collagen content in retinal substrates.

<p>Western blot stained for type VII collagen. A 290 kDa band appeared in samples (directly) placed in reducing agent buffer (group/lane <b>A</b>). Omitting the use of reducing agents resulted in fading of the 290 kDa band (group/lane <b>B</b>). When samples were incubated in 30 units/ml collagenase, the 290 kDa band disappeared completely, while a 145 kDa band appeared (group/lane <b>C</b>).</p

Confocal microscopy image of retinal section.

<p>Type VII collagen-positive vesicles (LH7.2; green) around two cell nuclei (white arrowheads) in the inner plexiform layer (IPL). Note filamentous GFAP (polyclonal; red) labeling within the nerve fiber layer and around blood vessels. Within the lumen of blood vessels, autofluorescent erythrocytes (*) are seen. They can be discriminated from the corpus amylaceum (white arrow) in terms of fluorescence intensity, size and location. The inner limiting membrane (ILM) directly above the left blood vessel shows some focal positivity for type VII collagen labeling. A control section does not show any Col VII or GFAP labeling. GCL ganglion cell layer. (400x, inset 960x)</p

Immunohistochemical analysis at the vitreoretinal junction of a paraffin-embedded section.

<p>Anti-type VII collagen labeling with monoclonal LH7.2. This donor sample contains a lot of cells within the ganglion cell layer (GCL) that have Col VII-positive vesicles in their cytoplasm. Four corpora amylacea reside in the inner plexiform layer (IPL). (400x)</p

Retinal type VII collagen distribution.

<p>Immunohistochemical analysis at the vitreoretinal junction of a paraffin-embedded, serially sectioned (3–4 <i>μ</i>m) human donor eye evaluated by light microscopy. Anti-type VII collagen labeling with monoclonal LH7.2 (sections 1, 3 & 4) and periodic acid-Schiff (sections 2 & 5). Type VII collagen is present in astrocytic corpora amylacea. At least three corpora amylacea were sliced in this series, consecutively visualized in sections 1–2 (white arrows), sections 2–3 (black arrows) and sections 3–5 (black arrowheads). Corpora amylacea reside in the ganglion cell layer and inner plexiform layer (IPL). Type VII collagen is also present in small vesicles, clustered near large nuclei in the inner retina (white arrowhead). The vesicles reside outside the nucleus, and within the cytoplasm (sections 1, 2 & 3 compared). The cytoplasm of the type VII collagen yielding cell type seems more extensive than that of most other cells residing in the ganglion cell layer (GCL). Furthermore, their nuclei are larger and appear less dense in PAS (or hematoxylin) stains. The inner limiting membrane (ILM) does not label visibly for type VII collagen. Negative control section (NC) shows no labeling. (200 x)</p