Distribution of the collagen IV isoforms in human Bruch’s membrane
Department of Ophthalmology, University of California, Davis, CA, USAq/#r*, 百拇医药
2 Department of Molecular Biology and Biochemistry, Okayama University Medical School, Okayama, Japanq/#r*, 百拇医药
3 Wilmer Eye Institute, Johns Hopkins Medical Institutes, Baltimore, MD, USAq/#r*, 百拇医药
Accepted for publication 22 August 2002q/#r*, 百拇医药
ABSTRACTq/#r*, 百拇医药
Aims: To determine the distribution of the {alpha} 1 to {alpha} 6 chains of type IV collagen in Bruch’s membrane of the human posterior pole.q/#r*, 百拇医药
Methods: Cryosections (10 µm) from 18 human eyes (20 months to 83 years old) were acid treated, blocked with 10% normal goat serum, incubated for 1 hour with monoclonal antibodies against type IV collagen isoform specific peptides at 1:75 dilution, and visualised with an ABC staining kit.q/#r*, 百拇医药
Results: In Bruch’s membrane, the {alpha} 1(IV) and {alpha} 2(IV) chains were identified in retinal pigment epithelial (10/18 = 55%) and choriocapillaris basement membranes (18/18 = 100%); the {alpha} 3(IV), {alpha} 4(IV), and {alpha} 5(IV) chains were also found in the retinal pigment epithelial basement membrane (13/18 = 72%). In the choroid, the {alpha} 1(IV) and {alpha} 2(IV) chains were detected in the blood vessels (18/18=100%). The {alpha} 6(IV) chain was not identified in any sections.
Conclusion: The heterogeneous distribution of {alpha} 1–2(IV) and {alpha} 3–5(IV) in Bruch’s membrane could give insights into the function of this structure in health, ageing, and diseases such as age related macular degeneration.k4!1g, 百拇医药
Keywords: basal deposits; Bruch’s membrane; collagen IV; isoformsk4!1g, 百拇医药
The basement membrane is a specialised extracellular matrix produced by several cell types that provides mechanical support, divides tissues into compartments, serves as a selective molecular sieve, and influences cell phenotype.1 Type IV collagen is one of the major components of the basement membrane. Six genetically distinct type IV collagen chains, {alpha} 1(IV)-{alpha} 6(IV), have been identified. Each type IV collagen molecule is a trimer composed of three {alpha} chains which form heterotrimeric molecules of {alpha} 1–2(IV), {alpha} 3–5(IV), and {alpha} 5–6(IV) chains.2,3 The different collagen IV molecules have distinct topographical locations and functions. For example, {alpha} 1–2(IV) collagen is found in blood vessels with ubiquitous distribution where it functions to maintain the integrity of the vascular wall during exposure to varying blood pressures.2 {alpha} 3–5(IV) collagen has a more limited distribution. In the kidney, it is confined to the glomerular basement membrane where it is essential for the long term maintenance of the structure and filtration of the glomerulus.4–7 Finally, {alpha} 5–6(IV) collagen has recently been found in smooth muscle basement membranes.8
Bruch’s membrane is a pentalaminar structure composed of the RPE basement membrane on the inner surface of the membrane, two collagenous zones divided by a middle elastic layer, and the choriocapillaris basement membrane on the outer surface of the membrane. The collagen IV isoform distribution in the neurosensory retina has been previously well characterised.9 While a number of studies have investigated the collagen IV expression pattern in Bruch’s membrane, these studies for the most part have been limited by the use of anti-collagen IV type 1–2 antibodies.10–14 Owing to the autofluorescence of Bruch’s membrane which limits using immunofluorescent techniques, the inherent structural complexity of Bruch’s membrane, and previously unrecognised isoforms of collagen IV, the pattern profile of the six type IV isoforms in Bruch’s membrane remains largely unknown.9,13,15 Identifying the specific collagen IV isoforms expressed in Bruch’s membrane could provide functional insights into this structure as well as determine a role in ageing and disease. For example, {alpha} 3–5(IV) collagen is associated with cell differentiation since it is expressed by highly differentiated central corneal epithelium, but not by undifferentiated limbal stem cells.16 Alternatively, Petitclerc et al demonstrated that {alpha} 3(IV) collagen contains anti-angiogenic properties.17 Thus, regions of Bruch’s membrane that are devoid of {alpha} 3(IV) collagen could be susceptible to developing choroidal neovascular membranes. The purpose of this study was to define the expression pattern of the collagen IV isoforms in Bruch’s membrane of the human globe by using a collection of isoform specific monoclonal antibodies which recognise epitopes localised to the C terminal non-collagenous globular domains of {alpha} 1–6(IV) chains.18
MATERIALS AND METHODS(9z3a5, http://www.100md.com
Tissue(9z3a5, http://www.100md.com
Human eyes (20 months to 83 years old) were obtained from the Sierra Eye and Tissue Bank (Sacramento, CA, USA) within 21 hours after death and placed in phosphate buffered saline (PBS) pH 7.4 in 4% paraformaldehyde at 4°C. The anterior segment was removed and the posterior pole was examined under a dissecting microscope and categorised according to the protocol in the ALARM study.19 The macular region, which was a 6 x 6 mm calotte centred around the fovea, was cryoprotected using the technique of Barthel and Raymond.20 Briefly, calottes were rinsed three times in 1X PBS containing 5% sucrose (w/v) for 10 minutes at 4°C and were progressively infiltrated with sucrose by 30 minute incubations at 4°C in PBS containing 10%, 12.5%, and 15% sucrose (w/v). Finally, calottes were infiltrated overnight in 1X PBS containing 20% sucrose (w/v) at 4°C.(9z3a5, http://www.100md.com
Tissue embedding
Calottes were first infiltrated in a 2:1 sucrose 20% (w/v):OCT compound (Miles, Inc, Elkhart, IN, USA) mixture for 30 minutes at room temperature according to the method of Barthel and Raymond.20 Each calotte was then embedded in fresh 2:1 sucrose 20% (w/v):OCT mixture and frozen by immersion in isopentane (Aldrich Chemical Co, Inc, Milwaukee, WI, USA) chilled with dry ice. All tissue blocks were stored at -80°C for later use.46itn, 百拇医药
Immunohistochemistry46itn, 百拇医药
Serial cryosections (10 µm) oriented parallel to the optic nerve-fovea plane were fixed with 4% paraformaldehyde for 10 minutes, acetone for 10 minutes, and 0.1 M KCl-HCl (pH 1.5) for 10 minutes. Sections were treated with 3% H2O2 in methanol for 5 minutes followed by blocking with 10% normal goat serum (Vector Laboratories, Inc, Burlingame, CA, USA) for 10 minutes. Sections were incubated with rat monoclonal antibodies against type IV collagen isoform specific peptides ({alpha} 1, H11; {alpha} 2, H22; {alpha} 3, H31; {alpha} 4, H43; {alpha} 5, H53; {alpha} 6, H63)18 at 1:75 dilution for 1 hour followed by goat anti-rat biotinylated secondary antibody (1:500; ICN Pharmaceuticals, Inc, Aurora, OH, USA) for 30 minutes. The reaction was amplified using an ABC kit for 30 minutes (Vector Laboratories, Inc), and visualised with VIP reagent (Vector Laboratories, Inc) for 5 minutes. The sections were counterstained with methyl green (Vector Laboratories, Inc). A purified rat IgG (ICN Pharmaceuticals, Inc) was used for controls, and results were confirmed with a second set of monoclonal anti-collagen IV isoform antibodies ({alpha} 1, H12; {alpha} 2, H25; {alpha} 3, H32; {alpha} 4, H44; {alpha} 5, H52; {alpha} 6, H66).18
Photography]mg3%, 百拇医药
Specimens were examined with an Olympus BH-2 microscope (Olympus Optical Co, Ltd, Tokyo, Japan) with a Kontron Elektronik ProgRes 3012 charged coupled device camera (Kontron Elektronik GmbH, Eching, Germany). Digitalised images were captured through the ProgRes digital camera plug-in directly to Adobe Photoshop 5.0 (Adobe Systems, Inc, Mountain View, CA, USA).]mg3%, 百拇医药
Statistical analysis]mg3%, 百拇医药
A specific isoform was designated "expressed" when specific labelling was either intermittently or uniformly distributed in the sections examined. The Fisher’s exact test was used to test the null hypothesis that the expression of the individual isoform in the fundus was due to chance. The McNemar’s test was used to determine the probability that differences in expression between different collagen isoforms were due to chance. The expression patterns observed in this study were considered significant when the probability calculated from the above tests was p<0.05.
RESULTS.@t#ds, 百拇医药
Eighteen human eyes were obtained from donors ranging in age from 20 months to 83 years old. One eye was from a 20 month old, three eyes were from donors 50–59 years old, 11 eyes were from donors 60–69 years old, and three eyes were from donors 70–83 years old..@t#ds, 百拇医药
Table 1 shows the isoform specific expression profile of collagen IV in the human posterior pole. Strong immunostaining for the {alpha} 1–2(IV) and {alpha} 3–5(IV) chains was observed in Bruch’s membrane. In the RPE basement membrane, there was a slight predominance of immunostaining for {alpha} 3–5(IV) (13/18 eyes = 72%) compared to {alpha} 1–2(IV) (10/18 eyes = 55%), although this difference was not significant (p = 0.24, McNemar’s test) (Fig 1). No age related distribution of either {alpha} 1–2(IV) or {alpha} 3–5(IV) isoforms was identified. {alpha} 1–2(IV) were identified in the choriocapillaris basement membrane in 100% (18/18 eyes) while {alpha} 3–5(IV) were observed in 28% (5/18 eyes; p = 0.001, McNemar’s test). Mild immunostaining for the collagen {alpha} 1–5(IV) isoforms was observed in the middle layers of Bruch’s membrane. In the choroid, {alpha} 1(IV) and {alpha} 2(IV) were detected in the blood vessels (18/18 eyes = 100%). Mild staining was seen in the choroidal stroma with antibodies to {alpha} 1(IV) to {alpha} 5(IV). Collagen IV {alpha} 6 expression was not seen anywhere in the posterior segment with either H63 or H66 antibody.
fig.ommitteed;*cul2, 百拇医药
Table 1 Distribution of {alpha} 1–6(IV) collagen in Bruch’s membrane and choroid of human globes detected with isoform specific anti-collagen IV monoclonal antibodies;*cul2, 百拇医药
fig.ommitteed;*cul2, 百拇医药
Figure 1 Distribution of collagen IV isoforms in Bruch’s membrane and choroid. (A) From an 83 year old female showing immunostaining for {alpha} 3(IV) in the RPE basement membrane (black arrowheads) and minimal staining in the choriocapillaris basement membrane (red arrowheads). Similar staining patterns were seen for {alpha} 4–5(IV) (data not shown). (B) From a 64 year old male with staining for {alpha} 1(IV) predominantly in the choriocapillaris basement membrane (red arrowheads) and no staining in the RPE basement membrane (black arrowheads). Choroidal blood vessels also stain for {alpha} 1(IV). Similar staining was seen for {alpha} 2(IV) (data not shown). (C) In contrast, no staining in the same 64 year old male is seen with the rat IgG control. Bar = 10 µm.;*cul2, 百拇医药
Two eyes contained basal deposits and drusen in Bruch’s membrane. In both eyes, two labelling patterns for collagen IV were observed. The more common pattern was an interruption of staining for all of the {alpha} (IV) isoforms in basal deposits and drusen (Fig 2A). Less common was a continuation of the RPE basement membrane, which stained for both {alpha} 1–2(IV) and {alpha} 3–5(IV), through the deposits (Fig 2B.
fig.ommitteedpu-[n#n, http://www.100md.com
Figure 2 Expression pattern of collagen IV in Bruch’s membrane containing basal deposits. (A) The RPE basement membrane (black arrowhead) which stains for {alpha} 3(IV), is interrupted in an area of a basal deposit (* * *) in an 83 year old female donor. The RPE basement membrane is also identified adjacent to the RPE cell overlying the basal deposit (red arrowhead). (B) In a different section from the same donor, the RPE basement membrane (black arrowheads) is lightly stained in a basal deposit that spans the entire section. The choriocapillaris basement membrane (red arrowheads) is prominently stained with the anti-{alpha} 1(IV) antibody. Bar = 10 µm.pu-[n#n, http://www.100md.com
DISCUSSIONpu-[n#n, http://www.100md.com
Our immunohistochemical studies identified a heterogeneous distribution of the collagen IV isoforms in the posterior fundus. In Bruch’s membrane, the RPE basement membrane stained for both {alpha} 1–2(IV) and {alpha} 3–5(IV) while the choriocapillaris basement membrane stained primarily for {alpha} 1–2(IV). Choroidal blood vessels, like those in the neurosensory retina,9 stained for {alpha} 1–2(IV) which is typical for endothelial basement membranes.2 An unusual finding was the presence, on a limited basis, of {alpha} 3–5(IV) in the choriocapillaris basement membrane.
The co-expression of {alpha} 1–2(IV) and {alpha} 3–5(IV) collagen at least partially explains the inconsistent results obtained from previous immunohistochemistry studies of Bruch’s membrane with different type IV collagen antibodies. Previous studies have used polyclonal antibodies raised against type IV collagen isolated from human placenta or EHS sarcoma, both of which contain {alpha} 1–2(IV) collagen.2 Using an anti-collagen IV antibody raised against placenta, Marshall et al showed that type IV collagen was absent from the RPE basement membrane, but was present in the choriocapillaris basement membrane.13,21 Likewise, in the rat, Lin and Essner found greater expression of collagen IV in the choriocapillaris than the RPE basement membrane with their polyclonal anti-collagen IV antibodies.15 Our study suggests that these different staining characteristics could be explained by the mixture of both {alpha} 1–2(IV) and {alpha} 3–5(IV) collagen in the RPE basement membrane compared to the choriocapillaris basement membrane. Kleppel et al identified a signal in the inner portion of Bruch’s membrane using a monoclonal antibody generated against the non-collagenous domain of type IV collagen isolated from human kidney glomerular basement membranes, which probably represents {alpha} 3–5(IV).10,11 In their study, the exact inner layer of Bruch’s membrane was not identified. Our results extend the observations by Kleppel et al by precisely identifying both the specific {alpha} 3–5(IV) isoforms and localising their distribution to the RPE basement membrane.
The limited sample size and elderly donor age prevent us from drawing any conclusions regarding the isoform distribution as a function of age. However, two eyes contained age related basal deposits in Bruch’s membrane for which two unique staining patterns were observed. The more common finding was an interruption of labelling for any of the collagen IV isoforms. These interruptions in staining could represent degraded areas of RPE basement membrane, an artefact related to tissue processing, or epitope masking from macromolecules within basal deposits. Alternatively, basement membrane proteins develop, with age, an increase in the number of cross links either from oxidative stress or advanced glycation end products, which could shield the epitopes from our antibody set.22–24in;n!, 百拇医药
The patchy distribution of the {alpha} 1–2(IV) and (3–5)IV isoform staining pattern could influence changes that develop to Bruch’s membrane. For example, {alpha} 3–5(IV) is more resistant to proteolysis than other collagen IV isoforms.22,25 Regions that contain {alpha} 3–5(IV)—for example, by being relatively resistant to proteolysis, could promote matrix protein expansion from reduced degradation to promote early basal deposit formation during ageing.13,21,26 Alternatively, Petitclerc et al demonstrated that {alpha} 3(IV) contains anti-angiogenic properties.17 Focal loss of {alpha} 3(IV) or regions of relatively less {alpha} 3–5(IV) collagen could increase susceptibility to choroidal neovascular membrane formation. With the potential differences in function among the different isoforms, further study is necessary to define its role in health and disease associated with Bruch’s membrane.
ACKNOWLEDGEMENTSd#6}?6, 百拇医药
We thank Yoshikazu Sado, PhD, for his generous donation of the collagen IV antibodies and The Sierra Eye and Tissue Bank, Sacramento, CA, USA.d#6}?6, 百拇医药
Grant support: National Eye Institute EY14005 (JTH), RPB Manpower Award (JTH), an unrestricted RPB grant, and a gift from the Friends of Macular Degeneration Society (JTH), grant in aid for International Scientific Research (Joint Research, 11694280) of Ministry of Education, Science, Sports, and Culture of Japan (YN).d#6}?6, 百拇医药
Proprietary interest: None.d#6}?6, 百拇医药
REFERENCESd#6}?6, 百拇医药
Schittny JC, Yurchenco PD. Basement membranes: molecular organization and function in development and disease. Curr Opin Cell Biol 1989;1:983–8.d#6}?6, 百拇医药
Sado Y, Kagawa M, Naito I, et al. Organization and expression of basement membrane collagen IV genes and their roles in human disorders. J Biochem (Tokyo) 1998;123:767–76.d#6}?6, 百拇医药
Hudson BG, Reeders ST, Tryggvason K. Type IV collagen: structure, gene organization, and role in human diseases. Molecular basis of Goodpasture and Alport syndromes and diffuse leiomyomatosis. J Biol Chem 1993;268:26033–6.
Boutaud A, Borza DB, Bondar O, et al. Type IV collagen of the glomerular basement membrane. Evidence that the chain specificity of network assembly is encoded by the noncollagenous NC1 domains. J Biol Chem 2000;275:30716–24.?, http://www.100md.com
Harvey SJ, Zheng K, Sado Y, et al. Role of distinct type IV collagen networks in glomerular development and function. Kidney Int 1998;54:1857–66.?, http://www.100md.com
Gunwar S, Ballester F, Noelken ME, et al. Glomerular basement membrane. Identification of a novel disulfide-cross-linked network of alpha3, alpha4, and alpha5 chains of type IV collagen and its implications for the pathogenesis of Alport syndrome. J Biol Chem 1998;273:8767–75.?, http://www.100md.com
Ninomiya Y, Kagawa M, Iyama K, et al. Differential expression of two basement membrane collagen genes, COL4A6 and COL4A5, demonstrated by immunofluorescence staining using peptide-specific monoclonal antibodies. J Cell Biol 1995;130:1219–29.?, http://www.100md.com
Borza DB, Bondar O, Ninomiya Y, et al. The NC1 domain of collagen IV encodes a novel network composed of the alpha 1, alpha 2, alpha 5, and alpha 6 chains in smooth muscle basement membranes. J Biol Chem 2001;276:28532–40.
Ljubimov AV, Burgeson RE, Butkowski RJ, et al. Basement membrane abnormalities in human eyes with diabetic retinopathy. J Histochem Cytochem 1996;44:1469–79.]2$2, http://www.100md.com
Kleppel MM, Santi PA, Cameron JD, et al. Human tissue distribution of novel basement membrane collagen. Am J Pathol 1989;134:813–25.]2$2, http://www.100md.com
Kleppel MM, Michael AF. Expression of novel basement membrane components in the developing human kidney and eye. Am J Anat 1990;187:165–74.]2$2, http://www.100md.com
Das A, Frank RN, Zhang NL, et al. Ultrastructural localization of extracellular matrix components in human retinal vessels and Bruch’s membrane. Arch Ophthalmol 1990;108:421–9.]2$2, http://www.100md.com
Marshall GE, Konstas AG, Reid GG, et al. Collagens in the aged human macula. Graefes Arch Clin Exp Ophthalmol 1994;232:133–40.]2$2, http://www.100md.com
Ishizaki M, Westerhausen-Larson A, Kino J, et al. Distribution of collagen IV in human ocular tissues. Invest Ophthalmol Vis Sci 1993;34:2680–9.]2$2, http://www.100md.com
Lin WL, Essner E. Differential localization of type IV collagen in Bruch’s membrane of the eye: comparison of two polyclonal antibodies. Cell Mol Biol (Noisy-le-grand) 1994;40:851–8.
Ljubimov AV, Burgeson RE, Butkowski RJ, et al. Human corneal basement membrane heterogeneity: topographical differences in the expression of type IV collagen and laminin isoforms. Lab Invest 1995;72:461–73.*, 百拇医药
Petitclerc E, Boutaud A, Prestayko A, et al. New functions for non-collagenous domains of human collagen type IV. Novel integrin ligands inhibiting angiogenesis and tumor growth in vivo. J Biol Chem 2000;275:8051–61.*, 百拇医药
Sado Y, Kagawa M, Kishiro Y, et al. Establishment by the rat lymph node method of epitope-defined monoclonal antibodies recognizing the six different alpha chains of human type IV collagen. Histochem Cell Biol 1995;104:267–75.*, 百拇医药
Curcio CA, Medeiros NE, Millican CL. The Alabama Age-Related Macular Degeneration Grading System for donor eyes. Invest Ophthalmol Vis Sci 1998;39:1085–96.*, 百拇医药
Barthel LK, Raymond PA. Improved method for obtaining 3-microns cryosections for immunocytochemistry. J Histochem Cytochem 1990;38:1383–8.*, 百拇医药
Marshall GE, Konstas AG, Reid GG, et al. Type IV collagen and laminin in Bruch’s membrane and basal linear deposit in the human macula. Br J Ophthalmol 1992;76:607–14.
Mott JD, Khalifah RG, Nagase H, et al. Nonenzymatic glycation of type IV collagen and matrix metalloproteinase susceptibility. Kidney Int 1997;52:1302–12.}pw^gvu, 百拇医药
Riedle B, Kerjaschki D. Reactive oxygen species cause direct damage of Engelbreth-Holm-Swarm matrix. Am J Pathol 1997;151:215–31.}pw^gvu, 百拇医药
Handa JT, Verzijl N, Matsunaga H, et al. Increase in the advanced glycation end product pentosidine in Bruch’s membrane with age. Invest Ophthalmol Vis Sci 1999;40:775–9.}pw^gvu, 百拇医药
Kahsai TZ, Enders GC, Gunwar S, et al. Seminiferous tubule basement membrane. Composition and organization of type IV collagen chains, and the linkage of alpha3(IV) and alpha5(IV) chains. J Biol Chem 1997;272:17023–32.}pw^gvu, 百拇医药
Loffler KU, Lee WR. Basal linear deposit in the human macula. Graefes Arch Clin Exp Ophthalmol 1986;224:493–501.(L Chen N Miyamura Y Ninomiya and J T Handa)
2 Department of Molecular Biology and Biochemistry, Okayama University Medical School, Okayama, Japanq/#r*, 百拇医药
3 Wilmer Eye Institute, Johns Hopkins Medical Institutes, Baltimore, MD, USAq/#r*, 百拇医药
Accepted for publication 22 August 2002q/#r*, 百拇医药
ABSTRACTq/#r*, 百拇医药
Aims: To determine the distribution of the {alpha} 1 to {alpha} 6 chains of type IV collagen in Bruch’s membrane of the human posterior pole.q/#r*, 百拇医药
Methods: Cryosections (10 µm) from 18 human eyes (20 months to 83 years old) were acid treated, blocked with 10% normal goat serum, incubated for 1 hour with monoclonal antibodies against type IV collagen isoform specific peptides at 1:75 dilution, and visualised with an ABC staining kit.q/#r*, 百拇医药
Results: In Bruch’s membrane, the {alpha} 1(IV) and {alpha} 2(IV) chains were identified in retinal pigment epithelial (10/18 = 55%) and choriocapillaris basement membranes (18/18 = 100%); the {alpha} 3(IV), {alpha} 4(IV), and {alpha} 5(IV) chains were also found in the retinal pigment epithelial basement membrane (13/18 = 72%). In the choroid, the {alpha} 1(IV) and {alpha} 2(IV) chains were detected in the blood vessels (18/18=100%). The {alpha} 6(IV) chain was not identified in any sections.
Conclusion: The heterogeneous distribution of {alpha} 1–2(IV) and {alpha} 3–5(IV) in Bruch’s membrane could give insights into the function of this structure in health, ageing, and diseases such as age related macular degeneration.k4!1g, 百拇医药
Keywords: basal deposits; Bruch’s membrane; collagen IV; isoformsk4!1g, 百拇医药
The basement membrane is a specialised extracellular matrix produced by several cell types that provides mechanical support, divides tissues into compartments, serves as a selective molecular sieve, and influences cell phenotype.1 Type IV collagen is one of the major components of the basement membrane. Six genetically distinct type IV collagen chains, {alpha} 1(IV)-{alpha} 6(IV), have been identified. Each type IV collagen molecule is a trimer composed of three {alpha} chains which form heterotrimeric molecules of {alpha} 1–2(IV), {alpha} 3–5(IV), and {alpha} 5–6(IV) chains.2,3 The different collagen IV molecules have distinct topographical locations and functions. For example, {alpha} 1–2(IV) collagen is found in blood vessels with ubiquitous distribution where it functions to maintain the integrity of the vascular wall during exposure to varying blood pressures.2 {alpha} 3–5(IV) collagen has a more limited distribution. In the kidney, it is confined to the glomerular basement membrane where it is essential for the long term maintenance of the structure and filtration of the glomerulus.4–7 Finally, {alpha} 5–6(IV) collagen has recently been found in smooth muscle basement membranes.8
Bruch’s membrane is a pentalaminar structure composed of the RPE basement membrane on the inner surface of the membrane, two collagenous zones divided by a middle elastic layer, and the choriocapillaris basement membrane on the outer surface of the membrane. The collagen IV isoform distribution in the neurosensory retina has been previously well characterised.9 While a number of studies have investigated the collagen IV expression pattern in Bruch’s membrane, these studies for the most part have been limited by the use of anti-collagen IV type 1–2 antibodies.10–14 Owing to the autofluorescence of Bruch’s membrane which limits using immunofluorescent techniques, the inherent structural complexity of Bruch’s membrane, and previously unrecognised isoforms of collagen IV, the pattern profile of the six type IV isoforms in Bruch’s membrane remains largely unknown.9,13,15 Identifying the specific collagen IV isoforms expressed in Bruch’s membrane could provide functional insights into this structure as well as determine a role in ageing and disease. For example, {alpha} 3–5(IV) collagen is associated with cell differentiation since it is expressed by highly differentiated central corneal epithelium, but not by undifferentiated limbal stem cells.16 Alternatively, Petitclerc et al demonstrated that {alpha} 3(IV) collagen contains anti-angiogenic properties.17 Thus, regions of Bruch’s membrane that are devoid of {alpha} 3(IV) collagen could be susceptible to developing choroidal neovascular membranes. The purpose of this study was to define the expression pattern of the collagen IV isoforms in Bruch’s membrane of the human globe by using a collection of isoform specific monoclonal antibodies which recognise epitopes localised to the C terminal non-collagenous globular domains of {alpha} 1–6(IV) chains.18
MATERIALS AND METHODS(9z3a5, http://www.100md.com
Tissue(9z3a5, http://www.100md.com
Human eyes (20 months to 83 years old) were obtained from the Sierra Eye and Tissue Bank (Sacramento, CA, USA) within 21 hours after death and placed in phosphate buffered saline (PBS) pH 7.4 in 4% paraformaldehyde at 4°C. The anterior segment was removed and the posterior pole was examined under a dissecting microscope and categorised according to the protocol in the ALARM study.19 The macular region, which was a 6 x 6 mm calotte centred around the fovea, was cryoprotected using the technique of Barthel and Raymond.20 Briefly, calottes were rinsed three times in 1X PBS containing 5% sucrose (w/v) for 10 minutes at 4°C and were progressively infiltrated with sucrose by 30 minute incubations at 4°C in PBS containing 10%, 12.5%, and 15% sucrose (w/v). Finally, calottes were infiltrated overnight in 1X PBS containing 20% sucrose (w/v) at 4°C.(9z3a5, http://www.100md.com
Tissue embedding
Calottes were first infiltrated in a 2:1 sucrose 20% (w/v):OCT compound (Miles, Inc, Elkhart, IN, USA) mixture for 30 minutes at room temperature according to the method of Barthel and Raymond.20 Each calotte was then embedded in fresh 2:1 sucrose 20% (w/v):OCT mixture and frozen by immersion in isopentane (Aldrich Chemical Co, Inc, Milwaukee, WI, USA) chilled with dry ice. All tissue blocks were stored at -80°C for later use.46itn, 百拇医药
Immunohistochemistry46itn, 百拇医药
Serial cryosections (10 µm) oriented parallel to the optic nerve-fovea plane were fixed with 4% paraformaldehyde for 10 minutes, acetone for 10 minutes, and 0.1 M KCl-HCl (pH 1.5) for 10 minutes. Sections were treated with 3% H2O2 in methanol for 5 minutes followed by blocking with 10% normal goat serum (Vector Laboratories, Inc, Burlingame, CA, USA) for 10 minutes. Sections were incubated with rat monoclonal antibodies against type IV collagen isoform specific peptides ({alpha} 1, H11; {alpha} 2, H22; {alpha} 3, H31; {alpha} 4, H43; {alpha} 5, H53; {alpha} 6, H63)18 at 1:75 dilution for 1 hour followed by goat anti-rat biotinylated secondary antibody (1:500; ICN Pharmaceuticals, Inc, Aurora, OH, USA) for 30 minutes. The reaction was amplified using an ABC kit for 30 minutes (Vector Laboratories, Inc), and visualised with VIP reagent (Vector Laboratories, Inc) for 5 minutes. The sections were counterstained with methyl green (Vector Laboratories, Inc). A purified rat IgG (ICN Pharmaceuticals, Inc) was used for controls, and results were confirmed with a second set of monoclonal anti-collagen IV isoform antibodies ({alpha} 1, H12; {alpha} 2, H25; {alpha} 3, H32; {alpha} 4, H44; {alpha} 5, H52; {alpha} 6, H66).18
Photography]mg3%, 百拇医药
Specimens were examined with an Olympus BH-2 microscope (Olympus Optical Co, Ltd, Tokyo, Japan) with a Kontron Elektronik ProgRes 3012 charged coupled device camera (Kontron Elektronik GmbH, Eching, Germany). Digitalised images were captured through the ProgRes digital camera plug-in directly to Adobe Photoshop 5.0 (Adobe Systems, Inc, Mountain View, CA, USA).]mg3%, 百拇医药
Statistical analysis]mg3%, 百拇医药
A specific isoform was designated "expressed" when specific labelling was either intermittently or uniformly distributed in the sections examined. The Fisher’s exact test was used to test the null hypothesis that the expression of the individual isoform in the fundus was due to chance. The McNemar’s test was used to determine the probability that differences in expression between different collagen isoforms were due to chance. The expression patterns observed in this study were considered significant when the probability calculated from the above tests was p<0.05.
RESULTS.@t#ds, 百拇医药
Eighteen human eyes were obtained from donors ranging in age from 20 months to 83 years old. One eye was from a 20 month old, three eyes were from donors 50–59 years old, 11 eyes were from donors 60–69 years old, and three eyes were from donors 70–83 years old..@t#ds, 百拇医药
Table 1 shows the isoform specific expression profile of collagen IV in the human posterior pole. Strong immunostaining for the {alpha} 1–2(IV) and {alpha} 3–5(IV) chains was observed in Bruch’s membrane. In the RPE basement membrane, there was a slight predominance of immunostaining for {alpha} 3–5(IV) (13/18 eyes = 72%) compared to {alpha} 1–2(IV) (10/18 eyes = 55%), although this difference was not significant (p = 0.24, McNemar’s test) (Fig 1). No age related distribution of either {alpha} 1–2(IV) or {alpha} 3–5(IV) isoforms was identified. {alpha} 1–2(IV) were identified in the choriocapillaris basement membrane in 100% (18/18 eyes) while {alpha} 3–5(IV) were observed in 28% (5/18 eyes; p = 0.001, McNemar’s test). Mild immunostaining for the collagen {alpha} 1–5(IV) isoforms was observed in the middle layers of Bruch’s membrane. In the choroid, {alpha} 1(IV) and {alpha} 2(IV) were detected in the blood vessels (18/18 eyes = 100%). Mild staining was seen in the choroidal stroma with antibodies to {alpha} 1(IV) to {alpha} 5(IV). Collagen IV {alpha} 6 expression was not seen anywhere in the posterior segment with either H63 or H66 antibody.
fig.ommitteed;*cul2, 百拇医药
Table 1 Distribution of {alpha} 1–6(IV) collagen in Bruch’s membrane and choroid of human globes detected with isoform specific anti-collagen IV monoclonal antibodies;*cul2, 百拇医药
fig.ommitteed;*cul2, 百拇医药
Figure 1 Distribution of collagen IV isoforms in Bruch’s membrane and choroid. (A) From an 83 year old female showing immunostaining for {alpha} 3(IV) in the RPE basement membrane (black arrowheads) and minimal staining in the choriocapillaris basement membrane (red arrowheads). Similar staining patterns were seen for {alpha} 4–5(IV) (data not shown). (B) From a 64 year old male with staining for {alpha} 1(IV) predominantly in the choriocapillaris basement membrane (red arrowheads) and no staining in the RPE basement membrane (black arrowheads). Choroidal blood vessels also stain for {alpha} 1(IV). Similar staining was seen for {alpha} 2(IV) (data not shown). (C) In contrast, no staining in the same 64 year old male is seen with the rat IgG control. Bar = 10 µm.;*cul2, 百拇医药
Two eyes contained basal deposits and drusen in Bruch’s membrane. In both eyes, two labelling patterns for collagen IV were observed. The more common pattern was an interruption of staining for all of the {alpha} (IV) isoforms in basal deposits and drusen (Fig 2A). Less common was a continuation of the RPE basement membrane, which stained for both {alpha} 1–2(IV) and {alpha} 3–5(IV), through the deposits (Fig 2B.
fig.ommitteedpu-[n#n, http://www.100md.com
Figure 2 Expression pattern of collagen IV in Bruch’s membrane containing basal deposits. (A) The RPE basement membrane (black arrowhead) which stains for {alpha} 3(IV), is interrupted in an area of a basal deposit (* * *) in an 83 year old female donor. The RPE basement membrane is also identified adjacent to the RPE cell overlying the basal deposit (red arrowhead). (B) In a different section from the same donor, the RPE basement membrane (black arrowheads) is lightly stained in a basal deposit that spans the entire section. The choriocapillaris basement membrane (red arrowheads) is prominently stained with the anti-{alpha} 1(IV) antibody. Bar = 10 µm.pu-[n#n, http://www.100md.com
DISCUSSIONpu-[n#n, http://www.100md.com
Our immunohistochemical studies identified a heterogeneous distribution of the collagen IV isoforms in the posterior fundus. In Bruch’s membrane, the RPE basement membrane stained for both {alpha} 1–2(IV) and {alpha} 3–5(IV) while the choriocapillaris basement membrane stained primarily for {alpha} 1–2(IV). Choroidal blood vessels, like those in the neurosensory retina,9 stained for {alpha} 1–2(IV) which is typical for endothelial basement membranes.2 An unusual finding was the presence, on a limited basis, of {alpha} 3–5(IV) in the choriocapillaris basement membrane.
The co-expression of {alpha} 1–2(IV) and {alpha} 3–5(IV) collagen at least partially explains the inconsistent results obtained from previous immunohistochemistry studies of Bruch’s membrane with different type IV collagen antibodies. Previous studies have used polyclonal antibodies raised against type IV collagen isolated from human placenta or EHS sarcoma, both of which contain {alpha} 1–2(IV) collagen.2 Using an anti-collagen IV antibody raised against placenta, Marshall et al showed that type IV collagen was absent from the RPE basement membrane, but was present in the choriocapillaris basement membrane.13,21 Likewise, in the rat, Lin and Essner found greater expression of collagen IV in the choriocapillaris than the RPE basement membrane with their polyclonal anti-collagen IV antibodies.15 Our study suggests that these different staining characteristics could be explained by the mixture of both {alpha} 1–2(IV) and {alpha} 3–5(IV) collagen in the RPE basement membrane compared to the choriocapillaris basement membrane. Kleppel et al identified a signal in the inner portion of Bruch’s membrane using a monoclonal antibody generated against the non-collagenous domain of type IV collagen isolated from human kidney glomerular basement membranes, which probably represents {alpha} 3–5(IV).10,11 In their study, the exact inner layer of Bruch’s membrane was not identified. Our results extend the observations by Kleppel et al by precisely identifying both the specific {alpha} 3–5(IV) isoforms and localising their distribution to the RPE basement membrane.
The limited sample size and elderly donor age prevent us from drawing any conclusions regarding the isoform distribution as a function of age. However, two eyes contained age related basal deposits in Bruch’s membrane for which two unique staining patterns were observed. The more common finding was an interruption of labelling for any of the collagen IV isoforms. These interruptions in staining could represent degraded areas of RPE basement membrane, an artefact related to tissue processing, or epitope masking from macromolecules within basal deposits. Alternatively, basement membrane proteins develop, with age, an increase in the number of cross links either from oxidative stress or advanced glycation end products, which could shield the epitopes from our antibody set.22–24in;n!, 百拇医药
The patchy distribution of the {alpha} 1–2(IV) and (3–5)IV isoform staining pattern could influence changes that develop to Bruch’s membrane. For example, {alpha} 3–5(IV) is more resistant to proteolysis than other collagen IV isoforms.22,25 Regions that contain {alpha} 3–5(IV)—for example, by being relatively resistant to proteolysis, could promote matrix protein expansion from reduced degradation to promote early basal deposit formation during ageing.13,21,26 Alternatively, Petitclerc et al demonstrated that {alpha} 3(IV) contains anti-angiogenic properties.17 Focal loss of {alpha} 3(IV) or regions of relatively less {alpha} 3–5(IV) collagen could increase susceptibility to choroidal neovascular membrane formation. With the potential differences in function among the different isoforms, further study is necessary to define its role in health and disease associated with Bruch’s membrane.
ACKNOWLEDGEMENTSd#6}?6, 百拇医药
We thank Yoshikazu Sado, PhD, for his generous donation of the collagen IV antibodies and The Sierra Eye and Tissue Bank, Sacramento, CA, USA.d#6}?6, 百拇医药
Grant support: National Eye Institute EY14005 (JTH), RPB Manpower Award (JTH), an unrestricted RPB grant, and a gift from the Friends of Macular Degeneration Society (JTH), grant in aid for International Scientific Research (Joint Research, 11694280) of Ministry of Education, Science, Sports, and Culture of Japan (YN).d#6}?6, 百拇医药
Proprietary interest: None.d#6}?6, 百拇医药
REFERENCESd#6}?6, 百拇医药
Schittny JC, Yurchenco PD. Basement membranes: molecular organization and function in development and disease. Curr Opin Cell Biol 1989;1:983–8.d#6}?6, 百拇医药
Sado Y, Kagawa M, Naito I, et al. Organization and expression of basement membrane collagen IV genes and their roles in human disorders. J Biochem (Tokyo) 1998;123:767–76.d#6}?6, 百拇医药
Hudson BG, Reeders ST, Tryggvason K. Type IV collagen: structure, gene organization, and role in human diseases. Molecular basis of Goodpasture and Alport syndromes and diffuse leiomyomatosis. J Biol Chem 1993;268:26033–6.
Boutaud A, Borza DB, Bondar O, et al. Type IV collagen of the glomerular basement membrane. Evidence that the chain specificity of network assembly is encoded by the noncollagenous NC1 domains. J Biol Chem 2000;275:30716–24.?, http://www.100md.com
Harvey SJ, Zheng K, Sado Y, et al. Role of distinct type IV collagen networks in glomerular development and function. Kidney Int 1998;54:1857–66.?, http://www.100md.com
Gunwar S, Ballester F, Noelken ME, et al. Glomerular basement membrane. Identification of a novel disulfide-cross-linked network of alpha3, alpha4, and alpha5 chains of type IV collagen and its implications for the pathogenesis of Alport syndrome. J Biol Chem 1998;273:8767–75.?, http://www.100md.com
Ninomiya Y, Kagawa M, Iyama K, et al. Differential expression of two basement membrane collagen genes, COL4A6 and COL4A5, demonstrated by immunofluorescence staining using peptide-specific monoclonal antibodies. J Cell Biol 1995;130:1219–29.?, http://www.100md.com
Borza DB, Bondar O, Ninomiya Y, et al. The NC1 domain of collagen IV encodes a novel network composed of the alpha 1, alpha 2, alpha 5, and alpha 6 chains in smooth muscle basement membranes. J Biol Chem 2001;276:28532–40.
Ljubimov AV, Burgeson RE, Butkowski RJ, et al. Basement membrane abnormalities in human eyes with diabetic retinopathy. J Histochem Cytochem 1996;44:1469–79.]2$2, http://www.100md.com
Kleppel MM, Santi PA, Cameron JD, et al. Human tissue distribution of novel basement membrane collagen. Am J Pathol 1989;134:813–25.]2$2, http://www.100md.com
Kleppel MM, Michael AF. Expression of novel basement membrane components in the developing human kidney and eye. Am J Anat 1990;187:165–74.]2$2, http://www.100md.com
Das A, Frank RN, Zhang NL, et al. Ultrastructural localization of extracellular matrix components in human retinal vessels and Bruch’s membrane. Arch Ophthalmol 1990;108:421–9.]2$2, http://www.100md.com
Marshall GE, Konstas AG, Reid GG, et al. Collagens in the aged human macula. Graefes Arch Clin Exp Ophthalmol 1994;232:133–40.]2$2, http://www.100md.com
Ishizaki M, Westerhausen-Larson A, Kino J, et al. Distribution of collagen IV in human ocular tissues. Invest Ophthalmol Vis Sci 1993;34:2680–9.]2$2, http://www.100md.com
Lin WL, Essner E. Differential localization of type IV collagen in Bruch’s membrane of the eye: comparison of two polyclonal antibodies. Cell Mol Biol (Noisy-le-grand) 1994;40:851–8.
Ljubimov AV, Burgeson RE, Butkowski RJ, et al. Human corneal basement membrane heterogeneity: topographical differences in the expression of type IV collagen and laminin isoforms. Lab Invest 1995;72:461–73.*, 百拇医药
Petitclerc E, Boutaud A, Prestayko A, et al. New functions for non-collagenous domains of human collagen type IV. Novel integrin ligands inhibiting angiogenesis and tumor growth in vivo. J Biol Chem 2000;275:8051–61.*, 百拇医药
Sado Y, Kagawa M, Kishiro Y, et al. Establishment by the rat lymph node method of epitope-defined monoclonal antibodies recognizing the six different alpha chains of human type IV collagen. Histochem Cell Biol 1995;104:267–75.*, 百拇医药
Curcio CA, Medeiros NE, Millican CL. The Alabama Age-Related Macular Degeneration Grading System for donor eyes. Invest Ophthalmol Vis Sci 1998;39:1085–96.*, 百拇医药
Barthel LK, Raymond PA. Improved method for obtaining 3-microns cryosections for immunocytochemistry. J Histochem Cytochem 1990;38:1383–8.*, 百拇医药
Marshall GE, Konstas AG, Reid GG, et al. Type IV collagen and laminin in Bruch’s membrane and basal linear deposit in the human macula. Br J Ophthalmol 1992;76:607–14.
Mott JD, Khalifah RG, Nagase H, et al. Nonenzymatic glycation of type IV collagen and matrix metalloproteinase susceptibility. Kidney Int 1997;52:1302–12.}pw^gvu, 百拇医药
Riedle B, Kerjaschki D. Reactive oxygen species cause direct damage of Engelbreth-Holm-Swarm matrix. Am J Pathol 1997;151:215–31.}pw^gvu, 百拇医药
Handa JT, Verzijl N, Matsunaga H, et al. Increase in the advanced glycation end product pentosidine in Bruch’s membrane with age. Invest Ophthalmol Vis Sci 1999;40:775–9.}pw^gvu, 百拇医药
Kahsai TZ, Enders GC, Gunwar S, et al. Seminiferous tubule basement membrane. Composition and organization of type IV collagen chains, and the linkage of alpha3(IV) and alpha5(IV) chains. J Biol Chem 1997;272:17023–32.}pw^gvu, 百拇医药
Loffler KU, Lee WR. Basal linear deposit in the human macula. Graefes Arch Clin Exp Ophthalmol 1986;224:493–501.(L Chen N Miyamura Y Ninomiya and J T Handa)