Lack of collagen XVIII/endostatin results in eye abnormalities

Mice lacking collagen XVIII and its proteolytically derived product endostatin show delayed regression of blood vessels in the vitreous along the surface of the retina after birth and lack of or abnormal outgrowth of retinal vessels. This suggests that collagen XVIII/endostatin is critical for normal blood vessel formation in the eye. All basement membranes in wild-type eyes, except Descemet's membrane, showed immunogold labeling with antibodies against collagen XVIII. Labeling at sites where collagen fibrils in the vitreous are connected with the inner limiting membrane and separation of the vitreal matrix from the inner limiting membrane in mutant mice indicate that collagen XVIII is important for anchoring vitreal collagen fibrils to the inner limiting membrane. The findings provide an explanation for high myopia, vitreoretinal degeneration and retinal detachment seen in patients with Knobloch syndrome caused by loss-of-function mutations in collagen XVIII.     

Basement membrane stimulates the polarized distribution of integrins but not the Na,K-ATPase in the retinal pigment epithelium.

The basement membrane stimulates the differentiation and polarity of simple transporting epithelia. We demonstrated for the retinal pigment epithelium (RPE) of chicken embryos that polarity develops gradually. Although the RPE and an immature basement membrane are established on embryonic day 4 (E4), the distribution of the Na,K-ATPase and a family of basement membrane receptors containing the beta 1 subunit of integrin is nonpolarized. The percentage of polarized cells increases gradually until cells in all regions of the epithelium are polarized on E11. During this time, the basement membrane increases in size and complexity to form Bruch's membrane. To study the ability of the basement membrane to stimulate the polarized distribution of the beta 1 integrins or the Na,K-ATPase, RPE was harvested from E7, E9, or E14 embryos and cultured on Bruch's membrane isolated (in association with the choroid) from E14 embryos. As a control, the RPE was plated on the side of the choroid lacking a Bruch's membrane. The distribution of the beta 1 integrins and the Na,K-ATPase was determined by indirect immunofluorescence. Bruch's membrane stimulated the polarized distribution of the beta 1 integrins regardless of the developmental age of the RPE even though E7 RPE is nonpolarized in vivo. To examine the role of individual matrix components, RPE was plated on matrix-coated filters. The polarized distribution of the beta 1 integrins was stimulated by laminin, collagen IV, and Matrigel but not by fibronectin. Interestingly, laminin and collagen IV are present in the basement membrane on E4 when RPE is not polarized in vivo. Under no circumstances was the distribution of the Na,K-ATPase polarized. These data indicate that the basement membrane influences the distribution of a subset of plasma membrane proteins but that other factors are required for full polarity.     

Basement Membrane Zone Collagens XV and XVIII/Proteoglycans Mediate Leukocyte Influx in Renal Ischemia/Reperfusion

Collagen type XV and XVIII are proteoglycans found in the basement membrane zones of endothelial and epithelial cells, and known for their cryptic anti-angiogenic domains named restin and endostatin, respectively. Mutations or deletions of these collagens are associated with eye, muscle and microvessel phenotypes. We now describe a novel role for these collagens, namely a supportive role in leukocyte recruitment. We subjected mice deficient in collagen XV or collagen XVIII, and their compound mutant, as well as the wild-type control mice to bilateral renal ischemia/reperfusion, and evaluated renal function, tubular injury, and neutrophil and macrophage influx at different time points after ischemia/reperfusion. Five days after ischemia/reperfusion, the collagen XV, collagen XVIII and the compound mutant mice showed diminished serum urea levels compared to wild-type mice (all p<0.05). Histology showed reduced tubular damage, and decreased inflammatory cell influx in all mutant mice, which were more pronounced in the compound mutant despite increased expression of MCP-1 and TNF-α in double mutant mice compared to wildtype mice. Both type XV and type XVIII collagen bear glycosaminoglycan side chains and an in vitro approach with recombinant collagen XVIII fragments with variable glycanation indicated a role for these side chains in leukocyte migration. Thus, basement membrane zone collagen/proteoglycan hybrids facilitate leukocyte influx and tubular damage after renal ischemia/reperfusion and might be potential intervention targets for the reduction of inflammation in this condition.     

The collagen type XVIII endostatin domain is co-localized with perlecan in basement membranes in vivo.

The C-terminal globular endostatin domain of collagen type XVIII is anti-angiogenic in a variety of experimental tumor models, and clinical trials to test it as an anti-tumor agent are already under way. In contrast, many of its cell biological properties are still unknown. We systematically localized the mRNA of collagen type XVIII with the help of in situ hybridization (ISH) and detected it in epithelial and mesenchymal cells of almost all organ systems throughout mouse development. Light and electron microscopic immunohistochemistry (IHC) revealed that the endostatin domain is a widespread component of almost all epithelial basement membranes in all major developing organs, and in all basement membranes of capillaries and blood vessels. Furthermore, quantitative immunogold double labeling demonstrated a co-localization of 50% of the detected endostatin domain together with perlecan in basement membranes in vivo. We conclude that the endostatin domain of collagen type XVIII plays a role, even in early stages of mouse development, other than regulating angiogenesis. In the adult, the endostatin domain could well be involved in connecting collagen type XVIII to the basement membrane scaffolds. At least in part, perlecan appears to be an adaptor molecule for the endostatin domain in basement membranes in vivo.     

Regulated expression of apolipoprotein E by human retinal pigment epithelial cells

In early age-related macular degeneration (AMD), lipid-containing deposits (drusen) accumulate in Bruch's membrane underlying the retinal pigment epithelium (RPE). Recent studies indicate that apolipoprotein E (apoE) may play a role in lipid trafficking in AMD. Compared with the apoE3 allele, the apoE4 and apoE2 alleles are associated with decreased and increased risk for AMD, respectively; drusen contain high levels of apoE, and apoE null mice develop lipid deposits in Bruch's membrane similar to those observed in AMD. Primary cultures of human RPE cells expressing the apoE3 allele were grown on Transwell culture plates. Western blotting, ELISA assay, and mass spectrometry confirmed that apoE3 was secreted into the apical and basal chambers and that secretion was upregulated by thyroid hormone, 9-cis-retinoic acid, and 22(R)-hydroxycholesterol. In addition, basally secreted apoE associated with exogenously added HDL. These results indicate that apoE secretion can be regulated by specific hormones and that apoE associates with HDL. The findings are consistent with a role for apoE in lipid trafficking through Bruch's membrane and may be relevant to AMD.     

Endostatin binds biglycan and LDL and interferes with LDL retention to the subendothelial matrix during atherosclerosis

Retention of lipoproteins to proteoglycans in the subendothelial matrix (SEM) is an early event in atherosclerosis. We recently reported that collagen XVIII and its proteolytically released fragment endostatin (ES) are differentially depleted in blood vessels affected by atherosclerosis. Loss of collagen XVIII/ES in atherosclerosis-prone mice enhanced plaque neovascularization and increased the vascular permeability to lipids by distinct mechanisms. Impaired endothelial barrier function increased the influx of lipoproteins across the endothelium; however, we hypothesized that enhanced retention might be a second mechanism leading to the increased lipid content in atheromas lacking collagen XVIII. We now demonstrate a novel property of ES that binds both the matrix proteoglycan biglycan and LDL and interferes with LDL retention to biglycan and to SEM. A peptide encompassing the alpha coil in the ES crystal structure mediates the major blocking effect of ES on LDL retention. ES inhibits the macrophage uptake of biglycan-associated LDL indirectly by interfering with LDL retention to biglycan, but it has no direct effect on the macrophage uptake of native or modified lipoproteins. Thus, loss of ES in advanced atheromas enhances lipoprotein retention in SEM. Our data reveal a third protective role of this vascular basement membrane component during atherosclerosis.     

Retinal pigment epithelium response to oxidant injury in the pathogenesis of early age-related macular degeneration

Age-related macular degeneration (AMD) represents the leading cause of vision loss in the elderly. Accumulation of lipid- and protein-rich deposits under the retinal pigment epithelium (RPE) heralds the onset of early AMD, but the pathogenesis of subretinal deposit formation is poorly understood. Numerous hypothetical models of deposit formation have been proposed, including hypotheses for a genetic basis, choroidal hypoperfusion, abnormal barrier formation, and lysosomal failure. This review explore the RPE injury hypothesis, characterized by three distinct stages (1) Initial RPE oxidant injury, caused by any number of endogenous or exogenous oxidants, results in extrusion of cell membrane "blebs," together with decreased activity of matrix metalloproteinases (MMPs), promoting bleb accumulation under the RPE as basal laminar deposits (BLD). (2) RPE cells are subsequently stimulated to increase synthesis of MMPs and other molecules responsible for extracellular matrix turnover (i.e., producing decreased collagen), affecting both RPE basement membrane and Bruchs membrane (BrM). This process leads to progression of BLD into basal linear deposits (BLinD) and drusen by admixture of blebs into BrM, followed by the formation of new basement membrane under the RPE to trap these deposits within BrM. We postulate that various hormones and other plasma-derived molecules related to systemic health cofactors are implicated in this second stage. (3) Finally, macrophages are recruited to sites of RPE injury and deposit formation. The recruitment of nonactivated or scavenging macrophages may remove deposits without further injury, while the recruitment of activated or reparative macrophages, through the release of inflammatory mediators, growth factors, or other substances, may promote complications and progression to the late forms of the disease.     

Collagen XVIII/endostatin structure and functional role in angiogenesis

The angiogenesis inhibitor endostatin is a 20 kDA C-terminal fragment of collagen XVIII, a proteoglycan/collagen found in vessel walls and basement membranes. The endostatin fragment was originally identified in conditioned media from a murine endothelial tumor cell line. Endostatin inhibits endothelial cell migration in vitro and appears to be highly effective in murine in vivo studies. The molecular mechanisms behind the inhibition of angiogenesis have not yet been elucidated. Studies of the crystal structure of endostatin have shown a compact globular fold, with one face particularly rich in arginine residues acting as a heparin-binding epitope. It was initially suggested that zinc binding was essential for the antiangiogenic mechanism but later studies indicate that zinc has a structural rather than a functional role in endostatin. The generation of endostatin or endostatin-like collagen XVIII fragments is catalyzed by proteolytic enzymes, including cathepsin L and matrix metalloproteases, that cleave peptide bonds within the protease-sensitive hinge region of the C-terminal domain. The processing of collagen XVIII to endostatin may represent a local control mechanism for the regulation of angiogenesis.     

Collagen XVIII and corneal reinnervation following keratectomy

The significance of collagen XVIII in the regulation of corneal reinnervation remains largely unknown. We used whole-mount immunoconfocal microscopy to localize collagen XVIII to the nerve basement membrane of wild-type (WT) mouse corneas. Transmission electron microscopy showed corneal nerve disorganization in collagen XVIII knockout mice (col18a1(-/-)). Antibody 2H3-specific neurofilament colocalized with collagens XVIII and IV and laminin-2 in WT mouse corneas, but did not colocalize with collagen IV and laminin-2 in col18a1(-/-) mouse corneas. Following keratectomy, col18a1(-/-) mice displayed decreased corneal neurite extension compared to WT mice. Our data indicate that collagen XVIII may play an important role in corneal reinnervation after wounding.     

Endostatins derived from collagens XV and XVIII differ in structural and binding properties, tissue distribution and anti-angiogenic activity

Endostatin is a fragment of the C-terminal domain NC1 of collagen XVIII that inhibits angiogenesis and tumor growth. We report the characterization of a collagen XV endostatin analogue and its parent NC1 domain, obtained by recombinant expression in mammalian cells. Both NC1 domains contain a trimerization domain, a hinge region that is more sensitive to proteolysis in collagen XVIII and the endostatin domain. Unlike endostatin-XVIII, endostatin-XV does not bind zinc or heparin, which is explained by the crystal structure of endostatin-XV. The collagen XV and XVIII fragments inhibited chorioallantoic membrane angiogenesis induced by basic fibroblast growth factor (FGF-2) or vascular endothelial growth factor (VEGF), but there are striking differences depending on which cytokine is used and whether free endostatins or NC1 domains are applied. The collagen XV and XVIII fragments showed a similar binding repertoire for extracellular matrix proteins. Differences were found in the immunohistological localization in vessel walls and basement membrane zones. Together, these data indentify endostatin-XV as an angiogenesis inhibitor, which differs from endostatin-XVIII in several important functional details.     

Lack of collagen XVIII long isoforms affects kidney podocytes, whereas the short form is needed in the proximal tubular basement membrane

Collagen XVIII is characterized by three variant N termini, an interrupted collagenous domain, and a C-terminal antiangiogenic domain known as endostatin. We studied here the roles of this collagen type and its variant isoforms in the mouse kidney. Collagen XVIII appeared to be in a polarized orientation in the tubular basement membranes (BMs), the endostatin domain embedded in the BM, and the N terminus residing at the BM-fibrillar matrix interface. In the case of the glomerular BM (GBM), collagen XVIII was expressed in different isoforms depending on the side of the GBM. The orientation appeared polarized here, too, both the endothelial promoter 1-derived short variant of collagen XVIII and the epithelial promoter 2-derived longer variants having their C-terminal endostatin domains embedded in the BM and the N termini at the respective BM-cell interfaces. In addition to loosening of the proximal tubular BM structure, the Col18a1(-/-) mice showed effacement of the glomerular podocyte foot processes, and microindentation studies showed changes in the mechanical properties of the glomeruli, the Col18a1(-/-) glomeruli being ～30% softer than the wild-type. Analysis of promoter-specific knockouts (Col18a1(P1/P1) and Col18a1(P2/P2)) indicated that tubular BM loosening is due to a lack of the shortest isoform, whereas the glomerular podocyte effacement was due to a lack of the longer isoforms. We suggest that lack of collagen XVIII may also have disparate effects on kidney function in man, but considering the mild physiological findings in the mutant mice, such effects may manifest themselves only late in life or require other compounding molecular changes.     

Endostatin's heparan sulfate-binding site is essential for inhibition of angiogenesis and enhances in situ binding to capillary-like structures in bone explants

The functional role of endostatin's affinity for heparan sulfates was addressed using an ex vivo bone angiogenesis model. Capillary-like sprouts showed prominent expression of collagen XVIII/endostatin. Outgrowth of endothelial cells was not altered in the absence of collagen XVIII but inhibited by the addition of recombinant endostatin. Mutant non-heparan sulfate binding endostatin and the collagen XV endostatin homologue were ineffective. The ability of mutant endostatin to bind to capillary structures was reduced when compared to endostatin. Endostatin-XV completely failed to bind to endothelial cells. Our data indicate that endostatin's angiostatic function is heparan sulfate-dependent, and that in situ-binding of endostatin to endothelial cells is increased by heparan sulfates.     

The multiple functions of collagen XVIII in development and disease

Collagen XVIII is a heparan sulphate proteoglycan which is expressed ubiquitously in different basement membranes throughout the body. Its C-terminal fragment, endostatin, has been found to inhibit angiogenesis and tumor growth by restricting endothelial proliferation and migration and inducing apoptosis of endothelial cells. Collagen XVIII has three variants, of which the shortest one is found in most vascular and epithelial BM structures, whereas the longer variants are found especially in the liver. The longest or frizzled variant has a cysteine-rich domain in its N-terminus that has been shown to inhibit Wnt signaling in vitro. The presence of collagen XVIII homologues in organisms such as C. elegans, Xenopus laevis, zebrafish and chick suggests a fundamental role for this BM collagen. Mutations in the collagen XVIII gene lead to the Knobloch syndrome, which is characterized by high myopia, vitreoretinal degeneration with retinal detachment, macular abnormalities and occipital encephalocele. Mice lacking collagen XVIII also show several ocular abnormalities. This suggests that in physiological conditions collagen XVIII is mostly needed for the proper development of the eye. Moreover, it appears to be needed for the structural stability of basement membranes in several other organs, and increasing evidence shows its importance for other organs in non-physiological situations such as atherosclerosis, glomerulonephritis or other type of tissue damage. This review focuses on clarifying the roles of collagen XVIII and its variants and domains in various physiological and pathological conditions.     

Tumor suppression by collagen XV is independent of the restin domain

Non-fibrillar collagen XV is a chondroitin sulfate modified glycoprotein that is associated with the basement membrane zone in many tissues. Its precise functions remain to be fully elucidated though it clearly plays a critical role in the structural integrity of the extracellular matrix. Loss of collagen XV from the basement membrane zone precedes invasion of a number of tumor types and we previously showed that collagen XV functions as a dose-dependent suppressor of tumorigenicity in cervical carcinoma cells. The carboxyl terminus of another non-fibrillar collagen (XVIII) is cleaved to produce endostatin, which has anti-angiogenic effects and thus may act as a tumor suppressor in vivo. Since collagen XV has structural similarity with collagen XVIII, its C-terminal restin domain could confer tumor suppressive functions on the molecule, though our previous data did not support this. We now show that expression of collagen XV enhances the adhesion of cervical carcinoma cells to collagen I in vitro as does the N-terminus and collagenous regions of collagen XV, but not the restin domain. Destruction of a cysteine residue in the collagenous region that is critical for intermolecular interactions of collagen XV abolished the enhanced adhesion to collagen I. Finally, we demonstrate that unlike full length collagen XV, expression of the restin domain alone does not suppress tumorigenicity of cervical carcinoma cells in vivo; hence, this process is dependent on functions and interactions of other parts of the protein.     

Demonstration of microfibrils in Bruch's membrane of the eye

The cationic dyes ruthenium red and alcian blue were used to visualize a population of microfibrils in Bruch's membrane, a compound basement membrane located in the uveal tract of the eye between the retinal pigment epithelium and choriocapillaris. Microfibrils were tubular structures, 10-12 nm in diameter, that showed a characteristic beaded pattern. The majority of microfibrils appeared as a dense mantle around the layer of amorphous elastin. Microfibrils and collagen fibers were also present as a loosely organized meshwork in the collagenous zone of the membrane. Microfibrils were also seen along the basal surface of the retinal pigment epithelium where they appeared to insert into the substance of the basal lamina. Ruthenium red staining of microfibrils was not abolished by prior exposure of tissue to several kinds of degradative enzymes. The findings suggest that the elastic properties of Bruch's membrane may depend on both the elastin and microfibrillar components.     

Role of hypoxia and extracellular matrix-integrin binding in the modulation of angiogenic growth factors secretion by retinal pigmented epithelial cells.

The retinal pigmented epithelium (RPE) is a monolayer of polarized cells located between retinal photoreceptors and blood vessels of the choroid. The basal surface of RPE cells rests on Bruch's membrane, a complex extracellular matrix structure which becomes abnormal in several disease processes, including age-related macular degeneration (AMD). Ruptures or abnormalities in Bruch's membrane are frequently accompanied by choroidal neovascularization. Disturbed interaction of RPE cells with their extracellular matrix (ECM) could play a role in this process. The present study was undertaken to examine the complex interactions between hypoxia, integrin, and ECM in the regulation of RPE functions. Antibody blocking experiments demonstrated that RPE cell adhesion to vitronectin is mediated primarily through alphavbeta5 and adhesion to fibronectin occurs through alpha5beta1. RPE adhesion to immobilized laminin demonstrated highest level of non-RGD-mediated adhesion as compared to that with collagen IV or the RGD matrices such as vitronectin (alphavalpha5) , fibronectin (alpha5beta1), or thrombospondin (alpha5beta1 + alphavbeta5). Addition of soluble vitronectin, or fibrinogen to RPE cell cultures resulted in a small to moderate increase in VEGF and FGF2 in the media, while each of these growth factors was dramatically increased after addition of thrombospondin 1 (TSP1). In contrast, soluble fibronectin resulted in differential upregulation of VEGF but not FGF2. Similarly, immobilized TSP1 resulted in differential greater upregulation in VEGF but not FGF2 release from RPE as compared to other ECMs under either normoxic or hypoxic conditions. Additionally, hypoxia resulted in a time-dependent increase in VEGF, but not FGF2 release in the media. RPE cells grown on TSP1-coated plates showed increased VEGF and FGF2 in their media compared to cells grown on plates coated with type IV collagen, laminin, vitronectin, or fibronectin. The TSP1-induced increase in secretion of growth factors was partially blocked by anti-alpha5beta1, anti-alphavbeta3, and anti-alphavbeta5 antibodies indicating that it may be mediated in part by TSP1 binding to those integrins. These data suggest that alterations in oxygen levels (hypoxia/ischemia) and ECM of RPE cells, a prominent feature of AMD, can cause increased secretion of angiogenic growth factors that might contribute to the development of choroidal neovascularization. These data also suggest the potential modulatory role of VEGF release from RPE by ECM and alphavbeta5 and alpha5beta1 integrins.     

Conditional Ablation of the Choroideremia Gene Causes Age-Related Changes in Mouse Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) is a pigmented monolayer of cells lying between the photoreceptors and a layer of fenestrated capillaries, the choriocapillaris. Choroideremia (CHM) is an X-linked progressive degeneration of these three layers caused by the loss of function of Rab Escort protein-1 (REP1). REP1 is involved in the prenylation of Rab proteins, key regulators of membrane trafficking. To study the pathological consequences of chronic disruption of membrane traffic in the RPE we used a cell type-specific knock-out mouse model of the disease, where the Chm/Rep1 gene is deleted only in pigmented cells (Chm^Flox, Tyr-Cre+). Transmission electron microscopy (TEM) was used to quantitate the melanosome distribution in the RPE and immunofluorescent staining of rhodopsin was used to quantitate phagocytosed rod outer segments in retinal sections. The ultrastructure of the RPE and Bruch’s membrane at different ages was characterised by TEM to analyse age-related changes occurring as a result of defects in membrane traffic pathways. Chm/Rep1 gene knockout in RPE cells resulted in reduced numbers of melanosomes in the apical processes and delayed phagosome degradation. In addition, the RPE accumulated pathological changes at 5–6 months of age similar to those observed in 2-year old controls. These included the intracellular accumulation of lipofuscin-containing deposits, disorganised basal infoldings and the extracellular accumulation of basal laminar and basal linear deposits. The phenotype of the Chm^Flox, Tyr-Cre+ mice suggests that loss of the Chm/Rep1 gene causes premature accumulation of features of aging in the RPE. Furthermore, the striking similarities between the present observations and some of the phenotypes reported in age-related macular degeneration (AMD) suggest that membrane traffic defects may contribute to the pathogenesis of AMD.     

The NC1/endostatin domain of Caenorhabditis elegans type XVIII collagen affects cell migration and axon guidance

Type XVIII collagen is a homotrimeric basement membrane molecule of unknown function, whose COOH-terminal NC1 domain contains endostatin (ES), a potent antiangiogenic agent. The Caenorhabditis elegans collagen XVIII homologue, cle-1, encodes three developmentally regulated protein isoforms expressed predominantly in neurons. The CLE-1 protein is found in low amounts in all basement membranes but accumulates at high levels in the nervous system. Deletion of the cle-1 NC1 domain results in viable fertile animals that display multiple cell migration and axon guidance defects. Particular defects can be rescued by ectopic expression of the NC1 domain, which is shown to be capable of forming trimers. In contrast, expression of monomeric ES does not rescue but dominantly causes cell and axon migration defects that phenocopy the NC1 deletion, suggesting that ES inhibits the promigratory activity of the NC1 domain. These results indicate that the cle-1 NC1/ES domain regulates cell and axon migrations in C. elegans.     

Chronic cigarette smoke causes oxidative damage and apoptosis to retinal pigmented epithelial cells in mice

The purpose of this study was to determine whether mice exposed to chronic cigarette smoke develop features of early age-related macular degeneration (AMD). Two month old C57Bl6 mice were exposed to either filtered air or cigarette smoke in a smoking chamber for 5 h/day, 5 days/week for 6 months. Eyes were fixed in 2.5% glutaraldehyde/2% paraformaldehyde and examined for ultrastructural changes by transmission electron microscopy. The contralateral eye was fixed in 2% paraformaldehyde and examined for oxidative injury to the retinal pigmented epithelium (RPE) by 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-OHdG) immunolabeling and apoptosis by TUNEL labeling. Mice exposed to cigarette smoke had immunolabeling for 8-OHdG in 85+/-3.7% of RPE cells counted compared to 9.5+/-3.9% in controls (p<0.00001). Bruch membrane was thicker in mice exposed to smoke (1086+/-332 nm) than those raised in air (543+/-132 nm; p = 0.0069). The two most pronounced ultrastructural changes (severity grading scale from 0-3) seen were a loss of basal infoldings (mean difference in grade = 1.98; p<0.0001), and an increase in intracellular vacuoles (mean difference in grade = 1.7; p<0.0001). Ultrastructural changes to Bruch membrane in cigarette-smoke exposed mice were smaller in magnitude but consistently demonstrated significantly higher grade injury in cigarette-exposed mice, including basal laminar deposits (mean difference in grade = 0.54; p<0.0001), increased outer collagenous layer deposits (mean difference in grade = 0.59; p = 0.002), and increased basal laminar deposit continuity (mean difference in grade = 0.4; p<0.0001). TUNEL assay showed a higher percentage of apoptotic RPE from mice exposed to cigarette smoke (average 8.0+/-1.1%) than room air (average 0+/-0%; p = 0.043). Mice exposed to chronic cigarette smoke develop evidence of oxidative damage with ultrastructural degeneration to the RPE and Bruch membrane, and RPE cell apoptosis. This model could be useful for studying the mechanism of smoke induced changes during early AMD.