Chapter 20 Structure, Function, and Pathology of Bruch’s Membrane
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Introduction, history, embryology
Bruch’s membrane is a thin (2–4 µm), acellular, five-layered extracellular matrix located between the retina and choroid.1,2 It extends anteriorly to the ora serrata, interrupted only by the optic nerve. Tissue resembling Bruch’s membrane is visible anterior to the ora serrata extending forward to the pigmented epithelium of the ciliary body. Bruch’s membrane lies between the metabolically active retinal pigment epithelium (RPE) and a capillary bed (choriocapillaris) and thus serves two major functions as the substratum of the RPE and a vessel wall. It has major clinical significance because of its involvement in age-related macular degeneration (AMD) and other chorioretinal diseases.
Early history
Carl Ludwig Wilhelm Bruch first isolated the “lamina vitrea” that we now know as Bruch’s membrane, and described it in his 1844 doctoral thesis,3,4 where he also first described the tapetum found in many mammals. By light microscopy, Bruch’s membrane appeared transparent with little internal structure. Later studies by Smirnow5 divided this membrane into an outer elastic layer (first described by Sattler in 1877) and an inner cuticular layer, separated by a dense plexus of very fine elastic fibers.6,7
Development of Bruch’s membrane
The bipartite character of Bruch’s membrane arises from the embryology of its tissue. When the optic cup invaginates and folds, its inner layer forms the neural retina, and its outer layer forms the RPE. The RPE lies in contact with mesenchyme. At this apposition, Bruch’s membrane forms by 6–7 weeks’ gestation. Thus, its inner layer is composed of ectodermal tissue and its outer layer is composed of mesodermal tissue. At the border of two layers, the elastic layer forms last, becoming histologically visible by 11–12 weeks.8–10
The collagen that fills the extracellular space and the later-appearing elastin appear to be made by invading fibroblasts and the filopodia of endothelial cells lining the adjacent choriocapillaris. The two basal laminas are produced by their associated cell layers.11 In addition to collagen IV subunits specific to specialized basal lamina, RPE expresses genes for structural collagen III and angiostatic collagen XVIII in a developmentally regulated manner linked to photoreceptor maturation.12
By week 13, fenestrations are apparent in the endothelium facing Bruch’s membrane,10 indicating that, at this stage, transport across this tissue may be functional. Choroidal endothelial cells originate from paraocular mesenchyme. Development of the choroidal vasculature, and Bruch’s as part of it, depends on differentiated RPE and its production of inductive signals, including basic fibroblast growth factor and vascular endothelial growth factor (VEGF).13
Structure of Bruch’s membrane in the young adult eye
Hogan’s five-layer nomenclature for Bruch’s membrane14 is commonly used. Gass proposed a three-layer system that did not include the cellular basal laminas as part of Bruch’s proper.15 These layers are shown in Fig. 20.1 and their constituents are given in Table 20.1.
Table 20.1 Structural and molecular components of Bruch’s membrane
| Layer (common abbreviation) | Component; age change | References |
|---|---|---|
| Basal laminar deposit (BlamD) | + Fibronectin, laminin, IV α4–5, VI, endostatin, EFEMP1 | 164, 167, 206–209 |
| RPE basal lamina (RPE-BL) | IV α1–5, V, laminins 1, 5, 10, and 11, nidogen-1, heparan sulfate, chondroitin sulfate | 18, 19, 21, 66, 210, 211 |
| Lipid wall/basal linear deposit (BlinD) | + Lipoproteins | 38, 39, 212 |
| Inner collagenous layer (ICL) | I, III, V, fibronectin, chondroitin sulfate, dermatan sulfate, lipoproteins ↑, apoE, heme, clusterin, vitronectin | 34, 35, 38, 39, 50, 66, 146, 152, 210, 213–215 |
| Elastic layer (EL) | Elastin ↑, calcium phosphate ↑ | 14, 66–68, 210, 216 |
| Outer collagenous layer (OCL) | I, III, V, fibulin-5, fibronectin, chondroitin sulfate, dermatan sulfate, lipoproteins ↑, apoE, clusterin | 21, 39, 50, 152, 210, 215, 217 |
| ChC-basal lamina | IV α1, 2, V, VI, laminin, heparan sulfate, chondroitin sulfate, endostatin | 18, 208, 210, 211, 218 |
| Bruch’s, throughout or layer not specified | I ↑, collagen solubility ↓, perlecan, MMP-2 ↑, MMP-9 ↑, TIMP-2; TIMP-3 ↑, pentosidine ↑, CML ↑, GA-AGE ↑, RGR-d, apoB, oxidized apoB-100, 7-KCh, LHP, HHE ↑, DHP-lys ↑, C3d ↑, C5b-9 ↑, pentraxin-3 ↑, thrombospondin-1, zinc | 62, 66, 138, 139, 147, 218–230 |
Table shows definitely localized components. Most determinations were made in macula. Studies showing histochemical/immunohistochemical verification of biochemistry and ultrastructural validation of structures identified by light microscopy techniques were given greater weight. Localizations were assigned to specific layers if immunogold-electron microscopy or high-magnification confocal microscopy images were available. Roman numerals denote collagens. Components are ordered within each layer: structural components, lipoproteins, extracellular matrix and its regulation, modified lipids and proteins, complement/immunity, cellular response/activity, metals. Known changes with advancing age are bold with an arrow indicating direction of change. New additions with age are shown with a plus (+). Plain text means no change or not tested.
CML, carboxymethyl-lysine226; 7-KCh, 7 keto-cholesterol229; GA-AGE, glycolaldehyde-derived advanced glycation end products221; HHE, 4-hydroxyhexenal66,218; DHP-lys, dihydropyridine lysine.66
RPE basal lamina (RPE-BL)
This ∼0.15-µm-thick layer is a meshwork of fine fibers like other basal laminas in the body.16,17 The RPE-BL resembles that of the choriocapillaris endothelium but does not contain collagen VI. The RPE-BL contains collagen IV α3–5,18 like that of kidney glomerulus, another organ with specialized filtration and transport functions. The RPE synthesizes specific laminins that preferentially adhere Bruch’s membrane to the RPE through interaction with integrins.19
Inner collagenous layer (ICL)
The ICL is ∼1.4 µm thick and contains 70-nm-diameter fibers of collagens I, III, and V in a multilayered criss-cross, parallel to the plane of Bruch’s membrane.1 The collagen grid is associated with interacting molecules, particularly the negatively charged proteoglycans chondroitin sulfate and dermatan sulfate.20,21
Elastic layer (EL)
The EL consists of stacked layers of linear elastin fibers, crisscrossing to form a 0.8-µm-thick sheet with interfibrillary spaces of ∼1 µm. This sheet extends from the edge of the optic nerve to the ciliary body pars plana.1 In addition to elastin fibers, the EL contains collagen VI, fibronectin, and other proteins, and collagen fibers from the ICL and outer collagenous layer (OCL) can cross the EL. Some EL elastin fibers are said to cross the tissue space between the choriocapillaris and join bundles of choroidal elastic tissue.22 The EL confers biomechanical properties, vascular compliance, and antiangiogenic barrier functions. It is more discontinuous in the macula, perhaps explaining why choroidal neovascularization (CNV) is more prominent there.23 This concept is supported by the extensive laser-induced neovascularization in mice deficient in lysyl oxidase-like 1, an enzyme required for elastin polymerization.24
Outer collagenous layer
The OCL contains many of the same molecular components as the ICL, and the collagen fibrils running parallel to the choriocapillaris additionally form prominent bundles. This layer, unlike the ICL, has periodic outward extensions between individual choriocapillary lumens called intercapillary pillars, where thickness cannot be determined due to the lack of a boundary. Between pillars, OCL thickness can range from 1 to 5 µm.25
Choriocapillaris basal lamina (ChC-BL)
This 0.07-µm-thick layer is discontinuous with respect to Bruch’s membrane due to the interruptions of the intercapillary pillars of the choroid. It is continuous with respect to the complex network of spaces defined by the choriocapillary lumens because the basal lamina envelops the complete circumference of the endothelium. A remarkable structural feature of the adjacent choriocapillary endothelium is fenestrations that are permeable to macromolecules (Fig. 20.2).26 This basal lamina may inhibit endothelial cell migration into Bruch’s membrane, as do basal laminas associated with retinal capillaries.27
Bruch’s membrane in an aged eye
Aging is the largest risk factor for developing AMD,28 and Bruch’s membrane undergoes significant age-related changes. Identification of factors predisposing to disease progression is a priority. This task has been challenged by difficulty imposed by the thinness of the tissue, and the closely integrated functions of RPE, Bruch’s, and choriocapillaris. Current opinion holds that RPE and Bruch’s membrane age in concert, and normal Bruch’s membrane aging transforms insidiously into AMD pathology.1,16,17,29 This section covers aging, to inform the following section on function.
Lipid accumulation: Bruch’s membrane lipoproteins
Early electron microscopists described aged Bruch’s membrane as being filled with debris, including amorphous electron-dense material, membrane fragments, vesicles, and calcification.1,25 Debris deposition in ICL and OCL begins in the second decade in the macula and is delayed in equatorial regions, a regional lag also reported for individual components.30 Identifying this material has been a fruitful approach to understanding antecedents of disease.
Most prominent among the changes in Bruch’s membrane is a profound accumulation of lipids. Clinical observations on fluid-filled RPE detachments in older adults led to Bird and Marshall’s hypothesis that a lipophilic barrier in Bruch’s blocked a normal, outwardly directed fluid efflux from the RPE31 (as opposed to leakage from CNV). This hypothesis motivated a seminal histochemical study by Pauleikhoff et al.32 that demonstrated oil red O-binding material (esterified cholesterol (EC), triglyceride (TG), fatty acid) localized exclusively to Bruch’s membrane, unlike other stains. This lipid was absent <30 years, variably present at 31–60 years, and abundant at ≥61 years.33,34 A specific fluorescent marker, filipin, which binds the 3β-hydroxy group of sterols to reveal unesterified (free) cholesterol (UC) or EC depending on tissue pretreatment,35 indicated that EC is a prominent component of the oil red O-binding deposition.35 Macular EC rose linearly from near zero at age 22 years to reach high and variable levels in aged donors. EC was detectable in periphery at ∼1/7 macular levels and increased significantly with age. Hot-stage polarizing microscopy34 similarly demonstrated prominent age-related increases in EC in Bruch’s membrane, manifest as liquid crystals (“Maltese crosses”) when examined through a polarizing filter. Few birefringent crystals signifying the neutral lipid TG were found.
Histochemical, ultrastructural, biochemical, gene expression, and cell biological evidence now indicate that the EC-rich material accumulating with age in Bruch’s membrane is a lipoprotein-containing apolipoprotein B, assembled by the RPE.36 This process, ongoing throughout life yet first revealed by aging, has implications for the formation of AMD-specific lesions, intraocular transport, RPE physiology, nutrition of outer retina, and maintenance of photoreceptor health. In 1926, Verhoeff and Sisson speculated that lipid deposition might precede Bruch’s membrane basophilia and fragmentation, common in older eyes due to “lime salts [calcification] in the elastic layer.”37
Ultrastructural studies described in Bruch’s membrane of older eyes36 numerous small (<100 nm), round, electron-lucent vesicular profiles, implying aqueous interiors. Lipid-preserving preparation techniques together with extraction studies show that these so-called vesicles are actually solid, lipid-containing particles, now considered lipoproteins (Fig. 20.3B). These methods include postfixation in osmium paraphenylenediamine (OTAP)35 and, most strikingly, quick-freeze/deep-etch (QFDE), a freeze fracture method with an etching step to remove frozen water.38–40 Particles vary in size from 60 to 100 nm but could be as large as 300 nm, occasionally appearing to coalesce (Fig. 20.3).
Lipoprotein particles are first seen among fibrils of the elastic layer in early adulthood, extending inward ultimately to fill most of the open space of the ICL by the seventh decade of life.40 Most fatefully, a new layer, the lipid wall,38 then forms with solid particles stacked 3–4 deep occupying nearly 100% of a space between RPE basal lamina and OCL of many older eyes. The lipid wall displaces ICL collagen fibrils that anchor the RPE basal lamina (Fig. 20.3). It is considered a precursor to basal linear deposits, a specific lesion of AMD (see below).
Lipoprotein composition can provide clues to sources of its components.41 When isolated (Fig. 20.4A), Bruch’s membrane lipoproteins are found to be EC-enriched (EC/total cholesterol = 0.56; EC/TG = 4–11; Fig. 20.4B). For comparison, hepatic very-low-density lipoprotein (VLDL), of similar diameter, is TG-rich. An early report of TG-enriched Bruch’s membrane neutral lipid42 was not replicated. Abundant EC points to the only mechanism by which neutral lipids are released directly from cells, an apoB-containing lipoprotein, like hepatic VLDL or intestinal chylomicrons. Significantly, RPE expresses the apoB gene and protein, along with microsomal triglyceride transfer protein (MTP), required for apoB lipidation and secretion. Lack of functional MTP is the basis of abetalipoproteinemia, a rare inherited disorder that includes a pigmentary retinopathy.43,44 The combination of apoB and MTP within native RPE marks these cells as constitutive lipoprotein secretors.45 Secretion of full-length apoB has been demonstrated in rat-derived and human-derived RPE cell lines.46,47 Consistent with an RPE origin, particles first appear in the elastic layer of Bruch’s membrane and fill in towards the RPE.39
Fig. 20.4 Bruch’s membrane lipoprotein composition. (A) Lipoprotein particles isolated from Bruch’s membrane are large and spherical; negative stain.153 (Source: Li C-M, Chung BH, Presley JB, et al. Lipoprotein-like particles and cholesteryl esters in human Bruch’s membrane: initial characterization. Invest Ophthalmol Vis Sci 2005;46:2576–86). Bar = 50 nm. (B) Bruch’s membrane lipoprotein composition inferred from direct assay,50,153 (Sources: Wang L, Li C-M, Rudolf M, et al. Lipoprotein particles of intra-ocular origin in human Bruch membrane: an unusual lipid profile. Invest Ophthalmol Vis Sci 2009;50:870–7) (Li C-M, Chung BH, Presley JB, et al. Lipoprotein-like particles and cholesteryl esters in human Bruch’s membrane: initial characterization. Invest Ophthalmol Vis Sci 2005;46:2576–86), druse composition, and retinal pigment epithelium gene expression.139,154 (Sources: Malek G, Li C-M, Guidry C, et al. Apolipoprotein B in cholesterol-containing drusen and basal deposits in eyes with age-related maculopathy. Am J Pathoi 2003;162:413–25) and (TG, Li C-M, Clark ME, Chimento MF, et al. Apolipoprotein localization in isolated drusen and retinal apolipoprotein gene expression. Invest Ophthalmol Vis Sci 2006;47:3119–28) TG, triglyceride; EC, esterified cholesterol; UC, unesterified cholesterol; PL, phospholipid; Apo, apolipoproteins. The question mark signifies that not all apolipoproteins are known.
Indirect evidence that Bruch’s membrane lipoproteins are of intraocular origin also emerges from the epidemiologic literature. If the EC deposition in Bruch’s membrane and AMD-associated lesions were a manifestation of systemic perifibrous lipid and atherosclerosis, then a strong positive correlation between disease status and plasma lipoprotein levels, like that documented for coronary artery disease,48 might be expected but has not emerged.49
Identifying the upstream sources of Bruch’s membrane lipoprotein constituents is essential for understanding the biological purpose of this pathway and the prospects for eventual clinical exploitation. Studies using isolated lipoproteins from Bruch’s membrane50 and Bruch’s membrane choroid EC51 report a high mole percentage of linoleate (>40%) and low docosahexaenoate (<1%) for all lipid classes.52 This composition strongly points away from photoreceptor outer segments (35% docosahexaenoate in membrane phospholipids) as an upstream source, as long postulated,53,54 and towards plasma lipoproteins (45–55% linoleate in all lipid classes). These data have been interpreted to signify that plasma lipoproteins are major contributors upstream to an apoB lipoprotein of RPE origin. In contrast, the sources of UC in Bruch’s lipoproteins are not yet known and could be outer segments, plasma lipoproteins, endogenous synthesis, or a combination.
Lipoproteins may thus be assembled from several sources, including outer segments, remnant components from the photoreceptor nutrient supply system, and endogenous synthesis. According to this model,52 plasma lipoproteins serve as vehicles for delivery of lipophilic nutrients (carotenoids,55 vitamin E, and cholesterol56) to photoreceptors by RPE, which has functional receptors for low-density lipoprotein (LDL) and high-density lipoprotein.57,58 Nutrients are stripped from these lipoproteins by the RPE for delivery to the photoreceptors, and the remnants are repackaged for secretion into Bruch’s membrane as part of apoB-containing lipoproteins, where they begin to accumulate during age and become toxically modified to instigate inflammation in AMD.
Other aging changes
Bruch’s membrane thickens throughout adulthood (20–100 years) two- to threefold under the macula and becoming more variable between individuals at older ages.25,59,60 Equatorial Bruch’s membrane changes little while Bruch’s membrane near the ora serrata increases twofold during this time.25 In the macula, the OCL thickens more prominently than the ICL.61 A large ultrastructural study of 121 human donor eyes demonstrated that the macular EL is 3–6 times thinner than peripheral EL23 at all ages.
Unbalanced regulation of extracellular matrix molecules and their modulator matrix are thought to result in Bruch’s membrane thickening. Increased histochemical reactivity for glycoconjugates, glycosaminoglycans (GAGs), collagen, and elastin is seen in the macula relative to equator and near the ora serrata.25 Collagen solubility declines with age.62 Matrix metalloproteinases MMP-2 and MMP-3 increase with age, as does a potent tissue inhibitor of metalloproteinases, TIMP-3. TIMP-3 immunoreactivity reaches adult levels at 30 years of age near vasculature in lung, kidney, and in Bruch’s membrane, signifying the end of developmental organogenesis.63 The reduction or absence of TIMP-3 is proangiogenic, as this protein not only regulates metalloproteinases during the normal turnover of Bruch’s membrane matrix components, but it also binds to VEGF.64,65
The EL thickens with age but decreases relative to overall thickening of Bruch’s membrane.23 Thus elastin referenced to other Bruch’s constituents, as detected by Raman spectroscopy, decreases with age.66 Similar arguments can be made for collagen III and IV. A prominent age change,67 noted early,37 is calcification and ensuing brittleness. This process involves fine deposition of electron-dense particulate matter,14 confirmed as calcium phosphate68 on individual elastin fibrils.
Long-lived proteins like collagens are modified in vivo by nonenzymatic Maillard and free radical reactions to yield advanced glycation end products (AGEs) and the formation of lipid-derived reactive carbonyl species like 4-hydroxyhexenal and linoleate hydroperoxide, collectively called age-related lipoperoxidation end products (ALEs). Accumulation of AGEs and ALEs, characteristic of diabetes and atherosclerosis, also occurs in aging Bruch’s membrane (Table 20.1). Finally, other components more prominent in aged eyes include complement components C3d, C5b-9, and pentraxin-3, a homolog of the acute-phase respondent C-reactive protein. Thus, at the molecular level, aging Bruch’s membrane contains evidence of many biological activities, including remodeling, oxidative damage, and inflammation, in addition to lipoprotein accumulation.
Function of Bruch’s membrane
As a vessel wall of the choroid, Bruch’s membrane’s primary function is structural, like other vessel walls. Its architecture is similar to vascular intima, with a subendothelial extracellular matrix and elastic layer corresponding to the internal elastic lamina. The abluminal surface of Bruch’s differs from other vessel walls in that it abuts a basal lamina, that of the RPE. The luminal surface faces a fenestrated vascular endothelium and basal lamina, making Bruch’s membrane structurally analogous to the renal glomerulus and providing a basis for commonality between retinal and kidney disease.69–71 The importance of fluid and macromolecular transport across the renal glomerulus is well known.72 Transport is a second important function of Bruch’s membrane.
Structural role of Bruch’s membrane
Bruch’s membrane encircles more than half the eye and stretches with the corneoscleral envelope as intraocular pressure (IOP) increases. It therefore withstands this stretch and returns to its original shape when IOP decreases. This tissue also stretches to accommodate changes in choroidal blood volume. Finally, the choroid (and Bruch’s membrane with it) may act as a spring that pulls the lens during accommodation.73,74 For these reasons, then, Bruch’s membrane requires elasticity. Marshall and Hussain’s group estimated the modulus of elasticity in Bruch’s membrane choroid preparations to be 7–19 MPa.75 These values are similar to those of sclera (although sclera is much thicker and thus can support more load), consistent with the notion that Bruch’s membrane contributes to load bearing. After early adulthood, the modulus of elasticity of human Bruch’s membrane–choroid complex increases (P < 0.001) at a rate of ∼1% per year. Bruch’s membrane stiffness in AMD eyes does not differ from age-matched normals.76
Transport role of Bruch’s membrane
Hydraulic conductivity of Bruch’s membrane
GAGs are concentrated in the interphotoreceptor matrix77,78 and corneal stroma.79 In both locations, these highly charged macromolecules maintain geometric fidelity essential for vision (periodic collagen spacing for corneal transparency, orderly photoreceptor spacing for visual sampling78,80,81). GAGs generate significant swelling pressure (up to 50 mmHg in cornea).82,83 Without a mechanism to maintain tissue deturgescence, GAGs would imbibe fluid, swell, destroy tissue geometry, and interfere with visual function. Corneal endothelium forestalls swelling by continuously pumping fluid out. This function is accomplished for retina by the RPE, and its failure can lead to retinal detachment. A driving force adequate to overcome the collective flow resistance of RPE, Bruch’s membrane, and choriocapillaris endothelium is provided by a gradient in fluid pressure and oncotic pressure (the osmotic pressure generated by plasma proteins). This balance is embodied by Starling’s law that characterizes the relationship between fluid flux (q = flow per unit area; positive when flow is out of the blood vessel) across a capillary vessel wall and the forces driving this flow:
(equation 1)We can estimate the magnitude of ΔP – σΔΠ using measured value of q and Lp. The fluid pumping rate by human RPE has been measured as q = 11 µL/h/cm2, similar to that in other animals (Table 20.2). The hydraulic conductivity of macular Bruch’s membrane/choroid of healthy young humans ranges from 20 to 100 × 10−10 m/s/Pa.84 Then, using q = 11 µL/h/cm2 and Lp = 50 × 10−10 m/s/Pa, we can calculate that the magnitude of (ΔP − σΔΠ) necessary to drive this flow through Bruch’s membrane is roughly 0.05 mmHg. (This does not include the flow resistance of choriocapillaris endothelium, which is not measured when Lp of a Bruch’s membrane/choroidal preparation is determined. For this highly fenestrated endothelium, Lp can be estimated as roughly 25 × 10−10 m/s/Pa,85 which does not affect our conclusions below.)
Table 20.2 Retinal pigment epithelium (RPE) fluid pumping rates
| Species | Fluid transport rate across RPE (µL/h/cm2) | References |
|---|---|---|
| Frog | 4.8–7.6 | 231, 232 |
| Rabbit | 12 ± 4 | 233, 234 |
| Canine | 6.4 | 235 |
| Primate* | 14 ± 3 | 236, 237 |
| Human | 11 | 238 |
RPE pumping rates were measured by readsorption of subretinal fluid or by direct measurement in culture.
* Cantrill and Pederson236 measured a much higher transport rate than that reported here, but used fluorescein as a tracer which likely does not track fluid flow due to its high diffusion coefficient.
σ can be roughly estimated by assuming that the fluid in the suprachoroidal space is in equilibrium with blood in the choroid. Using measurements of fluid pressure and of the plasma protein concentration (to estimate oncotic pressure) inside and outside the choriocapillaris,86–88 equation (1) can be used to find σ ≈ 0.5.
Allowing that Πcc = 27 mmHg,86 Pcc = IOP + 8 mmHg,87 and assuming that ΠRPE = 0 mmHg (fluid pumped by the RPE is assumed protein-free) and PRPE = IOP (assuming no pressure is generated by the RPE above that necessary for crossing Bruch’s), we find that ΔP – σΔΠ is approximately –5.5 mmHg pulling fluid into the choroid. Thus, in normal young adults, oncotic pressure within the choroid is more than sufficient to adsorb all the fluid pumped by the RPE. We can also use equation (1) to calculate that the lowest value of Lp that still adsorbs fluid pumped by the RPE without generating an elevated pressure at the RPE basal surface is Lp > 0.4 × 10−10 m/s/Pa.
Experiments using laser ablation of Bruch’s membrane/choroid explants allowed Starita et al.89 to conclude that the ICL was responsible for most of the flow resistance in Bruch’s membrane. Attempts to localize further the flow resistance using morphometric methods are complicated by first, stereological issues90 and second, the loss of ultrastructural fidelity from connective tissue conventionally processed for electron microscopy.38 Failure to appreciate the former difficulty can lead to unphysiologically low estimates for tissue porosity and thereby hydraulic conductivity.1
Age-related changes in hydraulic conductivity and disease
Fisher was the first to measure Lp of human Bruch’s membrane,91 finding that Lp decreased significantly with age. However, his values for Lp of Bruch’s membrane and other tissues are much lower than those found by later investigators.85,92,93 Marshall and Hussain’s group carefully revisited these measurements using Bruch’s membrane/choroid with RPE removed, a preparation that was simpler to create. They showed using laser ablation that the flow resistance of these preparations was entirely due to Bruch’s membrane.89 They also found that flow rate increased linearly with driving pressure, indicating that Lp of Bruch’s membrane is relatively insensitive to pressure up to 25 mmHg.
They reported that Lp of macular Bruch’s membrane exhibited a dramatic, exponential decline throughout life (Fig. 20.5), dropping from 130 × 10−10 m/s/Pa in young children to 0.52 × 10−10 m/s/Pa in old age. Lp of macular Bruch’s membrane dropped more rapidly with age than did that of the periphery, consistent with an accelerated process occurring in the macula.1,84,94,95 Note that the lowest value measured for Lp of Bruch’s membrane in normal eyes is similar to the calculated minimum value of Lp that allows complete fluid resorption (0.4 × 10−10 m/s/Pa; see above). Marshall and Hussain’s group reached similar conclusions regarding this process.94
Determining Lp of Bruch’s membrane in isolated macular samples of AMD eyes is difficult due to scar formation and other changes.94 However, Marshall and Hussein’s group showed that, in the periphery, Lp of Bruch’s membrane is decreased in AMD eyes as compared to age-matched normal eyes (Fig. 20.5).94 Assuming that similar processes occur in macular Bruch’s membrane due to the profound lipid accumulation in this region, then in diseased eyes, the RPE must generate higher pressures at its basal surface to drive fluid into the choriocapillaris, with further pathological consequences.31 Above an unknown threshold level, higher pressure will cause the RPE-BL to separate from the ICL, leading to RPE detachment and fluid accumulation, as seen in 12–20% of AMD patients.94
What causes the dramatic age-related decrease in Lp of Bruch’s membrane? It is natural to suspect the age-related lipid accumulation. In fact, McCarty et al.96 showed that lipid particles trapped in an extracellular matrix can generate very significant flow resistance, more than would be expected based simply on their size and number. However, Marshall and Hussain’s group observed that most of the marked change in Lp occurred before age 40 (Fig. 20.6A) while the increase in Bruch’s membrane lipid content occurred largely after this age. They thus concluded that other age-related changes must be responsible for changes in Lp.1,84
Fig. 20.6 (A) Hydraulic conductivity of human macular Bruch’s membrane/choroidal preparations as a function of age, as compared to lipid accumulation in human macular Bruch’s membrane; lines as exponential fits to the data. (B) Hydraulic resistivity of human macular Bruch’s membrane/choroidal preparations as a function of age,1 as compared to esterified cholesterol accumulation in human macular Bruch’s membrane35; lines are exponential fits to the data (the fits nearly overlie one another).
(Modified with permission from Marshall J, Hussain AA, Starita C, et al, editors. The retinal pigment epithelium: function and disease. New York: Oxford University Press; 1998. pp. 669–92.)
A different conclusion can be reached from examining age-effects on flow resistivity the inverse of Lp. Resistivity increases from a low of roughly R = 108 Pa/m/s for young individuals to R = 1010 Pa/m/s for aged persons. Thus, when hydraulic conductivity Lp drops from roughly 100 × 10−10 m/s/Pa to 25 × 10−10 m/s/Pa between birth and 40 years of age, 75% of its total possible decrease, resistivity R increases from 1 × 108 Pa/m/s to 4 × 108 Pa/m/s, only 4% of the ultimate increase. Simply put, hydraulic conductivity drops more rapidly with age at young ages because its value is high to start with. Fig. 20.6B plots resistivity and histochemically detected EC against age for Bruch’s membrane.97 The agreement between the trends and the fits to the data is striking. This is strong evidence that the increasing lipid content and progressively hydrophobic character of Bruch’s membrane are responsible for impairing fluid transfer with age, as postulated.31 The strong correlation between flow resistivity of Bruch’s membrane and lipid content was likewise found by Marshall and Hussain’s group.1,84,95 Laser ablation studies localizing flow resistance to the ICL89 further support this conclusion, because lipids accumulate prominently in the ICL with aging.39 Further, more laser pulses were required to abolish flow resistance in the oldest eyes, consistent with presence of a lipid wall, requiring prior removal.
Thus, it appears that decreased Lp and increased resistivity of Bruch’s membrane with aging are closely related to the age-related accumulation of lipids, primarily EC. Lipids accumulate more rapidly in the macular Bruch’s membrane than in the periphery.35,98 Thus, Lp of the macula decreases more rapidly with age than it does in the periphery.
Permeability of Bruch’s membrane to solute transport
Along with bulk fluid flow, there is significant transport of individual molecular species across Bruch’s membrane, including dissolved gases, nutrients, cytokines, and waste products driven by passive diffusion. Flow crossing Bruch’s membrane is too slow to influence this process. This can be seen through calculation of the Peclet number, the relative magnitude of convection of a species due to bulk flow to that of diffusion99:
(equation 2)where V is the velocity of the flow, L is the transport path length, and D0 the free diffusion coefficient of the species being transported. (The free diffusion coefficient in saline is used rather than its value in tissue, since the species carried by flow is constrained to the same extent by the tissue as is its diffusion). Using the RPE pumping rate (Table 20.2) for V, Bruch’s membrane thickness (average of 3 µm59) for L, and a range of diffusion coefficients of species crossing Bruch’s membrane (2 × 10−7 cm2/s for LDL to 2 × 10−5 cm2/s for oxygen99,100), we find that the Peclet number ranges in value from 5 × 10−5 to 5 × 10−3. Thus, convection is negligible in transporting species across Bruch’s membrane under physiological conditions.
(equation 3)The transport of amino acids,101 serum proteins,102 drugs,103 and LDL104 across Bruch’s membrane has been examined. There are technical challenges to these experiments. First, as indicated in equation (3), diffusional flux depends on the length of the tissue. Since the diffusion coefficient of the transported species is likely different in Bruch’s membrane than in the choroid in a combined preparation, but the path lengths of both tissue components are usually not determined, it is difficult to use the measured values to determine absolute values of permeability. Instead the more easily measured flux rate (j: see equation 3) is usually presented. Second, since larger macromolecules used in diffusion studies are hindered in their passage into the tissue, an oncotic pressure could develop, generating a fluid flow in the opposite direction of transport and thus complicating the results. Nonetheless, useful comparative results can be generated.
The transport rate across human Bruch’s membrane declines linearly with age for all molecules measured. Amino acids exhibited permeabilities of 0.6 × 10−4 cm/s (phenylalanine) to 1.2 × 10−4 cm/s (glycine) for young Bruch’s membrane and exhibited a modest decline (twofold or less) with aging.101 Serum proteins decrease more markedly, dropping from 3.5 × 10−6 cm/s in the first decade to 0.2 × 10−6 cm/s in the ninth decade, a >10-fold decrease.102 In particular, proteins larger than 100 kDa have significantly decreased flux through Bruch’s membrane of older individuals. Macular Bruch’s membrane showed a steeper decrease with age than did the periphery.105 Permeability was reduced in eyes with AMD relative to age-matched normal eyes.105
Decreased permeability of Bruch’s membrane to transport is likely due to a decrease in diffusion coefficients, especially for the larger species affected by interaction with extracellular matrix and lipoproteins. As indicated in equation (3), increased path length due to age-related thickening of Bruch’s membrane59 could also have a significant effect.
An original proposal of a molecular weight exclusion limit to Bruch’s membrane macromolecule transport of 66–200 kDa101,102 has been questioned by more recent work suggesting that, if such a limits exists, it is much higher.105 Because of the importance of lipoproteins in transporting lipophilic nutrients to the RPE for ultimate use by the photoreceptors, and also because lipoproteins accumulate with age in Bruch’s membrane, Cankova et al.104 specifically examined the reflection coefficient of bovine Bruch’s membrane to plasma LDL. They measured a reflection coefficient of 0.58 (compared to a reflection coefficient of arterial endothelium to LDL of 0.998 and arterial intima to LDL of 0.827106). Thus, while LDL did not pass freely through Bruch’s membrane, it could nonetheless pass. Hussain et al.105 also concluded that particles as large as LDL could cross Bruch’s membrane. Accordingly, RPE cells have been shown to internalize plasma LDL from the choroid.56,107,108
These considerations are relevant not only to understanding mass transfer between the choriocapillaris and the RPE, but also for transscleral drug delivery strategies, including antiangiogenic agents for treating AMD and steroids for treating diabetic retinopathy.109,110 Lipophilic solutes are significantly hindered in their transport by Bruch’s membrane/choroid,103 while hydrophilic moieties are blocked by the RPE.111
Summary and implications
Bruch’s membrane’s physiological roles are structural and facilitating transport. Transport across Bruch’s membrane is increasingly hindered with age, due at least partly to the marked age-related accumulation of EC-rich lipoproteins in this tissue, impeding pumping of fluid from RPE.94 A ≥90% decrease in transport of some species from the choroid102,105 may include lipophilic essentials delivered by lipoproteins. This decline in transport capability is thought to have functional consequences for photoreceptors.112 A well-characterized change occurring through the lifespan of individuals with healthy maculas is slowed dark adaptation,113 attributed to impaired translocation of retinoids across the RPE–Bruch’s interface. This slowing, worse in AMD patients,114,115 can be partly ameliorated by short-term administration of high-dose vitamin A,116 presumably overcoming the translocation deficit via mass action.
Pathology of Bruch’s membrane
AMD lesions
In aging and AMD, characteristic extracellular lesions accumulate in tissue compartments anterior to the ICL. Known as drusen and basal deposits,29,117 these lipid-containing aggregations ultimately impact RPE and photoreceptor health by impairing transport, causing inflammation, and predisposing to CNV (Fig. 20.7). Basal linear deposit (BlinD) forms consequent to lipoprotein accumulation in Bruch’s membrane and formation of the lipid wall, likely involving oxidation of individual lipid classes and local inflammation. Drusen could form by similar mechanisms, plus lipoprotein aggregation and other undefined processes that cause the distinctive dome shape of these lesions. Basal laminar deposit (BlamD) forms in parallel with lipid deposition in Bruch’s and may indicate RPE stressed by it. We begin by discussing drusen, due to their importance in AMD.
Drusen
In a fundus view, drusen are yellow-white deposits 30–300 µm in diameter posterior to the RPE. By optical coherence tomography, they appear as variably hyporeflective spaces in the same location.118 Histologically, drusen are focal, domed lesions between the RPE basal lamina and the ICL, i.e., in the same sub-RPE tissue compartment as the lipid wall and BlinD. Found in most older adults,67,119 drusen are more numerous in peripheral retina than in macula.120–122 Drusen are typically classified as “hard” and “soft” by the appearance of their borders. Soft drusen confer high risk of advanced disease123–126 and, importantly, are found only in the macula.122 Other rare druse types exist and are less well characterized.127
In separate 1854 publications, Donders (a Dutch ophthalmologist) and Wedl (an Austrian pathologist) described “colloid bodies” (Colloidkugeln) or “hyaline deposits” on the inner surface of the choroid in older or diseased human eyes3,128,129 (translated by Busk). Both authors interpreted the droplets that filled these deposits as “fat globules.” The term “drusen” originated with Müller in 1856, from the German word for the geological term geode (not to be confused with Drüse, meaning gland).130 The name drusen was adopted by English writers early in the 20th century,131 yet “colloid body” was used by Holloway and Verhoeff into the 1920s.132 The basis of the fatty content emerged slowly. Lauber133,134 noted that deposits between the lamina vitrea and the RPE were sudanophilic in 1924. Wolter and Falls135 stated that “hyaline bodies [drusen] … stain reddish with … oil red O” in 1962.
Extant theories for druse formation, extending back to their discovery,130 fit into two general categories: transformation of the overlying RPE and deposition of materials on to Bruch’s membrane. The latter is now accepted.129,135 The RPE has been implicated as a source of many druse components, via budding of membrane-bound packets of cytoplasm or secretion. The contribution of plasma-derived components, in contrast, has not been well characterized. The existence of druse subregions additionally suggests remodeling in the extracellular compartment, such as cellular invasion and enzymatic activity23,136–138 and uplifting of the lipid wall.35,139
Most prominent among druse constituents are lipids, an observation made by their earliest discoverers. All drusen contain EC and UC, in addition to phosphatidylcholine, other phospholipids, and ceramides.34,35,137,139–142 Extractable lipids account for ≥40% of hard druse volume143 and likely more for macular soft drusen.139 This includes large EC-rich lakes in soft drusen (Fig. 20.8), reminiscent of atherosclerotic plaques.144 Only half of macular drusen take up hydrophilic fluorescein in angiography,145 possibly reflecting differing proportions of polar and neutral lipids in individual lesions.141 Discrete nonlipid components in some drusen include amyloid assemblies and granules of lipofuscin or melanin, indicating cellular origin (Table 20.3, online). Other constituents present in all drusen include vitronectin, TIMP-3, complement factor H, complement components C3 and C8, crystallins, and zinc.23,136,138,146–150
Apolipoprotein immunoreactivity appears in drusen with high frequency (100%, apoE; >80% apoB; 60%, A–I).139,151–154 Plasma-abundant apoC-III is present in fewer drusen than plasma-sparse apoC-I, indicating a specific retention mechanism for plasma-derived apolipoproteins or an intraocular source. Importantly, hard drusen contain many solid, Folch-extractable electron-dense particles of the same diameter as the lipoproteins that accumulate with age in Bruch’s membrane. These observations together with the appearance of membranous debris in soft drusen (below) make an RPE-secreted apoB-containing lipoprotein particle an efficient mechanism to place multiple lipids and apolipoproteins within lesion compartments.
The principal lipid-containing component of soft drusen and BlinD was called “membranous debris” by the Sarks.123,155,156 These lesions are richer in histochemically detectable UC than surrounding cellular membranes.141,142 By transmission electron microscopy following osmium tetroxide postfixation, membranous debris appears as variably sized, contiguous coils of uncoated membranes consisting of uni- or multilamellar electron-dense lines, denser than cellular membranes, surrounding an electron-lucent center (Fig. 20.1). Since conventional ultrastructural preparation methods can remove lipids, the building blocks of membranous debris are more plausibly the UC-rich exteriors of lipoproteins (native and fused) whose neutral lipid interiors are not well preserved in postmortem tissue.39,153,157 Rather than vesicles, then, membranous debris is likely aggregated or fused particles that could collectively account for the abundant EC in sub-RPE deposits. EC abundance and ultrastructural evidence for solid, nonvesicular particles suggest that the major lipid-containing component of AMD-specific lesions can be called “lipoprotein-derived debris” rather than membranous debris.
Basal linear deposit
BlinD is a thin (0.4–2 µm) layer located in the same sub-RPE compartment as soft drusen. BlinD is not visible clinically except as associated with other pathology. By OTAP and QFDE, BlinD is rich in solid lipoprotein particles and lipid pools (Fig. 20.9A, C). BlinD and soft drusen are considered alternate forms (layer and lump) of the same entity158 and may interchange over time. Soft drusen are oily, difficult to isolate individually, and are biomechanically more fragile than hard drusen,122 properties applicable to BlinD by inference. Both lesions could thus be permissive to invading capillaries of type I CNV.159,160 ApoE and apoB are present in BlinD and its precursor, the lipid wall.139,151,152 Transitional morphologies between lipid wall and BlinD have been reported.161
Basal laminar deposit
BlamD forms small pockets between the RPE and the RPE-BL in many older normal eyes or a continuous layer as thick as 15 µm in AMD eyes142,156,162 (Fig. 20.7). Some authors consider a continuous layer of BlamD a histological definition of AMD.163 Ultrastructurally, BlamD resembles basement membrane material (Fig. 20.9B), containing laminin, fibronectin, type IV and type VI collagen.164–167 The latter is a distinctive banded material with 120 nm periodicity, called wide- or long-spacing collagen, which also appears in other ocular locations like epiretinal membranes. Thick BlamD, associated with advanced AMD risk,156 contains histochemically detectable lipid, including UC and EC141,142 and is a classically described site for membranous debris (Fig. 20.1C). By lipid-preserving methods, solid particles are seen in BlamD (Fig. 20.9A, B). Especially enriched in basal mounds156 (Fig. 20.7), lipoprotein-derived debris in BlamD may be considered as retained in transit from the RPE to BlinD and/or drusen.139,141,142 Morphologically heterogeneous BlamD also contains vitronectin, MMP-7, TIMP-3, C3, and C5b-9,162 EC, and UC.142 Evoked in numerous mouse models of aging, stress, and genetic manipulation, BlamD is a reliable marker of RPE stress.168
Subretinal drusenoid debris
Hypotheses of druse formation must eventually also account for subretinal drusenoid debris (SDD). Located adjacent to RPE in the subretinal space, SDD was first described in AMD eyes by the Sarks.155 Ultrastructurally similar to soft drusen, these deposits are enriched in UC, apoE, vitronectin, and complement factor H, and, like drusen, they lack markers for photoreceptors, Müller cells, and RPE apical processes.142,169 Clinically this material is called reticular drusen in a fundus view170 and subretinal drusenoid debris in a cross-sectional view.171 Conferring lower risk for advanced AMD than conventional drusen,172 SDD appear in up to 60% of geographic atrophy eyes,172,173 appearing as focal deposits near the fovea and part of large sheets elsewhere in the macula.174 This coherent morphology suggests a specific formative process, possibly involving microglia resident in that compartment.175,176
Summary
Levels of significance ascribed to molecules sequestered in drusen (Table 20.3, online), and by inference, BlinD, include toxicity to the overlying RPE, stigmata of formative processes (extrusion of cellular materials, secretion, extracellular enzymatic processing, cellular activity), and markers of a diffusely distributed disease process affecting RPE and Bruch’s membrane. Additional significance can be ascribed to these lesions as physical objects that increase path length between choriocapillaries and retina and provide a biomechanically unstable cleavage plane between RPE BL and ICL.
Response-to-retention hypothesis of AMD
The parallels between the pathology of arterial intima of large arteries and that of Bruch’s membrane are striking. Both diseases feature cholesterol-rich lesions in subendothelial compartments within the systemic circulation, involving many of the same molecules and biological processes at multiple steps, as long anticipated.177,178 According to the response-to-retention theory of atherosclerosis, plasma lipoproteins cross the vascular endothelium of large arteries, and bind to extracellular matrix. By itself, this process is not pathological. However, lipoprotein components become modified via oxidative and nonoxidative processes, and launch numerous downstream deleterious events, including inflammation, macrophage recruitment, and neovascularization, leading to disease.179,180 Parallel with apoB lipoprotein-instigated disease in arterial intima, an intraocular response to retention involving the RPE and Bruch’s membrane in aging and AMD would begin with age-related accumulation of lipoproteins of local origin. Oxidation, perhaps driven by reactive oxygen species from adjacent RPE mitochondria, would then initiate a pathological process resembling that in the vascular system with inflammation-driven downstream events including complement activation and structurally unstable lesions.36
Neovascular AMD
CNV, the major sight-threatening complication of AMD, involves angiogenesis along vertical and horizontal vectors: vertically across Bruch’s membrane, and laterally external to the RPE (type 1 CNV181), laterally within the subretinal space (type 2 CNV), or further anteriorly into the retina (type 3 CNV).181–183 Of 40+ conditions involving CNV, AMD is the most prevalent, followed by ocular histoplasmosis,181 and including angioid streaks (see below). CNV is a multifactorial nonspecific wound-healing response to various specific stimuli, involving VEGF stimulation of choriocapillaris endothelium, compromise to Bruch’s membrane, and participation of macrophages.181 Impaired transport across Bruch’s membrane in AMD increasingly isolates the RPE from its metabolic source in the choriocapillaries and enhances the challenge in waste product disposal. VEGF released by RPE as a stress signal initiates an angiogenic response by the endothelium. However, Bruch’s membrane compromise is essential for CNV to proceed, as evidenced by intrachoroidal neovascularization without CNV in a mouse overexpressing VEGF in the setting of an intact Bruch’s membrane.184
Bruch’s membrane in a state of compromise can be breached easily by new vessels in AMD. It is notable that the EL is thinner and more interrupted in eyes with neovascular AMD.23 The length of gaps in the EL is greater in eyes with early AMD and any CNV.23 In paired donor eyes with and without CNV secondary to AMD, progressed eyes are distinguished by calcification and breaks in Bruch’s membrane.185 In contrast, calcification in a small number of geographic atrophy eyes is unremarkable.186
BlinD furthers this process by presenting a horizontal cleavage plane for vessel formation to exploit. The lipid-rich composition, relative lack of structural elements like collagen fibrils, lesion biomechanical instability,122 and proinflammatory, proangiogenic compounds like 7-ketocholesterol and linoleate hydroperoxide160,181,187 likely promote vessel growth in this plane.160
Angioid streaks (ABCC6, MTP genes)
Angioid streaks are ruptures in Bruch’s membrane associated with multiple disorders, caused by excess calcification of the elastic layer188 and often accompanied by CNV. They are prominent ocular manifestation of pseudoxanthoma elasticum (PXE), a systemic connective tissue disorder. PXE patients harbor mutations of a hepatically expressed lipid transporter ABCC6.189 Clinical presentation includes, in addition to streaks and CNV, peau d’orange (flat, yellow, drusen-like lesions), optic nerve head drusen, outer retinal tubulations, subretinal fluid, and pigmentary changes.190 PXE clinical manifestations are believed to be related to ectopic mineralization of nonhepatic tissues, suggesting a defect in the transport of antimineralization agents.191
Angioid streaks are associated with abetalipoproteinemia,192–195 an extremely rare disorder with low plasma apoB-containing lipoproteins, acanthocytosis of erythrocytes, neuropathy, and pigmentary retinopathy. It is historically attributed to lack of lipophilic vitamins delivered by plasma LDL.44 The RPE is now known to express the abetalipoproteinemia gene (MTP),46 which cotranslationally lipidates apoB (see above). How MTP deficiency leads to angioid streaks is unknown. The finding, however, highlights that lack of apoB lipoproteins has negative consequences for Bruch’s membrane health, just as an excess of retained apoB lipoproteins has negative consequences via lesion formation and impaired transport (see above). Good chorioretinal function thus requires an optimal balance between these extremes.
Thick basal laminar deposits (TIMP-3, CTRP5, EFEMP1 genes)
Three autosomal dominant inherited disorders with adult onset – Sorsby fundus dystrophy, late-onset retinal degeneration (LORD) and malattia leventinese-Doyne honeycomb retinal dystrophy (ML-DH) – share phenotypic similarities with AMD and provide mechanistic support for many aspects of Bruch’s membrane physiology and pathophysiology, discussed above. All three conditions result from mutations in genes encoding extracellular matrix proteins or their regulators (Sorsby, TIMP3196; LORD – CTRP5197; and ML-DH, EFEMP1198). All three can progress to CNV to varying degrees (Sorsby > LORD > ML-DH). All three have visual dysfunction, especially rods, attributed to a nutritional night blindness that is responsive to short-term administration of high-dose vitamin A in Sorsby and LORD.199–201 Sorsby and LORD are notable for thick BlamD and areas of RPE atrophy202 and may involve macula and periphery, while ML-DH is notable for radially distributed drusen and peripapillary deposits. In Sorsby eyes mutant TIMP-3 localizes to BlamD. In ML-DH, EFEMP1 localizes to BlamD and not to the pathognomonic drusen themselves, suggesting an important role of BlamD in druse formation.
BlamD in Sorsby and LORD, like that in AMD, is notably rich in oil red O-binding lipid.203–205 The significance of these findings was unclear until a model of a Bruch’s membrane lipoprotein was articulated. In LORD eyes,205 deposits contain EC, UC, and apoB, and lipid-preserving ultrastructural methods revealed solid electron-dense particles tracking in intersecting networks across the BlamD. In hindsight, these may represent native lipoproteins in transit from RPE to the choriocapillaris rather than depositions/aggregations of plasma LDL, as originally speculated. Lipid particle disposition within these thick deposits has been replicated in a mouse model expressing the R345W EFEMP1 mutation.168
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