Adhesion Failures Determine the Pattern of Choroidal Neovascularization in the Eye: A Computer Simulation Study

Choroidal neovascularization (CNV) of the macular area of the retina is the major cause of severe vision loss in adults. In CNV, after choriocapillaries initially penetrate Bruch's membrane (BrM), invading vessels may regress or expand (CNV initiation). Next, during Early and Late CNV, the expanding vasculature usually spreads in one of three distinct patterns: in a layer between BrM and the retinal pigment epithelium (sub-RPE or Type 1 CNV), in a layer between the RPE and the photoreceptors (sub-retinal or Type 2 CNV) or in both loci simultaneously (combined pattern or Type 3 CNV). While most studies hypothesize that CNV primarily results from growth-factor effects or holes in BrM, our three-dimensional simulations of multi-cell model of the normal and pathological maculae recapitulate the three growth patterns, under the hypothesis that CNV results from combinations of impairment of: 1) RPE-RPE epithelial junctional adhesion, 2) Adhesion of the RPE basement membrane complex to BrM (RPE-BrM adhesion), and 3) Adhesion of the RPE to the photoreceptor outer segments (RPE-POS adhesion). Our key findings are that when an endothelial tip cell penetrates BrM: 1) RPE with normal epithelial junctions, basal attachment to BrM and apical attachment to POS resists CNV. 2) Small holes in BrM do not, by themselves, initiate CNV. 3) RPE with normal epithelial junctions and normal apical RPE-POS adhesion, but weak adhesion to BrM (e.g. due to lipid accumulation in BrM) results in Early sub-RPE CNV. 4) Normal adhesion of RBaM to BrM, but reduced apical RPE-POS or epithelial RPE-RPE adhesion (e.g. due to inflammation) results in Early sub-retinal CNV. 5) Simultaneous reduction in RPE-RPE epithelial binding and RPE-BrM adhesion results in either sub-RPE or sub-retinal CNV which often progresses to combined pattern CNV. These findings suggest that defects in adhesion dominate CNV initiation and progression.

CNV is usually limited to the sub-RPE space (i.e. between the RPE and BrM) and sub-retinal space (i.e. between the RPE and photoreceptors), though anastomosis of CNV capillaries with the inner-retinal vasculature occasionally occurs [7].

Oxygen Transport and Metabolism in the Retina
Two capillary beds supply oxygen and nutrients to most regions of the retina and remove waste products, the inner-retinal capillaries and the choriocapillaris (CC). The CC supplies more than 90 percent of the oxygen to the photoreceptors in dark-adapted conditions and almost 100 percent in light-adapted conditions [8]. The inner retinal capillaries supply oxygen to the inner layers of the retina, maintaining the oxygen partial pressure (PO 2 ) almost constant in both light and dark-adapted conditions [8,9]. The normal oxygen concentration at the OLM varies slightly, depending on the in-layer distance from the fovea.
The PIS is packed with mitochondria, so photoreceptors have the highest oxygen consumption rates of any cells in the human body. The metabolic activity and oxygen concentration of the photoreceptors depend on the intensity of light they receive. In dark-adapted conditions, photoreceptors consume oxygen at about twice their light-adapted rates.

Adhesion Properties of the RPE, POS and PIS
The lipid bilayer forming the cell membrane is flimsy and cannot, by itself, transmit large forces from cell to cell or from cell to extracellular matrix (ECM). Anchoring junctions solve this problem by forming strong membrane-spanning structures that tether inside the cell to the tension-bearing filaments of the cytoskeleton. RPE cells are apicobasally polarized. On the lateral surfaces of RPE cells, two bands of epithelial adhesion junctions connect to neighboring RPE cells ( Figure 2). On the apical-most lateral surfaces of RPE cells, a band of tight junctions (TJs) (zonula occludens, ZO, Mesh) seal adjacent RPE cells together, forming the outer blood-retina (oBRB) barrier and restricting transport of material (e.g. albumin) into and out of the retina [10][11][12]. On the lateral surfaces of RPE cells, basal to the TJs, adherens junctions (AJs) form another junctional band that goes all the way around each cell and mechanically connects the cytoskeleton of each RPE cell to the neighboring RPE cells, giving structural integrity to the RPE. RPE cells form AJs predominantly via N-cadherins [13,14] (RPE cells also produce a small amount of E-cadherin, the most common adhesion molecule in the AJs of most other epithelial tissues). In addition to these junctional bands, desmosome plaques (DPs) and gap junctions distributed on the lateral surfaces of the RPE cells connect neighboring RPE cells. Desmosomes are crucial for tissue integrity, resist calcium-depletion in developed tissue and help to resist shearing forces. When we refer to epithelial junctions without further qualification, we refer to the ensemble of junctional structures that participate in RPE-RPE adhesion, including TJs, AJs and DPs. The basal surfaces of RPE cells adhere to the very thin basal laminae of the RBaMs so strongly that the basal laminae behave like extensions of the cells' plasma membranes [15]. Intergrins mediate RPEbasal lamina adhesion. The RBaMs attach to the inner collagenous layer of BrM via microfibrils passing through both the elastic layer of BrM and the RBaMs [15,16]. When we refer to the RPE-BrM complex, we refer to the RPE-RBaM-BrM ensemble. Soft drusen reduce the adhesion between the RBaMs and the inner collagenous zone (Figure 1) of BrM and correlate with localized detachment of the RPE from BrM [6,[17][18][19]. Age-related modifications of BrM, especially soft drusen, also inhibit reattachment of transplanted RPE cells to BrM [20,21].
Photoreceptors pass spent photo-sensitive disks to the apical processes of RPE cells. This apical contact attaches photoreceptors to the RPE [22][23][24] more weakly than would RPE-RPE epithelial adhesion or attachment of RBaMs to BrM, so detachment of photoreceptors from the RPE (retinal detachment) due to impact is more likely than RPE tears (which break RPE-RPE epithelial junctions) or RPE detachment (which breaks RBaM-BrM attachment). Disruptions of RPE-POS contact affect not only the integrity of the oBRB, they also induce pathological cell growth and division in the RPE [25][26][27], disrupting the RPE epithelial structure and preventing successful therapeutic retinal reattachment (see Table 1).
Photoreceptors have limited or no motility and are held together in constant positions by multiple ECM components in the outer retina and OLM, ensuring consistant positional mapping of the visual field to the photoreceptors and the corresponding neurons in the visual cortex (somatotopic mapping). This somatotopic consistency is crucial to the development and maintenance of high-resolution visual perception.

Angiogenic and Antiangiogenic factors
Since laterally adjacent RPE cells form tight junctions, factors secreted by the photoreceptors on the apical side of the RPE do not pass through an intact RPE epithelial sheet to affect the choriocapillaris or CNV capillaries. The RPE secretes two VEGF-A isoforms from its basolateral surfaces to maintain the CC. VEGF-A 120 diffuses freely and does not bind to heparin-sulfate. More than 75% of RPE-derived VEGF is the VEGF-A 165 isoform [28] which has a weak affinity for heparin-sulfate, allowing it to diffuse across BrM while remaining resident long enough to bind to the VEGF receptors of the CC (otherwise the constant fluid flow from the vitreous humor to the CC and the high rate of CC blood flow would elute the VEGF before it bound to the EC's VEGF receptors). Mutant mice producing only VEGF-A 188 , which binds strongly to extracellular matrix and therefore has a short diffusion length, develop a normal CC but suffer CC atrophy and RPE and BrM abnormalities, leading to RPE loss and dramatic choroidal remodeling beginning at 7 months [29], suggesting that RPE-derived free-diffusing VEGF-A isoforms are necessary to maintain the choriocapillaris. These VEGF isoforms may also help support other retinal cell types. When we refer to VEGF-A without qualifications, we mean VEGF-A 165 , which appears to play the dominant role in CNV. In addition to RPE-derived VEGF-A, ECs, in general, produce multiple isoforms of VEGF-A, among which short-diffusing isoforms can serve as autocrine chemoattractants, playing a key role in capillary patterning (for a detailed discussion of capillary patterning mechanisms, see [30,31]). In many cases, ECs can only sense ECM-bound isoforms of VEGF-A when they are released from the ECM by matrix-degrading enzymes.
Since the retina is the most metabolically active tissue in the body, the density of capillaries in the CC is unusually high. The CC has small inter-capillary distances (~ 20 μm) [32,33] compare to typical inter-capillary distances (~ 100 μm to 200 μm). Since RPE-derived VEGF is essential for to maintain this dense population of ECs in the CC, we hypothesize that RPE-derived VEGF secretion must be substantial. The denser population of ECs consuming RPE-derived VEGF globally balances the higher secretion rate of RPE-derived VEGF. However, because of inhomogeneities in the CC-BrM-RPE complex [16], we hypothesize that RPE-derived VEGF does not diffuse uniformly, producing relatively high local concentrations of RPE-derived VEGF, sufficient to maintain a substantial population of activated ECs in the CC even in the healthy retina.
RPE cells also produce the antiangiogenic pigment-epithelium-derived factor (PEDF). At homeostasis, proangiogenic and antiangiogenic factors balance. In the aged human retina, PEDF has a spatial distribution similar to that of VEGF-A [34].
Numerous other diffusible pro and antiangiogenic factors [35,36] may help modulate capillary behavior, but we do not consider them in this paper (see also the Inflammation subsection below). While angiogenesis requires proangiogenic factors to dominate antiangiogenic factors, normal angiogenesis requires the factors to remain in rough balance. In pathological situations, when the levels of proangiogenic factors are too large relative to antiangiogenic factors, the resulting vessels do not mature and remain leaky and insufficient at oxygen and nutrient transport.

Angiogenesis and BrM Degradation
High levels of VEGF-A activate normally quiescent endothelial cells in blood vessels. Via a Delta/Notch contact-inhibition selection mechanism, small populations of these activated ECs become tip cells which lead angiogenic sprouts up gradients of VEGF-A [37,38]. A morphologically distinct population of activated ECs called stalk cells form the body of these angiogenic sprouts. While tip and stalk cells are distinct at any instant, they can dynamically exchange identities [39]. Macrophages and other immune cells can substitute for tip cells in their pathfinding role. Tip cells have very low rates of proliferation, while stalk cells proliferate at moderate rates.

Inflammation
Inflammation and immune cells play major roles in CNV. Macrophages in normal retina remove debris accumulated in BrM [55], helping to maintain normal retinal structure and function. However, chronic or excessive acute inflammation can promote CNV initiation and impair the integrity of the RPE, promoting CNV progression. Irregularities in regulation of the complement cascade and overactivity of immune cells may perturb RPE cells, causing them to form more basal deposits (both soft and hard drusen), which in turn induce a stronger immune response which can initiate angiogenesis [56,57]. Angiopoietin-2, a key proangiogenic inflammatory factor, activates quiescent ECs with a response modulated by local VEGF concentrations and increases ECs' directional motility. Angiopoietin-1, on the other hand, inhibits activation of ECs and helps newly-formed vessels mature [58]. Pro-inflammatory cytokines, e.g., tumor necrosis factor alpha (TNF-α) and interleukin-1β and -8, result in extensive breakdown of both the oBRB and inner blood-retinal barrier (iBRB) which separates inner-retinal capillaries from the outer retina [12,[59][60][61][62].
Inflammation may affect early and late-stage CNV by weakening RPE-RPE and RPE-POS adhesion. Inflammation also impairs the juxtacrine Delta/Notch inhibitory signaling which couples adjacent ECs and normally promotes ECs quiescence and inhibits tip-cell selection, increasing EC activation [63].