3Retinal biochemistry, physiology, and cell biology

INTRODUCTION

The vitreous, the vasculature of the retina, macular pigments, phototransduction, retinal pigment epithelium (RPE), the Bruch’s membrane (BM), and the extracellular matrix (ECM) all play an important role in the normal function of the retina, as well as in diseases. Understanding the pathophysiology allows us to target treatment. As ocular angiogenesis, immunity, and inflammation are covered elsewhere, those subjects will not be discussed in this chapter.

VITREOUS BIOCHEMISTRY

Vitreous is an important ocular structure in normal physiology and pathologic conditions of the posterior segment.1 It is a dilute meshwork of collagen fibrils interspersed with extensive arrays of hyaluronan molecules.

Hyaluronan is a major macromolecule of vitreous. It is a long, unbranched polymer of repeating disaccharide linked by glycosidic bonds. Hyaluronan is covalently linked to a protein core: the ensemble is called proteoglycan. Vitreous also contains collagen type II, a hybrid of types V/XI, and type IX collagen in a molar ratio of 75 : 10 : 15, respectively. Vitreous collagens are organized into fibrils, with type V/XI residing in the core, type II collagen surrounding the core, and type IX collagen on the surface of the fibril.

The collagen fibrils provide a scaffold-like structure by the hydrophilic hyaluronan. If collagen is removed, the remaining hyaluronan forms a viscous solution; if hyaluronan is removed, the gel shrinks but is not destroyed. The chondroitin sulfate chains of type IX collagen bridge between adjacent collagen fibrils in a ladder-like configuration spacing them apart. Such spacing is necessary for vitreous transparency: keeping vitreous collagen fibrils separated by at least one wavelength of incident light minimizes light scattering, allowing unhindered transmission of light to the retina.

VITREOUS DEGENERATION WITH AGING

During aging, substantial alterations take place in the vitreous body. The gel-to-liquid ratio is reduced with age. In the posterior vitreous, large pockets of liquid vitreous, recognized clinically as “lacunae,” are formed. By the age of 80–90 years, more than half the vitreous body is liquid.

Strong adhesion exists between the posterior vitreous cortex and the internal limiting membrane (ILM) of the retina, primarily at the vitreous base and at the posterior pole. Vitreoretinal adhesion appears to be strongest at the disc, fovea, and along retinal blood vessels. With age, there is weakening of vitreoretinal adhesion, most likely due to biochemical alterations in the ECM at the vitreoretinal interface.

These biochemical changes may play a role in the observed weakening of the vitreoretinal interface during aging. Identifying the molecular nature of these changes may provide opportunities for pharmacologic lysis of vitreoretinal adhesion.

PHYSIOLOGICAL AND PATHOLOGICAL CHANGES IN THE VITREORETINAL INTERFACE

Posterior vitreous detachment (PVD) results from weakening of the adhesion between the posterior vitreous cortex and the ILM, in conjunction with liquefaction within the vitreous body. Weakening of the posterior vitreous cortex/ILM adhesion at the posterior pole allows liquid vitreous to enter the retrocortical space via the prepapillary hole. Volume displacement from the central vitreous to the preretinal space causes the observed collapse of the vitreous body.

PVD may exert traction at the vitreoretinal adhesion, leading to hemorrhage, retinal tears, and detachment. Focal attachment in the foveal area can cause vitreomacular traction, with associated diffuse macular edema. Proliferative diabetic retinopathy can be greatly aggravated by anomalous PVD. Effects upon vitreous involve posterior vitreoschisis, where splitting of the posterior vitreous cortex and forward displacement of the vitreous body leave the outer layer of the split posterior vitreous cortex still attached to the retina. This can result in epiretinal membrane, and contribute to macular holes and tractional retinal detachment.

BLOOD–RETINAL BARRIER

As in the central nervous system, the retina is protected against the invasion of circulating macromolecules by cell specialized barriers. Endothelial and epithelial cells display tight junctions forming zonulae occludens which block the transit of such molecules. Disruptions of these barriers are due to alterations in those lining cells affecting the physiologic properties of permeability and transport or the intercellular relationship represented by the tight junctions.

In the eye, there are two systems of barriers: the blood retina–vitreous barrier (BRB) in the posterior segment and the blood–aqueous barrier in the anterior segment. In the ciliary body, nonpigmented cells have zonulae occludens which restrict macromolecule traffic through the fenestrated blood vessels and the iris vessels are continuous with endothelial cells adjoined by tight junctions. Similarly, in the retina, the pigmented retinal epithelium tight junctions block the transit of macromolecules that cross the fenestrated choriocapillaries. Retinal vessels are continuous with endothelial cells displaying tight junctions that perform the barrier function. In the retinal periphery there is a region in the ora serrata margin, where the blood–aqueous barrier joins the BRB.

The maintenance of the retinal milieu microenvironment is essential to preserve visual function. The access of blood-borne molecules in eye tissues and spaces is controlled by cellular transport mechanisms. The BRB is required to maintain retinal homeostasis. Imbalance in the supply and demand of oxygen and nutrients across the BRB leads to pathological angiogenesis. A series of ocular pathologies are accompanied by breakdown of barriers affecting the transport system. Increased vascular permeability underlies the pathology of retinopathy of prematurity, diabetic retinopathy, and age-related macular degeneration. From the pharmacological point of view, the barrier concept has implications on the effectiveness of drug delivery to the retina.2

Biology Cell and Physiology, Biochemistry,•tinal3 rchapteR

Figure 3.1  Electron micrograph of tight junction between two nonpigmented cells (*) and pigmented and nonpigmented cells at the ora serrata margin (arrow) (chicken eye).

TIGHT JUNCTIONS

Tight junctions play an important role in controlling flux across both endothelial and epithelial cells and maintaining the desirable retinal hydration, ionic and solutes balance. They provide both a barrier function restricting the paracellular flux of molecules and a fence function maintaining apical and basolateral membrane composition within a cell. Apparently these junctions may not be necessary to establish cell polarity

The structure of tight junctions is revealed by electron microscopy of ultrathin tissue sections as adjacent cell areas presenting discontinuous fusing points of the outer-membrane leaflets. The intercellular space remains obliterated (Figure 3.1). Freeze-fracture replicas corresponding to the same region reveal the tight junction as a network of anastomosing strands of particles, as seen in the P face (protoplasmic aspect of the intramembrane domain) or complementary grooves in the E face (exoplasmic view) (Figure 3.2).

The molecular components of tight junctions reveal a highly complex assembly of proteins. More than 40 proteins had been found in association with tight junctions, not only functioning as structural components of the paracellular barrier but participating in the regulation of gene expression and cell proliferation. The most studied proteins, particularly related to the BRB, are the ZO-1 (zonula occludens), claudins, occludins, and junction adhesion molecules (JAM). They were extensively demonstrated in epithelia and endothelia, including immunohistochemical location. Claudin and occludin are transmembrane proteins cross-linked with ZO-1, which acts as a scaffold, linking the integral proteins of the tight junction to the actin cytoskeleton, and JAM is suggested to participate as the ultimate sealing molecule of the intercellular space. Other molecules are involved in cell signaling through their association with kinases and Ras or in vesicular trafficking. These tight junction components have been shown to affect several signaling and transcriptional pathways, including self-regulation, changing the

Figure 3.2  Freeze-fracture replica of epithelial tight junction (chicken ciliary body).

expression of structural tight junction proteins which are associated with several disease conditions.3

Changes in tight junction proteins have been observed in a wide variety of retinal diseases associated with loss of the BRB. These changes include not only alteration in the content of junctional proteins but also their redistribution and phosphorylation.

BLOOD–RETINA BARRIER DISRUPTION

Many factors contribute to BRB disruption, including inflammatory mediators, neovascularization, vascular endothelial growth factor, and others. Early breakdown of the BRB can be detected in diabetic retinopathy by fluorescein leakage into the vitreous with fluorometric methods.

The passive diffusion permeability of a barrier depends on the physicochemical properties of a substance; its molecular weight and lipophilicity determine bioavailability for a tissue. Tightness of a barrier is evaluated by measuring the transepithelial or transendothelial resistance of cultured cells. In vitro model systems represent useful tools for predicting the permeability for certain drugs and molecules.4

To reach their targets, therapeutic agents need to cross epithelial and endothelial linings. This is possible by the transcellular and paracellular pathways. The former is employed by lipophilic drugs and by molecules selectively transported by vesicles, channels, pumps, and carriers present in the plasma membrane. Hydrophilic molecules cannot cross biological membranes. Therefore their transport is significantly enhanced if they move through the paracellular pathway. Transit through this route is regulated by tight junctions. To enhance drug delivery across epithelial and endothelial barriers it is necessary to open the paracellular route in a reversible manner. Some patented inventions were designed to alter the tight junctions with peptides homologous to the external loops of integral proteins, antisense oligonucleotides and