Ординатура / Офтальмология / Английские материалы / The Retina and its Disorders_Besharse, Bok_2011

Structure and Function

Tissue Level

The distinctive structure of the outer blood–retinal barrier leads to unique functions. Unlike other epithelia and endothelia, the RPE separates two solid tissues – the choroid and the neural retina. Early in embryogenesis, the apical surface of the RPE borders a fluid-filled lumen, the lumen of the optic vesicle. As development proceeds, this lumen is reduced to a potential space known as the subretinal space (Figure 2). In this space, the microvilli of the RPE’s apical pole interdigitate with the outer segments of the photoreceptor cells. The intimate contact of the RPE and photoreceptors allows retinoids of the visual cycle to readily shuttle back and forth across the subretinal space, and allows disk membranes shed by the photoreceptors to be phagocytosed

by the RPE. The ionic composition of the subretinal space is carefully regulated to support the functions of the photoreceptors and the RPE. To this end, the RPE absorbs water. Water continuously enters the retina from the inner vascular bed and vitreous and is transported by the RPE into the choroid for removal by the choroidal circulation. Failure of this process results in retinal edema and even retinal detachment. Contrast this with another region of the blood–brain barrier, the epithelium of the choroid plexus. The epithelium of the choroid plexus is also derived from the neuroepithelium, but in this case, the lumen of the neural tube expands to form the ventricular system. Rather than absorb fluid, the choroid plexus secretes copious volumes of cerebral spinal fluid. To understand the functional differences between the RPE and the epithelium of the choroid plexus, we need to look more closely at the structure of the barrier.

Retina

Subretinal space

RPE

Bruch’s membrane

Lateral membranes enlarged

kisses

Connector to:

-Neural tube

-3rd ventricle (with optic nerve)

RPE

Retina

Subretinal space

Cellular Level

The RPE is a monolayer of cells that are joined by a complex of junctions known as the apical junctional complex. The apical junctional complex encircles each cell to bind the monolayer together much like the plastic rings that hold together a six-pack of canned beverages. The complex consists of three junctions (tight, adherens, and gap) whose functions are intertwined. Adherens junctions bind neighboring cells together. Tight junctions form a partially occluding seal that semiselectively retards diffusion through the paracellular spaces of the monolayer (Figure 1). Both junctions regulate proliferation, cell size, and the polarized distribution of plasma membrane proteins. The junctions of the complex work in concert, but we focus on how tight junctions contribute to barrier function.

Tight junctions form a barrier between the apical and basolateral poles of the cell. They work in conjunction with intracellular trafficking pathways to create and maintain an apicobasal polarity that is essential for the blood–retinal barrier to function. Unlike most epithelia, the Na,K-ATPase (ATP, adenosine triphosphate) is enriched in the apical membrane rather than localized to the basolateral membrane. Although this initial discovery suggested that the RPE is an upside-down epithelium, it is now known that only a few RPE proteins have an atypical distribution. What is crucial for RPE function is the distribution of membrane channels and transporters. The RPE and the epithelium of the choroid plexus both have an apical Na,K-ATPase that provides the energy for vectorial transport. It is the distribution of the various ion channels and transporters that determines whether the RPE absorbs water or the epithelium of the choroid plexus secretes it. Briefly, the polarized distribution of transporters in the RPE results in the active transport of chloride from the apical to basal side of the cell. As a result, the apical side of the monolayer has a positive charge relative to the basal side. Sodium and potassium are transported down this electrical gradient to balance the chloride transport. The osmotic gradient that results pulls water in the apical to basal direction. The polarized distribution of the various channels and transporters differs in the epithelium of the choroid plexus to support the opposite, basal to apical, transport of water. There are general housekeeping mechanisms that recognize the targeting signals encoded in a protein’s structure to deliver it to the correct membrane. In some cases, different tissuespecific isoforms encode different targeting signals; in others, tissue-specific variations in the targeting machinery give each epithelium its unique character.

The vectorial transport mechanism outlined above would have little effect, if transepithelial ion gradients were dissipated by the paracellular pathway. Early microscopists believed that a zonular band of junctions occluded the paracellular space by completely encircling each cell. Their

name for this junction, zonula occuldens or tight junction, is misleading because the junction is selectively leaky. The degree of leakiness, and selectivity for certain ions, not only varies among epithelia, but is essential for epithelial function as well. The permeability and semiselectivity of the junctions are matched to transcellular transport mechanism. For example, RPE tight junctions need to be leakier to sodium than to chloride, because RPE pumps chloride across the cell and needs a leak for cations to passively follow the chloride flux. The inadequate capacity for sodium and potassium to cross the cell is ameliorated by the sodiumselective leak through the tight junction.

A compelling example of how transcellular transport is matched to tight junction selectivity is provided by the kidney. The diuretic hormone, aldosterone, changes the flux of water by acting simultaneously on a membrane sodium channel and a tight junction protein that regulates sodium selectivity. In the retina, the volume and composition of the subretinal space vary with the day/night cycle. It remains to be investigated how the properties of tight junctions and membrane transporters are coordinated to manage the subretinal space during this cycle.

Molecular Level

Proteins of the apical junctional complex fall into transmembrane, adaptor, and signaling/regulatory categories. Adaptors link the transmembrane proteins to signaling proteins and a cortical band of actin filaments. Besides known members of the tight junction (Figure 3), proteomics suggest that over 912 proteins are associated with the tight junction. The adaptor proteins express multiple copies of PDZ domains. This protein-binding domain of approximately 100 amino acids takes its name from the three proteins that defined this class of protein-binding domain: postsynaptic density protein 95 (PSD-95), disks large, and zonula occludins 1 (ZO-1). PDZs are the largest family of protein-binding domains and form the basis of many protein complexes. Each adaptor protein expresses

Apical membranes

Transmembrane proteins

24 claudins, 3 JAMs, occludin, CAR, CRB3

Adaptor proteins

ZO-1, -2, -3; MAGI-1, -3; MUPP1,

Par 3,6; AF-6, Pals 1, PATJ

Figure 3 Composition of the tight junctions.

multiple homologs of PDZ domains that have distinct, but sometimes overlapping binding specificities. Together with other protein-binding motifs, for example, SRC homology 3 domain, guanylate kinase, bi-tryptophan domain that binds proline-rich peptides, Dilute domain, and Phox and Bem1P domain, the known adaptor proteins have the capacity to bind the large number of regulatory proteins that proteomics suggests. A junction of such complexity would be inconsistent with the original view of the tight junction as a static barrier.

The tight junction is a highly dynamic structure with the potential to rapidly respond to environmental stimuli. Photobleaching studies demonstrate that ZO-1 and occludin, a regulator of permeability, rapidly associate and dissociate from the junction. Occludin has a very short half-life and its degradation is regulated by endocytic

and ubiquitin pathways. Therefore, the cell exerts a fine control over the properties of the tight junction that can rapidly respond to changes in the environment. This flexibility extends to the claudin family of transmembrane proteins. Membrane proteins typically have a half-life on the order of days, but the claudins that have been studied have a half-life of 4–12 h. Claudins form the anastomosing network of strands that are observed by freeze-fracture electron microscopy (Figure 4) and schematized in Figure 1. There are 24 or more claudins. Each epithelium expresses a subset of claudins. The subset of claudins, expressed and localized to the tight junction, determines the selectivity and permeability of the junction. In the kidney example given above, aldosterone decreases the expression of claudin 4 to increase the sodium leak through the tight junctions.

Although the basic structure of the outer blood–retinal barrier is conserved among species, there are speciesspecific variations in the composition of the subretinal space, the properties of transmembrane transport, and the composition of the tight junctions. In human RPE, the principal claudins appear to be claudins 3, 10, and 19. Claudin 19 has been linked to kidney disease and visual impairment. By contrast, chick RPE expresses primarily claudins 1 and 20 with lesser amounts of claudins 2, 4L2, 5, and 12. Claudins 1 and 3 are fairly ubiquitous with most epithelia expressing one or the other. Claudin 2 increases sodium permeability in some contexts. However, the study of how claudins affect permeability is in its infancy particularly in regard to the effects of cellular context on function.

Regulation of RPE Tight Junctions

Clues from Embryonic Maturation

What is a mature, differentiated RPE monolayer? Which markers and how many should we use to render this judgment? Might enhancing the expression of some markers in a culture experiment lessen the expression of others? If cellular pathways form an integrated web, would overor underexpression of a protein have deleterious effects? A default path for human embryonic stem cells appears to be an RPE-like cell, but those cells lack some RPE proteins and express non-RPE proteins. The RPE is the first retinal cell to form during development, but despite its undeniable RPE character, the early RPE cell is only partially differentiated. It will undergo many transformations, as the neural retina and choroid differentiate on either side of it. The RPE is very plastic. Depending upon pathology or culture conditions, one can observe many partially differentiated states, or even transform RPE to other phenotypes. This may be the reason why RPE transplants fail when retinal degeneration is advanced. Dedifferentiation would be a normal response of healthy RPE to this abnormal environment. In support of this hypothesis, RPE transplants were most successful when the RPE and neural retina were cotransplanted. In some ways, culture is like a disease state where a degenerate neural retina and choroid no longer send RPE the signals that maintain key functions. In the chick culture model described, we found that retinal secretions promote RPE differentiation over the course of days rather than the months required without the retina. More work will be needed to determine whether these findings apply to mammalian eyes.

A study of chick embryonic development illustrates the maturation process. We can describe early, intermediate, and late phases of development. The early phase extends from the time that the RPE forms on embryonic day 3

(E3) until the inner segments of photoreceptors protrude the outer limiting membrane on E9. The intermediate phase extends from E9 to E15, when photoreceptors begin to elaborate outer segments. The late phase extends from E15 until hatching on E21. During the intermediate phase, the layers of Bruch’s membrane gradually form. Fenestrations in the walls of the choroidal capillaries begin to form in the intermediate phase, but are not fully elaborated until the middle of the late phase. In parallel with the formation of fenestrations in the capillaries, infoldings of the basolateral membrane begin to form in the intermediate phase, but are not completely elaborated until the middle of the late phase. As the basolateral membranes elaborate infoldings, apical membrane microvilli elongate in coordination with the elongation of photoreceptor inner and outer segments. Some plasma membrane proteins have a distribution that is polarized between the apical and basolateral membranes as early as E7. Nevertheless, some proteins become polarized later in development in parallel with the morphological changes of the apical and basolateral membranes. Examples include basigin, monocarboxylate transporters, and the Na,K-ATPase. This coordination of the development of photoreceptors, RPE plasma membranes, Bruch’s membrane, and the choriocapillaris is conserved between chickens and mammals. It is in the context of this maturing environment that the RPE completes its differentiation.

The RPE is the first retinal layer to overtly differentiate. Despite an epithelial morphology and the expression of RPE markers, early embryonic RPE is still immature. Between E7 and E18 of chick development, 40% of the transcriptome changes with substantial effects on the extracellular matrix, junctional complexes, cell surface receptors, signal transduction pathways, cytoskeleton, regulators of gene expression, and transmembrane transport proteins. Some genes turned on and others off, but most changed their level of expression relative to one another. These data suggest why RPE cultures that express the same tissue-specific markers can function so differently. Without direction from the neural retina or choroid to establish balanced gene expression, cultured RPE can adopt the range of behaviors it displays during development in vivo.

Assembly of Tight Junctions during

Differentiation

Most studies of the assembly of the apical junctional complex were performed in mouse blastocysts or in culture using kidney or intestinal cell lines. The rapid kinetics of assembly in these models makes it difficult to parse the role of the putative assembly proteins. The consensus is that a primordial adherens junction forms first, followed by the segregation of tight junction components into a nascent tight junction. Nectin, E-cadherin, and junctional

adhesion molecule-A (JAM-A) trigger the formation of the primordial adherens junction. These transmembrane proteins form homodimers with their counterparts on the neighboring cell. Their cytoplasmic domains crystallize a complex of proteins by binding an adaptor protein. For example, JAM-A binds the adaptors, AF-6 (Afadin), PAR3 (Partitioning defective-3 homologue), and ZO-1. Their multiple protein-binding domains enable these adaptors to assemble a complex. For example, PAR-3 binds PAR-6 (Partitioning defective-6 homologue) and the atypical protein kinase C, which contributes to cell polarity. JAM-A also localizes ZO-1 to the apical side of the complex to initiate the formation of a tight junction. The tight junction transmembrane proteins, occludin and claudin, bind this nascent complex. Anti-JAM-A antibodies block the formation of tight junctions, as evidenced by the mislocalization of occludin and a low transepithelial electrical resistance (TER). Nevertheless, the adherens junction did form, as evidenced by the localization of E-cadherin and ZO-1. Taken together, these data suggest that JAM-A may play a role in assembling adherens junctions, but plays a more critical role in assembling tight junctions.

As the apical junctional complex assembles slowly during normal RPE development, the process may be studied in greater detail. In chick, primordial adherens junctions are already present in the neuroepithelium that forms the RPE on E3, days before rudimentary tight junctions begin to form on E7. Many of the proteins described above in the assembly of the apical junctional complex are present at this time, but the adherens junction will remodel throughout development both morphologically and molecularly. In each phase of development, different cadherins will appear and disappear. The early phase includes many tight-junctional proteins: ZO-1, occludin, and the assembly proteins AF-6, JAM-A, PAR3, and PAR6. Nonetheless, tight junctions are absent until claudins expression begins on E7 and short, sparse tight-junctional strands begin to appear (Figure 4). During the intermediate phase, tightjunctional strands grow in number and length to gradually coalesce into a complete network that encircles each cell. The tight junction first becomes functional between E10 and E12 (defined by the ability to block the transepithelial diffusion of horseradish peroxidase). During the late phase, structural modifications of the tight junctions continue. Like the adherens junctions, these morphological changes are accompanied by molecular changes. Some claudin messenger RNAs (mRNAs) appeared early, but others appeared during the intermediate or late phases. The expression of some of the early-appearing claudins decreased during the late phase. During the intermediate phase, there was a switch in the expression of ZO-1 isoforms, an event also observed during tight-junction formation in pre-implanta- tion embryos. ZO-3 did not appear until the late phase of development. Although changes in protein expression parallel gene expression to some extent, it appears that the

claudins and ZO proteins are also regulated by effects on protein stability and subcellular localization.

The molecular and morphological changes in the apical junctional complex imply the function of the outer blood–retinal barrier changes during this long maturation process. The changes in claudin expression imply changes in the selectivity and permeability of the tight junctions. It would be reasonable to expect that this would be coupled with changes in transepithelial transport. Among the changes in the transcriptome, many involve membrane transporters. Several should be mentioned, because there is also physiological and cell biological data to corroborate the changes in gene expression. Changes in the expression and polarized distribution of the monocarboxylate transporters and Na,K-ATPase have already been mentioned. Early in development, several facilitated glucose transporters are expressed, but more are expressed later in development, including a sodium-coupled glucose transporter. The appearance of the latter transporters corresponds to the time that the tight junctions become relatively impermeable to glucose. These changes are essential because the retina has a high demand for glucose. It appears that housekeeping transporters that are sufficient for the RPE’s individual needs are replaced by a transcellular, active transport mechanism at the time the blood–retinal barrier forms. These conclusions are based on studies of a primary cell culture model of RPE maturation.

Culture Models to Study Regulation of the Outer Blood–Retinal Barrier

RPE can be cultured on matrix-coated filters that are suspended in a culture dish (Figure 5). This architecture separates the media compartment into apical and basal chambers. The cultures spontaneously polarize with the basal membrane against the filter substrate and the apical microvilli projecting into the upper chamber. The culture architecture allows the cells to feed from the basolateral membranes, as in vivo. By contrast, plastic-grown cells need to feed from the apical membranes. Another advantage is that it is easy to measure barrier function by placing tracers in one medium chamber and measuring their flux

Figure 5 RPE cell culture.

across the monolayer into the opposite chamber. Further, electrodes may be placed to measure a TER. This flexibility is important because selectivity and permeability of tight junctions are regulated semi-independently. The TER is commonly used to assess tight junctions, but TER is an amalgam of transcellular and paracellular resistances to a current that is carried by all the ions of the extracellular space. The TER often approximates the resistance of the tight junctions, because the transjunctional current is often much greater than the transcellular current. Junctions of epithelia with a similar TER can differ in their ion selectivity. Therefore, it is valuable to measure ion fluxes directly. Further, the permeation of mannitol (a small organic tracer) can be modulated independently of TER, and vice versa. Accordingly, multiple assays of barrier function are required for a full assessment.

Although it is relatively easy to culture RPE from many species, including human adults, it is difficult to establish cultures that form an effective barrier. A good example of the problems encountered is the human-derived ARPE19 cell line. This spontaneously transformed cell line has many RPE-specific properties, but its tight junctions are immature. Many claudins were expressed that are undetected in native tissue, and some of the claudins expressed in vivo were undetected in ARPE19. By modifying culture conditions, barrier function, morphology, and melanin expression could be improved. Although claudin expression was affected somewhat, large differences between native RPE and ARPE19 remained. Like the chick studies described above, genomic analyses of native and cultured human RPE have become or are becoming available, which will allow a molecular definition that can compare native to cultured RPE.

Several culture systems have been devised that allow RPE to form a barrier that resembles native RPE. The most highly differentiated are primary or secondary cultures that were isolated from the intermediate phase of RPE development. This stage corresponds to E14 in the chick, postnatal day 5 in rat, and 18–22 weeks gestation in the human. Notably, human fetal RPE appears to have a broader window, as cultures isolated from 13-week fetuses also have excellent properties. Each culture relies on highly specialized medium that includes low amounts, or no, serum. In some cases, the medium appears to remove the need for choroidal or retinal stimulation, but these cultures require 1–2 months in culture to fully mature. Rat and chick cultures that were maintained in serum-free medium were very sensitive to the addition of serum to the apical medium chamber, as might be seen in pathology. For each species, apical serum decreased barrier function, as measured by the TER.

In contrast to the other models, the chick model was designed to study tissue interactions. It used primary cultures that formed incomplete tight junctions with a low TER in a serum-free medium. However, the cultures were

very sensitive to retinal secretions. A medium conditioned by the organ culture of neural retinas induced the formation of complete tight junctions with a TER that was similar to native RPE. Reconstitution experiments showed that contact with the neural retina was required for the proper polarized distribution of the Na,K-ATPase and certain integrins. A central finding was that retinal interactions promoted differentiation on a timescale of days rather than months. Besides cellular junctions, the retinal conditioned medium affected the expression of genes related to the visual cycle, phagocytosis, cytoskeleton, and transmembrane transport. Besides gene expression, retinal conditioned medium affects the half-life and subcellular localization of claudins and ZO proteins. By regulating membrane transporters and tight junctions, the retina and RPE appear to collaborate in regulating the subretinal space. This is an area of research that needs to be explored in greater detail.

RPE in the Larger Context of Ocular

Biology and Disease

Many diseases are coming to be viewed as a low-grade inflammatory process, including age-related macular degeneration. Investigations of inflammatory diseases of the intestine (Crohn’s) and central nervous system (multiple sclerosis) demonstrate that disease can affect the tight junctions in part through the action of inflammatory cytokines. Certainly, disease also affects the membrane transporters of epithelia and endothelia. In the renal field, there has been progress in understanding the interrelationships of tight junctions and membrane transporters and how they are coordinately regulated. The retina field lags behind, but the availability of good culture models and the advances in genomics and systems biology hold great promise for the future.

See also: Breakdown of the RPE Blood–Retinal Barrier; Phototransduction: The Visual Cycle; Retinal Pigment Epithelium: Cytokine Modulation of Epithelial Physiology.

Further Reading

Bradbury, M. W. B. (1979). The Concept of a Blood–Brain Barrier. New York: Wiley.

Burke, J. M. (2008). Epithelial phenotype and the RPE: Is the answer blowing in the Wnt? Progress in Retinal and Eye Research 27(6): 579–595.

Cereijido, M. and Anderson, J. M. (eds.) (2001). Tight Junctions. Boca Raton, FL: CRC Press.

Cereijido, M., Contreras, R. G., Shoshani, L., Flores-Benitez, D., and Larre, I. (2008). Tight junction and polarity interaction in the transporting epithelial phenotype. Biochimica et Biophysica Acta 1778: 770–793.

Grunwald, G. B. (1996). Cadherin cell adhesion molecules in retinal development and Pathology. Progress in Retinal Eye Research 15: 363–392.

Guillemot, L., Paschoud, S., Pulimeno, P., Foglia, A., and Citi, S. (2008). The cytoplasmic plaque of tight junctions: A scaffolding and signalling center. Biochimica et Biophysica Acta 1778: 601–613.

Klimanskaya, I., Hipp, J., Rezai, K. A., et al. (2004). Derivation and comparative assessment of retinal pigment epithelium from human embryonic stem cells using transcriptomics. Cloning and Stem Cells 6: 217–245.

Le Moellic, C., Boulkroun, S., Gonzalez-Nunez, D., et al. (2005). Aldosterone and tight junctions: Modulation of claudin-4 phosphorylation in renal collecting duct cells. American Journal of Physiology – Cell Physiology 289: C1513–C1521.

Radtke, N. D., Aramant, R. B., Petry, H. M., et al. (2008). Vision improvement in retinal degeneration patients by implantation of retina

together with retinal pigment epithelium. American Journal of Ophthalmology 146: 172–182.

Rajasekaran, S. A., Beyenbach, K. W., and Rajasekaran, A. K. (2008). Interactions of tight junctions with membrane channels and transporters. Biochimica et Biophysica Acta 1778: 757–769.

Rizzolo, L. J. (2007). Development and role of tight junctions in the retinal pigment epithelium. International Review of Cytology 258: 195–234.

Strauss, O. (2005). The retinal pigment epithelium in visual function.

Physiological Reviews 85: 845–881.

Van Itallie, C. M. and Anderson, J. M. (2006). Claudins and epithelial paracellular transport. Annual Review of Physiology 68: 403–429.

Wilt, S. D. and Rizzolo, L. J. (2001). Unique aspects of the blood–brain barrier. In: Anderson, J. M. and Cereijido, M. (eds.) Tight Junctions, pp. 415–443. Boca Raton, FL: CRC Press.

Glossary

Cotton-wool spots (CWSs) – Also known as soft exudates, these may be found in nonproliferative diabetic retinopathy (NPDR). They are composed of accumulations of neuronal debris within the retinal nerve fiber layer, and result from disruption and stasis of axoplasmic flow.

Diabetes control and complications

trial (DCCT) – A study that contributed to our understanding that intensive glycemic control is associated with a reduced risk of newly diagnosed retinopathy and a reduced progression of existing retinopathy in people with diabetes.

Diabetes mellitus (DM) – A metabolic disorder characterized by sustained hyperglycemia secondary to lack or diminished efficacy of endogenous insulin.

Diabetic retinopathy (DR) – A retinal disease consequent to development of DM.

Insulin-dependent diabetes mellitus (IDDM) –

A term sometimes used to refer to type I diabetes.

Intraretinal microvascular abnormalities (IRMAs) – The tortuous, hypercellular micro vessels that develop in NPDR.

New vessels elsewhere (NVE) – The neovascularization of the retina found greater than one disk diameter from the optic nerve head.

New vessels on disk (NVD) – The neovascularization on or within one disk diameter of the optic nerve head.

Non-insulin-dependent diabetes mellitus (NIDDM) – An older term for type II diabetes or adultonset diabetes.

Nonproliferative diabetic retinopathy (NPDR) –

DR characterized by intraretinal microvascular changes which precede the proliferative phase.

Optical coherence tomography (OCT) –

A noninvasive imaging technique that permits analysis of retinal structure in the living eye.

Proliferative diabetic retinopathy (PDR) – DR characterized by the presence of retinal neovascularization.

The early treatment diabetic retinopathy study (ETDRS) – A large study of progression and treatment of DR.

United Kingdom Prospective Diabetes Study (UKPDS) – A study that contributed to our

understanding that intensive glycemic control is associated with a reduced risk of newly diagnosed retinopathy and a reduced progression of existing retinopathy in people with diabetes.

Vascular endothelial growth factor (VEGF) –

A growth factor that promotes development of endothelial cells.

Background

Diabetes mellitus (DM) is a metabolic disorder characterized by sustained hyperglycemia secondary to lack or diminished efficacy of endogenous insulin. The terminology used for classification of different types of diabetes is evolving and can be a source of confusion. Traditionally, there have been two types of DM: type I diabetes and type II diabetes (these terminologies are used throughout the remainder of this article). Immune-mediated diabetes is the latest, and perhaps most descriptive, terminology applied to type I diabetes. Autoimmune destruction of insulin-producing pancreatic islet cells is postulated as instrumental in the pathogenesis of type I diabetes. Previous terminology used for type I diabetes included insulin-dependent diabetes mellitus (IDDM), and juvenileonset diabetes. Type II diabetes is characterized by relative deficiencies of insulin and/or peripheral insulin resistance. It was previously known as non-insulin-dependent diabetes mellitus (NIDDM) or adult-onset diabetes.

DM is a common medical problem and is a major global source of morbidity and mortality. The incidence of DM is thought to be increasing throughout the world in part due to an increasing incidence of obesity and sedentary lifestyles. In the United States, it is estimated that 7.8% of the total population has DM, and it causes a vast array of long-term systemic complications which have a significant impact on both quality and quantity of life. Patients with DM have heart disease, death rates, and stroke rates that are 2–4 times higher than adults without diabetes, and DM is the leading cause of end-stage renal disease in the United States. Further, people with diabetes are more susceptible to many other illnesses and, once acquired, often have worse prognoses (e.g., pneumonia).

From an ophthalmic standpoint, DM causes numerous complications. Chief among these complications are diabetic retinopathy (DR), unstable refractions, accelerated

cataracts, rubeosis iridis, which can lead to neovascular glaucoma, cranial nerve palsies, reduced corneal sensitivity, papillopathy, and poor wound healing. The incidence of blindness is 25 times higher in patients with diabetes than in the general population. Furthermore, DR is the most common cause of blindness in patients aged 20–74 years, accounting for 12 000–24 000 new cases of blindness in the United States each year.

Risk Factors for DR

The prevalence of DR in the diabetic population increases with the duration of diabetes and patient age. Studies have shown that after 20 years of diabetes, nearly 99% of patients with type I DM and approximately 60% of patients with type II DM have some degree of DR (Figure 1). DR rarely develops in children younger than 10 years of age, regardless of the duration of diabetes. The risk of DR increases after puberty. Approximately 5% of type II diabetics have DR at presentation; this observation is a reflection of the typically insidious onset of hyperglycemia in type II diabetes many years before the diagnosis is firmly established.

In addition to duration of DM, other risk factors for the development of DR include poor glycemic control, the type of diabetes (type 1 more than type 2), and the presence or absence of associated conditions such as hypertension, smoking, dyslipidemia, nephropathy, and pregnancy. The Diabetes Control and Complications Trial (DCCT) and

Duration of diabetes

Figure 1 Incidence of diabetic retinopathy (DR) increases over time. Duration of DM is directly associated with an increased prevalence of DR in people with both type I and type II DM. The figure represents the percent of diabetic patients with retinopathy according to duration of disease in patients under the age of 30 years who were treated with insulin (primarily type I diabetics) and patients over the age of 30 years who were not treated with insulin (primarily type II diabetics). Retinopathy increased over time in both groups, affecting virtually all patients with

type I diabetes by 20 years. The increased incidence in type II diabetes at 3 years is likely secondary to the difficulty in determining the exact time of onset of type II DM. Data from Klein, R., Klein, B. E., Moss, S. E., Davis, M. D. and DeMets, D. L. (1984). The Wisconsin Epidemiologic Study of Diabetic Retinopathy: III. Prevalence and risk of diabetic retinopathy when age at diagnosis is 30 or more years. Archives of Ophthalmology 102: 527.

the United Kingdom Prospective Diabetes Study (UKPDS) demonstrated that intensive glycemic control is associated with a reduced risk of newly diagnosed retinopathy and a reduced progression of existing retinopathy in people with DM (type I in DCCT (Figure 2) and type II in UKPDS). According to the DCCT, intensive insulin therapy reduced the incidence of new cases of DR by as much as 76% compared with conventional therapy. The UKPDS found similar results in type II diabetics; each 1% point reduction in glycosylated hemoglobin was associated with a 37% reduction in development of retinopathy. Further, the UKPDS showed that control of hypertension was also beneficial in reducing progression of DR. Pregnancy is occasionally associated with rapid progression of DR; thus, women with diabetes who become pregnant require more frequent evaluation of the retina. Pregnant women without any DR are at a 10% risk of developing nonproliferative diabetic retinopathy (NPDR) during their pregnancy. Of those with preexisting NPDR, 4% progress to proliferative retinopathy (Figure 3).

Pathogenesis

The pathogenesis of DR is the current subject of intense research. It is theorized that exposure to chronic hyperglycemia results in a number of biochemical and physiologic alterations that ultimately produce retinal vascular changes and subsequent retinal injury and ischemia. The list of hematologic and biochemical abnormalities theorized to play a role in the development of DR

Figure 2 Intensive glycemic control slows progression of retinopathy. Cumulative incidence of progressive retinopathy in patients with type 1 diabetes and early nonproliferative retinopathy who were treated with either conventional or intensive insulin therapy for 9 years. Intensive glycemic control reduced the risk of DR progression over time by 54%, although intensive therapy was associated with transient worsening in the first year ( p < 0.001). Data from Diabetes Control and Complications Trial Research Group (1993). The effect of intensive treatment of diabetes on the development and progression of long-term complications in insulin-dependent diabetes mellitus. The New England Journal of Medicine

329: 977.

Glycosylated hemoglobin (%)

Figure 3 Progression of DR in relation to glycemic control. The figure shows the rate of progression of retinopathy in patients with type 1 diabetes according to mean glycosylated hemoglobin values (solid line). Better glycemic control was associated with slower rates of DR progression. The dashed lines represent the 95% confidence intervals. Data from Diabetes Control and Complications Trial Research Group (1993). The effect of intensive treatment of diabetes on the development and progression of long-term complications in insulin-dependent diabetes mellitus. The New England Journal of Medicine

329: 977.

includes the following: impairment of retinal blood vessel autoregulation, the occurrence of retinal microthrombosis and subsequent ischemia, accumulation of advanced glycosylation end products, and damage caused by reactive oxygen species. The role that growth factors (e.g., vascular endothelial growth factor (VEGF)) play in the formation of DR is discussed later. There have also been recent considerations of categorizing DR as an inflammatory disease. Trials investigating anti-inflammatory agents for prevention or treatment of DR in humans are ongoing.

Specific retinal vascular changes theorized to be instrumental in DR include the loss of pericytes, basement membrane thickening, and impaired endothelial cell function. The walls of retinal capillaries consist of endothelial cells and pericytes and are devoid of smooth muscle and elastic tissue. Endothelial cells form a single layer on a basement membrane and are linked by tight junctions that form the inner blood–retinal barrier. Pericytes are found external to the endothelial cells and have pseudopodial processes that envelop the capillary. It is believed that pericytes have contractile properties and are thought to participate in autoregulation of the microvascular capillary circulation (analogous in function to the smooth muscle found in larger arteries). The classic histologic finding of early DR in the human retina is the loss of microvascular pericytes; however, the exact mechanism by which pericytes are preferentially lost early in DR is

unknown. Thickening of the retinal capillary basement membrane is another well-known lesion found in DR. In addition to basement membrane thickening, patients with DR are also found to have vacuolization and deposition of fibrillar collagen in their basement membranes. Similar to the loss of pericytes, the exact biochemical events that lead to basement membrane alterations in DR are not fully evident. Several studies implicate the sorbitol pathway in this process. The sorbitol pathway is the name given to the sequence of reactions that convert glucose to fructose involving the enzymes aldose reductase and sorbitol dehydrogenase. In this pathway, glucose is reduced first to sorbitol, which is then oxidized to fructose. However, since the latter reaction occurs slowly in many cells, sorbitol may build to high, and possibly, toxic concentrations. The toxicity of sorbitol is theorized to perhaps lead to basement membrane alterations. The final vascular change that appears to be instrumental in DR is the loss of endothelial cell function and the subsequent breakdown of the blood–retinal barrier. One possible cause of the blood–retinal barrier breakdown is opening of the tight junctions (zonulae occludentes) between adjacent microvascular endothelial cell processes. Several proteins are known to be involved with tight junction function, namely ZO-1, occluding and claudin. Studies have shown that high glucose levels appear to inhibit ZO-1 expression. Further experiments have shown reduced expression and anatomical distribution of occludin in experimental diabetes. Finally, studies have shown that intravitreal injections of the growth factor VEGF in rats increased production of nitric oxide (NO) and increased phosphorylation of ZO-1 and occludin, changes that result in increased breakdown of the blood–retinal barrier.

Advanced stages of DR are marked by the proliferation of new blood vessels. Retinal neovascularization is a devastating process that can lead to blindness in DR. Neovascularization develops through angiogenesis, in which capillaries develop from preexisting blood vessels. In DR, the regulatory mechanisms of angiogenesis can become compromised, which leads to uncontrolled endothelial cell division. On a molecular level, angiogenesis is a complicated pathway involving interplay between a number of angiogenic messengers, proteolytic enzymes, and the preexisting vessels themselves. The common final product in angiogenesis is activation of vascular endothelial cells. Several endothelial cell mitogens have been isolated and studied, including VEGF, platelet-derived growth factor (PDGF), insulin-like growth factor (IGF), basic fibroblast growth factor (bFGF), protein kinase C (PKC), antiopoietins, integrins, and ephrins. VEGF is commonly considered the most potent angiogenic factor, and some of the other molecules may act indirectly through VEGF. As the activated endothelial cells proliferate, they secrete proteolytic enzymes that degrade the parent vessel’s basement