Ординатура / Офтальмология / Английские материалы / Diabetic Retinopathy_Lang_2007

retinopathy and holds promise to improve the visual prognosis in patients with diabetic retinopathy.

The book provides an update of new insights into the pathogenesis, diagnosis and especially the treatment of diabetic retinopathy and gives detailed information about latest research achievements. Therefore, it is suitable for general ophthalmologists, retina specialists and diabetologists. It provides a collection of latest findings in diabetic retinopathy by excellent authors, and therefore, deserves the attention of everyone who is interested in this subject.

I want to thank all the coworkers for their great efforts in passing on their profound knowledge. The contents of the book will not only advance our understanding of diabetic retinopathy based on the provided knowledge, but also improve our diagnosis and treatment strategies in the permanent efforts to help the numerous patients who suffer from diabetic retinopathy and are threatened by visual problems.

Prof. Dr. Gabriele E. Lang, Ulm

Early stages of vascular dysfunction are characterized by a breakdown of the blood-retinal barrier in both humans and rodent models of experimental diabetes. Breakdown of the blood-retinal barrier can be observed prior to latestage vascular alterations leading to proliferative diabetic retinopathy.

Blood-retinal barrier breakdown contributes to macular edema, which occurs in over 25% of people with diabetes and correlates highly with visual impairment in people with diabetic retinopathy [1]. Treatment by laser coagulation is limited to focal edema, but is controversial in diffuse edema and proven to be ineffective in ischemic diabetic maculopathy.

Breakdown of the Blood-Retinal Barrier

Although changes to retinal blood flow may partially explain the extravasation of fluid, the most important mechanism is the breakdown of the bloodretinal barriers [2].

The movement of water through the blood-retinal barrier appears to have two dominant components: a passive (bidirectional) transport and an active transport directed from the retina to the blood. Theoretically, macular edema develops when the inflow of fluid into the retina exceeds the outflow. Passive transport (permeability) of fluorescein has been shown to increase in relation to the progression of retinopathy [3, 4]. In vitro studies of isolated retinal pigment epitheliumchoroid preparations showed that the outward active transport of fluorescein is substantially greater than the passive transport and that this transport is inhibited by metabolic (oubain) and competitive inhibitors (probenecid) [5–8]. Unlike active transport, passive permeability is related to the degree of retinopathy in that eyes with severe nonproliferative diabetic retinopathy have a passive permeability that is significantly increased compared with moderate retinopathy. The active resorptive functions of the blood-retinal barrier in diabetes are likely to be increased to counteract edema formation, although the increase is too little [9].

Besides the retinal pigment epithelium (outer blood-retinal barrier), the vascular endothelium (inner blood-retinal barrier) forms the main barrier against the passage of macromolecules and circulating cells from blood to the extracelluar space. In diabetes, the endothelial cell loss of the retinal vessels is likely to account for the majority of the early blood-retinal barrier breakdown and is the initial site of damage. Passive permeability through the endothelium can be increased by three general mechanisms: (1) increased transcellular transport, (2) dysfunction of the intercellular junctions, and (3) increased endothelial cell destruction.

Although other factors such as impairment of the perivascular supporting cells might influence vascular permeability, the primary damage is likely to predominantly affect endothelial cells.

Transcellular Transport

Besides an increase in vascular permeability through endothelial cell death and alterations in the cell-cell junctions, direct transport via pinocytosis is potentially involved in the increased diabetic vascular leakage [10, 11]. Despite the fact that pinocytic transport is critically involved in transepithelial fluid exchange, its regulation in diabetic retinopathy has not been investigated in the context of the molecular factors involved, as outlined above.

Distinct growth factors are causally related to neovascularization and/ or vascular leakage: the disruption of endothelial integrity leads to retinal ischemia and vascular endothelial growth factor (VEGF)-mediated iris and retinal neovascularization [12–14]. VEGF is 50,000 times more potent than histamine in causing vascular permeability [15–20]. Previous work has shown that retinal VEGF levels correlate with diabetic blood-retinal barrier breakdown in rodents [21, 22] and humans [23]. Flt-1(1-3Ig)Fc, a soluble VEGF receptor, reverses early diabetic blood-retinal barrier breakdown and diabetic leukostasis in a dose-dependent manner [14]. Early blood-retinal barrier breakdown localizes, in part, to retinal venules and capillaries of the superficial inner retinal circulation [24] and can be sufficiently reduced by VEGF inhibition. Although VEGF is only one of the molecules involved in the various cytokine cascades, it is likely to be one of the most efficient therapeutic targets. Ongoing clinical studies investigate the efficacy of VEGF inhibition on diabetic macular edema.

Due to the current knowledge, VEGF causes vascular hyperpermeability by opening interendothelial junctions and induction of fenestrations and vesiculo-vacuolar organelles. As for the blood-retinal barrier endothelium, other cellular mechanisms may translate increased permeability caused by VEGF [25]. In these leaky blood vessels, the number of pinocytotic vesicles at the endothelial luminal membrane is significantly higher, and these pinocytotic vesicles transport plasma immunoglobulin G. By electron microscopy, no fenestrations or vesicles were found in the endothelial cells of the VEGFaffected eyes.

Intercellular Junctions and Their Alterations in

Diabetic Retinopathy

Endothelial cells are important constituents of the vasculature and essential for the separation of blood from the surrounding tissues. They also control the passage of proteins and cells from the blood stream into these tissues, either by using a specialized transcellular vesicle transport system or by selective opening and closing of intercellular junctions.

It is likely that inflammatory agents increase permeability by binding to specific receptors that transduce intercellular signals, which in turn cause cytoskeletal reorganization and coordinated widening of the interendothelial contacts. Endothelial junctions also regulate leukocyte extravasation upon inflammatory stimuli. Once leukocytes have adhered to the endothelium, a coordinated opening of interendothelial cell junctions occurs without the loss of its barrier function. Under diabetic conditions, inflammatory mediators may cause aberrant opening of intercellular contacts, now also resulting in loss of barrier function and thus vascular leakage. However, both the regulation and the structural composition of retinal endothelial junctions and their alterations in diabetic retinopathy are largely unknown.

Composition of Intercellular Junctions in the Retina

Intercellular junctions of vascular endothelial cells consist of tight junctions, adherens junctions and gap junctions. Depending on the type of vessel, there is a fourth structure called ‘complexus adherents’ or ‘syndesmosome’ consisting of a mixture of adherens junction components and desmosomal components [26]. Although endothelial cells are polarized, tight junctions are not only found at the interface between the apical and basolateral membrane domains, as observed in simple epithelia, but are often intermingled with the adherens junctions all along the cleft [27]. The molecular composition and complexity of intercellular junctions varies along the vasculature. More complex and distinct structures are formed in those cells with an increased barrier function, such as the blood-retinal barrier [28].

Both tight junctions and adherens junctions consist of transmembrane molecules, which connect cells with each other and are linked to cytoskeletal linker molecules [29, 30]. In addition, a variety of regulatory molecules are also found at these sites. These are most likely important for the regulated interaction with the cytoskeleton and for communicating alterations in adhesion.

Alterations in Intercellular Junctions in Diabetic Retinopathy

One of the first clinical manifestations in diabetic retinopathy is vascular leakage, indicating that disturbance of the blood-retinal barrier is an early event. Even though tracer studies have shown a disturbance in the paracellular pathway [31], relatively little is known about how the molecular junctional components are affected. In the streptozotocin-induced diabetic rat model, occludin distribution and amounts are altered correlating with an increase in paracellular permeability [31].

In humans, there is one report on decreased expression of vascular endothelial (VE)-cadherin in the diabetic retina [32]. Alterations induced by diabetes have also been described in the placental vasculature where ZO-1,

VE-cadherin, -catenin and occludin showed reduced staining at the junctions correlating with an increase in tyrosine phosphorylation [33].

Regulation of Junctional Stability, Strength and Permeability

Intercellular junctions are dynamic entities even under steady-state conditions and can exchange molecular components and interactions without losing cell-cell contact or barrier function.

VEGF can induce phosphorylation of the tight junctional proteins occludin and ZO-1 [34]. In the diabetic retina, VEGF is strongly upregulated and has been implicated as a mediator of vascular leakage and neovascularization. At present, it is not known how the VE-cadherin/VEGF receptor interaction and its signaling pathway is altered in diabetic retinopathy.

Diabetes is a consequence of insulin deficiency or insulin resistance. Thus, diabetic retinopathy may be caused directly by absent or aberrant insulin receptor signaling or it may result from secondary effects, since insulin signaling affects a diverse range of downstream pathways. Specific deletion of either the insulin receptor or its close relative the insulin-like growth factor receptor in vascular endothelial cells did not result in any obvious decrease in vascular integrity, nor did it seem to compromise the blood-brain barrier, arguing for the latter situation [35]. However, a direct effect cannot be ruled out because of overlapping functions of the insulin receptor and insulin-like growth factor receptor.

Matrix Changes Affect Formation of Edema in the

Diabetic Retina

Degradation of the extracellular matrix affects endothelial cell function at many levels, causing endothelial cell liability which is required for cellular invasion and proliferation, or influencing the cellular resistance and therefore the vascular permeability. The degradation and modulation of the extracellular matrix is exerted by matrix metalloproteinases (MMPs), a family of zinc-binding, calcium-dependent enzymes [36, 37]. Elevated expression of MMP-9 and MMP-2 has been shown in diabetic neovascular membranes [38, 39], although a direct effect of glucose on MMP-9 expression in vascular endothelial cells could not be shown [40].

It is likely that MMPs participate at various stages during the course of the blood-retinal barrier dysfunction and breakdown. Their actions include early changes of the endothelial cell resistance that influence intercellular junction formation and function [41] and active participation in the endothelial and pericyte cell death [42] occurring late in the course of the disease.

Extracellular matrix and basement membrane components are required for cellular adhesion, migration and differentiation. They also play a particular role in the delimiting of tissue boundaries and the formation of vascular and neural networks. Changes in the deposition of the extracellular matrix during diabetes mellitus are well established. However, the molecular composition of the basement membranes in the normal and diabetic retina is only poorly described. Furthermore, alterations in the repertoire of cellular receptors for basement membrane components and the effects these changes may have upon the cells of the retina have not been investigated.

Basement membranes are specialized extracellular matrices found underlying all epithelia and endothelia and surround many mesenchymal cell types. Besides separating tissues, they have important roles in axonal guidance and neuronal migration and survival, as well as synapse formation [43], both by acting directly upon specific cell receptors and by acting as a reservoir for many growth factors, in particular the fibroblast growth factor and transforming growth factorfamily [44].

All basement membranes are formed by members of three ubiquitous protein families, i.e. laminins, nidogens and collagen IV, and by the proteoglycan perlecan. Basement membrane variability is derived from the fact that there are 15 laminin isoforms [45], 6 collagen IV chains and 2 members of the nidogen family. These proteins are often expressed in a highly regulated developmental and temporal manner and vary in their use of cellular receptors. Further basement membrane diversity is produced by the presence of more restricted proteins which may be integral basement membrane proteins, such as collagen XVIII [46], or associated with the basement membrane, such as matrilin-2 [47]. Basement membranes are found in three regions of the retina: in Bruch’s membrane underlying the pigment epithelium and separating it from the choroid, in the vitroretinal border as the inner limiting membrane, and in the endothelial basement membrane forming part of the blood-retinal barrier.

Cellular Interaction and Its Relevance to Vascular Leakage

Leukocyte infiltration of retinal tissue characterizes many inflammatory diseases such as diabetes, pars planitis or choroidal inflammatory diseases. In diabetes, activated leukocytes adhere to the retinal vascular endothelium [12, 48]. Increased leukostasis is one of the first histological changes in diabetic retinopathy and occurs prior to any apparent clinical pathology. Adherent leukocytes play a crucial role in diabetic retinopathy by directly inducing endothelial cell death in capillaries [49], causing vascular obstruction and vascular leakage. Endothelial cell death precedes the formation of acellular capillaries [48].

However, over time, acellular capillaries prevail and become widespread. Although the mechanism of this destructive process remains elusive, it is clear that the interaction between altered leukocytes and endothelial cells and the subsequent endothelial damage represents a crucial pathogenic step [12, 49–51].

Previous studies of vascular casts in diabetic retinopathy have suggested that the loss of pericytes represents the earliest histologically visible alteration [52, 53]. Interaction between pericytes and endothelial cells is important in the maturation, remodeling and maintenance of the vascular system via the secretion of growth factors and/or modulation of the extracellular matrix [54]. There is also evidence that pericytes are involved in the transport across the bloodretinal barrier and the regulation of vascular permeability.

Knowledge on vascular plasticity has greatly increased in the past years. It is likely that in the diabetic retina, repair mechanims take place as well. However, cell differentiation and recruitment to the vessel walls are likely to be altered under diabetic conditions.

Adult bone marrow (BM) contains cells capable of differentiating along hematopoietic (Lin ) or nonhematopoietic (Lin ) lineages. Lin hematopoietic stem cells have recently been shown to contain a population of endothelial precursor cells (EPCs) with the capacity to form blood vessels [55, 56].

In a crucial set of experiments, adult mice were durably engrafted with hematopoietic stem cells isolated from transgenic mice expressing green fluorescent protein after which retinal ischemia was induced to promote neovascularization [57]. In this model, self-renewing adult hematopoietic stem cells had functional hemangioblast activity, i.e. they could clonally differentiate into all hematopoietic cell lineages as well as into endothelial cells that revascularize adult retina.

Using green fluorescent protein chimeric mice, it was further demonstrated that laser injury of the choroidal vasculature was sufficient to induce stem cell recruitment and subsequent formation of choroidal neovascularization. Green fluorescent protein-positive cells formed part of the functional vasculature in the choroid as early as 1 week after injury and remained present during follow-up.

Furthermore, it was shown that intravitreally injected Lin BM cells selectively target retinal astrocytes, cells that serve as a template for both developmental and injury-associated retinal angiogenesis. When Lin BM cells were injected into neonatal mouse eyes, they extensively and stably incorporated into forming retinal vasculature [58]. When EPC-enriched hematopoietic stem cells were injected into the eyes of neonatal rd/rd mice, whose vasculature ordinarily degenerates with age, they rescued and maintained a normal vasculature. In contrast, normal retinal angiogenesis was inhibited when EPCs expressing a potent angiostatic protein were injected.

In diabetes, it was demonstrated that BM-derived EPCs are recruited to the pancreas in response to islet injury. EPC-mediated neovascularization of the pancreas could in principle be exploited to facilitate the recovery of nonterminally injured -cells or to improve the survival and/or function of islet allografts [59].

Taken together, these studies emphasize the likelihood of an EPC involvement in repair mechanisms of the diabetic vasculature. Besides, in increased stem cell recruitment, these cells and their potential to differentiate might be altered in diabetes. Although likely, alterations in stem cell recruitment and differentiation in diabetes have not yet been investigated in detail.

Endothelial Cell Damage and Apoptosis in the Diabetic Retina

Blood-retinal barrier breakdown is at least in part due to endothelial cell damage and apoptosis. The proapoptotic molecule Fas ligand (FasL) induces apoptosis in cells that carry its receptor Fas (CD95) [60]. There is evidence that FasL is expressed on vascular endothelium where it functions to inhibit leukocyte extravasation. The expression of FasL on vascular endothelial cells might thus prevent detrimental inflammation by inducing apoptosis in leukocytes as they attempt to enter the vessel. In fact, during inflammation and ensuing tumor necrosis factorrelease, the endothelium not only upregulates several adhesion molecules [61], but also downregulates FasL and allows leukocyte adherence, survival and thus migration to sites of infection and wounding. In experimental diabetic retinopathy, inhibition of Fas-mediated apoptotic cell death reduces vascular leakage [50]. However, diabetic endothelial cell death, as to the cumulative damage during the diabetic course, might play an increasing role in diabetic vascular leakage, and thus, in diabetic maculopathy.

Conclusion

Diabetic macular disease is considered a structural alteration to the macula in any of the following manners:

•collection of intraretinal fluid in the macula with or without exudates (lipids) and with or without cystoid changes;

•nonperfusion of parafoveal capillaries with or without intraretinal fluid;

•traction in the macula by fibrous tissue proliferation that is dragging the retinal tissue causing surface wrinkling, or detachment of the macula;

•intraretinal or preretinal hemorrhage in the macula;

•lamellar or full-thickness retinal hole formation;

•a combination of the above.

Early intervention in macular edema is undoubtedly advantageous, as the risk of ultrastructural alterations induced by a persistent macular edema increases with time. It is well known that with time, the central avascular zone and the areas of ischemia are likely to increase. The current hope to treat even ischemic maculopathy pharmacologically will largely depend on the long-term results with e.g. anti-VEGF therapies or intravitreal steroids. Currently, we are only at the edge of understanding diabetic macular edema at a molecular level, but it becomes clear that only a thorough investigation of the pathogenesis of diabetic retinal vascular leakage will help to identify new and potentially more efficient targets for intervention and prophylaxis of diabetic macular edema.

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