Molecular basis of the inner blood-retinal barrier and its breakdown in diabetic macular edema and other pathological conditions

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Abstract

Breakdown of the inner endothelial blood-retinal barrier (BRB), as occurs in diabetic retinopathy, age-related macular degeneration, retinal vein occlusions, uveitis and other chronic retinal diseases, results in vasogenic edema and neural tissue damage, causing loss of vision. The central mechanism of altered BRB function is a change in the permeability characteristics of retinal endothelial cells caused by elevated levels of growth factors, cytokines, advanced glycation end products, inflammation, hyperglycemia and loss of pericytes. Subsequently, paracellular but also transcellular transport across the retinal vascular wall increases via opening of endothelial intercellular junctions and qualitative and quantitative changes in endothelial caveolar transcellular transport, respectively. Functional changes in pericytes and astrocytes, as well as structural changes in the composition of the endothelial glycocalyx and the basal lamina around BRB endothelium further facilitate BRB leakage. As Starling's rules apply, active transcellular transport of plasma proteins by the BRB endothelial cells causing increased interstitial osmotic pressure is probably the main factor in the formation of macular edema. The understanding of the complex cellular and molecular processes involved in BRB leakage has grown rapidly in recent years. Although appropriate animal models for human conditions like diabetic macular edema are lacking, these insights have provided tools for rational design of drugs aimed at restoring the BRB as well as for design of effective transport of drugs across the BRB, to treat the chronic retinal diseases such as diabetic macular edema that affect the quality-of-life of millions of patients.

Introduction

Retinal vascular leakage from loss of function of the blood–retinal barrier (BRB) and subsequent macular edema are the main causes of visual loss and blindness in major eye diseases such as diabetic retinopathy (DR), age-related macular degeneration (AMD), retinal vein occlusion and uveitis (Fig. 1). Despite recent advances, there is still a fundamental lack of understanding of the cellular mechanisms underlying both the function of the BRB in physiological conditions as well as its dysfunction in pathological conditions. However, it has become clear that the previously prevailing concept that BRB loss is the result of unspecified endothelial cell damage' has become obsolete. It should be replaced by the notion that dynamic adaptations of endothelial cells and other cell types involved in the BRB underlie vascular leakage in retinal disease.

A complex dual vascular system provides oxygen and nutrients to the metabolically highly active neural retina, a tissue that has a higher oxygen consumption per unit weight of tissue than any other human tissue (Arden et al., 2005). The choriocapillaris provides blood supply to the photoreceptors in the outer retina, while capillaries sprouting from the central retinal artery provide oxygen and nutrients to the inner retina. These two distinct vascular beds not only differ in embryonic origin, but also in their properties and functions in the adult eye. The endothelium of choroidal capillaries is highly fenestrated and permeable. The capillaries in the inner retina have a continuous endothelium with a barrier function and are organized in two parallel layers, whereas the outer retina is completely avascular.

Retinal neural tissue is protected from potentially harmful molecules in the circulation by the inner BRB that regulates the entry of molecules into the inner retina. To complete this protective environment of the ocular interior, other blood-ocular barriers are formed by retinal pigment epithelium (outer BRB), epithelium of ciliary processes (blood-aqueous barrier), the capillaries of the optic nerve, with the exception of the pre-laminar part (Hofman et al., 2001b), and by endothelial cells of capillaries of the iris and ciliary muscle (Cunha-Vaz, 1997; Hofman et al., 2001a; Raviola, 1977). In this review, the focus is on the inner BRB, as other components of the blood-ocular barriers have been adequately discussed elsewhere (Freddo, 2012; Rizzolo et al., 2011; Simó et al., 2010).

The intercellular spaces between retinal endothelial cells that form the BRB are sealed by elaborate tight junctions, and the cells themselves lack fenestrations and have few pinocytotic vesicles (Bradbury, 1985; Raviola, 1977) (Fig. 2). These features of the BRB, which are comparable to those of the blood-brain barrier (BBB) endothelium, result in high transendothelial electrical resistance (TEER) and restricted paracellular permeability. The resistance of the BRB is not exactly known, but is likely similar to that of the BBB, with a TEER of 1500–2000 Ω·cm2 (Butt et al., 1990). In comparison, human placental endothelial cells have a TEER of 22–52 Ω·cm2 (Jinga et al., 2000), which permits rapid paracellular exchange of nutrients and waste products between mother and fetus, whereas urinary bladder epithelium has a resistance of 6000–30,000 Ω·cm2, necessary to protect underlying tissues against the toxic urine and to preserve urine hyperosmolarity (Negrete et al., 1996). The neural retina is highly vulnerable and any vascular change leading to reduced barrier properties can be detrimental for visual function. In recent years, increasing insight has been gained in the molecular mechanisms of pathological BRB breakdown. In the present review, structural, cellular, molecular and mechanistic aspects of BRB functions and their loss in eye diseases are discussed.

Most cellular and molecular knowledge on the mechanisms of BRB breakdown comes from rodent and in vitro models. Although much progress has been achieved by the development of these models, it must be stressed that the extrapolation of this knowledge to the mechanisms involved in BRB dysfunction in human eye disease must be approached with caution. In fact, a huge knowledge gap exists between the descriptive data from the human clinical manifestations of ocular pathologies such as diabetic macular edema (DME), uveitis and venous occlusions and the knowledge gained from animal models and in vitro systems that endeavor to mimic these pathologies. On the clinical side, it is often difficult to make a clear-cut definition of the disease and from the research side it is difficult to mimic the pathology that is observed in the clinic. The success of drugs in the treatment of DME, such as anti-vascular endothelial growth factor (VEGF) therapies or corticosteroids has lead to hypotheses that point to the involvement of VEGF or inflammation, but the exact mechanisms have only partly been resolved. For example, no animal model thus far is able to mimic what really happens in the human retina in the context of DR or DME. The same applies for in vitro models of the BRB. Although these models provide a very useful tool for high-throughput testing and functional analysis of individual proteins, cells in culture rapidly lose their BRB properties, such as their high number of tight junctions and low number and specific cellular distribution of caveolae. Furthermore, it is virtually impossible to mimic the effects of chronic hyperglycemia in an in vitro system. Thus, in these models as well, the translation of insights obtained to understanding pathophysiology of human disease remains troublesome. Bearing this in mind, it is clear that the interpretation of results from in vivo and in vitro studies must be done with great care. Nevertheless, it is to be expected that new information from existing and novel models and confirmatory and complementary studies in human tissues and in patients will eventually close the current knowledge gap and will lead to the detailed understanding of the mechanisms of BRB breakdown in ocular pathologies.

Section snippets

Clinical pathology

In human ocular disorders with macular edema such as DR, AMD, retinal vein occlusion and uveitis, increased retinal vascular permeability is involved. As in other vascular beds, the rules of Starling determine water homeostasis and edema formation in the retina. Net water transport over the endothelium is determined by the sum of hydrostatic pressure and osmotic pressure of the luminal and extraluminal compartments. Increased osmotic pressure of the interstitial compartment due to leakage of

Endothelial cells

The specific structural properties of the retinal vasculature provide the basis of the BRB function. Retinal capillaries that form the BRB consist of a single layer of tightly adherent endothelial cells, a basal lamina (BL) and surrounding pericytes, astrocytes and microglia (Fig. 3). This complex is called the neurovascular unit. Selectively regulated transport of molecules is possible across this barrier. Two pathways serve this purpose, the paracellular pathway which is regulated by dynamic

Mediators of BRB dysfunction and increased BRB permeability

Hyperglycemia, hypoxia, oxidative stress and/or inflammation are the main underlying processes in the human ocular diseases where dysfunction of the BRB is a major cause of loss of vision. In the later sections, their effects on BRB function and their use as an intervention target are discussed.

Mechanisms of barrier breakdown

In BRB loss and leakage in pathological conditions, the specialized properties of the neurovascular unit regulating barrier integrity are altered. The endothelial cells of the normal BRB possess well-developed intracellular tight junctions, have few caveolar vesicles, located at their abluminal side, and express selective transporters, and all of these properties may be changed in BRB loss, but a true understanding of these mechanisms in human disease is still lacking. The tight junctions and

Therapeutic modulation of the BRB

The present treatment modalities for macular edema are laser therapy in DR and branch retinal vein occlusion, and intravitreal injections with corticosteroids and anti-VEGF agents (reviewed in Wenick and Bressler, 2012; Witkin and Brown, 2011) in several conditions. Corticosteroids and anti-VEGF agents have shown efficacy in reducing macular edema, suggesting involvement of VEGF-A and/or inflammatory mechanisms in the underlying pathophysiology. However, as indicated above, the exact working

Concluding remarks and directions for future research

The current literature on the molecular basis of the inner BRB and its breakdown in DME and other pathological conditions shows that in addition to paracellular transport, evidence for an important additional or even predominant role of transcellular transport across the endothelial cells is substantial.

Macular edema, as the main clinical result of altered BRB permeability, is still a major cause of blindness as a final common pathway of a large variety of ocular diseases. Our understanding of

Contribution of each author

Dr Ingeborg Klaassen designed the content of the review and was the first author of all versions of the manuscript including the final version.

Prof. Dr. Cornelis Van Noorden adviced in the design of the content of the review from a cell biological point of view and corrected and redesigned all versions of the manuscript including the final version.

Prof. Dr. Reinier Schlingemann adviced in the design of the content of the review from a clinical/pathological point of view and corrected and

Acknowledgements

We thank Kees Hoeben for his help with electron microscopy work and Monique Arendse for help with preparing the manuscript. This study was supported by grants of the Diabetes Fonds Nederland (Grant 1999.050) and by grants of the Landelijke Stichting voor Blinden en Slechtzienden (LSBS); Stichting Blinden-Penning; Stichting Oogfonds Nederland; Vereniging Blindenbelangen Rotterdam; Stichting Blindenhulp; Prof. Hoppenbrouwers Fonds, Stichting Nederlands Oogheelkundig Onderzoek (SNOO). Fig. 7 is

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