Microvascular leakage in diabetic retinopathy

Background

Diabetic macular edema (DME) is a major cause of loss of vision in patients with diabetic retinopathy (DR), in particular in non-proliferative DR (NPDR) in type 2 diabetes mellitus (DM). In the normal retina, the neural tissues are protected by the blood-retinal barrier (BRB), which is formed by specialized endothelial cells which have inter-endothelial tight junctions and an array of specific transcellular transport mechanisms. DME is characterized by focal profuse vascular leakage and swelling of the retina, caused by loss of the BRB in the retinal vasculature near areas of ischemia. Despite the introduction of laser treatment which may arrest further visual loss in some patients, DME is still the most important cause of blindness in DM.

Interestingly, also in PCDR subtle changes in the BRB occur and lead to diffusively increased retinal vascular permeability, a change which could be a crucial step in the progression to NPDR. Therefore, understanding of the pathogenesis of BRB loss in these two stages of DR may allow development of more effective therapeutic strategies to treat DME and possibly even open avenues to prevent progression from PCDR to NPDR.

Two main cellular mechanisms have been proposed to cause BRB loss in DR: increased paracellular transport caused by changes in inter-endothelial tight junction integrity, and increased transendothelial transport mediated by caveolae. Caveolae have been studied to a limited extent, since the molecular constituents of these transport vesicles were only identified recently. Most studies investigating increased permeability in the retina have focused on paracellular transport and tight junction integrity.

In recent years, the molecular content of caveolae has been elucidated allowing studies into their functional significance in health and disease. Caveolin-1 (Cav-1) was shown to be essential for the presence and function of caveolae, and plasmalemma vesicle protein 1 (PLVAP or PV-1) was very recently identified as the target for PAL-E, an antibody developed more than 20 years ago by our group [1]. We want to investigate what the relative contribution is of transcellular transport as compared to paracellular transport.

Preliminary Research

In previous studies we have found several lines of evidence that caveolae play an important role in BRB loss in DR, in addition to paracellular transport:

  PLVAP (PAL-E), which is absent from normal blood-brain barrier endothelium, is associated in the brain and eye with barrier disruption, such as in tumors, acute ischemia and DR [1-10].

We have shown increased expression in human leaky retinal vessels of PLVAP [4], and showed that PLVAP is associated with caveolae by immunoelectron microscopy [1; Fig.1].

Fig.1. Immunolabeling of endothelial cells by antibody PAL-E. Note the particles on the exterior of endothelial vesicles (arrowheads and inset). x78,000; Inset x175,000. Schlingemann et al. 1985 [1].

In ultrastructural studies of experimental VEGF-induced retinopathy in monkeys, we observed an altered distribution of caveolae in endothelial cells in association with vascular leakage caused by VEGF. In healthy capillaries of the BRB, caveolae were located mainly at the abIuminal side of endothelial cells, whereas endothelium of leaky vessel showed caveolae mainly at the luminal side [10].

  In VEGF-induced retinopathy in monkeys, we found that PLVAP expression is induced by VEGF in vivo [10], whereas in cultured endothelial cells, PLVAP expression is also induced by VEGF [11].

  In molecular studies of human and rodent DR, we have shown increased mRNA levels of caveolin-1 (Cav-1) and PLVAP in the diabetic retina [11]. These findings suggest that these proteins may have functional relevance for vascular leakage in DR.

References:

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