A three-pore model describes transport properties of bovine retinal endothelial cells in normal and elevated glucose

Diabetic retinopathy (DR) is the leading cause of blindness among working-age adults, but the mechanisms by which diabetes leads to retinal microvascular complications is still not completely understood. The initial stage of DR is marked by an elevated permeability of the blood vessels of the retina1 and subsequent plasma leakage to the interstitial space. Elevated glucose is believed to contribute to various vascular dysfunctions2,3—among them, loss of microvascular barrier integrity.4,5 However, an increase in VEGF that occurs even before morphologic abnormalities are observed has been described in patients with DR.6,7 The relative contribution of the direct effect of glucose compared with changes in cytokine expression on the inner blood–retinal barrier (iBRB) remains under investigation. Expression and organization of a well-developed tight junction (TJ) complex in the inner retinal capillaries contributes to the formation of the iBRB. Immunohistochemical staining for albumin performed in patients at different stages of DR has shown that these vessels are the primary site of the vascular leakage that results in retinal edema.1 However, while changes in lipid, albumin, and fluorescein accumulation in the retina are routinely observed as an indication of altered endothelial permeability in patients with diabetic retinopathy, little has been done to characterize the various routes of transport that may affect vascular permeability. Transport of molecules across the vascular endothelium may occur by transcellular pathways, including specific transporters or by paracellular transport, which includes transport across the junctional complex, across a broken junctional complex, or across a large gap caused by cell death or cell division.8 The two principal mechanisms driving paracellular transport of molecules and fluid across the endothelium are diffusion, which is movement from regions of higher concentration to regions of lower concentration, and convection, a mechanism of transport that results from the bulk motion of fluid typically driven by a pressure gradient. Molecular motion may differ from local fluid motion, because the solute molecules also diffuse simultaneously.9 Thus, oncotic pressure and hydrostatic pressure drive both fluid and solute transport, but the availability of various routes of transport controls the rate of flux across the endothelium. Information specific to the transport pathways of the iBRB is essential to understanding vascular permeability in DR; however, this information is difficult to obtain with in vivo models. Previous in vitro results suggested that hyperglycemia increases BREC diffusive permeability to small solutes,10,11 but more recent data on human retinal endothelial cells (HRECs) have suggested that upregulation of several cytokines causes endothelial dysfunction and not hyperglycemia, per se.12 Moreover, experiments in human retinal pigment epithelial cells (HRPECs) have demonstrated that hyperglycemia reduces the diffusive flux of both 40- and 70-kDa dextran, with no changes in TJs. In animal studies, increased permeability is normally observed anywhere from 1 to 6 months after streptozotocin-induced hyperglycemia,13 but this may be secondary to an increase in cytokines, such as IL-1 and TNF-α, and growth factors, such as VEGF. In the present study, we examined an in vitro model system of retinal endothelial cells to quantify fluid and solute transport after both elevated glucose and VEGF treatments. Results from this analysis allowed the development of a three-pore model—namely, transcellular transport through vesicles, broken TJs, and large pores, also termed leaky junctions. A determination of the fractional transport through each pore for various size solutes was calculated. Hyperglycemia was shown to have no effect on water or solute transport across the endothelium, whereas VEGF increased permeability in both low- and high-glucose concentrations. The data suggest that 6 days of high glucose have little direct effect on endothelial permeability and suggest that hyperglycemia in diabetes alters permeability indirectly through cytokine production.