Altered expression of genes related to blood–retina barrier disruption in streptozotocin-induced diabetes

Abstract

Disruption of the blood–retina barrier (BRB) is an early phenomenon in preclinical diabetic retinopathy (PCDR). Two vascular permeability pathways may be affected, the paracellular pathway involving endothelial cell tight junctions, and the endothelial transcellular pathway mediated by endocytotic vesicles (caveolae). The relative contribution of both pathways to vascular permeability in PCDR is unknown. We compared transcription levels in entire rat retina of genes related to these pathways between control conditions and after 6 and 12 weeks of streptozotocin-induced diabetes, as well as in bovine retinal endothelial cells (BRECs) exposed to VEGF and bovine retinal pericytes (BRPCs), using real-time quantitative RT-PCR. To confirm endothelial-specificity, immunohistochemical staining was performed in rat retina, and mRNA transcript levels were compared between BRECs and BRPCs. mRNA and protein of most paracellular transport-related genes were specifically expressed by retinal endothelial cells, whereas vesicle transport-related mRNA and proteins were present in various retinal cell types, including endothelial cells. Expression of selected endothelial cell tight junction genes and particularly that of occludin and claudin-5 was reduced in the diabetic retina and in BRECs after exposure to VEGF. Expression of 6 out of 11 vesicular transport-related genes was upregulated after induction of diabetes. Of these, only plasmalemma vesicle-associated protein (PV-1) was exclusively expressed in BRECs and not in BRPCs. PV-1 transcription was markedly induced in diabetic retina and by VEGF in BRECs. Caveolin-1 immunostaining was primarily found in the retinal vasculature, and its mRNA levels in BRECs were highly abundant and VEGF-inducible. Whereas the endothelial tight junction genes occludin and claudin-5 showed a transient downregulation, we observed long-term upregulation in diabetic retina and VEGF-induced expression in BRECs of the vesicular transport-related genes caveolin-1 and PV-1. The altered gene expression profiles observed in this study suggest a transient induction of the paracellular pathway and prolonged involvement of transcellular endothelial transport mechanisms in the increased permeability of retinal capillaries in PCDR.

Introduction

Blood–retinal barrier (BRB) loss is crucial in the pathogenesis of diabetic retinopathy. In preclinical DR it occurs early as diffuse retinal vascular permeability and may represent an important mechanism accelerating the early retinal changes caused by diabetes (Frank, 2004). In the later clinical stage of DR, diabetic macular edema from focal profuse vascular leakage due to BRB loss is the major cause of loss of vision in patients with DR, particularly in non-proliferative DR in type 2 diabetes mellitus (Fong et al., 2004).

Two mechanisms have been proposed as possible causes of vascular leakage in DR; i.e., increased paracellular leakage due to changes in integrity of endothelial tight junctions, and increased transendothelial transport mediated by caveolae (Antonetti et al., 1998, Antonetti et al., 1999, Barber and Antonetti, 2003, Brankin et al., 2005, Cunha-Vaz, 1980, Hofman et al., 2000, Vinores et al., 1993). Retinal vascular permeability has been associated with the presence of extravascular albumin (Schlingemann et al., 1999, Vinores et al., 1998), decreased tight junctional staining (Antonetti et al., 1998, Antonetti et al., 1999, Barber and Antonetti, 2003, Brankin et al., 2005) and upregulation of the number of intra-endothelial pinocytotic vesicles (Cunha-Vaz, 1980, Hofman et al., 2000, Vinores et al., 1993). Leaky endothelial tight junctions have been demonstrated in retinas of diabetic rabbits (Vinores et al., 1998), retinas of rats with streptocotozin (STZ)-induced diabetes and in rats after intraocular injection with VEGF (Antonetti et al., 1999, Barber and Antonetti, 2003). However, few or no leaky endothelial tight junctions have been observed in retinas of diabetic humans and in retinas of rats with galactose-induced diabetes (Vinores and Campochiaro, 1989).

To establish the possible contribution of each of these mechanisms to retinal vascular leakage, we investigated in rat retina the expression of a set of relevant endothelial tight junction genes and vesicular transport-related genes and determined their altered expression in STZ-induced diabetes.

Ten tight junction proteins, occludin, claudin-1, -5 and -12, junction adhesion molecule (JAM)1, -2 and -3, endothelial cell-selective adhesion molecule (ESAM), poliovirus receptor-related 1 (PVRL1, also known as nectin) and zonula occludens (ZO-1) were selected (Fig. 1, Table 3) (Dejana, 2004, Harhaj and Antonetti, 2004). Of these, occludin, claudin-5 and ZO-1 have been studied previously in the context of BRB disruption, demonstrating reduced protein levels and reduced phosphorylation of occludin and ZO-1 in diabetes and vascular endothelial growth factor (VEGF)-induced vascular permeability (Antonetti et al., 1999, Barber and Antonetti, 2003). Only sparse data is available on the presence and distribution patterns of other tight junction proteins. Three adherens junction proteins, VE-cadherin, β-catenin and N-cadherin were selected (Fig. 1, Table 3) (Dejana, 2004). VE-cadherin and β-catenin, which form a complex in adherens junctions, were found to be essential in endothelial cell survival (Carmeliet et al., 1999). Their protein expression is decreased after exposure to VEGF (Kevil et al., 1998, Wright et al., 2002), and albumin-derived advanced glycation end products (Otero et al., 2001). Tyrosine phosphorylation of this complex is associated with increased permeability (Esser et al., 1998). Furthermore, decreased VE-cadherin protein expression was observed in a patient with DR (Davidson et al., 2000) and diabetes-induced reduction in VE-cadherin expression was reported to be induced by proteolytic degradation (Navaratna et al., 2007). N-cadherin is a cell–cell adhesion molecule that is involved in binding of endothelial cells and pericytes (Gerhardt et al., 1999), but was also found to be expressed in neural cells of the retina (Balsamo et al., 1991, Gerhardt et al., 1999, Matsunaga et al., 1988).

Eleven vesicular transport-related proteins were selected based on their presence in endothelial cells and/or their interaction with caveolin-1 (Fig. 1, Table 3). Plasmalemma vesicle-associated protein (PLVAP also known as PV-1 or PAL-E) is an endothelial cell-specific pinocytotic vesicle component (Schlingemann et al., 1985, Stan et al., 2001, Niemela et al., 2005) that is upregulated in human DR (Schlingemann et al., 1999) and monkey retina after VEGF treatment (Hofman et al., 2000, Hofman et al., 2001a). Caveolin-1 is an essential component of caveolae (Drab et al., 2001). Diabetes-induced rat caveolin-1 overexpression in lung alveolae is accompanied by elevated permeability due to increased transcytosis, whereas paracellular transport remains unchanged (Pascariu et al., 2004). Dynamin 2, which directly interacts with caveolin-1 (Yao et al., 2005), plays a regulatory role in VEGFR2-mediated endothelial signaling (Bhattacharya et al., 2005). Dynamin-1 and -2 are found in the retina (Sherry and Heidelberger, 2005, Sontag et al., 1994), and bind PACSIN2 (also known as syndapin-II; Kessels et al., 2006). PACSIN2, NSF, SNAP25 and VAMP (vesicle-associated membrane protein)-1 and -2 are all part of the SNARE-complex and both VAMPs are expressed in the mouse retina (Sherry et al., 2003). Flotillins-1 and -2 are integral membrane proteins of caveolae which have been identified in mouse retina (Lang et al., 1998).

Leakage of retinal vessels is an early step in DR but the mechanisms involved are not fully understood. Therefore, we analyzed the presence and regulation of mRNA expression of endothelial paracellular and transcellular pathway-related genes by real-time quantitative RT-PCR in entire retinas of control and diabetic rats and cultured bovine retinal endothelial cells (BRECs) with or without VEGF stimulation and pericytes (BRPCs). Furthermore, the location of protein in the rat retina was investigated by immunohistochemistry.