Diabetic retinopathy (DR) is one of the major complications of diabetes mellitus and the main cause of blindness in industrialized countries. Events in the genesis and early development of DR include the loss of retinal pericytes, thickening of the capillary basement membrane, and increased leukocyte adhesion to retinal endothelial cells (RECs). It is now a generally accepted concept that the pathogenesis of DR is driven by deregulated expression of growth factors. Of these, vascular endothelial growth factor (VEGF) is considered most important because its levels are substantially elevated in the vitreous fluid and in the retinal vasculature of patients with DR.
1–3 Although different VEGF-binding receptors have been identified, its effects on cellular proliferation, migration, and permeability are mainly mediated through VEGF receptor 2.
4,5 VEGF is also an activator of several isoforms of serine/threonine-specific protein kinase C (PKC) in endothelial cells (ECs), and it seems possible that pathogenic processes depend on this pathway.
6–9 The PKC family includes several members that differ in their structure, required cofactors, and substrate specificity. They can be divided into three subgroups: conventional (α, βI/II, and γ), novel (δ, ε, and θ), and atypical (ζ and ι/λ) PKC. Activation of PKC by hyperglycemia, especially of isoform β, has been implicated in the processes deregulated in DR.
6,8,10 Hyperglycemia also results in upregulated expression of VEGF and components of the extracellular matrix that contribute to elevated permeability of RECs, probably leading to macular edema in DR.
6,11–13 Several in vitro studies showed that altered permeability of ECs induced by VEGF
165 was associated with changed cellular localization, expression, and/or modification of tight junction (TJ) proteins.
11–17 These complexes of membrane proteins like claudins or occludin and membrane-associated proteins (e.g., ZO-1) are crucial determinants of paracellular signaling of ECs.
18,19 Site-specific phosphorylation of occludin induced by short-term treatment with VEGF
165 of bovine retinal endothelial cells (BRECs) may interfere with TJ assembly.
14,16,20 However, in these previous investigations, the long-term effects of VEGF that should be more relevant in the pathologic situation in vivo were neglected. We showed that long-term treatment of immortalized (i)BRECs with VEGF
165 indeed resulted in delocalization of the TJ proteins occludin and claudin-1 from the plasma membrane. This result was not associated with a change in expression or a modification of occludin, but expression of claudin-1 was markedly reduced.
17 Claudin-1 is expressed by microvascular endothelial cells of the brain and retina, although at a lower level than other claudins.
17,21,22 It is an essential part of TJ in the mammalian epidermal barrier and claudin-1-deficient mice die soon after birth.
23 In human Caco-2 cells, claudin-1 is phosphorylated at serine/threonine residues, and PKCθ is involved in the regulation of its expression and cellular localization.
24 Based on these previous observations, we studied whether PKC is involved in long-term effects of VEGF
165 on claudin-1 and permeability of iBRECs, and whether the presence of claudin-1 is important in proper barrier function in these cells.