Reduced Connexin 43 Expression and Its Effect on the Development of Vascular Lesions in Retinas of Diabetic Mice

Gap junctions are membrane channels that allow the transfer of ions and small molecules <1 kDa1 between adjacent cells for the maintenance of vascular homeostasis.2,3 Each of these channels is composed of two connexin hemichannels that couple with one another to form a functional gap junction channel.4 The presence of gap junctions in the retina and an extensive cell-to-cell coupling within the retinal microvessels have been well documented.5 Importantly, an abundant expression of Cx43 in the retinal endothelial cells and pericytes6 suggests substantial gap junction coupling in the retinal vascular cells. Studies investigating gap junctions between pericytes and endothelial cells in culture identified the junctional transfer of small molecules.7 Examination of human retinal capillaries has revealed that the basement membrane interposed between endothelial cells and pericytes is absent at specific points to permit cell membrane contacts8 that could facilitate gap junction activity between these cells. Abnormal connexin expression in diabetes is associated with the development of various complications involving the skin,9 kidneys,10 bladder,11 perineurium,12 lens,13 heart,14 and other tissues; however, its role in the development of vascular lesions in the diabetic retina remains unknown. Although hyperglycemia is known to induce apoptosis and trigger retinal vascular cell loss, a hallmark of background DR, the mechanisms underlying hyperglycemia-induced apoptosis are not completely understood. Increasing evidence indicates that compromised gap junction activity can have serious consequences, including the initiation of apoptosis and the breakdown of vascular homeostasis. Gap junction-mediated exchange of small molecules, such as nucleotides, cAMP, IP3, and Ca2+, plays a critical role in cell function and viability.15 Studies investigating the role of reduced gap junction intercellular communication (GJIC) activity on cell survival or apoptosis have reported that the downregulation of Cx43 expression increases apoptosis in rat ventricular myocytes16 and microvascular endothelial cells.17 Increased apoptosis was reported in Cx43± mice in which astrocytic gap junctional communication was decreased.18 Further studies with Cre mice with floxed Cx43 lacking the Cx43 gene in astrocytes showed increased apoptosis in cells representing penumbral lesions.19 Similarly, osteoblast cells derived from Cx43-null mice exhibited increased apoptosis.20 Taken together, these studies suggest that an alteration in Cx43 expression profoundly affects cell survival. Our previous studies have shown that high glucose downregulates Cx43 expression in microvascular endothelial cells and retinal pericytes, with concomitant reductions in GJIC activity.21,22 Recently, we have shown that high glucose-induced downregulation of Cx43 expression is an early trigger for inducing apoptosis in retinal endothelial cells.17 In line with our findings, other reports have indicated high glucose-induced Cx43 downregulation in retinal endothelial cells in vitro.23 Studies investigating pericytes isolated from diabetic rat retinas have shown a dramatic reduction in cell-to-cell coupling.5 Although these studies demonstrate that high glucose-induced downregulation of Cx43 expression and GJIC promotes apoptosis in vitro, it is unknown whether diabetes promotes similar changes in vivo in the retina, contributing to retinal vascular cell loss and the breakdown of retinal vascular homeostasis associated with DR. In this study, we investigated whether diabetes-induced downregulation of Cx43 expression promotes characteristic microvascular cell loss in two animal models, the streptozotocin-induced diabetic mouse and the Cx43± mouse.