This is the first report indicating that diabetes reduces Cx43 expression in the retina and that a reduction in Cx43 expression promotes the development of AC and PL similar to the development seen in DR. Increases in the numbers of AC and PL in the diabetic mice were strikingly similar to those observed in the Cx43± mice, suggesting that a 50% decrease in Cx43 expression is sufficient to induce the development of AC and PL, as seen in diabetes. Data from TUNEL assay confirmed that the retinal vascular cell loss observed in the Cx43± mice resulted from apoptosis, as seen in the retinas of diabetic mice.
The current findings are supported by previous in vitro studies showing that high glucose reduces Cx43 gene expression in retinal endothelial cells and pericytes.21,22
More recently, Cx43 downregulation and a concomitant reduction in GJIC activity was shown to be sufficient to induce apoptosis.17
In line with our previous findings, a study showed that high glucose-induced Cx43 downregulation can occur through increased phosphorylation and elevated proteasomal degradation of Cx43.23
Additionally, cell-to-cell coupling in freshly isolated retinal pericytes was shown to be reduced in diabetic rats.5
Although these studies provide valuable information related to reduced Cx43 expression, decreased GJIC activity, and its damaging effects, the current findings provide evidence that diabetes-induced Cx43 downregulation can promote vascular cell loss in the retina. This implicates hyperglycemia-induced inhibition of Cx43 expression as a potential mechanism contributing to the development of AC and PL in diabetes.
Although altered Cx43 levels play a significant role in the development of complications in vascular diseases, their involvement in the pathogenesis of DR is unclear. In prehypertensive rats, the aortic endothelium exhibits decreased Cx43 expression and contributes to accelerated vascular cell wall remodeling.28
In late stages of human coronary atherosclerosis, Cx43 is downregulated, and this decline parallels the accumulation of extracellular matrix material.29
Interestingly, we have observed that high glucose-induced overexpression of extracellular matrix components regulates Cx43 expression.30
Although Cx43 is known to be widely expressed in the normal retina, its involvement in the development of vascular lesions in the diabetic retina has been unclear. In diabetes, differential expression of Cx43 has been reported in several organs and cell types: decreased Cx43 expression in delayed wound healing in the diabetic skin,9
marked decreases in Cx43 in endothelial cells related to compromised renal blood flow in diabetes,10
and time-dependent and cell-specific decreases in Cx43 in urinary bladder dysfunction in diabetes.11
Taken together, these reports and our current findings indicate that decreased Cx43 expression plays an important role in various pathologic conditions, including retinal vascular cell loss associated with DR.
It is unknown whether reduced connexin expression in the retina may affect other cellular events associated with the development of DR. The docking of connexin hemichannels between adjacent cells is facilitated by cadherin proteins.31
In nonproliferative DR, cadherin-5 expression in the retina is reduced,32
which may compromise interactions between cadherin and the cytoplasmic loop domain of Cx43.33
Such altered interactions could compromise barrier characteristics in the retinal vessels. Additionally, because blood-retinal barrier (BRB) breakdown in diabetes involves the decreased expression of tight junction protein ZO-1,34
which can alter Cx43 function35
through interaction with the COOH-terminal of Cx43,36
it is possible that in diabetes, BRB breakdown involves both tight junctions and gap junctions.
Although hyperglycemia is known to trigger apoptosis and to play a prominent role in retinal vascular cell loss, it is unclear through which mechanism hyperglycemia induces apoptosis. Increasing evidence suggests that cell death or cell survival modulators exchanged through gap junctions influence apoptotic events. However, despite several studies investigating the role of connexin in mediating apoptosis, the findings remain inconclusive largely because of the complexity involving the exchange of both cell death and cell survival signals that can pass through these channels. Additionally, the exchange of these modulators could be influenced by the cell type, the cellular context, and the impact connexin proteins have beyond their channel function on the cell death process. For example, connexin hemichannel–mediated alteration in signal transduction can affect mitochondrial functioning and could contribute to cell death. Taken together, these findings suggest that under high glucose or hyperglycemic conditions, a build-up of proapoptotic messengers including IP3, glutamate, and NAD+, among others, is accelerated because their ability to leave the cell is thwarted by reduced connexin channels. Our findings provide new evidence that diabetes-induced downregulation of Cx43 may disturb vascular homeostasis and that it promotes apoptosis in DR. Strategies to protect retinal vascular cells by regulating cell-to-cell communication in diabetes may have therapeutic value.