We sought to investigate whether HG condition has an effect on mitochondrial morphology and function in retinal pericytes and whether such changes are associated with increased apoptosis. Pericyte loss is a well-documented pathology of the retinal capillaries during the early stages of nonproliferative diabetic retinopathy.6
Understanding the mechanisms by which hyperglycemia accelerates apoptosis of retinal pericytes may help identify novel targets for therapeutic intervention. The results from this present study indicate that exposure to HG caused a fragmentation in retinal pericytes, and prolonged HG caused mitochondrial membrane potential and oxygen consumption to decrease, along with decreased extracellular acidification. Importantly, the mitochondrial dysfunction seen with prolonged HG exposure appeared to underlie increased apoptosis of the retinal pericytes.
Our results demonstrate for the first time that HG condition can cause dramatic changes to mitochondrial morphology in retinal pericytes. This mitochondrial fragmentation was similar to that observed in retinal endothelial cells grown in an HG condition.34
The retinal pericytes also displayed a bi-phasic mitochondrial fragmentation, a rapid fragmentation in response to HG from which the pericytes recovered and a persistent fragmentation that occurred after 48 hours of HG. Bi-phasic mitochondrial fragmentation has been reported in other studies, and it has been shown that mitochondrial fission in response to acute HG exposure influences ROS overproduction.24,25
Although upregulation of the ROS level is closely associated with the first round of mitochondrial fragmentation and the recovery of morphology reduces ROS levels, the exact implication of these cellular events is currently unknown. We speculate that the morphology recovery is an attempt by the cells to recover against oxidative stress through regaining mitochondrial structural integrity; however, the cells also ultimately succumb to the HG insult and permanent mitochondrial fragmentation. Permanent mitochondrial fragmentation in retinal pericytes exposed to HG condition may play a significant role in promoting mitochondrial dysfunction and subsequent apoptosis, leading to pericytes loss in diabetic retinopathy.
Recent research has demonstrated how maintenance of mitochondrial morphology is critical for regulating mitochondrial metabolism, ROS production, and induction of apoptosis.35
Our previous report on retinal endothelial cells demonstrated how fragmentation of the mitochondrial network could compromise the metabolic capacity of the mitochondria in these cells when grown in an HG condition.34
Similarly, our data also indicate that HG-induced mitochondrial fragmentation may play a role in decreasing mitochondrial oxygen consumption. Mitochondrial membrane potential was similarly decreased and displayed significant heterogeneity within individual cells. The observed mitochondrial membrane potential heterogeneity may represent a functional manifestation of the more disconnected mitochondrial network, which ultimately may influence apoptosis.36
Consequently, mitochondrial morphology changes in both retinal endothelial cells and pericytes may exacerbate mitochondrial functional changes and the progression to apoptosis in an HG or diabetic condition.
The cause–effect relationship of mitochondrial fragmentation has recently been reported in endothelial cells derived from diabetic patients.37
Mitochondrial fragmentation was studied in the context of hyperglycemia in vascular cells, and the results showed increased mitochondrial fission as a contributing mechanism for endothelial dysfunction in a diabetic condition. In particular, Fis1
, a gene responsible for regulating mitochondrial fission, was overexpressed in the diabetic endothelial cells and was concomitant with mitochondrial fragmentation. Furthermore, when siRNA was used to prevent HG-induced Fis1
overexpression in a tissue culture model of diabetes, the mitochondrial fragmentation was observed to be significantly reduced, and ROS upregulation, a hallmark of diabetes, was also inhibited. In this study, we are reporting that HG induces mitochondrial fragmentation in retinal pericytes and that the morphology change is associated with mitochondrial dysfunction and subsequent apoptosis of the pericytes.
Since mitochondrial oxygen consumption is compromised in retinal pericytes exposed to HG, we assessed whether extracellular acidification was altered to analyze any changes in glycolytic levels of the cells. In retinal endothelial cells, we reported an increase in extracellular acidification levels, possibly to compensate for HG-induced decreased mitochondrial oxygen consumption. Interestingly, the retinal pericytes displayed a significant decrease in extracellular acidification. This inability to compensate for HG-induced decrease in mitochondrial oxygen consumption may indicate an increased susceptibility of the retinal pericytes to the HG insult. In addition, the differences seen between the pericytes and endothelial cells may be explained by differential transport of glucose between the two cell types. Previous reports have shown that HG downregulates Glut1 activity in pericytes, but not in endothelial cells.38
Thus, retinal pericytes and endothelial cells differentially alter extracellular acidification levels and possibly glycolytic levels in response to HG. Further research is needed to identify the mechanisms and consequences for the observed disparity.
The findings of this study indicate a novel step in the pathway for accelerated apoptosis in retinal pericytes, including a direct effect of HG on mitochondrial morphology. Permanent mitochondrial fragmentation, decreased mitochondrial metabolic capacity and extracellular acidification are observed in the pericytes grown in HG, along with increased apoptosis. The results provide further understanding of HG-induced mitochondrial dysfunction and how it may contribute to apoptosis of retinal pericytes. Preventing mitochondrial morphology changes in response to hyperglycemic environment may represent a novel therapeutic target for preventing pericyte loss in the early stages of diabetic retinopathy.