In this study, we describe a novel mechanism of pericyte loss in experimental diabetic retinopathy. We demonstrated that pericyte loss in diabetic retinopathy is restricted to a subset of pericytes located on straight capillaries. Hyperglycemia-induced loss of pericytes, predominantly on straight capillaries, is accompanied by increased numbers of pericytes, migrating from the same location into the perivascular position. Increased numbers of migrating pericytes are LacZ negative. Furthermore, we show that Ang-2 is crucial for pericyte migration, since Ang-2–deficient mice completely lack hyperglycemia-induced pericyte migration, whereas overexpression of Ang-2 mimics increased pericyte migration observed in diabetic retinas.
Experimental long-term hyperglycemia enhances pericyte detachment, leading to reduced pericyte coverage of retinal capillaries. Growing evidence suggests that pericyte detachment and migration from underlying vessels into the perivascular parenchym is a general feature of pericytes in response to different kinds of stress inducers. In brain capillaries, pericytes migrate from capillaries as a result of ischemia, hypoxia, or injury (40
). In response to traumatic brain injury, ~40% of capillary pericytes migrated from their microvascular location and remained in a perivascular position, while remaining pericytes on capillaries displayed signs of degeneration. In tracheal capillaries, blockage of VEGF signaling resulted in migration of pericytes from the regressing capillaries onto surviving vessels, leaving behind empty basement membrane tubes, comparable to those observed in diabetic retinopathy (42
). These data show that pericyte attachment is actively modulated depending on environmental conditions.
In the diabetic retina, pericyte loss is one of the earliest and most characteristic morphological changes. Our previous data revealed the importance of the Ang-2/Tie-2 system in the regulation of vascular morphological changes in the diabetic retina. In diabetic rat retina, Ang-2 is upregulated before pericyte loss becomes evident, and increased expression of Ang-2 correlates with the depletion of perivascular cells (33
). Intravitreal injection of Ang-2 results in pericyte dropout, and Ang-2 haploinsufficiency appears to protect against diabetes-induced pericyte dropout. Now, we show that Ang-2 regulates pericyte migration in experimental diabetic retinopathy. In loss of function experiments, we show that Ang-2 is required for hyperglycemia-induced migration of pericytes, since Ang-2–deficient mice lack increased migration of pericytes compared with diabetic WT mice. In addition, gain of function experiments showed that overexpression of Ang-2 enhances pericyte migration in nondiabetic and diabetic animals. The extent of pericyte migration, induced by Ang-2 overexpression in nondiabetic retinas, was similar to the one observed in WT diabetic animals, highlighting the importance of the Ang-Tie system in cellular crosstalk and its involvement in the earliest stages of diabetic retinopathy. Nevertheless, we found a trend for a hyperglycemia-induced increase in pericyte migration in Ang-2–overexpressing and –deficient retinas, suggesting that other factors than Ang-2 may be of some importance for pericyte migration. Recent observations in microvascular endothelial cells revealed that high glucose increases the expression of Ang-2 through modification of corepressor mSim3A by methylglyoxal, an important intracellular advanced glycation end product (AGE) (43
). Because methylglyoxal is the predominant intracellular AGE in the diabetic retina, this study links biochemical changes of diabetes to increased activation of the Ang-2/Tie-2 system and therefore to our observation that hyperglycemia enhances detachment and migration of pericytes from microvasculature, resulting in the typical pericyte loss of diabetic retinopathy.
Moreover, our data demonstrate that hyperglycemia-induced loss of pericytes is not equal in retinal pericyte subpopulations, such as it is restricted to pericytes located on straight capillaries of the retinal microvasculature. Straight capillaries are the only sites from which pericytes migrate. According to our observations, hyperglycemia reduces the number of S-PCs by ~460 cells per mm2 of capillary area after 6 months of hyperglycemia and simultaneously increases the numbers of migrating pericytes by ~80 cells per mm2 of capillary area. The gap between the numbers of lost and migrating pericytes is partly explained by the digestion process, since this eliminates pericytes that are completely dissociated. Furthermore, the retinal digest preparations represent snapshots of the permanently remodeling vasculatures. The quantitative contribution of pericyte migration to pericyte loss in experimental diabetic retinopathy and the destiny of dissociated pericytes remain to be established.
LacZ in XLacZ mice labels ~52% of all pericytes and is therefore a restricted pericyte marker, such as others. Nevertheless, quantitation of total pericyte numbers and the expression of LacZ showed that only LacZ-positive pericytes are significantly reduced in diabetic retinas. Therefore, quantification of LacZ-expressing pericytes may provide a simplified method for the quantification of pericyte loss in experimental diabetic retinopathy. Furthermore, we showed that increased numbers of migrating pericytes rise from pericytes that are LacZ negative. This is in agreement with published data showing that proliferation and migration of LacZ-expressing cells are associated with transgene downregulation and vessel injury leads to changes in marker expression in pericyte subpopulations (29
The causes of early pericyte loss in diabetic retinopathy have not been fully delineated. Numerous studies demonstrated that increased levels of glucose and AGEs trigger pericyte death in cell culture and that pericyte apoptosis is increased in human diabetic retinas as well as in experimental diabetic retinopathy (44
). However, pericyte apoptosis fails to explain the total extent of pericyte loss and its time course in experimental diabetic retinopathy. In animal models of diabetic retinopathy, pericyte apoptosis has been detected at later stages, after more than 6 months of hyperglycemia (31
). Not compatible with this observation is that significant pericyte loss is already detectable after 3 months of experimental diabetes (33
). Pericyte migration, as an alternative or additional mechanism of pericyte loss in diabetic retinopathy, offers a possible explanation for the discrepancy between total extent of pericyte loss and published data of pericyte apoptosis in the diabetic retina.
In summary, our data provide morphological evidence that pericyte migration represents a novel mechanism of pericyte loss in the diabetic retina. We further show that this mechanism is regulated by signaling via the Ang-2/Tie-2 pathway. The exact mechanism underlying this process and the destiny of resting and migrating pericytes remain to be investigated.