of the angiopoietin-Tie system in diabetic pericyte loss, the expression of Ang-1 and -2 was studied in a diabetic rat model, in which the onset of pericyte dropout is exactly known. It was found that Ang-2 is 37-fold upregulated prior to the onset of pericyte dropout. Injection of recombinant Ang-2 into the vitreous of nondiabetic rats reproduced pericyte dropout within days, and the 25% pericyte loss of diabetic C56BL6/J mice after 6 months of diabetes was abolished in a diabetic mouse with a 50% reduction of Ang-2 gene dose (101). Constitutive overexpression of Ang-2 in photoreceptor cells induces reduced pericyte coverage in the deep capillary layers of the retina (102). Diabetes aggravates the pericyte deficiency and promotes acellular capillaries over time, similarly to the changes in PDGF-B+/− mice. These data suggest that hyperglycemic regulation of angiopoietin-2 plays a crucial role in the survival of pericytes in the diabetic retina and the subsequent development of pathology. This notion leads to the question of the regulation of growth factors such as angiopoietin-2 by glucose and its intracellular metabolites. This will be addressed later.

VEGF-VEGFR2

VEGF is a major determinant of retinal vascular development and responses to injury. The murine VEGF gene is alternatively transcribed to yield the VEGF120, VEGF164, and VEGF188 isoforms, which differ in their potential to bind to heparan sulfate. While the VEGF164 mouse yielded regular pericyte-recruiting patterns in all vascular beds, the VEGF120 mouse had major defects in pericyte recruitment to capillaries and arterioles, but not to venules. As signs of impaired vessel maturation and patency, extravasations of red blood cells were also observed. Endothelial cells provide pericyte-recruiting factors like PDGF-B, and VEGF120 mice had lower PDGF-B expression suggesting that the defect was secondary to the endothelial defect. Endothelial cells of larger vessels express VEGF-R1, but the affinity of VEGF120 to this receptor is lower than other VEGF isoforms. It is thus unclear whether this axis contributes significantly to pericyte recruitment to capillaries in the retina. The 188 isoform of VEGF did not yield a defect in pericyte recruitment to capillaries. In essence, VEGF may play a significant role in pericyte recruitment, but the specific and predominant mechanism working in the eye, and in particular in the capillary network, may be difficult to dissect (103).

IGF-1/IGF-R

The growth hormone-IGF-1-IGF-1 receptor system has been involved in the pathogenesis of proliferative diabetic retinopathy since the early days when pituitary ablation was the only feasible approach. However, the question of a possible involvement in incipient changes was only addressed recently by Ruberte et al. They used a mouse overexpressing IGF-1 in several organs, including the retina. The transgene was found in the outer nuclear layer and in photoreceptors. After 2 months of paracrine IGF-1 exposure, quantitative retinal morphometry in retinal digest preparations revealed an almost 50% reduction of pericyte numbers, while the numbers of endothelial cells remained unexpectedly unchanged. Of note, acellular capillaries were more numerous already at this early time point. In analogy to cells exposed to high glucose in the presence of low IGF-1 the authors claimed a cooperative effect of glucose and IGF-1 on the observed ocular alterations (104).