Endothelial progenitor cells (EPCs) mobilized and activated by neurotrophic factors may contribute to pathologic neovascularization in diabetic retinopathy

Abstract

Diabetic retinopathy is characterized by pathological retinal neovascularization. Accumulating evidence has indicated that high levels of circulating endothelial progenitor cells (EPCs) are an important risk factor for neovascularization. Paradoxically, the reduction and dysfunction of circulating EPCs has been extensively reported in diabetic patients. We hypothesized that EPCs are differentially altered in the various vasculopathic complications of diabetes mellitus, exhibiting distinct behaviors in terms of angiogenic response to ischemia and growth factors and potentially playing a potent role in motivating vascular precursors to induce pathological neovascularization. Circulating levels of EPCs from diabetic retinopathy patients were analyzed by flow cytometry and by counting EPC colony-forming units, and serum levels of neurotrophic factors were measured by enzyme-linked immunosorbent assay. We found increased levels of nerve growth factor and brain-derived neurotrophic factor in the blood of diabetic retinopathy patients; this increase was correlated with the levels of circulating EPCs. In addition, we demonstrated that retinal cells released neurotrophic factors under hypoxic conditions to enhance EPC activity in vitro and to increase angiogenesis in a mouse ischemic hindlimb model. These results suggest that neurotrophic factors may induce neoangiogenesis through EPC activation, leading to the pathological retinal neovascularization. Thus, we propose that neovascularization in the ischemic retina might be regulated by overexpression of neurotrophic factors.

Figure 1

Increased circulating EPC levels in DR group versus controls shown by flow cytometry analysis. Representative flow cytometry patterns from (A) a normal control and (B) a DR patient. EPCs were defined with positive expression for both CD133-allophycocyanin (forward scatter) and CD34-Pe-cy7 (side scatter). P4 (window) represents CD34 AC133 double positive populations. The graph below the photographs displays the statistical analysis comparison, which indicates an increased percentage of EPCs in DR group (DR: 0.093 ± 0.052%, vs. control: 0.045 ± 0.02%, n = 20, *P < 0.01).

Figure 2

Representative EPC-CFU. A: Phase contrast microscopy showing the early EPC-CFU in culture. The colony is seen as a central cluster of round cells with multiple spindle-shaped cells radiating from the periphery. B: VEGFR2-positive staining (red) seen by confocal microscopy.

Figure 3

NGF and BDNF serum levels. A: Increased NFG level in DR group as compared with other groups: 562.45 ± 125.78 (DR) versus 248.47 ± 102.32 (PAD) versus 243.56 ± 98.80 (control). B: Increased BDNF level in DR group as compared with other groups: 30.59 ± 9.93 (DR) versus 13.71 ± 4.19 (PAD) versus 16.76 ± 5.36 (control). *P < 0.05.

Figure 4

Two-chamber co-culture with retinal cells under hypoxic conditions enhances EPC cell migration. In a transwell co-culture system under hypoxic conditions with an 8 μm hole-size (purple) filter, many cells (yellow) stayed in the upper chamber (A) and few cells passed through the filter to the lower chamber (C) when co-cultured for 6 hours with HMVECs. In contrast, after co-culture with retinal cells, many more EPCs passed through the holes into the lower chamber (D) and fewer cells stay in the upper chamber (B).

Figure 5

Retinal-cell–conditioned medium enhances EPC differentiation and tube formation in culture. Enhanced EPC cell differentiation is shown by representative immunostaining in the groups supplemented with retinal-cell–conditioned medium (A), as compared with controls without added conditioned medium (B). Red: VEGFR2 staining; green: VE-cadherin staining; blue: nuclear staining. Yellow staining denotes overlap of green and red. Tube formation of EPCs stimulated by retinal-cell-conditioned culture medium (C), as compared with controls (D).

Figure 6

Increase in neurotrophin expression in retinal glia cell culture medium induced by hypoxia. Increased neurotrophin expression under hypoxic conditions included NGF, BDNF, GNDF (NGF: 545.68 ± 120.70 pg/ml vs. 712.90 ± 116.78; pg/ml; BDNF: 292.00 ± 92.08 pg/ml vs. 610.03 ± 148.42 pg/ml; GNDF: 28.94 ± 8.29 pg/ml vs. 71.07 ± 13.44 pg/ml; as compared with the control cultures at normoxic conditions. *P < 0.01.

Figure 7

By 20 days after injection, retinal-cell–conditioned culture medium versus control treatment was associated with improved perfusion to the ischemic limb and increase in capillary density in ischemic gastrocnemius muscle. A: Representative photograph of the laser scan perfusion image. Changes in the perfusion ratio were calculated as the perfusion ratio at follow-up (20 days postinjection) minus the ratio preinjection (10 days post surgery, labeled day 0). Graphs to the right of photographs represent the quantitative assessments with n = 10/group for comparison. There was a marked increase in limb perfusion ratios on day 20 after injection with retinal-cell–conditioned medium versus control treatment (n = 10/group *P < 0.01.) B: Left, a representative CD31 immunohistochemistry staining for capillary density; red dots indicate endothelial cells (CD31 positive cells marking capillaries) (magnification ×200). Graphs to the right of photographs represent the quantitative assessments with n = 10/group for all comparisons. Capillary density was increased with conditioned medium versus control treatment, *P < 0.01.