Human CD133+ progenitor cells promote the healing of diabetic ischemic ulcers by paracrine stimulation of angiogenesis and activation of Wnt signaling

Abstract

We evaluated the healing potential of human fetal aorta-derived CD133(+) progenitor cells and their conditioned medium (CD133(+) CCM) in a new model of ischemic diabetic ulcer. Streptozotocin-induced diabetic mice underwent bilateral limb ischemia and wounding. One wound was covered with collagen containing 2x10(4) CD133(+) or CD133(-) cells or vehicle. The contralateral wound, covered with only collagen, served as control. Fetal CD133(+) cells expressed high levels of wingless (Wnt) genes, which were downregulated following differentiation into CD133(-) cells along with upregulation of Wnt antagonists secreted frizzled-related protein (sFRP)-1, -3, and -4. CD133(+) cells accelerated wound closure as compared with CD133(-) or vehicle and promoted angiogenesis through stimulation of endothelial cell proliferation, migration, and survival by paracrine effects. CD133(+) cells secreted high levels of vascular endothelial growth factor (VEGF)-A and interleukin (IL)-8. Consistently, CD133(+) CCM accelerated wound closure and reparative angiogenesis, with this action abrogated by co-administering the Wnt antagonist sFRP-1 or neutralizing antibodies against VEGF-A or IL-8. In vitro, these effects were recapitulated following exposure of high-glucose-primed human umbilical vein endothelial cells to CD133(+) CCM, resulting in stimulation of migration, angiogenesis-like network formation and induction of Wnt expression. The promigratory and proangiogenic effect of CD133(+) CCM was blunted by sFRP-1, as well as antibodies against VEGF-A or IL-8. CD133(+) cells stimulate wound healing by paracrine mechanisms that activate Wnt signaling pathway in recipients. These preclinical findings open new perspectives for the cure of diabetic ulcers.

Figure 1

Accelerated wound closure in the presence of CD133⁺ cells (A, middle) is associated with higher temporary wound capillarization at day 7 (B). Capillary density declines to levels of wounds treated with CD133⁻ cells or collagen gel alone by day 14 (C) (n=12 mice per group). *P<0.05 vs collagen, +P<0.05 vs CD133⁻ cells. Bar=20 μm.

Figure 2

Endothelial cell proliferation is stimulated in diabetic wounds by CD133⁺ cells (A), whereas apoptosis is reduced (B). CD133⁻ cells did not influence endothelial apoptosis or proliferation (n=5 per group). *P<0.05 vs collagen, +P<0.05 vs CD133⁻ cells. Bar=10 μm.

Figure 3

Cytometric bead array indicated higher secretion of interleukins (A), GFs (B), and chemokines (C) by CD133⁺ cells as compared to CD133⁻ cells (n=4 per group). **P<0.01, ***P<0.005 vs CD133⁻ cells.

Figure 4

Administration of CD133⁺ CCM facilitated wound closure in diabetic animals over a time course of 14 days, whereas CD133⁻ CCM was ineffective (A). Wound closure was accompanied by temporarily higher capillarization of wounds treated with CD133⁺ CCM on day 7 (B and D). Capillary density decreased to levels only slightly above wounds treated with collagen alone by day 14 (C). CD133⁻ CCM did not accelerate wound closure or stimulate capillarization at any time point (A through C) (n=6 per group). **P<0.01 vs collagen, ++P<0.01 vs CD133⁻ cells. Bar=10 μm.

Figure 5

Treatment of HUVECs with CD133⁺ CCM enhanced network formation (A), gap closure (B), and chemotaxis (C), whereas CD133⁻ CCM was less potent. CD133⁺ CCM blunted HG-induced apoptosis (D). Data are means±SEM. **P<0.01, ***P<0.005 vs nonconditioned medium (NCCM), $P<0.05 vs CD133⁻ cells, ##P<0.01 vs normal glucose.

Figure 6

Wound closure, facilitated by unmodified CD133⁺ CCM, is retarded as compared to vehicle, when Wnt antagonist sFRP-1 is added (A). Treatment of wounds with sFRP-1 in the absence of CD133⁺ CCM did not modulate wound closure (A). Likewise, capillarization of wounds was supported by CD133⁺ CCM only in the absence of sFRP-1 (B) (n=7 per group). *P<0.05, **P<0.01, ***P<0.005 vs NCCM, +++P<0.005 vs CD133⁺ CCM.