Role of endothelial progenitor cells and inflammatory cytokines in healing of diabetic foot ulcers

Abstract

Background: To evaluate changes in endothelial progenitor cells (EPCs) and cytokines in patients with diabetic foot ulceration (DFU) in association with wound healing.

Methods: We studied healthy subjects, diabetic patients not at risk of DFU, at risk of DFU and with active DFU. We prospectively followed the DFU patients over a 12-week period. We also investigated similar changes in diabetic rabbit and mouse models of wound healing.

Results: All EPC phenotypes except the kinase insert domain receptor (KDR)(+)CD133(+) were reduced in the at risk and the DFU groups compared to the controls. There were no major EPC differences between the control and not at risk group, and between the at risk and DFU groups. Serum stromal-cell derived factor-1 (SDF-1) and stem cell factor (SCF) were increased in DFU patients. DFU patients who healed their ulcers had lower CD34(+)KDR(+) count at visits 3 and 4, serum c-reactive protein (CRP) and granulocyte-macrophage colony-stimulating factor (GM-CSF) at visit 1, interleukin-1 (IL-1) at visits 1 and 4. EPCs tended to be higher in both diabetic animal models when compared to their non-diabetic counterparts both before and ten days after wounding.

Conclusions: Uncomplicated diabetes does not affect EPCs. EPCs are reduced in patients at risk or with DFU while complete wound healing is associated with CD34(+)KDR(+) reduction, suggesting possible increased homing. Low baseline CRP, IL-1α and GM-CSF serum levels were associated with complete wound healing and may potentially serve as prognostic markers of DFU healing. No animal model alone is representative of the human condition, indicating the need for multiple experimental models.

Figure 1. An example of flow cytometric analysis of human peripheral blood sorted on CD45^dim cells in a patient with DFU (A) and a healthy control subject (B).

The triple positive phenotype (CD34⁺/KDR⁺/CD133⁺) was determined by gating the CD133⁺ cells on the CD34⁺/KDR⁺. 1.000.000 events per sample were acquired and the counts for each phenotype are shown in the picture. Smaller counts in all phenotypew were observed in the diabetic patient (A) when compared to the healthy subject (B) in the double and triple measurements.

Figure 2. Changes in EPCs measurements during the four study visits between the patients who did not heal their ulcers (NH) and those who did (H).

There were no differences in all EPC measurements at baseline but patients who healed their ulcers had lower CD34⁺KDR⁺ counts at visits 3 and 4 and CD34⁺CD133⁺ at visit 4. Data are presented as the median and interquartile range box.

Figure 3. Changes in CRP (3a), IL-1a (3B) and GM-CSF (3C) during the four study visits between the patients who did not heal their ulcers (NH) and those who did (H).

Data are presented as the median and interquartile range box. Patients who healed their ulcers had lower serum CRP and GM-CSF at visit 1 and lower IL-1α at visits 1 and 4.

Figure 4. A and B: Forearm skin biopsy staining for SDF-1in a diabetic patient (Figure 4A) and a healthy control subject (Figure 4B), (frozen sections, x100).

SDF-1 was expressed by stromal cells (black arrows) and endothelial cells (red arrows) and the staining pattern was mostly cytoplasmic and occasionally nuclear in cases of increased expression. The number of stained stromal cells and the intensity of staining were increased in in diabetic patients while no difference was found in the number of stained endothelial cells. C and D: Foot skin staining for CXCR4 in a diabetic patient (Figure 4C) and a healthy control subject (Figure 4D) (frozen sections, x200). CXCR4 was expressed by stromal cells (black arrows), endothelial cells (red arrows) and epithelial cells (blue arrows) and the staining pattern was mostly membranar and cytoplasmic. The intensity of staining was higher in in the diabetic group (p<0.05) but no differences were observed between the two groups in the number of positive stromal and endothelial cells (p=NS).