Association of increasing burn severity in mice with delayed mobilization of circulating angiogenic cells

Abstract

Objective: To perform a systematic exploration of the phenomenon of mobilization of circulating angiogenic cells (CACs) in an animal model. This phenomenon has been observed in patients with cutaneous burn wounds and may be an important mechanism for vasculogenesis in burn wound healing.

Design: We used a murine model, in which burn depth can be varied precisely, and a validated culture method for quantifying circulating CACs.

Setting: Michael D. Hendrix Burn Research Center, Baltimore, Maryland.

Participants: Male 129S1/SvImJ mice, aged 8 weeks, and 31 patients aged 19-59 years with burn injury on 1% to 64% of the body surface area and evidence of hemodynamic stability.

Main outcome measures: Burn wound histological features, including immunohistochemistry for blood vessels with CD31 and alpha-smooth muscle actin antibodies, blood flow measured with laser Doppler perfusion imaging, and mobilization of CACs into circulating blood measured with a validated culture technique.

Results: Increasing burn depth resulted in a progressive delay in the time to mobilization of circulating CACs and reduced mobilization of CACs. This delay and reduction in CAC mobilization was associated with reduced perfusion and vascularization of the burn wound tissue. Analysis of CACs in the peripheral blood of the human patients, using a similar culture assay, confirmed results previously obtained by flow cytometry, that CAC levels peak early after the burn wound.

Conclusion: If CAC mobilization and wound perfusion are important determinants of clinical outcome, then strategies designed to augment angiogenic responses may improve outcome in patients with severe burn wounds.

Trial registration: ClinicalTrials.gov NCT00796627.

Figure 1. Histological analysis

(a) Eschar. Left panel: Photomicrograph shows an eschar (arrow) with inflammatory infiltrate at 21 days after burn wounding. Right panel: Bar graph shows the percentage of wounds with eschar on day 21. (b) Scar thickness. Left panel: Measurement of scar thickness in a hematoxylin- and eosin-stained section. Right panel: Bar graph shows mean (± SEM) scar thickness on day 21 (*P<0.05, ANOVA with Tukey test, n = 12).

Figure 1. Histological analysis

(a) Eschar. Left panel: Photomicrograph shows an eschar (arrow) with inflammatory infiltrate at 21 days after burn wounding. Right panel: Bar graph shows the percentage of wounds with eschar on day 21. (b) Scar thickness. Left panel: Measurement of scar thickness in a hematoxylin- and eosin-stained section. Right panel: Bar graph shows mean (± SEM) scar thickness on day 21 (*P<0.05, ANOVA with Tukey test, n = 12).

Figure 2. Analysis of circulating angiogenic cells (CACs)

Analysis of CACs in the peripheral blood of mice subjected to burns of increasing duration. Mouse peripheral blood mononuclear cells cultured in the presence of endothelial growth factors were stained with FITC-lectin (green) and DiI-acetylated LDL (red). (a) Mice were subjected to 4-second (b), 6-second (c), and 8-second (d) burns and peripheral blood was analyzed for the presence of CACs on the indicated day after burn wounding (day 0, non-burned controls). *P<0.05 compared to day 0 (Student’s t test).

Figure 3. Analysis of wound blood flow

Laser Doppler perfusion imaging was performed on days 3, 7, and 14 after burning. Mean (± SEM) blood flow (*P<0.01, ANOVA with Tukey test, n = 8 for each group).

Figure 4. Immunohistochemical analysis of wound vascularization

(a–b) CD31 staining. (a) Bar graph shows the mean (± SEM) vessel counts per 200× field (*P<0.01, ANOVA with Tukey test, n = 8 for each group). (b) The central portion of a 4 and 8 second burn wound on day 21 stained with an antibody against CD31 (PECAM-1), which is expressed by vascular endothelial cells. (c–d) SMA staining. (c) Bar graph shows the mean (± SEM) number of vessels (*P<0.05, ANOVA with Tukey test, n = 8 for each group). (d) The central portion of a 4 and 8 second burn wound on day 21 stained with an antibody against smooth muscle actin (SMA), which is expressed by vascular pericytes and smooth muscle cells.

Figure 4. Immunohistochemical analysis of wound vascularization

(a–b) CD31 staining. (a) Bar graph shows the mean (± SEM) vessel counts per 200× field (*P<0.01, ANOVA with Tukey test, n = 8 for each group). (b) The central portion of a 4 and 8 second burn wound on day 21 stained with an antibody against CD31 (PECAM-1), which is expressed by vascular endothelial cells. (c–d) SMA staining. (c) Bar graph shows the mean (± SEM) number of vessels (*P<0.05, ANOVA with Tukey test, n = 8 for each group). (d) The central portion of a 4 and 8 second burn wound on day 21 stained with an antibody against smooth muscle actin (SMA), which is expressed by vascular pericytes and smooth muscle cells.

Figure 4. Immunohistochemical analysis of wound vascularization

(a–b) CD31 staining. (a) Bar graph shows the mean (± SEM) vessel counts per 200× field (*P<0.01, ANOVA with Tukey test, n = 8 for each group). (b) The central portion of a 4 and 8 second burn wound on day 21 stained with an antibody against CD31 (PECAM-1), which is expressed by vascular endothelial cells. (c–d) SMA staining. (c) Bar graph shows the mean (± SEM) number of vessels (*P<0.05, ANOVA with Tukey test, n = 8 for each group). (d) The central portion of a 4 and 8 second burn wound on day 21 stained with an antibody against smooth muscle actin (SMA), which is expressed by vascular pericytes and smooth muscle cells.

Figure 4. Immunohistochemical analysis of wound vascularization

(a–b) CD31 staining. (a) Bar graph shows the mean (± SEM) vessel counts per 200× field (*P<0.01, ANOVA with Tukey test, n = 8 for each group). (b) The central portion of a 4 and 8 second burn wound on day 21 stained with an antibody against CD31 (PECAM-1), which is expressed by vascular endothelial cells. (c–d) SMA staining. (c) Bar graph shows the mean (± SEM) number of vessels (*P<0.05, ANOVA with Tukey test, n = 8 for each group). (d) The central portion of a 4 and 8 second burn wound on day 21 stained with an antibody against smooth muscle actin (SMA), which is expressed by vascular pericytes and smooth muscle cells.

Figure 5. Analysis of burn wound closure

The wound area was measured by computer-assisted planimetry on day 21. The mean (± SEM) wound area is plotted in the bar graph (*P<0.001, ANOVA with Tukey test, n = 8 for each group).

Figure 6. CAC levels in burn patients at intervals after injury

(a) Serial analysis of CACs in burn patients. CACs were quantified in blood samples (n = 7, 15, 7, 5, and 8 for the sequential time periods studied; P<0.05, ANOVA). (b) Comparison of samples drawn in the first 48 hours after injury with later samples. The data were pooled into two groups (*P<0.001, Student’s t test, n = 22 samples in the <48 hours group and n = 20 in the >48 hours group).