Burn injury-induced alterations in wound inflammation and healing are associated with suppressed hypoxia inducible factor-1alpha expression

Abstract

A major complication associated with burn injury is delayed wound healing. While healing of the burn injury site is essential, healing of distal injury sites caused by surgical interventions and other processes also is important. The impact of burn injury on healing of these distal wound sites is not understood clearly. To study this, mice were subjected to major burn injury or a sham procedure. Immediately following, excisional wounds were made on the dorsal surface caudal to the burn site and wound closure was monitored over a 7-d period by planimetry. In a second series of experiments, plasma and excisional wounds were collected for in vitro analysis of cyto- and chemokine levels, L-arginine metabolism, and hypoxia-inducible factor (HIF)-1alpha expression. At 1-7 d post-injury, a significant inflammatory response was evident in both groups, but the healing process was delayed in the burn-injured mice. At 3 d post-injury, wound levels of tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and keratinocyte-derived chemokine were suppressed in the burn group. This difference in the wound inflammatory response was independent of changes in L-arginine metabolism (nitrate levels, inducible nitric oxide synthase expression, arginase activity), but correlated with a marked reduction in HIF-1alpha protein levels. In conclusion, these findings suggest that HIF-1alpha and the inflammatory response play a significant role in wound healing, and reduced levels of HIF-1alpha contribute to the impaired healing response post-burn.

Figures

Figure 1

Plasma cytokine, chemokines, and nitrite/nitrate levels. Mice were subjected to an excisional wound only (EW) or a dual injury of thermal injury with excisional wounding (Burn/EW) and plasma samples collected at 1–7 d post-injury. Plasma levels of cytokines [A–D], chemokines [E,F], and nitrite/nitrate [G] were determined as described in the Materials and Methods section. Data are mean ± SEM; n = 3–7 mice/group. *P < 0.05 as compared with EW group.

Figure 2

Time course of wound closure. Mice were subjected to an excisional wound only (EW) or a dual injury of thermal injury with excisional wounding (Burn/EW) and wound closure was determined 1–7 d post-injury as described in the Materials and Methods section. Data are mean ± SEM; n = four mice/group. *P < 0.05 as compared with EW group.

Figure 3

Wound closure at 3 d post-injury. Mice were subjected to an excisional wound only (EW) or a dual injury of thermal injury with excisional wounding (Burn/EW). Digital photographs were taken of representative animals at 3 d post-injury.

Figure 4

Excisional wound cytokine and chemokine levels. At 3 d post-injury, excisional wounds were collected from mice receiving an excisional wound only (EW) or a dual injury of thermal injury with excisional wounding (Burn/EW). Wound homogenates were generated and cytokine and chemokine levels were determined as described in the Materials and Methods section. Levels for TNF-α IL-6, IL-10, KC, MCP-1, and TGF-β were determined. Data are mean ± SEM; n = four mice/group. *P< 0.05 as compared with EW group.

Figure 5

Excisional wound L-arginine metabolism. At 3 d post-injury, excisional wounds were collected from mice receiving an excisional wound only (EW) or a dual injury of thermal injury with excisional wounding (Burn/EW). Wound homogenates were assessed for various parameters of L-arginine metabolism: [A] nitrite/nitrate levels; [B] iNOS protein expression and; [C] arginase activity as described in the Materials and Methods section. Blots are of representative experiments. Data are mean ± SEM; n = four mice/group.

Figure 6

Excisional wound HIF-1α protein expression. At 3 d post-injury excisional wounds were collected from mice receiving an excisional wound only (EW) or a dual injury of thermal injury with excisional wounding (Burn/EW). Wound homogenates were assessed for HIF-1α expression by ELISA [A] and Western blot [B] as described in the Materials and Methods section. Blots are of representative experiments. Arrow indicates band of interest. Data are mean ± SEM; n = four mice/group. *P < 0.05 as compared with EW group.