Enhanced Survival and Accelerated Perfusion of Skin Flap to Recipient Site Following Administration of Human α1-Antitrypsin in Murine Models

Abstract

Objective: Skin flaps are routinely used in reconstructive surgery yet remain susceptible to ischemia and necrosis. Distant flaps require lengthy time to detach causing patient discomfort. Human α1-antitrypsin (hAAT) is a clinically available serum glycoprotein. hAAT was shown to support mature vessel formation and enhance tissue survival following ischemia-reperfusion injuries. The purpose of the presented study was to examine the effect of hAAT on skin flap survival and distant "tube" flap perfusion through its recipient site. Approach: Random-pattern skin flaps were performed on mice treated with clinical-grade hAAT using three unique routes of administration (transgenic, i.p. and s.c. infiltration); necrotic area and tissue perfusion were assessed. Blockade of vascular endothelial growth factor (VEGF) and nitric oxide synthase (NOS) were used to explore aspects of mechanism of action. A distant tube flap model was performed to examine time to perfusion. Results: hAAT-treated mice displayed approximately two-fold smaller necrotic flap areas versus controls across all hAAT administration routes. Flaps displayed greater perfusion as early as 3 days postsurgery (64.6% ± 4.0% vs. 43.7% ± 1.7% in controls; p = 0.007). hAAT-mediated flap survival was prominently NOS dependent, but only partially VEGF dependent. Finally, distant flaps treated with hAAT displayed significantly earlier perfusion versus controls (mean 9.6 ± 1.6 vs. 13.1 ± 1.0 days; p = 0.0005). Innovation: The established safety record of hAAT renders it an attractive candidate toward improving skin flap surgery outcomes, particularly during VEGF blockade. Conclusions: hAAT treatment enhances survival and accelerates perfusion of skin flaps in animal models in a NOS-dependent manner, partially circumventing VEGF blockade. Further mechanistic studies are required.

Keywords: VEGF; inflammation; nitric oxide synthase; revascularization; wound healing.

Eldad Silberstein, MD

Figure 1.

Illustration of the two flap models used in the study. Design and orientation of flap models. Dashed lines, sutures. (A) Random flap, (B) distant tube flap. The clamp is positioned around the lateral thoracic pedicle during transient ischemic conditioning. Color images are available online.

Figure 2.

Random-pattern skin flap survival in the presence of hAAT. Caudally based random-pattern skin flap was performed, and flap survival was assessed 7 days after surgery. Comparison of three hAAT administration routes and sources. (A) Transgenic hAAT expression. WT mice (n = 10); hAAT^+/+, hAAT-overexpressing transgenic mice (n = 10). Percent necrotic area. Data are representative of two independent experiments. (B) Systemic hAAT administration. Single injection of human serum albumin (75 mg/kg, n = 7) or hAAT (60 mg/kg, n = 10); Percent necrotic area. Data are representative of three independent experiments. (C) Local hAAT administration. Subcutaneous infiltration along flap borders of PBS (100 μL, n = 7), human serum albumin (125 μg/100 μL PBS, n = 9), or hAAT (100 μg/100 μL PBS, n = 9). Data pooled out of three independent experiments. All graphs show Mean ± SD, ***p < 0.001, ****p < 0.0001 between indicated groups by nonparametric Mann–Whitney test. (D) Macroscopic and histological analysis. Representative images of s.c.-treated flaps. White, demarcation line for histological sectioning; red, approximated necrotic area borders. Right, paired representative histological sections. H&E staining, × 10 magnification; stitched images rendered from three microscope view fields per sample. hAAT, human α1-antitrypsin; PBS, phosphate-buffered saline; H&E, Hematoxylin and Eosin; WT, wild-type. Color images are available online.

Figure 3.

In vivo perfusion assessment of random-pattern flap. A caudally based random-pattern skin flap was performed, and flap perfusion was assessed 3 days after surgery. Comparison between s.c. human serum albumin (125 μg/100 μL PBS, n = 7) and hAAT (100 μg/100 μL PBS, n = 7) administered at the time of surgery. Fluorescein uptake (4 mg/kg, i.p.), IVIS imaging. (A) Relative flap area fluorescent counts, day 0 set at 100%. Mean ± SEM, **p < 0.01 between treatment groups per time point by nonparametric Mann–Whitney test. (B) Representative flap images. Fluorescent image superimposed on photograph. Dashed line, flap area. IVIS, in vivo imaging system; VEGF, vascular endothelial growth factor. Color images are available online.

Figure 4.

Effect of hAAT on flap survival during VEGF inhibitor treatment. A caudally based random-pattern skin flap was raised, and flap survival was assessed 7 days after surgery. Comparison between WT and hAAT^+/+ mice, (A) Mice treated with either a single dose of anti-VEGF antibody immediately after surgery (Bevacizumab, 5 mg/kg, n = 5 per group) or with PBS (CT). Percent necrotic area. Mean ± SD, *p < 0.05, **p < 0.01 between indicated groups by nonparametric Mann–Whitney test. Data are representative of three independent experiments. (B) Mice treated with either of NOS inhibitor, L-NAME (10 mg/kg/day; n = 6); or NOS inhibitor, L-NIL (3 mg/kg/day; n = 6); or PBS (CT). Percent necrotic area. Mean ± SD, *p < 0.05, **p < 0.01 between indicated groups using nonparametric Mann–Whitney test. Data are representative of three independent experiments.

Figure 5.

Perfusion of flap by recipient site in a distant “tube” flap model. A “tube” flap was performed and treated with distal tip s.c. human serum albumin (125 μg/100 μL PBS, n = 8) and hAAT (100 μg/100 μL PBS, n = 8) administered at the time of surgery. Flaps were either “trained” by daily applications of a microvascular clamp to flap pedicle (10-min ischemia time, 5-min reperfusion time; × 3) or not trained. (A) Time to perfusion through distal tip. Flap perfusion was defined as positive fluorescein signal obtained from flap while pedicle was clamped. Scatter box-plot of measured perfusion day. Min to Max box plot, middle line states Mean ± SD, **p < 0.01, ***p < 0.001 using nonparametric Mann–Whitney test between indicated groups. (B) Representative images, nontrained day 8 distant flaps, with and without pedicle clamping. Fluorescent image merged with photograph. Color images are available online.