Overexpressed HIF-2α in Endothelial Cells Promotes Vascularization and Improves Random Pattern Skin Flap Survival

Abstract

Background: The local skin flap procedure is very useful for reconstruction. However, flap necrosis caused by circulatory failure can occur at its distal portion. Hypoxia-inducible factors (HIFs) in endothelial cells (ECs) help to maintain ECs and promote vascularization, and HIF-2α is abundantly expressed in ECs. However, the mechanisms of action of HIF-2α in ECs are not yet fully understood. The aim of this study was to evaluate the in vivo effects of overexpression of HIF-2α in ECs on skin flap survival.

Methods: A random pattern skin flap (1.0 × 3.0 cm) was elevated on the dorsum of transgenic mice (Tg mice) with EC-specific HIF-2α conditional overexpression and wild-type littermate control mice (n = 6). Flap survival was evaluated on postoperative day 7. Tissue samples from the skin flaps were harvested and analyzed using Western blotting, quantitative reverse transcriptase-polymerase chain reaction, and immunohistochemistry.

Results: The HIF-2α mRNA and protein levels were significantly increased in the Tg mice when compared with control mice. Tg mice had significantly increased skin flap survival areas (72.0% ± 2.7%) when compared with wild-type mice (45.7% ± 1.1%). Moreover, histological examination revealed an increase in the subcutaneous blood vessel counts in the Tg mice.

Conclusions: Specific overexpression of HIF-2α in ECs promoted vascularization and enhanced skin flap survival in vivo in a mouse model.

Fig. 1.

A, Schematic representation of the LSL-HIF-2α transgenic mouse gene. Set3 (for2α) and td Tomato (solid triangles) indicate the sites of PCR primers used for mouse genotyping. B, Quantitative RT-PCR-based genotyping for td Tomato. Y axis shows relative mRNA level. PCR was performed with primers td Tomato. C, Quantitative RT-PCR-based genotyping for HIF-2α. Y axis shows relative copy number of HIF-2α to Arnt gene. PCR was performed with primers Set3 (for2α). Transgene copy number in 2 transgenic mice lines was compared. D, HIF-1α and HIF-2α mRNA levels in the lung. High-copy HIF-2α transgenic lines maintained under normoxic (21% O₂) conditions. Data in bar graphs are presented as means ± SEM (n = 6). **P < 0.01. E and F, Western blot analysis of HIF-1α and HIF-2α protein in the lung. High-copy HIF-2α transgenic lines maintained under hypoxic (10% O₂, 90% N₂) conditions. β-Actin was used as a loading control. Values are means ± SEM (n = 6). *P < 0.05. RT-PCR indicates reverse transcriptase-polymerase chain reaction; Tg, endothelial cell–specific HIF-2α conditional overexpression transgenic mice.

Fig. 2.

Comparison of flap viability between the control mice and the Tg mice. A, Low-copy HIF-2α transgenic lines. B, High-copy HIF-2α transgenic lines. Flap survival was evaluated on postoperative day 7. Data in bar graphs are presented as percentages of flap survival ± SEM (n = 6). ***P < 0.001.

Fig. 3.

Expression of HIF-2α protein in a mouse skin flap model. A and B, Western blot analyses were performed on tissue lysates from dorsal skin taken preoperatively and from the proximal parts of the skin flaps on postoperative day 7. High-copy HIF-2α transgenic lines and wild-type littermate control mice were subjected to analysis. β-Actin was used as a loading control. Values are means ± SEM (n = 6). *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 4.

HIFs, VEGF, and PHD mRNA levels in the mouse skin flap model. Transcript levels of angiogenesis-related factors were assessed by quantitative RT-PCR analysis. A, Total RNA was isolated from dorsal skin taken before operation. Values are means ± SEM (n = 6). B and C, Total RNA was isolated from the proximal parts of the skin flaps on postoperative day 1 and 7. Data in bar graphs are presented as means ± SEM (n = 6). *P < 0.05, **P < 0.01, ***P < 0.001. RT-PCR indicates reverse transcriptase-polymerase chain reaction.

Fig. 5.

Effect of specific HIF-2α overexpression in ECs on postoperative angiogenesis and vasculature in mouse subcutaneous plexus. Section of proximal portions of skin flaps preoperatively (A) and on postoperative day 7 (B) were stained with hematoxylin and eosin (HE) and rhodamine-lectin. C, The number of lectin-positive (red) vessels was counted per high-power field. Scale bar indicates 200 μm. ***P < 0.001.