Angiogenesis in wounds treated by microdeformational wound therapy

Abstract

Background: Mechanical forces play an important role in tissue neovascularization and are a constituent part of modern wound therapies. The mechanisms by which vacuum assisted closure (VAC) modulates wound angiogenesis are still largely unknown.

Objective: To investigate how VAC treatment affects wound hypoxia and related profiles of angiogenic factors as well as to identify the anatomical characteristics of the resultant, newly formed vessels.

Methods: Wound neovascularization was evaluated by morphometric analysis of CD31-stained wound cross-sections as well as by corrosion casting analysis. Wound hypoxia and mRNA expression of HIF-1α and associated angiogenic factors were evaluated by pimonidazole hydrochloride staining and quantitative reverse transcription-polymerase chain reaction (RT-PCR), respectively. Vascular endothelial growth factor (VEGF) protein levels were determined by western blot analysis.

Results: VAC-treated wounds were characterized by the formation of elongated vessels aligned in parallel and consistent with physiological function, compared to occlusive dressing control wounds that showed formation of tortuous, disoriented vessels. Moreover, VAC-treated wounds displayed a well-oxygenated wound bed, with hypoxia limited to the direct proximity of the VAC-foam interface, where higher VEGF levels were found. By contrast, occlusive dressing control wounds showed generalized hypoxia, with associated accumulation of HIF-1α and related angiogenic factors.

Conclusions: The combination of established gradients of hypoxia and VEGF expression along with mechanical forces exerted by VAC therapy was associated with the formation of more physiological blood vessels compared to occlusive dressing control wounds. These morphological changes are likely a necessary condition for better wound healing.

Figure 1

Diagram of the VAC mouse model used in the current study. The VAC device did not affect ambulation, neither well being of the treated animals.

Figure 2

Morphometric analysis of CD31 stained wound cross sections. VAC treatment is associated with the formation of small endothelial vessels and higher vessel density, whereby occlusive dressing control wounds are associated with large dilatated vessels.

Figure 3

Corrosion casting analysis of control wounds. A. Change of normal arborized vasculature towards a chaotic bloated, densely packed, tufts of vessels. B. Dilated vessel formations, abnormal tortousity of vessels, wide alterations of vessel diameters. C. Densely packed vessel formations, tumor-like vasculature, deregulation of normal vessel arborization, blind-ending dilated occluded vessels.

Figure 4

Corrosion casting analysis of VAC treated wounds. A. Hypervascularized wound margin, vessel wall. B. Directionalization of vessels: from small vessels branches to tortous low densely packed vessel formations that pursuit the wound centre. C. Arborized vessel branches with vessel hierarchy, small vessel diameter in clearly defined vessel network, directionalized.

Figure 5

Pimonidazole hydrochloride staining of wound cross sections of animals sacrificed at Day 3 after wounding shows control wounds (C) presenting with a diffuse hypoxia of the granulation tissue and interstitial spaces. VAC treated wounds (A and B) instead present with a superior oxygenation of the wound bed, with few hypoxic macrophages (B) located at the VAC-Foam interface. This Hypoxic gradient is associated with the formation of a VEGF gradient as assessed by western blot analysis (D). VEGF protein levels are higher at the VAC-Foam interface if compared to the wound bed. VEGF dimers are increased in the wound bed of VAC treated wounds.

Figure 6

mRNA profile of HIF-1α and related angiogenic factors and receptors during the time course of wound healing as assessed by quantitative RT-PCR in tissues harvested from the wound bed. In both groups mRNA expression of HIF-1α, VEGF-A, PDGF-BB and VEGF-R2 showed a progressive increase over the observation time, whereby control wounds were associated with a 15 fold increased expression of HIF-1α and 5 fold increased expression of VEGF-A.

Figure 7

The formation of a directionalized outgrowth of more functional vessels was associated with improved wound closure in VAC treated wounds.