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Vascular endothelial growth factor-C gene therapy restores lymphatic flow across incision wounds

ANNE SAARISTO^*,,1, TUOMAS TAMMELA^*,1, JARI TIMONEN, SEPPO YLA-HERTTUALA, ERKKI TUKIAINEN, SIRPA ASKO-SELJAVAARA and KARI ALITALO^*,2

^* Molecular/Cancer Biology Laboratory and Ludwig Institute for Cancer Research, Biomedicum Helsinki and Helsinki University Central Hospital, University of Helsinki, Helsinki, Finland;
Department of Surgery, Paijat-Hame Central Hospital, Lahti, Finland;
Department of Plastic Surgery, Helsinki University Central Hospital, Helsinki, Finland; and
A. I. Virtanen Institute, Department of Medicine and Gene Therapy Unit, University of Kuopio, Kuopio, Finland

²Correspondence: Molecular/Cancer Biology Laboratory, Biomedicum, P.O.B. 63 (Haartmaninkatu 8), University of Helsinki, Helsinki 00014, Finland. E-mail: kari.alitalo{at}helsinki.fi

SPECIFIC AIMS

Lymphatic insufficiency, edema, and poor blood perfusion are common problems in reconstructive surgery. We aimed at rebuilding the lymphatic vessel network severed after surgical incision by introducing adenoviral vascular endothelial growth factor C (VEGF-C) gene transfer to the edges of a cutaneous flap. The feasibility of this form of therapy was assessed by assaying drainage of lymphatic fluid from the flap to axillary lymph nodes across the incision wound, and by quantification of lymphatic vessels in the wound area.

PRINCIPAL FINDINGS

1. Adenoviral VEGF-C and VEGF-C156S restore drainage of lymphatic fluid across the incision wound
Lymphatic vessel function is lost after surgical incision. VEGF-C is the first growth factor that was found capable of directly inducing growth of new lymphatic vessels via VEGFR-3, the principal mediator of lymphangiogenic signals. In this study, we have used adenoviral gene transfer of VEGF-C or VEGF-C156S, a mutant ligand specific for VEGFR-3, in an epigastric skin flap model in order to restore lymphatic vessel function in the flap. Mice were analyzed 2 wk to 2 months after the operation. When FITC-conjugated dextran was injected into the cranial edge of the flap that had been transduced with adenoviral VEGF-C or VEGF-C156S, a network of dextran-containing lymphatic vessels was detected, and some of these vessels drained fluid across the incision wound (Fig. 1 A, B). In AdLacZ-treated control samples, only a few functional lymphatic vessels were present (Fig. 1C ). Two wk after the operation, FITC-dextran drainage into axillary lymph nodes was detected in 75–80% of VEGF-C- or VEGF-C156S-treated mice and at later time points, in 100% of the mice (Fig. 1D, E ). In contrast, corresponding figures were 12.5% and 20–33% in the AdLacZ control group.

View larger version (48K): [in this window] [in a new window]   Figure 1. Epigastric skin flap model. Wound margins (blue line), injection site for adenoviruses (red arrows), skin area analyzed (green dashed line), and assayed axillary lymph nodes (black dashed line) are indicated. A–C) Microlymphangiography was performed to test the function of the lymphatic vessels in the flap. Fluorescent FITC-dextran was injected intradermally into the cranial flap region (white dashed line), in the immediate vicinity of the injection site of the adenovirus (red line). A–C) The blue dashed line in marks the site of the wound. Note prominent lymphatic vasculature near the flap border in the VEGF-C- and VEGF-C156S-treated mice (arrows, A, B), while LacZ- infected mice show only few functional lymphatic vessels in the region. D–F) Analysis of the axillary lymph nodes after FITC-dextran injection. Note accumulation of fluorescent dye in the axillary lymph nodes only in the mouse treated with adenoviral VEGF-C or VEGF-C156S (arrows). Bar graph shows the percentage of mice in which fluorescence was detected in the axillary lymph nodes at different time points in the 3 study groups.

2. Treatment with AdVEGF-C and AdVEGF-C156S results in increased lymphatic vessel density in the flap margin
Immunohistochemical analysis of the cranial flap margins demonstrated numerous large VEGFR-3 positive lymphatic vessels near the incision area in AdVEGF-C- or AdVEGF-C156S-treated mice. In contrast, we found only few small lymphatic vessels around the incision area in AdLacZ infected mice. When adjacent tissue sections were stained for the blood vessel marker PECAM-1, no significant differences were evident between the study groups. Quantification of lymphangiogenic and angiogenic responses by counting the number of VEGFR-3 or PECAM-1 positive vessels, respectively, indicated that AdVEGF-C and AdVEGF-C156S induced a significant increase in the number of lymphatic vessels in comparison to the AdLacZ control (Fig. 2 A), but a number of lymphatic vessels regressed after cessation of adenoviral expression. In contrast, in control samples, the number of VEGFR-3 positive lymphatic vessels slowly increased during the follow-up period (Fig. 2A ). However, even when analyzed at the 2-month time point, lymphatic vessels in the incision area were 1.8-fold more numerous in AdVEGF-C-treated mice than in AdLacZ- treated controls. Quantification of PECAM-1 positive vessels indicated a small increase in the number of blood vessels in AdVEGF-C-treated flaps when compared with VEGF-C156S- or AdLacZ-treated flaps. The blood vessel differences were not statistically significant (Fig. 2B ).

View larger version (26K): [in this window] [in a new window]   Figure 2. Analysis of lymphatic and blood vessels at the flap margin. A, B) Quantification of the lymphangiogenic and angiogenic responses in the adenoviral vector-treated samples. The VEGFR-3 positive lymphatic vessels (A) and PECAM-1 positive blood vessels (B) were counted under a high power microscope in samples taken at different time points. P values are indicated.

CONCLUSIONS AND SIGNIFICANCE

Pro-angiogenic gene therapy has shown great promise in the treatment of cardiovascular ischemic diseases. Ischemia and edema also cause problems in reconstructive surgery, particularly in high-risk patients who have diabetes or systemic peripheral vascular problems. There is typically postoperative swelling, sometimes exacerbated by infection, and these may eventually lead to necrosis if tissue perfusion is not adequate. The main cause of flap necrosis and failure in operations of this type is considered to be arterial or venous insufficiency, but lymphatic insufficiency also contributes to edema and poor blood perfusion in the flaps. The flap artery and vein(s) are surgically reconstructed, but lymphatic vessel function is lost after surgical incision. Impairment of lymphatic function results in tissue edema, insufficient blood perfusion, impaired immune function, and fibrosis.

Our present study shows that pro-lymphangiogenic VEGF-C or VEGF-C156S therapy can be used to reconstruct the lymphatic vessel network severed by the incision wound in free flap operations. Drainage of the FITC-dextran tracer across the surgical wound to the axillary lymph nodes improved in growth factor-treated mice at all time points (Fig. 3 ). This finding suggests that lymphatic function recovered rapidly in the treatment group. In the VEGF-C treated mice, improved lymphatic vessel function persisted, despite the fact that adenoviral VEGF-C gene expression was down-regulated and part of the generated lymphatic vessels regressed during the follow-up period. These data suggest that those lymphatic vessels that become functional remain stable in the tissue even without continuous growth factor stimulation. Presumably, nonfunctional vessels are pruned when growth factor overexpression is lost. Although spontaneous lymphangiogenesis also took place in control mice, the response was in most cases insufficient and the vasculature remained nonfunctional. VEGF-C stimulus thus appears to be helpful in restoring the function of the lymphatic vasculature.

View larger version (60K): [in this window] [in a new window]   Figure 3. Schematic representation of lymphatic function after surgery. In untreated control animals, repair of the lymphatic network in the incision area is incomplete and insufficient for proper lymphatic drainage across the flap margin to lymph nodes. Mice treated with adenoviral VEGF-C or VEGF-C156S display an increase in number of lymphatic vessels in the wound area, which facilitates flow of lymph through the wound to lymph nodes.

Several other groups have reported that different pro-angiogenic molecules, such as VEGF, FGF-2, and Ang1, enhance flap survival. Pro-lymphangiogenic therapy has previously been used in animal models of lymphedema, but not in the setting of reconstructive surgery or wound healing. According to clinical studies, tissue edema in free flaps is most severe 2 wk after the operation; thereafter the thickness of the flap gradually returns to normal. The adenoviral gene transfer used in this study provides local and transient transgene expression, which should be safe for possible human trials. In general, adenoviruses expressing VEGFs have been abundantly used in human clinical trials for treatment of a variety of conditions in a number of different tissue types. Pro-lymphangiogenic VEGF-C gene therapy may reduce or prevent tissue edema and promote vascular perfusion in other forms of postoperative edema.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-1592fje;

¹ These authors contributed equally to this work.

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