VEGF-C gene therapy augments postnatal lymphangiogenesis and ameliorates secondary lymphedema

Although lymphedema is a common clinical condition, treatment for this disabling condition remains limited and largely ineffective. Recently, it has been reported that overexpression of VEGF-C correlates with increased lymphatic vessel growth (lymphangiogenesis). However, the effect of VEGF-C–induced lymphangiogenesis on lymphedema has yet to be demonstrated. Here we investigated the impact of local transfer of naked plasmid DNA encoding human VEGF-C (phVEGF-C) on two animal models of lymphedema: one in the rabbit ear and the other in the mouse tail. In a rabbit model, following local phVEGF-C gene transfer, VEGFR-3 expression was significantly increased. This gene transfer led to a decrease in thickness and volume of lymphedema, improvement of lymphatic function demonstrated by serial lymphoscintigraphy, and finally, attenuation of the fibrofatty changes of the skin, the final consequences of lymphedema. The favorable effect of phVEGF-C on lymphedema was reconfirmed in a mouse tail model. Immunohistochemical analysis using lymphatic-specific markers: VEGFR-3, lymphatic endothelial hyaluronan receptor-1, together with the proliferation marker Ki-67 Ab revealed that phVEGF-C transfection potently induced new lymphatic vessel growth. This study, we believe for the first time, documents that gene transfer of phVEGF-C resolves lymphedema through direct augmentation of lymphangiogenesis. This novel therapeutic strategy may merit clinical investigation in patients with lymphedema.

Lymphedema is defined as the progressive accumulation of protein-rich fluid in the interstitial spaces that results from an anatomic or functional obstruction in the lymphatic system (1). While primary lymphedema occurs infrequently on a hereditary or idiopathic basis, secondary lymphedema is common worldwide, primarily due to the increase in radical surgery and radiotherapy for cancer in developed countries and infectious disease (filariasis) in developing countries (2, 3). Despite substantial advances in both surgical and conservative techniques, therapeutic options for management of lymphedema are limited (3, 4). Pathophysiologically, restoration of the lymph-transporting capacity would appear to represent the optimal treatment for lymphedema. However, no means for accomplishing new lymphatic channel development currently exists. Growth of new lymphatic vessels (lymphangiogenesis) in healthy animals is rapid. The best example of the natural recovery of lymphatic drainage in animals is the complete restoration of lymphatic flow after limb reimplantation (5–9). Therefore the primary difficulty found in lymphedema animal models is to develop a method to sustain lymphedema long enough to allow evaluation of therapies.

Recent molecular studies have begun to elucidate the basis for lymphangiogenesis that can be stimulated by various cytokines, including VEGF-C (VEGF-2) (10, 11). VEGF-C, the first ligand to be discovered for VEGFR-3 (Flt4), is a member of the VEGF family of polypeptide growth factors. VEGF-C binds to endothelial cell receptors VEGFR-2 (Flk1) and VEGFR-3 (12–15). Although VEGFR-3 plays a critical role for both vascular and lymphatic endothelial cell development, its expression becomes limited to the lymphatic endothelium beginning in the late stages of development (16–18). Overexpression of VEGF-C cDNA in the skin of transgenic mice induced lymphatic endothelial cell proliferation and hyperplasia of the lymphatic vasculature, and recombinant VEGF-C specifically stimulated lymphangiogenesis in chorioallantoic membrane (11, 19). Recently, direct evidence of the link between VEGFR-3 and lymphedema has been found: it has been reported that human hereditary lymphedema is associated with heterozygous missense mutation of the Flt4 gene, which leads to insufficient VEGFR-3 signaling (20, 21). Recently, it was demonstrated that subcutaneous injection of adenovirus or adeno-associated virus encoding VEGF-C could generate lymphatic vessels in the skin of normal mice (22) and in a mouse model (chy mouse) of primary lymphedema (23). Although these studies showed that VEGF-C could induce lymphangiogenesis in vivo, they failed to show that this VEGF-C–induced lymphangiogenesis could improve overall lymphatic vascular dysfunction and prevent chronic changes accompanied by lymphedema, which are the key determinants of whether VEGF-C can be used as a therapeutic option to treat human lymphedema.

Accordingly, first we sought to establish reliable animal models of secondary lymphedema. The two animal models used here provided complementary measurements: the rabbit ear had the advantage of size, which is conducive to direct measurements and lymphoscintigraphy, whereas the mouse tail had advantages for immunohistochemistry owing to the availability of lymphatic vessel–specific Ab’s. Next, using these animal models, we investigated whether local transfer of naked plasmid DNA encoding human VEGF-C (phVEGF-C) could promote lymphangiogenesis and improve physical, functional, and pathologic aspects of lymphedema.

VEGF-C gene therapy induces remission of lymphedema. Seeking to establish an appropriate animal model, initial experiments using young (6–8 months old) rabbits showed expedited regression of lymphedema such that we could not properly evaluate the effect of gene transfer. In the older rabbits (3–4 years old), a substantial degree of lymphedema developed and was sustained for more than 12 weeks.

To investigate the effect of phVEGF-C gene transfer on this lymphedema model, we measured ear thickness and volume over a 12-week period. Ear thickness was consistently smaller in the VEGF-C group than in the saline group. The difference between the groups was statistically significant beginning at 2 weeks and was maintained for the duration of the study (2 weeks: 4.5 ± 0.3 vs. 5.4 ± 0.2 mm, P < 0.05; 3 weeks: 3.9 ± 0.2 vs. 4.6 ± 0.3 mm, P < 0.05; 8 weeks: 2.8 ± 0.2 vs. 3.6 ± 0.3 mm, P < 0.05; 10 weeks: 2.6 ± 0.2 vs. 3.5 ± 0.3 mm, P < 0.05; 12 weeks: 2.4 ± 0.2 vs. 3.3 ± 0.2 mm, P < 0.01) (Figure 1c). Similarly, ear volume was consistently smaller in the VEGF-C group than in the saline group for the duration of the study (2 weeks: 33.1 ± 2.2 vs. 38.2 ± 1.5 ml, P < 0.05; 3 weeks: 29.4 ± 1.5 vs. 34.1 ± 1.0 ml, P < 0.05; 4 weeks: 26.5 ± 1.8 vs. 31.7 ± 1.7 ml, P < 0.05; 8 weeks: 18.3 ± 2.3 vs. 26.3 ± 1.9 ml, P < 0.05; 10 weeks: 17.1 ± 2.1 vs. 25.5 ± 1.8 ml, P < 0.01; 12 weeks: 14.6 ± 2.3 vs. 24.5 ± 1.3 ml, P < 0.01) (Figure 1d). The VEGF-C group had significantly thinner skin than the saline group at 8 weeks (2.8 ± 0.2 vs. 3.8 ± 0.2 mm, P < 0.05).

Lymphoscintigraphy demonstrates enhanced lymphatic drainage after VEGF-C gene transfer. In normal ears, lymphatic flow assumes a linear pattern, and the draining LNs are clearly visible at the base of the skull. (Figure 2b). Imaging performed at day 1 after surgery showed successful surgical blockade of lymphatic egress in all animals (Figure 2, c and e). Follow-up lymphoscintigraphy at 4, 8, and 12 weeks showed dynamic changes of radiotracer clearance from the operated ears that was more efficient in the phVEGF-C–transfected ear than in the saline-injected ear. Images at 12 weeks revealed that the saline-injected ear still showed a dermal backflow pattern with faint visualization of LNs, while the phVEGF-C–transfected ear shows a linear pattern of lymphatic drainage and clear visualization of LNs (Figure 2, d and f).

Quantification of lymphatic drainage (Figure 2, g and h) over the study period revealed consistently lower retention of radioactivity in the VEGF-C group than in the saline group; this achieved statistical significance at 8 weeks (radioactivity index, 3.8 ± 0.4 vs. 5.0 ± 0.5, P < 0.05) and 12 weeks (radioactivity index, 2.2 ± 0.3 vs. 4.2 ± 0.4, P < 0.05) (Figure 2i).

Transgene expression of phVEGF-C in a rabbit ear model. To assess transgene expression of injected phVEGF-C in ear skin, we performed Western blotting of VEGF-C protein. In our experiments, two different bands were detected using two anti–VEGF-C Ab’s. A 58-kDa band corresponds to the earliest processed form, while a 31-kDa band represents the major secreted form of VEGF-C polypeptides. Densitometric analysis of multiple experiments revealed that expression of the 58-kDa VEGF-C isoform in the phVEGF-C–transfected bridge was 2.6 times and 3.2 times higher than that in the saline-injected and the normal skin, respectively (P < 0.01) (Figure 3a). Expression of the 31-kDa VEGF-C isoform in the phVEGF-C–transfected bridge was 3.6 times and 3.9 times higher than that in the saline-injected and the normal skin, respectively (P < 0.01) (Figure 3b).

Gene transfer of phVEGF-C increases VEGFR-3 expression. A partial 470-bp rabbit VEGFR-3 cDNA was cloned by RT-PCR using degenerate oligonucleotide primers (GenBank accession number AF453570). The amino acid sequence displayed 92.9%, 93.6%, and 94.3% identity with human, bovine, and mouse VEGFR-3 (Figure 3c). We investigated VEGFR-3 expression using RT-PCR, revealing a nearly 1.7-fold induction of VEGFR-3 mRNA levels by VEGF-C compared with saline control (P < 0.01, Figure 3g).

Gene transfer of phVEGF-C increases phosphorylation of VEGFR-3 in a mouse tail model. We investigated the effect of phVEGF-C gene transfer on the tyrosyl phosphorylation of VEGFR-3 and VEGFR-2 by immunoprecipitation with anti-phosphotyrosine Ab followed by Western blot analysis with anti–VEGFR-3 and anti–VEGFR-2 Ab’s, respectively. Phosphorylated VEGFR-3 (195 kDa) in the phVEGF-C–transfected samples was 1.6 times and 1.8 times higher than in the samples from the saline and LacZ groups, respectively (P < 0.05) (Figure 3h). Gene transfer of phVEGF₁₆₅ did not increase phosphorylated VEGFR-3 compared with the controls. Phosphorylated VEGFR-2 in the phVEGF₁₆₅-transfected sample was 2.0, 1.8, and 1.6 times higher (235-kDa band) than in the samples of the saline, LacZ, and VEGF-C groups, respectively (P < 0.05) (Figure 3i). Phosphorylated VEGFR-2 was slightly higher in the phVEGF-C–transfected samples than in the control groups (saline and LacZ) but was not statistically significant.

Gene transfer of phVEGF-C improves lymphedema in a mouse tail model. To determine whether the effect of phVEGF-C could be reproduced in another lymphedema model, similar experiments were performed in a mouse tail model (30) (Figure 4a). In the VEGF-C group, compared with the saline, LacZ, and VEGF₁₆₅ groups, the tail thickness was consistently smaller beginning at 3 weeks (3 weeks: 4.05 ± 0.08 vs. 4.37 ± 0.07, 4.32 ± 0.08, and 4.30 ± 0.07 mm, P < 0.05; 4 weeks: 4.01 ± 0.09 mm vs. 4.42 ± 0.08, 4.31 ± 0.08, and 4.28 ± 0.07 mm, P < 0.05; 5 weeks: 4.00 ± 0.07 mm vs. 4.35 ± 0.08, 4.28 ± 0.08, and 4.28 ± 0.08 mm, P < 0.05), respectively.

Figure 4

(a) Gene transfer of phVEGF-C decreases lymphedema in a mouse tail model of lymphedema. Tail thickness was significantly greater in the operated tail than in the unoperated tail during the entire 5 weeks. In the VEGF-C group, compared with the saline, LacZ, and VEGF₁₆₅ groups, the tail thickness was significantly smaller at 3–5 weeks (*P < 0.05). No-op, no operation. (b–m) phVEGF-C induces lymphangiogenesis in a mouse tail model of lymphedema. (b–k) Immunohistochemistry using markers of lymphatic endothelium, LYVE-1 (b–f), and VEGFR-3 (g–k), in normal (b and g) and operated (3 weeks after lymphedema surgery) skin sections from the saline (c and h), LacZ (d and i), VEGF-C (e and j), and VEGF₁₆₅ (f and k) groups. Lymphatic vessels are seen as brown color (black arrows). Note the abundance of hyperplastic lymphatic vessels in phVEGF-C–transfected sections (e and j). l and m show quantification of LYVE-1– and VEGFR-3–positive lymphatic vessels. Compared with normal and control (saline, LacZ, and VEGF₁₆₅) groups, the VEGF-C group showed significantly higher lymphatic vessel density. *P < 0.05 vs. normal; **P < 0.01 vs. LE-saline and LE-LacZ. Scale bar, 100 μm.

Gene transfer of phVEGF-C promotes lymphatic vessel growth in a mouse tail model. The VEGF-C group showed higher density of LYVE-1–positive lymphatic vessels than the other groups (VEGF-C, 85 ± 7 per mm²; saline, 38 ± 4 per mm²; LacZ, 42 ± 5 per mm²; VEGF₁₆₅, 46 ± 5 per mm², P < 0.01) (Figure 4, b–f and l). Skin sections stained with VEGFR-3 Ab showed similar results (VEGF-C, 81 ± 8 per mm²; saline, 37 ± 4 per mm²; LacZ, 43 ± 5 per mm²; VEGF₁₆₅, 44 ± 5 per mm², P < 0.01) (Figure 4, g–k and m). After phVEGF-C transfection, lymphatic vessels appeared hyperplastic (Figure 4, e and j). In sections at 3-week follow-up, the number of lymphatic vessels containing Ki-67⁺ nuclei was 2.5 times higher in the VEGF-C group than in the saline or LacZ groups (VEGF-C, 58 ± 7 per mm²; saline, 23 ± 3 per mm²; LacZ, 26 ± 4 per mm², P < 0.01) (Figure 5, a–j).

Figure 5

(a–j) phVEGF-C induces proliferation of lymphatic endothelial cells. Double immunohistochemistry using LYVE-1 and Ki-67 in active lymphangiogenesis site from skin sections. In a, d, and g, LYVE-1 staining of lymphatic vessels (arrows) in the dermis. In b, e, and h, green fluorescence (white arrowheads) depicts the nuclear staining of Ki-67. In c, f, and i, double fluorescence (yellow arrowheads) demonstrates Ki-67⁺ nuclei (green) in lymphatic vessels (red). Lymphatic vessels in normal skin (c) are shown negative for Ki-67. In the LacZ group, some of the lymphatic vessels contain Ki-67⁺ nuclei (f). White arrows in f show Ki-67^– lymphatic vessels. In phVEGF-C–transfected skin, most of the LYVE-1–positive lymphatic vessels are positive for Ki-67 (i), indicating that active cell division occurs in the lymphatic vessels. (j) Number of Ki-67⁺ nuclei are 2.5 times higher in the VEGF-C group. *P < 0.01 compared with normal; **P < 0.01 compared with saline and LacZ. Scale bar, 100 μm. (k–u) phVEGF-C does not increase capillary density in two animal models of lymphedema. Immunohistochemistry with CD31 (PECAM-1) in a rabbit ear (k–n) and a mouse tail (p–t) model of lymphedema on skin sections from the normal (k and p), saline (l and q), LacZ (m and r), VEGF-C (n and s), and VEGF₁₆₅ (t) groups. Vascular endothelial cells are stained red (black arrows). o and u show quantification of capillary density. Only the VEGF₁₆₅ group in the mouse tail model demonstrated significantly higher capillary density than the other groups. *P < 0.01 vs. saline, LacZ, and VEGF-C. Scale bar, 100 μm.

Blood capillary density analysis. Rabbit ear and mouse tail skins were stained for an endothelial cell marker, CD31 (36). In the rabbit lymphedema model, capillary density was not significantly different among the saline (193 ± 18 per mm²), LacZ (198 ± 22 per mm²), or VEGF-C (201 ± 20 per mm²) groups (Figure 5, k–o). Similar findings were observed in the capillary density of operated mouse tail groups (saline, 172 ± 18 per mm²; LacZ, 181 ± 19 per mm²; VEGF-C, 189 ± 20 per mm², P value not significant) (Figure 5b, F–K). However, the VEGF₁₆₅ group (302 ± 27 per mm²) showed significantly higher capillary density than the saline, LacZ, or VEGF-C groups (P < 0.01).

Chronic lymphedema is a disabling condition characterized by thickening of the skin due to fibrofatty deposition in underlying tissues as well as disfiguring swelling of affected limbs. In most cases of secondary lymphedema in humans, depletion of lymphatic vessels is the culprit in its pathogenesis (1–4). Here we show that phVEGF-C gene therapy, by promoting lymphangiogenesis, favorably modulates all the phenotypic changes associated with secondary lymphedema. We believe the present study is the first to document improvement in the clinical and pathologic features of lymphedema resulting from enhancement of lymphatic drainage by phVEGF-C gene therapy.

In two animal models, we demonstrate significant attenuation of lymphedema by phVEGF-C gene transfer. The effect prevailed over the chronic phase as well as the acute phase of lymphedema. This improvement was also confirmed in histologic sections, which reflect chronic fibrofatty changes more accurately. Prevention or reduction of fibrotic change is one of the most important goals of therapy for lymphedema, since this secondary change can drive lymphedema into a vicious cycle by increasing interstitial solid pressure (by fibrofatty deposition) and thus collapsing already reduced or impaired lymphatic vessels (3–5, 24, 25). That the improvement in the physical indices of lymphedema was the actual result of improved lymphatic drainage was documented by quantitative lymphoscintigraphy. Although VEGF-C plasmid transgene expression is usually limited to less than 30 days (27), our results indicate that once the lymphatic connection is reestablished, the recovery of drainage function can be maintained.

VEGF-C protein expression was documented in situ after phVEGF-C gene transfer. We detected the partially processed form (58 kDa) and major secreted form (31 kDa) of VEGF-C (37, 38) after local gene delivery. VEGF-C is produced as a 61-kDa prepropeptide form which undergoes multistep proteolytic maturation. The secreted 31-kDa form predominantly activates VEGFR-3 (37). VEGFR-3 mRNA expression was very low in normal skin, slightly higher in the saline-injected experimental ears, and strongly upregulated following phVEGF-C gene transfer. We also directly measured the number of lymphatic vessels using lymphatic-specific markers. LYVE-1 or VEGFR-3 staining confirmed augmentation of lymphangiogenesis in phVEGF-C–transfected mouse tails. The hyperplastic nature of proliferating lymphatic vessels was consistent with previous reports (19, 22). Ki-67 staining documented that proliferating lymphatic endothelial cells exist in more than half the lymphatic vessels in phVEGF-C–transfected skin, suggesting a potent lymphangiogenic effect of phVEGF-C.

As VEGF-C is also known to activate VEGFR-2 and thus to induce angiogenesis in vitro and in ischemic tissues (15, 32), we evaluated capillary density from both animal models and found it was slightly higher in the VEGF-C group but not significantly different from that of the saline or LacZ control groups. The apparent absence of fully processed 21-kDa product of VEGF-C, which has potent angiogenic activity, and thus the weak activation of VEGFR-2 phosphorylation, could explain the lack of obvious angiogenesis in these animal models. These findings are compatible with previous reports that claimed no discernible angiogenesis in transgenic mice overexpressing VEGF-C in the skin (19) and in normal mouse skin that was transfected with adeno–VEGF-C (22). Physiologic function of any ligand is dependent on the temporal and spatial expression of its specific receptors. In the case of VEGF-A–induced angiogenesis, the absence of ischemia-induced regional upregulation of VEGFR-2 has been shown to result in nullification of the angiogenic effect of transient overexpression of VEGF-A (39, 40). To further address the concern that increased angiogenesis might improve lymphedema, we investigated the effect of VEGF-A (phVEGF₁₆₅) plasmid gene transfer in the mouse tail model and found that augmenting angiogenesis but not lymphangiogenesis did not improve lymphedema.

The potential clinical relevance and limitations of our study derive from certain features of the design and the findings. First, the models are pathophysiologically similar to the secondary forms of human lymphedema. The models we used do not represent the entire spectrum of lymphedema found in humans, especially the primary form of lymphedema, which is an inherited developmental disorder of the lymphatic system. However, pathophysiologically our models are approximations of secondary lymphedema, which comprises most cases of lymphedema and results primarily from surgical removal of lymphatic vessels and lymph nodes in industrialized countries (1–4). Additionally, in our models, we used gene therapy in an acute/subacute stage of lymphedema. Therefore, whether this gene therapy can be effective in chronic cases is uncertain. As there are concerns about the potential enhancement of tumor growth and metastasis by VEGF-C in tumor models (41), we need to consider the potential advantages and dangers of using local VEGF-C therapy in patients with lymphedema caused by cancer treatment. This issue can be resolved after performing experiments adopting tumor implantation and treatment such as a combination of surgery, chemotherapy or radiation, and local VEGF-C gene transfer. Third, the approach of gene therapy using naked plasmid DNA (phVEGF-C) has been used in early clinical trials and has an accumulating record of safety (42). Finally, to the best of our knowledge, this study represents the first experimental proof of a beneficial effect of VEGF-C gene therapy on lymphedema per se. Our findings clearly indicate a favorable effect of phVEGF-C–induced lymphangiogenesis on lymphedema and thus represent a novel therapeutic paradigm for the treatment of this otherwise difficult-to-manage condition.

This paper is dedicated to Jeffrey M. Isner, who passed away on October 31, 2001. We would like to gratefully acknowledge his inspirational leadership. We gratefully acknowledge M. Neely, I. Johnson, and T. Shiojima for their excellent secretarial assistance. This study was supported in part by NIH grants HL-53354, HL-60911, HL-63414, HL-63695, and HL-66957, and by the Shaughnessy Center for Clinical Genetics, Boston, Massachusetts, USA. Young-sup Yoon is the recipient of a fellowship from the American Heart Association, New England Affiliate.

Conflict of interest: The authors have declared that no conflict of interest exists.

Nonstandard abbreviations used: naked plasmid DNA encoding human VEGF-C (phVEGF-C); coefficient of variation (cv); lymphatic endothelial hyaluronan receptor–1 (LYVE-1); platelet endothelial cell adhesion molecule–1 (PECAM-1).

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