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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online October 18, 2002 as doi:10.1096/fj.02-0401fje. |
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Division of Cardiovascular Medicine, Falk Cardiovascular Research Center, Stanford University School of Medicine, Stanford, California, USA;
Derald H. Ruttenberg Cancer Center, Mount Sinai School of Medicine, New York, New York, USA;
Diagnostic Radiology and Nuclear Medicine, Stanford University School of Medicine, Stanford, California, USA; and
Haartman Institute, University of Helsinki, Helsinki, Finland
2Correspondence: Division of Cardiovascular Medicine, Falk Cardiovascular Research Center, Stanford University School of Medicine, Stanford, CA 94305, USA. E-mail: srockson{at}cvmed.stanford.edu
SPECIFIC AIM
Chronic postsurgical lymphedema, typically acquired as an iatrogenic consequence of therapy for breast or pelvic malignancies, is an ideal candidate for targeted gene therapy to elicit therapeutic, regenerative growth of lymphatic vasculature. Accordingly, we developed a model for this disorder in the rabbit ear in order to investigate the therapeutic potential for lymphangiogenesis with exogenously administered human recombinant VEGF-C.
PRINCIPAL FINDINGS
1. Segmental surgical resection of the lymphatic vasculature at the base of a single ear was well tolerated in all experimental subjects and yielded chronic, stable edema in 16 of the 18 subjects
The increase in ear volume as measured by water displacement volume averaged 56% (range, 12.5–115%). When compared with volumes of the nonoperated ears, these increases in volume were highly significant (P<0.0001). Of these 16 rabbits, 8 were treated with a single 100 µg dose of recombinant VEGF-C; the remaining 8 received a comparable volume of injected saline and represented the untreated control state.
2. VEGF-C enhances lymphatic function in lymphedema
We measured the effect of treatment on dynamic lymphatic function with serial radiocontrast lymphoscintigraphy. Qualitative assessment of the static lymphoscintigraphic images disclosed accumulation of radiotracer in the saline-treated edematous ears. In contrast, VEGF-C therapy of lymphedema produced substantial reductions in the degree of dermal backflow, evident in a reduction both in the density and expanse of the retained radionuclide. The degree of radionuclide retention in the tissues, inversely related to the clearance of the imaging agent, is termed ‘dermal backflow’. The reduction in dermal backflow seen in VEGF-C treated ears was significant (P<0.01) when compared with saline-treated lymphedema.
3. In experimental chronic lymphedema, lymphatic vessels disappeared substantially from the upper dermis and were seen mostly in the lower dermis
In contrast, the area occupied by blood vessels was generally increased (P<0.01). We analyzed vascularity through double immunofluorescent staining. We used antibodies to PAL-E, a specific marker of blood vessels, and CD31, ubiquitously expressed in endothelial cells (lymphatic and blood vessels), to visualize lymphatic (CD31+/PAL-E-) and blood (CD31+/PAL-E+) vasculature.
4. Exogenous VEGF-C administration produced immunohistochemical evidence of a lymphangiogenic response
After VEGF-C treatment, there was a significant reproducible increase in lymphatic vascularity compared with the saline-treated lymphedema specimens (P<0.01; Fig. 1
). Overall, blood vascularity of VEGF-C-treated specimens was not significantly different from that of normal skin.
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5. Light microscopy of untreated experimental lymphedema revealed thickening of the skin and subcutaneous (s.c.) tissues
There was evidence of a highly thickened epidermis, a highly cellular dermis, and an overall increase in thickness of the tissues. Thickness of the epidermis in lymphedema increased 
three- to fourfold and there was evidence of parakeratosis (Fig. 2
). These changes are consistent with the cutaneous patterns observed in human chronic lymphedema.
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6. VEGF-C restores normal tissue architecture
After VEGF-C administration, the histology closely resembles that of normal ears: the thickness of the epidermis is normalized and there is an overall decrease in cellularity of the dermis.
7. Immunohistochemistry confirms the salutary effect of VEGF-C treatment on tissue hypercellularity
Cutaneous thickening of the lymphedematous tissues is characterized by a dramatically increased cellular density in the dermis compared with the skin of the normal ears (P<0.001; Fig. 1
). Strikingly, treatment with VEGF-C restored the cellularity of the dermis to normal levels.
CONCLUSION AND SIGNIFICANCE
In this model of chronic postsurgical lymphatic insufficiency, which functionally and histologically simulates the human disease state of secondary lymphedema, VEGF-C administration had a marked lymphangiogenic effect. The resultant improvement in lymphoscintigraphically documented fluid transport is accompanied not only by the expected immunohistochemical changes of a lymphangiogenic response, but also by a significant normalization of the cellular architecture in the treated tissues. We have thus used the rabbit ear model of chronic acquired lymphedema to effectively demonstrate the feasibility of eliciting a lymphangiogenic response to exogenously administered VEGF-C.
Our observations serve to complement recent reports of therapeutic lymphangiogenesis in a mouse model of primary (hereditary) lymphedema in which an inactivating VEGFR-3 mutation in the germ line is accompanied by swelling of the limbs and hypoplastic cutaneous lymphatic vessels. Virus-mediated VEGF-C gene therapy in these lymphedematous mice leads to the generation of new, functional lymphatic vessels. Observations in the Chy mouse provide a paradigm for gene therapy of diseases associated with mutant receptors related to lymphatic development. The current investigation extends such observations to the realm of acquired lymphatic insufficiency, where the lymphangiogenic response to vascular injury is inadequate to circumvent the structural consequences of lymph stasis.
The demonstration of effective lymphangiogenesis in this model significantly enhances the prospects for growth factor-mediated therapies to a proportionately larger segment of the diseased population than is represented by Milroy’s disease alone. Perhaps of greatest interest is the observation that VEGF-C-induced lymphangiogenesis invokes a tissue remodeling response, with marked reversal of the hypercellularity that characterizes the untreated lymphedematous state. In the human clinical condition of lymphedema, it is the chronic architectural derangement in the skin and s.c. tissues that pose the greatest source of morbidity and dysfunction for the patient. The seemingly inexorable progression to s.c. fibrosis has so far lacked efficacious avenues for prevention or therapy.
This study represents a first observation that therapeutic lymphangiogenesis with exogenous VEGF-C will not only restore lymphatic function in chronic, acquired lymphedema but will also have a profound restorative effect on tissue cellularity and architecture. These observations heighten the expectation that molecular therapeutics for lymphatic insufficiency will have applicability to the reversal of both fluid accumulation and structural alteration in these disorders. Delineation of the mechanisms that promote such changes will require intensive further investigation. Additional study of this model, and its responsiveness to therapeutic lymphangiogenesis should entail a detailed characterization of the dose-response relationships, the temporal nature and durability of the therapeutic response, and further identification and characterization of the labile cellular populations observed in the skin before and after successful therapeutic lymphangiogenesis.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0401fje; to cite this article, use FASEB J. (October 18, 2002) 10.1096/fj.02-0401fje 
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