Lymphangiogenesis is considered a promising approach for increasing fluid drainage during secondary lymphedema. However, organization of lymphatics into functional capillaries may be dependent upon interstitial flow (IF). The present study was undertaken to determine the importance of lymphangiogenesis for lymphedema resolution. We created a lymphatic obstruction that produces lymphedema in mouse tail skin. The relatively scar-free skin regeneration that occurred across the obstruction allowed the progression of lymphangiogenesis to be observed and compared with the evolution of lymphedema. The role of vascular endothelial growth factor-C (VEGF-C)/VEGF receptor (VEGFR)-3 signaling in lymphedema resolution was investigated by exogenous administration of VEGF-C or neutralizing antibodies against VEGFR-3. VEGF-C protein improved lymphedema at 15 days [reducing dermal thickness from 742 +/- 105 to 559 +/- 141 microm with 95% confidence intervals (CIs), P < 0.05] without increasing lymphatic capillary coverage (11.6 +/- 6.4% following VEGF-C treatment relative to 9.6 +/- 6.2% with 95% CIs, P > 0.50). Blocking VEGFR-3 signaling did not inhibit lymphedema resolution at 25 days (dermal thickness of 462 +/- 127 microm following VEGFR-3 inhibition relative to 502 +/- 87 microm with 95% CIs) or inhibit IF, although VEGFR-3 blocking prevented lymphangiogenesis (reducing lymphatic coverage to 0.2 +/- 0.7% relative to 8.7 +/- 7.3% with 95% CIs, P < 0.005). A second mouse tail lymphedema model was employed to investigate the ability of VEGF-C to increase fluid drainage across a scar. We found that neither neutralization of VEGFR-3 nor administration of VEGF-C affected the course of skin swelling over 25 days. These findings suggest that resolution of lymphedema in the mouse tail skin may be more dependent upon IF and regeneration of the extracellular matrix across the obstruction than lymphatic capillary regeneration.

Vascular endothelial growth factor (VEGF)-C is necessary for lymphangiogenesis, and excess VEGF-C has been shown to be ameliorative for edema produced by lymphatic obstruction in experimental models. However, it has recently been shown that edema can resolve in the mouse tail even in the complete absence of capillary lymphangiogenesis when distal lymph fluid crosses the regenerating wound site interstitially. This finding has raised questions about the action of VEGF-C/VEGF receptor (VEGFR) signaling during the resolution of experimental edema. Here, the roles of VEGFR-2 and VEGFR-3 signaling in edema resolution were explored. It was found that edema resolved following neutralization of either VEGFR-2 or VEGFR-3 in the mouse tail skin, which inhibited lymphangiogenesis. Neutralization of either VEGFR-2 or VEGFR-3 reduced angiogenesis at the site of obstruction at day 10 (9.2 ± 1.2% and 11.5 ± 1.0% blood capillary coverage, respectively) relative to controls (14.3 ± 1.5% blood capillary coverage). Combined VEGFR-2/-3 neutralization more strongly inhibited angiogenesis (6.9 ± 1.5% blood capillary coverage), leading to a reduced wound repair of the lymphatic obstruction and extended edema in the tail skin. In contrast, improved tissue repair of the obstruction site increased edema resolution. Macrophages in the swollen tissue were excluded as contributing factors in the VEGFR-dependent extended edema. These results support a role for VEGFR-2/-3-combined signaling in the resolution of experimental edema that is lymphangiogenesis independent.

Lymphatic dysfunction in lymphedema results in chronic accumulation of interstitial fluid and life-long tissue swelling. In the absence of restored lymphatic drainage via adequate lymphangiogenesis, the interstitial environment can remodel in ways that decrease the elevated interstitial stress. Presently, relatively little is known about the glycosaminoglycans (GAGs) that become upregulated in the interstitium during lymphedema. We employed a mouse tail model of acute lymphedema that reproduces important features of the chronic human condition to establish a relationship between hyaluronan (HA) and sulfated GAG concentration with tissue swelling. We found that HA was upregulated by tissue injury at day 5 and became upregulated again by skin swelling (HA content increasing by 27% relative to controls at days 15 and 20). Surprisingly, the second phase of HA expression was associated with the declining phase of the tail skin swelling (tail diameter significantly decreasing by 17% from day 10 peak to day 20), demonstrating that HA is upregulated by tissue swelling and may help to counteract the edema in the mouse tail. This finding was confirmed by intradermal injection of an HA degrading enzyme (hyaluronidase) to the swollen tail, which was found to worsen the tail swelling. Sulfated GAGs, including chondroitin sulfate (CS), were not regulated by tissue swelling. The results demonstrate that HA, but not sulfated GAGs, is upregulated in the interstitium by acute tissue swelling. We speculate that HA expression during lymphedema may be part of a natural adaptive mechanism of the interstitial environment to reduce capillary filtration and increase interstitial fluid outflow following lymphatic obstruction and fluid accumulation.

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