Figure 1.
Tail volume response to pharmacotherapy.
Changes in tail volume are expressed as a percentage of the volume on day 0. By day 7, lymphedema (LY) mice demonstrate significant increase in tail volume. NSAID-treated lymphedema (LY-NSAID) mice were significantly less edematous than normal controls (NL) or sham surgery control (SH) mice. Conversely, sTNF-R1-treated lymphedema (LY-sTNF-R1) mice were significantly more edematous. *P<0.05 vs LY, †P<0.05 vs LY-NSAID.
Figure 2.
Histological responses to pharmacotherapy.
Tail sections were harvested 16 mm from the base of the tail, stained with hematoxylin/eosin, and examined by light microscopy. (A) Representative histology. Specimens from normal control (NL) mice, sham surgery control (SH) mice, and lymphedema (LY) mice treated with either PBS, ketoprofen (NSAID), or the TNF-α inhibitor sTNFR-1. Untreated LY show hyperkeratosis, epidermal spongiosis and edema, irregularity of the epidermal/dermal junction, elongation of the dermal papillae, and a 2- to 3-fold expansion of tissue between the bone and the epidermis. There are numerous dilated microlymphatics and increased cellularity, including a large infiltration of neutrophils. Treatment with NSAID normalizes these pathological findings whereas treatment with sTNFR-1 exacerbates the pathology. (B) Quantification of epidermal thickness (ET). Changes in ET are expressed as a percentage of the average ET of NL. ET of NSAID-treated lymphedema (LY-NSAID) mice was significantly reduced compared to untreated LY mice (P<0.0005) and were not significantly different than NL or SH control mice. ET of sTNF-R1 treated lymphedema (LY-sTNF-R1) mice was significantly increased compared to untreated LY mice (P<0.05). *P<0.05 vs LY, †P<0.0005 vs LY-NSAID.
Figure 3.
Targeted gene expression analysis by quantitative real-time PCR.
Fold-changes of gene expression are relative to normal (NL) controls on day 11. NSAID treatment significantly induced the expression of TNF-α, MCP-1, VEGF-C, VEGFR-3 and Prox1 in lymphedema (LY) mice. The TNF-α inhibitor sTNF-R1 downregulated TNF-α but did not affect other genes. *P<0.05 vs LY, †P<0.05 vs LY-NSAID.
Figure 4.
TNF-α levels in tissue homogenates of tail skin.
Median fluorescence intensity was used to assess the relative tissue concentrations of TNF-α. TNF-α levels were significantly higher in mice with NSAID-treated lymphedema (LY-NSAID) (P<0.005) than in normal controls or mice with untreated or sTNF-R1-treated lymphedema (LY-sTNF-R1). *P<0.005 vs untreated lymphedema, †P<0.005 vs LY-NSAID.
Figure 5.
Inflammatory cytokine levels in tissue homogenates of tail skin.
Median fluorescence intensity was used to assess the relative tissue concentrations of MCP-1, MCP-3, MIP1a, Eotaxin, and VEGF-A. MCP-1 levels were elevated in mice with lymphedema (LY) and further increased by both ketoprofen and sTNF-R1 anti-inflammatory treatments. MCP-3 levels were also elevated in lymphedema but were significantly reduced by both treatments. A similar pattern was observed for macrophage inflammatory protein 1a (MIP1a). Like TNF-α, eotaxin levels were reduced by NSAID therapy and decreased by sTNF-R1. No effect on the blood vascular growth factor VEGF-A was observed. *P<0.04 vs to LY, †P = 0.05 vs LY, ‡P<0.05 vs LY-NSAID.
Figure 6.
Model of inflammation and lymphedema.
Loss of lymphatic vascular integrity leads to diminished lymph transport, which promotes both edema and inflammation. TNF-α, a potent mediator of inflammation, is also a known inducer of the pro-lymphangiogenic factor, VEGF-C. Both ketoprofen and pegsunercept have general inhibitory effects on inflammation, ketoprofen promotes endogenous repair mechanisms mediated by VEGF-C and VEGFR-3by simultaneous inducing TNF-α. In contrast, pegsunercept directly inhibits TNF-α and therefore exacerbates the disease state by disrupting pro-lymphangiogenic processes driven by VEGF-C and VEGFR-3.