Hydrolysis of Hyaluronic Acid in Lymphedematous Tissue Alleviates Fibrogenesis via TH1 Cell-Mediated Cytokine Expression

Abstract

Although surgery and radiation are beneficial for treating cancer, they can also lead to malfunctions of the lymphatic system such as secondary lymphedema. This abnormality of the lymphatic system is characterized by severe swelling, adipogenesis, inflammation, and fibrosis in the lymphedematous region. Moreover, the proliferation of fibrotic tissue in the lymphedematous region generates edema that is no longer spontaneously reversible. No treatment for fibrosis has been validated in patients with lymphedema. In our efforts to develop a therapeutic agent for lymphedema fibrosis, we used a newly established mouse hind limb model. Previous studies have demonstrated that hyaluronic acid accumulates in the lymphedematous region. Thus, we challenged mice with of hyaluronidase (HYAL), with the aim of reducing fibrogenesis. After subcutaneous injections in the lymphedematous mouse leg every two days, the volume of lymphedema had reduced significantly by 7 days post-operation. Histochemical analysis indicated that collagen accumulation and myofibroblast differentiation were decreased in epidermal tissues after HYAL injection. Moreover, it was associated with upregulation of interferon-gamma, increased numbers of Th1 cells, and downregulation of interleukin-4 and interleukin-6 in the lymphedematous region and spleen. These results indicate that hydrolysis of hyaluronic acid can boost an anti-fibrotic immune response in the mouse lymphedema model.

Figure 1

Analysis of the mouse model of lymphedema. Images of the right mouse leg at 3 days postsurgery. (A) Sham-operated mouse (without lymphatic damage) and mouse with lymphedema. (B,C) Cross-sectional mouse leg histology. Proximal slides were visualized via Masson’s trichrome staining. Scale bar = 5000 μm. Collagen is denoted by blue staining. Percentages of fibrotic areas (blue areas) were determined using Image J software. (D) Western blot analysis of the lymphedema markers VEGFR-3 and LYVE-1, alpha-smooth muscle actin, and GAPDH as a loading control. (E) Quantitative real-time PCR analysis of the expression of various hyaluronic acid synthases.

Figure 2

Subcutaneous injection of hyaluronidase alleviates lymphedema in a mouse model. At 7 days postsurgery, PBS-injected mice with lymphedema (PBS-LE) and hyaluronidase-injected mice with lymphedema (HYAL-LE) were sacrificed. Cross-sections of the legs with edema were then analyzed. (A,B,C) Representative Masson’s trichrome staining of fibrotic areas. Scale bar = 500 μm. (D,E) Image J analysis of dermal thickness and fibrotic (blue) areas. Data are presented as means ± SEMs. *P < 0.05, **P < 0.005, ***P < 0.001. (F) Schematic diagram of the PBS and hyaluronidase injection schedule. •, hyaluronidase and PBS injection; ■, time of sacrifice.

Figure 3

Hyaluronidase treatment restores dilated lymphatic vessels to round lymphatic vessels and downregulates the expression of fibrosis markers. Histological analysis of the effects of hyaluronidase on fibrosis in lymphedema. (A,B) Immunofluorescence staining of LYVE-1, VEGFR-3, and alpha-smooth muscle actin in proximal mouse leg sections. Scale bar = 100 μm. Regions of LTVE-1 and VEGFR-3 colocalization in the lymphatic vessels are marked with yellow arrows. (C,D) Immunohistochemistry analysis of RAC1 and CTGF in the dermis layer of the mouse leg. Scale bar = 500 μm.

Figure 4

Hyaluronidase injection induces anti-fibrotic responses. Molecular analysis of mouse leg lysates. (A–C) Quantitative real-time PCR analysis of VEGF-D, fibronectin, MMP3, MMP9, α-SMA and HABP2. (A) Increased expression levels of MMP3 and MMP9 were observed, (B) Whereas decreased expression levels of VEGF-D, and fibronectin were observed in the hyaluronidase-injected mice. (C) The expression of α-SMA was decreased with hyaluronidase treatment, but HABP2 expression was increased. Data are presented as means ± SEMs. *P < 0.05, ***P < 0.001.

Figure 5

Subcutaneous hyaluronidase injection alters cytokine expression. Analysis of cytokine expression in total mouse leg tissue. (A) Quantitative real-time PCR analysis of IL-6 mRNA expression. Data are presented as means ± SEMs. (B) ELISA analysis of the levels of TGF-β, IL-4, IFN-γ, and IL-12. Data are expressed as median optical intensities. Data are presented as means ± SEMs. (C) Representative western blot of IFN-γ and CD44 expression. **P < 0.01, ***P < 0.001.

Figure 6

Hyaluronidase injection significantly alters splenocyte T cell populations. Analysis of cytokine expression and T_h cell population analysis in total mouse spleen tissue. (A) ELISA analysis of the levels of TGF-β, IL-4, IFN-γ on spleen. Data are expressed as median optical intensities. Data are presented as means ± SEMs. (B) Representative flow cytometry results of T_H1 and T_H2 cell populations. Data are presented as means ± SEMs. (C,D) Quantitative real-time PCR analysis of IL-6 and HABP2 mRNA expression. Data are presented as means ± SEMs. *P < 0.05, **P < 0.005, ***P < 0.001.