TGF-beta 1 mediates pathologic changes of secondary lymphedema by promoting fibrosis and inflammation

Background: Secondary lymphedema is a common complication of cancer treatment, and previous studies have shown that the expression of transforming growth factor-beta 1 (TGF-beta 1), a pro-fibrotic and anti-lymphangiogenic growth factor, is increased in this disease. Inhibition of TGF-beta 1 decreases the severity of the disease in mouse models; however, the mechanisms that regulate this improvement remain unknown. Methods: Expression of TGF-beta 1 and extracellular matrix molecules (ECM) was assessed in biopsy specimens from patients with unilateral breast cancer-related lymphedema (BCRL). The effects of TGF-beta 1 inhibition using neutralizing antibodies or a topical formulation of pirfenidone (PFD) were analyzed in mouse models of lymphedema. We also assessed the direct effects of TGF-beta 1 on lymphatic endothelial cells (LECs) using transgenic mice that expressed a dominant-negative TGF-beta receptor selectively on LECs (LECDN-RII). Results: The expression of TGF-beta 1 and ECM molecules is significantly increased in BCRL skin biopsies. Inhibition of TGF-beta 1 in mouse models of lymphedema using neutralizing antibodies or with topical PFD decreased ECM deposition, increased the formation of collateral lymphatics, and inhibited infiltration of T cells. In vitro studies showed that TGF-beta 1 in lymphedematous tissues increases fibroblast, lymphatic endothelial cell (LEC), and lymphatic smooth muscle cell stiffness. Knockdown of TGF-beta 1 responsiveness in LECDN-RII resulted in increased lymphangiogenesis and collateral lymphatic formation; however, ECM deposition and fibrosis persisted, and the severity of lymphedema was indistinguishable from controls. Conclusions: Our results show that TGF-beta 1 is an essential regulator of ECM deposition in secondary lymphedema and that inhibition of this response is a promising means of treating lymphedema.