Hyaluronan-induced VEGF-C promotes fibrosis-induced lymphangiogenesis via Toll-like receptor 4-dependent signal pathway

https://doi.org/10.1016/j.bbrc.2015.09.023Get rights and content

Highlights

  • Hyaluronan (HA) accumulation is correlated with lymphangiogenesis in the obstructed kidney.

  • HA potentiates VEGF-C expression in bone marrow-derived macrophages.

  • Lymphangiogenesis in obstructed kidney is regulated by a TLR4-dependent signaling pathway.

Abstract

Hyaluronan (HA), a component of the extracellular matrix, modulates cellular behavior including angiogenesis. However, little is known about the effect of HA on lymphangiogenesis in fibrosis model. In this study, we investigated the roles of HA in lymphangiogenesis of unilateral ureteral obstruction (UUO). We found that HA cooperated synergistically with vascular endothelial cell growth factor-C to stimulate capillary-like tube formation and increase migration of cells in a haptotaxis assay. Accumulation of HA in the cortical interstitial space was positively correlated with the number of lymphatic vessels after UUO. Depletion of macrophages with clodronate decreased UUO-induced HA accumulation and lymphangiogenesis. Additionally, hyaluronan synthase (HAS) mRNA expression and HA production were increased in bone marrow-derived macrophages upon stimulation with TGF-β1. Transfer of mHAS2 and mHAS3 knock-down CD11b-positive macrophages to SCID mice resulted in a partial decrease in UUO-induced lymphangiogenesis. HA increased expression of vascular endothelial cell growth factor-C in macrophages. Vascular endothelial cell growth factor-C expression and LYVE-1-positive lymphatic area was significantly lower in the UUO-kidney from TLR4 null mice than that from TLR4 wild-type mice. Collectively, these results suggest that HA increases lymphangiogenesis in renal fibrosis model and also stimulates vascular endothelial cell growth factor-C production from macrophages through Toll-like receptor 4-dependent signal pathway.

Introduction

Tissue injury results in inflammatory cell infiltration and extracellular matrix remodeling with accumulation of hyaluronan (HA) [1]. HA, a glycosaminoglycan found throughout the extracellular matrix, displays several biological roles including water homoeostasis, tumor metastasis, wound healing, and modulation of inflammation [2]. HA has also been shown to be involved in monocyte activation [3], leukocyte adhesion to the endothelium [4], and vascular angiogenesis [5]. Angiogenic effect of HA appears to depend on HA concentration and molecular size [6]. In the kidney, HA is normally expressed only on papillae. Increased expression of HA in the cortex is related to several renal pathologic conditions including acute ischemic injury [7], interstitial nephritis [8], acute human kidney graft rejection [9], cyclosporine toxicity [10], and IgA nephropathy [11]. HA accumulation in the fibrotic rat renal cortex after ureteral obstruction has also been reported [12].

Lymphangiogenesis, which refers to the formation of new lymphatic vessels, is associated with many pathological conditions such as tumor metastasis, wound healing, and inflammation. Renal lymphangiogenesis has been demonstrated in a rat remnant kidney model and human transplanted kidney [13], [14]. We have previously demonstrated that vascular endothelial growth factor (VEGF)-C is a lymphangiogenic factor associated with lymphangiogenesis in the fibrotic kidney in a mouse model of ureteral obstruction [14]. Recently, it has been shown that low molecular weight HA induces lymphangiogenesis in vitro [15]. Lymphangiogenesis may be a feature of renal fibrosis after ureteral obstruction, and fibrosis in a unilateral ureteral obstructed (UUO) kidney is characterized by excessive accumulation of HA. However, little is known about the effects of HA on lymphangiogenesis in ureteral obstruction-induced renal fibrosis.

Based on the available information, we hypothesized that HA has a role in UUO-induced lymphangiogenesis. Thus, our aim in this study was to evaluate the lymphangiogenic effect of HA in a mouse model of UUO-induced renal fibrosis and to determine how HA regulates VEGF-C production in macrophages.

Section snippets

Materials and methods

Detailed methods are available in Supplementary information.

HA induces in vitro lymphangiogenesis of hLECs

It has been demonstrated that HA induces proliferation, migration and capillary tube formation in lymphatic endothelial cells [15]. We further evaluated the in vitro lymphangiogenic effect of HA. To assess sprouting of hLECs from mouse thoracic ducts, we performed a lymphatic ring assay. HA increased the number of LEC sprouts compared to the control buffer (Supplementary Fig. S1a and S1b). Haptotaxis refers to cell migration along a chemoattractant gradient associated with the surface. Collagen

Discussion

HA accumulation in tissue has been demonstrated after tissue injury. HA content has been shown to be increased in bleomycin-induced lung fibrosis and a pulmonary ischemic model due to decreased HA degradation or increased HA synthesis [22]. HA levels in the liver and serum are increased in liver cirrhosis [23]. HA levels are also increased in the kidney in several pathologic conditions including acute ischemic injury [7], cyclosporine toxicity [10], and the UUO rat kidney [12]. However, the

Acknowledgments

This work was supported by the National Research Foundation of Korea (NRF) funded by the Korean government (NRF-2014R1A1A4A01003832; Park S.K.), research funds of Chonbuk National University in 2013 (Lee A.S.) and by a grant (CUHBRI-2012- 02-003) from CNUH-BRI (Kim W.) and MRC (No.2008-0062279; Kim W).

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    Jung YJ and Lee AS contributed equally to this work.

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