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Hyaluronan-induced VEGF-C promotes fibrosis-induced lymphangiogenesis via Toll-like receptor 4-dependent signal pathway
Biochemical and Biophysical Research Communications 466 (2015) 339e345
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Hyaluronan-induced VEGF-C promotes fibrosis-induced lymphangiogenesis via Toll-like receptor 4-dependent signal pathway Yu Jin Jung a, e, 1, Ae Sin Lee a, f, 1, Tung Nguyen-Thanh a, Kyung Pyo Kang a, e, Sik Lee a, e, Kyu Yun Jang b, Myung Ki Kim c, e, Sun Hee Kim d, Sung Kwang Park a, e, **, Won Kim a, e, * a
Department of Internal Medicine, Chonbuk National University Medical School, Jeonju, Republic of Korea Department of Pathology, Chonbuk National University Medical School, Jeonju, Republic of Korea Department of Urology, Chonbuk National University Medical School, Jeonju, Republic of Korea d Department of Physiology, Chonbuk National University Medical School, Jeonju, Republic of Korea e Research Institute of Clinical Medicine of Chonbuk National University-Biomedical Research Institute of Chonbuk National University Hospital, Jeonju, Republic of Korea f Korea Food Research Institute, Seongnam, Republic of Korea b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 August 2015 Accepted 5 September 2015 Available online 8 September 2015
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-b1. Transfer of mHAS2 and mHAS3 knock-down CD11b-positive macrophages to SCID mice resulted in a partial decrease in UUOinduced 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. © 2015 Elsevier Inc. All rights reserved.
Keywords: Hyaluronan Lymphatic endothelial cells Lymphangiogenesis Kidney fibrosis Macrophages Toll-like receptor 4
1. 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
* Corresponding author. Department of Internal Medicine, Chonbuk National University Medical School 20, Gunji-ro, Jeonju, 560-180, Republic of Korea. ** Corresponding author. Department of Internal Medicine, Chonbuk National University Medical School 20, Gunji-ro, Jeonju, 560-180, Republic of Korea. E-mail addresses: [email protected] (S.K. Park), [email protected] (W. Kim). 1 Jung YJ and Lee AS contributed equally to this work. http://dx.doi.org/10.1016/j.bbrc.2015.09.023 0006-291X/© 2015 Elsevier Inc. All rights reserved.
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
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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 obstructioninduced 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. 2. Materials and methods Detailed methods are available in Supplementary information.
2.5. Quantitative real-time RT-PCR Quantitative real-time reverse transcription polymerase chain reaction (quantitative real-time RT-PCR) of mouse hyaluronan synthase (mHAS)1, mHAS2, and mHAS3 from kidney, BMDMs was performed with preciously describe methods (Supplementary table 1) [14]. 2.6. Immunoblotting Immunoblotting was performed as described previously [18]. BMDMs were homogenized, and a VEGF-C antibody (Abcam, Cambridge, MA), phosphor-ERK1/2, ERK1, phopsho-Akt, Akt (Cell signaling technology, Beverly, MA) were used. 2.7. Lymphatic endothelial cell tube formation assay In vitro tube formation assay was performed using a threedimensional culture of hLECs on Matrigel gel (SigmaeAldrich) as described previously [18]. 2.8. Haptotaxis assay
2.1. Animal experiments: UUO model Animal studies were reviewed and approved by the Institutional Animal Care and Use Committee of Chonbuk National University. Male C57BL/6 mice (18e20 g body weight) were purchased from Orient Bio Inc. (Seoul, Korea) and ureteral ligation was performed as described previously [14]. C3H/HeN (TLR4 wild-type) and C3H/HeJ (TLR4 null) mice were purchased from Japan SLC Inc. (Shizuoka, Japan). Kidney samples were collected 1 and 2 w after ureteral obstruction. 2.2. Cells and reagents To obtain bone marrow-derived macrophages (BMDMs), bone marrow was isolated from femurs and tibias of male C57BL/6 mice as described previously [14]. Human lymphatic endothelial cells (hLECs) were obtained from PromoCell (Heidelberg, Germany) and recombinant human VEGF-C was purchased from R&D Systems (Minneapolis, MN). TGF-b1, PD98059 (10 mmol/L), LY294002 (10 mmol/L), and wortmannin (30 nmol/L) were purchased from SigmaeAldrich (St Louis, MO, USA). We incubated BMDMs with MyD88 inhibitory peptide (Novus Biologicals, Littleton, CO) before stimulating with HA. 2.3. Immunofluorescence staining Immunofluorescence staining was performed as described previously [14]. Anti-mouse F4/80 (eBioscience, San Diego, CA), and anti-VEGF-C (Invitrogen, Carlsbad, CA) antibodies were used to stain frozen sections of mouse kidney. For HA detection, kidney sections were incubated for 1 h with a biotinylated HA-binding protein (HABP; Calbiochem, San Diego, CA). 2.4. Depletion of macrophages with clodronate treatment Clodronate was purchased from Sigma (St Louis), and liposomeencapsulated clodronate was prepared according to a previously described method [16]. For systemic depletion of macrophages, clodronate liposomes (CDL, 50 mg/kg) were administered through the peritoneum 1 day before operation and once every two days until kidney harvest [17].
To evaluate migration of hLECs along a gradient of ECM-bound chemoattractants, we used a haptotaxis assay (Cell Biolabs, San Diego, CA) based on a Boyden chamber precoated on the underside with collagen I. Migratory cells were quantified using either a colorimetric or fluorescence plate reader. 2.9. Three-dimensional lymphatic-ring assay Thoracic ducts were harvested as described previously [19]. HA (10, 50, or 100 mg/mL), control buffer, and recombinant VEGF-A or VEGF-C (R&D Systems, Minneapolis, MN) were added to the culture medium. 2.10. Enzyme-linked immunosorbent assay HA was measured using an enzyme-linked immunosorbent assay kit (Quantkine ELISA-hyaluronan, R&D Systems) in accordance with the manufacturer's protocol. 2.11. Statistics Results are presented as means ± SD. The level for statistical significance was defined as P < 0.05. A non-parametric KruskaleWallis one-way analysis of variance of ranks was applied for multiple intergroup comparisons followed by Dunn's test to identify significant differences from the control group. Statistical correlation between the HA level and number of lymphatic vessels was evaluated by examining Pearson's correlation coefficients. 3. Results 3.1. 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 I is one of the main components in renal fibrosis after ureteral obstruction
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[20]. Therefore, we examined whether HA stimulates collagen-I associated migration of hLECs using a haptotaxis assay kit. HA significantly increased the number of hLECs that migrated to a collagen-coated surface (Supplementary Fig. S1c). Furthermore, we evaluated whether HA (MW 35e75 kD) and VEGF-C display a synergistic effect on lymphangiogenesis in vitro. Treatment of hLECs with HA increased tube formation compared to control buffer-treated cells and the numbers of tube formed after treatment with HA and VEGF-C was significantly higher than the number of tubes that formed after treatment with HA or VEGF-C alone (Supplementary Fig. S1d and S1e). HA significantly increased proliferation of hLECs compared to the control buffertreated cells (Supplementary Fig. S1f). 3.2. HA accumulation is correlated with the number of LYVE-1positive lymphatic vessels in the UUO kidney We previously demonstrated that lymphangiogenesis is increased in the fibrotic kidney [14]. Thus, we examined the role of HA in UUO-induced renal lymphangiogenesis. First, to determine whether HA is accumulated in the UUO kidney of a mouse model, we stained kidney sections of sham- and UUO-operated mice using a HA-binding protein that detects HA greater than or equal 20 kD in the molecular weight. Immunohistochemical staining revealed scanty expression of HA in the renal cortex in sham-operated mice. In contrast, HA accumulation in the interstitial space of the renal cortex was increased at 1 and 2 w after UUO (Supplementary Fig. S2a and S2b). HA expression was substantially higher in
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obstructed renal cortices than normal renal cortices of human kidneys (Supplementary Fig. S2c, S2d, and S2e). To determine whether the accumulation of HA was correlated with the change in vascular density of LYVE-1-positive lymphatic cells, kidney sections from sham- and UUO-operated mice were stained with a LYVE-1 antibody and HA-binding protein. In sham-operated kidneys, LYVE-1-positive lymphatic vessels were found only around renal arterioles in renal cortex. In UUO kidneys, LYVE-1-positive lymphatic vessels were located in the HA-positive area (Fig. 1a). We also found that the HA-positive area was positively correlated with the number of LYVE-1-positive lymphatic vessels in the UUO kidney (Fig. 1b). 3.3. Expression of mHAS2 and mHAS3 mRNA is increased in the UUO kidney HA is synthesized by HAS1, HAS2, and HAS3; we therefore evaluated the mRNA expression of mHAS1, mHAS2, and mHAS3 in sham- and UUO-operated kidneys by quantitative real-time RTPCR. mHAS1 mRNA expression in UUO-operated kidney was increased significantly by 2.5-fold at 1 w over sham-operated kidneys but somewhat decreased at 2 w (Fig. 1c). The increase in mRNA levels of mHAS2 and mHAS3 in UUO-operated kidney was sustained up to 2 w. To evaluate changes in the level of HA in the kidney, tissue levels of HA were measured using an ELISA kit that measures HA greater than or equal to 35 kD in size. We observed an 8-fold increase in total HA at 1 w after UUO, and the levels of HA remained elevated up to 2 w after UUO (Fig. 1d).
Fig. 1. HA levels is correlated with the number of LYVE-1-positive lymphatic vessels in UUO. (A) Sham-operated (Sham) and ureteral obstructed kidneys (UUO) obtained 2 w after the operation were double immunostained for lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) and hyaluronan (HA). Scale bar ¼ 50 mm B) Correlation between the number of LYVE-1-positive lymphatic vessels and HA-positive area (n ¼ 7 in each group). (C) Analysis of the mRNA expression of mouse hyaluronan synthase (mHAS)1, mHAS2, and mHAS3 by quantitative real-time RT-PCR in sham-operated and UUO kidneys (1 and 2 w). Fold-changes in mHAS1, mHAS2, and mHAS3 mRNA expression are shown relative to sham-operated kidneys. n ¼ 6 per group. (D) Tissue levels of HA in the kidney. HA was quantified by ELISA. n ¼ 5 per group. Bars represent mean ± SD. *P < 0.05 versus sham; ***P < 0.001 versus sham.
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3.4. TGF-b1 increases mRNA expression of mHAS1, mHAS2, and mHAS3 as well as HA production in bone marrow-derived macrophages TGF-b1 is a cytokine linked to renal fibrosis and decreased function of the kidney [21]. We therefore explored whether HA is produced by macrophages after stimulation with TGF-b1. To gain insight into the effects of TGF-b1 on mHAS1, mHAS2, and mHAS3 mRNA expression, we treated BMDMs with TGF-b1. Treatment of BMDMs with TGF-b1 increased mHAS2 and mHAS3 mRNA expression (Supplementary Fig. S3a). ELISA data showed that HA production was significantly increased in TGF-b1-treated BMDMs compared
to those in control bufferetreated BMDMs (Supplementary Fig. S3b). Treatment of BMDMs with TGF receptor inhibitors (SD208, and SB431542) significantly decreased TGF-b1-induced HA production (Supplementary Fig. S3c). We also found that HA stimulated proliferation of BMDMs (Supplementary Fig. S3d). 3.5. Macrophage depletion with clodronate decreases UUO-induced HA accumulation and lymphangiogenesis We explored the role of macrophages in HA accumulation in UUO kidneys by depleting the cells with clodronate liposomes. Treatment with clodronate liposomes reduced the number of F4/
Fig. 2. Clodronate liposomes markedly reduce number of F4/80-positive macrophages and lymphangiogenesis induced by ureteral obstruction. (A) Immunofluorescence staining of F4/80 and HA in the kidney after depletion of macrophages. Clodronate liposome (CDL) at a concentration of 50 mg/kg was administered 1 day before the operation and every second day before harvesting kidneys. Empty control liposomes (CLs) were injected as a control. Kidneys from mice that underwent sham or UUO operation were collected 1 week after the operation. Tissues were fixed in 4% formaldehyde solution, and kidney sections were then stained with a F4/80 antibody and HA-binding protein. (B) Number of F4/ 80-positive macrophages per unit area of the kidney after treatment with CDL or CL. Number of F4/80-positive macrophages in sham- or UUO-operated kidneys was measured in each unit area. n ¼ 4 per group. (C) HA levels in the kidney were quantified by ELISA after treatment with CDL or CL. n ¼ 5 per group. (D) Immunofluorescence staining of LYVE-1 and HA in the kidney after depletion of macrophages. (e) Quantification of the number of LYVE-1-positive vessels per unit area of sham or UUO kidney after treatment with CDL or CL. n ¼ 5 per group. Bars represent mean ± SD. **P < 0.01 versus sham þ CL; ***P < 0.001 versus sham þ CL; þþP < 0.01 versus UUO þ CL.
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80-positive macrophages in the kidney by approximately 92% and also markedly suppressed UUO-induced HA expression (Fig. 2a and b). ELISA data also showed that HA levels in clodronate-treated kidneys were significantly lower than control buffer-treated kidneys after UUO operation (Fig. 2c). Depletion of macrophages by clodronate significantly reduced UUO-induced renal lymphangiogenesis in the kidney (Fig. 2d and e). 3.6. Transfer of mHAS2 and mHAS3 knock-down CD11b-positive BMDMs to severe combined immunodeficiency (SCID) partially decreases UUO-induced lymphangiogenesis Because expression of mHAS2 and mHAS3 in BMDMs is induced by treatment with TGF-b1, we evaluated the effect of mHAS2 and mHAS3 knock-down in CD11b-positive BMDMs on HA-induced lymphangiogenesis after UUO using mHAS2- and mHAS3-specific short hairpin RNA lentiviral vectors (mHAS2 and mHAS3 shRNA). We confirmed that mHAS2 and mHAS3 mRNA expression was suppressed in CD11b-positive BMDMs (Supplementary fig. 4a). VEGF-C mRNA expression was significantly decreased in macrophage after knockdown of HAS 2 or 3 (Supplementary Fig. S4b). Before transfer of Has2/Has3 knockdown CD11b-positive BMDMs into SCID mice, BMDMs were labeled with red fluorescent dye (1,10 dioctadecyl-3,3,30 ,30 -tetramethylindocarbocyanine perchlorate; Dil). Confocal microscopic findings showed that the number of Dilpositive BMDMs transfected with control-shRNA was not significantly different from the number of BMDMs transfected with HAS2 and HAS3-sh RNA in UUO kidney (Supplementary fig. 4c and 4d). The mRNA expression of VEGF-C was decreased in UUO-operated kidneys after transfer of mHAS2 and mHAS3 shRNA-transfected BMDMs compared with UUO-operated kidneys after transfer of non-target shRNA-transfected BMDMs (Supplementary Fig. S4e). Adaptive transfer of mHAS2 and mHAS3 shRNA-transfected BMDMs partially decreased UUO-induced lymphangiogenesis (Supplementary Fig. S4f and S4g). 3.7. HA potentiates VEGF-C expression in BMDMs We evaluated whether stimulation of macrophages with HA increases the expression of VEGF-C protein in BMDMs. Immunoblot
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analyses showed that HA increased VEGF-C expression in BMDMs in a time-dependent manner (Fig. 3a and b). 3.8. HA-induced VEGF-C expression and UUO-induced lymphangiogenesis are regulated by a TLR4-dependent signaling pathway To examine whether HA-induced VEGF-C production in BMDMs is linked to the TLR4 signaling pathway, we harvested BMDMs from C3H/HeJ (TLR4 null) mice and treated the cells with various concentrations of HA. Western blot analyses showed that VEGF-C expression was dramatically decreased in BMDMs from TLR4 null mice compared to BMDMs from C3H/HeN (TLR4 wild-type) mice (Fig. 4a). Furthermore, a MyD88 (major adaptor molecule for all TLRs) inhibitory peptide, which inhibits homodimerization of MyD88, suppressed HA-induced VEGF-C expression in BMDMs (Fig. 4b). We explored whether VEGF-C expression and LYVE-1positive area were changed in TLR4 null mice. Western blot analyses showed that VEGF-C expression was decreased in the kidney from TLR4 null mice compared to that from TLR4 wild-type mice at 1 w after UUO (Fig. 4c). ERHR-3-positive macrophage infiltration in kidney from TLR4 null mice was significantly decreased compared to kidney from control mice (Fig. 4d). Immunofluorescence data showed that LYVE-1-positive area was significantly lower in the UUO-kidney from TLR4 null mice than that from TLR4 wild-type mice (Fig. 4e and f). 4. Discussion HA accumulation in tissue has been demonstrated after tissue injury. HA content has been shown to be increased in bleomycininduced 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 role of HA in lymphatic vessel remodeling in kidney diseases has not been determined. In the present study, we demonstrated that HA was produced by macrophages after stimulation with TGF-b1. Transfer of mHAS2/
Fig. 3. HA potentiates VEGF-C expression (A and B) Immunoblot analyses of VEGF-C expression by BMDMs. BMDMs were collected and treated with HA (1, 10, 50, and 100 mg/mL; molecular-weight HA 35e75 kD) for 24 h (A). BMDMs were incubated with HA (50 mg/mL) for 6, 12, 24, and 48 h (B). Blots (top) were probed with anti-VEGF-C. The membrane was stripped and reprobed with an anti-actin antibody to control for protein loading in each lane. n ¼ 4 per group. N ¼ 5 per group. Bars represent mean ± SD. *P < 0.05 versus BMDMs treated with control buffer (CB).
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mHAS3-knockdown macrophages to SCID mice suppressed UUOinduced lymphangiogenesis. We also found that HA increased VEGF-C production through a TLR4-MyD88 signaling pathway in BMDMs. HA is recognized by several receptors, such as CD44, Rhamm (CD168), hyaluronic acid receptor for endocytosis (HARE), LYVE-1, and TLRs [24]. The TLR2/TLR4-MyD88 signaling pathway has been linked to the suppressive effect of HA oligosaccharides on chemokine production and progression of kidney disease [25,26]. Park
et al. [27] have demonstrated that HA promotes angiogenesis through RHAMM-TGFbRI signaling in vascular endothelial cells. In this study, we demonstrated that HA increased the production of VEGF-C in BMDMs through the TLR4 and MyD88-dependent signaling pathway. Our data reveal for the first time that the TLR4-MyD88 signaling axis is involved in HA-induced VEGF-C production in the kidney. Recently, Wu et al. [15] have demonstrated that low molecular weight HA increases proliferation, migration, and capillary-like
Fig. 4. TLR4-dependent signal mechanism is associated with HA-induced lymphangiogenesis. (A) BMDMs were collected from C3H/HeN and C3H/HeJ mice and then treated with HA (1, 10, 50, and 100 mg/mL; molecular-weight HA 35e75 kD) for 24 h and immunoblot analysis was performed. Blots (top) were probed with anti-VEGF-C. The membrane was stripped and reprobed with an anti-actin antibody to control for protein loading in each lane. n ¼ 4 per group. *P < 0.05 versus CB; **P < 0.01 versus CB; þP < 0.05 versus BMDMs from C3H/HeN mice treated with HA. (B) BMDMs were treated with a MyD88 inhibitory peptide (100 mmol/L) and immunoblot analysis was performed. Blots (top) were probed with anti-VEGF-C. The membrane was stripped and reprobed with an anti-actin antibody to control for protein loading in each lane. n ¼ 4 per group. **P < 0.01 versus CB; ## P < 0.01 versus BMDMs treated with HA. (C) Immunoblot analysis of VEGF-C in the kidney from sham-operated or UUO mice. n ¼ 4 per group. **P < 0.01 versus sham-operated C3H/HeN mice; $$ P < 0.01 versus UUO-operated C3H/HeN mice. (D) Number of ERHR3-positive macrophages per unit area of the kidney from C3H/HeN and C3H/HeJ mice. Kidneys were harvested from C3H/HeN and C3H/HeJ mice 1 w after sham- or UUO operation. Number of ERHR-positive macrophages in sham- or UUO-operated kidneys was measured in each unit area (400x). n ¼ 4 per group. ***P < 0.001 versus sham-operated C3H/HeN mice; $$$P < 0.001 versus UUO-operated C3H/HeN mice. (E) Immunofluorescence staining of LYVE-1 in the kidney. Kidneys were harvested from C3H/HeN and C3H/HeJ mice 1 w after sham- or UUO operation. (F) Density of LYVE-1-positive area per unit area of sham or UUO kidney from C3H/HeN and C3H/HeJ mice. n ¼ 4 per group. Bars represent mean ± SD *P < 0.05 versus sham-operated C3H/HeN mice; $ P < 0.05 versus UUO-operated C3H/HeN mice.
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tube formation through LYVE-1 in LECs. They have also demonstrated that HA increases phosphorylation of protein kinase C a/bII and ERK1/2 [15]. Consistent with the results, we confirmed that HA increased capillary-like tube formation and proliferation. Furthermore, we showed that HA collaborated synergistically with VEGF-C, thereby promoting capillary-like tube formation and sprouting of hLECs. We used a Boyden Chamber precoated with collagen I in our haptotaxis assay, because collagen I is the main component of renal fibrosis after UUO. HA treatment increased the number of migrating cells in the haptotaxis assay. Because macrophages can be a source of VEGF-C production, decreased macrophage infiltration in the UUO kidneys of TLR4 null mice may be related to the observed reduction of UUO-induced lymphangiogenesis. In this study, we showed that TGF-b1-induced HA from macrophage induces lymphangiogenesis in UUO mouse model. Taken together, our results suggest that manipulation of lymphangiogenesis using HA could be a potentially valuable treatment strategy for fibrotic conditions and lymphatic cancer metastasis.
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Acknowledgments
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This work was supported by the National Research Foundation of Korea (NRF) funded by the Korean government (NRF2014R1A1A4A01003832; Park S.K.), research funds of Chonbuk National University in 2013 (Lee A.S.) and by a grant (CUHBRI-201202-003) from CNUH-BRI (Kim W.) and MRC (No.2008-0062279; Kim W).
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Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2015.09.023.
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Hyaluronan-induced VEGF-C promotes fibrosis-induced lymphangiogenesis via Toll-like receptor 4-dependent signal pathway Yu Jin Jung a, e, 1, Ae Sin Lee a, f, 1, Tung Nguyen-Thanh a, Kyung Pyo Kang a, e, Sik Lee a, e, Kyu Yun Jang b, Myung Ki Kim c, e, Sun Hee Kim d, Sung Kwang Park a, e, **, Won Kim a, e, * a
Department of Internal Medicine, Chonbuk National University Medical School, Jeonju, Republic of Korea Department of Pathology, Chonbuk National University Medical School, Jeonju, Republic of Korea Department of Urology, Chonbuk National University Medical School, Jeonju, Republic of Korea d Department of Physiology, Chonbuk National University Medical School, Jeonju, Republic of Korea e Research Institute of Clinical Medicine of Chonbuk National University-Biomedical Research Institute of Chonbuk National University Hospital, Jeonju, Republic of Korea f Korea Food Research Institute, Seongnam, Republic of Korea b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 August 2015 Accepted 5 September 2015 Available online 8 September 2015
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-b1. Transfer of mHAS2 and mHAS3 knock-down CD11b-positive macrophages to SCID mice resulted in a partial decrease in UUOinduced 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. © 2015 Elsevier Inc. All rights reserved.
Keywords: Hyaluronan Lymphatic endothelial cells Lymphangiogenesis Kidney fibrosis Macrophages Toll-like receptor 4
1. 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
* Corresponding author. Department of Internal Medicine, Chonbuk National University Medical School 20, Gunji-ro, Jeonju, 560-180, Republic of Korea. ** Corresponding author. Department of Internal Medicine, Chonbuk National University Medical School 20, Gunji-ro, Jeonju, 560-180, Republic of Korea. E-mail addresses: [email protected] (S.K. Park), [email protected] (W. Kim). 1 Jung YJ and Lee AS contributed equally to this work. http://dx.doi.org/10.1016/j.bbrc.2015.09.023 0006-291X/© 2015 Elsevier Inc. All rights reserved.
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
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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 obstructioninduced 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. 2. Materials and methods Detailed methods are available in Supplementary information.
2.5. Quantitative real-time RT-PCR Quantitative real-time reverse transcription polymerase chain reaction (quantitative real-time RT-PCR) of mouse hyaluronan synthase (mHAS)1, mHAS2, and mHAS3 from kidney, BMDMs was performed with preciously describe methods (Supplementary table 1) [14]. 2.6. Immunoblotting Immunoblotting was performed as described previously [18]. BMDMs were homogenized, and a VEGF-C antibody (Abcam, Cambridge, MA), phosphor-ERK1/2, ERK1, phopsho-Akt, Akt (Cell signaling technology, Beverly, MA) were used. 2.7. Lymphatic endothelial cell tube formation assay In vitro tube formation assay was performed using a threedimensional culture of hLECs on Matrigel gel (SigmaeAldrich) as described previously [18]. 2.8. Haptotaxis assay
2.1. Animal experiments: UUO model Animal studies were reviewed and approved by the Institutional Animal Care and Use Committee of Chonbuk National University. Male C57BL/6 mice (18e20 g body weight) were purchased from Orient Bio Inc. (Seoul, Korea) and ureteral ligation was performed as described previously [14]. C3H/HeN (TLR4 wild-type) and C3H/HeJ (TLR4 null) mice were purchased from Japan SLC Inc. (Shizuoka, Japan). Kidney samples were collected 1 and 2 w after ureteral obstruction. 2.2. Cells and reagents To obtain bone marrow-derived macrophages (BMDMs), bone marrow was isolated from femurs and tibias of male C57BL/6 mice as described previously [14]. Human lymphatic endothelial cells (hLECs) were obtained from PromoCell (Heidelberg, Germany) and recombinant human VEGF-C was purchased from R&D Systems (Minneapolis, MN). TGF-b1, PD98059 (10 mmol/L), LY294002 (10 mmol/L), and wortmannin (30 nmol/L) were purchased from SigmaeAldrich (St Louis, MO, USA). We incubated BMDMs with MyD88 inhibitory peptide (Novus Biologicals, Littleton, CO) before stimulating with HA. 2.3. Immunofluorescence staining Immunofluorescence staining was performed as described previously [14]. Anti-mouse F4/80 (eBioscience, San Diego, CA), and anti-VEGF-C (Invitrogen, Carlsbad, CA) antibodies were used to stain frozen sections of mouse kidney. For HA detection, kidney sections were incubated for 1 h with a biotinylated HA-binding protein (HABP; Calbiochem, San Diego, CA). 2.4. Depletion of macrophages with clodronate treatment Clodronate was purchased from Sigma (St Louis), and liposomeencapsulated clodronate was prepared according to a previously described method [16]. For systemic depletion of macrophages, clodronate liposomes (CDL, 50 mg/kg) were administered through the peritoneum 1 day before operation and once every two days until kidney harvest [17].
To evaluate migration of hLECs along a gradient of ECM-bound chemoattractants, we used a haptotaxis assay (Cell Biolabs, San Diego, CA) based on a Boyden chamber precoated on the underside with collagen I. Migratory cells were quantified using either a colorimetric or fluorescence plate reader. 2.9. Three-dimensional lymphatic-ring assay Thoracic ducts were harvested as described previously [19]. HA (10, 50, or 100 mg/mL), control buffer, and recombinant VEGF-A or VEGF-C (R&D Systems, Minneapolis, MN) were added to the culture medium. 2.10. Enzyme-linked immunosorbent assay HA was measured using an enzyme-linked immunosorbent assay kit (Quantkine ELISA-hyaluronan, R&D Systems) in accordance with the manufacturer's protocol. 2.11. Statistics Results are presented as means ± SD. The level for statistical significance was defined as P < 0.05. A non-parametric KruskaleWallis one-way analysis of variance of ranks was applied for multiple intergroup comparisons followed by Dunn's test to identify significant differences from the control group. Statistical correlation between the HA level and number of lymphatic vessels was evaluated by examining Pearson's correlation coefficients. 3. Results 3.1. 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 I is one of the main components in renal fibrosis after ureteral obstruction
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[20]. Therefore, we examined whether HA stimulates collagen-I associated migration of hLECs using a haptotaxis assay kit. HA significantly increased the number of hLECs that migrated to a collagen-coated surface (Supplementary Fig. S1c). Furthermore, we evaluated whether HA (MW 35e75 kD) and VEGF-C display a synergistic effect on lymphangiogenesis in vitro. Treatment of hLECs with HA increased tube formation compared to control buffer-treated cells and the numbers of tube formed after treatment with HA and VEGF-C was significantly higher than the number of tubes that formed after treatment with HA or VEGF-C alone (Supplementary Fig. S1d and S1e). HA significantly increased proliferation of hLECs compared to the control buffertreated cells (Supplementary Fig. S1f). 3.2. HA accumulation is correlated with the number of LYVE-1positive lymphatic vessels in the UUO kidney We previously demonstrated that lymphangiogenesis is increased in the fibrotic kidney [14]. Thus, we examined the role of HA in UUO-induced renal lymphangiogenesis. First, to determine whether HA is accumulated in the UUO kidney of a mouse model, we stained kidney sections of sham- and UUO-operated mice using a HA-binding protein that detects HA greater than or equal 20 kD in the molecular weight. Immunohistochemical staining revealed scanty expression of HA in the renal cortex in sham-operated mice. In contrast, HA accumulation in the interstitial space of the renal cortex was increased at 1 and 2 w after UUO (Supplementary Fig. S2a and S2b). HA expression was substantially higher in
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obstructed renal cortices than normal renal cortices of human kidneys (Supplementary Fig. S2c, S2d, and S2e). To determine whether the accumulation of HA was correlated with the change in vascular density of LYVE-1-positive lymphatic cells, kidney sections from sham- and UUO-operated mice were stained with a LYVE-1 antibody and HA-binding protein. In sham-operated kidneys, LYVE-1-positive lymphatic vessels were found only around renal arterioles in renal cortex. In UUO kidneys, LYVE-1-positive lymphatic vessels were located in the HA-positive area (Fig. 1a). We also found that the HA-positive area was positively correlated with the number of LYVE-1-positive lymphatic vessels in the UUO kidney (Fig. 1b). 3.3. Expression of mHAS2 and mHAS3 mRNA is increased in the UUO kidney HA is synthesized by HAS1, HAS2, and HAS3; we therefore evaluated the mRNA expression of mHAS1, mHAS2, and mHAS3 in sham- and UUO-operated kidneys by quantitative real-time RTPCR. mHAS1 mRNA expression in UUO-operated kidney was increased significantly by 2.5-fold at 1 w over sham-operated kidneys but somewhat decreased at 2 w (Fig. 1c). The increase in mRNA levels of mHAS2 and mHAS3 in UUO-operated kidney was sustained up to 2 w. To evaluate changes in the level of HA in the kidney, tissue levels of HA were measured using an ELISA kit that measures HA greater than or equal to 35 kD in size. We observed an 8-fold increase in total HA at 1 w after UUO, and the levels of HA remained elevated up to 2 w after UUO (Fig. 1d).
Fig. 1. HA levels is correlated with the number of LYVE-1-positive lymphatic vessels in UUO. (A) Sham-operated (Sham) and ureteral obstructed kidneys (UUO) obtained 2 w after the operation were double immunostained for lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) and hyaluronan (HA). Scale bar ¼ 50 mm B) Correlation between the number of LYVE-1-positive lymphatic vessels and HA-positive area (n ¼ 7 in each group). (C) Analysis of the mRNA expression of mouse hyaluronan synthase (mHAS)1, mHAS2, and mHAS3 by quantitative real-time RT-PCR in sham-operated and UUO kidneys (1 and 2 w). Fold-changes in mHAS1, mHAS2, and mHAS3 mRNA expression are shown relative to sham-operated kidneys. n ¼ 6 per group. (D) Tissue levels of HA in the kidney. HA was quantified by ELISA. n ¼ 5 per group. Bars represent mean ± SD. *P < 0.05 versus sham; ***P < 0.001 versus sham.
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3.4. TGF-b1 increases mRNA expression of mHAS1, mHAS2, and mHAS3 as well as HA production in bone marrow-derived macrophages TGF-b1 is a cytokine linked to renal fibrosis and decreased function of the kidney [21]. We therefore explored whether HA is produced by macrophages after stimulation with TGF-b1. To gain insight into the effects of TGF-b1 on mHAS1, mHAS2, and mHAS3 mRNA expression, we treated BMDMs with TGF-b1. Treatment of BMDMs with TGF-b1 increased mHAS2 and mHAS3 mRNA expression (Supplementary Fig. S3a). ELISA data showed that HA production was significantly increased in TGF-b1-treated BMDMs compared
to those in control bufferetreated BMDMs (Supplementary Fig. S3b). Treatment of BMDMs with TGF receptor inhibitors (SD208, and SB431542) significantly decreased TGF-b1-induced HA production (Supplementary Fig. S3c). We also found that HA stimulated proliferation of BMDMs (Supplementary Fig. S3d). 3.5. Macrophage depletion with clodronate decreases UUO-induced HA accumulation and lymphangiogenesis We explored the role of macrophages in HA accumulation in UUO kidneys by depleting the cells with clodronate liposomes. Treatment with clodronate liposomes reduced the number of F4/
Fig. 2. Clodronate liposomes markedly reduce number of F4/80-positive macrophages and lymphangiogenesis induced by ureteral obstruction. (A) Immunofluorescence staining of F4/80 and HA in the kidney after depletion of macrophages. Clodronate liposome (CDL) at a concentration of 50 mg/kg was administered 1 day before the operation and every second day before harvesting kidneys. Empty control liposomes (CLs) were injected as a control. Kidneys from mice that underwent sham or UUO operation were collected 1 week after the operation. Tissues were fixed in 4% formaldehyde solution, and kidney sections were then stained with a F4/80 antibody and HA-binding protein. (B) Number of F4/ 80-positive macrophages per unit area of the kidney after treatment with CDL or CL. Number of F4/80-positive macrophages in sham- or UUO-operated kidneys was measured in each unit area. n ¼ 4 per group. (C) HA levels in the kidney were quantified by ELISA after treatment with CDL or CL. n ¼ 5 per group. (D) Immunofluorescence staining of LYVE-1 and HA in the kidney after depletion of macrophages. (e) Quantification of the number of LYVE-1-positive vessels per unit area of sham or UUO kidney after treatment with CDL or CL. n ¼ 5 per group. Bars represent mean ± SD. **P < 0.01 versus sham þ CL; ***P < 0.001 versus sham þ CL; þþP < 0.01 versus UUO þ CL.
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80-positive macrophages in the kidney by approximately 92% and also markedly suppressed UUO-induced HA expression (Fig. 2a and b). ELISA data also showed that HA levels in clodronate-treated kidneys were significantly lower than control buffer-treated kidneys after UUO operation (Fig. 2c). Depletion of macrophages by clodronate significantly reduced UUO-induced renal lymphangiogenesis in the kidney (Fig. 2d and e). 3.6. Transfer of mHAS2 and mHAS3 knock-down CD11b-positive BMDMs to severe combined immunodeficiency (SCID) partially decreases UUO-induced lymphangiogenesis Because expression of mHAS2 and mHAS3 in BMDMs is induced by treatment with TGF-b1, we evaluated the effect of mHAS2 and mHAS3 knock-down in CD11b-positive BMDMs on HA-induced lymphangiogenesis after UUO using mHAS2- and mHAS3-specific short hairpin RNA lentiviral vectors (mHAS2 and mHAS3 shRNA). We confirmed that mHAS2 and mHAS3 mRNA expression was suppressed in CD11b-positive BMDMs (Supplementary fig. 4a). VEGF-C mRNA expression was significantly decreased in macrophage after knockdown of HAS 2 or 3 (Supplementary Fig. S4b). Before transfer of Has2/Has3 knockdown CD11b-positive BMDMs into SCID mice, BMDMs were labeled with red fluorescent dye (1,10 dioctadecyl-3,3,30 ,30 -tetramethylindocarbocyanine perchlorate; Dil). Confocal microscopic findings showed that the number of Dilpositive BMDMs transfected with control-shRNA was not significantly different from the number of BMDMs transfected with HAS2 and HAS3-sh RNA in UUO kidney (Supplementary fig. 4c and 4d). The mRNA expression of VEGF-C was decreased in UUO-operated kidneys after transfer of mHAS2 and mHAS3 shRNA-transfected BMDMs compared with UUO-operated kidneys after transfer of non-target shRNA-transfected BMDMs (Supplementary Fig. S4e). Adaptive transfer of mHAS2 and mHAS3 shRNA-transfected BMDMs partially decreased UUO-induced lymphangiogenesis (Supplementary Fig. S4f and S4g). 3.7. HA potentiates VEGF-C expression in BMDMs We evaluated whether stimulation of macrophages with HA increases the expression of VEGF-C protein in BMDMs. Immunoblot
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analyses showed that HA increased VEGF-C expression in BMDMs in a time-dependent manner (Fig. 3a and b). 3.8. HA-induced VEGF-C expression and UUO-induced lymphangiogenesis are regulated by a TLR4-dependent signaling pathway To examine whether HA-induced VEGF-C production in BMDMs is linked to the TLR4 signaling pathway, we harvested BMDMs from C3H/HeJ (TLR4 null) mice and treated the cells with various concentrations of HA. Western blot analyses showed that VEGF-C expression was dramatically decreased in BMDMs from TLR4 null mice compared to BMDMs from C3H/HeN (TLR4 wild-type) mice (Fig. 4a). Furthermore, a MyD88 (major adaptor molecule for all TLRs) inhibitory peptide, which inhibits homodimerization of MyD88, suppressed HA-induced VEGF-C expression in BMDMs (Fig. 4b). We explored whether VEGF-C expression and LYVE-1positive area were changed in TLR4 null mice. Western blot analyses showed that VEGF-C expression was decreased in the kidney from TLR4 null mice compared to that from TLR4 wild-type mice at 1 w after UUO (Fig. 4c). ERHR-3-positive macrophage infiltration in kidney from TLR4 null mice was significantly decreased compared to kidney from control mice (Fig. 4d). Immunofluorescence data showed that LYVE-1-positive area was significantly lower in the UUO-kidney from TLR4 null mice than that from TLR4 wild-type mice (Fig. 4e and f). 4. Discussion HA accumulation in tissue has been demonstrated after tissue injury. HA content has been shown to be increased in bleomycininduced 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 role of HA in lymphatic vessel remodeling in kidney diseases has not been determined. In the present study, we demonstrated that HA was produced by macrophages after stimulation with TGF-b1. Transfer of mHAS2/
Fig. 3. HA potentiates VEGF-C expression (A and B) Immunoblot analyses of VEGF-C expression by BMDMs. BMDMs were collected and treated with HA (1, 10, 50, and 100 mg/mL; molecular-weight HA 35e75 kD) for 24 h (A). BMDMs were incubated with HA (50 mg/mL) for 6, 12, 24, and 48 h (B). Blots (top) were probed with anti-VEGF-C. The membrane was stripped and reprobed with an anti-actin antibody to control for protein loading in each lane. n ¼ 4 per group. N ¼ 5 per group. Bars represent mean ± SD. *P < 0.05 versus BMDMs treated with control buffer (CB).
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mHAS3-knockdown macrophages to SCID mice suppressed UUOinduced lymphangiogenesis. We also found that HA increased VEGF-C production through a TLR4-MyD88 signaling pathway in BMDMs. HA is recognized by several receptors, such as CD44, Rhamm (CD168), hyaluronic acid receptor for endocytosis (HARE), LYVE-1, and TLRs [24]. The TLR2/TLR4-MyD88 signaling pathway has been linked to the suppressive effect of HA oligosaccharides on chemokine production and progression of kidney disease [25,26]. Park
et al. [27] have demonstrated that HA promotes angiogenesis through RHAMM-TGFbRI signaling in vascular endothelial cells. In this study, we demonstrated that HA increased the production of VEGF-C in BMDMs through the TLR4 and MyD88-dependent signaling pathway. Our data reveal for the first time that the TLR4-MyD88 signaling axis is involved in HA-induced VEGF-C production in the kidney. Recently, Wu et al. [15] have demonstrated that low molecular weight HA increases proliferation, migration, and capillary-like
Fig. 4. TLR4-dependent signal mechanism is associated with HA-induced lymphangiogenesis. (A) BMDMs were collected from C3H/HeN and C3H/HeJ mice and then treated with HA (1, 10, 50, and 100 mg/mL; molecular-weight HA 35e75 kD) for 24 h and immunoblot analysis was performed. Blots (top) were probed with anti-VEGF-C. The membrane was stripped and reprobed with an anti-actin antibody to control for protein loading in each lane. n ¼ 4 per group. *P < 0.05 versus CB; **P < 0.01 versus CB; þP < 0.05 versus BMDMs from C3H/HeN mice treated with HA. (B) BMDMs were treated with a MyD88 inhibitory peptide (100 mmol/L) and immunoblot analysis was performed. Blots (top) were probed with anti-VEGF-C. The membrane was stripped and reprobed with an anti-actin antibody to control for protein loading in each lane. n ¼ 4 per group. **P < 0.01 versus CB; ## P < 0.01 versus BMDMs treated with HA. (C) Immunoblot analysis of VEGF-C in the kidney from sham-operated or UUO mice. n ¼ 4 per group. **P < 0.01 versus sham-operated C3H/HeN mice; $$ P < 0.01 versus UUO-operated C3H/HeN mice. (D) Number of ERHR3-positive macrophages per unit area of the kidney from C3H/HeN and C3H/HeJ mice. Kidneys were harvested from C3H/HeN and C3H/HeJ mice 1 w after sham- or UUO operation. Number of ERHR-positive macrophages in sham- or UUO-operated kidneys was measured in each unit area (400x). n ¼ 4 per group. ***P < 0.001 versus sham-operated C3H/HeN mice; $$$P < 0.001 versus UUO-operated C3H/HeN mice. (E) Immunofluorescence staining of LYVE-1 in the kidney. Kidneys were harvested from C3H/HeN and C3H/HeJ mice 1 w after sham- or UUO operation. (F) Density of LYVE-1-positive area per unit area of sham or UUO kidney from C3H/HeN and C3H/HeJ mice. n ¼ 4 per group. Bars represent mean ± SD *P < 0.05 versus sham-operated C3H/HeN mice; $ P < 0.05 versus UUO-operated C3H/HeN mice.
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tube formation through LYVE-1 in LECs. They have also demonstrated that HA increases phosphorylation of protein kinase C a/bII and ERK1/2 [15]. Consistent with the results, we confirmed that HA increased capillary-like tube formation and proliferation. Furthermore, we showed that HA collaborated synergistically with VEGF-C, thereby promoting capillary-like tube formation and sprouting of hLECs. We used a Boyden Chamber precoated with collagen I in our haptotaxis assay, because collagen I is the main component of renal fibrosis after UUO. HA treatment increased the number of migrating cells in the haptotaxis assay. Because macrophages can be a source of VEGF-C production, decreased macrophage infiltration in the UUO kidneys of TLR4 null mice may be related to the observed reduction of UUO-induced lymphangiogenesis. In this study, we showed that TGF-b1-induced HA from macrophage induces lymphangiogenesis in UUO mouse model. Taken together, our results suggest that manipulation of lymphangiogenesis using HA could be a potentially valuable treatment strategy for fibrotic conditions and lymphatic cancer metastasis.
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Acknowledgments
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This work was supported by the National Research Foundation of Korea (NRF) funded by the Korean government (NRF2014R1A1A4A01003832; Park S.K.), research funds of Chonbuk National University in 2013 (Lee A.S.) and by a grant (CUHBRI-201202-003) from CNUH-BRI (Kim W.) and MRC (No.2008-0062279; Kim W).
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Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2015.09.023.
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