The Roles And Mechanisms Of Macrophages Participating Lymphangiogenesis In Renal Fibrosis

Inflammation plays a crucial role in the occurrence and development of fibrosis, which finally results in end-stage renal disease (ESRD). As one part of inflammation, Macrophages play an important role in the occurrence and development of fibrosis, and the mechanism of macrophage involvement in the development of fibrosis is very complex. Lymphangiogenesis is a new focus in the field of inflammation, involving tumor migration, transplant rejection and wound healing. Recent studies have revealed the association of lymphangiogenesis with renal fibrosis. It is also reported that macrophages are involved in lymphangiogenesis through direct and indirect mechanisms in various other tissues. On the other hand, autophagy contributes to macrophage polarization. And autophagy can be regulated by VEGF-C in a recent study about cancer cells. Therefore, we hypothesized that there was a close relationship between macrophages in the lymphatic vessel of renal fibrosis, and the mechanism was related to autophagy.In this study, we demonstrated that lymphangiogenesis existed in renal fibrosis model and was positively correlated with fibrosis degree and macrophage infiltration. We also showed that, compared to rest macrophages (MO) and alternatively activated macrophages (M2), classically activated macrophages (M1) predominantly transdifferentiated into lymphatic endothelial cells (LEG). In the meantime, VEGF-C increased M1 polarization further and its transdifferentiation into LEC by activating VEGFR3. It was suggested that VEGF-C/VEGFR3 pathway activation downregulated macrophage autophagy and sequentially regulated its phenotype. And induction of autophagy in macrophages by rapamycin decreased Ml polarization and its differentiation into LEC.These results suggest that, in renal fibrosis microenviroroment, VEGF-C promotes macrophages polarization to M1 by suppressing macrophage autophagy, then increased the transdifferentiation of Ml into LEC.Part 1 The relationship between lymphangiogenesis and macrophages infiltration in renal fibrosisObjective To investigate lymphangiogenesis in renal fibrosis model and its relationship with fibrosis degree and macrophages infiltrationMethods Mice fibrosis model was established by unilateral ureteral obstruction (UUO) and adriamycin injection (ADR). Masson staining wad used to observe renal fibrosis. Immunohistochemistry (IHC), Western blot(WB) and realtime PCR were conducted for a-SMA, Collagen 1, PDGFR-β、F4/80, LYVE-1, Prox-1 and VEGF-C expression. Co-expression of LYVE-1 and F4/80 was tested by immunofluorescence (IF). Depletion of macrophages in UUO was achieved by clodronate liposome injection, the control is liposome PBS and PBS.Results In Sham and control group, Masson showed no collagen deposition in renal interstitium, and there is rare expression of a-SMA, Collagen 1, PDGFR-β, F4/80, LYVE-1, Prox-1 and VEGF-C. In UUO and ADR group, massive collagen deposition was observed in renal interstitium, and the expression of a-SMA, Collagen 1, PDGFR-β、F4/80, LYVE-1, Prox-1 and VEGF-C were significantly increased. Quantification and correlation analysis of IHC for a-SMA、F4/80、LYVE-1 and VEGF-C suggested that lymphangiogenesis was positively correlated with renal fibrosis degree and macrophage infiltration.Depletion of macrophages in UUO caused a significant reduction of a-SMA, Collagen1, PDGFR-β, F4/80, LYVE-1, Prox-1 and VEGF-C. At the same time, co-localization of F4/80 and LYVE-1 were found existed in UUO and ADR mice.Conclusions In renal fibrosis, lymphangiogenesis was positively correlated with renal fibrosis degree and macrophage numbers, and infiltrated macrophages may transdifferentiate into LEC directly, forming rymph vessels.Part 2 Macrophages transdifferentiate into lymphatic endothelial cells via VEGF-C/VEGFR3 pathway in renal fibrosisObjective To verify the ability and mechanism of macrophages differentiating into lymphatic endothelial cells.Methods After isolation and conditioned culture, primary macrophages were induced to rest macrophages (MO) with PBS, classical activated macrophages (M1) with IFN-y/LPS and alternatively activated macrophages (M2) with IL-4/IL-13. Primary macrophages were identified by morphobgy observation, WB for iNOS and Arginase, realtime PCR for iNOS, Arginase, TNF-, CD206, YM-1 and FIZZ-1, flow cytometry for Dectin-1, MHC-II and CD86, IF for iNOS and CD206. After M0/M1/M2 were stimulated by VEGF-C, transfected by VEGF-C SiRNA with Lqwfectamine RNAimax, and treated with VEGFR3 inhibitor SARI31675, the samples were tested for LYVE-1, Prox-1, Podoplanin, VEGFR3, VEGF-C, iNOS, CD206 and Arginase in different methods. M0/M1/M2 were seeded in growth factors reduced Matrigel and cultured with EBM-2(containing VEGF-C or not) as three dimensional culture in vitro. On the hand, M1/M2 were labeled with Dil and transfer to fibrosis model by tail vein injection.Then frozen sections were used to observe Dil-macrophages and their co-localization with F4/80 orLYVE-1.Results Bone marrow derived macrophages were successfully isolated and cultured. WB, IF and realtime PCR showed that M1 expressed more LYVE-1, Prox-1 and Podoplanin than M0/M2. VEGF-C upregulated NOS, LYVE-1, Prox-1 and Podoplanin in Ml, this effect could be diminished by Si-VEGF-C or SAR131675. Compared to M0/M2, M1 formed more tube-like structures in Matrigel. Adoptive transfer of DiI-M1 also formed more tube-like structures in vivo than D3-M0/M2.Conclusions From the results of these studies, we determined that macrophages, especially M1,can be transdifferentiated into LEC. VEGF-C/VEGFR3 signaling promoted M1 polarization and their transdifferentiation ability.Part 3 The mechanism of VEGF-C/VEGFR3 signal modulating macrophage autophagy and then promoting its transdifferentiation into lymphatic endothelial cellsObjective To explore the role and mechanism of VEGF-C mediated autophagy modulating the transdifferentiation of macrophages into lymphatic endothelial cells.Methods In vitro, M0/M1/M2 were added VEGF-C.M1 were transfected by Si-VEGF-C with Lipofectamine RNAimax and stimulated with VEGFR3 inhibitor SAR131675. IF and WB were conducted for LC3 and p62. On the other hand, M0/M1/M2 were stimulated with autophagy inducer rapamycin, and the expression of LC3, p62, iNOS, Arginase, LYVE-1, Prox-1 and Podoplanin were tested by WB. In vivo, UUO and ADR mice were detected for F4/80 and LC3 co-localization by IF. UUO mice were also treated by rapamycin, the kidney tissues were acquired for flow cytometry (F4/80, CD11b, CD86, MHC-II, and Podoplanin).Results In UUO and ADR kidney tissue, autophagy occurred in macrophages, and the level was varied. In vitro results suggested that M1 expressed lower LC3-II and higher p62 than M0/M2. VEGF-C decreased LC3-II and increased p62 in M0/M1/M2. Si-VEGF-C and SARI 31675 eliminated the impact of VEGF-C on Ml. And rapamycin reversed the effects of VEGF-C on M0/M1/M2. In vivo, compared with UUO group, rapamycin decreased the percentage of F4/80+CDllb+CD86+MHC-II+Ml and F4/80+CDllb+Podoplanin+cells in F4/80+CDllb+macrophages.Conclusions VEGF-C/VEGFR3 signaling activation downregulated macrophages autophagy, and then promoted Ml polarization, finally raised the ability of Ml differentiation into LEC. Our results reveal that macrophages participate lymphangiogenes is in renal fibrosis through direct transdifferentiation; the mechanism is associated with macrophage polarization modulated by VEGF-C/VEGFR3 mediated autophagy.