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Abstract

Lymphatic vessels play a pivotal role in tissue homeostasis and repair, yet their function in periodontal healing remains poorly defined. In this study, we investigated the spatial dynamics and functional significance of lymphatics during periodontal wound repair using a mouse model of ligature-induced periodontitis. By combining Prox1-tdTomato transgenic reporter mice with advanced tissue clearing and light sheet microscopy, we generated high-resolution 3-dimensional images of gingival lymphatic networks under both physiological and repair conditions. Our observations revealed that lymphatic vessels undergo active and spatially organized reassembly during the healing phase. Notably, we found a region-specific increase in lymphatic branching and density in the marginal gingiva, suggesting localized lymphangiogenesis. Time course analysis confirmed that this lymphatic remodeling peaks during the granulation tissue formation phase. We further identified epithelial upregulation of Vegfc during the repair phase and demonstrated that local administration of vascular endothelial growth factor C (VEGF-C) C156S, a VEGFR-3–specific agonist, enhanced lymphatic vessel function and alveolar bone repair. To explore the underlying molecular mechanisms, we successfully established, for the first time, primary cultures of gingiva-derived lymphatic endothelial cells (LECs). Comparative transcriptomic analysis revealed distinct signatures of gingival LECs compared to lymph node or dermal LECs. These cells maintained typical LEC characteristics in vitro and responded robustly to VEGF-C stimulation. RNA sequencing and functional apoptosis assays revealed significant modulation of apoptosis-related genes, including downregulation of Ell3 and upregulation of Siah1b, suggesting a survival-promoting role for VEGF-C signaling in LECs. Collectively, our findings highlight a previously underappreciated regenerative role of lymphatic vessels in the periodontium and establish lymphangiogenic activation as a potential therapeutic strategy for enhancing periodontal repair. The successful culture of primary gingival LECs further provides a novel platform for mechanistic studies in oral lymphatic biology.

Keywords

lymphangiogenesis vascular endothelial growth factor receptor-3 periodontitis cell survival alveolar bone loss

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References

Abe

Hajishengallis

2013. Optimization of the ligature-induced periodontitis model in mice. J Immunol Methods. 394(1–2):49–54. https://doi.org/10.1016/j.jim.2013.05.002

Alitalo

2011. The lymphatic vasculature in disease. Nat Med. 17(11):1371–1380. https://doi.org/10.1038/nm.2545

Biswas

, et al. 2023. Lymphatic vessels in bone support regeneration after injury. Cell. 186(2):382–397.e24. https://doi.org/10.1016/j.cell.2022.12.031

Butler

Dagenais

Rockson

Glover

. 2007. A novel VEGFR3 mutation causes Milroy disease. Am J Med Genet A. 143A(11):1212–1217. https://doi.org/10.1002/ajmg.a.31703

Frye

, et al. 2018. Matrix stiffness controls lymphatic vessel formation through regulation of a GATA2-dependent transcriptional program. Nat Commun. 9(1):1511. https://doi.org/10.1038/s41467-018-03959-6

Goto

, et al. 2022. Lymphatics and fibroblasts support intestinal stem cells in homeostasis and injury. Cell Stem Cell. 29(8):1246–1261.e6. https://doi.org/10.1016/j.stem.2022.06.013

Gur-Cohen

, et al. 2019. Stem cell-driven lymphatic remodeling coordinates tissue regeneration. Science. 366(6470):1218–1225. https://doi.org/10.1126/science.aay4509

Hossain

, et al. 2024. Vascular endothelial growth factor C (VEGF-C) sensitizes lymphatic endothelial cells to oxidative-stress-induced apoptosis through DNA damage and mitochondrial dysfunction: implications for lymphedema. Int J Mol Sci. 25(14):7828. https://doi.org/10.3390/ijms25147828

, et al. 2024. Lymphatic vessel: origin, heterogeneity, biological functions, and therapeutic targets. Signal Transduct Target Ther. 9(1):9. https://doi.org/10.1038/s41392-023-01723-x

10.

Huggenberger

, et al. 2010. Stimulation of lymphangiogenesis via VEGFR-3 inhibits chronic skin inflammation. J Exp Med. 207(10):2255–2269. https://doi.org/10.1084/jem.20100559

11.

Iwayama

, et al. 2022. Plap-1 lineage tracing and single-cell transcriptomics reveal cellular dynamics in the periodontal ligament. Development. 149(19):dev.201203. https://doi.org/10.1242/dev.201203

12.

Karkkainen

, et al. 2000. Missense mutations interfere with VEGFR-3 signalling in primary lymphoedema. Nat Genet. 25(2):153–159. https://doi.org/10.1038/75997

13.

Kitamoto

, et al. 2020. The intermucosal connection between the mouth and gut in commensal pathobiont-driven colitis. Cell. 182(2):447–462.e14. https://doi.org/10.1016/j.cell.2020.05.048

14.

Kitamoto

Kamada

2022. Periodontal connection with intestinal inflammation: microbiological and immunological mechanisms. Periodontol 2000. 89(1):142–153. https://doi.org/10.1111/prd.12424

15.

Koning

Mebius

RE.

2020. Stromal cells and immune cells involved in formation of lymph nodes and their niches. Curr Opin Immunol. 64:20–25. https://doi.org/10.1016/j.coi.2020.03.003

16.

Kuonqui

, et al. 2025. Regulation of VEGFR3 signaling in lymphatic endothelial cells. Front Cell Dev Biol. 13:1527971. https://doi.org/10.3389/fcell.2025.1527971

17.

Liu

, et al. 2020. Lymphoangiocrine signals promote cardiac growth and repair. Nature. 588(7839):705–711. https://doi.org/10.1038/s41586-020-2998-x

18.

Mäkinen

, et al. 2001. Isolated lymphatic endothelial cells transduce growth, survival and migratory signals via the VEGF-C/D receptor VEGFR-3. EMBO J. 20(17):4762–4773. https://doi.org/10.1093/emboj/20.17.4762

19.

Marchesan

, et al. 2018. An experimental murine model to study periodontitis. Nat Protoc. 13(10):2247–2267. https://doi.org/10.1038/s41596-018-0035-4

20.

Matsumoto

, et al. 2019. Advanced CUBIC tissue clearing for whole-organ cell profiling. Nat Protoc. 14(12):3506–3537. https://doi.org/10.1038/s41596-019-0240-9

21.

Mkonyi

Bletsa

Fristad

Wiig

Berggreen

2010. Importance of lymph vessels in the transcapillary fluid balance in the gingiva studied in a transgenic mouse model. Am J Physiol Heart Circ Physiol. 299(2):H275-83. https://doi.org/10.1152/ajpheart.01199.2009

22.

Mkonyi

, et al. 2012. Lymphangiogenesis is induced during development of periodontal disease. J Dent Res. 91(1):71–77. https://doi.org/10.1177/0022034511424747

23.

Niec

, et al. 2022. Lymphatics act as a signaling hub to regulate intestinal stem cell activity. Cell Stem Cell. 29(7):1067–1082.e18. https://doi.org/10.1016/j.stem.2022.05.007

24.

Oliver

Kipnis

Randolph

Harvey

NL.

2020. The lymphatic vasculature in the 21st century: novel functional roles in homeostasis and disease. Cell. 182(2):270–296. https://doi.org/10.1016/j.cell.2020.06.039

25.

Papadakou

, et al. 2017. Role of hyperplasia of gingival lymphatics in periodontal inflammation. J Dent Res. 96(4):467–476. https://doi.org/10.1177/0022034516681762

26.

Raica

, et al. 2015. Mast cells stimulate lymphangiogenesis in the gingiva of patients with periodontal disease. In Vivo. 29(1):29–34.

27.

Saaristo

, et al. 2002. Lymphangiogenic gene therapy with minimal blood vascular side effects. J Exp Med. 196(6):719–730. https://doi.org/10.1084/jem.20020587

28.

Shang

Liang

Kapron

Liu

2019. Pathophysiology of aged lymphatic vessels. Aging (Albany NY). 11(16):6602–6613. https://doi.org/10.18632/aging.102213

29.

Stritt

Koltowska

Mäkinen

2021. Homeostatic maintenance of the lymphatic vasculature. Trends Mol Med. 27(10):955–970. https://doi.org/10.1016/j.molmed.2021.07.003

30.

Susaki

, et al. 2014. Whole-brain imaging with single-cell resolution using chemical cocktails and computational analysis. Cell. 157(3):726–739. https://doi.org/10.1016/j.cell.2014.03.042

31.

Ushijima

Inoue

Domon

Yoshida

2008. Distribution and organization of lymphatic vessels in the mouse gingiva: an immunohistochemical study with LYVE-1. Arch Oral Biol. 53(7):652–658. https://doi.org/10.1016/j.archoralbio.2008.01.012

32.

Wei

W-F

, et al. 2023. Cancer-associated fibroblast-derived PAI-1 promotes lymphatic metastasis via the induction of EndoMT in lymphatic endothelial cells. J Exp Clin Cancer Res. 42(1):160. https://doi.org/10.1186/s13046-023-02714-0

33.

Yeo

Angeli

2017. Bidirectional crosstalk between lymphatic endothelial cell and T cell and its implications in tumor immunity. Front Immunol. 8:83. https://doi.org/10.3389/fimmu.2017.00083

34.

Zhang

, et al. 2021. Targeting local lymphatics to ameliorate heterotopic ossification via FGFR3-BMPR1a pathway. Nat Commun. 12(1):4391. https://doi.org/10.1038/s41467-021-24643-2

35.

Zolla

, et al. 2015. Aging-related anatomical and biochemical changes in lymphatic collectors impair lymph transport, fluid homeostasis, and pathogen clearance. Aging Cell. 14(4):582–594. https://doi.org/10.1111/acel.12330

VEGF-C–Driven Lymphatic Survival Signaling Promotes Periodontal Repair

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