Later stages of secondary lymphedema are associated with the massive deposition of adipose tissue (AT). The factors driving lymphedema-associated AT (LAT) expansion in humans remain rather elusive. We hypothesized that LAT expansion could be based on alterations of metabolic, adipogenic, immune and/or angiogenic qualities of AT. AT samples were acquired from upper limbs of 11 women with unilateral breast cancer-related lymphedema and 11 healthy women without lymphedema. Additional control group of 11 female breast cancer survivors without lymphedema was used to assess systemic effects of lymphedema. AT was analysed for adipocyte size, lipolysis, angiogenesis, secretion of cytokines, immune and stem cell content and mRNA gene expression. Further, adipose precursors were isolated and tested for their proliferative and adipogenic capacity. The effect of undrained LAT- derived fluid on adipogenesis was also examined. Lymphedema did not have apparent systemic effect on metabolism and cytokine levels, but it was linked with higher lymphocyte numbers and altered levels of several miRNAs in blood. LAT showed higher basal lipolysis, (lymph)angiogenic capacity and secretion of inflammatory cytokines when compared to healthy AT. LAT contained more activated CD4+ T lymphocytes than healthy AT. mRNA levels of (lymph)angiogenic markers were deregulated in LAT and correlated with markers of lipolysis. In vitro, adipose cells derived from LAT did not differ in their proliferative, adipogenic, lipogenic and lipolytic potential from cells derived from healthy AT. Nevertheless, exposition of preadipocytes to LAT-derived fluid improved their adipogenic conversion when compared with the effect of serum. This study presents results of first complex analysis of LAT from upper limb of breast cancer survivors. Identified LAT alterations indicate a possible link between (lymph)angiogenesis and lipolysis. In addition, our in vitro results imply that AT expansion in lymphedema could be driven partially by exposition of adipose precursors to undrained LAT-derived fluid.

2nd Internal Medicine Department Kralovske Vinohrady University Hospital Prague 10 Czech Republic

Department of Pathophysiology Centre for Research On Nutrition Metabolism and Diabetes 3rd Faculty of Medicine Charles University Ruská 87 100 00 Prague 10 Czech Republic

Department of Radiotherapy and Oncology Kralovske Vinohrady University Hospital Prague 10 Czech Republic

Department of Surgery 2nd Faculty of Medicine Charles University and Motol University Hospital Prague 5 Czech Republic

Franco Czech Laboratory for Clinical Research on Obesity 3rd Faculty of Medicine Prague 10 Czech Republic

Zobrazit více v PubMed

Brorson H, Ohlin K, Olsson G, Karlsson MK. Breast cancer-related chronic arm lymphedema is associated with excess adipose and muscle tissue. Lymphat. Res. Biol. 2009;7(1):3–10. doi: 10.1089/lrb.2008.1022. PubMed DOI

Mehrara BJ, Greene AK. Lymphedema and obesity: is there a link? Plast. Reconstr. Surg. 2014;134(1):154e–160e. doi: 10.1097/PRS.0000000000000268. PubMed DOI PMC

Szolnoky G, Dobozy A, Kemeny L. Towards an effective management of chronic lymphedema. Clin. Dermatol. 2014;32(5):685–691. doi: 10.1016/j.clindermatol.2014.04.017. PubMed DOI

Karkkainen MJ, Saaristo A, Jussila L, Karila KA, Lawrence EC, Pajusola K, Bueler H, Eichmann A, Kauppinen R, Kettunen MI, et al. A model for gene therapy of human hereditary lymphedema. Proc. Natl. Acad. Sci. USA. 2001;98(22):12677–12682. doi: 10.1073/pnas.221449198. PubMed DOI PMC

Harvey NL, Srinivasan RS, Dillard ME, Johnson NC, Witte MH, Boyd K, Sleeman MW, Oliver G. Lymphatic vascular defects promoted by Prox1 haploinsufficiency cause adult-onset obesity. Nat. Genet. 2005;37(10):1072–1081. doi: 10.1038/ng1642. PubMed DOI

Li Y, Zhu W, Zuo L, Shen B. The role of the mesentery in Crohn's disease: the contributions of nerves, vessels, lymphatics, and fat to the pathogenesis and disease course. Inflamm. Bowel Dis. 2016;22(6):1483–1495. doi: 10.1097/MIB.0000000000000791. PubMed DOI

Nougues J, Reyne Y, Dulor JP. Differentiation of rabbit adipocyte precursors in primary culture. Int J Obes. 1988;12(4):321–333. PubMed

Aschen S, Zampell JC, Elhadad S, Weitman E, De Brot M, Mehrara BJ. Regulation of adipogenesis by lymphatic fluid stasis: part II. Expression of adipose differentiation genes. Plast. Reconstr. Surg. 2012;129(4):838–847. doi: 10.1097/PRS.0b013e3182450b47. PubMed DOI PMC

Zampell JC, Aschen S, Weitman ES, Yan A, Elhadad S, De Brot M, Mehrara BJ. Regulation of adipogenesis by lymphatic fluid stasis: part I Adipogenesis, fibrosis, and inflammation. Plast. Reconstr. Surg. 2012;129(4):825–834. doi: 10.1097/PRS.0b013e3182450b2d. PubMed DOI PMC

Levi B, Glotzbach JP, Sorkin M, Hyun J, Januszyk M, Wan DC, Li S, Nelson ER, Longaker MT, Gurtner GC. Molecular analysis and differentiation capacity of adipose-derived stem cells from lymphedema tissue. Plast. Reconstr. Surg. 2013;132(3):580–589. doi: 10.1097/PRS.0b013e31829ace13. PubMed DOI PMC

Zampell JC, Yan A, Elhadad S, Avraham T, Weitman E, Mehrara BJ. CD4(+) cells regulate fibrosis and lymphangiogenesis in response to lymphatic fluid stasis. PLoS ONE. 2012;7(11):e49940. doi: 10.1371/journal.pone.0049940. PubMed DOI PMC

Rutkowski JM, Moya M, Johannes J, Goldman J, Swartz MA. Secondary lymphedema in the mouse tail: Lymphatic hyperplasia, VEGF-C upregulation, and the protective role of MMP-9. Microvasc. Res. 2006;72(3):161–171. doi: 10.1016/j.mvr.2006.05.009. PubMed DOI PMC

Tashiro K, Feng J, Wu SH, Mashiko T, Kanayama K, Narushima M, Uda H, Miyamoto S, Koshima I, Yoshimura K. Pathological changes of adipose tissue in secondary lymphedema. Br. J. Dermatol. 2016;177:158. doi: 10.1111/bjd.15238. PubMed DOI

Ly CL, Kataru RP, Mehrara BJ. Inflammatory manifestations of lymphedema. Int. J. Mol. Sci. 2017;18(1):171. doi: 10.3390/ijms18010171. PubMed DOI PMC

Rojas-Rodriguez R, Gealekman O, Kruse ME, Rosenthal B, Rao K, Min S, Bellve KD, Lifshitz LM, Corvera S. Adipose tissue angiogenesis assay. Methods Enzymol. 2014;537:75–91. doi: 10.1016/B978-0-12-411619-1.00005-7. PubMed DOI PMC

Schmitz KH, Troxel AB, Dean LT, DeMichele A, Brown JC, Sturgeon K, Zhang Z, Evangelisti M, Spinelli B, Kallan MJ, et al. Effect of home-based exercise and weight loss programs on breast cancer-related lymphedema outcomes among overweight breast cancer survivors: The WISER Survivor Randomized Clinical Trial. JAMA Oncol. 2019;5:1605. doi: 10.1001/jamaoncol.2019.2109. PubMed DOI PMC

Severo JS, Morais JBS, Beserra JB, Dos Santos LR, de Sousa Melo SR, de Sousa GS, de Matos Neto EM, Henriques GS, do Nascimento Marreiro D. Role of zinc in zinc-alpha2-glycoprotein metabolism in obesity: a review of literature. Biol. Trace. Elem. Res. 2020;193(1):81–88. doi: 10.1007/s12011-019-01702-w. PubMed DOI

Haider N, Larose L. Harnessing adipogenesis to prevent obesity. Adipocyte. 2019;8(1):98–104. doi: 10.1080/21623945.2019.1583037. PubMed DOI PMC

Martin EC, Qureshi AT, Llamas CB, Burow ME, King AG, Lee OC, Dasa V, Freitas MA, Forsberg JA, Elster EA, et al. Mirna biogenesis pathway is differentially regulated during adipose derived stromal/stem cell differentiation. Adipocyte. 2018;7(2):96–105. PubMed PMC

Chen Z, Lai TC, Jan YH, Lin FM, Wang WC, Xiao H, Wang YT, Sun W, Cui X, Li YS, et al. Hypoxia-responsive miRNAs target argonaute 1 to promote angiogenesis. J. Clin. Invest. 2013;123(3):1057–1067. doi: 10.1172/JCI65344. PubMed DOI PMC

Jha SK, Rauniyar K, Jeltsch M. Key molecules in lymphatic development, function, and identification. Ann. Anat. 2018;219:25–34. doi: 10.1016/j.aanat.2018.05.003. PubMed DOI

Conrad C, Niess H, Huss R, Huber S, von Luettichau I, Nelson PJ, Ott HC, Jauch KW, Bruns CJ. Multipotent mesenchymal stem cells acquire a lymphendothelial phenotype and enhance lymphatic regeneration in vivo. Circulation. 2009;119(2):281–289. doi: 10.1161/CIRCULATIONAHA.108.793208. PubMed DOI

Hamik A, Wang B, Jain MK. Transcriptional regulators of angiogenesis. Arterioscler. Thromb. Vasc. Biol. 2006;26(9):1936–1947. doi: 10.1161/01.ATV.0000232542.42968.e3. PubMed DOI

Moon HE, Ahn MY, Park JA, Min KJ, Kwon YW, Kim KW. Negative regulation of hypoxia inducible factor-1alpha by necdin. FEBS Lett. 2005;579(17):3797–3801. doi: 10.1016/j.febslet.2005.05.072. PubMed DOI

Redondo PAG, Gubert F, Zaverucha-do-Valle C, Dutra TPP, Ayres-Silva JP, Fernandes N, de Souza AAP, Loizidou M, Takiya CM, Rossi MID, et al. Lymphatic vessels in human adipose tissue. Cell Tissue Res. 2020;379(3):511–520. doi: 10.1007/s00441-019-03108-5. PubMed DOI

Souma T, Thomson BR, Heinen S, Carota IA, Yamaguchi S, Onay T, Liu P, Ghosh AK, Li C, Eremina V, et al. Context-dependent functions of angiopoietin 2 are determined by the endothelial phosphatase VEPTP. Proc. Natl. Acad. Sci. USA. 2018;115(6):1298–1303. doi: 10.1073/pnas.1714446115. PubMed DOI PMC

Halin S, Rudolfsson SH, Doll JA, Crawford SE, Wikstrom P, Bergh A. Pigment epithelium-derived factor stimulates tumor macrophage recruitment and is downregulated by the prostate tumor microenvironment. Neoplasia. 2010;12(4):336–345. doi: 10.1593/neo.92046. PubMed DOI PMC

Yuan Y, Arcucci V, Levy SM, Achen MG. Modulation of immunity by lymphatic dysfunction in lymphedema. Front. Immunol. 2019;10:76. doi: 10.3389/fimmu.2019.00076. PubMed DOI PMC

Ogata F, Fujiu K, Matsumoto S, Nakayama Y, Shibata M, Oike Y, Koshima I, Watabe T, Nagai R, Manabe I. Excess lymphangiogenesis cooperatively induced by macrophages and CD4(+) T cells drives the pathogenesis of lymphedema. J. Invest. Dermatol. 2016;136(3):706–714. doi: 10.1016/j.jid.2015.12.001. PubMed DOI

Borg ML, Andrews ZB, Duh EJ, Zechner R, Meikle PJ, Watt MJ. Pigment epithelium-derived factor regulates lipid metabolism via adipose triglyceride lipase. Diabetes. 2011;60(5):1458–1466. doi: 10.2337/db10-0845. PubMed DOI PMC

Notari L, Baladron V, Aroca-Aguilar JD, Balko N, Heredia R, Meyer C, Notario PM, Saravanamuthu S, Nueda ML, Sanchez-Sanchez F, et al. Identification of a lipase-linked cell membrane receptor for pigment epithelium-derived factor. J. Biol. Chem. 2006;281(49):38022–38037. doi: 10.1074/jbc.M600353200. PubMed DOI

Zechner R, Zimmermann R, Eichmann TO, Kohlwein SD, Haemmerle G, Lass A, Madeo F. FAT SIGNALS–lipases and lipolysis in lipid metabolism and signaling. Cell Metab. 2012;15(3):279–291. doi: 10.1016/j.cmet.2011.12.018. PubMed DOI PMC

Miller NE, Michel CC, Nanjee MN, Olszewski WL, Miller IP, Hazell M, Olivecrona G, Sutton P, Humphreys SM, Frayn KN. Secretion of adipokines by human adipose tissue in vivo: partitioning between capillary and lymphatic transport. Am. J. Physiol. Endocrinol. Metab. 2011;301(4):E659–667. doi: 10.1152/ajpendo.00058.2011. PubMed DOI

Wong BW, Wang X, Zecchin A, Thienpont B, Cornelissen I, Kalucka J, Garcia-Caballero M, Missiaen R, Huang H, Bruning U, et al. The role of fatty acid beta-oxidation in lymphangiogenesis. Nature. 2017;542(7639):49–54. doi: 10.1038/nature21028. PubMed DOI

van Hall G, Steensberg A, Sacchetti M, Fischer C, Keller C, Schjerling P, Hiscock N, Moller K, Saltin B, Febbraio MA, et al. Interleukin-6 stimulates lipolysis and fat oxidation in humans. J. Clin. Endocrinol. Metab. 2003;88(7):3005–3010. doi: 10.1210/jc.2002-021687. PubMed DOI

Planck T, Parikh H, Brorson H, Martensson T, Asman P, Groop L, Hallengren B, Lantz M. Gene expression in Graves' ophthalmopathy and arm lymphedema: similarities and differences. Thyroid: Off J. Am. Thyroid Assoc. 2011;21(6):663–674. doi: 10.1089/thy.2010.0217. PubMed DOI

Saupe F, Schwenzer A, Jia Y, Gasser I, Spenle C, Langlois B, Kammerer M, Lefebvre O, Hlushchuk R, Rupp T, et al. Tenascin-C downregulates wnt inhibitor dickkopf-1, promoting tumorigenesis in a neuroendocrine tumor model. Cell Rep. 2013;5(2):482–492. doi: 10.1016/j.celrep.2013.09.014. PubMed DOI

Catalan V, Gomez-Ambrosi J, Rodriguez A, Ramirez B, Rotellar F, Valenti V, Silva C, Gil MJ, Salvador J, Fruhbeck G. Increased tenascin C and Toll-like receptor 4 levels in visceral adipose tissue as a link between inflammation and extracellular matrix remodeling in obesity. J. Clin. Endocrinol. Metab. 2012;97(10):E1880–1889. doi: 10.1210/jc.2012-1670. PubMed DOI PMC

Sawane M, Kajiya K, Kidoya H, Takagi M, Muramatsu F, Takakura N. Apelin inhibits diet-induced obesity by enhancing lymphatic and blood vessel integrity. Diabetes. 2013;62(6):1970–1980. doi: 10.2337/db12-0604. PubMed DOI PMC

Laforest S, Michaud A, Paris G, Pelletier M, Vidal H, Géloën A, Tchernof A. Comparative analysis of three human adipocyte size measurement methods and their relevance for cardiometabolic risk. Obesity. 2017;25(1):122–131. doi: 10.1002/oby.21697. PubMed DOI

Hsieh PN, Fan L, Sweet DR, Jain MK. The Kruppel-like factors and control of energy homeostasis. Endocr. Rev. 2019;40(1):137–152. doi: 10.1210/er.2018-00151. PubMed DOI PMC

Zaragosi LE, Wdziekonski B, Villageois P, Keophiphath M, Maumus M, Tchkonia T, Bourlier V, Mohsen-Kanson T, Ladoux A, Elabd C, et al. Activin a plays a critical role in proliferation and differentiation of human adipose progenitors. Diabetes. 2010;59(10):2513–2521. doi: 10.2337/db10-0013. PubMed DOI PMC

Rossmeislova L, Malisova L, Kracmerova J, Tencerova M, Kovacova Z, Koc M, Siklova-Vitkova M, Viquerie N, Langin D, Stich V. Weight loss improves the adipogenic capacity of human preadipocytes and modulates their secretory profile. Diabetes. 2013;62(6):1990–1995. doi: 10.2337/db12-0986. PubMed DOI PMC

Caso G, McNurlan MA, Mileva I, Zemlyak A, Mynarcik DC, Gelato MC. Peripheral fat loss and decline in adipogenesis in older humans. Metabolism. 2013;62(3):337–340. doi: 10.1016/j.metabol.2012.08.007. PubMed DOI PMC

Tchoukalova Y, Koutsari C, Jensen M. Committed subcutaneous preadipocytes are reduced in human obesity. Diabetologia. 2007;50(1):151–157. doi: 10.1007/s00125-006-0496-9. PubMed DOI

Clement CC, Santambrogio L. The lymph self-antigen repertoire. Front. Immunol. 2013;4:424. doi: 10.3389/fimmu.2013.00424. PubMed DOI PMC

Escobedo N, Proulx ST, Karaman S, Dillard ME, Johnson N, Detmar M, Oliver G. Restoration of lymphatic function rescues obesity in Prox1-haploinsufficient mice. JCI Insight. 2016;1:2. doi: 10.1172/jci.insight.85096. PubMed DOI PMC

Engin AB. MicroRNA and adipogenesis. Adv. Exp. Med. Biol. 2017;960:489–509. doi: 10.1007/978-3-319-48382-5_21. PubMed DOI

Thomou T, Mori MA, Dreyfuss JM, Konishi M, Sakaguchi M, Wolfrum C, Rao TN, Winnay JN, Garcia-Martin R, Grinspoon SK, et al. Adipose-derived circulating miRNAs regulate gene expression in other tissues. Nature. 2017;542(7642):450–455. doi: 10.1038/nature21365. PubMed DOI PMC

Aldrich MB, Guilliod R, Fife CE, Maus EA, Smith L, Rasmussen JC, Sevick-Muraca EM. Lymphatic abnormalities in the normal contralateral arms of subjects with breast cancer-related lymphedema as assessed by near-infrared fluorescent imaging. Biomed. Opt. Express. 2012;3(6):1256–1265. doi: 10.1364/BOE.3.001256. PubMed DOI PMC

Arner P, Andersson DP, Backdahl J, Dahlman I, Ryden M. Weight Gain and Impaired Glucose Metabolism in Women Are Predicted by Inefficient Subcutaneous Fat Cell Lipolysis. Cell Metab. 2018;28(1):45–54 e43. doi: 10.1016/j.cmet.2018.05.004. PubMed DOI

Carpenter AE, Jones TR, Lamprecht MR, Clarke C, Kang IH, Friman O, Guertin DA, Chang JH, Lindquist RA, Moffat J, et al. Cell Profiler: image analysis software for identifying and quantifying cell phenotypes. Genome Biol. 2006;7(10):R100. doi: 10.1186/gb-2006-7-10-r100. PubMed DOI PMC

Čížková T, Štěpán M, Daďová K, Ondrůjová B, Sontáková L, Krauzová E, Matouš M, Koc M, Gojda J, Kračmerová J, et al. Exercise training reduces inflammation of adipose tissue in the elderly: cross-sectional and randomized interventional trial. J. Clin. Endocrinol. Metab. 2020;105:e4510. doi: 10.1210/clinem/dgaa630. PubMed DOI

Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods. 2012;9(7):676–682. doi: 10.1038/nmeth.2019. PubMed DOI PMC

Malisova L, Kovacova Z, Koc M, Kracmerova J, Stich V, Rossmeislova L. Ursodeoxycholic acid but not tauroursodeoxycholic acid inhibits proliferation and differentiation of human subcutaneous adipocytes. PLoS ONE. 2013;8(12):e82086. doi: 10.1371/journal.pone.0082086. PubMed DOI PMC

Brezinova M, Cajka T, Oseeva M, Stepan M, Dadova K, Rossmeislova L, Matous M, Siklova M, Rossmeisl M, Kuda O. Exercise training induces insulin-sensitizing PAHSAs in adipose tissue of elderly women. Biochim. Biophys. Acta Mol. Cell Biol. Lipids. 2019;1865:158576. doi: 10.1016/j.bbalip.2019.158576. PubMed DOI

Chong J, Xia J. Using MetaboAnalyst 4.0 for metabolomics data analysis, interpretation, and integration with other omics data. Methods Mol. Biol. 2020;2104:337–360. doi: 10.1007/978-1-0716-0239-3_17. PubMed DOI