VEGF ‐C is required for intestinal lymphatic vessel maintenance and lipid absorption

• DOI: 10.15252/emmm.201505731
• Publication Date: 2015-10-13T17:58:10Z
• Authors: Harri Nurmi; Pipsa Saharinen; Georgia Zarkada; Wei Zheng; Marius R Robciuc; Kari Alitalo
• Keywords: Lymphatic vessel; Lymphatic Endothelium

Report12 October 2015Open Access VEGF-C is required for intestinal lymphatic vessel maintenance and lipid absorption Harri Nurmi Wihuri Research Institute Translational Cancer Biology Program, Biomedicum Helsinki, University of Finland Search more papers by this author Pipsa Saharinen Georgia Zarkada Wei Zheng Marius R Robciuc Kari Alitalo Corresponding Author Information Nurmi1, Saharinen1, Zarkada1, Zheng1, Robciuc1,‡ 1,‡ 1Wihuri ‡These authors contributed equally to work *Corresponding author. Tel: [phone], E-mail: [email protected] EMBO Mol Med (2015)7:1418-1425https://doi.org/10.15252/emmm.201505731 PDFDownload PDF article text main figures. Peer ReviewDownload a summary the editorial decision process including letters, reviewer comments responses feedback. ToolsAdd favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Vascular endothelial growth factor C (VEGF-C) binding its tyrosine kinase receptor VEGFR-3 drives during development in pathological processes. Although VEGF-C/VEGFR-3 pathway provides target treatment cancer lymphedema, physiological functions adult vasculature are unknown. We show here that necessary perinatal lymphangiogenesis, but only intestine. Following Vegfc gene deletion mice, vessels, lacteal underwent gradual atrophy, which was aggravated when also Vegfd deleted. expressed subset smooth muscle cells adjacent lacteals villus wall. The Vegfc-deleted mice showed defective increased fecal excretion dietary cholesterol fatty acids. When fed high-fat diet, Vegfc-deficient were resistant obesity had improved glucose metabolism. Our findings indicate lymphangiogenic factors provide trophic dynamic regulation vasculature, could be especially important adiposity Synopsis A new mouse model allows effective, timed long lasting vascular Cre-Lox system highlights relevance absorption. halted vessels at all stages development. In adults with normally developed system, deficiency specifically induced atrophy. Lymphatic atrophy intestine no impact on animal welfare reduced uptake high fat diet-induced obesity. Introduction regulate tissue fluid homeostasis, immune cell trafficking, absorption, their malfunction leads chronic edema impaired (Cueni Detmar, 2008; Alitalo, 2011; Koltowska et al, 2013). Lymphangiogenesis occurs processes such as inflammation tumor metastasis inhibitors receptors currently clinical trials human patients (Alitalo, 2011). guided primarily VEGF-C-mediated activation VEGFR-3, VEGF (Makinen 2001; Karkkainen 2004). absence VEGF-C, arrested initial spouting from embryonic veins (Karkkainen VEGF-D, second ligand, cannot compensate development, it induces lymphangiogenesis overexpressed results mild skin (Rissanen 2003; 2004; Paquet-Fifield 2013; Astin 2014). During postnatal period, continue expand, intestine, grow into villi facilitate fat-rich milk (Kim 2007). Lipid allowed Gaspare Aselli discover content milky 17th century (Dixon, 2010). Recent studies have shown actively involved transport small epithelium further blood circulation, although detailed mechanisms not been elucidated several genetic models Vegfr3 hypomorphic Chy missense point mutation Vegfr3, lipid-rich chylous ascites develops after birth resolves before weaning 2001, Haiko 2008). However, possible function known. Here we studied role growth, maintenance, neonatal using effective (Aspelund demonstrate has crucial Results Discussion arrests developing gene-deleted embryos, sprouting major day (E) 10.5 embryos die between E15.5 E17.5 To study how loss affects last trimester fetal crossed Vegfcflox/flox 2014) expressing universal deletor R26Cre-ERT2 (Ventura delete R26Cre-ERT2;Vegfcflox/flox pregnant females injected 4-OH tamoxifen E12.5 E13.5. Analysis E18.5 indicated (VCiΔR26) lack mesenteric either completely (data shown) or exhibit fragments blind-ended stubs extend toward wall (Fig 1A B). Deletion E14.5 blocked maturation collecting vessels. lacked valves much thinner than wild-type (WT) 1C D). addition, failed develop VCiΔR26 whereas affected 1E–H). Figure 1. miceMice received figure. Immunofluorescence analyses performed (A–H) (I–L) pups, (M–U) adults. A–D. Mesenteric (PECAM1, red) (LYVE1, green; PROX1, gray). Asterisks valves, arrow indicates stub, arrowhead an isolated fragment E–H. Blood green) (VEGFR-3, I, J. Detection (arrows) P6. K, L. LYVE1 staining M–T. mice. Genotypes lengths (U). U. Quantification areas (M–T). Length i∆Vegfc months (mo), VEGF-D genotype. Data represented mean ± SEM. Significant differences determined one-way ANOVA Bonferroni post hoc analysis compared WT (M). *P = 0.003, **P 0.001, ***P 0.0008, ♯P 0.0002, §P 0.0001. information: Scale bars: 200 μm 400 (K–T). n 25 (M); 5 (N); 8 (O); 7 (P); 6 (Q); (R); (S); 3 (T). Download figure PowerPoint Lacteal lipids order determine whether formation deleted daily injections pups 1 (P1) P4. Upon necropsy P6, observed littermates 1I J). cross sections P6 revealed 1K L Appendix Fig S1A wall, intrauterine expand upon period (Appendix S1C–H). density S1I J), changes S1K L). Taken together, these developmental early lymphangiogenesis. needed us induce deletion, administered starting weeks age. Real-time PCR tissues mRNA levels 2–15% Table S1). Notably, whole-mount undergo 1M–U). Shorter already S2A), months, strikingly shorter, containing number cells, suggesting due S2B). Thus, started then progressed network contrast, even effectively there lymph nodes, ears, trachea S3A–E). relies principally signaling, while other known ligand signaling Vegfc−/− Yet, found (Veikkola Baluk 2005). Constitutive effect agreement previous reports 1U) (Baldwin Surprisingly, however, conditional Vegfd−/− resulted severe alone These data primary signal can partially VEGF-C. Because did observe expression (WT 1.02 SEM 0.14 vs. 0.98 0.16), likely bioactive compensates Next, sought identify types express Previous our laboratory arterial (SMCs) both humans (Partanen 2000; Paavonen 2002; define receptors, β-Gal intestines Vegfc/LacZ 2004), Vegfr3/LacZ (Dumont 1998), Vegfr2/LacZ (Shalaby 1995) weak comparison VEGFR-2 stainings, respectively 2A Higher resolution combined immunohistochemistry demonstrated SMCs, inner circular layer muscle, SMC fibers 2C). villi, most prominent LYVE1-counterstained 2D). pattern, analyzed PECAM-1 counterstaining 2E), confirmed SMCs. Whole-mount confocal microscopy close contact basal part where detected 2D F). suggest SMCs source maintain architecture It should noted contractility suggested contraction around normal conditions (Hosoyamada Sakai, 2005, 2007; Breslin Gogineni 2. Smooth Overview section stained nuclear red highlighting location higher magnification images (B–F); (I) entire (II) base. pattern (Ctrl), (VC), (VR-3), (VR-2) representing base reaction actin (SMA) peroxidase staining. Surface image β-Gal-stained (left) PECAM1 (right). (LYVE1), capillaries (PECAM1), (SMA). Arrows SMC, arrowheads villus, asterisks highlight 50 μm, except (C) inset μm. dependent Rosa26CreERT2;Vegr3flox/flox (R3iΔR26) (Haiko As expected, regression, similar S4A altered, via stabilize plexus. previously inhibitor cediranib inhibits adenoviral delivery (Heckman administration sunitinib doses block (Ki: 17 nM/VEGFR-2 9 nM) (Faivre 2007) significantly S4C–E), line report (Kamba 2006). appear VEGFR inhibition. reduces inducing resistance oral tolerance test earlier. clearance triglycerides plasma injection Triton WR 1339, lipoprotein lipase activity (Otway Robinson, 1967). triglyceride serum oil bolus S5A). correlate exclude involvement effects deletion. reduction diet (HFD). Initial experiments 129SV/C57Bl/6J mixed background reveal body weight, metabolism S5B C). Interestingly, background, levels, free acid (FFA) indicating S5D). HFD feeding pure C57Bl/6J established 8-week-old male 4 later. gained less weight better littermates, independently concurrent 3A–C S5E At HFD, very low amounts chyle one out 16 two five Vegfc- plus Vegfd-deleted leakage. Body composition significant total percentage, lean 3D S5G). accumulation explained caloric intake, since food consumption 3E). expected basis feces 3F–H). No difference regular majority calories derived carbohydrate. This result 3. Intestinal regression obesityTwo-month-old (HFD) seven analysis. change average fold weight. 16, WT; 6, Vegfd−/−; VCiΔR26; 5, VCiΔR26. comparisons HFD. WT. 0.004; 0.003. Glucose (GTT) six unpaired two-tailed t-test. 0.014; 0.041. Total percentage measurements weights visceral (VF) subcutaneous (SF) time necropsy. 0.006; 0.001; #P 0.008; 0.006. each group. Food fifth week 9, 10, immunofluorescence length (solid striped color bars, respectively) LYVE1+ area (F). 0.0002; 0.00007. Free 0.007. 100 (villi) 300 (intestinal wall). Use newly targeted quiescent state require signals structure function. unexpected finding blockade may therapeutic opportunities. specific phase I additional benefit cardiovascular disease reducing excess lipids. Further address employing blocking therapeutics. Materials Methods Study approval National Animal Experiment Board approved involving use Mice lines Vegfcfl/fl 2014), Vegfr3fl/fl 2008), Rosa26-CreERT2 2007), 2005), described previously. used 129SV backcrossing strain least generations. For induction Cre-mediated recombination mother days consecutive intragastric (4-OHT) (Sigma) (25 mg/ml dissolved μl ethanol/olive oil). P1 P4 4-OHT ethanol). Recombination (7–8 old) done (Sigma, corn mg/ml, μl) days. Detailed experiment information Supplementary Methods. Statistics Quantitative groups t-test followed multiple comparisons. Values P-value < 0.05 considered significant. Acknowledgements thank Jarmo Koponen, Essi Salminen, Maija Atuegwu, Mari Jokinen, Tapio Tainola, Kirsi Mänttäri technical assistance, personnel Imaging Unit service Laboratory Center Helsinki housing. supported Leducq Transatlantic Network Excellence Lymph Vessels Obesity Cardiovascular Disease (grant no: 11CVD03), Marie Curie ITN Vessel consortium 317250) Seventh Framework Program European Union, Academy Centre 2014-2019 271845), Council (ERC-2010-AdG-268804); Finnish Organizations, Sigrid Juselius Foundation, Foundation Ida Montin (to H.N.). contributions HN MRR designed experiments, acquisition, interpretation data, wrote manuscript; PS produced mice; GZ WZ experiments; KA conducted scientific direction, manuscript. Conflict interest declare they conflict interest. paper Problem Specific treatment, long-term inhibition pathways still incompletely Preclinical follow-up now novel system. eliminates confounding defects models. timed, long-lasting Vegfc. Regression protected obesogenic excessive uptake. Impact Long-term well tolerated opportunities diseases. Supporting (PDF document, 13.7 MB) Review Process File 470.5 KB) References K (2011) disease. Nat 17: 1371–1380CrossrefCASPubMedWeb Science®Google Scholar Aspelund A, Tammela T, Antila S, H, Leppanen VM, G, Stanczuk L, Francois M, Makinen P al (2014) Schlemm's canal VEGF-C/VEGFR-3-responsive lymphatic-like vessel. J Clin Invest 124: 3975–3986CrossrefCASPubMedWeb JW, Haggerty MJ, Okuda KS, Le Guen Misa JP, Tromp Hogan BM, Crosier KE, zebrafish facial sprouting. Development 141: 2680–2690CrossrefCASPubMedWeb Baldwin ME, Halford MM, Roufail Williams RA, Hibbs ML, Grail D, Kubo Stacker SA, Achen MG (2005) D dispensable Cell Biol 25: 2441–2449CrossrefCASPubMedWeb P, Ator E, Lyubynska N, MG, Hicklin DJ, Jeltsch Petrova TV, Pytowski B, SA Pathogenesis persistent hyperplasia airway inflammation. 115: 247–257CrossrefCASPubMedWeb Gaudreault Watson KD, Reynoso R, Yuan SY, Wu MH (2007) factor-C stimulates pump receptor-3-dependent mechanism. Am Physiol Heart Circ 293: H709–H718CrossrefCASPubMedWeb Cueni LN, Detmar M (2008) health Lymphat Res 6: 109–122CrossrefPubMedGoogle Dixon JB (2010) transport: sewer subway? Trends Endocrinol Metab 21: 480–487CrossrefCASPubMedWeb Dumont Jussila Taipale J, Lymboussaki Mustonen Pajusola Breitman (1998) failure deficient receptor-3. Science 282: 946–949CrossrefCASPubMedWeb Faivre Demetri Sargent W, Raymond E Molecular efficacy future Rev Drug Discov 734–745CrossrefCASPubMedWeb Caunt Crow Lee CV, Fuh van Bruggen Ye Weimer RM (2013) Inhibition modulates distal remodeling secondary metastasis. PLoS ONE 8: e68755CrossrefCASPubMedWeb Keskitalo equivalent embryos. 28: 4843–4850CrossrefCASPubMedWeb Heckman CA, Holopainen Wirzenius Yla-Herttuala Wedge SR, Jurgensmeier JM, blocks ligand-induced receptor-3 68: 4754–4762CrossrefCASPubMedWeb Hosoyamada Y, Sakai T Structural mechanical crypts rat intestine: integrative reevaluation ultrastructural Anat Embryol 210: 1–12CrossrefPubMedWeb Mechanical components special reference axial villi. Arch Histol Cytol 70: 107–116CrossrefPubMedWeb Kamba Tam BY, Hashizume Haskell Sennino Mancuso MR, Norberg SM, O'Brien Davis RB, Gowen LC (2006) VEGF-dependent plasticity fenestrated microvasculature. 290: H560–H576CrossrefCASPubMedWeb Sainio Partanen Jackson DG, Talikka Rauvala H (2004) first veins. Immunol 5: 74–80CrossrefCASPubMedWeb Saaristo Karila KA, Lawrence EC, Bueler Eichmann Kauppinen Kettunen MI (2001) therapy hereditary lymphedema. Proc Natl Acad Sci USA 98: 12677–12682CrossrefCASPubMedWeb Kim Sung HK, Koh GY Dev Dyn 236: 2020–2025Wiley Online LibraryCASPubMedWeb Betterman KL, Harvey NL, BM Getting about: emergence morphogenesis vertebrate vasculature. 140: 1857–1870CrossrefCASPubMedWeb Veikkola Mustjoki Karpanen Catimel Nice Wise Mercer Kowalski Kerjaschki Isolated transduce survival migratory VEGF-C/D VEGFR-3. 20: 4762–4773Wiley Otway Robinson DS (1967) non-ionic detergent (Triton 1339) removal acids rat. 190: 309–319Wiley Mandelin Li TF, Ristimaki Konttinen YT (2002) healthy arthritic synovium. Rheumatol 29: 39–45CASPubMedWeb Levy Sato Shayan Karnezis Davydova Nowell CJ, Ma GZ, Zhang YF factor-d caliber lymphatics dermis. Dermatol 133: 2074–2084CrossrefCASPubMedWeb TA, Arola Ora Miettinen (2000) neuroendocrine receptor, tissues. FASEB 14: 2087–2096Wiley Rissanen TT, Markkanen JE, Gruchala Heikura Puranen MI, Kholova (2003) strongest angiogenic effector among VEGFs delivered skeletal adenoviruses. 92: 1098–1106CrossrefCASPubMedWeb Shalaby F, Rossant Yamaguchi TP, Gertsenstein XF, Schuh AC (1995) Failure blood-island vasculogenesis Flk-1-deficient Nature 376: 62–66CrossrefCASPubMedWeb Thurston McDonald DM, Signalling sufficient transgenic 1223–1231Wiley Ventura Kirsch McLaughlin Tuveson DA, Grimm Lintault Newman Reczek EE, Weissleder Jacks Restoration p53 tumour vivo. 445: 661–665CrossrefCASPubMedWeb ArticleNext Article Read MoreAbout coverClose modalView large imageVolume 7,Issue 11,November 2015Cover: month's cover " absorption" Nurmi, R. Robciuc, colleagues. composite shows (green) surrounding (red) within Green autofluorescence enhanced border enterocytes. © Cover Finland. Volume 7Issue 111 November 2015In issue FiguresReferencesRelatedDetailsLoading ...