METABOLISM

Lacteal junction zippering protectsagainst diet-induced obesityFeng Zhang1, Georgia Zarkada1, Jinah Han1, Jinyu Li1, Alexandre Dubrac1,Roxana Ola1,2, Gael Genet1, Kevin Boyé1, Pauline Michon1,3, Steffen E. Künzel1,Joao Paulo Camporez4, Abhishek K. Singh5, Guo-Hua Fong6, Michael Simons1,Patrick Tso7, Carlos Fernández-Hernando5, Gerald I. Shulman4,8,William C. Sessa9, Anne Eichmann1,3,8*

Excess dietary lipid uptake causes obesity, a major global health problem. Enterocyte-absorbed lipids are packaged into chylomicrons, which enter the bloodstream throughintestinal lymphatic vessels called lacteals. Here, we show that preventing lactealchylomicron uptake by inducible endothelial genetic deletion of Neuropilin1 (Nrp1) andVascular endothelial growth factor receptor 1 (Vegfr1; also known as Flt1) renders miceresistant to diet-induced obesity. Absence of NRP1 and FLT1 receptors increased VEGF-Abioavailability and signaling through VEGFR2, inducing lacteal junction zippering andchylomicron malabsorption. Restoring permeable lacteal junctions by VEGFR2 andvascular endothelial (VE)–cadherin signaling inhibition rescued chylomicron transport inthe mutant mice. Zippering of lacteal junctions by disassembly of cytoskeletal VE-cadherinanchors prevented chylomicron uptake in wild-type mice. These data suggest thatlacteal junctions may be targets for preventing dietary fat uptake.

Dietary fats are absorbed by enterocytesand incorporated into triglyceride-rich lipo-proteins, called chylomicrons. Nearly alldietary lipids are transported in chylomi-crons from the intestine to tissues via the

lymphatic system. Chylomicrons enter the lym-phatics through lacteals, specialized lymphaticcapillaries located in the center of the intestinalvilli. From there, they are transported throughthemesenteric lymphatic vessels into the thorac-ic duct, which drains into the venous circula-tion, and become metabolized by the liver (1–3).Preventing lacteal growth in mice by condi-

tional deletion of the genes encoding VEGF-Cor Delta-like 4 (DLL4) renders mice resistant tohigh-fat diet (HFD)–induced obesity, providingexperimental evidence that lacteals could be tar-geted to prevent obesity (4, 5). The cellular mech-anisms controlling chylomicron entry into thelacteals are poorly understood; some studies sug-

gested that chylomicrons enter through junc-tions between lymphatic endothelial cells (LECs)lining the lacteals (5–7), but other studies haveshown that they pass through LECs by trans-cytosis (3). The molecular mechanisms control-ling lacteal chylomicron uptake are currentlyunknown. Here we show that chylomicron up-take is controlled by VEGF-A signaling, throughmodulation of lacteal junctions. Lacteals respondto high VEGF-A levels by zippering up their junc-tions, which impairs chylomicron passage andrenders mice resistant to diet-induced obesity.FLT1 and NRP1 are VEGF-A receptors that

were previously implicated in metabolism regu-lation, but how they function in endothelium inthis context remained undefined (fig. S1). Wetherefore generated pan-endothelial deletionsof Nrp1 and Flt1 by crossingNrp1 fl/fl and Flt1 fl/fl

mice to Cdh5-CreERT2 mice, where gene deletioncan be induced by tamoxifen (TAM) in cells ex-pressing the vascular endothelial cadherin pro-moter (hereafter referred to as Nrp1;Flt1iECkomice). Adult 5-week-old males and Cre-negativelittermate controls received TAM to induce ef-ficient gene deletion in the endothelium, as con-firmed byWestern blot and real-time quantitativepolymerase chain reaction on purified endothelialcells (fig. S2). HFD feeding was initiated at week 8and continued for 16 weeks (wk) (fig. S2). Controlmice doubled their body weight after 16wk ofHFD feeding, whereasNrp1;Flt1iECkomice gainedlittle weight (Fig. 1, A to C). Noweight differenceswere observed in mice on a normal chow diet(Fig. 1D). The weight of Nrp1iECko mice wassimilar to that of littermate controls (Fig. 1E),whereas weight gain in Flt1 single mutants wasslightly reduced, and attributed to increased adi-pose tissue angiogenesis (8, 9) (Fig. 1F), as de-scribed previously.

HFD-fedNrp1;Flt1iECkomice had reduced fatand increased lean body mass ratios when com-pared to control littermates, and their body com-position resembled that of lean control mice on anormal chow diet (Fig. 1G and fig. S3). In contrastto controls, Nrp1;Flt1iECko mice had reducedliver triglyceride content and did not develophepatic steatosis after 8wk of HFD feeding (Fig.1H and fig. S4). Nrp1;Flt1iECko and control miceshowed similar food and water consumption,physical activity, O2 consumption, CO2 produc-tion, and energy expenditure after 2wk and 8wkof HFD feeding (Fig. 1I and fig. S5). Glucosetolerance in Nrp1;Flt1iECko mice was improvedcompared to controls (Fig. 1J).Plasma triglycerides, total cholesterol, and high-

density lipoprotein cholesterol (HDL) concen-trations were reduced in Nrp1;Flt1iECko micewhen compared to controls after 16wk on aHFD (Fig. 2A), indicating that resistance to diet-induced obesity was either due to defective lipiduptake into the bloodstream, reduction in very-low-density lipoprotein (VLDL) production, orenhanced VLDL and chylomicron catabolism.To determine if lipid uptake was affected, wemeasured plasma triglyceride uptake after intra-gastric gavage with olive oil in the presence orabsence of the lipoprotein lipase (LPL) inhib-itor TritonWR1339. As expected, circulating tri-glyceride concentrations increased in controls inboth conditions, but the effect was abrogated inNrp1;Flt1iECko mice (Fig. 2, B and C). Similarresults were obtained when we injected the micewith the LPL inhibitor Poloxamer 407 and fedthem via gavage with oil mixed with [3H]-triolein(Fig. 2D), indicating that absence of NRP1 andFLT1 attenuates fat absorption. Bomb calorimetryshowed increased calorie content in the feces ofNrp1;Flt1iECko mice, suggesting that part of thelipid is not absorbed but rather is cleared in thefeces (Fig. 2E). Histological analysis revealedthe presence of oil-red-O–labeled lipid particlesin the colon ofNrp1;Flt1iECko mice, whereas lesslipid was present in the colon of control mice(fig. S6). We confirmed reduced intestinal lipiduptake in Nrp1;Flt1iECkomice by analyzing post-natal day 7 (P7) mesenteries, after neonatal genedeletion. Chylomicrons were present in the mes-enteric lymphatic vessels of control mice andsingle Nrp1 or Flt1 knockout mice, but few lym-phatic vessels containing milky chyle could beseen inNrp1;Flt1iECko pups (Fig. 2F and fig. S7).Neonatal Nrp1;Flt1iECko mice had decreasedweight gain (fig. S8), which is consistent withlipid malabsorption.Expression of key enzymes and components

required for chylomicron assembly, includingMtp, ApoB, Sar1b, Plagl2, and fatty acid trans-porters (CD36 and Fabps), was similar in thejejunumofNrp1;Flt1iECko and controlmice, indi-cating that the enterocytes devoid of both Nrp1and Flt1 can assemble and secrete chylomicrons(fig. S9). Transmission electronmicroscopy (TEM)showed similar morphology of intestinal enter-ocytes and the presence of chylomicrons withinenterocytes and in the interstitial space betweencapillaries and lacteals regardless of genotype

RESEARCH

Zhang et al., Science 361, 599–603 (2018) 10 August 2018 1 of 5

1Cardiovascular Research Center, Yale University School ofMedicine, New Haven, CT 06510-3221, USA. 2Department ofBasic, Preventive and Clinical Science, University ofTransylvania, 500019 Brasov, Romania. 3INSERM U970, ParisCardiovascular Research Center, 75015 Paris, France.4Department of Internal Medicine, Yale University School ofMedicine, New Haven, CT, USA. 5Departments ofComparative Medicine and Pathology, Vascular Biology andTherapeutics Program and Integrative Cell Signaling andNeurobiology of Metabolism Program, Yale University Schoolof Medicine, New Haven, CT, USA. 6Department of CellBiology, University of Connecticut Health Center, Farmington,CT, 06030-3501, USA. 7Department of Pathology andLaboratory Medicine, Metabolic Diseases Institute, Universityof Cincinnati, Cincinnati, OH 45237-0507, USA. 8Departmentof Cellular and Molecular Physiology, Yale University Schoolof Medicine, New Haven, CT, USA. 9Department ofPharmacology, Vascular Biology and Therapeutics Program,Yale University School of Medicine, New Haven, CT, USA.*Corresponding author. Email: anne.eichmann@yale.edu

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(Fig. 2G and fig. S10). TEM also revealed the pres-ence of lacteal LECs but notably, the lacteal lumenwas almost completely lacking chylomicrons inNrp1;Flt1iECko animals, whereas numerous chy-lomicronswere presentwithin the lacteal lumen incontrols (Fig. 2G and fig. S10). High-magnificationviews showed many open junctions betweenLECs in controls, but more closed LEC junctionsinNrp1;Flt1iECkomice (Fig. 2Gand fig. S10). Thesedata are consistent with the concept that alteredlacteal junctions result in defective lacteal chylo-micron uptake, which limits weight gain in theabsence of both NRP1 and FLT1.Immunostaining of lacteal junctions with VE-

cadherin confirmed that Nrp1;Flt1iECko miceexhibited fewer button-like LEC junctions anddeveloped more impermeable zipper junctionscompared to controls (Fig. 3, A and B). Lactealsdevoid of both Nrp1 and Flt1 were of similarlength, but showed reduced width compared tothose lacking either Nrp1 and Flt1 alone, or con-trol animals (fig. S11). VE-cadherin staining alsorevealed enlarged villus capillaries and disruptedblood endothelial cell (BEC) junctions, leading toleakage of intravenously injected fluorescentdextran in Nrp1;Flt1iECkomice (Fig. 3, C and D,and fig. S12). Postnatal or adult deletion of bothNrp1 and Flt1 caused altered vascular morphol-ogy and villus edema, which was surprisinglywell tolerated and did not compromise vascularor enterocyte ultrastructure even after prolongedHFD feeding (fig. S10). Staining for pericytes,smooth muscle cells, and macrophages and anal-ysis of inflammatory marker expression revealedno changes between controls and Nrp1;Flt1iECkomutants (fig. S13), suggesting aprimary effect uponvillus vasculature by the absence of Nrp1 and Flt1.Loss of junctional VE-cadherin staining in

BECs is a hallmark of increased VEGF-A signal-ing (10), and FLT1 is a known VEGF-A decoyreceptor (11). To test if loss of NRP1 and FLT1increased intestinal VEGF-A signaling, we probedlysates from control and Nrp1;Flt1iECko tissueswith antibodies against phosphorylated and totalVEGFR2. Activation of both tyrosine-1173 (Y1173)and Y949 signalingwas significantly increased inthe absence of both Nrp1 and Flt1 when com-pared to control littermates or single Flt1iECkomice (Fig. 3E and fig. S14), indicating that NRP1enhances FLT1 decoy function. NRP1 also acts asa VEGFR2 co-receptor to activate downstreamERK signaling (12), and ERK activation was ac-cordingly deficient in most Nrp1;Flt1iECko tis-sues (fig. S14).Loss of Flt1 has been shown to induce adipose

tissue browning (8, 9), and we observed increasedvascular density in white adipose tissue (WAT)but not in brown adipose tissue (BAT) in Nrp1;Flt1iECkomice (fig. S15). Expression levels of themitochondrial uncoupling protein UCP1 wereonly slightly increased in Nrp1;Flt1iECko epidid-ymalWAT andwere unchanged in subcutaneousWAT and BAT (fig. S15). Thus, adipose tissuebrowning is unlikely to account for resistance todiet-induced obesity in Nrp1;Flt1iECko animals.VEGF-A is known to increase blood-vascular

permeability (13, 14), and lacteals express VEGFR2

(fig. S16), but effects of VEGF-A on lacteal junc-tions have previously not been investigated, tothe best of our knowledge. Intravenous VEGF-Ainjection disrupted BEC junctions after 30 minas expected, but also increased the number ofzipper-like junctions in lacteals (Fig. 3, F and G).Intravenous injection of the lymphatic growthfactor VEGF-C also increased junction zippering,but to a lesser extent than VEGF-A, whereas amutant VEGF-C-156S protein that cannot bindVEGFR2 (15) had no effect (Fig. 3G and fig. S16).In vitro, treatment of starved confluent LECswithVEGF-A and VEGF-C, but not VEGF-C-156S, pro-moted the appearance of straighter VE-cadherin–lined junctions and decreased actin stress fiberanchoring to perpendicular arranged VE-cadherin(fig. S16). These data suggested that increasedVEGF-A–VEGFR2 signaling in Nrp1;Flt1iECkovilli might cause defective chylomicron uptakevia lacteal junction zippering.

Intestinal villi show high levels of Vegf-a ex-pression, lower levels of Vegf-c, and little expres-sion of Vegf-b (fig. S17). Expression of Flt1 isincreased at birth, concomitantly with the ex-pression of Mtp and ApoB, a known inducer ofFlt1 (16) (fig. S17). RNA sequencing showed thatmature intestinal BECs express four times asmany copies of Nrp1 when compared to Flt1(fig. S17), consistent with the concept that chy-lomicrons induce an increase of FLT1, whichfunctions together with NRP1 to prevent exces-sive VEGF-A signaling, thereby allowing lactealjunctionmaturation and chylomicron absorption.Cdh5-CreERT2 mediates gene deletion in both

intestinal BECs and LECs, raising the possibilitythat NRP1;FLT1 function might be required inboth compartments in a cell-autonomous man-ner. We therefore generated LEC-specific Nrp1and Flt1 deletions using Prox1-CreERT2(BAC)

mice. Prox1-CreERT2(BAC) specifically recombined

Zhang et al., Science 361, 599–603 (2018) 10 August 2018 2 of 5

Fig. 1. Nrp1;Flt1iECko mice are resistant to diet-induced obesity. (A) Nrp1;Flt1iECko and Cre-negative littermate control (Ctrl) mice after 16-week (wk) HFD. (B and C) Growth curves (B) andweight gain (C) of Ctrl (n = 8) and Nrp1;Flt1iECko (n = 11) mice after 16wk HFD feeding. Data aremean ± SEM. ***p < 0.001. Mann-Whitney U test. (D to F) Weight gain of normal chow (NC)- or HFD-fed mice (n = 4 to 11 per group). Data are mean ± SEM. ***p < 0.001. Mann-Whitney U test. (G) Bodycomposition of mice (n = 7 to 8 per group) after 8wk NC or HFD. Data are mean ± SEM. *p < 0.05,***p < 0.001. Mann-Whitney U test. (H) Quantification of BODIPY staining intensity of livercryosections (left) and liver triglyceride content (right) from 8wk HFD-fed mice (n = 4 to 5 pergroup). Data are mean ± SEM. *p < 0.05, ***p < 0.001. Mann-Whitney U test. (I) Calorie intake,energy expenditure, and activity of Ctrl (n = 7) and Nrp1;Flt1iECko (n = 5) mice after 2wk on HFD.Data represent 24 hours (hrs), light hrs (7:00 a.m. to 7:00 p.m.) and dark hrs (7:00 p.m. to7:00 a.m.) average, normalized to lean body mass (LBM). Error bars: SEM. (J) Intraperitonealglucose tolerance test in HFD-fed Ctrl (n = 10) and Nrp1;Flt1iECko (n = 6) mice. Data aremean ± SEM. *p < 0.05; **p<0.01. Mann-Whitney U test.

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Fig. 2. Endothelial Nrp1;Flt1 deletionprevents lacteal chylomicron absorption.(A) Plasma lipid profile in 6-hours fastedCtrl (n = 6) and Nrp1;Flt1iECko mice(n = 6) after 16wk HFD. Data aremean ± SEM. **p < 0.01. Mann-WhitneyU test. (B and C) Plasma triglyceride contentin NC-fed adult mice after gavage with 200 mlof olive oil. All mice (n = 8 to 10 per group)received 6 hours of fasting, and mice in(C) received Triton WR1339 (0.5 g/kg)intraperitoneally (i.p.) 30 min beforegavage. Data are mean ± SEM. **p < 0.01,***p < 0.001. Mann-Whitney U test. (D) Plasma3H CPM (counts per minute) in NC-fedadult mice after gavage with 3H-triolein–containing lipid. Mice (n = 5 per group)were fasted for 6 hours and injected i.p.with poloxamer 407 (1 g/kg) 30 min beforegavage. Data are mean ± SEM. *p < 0.05;**p < 0.01, ***p < 0.001. Mann-Whitney U test.(E) Fecal bomb calorimetry analysis of 2wkHFD-fed mice (n = 8 per group). Data aremean ± SEM. ***p < 0.001. Mann-WhitneyU test. (F) Quantifications of chyle-filledlymphatics in mesenteries of P7 mice afterinjection with TAM (3 × 100 mg) at P2 toP4. Each symbol represents one mouse(n = 17 to 24 per group). Error bars: SEM.***p < 0.001. Mann-Whitney U test. (G) Transmission electron microscopy of jejunum central lacteals in P13 mice. LL: lacteal lumen; LEC:lymphatic endothelial cell; CM: chylomicron. JNC: junction. The closed LEC junction and empty lacteal lumen are apparent in Nrp1;Flt1iECko mice.

Fig. 3. Increased VEGF-A signalingalters villus endothelial junctions.(A) VE-Cad and LYVE-1 staining of whole-mounted jejunum lacteals from P13 toP18 mice after postnatal TAM administration.(B) Quantification of zipper-like lactealjunctions in (A). Each symbol representsone lacteal (7 mice per group). Data aremean ± SEM. ***p < 0.001. Mann-WhitneyU test. (C) VE-Cad staining of whole-mountedjejunum BEC junctions from P7 mice.(D) Quantification of dextran leakage inP11 mice after Rhodamine-dextranand Alexa 647-IsoB4 intravenous (i.v.)injection for 10 min. Each symbol representsone villus (5 to 6 mice per group). Dataare mean ± SEM. ***, p < 0.001. Mann-WhitneyU test. (E) Western blots and quantificationsof VEGFR2 phosphorylation in P7 jejunumlysates. n = 5 to 6 mice per group.Data are mean ± SEM. **p < 0.01. Mann-Whitney U test. (F and G) VE-Cad staining of villusBEC junctions and quantification of zipper-likelacteal junctions in jejunum from P18 to P21wild-type mice 30 min after injection of growthfactors or phosphate-buffered saline (PBS).Each symbol represents one lacteal (4 to6 mice per group). Data are mean ± SEM.n.s., not significant; **p < 0.01, ***p < 0.001.Mann-Whitney U test.

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mesenteric and lacteal LECs, but loss ofNrp1;Flt1in LECs had no detectable effects on postnatalmesenteric chyle uptake or on weight gain inadult HFD-fed mice (fig. S18). Lymphatic button-like junctions were present and responded toVEGF-A by forming zippers (fig. S18). Togetherwith highly enriched expression of Nrp1 andFlt1 in intestinal BECs compared to LECs (17),these data suggest that NRP1 and FLT1 func-tion in villus BECs to antagonize VEGF-A–VEGFR2signaling on lacteal LECs.The effect of Nrp1;Flt1 deletion on LEC junc-

tions was tissue-specific, as the junction mor-phology in initial lymphatics in the skin anddiaphragmofNrp1;Flt1iECkomicewas similar tothat of controls (fig. S19). Intravenous VEGF-A,but not VEGF-C or VEGF-C-156S, modestly in-creased dermal initial lymphatic junction zipper-ing (fig. S19). However, intradermal injection ofVEGF-A or VEGF-C robustly increased dermalLEC junction zippering, whereas VEGF-C-156Sdid not (fig. S19). High VEGFR2 signaling maytherefore be a general inducer of lymphatic junc-tion zippering, which is antagonized by NRP1;FLT1 decoys in the small intestine and via othermechanisms in other tissues.If enhanced VEGF-A signaling accounted for

defective chylomicron uptake in Nrp1;Flt1iECkomice, blocking VEGFR2 function should rescuethis process. Indeed, intraperitoneal injection ofthe VEGFR2 blocking antibody DC101 (18) de-creasedVEGFR2 phosphorylation, and increaseduptake of mesenteric chyle in neonates 6 hoursafter treatment (Fig. 4, A and B, and fig. S20).Dilation of villus capillaries was reduced, andwidth of lacteals increased (fig. S20). DC101-treated double mutants showed less dextran leakfrom intestinal capillaries (fig. S20), had moreLEC button-like junctions and fewer zippers,and had higher plasma lipid concentrations inthe bloodstream when compared to untreatedNrp1;Flt1iECko animals (Fig. 4C-D).We next determined if changes in lacteal junc-

tions were sufficient to impair chylomicron up-take. Button-like junctions develop postnatallyby transformation of zipper-like junctions in apoorly characterized mechanism that involvesangiopoietin2 (ANGPT2) and changes in plasmamembrane localization of VE-cadherin andLYVE-1 (19–21). LYVE-1 immunostaining andprotein abundances of LYVE-1 and ANGPT2 werecomparable between Nrp1;Flt1iECko mutantsand controls, and inhibition of matrix metal-loproteinase (MMP)–mediated LYVE-1 cleavage(22) had no effect on mesenteric chylomicronuptake (fig. S21). To test VE-cadherin function,we used the BV13 antibody, which disrupts endo-thelial adherens junctions in BECs and LECs (19).Intraperitoneal injection of BV13 (10 mg/g) intopostnatal mice rescued lacteal chylomicron uptakeinto mesenteric lymphatics in Nrp1;Flt1iECkomutants and disrupted LEC junctions (Fig. 4, Eand F, and fig. S22). Notably, BV13 treatmentdisrupted capillary BEC adherens junctions butdid not affect chylomicron uptake in controlmice (fig. S22). This result indicates that disrup-tion of BEC junctions per se has no effect on

Zhang et al., Science 361, 599–603 (2018) 10 August 2018 4 of 5

Fig. 4. Lacteal zipper junctions prevent chylomicron absorption. (A to D) DC101 rescues fatabsorption in Nrp1;Flt1iECko mice. (A) Experimental timeline of TAM injection and DC101 treatment(30 mg/g, i.p., for 6 hours) and mesenteries of P7 Nrp1;Flt1iECko mice with or without DC101.(B) Quantification of chyle-filled lymphatics in (A). Each symbol represents one mouse (n = 11to 24 per group). Data are mean ± SEM. n.s., not significant; ***p < 0.001. Mann-Whitney U test.(C) Quantification of zipper-like lacteal junctions in P13 to P18 Nrp1;Flt1iECko jejunum with or withoutDC101. Each symbol represents one lacteal (4 to 7 mice per group). Data are mean ± SEM. n.s., notsignificant; ***p < 0.001. Mann-Whitney U test. (D) Plasma triglyceride content in NC-fed adultmice after 6 hours of fasting and lipid gavage. All mice (n = 5 to 8 per group) received TritonWR1339(0.5 g/kg, i.p., 30 min before gavage) and some received DC101 (30 mg/g, i.p., 4 hours beforegavage). Data are mean ± SEM. *p < 0.05, **p < 0.01. Mann-Whitney U test. (E and F) BV13rescues chylomicron uptake in Nrp1;Flt1iECko mice. (E) Quantification of chyle-filled lymphatics inP7 mice with or without BV13 treatment (10 mg/g, i.p., for 4 hours). Each symbol represents one mouse(n = 5 per group). Data show mean ± SEM. n.s., not significant; **p < 0.01. Mann-Whitney U test.(F) VE-Cad and LYVE-1 staining of jejunum lacteals from P13 Nrp1;Flt1iECkomice with or without BV13.(G to I) ROCK inhibition prevents chylomicron uptake in wild-type mice. (G) Quantification of chyle-filled lymphatics in mesenteries of P10 mice treated with Y27632 (20 mg/g, i.p., for 4 hours)or PBS. Each symbol represents one mouse (n = 5 per group). Data are mean ± SEM. *p < 0.05.Mann-Whitney U test. (H) Quantification of zipper-like lacteal junctions in P13 to P15 mice treated withY27632 or PBS. Each symbol represents one lacteal (4 mice per group). Data are mean ± SEM.***p < 0.001. Mann-Whitney U test. (I) TEM analysis of lacteals in P13 mice with or without Y27632treatment. LL: lacteal lumen; LEC: lymphatic endothelial cell; CM: chylomicron. JNC: junction.(J) Model of NRP1;FLT1 effects on BECs and LECs in intestinal villi. VEGF-A binding to NRP1;FLT1on BECs limits VEGF-A bioavailability for VEGFR2, resulting in continuous and discontinuouscell junctions in BECs and LECs, respectively. Discontinuous button-like LEC junctions allowlacteal chylomicron uptake. NRP1;FLT1 are highly expressed only on BECs. Increased VEGF-Aconcentrations or Nrp1;Flt1 deletion in BECs result in excessive VEGFR2 activation, which disrupts BECjunctions while zippering up lacteal LEC junctions, thereby preventing chylomicron uptake.This phenotype can be rescued by inhibition of VEGFR2 signaling with DC101.

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chylomicron transport and that lacteal LECjunctions, but not capillary BEC junctions, con-trol chylomicron uptake.In a complementary approach, we treated

Nrp1;Flt1iECko mice with dexamethasone, adrug that induces zipper–to–button-like junctiontransition in tracheal lymphatics (21). Short-termdexamethasone treatment decreased the numberof zipper-like junctions and increased chyle up-take in mesenteries of a subset ofNrp1;Flt1iECkomice (fig. S23).Nrp1;Flt1iECkomutant BEC junc-tions were unaffected by dexamethasone, buttreated mutant animals exhibited fewer zipper-like junctions (fig. S23), confirming that LEC butnot BEC junctional changes correlate with chy-lomicron uptake.We reasoned that junctional changes between

continuous VE-cadherin–lined zippers and dis-continuous buttons could depend on cytoskeletalVE-cadherin anchoring, which can be inhibitedin BECs by antagonizing Rho guanosine triphos-phatase (GTPase) signaling (23). Indeed, theROCK inhibitor Y27632 induced straighter junc-tions of cultured LECs in vitro (fig. S24), andshort-term treatment of wild-type mice withY27632 reduced chylomicron transport to mes-enteric lymphatics and increased lacteal junc-tion zippering, without affecting BEC junctions(Fig. 4, G andH, and fig. S24). TEM showed openlacteal junctions and chylomicrons in the lacteallumen of untreated mice, but closed junctionsand almost no chylomicrons in the lacteal lumenin Y27632-treated mice (Fig. 4I).Together, our findings demonstrate that high

VEGF-A signaling has opposing effects on bloodand lymphatic vessels: opening capillary cell-cell junctions via well-studied pathways (10, 24),but closing lymphatic junctions via transforma-tion of buttons into zippers. We show that LECVEGFR2 activation inhibits VE-cadherin cyto-skeletal anchoring, which can be mimicked byROCK inhibition in vitro and in vivo. Cyto-skeletal VE-cadherin anchoringmight generatepulling forces that help maintain lacteal buttonjunctions open. Notably, we show that short-

term effects of VEGF-A gain of function (overhours instead of days) (25) is sufficient to func-tionally switch junction morphology and lipiduptake. This reveals an intestinal-specific lac-teal barrier that could be closed on demand toprevent lipid uptake (Fig. 4J). Thus, NRP1 andFLT1 appear to function collaboratively as adouble decoy receptor system in intestinal BECsto limit VEGF-A signaling (Fig. 4J). VEGF-A–VEGFR2 signaling might provide a LEC growthsignal together with VEGF-C–VEGFR3 (21, 26).In addition, VEGFR2 signaling acts as a physio-logical inhibitor of lacteal maturation, whoseactivity must be dampened to allow acquisitionof transport function. This mechanism may alsoapply to other tissues, with implications foredema prevention. Full knowledge of the mech-anism responsible for LEC junction zipperingmay hold promise for identifying moleculartargets for the treatment of obesity. Of note,ROCK inhibitors are clinically approved for treat-ment of cerebral vasospasm in some countriesand have been shown to improve metabolismand decrease obesity in rodent models (27–30).Further studies of the effects of such drugs inobesity prevention, aswell as of potential adverseeffects of lacteal junction zippering on intestinalhealth, may be warranted.

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ACKNOWLEDGMENTS

We thank Yale Mouse Metabolic Phenotyping Center, YaleElectron Microscopy Facility, and Cincinnati Mouse MetabolicPhenotyping Center for assistance; Y. Zhao for ANCOVAanalysis; and K. Alitalo, J.-L. Thomas, and S. Lee for discussion.Funding: This study was supported by grants from NHLBI(1R01HLI125811), NEI (1R01EY025979-01, P30 EY026878), andAgence nationale de la recherche (ANR Brainwash) to A.E. F.Z.was supported by a T32 grant from NHLBI (2T32HL7950-16A1).G.Z. was supported by an EMBO Long-Term fellowship (ALTF87-2016) and a T32 grant from NHLBI (2T32HL007974-16).A.D. and R.O. were supported by AHA postdoctoral fellowships14POST20380207 and 15POST25560114, respectively. M.S.was supported by NIH grants R01HL053793 and P01HL107205.Author contributions: F.Z., G.Z., J.H., J.L., A.D., R.O., G.G., K.B.,P.M., S.E.K., J.P.C., and A.K.S. performed experiments andanalyzed data. A.E. and F.Z. designed experiments, analyzeddata, and wrote the manuscript. G.-H.F., M.S., P.T., C.F.H., W.C.S.,and G.I.S. provided reagents or expertise. Competing interests:A.E. and F.Z. are inventors on a patent application (U.S. ProvisionalApplication no. 62/686,140) submitted by Yale University thatcovers compositions and methods for preventing dietary lipiduptake and obesity. The authors declare no additional competinginterests. Data and materials availability: All data are availablein the manuscript or the supplementary materials.

SUPPLEMENTARY MATERIALS

www.sciencemag.org/content/361/6402/599/suppl/DC1Materials and MethodsFigs. S1 to S24Table S1References (31–51)

11 September 2017; resubmitted 4 April 2018Accepted 27 June 201810.1126/science.aap9331

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Lacteal junction zippering protects against diet-induced obesity

Fernández-Hernando, Gerald I. Shulman, William C. Sessa and Anne EichmannSteffen E. Künzel, Joao Paulo Camporez, Abhishek K. Singh, Guo-Hua Fong, Michael Simons, Patrick Tso, Carlos Feng Zhang, Georgia Zarkada, Jinah Han, Jinyu Li, Alexandre Dubrac, Roxana Ola, Gael Genet, Kevin Boyé, Pauline Michon,

DOI: 10.1126/science.aap9331 (6402), 599-603.361Science 

, this issue p. 599; see also p. 551Sciencesubsequently rendered resistant to weight gain if NRP1 and FLT1 were inactivated.open junctions to closed, zipped structures (see the Perspective by McDonald). Mice that were fed a high-fat diet weregrowth factor receptor 1 (VEGFR1, also known as FLT1), are required to convert the entry spaces between lacteals from

report that two endothelial cell receptors, neuropilin-1 (NRP1) and vascular endothelialet al.passage occurs. Zhang into the body. Lacteals are lymphatic vessels that act as the highway for chylomicron transport, but it is unclear how

Chylomicrons are specialized particles that carry dietary fats from the intestine to the bloodstream for absorptionZipping up obesity

ARTICLE TOOLS http://science.sciencemag.org/content/361/6402/599

MATERIALSSUPPLEMENTARY http://science.sciencemag.org/content/suppl/2018/08/08/361.6402.599.DC1

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http://science.sciencemag.org/content/361/6402/599#BIBLThis article cites 51 articles, 20 of which you can access for free

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