Title: Hypercholesterolemic Mice Exhibit Lymphatic Vessel Dysfunction and Degeneration
Abstract: Lymphatic vessels are essential for lipid absorption and transport. Despite increasing numbers of observations linking lymphatic vessels and lipids, little research has been devoted to address how dysregulation of lipid balance in the blood, ie, dyslipidemia, may affect the functional biology of lymphatic vessels. Here, we show that hypercholesterolemia occurring in apolipoprotein E-deficient (apoE−/−) mice is associated with tissue swelling, lymphatic leakiness, and decreased lymphatic transport of fluid and dendritic cells from tissue. Lymphatic dysfunction results in part from profound structural abnormalities in the lymphatic vasculature: namely, initial lymphatic vessels were greatly enlarged, and collecting vessels developed notably decreased smooth muscle cell coverage and changes in the distribution of lymphatic vessel endothelial hyaluronic acid receptor-1 (LYVE-1). Our results provide evidence that hypercholesterolemia in adult apoE−/− mice is associated with a degeneration of lymphatic vessels that leads to decreased lymphatic drainage and provides an explanation for why dendritic cell migration and, thus, immune priming, are compromised in hypercholesterolemic mice. Lymphatic vessels are essential for lipid absorption and transport. Despite increasing numbers of observations linking lymphatic vessels and lipids, little research has been devoted to address how dysregulation of lipid balance in the blood, ie, dyslipidemia, may affect the functional biology of lymphatic vessels. Here, we show that hypercholesterolemia occurring in apolipoprotein E-deficient (apoE−/−) mice is associated with tissue swelling, lymphatic leakiness, and decreased lymphatic transport of fluid and dendritic cells from tissue. Lymphatic dysfunction results in part from profound structural abnormalities in the lymphatic vasculature: namely, initial lymphatic vessels were greatly enlarged, and collecting vessels developed notably decreased smooth muscle cell coverage and changes in the distribution of lymphatic vessel endothelial hyaluronic acid receptor-1 (LYVE-1). Our results provide evidence that hypercholesterolemia in adult apoE−/− mice is associated with a degeneration of lymphatic vessels that leads to decreased lymphatic drainage and provides an explanation for why dendritic cell migration and, thus, immune priming, are compromised in hypercholesterolemic mice. Lymphatic vessels are essential for maintaining tissue fluid balance, facilitating immune cell trafficking from the periphery to lymph nodes, and absorbing lipoprotein from the gut and from tissue adipocytes.1Nanjee MN Cooke CJ Wong JS Hamilton RL Olszewski WL Miller NE Composition and ultrastructure of size subclasses of normal human peripheral lymph lipoproteins: quantification of cholesterol uptake by HDL in tissue fluids.J Lipid Res. 2001; 42: 639-648PubMed Google Scholar, 2Swartz MA The physiology of the lymphatic system.Adv Drug Deliv Rev. 2001; 50: 3-20Crossref PubMed Scopus (524) Google Scholar Although these three functions are likely to be interconnected, their interdependence has not been well established. When lymphatic vessels are malformed or dysfunctional, swelling occurs, which, when chronic, can lead to dermal lipid accumulation3Schirger A Harrison E Janes J Idiopathic lymphedema. Review of 131 cases.JAMA. 1962; 182: 14-22Crossref PubMed Scopus (44) Google Scholar, 4Rutkowski 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: 161-171Crossref PubMed Scopus (172) Google Scholar and impaired immune responses.5Ruocco V Schwartz RA Ruocco E Lymphedema: an immunologically vulnerable site for development of neoplasms.J Am Acad Dermatol. 2002; 47: 124-127Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, 6Harwood CA Mortimer PS Causes and clinical manifestations of lymphatic failure.Clin Dermatol. 1995; 13: 459-471Abstract Full Text PDF PubMed Scopus (42) Google Scholar In mice heterozygous for the homeobox gene Prox1, which is essential for the development of lymphatic vasculature, abnormal lymph leakage correlated with disrupted integrity of the lymphatic vasculature, particularly in the valves. This leakage leads to increased lipid storage in adipocytes and adipogenesis.7Harvey 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: 1072-1081Crossref PubMed Scopus (439) Google Scholar Conversely, patients with lipedema, a vascular disease characterized by edema and increased subcutaneous adipose tissue in the legs, present with microlymphatic aneurysms8Amann-Vesti BR Franzeck UK Bollinger A Microlymphatic aneurysms in patients with lipedema.Lymphology. 2001; 34: 170-175PubMed Google Scholar and functional lymphatic alterations similar to those found in patients with lymphedema.9Bilancini S Lucchi M Tucci S Eleuteri P Functional lymphatic alterations in patients suffering from lipedema.Angiology. 1995; 46: 333-339Crossref PubMed Scopus (93) Google Scholar Despite these clinical data linking lipid homeostasis to lymphatic vessels, little basic research has been devoted to address how dysregulation of the lipid balance in the blood, ie, dyslipidemia, may affect the functional biology of lymphatic vessels. Dyslipidemia that includes elevated levels of total cholesterol in the form of low-density lipoprotein (hypercholesterolemia) with correspondingly decreased levels of high-density lipoprotein is a major risk factor for atherosclerosis and a common clinical feature of autoimmune diseases10Burger D Dayer JM High-density lipoprotein-associated apolipoprotein A-I: the missing link between infection and chronic inflammation?.Autoimmun Rev. 2002; 1: 111-117Crossref PubMed Scopus (175) Google Scholar and certain cancers.11Sako A Kitayama J Kaisaki S Nagawa H Hyperlipidemia is a risk factor for lymphatic metastasis in superficial esophageal carcinoma.Cancer Lett. 2004; 208: 43-49Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar Dyslipidemia in mice is associated with significant systemic manifestations that lead to marked inflammatory changes in many tissues, especially skin.12van Ree JH Gijbels MJ van den Broek WJ Hofker MH Havekes LM Atypical xanthomatosis in apolipoprotein E-deficient mice after cholesterol feeding.Atherosclerosis. 1995; 112: 237-243Abstract Full Text PDF PubMed Scopus (39) Google Scholar, 13Zabalawi M Bharadwaj M Horton H Cline M Willingham M Thomas MJ Sorci-Thomas MG Inflammation and skin cholesterol in LDLr−/−, apoA-I−/− mice: link between cholesterol homeostasis and self-tolerance?.J Lipid Res. 2007; 48: 52-65Crossref PubMed Scopus (37) Google Scholar, 14Feingold KR Elias PM Mao-Qiang M Fartasch M Zhang SH Maeda N Apolipoprotein E deficiency leads to cutaneous foam cell formation in mice.J Invest Dermatol. 1995; 104: 246-250Crossref PubMed Scopus (30) Google Scholar, 15Angeli V Llodra J Rong JX Satoh K Ishii S Shimizu T Fisher EA Randolph GJ Dyslipidemia associated with atherosclerotic disease systemically alters dendritic cell mobilization.Immunity. 2004; 21: 561-574Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar Cholesterol accumulation in the skin also occurs in humans with severe familial disease,16Yamamoto A Kamiya T Yamamura T Yokoyama S Horiguchi Y Funahashi T Kawaguchi A Miyake Y Beppu S Ishikawa K Matsuzawa Y Takaichi S Clinical features of familial hypercholesterolemia.Arteriosclerosis. 1989; 9: I66-I74PubMed Google Scholar and it is well established that dyslipidemia not only promotes atherosclerotic disease but causes a systemic dysfunction of the blood vasculature in man and animal models.17Shimokawa H Primary endothelial dysfunction: atherosclerosis.J Mol Cell Cardiol. 1999; 31: 23-37Abstract Full Text PDF PubMed Scopus (364) Google Scholar Here, we show, using mice lacking apolipoprotein E (apoE−/−) as a model of dyslipidemia, that this systemic dysfunction extends to the lymphatic vasculature as well, because lymphatic vessel integrity and transport function declined as hypercholesterolemia advanced. Male wild-type and apoE−/− mice fully backcrossed on the C57BL/6 background were obtained from The Jackson Laboratory (Bar Harbor, ME). Both wild-type and apoE−/− mice were maintained on a chow diet (18% protein and >5% fat, Harlan Teklad, Madison, WI) when stated or otherwise switched at 6 weeks of age to a high-fat diet (21% milk fat and 0.15% cholesterol; Harlan Teklad) for 10 to 13 weeks or for an additional 24 to 29 weeks, corresponding to 16 to 19 and 30 to 35 weeks of age, respectively. All studies were approved by the institutional animal care and use committees at Mt. Sinai School of Medicine, National University of Singapore, and the Veterinary Authorities of the Canton Vaud. Total plasma cholesterol was measured with a commercial kit (BioVision, Mountain View, CA). Cryosections (6 to 8 μm) from skin were prepared as described previously.18Angeli V Ginhoux F Llodra J Quemeneur L Frenette PS Skobe M Jessberger R Merad M Randolph GJ B cell-driven lymphangiogenesis in inflamed lymph nodes enhances dendritic cell mobilization.Immunity. 2006; 24: 203-215Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar For histological analysis, sections were stained with hematoxylin and eosin or oil red O. For immunohistochemistry, primary antibodies used included rabbit anti-lymphatic vessel endothelial hyaluronic acid receptor-1 (LYVE-1) polyclonal antibody (Upstate Biotechnology, Charlottesville, VA), hamster anti-podoplanin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and biotinylated anti-CD45 antibodies. Cy3- or Alexa 647-conjugated anti-rabbit (Jackson ImmunoResearch Laboratories, West Grove, PA) and Alexa 488-conjugated anti-hamster (Invitrogen, Carlsbad, CA) antibodies and Cy3-conjugated streptavidin were used for detection. Sections were counterstained with 4,6-diamidino-2-phenylindole for cell nuclei visualization and mounted for analysis. Whole-mount immunohistochemical analysis of ear skin and intestine was performed as described previously.7Harvey 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: 1072-1081Crossref PubMed Scopus (439) Google Scholar Mice were perfused with 4% paraformaldehyde, and tissue was dissected and further fixed in 4% paraformaldehyde overnight at 4°C. Then tissues were incubated in blocking solution 0.5% bovine serum albumin and 0.3% Triton X-100 in PBS overnight at 4°C and finally incubated with anti-LYVE-1 and anti-podoplanin antibodies followed by Cy3- or Alexa 647-conjugated anti-rabbit and Alexa 488-conjugated anti-hamster antibodies. In some experiments, whole mounts were also stained with Cy3-conjugated anti-smooth muscle actin (Sigma-Aldrich, St. Louis, MO) or with anti-rat CD31 (PECAM-1; BD Biosciences, San Jose, CA) revealed by Cy3-conjugated anti-rat antibody (Jackson ImmunoResearch) to analyze smooth muscle cell coverage and valves on collecting vessels, respectively.19Dellinger MT Hunter RJ Bernas MJ Witte MH Erickson RP Chy-3 mice are Vegfc haploinsufficient and exhibit defective dermal superficial to deep lymphatic transition and dermal lymphatic hypoplasia.Dev Dyn. 2007; 236: 2346-2355Crossref PubMed Scopus (36) Google Scholar Specimens were viewed with a fluorescence (Axio imager.Z1, Axiocam HRM camera; Carl Zeiss Micro Imaging, Inc., Jena, Germany) or confocal microscope (Leica TCS SP5; Leica Microsystems, Inc., Deerfield, IL) using LAS AF confocal software (version 1.8.2; Leica Microsystems, Inc.). To determine the diameter of LYVE-1+ vessels, images of representative whole mounts stained for LYVE-1 were acquired on a Zeiss fluorescence microscope with a Zeiss MRm camera, and computer-assisted morphometric analysis of initial lymphatic vessels was performed using Metamorph 6.3 software (Molecular Devices, Toronto, ON, Canada). Linear vessel segments were outlined using a freehand drawing tool and Wacom monitor; vessel junctions were avoided. The average vessel diameter was then calculated from each highlighted vessel by the software; 10 images were used from each mouse (n = 5 to 7 for each condition). Evans blue (1% w/v) dye was injected at the inner surface of the rim of the ear from anesthetized mice using a 10-μl Hamilton syringe, a standard method to macroscopically visualize cutaneous lymphatic vessels and lymphatic drainage.20Kajiya K Hirakawa S Detmar M Vascular endothelial growth factor-A mediates ultraviolet B-induced impairment of lymphatic vessel function.Am J Pathol. 2006; 169: 1496-1503Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar Mouse ears were photographed within 1 minute after dye injection. The functional uptake of the initial lymphatic vessels in the tail was determined by adapting the technique of fluorescence microlymphangiography.4Rutkowski 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: 161-171Crossref PubMed Scopus (172) Google Scholar, 21Swartz MA Berk DA Jain RK Transport in lymphatic capillaries. I. Macroscopic measurements using residence time distribution theory.Am J Physiol. 1996; 270: H324-H329PubMed Google Scholar, 22Swartz MA Kaipainen A Netti PA Brekken C Boucher Y Grodzinsky AJ Jain RK Mechanics of interstitial-lymphatic fluid transport: theoretical foundation and experimental validation.J Biomech. 1999; 32: 1297-1307Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar In brief, a 30-gauge needle catheter containing 0.9% NaCl with 1% fluorescein isothiocyanate-conjugated dextran (70 kDa) was placed intradermally into the tip of the tail. The catheter was attached to a low-pressure reservoir that permitted changes of infusion pressures of 40, 45, 50, and 55 cm H2O. The low infusion pressure allowed uptake into the lymphatic capillaries without deformation of the vessels or changes in their function due to high pressure. The fluorescent dextran, once in the tail, either travels through the interstitial space (linearly with pressure change) or is taken up and transported by the lymphatic capillaries. The infusion flow rate was continually monitored; then pressures were changed and flow rates were monitored for 30 minutes per pressure setting. By also monitoring the diffusive front of fluorescence at each infusion pressure, using a fluorescence microscope, the hydraulic conductivity of the matrix and than that of the lymphatic conductance were calculated.21Swartz MA Berk DA Jain RK Transport in lymphatic capillaries. I. Macroscopic measurements using residence time distribution theory.Am J Physiol. 1996; 270: H324-H329PubMed Google Scholar, 22Swartz MA Kaipainen A Netti PA Brekken C Boucher Y Grodzinsky AJ Jain RK Mechanics of interstitial-lymphatic fluid transport: theoretical foundation and experimental validation.J Biomech. 1999; 32: 1297-1307Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar For adoptive transfer of dendritic cells (DCs), spleens from CD45.1+ wild-type or CD45.2+apoE−/− congenic mice, respectively, were isolated and digested with collagenase D as described previously for lymph nodes.23Robbiani DF Finch RA Jager D Muller WA Sartorelli AC Randolph GJ The leukotriene C4 transporter MRP1 regulates CCL19 (MIP-3β, ELC)-dependent mobilization of dendritic cells to lymph nodes.Cell. 2000; 103: 757-768Abstract Full Text Full Text PDF PubMed Scopus (417) Google Scholar Then CD11c+ DCs were isolated using a magnetic microbead-based, positive-selection kit (Miltenyi Biotech, Gladbach, Germany). Wild-type and apoE−/− DCs were mixed in a nearly equal ratio, and this “preinjection” ratio (CD45.1+ DC frequency/CD45.2+ DC frequency) was recorded by fluorescence-activated cell sorting. Mixed DC suspensions (8 × 105 cells) were injected into each side of the scapular skin of wild-type CD45.1 × CD45.2 F1 recipient mice, using at least four recipient mice per experiment. After 36 hours, cell suspensions from brachial lymph nodes were stained for CD11c and CD45.1 or CD45.2. To obtain a “postinjection” ratio of DCs, total CD11c+ cells were gated, CD45.1+CD45.2+ double-positive host cells were ignored, and the frequency of CD45.1+ and CD45.2+ single-positive DCs was determined. These frequencies were used to tally the postinjection ratio of DCs (wild-type CD45.1+ cells/apoE−/− CD45.2+ cells). In other experiments (Figure 1C), CD45.1+ wild-type DCs were injected into CD45.2+ wild-type or CD45.2+apoE−/− recipient mice, and the total number of transferred DCs that migrated into lymph nodes was determined by multiplying the percentage of CD11c+ congenic DCs observed in the lymph node suspension by the number of total lymph node cells. Statistical analysis was performed with a two-tailed nonparametric Mann-Whitney U test for single comparisons or analysis of variance with a post hoc test using Bonferroni’s method for multiple comparisons. P < 0.05 was considered significant. As a constituent of triglyceride-rich plasma lipoproteins, apoE serves as an important ligand for lipoprotein recognition and clearance by lipoprotein receptors. Consequently, homozygous deletion of the apoE gene in mice results in a pronounced increase in the plasma levels of cholesterol.24Breslow JL Mouse models of atherosclerosis.Science. 1996; 272: 685-688Crossref PubMed Scopus (573) Google Scholar, 25Reddick RL Zhang SH Maeda N Atherosclerosis in mice lacking apo E. Evaluation of lesional development and progression.Arterioscler Thromb. 1994; 14: 141-147Crossref PubMed Scopus (551) Google Scholar, 26Nakashima Y Plump AS Raines EW Breslow JL Ross R ApoE-deficient mice develop lesions of all phases of atherosclerosis throughout the arterial tree.Arterioscler Thromb. 1994; 14: 133-140Crossref PubMed Google Scholar As reported previously,27Lin RY Choudhury RP Cai W Lu M Fallon JT Fisher EA Vlassara H Dietary glycotoxins promote diabetic atherosclerosis in apolipoprotein E-deficient mice.Atherosclerosis. 2003; 168: 213-220Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar apoE-deficient mice (apoE−/−) fed a standard chow diet typically developed spontaneous hypercholesterolemia, and their plasma cholesterol reached 600 to 900 mg/dl at and from 6 weeks of age (Figure 1A). In contrast, age-, sex-, and diet-matched wild-type mice had cholesterol levels of <100 mg/dl (Figure 1A). Consistent with other reports,24Breslow JL Mouse models of atherosclerosis.Science. 1996; 272: 685-688Crossref PubMed Scopus (573) Google Scholar, 28Nakagami H Osako MK Takami Y Hanayama R Koriyama H Mori M Hayashi H Shimizu H Morishita R Vascular protective effects of ezetimibe in ApoE-deficient mice.Atherosclerosis. 2009; 203: 51-58Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar a high-cholesterol, high-fat diet significantly accelerated and exacerbated the accumulation of blood cholesterol in apoE−/− mice, with levels rising to 2 to 3 times that of apoE−/− mice fed a chow diet (Figure 1A). Previously, we demonstrated that apoE−/− mice from 16 weeks of age exhibit leukocyte accumulation in skin and compromised migration of endogenous skin DCs into draining lymph nodes.15Angeli V Llodra J Rong JX Satoh K Ishii S Shimizu T Fisher EA Randolph GJ Dyslipidemia associated with atherosclerotic disease systemically alters dendritic cell mobilization.Immunity. 2004; 21: 561-574Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar However, in this latter report, we did not determine whether the defect developed autonomously within DCs or whether and how environmental conditions (eg, lymphatic drainage) contributed. To address this point, we adoptively transferred splenic CD11c+ cells, highly enriched in DCs (supplemental Figure S1, http://ajp.amjpathol.org), derived from CD45.2 wild-type and CD45.1 hypercholesterolemic apoE−/− mice into skin of wild-type CD45.1/2 F1 recipient mice, or, conversely, CD11c+ splenic DCs derived from CD45.2 wild-type mice were transferred into wild-type or apoE−/− congenic CD45.1 recipient mice fed either a chow or high-fat diet. Migration of the transferred cells to the skin-draining lymph node was analyzed 36 hours later. When injected into wild-type mice with a low-cholesterol environment, the migration of apoE−/− DCs was similar to that of wild-type DCs, as assessed by coinjecting wild-type and apoE−/− CD11c+ DCs in an equal ratio and assaying their ratio within draining lymph nodes (Figure 1B). Thus, neither the lack within DCs of the apoE gene nor their preconditioning in a hypercholesterolemic environment directly affected DC migration from skin into lymphatic vessels. In contrast, wild-type DCs injected into apoE−/− mice showed substantially decreased migration compared with their migration in wild-type recipients, regardless of their diet (Figure 1C). These findings reveal that the impaired DC migration to lymph nodes that occurs during hypercholesterolemia results from modifications within the environment of the migrating DCs, rather than effects on DCs themselves. Because lymphatic vessels are essential for trafficking of DCs,29Randolph GJ Angeli V Swartz MA Dendritic-cell trafficking to lymph nodes through lymphatic vessels.Nat Rev Immunol. 2005; 5: 617-628Crossref PubMed Scopus (877) Google Scholar we hypothesized that these environmental modifications might include alterations in the lymphatic vasculature. Consistent with this hypothesis, we found that apoE−/− mice exhibited tissue swelling, which can result from the impaired lymphatic drainage of interstitial fluid. Tail diameters from apoE−/− mice were larger than those of age-, sex-, and diet-matched wild-type controls (Table 1). At 16 weeks of age, swelling of the tail was visible macroscopically in apoE−/− mice fed a chow or high-fat diet (Figure 2A). Hematoxylin and eosin stains of tail skin sections from apoE−/− mice and matched controls revealed the appearance of numerous “open” areas that probably result from the accumulation of extracellular fluid20Kajiya K Hirakawa S Detmar M Vascular endothelial growth factor-A mediates ultraviolet B-induced impairment of lymphatic vessel function.Am J Pathol. 2006; 169: 1496-1503Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 30Kajiya K Sawane M Huggenberger R Detmar M Activation of the VEGFR-3 pathway by VEGF-C attenuates UVB-induced edema formation and skin inflammation by promoting lymphangiogenesis.J Invest Dermatol. 2009; 129: 1292-1298Crossref PubMed Scopus (81) Google Scholar in the dermis of 20-week-old apoE−/− mice fed either a chow or high-fat diet compared with wild-type mice (Figure 2B). In addition, this dermal edema in apoE−/− mice was associated with a notably increased number of CD45+ leukocytes (Figure 2C). Swelling in the footpad of apoE−/− mice fed a chow or high-fat diet was also evident by 20 weeks of age (Figure 2D), and oil red O staining revealed that this footpad swelling was, in part, associated with lipid deposition (Figure 2E). This last finding is consistent with a previous study reporting cholesterol accumulation in skin from atherosclerotic mice.14Feingold KR Elias PM Mao-Qiang M Fartasch M Zhang SH Maeda N Apolipoprotein E deficiency leads to cutaneous foam cell formation in mice.J Invest Dermatol. 1995; 104: 246-250Crossref PubMed Scopus (30) Google ScholarTable 1Tail diameters measured 4 cm from the tip, in wild-type and apoE−/− mice at 6 and 16 weeks of ageStrainTail diameter (mm)6 weeks of age16 weeks of age fed a chow diet16 weeks of age fed a high-fat dietWild-type2.32 ± 0.062.40 ± 0.042.36 ± 0.08apoE−/−2.23 ± 0.052.55 ± 0.11*Significant differences from wild-type controls: P < 0.0001 (n = 3 to 11).2.56 ± 0.08*Significant differences from wild-type controls: P < 0.0001 (n = 3 to 11).Some mice were fed a high-fat diet from 6 weeks of age. Values are shown as mean ± SD.* Significant differences from wild-type controls: P < 0.0001 (n = 3 to 11). Open table in a new tab Some mice were fed a high-fat diet from 6 weeks of age. Values are shown as mean ± SD. We next specifically assessed the lymphatic transport of macromolecules in apoE−/− mice. As a first approach, we examined the transport of Evans blue dye, which binds interstitial proteins such as albumin and is taken up into the lymphatic vessels, after intradermal injection into the ear. In 16-week-old wild-type mice fed a high-fat diet, we could visualize fine, distinct lymphatic capillaries as the dye was taken up into the vessels and transported toward the base of the ear (Figure 3A). In contrast, when injected into the ear of 16-week-old apoE−/− mice fed a high-fat diet, dye was poorly taken up into lymphatic capillaries and appeared to leak out into the surrounding interstitial space such that lymphatic capillaries could not be clearly delineated (Figure 3B). Because lymph leakage has typically been associated with dysfunctional valves,7Harvey 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: 1072-1081Crossref PubMed Scopus (439) Google Scholar, 31Schmid-Schonbein GW Microlymphatics and lymph flow.Physiol Rev. 1990; 70: 987-1028PubMed Google Scholar this observation suggested that lymphatic vessels themselves were dysfunctional in the hypercholesterolemic mice. We next quantified the functional uptake of the initial lymphatic vessels in tail skin, which exhibit a strikingly regular hexagonal network that is easily visualized by fluorescence microlymphangiography after fluorescent dextran, used as a lymphatic tracer, is introduced into the tip of the tail at constant pressure.22Swartz MA Kaipainen A Netti PA Brekken C Boucher Y Grodzinsky AJ Jain RK Mechanics of interstitial-lymphatic fluid transport: theoretical foundation and experimental validation.J Biomech. 1999; 32: 1297-1307Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar By comparing how flow into the dermis changes with infusion pressure, and simultaneously how tracer movement in the dermis and into the lymphatic capillaries changes with infusion pressure, we can derive an in situ measurement of hydraulic conductivity (representing how easily fluid can move through the extracellular matrix) and lymphatic conductance (representing how readily lymphatics drain interstitial fluid for a given tissue fluid pressure). This method has demonstrated utility for the detection of impaired lymphatic function in established models of lymphedema.4Rutkowski 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: 161-171Crossref PubMed Scopus (172) Google Scholar, 22Swartz MA Kaipainen A Netti PA Brekken C Boucher Y Grodzinsky AJ Jain RK Mechanics of interstitial-lymphatic fluid transport: theoretical foundation and experimental validation.J Biomech. 1999; 32: 1297-1307Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar In young, 4- to 5-week-old apoE−/− mice (Figure 3C), we observed no difference in lymphatic conductance or hydraulic conductivity relative to that in age-matched wild-type controls (Figure 3C). In contrast, the capacity of lymphatics to take up the injected fluorescent tracer in apoE−/− mice was reduced by approximately 50% in apoE−/− mice fed a high-fat diet and examined at 16–18 weeks of age (Figure 3D, left panel). This defect was not associated with any changes in tissue structure as seen by the hydraulic conductivity (Figure 3D, right panel). However, apoE−/− mice fed a chow diet did not show a significant defect in lymphatic conductance (Figure 3D, left panel). Because DC migration is dramatically and similarly compromised in apoE−/− mice fed either a chow or a high-fat diet, it was possible that other defects in lymphatic vessels contributed to the impaired DC migration in apoE−/− mice, in addition to decreased lymphatic conductance within the initial lymphatic capillary plexus measured here. Thus, we further investigated the morphology and the structure of initial and collecting lymphatic vessels. Because lymphedematous tissue often displays hyperplastic lymphatic vessels,4Rutkowski 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: 161-171Crossref PubMed Scopus (172) Google Scholar we investigated the morphology of initial lymphatic vessels that lie in the subepidermal region using immunofluorescence with LYVE-1 and podoplanin (supplemental Figure S2, see http://ajp.amjpathol.org). At 6 weeks of age, initial lymphatic vessels in tail skin from wild-type and apoE−/− mice appeared similarly normal, ie, in a partially collapsed state (Figure 4A), consistent with the expected slow passage of fluid through resting tissue. In contrast, in 16-week-old apoE−/− mice, the majority of initial lymphatic vessels were notably dilated with open lumens compared with those in age-matched wild-type mice (Figure 4, B–D). Consistent with the exacerbating effects of the high-fat diet on blood cholesterol levels, we found that this high-fat diet furthered the enlargement of initial lymphatic vessels in apoE−/− mice compared with that in apoE−/− mice fed a chow diet (Figure 4, C and D). In wild-type mice fed a high-fat diet, the size of the initial lymphatic vessels was generally similar to that in young or 16-week-old wild-type mice fed a chow diet