Obesity but not high-fat diet impairs lymphatic function

Abstract

Background/objectives: High-fat diet (HFD)-induced obesity has significant negative effects on lymphatic function, but it remains unclear whether this is a direct effect of HFD or secondary to adipose tissue deposition.

Methods: We compared the effects of HFD on obesity-prone and obesity-resistant mice and analyzed lymphatic function in vivo and in vitro.

Results: Only obesity-prone mice had impaired lymphatic function, increased perilymphatic inflammation and accumulation of lipid droplets surrounding their lymphatic endothelial cells (LECs). LECs isolated from obesity-prone mice, in contrast to obesity-resistant animals, had decreased expression of VEGFR-3 and Prox1. Exposure of LECs to a long-chain free fatty acid increased cellular apoptosis and decreased VEGFR-3 expression, while inhibition of intracellular inhibitors of VEGFR-3 signaling pathways increased cellular viability.

Conclusions: Collectively, our studies suggest that HFD-induced obesity decreases lymphatic function by increasing perilymphatic inflammation and altering LEC gene expression. Reversal of diminished VEGFR-3 signaling may rescue this phenotype and improve lymphatic function.

Figure 1

HFD results in decreased lymphatic vascular density in obesity-prone mice. (a) Representative H&E-stained sections of back skin from NCD- or HFD-fed C57BL/6J, BALB/cJ and MSTN^ln mice (scale bar=200 μm; bracket surrounds subcutaneous adipose tissues). (b) Representative immunofluorescent localization of LYVE-1⁺ vessels in upper limb tissues of NCD- or HFD-fed mice in all groups (scale bar=100 μm). (c) Upper panel: Body weights of mice on NCD (open circles) and HFD (filled circles) in all groups (n=5/group). C57BL/6J HFD vs NCD (*P<0.0001). BALB/cJ HFD vs NCD (*P<0.05), MSTN^ln mice had no significant difference. Middle panel: Quantification of subcutaneous soft tissue thickness in NCD- and HFD-fed mice in all groups (n=5–10/group). C57BL/6J HFD vs NCD (*P<0.0001). MSTN^ln and BALB/cJ mice show no significant difference between NCD and HFD groups. Lower panel: Quantification of upper limb LYVE-1⁺ lymphatic vessel density per high powered field (HPF) and quadrant in NCD- and HFD-fed mice in all groups (n=5 animals × 4HPF/group). C57BL/6J HFD vs NCD (*P<0.0001). MSTN^ln and BALB/cJ mice show no significant difference between NCD and HFD groups.

Figure 2

Obesity impairs lymphatic transport of macromolecules to draining lymph nodes. (a) Representative hindlimb ^99mTc heat maps for NCD- and HFD-fed C57BL/6J, BALB/cJ and MSTN^ln mice. White arrows indicate the uptake in the inguinal lymph nodes (n=4/group). (b) Quantification of the rate of ^99mTc uptake in inguinal lymph nodes following hindlimb injection. Data are presented as fold change relative to their NCD controls in each group (n=16/group). C57BL/6J HFD vs NCD (*P<0.0005). MSTN^ln and BALB/cJ had no significant difference between NCD and HFD groups. (c) Quantification of peak nodal uptake of ^99mTc in inguinal lymph nodes following hindlimb injection. Data are presented as fold change relative to their NCD controls in each group (n=4/group). C57BL/6J HFD vs NCD (*P<0.05). MSTN^ln and BALB/cJ had no significant difference between NCD and HFD groups. (d) Representative Prussian blue-stained histological cross-sections of axillary lymph nodes harvested from NCD- or HFD-fed mice in all groups (scale bar=500 μm). (e) Quantification of Prussian blue staining as a percentage of the total lymph-node area in NCD- and HFD-fed mice (n=3–9/group). C57BL/6J HFD vs NCD (*P<0.05). MSTN^ln and BALB/cJ had no significant difference between NCD and HFD groups. (f) Quantification of migrating CD45.1⁺ DCs (MHCII⁺/CD11C⁺) in inguinal and popliteal lymph nodes of NCD- (open circles) and HFD- (filled circles) fed mice in all groups (n=5/group). Data are presented as fold change from respective NCD-fed controls. C57BL/6J HFD vs NCD (*P<0.05). MSTN^ln and BALB/cJ had no significant difference between NCD and HFD groups. (g) Representative dot plot graphs of inguinal/popliteal lymph node flow cytometry analyzing migrating CD45.1⁺ DCs in all groups. Red square surrounds DC population.

Figure 3

Obesity results in local tissue and perilymphatic inflammation. (a) Representative immunofluorescence co-localization (upper panel) of CD3⁺ cells (green) and LYVE-1⁺ vessels (red) in ear skin whole mounts harvested from NCD- and HFD-fed C57BL/6J and MSTN^ln mice. Blue stain is DAPI (scale bar=30 μm). Quantification of perilymphatic CD3⁺ cells in skin ear cross-sections of NCD- (open circles) and HFD- (filled circles) fed mice in all groups (lower panel). C57BL/6J HFD vs NCD (*P<0.0001, n=4 quadrants × 4–5 animals/group). MSTN^ln and BALB/cJ mice showed no significant difference between NCD and HFD groups. (b) Representative immunofluorescence co-localization (upper panel) of CD11b⁺ cells (green) and LYVE-1⁺ vessels (red) in ear skin whole mounts harvested from NCD- and HFD-fed C57BL/6J and MSTN^ln mice. Blue stain is DAPI (scale bar=30 μm). Quantification of perilymphatic CD11b⁺ cells in skin ear cross-sections of mice fed NCD (open circles) or HFD (filled circles) in all groups (lower panel). C57BL/6J HFD vs NCD (*P<0.0001, n=4 quadrants × 5 animals/group). MSTN^ln and BALB/cJ mice showed no significant difference between NCD and HFD groups. (c) Quantification of VEGF-C protein levels in serum harvested from lean and obese C57BL/6J mice (n=10/group, *P<0.05). (d) Quantification of VEGF-C protein levels in back skin tissues harvested from lean and obese C57BL/6J mice (n=6–10/group, *P<0.003). (e) Quantification of TNF-α protein levels in back skin tissues harvested from lean and obese C57BL/6J mice (n=4–6/group, *P<0.05). (f) Quantification of IL-1β protein levels in back skin tissues harvested from lean and obese C57BL/6J mice (n=5/group, *P<0.03).

Figure 4

Obesity downregulates expression of lymphatic markers in LECs. (a) Quantification of mRNA expression in LECs sorted from NCD- and HFD-fed mice skin tissues for all groups, demonstrating relative expression of Prox1, VEGFR-3, CCL21, ICAM-1 and Bax (n=4–6/group). Data are presented as fold change relative to the NCD-fed controls in all groups. Note the downregulation of Prox1, VEGFR-3 and CCL21 (*P<0.01 for all) and the upregulation of ICAM-1 (*P<0.05) and Bax (*P<0.01) in obese C57BL/6J when compared with their lean controls. (b) Representative immunofluorescent localization of LYVE-1⁺(red) and CCL21⁺(white) in NCD- and HFD-fed mice in all groups (DAPI is blue; scale bar=25 μm). (c) Quantification of CCL21 protein levels in hindlimb tissues harvested from lean and obese C57BL/6J mice (n=6–8/group, *P<0.01). (d) Representative low and high power (dashed box shown in lower right inset) immunofluorescent localization of LYVE-1⁺ cells (red) and Prox1 (top, white), VEGFR-3 (middle, white) and p-AKT (bottom, white) (DAPI nuclear stain is blue; scale bars=50 and 25 μm).

Figure 5

Obesity results in accumulation of large lipid droplets around lymphatic vessels. (a) Representative low (left) and high power (right) immunohistological localization of CD45⁺ cells (brown) surrounding adipocytes (white) in back skin of obese C57BL/6J mice (scale bars=100 and 25 μm, respectively). (b) Representative immunofluorescent localization (left) and orthoslice (right) from daughter region (white dotted square) of LYVE⁺ cells (red), BODIPY⁺ lipids (green) and nuclear DAPI stain (blue), in whole mount ear tissues of NCD- or HFD-fed C57BL/6J mice (scale bar=20 μm). (c) Representative bright-field images showing LECs (top) and ASCs (bottom) cultured in media containing increasing concentrations of SA for 12 h. (d) Quantification of caspase-3 activity in LECs (circles) and ASCs (squares) after treatment with SA for 12 h (n=3–5/group, *P<0.0001). (e) Representative immunofluorescent localization of VEGFR-3 expression (green) in LECs cultured with 10 μm of SA for 12 h (DAPI nuclear stain is shown in blue, scale bar=25 μm). (f) Quantification of VEGFR-3 expression intensity in LECs cultured in media (open circle) or media with 10 μm SA (closed circle) for 12 h (n=14/group, *P<0.0001). (g) Quantification of caspase-3 activity in LECs cultured with media containing SA (circle) or SA with 0.3 nm PTENi (square) for 12 h (n=3/group, *P<0.01). (h) Quantification of LEC viability treated with media containing SA or SA with 0.3 nm PTENi for 12 h (n=12/group, *P<0.0001).

Figure 6

Exposure of LECs to SA in vitro downregulates expression of VEGFR-3 and its downstream mediators. (a, b) Representative western blot images (a) for VEGFR-3, p-AKT, p-eNOS and quantification (b) normalized to GAPDH expression of total cellular protein harvested from LECs treated with 1 μm SA, 10 μm SA+0.3 nm PTENi, PTENi (0.3 nm), SA+100 ng ml⁻¹ VEGF-C, VEGF-C (100 ng ml⁻¹), SA+100 nm insulin or insulin (100 nm) (n=4–5/group, *P<0.05 for all antibodies). (c) Left panels: Representative immunofluorescent co-localization of VEGFR-3 (green) and Prox1, Ki67 and p-AKT (all shown in pink) in LECs cultured in control media, media containing 10 μm SA, media containing 10 μm SA with PTENi (0.3 nm), 10 μm SA with VEGF-C (100 ng ml⁻¹) or 10 μm SA with insulin (100 nm) for 12 h (scale bar=25 μm). Right panels: Quantification of expression of Prox1, Ki67 and p-AKT in groups shown in the left panels (n=10–15/group, *P<0.0005).