Oxidized Low-Density Lipoprotein Drives Dysfunction of the Liver Lymphatic System

Abstract

Background and aims: As the incidence of nonalcoholic steatohepatitis (NASH) continues to rise, understanding how normal liver functions are affected during disease is required before developing novel therapeutics which could reduce morbidity and mortality. However, very little is understood about how the transport of proteins and cells from the liver by the lymphatic vasculature is affected by inflammatory mediators or during disease.

Methods: To answer these questions, we utilized a well-validated mouse model of NASH and exposure to highly oxidized low density lipoprotein (oxLDL). In addition to single cell sequencing, multiplexed immunofluorescence and metabolomic analysis of liver lymphatic endothelial cells (LEC)s we evaluated lymphatic permeability and transport both in vitro and in vivo.

Results: Confirming similarities between human and mouse liver lymphatic vasculature in NASH, we found that the lymphatic vasculature expands as disease progresses and results in the downregulation of genes important to lymphatic identity and function. We also demonstrate, in mice with NASH, that fluorescein isothiocyanate (FITC) dextran does not accumulate in the liver draining lymph node upon intrahepatic injection, a defect that was rescued with therapeutic administration of the lymphatic growth factor, recombinant vascular endothelial growth factor C (rVEGFC). Similarly, exposure to oxLDL reduced the amount of FITC-dextran in the portal draining lymph node and through an LEC monolayer. We provide evidence that the mechanism by which oxLDL impacts lymphatic permeability is via a reduction in Prox1 expression which decreases lymphatic specific gene expression, impedes LEC metabolism and reorganizes the highly permeable lymphatic cell-cell junctions which are a defining feature of lymphatic capillaries.

Conclusions: We identify oxLDL as a major contributor to decreased lymphatic permeability in the liver, a change which is consistent with decreased protein homeostasis and increased inflammation during chronic liver disease.

Keywords: Inflammation; Lymphangiogenesis; Oxidized LDL; Permeability; VEGFC.

Graphical abstract

Figure 1

LVD increases as patients progress through stages of NASH. (A) Representative images of biopsies from patients in Table 1 ranging from stage 0 to IV of fibrosis using the Brunt staging method. Histological sections were stained with the PDPN (D2-40) antibody and visualized with DAB (brown). Sections were then counterstained with hematoxylin to visualize nuclei. Scale bar is 100 μm. Inset is the indicated zoomed in region. Arrows point to representative lymphatic vessels. (B) Lymphatic vessel density (area of the lymphatics divided by the area of the defined tissue area) based on areas of fibrosis were evaluated from 7 to 10 patients per stage. P values were calculated using a 1-way analysis of variance. Actual P values are shown.

Figure 2

Liver lymphatic vessels increase in frequency during disease. (A) Mice were fed a control or HFHC diet for 24 weeks. Staining of liver sections was performed with DAPI (white), LYVE-1 (cyan), PDPN 8.8.1 (red), and CK19 (yellow). Scale bar is 100 μm. Shown is a representative image from 1 experiment with 5–7 mice per group repeated 2 additional times with similar results and statistical values. (B) Lymphatic vessels were designated as PDPN+CK19– structures with at least 2 nuclei per vessel. LVD was calculated as described in the Materials and Methods. (C) Number of Ki67+ lymphatic vessels per area of tissue over a 12-week to 24-week period. Few to no Ki67+ lymphatic vessels were detected prior to feeding HFHC diet. Three mice per time point were evaluated. Experiment was repeated with similar results. (D) Mean fluorescence intensity as a measure of protein levels of PDPN and LYVE-1 using inform software in indicated group acquired from images in panels A and B. (E) Staining and quantification of liver sections from control, 12-week-old mdr2–/– mice (a model of primary sclerosing cholangitis) and mice gavaged with ethanol (2% v/v) for 5 weeks (Lieber-DeCarli diet model). Sections were stained with PDPN, CK19, and DAPI. Scale bar is 20 μm. Statistical analysis was performed using an unpaired t test in which ∗P < .05. ∗∗P < .01, and ∗∗∗P < .001. ALD, alcoholic liver disease; BD, bile duct; LV, lymphatic vessel.

Figure 3

HFHC diet induces liver disease and increases cholesterol levels in mice. (A) Representative hematoxylin and eosin staining from control mice or mice fed an HFHC diet for 17 weeks (HFHC: 60% fat and 2% cholesterol). Scale bar is 20 μm. (B) Inflammation score (left) and steatosis score (right) for control or HFHC diet–fed mice. (C) Analysis of AST or ALT levels in the serum of mice fed either a control or HFHC diet for 17 weeks. (D) Quantitation of high-density lipoprotein (HDL), LDL, and total cholesterol (free and cell associated) by enzyme-linked immunosorbent assay from the liver or serum of mice fed a control or HFHC diet. (E) Whole-liver RT-qPCR of VEGFC transcript from control of HFHC diet. Shown are data from 1 independent experiment repeated at least 3 additional times. Statistical analysis was performed using an unpaired t test in which ∗P < .05, ∗∗P < .01, and ∗∗∗∗P < .001.

Figure 4

Single-cell sequencing of liver LECs identifies transcriptional changes associated with HFHC diet–induced liver disease. (A) Gating strategy used for flow sorting of endothelial cells from control- or HFHC-fed mice. Nonparenchymal cells from the liver were isolated and stained for indicated markers after running through a live/dead selection. All CD31 positive cells were sorted into a single tube and then processed for single-cell RNA sequencing. (B) Heatmap of genes typically associated with LECs are shown as a validation of our cell classification. Yellow is genes upregulated and purple is genes downregulated. Shown are cells from control mice. (C) Uniform manifold approximation and projection (UMAP) plot to visualize the similarities between cell types acquired using the 10x Genomics single cell sequencing platform. Cells were classified using clustifyr . Inset box shows endothelial cell populations acquired. Shown are cells from control and HFHC mice. (D) Gene expression data from LECs only were entered into Ingenuity software for pathway analysis. Shown are pathways with z-scores ≥2 in LECs from HFHC diet–fed compared with control diet–fed. Green color represents highly activated pathways while red scores represent the downregulation of pathways. Data in figure are combined data from 2–5 mice per capture with 4 independent captures acquired by the 10x Genomics 3′ kit following cell sorting. FSC-H, forward scatter-height; FSC-W, forward scatter-width; HSC, hepatic stellate cell; IM, infiltrating macrophage; KC, Kupffer cell; SSC-W, side scatter-width.

Figure 5

Liver LECs from mice fed an HFHC diet downregulate lymphatic genes and upregulate metabolic pathways. Violin plot of gene expression by (A) LECs, (B) LSECs, and (C) PECs. Shown are changes in gene expression in LECs from the livers of mice fed HFHC diet compared with Control diet. (D) Ridge plot showing that the distribution of per-cell Vegfr2/Vegfr3 ratios differs between the LECs from control- and HFHC-fed animals. A cell with a score of 0 in this metric has the average Vegfr2/Vegfr3 ratio, while a cell with a positive score has a higher than average Vegfr2/Vegfr3 ratio. (E) Cell cycle analysis of LECs (control/HFHC) based on gene expression from single cell sequencing. (F) Uniform manifold approximation and projection (UMAP) representation of VegfC expression in single cells where purple indicates increased expression. Cells from control and HFHC livers are shown as in Figure 4C. Exact P values are shown.

Figure 6

VEGFc (cys156ser) regulates lymphangiogenesis but not cholangiocyte or LSEC expansion in the liver. (A) Immunofluorescence staining and quantification of liver sections from indicated groups. Treatment with rVEGFC occurred at 18.5 weeks after initiation of diet and consisted of 2.5 weeks of treatment every other day. Sections were stained with PDPN, CK19, and DAPI. Scale bar is 50 μm. (B) Quantification of LVD was performed using inForm software and based on PDPN+CK19– vessels. (C) Quantification of cholangiocyte number was performed using ImageJ software and based on PDPN–CK19+ ductal structures. Statistical analysis was performed as in panel B. Shown are representative images and combined data from 2 independent experiments with 3–5 mice per group. (D) Six-week-old mice were fed a control or HFHC diet for 15 weeks before 3 weeks of rVEGFC or vehicle treatment. At 18 weeks, mice were euthanized and livers were harvested and digested for flow cytometry as in Finlon et al. Indicated markers were used to identify LECs and LSECs and frequency of Ki67+ cells from each type were calculated. Experiment quantified with 4–6 mice per group from 2 independent experiments. Statistical analysis was performed using a Student’s t test in which ∗P < .05 and ∗∗P < .01.

Figure 7

Liver lymphatic transport is impaired in HFHC-fed mice and rescued by rVEGFC. (A) Mice were fed with or without administration of rVEGFC (cys156ser). Prior to sacrifice, mice were anesthetized and livers were injected with 5 μL of 500-kD FITC-dextran into each of 3 liver lobes. Five minutes after administration of last injection, the portal-draining LN (PLN) and inguinal lymph nodes were removed. (B) Amount of FITC-labeled dextran was measured in portal at 5 minutes. Shown is the fold increase in FITC reading from portal LN to the inguinal lymph node from the same mouse. Fold increase over uninjected in labeled FITC-dextran from (C) the inguinal LN or (D) plasma. (E) Quantification of drainage of 70-kD FITC-dextran to the portal LN at 5 minutes in control or HFHC diet fed mice. (F) Mean fluorescence intensity of PDPN protein quantified from analyzed sections using InForm software as in Figure 2D. (G) Quantification of liver inflammation score by a pathologist blinded to the samples as in Lanaspa et al. Shown are combined data from 2 independent experiments with 3–6 mice per group. Statistical analysis in figure was performed using an unpaired t test in which ∗P < .05 and ∗∗∗P < .001. CLN, celiac lymph node; dLN, draining lymph node; MLN, mesenteric lymph node; PV, portal vein.

Figure 8

Intravenous injection of oxLDL results in decreased lymphatic drainage. (A) Quantification of oxLDL in the liver tissue of mice fed either a control- or HFHC-fed diet for 22 weeks. Shown are combined data from 2 independent experiments with 3–6 mice per group. (B) Experimental design for FITC-dextran assay after oxLDL administered intravenously for 2 weeks (every other day for 7 doses). (C) As in Figure 7, amount of FITC-dextran in the portal LN of mice injected with PBS given the HFHC diet for 24 weeks or the 7 doses of oxLDL over 2 weeks. Data normalized to nondraining inguinal lymph node from same mouse. (D) Representative hematoxylin and eosin images from mice injected with PBS (vehicle) or oxLDL for 2 weeks. No liver injury was observed in any of the mice. Scale bar is 100 μm. (E) RT-qPCR for indicated genes from livers of mice given oxLDL systemically. (F) 500-kD FITC-dextran drainage assay performed in mice given 150-μg native LDL systemically 7 times over 2 weeks rather than oxLDL as in panel B. Shown is representative data from 2 experiments with 3–5 mice per group. OxLDL without HFHC group was performed 1 additional time with 3–6 mice per group with similar results and P values. Statistical analysis was performed using an unpaired t test in which ∗P <.05, ∗∗P < .01 and ∗∗∗P < .001.

Figure 9

LECs take up oxLDL in vivo and oxLDL treatment in vitro changes LEC metabolism. (A) Gating strategy for liver LEC and LSEC after DIL (1,1’-Dioctadecyl-3,3,3’,3-tetramethylindocarbocyanine perchlorate)-labeled oxLDL injection (150 μg intravenously; Kalen Biomedical, Germantown, MD). Shown in the last plot is oxLDL at day 6 in LECs in which the gray histogram is oxLDL injection and white is PBS injection. (B) Amount of oxLDL in either LECs (white) or LSECs (gray) at day 2 or day 6 postinjection. Experiment was performed 4 times with similar results. Shown are representative data from 1 experiment that was repeated at least 2 more times with similar results and P values. (C) Overview of the tricarboxylic acid (TCA) cycle in which bolded metabolites are altered after treatment with oxLDL. (D) Changes in bolded metabolites from panel C. Metabolite abundance was measured using mass spectrometry in hLECs grown to 80% confluency on a thin layer of matrigel and treated for 24 hours with either vehicle (PBS) (blue) or 100-μg/mL of oxLDL (in PBS) (red) using mass spectrometry. (E) Heatmap and hierarchical clustering of changes in metabolites from independent experimental replicates. Experiment was repeated 2 independent times with 3 technical replicates per group. P values were calculated using a 1-way analysis of variance with multiple comparisons in which ∗P < .05 and ∗∗P < .01.

Figure 10

OxLDL treatment changes LEC permeability and VE-cadherin expression. (A) A total of 6–10,000 hLECs were plated onto a transwell with 0.4-μm pores and allowed to form a basement membrane for 3 days. After 3 days, the hLECs were treated with oxLDL for 1 hour or 24 hours prior to the addition of 500-kD FITC-Dextran to the top well. Hanks’ balanced salt solution in the bottom well was removed at indicated time points and the amount of FITC-dextran in the bottom well was measured. Linear regression was performed and determined that the slope of the line was significantly different with a P value of <.0001 as indicated by 4 stars. Data shown are from 1 independent experiment with at least 3 replicates per group. Experiment was repeated at least 2 additional times with similar results and P values. (B) Transwell inserts were removed from the plate and stained with VE-cadherin (red) and DAPI (blue). Shown are junctions between cells in which the scale bar on the larger images is 50 μm and the white box is zoomed in and on the right. Scale bar on zoomed out image is 10 μm. Quantification of VE-cadherin pixel intensity from 3 replicates normalized to the PBS control is shown. (C) Protein expression of VE-cadherin was calculated from cells treated with oxLDL as in panel B for either 4 or 24 hours. Shown is Western blot analysis using antibodies against VE-cadherin and GAPDH (loading control). Western blot was performed 3 times and quantification is shown from replicates normalized to time 0. (D) Vegfr2/Vegfr3 transcript ratio assessed by RT-qPCR of hLECs treated with 100 μg oxLDL or PBS for 24 hours. Experiment was repeated 3 times with similar results. (E) A total of 6–10,000 hLECs were seeded on a transwell as in panel A and treated with 1-ng TNFα (10 ng/mL) in 100-μL PBS (vehicle) and the amount of FITC-dextran recovered from the bottom well was measured every 30 minutes over a 3-hour period. Shown is fold change from time 0 before FITC was added to the top well. Shown are data from 1 experiment with 3 technical replicates. Experiment was repeated at least twice with similar results. Linear regression was performed and determined that the slope of the line was significantly different with a P value of <.0001 as indicated by 4 stars. (F) As in panel A, 0.4-μm transwell plated with a confluent layer (∼10,000 cells for 2–3 days) of HUVECs. Cells were treated with either PBS (black) or oxLDL (100 μg) (blue) for 24 hours prior to the addition of 500-kD FITC-dextran. At indicated timepoints, FITC-dextran was recovered from the bottom well and read on a fluorescent plate reader. Differences were not significant. (G) Cells from panel F were stained with VE-cadherin and fluorescence intensity was calculated as described previously. (H) Cells treated as in panels F and G were removed from the plate and RNA was isolated for quantification of relative levels of transcript based on amount of complementary DNA made. Fold increase over PBS is shown for indicated genes. (I) Ratio of Vegfr2/Vegfr3 transcript in HUVECs following treatment with either PBS or oxLDL. Experiment was repeated at least twice with similar results. ∗P < .05.