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. 2020 Dec:42:101081.
doi: 10.1016/j.molmet.2020.101081. Epub 2020 Sep 14.

Regulation of lymphatic function and injury by nitrosative stress in obese mice

Affiliations

Regulation of lymphatic function and injury by nitrosative stress in obese mice

Sonia Rehal et al. Mol Metab. 2020 Dec.

Abstract

Objective: Obesity results in lymphatic dysfunction, but the cellular mechanisms that mediate this effect remain largely unknown. Previous studies in obese mice have shown that inducible nitric oxide synthase-expressing (iNOS+) inflammatory cells accumulate around lymphatic vessels. In the current study, we therefore tested the hypothesis that increased expression of iNOS results in nitrosative stress and injury to the lymphatic endothelial cells (LECs). In addition, we tested the hypothesis that lymphatic injury, independent of obesity, can modulate glucose and lipid metabolism.

Methods: We compared the metabolic changes and lymphatic function of wild-type and iNOS knockout mice fed a normal chow or high-fat diet for 16 weeks. To corroborate our in vivo findings, we analyzed the effects of reactive nitrogen species on isolated LECs. Finally, using a genetically engineered mouse model that allows partial ablation of the lymphatic system, we studied the effects of acute lymphatic injury on glucose and lipid metabolism in lean mice.

Results: The mesenteric lymphatic vessels of obese wild-type animals were dilated, leaky, and surrounded by iNOS+ inflammatory cells with resulting increased accumulation of reactive nitrogen species when compared with lean wild-type or obese iNOS knockout animals. These changes in obese wild-type mice were associated with systemic glucose and lipid abnormalities, as well as decreased mesenteric LEC expression of lymphatic-specific genes, including vascular endothelial growth factor receptor 3 (VEGFR-3) and antioxidant genes as compared with lean wild-type or obese iNOS knockout animals. In vitro experiments demonstrated that isolated LECs were more sensitive to reactive nitrogen species than blood endothelial cells, and that this sensitivity was ameliorated by antioxidant therapies. Finally, using mice in which the lymphatics were specifically ablated using diphtheria toxin, we found that the interaction between metabolic abnormalities caused by obesity and lymphatic dysfunction is bidirectional. Targeted partial ablation of mesenteric lymphatic channels of lean mice resulted in increased accumulation of iNOS+ inflammatory cells and increased reactive nitrogen species. Lymphatic ablation also caused marked abnormalities in insulin sensitivity, serum glucose and insulin concentrations, expression of insulin-sensitive genes, lipid metabolism, and significantly increased systemic and mesenteric white adipose tissue (M-WAT) inflammatory responses.

Conclusions: Our studies suggest that increased iNOS production in obese animals plays a key role in regulating lymphatic injury by increasing nitrosative stress. In addition, our studies suggest that obesity-induced lymphatic injury may amplify metabolic abnormalities by increasing systemic and local inflammatory responses and regulating insulin sensitivity. These findings suggest that manipulation of the lymphatic system may represent a novel means of treating metabolic abnormalities associated with obesity.

Keywords: Inflammation; Lymphatic function; Metabolism; Nitrosative stress; Obesity; iNOS.

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Figures

Figure 1
Figure 1
iNOS mediates mesenteric lymphatic vessel dysfunction in obese mice. All panels compare wild-type (WT) and iNOS KO lean and high-fat diet-induced obese mice. (A) Representative graphs of lymphatic vessel pulsation in popliteal lymphatic vessels measured using indocyanine green (ICG)-based lymphangiography. (B) Quantification of lymphatic vessel packet frequency. (C) Dendritic cell trafficking assessed using the FITC painting assay. (D) Visualization of collecting lymphatic vessels in mesenteric white adipose tissue (M-WAT) by oral gavage-administered BODIPY (FL-C16). Inset, 2x magnification showing dilated, leaky lymphatic vessels. Scale bar, 500 μm. (E) Quantification of M-WAT lymphatic vessel diameter and (F) BODIPY leakage into the extraluminal space. N = 5/group, mean ± SD, two-way ANOVA with Sidak multiple comparison test (∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 ∗∗∗∗P < 0.0001).
Figure 2
Figure 2
Obesity-induced iNOS generates reactive nitrogen species in mesenteric fat and iNOS alter gene expression in lymphatic endothelial cells. All panels compare WT and iNOS KO lean and high-fat diet-induced obese mice except panels D and E. (A) Representative microscopic images of M-WAT containing lymphatic vessels, immunostained for VEGFR-3 and iNOS (scale bar, 100 μm). (B) Quantification of fluorescent iNOS signal. (C) qPCR for iNOS gene expression, from M-WAT tissue, in NCD and HFD mice. (D) Representative microscopic images of M-WAT containing lymphatic vessels, immunostained for iNOS, podoplanin, and CD11b (scale bar, 30 μm). (E) Quantification of iNOS+ cells in M-WAT. Representative flow cytometry dot plots showing gating for CD45 and CD45+ cells among iNOS+ cells and CD45+/iNOS+ cells among CD11b/F480. (F) Representative microscopic images of M-WAT containing lymphatic vessels, immunostained for alpha smooth muscle actin (α-SMA) and nitrosylated tyrosine (n-tyrosine) (scale bar, 100 μm). (G) Quantification of fluorescent n-tyrosine signal. (H) Peroxynitrite production in serum. (I–N) qPCR quantification of gene expression in LECs from mesenteric lymph nodes for the lymphatic markers (I) vascular endothelial growth factor receptor 3 (VEGFR-3), (J) podoplanin, (K) PROX-1, and the antioxidant enzymes (L) glutathione peroxidase (GPX), (M) superoxide dismutase (SOD), and (N) catalase (CAT). N = 4–5/group, mean ± SD, two-way ANOVA with Sidak multiple comparison test. (∗∗P < 0.01, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001).
Figure 3
Figure 3
LECs are highly sensitive to nitrosative stress compared with HUVECs. (A) Representative phase-contrast images of Matrigel-based tubule formation by cultured human dermal LECs (LECs) and human umbilical vein endothelial cells (HUVECs) in the presence or absence of 100 μM DETA NONOate (scale bar, 500 μm). (B) Quantification of branch points generated in tubule formation assay by LECs and HUVECs in the presence or absence of 500 μM DETA NONOate. (C) Dose–response curves for late apoptosis in LECs and HUVECs with or without DETA NONOate. (D) Dose–response curves for the effects of DETA NONOate on cell proliferation in LECs and HUVECs. (E) Quantification of LEC monolayer leakage of high molecular weight FITC in the presence or absence of 100 μM DETA NONOate. (F–K) Gene expression via qPCR for the following lymphatic markers following treatment with or without DETA NONOate and VEGF-C: (F) PROX-1, (G) podoplanin, (H) VEGFR-3, (I) LYVE1, and the antioxidant enzymes (J) GPX and (K) SOD in LECs. A-E, N = 5/group; F–K, N = 3–4; mean ± SD, two-way ANOVA with Sidak multiple comparison test or unpaired Student's t test. (∗P < 0.05, ∗∗∗P < 0.001).
Figure 4
Figure 4
Lymphatic injury causes insulin resistance, dyslipidemia, and gene expression changes in adipose tissue. All panels compare WT and lymphatic-ablated (DT-treated [IP unless otherwise indicated] FLT4-cre+/−-DTR+/−) mice. (A) Quantification of LECs in intestinal tissue. Representative flow cytometry dot plots showing gating for CD45CD31+podoplanin+ positive cells. (B–C) Tolerance of IP (B) glucose and (C) insulin. (D) Serum insulin. (E–J) qPCR-measured expression in epididymal fat of (E) the glucose transporter GLUT4, (F) IRS, (G) CCAAT/enhancer-binding protein alpha (C/EBP-α), (H) leptin, (I) adiponectin, and (J) peroxisome proliferator-activated receptor gamma (PPAR-γ). (K) ELISA-measured protein expression of phosphorylated IRS-1 (S307) relative to total IRS-1 in epididymal fat. (L) Insulin resistance calculated using homeostatic model assessment (HOMA-IR). (M–R) Serum lipid profiles of WT and FLT4-cre+/−-DTR+/− mice for (M) total cholesterol, (N) HDL, (O) triglycerides, and (P) LDL. (Q–R) ELISA quantification of serum adipocytokines, (Q) leptin, and (R) adiponectin. N = 4–5/group, mean ± SD, unpaired Student t-test. (∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001).
Figure 5
Figure 5
Lymphatic injury causes systemic and adipose tissue inflammation. All panels compare WT and lymphatic-ablated (DT-treated [IP unless otherwise indicated] FLT4-cre+/−-DTR+/−) mice. ELISA quantification of the inflammatory cytokines IFN-γ, IL-6, IL-1β, and TNF-α in (A) serum (B) visceral adipose tissue (VAT), (C) subcutaneous adipose tissue (SAT), and (D) liver. N = 5/group, mean ± SD, unpaired Student t-test. (∗P < 0.05, ∗∗P < 0.01).
Figure 6
Figure 6
Lymphatic injury causes nitrosative stress in mesenteric tissues. A-I, WT vs. lymphatic-ablated (DT-treated [IP unless otherwise indicated] FLT4-cre+/−-DTR+/−) mice. (A) Visualization of collecting lymphatic vessels in mesenteric white adipose tissue (M-WAT) by oral gavage-administered BODIPY (FL-C16). Inset, 2x magnification showing dilated, leaky lymphatic vessels. Scale bar, 500 μm. (B) Quantification of M-WAT lymphatic vessel diameter and (C) BODIPY leakage into the extraluminal space. (D) Representative confocal microscopic images of whole mount M-WAT containing lymphatic vessels, immunostained for iNOS and podoplanin and counterstained with DAPI (scale bar, 80 μm). (E–F) Quantification of iNOS+ cells in M-WAT; (E) representative flow cytometry dot plot and (F) as a percentage of live cells. (G) Representative confocal microscopic images of whole mount M-WAT containing lymphatic vessels, immunostained for alpha smooth muscle actin (α-SMA) and nitrosylated tyrosine (n-tyrosine) (scale bar, 50 μm). (H) Quantification of fluorescent n-tyrosine. (I) Peroxynitrite content in mesenteric tissues. J-O, comparison of WT and FLT4-cre+/−-DTR+/− mice treated with 1400 W or vehicle (PBS). (J) Tolerance of IP-injected glucose bolus. (K–O) Serum lipid levels of (K) total cholesterol, (L) HDL (M) triglycerides, (N) LDL, and (O) serum insulin. N = 4–5; mean ± SD, two-way ANOVA with Sidak multiple comparison test or unpaired Student's t-test. (∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001).
Figs1
Figs1
Supplementary Figure 1. Body composition of NCD-fed (lean) and HFD-fed (obese) WT and iNOS KO mice. All panels compare WT and iNOS KO lean and high-fat diet-induced obese mice. (A) Weight gain over 14 weeks of NCD (lean) or HFD (obese) feeding. (B) Gross representative images of body cavities exposing outlined epididymal fat pads. (C-E) Quantification of (C) food consumption and (D) inguinal and (E) reproductive (epididymal) fat pad weights. N = 5/group, mean ± SD, two-way ANOVA with Sidak multiple comparison test or unpaired Student's t-test. (∗∗∗∗P < 0.0001).
Figs2
Figs2
Supplementary Figure 2. iNOS contributes to obesity-induced glucose and insulin impairment. All panels compare WT and iNOS KO lean and high-fat diet-induced obese mice. (A) Oral glucose tolerance test (OGTT). (B-E) Quantification of (B) serum insulin, (C) Insulin resistance calculated using homeostatic model assessment (HOMA-IR), and (D) GLUT4mRNA and (E) S307 phosphorylation of insulin receptor substrate-1 (IRS-1) in adipose tissue. (F) Quantification of serum lipids: total cholesterol, HDL, LDL, and triglycerides. (G-H) ELISA quantification of (G) serum leptin and (H) serum adiponectin. N = 5/group, mean ± SD, two-way ANOVA with Sidak multiple comparison test or unpaired Student's t test. (∗P < 0.05, ∗∗P < 0.01, ∗∗∗∗P < 0.0001).
Figs3
Figs3
Supplementary Figure 3. DETA NONOate causes irreversible loss of LEC viability and changes in gene expression. (A) Timeline of DETA NONOate treatment and media replacement for experiment in B. (B) Quantification of viable cells following treatment with DETA NONOate for 1, 3, 6, and 24 h compared to untreated cells by MTT assay performed at 24 h. (C) Timeline of DETA NONOate treatment and media replacement for experiments in D-G. (D-G) qPCR quantification of LEC gene expression of (D) Prox1, (E) podoplanin, (F) VEGFR-3, and (G) LYVE1 in LECs treated with DETA NONOate for 48 h or 24 h and replaced with fresh media in comparison with untreated LECs. N = 3–4/group, mean ± SD, two-way ANOVA with Sidak multiple comparison test or unpaired Student's t test. (∗P < 0.05, ∗∗P < 0.01, ∗∗∗∗P < 0.0001).
Figs4
Figs4
Supplementary Figure 4. N-acetylcysteine treatment of LECs partially reverses NO-induced toxicity. All panels compare LECs treated with 500 μM of DETA NONOate, 5 mM of n-acetyl cysteine (NAC), both, or media alone (control). (A) Representative phase contrast images showing Matrigel-based tubule formation. (B) Quantification of branch points produced in tubule formation assay. (C) Quantification of apoptosis. (D) VEGFR-3 mRNA expression by qPCR. N = 4–5/group, mean ± SD, two-way ANOVA with Sidak multiple comparison test (∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001).
Figs5
Figs5
Supplementary Figure 5. Representative gating strategy for intestinal tissue flow analysis for LECs. All panels compare WT and lymphatic-ablated (DT-treated [IP unless otherwise indicated] FLT4-cre+/--DTR+/-) mice. Stained single-cell suspensions of intestinal tissue were first gated on 4′,6-diamidino-2-phenylindole (DAPI)-negative dead cells and later gated on for singlet cells. From the singlet cells, CD45-negative cells were gated and looked for podoplanin and CD31 double-positive LECs.
Figs6
Figs6
Supplementary Figure 6. Lymphatic ablation in FLT4-cre+/--DTR+/-mice is strictly limited to lymphatic vasculature. Representative images of (A) ear skin and (B) small intestine, immunostained for LYVE-1 (red) and iNOS (green) and counterstained with DAPI (blue). In FLT4-cre+/--DTR+/- mice, DT was injected into the ear skin in (A) and IP in (B). White arrows indicate disrupted lymphatic vessels and iNOS accumulation (scale bar, 30 μm). (C-G) Quantification of (C) food intake, (D) body weight (E), average weight gain, (F) reproductive fat pad weight, and (G) inguinal fat pad weight of DT-treated FLT4-cre+/--DTR+/- mice compared with wild-type controls. N = 4–5/group, mean ± SD, unpaired Student's t-test.
Figs7
Figs7
Supplementary Figure 7. Ablation of mesenteric lymphatic vessels does not affect intestinal villi or liver architecture or liver function. All panels compare WT and lymphatic-ablated (DT-treated [IP unless otherwise indicated] FLT4-cre+/--DTR+/-) mice. (A-B)) H&E images showing (A) villus and (B) liver architecture (upper low; lower high magnification). (C-L) Serum quantification of liver function indicators (C) alkaline phosphatase, (D) alanine transaminase, (E) aspartate transaminase, (F) total protein, (G) albumin, (H) globulin, (I) lactate dehydrogenase, (J) total bilirubin, (K) indirect bilirubin, and (L) albumin to globulin ratio. N = 5/group, mean ± SD, unpaired Student's t-test.

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