Chronic high-fat diet impairs collecting lymphatic vessel function in mice

Abstract

Lymphatic vessels play an essential role in intestinal lipid uptake, and impairment of lymphatic vessel function leads to enhanced adipose tissue accumulation in patients with lymphedema and in genetic mouse models of lymphatic dysfunction. However, the effects of obesity on lymphatic function have been poorly studied. We investigated if and how adipose tissue accumulation influences lymphatic function. Using a lymphatic specific tracer, we performed in vivo near-infrared (NIR) imaging to assess the function of collecting lymphatic vessels in mice fed normal chow or high-fat diet (HFD). Histological and whole mount analyses were performed to investigate the morphological changes in initial and the collecting lymphatic vessels. HFD was associated with impaired collecting lymphatic vessel function, as evidenced by reduced frequency of contractions and diminished response to mechanostimulation. Moreover, we found a significant negative correlation between collecting lymphatic vessel function and body weight. Whole mount analyses showed an enlargement of contractile collecting lymphatic vessels of the hind limb. In K14-VEGF-C mice, HFD resulted in a reduced spreading of the tracer within dermal lymphatic vessels. These findings indicate that adipose tissue expansion due to HFD leads to a functional impairment of the lymphatic vasculature, predominantly in collecting lymphatic vessels.

Figure 1. Reduced water content and increased adipose tissue after HFD.

Significantly increased body weight (A) and reduced skin water content in back skin (B) after 17 weeks of HFD. Hematoxylin stainings (C,D) and quantitative image analyses (E) revealed a significantly increased thickness of the adipose tissue, but not of the epidermis or dermis, in the tail skin of C57BL/6J:ICR HFD mice. Immunofluorescence stains for podoplanin (F,G; red; lymphatic vessels) and Meca32 (H,I; green; blood vessels) and quantitative image analyses (J) revealed a reduction of the average dermal lymphatic vessel size, but not blood vessel size, after HFD. Scale bars  =  100 μm. Data represent mean ± SD. *P≤0.05, **P≤0.01, ***P≤0.001.

Figure 2. Impaired collecting lymphatic vessel function after 17 weeks of HFD in FVB mice.

NIR-imaging (A) was performed after intradermal injection of 5 μL of 25 μM P20D680, a tracer specific for uptake into lymphatic vessels. Collecting lymphatic vessel contractility analysis was performed on the afferent vessels near the popliteal LN, while the response to mechanostimulation of the injection site by a cotton swab was assessed on the collecting lymphatic vessels of the lower limb. Normal (B) and irregular (C) contraction patterns in chow and HFD mice, respectively. Linear regression analysis (D) shows a significant negative correlation between weight and contraction frequencies. Normal (E) and impaired response (F) to mechanostimulation. Mice on HFD showed reduced response to mechanostimulation (G) as compared to mice on chow diet. **P≤0.01.

Figure 3. Whole mount analysis revealed enlarged collecting lymphatic vessels of the hind limb.

CD31 (A, green) and αSMA (B, red) stainings of collecting lymphatic vessels from chow diet fed mice were compared to CD31 (D) and αSMA (E) stainings of collecting lymphatic vessels from HFD fed mice. The collecting lymphatic vessels were observed to be enlarged in the HFD fed mice. CD31 (G, green) and αSMA (H, red) stainings of non-contractile ear collecting lymphatic vessels from chow diet fed mice were compared to CD31 (J, green) and αSMA (K, red) stainings from HFD fed mice. No major differences were observed. Merged channels are shown in (C, F, I and L). Scale bars  =  50 μm.

Figure 4. Impaired collecting lymphatic vessel function and reduced spread of tracer in dermal lymphatic vessels after 17 weeks of HFD in K14-VEGF-C mice.

NIR-imaging was performed after intradermal injection of 5 μL of 25 μM P20D680. Normal (A) and irregular (B) contraction patterns in chow and HFD mice, respectively. Linear regression analysis (C) shows a significant negative correlation between weight and contraction frequencies. High (D) and low (E) spreading of tracer within dermal lymphatic vessels in chow and HFD fed mice, respectively, after 15 minutes of imaging. Scale bars  =  2 mm. Quantification (F) shows lower percentage of tracer-perfused lymphatic vessels of the skin in the lower limb of mice on HFD as compared to chow diet. *P≤0.05.