Blunted flow-mediated responses and diminished nitric oxide synthase expression in lymphatic thoracic ducts of a rat model of metabolic syndrome

Abstract

Shear-dependent inhibition of lymphatic thoracic duct (TD) contractility is principally mediated by nitric oxide (NO). Endothelial dysfunction and poor NO bioavailability are hallmarks of vasculature dysfunction in states of insulin resistance and metabolic syndrome (MetSyn). We tested the hypothesis that flow-dependent regulation of lymphatic contractility is impaired under conditions of MetSyn. We utilized a 7-wk high-fructose-fed male Sprague-Dawley rat model of MetSyn and determined the stretch- and flow-dependent contractile responses in an isobaric ex vivo TD preparation. TD diameters were tracked and contractile parameters were determined in response to different transmural pressures, imposed flow, exogenous NO stimulation by S-nitro-N-acetylpenicillamine (SNAP), and inhibition of NO synthase (NOS) by l-nitro-arginine methyl ester (l-NAME) and the reactive oxygen species (ROS) scavenging molecule 4-hydroxy-tempo (tempol). Expression of endothelial NO synthase (eNOS) in TD was determined using Western blot. Approximately 25% of the normal flow-mediated inhibition of contraction frequency was lost in TDs isolated from MetSyn rats despite a comparable SNAP response. Inhibition of NOS with l-NAME abolished the differences in the shear-dependent contraction frequency regulation between control and MetSyn TDs, whereas tempol did not restore the flow responses in MetSyn TDs. We found a significant reduction in eNOS expression in MetSyn TDs suggesting that diminished NO production is partially responsible for impaired flow response. Thus our data provide the first evidence that MetSyn conditions diminish eNOS expression in TD endothelium, thereby affecting the flow-mediated changes in TD lymphatic function.

Keywords: lymph flow; lymphatic endothelial cells; lymphatic vessel contraction; metabolic syndrome; nitric oxide synthase.

Fig. 1.

Representative diameter tracings from paired albumin supplemented physiological saline solution (APSS) and l-nitro-arginine methyl ester (l-NAME) responses in control with the corresponding pressure (P) and flow. Representative diameter traces over a 1-min period for lymphatic thoracic ducts (TDs) isolated from control (A) and metabolic syndrome (MetSyn) rats (C) and their responses to l-NAME (B and D) respectively. Schematic of a pressure step protocol used in this study (E) for the traces displayed.

Fig. 2.

Effects of pressure (P) and imposed flow (F) on lymphatic TD function from control and MetSyn rats. TD contractions per min were counted at each experimental pressure and imposed flow (A and B, respectively). Ejection fraction (EF; C) and normalized vessel tone (D) for control and MetSyn TDs exposed to imposed flow were calculated as described in materials and methods. Data are presented as means ± SE; n = 23 for A; n = 20 for B–D. *P < 0.05, significance comparing MetSyn to control values at the same pressure by two-way ANOVA.

Fig. 3.

The effect of exogenous nitric oxide (NO) on lymphatic TD function in control and MetSyn rats. Frequency (A), EF (B), and vessel tone (C) were determined at each experimental pressure for control and MetSyn TDS. Data are presented as means ± SE; n = 9 for control; n = 7 for MetSyn. *P < 0.05, and 0.05 < P̂ < 0.10, significance comparing within each cohort at each experimental pressure by two-way ANOVA.

Fig. 4.

The effect of nitric oxide synthase (NOS) inhibition with l-NAME on lymphatic TD imposed flow responses in control and MetSyn rats. TDs were equilibrated for 20 min in 100 μM l-NAME and their responses to imposed flow were recorded and analyzed for Frequency (A) and vessel tone (B). Data are presented as means ± SE; n = 7 for MetSyn; n = 10 for control. *P < 0.05, significance comparing within the cohort by two-way ANOVA.

Fig. 5.

Differences between control and MetSyn TD function in response to flow are abolished when NOS is inhibited but not when reactive oxygen species (ROS) is inhibited. Frequency was normalized within each cohort to zero flow (F = 0) to compare the role for NO and ROS in the MetSyn TD flow response. MetSyn TDs displayed blunted flow-mediated inhibition of contractility (A). Inhibition of NOS with 100 μM l-NAME abolishes the differences in normalized frequency (B). Inhibition of ROS with 1 mM tempol did not restore flow-mediated inhibition in MetSyn TD (C). Data are presented as mean ± SE; n = 20 for control and MetSyn (A); n = 7 for control and 10 for MetSyn for the l-NAME response (B); and n = 6 for both control and MetSyn for the tempol response (C). *P < 0.05, significance by two-way ANOVA.

Fig. 6.

Reduced expression of eNOS in TDs isolated from MetSyn rats. Proteins were isolated from TDs isolated from control and MetSyn rats and used for Western blots to assess enothelial NOS (eNOS) expression. β-Actin (βAct) was used as a loading control. A: a representative Western blot; C, control sample; M, MetSyn sample; O, omitted sample in the analysis due to low amount of protein loading. B: quantitative analysis of eNOS expression. The eNOS-to-βAct ratios were normalized to control values. The values obtained from 3 different blots were used for the analysis. Data are presented as means ± SE; n = 5 for MetSyn and 6 for control. *P < 0.05, significance by two-way ANOVA.