Macrophage Polarization And Nitric Oxide Mechanisms In Lymphatic Dysfunction In A Rat Model Of Metabolic Syndrome

Metabolic syndrome (MetSyn) is the clustering of multiple metabolic disorders that further increase the risk for cardiovascular disease and has been recently linked to poor lymphatic function. The lymphatic system plays a crucial role in maintaining oncotic balance and returning excess fluid and macromolecules back to the blood circulation. In this dissertation we addressed the role of macrophage polarization and nitric oxide mechanisms in lymphatic dysfunction in a rat model of MetSyn. We hypothesized that mesenteric lymphatic vessel dysfunction would be associated with a polarization switch of resident macrophages after induction of peritonitis or metabolic syndrome. We used an intra-peritoneal injection of lipopolysaccharide (LPS) to simulate peritonitis in the rat and a seven-week high fructose-feeding regime to induce the MetSyn. We distinguished macrophage polarization and recruitment to the lymphatic collecting vessels using immunofluorescence and a combination of CD163, CD206, and major histocompatibility complex II (MHCII) expression. We determined the intrinsic mesenteric lymphatic contractility using the isolated mesenteric lymphatic vessel isobaric preparation. LPS-induced peritonitis increased the macrophage accumulation two fold and increased both CD163+CD206+ and CD163-CD206+ cell populations and had severely impaired lymphatic contractility. We also found evidence for a phenotype switch from CD163+MHCII- M2 macrophages to a M1 skewed CD163+MHCII+ phenotype in the MetSyn rats and impaired lymphatic contractility. Additionally, cultured lymphatic endothelial and muscle cells were found to express macrophage maturation and expansion markers in response to LPS stimulation. We also examined the role of nitric oxide in the contractile regulation of lymphatic thoracic ducts isolated from MetSyn rats. We found a reduced flow-dependent inhibition of contractility in metabolic syndrome thoracic ducts despite a normal response to the exogenous nitric oxide donor S-nitro-N-acetylpenicillamine (SNAP). The reactive oxygen species scavenging agent 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL) did not restore flow sensitivity, however control vessels treated with the nitric oxide synthase inhibitor L-NG-nitro arginine methyl ester (LNAME) had comparable flow inhibition to MetSyn thoracic ducts. Western blots of thoracic ducts revealed a 60% reduction in the expression of eNOS, which can explain the loss of shear sensitivity. Thus, this study demonstrates the mechanisms that underlie lymphatic dysfunction in the MetSyn

Developmental progression of lymphatic valve morphology and function

Introduction: The bileaflet valves found in collecting lymphatic vessels and some veins are essential for maintaining a unidirectional flow, which is important for lymphatic and venous function. Under an adverse pressure gradient, the two leaflets tightly overlap to prevent backflow. Valves are proposed to share four main stages of development, based on images obtained from randomly oriented valves in fixed mouse embryos, with the best structural views obtained from larger venous valves. It is not known at what stage lymphatic valves (LVs) become functional (e.g., able to oppose backflow), although a requirement for stage 4 is presumed.Methods: To gain an insight into this sequence of events for LVs, we used Prox1CreERT2:Foxo1fl/fl mice and Foxc2CreERT2:Foxo1fl/fl mouse models, in which deletion of the valve repressor factor Foxo1 promotes the development of new LVs in adult lymphatic vessels. Both strains also contained a Prox1eGFP reporter to image the lymphatic endothelium. Mesenteric collecting lymphatic vessels were dissected, cannulated, and pressurized for ex vivo tests of valve function. LVs at various stages (1–4 and intermediate) were identified in multi-valve segments, which were subsequently shortened to perform the backleak test on single valves. The GFP signal was then imaged at high magnification using a confocal microscope. Z-stack reconstructions enabled 1:1 comparisons of LV morphology with a quantitative measurement of back leak.Results: As expected, LVs of stages 1–3 were completely leaky in response to outflow pressure elevation. Stage 4 valves were generally not leaky, but valve integrity depended on the Cre line used to induce new valve formation. A high percentage of valves at leaflet an intermediate stage (3.5), in which there was an insertion of a second commissure, but without proper luminal alignment, effectively resisted back leak when the outflow pressure was increased.Discussion: Our findings represent the first 3D images of developing lymphatic valves and indicate that valves become competent between stages 3 and 4 of development

Inhibition of myosin light chain phosphorylation decreases rat mesenteric lymphatic contractile activity

Muscular lymphatics use both phasic and tonic contractions to transport lymph for conducting their vital functions. The molecular mechanisms regulating lymphatic muscle contractions are not well understood. Based on the well-established finding that the phosphorylation of myosin light chain 20 (MLC20) plays an essential role in blood vessel smooth muscle contraction, we investigated if phosphorylated MLC20 (pMLC20) would modulate the tonic and/or phasic contractions of lymphatic muscle. The effects of ML-7, a MLC kinase inhibitor (1–10 μM), were tested on the contractile parameters of isolated and cannulated rat mesenteric lymphatics during their responses to the known modulators, pressure (1–5 cmH2O) and substance P (SP; 10−7 M). Immunohistochemical and Western blot analyses of pMLC20 were also performed on isolated lymphatics. The results showed that 1) increasing pressure decreased both the lymphatic tonic contraction strength and pMLC20-to-MLC20 ratio; 2) SP increased both the tonic contraction strength and phosphorylation of MLC20; 3) ML-7 decreased both the lymphatic tonic contraction strength and pMLC20-to-MLC20 ratio; and 4) the increase in lymphatic phasic contraction frequency in response to increasing pressure was diminished by ML-7; however, the phasic contraction amplitude was not significantly altered by ML-7 either in the absence or presence of SP. These data provide the first evidence that tonic contraction strength and phasic contraction amplitude of the lymphatics can be differentially regulated, whereby the increase in MLC20 phosphorylation produces an activation in the tonic contraction without significant changes in the phasic contraction amplitude. Thus, tonic contraction of rat mesenteric lymphatics appears to be MLC kinase dependent