Flow-dependent regulation of rat mesenteric lymphatic vessel contractile response requires activation of endothelial TRPV4 channels.

OBJECTIVES: The objective of our study is to evaluate the involvement of the transient receptor potential vanilloid 4 (TRPV4) in the alteration of lymphatic pumping in response to flow and determine the signaling pathways involved. METHODS: We used immunofluorescence imaging and western blotting to assess TRPV4 expression in rat mesenteric lymphatic vessels. We examined inhibition of TRPV4 with HC067047, nitric oxide synthase (NOS) with L-NNA and cyclooxygenases (COXs) with indomethacin on the contractile response of pressurized lymphatic vessels to flow changes induced by a stepwise increase in pressure gradients, and the functionality of endothelial TRPV4 channels by measuring the intracellular Ca2+ response of primary lymphatic endothelial cell cultures to the selective agonist GSK1016790A. RESULTS: TRPV4 protein was expressed in both the endothelial and the smooth muscle layer of rat mesenteric lymphatics with high endothelial expression around the valve sites. When maintained under constant transmural pressure, most lymphatic vessels displayed a decrease in contraction frequency under conditions of flow and this effect was ablated through inhibition of NOS, COX or TRPV4. CONCLUSIONS: Our findings demonstrate a critical role for TRPV4 in the decrease in contraction frequency induced in lymphatic vessels by increases in flow rate via the production and action of nitric oxide and dilatory prostanoids.

Increased contractile activity and dilation of popliteal lymphatic vessels in the TNF-α-overexpressing TNFΔARE/+ arthritic mouse.

AIMS: TNF-α acute treatment has been found to disrupt lymphatic drainage in the setting of arthritis through the NF-kB-iNOS- signaling pathway. We examined whether popliteal lymphatic vessels (pLVs) contractile activity was altered in 12- and 24- week-old females of an arthritic mouse model overexpressing TNF-α (TNFΔARE/+). MAIN METHODS: pLVs were prepared for intravital imaging to measure lymph flow speed, and ex vivo functional responses to a stepwise increase in transmural pressure in the absence or presence of the non-selective NOS inhibitor (L-NNA) or the selective iNOS inhibitor (1400W) were compared between TNFΔARE/+ and WT mice. Total eNOS (t-eNOS) and eNOS phosphorylated at ser1177 (p-eNOS) were evaluated by western blotting. KEY FINDINGS: In vivo imaging revealed a significantly increase in lymph flow speed in TNFΔARE/+ mice in comparison to WT at both ages. Pressure myography showed an increase in contraction frequency, diameters and fractional pump flow at both ages, whereas amplitude and ejection fraction were significantly decreased in older TNFΔARE/+ mice. Additionally, contraction frequency was increased in the presence of 1400W, and systolic diameter was abolished with L-NNA in TNFΔARE/+ mice compared to WT. Significant increases in p-eNOS expression and neutrophil recruitment (MPO activity) were observed in TNFΔARE/+ mice compared to WT. SIGNIFICANCE: Our data reveal functional changes in pLVs, especially in advanced stage of arthritis. These alterations may be related to eNOS and iNOS response, which can affect drainage of the inflammatory content from the joints.

Lymph-derived chemokines direct early neutrophil infiltration in the lymph nodes upon Staphylococcus aureus skin infection.

A large number of neutrophils infiltrate the lymph node (LN) within 4 h after Staphylococcus aureus skin infection (4 h postinfection [hpi]) and prevent systemic S. aureus dissemination. It is not clear how infection in the skin can remotely and effectively recruit neutrophils to the LN. Here, we found that lymphatic vessel occlusion substantially reduced neutrophil recruitment to the LN. Lymphatic vessels effectively transported bacteria and proinflammatory chemokines (i.e., Chemokine [C-X-C motif] motif 1 [CXCL1] and CXCL2) to the LN. However, in the absence of lymph flow, S. aureus alone in the LN was insufficient to recruit neutrophils to the LN at 4 hpi. Instead, lymph flow facilitated the earliest neutrophil recruitment to the LN by delivering chemokines (i.e., CXCL1, CXCL2) from the site of infection. Lymphatic dysfunction is often found during inflammation. During oxazolone (OX)-induced skin inflammation, CXCL1/2 in the LN was reduced after infection. The interrupted LN conduits further disrupted the flow of lymph and impeded its communication with high endothelial venules (HEVs), resulting in impaired neutrophil migration. The impaired neutrophil interaction with bacteria contributed to persistent infection in the LN. Our studies showed that both the flow of lymph from lymphatic vessels to the LN and the distribution of lymph in the LN are critical to ensure optimal neutrophil migration and timely innate immune protection in S. aureus infection.

The Symbiotic Effect of a New Nutraceutical with Yeast ß-Glucan, Prebiotics, Minerals, and Silybum marianum (Silymarin) for Recovering Metabolic Homeostasis via Pgc-1α, Il-6, and Il-10 Gene Expression in a Type-2 Diabetes Obesity Model.

The use of natural products and derivatives for the prevention and control of non-communicable chronic diseases, such as type-2 diabetes (T2D), obesity, and hepatic steatosis is a way to achieve homeostasis through different metabolic pathways. Thus, male C57BL/6 mice were divided into the following groups: high-fat diet (HFD) vehicle, HFD + Supplemented, HFD + Supplemented_S, and isolated compounds. The vehicle and experimental formulations were administered orally by gavage once a day over the four weeks of the diet (28 consecutive days). We evaluated the energy homeostasis, cytokines, and mitochondrial gene expression in these groups of mice. After four weeks of supplementation, only the new nutraceutical group (HFD + Supplemented) experienced reduced fasting glycemia, insulin, HOMA index, HOMA-ß, dyslipidemia, ectopic fat deposition, and hepatic fibrosis levels. Additionally, the PPARγ coactivator 1 α (Pgc-1α), interleukin-6 (Il-6), and interleukin-10 (Il-10) gene expression were augmented, while hepatic steatosis decreased and liver parenchyma was recovered. The glutathione-S-transferase activity status was found to be modulated by the supplement. We discovered that the new nutraceutical was able to improve insulin resistance and hepatic steatosis mainly by regulating IL-6, IL-10, and Pgc-1α gene expression.

Endothelial dysfunction in rats with ligature-induced periodontitis: Participation of nitric oxide and cycloxygenase-2-derived products.

OBJECTIVES: Considering the evident relationship between periodontitis and cardiovascular diseases in humans, we aimed to study the in vitro vascular reactivity of aorta rings prepared from rats with ligature-induced periodontitis. METHODS: Seven days after the induction of unilateral periodontitis, the animals were euthanised; rings were prepared from the descending abdominal aortas and mounted in tissue baths for the in vitro measurement of the isometric force responses to norepinephrine (NE) and acetylcholine (ACh), as well as in the presence of inhibitors of nitric oxide synthase (NOS) and cycloxygenase (COX) isoenzymes. Aortic COX and NOS gene expressions were analysed by RT-PCR, as well as protein COX-2 expression by Western blot. RESULTS: Periodontitis resulted in significant alveolar bone loss and did not affect arterial pressure. However, both NE-induced contraction and ACh-induced relaxation were significantly decreased and related to the presence of endothelium. Diminished eNOS and augmented COX-2 and iNOS expressions were found in the aortas from rats with periodontitis, and the pharmacological inhibition of COX-2 or iNOS improved the observed vasomotor deficiencies. CONCLUSIONS: We can thus conclude that periodontitis induces significant endothelial dysfunction in rat aorta which is characterized by decreased eNOS expression and mediated by upregulated iNOS and COX-2 products.