Key pointsr Lymph transport is promoted by lymphatic pumping, a robust phasic contractile activity of the collecting lymphatic vessels. This contractile function, critical for tissue fluid homeostasis and immune cell transport to lymph nodes, is regulated by the amount of lymph entering the vessels and subsequent distension of the vessel wall.r While lymphatic pumping relies on influx of Ca 2+ through voltage-dependent Ca 2+ channels, characterization of these channels and details of their contribution to the regulation of stretch-activated contractions are lacking.r Here we report the expression of L-and T-type Ca 2+ channels in rat mesenteric lymphatic vessels and their differential role in regulating strength and frequency of lymphatic contractions.r This study fosters our knowledge on the mechanisms that drive stretch-activated lymphatic contractions. It may help in providing a basis to developing agents able to enhance lymphatic function, which could be of therapeutic benefit during lymphatic impairment such as lymphoedema.Abstract Lymph drainage maintains tissue fluid homeostasis and facilitates immune response. It is promoted by phasic contractions of collecting lymphatic vessels through which lymph is propelled back into the blood circulation. This rhythmic contractile activity (i.e. lymphatic pumping) increases in rate with increase in luminal pressure and relies on activation of nifedipine-sensitive voltage-dependent Ca 2+ channels (VDCCs). Despite their importance, these channels have not been characterized in lymphatic vessels. We used pressure-and wire-myography as well as intracellular microelectrode electrophysiology to characterize the pharmacological and electrophysiological properties of L-type and T-type VDCCs in rat mesenteric lymphatic vessels and evaluated their particular role in the regulation of lymphatic pumping by stretch. We complemented our study with PCR and confocal immunofluorescence imaging to investigate the expression and localization of these channels in lymphatic vessels. Our data suggest a delineating role of VDCCs in stretch-induced lymphatic vessel contractions, as the stretch-induced increase in force of lymphatic vessel contractions was significantly attenuated in the presence of L-type VDCC blockers nifedipine and diltiazem, while the stretch-induced increase in contraction frequency was significantly decreased by the T-type VDCC blockers mibefradil and nickel. The latter effect was correlated with a hyperpolarization. We propose that activation of T-type VDCCs depolarizes membrane potential, regulating the frequency of lymphatic contractions via opening of L-type VDCCs, which drive the strength of contractions.

Background and purpose: Rhythmical transient constrictions of the lymphatic vessels provide the means for efficient lymph drainage and interstitial tissue fluid balance. This activity is critical during inflammation, to avoid or limit oedema resulting from increased vascular permeability, mediated by the release of various inflammatory mediators. In this study, we investigated the mechanisms by which prostaglandin E2 (PGE2) and prostacyclin modulate lymphatic contractility in isolated guinea pig mesenteric lymphatic vessels. Experimental approach: Quantitative RT-PCR was used to assess the expression of mRNA for enzymes and receptors involved in the production and action of PGE2 and prostacyclin in mesenteric collecting lymphatic vessels. Frequency and amplitude of lymphatic vessel constriction were measured in the presence of these prostaglandins and the role of their respective EP and IP receptors assessed. Key results: Prostaglandin E2 and prostacyclin decreased concentration-dependently the frequency, without affecting the amplitude, of lymphatic constriction. Data obtained in the presence of the EP4 receptor antagonists, GW627368x (1 mM) and AH23848B (30 mM) and the IP receptor antagonist CAY10441 (0.1 mM) suggest that PGE2 predominantly activates EP4, whereas prostacyclin mainly stimulates IP receptors. Inhibition of responses to either prostaglandin with H89 (10 mM) or glibenclamide (1 mM) suggested a role for the activation of protein kinase A and ATP-sensitive K + channels. Conclusions and implications: Our findings characterized the inhibition of lymphatic pumping induced by PGE2 or prostacyclin in guinea pig mesenteric lymphatics. This action is likely to impair oedema resolution and to contribute to the pro-inflammatory actions of these prostaglandins.

Objective Mesenteric lymphatic vessels pumping, important to propel lymph and immune cells from the intestinal interstitium to the mesenteric lymph nodes, is compromised during intestinal inflammation. The objective of this study was to test the hypothesis that the pro-inflammatory cytokine tumor necrosis factor alpha (TNF-α), is a significant contributor to the inflammation-induced lymphatic contractile dysfunction, and to determine its mode of action. Methods Contractile parameters were obtained form isolated rat mesenteric lymphatic vessels mounted on a pressure myograph after 24-h incubation with or without TNF-α. Various inhibitors were administered and quantitative real-time PCR, western blotting and immunofluorescence confocal imaging were applied to characterize the mechanisms involved in TNF-α actions. Results Vessel contraction frequency was significantly decreased after TNF-α treatment and could be restored by selective inhibition of NF-кB, iNOS, guanylate cyclase and ATP-sensitive K+ channels. We further demonstrated that NF-кB inhibition also suppressed the significant increase in iNOS mRNA observed in TNF-α-treated lymphatic vessels and that TNF-α treatment favor the nuclear translocation of the p65 NF-κB subunit. Conclusions These findings suggest that TNF-α decreases mesenteric lymphatic contractility by activating the NF-κB - iNOS signaling pathway. This mechanism could contribute to the alteration of lymphatic pumping reported in intestinal inflammation.

Inflammatory bowel disease (IBD) has a complex pathophysiology with limited treatments. Structural and functional changes in the intestinal lymphatic system have been associated with the disease, with increased risk of IBD occurrence linked to a history of acute intestinal injury. To examine the potential role of the lymphatic system in inflammation recurrence, we evaluated morphological and functional changes in mouse mucosal and mesenteric lymphatic vessels, and within the mesenteric lymph nodes during acute ileitis caused by a 7-day treatment with dextran sodium sulfate (DSS). We monitored whether the changes persisted during a 14-day recovery period and determined their potential consequences on dendritic cell (DC) trafficking between the mucosa and lymphoid tissues. DSS administration was associated with marked lymphatic abnormalities and dysfunctions exemplified by lymphangiectasia and lymphangiogenesis in the ileal mucosa and mesentery, increased mesenteric lymphatic vessel leakage, and lymphadenopathy. Lymphangiogenesis and lymphadenopathy were still evident after recovery from intestinal inflammation and correlated with higher numbers of DCs in mucosal and lymphatic tissues. Specifically, a deficit in CD103 DCs observed during acute DSS in the lamina propria was reversed and further enhanced during recovery. We concluded that an acute intestinal insult caused alterations of the mesenteric lymphatic system, including lymphangiogenesis, which persisted after resolution of inflammation. These morphological and functional changes could compromise DC function and movement, increasing susceptibility to further gastrointestinal disease. Elucidation of the changes in mesenteric and intestinal lymphatic function should offer key insights for new therapeutic strategies in gastrointestinal disorders such as IBD. NEW & NOTEWORTHY Lymphatic integrity plays a critical role in small intestinal homeostasis. Acute intestinal insult in a mouse model of acute ileitis causes morphological and functional changes in mesenteric and intestinal lymphatic vessels. While some of the changes significantly regressed during inflammation resolution, others persisted, including lymphangiogenesis and altered dendritic cell function and movement, potentially increasing susceptibility to the recurrence of gastrointestinal inflammation.