High-speed lymphocyte tracking provided the spatial and temporal resolution to measure lymphocyte flux throughout the phasic contraction. Poiseuille flow was a reasonable model for estimating wall shear stress through most of the phasic contraction cycle of the intervalvular lymphatic segments. Shear rate was low but had large variations in magnitude compared to that seen in blood vessels.

Key Words: CCL21 Ⅲ ICAM-1 Ⅲ inflammation Ⅲ lymphedema Ⅲ in vitro Ⅲ overhydration I mmune functions of lymphatic endothelium include the transport of interstitial fluid to the lymph node, which provides constant sampling of peripheral antigens to antigenpresenting cells such as dendritic cells (DCs), macrophages, and B cells residing in the lymphatic endothelium, 1,2 and cell transport from the periphery to the lymph node. 3 These processes are important both for inducing an adaptive immune response as well as for maintaining tolerance to self-antigens. 4 Despite its importance, the active regulation of fluid and cell transport by lymphatic endothelium is largely unexplored. We asked how the lymphatic endothelium might sense and respond to its local physical environment to regulate these functions, particularly with respect to inflammation and tissue injury.Arguably, the first events in tissue injury and inflammation include the rapid release of mediators that increase the permeability of the local blood vessels, which leads to influx of plasma fluid and proteins into the interstitium, elevated interstitial fluid pressure, and increased lymph flow. [5][6][7][8] This is followed by the release of inflammatory cytokines such as tumor necrosis factor (TNF)-␣. Recently, several inflammatory cues including TNF-␣, TNF-␤, and interleukin-1, as well as pathogenic signals such as bacterial lipopolysaccharide (LPS), were shown to influence immune cell traffic into lymphatics by modulating lymphatic endothelial expression of intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule-1 (VCAM-1), and selectin (endothelial adhesion molecule 1 or CD62E), 9 -11 which are used by DCs for transmigration into lymphatics.Whereas the expression of such inflammatory cytokines following tissue injury can take several hours, 12 heightened lymph flow increases almost immediately. [5][6][7][8] We hypothesized that heightened transmural flow could act as an immediate cue for signaling injury or inflammatory conditions to lymphatic endothelium, driving changes in lymphatic endothelial transport functions to modulate fluid and DC trafficking to the draining lymph node. This could potentially act alone or in concert with inflammatory cytokines. Other recent evidence points to fluid shear stress as a modulator of nitric oxide release and lymphatic pump function in contractile lymphatics, 13,14 regulate leukocyte adhesion and transmigration events in blood endothelium. 15,16 However, the role of transmural flow on lymphatic endothelium and its transport functions has not been examined to date, and whereas leukocytes exiting blood vessels experience a high-shear environment before and during their transmigration, leukocytes entering lymphatics do not experience such conditions in the basal interstitium. Because lymphatic capillaries play a different role in inflammation than blood capillaries, their functional responses to environmental cues need to be separately investigated.Here, using both in vivo and in vitro systems, we d...

Lymphatic vasculature regulates fluid homeostasis by returning interstitial fluid to blood circulation. Lymphatic endothelial cells (LECs) are the building blocks of the entire lymphatic vasculature. LECs originate as a homogeneous population of cells predominantly from the embryonic veins and undergo stepwise morphogenesis to become the lymphatic capillaries, collecting vessels or valves. The molecular mechanisms underlying the morphogenesis of the lymphatic vasculature remain to be fully understood. Here we show that canonical Wnt/β-catenin signaling is necessary for lymphatic vascular morphogenesis. Lymphatic vascular-specific ablation of β-catenin in mice prevents the formation of lymphatic and lymphovenous valves. Additionally, lymphatic vessel patterning is defective in these mice, with abnormal recruitment of mural cells. We found that oscillatory shear stress (OSS), which promotes lymphatic vessel maturation, triggers Wnt/β-catenin signaling in LECs. In turn, Wnt/β-catenin signaling controls the expression of several molecules, including the lymphedema-associated transcription factor FOXC2. Importantly, FOXC2 completely rescues the lymphatic vessel patterning defects in mice lacking β-catenin. Thus, our work reveals that mechanical stimulation is a critical regulator of lymphatic vascular development via activation of Wnt/β-catenin signaling and, in turn, FOXC2.

It is well documented that cells secrete exosomes, which can transfer biomolecules that impact recipient cells’ functionality in a variety of physiologic and disease processes. The role of lymphatic drainage and transport of exosomes is as yet unknown, although the lymphatics play critical roles in immunity and exosomes are in the ideal size-range for lymphatic transport. Through in vivo near-infrared (NIR) imaging we have shown that exosomes are rapidly transported within minutes from the periphery to the lymph node by lymphatics. Using an in vitro model of lymphatic uptake, we have shown that lymphatic endothelial cells actively enhanced lymphatic uptake and transport of exosomes to the luminal side of the vessel. Furthermore, we have demonstrated a differential distribution of exosomes in the draining lymph nodes that is dependent on the lymphatic flow. Lastly, through endpoint analysis of cellular distribution of exosomes in the node, we identified macrophages and B-cells as key players in exosome uptake. Together these results suggest that exosome transfer by lymphatic flow from the periphery to the lymph node could provide a mechanism for rapid exchange of infection-specific information that precedes the arrival of migrating cells, thus priming the node for a more effective immune response.