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.

Given the known mechanosensitivity of the lymphatic vasculature, we sought to investigate the effects of dynamic wall shear stress (WSS) on collecting lymphatic vessels while controlling for transmural pressure. Using a previously developed ex vivo lymphatic perfusion system (ELPS) capable of independently controlling both transaxial pressure gradient and average transmural pressure on an isolated lymphatic vessel, we imposed a multitude of flow conditions on rat thoracic ducts, while controlling for transmural pressure and measuring diameter changes. By gradually increasing the imposed flow through a vessel, we determined the WSS at which the vessel first shows sign of contraction inhibition, defining this point as the shear stress sensitivity of the vessel. The shear stress threshold that triggered a contractile response was significantly greater at a transmural pressure of 5 cmH2O (0.97 dyne/cm(2)) than at 3 cmH2O (0.64 dyne/cm(2)). While contraction frequency was reduced when a steady WSS was applied, this inhibition was reversed when the applied WSS oscillated, even though the mean wall shear stresses between the conditions were not significantly different. When the applied oscillatory WSS was large enough, flow itself synchronized the lymphatic contractions to the exact frequency of the applied waveform. Both transmural pressure and the rate of change of WSS have significant impacts on the contractile response of lymphatic vessels to flow. Specifically, time-varying shear stress can alter the inhibition of phasic contraction frequency and even coordinate contractions, providing evidence that dynamic shear could play an important role in the contractile function of collecting lymphatic vessels.

Lymphedema, a disfiguring condition characterized by an asymmetrical swelling of the limbs, is suspected to be caused by dysfunctions in the lymphatic system. A possible source of lymphatic dysfunction is the reduced mechanosensitivity of lymphangions, the spontaneously contracting units of the lymphatic system. In this study, the entrainment of lymphangions to an oscillatory wall shear stress (OWSS) is characterized in rat thoracic ducts in relation to their shear sensitivity. The critical shear stress above which the thoracic ducts show a substantial inhibition of contraction was found to be significantly negatively correlated to the diameter of the lymphangion. The entrainment of the lymphangion to an applied OWSS was found to be significantly dependent on the difference between the applied frequency and the intrinsic frequency of contraction of the lymphangion. The strength of the entrainment was also positively correlated to the applied shear stress when the applied shear was less than the critical shear stress of the vessel. The ejection fraction and fractional pump flow were also affected by the difference between the frequency of the applied OWSS and the vessel's intrinsic contraction frequency. The results suggest an adaptation of the lymphangion contractility to the existing oscillatory shear stress as a function of its intrinsic contractility and shear sensitivity. These adaptations might be crucial to ensure synchronized contraction of lymphangions through mechanosensitive means and might help explain the lymphatic dysfunctions that result from impaired mechanosensitivity.

Key points We present the first in vivo evidence that lymphatic contraction can entrain with an external oscillatory mechanical stimulus. Lymphatic injury can alter collecting lymphatic contractility, but not much is known about how its mechanosensitivity to external pressure is affected, which is crucial given the current pressure application methods for treating lymphoedema. We show that oscillatory pressure waves (OPW), akin to intermittent pneumatic compression (IPC) therapy, optimally entrain lymphatic contractility and modulate function depending on the frequency and propagation speed of the OPW. We show that the OPW‐induced entrainment and contractile function in the intact collecting lymphatics are enhanced 28 days after a contralateral lymphatic ligation surgery. The results show that IPC efficacy can be improved through proper selection of OPW parameters, and that collecting lymphatics adapt their function and mechanosensitivity after a contralateral injury, switching their behaviour to a pump‐like configuration that may be more suited to the altered microenvironment. Abstract Intermittent pneumatic compression (IPC) is commonly used to control the swelling due to lymphoedema, possibly modulating the collecting lymphatic function. Lymphoedema causes lymphatic contractile dysfunction, but the consequent alterations in the mechanosensitivity of lymphatics to IPC is not known. In the present work, the spatiotemporally varying oscillatory pressure waves (OPW) generated during IPC were simulated to study the modulation of lymphatic function by OPW under physiological and pathological conditions. OPW with three temporal frequencies and three propagation speeds were applied to rat tail collecting lymphatics. The entrainment of the lymphatics to OPW was significantly higher at a frequency of 0.05 Hz compared with 0.1 Hz and 0.2 Hz (P = 0.0054 and P = 0.014, respectively), but did not depend on the OPW propagation speed. Lymphatic function was significantly higher at a frequency of 0.05 Hz and propagation speed of 2.55 mm/s (P = 0.015). Exogenous nitric oxide was not found to alter OPW‐induced entrainment. A contralateral lymphatic ligation surgery was performed to simulate partial lymphatic injury in rat tails. The intact vessels showed a significant increase in entrainment to OPW, 28 days after ligation (compared with sham) (P = 0.016), with a similar increase in lymphatic transport function (P = 0.0029). The results suggest an enhanced mechanosensitivity of the lymphatics, along with a transition to a pump‐like behaviour, in response to a lymphatic injury. These results enhance our fundamental understanding of how lymphatic mechanosensitivity assists the coordination of lymphatic contractility and how this might be leveraged in IPC therapy.

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Clinical manifestations of severe COVID-19 include coagulopathies that are exacerbated by the formation of neutrophil extracellular traps (NETs). Here, we report that pulmonary lymphatic vessels, which traffic neutrophils and other immune cells to the lung-draining lymph node (LDLN), can also be blocked by fibrin clots in severe COVID-19. Immunostained tissue sections from COVID-19 decedents revealed widespread lymphatic clotting not only in the lung, but notably in the LDLN, where the extent of clotting correlated with the presence of abnormal, regressed, or missing germinal centers. it strongly correlated with the presence of intralymphatic NETs. In mice, TNFα induced intralymphatic fibrin clots, and this could be inhibited by DNAse 1, which degrades NETs. In vitro, TNF induced lymphatic endothelial cell upregulation of ICAM-1 and CXCL8 among other neutrophil-recruiting factors as well as thrombomodulin downregulation. Furthermore, in decedents, lymphatic clotting in LDLNs. In a separate cohort of hospitalized patients, serum levels of MPO-DNA (a NET marker) inversely correlated with antiviral antibody titers, but D-dimer levels, indicative of blood thrombosis, did not correlate with either. In fact, patients with high MPO-DNA but low D-dimer levels generated poor anti-viral antibody titers. This study introduces lymphatic coagulation in lungs and LDLNs as a clinical manifestation of severe COVID-19 and suggests the involvement of NETosis of lymphatic-trafficking neutrophils. It further suggests that lymphatic clotting may correlate with impaired formation or maintenance of germinal centers necessary for robust antiviral antibody responses, although further studies are needed to determine whether and how lymphatic coagulation impacts adaptive immune responses.

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The lymphatic system has been proposed to play a crucial role in preventing the development and progression of osteoarthritis (OA). As OA develops and progresses, inflammatory cytokines and degradation by-products of joint tissues build up in the synovial fluid (SF) providing a feedback system to exacerbate disease. The lymphatic system plays a critical role in resolving inflammation and maintaining joint homeostasis, however, there is evidence that the lymphatics can become dysfunctional during OA. We hypothesized that the functional mechanics of lymphatic vessels (LVs) draining the joint could be directly compromised due to factors within SF derived from OA patients (OASF). Here, we utilized OASF and SF derived from healthy individuals (HSF) to investigate effects of SF entering the draining lymph on migration of lymphatic endothelial cells (LECs) in vitro, and lymphatic contractility of rat femoral LVs (RFLVs) ex vivo. Dilutions of OASF and HSF led to an increased migratory response in vitro compared to LECs treatment with media without serum. Ex vivo, OASF and HSF treatments were administered within the lumen of isolated LVs under controlled pressures. OASF treatment transiently enhanced the RFLVs tonic contractions while phasic contractions were significantly reduced after 1 hr of treatment and complete ceased after overnight treatment. HSF treatment on the other hand displayed a gradual decrease in lymphatic contractility (both tonic and phasic contractions). The observed variations after SF treatments suggest the pump function of LV draining the joint could be directly compromised in OA and thus might present a new therapeutic target.