Mechanobiology of lymphatic contractions.

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Contents lists available at ScienceDirect

Seminars in Cell & Developmental Biology journal homepage: www.elsevier.com/locate/semcdb

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Review

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Mechanobiology of lymphatic contractions Lance L. Munn ∗

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Q2 Department of Radiation Oncology, Massachusetts General Hospital, Boston, MA, United States

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Article history: Available online xxx

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Keywords: Lymphatic 13 Contraction 14 Mechanosensor 15 Mechanobiology 16 Stretch-activated channel Shear stress 17 18 Q4 Nitric oxide 11 12

The lymphatic system is responsible for controlling tissue fluid pressure by facilitating flow of lymph (i.e. the plasma and cells that enter the lymphatic system). Because lymph contains cells of the immune system, its transport is not only important for fluid homeostasis, but also immune function. Lymph drainage can occur via passive flow or active pumping, and much research has identified the key biochemical and mechanical factors that affect output. Although many studies and reviews have addressed how tissue properties and fluid mechanics (i.e. pressure gradients) affect lymph transport [1–3] there is less known about lymphatic mechanobiology. As opposed to passive mechanical properties, mechanobiology describes the active coupling of mechanical signals and biochemical pathways. Lymphatic vasomotion is the result of a fascinating system affected by mechanical forces exerted by the flowing lymph, including pressure-induced vessel stretch and flow-induced shear stresses. These forces can trigger or modulate biochemical pathways important for controlling the lymphatic contractions. Here, I review the current understanding of lymphatic vessel function, focusing on vessel mechanobiology, and summarize the prospects for a comprehensive understanding that integrates the mechanical and biomechanical control mechanisms in the lymphatic system. © 2015 Published by Elsevier Ltd.

Contents

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Lymphatic anatomy and network topology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lymphatic contractions: calcium dynamics, pacemaker potentials and mechanobiology of stretching . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Mechanical activation of contractions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lymphatic relaxation: endothelial derived relaxing factors and fluid shear stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Integrating lymphatic physiology using mathematical models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Lymphatic anatomy and network topology

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Blood microvasculature is somewhat permeable to water and 32 proteins, and a fraction of the circulating plasma extravasates 33 into tissue wherever there is a transmural pressure driving force. This can establish pressure gradients that extend from the blood 34 vessels to the lymphatic system, allowing for flow of plasma 35 36Q6 through the tissue, into the lymphatics. The process of flushing the extravascular space with plasma is likely useful for conditioning the 37 extracellular matrix and providing biomechanical signals to cells, 38 31Q5

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∗ Tel.: +1 6177264085. E-mail address: [email protected]

but it relies on proper pressure distribution in the tissue: if the pressure in the lymphatic bed is not lower than that in the surrounding tissue – at least transiently – then there is accumulation of pressure and fluid, resulting in edema. The lymphatic system is responsible for maintaining proper tissue-fluid balance [4] and organizing the immune response. Fluid from the tissue first enters blind-ending lymphatic capillaries, termed initial lymphatics (Fig. 1). The initial lymphatic walls are formed by overlapping endothelial cells that act as one-way valves to allow fluid into the vessels, but not out [5,6]. Initial lymphatics pass the fluid to collecting lymphatics, which constitute a system of variable-demand, distributed pumps [7]. Distinct compartments (lymphangions) within each collecting lymphatic vessel are defined by intraluminal luminal one-way valves [8–10]; lymphangions can

http://dx.doi.org/10.1016/j.semcdb.2015.01.010 1084-9521/© 2015 Published by Elsevier Ltd.

Please cite this article in press as: Munn LL. Mechanobiology of lymphatic contractions. Semin Cell Dev Biol (2015), http://dx.doi.org/10.1016/j.semcdb.2015.01.010

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Fig. 1. Lymphatic network topology and anatomy.

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drive flow via contractions of their muscle-invested walls. The collecting lymphatic vessels are arranged in a converging tree-like network, and lymph nodes are distributed throughout, so that lymph passes through them on the way back to the blood circulation [5]. However, active lymphatic pumping is not always operational, and deficient lymphatic pumping is involved in multiple clinical problems, including metabolic disorders [11], local immunocompromise [12,13], and lymphedema (fluid accumulation in tissue). The lymphatic system is also involved in cancer progression, as entry of metastatic cancer cells into the lymphatic system can result in lymph node metastases. Thus, the lymphatic system is central to a variety of pathological processes. A key question is how a connected network of pumps (lymphangions) can coordinate the contractions to move fluid efficiently back to the large veins. In the diverging arterial network of the cardiovascular system, flow is achieved by a single, central pump (the heart), and only relatively small (but important) local adjustments of diameters are able to achieve correct flow distribution to the capillaries. The inverse problem, which exists in the converging lymphatic network, is more difficult. One-way valves must be arranged appropriately, and individual contractions coordinated along vessels and at branch points so that the system does not “fight against itself”. Therefore, an overarching communication system might exist to provide the necessary coordination of the contractions. Although

many studies have delineated the various mechanical and chemical perturbations that affect phasic contractions, there are still questions about their role in organizing the contractions. 2. Lymphatic contractions: calcium dynamics, pacemaker potentials and mechanobiology of stretching To actively move fluid, the lymphangions have to contract, increasing local fluid pressure, which closes upstream – and opens downstream – valve. The contractions are driven by Ca2+ fluxes from extracellular and intracellular stores, which operate through myosin light chain kinase (MLCK) to allow binding of actin with the myosin light chain crossbridges, generating force [14–17]. To control lymphatic pumping on the larger scale, there needs to be synchronization of the calcium dynamics [18]. Coordination of the pumping should lead to a decrease in pressure pulses over the network, and indeed this is observed, especially with high lymph flow states. This implies that the active pumping is organized so that pressure pulses are smoothed as the lymph moves up the lymphatic tree [19]. Conceptually, there are many possible modes in which the contractions might synchronize. For example, in a series of lymphangions, all the odd numbered segments might contract at the same time that the even numbered segments are relaxing (Fig. 2A). This would serve to move the fluid effectively in “bucket brigade” style. Alternatively, two lymphangions might contract together, moving fluid downstream to the next pair of lymphangions,


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Fig. 2. Examples of lymphangion coordination. (A) Contractions might alternate in adjacent lymphangions. (B) It is also possible that two or more adjacent lymphangions contract and relax together. (C) If there is asymmetric contraction/relaxation in terms of the number of in-phase adjacent lymphangions, then the stroke volumes must not be the same (e.g., lymphangions 1 and 2 will have lower stroke volume than lymphangion 3).

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which are relaxing to accept the fluid (Fig. 2B). This scheme can be extended to even larger numbers of adjacent lymphangions in synchrony, with each ejecting and accepting equivalent volumes of fluid in a given cycle. But other modes are also conceivable. For example, two lymphangions might contract together, while only one of the downstream lymphangions dilates (Fig. 2C). This is possible only if the stroke volume of the relaxing compartment is larger than that of the individual contracting compartments, since it has to accept more fluid than if only a single upstream compartment were contracting. Although highly simplified, these examples illustrate the need for overarching control of the contractions, especially considering the additional complications at converging bifurcations, where the downstream branch accepts fluid from two upstream lymphangions. Thus, it is likely that some mechanism involving local feedback helps control the contractions and relaxations. Smooth muscle cell contractions are dependent on intracellular calcium, and the calcium concentrations are subject to multiple control mechanisms, summarized in Fig. 3. Ca2+ levels are normally low in the cytosol, but can undergo rapid changes due to influx through channels in the plasma membrane and sarcoplasmic reticulum. The contraction process starts as calcium fluctuations due to influx through channels are amplified to become spontaneous transient depolarizations (STDs). Similar to the Na+ /K+ system in nerve action potentials, it requires only a threshold level of Ca2+ in the cytosol to initiate the depolarization and cause a subsequent, larger calcium spike [20–23]. In the lymphatic muscle cells (LMCs), the Ca2+ influx appears to be through IP3 receptors [23], and STDs can be blocked by chelation of cytosolic Ca2+ [22]. These STDs can be modulated by activators or blockers of inositol 1,4,5-trisphosphate receptors (IP3Rs) or calcium-activated chloride channel (CaCC) blockers, so it is thought that Ca2+ release through IP3 receptors opens CaCC channels to generate STDs [23]. This process does not have to be present in all the muscle cells in the vessel wall, but can be driven by a subset of cells that act as pacemakers to initiate the contractions [24,25]. Firing of a pacemaker cell leads to propagation of the Ca2+ wave to adjacent cells, inducing a series of action potential-like spikes of calcium that cause the contractions. This mechanism of STDs is distinct from cardiac-like electrical pacemaker activity, which would require electrical control through nerve action potentials [22,25,26]. Certain lymphatic vessels have nerves in their walls that, when stimulated, induce an increase in pumping contraction frequency: experiments in bovine mesenteric vessels show that contraction is preceded by a single action potential [27], stimulated by the release of both ATP and noradrenaline

[28]. However, it remains to be seen how prevalent lymphatic innervation is or how such action potentials would be coordinated throughout the network. Although it is not clear how the calcium fluctuations progress to become STDs in an individual pacemaker cell, many biochemical pathways may be involved. Neurotransmitters such as noradrenaline, isoproterenol [28,29] and substance P [30,31] and inflammatory mediators such as histamine [13,17,20] have been shown to affect lymphatic contractions by affecting the calcium fluxes. Endothelial derived agents such as endothelin-1 (ET-1) can also enhance lymphatic vasomotion. ET-1 acts through IP3, directly affecting the IP3R-mediated release of Ca2+ [32]. 2.1. Mechanical activation of contractions Lymphatic vessel contractions are also influenced by mechanical signals. In the blood vasculature, resistance vessels respond to increased luminal pressure by constricting, a protective mechanism that prevents damage to the microvessels. The process is controlled by stretch-activation of calcium channels [33,34]. Lymphatic vessels have similar responses to increased pressure [35]. The effects are triggered by physical distension of the vessel wall, as they can be induced either by pressurizing the vessel or by stretching it with a mechanical device. For example, plasma dilution can be used to increase lymph production and the load on the lymphatic system. In response to plasma dilution, rat mesenteric collecting lymphatics respond with increased diastolic diameter, contraction frequency and amplitude, likely caused by intrinsic stretch-dependent mechanisms [19]. Because of the importance of wall stretch in lymphatic physiology, many groups have developed methodologies for studying vessel contractions under controlled conditions of preload and afterload [36–40]. In isolated mesenteric lymphatics, increasing luminal pressure without changing the axial pressure driving force increases both the frequency and force of the individual contractions [37,38] which are preceded by calcium transients [40]. This causes an increase in vessel output. However, at higher pressures, output decreases, likely due to pressure-limited stroke volume [39,41]. This autoregulation of contractions based on transmural pressure can increase lymph drainage in response to pressures caused by tissue edema or positional/gravitational changes. Interestingly, vessels isolated from various locations have different tension vs. length relationships. Applying isometric force to various rat lymphatic vessels (thoracic duct, cervical, and femoral lymph vessels), Gashev and coworkers [42] found similar tension


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Fig. 3. Control of calcium kinetics. Calcium enters the cytosol through ion channels (L-type, T-type, voltage gated, stretch-activated) in the plasma membrane or smooth endoplasmic reticulum (SER; SERCA, IP3 R) and acts through myosin light chain kinase (MLCK) to phosphorylate MLC, allowing formation of the myosin–actin crossbridges and cell contraction. Stretch-activated channels can also allow calcium to enter the cell. Calcium-activated chloride channels (CaCCs) can enhance depolarization during STD generation. Endothelial cells produce endothelial derived relaxing factors (EDRFs) such as histamine and NO in response to fluid shear. The EDRFs act on adjacent muscle cells through soluble guanylyl cyclase (sGC), cyclic GMP, protein kinase G (PKG) and myosin light chain phosphatase (MLCP) to reduce intracellular Ca2+ and can dephosphorylate MLC, causing relaxation.

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vs. length curves; all vessels generated near-maximal tensions over a relatively wide range of preloads, but there were differences in the highest pressure each vessel could withstand. This suggests that lymph vessels adapt their contractile force depending on their location in the lymphatic system. More evidence of distension-induced contractions has been documented using servo-controlled wire-myograph devices [43]. These devices allow isometric stretching of the vessel wall and measurement of the resulting contractions, independent of fluid pressure. Similar to increased fluid pressure, application of isometric stretch increases the amplitude and the frequency of phasic activity before declining at higher preloads [44], although the range of outputs differs significantly between the two methods [45]. In addition, the behavior is dependent on the rate of stress application: fast stretching induces bursts of higher frequency contractions [44]. In lymphatic smooth muscle cells, there are two types of voltage-dependent calcium channels (VDCCs) that appear to be important in regulating the phasic contractions: L-type (“longlasting”) and T-type (“transient) channels. It has recently been shown that these channels differentially regulate the strength and frequency of stretch-induced contractions. Nifedipine and diltiazem (which specifically block L-type channels) decreased the strength of contractions, while mibefradil and nickel (which block T-type channels) decreased contraction frequency [46]. Experiments such as described above suggest that the sensitivity of the calcium release mechanism is modulated by mechanical stresses. Our knowledge of mechanically activated channels lags behind that of voltage or ion-gated channels, but a number of candidates have been identified that could be involved in the lymphatic response to mechanical distension. Obvious candidates for mediating the stretch-induced changes in contractions are the stretch-activated ion channels (Fig. 4). Originally identified in nerve cells where they mediate processes such as sensation of touch, pain and hearing, stretch-activated channels

have received more attention recently in vasculature, where they are postulated to transduce fluid mechanical signals. It is known that endothelial [47–49] and smooth muscle cells [50,51] have stretch-activated ion channels that can initiate Ca2+ mobilization. These channels undergo conformational changes in response to membrane deformations. They have been implicated in the control of cell volume, mediating ion exchange as the channels are opened due to membrane stretch [52]. They are also involved in migration of epithelial cells, modulating local calcium fluxes at the cell trailing edge [53]. Stretch-activated channels can be identified and characterized using patch clamp studies in which the membrane is placed under

Fig. 4. Stretch activated calcium channels in the vessel wall change conformation in response to membrane tension, increasing calcium entry into the cell.


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tension by suction and the changes in ion flux measured. Much work on blood vessel endothelial cells [47,49] and smooth muscle cells [50,51] has concluded that calcium channels open more frequently under conditions of membrane stretch. Similar results are seen when endothelial cells are stretched on elastic substrates. Stretch allows Ca2+ entry from outside the cell, which then stimulates release of calcium from internal stores [48]. In endothelial cells, the increase in intracellular Ca2+ with stretch is dependent on Ca2+ in the external media and it can be blocked with gadolinium (Gd3+), which is specific for cation-selective stretch-activated channels [54]. In contrast, it is not affected by nifedipine, which blocks L-type voltage-gated calcium channels [48]. Although the identity of the mechanically responsive calcium channel in lymphatic vessels is not known, recent work has identified Piezo1 as an important stretch-sensitive channel in the endothelial cells of blood vessels. Piezo proteins transduce mechanical forces to cationic currents, and are evolutionarily conserved [55]. They can be blocked with the peptide GsMTx4 as well as gadolinium [56,57]. Also prominent in sensory neurons, Piezo1 proteins exist as subunits of Ca2+ -permeable channels, and are modulated by fluid forces [56]. They play an essential role in vascular development, as Piezo1-deficient endothelial cells have abnormal stress fibers and do not align with shear stress [58]. It remains to be seen whether Piezo proteins play a role in lymphatic contractions in response to stretch.

3. Lymphatic relaxation: endothelial derived relaxing factors and fluid shear stress In addition to the vasoactive agents that enhance lymphatic muscle contractions, there are mechanisms for damping the Ca2+ dynamics. The most well-studied is nitric oxide (NO), a vasodilator that acts at multiple points in the Ca2+ -contraction pathway. NO produced by the endothelium can quickly diffuse to the muscle cells to affect pumping [13,59–67]. It operates by modulating Ca2+ release and uptake [68], as well as the enzymes responsible for force production [69,70]. NO activates cytoplasmic guanylate cyclase in vascular smooth muscle to decrease vascular tone through cGMP-dependent mechanisms. NO decreases intracellular Ca2+ by inhibiting its entry from internal stores. This effects relaxation by decreasing the activity of myosin light chain kinase (Fig. 3). Specifically, NO can act through sGC and PKG to inhibit IP3 receptor channels, limiting Ca2+ influx. At the same time, it can activate SERCA and BKCa channels to increase Ca2+ outflux [59,67]. NO also directly induces relaxation by dismantling the myosin light chain crossbridges through PKG and myosin light chain phosphatase [71–73]. In general, elevated NO leads to dilated lymphatic vessels with decreased contraction frequency, consistent with the role of NO as a vasodilator able to blunt the Ca2+ response. Under normal conditions, the primary source of NO is the endothelium. Blood and lymphatic endothelial cells produce NO in response to fluid flow [63,67,74–77], and removal of the endothelium mimics reagents that block NO production [75]. Flow-induced NO is produced by eNOS in the endothelial cells [78], but during inflammation, NO can be produced by stromal cells via iNOS, independent of the endothelium. The resulting excess of NO can inhibit pumping [13], and iNOS-induced NO is also implicated the dysfunction of aging lymphatics [79]. Interestingly, it appears that NO is not the only endothelialderived relaxing factor (EDRF) important in lymphatic physiology. In isolated vessels, exposure to exogenous NO can reproduce the changes in pumping produced by flow, but blocking NO synthase does not completely abolish the effects of flow [80]. It has been shown that histamine may be another EDRF that compensates

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when NO is blocked [20]. In rat mesenteric vessels, histamine induces vessel relaxation through the H1 and H2 histamine receptors, and works in conjunction with NO through sGC to decrease tone and contraction frequency [81]. Only by blocking both NO and histamine can the relaxation be blocked [82]. It is appropriate that EDRFs are produced in response to fluid shear forces. They represent another mechanobiological feedback system that, together with stretch-activated calcium channels, can help control the phasic contractions. They provide a counterbalance to the calcium-induced contractions, maintaining vessel dilation in circumstances when low resistance is needed to allow pressuredriven flow [80]. Unfortunately, despite much research activity, relatively little is known about endothelial mechanobiology and the structures in the cells that transduce shear stress signals. Although channels such as Piezo1 might be involved, other structures such as adhesion molecules [83] and glycocalyx components [84,85] have been implicated in vessel mechanobiology. Furthermore, it is likely that cells in the lymphatic wall use distinct pathways to discriminate fluid shear stress and pressure-induced stretch, given the distinct responses to these stimuli. Thus, more research is needed to identify and characterize the mechanosensors involved in lymphatic physiology.

4. Integrating lymphatic physiology using mathematical models The complex behavior of the lymphatic system has led many groups to develop mathematical models to characterize and study its performance analytically. Some models have increased our understanding of the trade-off between active pumping and flow resistance and have explored various putative modes of pumping. Others have focused on basic properties of interstitial transport and lymphatic uptake. Lymph flow is not only driven by pumping collecting lymphatics, but also by pressure imbalances in the tissue. Swartz et al. used data from mouse tail lymphatics to numerically model the pressures and hydraulic conductivities that drive fluid into the lymphatics. They noted that the pressure driving forces are sensitive to tissue elasticity, which can decrease as tissue remodels during chronic edema [86]. More recent analyses have addressed the issue of initial lymphatic network topology, and how the shape of the network affects lymph drainage. Using homogenization theory, it is possible to show that most of the resistance to lymph flow occurs at the entrance to the initial lymphatics, and that the observed hexagonal arrangement of lymphatics in the mouse tail provides the optimum drainage in this tissue [87]. Some modeling efforts have addressed lymphatic pumping efficiency, valve function or vessel mechanics to quantify lymphatic performance. One category of models uses measured physical and functional properties of lymphatic vessels to simulate pumping. These models characterize the resulting performance of a single lymphatic vessel, represented by a number of contracting lymphangions in series. In general, input functions are imposed to drive flow or to cycle the contractions of individual lymphangions, and the output of the simulated vessel is calculated [88–90]. Such models can also be used to predict the conditions under which the system fails or to determine which parameters most strongly affect flow. For example, excessive back-pressure can prevent flow by causing valve leakage [91], and there is an optimum transmural pressure and number of lymphangion segments for a given length of vessel [92]. These models highlight the dual role of lymphatic vessels as conduits as well as pumps, and have shown that, in addition to contraction amplitude, the overall flow resistance through the vessel is a critical determinant of output [92]. Thus, the observed inhibition of contractions (which tend to increase flow resistance) by fluid flow is advantageous under conditions where


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pressure-driven flow is possible [93]. Key parameters in these models are the pressure-diameter relationship [94], the calcium force production (often a function of diameter) and the measured period of the contractions. When input into a fluid dynamics model, these can predict the lymph flow produced in a collecting lymphatic, and compare well with experimental results [95]. A more recent focus of experimental and modeling efforts has been the performance of the intraluminal valves during a contraction cycle. Rather than perfect one-way check valves, these structures allow some back flow, which appears to be dependent on many factors, including vessel diameter and axial pressure. Mathematical models that incorporate these subtleties of valve efficiency have been able to more accurately predict in vivo results [89]. As we have seen, lymphatic vessels can adjust their vasomotion to maintain fluid homeostasis. It is thought that they minimize contractions if existing fluid pressure gradients can drive flow, but actively pump to drive fluid otherwise [96]. This variable behavior has made it difficult to converge on a comprehensive, conceptual model of lymphatic function. Given that fluid shear stress and mechanical stretch play central roles in lymphatic function, it is likely that these vessels integrate physical signals to control lymph drainage. Indeed, in any control system, direct feedback between the quantity being controlled (fluid pressure and flow rate in this case) and the operation (lymphatic pumping) is needed. Furthermore, this control has to operate locally, but coordinate pumping over larger lengths of vessels. The nature of the feedback signals and the intramural communication are still areas of active investigation.

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5. Conclusions

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Mechanobiological control mechanisms can potentially explain the robust transport functions observed in the lymphatic system. They allow lymphatics to adapt to changes in the fluid environment to optimize lymph transport, automatically reacting to changes in tissue fluid pressures. It is interesting that many of the observed mechanism have analogs in the blood vasculature, but serve slightly different purposes in the lymphatic system. For example, EDRFs can relax both blood and lymphatic vessels, and they also react similarly to changes in pressure. Excess pressure in blood vessels can induce rapid constriction to limit flow to the microvasculature, and the same stretch-induced constriction appears to be one of the mechanical drivers of lymphatic phasic contractions. The periodic vasomotion of blood vessels is perhaps most closely related to lymphatic phasic contractions, but its purpose is not well-understood. Although not necessary for pumping blood, it may be involved in shuttling flow between capillary beds either randomly or in response to metabolic or thermal stimuli. The magnitudes of the fluid pressures and shear stresses vary significantly in the blood and lymphatic systems. For vessel stretch, the relevant quantity is the transwall pressure, which ranges from ∼150 mmHg in the aorta to ∼5–10 mmHg in the blood microvessels and collecting lymphatics [97,98]. However, mechanobiological mechanisms underlying the stretch response are sensitive to wall strain rather than fluid pressure. Furthermore, vessels with higher transwall pressures have more muscular walls, limiting the pressure-induced strain. Thus, it is possible that smooth muscle strains are similar throughout the blood and lymphatic systems. Shear stresses also vary somewhat throughout the blood vasculature, ranging from ∼10 to 20 dyn/cm2 in the large arteries and capillaries but increasing to as high as 100 dyn/cm2 in arterioles [99,100]. Lymphatic vessels lie at the low end of this range. Average shear rates in collecting lymphatic vessels have been reported as 0.64 ± 0.14 dyn/cm2 , with peaks of 4–12 dyn/cm2 [101], but can increase to 40 dyn/cm2 during edemagenic stress [102]. The large range of shear stresses in the blood circulation and the cyclic shear stress experienced by lymphatic endothelium during lymphatic

pumping raise the question of whether there is a floating threshold tuned to different levels in different vessels to determine EDRF production. It has also been proposed that it is not the absolute level of shear stress that determines endothelial mechanobiology, but the temporal changes in shear stress [103]. Thus, lymphatic endothelium may be more responsive by virtue of the dynamic shear stress during phasic contractions. The reliance of lymphatic pumping on mechanosensors provides additional opportunities for pharmacological intervention in lymphatic disorders. More research is needed to identify the mechanosensors that respond to stretch and shear stress in lymphatics so that drugs can be developed that specifically enhance or block these pathways. The hope would be to find targets that are specific to lymphatic mechanobiology with minimal toxicity to blood vessels and other tissues. Acknowledgment This work was funded in part by National Institutes of Health Q7 Grant NIH R01CA149285. References [1] Breslin JW. Mechanical forces and lymphatic transport. Microvasc Res 2014;96C:46–54. [2] Nipper ME, Dixon JB. Engineering the lymphatic system. Cardiovasc Eng Technol 2011;2:296–308. [3] Negrini D, Moriondo A. Lymphatic anatomy and biomechanics. J Physiol 2011;589:2927–34. [4] Thomas SN, Rutkowski JM, Pasquier M, Kuan EL, Alitalo K, Randolph GJ, et al. Impaired humoral immunity and tolerance in K14-VEGFR-3-Ig mice that lack dermal lymphatic drainage. J Immunol 2012;189:2181–90. [5] Mendoza E, Schmid-Schonbein GW. A model for mechanics of primary lymphatic valves. J Biomech Eng 2003;125:407–14. [6] Schmid-Schönbein GW. Microlymphatics and lymph flow. Physiol Rev 1990;70:987. [7] Roddie IC, Mawhinney HJ, McHale NG, Kirkpatrick CT, Thornbury K. Lymphatic motility. Lymphology 1980;13:166–72. [8] Gashev AA, Zawieja DC. Physiology of human lymphatic contractility: a historical perspective. Lymphology 2001;34:124–34. [9] Bazigou E, Wilson JT, Moore Jr JE. Primary and secondary lymphatic valve development: molecular, functional and mechanical insights. Microvasc Res Q8 2014. [10] Vittet D. Lymphatic collecting vessel maturation and valve morphogenesis. Microvasc Res 2014. [11] Zawieja SD, Wang W, Wu X, Nepiyushchikh ZV, Zawieja DC, Muthuchamy M. Impairments in the intrinsic contractility of mesenteric collecting lymphatics in a rat model of metabolic syndrome. Am J Physiol Heart Circ Physiol 2012;302:H643–53. [12] Elias RM, Johnston MG, Hayashi A, Nelson W. Decreased lymphatic pumping after intravenous endotoxin administration in sheep. Am J Physiol 1987;253:H1349–57. [13] Liao S, Cheng G, Conner DA, Huanga Y, Kucherlapati RS, Munn LL, et al. Impaired lymphatic contraction associated with immunosuppression. PNAS 2011;108:18784. [14] Ledvora RF, Barany M, Barany K. Myosin light chain phosphorylation and tension development in stretch-activated arterial smooth muscle. Clin Chem 1984;30:2063–8. [15] Nepiyushchikh ZV, Chakraborty S, Wang W, Davis MJ, Zawieja DC, Muthuchamy M. Differential effects of myosin light chain kinase inhibition on contractility, force development and myosin light chain 20 phosphorylation of rat cervical and thoracic duct lymphatics. J Physiol 2011;589:5415–29. [16] Wang W, Nepiyushchikh Z, Zawieja DC, Chakraborty S, Zawieja SD, Gashev AA, et al. Inhibition of myosin light chain phosphorylation decreases rat mesenteric lymphatic contractile activity. Am J Physiol Heart Circ Physiol 2009;297:H726–34. [17] Von Der Weid PY. Review article: lymphatic vessel pumping and inflammation – the role of spontaneous constrictions and underlying electrical pacemaker potentials. Aliment Pharmacol Ther 2001;15:1115–29. [18] McHale NG, Meharg MK. Co-ordination of pumping in isolated bovine lymphatic vessels. J Physiol 1992;450:503–12. [19] Benoit JN, Zawieja DC, Goodman AH, Granger HJ. Characterization of intact mesenteric lymphatic pump and its responsiveness to acute edemagenic stress. Am J Physiol 1989;257:H2059–69. [20] Fox JL, von der Weid PY. Effects of histamine on the contractile and electrical activity in isolated lymphatic vessels of the guinea-pig mesentery. Br J Pharmacol 2002;136:1210–8. [21] McHale NG, Allen JM, Iggulden HL. Mechanism of alpha-adrenergic excitation in bovine lymphatic smooth muscle. Am J Physiol 1987;252:H873–8.


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