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The lymphatic system has important roles in body fluid regulation, macromolecular homeostasis, lipid absorption, and immune function. To accomplish these roles, lymphatics must move fluid and its other contents (macromolecules, lipids/chylomicra, immune cells) from the interstitium through the lymphatics, across the nodes, and into the great veins. Thus, the principal task of the lymphatic vascular system is transport. The body must impart energy to the lymph via pumping mechanisms to propel it along the lymphatic network and use pumps and valves to generate lymph flow and prevent its backflow. The lymphatic system utilizes both extrinsic pumps, which rely on the cyclical compression and expansion of lymphatics by surrounding tissue forces, and intrinsic pumps, which rely on the intrinsic rapid/phasic contractions of lymphatic muscle. The intrinsic lymph pump function can be modulated by neural, humoral, and physical factors. Generally, increased lymph pressure/stretch of the muscular lymphatics activates the intrinsic lymph pump, while increased lymph flow/shear in the muscular lymphatics can either activate or inhibit the intrinsic lymph pump depending on the pattern and magnitude of the flow. To regulate lymph transport, lymphatic pumping and resistance must be controlled. A better understanding of these mechanisms could provide the basis for the development of better diagnostic and treatment modalities for lymphatic dysfunction.
The lymphatic system moves fluid from the interstitial spaces in the tissue parenchyma into the network of lymphatic vessels, through a series of lymph nodes into the postnodal lymph ducts that converge into the thoracic duct (for the lower half and upper left quadrant of the body) and right lymphatic duct (for the upper right quadrant of the body) before eventually emptying their lymph into the great veins. The unidirectional movement of lymph through this network is necessary for the transport of fluid, macromolecules, lipids, antigens, immune cells, and particulate matter. Thus, all of the important functions that the lymphatic system must accomplish to maintain body homeostasis depend on the controlled transport of lymph from the initial lymphatics to the great veins. Over the last 10 years, great strides have been made in the molecular and cellular processes that drive the formation and/or regeneration of the lymphatic vessels. This has greatly advanced our understanding of the developmental and remodeling processes that govern some of the structural considerations of the lymphatic system, particularly those in the initial lymphatic vessels where lymph is formed. Much less recent effort has been placed into the study of the lymphatic structures at any level beyond the most peripheral parts of the lymphatic network.
An understanding of the structure and function of the lymphatic architecture must go hand in hand if we are to develop a true appreciation of the impact of the lymphatic system in health and disease. When evaluating the lymphatic system it is crucial to remember that its principal purpose is the transport of lymph and it is by this regulated transport that ALL of the body's homeostatic functions that the lymphatic system participates in are served. Lastly but importantly, lymph transport includes not only the initial formation of lymph in the lymphatic capillaries but also the movement of lymph along the rest of the lymphatic network on its route to the veins.
Under steady states, most interstitial fluid pressures are either near atmospheric (i.e., near zero cm H2O relative pressure) or subatmospheric (−1 to −5cm H2O, i.e., negative relative pressures).1 Because of the great difficulty in performing accurate measures of pressure in the initial and early collecting lymphatics, there are not many measurements of pressures in the initial lymphatics. There are many more measures of pressures in the immediate downstream components of the lymphatic vascular network, the early lymphatic collecting vessels. However, in most instances the pressures measured in these vessels are on average slightly positive.2–7 Thus it would appear that the predominant fluid pressure gradient is one that does NOT favor fluid entering the initial lymphatic. So how does fluid enter the initial lymphatic structure to form lymph? There have been a few theories that have come about over the years to account for this process, but the data to support most of these theories have not been strong. One longstanding theory with good data to support it is that although the average prevailing pressure gradient between the interstitial fluid and the initial lymph opposes fluid entering the lymphatic, there are cyclical changes in both the interstitial fluid pressure and the lymph pressure in the initial lymphatic because of activities in either the tissue in which the lymphatic exists or the lymphatic itself that will produce a pressure gradient that transiently favors the formation of lymph.8–13
Anatomically, the initial lymphatics are composed of a layer of endothelial cells that have apparent transient gaps between parts of the adjacent endothelial cells. The endothelium of the initial lymphatics are unique in that they have valves not only across their lumen between the initial lymphatic bulb and the early collecting lymphatic plexus but also within in the walls of the initial lymphatics at these transient “gap sites” between adjacent lymphatic endothelial cells. Another unique characteristic is that the lymphatic endothelium is physically tethered into the surrounding tissue structure through the anchoring filaments.14–16 These unique structural characteristics coupled with the changing physical conditions in active tissues allow lymph to be formed when interstitial pressure is higher than the lymph pressure in the initial lymphatic. This opens the initial lymphatic valves and fluid enters the initial lymphatics through these valves.17–19
As the pressure profile across the initial lymphatic wall changes because of either tissue or lymphatic activities, to one that opposes lymph formation, these initial lymphatic valves close to prevent the movement of fluid back out of the initial lymphatic. Thus one gets transient lymph formation as a result of activity that drives the pressure gradient across the lymphatic wall towards one that favors lymph formation. Activities known to be important to this process are cyclical tissue deformations such as skeletal muscle contraction, heart contraction, gastrointestinal muscle contraction, breathing, and intrinsic contraction of the initial lymphatic. These same activities are also known to be responsible for creating local pressure gradients along the early lymphatic network that move fluid out of the initial lymphatic into the collecting vessels.10,11,13,20 Importantly, when either the intrinsic lymphatic contraction or the extrinsic tissue deformation enters a phase of relaxation or decompression, the initial lymphatic structure is “pulled open” because of the transmission of stored tissue energy through the anchoring filaments to the initial lymphatic wall. This can once again create a situation where the pressure gradients between the interstitium, the initial lymphatic, and the downstream collecting lymphatics transiently favor lymph formation.8–11
An analogous process is repeated along the lymphatic tree, moving lymph through the lymphatic network. However, since again the steady state fluid pressures along the lymphatic network are not conducive to the passive movement of fluid down a standing pressure gradient, as was shown decades ago in dogs (Table 1).21 The average lymph pressures in the more peripheral parts of the lymphatic network have lower pressures than do the final outflow tracts (thoracic duct and right lymphatic duct) of the lymphatic network and the average lymph pressures in the final outflow tracts are lower than the venous circulation into which they empty. Confounding this problem in animals that spend a significant part of their life in the upright position is the influence of gravitational forces on the lymph pressures.22,23 In the average height, upright human, there is a potential hydrostatic pressure gradient of −150cm H2O from the feet to the great veins of the neck. However, because this hydrostatic column of fluid in the lymphatic network is broken by the presence of the valves and nodal structures, the effective hydrostatic pressure gradient that opposes net central lymph flow is much less. Thus, lymph normally does not “drain” down the lymphatic network passively as is commonly described. Instead energy must be imparted to the lymph fluid in some fashion in order to transport it along the lymphatic network. In lower vertebrates, the lymphatic system utilizes a number of specialized “lymphatic hearts” situated in numerous locations within their bodies to drive lymph flow. Since humans and other mammals do not have the specialized lymphatic hearts that lower vertebrates do, we use a series of pumps and valves to overcome these prevailing pressure gradients and move lymph along the lymphatic network, through the nodes on to the great veins in the neck. Without the actions of these lymphatic pumps and valves, the proper function of the lymphatic system (i.e., transport) cannot occur. Consequently, to study the function/dysfunction of the lymphatic system, one must always consider the functions of the pumps and valves.
Once again, because the typical pressure gradients oppose central flow along the lymphatic network, to keep flow in the right direction, unidirectional valves are present.24–31 These important structures are a hallmark of the lymphatic network and their proper function depends upon some unique properties of the lymphatic.24,26,29,30,32,33 The function of the lymphatic valves is thought to be driven by the pressures and flow of fluid across them, given their unique structure. However, there is also some evidence for a possible role of specialized lymphatic muscle in the closure of these structures in some lymphatics.34 The critical role of these unique valves in lymph transport is highlighted in the development of lymphedema seen as a result of genetic mutations of the FOXC2 gene in mice and humans. FOXC2 is a genetic determinant of valves and loss of its normal function leads to lymphatic valvular failure and the development of lymphedema.35–37
The motion of a secondary lymphatic valve in an isolated rat mesenteric lymphatic throughout the intrinsic lymphatic contraction cycle can be seen in Fig. 1 and the supplemental video. The images show the vessel at the site of a lymphatic valve with the input (more peripheral section) oriented at the top of the images, the valve structure cut across the two leaflets (left and right highlighted in black) and the next downstream lymphatic sinus. Panels A–D depict the changes in the lymphatic structure due to the constriction of the lymphatic muscle during lymphatic pumping over an approximate 3-sec interval. As seen in panel A, the lymphatic is at rest (lymphatic diastole), the diameter of the lymphatic is ~105um at the arrow and the upper sections of the valve leaflets are sharply angled downward; the leaflets are partially open. Panel B is 1.1sec later and demonstrate the strong, rapid phasic contraction (lymphatic systole); the diameter at the arrow is now ~60μm and the valve leaflets are still sharply angled downward and are open. Panel C is 0.78sec later near the end of lymphatic systole; the diameter is similar in size to that in Panel B but the upper segments of the valve leaflets are “ballooned backwards” effectively closing the valve. Panel D represents the lymphatic 1.0sec later when the lymphatic is once again in lymphatic diastole and the valve leaflets are reopening.
A closer look at the lymphatic valve structure can be seen in Figure 2 and the supplemental video. These images were obtained in an isolated pressurized rat mesenteric lymphatic that was loaded with the intravital cellular fluorescent dye Cell Tracker Green. This dye will load into the cytoplasm of all living cells within the tissue. It was then imaged at a transmural pressure of 3cm H2O in calcium-free solution to stop the phasic contractions with a confocal/multiphoton microscope three-dimensionally at ~0.2μm intervals in the z axis and reconstructed in different orientations depicted in Panels A–D to display lymphatic structure. Panel A shows the vessel from the side with the more peripheral (input end) to the right. Panel B shows the lymphatic segment rotated 90 degrees with the input end facing the viewer; note the smooth transition to the lymphatic endothelial lined valve leaflets and the valvular opening into the sinus segment of the next lymphangion. The difference in cross-sectional areas of the end of the input segment, the outlet of the valve leaflets, and the beginning of the next lymphangion sinus is very dramatic (~ a 6-fold difference) and it is likely to play an important role in the hydrodynamics along the vessel. Panels C and D depict the segment rotated so as to show the opening of the output (next lymphangion sinus) and the backside of the valve leaflets also covered by lymphatic endothelial cells. Note the shape and structure of the valve leaflets with the slicker thicker trailing edges of the arch-shaped leaflets coming together to insert into the lymphatic wall (highlighted in the white rectangles). It is not known whether the cells that connect the insertion points of the valve leaflets into the muscular lymphatic wall are muscle or endothelial cells. The backside of the lymphatic leaflets appear to be the parts of the structure that bulge inward to oppose each other and form the seal of the valve at the end of the intrinsic lymphatic systole. In summary, the lymphatic valves are a hallmark of all lymphatic vessels that serve a number of important functions: They minimize lymph backflow when the pressure gradients are not conducive to central lymph flow. They help reduce the gravitational influence on lymph pressure by breaking up the hydrostatic lymph column. They also allow the sequential buildup (lymphangion by lymphangion) of lymph pressure to help overcome any opposing pressure gradients in a stepwise fashion. Lastly, they may be an anatomical site of fluid shear-sensation and therefore could play important roles in the production nitric oxide and the regulation of lymphatic contractions.
The lymphatic system uses lymph pumps (extrinsic and intrinsic) to provide the energy necessary to overcome the steady state opposing pressure gradients and propel lymph along the lymphatic network.6,23,25,38–43 Different motive forces, in conjunction with lymphatic valves, move lymph centrally. These motive forces can be categorized into two types dependent upon their source of energy; 1) the “intrinsic” lymph pump relies on the intrinsic rapid/phasic contractions of the lymphatic muscle to generate the needed forces, and 2) the “extrinsic” lymph pump that relies on the cyclical compression and expansion of lymphatics by surrounding extrinsic tissue forces to generate the pressures gradients to move lymph centrally. The extrinsic pumps are thought to predominate in the lymphatics of the heart, skeletal muscle, thorax, and the gut wall, while the intrinsic pumps are essential for lymph flow in most other lymphatic beds.
The collecting, transport, and some initial lymphatics possess layers of smooth muscle cells in their outer walls to generate and control the movement of lymph along the lymphatic network. The functional units within the muscular lymphatic vessels, called lymphangions, are arranged in series and separated by highly competent valves.40,44 This muscle layer is responsible for the regulation of lymphatic diameter and thus its compliance and resistance to generate and control lymph flow. Lymphatics that are not in a tissue that undergoes regular periodic changes in tissue pressure typically demonstrate phasic pumping contractions that generate the pressures that transiently drive lymph flow through the lymphatic network. Therefore, to regulate lymph transport function, the lymphatic contractions that drive pumping (strong, fast, brief contractions) and that alter flow-resistance (moderate, slow, long-lasting contractions) must both be controlled. Since in essence the lymphatics must work as both pumps and conduits, they have characteristics of both hearts and blood vessels.45–48 To achieve these divergent functions, local, neural, and humoral factors can modulate flow by altering the outflow resistance via tonic contraction/relaxation of the lymphatic muscle. Neural and humoral agents such as a-adrenergic agonists, prostanoids, natriuretic factors, bradykinin, substance P, and others modulate lymphatic tone, flow resistance, and thus lymphatic function.4,49–73 In addition, local physical factors such as stretch/pressure and shear/flow can also modulate lymphatic tone and function. It has been shown that lymphatics possess myogenic activity and that tonic contraction strength is modulated by stretch both in its magnitude and its temporal pattern.6,25,27,39,74–76 In all the lymphatic vessels we have studied in the rat, an increase in transmural pressure (i.e., increased stretch) results in a decrease in the strength of the tonic contraction when compared to the passive diameter.47
The intrinsic lymph pump generates lymph flow via the coordinated rapid strong contractions fashions and of lymphatic muscle cells.40,75,77–80 This type of contraction results in a rapid reduction of the lymphatic diameter, a decrease in lymphatic compliance, an increase in the local lymph pressure, closure of the upstream valve, opening of the downstream valve, and the ejection of lymph into the next downstream lymphangion. The phasic contractions are initiated by electrical pacemaker activity. While it is not yet clear exactly what type of cell the pacemaker is, it is located within the muscle layer of the lymphatic wall.81–88 While the electrophysiological properties of lymphatic muscle are key cellular regulators of the lymphatic contractile function, that subject is not the focus of this report but has recently been reviewed elsewhere.89 The intrinsic lymph pump can be analyzed using a cardiac cycle analogy42 where the phasic contractile cycle is divided into lymphatic diastolic and systolic periods. Lymphatic pumping function can then be evaluated using the phasic contraction frequency, ejection fraction, stroke volume, and lymph pump flow as can be seen in the lymphatic diameter tracing from a rat mesenteric lymphatic in Figure 3.7,42 Extending the cardiac pump analogy, the intrinsic lymph pump can be modulated via inotropic (i.e., changes in the strength of contraction) and/or chronotropic (i.e., mediated by changes in the contraction frequency) fashions and physical, neural, and humoral influences.4,6,25,39,50,56,63,65,67,90–98
Our laboratory group has focused much of our efforts analyzing the physical effects of stretch and shear on the intrinsic lymph pump and other contractile mechanisms. Elevated lymph pressure acting via an increase in the stretch of the lymphatic vessel is a classic activator of the lymph pump. Stretch of the lymphatic increases the lymphatic contraction frequency and initially increases the phasic contraction strength.6,25,44 However, further increases in pressure/stretch eventually produces a fall in the phasic contraction strength (inotropy), presumably as the ability to increase active contractile force begins to fail. This effect on inotropy is differentially expressed in lymphatics from different tissues47 and presumably is dependent on the typical tissue characteristics in that species and region. Although lymphatics from different species and tissues will achieve their maximum ability for intrinsic pumping at somewhat different pressures, they are all comparatively low, between 3 and 20cm H2O. The influence of pressure/stretch on the intrinsic lymph pump in rat mesenteric lymphatics, a more upstream peripheral lymphatic and rat thoracic duct, the final outflow tract of the lymphatic network, can be seen in Fig. 4 and the supplemental videos. For the rat thoracic duct, the peak intrinsic pump productivity was observed at a transmural pressure of ~3cm H2O, with no significant differences in pumping over a range of transmural pressures of 2–4cm. Transmural pressures beyond ~2–4cm H2O produced a weakening of the intrinsic pump. For the rat mesenteric lymphatics, maximum pumping occurred at a pressure of 5cm H2O, with no significant differences in pumping over the range of transmural pressures of 2–7cm. Inotropy of the rat mesenteric lymph pump declined at transmural pressures greater than 5–7cm H2O. These data indicate that optimal pumping in more peripheral lymphatics occurs at somewhat higher transmural pressures, presumably a reflection of the greater outflow resistance against which they must pump. For example, in these two lymphatics, the highest fractional pump flow was seen in the mesenteric lymphatics (6–8 volumes/min) at the optimal pressure levels and the lowest fractional pump flow (~2 volumes/min) was found in the thoracic duct.
Somewhat less well-studied in lymphatics are the effects of the other dominant physical factor—flow/shear. The magnitude and pattern of lymph flow is the result of numerous factors, lymph formation, extrinsic and intrinsic pumping, etc. Lymph flow will create shear forces that act on the lymphatic wall. These shear forces may alter lymphatic contractility in a fashion similar to those well-defined actions in blood vessels.99–101 Isolated bovine mesenteric lymphatics exposed to an imposed axial pressure gradient exhibited an inhibition of the intrinsic phasic lymph pump even at the low axial pressure gradients of ~3cm H2O, with further increases in the axial pressure gradient producing a complete inhibition of active pumping.102 However, the transmural pressure was not maintained constant in these experiments as the axial positive was increased, confounding the action of the physical forces. We have shown that increased lymph flow/shear in the muscular lymphatics can modulate intrinsic lymphatic pumping in a more controlled experimental protocol.91 We used isolated and perfused lymphatics from different regions of the body from the rat. In these experiments, we altered the imposed axial flow while maintaining a constant transmural pressure by changing the input and output pressures simultaneously to the same degree but in opposite directions. The imposed flow produced a strong inhibition of the active lymph pump in mesenteric lymphatics and especially the thoracic duct.91 These effects are shown in Table 2 and the accompanying supplemental videos. Shear due to the imposed flow gradient caused a loss of basal tonic contraction strength, reductions in the contraction frequency, amplitude of the phasic lymphatic contractions, ejection fraction, and fractional pump flow. The inhibition in the intrinsic pumping by imposed flow was significantly greater in the thoracic duct when compared to the mesenteric lymphatic. In the thoracic duct, an imposed flow gradient of 3–5cm H2O caused essentially complete cessation of the pumping activity. We hypothesize that this enhanced shear-sensitivity of the thoracic duct is due to its location in the lymphatic network—the final outflow path, where other pumps upstream (intrinsic and extrinsic) can drive lymph flow. We know that the inhibition of the intrinsic lymph pump by a relatively high steady state flow is dependent on the lymphatic endothelium, since removal of the endothelium eliminates this effect. We have also shown that this effect in these tissues is predominantly due to the production of nitric oxide (NO) since blockade of NO synthase (NOS) almost completely blocked this effect.91
However, one complication in these experiments was the issue of what level of imposed flow/shear should be used in these isolated vessel? Since at the time there were no measures of the lymph flow in these vessels in situ, we had to estimate the pressure gradient to apply based on our previous measures of the lymph pressure gradients associated with the intrinsic contractions. Later we developed and implemented methods whereby we could measure lymph flow in situ in the rat mesenteric lymphatics using hi-speed video lymphocyte-tracking techniques.103–105 By measuring lymphocyte velocity and lymphatic diameter throughout the lymphatic contractile cycle, we could better estimate the flow and shear profiles that these lymphatics are exposed to under normal and pathophysiological conditions. An example of the lymphocyte velocity and diameter profiles seen during intrinsic lymph pumping in situ in the rat mesenteric lymphatics can be seen in Fig. 5. The mean diameter of these lymphatics was ~105μm with phasic contractions amplitude of ~30%–40%. The lymphocyte density varied ~300–35,000cells/μL with a flux ~100–8000cells/minute. Based on simple Poiseuille flow models, the average lymph velocity was ~0.9mm/s with transient peaks up to 10-fold higher 2–9mm/s. The lymph velocity is ~180° out of phase with the phasic diameter changes with periods of flow reversal preceding each valve closure. The average calculated lymph flow is ~14μL/hour with average lymph shear stress of ~0.4–0.6dynes/cm2 and with shear stress peaks of 3–10dynes/cm2. Overall, this is a much more complex flow/shear pattern than what is typically exhibited in similar sized arterioles, with the lymphatic having greatly reduced averaged flow/shear but with much greater degree of change in flow/shear both in its magnitude and direction. Comparing these measured in situ values to what one would expect using the imposed axial pressure gradients in the isolated lymphatic experiments gives very similar peak velocities.104 However, one significant difference in the flow generated by an imposed axial pressure gradient in the isolated lymphatic experiments is that it is a much more of a steady flow/shear profile when compared to those measured in situ. How these flow/shear-induced effects may change when the magnitude and profile of flow/shear is altered is the focus of our current studies.
Additionally, the direction and magnitude of the pump modulation is dependent on the pattern and magnitude of the flow/shear as well as the sensitivity of the particular lymphatic region. We conducted a series of studies in rat thoracic duct using the blockade of eNOS with LNAME in isolated thoracic duct segments that were not exposed to any imposed axial pressure gradients.90 Thus, the only flow through these isolated lymphatics came about because of the intrinsic pumping activity. Blockade of NO in the thoracic duct increased the contraction frequency as we had seen earlier. The NO blockade also increased basal tone by 30%–50%, which altered lymphatic diastolic compliance and thus decreased lusitropy. This resulted in a decrease in the phasic contraction amplitude and ejection fraction. This implies that there is inherent NO activity in these vessels due to the flow generated by the intrinsic pumping and the inherent sensitivity of the vessel to shear. The phasic relatively low-level shear patterns generated by the intrinsic pump modulates intrinsic lymph pumping and increases the efficiency of the pump via NO-dependent mechanisms in distinction to the imposed steady flows.90 Thus it appears that there are substantial differences between the effects on the NO generated via a relatively high steady-state imposed flow versus the lower magnitude, oscillatory patterns of NO generated by flow via the intrinsic lymph pump. We propose that the imposed flow-dependent inhibition of the active lymph pump is a physiological mechanism that saves energy by decreasing or stopping intrinsic pumping when the lymphatics do not need to generate lymph flow because some other mechanism upstream is doing so. Inhibition of the intrinsic lymph pump under these conditions will also reduce lymph outflow resistance as a result of the net increase in average lymphatic diameter that occurs when strong phasic contractions are inhibited. This decrease in outflow resistance would ease the removal of fluid from the tissue producing the high lymph flows, thus facilitating the resolution of edema with a reduced energy cost. Thus, the influence of flow/shear on lymphatic pumping and contractile activity is complex and heavily intertwined in the accompanying changes in lymph pressure/stretch. Together, changes in these physical factors are critical regulators of lymphatic function. However, our understanding of these processes, as well as those of other neural and humoral processes, depends on our knowledge of BOTH the activities of the lymphatic endothelial cells and muscle cells and how these interactions generate/regulate lymphatic muscle contractions and thus function.
Impairment of lymph flow can result in a wide range of pathologies, including lymphedema, depressed immune function, impaired lipid metabolism, etc. Globally, lymphedema is on the World Health Organization's top ten list of debilitating diseases to conquer, with hundreds of millions patients affected by the lymphedema caused by parasitic lymphatic filariasis. In the United States, the most common lymphatic disease diagnosed is secondary lymphedema resulting from mastectomy, congestive heart failure, or reconstructive surgery, with millions of patients suffering from secondary lymphedema after surgical node resection alone.106 Most importantly, there are likely many other pathologies in which lymphatic dysfunction has important roles that have not yet been defined. Furthermore, our understanding of the relative roles of lymphatic structure versus function in lymphatic dysfunction is still minimal. Thus, although studies have provided some information on lymphatic pumping activity, the basic physical, cellular, and molecular regulation of lymphatic muscle contraction is still not well understood. In particular, our understanding of the molecular processes that regulate the tonic and phasic lymphatic muscle contractions is incomplete. A better understanding of these processes would provide the basis for the development of better diagnostic and treatment modalities for lymphatic disease.
This work was supported by grants from National Institutes of Health HL-07688, HL-75199, HL-080526, and HL085659.
The author would like to thank the following individuals who have helped conduct many of the studies described in this report: Eric Bridenbaugh, Gerry Cote, Ph.D., Michael Davis, Ph.D., Brandon Dixon, Ph.D. Anatoliy Gashev, M.D., Ph.D., Olga Gasheva, M.D., Jimmy Moore, Ph.D., Mariappan Muthuchamy, Ph.D., and Wei Wang, M.D.
Dr. Zwaieja has no conflicts of interest or financial ties to disclose.