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Scallan J, Huxley VH, Korthuis RJ. Capillary Fluid Exchange: Regulation, Functions, and Pathology. San Rafael (CA): Morgan & Claypool Life Sciences; 2010.

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Capillary Fluid Exchange: Regulation, Functions, and Pathology.

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Chapter 3The Lymphatic Vasculature

3.1. Anatomy and Nomenclature of the Lymphatic Vasculature

While initial study of the blood vascular system dates back to the sixth century BC, the lymphatic vasculature was not discovered until 1622 by Asellius. In stark contrast to the blood vasculature, the lymphatic circulatory system has been far less extensively studied. The reason for this is not its late discovery, but rather pertains to the prevailing misperception that the lymphatics represent a largely passive system for return of extravasated fluid and proteins to the systemic circulation and that no specific molecular markers had been identified to distinguish cells comprising this circulatory system from those in the blood vasculature until the last two decades [44]. As such, no common nomenclature for the lymphatic vasculature has developed, so one previously used for rat mesenteric lymphatics will be introduced next [231].

Interstitial fluid, formed from the extravasation of solute and fluid from the capillaries, enters blind-ended sacs composed only of an endothelial layer that is tethered to the interstitial matrix. These bulbous sacs (10–60 μm diameter) are called initial, or terminal, lymphatics. Initial lymphatics possess overlapping endothelial cells that behave collectively like a valve, only permitting unidirectional entry of fluid, solute, and cells into the lumen of these vessels (Figure 3.1). The fluid, thereafter referred to as lymph, next moves into lymphatic vessels of a similar diameter, termed microlymphatics (or lymphatic capillaries), consisting of an endothelial layer and basement membrane [267]. Since the phrase ‘lymphatic capillary’ is sometimes applied to initial lymphatics, and ‘lymphatic capillaries’ may be much larger than traditional capillaries carrying whole blood, this ambiguous phrase will be avoided hereafter. Microlymphatic vessels then carry lymph towards the larger collecting lymphatic vessels. Collecting lymphatics (50–200 μm diameter) are composed of endothelial cells, a basement membrane, lymphatic muscle cells, pericytes, and endothelial valves that prevent retrograde lymph flow [205,271]. Pericytes do not envelope the smaller initial and microlymphatics [205]. Valves are present in collecting lymphatics and differ from the initial lymphatic ‘valve’ in that they consist of two modified endothelial cell leaflets that meet in the vessel lumen, not unlike bicuspid valves of the larger mammalian venous system. The lymphatic muscle layer is unique in that it possesses both tonic and phasic contractile activity [232,271]. Phasic contractions of lymphatic muscle, referred to as spontaneous contractions, aid in propelling lymph along the intervalvular segments of the lymphatic vessel, called lymphangions. After passing through lymph nodes and then larger collecting lymphatic ducts, lymph is finally propelled to the thoracic duct, which empties into the left subclavian vein.

Figure 3.1. Expanding the interstitial fluid volume exerts radial tension on the anchoring filaments attached to endothelial cells of initial lymphatics, which increases luminal volume.

Figure 3.1

Expanding the interstitial fluid volume exerts radial tension on the anchoring filaments attached to endothelial cells of initial lymphatics, which increases luminal volume. This creates a small pressure difference that drives interstitial fluid into (more...)

The anatomy of collecting lymphatic vessels appears to be similar to that of comparable veins in that they both are low-pressure vessels vested with a muscle layer and intraluminal valves. In support of the theory of lymphatic development originally proposed by Sabin [227], one group has shown that lymphatic endothelial cells are derived directly from the cardinal vein [243]. Akin to the venules, numerous cytoplasmic vesicles have been reported in initial lymphatic endothelium [9,33,153,154,155,188], but a role for these vesicles in solute uptake has not been elucidated fully at present. However, whether lymphatic vessels possess other similar features – such as a glycocalyx – has not been established.

Although the preceding description is accurate for the mesenteric lymphatic vasculature, it is important to recognize that lymphatic vessel morphology varies greatly between organs. Since it is beyond the scope of this section to thoroughly summarize these varying anatomical features, the reader is directed to several excellent detailed reviews for this information [14,191,232]. Two of these reviews [14,232] summarize classical views on several aspects of the lymphatic vasculature, while the present chapter hopes to provide the reader with the most current perspectives, highlighting active areas of research and needed studies.

3.2. Lymph Formation

Lymph formation refers to the entry of fluid and protein into the initial lymphatics. The mechanisms responsible for this process are poorly understood, but two main hypotheses have been proposed. The first suggests that an osmotic gradient becomes established across the initial lymphatic wall through sieving of protein that then generates its own convective flow by pulling in protein-containing interstitial fluid against a concentration gradient [34]. Very little, if any, experimental support exists for this unlikely theory despite its original appearance nearly four decades ago. Therefore, the following discussion will focus on the second hypothesis, which relies upon a hydrostatic pressure gradient to fill the initial lymphatics.

As stated before, the initial lymphatics possess overlapping endothelial cells tethered to the tissue (Figure 3.1). Thus, when the tissue becomes hydrated it swells and pulls apart the endothelial cells to form pores ~2 μm in diameter that act like a nonselective one-way valve, trapping fluid, solute, and cells passively (Figure 3.1) [266]. Considering their unique structure, one would arrive at the logical conclusion that a pressure gradient across the interstitium may drive fluid and solute accumulation within the initial lymphatics. Few studies on interstitial pressure gradients have been performed, but each proposes a gradient between 0.2–0.8 cmH2O [99,278]. At first glance this pressure gradient seems small, but others have calculated that a pressure head of only 0.12 cmH2O is adequate to drive the capillary filtrate into the low resistance initial lymphatics [232]. The main problem with this hypothesis is that negative values of interstitial pressure are routinely measured [40,91]. Significant overlap of the simultaneously measured interstitial and initial lymphatic pressures was observed [40,100], depending on the superfusion solution and the time of measurement (immediately following exteriorization or 30 minutes later). Particularly interesting was that 30 minutes after exposure of the mesentery, superfused with oil to preserve natural tissue hydration, respective pressures in the interstitium and initial lymphatics were −0.2 and −0.25 mmHg [40]. Therefore, it is possible that a positive pressure gradient can allow fluid to enter the initial lymphatics even with a negative interstitial pressure. More current support for this hypothesis has been reported [182].

A passive interstitial pressure gradient, while sufficient, is not a complete description of every mechanism contributing to the formation of lymph. Pulsation of arteries was shown to aid in removal of interstitial tracer, which ceased after application of a steady arterial pressure [200]. Likewise, in the bat wing, cyclical dilation of the venules is a form of extrinsic pumping that also stimulates intrinsic spontaneous contractions of collecting lymphatics [63]. Other factors that increase local tissue pressure facilitate lymph formation such as respiration, muscle contraction (e.g., peristalsis, walking), elevated capillary filtration (e.g., venous hypertension, increased capillary permeability), and massage. Opposite to an increase in interstitial pressure, a variant of the hydrostatic pressure hypothesis posits that spontaneously contractile collecting lymphatic lymphangions, during their relaxation phase, are able to generate a suction force that draws interstitial fluid into the initial lymphatics [213]. Negative pressures produced by isolated bovine mesenteric collecting lymphatics under “low filling” states have been reported [78], but direct evidence for transmission of this suction to the initial lymphatics is needed.

Further support for the hydrostatic pressure gradient hypothesis is derived from studies demonstrating a positive correlation between interstitial pressure and lymph flow, which are discussed next.

3.3. Interstitial Fluid Pressure and its Influence on Lymph Flow

A convincing argument for an interstitial pressure gradient to drive lymph formation has been outlined in the previous section. The potential of the initial lymphatic lumen to collapse under a positive pressure difference is minimized by their unique anatomy. As noted above, initial lymphatic endothelial cells are tethered to the interstitium by anchoring filaments [156] responsible for holding the lumen open during conditions of increased tissue pressure or swelling, creating large (~2 μm diameter) interendothelial pores (Figure 3.1). The pores are a consequence of the punctate or “button”-like pattern of endothelial junctional adhesion proteins, in contrast to the contiguous expression of these molecules in blood vessel and collecting lymphatic endothelium [16]. Therefore, interstitial fluid is able to access the initial lymphatic lumen especially during edematous states when tissue pressure becomes positive.

Possibly as a result of the direct communication of the interstitial fluid and the lumen of the initial lymphatics, interstitial pressure and lymph flow are positively related. Several studies where tissue pressure was measured with the capsule technique provide direct evidence for this relationship [82,262]. Figure 3.2A from Taylor and coworkers [263], shows that the rise of lymph flow in the dog hind leg is steepest at tissue pressures of ~0−1 cmH2O and attains a sustained maximal value when tissue pressure reaches 2 cmH2O. The importance of this curve is that it maintains a constant interstitial volume due to the tight correlation between interstitial volume and interstitial pressure shown in Figure 3.2B [90]. Two mechanisms protecting against edema (i.e., edema safety factors) are evident from Figure 3.2, assuming that interstitial pressure is normally negative: 1) Since interstitial pressure must rise above 2 cmH2O for lymph flow to plateau, large changes in interstitial pressure can be accommodated before edema develops, and 2) An elevated lymph flow will quickly return the interstitial volume back to normal levels, as long as the excess volume does not exceed the capacity of the lymphatic circulation. Thus, a small increase in interstitial volume greatly increases its pressure, promoting lymph flow that acts to restore the interstitial volume to normal.

Figure 3.2. A, Relative increase in lymph flow versus tissue hydrostatic pressure (PT, mmHg) during edema produced by intravascular infusion of Ringer’s.

Figure 3.2

A, Relative increase in lymph flow versus tissue hydrostatic pressure (PT, mmHg) during edema produced by intravascular infusion of Ringer’s. B, Interstitial fluid volume (IFV in liters, L) versus PT. Reprinted from reference [260], with permission. (more...)

3.4. Lymphatic Solute Permeability

As summarized by Drinker [65], the main responsibility of the lymphatic circulaton is to be:

“…engaged steadily in returning blood proteins to the blood, and that in the absence of normal lymph function these substances will accumulate extravascularly.”

Therefore, because approximately 50% of the plasma proteins are filtered by the blood microvessels per day, and are not reabsorbed by the venules [159,178], the lymphatic vasculature alone is left with the task of returning these proteins to the blood [84,217]. Extravascular accumulation of plasma proteins, if unchecked, leads to the osmotic flow of water into the interstitium, producing edema. Further, Drinker [65] found that if thoracic duct lymph flow is diverted into a test tube, then the blood microvasculature “simply converts all the plasma to lymph.” Such a circumstance is not compatible with life and illustrates the importance of a properly functioning lymphatic circulation that returns protein and fluid to the blood.

Many studies have probed the ‘solute permeability’ of initial lymphatics in a qualitative fashion, meaning that absolute values of permeability were not measured directly. Instead, after injection of colloid or radiolabeled protein, inferences were made from visualizing tracer uptake, analyzing downstream lymph samples, or viewing histological sections. As previously stated, both interendothelial pores [154] and transendothelial vesicles [9,154] have been implicated in solute and fluid removal from the interstitium by initial lymphatics. The physiological role for vesicles in the lymphatic vasculature remains unknown, but their appearance may reflect the anatomic variation in lymphatic morphology [84]. Leak [154] favored the view that the interendothelial route was the major conduit for solute transport, whereas vesicles facilitated digestion of interstitial protein by the initial lymphatic endothelium. Generally, it is now believed that initial lymphatics are able to passively absorb particles, protein, cells, and fluid from the interstitium through large pores without regard for molecular size. Consequently, the lymph protein concentration of initial lymphatics probably approximates the protein concentration of the interstitium [261,285]. Much controversy still surrounds whether the protein concentration of lymph from collecting lymphatics is equal to that of interstitial fluid; i.e., whether collecting lymphatics possess the ability to concentrate solute [29,117,258,261].

Unlike initial lymphatic solute uptake, flux of solute across the collecting lymphatic walls depends on molecular size [167]. Studies performed by Mayerson [167] were initially aimed at answering questions regarding blood capillary permeability, using the lymphatic vaculature as a window into the interstitium, but became focused on how efficiently lymph is transported through the lymphatic circulation. By injecting a known amount of radiolabeled albumin directly into a canine limb collecting lymphatic duct (unknown diameter, but likely much larger than the peripheral collecting lymphatics, which average ~100 μm) and analyzing thoracic duct lymph, they were able to estimate the percent albumin lost across the vessel wall [201]. Not only is this method less sensitive than microfluorometric methods used today [108], but it almost certainly reflects the ‘permeability’ of the larger collecting lymphatic ducts and lymph nodes, not the prenodal microlymphatics or collecting lymphatic vessels. However, these studies were novel for the time and provided a first approximation of the size selectivity of the lymphatic ducts up to and including the thoracic duct. What Mayerson [167] discovered was that macromolecules equal to or greater than 6000 daltons (6 kDa) did not leave the larger ducts in ‘great’ quantity (albumin loss < 3%), while molecules smaller than 6 kDa escaped the vessels with ease. Simply put, large lymphatic ducts seemed to possess a relatively low permeability to large macromolecules and a higher permeability to molecules smaller than insulin.

Recently, a study of rat mesenteric collecting lymphatic solute flux largely confirmed Mayerson’s observations in that the isolated vessel segments retained FITC-labeled dextran molecules of 4, 12, and 70 kDa progressively as molecular size increased [194]. Another group determined that the permeability to bovine serum albumin of cultured lymphatic endothelial cell tubes (~100 μm diameter) was 14±7 x 10−7 cm · s−1 [208]. Conversely, a paper reporting rat mesenteric collecting lymphatic permeability to rat serum albumin (RSA) in vivo concluded that it did not significantly differ from venular RSA permeability (3.5 ± 1 x 10−7 cm · s−1 vs. 4.0 ± 1 x 10−7 cm · s−1, respectively), and that microlymphatic permeability to RSA was 12 x 10−7 cm · s−1 [231]. Three major implications are immediately apparent: 1) collecting lymphatics, at least in rat mesentery, exhibit a permeability to albumin no different from their embryological relative, the venules, supporting developmental work [243], 2) lymphatic endothelial cell tubes in culture that lack a basement membrane, lymphatic muscle, and pericytes presumably reflect microlymphatic versus collecting lymphatic permeability, warranting caution in data interpretation from cell culture studies, and 3) because lymph loses solute (and water) as it traverses the microlymphatics and collecting lymphatics, it is not identical to or representative of interstitial fluid. The last point must be emphasized, as lymph protein concentrations have been widely assumed to equal that of interstitial fluid. Indeed, lymph composition is further modified by nodal transit as evidenced by concentration differences in pre- vs postnodal lymph (which is the usual site for lymph collection for in vivo studies and often equated to interstitial fluid) [2,3,4,5]. Scallan and Huxley [231] presented evidence supporting the hypothesis that lymph is concentrated by collecting lymphatics via loss of water over solute. When the total protein and albumin concentration of plasma, interstitium, and collecting lymphatic lymph were measured simultaneously, lymph protein concentrations were significantly greater than interstitial protein concentrations. Consequently, these data show that solute flux is directed from the vessel lumen to the interstitium; i.e., collecting lymphatics leak solute (Figure 3.3). However, there is still a need for direct measures of collecting lymphatic hydraulic conductivity (or ‘water permeability coefficient’) to confirm that the concentration of solute occurs as a result of losing more water than solute from the vessel. The fact that lymph flow is inversely related to lymph protein concentration lends further support to this hypothesis [29].

But what does this mean in terms of edema? Lymphatic permeability may influence the effectiveness of the lymphatic edema safety factor given that vasoactive substances increasing collecting lymphatic permeability (to solute or water) may facilitate edema formation if most collecting lymphatics possess solute fluxes directed towards the interstitium. The implications of such findings in this newly revived area of research are that we will have to modify the conventional understanding of how lymphatic physiology affects fluid homeostasis.

3.5. Propulsion of Lymph by the Lymphatic Muscle Pump

The field of research examining the functional and molecular control of lymphatic contractility has been productive in the past two decades. One key discovery is that lymphatic muscle may act as a functional hybrid between smooth muscle and cardiac muscle because it contains molecular machinery from both cell types [181]. This has stimulated new hypotheses about how collecting lymphatics are able to independently regulate both tonic and phasic contractions.

Hydrostatic pressure increases progressively as lymph moves downstream into larger vessels of the lymphatic vasculature. On the contrary, peripheral veins experience a greater hydrostatic pressure than the downstream central veins, especially when standing, owing to the effects of gravity. Restated, the pressure gradient produced by the heart, in addition to the extrinsic venous pump, provides the driving force for venous return [14]. The lymphatic vasculature has no such pressure head (i.e., vis a tergo) so lymph does not – and cannot – drain passively through the lymphatic vessels, but requires propulsion. However, under edematous conditions, the interstitial pressure rises so that lymph may flow down a pressure gradient [209]. Lymph is transported throughout the lymphatic vasculature by intrinsic phasic contractions generated by the lymphatic muscle of collecting lymphatics that, along with valves, are necessary for unidirectional lymph flow. The spontaneous contractions are analogous to the cardiac contraction cycle consisting of a contraction and relaxation phase, stroke volume, and ejection fraction [20]. For comparison, the measured ejection fraction and contraction frequency of rat mesenteric collecting lymphatics were ~67% and 6 min−1, respectively [20]. Understanding the functional and molecular regulation of lymphatic spontaneous contractions is essential for developing therapeutic treatments for edema (and lymphedema) centered on augmenting contraction amplitude and/or frequency.

Since the walls of collecting lymphatics are vested with muscle cells, they are able to regulate their diameter and tone, therefore modulating lymph flow resistance. Several factors, both mechanical and chemical, are able to regulate collecting lymphatic tone [271]. Mechanical stimuli include lymph flow, shear stress, hydrostatic pressure, and temperature. Hydrostatic pressure has been shown to elicit a myogenic response in collecting lymphatic muscle (measured during the relaxation phase) analogous to the arteriolar myogenic response [56]. In this study, an elevation in hydrostatic pressure induced constriction, thus reducing the end diastolic diameter of isolated collecting lymphatics, similar to the arteriolar myogenic response to pressure. Interestingly, addition of the neuropeptide substance P to the superfusion bath potentiated this effect of pressure on tone. Chemical factors influencing collecting lymphatic tone include neurotransmitters, neuropeptides, hormones, and metabolites [12,58,212]. For example, substance P increases basal collecting lymphatic tone [12,58].

Similar to cardiac myocytes, length-tension curves have been determined for the perivascular muscle of collecting lymphatics, arterioles, and venules [20,286,287,288]. Wall tension and stress derived from these curves were found to be lowest in rat mesenteric lymphatics, while mesenteric veins possessed higher tension and stress, and that of mesenteric arteries was the highest. Functionally, this makes sense in that arterioles, the resistance vessels, constrict to regulate pressure and flow; venules and lymphatics possess lower hydrostatic pressures reflecting their roles as capacitance vessels. The same group estimated the optimal preload (or hydrostatic pressure) for collecting lymphatic tone during peak active force to be ~5–13 cmH2O, which compares well with measures of in vivo hydrostatic pressure [231,290].

Other research has focused on the regulation of collecting lymphatic phasic activity. Functional studies demonstrated that lymph flow inhibits spontaneous contraction frequency and amplitude of both collecting lymphatic and thoracic duct isolated vessels [79]. However, the conclusion was that in vivo total lymph flow (defined as passive flow plus contraction-generated flow) would not be diminished as expected. Instead, lack of pumping activity was suggested as a mechanism to reduce the outflow resistance in the presence of high passive flows [79,209].

Another vessel possessing spontaneous contractile activity besides the collecting lymphatics is the portal vein. Like the portal vein, collecting lymphatics were more sensitive to the rate of circumferential stretch than to the magnitude [57,123]. These characteristics are consistent with developmental work showing that lymphatic endothelial cells are derived from the cardinal vein [243]. In experiments where isolated mesenteric collecting lymphatics were exposed to pressure ramps of different rates, bursts of increased contraction frequency were observed with increasing hydrostatic pressure ramps while inhibition of contraction frequency was observed on the ramps decreasing in pressure [57]. When the effects of substance P on collecting lymphatic sensitivity to stretch were assessed, both contraction amplitude and frequency were enhanced under basal conditions and in response to elevations in pressure [12,58].

The molecular basis for lymphatic spontaneous contractions has just begun to be explored. Nitric oxide (NO) has been implicated in the modulation of collecting lymphatic spontaneous contractions [79]. Application of NO to the solution bathing isolated collecting lymphatics blunted spontaneous contraction frequency, amplitude, and ejection fraction in a fashion that imitated the effects of lymph flow. However, when the effect of elevated flow was investigated after the addition of the NO synthase inhibitor, L-NAME, NO did not fully explain the inhibition of contraction amplitude and frequency. NO involvement in the regulation of lymphatic spontaneous contractions is discussed in more detail in recent reviews [286,287].

Importantly, calcium has been studied in the context of lymphatic phasic activity. Several different Ca2+ channels have been identified in lymphatic muscle, including L-type and T-type channels [101]. A hypothesis for spontaneous transient depolarizations (STDs) in the generation of spontaneous contractions has been proposed [270,272]. At present, it is likely that summation of several STDs leads to a spontaneous contraction, with several ion channels appearing to be involved [270,272]. However, it is at present unclear whether a single STD or summation of several STDs leads to a spontaneous contraction. Another hypothesis states that pacemaker cells generate a current which spreads throughout the lymphatic muscle [190], although the precise location of these cells was made difficult by the diffusion of current [138]. A newer study, however, has identified a subpopulation of lymphatic muscle cells that may act as pacemaker cells [168]. While evidence for the sympathetic innervation of lymphatic muscle is abundant, its role in altering spontaneous contractions has not been fully investigated. Several current reviews cover this topic more thoroughly [169,268,270,286,287].

The benefits of studying the mechanisms of lymphatic contractions are obvious – during edema, when the lymphatic vessels appear overwhelmed, an increase in pumping efficiency would be expected to recover proper fluid balance. While this work has contributed to our knowledge of regulation and generation of lymph flow, a complete molecular understanding necessitates future studies.

3.6. Lymphangiogenesis

Lymphatic vessel development de novo has been alluded to in previous sections where recent experiments support the hypothesis proposed by Sabin [227]; i.e., that lymphatic endothelial cells bud directly off of the cardinal vein to form the primitive lymphatic vasculature [243]. This is in contrast to the alternative hypothesis [103] that lymphatic endothelium is formed solely from lymphangioblasts residing in the tissue. Lymphangiogenesis, commonly defined as any event that stimulates lymphatic vessel growth [250], has been a highly topical area of investigation for nearly two decades. Some aspects of the molecular pathways have been identified, but an understanding of their importance to lymphangiogenesis is still being pursued.

One molecule important for the commitment of endothelial cells to the lymphatic phenotype is Prox1, which was first shown in mice to be necessary for the development of the lymphatic vasculature [277]. Homozygous deletion of Prox1 resulted in embryos devoid of lymphatic vessels, which proved to be embryonically lethal [95]. Interestingly, an outbred line of Prox1+/− mice survived but developed adult-onset obesity, providing a link between malformed lymphatic vessels and visceral fat accumulation [95]. Very recently, it was demonstrated that down-regulation of Prox1 leads to the dedifferentiation of lymphatic endothelium into blood vessel endothelium, its default phenotype [124]. Because of its importance in determining endothelial cell identity, Prox1 is commonly used as a lymphatic marker, but is also expressed in the liver, pancreas, and brain. Another molecule used widely to identify lymphatic endothelium is lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1), which is expressed to a greater extent in initial versus collecting lymphatics in adult humans [267]. It is important to note that LYVE-1 is also expressed on infiltrating macrophages, as well as the sinusoidal endothelium of the liver and spleen, limiting its utility as a specific marker for lymphatic vessels [116]. Recent work suggests that this molecule does not play a role in lymphangiogenesis [116].

Much work has focused on vascular endothelial growth factors C and D (VEGF-C/D) as the prime regulators of lymphangiogenesis. Each molecule can bind to the receptors VEGFR-2, VEGFR-3, or to neuropilin-2 (Nrp2, expression limited to initial lymphatics) after proteolytic processing, which serves to increase receptor binding and specificity [127]. To further complicate the signaling mechanisms, VEGFR-2 and -3 can form homodimeric or heterodimeric receptor complexes to activate proliferation signals [61]. Additionally, Prox1 upregulates VEGFR-3 expression [277], suggestive of a role for VEGF-C in development. Binding of VEGF-C to VEGFR-3 induces transduction of proliferation and survival signals in cultured cells [165], adult tissues [112,119], and during development [129]. These cues then lead to migration and sprouting of lymphatic endothelial cells (LECs). Confirming its importance to lymphatic vasculogenesis, VEGF-C/VEGFR-3 signaling was necessary for the sprouting of LEC from the cardinal vein [129]. Surprisingly, mutation of the Nrp2 semaphorin receptor inhibits the formation of microlymphatics but not collecting lymphatics [284].

VEGF-C signaling is currently being explored for lymphedema therapy. Primary lymphedema, an inherited genetic disruption of the lymphatic vasculature, is known to result from mutations in the VEGFR-3 gene. The most common forms are Milroy’s disease and Meige’s disease, both of which are characterized by lymphatic vessel hypoplasia and fluid accumulation. A mouse model (Chy mouse) has been developed that mirrors Milroy’s disease and is reversed by the over-expression of VEGF-C [130]. No animal models exist for Meige’s disease, which has been estimated to account for nearly 94% of all primary lymphedema cases [220]. This has severely limited progress with regard to development of rational therapies. Secondary lymphedema is acquired after birth as a consequence of lymphatic vessel injury as a result of trauma, surgery, irradiation, infection, or cancer. This type of lymphedema usually results in the accumulation of protein-rich fluid in the tissues. However, breast cancer related lymphedema appears to be an exception to this general rule, being characterized by accumulation of a protein-poor exudate [17]. The mouse-tail model of secondary lymphedema has been examined with respect to VEGF-C therapy [38]. In this study, microlymphatic growth reversed the lymphedema and improved immune cell trafficking to normal levels. While these studies hold promise for therapeutic lymphangiogenesis, other molecules that may rescue lymphedema (discussed next) have yet to be fully investigated.

Like Prox1, the tyrosine kinase Syk as well as its adaptor protein SLP76 act to prevent the formation of anastomoses between lymphatics and the venous vessels. This notion is based on the fact that amino acid substitution mutations of these proteins promote the formation of communicating channels between the lymphatic and the venous systems [1]. Therefore, these two proteins are needed for the separation of the lymphatic and blood systems. The fact that Syk and SLP76 are also expressed in hematopoietic cells (but not endothelial cells) strongly suggests a novel role for hematopoietic cells in maintaining distinctly separate lymph and blood vasculatures.

Remodeling of the lymphatic network is a necessary component of vascular network maintenance. This includes pruning of microlymphatic networks, and recruitment of perivascular cells to collecting lymphatics (e.g., pericytes and muscle cells). The growth factor ephrinB2 appears to play a major role in these remodeling processes as evidenced by the fact that genetically engineered mice exhibiting defects in this protein possess hyperplasic lymphatics without intraluminal valves and are unable to prune microlymphatic networks [164].

The angiopoietin receptors, Tie1 and Tie2, are both expressed on lymphatic endothelial cells. Deletion of the gene for angiopoietin2 causes lymphatic hypoplasia, which was completely reversed by introducing the gene for angiopoietin1 in its place [74]. How angiopoietin (1 & 2) signaling ties into lymphangiogenesis is not well understood.

Thus far only the most pertinent molecules involved in lymphatic vascular development and growth have been discussed in detail. A large number of other molecules have been identified as potential mediators but their roles in lymphedema or lymphangiogenesis remain uncertain [11,121]. Further elucidation of their function could prove useful in developing novel therapies for lymphatic diseases.

3.7. Tumors and Lymphatic Metastasis

Traditionally, dissemination of cancer cells to organs distal to the primary tumor has been thought to be initiated only through tumor-associated blood vessels and not lymphatics within tumors. This concept likely arose as a consequence of the belief that intratumoral lymphatic vessels could not be penetrated by tumor cells (see [167]) or that they were collapsed in the high pressure environment within the tumor mass [157]. [A possible explanation for the former assumption is that blind reference to ‘the lymphatics’ in the literature – not specifying whether initial, micro-, or collecting – leads one to conclude erroneously that the properties of these three subtypes are identical. For example, initial lymphatics are almost freely permeable to solute and cells, while collecting lymphatics possess a relatively lower permeability.] However, it is now well established that initial and microlymphatic vessels invade tumors in a fashion similar to blood vessel neovascularization [244]. Two hypotheses have been proposed regarding tumor cell metastasis and the lymphatic vasculature. One favors passive absorption of free tumor cells into the lymphatic vasculature whereas the alternative hypothesis proposes that active signaling is involved [203]. Passive metastasis of tumor cells via the lymphatic circulation is certainly likely to occur, but current experiments are now probing the molecular mechanisms behind tumor cell metastasis, including tumor-associated lymphangiogenesis.

New research has provided support for the hypothesis invoking molecular regulation of tumor metastasis. An interesting mechanism has been described to explain chemotaxis of some cancer cells to the lymphatic circulation. Many breast cancer and melanoma cells express CCR7, the chemokine receptor for the ligand CCL21, constitutively secreted by the lymphatic vasculature. As a result, the cancer cells expressing CCR7 will chemotax towards the lymphatic circulation in response to CCL21 in vitro and in vivo [114]. In the same study, VEGF-C enhanced the secretion of CCL21 by cultured tumor cells. Coupled with the fact that many cancer cell types already express VEGF-C and -D [251], this is likely a primary mechanism by which cancer cells ‘find’ the lymphatic vasculature and promote lymph node metastasis. Further, a correlation between tumor-associated lymphangiogenesis and lymph node metastasis has been identified as a prognostic marker of disease [48]. However, whether or not lymphatic vessel density correlates with lymph node metastasis or a poor outcome has yet to be determined. Novel prognostic indicators of survival would be beneficial not only to the patient but also to the researcher as a guide to the mechanisms of tumor metastasis. Finally, other lymphatic growth factors have not enjoyed the attention VEGF-C has received [180], leading one to wonder whether other chemotactic or lymphangiogenic mechanisms exist.

3.8. Cessation of Lymph Flow and its Immunological Consequences

Continuous convection of interstitial fluid through the tissues and lymph through the lymphatic vessels is necessary for ensuring tissue health. When lymphatic vessels become dysfunctional due to either genetic or environmental processes, primary or secondary lymphedema will develop. A major consequence of any edema is tissue swelling, which serves to increase the distance molecules must diffuse to reach the cells in the tissue. Furthermore, immune cells monitoring the tissue have to cover longer distances before they reach the lymphatic circulation. In lymphedema where lymph flow is blocked, these immune cells become trapped in the tissue.

Antigen-presenting dendritic cells (APCs) recognize and bind antigens in the tissue to initiate the immune response, along with macrophages and natural killer cells. Once the antigen is bound, APCs express CCR7 allowing its chemotaxis towards the lymphatic vessels. Then the cell presents the antigen to T lymphocytes in the lymph nodes. The exact details and significance of these interactions are not fully understood at present [211,287].

When APC migration to the lymphatic vasculature is hindered, as occurs during lymphedema or cancer, their presence in the tissue may exacerbate local tissue inflammation [13]. Once APCs bind antigen they begin to produce inflammatory chemokines to attract other leukocytes and immature APCs [211]. This process exacerbates the local tissue inflammation observed in patients with lymphedema [121].

Several aspects of immune function have yet to be explained fully in the context of lymphedema. For example, it is unclear how APCs gain access to the luminal side of lymphatic vessels, but probably occurs through the initial lymphatics, most likely by passive flux into the lumen via the flow of interstitial fluid through the one-way endothelial valves. It is also possible that intravasation may occur through the endothelial junctions of the larger collecting lymphatics in a process analogous to leukocyte transmigration across postcapillary venular endothelium. It is not clear whether the constitutive expression of CCL21 on the collecting lymphatic endothelium is required for chemotaxis of the APC to the lymph node [211].

Copyright © 2010 by Morgan & Claypool Life Sciences.
Bookshelf ID: NBK53448

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