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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_3996_Библиотеки_им_академика_М_И_Перельмана
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By studying smaller, more segmented vessels of the guinea
pig mesentery, van Helden consistently recorded with a sharp
microelectrode small spontaneous transient depolarizations
(STDs) of the lymphatic muscle membrane potential. Because, either
individually or through summation, STDs underlie the larger action
potentials and muscle contractions, he proposed them as the lym-
phatic pacemaker [26]. This hypothesis is supported by a number
of observations reported in this original paper and subsequent stud-
ies [20,27–30]. STDs reflect the opening of Ca
2+
-activated inward
current carried by Cl
ions upon the “packeted” release of Ca
2+
from
IP
3
-sensitive stores within the muscle cells [25,29]. This hypothesis
that Cl
Ca
channels underlie STDs and participate in the lymphatic
pacemaking is also in agreement with the growing body of evidence
showing that Cl
Ca
channels are involved in agonist-induced smooth
muscle contraction [31,32].
The identity of Cl
Ca
channels has only recently been determined
as anoctamin-1 (ANO1), also known as TMEM16a. ANO1 has been
shown to be expressed in human, mouse, and rat lymphatics [33]
(von der Weid unpublished), and ANO1 current recorded from
lymphatic muscle cells acutely dissociated from mouse inguinal-
axillary collectors (SD Zawieja and MJ Davis, personal communica-
tion). The important role of ANO1 in lymphatic pumping was
further demonstrated by the strong reduction of lymphatic contrac-
tion frequency and a lack of response to increase in transmural pres-
sure after selective deletion of the Ano1 gene in mouse lymphatic
muscle cells (SD Zawieja and MJ Davis, personal communication).
However, action potentials were still occurring, although at a much
slower rate, again suggesting that other conductances, such as
those discussed earlier, are likely involved in the lymphatic pace-
maker. While it is important to keep in mind that different tissue
preparation or animal species may account for the variation in ion
channel contribution to the pacemaker mechanism, differences in
experimental conditions should also be considered. Notably, to
allow successful recordings of membrane potential and the pharma-
cological modulation of STD activity with sharp microelectrodes,
experiments were mostly performed on quiescent, unstimulated
vessel segments, displaying what we can consider true
“spontaneous contractile activity.” On the other hand, assessment
of the effects of these blockers on lymphatic pumping was typically
24 3. Lymphatic pumping and pathological consequences of its dysfunction
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performed on isolated vessels under pressure/stretch conditions
that may activate a pacemaker mechanism requiring activation of
a different set of ion channels than spontaneous pacemaking. It is
well known that the degree of distension or stretch of the lymphatic
vessel wall is the most important determining factor of the intrinsic
lymphatic pumping [34,35]. STD frequency was shown to increase
with stretch in rat mesenteric lymphatics mounted on a wire myo-
graph [36], and with intraluminal flow [20,37], other ion channels
may underlie the response to stretch or pressure.
Mechanisms of lymphatic contraction
In the absence of characterization of the contractile and regulatory
elements of the lymphatic muscle contractile apparatus, it had been
generally thought that lymphatic muscle cells were smooth muscle
cells. However, in a seminal study, Muthuchamy et al. [38] revealed
that lymphatic muscle contains both striated and smooth muscle
contractile elements and displays differences in both contractile
function and contractile machinery when compared with blood ves-
sels, demonstrating the uniqueness of the lymphatic muscle. Specif-
ically, mesenteric lymphatic muscle cells exhibit genotypic
and phenotypic characteristics of vascular, cardiac, and visceral
myocytes, as they express SMB smooth muscle myosin heavy chain
(with both SM1 and SM2 isoforms detected) and messages encoding
the fetal cardiac/skeletal slow-twitch muscle-specific β-MHC,
and four different actin isoforms, including cardiac α-actin, vascular
α-actin, enteric γ-actin, and skeletal α-actin, have also been detected
[38]. Preliminary data also demonstrate that lymphatic muscles
express striated muscle regulatory components, such as cardiac
troponin C and cardiac troponin T (MM Muthuchamy, personal
communication). This particular muscle phenotype likely provides
the strong contractile ability of the mesenteric vessels. Other func-
tional differences between lymphatic and vascular smooth muscles
have been demonstrated, including myofilament Ca
2+
sensitivity
and cooperativity [39] and lower active tension development [40].
Our understanding of the molecular mechanisms regulating tonic
and phasic contractions of lymphatic muscle also improved over the
last decade. Wang et al. [41] showed that while phosphorylation of
the myosin light chain 20 (MLC
20
), which triggers cross-bridge
25Origin and mechanisms of lymphatic pumping
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cycling and force development, primarily modulates mesenteric
lymphatic tonic contraction, it does not significantly affect the
amplitude of phasic contractions suggesting that tonic contraction
strength and phasic contraction amplitude of the lymphatics are dif-
ferentially regulated. Lymphatics exhibit a significant decrease in
tone in response to elevated pressure; in rat thoracic duct, it corre-
lated with a decrease in phosphorylation of MLC
20
at both of its
phosphorylation sites [42]. Using pharmacological tools, Hosaka
et al. [43] demonstrated the involvement of Rho kinase and myosin
phosphatase in lymphatic tonic contraction but maybe also in phasic
contractions (see also reviews by von der Weid and Muthuchamy
[44] and Chakraborty et al. [45]). Critically, mesenteric lymphatics
exhibit myogenic responses to changes in intraluminal pressures
and rate-sensitive contractile responses to stretch [46,47]. Thus,
thanks to these unique mechanical and contractile characteristics,
composed of both smooth and striated muscle regulatory mecha-
nisms, the lymphatic muscle can accomplish its essential function
in the maintenance of the normal lymph transport and in protection
against the formation of gross edema and other pathological
conditions.
Regulation of lymphatic pumping
Mechanical regulation
Regulation of the lymphatic pump is important to maintain fluid
balance in response to altered fluid load. Under basal condition, an
increased interstitial and intraluminal pressure results in a corre-
sponding rise in contraction frequency to a maximum, beyond
which flow drops due to a loss in stroke volume. As such, typical
profiles of lymphatic pumping in response to pressure appear as
bell-shaped curves and are observed in many animal species. All
lymphatics have an optimal pumping condition at relatively low
transmural pressures [34]. These pressures tend to be higher in more
peripheral lymphatic vessels, suggesting that more peripheral lym-
phatics can develop much higher pressures to prevail over the
greater outflow resistance [48]. For instance, mesenteric lymphatics
have the highest fractional pumping (6–8 volumes/min at optimal
pressure levels) [34].
26 3. Lymphatic pumping and pathological consequences of its dysfunction
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Lymph flow also affects the contraction frequency of lymphatic
vessels. Benoit et al. [34], measuring lymph flow in situ in rat mes-
enteric lymphatics by elevating lymph formation, found increased
parameters of active lymph contractility during periods of increased
lymph flow, while the pressure in the lymphatic network became
less pulsatile at high lymph flow states. Although such experiments
give important information on lymphatic contractile behavior, it is
difficult to clearly separate the effects of increased flow from the
effects of increased transmural pressure. A study on rat iliac micro-
lymphatics showed that increases in intraluminal perfusion corre-
spond to an increase in contraction frequency and a decrease in
the amplitude of contractions [49]. But during increases in imposed
flow in rat lymphatics, inhibition of both amplitude and frequency
of contraction was observed [50,51]. However, it is difficult to con-
clude that such imposed flow-dependent inhibition of the active
lymph pump decreases total lymph flow in vivo. At high levels of
lymph formation, passive lymph flow could become a greater driv-
ing force to move lymph than the active lymph pump. Flow-
dependent inhibition of the active lymph pump in such situations
could be a reasonable physiological mechanism to save metabolic
energy by temporarily decreasing or stopping contractions during
the time when the lymphatic does not need it. Inhibition of the
lymph pump under these circumstances could also reduce lymph
outflow resistance, as a result of a net increase in average lymphatic
diameter when contractions are inhibited. This reduction in outflow
resistance could ease the removal of fluid from the affected compart-
ment that is producing high lymph flows and thereby facilitate the
resolution of edema [50,52].
Chemical regulation
Lymphatic pumping is heavily modulated by factors present in
the lymph or in fluid surrounding the vessel, whether they are
released from nerves, cells present in the interstitium, the vessel
itself or from blood. These factors include endothelium-derived
mediators, neurotransmitters, and many circulating hormones
and inflammatory mediators (reviewed in von der Weid [53],
von der Weid and Rehal [54], and Pal et al. [55].
27Origin and mechanisms of lymphatic pumping
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Like blood vessels, lymphatic behavior is regulated by
endothelium-derived factors such as nitric oxide (NO), prostaglan-
dins, and histamine [56–59]. Both lymphatic tone and spontaneous
contractions are inhibited by NO produced as a result of shear stress
on the endothelium [51]. During the contraction-relaxation cycle,
shear stress increases with lymph flow during the contraction phase
of the cycle. This causes a rapid and transient production and release
of NO, which contributes to interrupt the contraction and promotes
vessel dilation [60]. NO inhibits the spontaneous contraction
through arresting calcium release associated with the pacemaker,
STDs [30], and activated ATP-sensitive K
+
channels to hyperpolar-
ize the lymphatic muscle [61]. Prostaglandins are among the most
important regulators of lymphatic vessel function. Although they
can be produced by surrounding tissues, these metabolites of arachi-
donic acid are also synthesized by the lymphatic endothelium,
which expresses cyclo-oxygenase 1 (COX1) [58,62–64]. Histamine
has more recently been demonstrated to be also produced by the
lymphatic endothelium and participate to the flow-dependent inhi-
bition of pumping [59]. Depending on species and vessel location,
histamine increases the frequency of lymphatic pumping via activa-
tion of H
1
receptors located on smooth muscle cells or decreases con-
traction frequency via H
2
receptor stimulation or via an indirect
effect mediated by the endothelium [27,65,66]. Histamine is also
predominant in mast cell granules and is the main mediator of
increased lymphatic pumping caused by mast cell activation, as
demonstrated in a milk-sensitized animal model, a model of food
allergy [67]. As will be discussed in “Changes during chronic
inflammation” section, other cells, such as macrophages or mast
cells, present in the vicinity of lymphatics in inflammatory situa-
tions, produce and release NO, prostaglandins, and histamine to
potentially alter lymphatic contractility [68].
Even if sparse in comparison with neighboring blood vessels,
innervation contributes to the regulation of lymphatic contractility
[69,70], and several neurotransmitters and neuromodulators have
been shown to modulate lymphatic contractility. Localized adrener-
gic and cholinergic nerve fibers were identified around initial and
collecting guinea pig mesenteric lymphatic vessels [70,71]. Norepi-
nephrine generally increases contraction frequency. This effect,
mediated via excitatory α-adrenoreceptor (both α
1
and α
2
receptor
28 3. Lymphatic pumping and pathological consequences of its dysfunction
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subtypes), can be modulated by an inhibitory action via
β-adrenoreceptor (β
1
and β
2
) stimulation, depending on the class
of adrenergic receptors expressed in the tissue [69,72–74].
Lymphatic alterations during disease
Many diseases present an inflammatory component. A subset of
these inflammatory diseases also have observable changes within
lymphatic structures that can have slight or drastic effects on the
affected party. Preclinical and clinical studies have shown changes
in the lymphatic vasculature in several disease types including those
affecting the skin, gut, and cancer microenvironments to mention
but a few. The manifestation of change can occur at any point
within the lymphatic circulatory system including structural
changes to initial lymphatics, collecting lymphatics, or even the
draining lymph node.
Lymphadenopathy
Lymphadenopathy is one of the most obvious changes to occur to
the lymphatic system during inflammation, infection, or disease.
Swelling of the lymph node is characterized by the infiltration of
peripheral immune cells to the lymph node cortex, expansion and
proliferation of the resident T cells and B cells within the node,
and fluid accumulation as a result of lymphatic net-outflow disrup-
tion [75]. The feature itself is a sign of an immune response, as anti-
gen surveillance, and response to such is the primary purpose of the
lymph node [76]. However, in chronic inflammatory conditions,
systemic inflammation, such as that found in murine models of
rheumatoid arthritis (RA), the lymphadenopathy can become
uncontrolled and result in node exhaustion and collapse [77]. The
regulation of lymph flow to the lymph node via the afferent lym-
phatic and away from the node through the efferent lymphatic
is regulated by fluid dynamics and pumping characteristics. It has
been demonstrated that insufficient flow to the lymph node through
dysfunctional pumping not only reduces the incidence of lymph-
adenopathy but also can result in localized tissue inflammation
resultant in inflammatory phenotypes such as elephantiasis [78].
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Transport of antigenic material to the lymph node “effector venue”
is paramount for a correct immune response. With the lymphatic
pump being an important part of this process, a correct flow is
critical in the system.
Lymphangiogenesis
Lymphangiogenesis, or proliferation of new lymphatic vessels, is
another feature of many tissues that are undergoing development or
have undergone an inflammatory insult (Reviewed in Halin and Det-
mar [79]). Sprouting of lymph vessels within a tissue is driven by vas-
cular endothelial growth factor C (VEGFC) and its receptor vascular
endothelial growth receptor 3 (VEGFR3) that, expressed on lym-
phatic endothelium, promotes proliferation and wound healing
[80,81]. The promotion of lymphatic architecture expansion is pre-
sumably in aid of fluid drainage and clearance of cells and materials
from the affected site although, functionally, this seldom occurs.
While the blood capillary form of angiogenesis is short lived within
a site, any lymphangiogenesis that occurs within the area is long
lived. The reason and effect of its presence are still debated to be ben-
eficial or detrimental, such as the long-last lymphatic alterations seen
within the small intestinal inflammation resultant from DSS treat-
ment in mice [82]. The analysis of lymphangiogenesis within differ-
ent tissues demonstrates a wide array of phenotypes depending on
how the system responds. While it is known that all new lymphatic
vessels branch from preexisting endothelium, the organization and
distribution of these new vessels are notably poor, leading many to
wonder whether they are actually beneficial or even functional. With
the discovery of markers of lymphatic vessel subtypes, differentia-
tion of initial and specialized lymphatic structures can be identified
through antibody labeling of LYVE-1, Prox1, VEGFR3, and podopla-
nin, further allowing investigation into the effects and purpose of
lymphangiogenesis within a disease.
Lymphangiectasia
As described earlier, collecting lymphatics propel large vol-
umes of fluid, cells, and metabolites, thanks to phasic contraction
of the muscle cells embedded in their wall. Inhibition of
30 3. Lymphatic pumping and pathological consequences of its dysfunction
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this activity leads to lymph stasis, which is typically accompanied
with dilation of the vessels. This dysfunction, known as
lymphangiectasia, can have many consequences on the surround-
ing tissue.
Dilation of the vessels can be caused by many means, as described
in “Chemical regulation” section (see also von der Weid and
Muthuchamy [44]), but an important contributor is NO. This gas-
eous molecule dilates lymphatic vessels within a matter of seconds
and halts pumping when present in high enough concentrations
[44,83].
Leaky vessels, along with the failure of lymphatic contractility,
primarily result in interstitial fluid accumulation, which has many
indirect consequences. Due to a reduction in fluid flow to the lymph
node, antigen transport from a sight of damage or injury is damp-
ened leading to an insufficient immune response [84]. In the latter
study, Liao et al. further demonstrated that iNOS-expressing
CD11b
+
Gr1
+
cells, while reliant on the lymphatics to propel them
toward the lymph node, also produce the NO responsible for vessel
dilation and pumping inhibition, thereby impeding the immune
response. The movement of fluid is also key in catabolite removal
and nutrient recycling; lymphangiectasia, therefore, can cause
a toxic accrual of metabolites promoting sterile inflammation.
The presence of sterile inflammation has been demonstrated
to improve lymphatic function after lymph node transfer in a toll-
like receptor-dependent fashion, providing a new area of research
into the regulation of contraction through innate immune cell
activation [85,86].
Lymphatic pumping in diseases
As mentioned, disturbed lymph flow can result in impaired
immune response, pathogen, and metabolite clearance. It is there-
fore feasible to suggest that the reduction in lymph clearance, seen
in many diseases, could be a contributing factor to its pathogenesis.
The loss of lymphatic pumping can occur in many diseases, some of
which have obvious signs of a reduced lymph clearance, such as
lymphedema, while others present less obvious signs but are inher-
ently still important.
31Lymphatic pumping in diseases
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Primary lymphedema
In primary lymphedema (congenital/hereditary), fluid collects as
a consequence of obstruction, malformation, or underdevelopment
of various lymphatic components. These dysfunctions can occur
within the initial lymphatics, the collecting lymphatics, or the lymph
node. Lymphedema usually affects regional drainage from extrem-
ities in the upper and lower limbs and can lead to swelling, skin
thickening and hardening, and pain. All features of lymphatic alter-
ations can be seen within lymphedema patients, including lymph-
adenopathy, lymphangiectasia within the affected limb/s, and
lymphangiogenesis within the tissue, specifically the skin dermis.
Research in the past two decades used advanced high-resolution
magnetic imaging to monitor changes within the lymphedematous
tissues of lymphedema patients, demonstrating many cutaneous
alterations within the dermis [87]. Almost 20 years later,
M Detmar’s group discovered that not only was prominent lym-
phatic hyperplasia evident but so was immune cell infiltration
and accumulation within the dermis of the murine tail-model of
lymphedema and was associated with tissue fibrosis, swelling,
and reduced wound healing efficacy, common phenomenon
observed in human counterparts [88].
Lymphedema itself can result from mutations in a variety of
genes including prox1, vegfr3, gata2, foxc2, and flt4, leading to
malformations of the vessels, valve formation, or cell-cell adhesion
[89–92]. FOXC2 having been identified in high incidence within
lymphedema-distichiasis has been shown to alter interactions
between LECs and pericytes resulting in the valve-defect phenotype
[93]. The VEGFR3 mutation prevalent in hereditary primary lymph-
edema such as Milroy’s disease is an amino-acid shift mutation that
inactivates the receptor preventing correct interaction and activa-
tion with VEGFC a nd VEGFD, key players in lymphatic g rowth
[94]. As the flow of lymph could be disrupted on many levels,
the consequence of any of these mutations alone or in collabora-
tion could be detrimental to fluid flow and lead to tissue fluid
accumulation. Little is documented about the morphological
changes within the tissue prior to the established disease; how-
ever, what is clear is a prominent alteration in collecting lymphatic
pumping efficacy [95– 97]. Collecting lymphatics play a central
32 3. Lymphatic pumping and pathological consequences of its dysfunction
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role in the onset of lymphedema, although the changes observed
can include occlusion of the vessel, dilation, loss of functional lym-
phatics, or fibrosis acting as a benign blockader of efficient con-
traction [98,99], a phenomenon that can n ow be observed within
patients through the use of indocyanine green fluorescence
lymphography [100].
Secondary lymphedema
While the etiology of primary forms of lymphedema can be
linked to lymphatic-associated genetic mutations, secondary
lymphedema, the more common forms of lymphedema, arises from
insults to the lymphatic architecture at the initial or collecting lym-
phatic level, or removal/disruption of the lymph node. It is common
for patients presenting with secondary lymphedema to have
enlarged, weak collecting vessels in the affected limb when com-
pared with the healthy bilateral controls suggesting issues in fluid
drainage and associated lymphangiectasia. However, whether
lymphedema precedes or follows, lymphatic dysfunction is still
debated as both lymph vessel ablation and fluid overload can both
affect function. While histological changes within human disease
states display altered lymphatic capillary size and density, transi-
tional studies in animal have been hampered by a lack of models
able to replicate all aspects of the disease and more specifically its
chronicity. However, while lasting only a few weeks, the edema
caused in the mouse tail by surgical ablation of a ring of skin, one
of the only useable and current model of animal lymphedema, cer-
tainly displays lymphangiectasia and many feature of the human
condition [101,102].
Lymphedema itself can affect all limbs in the upper and lower
body hemispheres and can quickly become a burden to those
afflicted, limiting movement. Visceral lymphatics can also be
affected although the occurrence is lower than peripheral. The cau-
sality of the disease can be a result of many anatomic changes,
including lymphatic hypoplasia or the absence of functional valves
through mutations in associated genes such as FOXC2 and others
mentioned earlier.
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