Lymphatic vessels in the uterine wall

Abstract

During pregnancy, maternal uterine blood vessels undergo dramatic vascular remodeling. However, until now, little was known about whether the lymphatic circulation undergoes similar changes and whether these vessels interact with placental cells that invade maternal tissue. Recent studies demonstrate that normally lymphatic vessels in the uterine wall are highly compartmentalized where their presence is mostly detected in deeper layers. In humans, this arrangement changes during pregnancy when extensive lymphangiogenesis occurs at the maternal-fetal interface. Placental cytotrophoblasts stimulate lymphatic growth in vivo and in vitro suggesting that they play a role in triggering pregnancy-induced decidual lymphangiogenesis. These data indicate that lymphatic vessels may have important functions at the implantation site during pregnancy.

Introduction

During pregnancy, the uterus has the remarkable ability to collaborate with the placenta to produce a hybrid organ that supports growth and development of the fetus. At this time, uterine tissues undergo dramatic changes that alter their cellular composition, a process that is controlled by complex interactions between maternal- and fetal-derived signals. Included in these changes are alterations in the vascular compartment. Initially, extensive angiogenesis occurs in the endometrium in preparation for implantation. Once pregnancy is established, placental cells from the fetus invade the uterine wall and remodel resident vasculature so that blood circulates through the intervillous space. Although these events have been well-described for some time (1), recent work has identified an additional component of this vascular remodeling program, which involves dramatic changes in the uterine lymphatic circulation.

Lymphatic vessels comprise a second vascular network, which functions in diverse processes such as interstitial fluid homeostasis, adaptive immunity, and digestion (2). Although anatomical and molecular analyses have suggested an absence of lymphatics in the endometrium (3–6), their presence in the uterine wall during pregnancy has only recently been addressed. It seems logical to propose a role for these structures at the maternal-fetal interface since, in other systems, dramatic changes in the vasculature are accompanied by lymphangiogenesis (lymphatic vessel growth) (7). This is thought to be important for regulating the increased vascular leakage that often occurs at sites of blood vessel angiogenesis. The great affinity of cytotrophoblasts for vascular endothelium raises the additional possibility that they could engage in a direct relationship with lymphatics. In fact, new data indicates that human pregnancy is associated with extensive lymphangiogenesis and suggest that placental cells play an active part in the process

Lymphatic vascular development in homeostasis and disease

The lymphatic circulation is a second vascular network that plays an important role in regulating interstitial fluid content and adaptive immunity (2). Its structure differs slightly from the blood vasculature. Flow is unidirectional and begins at blind-ending lymphatic capillaries that directly absorb extracellular fluid. These capillaries drain into pre-collecting and then collecting lymphatic vessels, the latter of which are invested with a smooth muscle layer and contain valves (2). The fluid, termed lymph, passes through the lymphatic network and intervening lymph nodes before being delivered to the blood via the thoracic and right lymphatic ducts.

Lymphatic vessels complement the blood vascular system in multiple ways. For example, during capillary exchange, fluid leaks from the blood into surrounding tissues where it is taken up by post-capillary venules. A portion of this fluid contains high molecular weight proteins and cannot pass freely back into the circulation. Lymphatic capillaries possess unique structural features that allow them to take up this protein-rich fluid, such as a discontinuous basal lamina and specialized, button-like cell-cell junctions (8). Defects in lymphatic function can result in a special type of swelling called lymphoedema, which results from either physical damage to local vessels or inherited genetic deficiencies that impair lymphangiogenesis.

The lymphatic circulation also plays an important role in organizing adaptive immune responses (9). Lymphocytes congregate in and circulate through lymph nodes, nodule-like structures intermittently spaced within lymphatic vessels. Circulating lymph filters through these nodes allowing lymphocytes to continually survey its contents. Foreign or pathogen-derived products will stimulate an immune response, which involves the various leukocyte populations that traffic between the blood and lymph nodes.

Dendritic cells are important players in immune responses initiated within lymph nodes. These cells are present in peripheral tissues and can be activated by pathogen-derived products and inflammatory signals, which upregulate phagocytosis and the expression of CCR7, the receptor for the chemokine CCL21 (10). Many lymphatic vessels constitutively express CCL21, which may allow them to attract activated dendritic cells providing access to local lymph nodes (11). Therefore, lymphoid tissue is critical in coordinating the cell-cell interactions that are needed to mount an efficient immune response. As a result, individuals with lymphatic defects are highly susceptible to chronic unresolved infections (12, 13). In this context, it is interesting to note that lymphatic vessels are excluded from several immune privileged sites including the brain and anterior chamber of the eye. In the latter instance, tolerance can be broken by inducing lymphatic growth and dendritic cell migration (14).

Knowledge of lymphatic vessel development and function has exploded over the past several years following the discovery of LYVE-1, a hyaluronic acid receptor specifically localized to lymphatic endothelial cells (2, 15, 16). Using this marker, the long-presumed theory that this vasculature arises from veins was confirmed. In mice, development of lymphatic vessels begins between embryonic day 9.0 and 9.5 when certain regions of the cardinal vein begin to express LYVE-1. Subsequently, expression of the transcription factor Prox1 is induced in a polarized manner and a subset of the LYVE-1/Prox1-positive cells bud off and migrate centrifugally in response to vascular endothelial growth factor-C (VEGF-C) produced by adjacent stromal cells (17, 18). These cells organize into primary lymph sacs, sprout, and eventually give rise to the mature lymphatic vascular system, which are distinguished from blood endothelial cells by expression of a cohort of molecules including LYVE-1, Prox-1, VEGFR3, Podoplanin, α9β1 integrins, and the chemokine CCL21.

In the adult, lymphangiogenesis involves sprouting from preexisting vessels and can occur at sites of injury and inflammation (19, 20). Recent evidence also supports an alternative pathway in which vessels form from CD11b+ macrophages that are recruited to specific sites (21). VEGF-C and Ang-2 can be involved; both molecules induce lymphatic endothelial cell hyperplasia and vessel formation when overexpressed in mice where the actions of Ang-2 appear to be upstream of VEGF-C (22–24). Tumors cells can also express VEGF-C and –D and, in mouse models of tumorigenesis, these molecules increase lymphangiogenesis and metastasis (25–27). In addition, in various human tumors, VEGF-C and -D levels are positively correlated with lymphatic invasion and metastasis, and, in some cases, poor prognosis (28). Together, these observations have stimulated great interest in developing methods of targeting lymphangiogenesis for the purpose of limiting tumor growth and metastasis.

Lymphatic vessel distribution in the uterus

Despite its proximity to the genital tract microbial flora, the outer endometrial layer of the uterus lacks a significant lymphatic circulation. This observation has been reported for mice (3), rats (4), rabbits (5), and humans (6) where lymphatic vessels are restricted to myometrial and serosal layers. The recent discovery of lymphatic-specific molecular markers has made it possible to systematically analyze human endometrial samples from both the proliferative and secretory phase of the menstrual cycle. We and others have used this strategy to determine that LYVE-1-positive lymphatic vessels are absent from the full thickness of the human endometrium at all stages of the menstral cycle (Table 1), but are numerous in the myometrial layer of these same samples (29)(P. Rogers, personal communication).

Table 1.

LYVE-1-positive lymphatic vessels in the uterine wall

	Donors (no.)	Average number of LYVE-1-positive vessels/section
Human
Non-pregnant
Endometrium
proliferative phase	6	0
secretory phase	11	0
Myometrium
proliferative phase	6	++a
secretory phase	8	++a
Pregnant
1st trimester decidua	10	4
2nd trimester
decidua basalis	12	5.4
decidua capsularis	5	>25
3rd trimester
decidua basalis	13	3.25
decidua capsularis	7	>30
Mouse
Pregnant
decidua	5	0
myometrium	5	++a

^aVessels were present but numbers were not quantified

Despite the absence of vessels expressing LYVE-1, Donoghue et al. (30) detected LYVE-1-negative vessels in the endometrium that express podoplanin, a different lymphatic-specific marker. Interestingly, the patterning of these vessels was highly regulated in that they were almost exclusively located in the endometrium basalis, the region directly adjacent to the myometrium, particularly surrounding spiral arterioles. Very few podoplanin-positive vessels were detected in the endometrium functionalis, the luminal portion shed during menstruation, which may be related to the amount of tissue oedema normally seem in this layer. The presence of podoplanin but not LYVE-1 appears to result from heterogeneous marker expression within the lymphatic vasculature, which may not be fully characterized for these recently discovered molecules. Nevertheless, all the data to date shows that the distribution of lymphatic vessels in the endometrium is highly regulated.

Thus far, the reasons for excluding lymphatics from portions of the uterine mucosa remain obscure but could include aspects of endometrial cycling. There may be a need to protect a female’s immune system from exposure to tissue remodeling products, which would include apoptotic cells and their contents, a possible source of autoantigens. At the same time, this arrangement could prevent production of antibodies against sperm, an important risk factor associated with infertility (31). Experiments in rodents suggest that excluding lymphatic vessels from the endometrium effectively separates it from systemic immunity. Dyes and leukocytes introduced into the myometrium normally drain into local lymph nodes while those injected into the endometrium remain at the injection site (4, 32). Further clues about the importance of regulating lymphatic vessels could come from pathological samples or animal models. For example, it would be interesting to determine their distribution in women with endometriosis or recurrent infertility. Also, inducing lymphatic growth in the mouse by expressing a lymphangiogenic molecule in the uterus could be informative, particularly with regard to its effect on fertility.

Given the strict regulation of lymphatic vessels in the uterine wall, we became interested in investigating their distribution in the decidua (pregnant endometrium). Interestingly, 18^th century anatomists described hyperplasia of pelvic lymphatic vessels during pregnancy (33). In addition, studies aimed at localizing angiogenic molecules to the maternal-fetal interface showed that human trophoblasts express various lymphangiogenic molecules. These include VEGF-C and angiopoietin-2 (Ang-2) (34,35), which are well-known triggers of lymphatic growth in other settings (18, 22–24, 36). These observations led us to explore whether lymphatic vessels continued to be excluded from certain regions of the uterine wall during pregnancy.

Our analyses, which involved immunostaining decidual tissue sections with LYVE-1, revealed that, in contrast to the non-pregnant state, lymphatics are prominent in the decidua (Table 1). The vessels were localized to all regions including the basalis (where the placenta attaches), the parietalis (opposite the placental attachment site), and capsularis (adjacent to amniotic membrane), of which the latter contains the highest density (Table 1). With regard to interactions with cytotrophoblasts, invasive cells were consistently detected in contact with lymphatic vessels, but, similar to veins, they did not enter the lumen and their endothelial layer was never remodeled.

Cytotrophoblasts activate lymphatic growth in vitro and in vivo

Invasive cytotrophoblasts express several lymphangiogenic molecules, suggesting that they may be involved in inducing lymphatic growth in the decidua. Consistent with this hypothesis, cytotrophoblast-conditioned medium from either first- or second-trimester cells induces lymphatic endothelial cell migration. This effect can be attributed to multiple proteins within the media including TNFa, FGF-2, and VEGF family members (29).

In addition to their activity in culture, cytotrophoblasts also stimulate lymphangiogenesis in vivo. This parameter was assayed using an in vivo model of human placental invasion in which chorionic villous explants were transplanted into SCID mice allowing analysis of cytotrophoblast invasion and vascular remodeling in a complex tissue environment (29). This in vivo model utilized two transplantation sites, the kidney capsule and mammary fat pad. Both locations supported cytotrophoblast differentiation, but each was amenable to distinct analysis with regard to invasion and vascular remodelling (29).

Histological analyses of murine tissues receiving villous transplants show that aspects of placental invasion can be studied using the mouse as a surrogate implantation site. In both the kidney and mammary fat pad, cytotrophoblasts differentiate down the invasive pathway, robustly invade, and disrupt resident blood vessels (37). In the mammary fat pad, histologically distinguishable arteries and veins show that the cells maintain their tropism for arterial vessels even outside the uterus. Interestingly, placental cells induce apoptosis specifically in arterial endothelial and smooth muscle cells despite interactions with the stroma and veins. Beneath the kidney capsule, cytotrophoblasts stimulate a progressive infiltration of LYVE-1-positive lymphatic endothelial cells that eventually form lumen-containing vessels. Neither decidual tissue or murine trophoblasts contain this activity when placed in the same location showing its specificity to cytotrophoblasts (29).

Together, in vitro and in vivo analyses support the hypothesis that placental cells directly contribute to the formation of lymphatic vessels at the maternal-fetal interface. However, they may not be acting alone. For example, uterine NK cells also express VEGF-C and Ang-2 (38, 39) and have haemeangiogenic properties in vivo (40). The latter study did not address the possibility of lymphatic growth in addition to blood vessels, but the two frequently occur concomitantly. The fact that NK cells from the endometrium and the decidua express the above molecules raises the possibility that another parameter only present during pregnancy is required for uterine lymphangiogenesis.

Lymphangiogenesis at the maternal-fetal interface

Our current understanding of lymphatic vessels in the uterine wall includes a description of their distribution throughout the menstrual cycle and during pregnancy as well as candidate cells and molecules that regulate their dynamic growth. Many other important questions remain, particularly their function at the maternal-fetal interface. In analogy with other systems, decidual lymphatic vessels could be important regulators of fluid homeostasis. Blood flow to the uterus is dramatically heightened as a consequence of vascular remodeling, especially near term, and could result in increased interstitial fluid accumulation. If not properly cleared, excess fluid could potentially harm the fetus.

Lymphatic vessels could also play a role in maternal-fetal immunity. During this time, the mother must balance protection of the fetus with tolerance of its hemiallogeneic tissues. Experiments performed in rodents suggest that the endometrium is sequestered from a female’s systemic immunity by restricting movement from it to local lymphatic tissue (4). Establishing a lymphatic circulation in the decidua could connect the endometrium to the mother’s immune system. This could enhance surveillance of the maternal-fetal interface possibly helping to combat infections, which are associated with preterm labor (41). Alternatively, lymphatic vessels could play a part in establishing maternal tolerance. Placental cells do not appear to enter the lymphatic circulation (29); however, dendritic cells, also numerous at the maternal-fetal interface, could traffic to regional lymph nodes for the purpose of presenting fetal antigens. CD56^bright NK cells may also traffic from the decidua since they are the most abundant subset of uterine leukocytes during pregnancy. Indeed, CD56^bright NK cells are found in secondary lymphoid tissue, but their function in this location, which is thought to be regulatory, is not well understood (42, 43).

Finally, it will be interesting to determine whether changes in the patterning of uterine lymphatics contribute to pathological conditions that affect both the pregnant and non-pregnant uterus. For example, ectopic lymphangiogenesis, which can break tolerance in other immune privileged sites (14), could participate in the pathogenesis of endometriosis. Conversely, a reduction in lymphatic vessels could be associated with dangerous fluid accumulation at the implantation site or within the amniotic cavity as well as compromise maternal-fetal immunity. Future studies are needed to test the role of lymphatic vessels during pregnancy, but whatever their function, they likely play a crucial role in the formation and maintenance of the maternal-fetal interface.