Gene expression profile of lymphatic endothelial cells

The lymphatic system was first described at around the same time as the blood circulation centuries ago, but the biological function elucidation of LECs (lymphatic endothelial cells) is far less than that of BVECs (blood vascular endothelial cells). Since the discovery of molecular markers for LECs and exploration of lymphatic role in tumour metastasis, more attention has been given to basic lymphatic research. Approx. 150 known genes were found to be expressed at the mRNA and protein levels by LECs. These molecules play an important role in lymphangiogenesis, signalling, tumour metastasis, immune function and fluid transport. This review provides a brief outline of gene expression profile of LECs and the molecular biological function, which will give the reader a better understanding about the mechanics of lymphatic function and some pathologies related to the lymphatic system such as lymphoedema, and facilitate advanced scientific research into lymphatic biology.     

Angiopoietin-2 promotes inflammatory lymphangiogenesis and its effect can be blocked by the specific inhibitor L1-10

Angiopoietin (Ang)-2, a ligand of the receptor tyrosine kinase Tie2, is known to be involved in the regulation of embryonic lymphangiogenesis. However, the role of Ang-2 in postnatal pathological lymphangiogenesis, such as inflammation, is largely unknown. We used a combination of imaging, molecular, and cellular approaches to investigate whether Ang-2 is involved in inflammatory lymphangiogenesis. We observed strong and continuous expression of Ang-2 on newly generated lymphatic vessels for 2 wk in sutured corneas of BALB/c mice. This expression was concurrent with an increased number of lymphatic vessels. TNF-α expression also increased, with peak TNF-α expression occurring before peak Ang-2 expression was reached. In vitro experiments showed that TNF-α stimulates Ang-2 and Tie2 and ICAM-1 expression on human lymphatic endothelial cells (LECs) and blood vascular endothelial cells (BECs). Ang-2 alone did not affect the biological behavior of LECs, whereas Ang-2 combined with TNF-α significantly promoted the proliferation of LECs but not BECs. In mouse models, blockade of Ang-2 with L1-10, an Ang-2-specific inhibitor, significantly inhibited lymphangiogenesis but promoted angiogenesis. These results clearly indicate that Ang-2 acts as a crucial regulator of inflammatory lymphangiogenesis by sensitizing the lymphatic vasculature to inflammatory stimuli, thereby directly promoting lymphangiogenesis. The involvement of Ang-2 in inflammatory lymphangiogenesis provides a strong rationale for the exploitation of anti-Ang-2 treatment in the prevention and treatment of tumor metastasis and transplant rejection.     

δ-Catenin Activates Rho GTPase,Promotes Lymphangiogenesis and Growth of Tumor Metastases

δ-catenin, an adherens junctions protein, is not only involved in early development, cell-cell adhesion and cell motility in neuronal cells, but it also plays an important role in vascular endothelial cell motility and pathological angiogenesis. In this study, we report a new function of δ-catenin in lymphangiogenesis. Consistent with expression of δ-catenin in vascular endothelial cells, we detected expression of the gene in lymphatic endothelial cells (LECs). Ectopic expression of δ-catenin in LECs increased cell motility and lymphatic vascular network formation in vitro and lymphangiogenesis in vivo in a Matrigel plug assay. Conversely, knockdown of δ-catenin in LECs impaired lymphangiogenesis in vitro and in vivo. Biochemical analysis shows that δ-catenin regulates activation of Rho family small GTPases, key mediators in cell motility. δ-catenin activates Rac1 and Cdc42 but inhibits RhoA in LECs. Notably, blocking of Rac1 activation impaired δ-catenin mediated lymphangiogenesis in a Matrigel assay. Consistently, loss of δ-catenin in mice inhibited the growth of tumor metastases. Taken together, these findings identify a new function of δ-catenin in lymphangiogenesis and tumor growth/metastasis, likely through modulation of small Rho GTPase activation. Targeting δ-catenin may offer a new way to control tumor metastasis.     

Lymphatic endothelial cells, lymphangiogenesis, and extracellular matrix

Exciting studies involving the molecular regulation of lymphangiogenesis in lymphatic-associated disorders (e.g., wound healing, lymphedema and tumor metastasis) have focused renewed attention on the intrinsic relationship between lymphatic endothelial cells (LECs) and extracellular matrix (ECM) microenvironment. ECM molecules and remodeling events play a key role in regulating lymphangiogenesis, and the "functionality"-relating molecules, especially hyaluronan, integrins, reelin, IL-7, and matrix metalloproteinases, provide the most fundamental and critical prerequisite for LEC growth, migration, tube formation, and survival, although lymphangiogenesis is directly or/and indirectly controlled by VEGF-C/-D/VEGFR- 3- Prox-1-, Syk/SLP76-, podoplanin/Ang-2/Nrp-2-, FOXC2-, and other signaling pathways in embryonic and pathological processes. New knowledge regarding the differentiation of initial lymphatics should enable improvements in understanding of a variety of cytokines, chemokines, and other factors. The lymphatic colocalization with histochemical staining by using the novel molecular markers (e.g., LYVE-1), along with subsequent injection technique with ferritin or some tracer, will reveal functional and structural features of newly formed and preexisting lymphatics. Growing recognition of the multiple functions of ECM and LEC molecules for important physiological and pathological events may be helpful in identifying the crucial changes in tissues subjected to lymph circulation and ultimately in the search for rational therapeutic approaches to prevent lymphatic-associated disorders.     

Phosphorylation of Akt and ERK1/2 is required for VEGF-A/VEGFR2-induced proliferation and migration of lymphatic endothelium

There is growing evidence that vascular endothelial growth factor-A (VEGF-A), a ligand of the receptor tyrosine kinases VEGFR1 and VEGFR2, promotes lymphangiogenesis. However, the underlying mechanisms by which VEGF-A induces the growth of lymphatic vessels remain poorly defined. Here we report that VEGFR2, not VEGFR1, is the primary receptor regulating VEGF-A-induced lymphangiogenesis. We show that specific inhibition of VEGF-A/VEGFR2 signaling with the fully human monoclonal antibody r84 significantly inhibits lymphangiogenesis in MDA-MB-231 tumors. In vitro experiments with primary human dermal lymphatic endothelial cells (LECs) demonstrate that blocking VEGF-A activation of VEGFR2, not VEGFR1, significantly inhibits VEGF-A-induced proliferation and migration of LECs. We show that VEGF-A stimulation of LECs leads to the phosphorylation of VEGFR2 (Tyr 951, 1054, 1059, 1175, and 1214) which subsequently triggers PKC dependent phosphorylation of ERK1/2 and PI3-K dependent phosphorylation of Akt. Additionally, we demonstrate that inhibitors that suppress the phosphorylation of ERK1/2 and Akt significantly block VEGF-A- induced proliferation and migration of LECs. Together, these results shed light on the mechanisms regulating VEGF-A-induced proliferation and migration of LECs, reveal that VEGFR2 is the primary signaling VEGF-A receptor on lymphatic endothelium, and suggest that therapeutic agents targeting the VEGF-A/VEGFR2 axis could be useful in blocking the pathological formation of lymphatic vessels.     

Alternatively activated RAW264.7 macrophages enhance tumor lymphangiogenesis in mouse lung adenocarcinoma

Tumor‐associated macrophages (TAMs) have been implicated in promoting tumor progression and invasion. The onset and maintenance of tumor angiogenesis and lymphangiogenesis also seem to be partly driven by a group of polarized alternatively activated macrophages (aaMphi) in lung adenocarcinoma. Here, the aaMphi and classically activated macrophages (caMphi) were obtained using RAW264.7 cells via IL‐4 and IFN‐γ + LPS treatment, respectively. Co‐inoculation of aaMphi with Lewis lung carcinoma (LLC) cells promoted tumor growth, increased lymph node metastasis, and reduced the survival in C57BL/6 mice bearing LLC. Furthermore, the effects of the activated macrophages on the lymphangiogenesis‐related properties of lymphatic endothelial cells (LECs) were investigated in vitro. When LECs were cultured in macrophages conditioned medium or in a co‐culture system of macrophages and LECs, aaMphi significantly promoted proliferation, migration, and tube‐like formation of LECs. We identified high VEGF‐C expression in aaMphi and low expression in caMphi as well as unactivated macrophages by ELISA and Western blotting. In LECs, co‐culture with aaMphi resulted in a significant increase of mRNA levels of specific lymphatic marker VEGF receptor‐3 and the homeobox gene Prox‐1, as well as lymphangiogenic factor VEGF‐C rather than VEGF‐D by quantitative RT‐PCR. Furthermore, enhanced LECs migration and capillary formation by co‐culture with aaMphi were significantly inhibited by rVEGF receptor‐3/Fc chimera. In conclusion, these data show that aaMphi play a critical role in tumor‐induced lymphangiogenesis through up‐regulating VEGF‐C and increasing lymphangiogenesis‐related behavior of LECs, which may contribute to lymphatic invasion in lung adenocarcinoma. J. Cell. Biochem. 107: 134–143, 2009. © 2009 Wiley‐Liss, Inc.     

Sirtuin 3 deficiency aggravates angiotensin II-induced hypertensive cardiac injury by the impairment of lymphangiogenesis

Lymphangiogenesis is possibly capable of attenuating hypertension-induced cardiac injury. Sirtuin 3 (SIRT3) is an effective mitochondrial deacetylase that has the potential to modulate this process; however, its role in hypertension-induced cardiac lymphangiogenesis to date has not been investigated. Our experiments were performed on 8-week-old wild-type (WT), SIRT3 knockout (SIRT3-KO) and SIRT3 overexpression (SIRT3-LV) mice infused with angiotensin II (Ang II) (1000 ng/kg per minute) or saline for 28 days. After Ang II infusion, SIRT3-KO mice developed a more severe cardiac remodelling, less lymphatic capillaries and lower expression of lymphatic marker when compared to wild-type mice. In comparison, SIRT3-LV restored lymphangiogenesis and attenuated cardiac injury. Furthermore, lymphatic endothelial cells (LECs) exposed to Ang II in vitro exhibited decreased migration and proliferation. Silencing SIRT3 induced functional decrease in LECs, while SIRT3 overexpression LECs facilitated. Moreover, SIRT3 may up-regulate lymphangiogenesis by affecting vascular endothelial growth factor receptor 3 (VEGFR3) and ERK pathway. These findings suggest that SIRT3 could promote lymphangiogenesis and attenuate hypertensive cardiac injury.     

Rapamycin inhibition of CFA-induced lymphangiogenesis in PLN is independent of mast cells

Elucidation of the events responsible for the interaction between lymphatic endothelial cells (LECs) and mast cells (MCs) may prove to be a valuable source for controlling lymphangiogenesis. In the present study, we compared immunohistochemical and RT-PCR findings of the popliteal lymph node (PLN) and footpad skin in C57BL/6J and WBB6F1 mice, the MC-deficient strain. The results indicated that MCs play certain role in complete Freund’s adjuvant-induced intranodal lymphangiogenesis. VEGF-A, VEGFR-2 and TNF-α were crucial factors in lymphangiogenesis both in the PLN and skin. Moreover, the in vivo administration of the specific mTOR inhibitor, rapamycin inhibited lymphangiogenesis independent of MCs in PLN rather than in the skin. Further study on anti-lymphangiogenic effect will contribute to our understanding of LEC and MC modulation in pathological lymphangiogenesis.     

Lymphangiogenesis in renal fibrosis arises from macrophages via VEGF-C/VEGFR3-dependent autophagy and polarization

Inflammation plays a crucial role in the occurrence and development of renal fibrosis, which ultimately results in end-stage renal disease (ESRD). There is new focus on lymphangiogenesis in the field of inflammation. Recent studies have revealed the association between lymphangiogenesis and renal fibrosis, but the source of lymphatic endothelial cells (LECs) is not clear. It has also been reported that macrophages are involved in lymphangiogenesis through direct and indirect mechanisms in other tissues. We hypothesized that there was a close relationship between macrophages and lymphatic endothelial progenitor cells in renal fibrosis. In this study, we demonstrated that lymphangiogenesis occurred in a renal fibrosis model and was positively correlated with the degree of fibrosis and macrophage infiltration. Compared to resting (M0) macrophages and alternatively activated (M2) macrophages, classically activated (M1) macrophages predominantly transdifferentiated into LECs in vivo and in vitro. VEGF-C further increased M1 macrophage polarization and transdifferentiation into LECs by activating VEGFR3. It was suggested that VEGF-C/VEGFR3 pathway activation downregulated macrophage autophagy and subsequently regulated macrophage phenotype. The induction of autophagy in macrophages by rapamycin decreased M1 macrophage polarization and differentiation into LECs. These results suggested that M1 macrophages promoted lymphangiogenesis and contributed to newly formed lymphatic vessels in the renal fibrosis microenvironment, and VEGF-C/VEGFR3 signaling promoted macrophage M1 polarization by suppressing macrophage autophagy and then increased the transdifferentiation of M1 macrophages into LECs.Subject terms: Lymphangiogenesis, End-stage renal disease     

Interaction between the extracellular matrix and lymphatics: Consequences for lymphangiogenesis and lymphatic function

The lymphatic system is important for body fluid balance as well as immunological surveillance. Due to the identification of new molecular markers during the last decade, there has been a recent dramatic increase in our knowledge on the molecular mechanisms involved in lymphatic vessel growth (lymphangiogenesis) and lymphatic function. Here we review data showing that although it is often overlooked, the extracellular matrix plays an important role in the generation of new lymphatic vessels as a response to physiological and pathological stimuli. Extracellular matrix-lymphatic interactions as well as biophysical characteristics of the stroma have consequences for tumor formation, growth and metastasis. During the recent years, anti-lymphangiogenesis has emerged as an additional therapeutic modality to the clinically applied anti-angiogenesis strategy. Oppositely, enhancement of lymphangiogenesis in situations of lymph accumulation is seen as a promising strategy to a set of conditions where few therapeutic avenues are available. Knowledge on the interaction between the extracellular matrix and the lymphatics may enhance our understanding of the underlying mechanisms and may ultimately lead to better therapies for conditions where reduced or increased lymphatic function is the therapeutic target.     

Th2 Cytokines Inhibit Lymphangiogenesis

Lymphangiogenesis is the process by which new lymphatic vessels grow in response to pathologic stimuli such as wound healing, inflammation, and tumor metastasis. It is well-recognized that growth factors and cytokines regulate lymphangiogenesis by promoting or inhibiting lymphatic endothelial cell (LEC) proliferation, migration and differentiation. Our group has shown that the expression of T-helper 2 (Th2) cytokines is markedly increased in lymphedema, and that these cytokines inhibit lymphatic function by increasing fibrosis and promoting changes in the extracellular matrix. However, while the evidence supporting a role for T cells and Th2 cytokines as negative regulators of lymphatic function is clear, the direct effects of Th2 cytokines on isolated LECs remains poorly understood. Using in vitro and in vivo studies, we show that physiologic doses of interleukin-4 (IL-4) and interleukin-13 (IL-13) have profound anti-lymphangiogenic effects and potently impair LEC survival, proliferation, migration, and tubule formation. Inhibition of these cytokines with targeted monoclonal antibodies in the cornea suture model specifically increases inflammatory lymphangiogenesis without concomitant changes in angiogenesis. These findings suggest that manipulation of anti-lymphangiogenic pathways may represent a novel and potent means of improving lymphangiogenesis.     

A hypothesis on the role of primitive macrophages in initial embryonic lymphatic development

Recent evidences have shown that macrophages are tightly related to pathological lymphangiogenesis. However, the effects which primitive macrophages take in embryonic lymphatic development remains unclear. Here, we postulate that the primitive macrophages may play an important role in initial embryonic lymphatic development. The possible mechanism: primitive macrophages induce BECs to transdifferentiate into LECs and initiate the budding, moreover, the lymph sacs are not only formed by LECs but also some scattered cells with macrophage characteristics preferentially located in the loose mesenchyme.     

Molecular regulation of lymphangiogenesis and targets for tissue oedema.

New insight has recently been obtained into the molecular mechanisms regulating the function of lymphatic endothelial cells. Vascular endothelial growth factors-C and -D have been shown to stimulate lymphangiogenesis, and their receptor VEGFR-3 has been linked to human hereditary lymphoedema, although there is evidence that other genes are also involved. These data suggest that it may become possible to stimulate lymphatic growth and function and to treat tissue oedema involved in many diseases.     

Endostatin suppresses colorectal tumor-induced lymphangiogenesis by inhibiting expression of fibronectin extra domain A and integrin α9

Endostatin is a natural occurring anti-angiogenic peptide and has been shown to inhibit tumor lymphangiogenesis by suppressing the expression of tumor-stimulating growth factors. We have previously shown that fibronectin alternative extra domain A (EDA) facilitates lymphangiogenesis of colorectal tumors. Since it is known that EDA interacts with integrin α9 in the lymphatic endothelial cells (LECs), we hypothesized that endostatin may target EDA-integrin α9 pathway to inhibit colorectal tumor-induced lymphangiogenesis. To test this hypothesis, we examined the effect of endostatin on EDA secreted by SW480 colorectal cancer cells and treated human LECs with different doses of endostatin in the presence of conditional medium from SW480 cells. We found that endostatin significantly reduced EDA secretion by SW480 cells and the expression of integrin α9 in LECs. Immunofluorescence studies showed that EDA and integrin α9 colocalized on the cell membrane of LECs and these colocalizations were dramatically reduced by endostatin. Co-immunoprecipitation studies demonstrated that EDA interacted with integrin α9 in LECs, and showed that endostatin treatment inhibited the formation of EDA-integrin α9 complex in LECs. Furthermore, we found that the arrangement and polarity of LEC cytoskeletons were destroyed by endostatin substantially, leading to a reduced formation of tube-like structures of LECs and a suppressed chemotaxis of LECs toward SW480 cells. Consistently, EDA and integrin α9 expressions as well as lymphangiogenesis were significantly suppressed by endostatin in colorectal cancer xenografts. In conclusion, our results suggest that endostatin reduces colorectal tumor-induced lymphangiogenesis, at least in part, by inhibiting EDA-integrin α9 pathway.     

Limited expression of reticulocalbin-1 in lymphatic endothelial cells in lung tumor but not in normal lung

Lymphatic endothelial cells in tumors (T-LECs) are considered to have different characteristics from LECs in non-tumor tissues (N-LECs). However, differences between the two types have not been well analyzed at molecular level. In this report, we performed differential proteome analysis of T-LEC and N-LEC models prepared by cultivation of LECs in tumor conditioned medium. By expression profiling of identified proteins using tissue microarrays, reticulocalbin-1 was found to be expressed in clinical specimen-derived T-LECs and lung cancer cells but not N-LECs. It is suggested that reticulocalbin-1 may be an important molecule in understanding T-LEC function and control of lymphatic metastasis.     

Maldevelopment of dermal lymphatics in Wnt5a-knockout-mice

Maintenance of tissue homeostasis and immune surveillance are important functions of the lymphatic vascular system. Lymphatic vessels are lined by lymphatic endothelial cells (LECs). By gene micro-array expression studies we recently compared human lymphangioma-derived LECs with umbilical vein endothelial cells (HUVECs). Here, we followed up on these studies. Besides well-known LEC markers, we observed regulation of molecules involved in immune regulation, acetylcholine degradation and platelet regulation. Moreover we identified differentially expressed WNT pathway components, which play important roles in the morphogenesis of various organs, including the blood vascular system. WNT signaling has not yet been addressed in lymphangiogenesis. We found high expression of FZD3, FZD5 and DKK2 mRNA in HUVECs, and WNT5A in LECs. The latter was verified in normal skin-derived LECs. With immunohistological methods we detected WNT5A in LECs, as well as ROR1, ROR2 and RYK in both LECs and HUVECs. In the human, mutations of WNT5A or its receptor ROR2 cause the Robinow syndrome. These patients show multiple developmental defects including the cardio-vascular system. We studied Wnt5a-knockout (ko) mouse embryos at day 18.5. We show that the number of dermal lymphatic capillaries is significantly lower in Wnt5a-null-mice. However, the mean size of individual lymphatics and the LEC number per vessel are greater. In sum, the total area covered by lymphatics and the total number of LECs are not significantly altered. The reduced number of lymphatic capillaries indicates a sprouting defect rather than a proliferation defect in the dermis of Wnt5a-ko-mice, and identifies Wnt5a as a regulator of lymphangiogenesis.     

Isolation and characterization of lymphatic microvascular endothelial cells from human tonsils

Human lymphatic endothelial cells (LECs) have isolated prevalently from human derma and tumors. As specialized lymphatic organs within the oropharynx, palatine tonsils are easily obtained and rich in lymphatic venules. Using a two-step purification method based on the sorting of endothelial cells with Ulex Europaeus Agglutinin 1 (UEA-1)-coated beads, followed by purification with monoclonal antibody D2-40, we successfully purified LECs from human palatine tonsils. The LECs were expanded on flasks coated with collagen type 1 and fibronectin for up to 8-10 passages and then analyzed for phenotypic and functional properties. Cultured cells retained the phenotypic pattern of the lymphatic endothelium of palatine tonsils and expressed functional VEGFR-3 molecules. In fact, stimulation with VEGFR-3 ligand, the vascular endothelium grow factor C, induced a marked increase in cell proliferation. Similarly to blood endothelial cells (BECs), LECs were able to form tube-like structure when seeded in Cultrex basement membrane extract. Comparative studies performed on LECs derived from palatine tonsils and iliac lymphatic vessels (ILVs), obtained with the same procedures, showed substantial discrepancies in the expression of various lymphatic markers. This points to the existence of micro- and macrovessel-derived LECs with different phenotypes, possibly involving different biological activities and functions. Palatine tonsil- and ILV-derived LECs may, therefore, represent new models for investigating function and biochemical properties of these lymphatic endothelia.