Lymphangiogenesis, Myeloid Cells and Inflammation

Lianping Xing; Rui-Cheng Ji

Disclosures

Expert Rev Clin Immunol. 2008;4(5):599-613. 

In This Article

Abstract and Lymphangiogenesis

The lymphatic system is essential for the maintenance of tissue fluid balance, immune surveillance and the absorption of fatty acids in the gastrointestinal tract. The lymphatic circulation is also a key player in disease processes such as cancer metastasis, lymphedema and various inflammatory disorders. With the identification of specific growth factors for lymphatic endothelial cells and markers that distinguish blood and lymphatic vessels, as well as the development of invivo imaging technologies that provide new tools to examine the lymphatic drainage function in real time, many advancements have been made in lymphatic vascular research during the past few years. Despite these significant achievements, our understanding of the role of lymphatics in disease processes other than cancer metastasis is still rather limited. The current review will focus on the recent progress made in studies of lymphatics in inflammatory disorders.

Lymphatic vessels are present in almost all tissues but are absent from avascular structures, such as the epidermis, hair, nails, cartilage and cornea. The lymphatic vascular system is composed of an extensive network of thin-walled capillaries that drain protein-rich lymph from the extracellular spaces. In contrast to blood vessels, these lymphatic capillaries lack a continuous basement membrane as well as surrounding pericytes or smooth muscle cells. Lymphatic endothelial cells (LECs) are connected to the surrounding extracellular matrix by specialized fibrillin-containing anchoring filaments.[1] When the interstitial fluid pressure is increased, these filaments exert tension on LECs, thereby widening the capillary lumens and opening overlapping cell junctions, which facilitate the uptake of fluid, macromolecules and cells. Major functions of the lymphatic system are conventionally considered to be the maintenance of tissue fluid homeostasis, mediation of the afferent immune response and transportation of absorbed proteins and fats from the gut to the systemic circulation.[2,3,4]

Studies of the lymphatic system have been hampered until recently by the lack of specific markers that distinguish blood from lymphatic vessels, knowledge regarding specific growth factors for LECs and technologies to monitor lymphatic drainage in small animals. However, the progress of recent research has led to significant advances in understanding lymphatic regulation and new functions of the lymphatic system in normal and diseased conditions.

Major advances in lymphatic research have been made possible by the establishment of defined cultures of blood vascular endothelial cells and LECs.[5,6,7,8] Using gene array analysis of LECs versus blood vascular endothelial cells, numerous previously unknown lineage-specific markers for blood and lymphatic vascular endothelium have been identified. Newly identified LEC-specific markers include the prospero-related homeobox1 (Prox1), the lymphatic vascular endothelial hyaluronan receptor (LYVE-1), the mucin-type transmembrane glycoprotein podoplanin and VEGF receptor (VEGFR)-3.[3] Some of these markers, such as LYVE-1, do not play an essential role in lymphatic development, as shown by the fact that deletion of the gene does not lead to abnormality of the lymphatic vascular.[9,10] By contrast, other markers are essential factors for embryonic development of lymphatic vasculature.

Prox1 is the first identified transcription factor essential for early lymphatic development. Prox1-/- embryos died at approximately embryonic day (E)11.5, when the budding of lymphatic vessels from veins occurs. Prox1 expression is required for the commitment of common vascular progenitor cells to LECs. Prox1-/- embryos do not coexpress any lymphatic markers, such as VEGFR-3 or LYVE-1. Instead, the mutant cells appear to have a blood vascular phenotype, as determined by their expression of laminin and CD34, indicating that Prox1 activity is essential for both maintenance of the budding of the venous endothelial cells and differentiation toward the lymphatic phenotype.[11] Adult Prox1± mice have increased fat apposition due to abnormal lymph leakage from mispatterned and ruptured lymphatic vessels, raising a possible link between impaired lymphatic drainage and obesity.[12]

Podoplanin- (also named T1α)-deficient mice (in a 129S/v genetic background) were originally generated in 2003 and these mice die at birth due to respiratory failure.[13] Podoplanin-/- animals have defects in lymphatic but not blood vessel pattern formation. The defects are associated with diminished lymphatic transport, congenital lymphedema and dilation of lymphatic vessels. Studies in cultured endothelial cells indicate that podoplanin promotes cell adhesion, migration and tube formation; whereas, small interfering RNA-mediated inhibition of podoplanin expression decreases LEC adhesion.[14] A second podoplanin-knockout mouse line (in a 50% 129S/v: 50% Swiss background) was reported in 2008. Interestingly, this podoplanin-knockout mouse model is characterized by increased embryonic and fetal death. Approximately 40% of the homozygote embryos die between stages E10 and E16. In addition, 50% of the neonatal mutant mice die within the first weeks of life. The cause of embryonic, fetal and neonatal death and the different phenotypes of two podoplanin-knockout mouse lines are presently not known.[15]

VEGFR-3 signals in LECs through a similar signaling transducing pathway as VEGFR-1 and VEGFR-2 in blood vascular endothelial cells, but it does not overlap with them. VEGFR-3 deletion causes embryonic death at E9.5. The defect is related to the remodeling of the primary vascular network, which occurs before the emergence of the lymphatic vessels.[16] During embryo development from E8.5 to E12.5, VEGFR-3 mRNA is expressed in angioblasts of head mesenchyme and the cardinal vein. At later stages, however, VEGFR-3 becomes restricted to the lymphatic vessels,[17,18] suggesting that VEGFR-3 may play a more important role in LECs. To support this, transgenic (Tg) mice carrying a soluble form of inhibitory VEGFR-3-Ig under the keratin-14 promoter, which directs transgene expression to the basal epidermal cells of the skin and neutralizes the activity of VEGF-C and VEGF-D, develop a lymphedema-like phenotype with normal blood vasculature.[19]

The most commonly used lymphatic markers in the literature include LYVE-1, Prox1, podoplanin and VEGFR-3. Lymphatic vessels do not express all these markers, which may be related to the stage of LEC differentiation and the microenvironment. The regulation of LEC marker expression is not clear and it is not known if external factors, such as cytokines and tissue injury, could affect the levels of these markers in LECs. Among these markers, the expression level of LYVE-1 is often unpredictable and is also unstable. Decreasing LYVE-1 expression is observed in LEC cell lines or primary LECs when they are cultured invitro.[20] A subset of inflammatory macrophages express Prox-1, podoplainin and VEGFR-3, but not LYVE-1.[21] Similarly, some LEC lines express all other LEC markers except LYVE-1.[22] High LYVE-1 levels are reported in lymphatic capillaries and the expression level is reduced in collecting lymphatic vessels.[20] The significance of changes in LYVE-1 expression levels is not clear. Since these LEC markers are not exclusively expressed by LECs[23] and they are often heterogeneously expressed in LECs between the initial and collecting lymph vessels,[20] more than one marker needs to be used for identification of LECs.

The lymphatic vessels are composed of primary valves, which localize at LEC junctions of collecting vessels and function to prevent fluid transport from the initial lymphatics back into the tissue space. Currently, the valves are identified by immunostaining with antiforkhead transcription factor (FOXC)-2 antibodies. The foxc2 gene is important for the normal development and maintenance of venous and lymphatic valves. Mutations in the foxc2 gene lead to lymphedema distichiasis, an inherited primary lymphedema due to lymphatic valve failure.[24] Another immunostaining phenotype of lymphatic valves is discontinuous expression of endothelial junction molecules, PECAM-1 and VE-cadherin, at the valve region, which may reflect the separation of local membrane regions between neighboring endothelial cells.[25]

A variety of factors affect lymphangiogenesis invitro and invivo. Members of the VEGF, platelet-derived growth factor (PDGF), fibroblast growth factor (FGF) and insulin-like growth factor (IGF) families have all been reported to influence lymphatics.[26] Among them, the most specific and potent LEC growth factors reported to date are VEGF-C and VEGF-D.[27,28] As with VEGF-A, -C and -D also induce proliferation, migration and growth of blood vascular endothelial cells by binding VEGFR-2.[26,29] However, unlike VEGF-A, they promote lymphangiogenesis through the VEGFR-3 signaling pathway.[30] This appears to be a nonredundant function, as VEGF-C-/- mice are embryonically lethal due to their lack of lymphatic vessels.[30] VEGF-C is normally expressed in the heart, lymph nodes (LNs), placenta and gut.[31] Under pathological conditions, VEGF-C is highly expressed by many other cell types, such as tumor cells and myeloid lineage cells, and plays a critical role in tumor and inflammation-related lymphangiogenesis.

The PDGF family of growth factors primarily serves the function of stabilizing vascular networks and plays a role in cancer metastases. Its members also act as lymphangiogenic factors. PDGF-BB stimulates MAP kinase activation activity and cell motility of primary LECs invitro and induces lymphatic vessel growth invivo. The expression of PDGF-BB in murine fibrosarcoma cells enhances tumor metastasis to LNs by promoting tumor lymphangiogenesis.[32] The overexpression of PDGF-BB recruits smooth muscle cells to newly formed lymphatic vessels and stimulates their maturation invivo.[33] These data suggest that targeting the PDGF/PDGF-receptor signaling pathway may provide a novel strategy to block tumor neoangiogenesis and lymphangiogenesis, thereby inhibiting tumor growth and metastasis.[34]

In a mouse corneal angiogenesis assay, FGF-2 stimulates both lymphangiogenesis and angiogenesis. FGF-2 upregulates VEGF-C expression in vascular endothelial and perivascular cells. Anti-VEGFR-3 neutralizing antibody inhibits FGF-2-induced lymphangiogenesis. Since the expression of both FGF-2 and VEGF-C is increased in cancer cells, anti-FGF may be a potential strategy for the inhibition of lymphatic metastasis in cancer therapy.[35]

Lymphatic endothelial cells express the receptors for IGFs. IGF-1 and IGF-2 induce lymphangiogenesis using the same mouse corneal angiogenesis assay described above. Interestingly, unlike FGF-2, whose effect on LECs can be blocked through inhibition of VEGFR-3,[35] IGF-1-induced lymphangiogenesis could not be blocked by a soluble VEGFR3-Ig.[36] Invitro, IGF-1 and IGF-2 significantly stimulate proliferation and migration of primary LECs, and induce phosphorylation of several intracellular signaling pathways, such as Akt, Src and ERK in these cells. Thus, IGFs might act as direct lymphangiogenic factors. Since members of the IGF ligand and receptor families are widely expressed in various types of solid tumors, IGF signaling molecules are likely to contribute to lymphatic metastasis.

Most of these studies used the mouse corneal angiogenesis assay, yet the cornea normally lacks blood and lymphatic vessels. Thus, the physiological and pathological roles of this growth factor-induced lymphangiogenesis need to be further investigated in other disease models where the lymphatic circulation presents under physiologic conditions.

In Vivo
Dynamic Measurement of the Lymphatic Function

One of the major lymphatic functions is to transport protein-rich lymph from the extracellular spaces to local draining LNs.[1] Removal of lymph from the diseased sites to local draining LNs may be beneficial or detrimental to the disease process, depending on the nature of the disease.[2,3] However, approaches to noninvasively and routinely measure lymphatic draining function invivo, especially in mice are lacking. This limits our investigation of the role of lymphatic flow and its molecular regulation using mouse models.

Previous imaging methods that have been used to study rodent lymphatics include whole body x-ray, magnetic resonance imaging (MRI), optical imaging, ultrasound and intravital microscopy. Engeset etal. first used x-ray radiography to image lymphatic drainage through intradermal injection of mercury in the mouse tail, which drained into the ischial LNs.[37] MRI lymphangiography with dendrimer-based macromolecular agents conjugated with gadolinium enabled the detection of deep LNs and lymphatic vessels in mice.[38,39,40] Leu etal. described the circumferential 'honeycomb' lymphatic structure using intravital microscopy and measured the lymph flow velocity in the superficial lymphatic network of the mouse tail after intradermal injection of FITC-dextran.[41] Fluorescent quantum dots for whole-body LN mapping and lymphatic drainage to nodes have also been reported.[42] Although all these technologies can visualize the path of a contrast agent within lymphatic vessels and measure the lymph flow rate, they require special instruments, highly trained personnel and are expensive, which limits their use as routine methods in laboratory animals.

Last year, Sharma etal. reported the utilization of indocyanine green (ICG), a near-infrared (NIR) fluorophore that has been used in clinical practice for more than 50years,[43] and NIR fluorescent imaging to examine the lymphatic drainage in swine.[44] Subsequently, they used the same approach in humans to image propulsive lymphatic trafficking from human breast tissue to the axillary lymph basin.[45] A similar procedure was also successfully applied in normal hairless mice.[46] The advantage of the NIR-ICG imaging technique is that it is dynamic and noninvasive, which allows researchers to longitudinally quantify rate, velocity and other properties of the lymphatic fluid in mice. Using the NIR-ICG imaging in TNF-Tg mice with established arthritis, we examined the lymphatic drainage capacity from the inflamed foot to local draining LNs; that is, popliteal LNs (PLNs) of these mice. The ICG fluorescent signal was identified in the PLN within 30min after intradermal injection of ICG into the footpad and disappeared gradually as the lymphatic flow moved to distant LNs. The ICG signal in the PLNs and the footpads disappeared on the next day in wild-type littermates but most signals remained in TNF-Tg mice (Figure1), suggesting impaired lymphatic trafficking and drainage away from the PLNs and footpads of the arthritic legs. Although several technical issues remain to be solved, such as reduction of animal variation and standardization of analytic methods, NIR-ICG may represent a new tool to investigate lymph draining function and its change in response to potential mediators or under pathologic conditions in mice. The NIR-ICG image has several advantages over other methods and it is simple, inexpensive, portable and easy to perform.

Figure 1.

Delayed lymphatic clearance from the popliteal LNs and footpad of TNF-Tg mice with established arthritis by ICG-NIR fluorescent imaging. A dose of 1 µg/10 µl ICG was intradermally injected into the footpad of a 6-month-old TNF-Tg mouse with severe ankle joint arthritis and a wild-type littermate. NIR imaging was 1, 24 and 48 h later. The intensity of ICG fluorescence in the popliteal LNs or injection sites at different time points after ICG injection in TNF-Tg and wild-type mice is shown. ICG fluorescence disappears rapidly from the popliteal LNs or injection sites of wild-type mice with time but remains in TNF-Tg mice, suggesting delayed lymphatic clearance in the arthritic leg. ICG = Indocyanine green; LN = Lymph node; NIR = Near infrared; Tg = Transgenic.

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