Molecular control of lymphangiogenesis

The lymphatic vasculature plays a critical role in the regulation of body fluid volume and immune function. Extensive research into the molecular mechanisms that control blood vessel growth has led to identification of molecules that also regulate development and growth of the lymphatic vessels. This is generating a great deal of interest in the molecular control of the lymphatics in the context of embryogenesis, lymphatic disorders and tumor metastasis. Studies in animal models carried out over the past three years have shown that the soluble protein growth factors, vascular endothelial growth factor (VEGF)-C and VEGF-D, and their cognate receptor tyrosine kinase, VEGF receptor-3 (VEGFR-3), are critical regulators of lymphangiogenesis. Furthermore, disfunction of VEGFR-3 has recently been shown to cause lymphedema. The capacity to induce lymphangiogenesis by manipulation of the VEGF-C/VEGF-D/VEGFR-3 signaling pathway offers new opportunities to understand the function of the lymphatic system and to develop novel treatments for lymphatic disorders.     

Microanatomy of the intestinal lymphatic system

The intestinal lymphatic system comprises two noncommunicating lymphatic networks: one containing the lacteals draining the villi and the connecting submucosal lymphatic network and one containing the lymphatics that drain the intestine muscular layer. These systems deliver lymph into a common network of collecting lymphatics originating near the mesenteric border. The intestinal lymphatic system serves vital functions in the regulation of tissue fluid homeostasis, immune surveillance, and the transport of nutrients; conversely, this system is affected by, and directly contributes to, disease processes within the intestine. Recent discoveries of specific lymphatic markers, factors promoting lymphangiogenesis, and factors selectively affecting the development of intestinal lymphatics, hold promise for unlocking the role of lymphatics in the pathogenesis of diseases affecting the intestine and for intestinal lymphatic selective therapies. Vital to progress in understanding how the intestinal lymphatic system functions is the integration of recent advances identifying molecular pathways for lymphatic growth and remodeling with advanced imaging modalities to observe lymphatic function and dysfunction in vivo.     

Lymphatic function is regulated by a coordinated expression of lymphangiogenic and anti-lymphangiogenic cytokines

Lymphangiogenic cytokines such as vascular endothelial growth factor-C (VEGF-C) are critically required for lymphatic regeneration; however, in some circumstances, lymphatic function is impaired despite normal or elevated levels of these cytokines. The recent identification of anti-lymphangiogenic molecules such as interferon-γ (IFN-γ), transforming growth factor-β1, and endostatin has led us to hypothesize that impaired lymphatic function may represent a dysregulated balance in the expression of pro/anti-lymphangiogenic stimuli. We observed that nude mice have significantly improved lymphatic function compared with wild-type mice in a tail model of lymphedema. We show that gradients of lymphatic fluid stasis regulate the expression of lymphangiogenic cytokines (VEGF-A, VEGF-C, and hepatocyte growth factor) and that paradoxically the expression of these molecules is increased in wild-type mice. More importantly, we show that as a consequence of T-cell-mediated inflammation, these same gradients also regulate expression patterns of anti-lymphangiogenic molecules corresponding temporally and spatially with impaired lymphatic function in wild-type mice. We show that neutralization of IFN-γ significantly increases inflammatory lymph node lymphangiogenesis independently of changes in VEGF-A or VEGF-C expression, suggesting that alterations in the balance of pro- and anti-lymphangiogenic cytokine expression can regulate lymphatic vessel formation. In conclusion, we show that gradients of lymphatic fluid stasis regulate not only the expression of pro-lymphangiogenic cytokines but also potent suppressors of lymphangiogenesis as a consequence of T-cell inflammation and that modulation of the balance between these stimuli can regulate lymphatic function.     

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.     

Vascular endothelial growth factor-C-mediated lymphangiogenesis promotes tumour metastasis

Metastasis is a frequent and lethal complication of cancer. Vascular endothelial growth factor-C (VEGF-C) is a recently described lymphangiogenic factor. Increased expression of VEGF-C in primary tumours correlates with dissemination of tumour cells to regional lymph nodes. However, a direct role for VEGF-C in tumour lymphangiogenesis and subsequent metastasis has yet to be demonstrated. Here we report the establishment of transgenic mice in which VEGF-C expression, driven by the rat insulin promoter (Rip), is targeted to beta-cells of the endocrine pancreas. In contrast to wild-type mice, which lack peri-insular lymphatics, RipVEGF-C transgenics develop an extensive network of lymphatics around the islets of Langerhans. These mice were crossed with Rip1Tag2 mice, which develop pancreatic beta-cell tumours that are neither lymphangiogenic nor metastatic. Double-transgenic mice formed tumours surrounded by well developed lymphatics, which frequently contained tumour cell masses of beta-cell origin. These mice frequently developed pancreatic lymph node metastases. Our findings demonstrate that VEGF-C-induced lymphangiogenesis mediates tumour cell dissemination and the formation of lymph node metastases.     

Normal lymphatic development and function in mice deficient for the lymphatic hyaluronan receptor LYVE-1

The hyaluronan receptor LYVE-1 is expressed abundantly on the surfaces of lymphatic vessels and lymph node sinus endothelial cells from early development, where it has been suggested to function both in cell adhesion/transmigration and as a scavenger for hyaluronan turnover. To investigate the physiological role(s) of LYVE-1, we generated mice in which the gene for the receptor was inactivated by replacement with a beta-galactosidase reporter. LYVE-1(-/-) mice displayed an apparently normal phenotype, with no obvious alteration in lymphatic vessel ultrastructure or function and no apparent change in secondary lymphoid tissue structure or cellularity. In addition, the levels of hyaluronan in tissue and blood were unchanged. LYVE-1(-/-) mice also displayed normal trafficking of cutaneous CD11c(+) dendritic cells to draining lymph nodes via afferent lymphatics and normal resolution of oxazolone-induced skin inflammation. Finally, LYVE-1(-/-) mice supported normal growth of transplanted B16F10 melanomas and Lewis lung carcinomas. These results indicate that LYVE-1 is not obligatory for normal lymphatic development and function and suggest either the existence of compensatory receptors or a role more specific than that previously envisaged.     

Role of lymphangiogenic factors in tumor metastasis

Nearly four centuries after the discovery of lymphatic vessels, the molecular mechanisms underlying their development are beginning to be elucidated. Vascular endothelial growth factor C (VEGF-C) and VEGF-D, via signaling through VEGFR-3, appear to be essential for lymphatic vessel growth. Observations from clinicopathological studies have suggested that lymphatic vessels serve as the primary route for the metastatic spread of tumor cells to regional lymph nodes. Recent studies in animal models have provided convincing evidence that tumor lymphangiogenesis facilitates lymphatic metastasis. However, it is not clear how tumor-associated lymphangiogenesis is regulated, and little is known about how tumor cells escape from the primary tumor and gain entry into the lymphatics. This review examines some of these issues and provides a brief summary of the recent developments in this field of research.     

Lymphangiogenesis and tumor metastasis

The lymphatic system transports interstitial fluid and macromolecules from tissues back to the blood circulation, and plays an important role in the immune response by directing the traffic of lymphocytes and antigen-presenting cells. The lymphatic system also constitutes one of the most important pathways of tumor dissemination. In many human cancers, increased expression of vascular endothelial growth factor-C (VEGF-C) is correlated with regional lymph node metastases. Experimental studies using transgenic mice overexpressing VEGF-C or xenotransplantation of VEGF-C-expressing tumor cells into immunodeficient mice have demonstrated a role for VEGF-C in tumor lymphangiogenesis and the subsequent formation of lymph node metastases. However, there is at present little evidence for lymphangiogenesis in human tumors and the relative importance of preexisting vs. newly formed lymphatics for metastasis in humans remains to be determined. Nonetheless, the striking correlation between the levels of VEGF-C in primary human tumors and lymph node metastases predicts its importance in cancer spread. Aside from promoting lymphangiogenesis, VEGF-C may also activate lymphatics to promote tumor cell chemotaxis, lymphatic intravasation and hence tumor cell dissemination.Work in the authors' laboratories was supported by grants from the Swiss National Science Foundation (no. 3100–064037.00) (to M.S.P), the Speaker's Fund for Biomedical Research (to M.S.) and the Peter Sharp Foundation (to M.S.). Parts of this review will be published in abbreviated form in Thrombosis and Haemostasis     

Lymphangiogenesis is required for pancreatic islet inflammation and diabetes

Lymphangiogenesis is a common phenomenon observed during inflammation and engraftment of transplants, but its precise role in the immune response and underlying mechanisms of regulation remain poorly defined. Here we showed that in response to injury and autoimmunity, lymphangiogenesis occurred around islets and played a key role in the islet inflammation in mice. Vascular endothelial growth factors receptor 3 (VEGFR3) is specifically involved in lymphangiogenesis, and blockade of VEGFR3 potently inhibited lymphangiogenesis in both islets and the draining LN during multiple low-dose streptozotocin (MLDS) induced autoimmune insulitis, which resulted in less T cell infiltration, preservation of islets and prevention of the onset of diabetes. In addition to their well-known conduit function, lymphatic endothelial cells (LEC) also produced chemokines in response to inflammation. These LEC attracted two distinct CX3CR1(hi) and LYVE-1(+) macrophage subsets to the inflamed islets and CX3CR1(hi) cells were influenced by LEC to differentiate into LYVE-1(+) cells closely associated with lymphatic vessels. These observations indicate a linkage among lymphangiogenesis and myeloid cell inflammation during insulitis. Thus, inhibition of lymphangiogenesis holds potential for treating insulitis and autoimmune diabetes.     

lyve1 expression reveals novel lymphatic vessels and new mechanisms for lymphatic vessel development in zebrafish

We have generated novel transgenic lines that brightly mark the lymphatic system of zebrafish using the lyve1 promoter. Facilitated by these new transgenic lines, we generated a map of zebrafish lymphatic development up to 15 days post-fertilisation and discovered three previously uncharacterised lymphatic vessel networks: the facial lymphatics, the lateral lymphatics and the intestinal lymphatics. We show that a facial lymphatic vessel, termed the lateral facial lymphatic, develops through a novel developmental mechanism, which initially involves vessel growth through a single vascular sprout followed by the recruitment of lymphangioblasts to the vascular tip. Unlike the lymphangioblasts that form the thoracic duct, the lymphangioblasts that contribute to the lateral facial lymphatic vessel originate from a number of different blood vessels. Our work highlights the additional complexity of lymphatic vessel development in the zebrafish that may increase its versatility as a model of lymphangiogenesis.     

Lymphatic ontogeny and effect of hypoplasia in developing lung

The pulmonary lymphatic vasculature plays a vital role in maintaining fluid homeostasis required for efficient gas exchange at capillary alveolar barriers and contributes to lung fluid clearance at birth. To further understanding of pulmonary lymphatic function at birth, lineage-tracing analysis of mouse lung was used. Lineage analysis confirmed that lymphatic endothelial cells (LEC) bud from extrapulmonary lymphatics and demonstrated that LEC migrate into developing lung along precise pathways. LEC cluster first in the primary bronchovascular region then along the secondary broncho-arterial regions and along veins. Small lymphatic vessels in distal lung develop from LEC that have migrated into lung mesenchyme from the extrapulmonary lymphatics. Finally, proximal and distal lymphatics remodel to form vessels with lumens in stereotypical locations. Loss of function analysis with lung-specific expression of a secreted form of the extracellular domain of vascular endothelial growth factor receptor-3 (dnR3) caused significant embryonic pulmonary lymphatic hypoplasia with fourfold reduction in distal LEC. Lung-specific expression of dnR3 did not affect blood vascular development, overall lung organogenesis or lymphatic development in other organs. Neonatal mice with pulmonary lymphatic hypoplasia developed respiratory distress with significantly increased mortality. During the transition to air breathing, lymphatic hypoplasia adversely affected fetal lung fluid clearance as determined by wet/dry weight analysis and morphometric analysis of bronchovascular cuffing and mesenchymal thickening. Surfactant synthesis was unaffected. Together, these data demonstrate that lung lymphatics develop autonomously and that pulmonary lymphatic hypoplasia is detrimental to survival of the neonate due to impaired lung fluid clearance.     

Lymphatic network and lymphangiogenesis in the gastric wall.

A family of growth factors highly specific for endothelial cells was identified more than 10 years ago, in which the receptor of vascular endothelial growth factor C (VEGFR-3) is implicated in the regulation of lymphatic development and regeneration. Comparative studies on the lymphatic network and lymphangiogenesis have been done mainly using combined 5'-nucleotidase (5'-Nase) enzyme and VEGFR-3 immunohistochemical approaches in adult and fetal gastric walls. Developing lymphatic networks represented fewer blind ends and branches than mature networks in whole-mount preparations. Many circular lymphatic-like structures exhibited VEGFR-3 expression and weak 5'-Nase activity in the early embryonic stage, showing visible morphological properties in the lymphatic endothelium. These newly formed lymphatics showed an obvious accumulation in the submucosa and serosa and a variation in the intensity of VEGFR-3 binding to endothelial cells among samples. A reaction product for anti-VEGFR-3 was found on the luminal surface of endothelial cells and on the membrane of some organelles and intraluminal lymphocytes. These findings indicate that an active proliferating feature of the clustered developing lymphatics may create a favorable environment for their sprouting and growth, which may serve as a functional requirement for lymph drainage in the region.     

Characteristics of Multi-Organ Lymphangiectasia Resulting from Temporal Deletion of Calcitonin Receptor-Like Receptor in Adult Mice

Adrenomedullin (AM) and its receptor complexes, calcitonin receptor-like receptor (Calcrl) and receptor activity modifying protein 2/3, are highly expressed in lymphatic endothelial cells and are required for embryonic lymphatic development. To determine the role of Calcrl in adulthood, we used an inducible Cre-loxP system to temporally and ubiquitously delete Calcrl in adult mice. Following tamoxifen injection, Calcrl^fl/fl/CAGGCre-ER™ mice rapidly developed corneal edema and inflammation that was preceded by and persistently associated with dilated corneoscleral lymphatics. Lacteals and submucosal lymphatic capillaries of the intestine were also dilated, while mesenteric collecting lymphatics failed to properly transport chyle after an acute Western Diet, culminating in chronic failure of Calcrl^fl/fl/CAGGCre-ER™ mice to gain weight. Dermal lymphatic capillaries were also dilated and chronic edema challenge confirmed significant and prolonged dermal lymphatic insufficiency. In vivo and in vitro imaging of lymphatics with either genetic or pharmacologic inhibition of AM signaling revealed markedly disorganized lymphatic junctional proteins ZO-1 and VE-cadherin. The maintenance of AM signaling during adulthood is required for preserving normal lymphatic permeability and function. Collectively, these studies reveal a spectrum of lymphatic defects in adult Calcrl^fl/fl/CAGGCre-ER™ mice that closely recapitulate the clinical symptoms of patients with corneal, intestinal and peripheral lymphangiectasia.     

Characterization of lymphangiogenesis in a model of adult skin regeneration

To date, adult lymphangiogenesis is not well understood. In this study we describe the evolution of lymphatic capillaries in regenerating skin and correlate lymphatic migration and organization with the expression of matrix metalloproteinases (MMPs), immune cells, the growth factors VEGF-A and VEGF-C, and the heparan sulfate proteogylcan perlecan, a key component of basement membrane. We show that while lymphatic endothelial cells (LECs) migrate and organize unidirectionally, in the direction of interstitial fluid flow, they do not sprout into the region but rather migrate as single cells that later join together into vessels. Furthermore, in a modified "shunted flow" version of the model, infiltrated LECs fail to organize into functional vessels, indicating that interstitial fluid flow is necessary for lymphatic organization. Perlecan expression on new lymphatic vessels was only observed after vessel organization was complete and also appeared first in the distal region, consistent with the directionality of lymphatic migration and organization. VEGF-C expression peaked at the initiation of lymphangiogenesis but was reduced to lower levels throughout organization and maturation. In mice lacking MMP-9, lymphatics regenerated normally, suggesting that MMP-9 is not required for lymphangiogenesis, at least in mouse skin. This study thus characterizes the process of adult lymphangiogenesis and differentiates it from sprouting blood angiogenesis, verifies its dependence on interstitial fluid flow for vessel organization, and correlates its temporal evolution with those of relevant environmental factors.     

Active interaction of human A375 melanoma cells with the lymphatics in vivo

We have used the avian chorioallantoic membrane (CAM) to study the interaction of tumor cells with the lymphatics in vivo. The vascular endothelial growth factor-C (VEGF-C) has been shown to be lymphangiogenic. We have therefore grown VEGF-C-expressing human A375 melanoma cells on the CAM. These tumors induced numerous lymphatics at the invasive front, and compressed or destroyed VEGF receptor (R)-3-positive lymphatics were observed within the solid tumors. The lymphatics in the CAM and in the A375 melanomas could also be demonstrated with an antibody against Prox 1, a highly specific marker of lymphatic endothelial cells. Proliferation studies revealed a BrdU labeling index of 11.6% of the lymphatic endothelial cells in the tumors and at their margins. A great number of melanoma cells invaded the lymphatics. Such interactions were not observed with VEGF-C-negative Malme 3 M melanoma cells. Lymphangiogenesis was inhibited to some extent when A375 melanoma cells were transfected with cDNA encoding soluble VEGFR-3 (sflt4), and the BrdU labeling index of the lymphatics in these tumors was 3.9%. Invasion of lymphatics and growth of blood vascular capillaries were not inhibited by the transfection. Therefore, tumor-induced lymphangiogenesis seems to be dependent to some extent on VEGF-C/flt4 interactions, but invasion of lymphatics seems to be a distinct mechanism.     

Soluble VEGFR-2: an antilymphangiogenic variant of VEGF receptors

The vascular endothelial growth factor (VEGF) family of secreted proteins and their receptors are major regulators of blood vessel development (hemangiogenesis) and lymphatic vessel development (lymphangiogenesis). VEGF acts through a complex system of receptor tyrosine kinases, which can be membrane bound or soluble. New data concerning the receptor system are still emerging, thus contributing to the complexity of the system. Very recently a soluble form of VEGFR-2, termed sVEGFR-2, which is a result of alternative splicing, has been discovered. Earlier, it has been shown that a secreted/soluble form of VEGFR-1, termed sVEGFR-1, is produced by alternative splicing and exerts an antihemangiogenic effect by binding VEGF-A. The newly discovered spliced variant of sVEGFR-2 binds the lymphangiogenic growth factor VEGF-C and thus inhibits VEGF-C-induced activation of VEGFR-3, consequently inhibiting lymphatic endothelial cell proliferation. Its inactivation in murine embryos permits hyperplasia of dermal lymphatics and invasion of lymphatics into the cornea. Tumor lymphangiogenesis seems to influence the metastatic behavior of malignant cells. A correlation has been found between the downregulation of sVEGFR-2 and the malignant progression of neuroblastoma, which is characterized by lymphogenic metastases in progressed stages. Data show that lymphangiogenesis is regulated by both activators and inhibitors, and its balance is crucial in health and disease.     

Hyaluronan promotes tumor lymphangiogenesis and intralymphantic tumor growth in xenografts

Hyaluronan （HA）, a high molecular weight glycosaminoglycan in the extracellular matrix, has been implicated in the promotion of malignant phenotypes, including tumor angiogenesis. However, little is known about the effect of HA on tumor-associated lymphangiogenesis. In this study, mouse hepatocellular carcinoma Hca-F cells combined with or without HA were injected subcutaneously into C3H/Hej mice, then angiogenesis and lymphangiogenesis of implanted tumors were examined by immunostaining for plateletendothelial cell adhesion molecule-1 and lymphatic vascular endothelial hyaluronan receptor-1 respectively. Interestingly, we found HA promotes tumor lymphangiogenesis and the occurrence of intratumoral lymphatic vessels, but has little effect on tumor angiogenesis. Moreover, HA also promotes intralymphatic tumor growth, although it is not sufficient to potentiate lymphatic metastasis. These results suggest that HA, which is elevated in most malignant tumor stroma, may also play a role in tumor progression by promoting lymphangiogenesis.     

An In Vivo Method to Quantify Lymphangiogenesis in Zebrafish

Background

Lymphangiogenesis is a highly regulated process involved in the pathogenesis of disease. Current in vivo models to assess lymphangiogenesis are largely unphysiologic. The zebrafish is a powerful model system for studying development, due to its rapid growth and transparency during early stages of life. Identification of a network of trunk lymphatic capillaries in zebrafish provides an opportunity to quantify lymphatic growth in vivo.

Methods and Results

Late-phase microangiography was used to detect trunk lymphatic capillaries in zebrafish 2- and 3-days post-fertilization. Using this approach, real-time changes in lymphatic capillary development were measured in response to modulators of lymphangiogenesis. Recombinant human vascular endothelial growth factor (VEGF)-C added directly to the zebrafish aqueous environment as well as human endothelial and mouse melanoma cell transplantation resulted in increased lymphatic capillary growth, while morpholino-based knockdown of vegfc and chemical inhibitors of lymphangiogenesis added to the aqueous environment resulted in decreased lymphatic capillary growth.

Conclusion

Lymphatic capillaries in embryonic and larval zebrafish can be quantified using late-phase microangiography. Human activators and small molecule inhibitors of lymphangiogenesis, as well as transplanted human endothelial and mouse melanoma cells, alter lymphatic capillary development in zebrafish. The ability to rapidly quantify changes in lymphatic growth under physiologic conditions will allow for broad screening of lymphangiogenesis modulators, as well as help define cellular roles and elucidate pathways of lymphatic development.