Characteristics of lymphatic endothelial cells in physiological and pathological conditions

Impairment of lymphatic structure and function, e.g., inadequate endothelial permeability and intercellular openings, abnormal lymphangiogenesis and overexpression for immunoreactive agents, will result in tumor metastasis, autoimmune response alteration and accumulation of interstitial fluid and proteins. Recently, several novel molecules have been identified that allow a more precise distinction between lymphatic and blood vascular endothelium. The differences in expression of endothelial markers on the lymphatic vessel strongly suggest the possibility that there will be important divergence in the differentiating and regenerating responses in lymphatic behavior to various pathological processes. Undoubtfully, molecular techniques would also lead to the definition of unique markers found on lymphatic endothelial cells (LECs) in lymphatic-associated diseases which are mostly involved in lymphangiogenesis. This review is mainly concentrated on the characteristics of LECs in diabetes, wound healing, lymphedema and tumor, especially in the experimental models that have offered insight into the LEC role in these diseases affecting the lymphatic system. Increased knowledge of the molecular signaling pathways driving lymphatic development and lymphangiogenesis should boost the impact of therapeutics on the diseases. Although the field about the mechanisms that control the formation and lineage-specific differentiation and function of lymphatic vessels has experienced rapid progress in the past few years, an understanding of the basis of the differences and their implications in the pathological conditions will require much more investigation.     

Walking the Line: A Fibronectin Fiber-Guided Assay to Probe Early Steps of (Lymph)angiogenesis

Angiogenesis and lymphangiogenesis are highly complex morphogenetic processes, central to many physiological and pathological conditions, including development, cancer metastasis, inflammation and wound healing. While it is described that extracellular matrix (ECM) fibers are involved in the spatiotemporal regulation of angiogenesis, current angiogenesis assays are not specifically designed to dissect and quantify the underlying molecular mechanisms of how the fibrillar nature of ECM regulates vessel sprouting. Even less is known about the role of the fibrillar ECM during the early stages of lymphangiogenesis. To address such questions, we introduced here an in vitro (lymph)angiogenesis assay, where we used microbeads coated with endothelial cells as simple sprouting sources and deposited them on single Fn fibers used as substrates to mimic fibrillar ECM. The fibers were deposited on a transparent substrate, suitable for live microscopic observation of the ensuing cell outgrowth events at the single cell level. Our proof-of-concept studies revealed that fibrillar Fn, compared to Fn-coated surfaces, provides far stronger sprouting and guidance cues to endothelial cells, independent of the tested mechanical strains of the Fn fibers. Additionally, we found that VEGF-A, but not VEGF-C, stimulates the collective outgrowth of lymphatic endothelial cells (LEC), while the collective outgrowth of blood vascular endothelial cells (HUVEC) was prominent even in the absence of these angiogenic factors. In addition to the findings presented here, the modularity of our assay allows for the use of different ECM or synthetic fibers as substrates, as well as of other cell types, thus expanding the range of applications in vascular biology and beyond.     

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.     

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.     

Hepatocyte growth factor promotes lymphatic vessel formation and function

The lymphatic vascular system plays a pivotal role in mediating tissue fluid homeostasis and cancer metastasis, but the molecular mechanisms that regulate its formation and function remain poorly characterized. A comparative analysis of the gene expression of purified lymphatic endothelial cells (LEC) versus blood vascular endothelial cells (BVEC) revealed that LEC express significantly higher levels of hepatocyte growth factor receptor (HGF-R). Whereas little or no HGF-R expression was detected by lymphatic vessels of normal tissues, HGF-R was strongly expressed by regenerating lymphatic endothelium during tissue repair and by activated lymphatic vessels in inflamed skin. Treatment of cultured LEC with HGF promoted LEC proliferation, migration and tube formation. HGF-induced proliferation of LEC did not require vascular endothelial growth factor receptor-3 activation, and HGF-induced cell migration was partially mediated via integrin alpha-9. Transgenic or subcutaneous delivery of HGF promoted lymphatic vessel formation in mice, whereas systemic blockade of HGF-R inhibited lymphatic function. These results identify HGF as a novel, potent lymphangiogenesis factor, and also indicate that HGF-R might serve as a new target for inhibiting pathological lymphangiogenesis.     

Lymphangiogenesis and Lymphatic Remodeling Induced by Filarial Parasites: Implications for Pathogenesis

Even in the absence of an adaptive immune system in murine models, lymphatic dilatation and dysfunction occur in filarial infections, although severe irreversible lymphedema and elephantiasis appears to require an intact adaptive immune response in human infections. To address how filarial parasites and their antigens influence the lymphatics directly, human lymphatic endothelial cells were exposed to filarial antigens, live parasites, or infected patient serum. Live filarial parasites or filarial antigens induced both significant LEC proliferation and differentiation into tube-like structures in vitro. Moreover, serum from patently infected (microfilaria positive) patients and those with longstanding chronic lymphatic obstruction induced significantly increased LEC proliferation compared to sera from uninfected individuals. Differentiation of LEC into tube-like networks was found to be associated with significantly increased levels of matrix metalloproteases and inhibition of their TIMP inhibitors (Tissue inhibitors of matrix metalloproteases). Comparison of global gene expression induced by live parasites in LEC to parasite-unexposed LEC demonstrated that filarial parasites altered the expression of those genes involved in cellular organization and development as well as those associated with junction adherence pathways that in turn decreased trans-endothelial transport as assessed by FITC-Dextran. The data suggest that filarial parasites directly induce lymphangiogenesis and lymphatic differentiation and provide insight into the mechanisms underlying the pathology seen in lymphatic filariasis.     

Extracellular Matrix in Neural Plasticity and Regeneration

The extracellular matrix (ECM) is a fundamental component of biological tissues. The ECM in the central nervous system (CNS) is unique in both composition and function. Functions such as learning, memory, synaptogenesis, and plasticity are regulated by numerous ECM molecules. The neural ECM acts as a non-specific physical barrier that modulates neuronal plasticity and axon regeneration. There are two specialized types of ECM in the CNS, diffuse perisynaptic ECM and condensed ECM, which selectively surround the perikaryon and initial part of dendritic trees in subtypes of neurons, forming perineuronal nets. This review presents the current knowledge about the role of important neuronal ECM molecules in maintaining the basic functions of a neuron, including electrogenesis and the ability to form neural circuits. The review mainly focuses on the role of ECM components that participate in the control of key events such as cell survival, axonal growth, and synaptic remodeling. Particular attention is drawn to the numerous molecular partners of the main ECM components. These regulatory molecules are integrated into the cell membrane or disposed into the matrix itself in solid or soluble form. The interaction of the main matrix components with molecular partners seems essential in molecular mechanisms controlling neuronal functions. Special attention is paid to the chondroitin sulfate proteoglycan 4, type 1 transmembrane protein, neural-glial antigen 2 (NG2/CSPG4), whose cleaved extracellular domain is such a molecular partner that it not only acts directly on neural and vascular cells, but also exerts its influence indirectly by binding to resident ECM molecules.

     

Platelet activation receptor CLEC-2 regulates blood/lymphatic vessel separation by inhibiting proliferation, migration, and tube formation of lymphatic endothelial cells

The platelet activation receptor CLEC-2 plays crucial roles in thrombosis/hemostasis, tumor metastasis, and lymphangiogenesis, although its role in thrombosis/hemostasis remains controversial. An endogenous ligand for CLEC-2, podoplanin, is expressed in lymphatic endothelial cells (LECs). We and others have reported that CLEC-2-deficiency is lethal at mouse embryonic/neonatal stages associated with blood-filled lymphatics, indicating that CLEC-2 is essential for blood/lymphatic vessel separation. However, its mechanism, and whether CLEC-2 in platelets is necessary for this separation, remains unknown. We found that specific deletion of CLEC-2 from platelets leads to the misconnection of blood/lymphatic vessels. CLEC-2(+/+) platelets, but not by CLEC-2(-/-) platelets, inhibited LEC migration, proliferation, and tube formation but had no effect on human umbilical vein endothelial cells. Additionally, supernatants from activated platelets significantly inhibited these three functions in LECs, suggesting that released granule contents regulate blood/lymphatic vessel separation. Bone morphologic protein-9 (BMP-9), which we found to be present in platelets and released upon activation, appears to play a key role in regulating LEC functions. Only BMP-9 inhibited tube formation, although other releasates including transforming growth factor-β and platelet factor 4 inhibited proliferation and/or migration. We propose that platelets regulate blood/lymphatic vessel separation by inhibiting the proliferation, migration, and tube formation of LECs, mainly because of the release of BMP-9 upon activation by CLEC-2/podoplanin interaction.     

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.     

Mesenchymal stem cells promote lymphangiogenic properties of lymphatic endothelial cells

Lymphatic metastasis is one of the main prognostic factors concerning long‐term survival of cancer patients. In this regard, the molecular mechanisms of lymphangiogenesis are still rarely explored. Also, the interactions between stem cells and lymphatic endothelial cells (LEC) in humans have not been well examined. Therefore, the main objective of this study was to assess the interactions between mesenchymal stem cells (MSC) and LEC using in vitro angiogenesis assays. Juvenile LEC were stimulated with VEGF‐C, bFGF, MSC‐conditioned medium (MSC‐CM) or by co‐culture with MSC. LEC proliferation was assessed using a MTT assay. Migration of the cells was determined with a wound healing assay and a transmigration assay. To measure the formation of lymphatic sprouts, LEC spheroids were embedded in collagen or fibrin gels. The LEC's capacity to form capillary‐like structures was assessed by a tube formation assay on Matrigel^®. The proliferation, migration and tube formation of LEC could be significantly enhanced by MSC‐CM and by co‐culture with MSC. The effect of stimulation with MSC‐CM was stronger compared to stimulation with the growth factors VEGF‐C and bFGF in proliferation and transmigration assays. Sprouting was stimulated by VEGF‐C, bFGF and by MSC‐CM. With this study, we demonstrate the potent stimulating effect of the MSC secretome on proliferation, migration and tube formation of LEC. This indicates an important role of MSC in lymphangiogenesis in pathological as well as physiological processes.     

In vitro assays using primary embryonic mouse lymphatic endothelial cells uncover key roles for FGFR1 signalling in lymphangiogenesis

Despite the importance of blood vessels and lymphatic vessels during development and disease, the signalling pathways underpinning vessel construction remain poorly characterised. Primary mouse endothelial cells have traditionally proven difficult to culture and as a consequence, few assays have been developed to dissect gene function and signal transduction pathways in these cells ex vivo. Having established methodology for the purification, short-term culture and transfection of primary blood (BEC) and lymphatic (LEC) vascular endothelial cells isolated from embryonic mouse skin, we sought to optimise robust assays able to measure embryonic LEC proliferation, migration and three-dimensional tube forming ability in vitro. In the course of developing these assays using the pro-lymphangiogenic growth factors FGF2 and VEGF-C, we identified previously unrecognised roles for FGFR1 signalling in lymphangiogenesis. The small molecule FGF receptor tyrosine kinase inhibitor SU5402, but not inhibitors of VEGFR-2 (SU5416) or VEGFR-3 (MAZ51), inhibited FGF2 mediated LEC proliferation, demonstrating that FGF2 promotes proliferation directly via FGF receptors and independently of VEGF receptors in primary embryonic LEC. Further investigation revealed that FGFR1 was by far the predominant FGF receptor expressed by primary embryonic LEC and correspondingly, siRNA-mediated FGFR1 knockdown abrogated FGF2 mediated LEC proliferation. While FGF2 potently promoted LEC proliferation and migration, three dimensional tube formation assays revealed that VEGF-C primarily promoted LEC sprouting and elongation, illustrating that FGF2 and VEGF-C play distinct, cooperative roles in lymphatic vascular morphogenesis. These assays therefore provide useful tools able to dissect gene function in cellular events important for lymphangiogenesis and implicate FGFR1 as a key player in developmental lymphangiogenesis in vivo.     

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.     

Targeting angiogenesis with compounds from the extracellular matrix

The extracellular matrix (ECM) is the central element of a pericellular network of bioactive molecules. It orchestrates molecular interactions, availability and activity, acting as a key regulator of cell functions and complex biological processes, including physiological and pathological angiogenesis. The ECM serves as a source of both stimulatory and inhibitory angiogenesis regulatory factors. The observation that several endogenous inhibitors of angiogenesis derive from the ECM proves its importance in physiological angiogenesis, and point to the ECM as a precious source of therapeutic agents for angiogenesis-driven diseases, including cancer growth and metastatic dissemination. This review focuses on the different approaches to exploit ECM molecules for designing tools for therapeutic inhibition or monitoring of pathological angiogenesis, with particular focus on antineoplastic therapy, and emphasis on peptides of ECM moieties and mimetic small molecules.     

Genetic dissection of tumor angiogenesis: are PlGF and VEGFR-1 novel anti-cancer targets?

Many proliferative diseases, most typically cancer, are driven by uncontrolled blood vessel growth. Genetic studies have been very helpful in unraveling the cellular and molecular players in pathological blood vessel formation and have provided opportunities to reduce tumor growth and metastasis. The fact that tumor vessels and normal blood vessels have distinct properties may help in designing more specific--and therefore safer--anti-angiogenic strategies. Such strategies may interfere with angiogenesis at the cellular or molecular level. Possible molecular targets include angiogenic growth factors and their receptors, proteinases, coagulation factors, junctional/adhesion molecules and extracellular matrix (ECM) components. Some anti-angiogenic drugs, i.e., vascular endothelial growth factor (VEGF) antibodies and VEGF receptor-2 (VEGFR-2) inhibitors, have progressed into clinical cancer trials. While the results of these trials support the potential of anti-angiogenic therapy to treat cancer, they also demonstrate the need for more effective and safer alternatives. Targeting placental growth factor (PlGF) or VEGFR-1 may constitute such an alternative since animal studies have proven their pleiotropic working mechanism and attractive safety profile. Together, these insights may bring anti-angiogenic drugs closer from bench to bedside.     

Extracellular matrix and growth factors in the pathogenesis of some craniofacial malformations

The normal development of cranial primordia and orofacial structures involves fundamental processes in which growth, morphogenesis, and cell differentiation take place and interactions between extracellular matrix (ECM) components, growth factors and embryonic tissues are involved. Biochemical and molecular aspects of craniofacial development, such as the biological regulation of normal or premature cranial suture fusion, has just begun to be understood, thanks mainly to studies performed in the last decade. Several mutations has been identified in both syndromic and non-syndromic craniosynostosis patients throwing new light onto the etiology, classification and developmental pathology of these diseases. In the more common craniosynostosis syndromes and other skeletal growth disorders, the mutations were identified in the genes encoding fibroblast growth factor receptor types 1-3 (FGFR1, 2 and 3) where they are dominantly acting and affect specific and important protein binding domain. The unregulated FGF signaling during intramembranous ossification is associated to the Apert and Crouzon syndrome. The non syndromic cleft of the lip and/or palate (CLP) has a more complex genetic background if compared to craniosynostosis syndrome because of the number of involved genes and type of inheritance. Moreover, the influence of environmental factor makes difficult to clarify the primary causes of this malformation. ECM represents cell environment and results mainly composed by collagens, fibronectin, proteoglycans (PG) and hyaluronate (HA). Cooperative effects of ECM and growth factors regulate regional matrix production during the morphogenetic events, connective tissue remodelling and pathological states. In the present review we summarize the studies we performed in the last years to better clarify the role of ECM and growth factors in the etiology and pathogenesis of craniosynostosis and CLP diseases.     

Tensile mechanical properties of three-dimensional type I collagen extracellular matrices with varied microstructure

The importance and priority of specific micro-structural and mechanical design parameters must be established to effectively engineer scaffolds (biomaterials) that mimic the extracellular matrix (ECM) environment of cells and have clinical applications as tissue substitutes. In this study, three-dimensional (3-D) matrices were prepared from type I collagen, the predominant compositional and structural component of connective tissue ECMs, and structural-mechanical relationships were studied. Polymerization conditions, including collagen concentration (0.3-3 mg/mL) and pH (6-9), were varied to obtain matrices of collagen fibrils with different microstructures. Confocal reflection microscopy was used to assess specific micro-structural features (e.g., diameter and length) and organization of component fibrils in 3-D. Microstructural analyses revealed that changes in collagen concentration affected fibril density while maintaining a relatively constant fibril diameter. On the other hand, both fibril length and diameter were affected by the pH of the polymerization reaction. Mechanically, all matrices exhibited a similar stress-strain curve with identifiable "toe," "linear," and "failure" regions. However the linear modulus and failure stress increased with collagen concentration and were correlated with an increase in fibril density. Additionally, both the linear modulus and failure stress showed an increase with pH, which was related to an increasedfibril length and a decreasedfibril diameter. The tensile mechanical properties of the collagen matrices also showed strain rate dependence. Such fundamental information regarding the 3-D microstructural-mechanical properties of the ECM and its component molecules are important to our overall understanding of cell-ECM interactions (e.g., mechanotransduction) and the development of novel strategies for tissue repair and replacement.     

PtdIns(3,4)P2 instigates focal adhesions to generate podosomes

Cell-to-extracellular matrix (ECM) adhesion plays important roles in various biological events, such as proliferation, differentiation and migration. Distinct from other types of adhesion structures (focal complexes, focal adhesions and so on), podosomes and invadopodia are thought to have additional functions beyond attachment, possibly including invasion into the ECM. For podosomes and invadopodia to invade into the ECM, molecules involved in adhesion, actin polymerization and ECM degradation must be recruited to sites of action. Our recent study demonstrated that podosomes form near newly formed focal adhesions via the minimally expressed phosphoinositide PtdIns(3,4) P2-mediated recruitment of the Tks5-Grb2 scaffold, followed by the accumulation of N-WASP. Although this study demonstrated details of molecular interplay during the transformation of focal adhesion, its regulation in the in vivo invasion process remains to be clarified. Here, we discuss the molecular bases of the transformation of focal adhesions to podosomes/invadopodia based on current understanding.Key words: podosome, invadopodium, focal adhesion, Tks5, PtdIns(3,4)P2, N-WASP     

The interplay between the proteolytic,invasive, and adhesive domains of invadopodia and their roles in cancer invasion

Invadopodia are actin-based protrusions of the plasma membrane that penetrate into the extracellular matrix (ECM), and enzymatically degrade it. Invadopodia and podosomes, often referred to, collectively, as “invadosomes,” are actin-based membrane protrusions that facilitate matrix remodeling and cell invasion across tissues, processes that occur under specific physiological conditions such as bone remodeling, as well as under pathological states such as bone, immune disorders, and cancer metastasis. In this review, we specifically focus on the functional architecture of invadopodia in cancer cells; we discuss here three functional domains of invadopodia responsible for the metalloproteinase-based degradation of the ECM, the cytoskeleton-based mechanical penetration into the matrix, and the integrin adhesome-based adhesion to the ECM. We will describe the structural and molecular organization of each domain and the cross-talk between them during the invasion process.