Taija Mäkinen

Ephrin-B2 controls VEGF-induced angiogenesis and lymphangiogenesis

In development, tissue regeneration or certain diseases, angiogenic growth leads to the expansion of blood vessels and the lymphatic vasculature. This involves endothelial cell proliferation as well as angiogenic sprouting, in which a subset of cells, termed tip cells, acquires motile, invasive behaviour and extends filopodial protrusions. Although it is already appreciated that angiogenesis is triggered by tissue-derived signals, such as vascular endothelial growth factor (VEGF) family growth factors, the resulting signalling processes in endothelial cells are only partly understood. Here we show with genetic experiments in mouse and zebrafish that ephrin-B2, a transmembrane ligand for Eph receptor tyrosine kinases, promotes sprouting behaviour and motility in the angiogenic endothelium. We link this pro-angiogenic function to a crucial role of ephrin-B2 in the VEGF signalling pathway, which we have studied in detail for VEGFR3, the receptor for VEGF-C. In the absence of ephrin-B2, the internalization of VEGFR3 in cultured cells and mutant mice is defective, which compromises downstream signal transduction by the small GTPase Rac1, Akt and the mitogen-activated protein kinase Erk. Our results show that full VEGFR3 signalling is coupled to receptor internalization. Ephrin-B2 is a key regulator of this process and thereby controls angiogenic and lymphangiogenic growth.

Vascular Endothelial Growth Factor Receptor-3

Cell-cell communication during vascular development and tumour angiogenesis seems to involve at least five endothelial cell-specific tyrosine kinase receptors belonging to two distinct subclasses: two receptors of the Tie family, and three vascular endothelial cell growth factor receptors, VEGFR-1, -2 and -3, originally named Fltl (Fms-like tyrosine kinase), KDR/Flk-1 (Kinase insert-domain containing receptor or fetal-liver kinase-1) and Flt4, respectively. VEGFRs are subclass-III receptor tyrosine kinases, homologous to the platelet-derived growth factor (PDGF)-receptor family, having seven immunoglobulin homology domains in the extracellular domain, and a tyrosine kinase intracellular domain split by a kinase insert sequence (for recent reviews, see Klagsbrun and D’Amore 1996; Folkman and D’Amore 1996; Mustonen and Alitalo 1995; Korpelainen and Alitalo, 1998, Claesson-WELSH, this book).

Vegfc is required for vascular development and endoderm morphogenesis in zebrafish

During embryogenesis, complex morphogenetic events lead endodermal cells to coalesce at the midline and form the primitive gut tube and associated organs. While several genes have recently been implicated in endoderm differentiation, we know little about the genes that regulate endodermal morphogenesis. Here, we show that vascular endothelial growth factor C (Vegfc), an angiogenic as well as a lymphangiogenic factor, is unexpectedly involved in this process in zebrafish. Reducing Vegfc levels using morpholino antisense oligonucleotides, or through overexpression of a soluble form of the VEGFC receptor, VEGFR‐3, affects the coalescence of endodermal cells in the anterior midline, leading to the formation of a forked gut tube and the duplication of the liver and pancreatic buds. Further analyses indicate that Vegfc is additionally required for the initial formation of the dorsal endoderm. We also demonstrate that Vegfc is required for vasculogenesis as well as angiogenesis in the zebrafish embryo. These data argue for a requirement of Vegfc in the developing vasculature and, more surprisingly, implicate Vegfc signalling in two distinct steps during endoderm development, first during the initial differentiation of the dorsal endoderm, and second in the coalescence of the anterior endoderm to the midline.

Vascular endothelial growth factor receptor 3 directly regulates murine neurogenesis.

Neural stem cells (NSCs) are slowly dividing astrocytes that are intimately associated with capillary endothelial cells in the subventricular zone (SVZ) of the brain. Functionally, members of the vascular endothelial growth factor (VEGF) family can stimulate neurogenesis as well as angiogenesis, but it has been unclear whether they act directly via VEGF receptors (VEGFRs) expressed by neural cells, or indirectly via the release of growth factors from angiogenic capillaries. Here, we show that VEGFR-3, a receptor required for lymphangiogenesis, is expressed by NSCs and is directly required for neurogenesis. Vegfr3:YFP reporter mice show VEGFR-3 expression in multipotent NSCs, which are capable of self-renewal and are activated by the VEGFR-3 ligand VEGF-C in vitro. Overexpression of VEGF-C stimulates VEGFR-3-expressing NSCs and neurogenesis in the SVZ without affecting angiogenesis. Conversely, conditional deletion of Vegfr3 in neural cells, inducible deletion in subventricular astrocytes, and blocking of VEGFR-3 signaling with antibodies reduce SVZ neurogenesis. Therefore, VEGF-C/VEGFR-3 signaling acts directly on NSCs and regulates adult neurogenesis, opening potential approaches for treatment of neurodegenerative diseases.

Intrinsic versus microenvironmental regulation of lymphatic endothelial cell phenotype and function.

Vascular endothelial cells are characterized by a high degree of functional and phenotypic plasticity, which is controlled both by their pericellular microenvironment and their intracellular gene expression programs. To gain further insight into the mechanisms regulating the endothelial cell phenotype, we have compared the responses of lymphatic endothelial cells (LECs) and blood vascular endothelial cells (BECs) to vascular endothelial growth factors (VEGFs). VEGFR‐3‐specific signals are sufficient for LEC but not BEC proliferation, as shown by the ability of the specific ligand VEGF‐C156S to stimulate cell cycle entry only in LECs. On the other hand, we found that VEGFR‐3 stimulation did not induce LEC cell shape changes typical of VEGFR‐2‐stimulated LECs, indicating receptor‐specific differences in the cytoskeletal responses. Genes induced via VEGFR‐2 also differed between BECs and LECs: angiopoietin‐2 (Ang‐2) was induced via VEGFR‐2 in BECs and LECs, but the smooth muscle cell (SMC) chemoattractant BMP‐2 was induced only in BECs. Both BECs and LECs were able to promote SMC chemotaxis, but contact with SMCs led to down‐regulation of VEGFR‐3 expression in BECs in a 3‐dimensional coculture system. This was consistent with the finding that VEGFR‐3 is down‐regulated in vivo at sites of endothelial cell‐pericyte/smooth muscle cell contacts. Collectively, these data show intrinsic cell‐specific differences of BEC and LEC responses to VEGFs and identify a pericellular regulatory mechanism for VEGFR‐3 down‐regulation in endothelial cells.—Veikkola, T., Lohela, M., Ikenberg, K., Mäkinen, T., Korff, T., Saaristo, A., Jeltsch, M., Augustin, H. G., Alitalo, K. Intrinsic versus microenvironmental regulation of lymphatic endothelial cell phenotype and function. FASEB J. 17, 2006–2013 (2003)

Ephb receptors and ephrin-B3 regulate axon guidance at the ventral midline of the embryonic mouse spinal cord

EphB receptors and their ephrin-B ligands are required for midline guidance decisions at several rostrocaudal levels of the developing CNS. In the embryonic vertebrate spinal cord, ephrin-B3 is localized to the floor plate (FP) at the ventral midline (VM), ephrin-B1 and ephrin-B2 are expressed in the dorsal spinal cord, and decussated EphB receptor-bearing commissural axons navigate between these ventral and dorsal ephrin-B domains. Despite these compelling expression patterns, the in vivo role(s) for EphB and ephrin-B proteins in regulating the guidance of spinal commissural axons has not been established. Here, we use DiI (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate) labeling to assess the pathfinding of commissural axons in the spinal cords of ephrin-B and EphB mutant mouse embryos. In mice lacking ephrin-B3 or multiple EphB receptors, a significant number of axons followed aberrant trajectories in the immediate vicinity of the VM. Furthermore, forked transverse commissural (FTC) axons, a unique class of commissural axons that continues to project in the transverse plane on the contralateral side of the FP, were present at a markedly higher frequency in ephrin-B3 and EphB mutants, compared with wild-type embryos. Neither the midline guidance errors nor excessive numbers of FTC axons were observed in the spinal cords of ephrin-B3lacz mice that express a truncated form of ephrin-B3, which is capable of forward but not reverse signaling. In contrast to the midline guidance defects observed in EphB and ephrin-B3 mutant embryos, wild-type-like contralateral projections were observed in mice lacking ephrin-B1 and/or ephrin-B2.

Effect of inflammatory cytokines on the expression of the vascular endothelial growth factor‐C

The development of the vascular system involves vasculogenesis and angiogenesis. In vasculogenesis the endothelium of blood vessels forms by in situ differentiation from precursor cells called angioblasts. During later embryogenesis and adult life the new blood vessels are formed mainly via angiogenesis, most commonly involving sprouting of capillaries from preexisting blood vessels (Hanahan & Folkman 1996). n nAngiogenesis is thus an important process in many physiological and pathological conditions such as female reproductive functions, wound repair, tumour growth and metastasis, and chronic inflammatory diseases (Hanahan & Folkman 1996). Vascular endothelial growth factor (VEGF), also known as vascular permeability factor or vasculotropin, is an important angiogenic agent and the most specific known endothelial cell growth factor (Ferrara & Davis-Smyth 1997). VEGF also induces vascular permeability, regulates production of proteases and their inhibitors, and promotes endothelial cell differentiation, movement, and survival (Ferrara & Davis-Smyth 1997). Several VEGF isoforms are produced by alternative splicing of a single gene of which VEGF121, VEGF145 and VEGF165 are secreted soluble proteins and VEGF189 remains bound at the cell surface (Ferrara & Davis-Smyth 1997). VEGF homodimers bind and signal through tyrosine kinase receptors VEGFR-1 (Flt-1) and VEGFR-2 (Flk-1/KDR) that are expressed by the endothelial cells. Recently Soker et al. (1998) reported binding of VEGF165 isoform also to neuropilin-1 which has been previously identified as a receptor for the collapsin/semaphorin family. Genetic disruption of VEGF and its receptors indicate that they are necessary for vasculogenesis and/or angiogenesis. Knock-out mice of VEGFR-1 have abnormal vascular organization (Fong et al. 1995) and VEGFR-2 deficient mice show complete inhibition of vascular development (Shalaby et al. 1995). In addition, heterozygous VEGF knock-out mice have impaired blood vessel formation (Carmeliet et al. 1996; Ferrara et al. 1996). n nVEGF-C (VEGF-related protein or VEGF-2) was initially isolated from conditioned media from PC-3 prostatic adenocarcinoma cells and cloned from PC-3 cell library (Joukov et al. 1996). The VEGF-C gene is 40 kb long and contains seven exons. Exons 3 and 4 are homologous with the VEGF gene, exons 5 and 7 encode cysteine-rich motifs, and exon 6 has motifs typical for the silk protein synthesized by the salivary gland of midge larvae. The 5′ untranslated region of the VEGF-C gene shows promoter activity in reporter gene assays and it contains putative binding sites for Sp-1, AP-2, and NF-κB transcription factors (Chilov et al. 1997). The human VEGF-C cDNA encodes a protein of 419 amino acids and the predicted molecular mass is 46.9 kD. VEGF-C is first synthesized as a preproprotein consisting N-terminal signal sequence, followed by N-terminal propeptide, the VEGF homology domain, and C-terminal propeptide. The major secreted VEGF-C form is a proteolytically cleaved homodimer. VEGF-C precursor protein has little activity, but the fully processed form binds and activates VEGFR-2 and VEGFR-3 (Flt-4) (Joukov et al. 1996). n nVEGF-C stimulates the migration of endothelial cells and increases vascular permeability (Joukov et al. 1996). However, unlike VEGF, it is relatively weak mitogen for blood vascular endothelial cells, but it stimulates proliferation of lymphatic endothelial cells (Joukov et al. 1997). VEGF-C mRNA is expressed at low levels in many tissues including lymph nodes, heart, placenta, skeletal muscle, ovary, and small intestine (Joukov et al. 1996) and it is a ligand for VEGFR-2 and VEGFR-3 (Joukov et al. 1996; Joukov et al. 1997). VEGFR-3 is expressed in most endothelial cells in early embryos, but later in development it becomes restricted to the venous compartment, and in adult tissues the expression of VEGFR-3 is restricted to the lymphatic endothelium (Kaipainen et al. 1995). Thus, VEGFR-3 is the first specific marker for the lymphatic endothelium and provides a new tool to investigate the lymphatic endothelial cell system, which has been less studied than the endothelial cells of blood vessels. Cardiovascular failure during embryonic development in VEGFR-3 knock out mice shows that VEGFR-3 has also a role in blood vessel formation (Dumont et al. 1998). n nThe interaction of VEGF-C and lymphatic vessels is evident in mice overexpressing VEGF-C gene under transcriptional control of the human keratin 14 promoter that directs the expression of the transgene to the basal cells of stratified squamous epithelia. These mice develop hyperplastic lymphatic vessels in the skin that have overlapping endothelial junctions, anchoring filaments in the vessel wall, and a discontinuous and even partially absent basement membrane, all characteristics typical for lymphatic vessels (Jeltsch et al. 1997). The network of lymphatic vessels had similar mesh sizes in both normal and transgenic mice, but the diameter of the vessels was twice as large in transgenic animals. Overexpression of VEGF-C induced endothelial cell proliferation that lead to hyperplasia, but not to sprouting of lymphatic vessels or blood vessel angiogenesis. In addition, VEGF-C has been shown to induce lymphangiogenic response in avian chorioallantoic membrane assay (Oh et al. 1997). n nOther members of the VEGF-family include placenta growth factor (PlGF) and more recently discovered members of the VEGF family VEGF-B (VEGF-related factor), VEGF-D (c-fos-induced growth factor) and VEGF-E. PlGF shares a 56% identity at the amino acid level with the PDGF-like region of VEGF (Maglione et al. 1991). PlGF and VEGF can form heterodimers that bind VEGFR-2 and induce endothelial cell proliferation and migration (DiSalvo et al. 1995 and Cao et al. 1996). However, PlGF homodimers that only bind VEGFR-1 do not induce growth of endothelial cells (Park et al. 1994). VEGF-B binds to VEGFR-1 and regulates urokinase type plasminogen activator and plasminogen activator inhibitor 1 expression and activity in endothelial cells (Olofsson et al. 1996, 1998). It is expressed in most tissues and the expression is especially high in the heart and skeletal muscle. VEGF-D is related relatively closely to VEGF-C. Similarly to VEGF-C it binds to VEGFR-2 and VEGFR-3 and is an endothelial cell mitogen (Achen et al. 1998). VEGF-D is most abundantly expressed in the heart, the lung, skeletal muscle, colon, and small intestine VEGF-E is viral homologue of VEGF that binds to VEGFR-2 (Ogawa et al. 1998; Wise et al. 1999). n nAngiogenesis is an important component of chronic inflammatory diseases such as rheumatoid arthritis (RA) and psoriasis (Folkman 1995). Blood vessels maintain the chronic inflammatory state by transporting inflammatory cells to the site of inflammation and supplying nutrients and oxygen to the proliferating tissue. The synovium in RA is characterized by formation of highly vascularized synovial tissue that invades and destroys the cartilage and the bone. Levels of VEGF have been found to be high in the synovial fluid of RA patients (Koch et al. 1994) and VEGF mRNA and protein are expressed by synovial lining cells, magrophages, fibroblasts, and smooth muscle cells in highly vascularized areas in the RA synovial tissue (Fava et al. 1994). Tumour necrosis factor (TNF)-α and interleukin (IL)-1 are proinflammatory cytokines that have an important role in inflammatory conditions and they may account for the majority of magrophage-derived angiogenic activity in RA (Szekanecz et al. 1998). n nIL-1 and TNF-α stimulate expression of VEGF-C in human lung fibroblasts and in human umbilical vein endothelial cells (HUVEC) (Ristimaki et al. 1998). This cytokine-induced expression of VEGF-C may have a role in inflammation by controlling the composition and pressure of interstitial fluid and by facilitating lymphocyte trafficking. Similarly, the expression of VEGF has been shown to be stimulated by IL-1 and/or TNF-α in several cell types including human synovial fibroblasts (Ben-Av et al. 1995), rat aortic smooth muscle cells (Li et al. 1995), keratinocytes (Frank et al. 1995), and human lung fibroblasts (Ristimaki et al. 1998). In addition, IL-1 and TNF-α induce VEGFR-2 mRNA in HUVECs (Ristimaki et al. 1998; Giraudo et al. 1998). All this suggests that both production of VEGF and VEGF-C and responsiveness of these growth factors via modulation of VEGFR-2 expression is under tight control facilitated by proinflammatory cytokines. Further, the anti-inflammatory glucocorticoid dexamethasone inhibits IL-1-induced VEGF and VEGF-C mRNA expression (Ristimaki et al. 1998). In addition to cytokines, VEGF-C mRNA levels are increased after stimulation by platelet-derived growth factor, epidermal growth factor, and transforming growth factor-β (Enholm et al. 1997). n nHypoxia, which is an important stimulus for angiogenesis and inducer of VEGF expression, does not induce VEGF-C expression (Ristimaki et al. 1998). Hypoxia induces VEGF expression by trascriptional activation via hypoxia-inducible factor-1 and by postranscriptional stabilization of the mRNA (Ikeda et al. 1995; Levy et al. 1995; Liu et al. 1995). Similarly, the mechanism of action of IL-1 on VEGF has been suggested to depend on both trascriptional and post-transcriptional regulation (Li et al. 1995). The rapid decay of the VEGF mRNA has been shown to be dependent on protein that binds to AU-rich instability motifs in 3′-untranslated region of VEGF mRNA (Levy et al. 1995) that are not present in the VEGF-C 3′-untranslated region. Indeed, expression of VEGF-C seems to be mainly regulated at the trascriptional level and not by stabilization of the mRNA (Enholm et al. 1997; Ristimaki et al. 1998). The upregulation of VEGF-C by proinflammatory cytokines may have an important role in inflammation by controlling composition and pressure of interstitial fluid and by facilitating lymphocyte trafficking.

Lymphatic Vascular Morphogenesis

Lymphatic vessels participate in tissue homeostasis and immune surveillance by draining excess fluid and immune cells from tissues to blood circulation. Impaired lymphatic function can lead to tissue swelling, or lymphoedema, and associated complications, such as chronic inflammation and fat accumulation. The critical role of lymphatic vessels in a number of pathological conditions, including tumour metastasis, has led to an interest in identifying signalling pathways regulating lymphatic vessel development and growth. Here, we review the current knowledge on the molecular mechanisms of lymphatic development and how lymphatic vasculature contributes to diseases.

Detection, Isolation and Culture of Lymphatic Endothelial Cells

Lymphatic vessels are essential for the maintenance of normal tissue fluid balance and immune surveillance, but they also provide a pathway for metastasis in many types of cancers (reviewed in Alitalo and Carmeliet 2002). In spite of the importance of lymphatic vessels in medicine, the cell biology of this part of the vascular system has received little attention until recently. Only few lymphatic endothelial cell lines have been available for molecular biological studies, and these were mainly derived from lymphatic tumors. However, the identification of lymphatic specific markers during the past few years and the isolation and maintenance of primary cultures of lymphatic endothelial cells have enabled studies of the molecular properties of these cells.