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). Angiogenesis 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). VEGF-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). VEGF-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). The 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). Other 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). Angiogenesis 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). IL-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). Hypoxia, 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.