Berndt Enholm

Comparison of VEGF, VEGF-B, VEGF-C and Ang-1 mRNA regulation by serum, growth factors, oncoproteins and hypoxia.

The vascular endothelial growth factor (VEGF) family has recently been expanded by the isolation of two additional growth factors, VEGF-B and VEGF-C. Here we compare the regulation of steady-state levels of VEGF, VEGF-B and VEGF-C mRNAs in cultured cells by a variety of stimuli implicated in angiogenesis and endothelial cell physiology. Hypoxia, Ras oncoprotein and mutant p53 tumor suppressor, which are potent inducers of VEGF mRNA did not increase VEGF-B or VEGF-C mRNA levels. Serum and its component growth factors, platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) as well as transforming growth factor-β (TGF-β) and the tumor promoter phorbol myristate 12,13-acetate (PMA) stimulated VEGF-C, but not VEGF-B mRNA expression. Interestingly, these growth factors and hypoxia simultaneously downregulated the mRNA of another endothelial cell specific ligand, angiopoietin-1. Serum induction of VEGF-C mRNA occurred independently of protein synthesis; with an increase of the mRNA half-life from 3.5 h to 5.5 – 6 h, whereas VEGF-B mRNA was very stable (T1/2>8 h). Our results reveal that the three VEGF genes are regulated in a strikingly different manner, suggesting that they serve distinct, although perhaps overlapping functions in vivo.

Adenoviral Expression of Vascular Endothelial Growth Factor-C Induces Lymphangiogenesis in the Skin

The growth of blood and lymphatic vasculature is mediated in part by secreted polypeptides of the vascular endothelial growth factor (VEGF) family. The prototype VEGF binds VEGF receptor (VEGFR)-1 and VEGFR-2 and is angiogenic, whereas VEGF-C, which binds to VEGFR-2 and VEGFR-3, is either angiogenic or lymphangiogenic in different assays. We used an adenoviral gene transfer approach to compare the effects of these growth factors in adult mice. Recombinant adenoviruses encoding human VEGF-C or VEGF were injected subcutaneously into C57Bl6 mice or into the ears of nude mice. Immunohistochemical analysis showed that VEGF-C upregulated VEGFR-2 and VEGFR-3 expression and VEGF upregulated VEGFR-2 expression at 4 days after injection. After 2 weeks, histochemical and immunohistochemical analysis, including staining for the lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1), the vascular endothelial marker platelet-endothelial cell adhesion molecule-1 (PECAM-1), and the proliferating cell nuclear antigen (PCNA) revealed that VEGF-C induced mainly lymphangiogenesis in contrast to VEGF, which induced only angiogenesis. These results have significant implications in the planning of gene therapy using these growth factors.

Adenoviral VEGF-C overexpression induces blood vessel enlargement, tortuosity, and leakiness but no sprouting angiogenesis in the skin or mucous membranes

Vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs) are important regulators of blood and lymphatic vessel growth and vascular permeability. The VEGF‐C/VEGFR‐3 signaling pathway is crucial for lymphangiogenesis, and heterozygous inactivating missense mutations of the VEGFR‐3 gene are associated with hereditary lymphedema. However, VEGF‐C can have potent effects on blood vessels because its receptor VEGFR‐3 is expressed in certain blood vessels and because the fully processed form of VEGF‐C also binds to the VEGFR‐2 of blood vessels. To characterize the in vivo effects of VEGF‐C on blood and lymphatic vessels, we have overexpressed VEGF‐C via adenovirus‐and adeno‐associated virus‐mediated transfection in the skin and respiratory tract of athymic nude mice. This resulted in dose‐dependent enlargement and tortuosity of veins, which, along with the collecting lymphatic vessels were found to express VEGFR‐2. Expression of angiopoietin 1 blocked the increased leakiness of the blood vessels induced by VEGF‐C whereas vessel enlargement and lymphangiogenesis were not affected. However, angiogenic sprouting of new blood vessels was not observed in response to AdVEGF‐C or AAV‐VEGF‐C. These results show that virally produced VEGF‐C induces blood vessel changes, including vascular leak, but its angiogenic potency is much reduced compared with VEGF in normal skin.—Saaristo, A., Veikkola, T., Enholm, B. Hytönen, M., Arola, J., Pajusola, K., Turunen, P., Jeltsch, M., Karkkainen, M. J., Kerjaschki, D., Bueler, H., Ylä‐Herttuala, S., Alitalo, K. Adenoviral VEGF‐C overexpression induces blood vessel enlargement, tortuosity, and leakiness but no sprouting angiogenesis in the skin or mucous membranes. FASEB J. 16, 1041–1049 (2002)

Lymphangiogenic Gene Therapy With Minimal Blood Vascular Side Effects

Recent work from many laboratories has demonstrated that the vascular endothelial growth factor-C/VEGF-D/VEGFR-3 signaling pathway is crucial for lymphangiogenesis, and that mutations of the Vegfr3 gene are associated with hereditary lymphedema. Furthermore, VEGF-C gene transfer to the skin of mice with lymphedema induced a regeneration of the cutaneous lymphatic vessel network. However, as is the case with VEGF, high levels of VEGF-C cause blood vessel growth and leakiness, resulting in tissue edema. To avoid these blood vascular side effects of VEGF-C, we constructed a viral vector for a VEGFR-3–specific mutant form of VEGF-C (VEGF-C156S) for lymphedema gene therapy. We demonstrate that VEGF-C156S potently induces lymphangiogenesis in transgenic mouse embryos, and when applied via viral gene transfer, in normal and lymphedema mice. Importantly, adenoviral VEGF-C156S lacked the blood vascular side effects of VEGF and VEGF-C adenoviruses. In particular, in the lymphedema mice functional cutaneous lymphatic vessels of normal caliber and morphology were detected after long-term expression of VEGF-C156S via an adeno associated virus. These results have important implications for the development of gene therapy for human lymphedema.

Vascular Endothelial Growth Factor-B Induces Myocardium-Specific Angiogenesis and Arteriogenesis via Vascular Endothelial Growth Factor Receptor-1– and Neuropilin Receptor-1–Dependent Mechanisms

Background— New revascularization therapies are urgently needed for patients with severe coronary heart disease who lack conventional treatment options. Methods and Results— We describe a new proangiogenic approach for these no-option patients using adenoviral (Ad) intramyocardial vascular endothelial growth factor (VEGF)-B186 gene transfer, which induces myocardium-specific angiogenesis and arteriogenesis in pigs and rabbits. After acute infarction, AdVEGF-B186 increased blood vessel area, perfusion, ejection fraction, and collateral artery formation and induced changes toward an ischemia-resistant myocardial phenotype. Soluble VEGF receptor-1 and soluble neuropilin receptor-1 reduced the effects of AdVEGF-B186, whereas neither soluble VEGF receptor-2 nor inhibition of nitric oxide production had this result. The effects of AdVEGF-B186 involved activation of neuropilin receptor-1, which is highly expressed in the myocardium, via recruitment of G-protein-&agr; interacting protein, terminus C (GIPC) and upregulation of G-protein-&agr; interacting protein. AdVEGF-B186 also induced an antiapoptotic gene expression profile in cardiomyocytes and had metabolic effects by inducing expression of fatty acid transport protein-4 and lipid and glycogen accumulation in the myocardium. Conclusions— VEGF-B186 displayed strikingly distinct effects compared with other VEGFs. These effects may be mediated at least in part via a G-protein signaling pathway. Tissue-specificity, high efficiency in ischemic myocardium, and induction of arteriogenesis and antiapoptotic and metabolic effects make AdVEGF-B186 a promising candidate for the treatment of myocardial ischemia.

Intravascular Adenovirus-Mediated VEGF-C Gene Transfer Reduces Neointima Formation in Balloon-Denuded Rabbit Aorta

BackgroundGene transfer to the vessel wall may provide new possibilities for the treatment of vascular disorders, such as postangioplasty restenosis. In this study, we analyzed the effects of adenovirus-mediated vascular endothelial growth factor (VEGF)-C gene transfer on neointima formation after endothelial denudation in rabbits. For comparison, a second group was treated with VEGF-A adenovirus and a third group with lacZ adenovirus. Clinical-grade adenoviruses were used for the study. Methods and ResultsAortas of cholesterol-fed New Zealand White rabbits were balloon-denuded, and gene transfer was performed 3 days later. Animals were euthanized 2 and 4 weeks after the gene transfer, and intima/media ratio (I/M), histology, and cell proliferation were analyzed. Two weeks after the gene transfer, I/M in the lacZ-transfected control group was 0.57±0.04. VEGF-C gene transfer reduced I/M to 0.38±0.02 (P <0.05 versus lacZ group). I/M in VEGF-A–treated animals was 0.49±0.17 (P =NS). The tendency that both VEGF groups had smaller I/M persisted at the 4-week time point, when the lacZ group had an I/M of 0.73±0.16, the VEGF-C group 0.44±0.14, and the VEGF-A group 0.63±0.21 (P =NS). Expression of VEGF receptors 1, 2, and 3 was detected in the vessel wall by immunocytochemistry and in situ hybridization. As an additional control, the effect of adenovirus on cell proliferation was analyzed by performing gene transfer to intact aorta without endothelial denudation. No differences were seen in smooth muscle cell proliferation or I/M between lacZ adenovirus and 0.9% saline–treated animals. ConclusionsAdenovirus-mediated VEGF-C gene transfer may be useful for the treatment of postangioplasty restenosis and vessel wall thickening after vascular manipulations.

Reevaluation of the Role of VEGF-B Suggests a Restricted Role in the Revascularization of the Ischemic Myocardium

Objective—The endogenous role of the VEGF family member vascular endothelial growth factor-B (VEGF-B) in pathological angiogenesis remains unclear. Methods and Results—We studied the role of VEGF-B in various models of pathological angiogenesis using mice lacking VEGF-B (VEGF-B−/−) or overexpressing VEGF-B167. After occlusion of the left coronary artery, VEGF-B deficiency impaired vessel growth in the ischemic myocardium whereas, in wild-type mice, VEGF-B167 overexpression enhanced revascularization of the infarct and ischemic border zone. By contrast, VEGF-B deficiency did not affect vessel growth in the wounded skin, hypoxic lung, ischemic retina, or ischemic limb. Moreover, VEGF-B167 overexpression failed to enhance vascular growth in the skin or ischemic limb. Conclusion—VEGF-B appears to have a relatively restricted angiogenic activity in the ischemic heart. These insights might offer novel therapeutic opportunities.

Vascular Endothelial Growth Factor-C: A Growth Factor for Lymphatic and Blood Vascular Endothelial Cells

The endothelial cells lining all vessels of the circulatory system have been recognized as key players in a variety of physiological and pathological settings. They act as regulators of vascular tone via the inducible nitric oxide system and in angiogenesis, the formation of blood vessels de novo. Aberrant regulation of endothelial cells contributes to tumor formation, atherosclerosis, and diseases such as psoriasis and rheumatoid arthritis. Among the most recently discovered growth factors for endothelial cells are newly isolated members of the platelet-derived growth factor/vascular endothelial growth factor (VEGF) family, VEGF-B, VEGF-C, and VEGF-D. VEGF-C is the ligand for the receptor tyrosine kinase VEGFR-3 (also known as Flt4), which is expressed predominantly in lymphatic endothelium of adult tissues, but a proteolytically processed form of VEGF-C can also activate VEGFR-2 of blood vessels. The lymphatic vessels have been known since the 17th century, but their specific roles in health and disease are still poorly understood. With the discovery of VEGF-C and its cognate receptor VEGFR-3, the regulation and functions of this important component of the circulatory system can be investigated.

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.