Showing papers by "Kari Alitalo published in 2004"

Vascular endothelial growth factor C is required for sprouting of the first lymphatic vessels from embryonic veins.

Abstract: Lymphatic vessels are essential for immune surveillance, tissue fluid homeostasis and fat absorption. Defects in lymphatic vessel formation or function cause lymphedema. Here we show that the vascular endothelial growth factor C (VEGF-C) is required for the initial steps in lymphatic development. In Vegfc-/- mice, endothelial cells commit to the lymphatic lineage but do not sprout to form lymph vessels. Sprouting was rescued by VEGF-C and VEGF-D but not by VEGF, indicating VEGF receptor 3 specificity. The lack of lymphatic vessels resulted in prenatal death due to fluid accumulation in tissues, and Vegfc+/- mice developed cutaneous lymphatic hypoplasia and lymphedema. Our results indicate that VEGF-C is the paracrine factor essential for lymphangiogenesis, and show that both Vegfc alleles are required for normal lymphatic development.

Defective valves and abnormal mural cell recruitment underlie lymphatic vascular failure in lymphedema distichiasis

Abstract: Lymphatic vessels are essential for the removal of interstitial fluid and prevention of tissue edema. Lymphatic capillaries lack associated mural cells, and collecting lymphatic vessels have valves, which prevent lymph backflow. In lymphedema-distichiasis (LD), lymphatic vessel function fails because of mutations affecting the forkhead transcription factor FOXC2. We report that Foxc2−/− mice show abnormal lymphatic vascular patterning, increased pericyte investment of lymphatic vessels, agenesis of valves and lymphatic dysfunction. In addition, an abnormally large proportion of skin lymphatic vessels was covered with smooth muscle cells in individuals with LD and in mice heterozygous for Foxc2 and for the gene encoding lymphatic endothelial receptor, Vegfr3 (also known as Flt4). Our data show that Foxc2 is essential for the morphogenesis of lymphatic valves and the establishment of a pericyte-free lymphatic capillary network and that it cooperates with Vegfr3 in the latter process. Our results indicate that an abnormal interaction between the lymphatic endothelial cells and pericytes, as well as valve defects, underlie the pathogenesis of LD.

Kaposi sarcoma herpesvirus–induced cellular reprogramming contributes to the lymphatic endothelial gene expression in Kaposi sarcoma

Abstract: The biology of Kaposi sarcoma is poorly understood because the dominant cell type in Kaposi sarcoma lesions is not known(1-4). We show by gene expression microarrays that neoplastic cells of Kaposi sarcoma are closely related to lymphatic endothelial cells (LECs) and that Kaposi sarcoma herpesvirus (KSHV)(5,6) infects both LECs and blood vascular endothelial cells (BECs) in vitro. The gene expression microarray profiles of infected LECs and BECs show that KSHV induces transcriptional reprogramming of both cell types. The lymphangiogenic molecules VEGF-D and angiopoietin-2 were elevated in the plasma of individuals with acquired immune deficiency syndrome and Kaposi sarcoma. These data show that the gene expression profile of Kaposi sarcoma resembles that of LECs, that KSHV induces a transcriptional drift in both LECs and BECs and that lymphangiogenic molecules are involved in the pathogenesis of Kaposi sarcoma.

Comparative Evaluation of FGF-2-, VEGF-A-, and VEGF-C-Induced Angiogenesis, Lymphangiogenesis, Vascular Fenestrations, and Permeability

Abstract: Several endothelial growth factors induce both blood and lymphatic angiogenesis. However, a systematic comparative study of the impact of these factors on vascular morphology and function has been lacking. In this study, we report a quantitative analysis of the structure and macromolecular permeability of FGF-2-, VEGF-A-, and VEGF-C-induced blood and lymphatic vessels. Our results show that VEGF-A stimulated formation of disorganized, nascent vasculatures as a result of fusion of blood capillaries into premature plexuses with only a few lymphatic vessels. Ultrastructural analysis revealed that VEGF-A-induced blood vessels contained high numbers of endothelial fenestrations that mediated high permeability to ferritin, whereas the FGF-2-induced blood vessels lacked vascular fenestrations and showed only little leakage of ferritin. VEGF-C induced approximately equal amounts of blood and lymphatic capillaries with endothelial fenestrations present only on blood capillaries, mediating a medium level of ferritin leakage into the perivascular space. No endothelial fenestrations were found in FGF-2-, VEGF-A-, or VEGF-C-induced lymphatic vessels. These findings highlight the structural and functional differences between blood and lymphatic vessels induced by FGF-2, VEGF-A, and VEGF-C. Such information is important to consider in development of novel therapeutic strategies using these angiogenic factors.

Preexisting Lymphatic Endothelium but not Endothelial Progenitor Cells Are Essential for Tumor Lymphangiogenesis and Lymphatic Metastasis

Abstract: Endothelial progenitor cells have been shown to contribute to angiogenesis in various tumor models. Here, we have studied the relative contributions of bone marrow (BM)-derived endothelial progenitors and pre-existing lymphatic vessels to tumor lymphangiogenesis. We did not find significant incorporation of genetically marked BM-derived cells in lymphatic vessels during tumor- or vascular endothelial growth factor C-induced lymphangiogenesis. The degree of tumor lymphangiogenesis correlated with lymphatic vessel density in the peritumoral area, and despite tumor lymphangiogenesis, lymphatic metastasis failed to occur in gene-targeted vascular endothelial growth factor C(+/-) mice that have hypoplasia of the lymphatic network. Our data demonstrate that during tumor lymphangiogenesis and cancer cell dissemination via the lymphatics, the newly formed lymphatic vessels sprout from the pre-existing local lymphatic network with little if any incorporation of BM-derived endothelial progenitor cells.

Origins of Serum Semicarbazide-Sensitive Amine Oxidase

Abstract: Semicarbazide-sensitive amine oxidases (SSAO) are enzymes that are capable of deaminating primary amines to produce aldehyde, ammonia, and hydrogen peroxide. This activity has been associated with vascular adhesion protein-1 (VAP-1) and is found in the serum, endothelium, adipose, and smooth muscle of mammals. Circulating SSAO activity is increased in congestive heart failure, diabetes, and inflammatory liver diseases. To investigate the origin of circulating SSAO activity, two transgenic mouse models were created with full-length human VAP-1 (hVAP-1) expressed on either endothelial (mTIEhVAP-1) or adipose tissues (aP2hVAP-1), with tie-1 and adipocyte P2 promoters, respectively. Under normal conditions a circulating form of hVAP-1 was found at high levels in the serum of mice with endothelium-specific expression and at low levels in the serum of mice with adipose specific expression. The level of circulating hVAP-1 in the transgenic mice varied with gender, transgene zygosity, diabetes, and fasting. Serum SSAO activity was absent from VAP-1 knockout mice and endothelial cell-specific expression of human VAP-1 restored SSAO activity to the serum of VAP-1 knockout mice. Together, these experiments show that in the mouse VAP-1 is the only source of serum SSAO, that under physiological conditions vascular endothelial cells can be a major source of circulating VAP-1 protein and SSAO, and that serum VAP-1 can originate from both endothelial cells and adipocytes during experimental diabetes. An increased endothelial cell capacity for lymphocyte binding and altered expression of redox-sensitive proteins was also associated with the mTIEhVAP-1 transgene. (Circ Res. 2004;95:50-57.)

Vegfc is required for vascular development and endoderm morphogenesis in zebrafish.

Abstract: 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-C gene therapy restores lymphatic flow across incision wounds

Abstract: Edema and insufficient blood perfusion are common problems in reconstructive surgery. The blood vasculature is reconstructed in microvascular flaps, whereas lymphatic vessel function is lost after surgical incision. Here, we demonstrate that vascular endothelial growth factor C (VEGF-C) gene transfer can be used to reconstruct a lymphatic vessel network severed by incision of skin flaps. We used adenoviral VEGF-C gene transfer at the edges of epigastric skin flaps in mice. Our results show that VEGF-C gene expression results in the formation of anastomoses between the lymphatic vessels of the skin flap and the surrounding lymphatic vasculature. Some spontaneous lymphangiogenesis also took place in the control mice, but the lymphatic vessels generated remained nonfunctional even 2 months postoperatively. In contrast, the VEGF-C treated mice demonstrated persistent lymphatic vessel function during the 2 month follow-up despite the transient nature of the adenoviral VEGF-C gene expression. The restoration of lymphatic function by VEGF-C in skin flaps provides new tools to promote vascular perfusion and to reduce tissue edema in skin and muscle flaps. These results have important implications for the prevention and treatment of surgically induced secondary lymphedema.

Specific Induction of tie1 Promoter by Disturbed Flow in Atherosclerosis-Prone Vascular Niches and Flow-Obstructing Pathologies

Abstract: Nonlaminar flow is a major predisposing factor to atherosclerosis. Yet little is known regarding hemodynamic gene regulation in disease-prone areas of the vascular tree in vivo. We have determined spatial patterns of expression of endothelial cell receptors in the arterial tree and of reporter gene constructs in transgenic animals. In this study we show that the endothelial cell-specific receptor Tie1 is induced by disturbed flow in atherogenic vascular niches. Specifically, tie1 expression in the adult is upregulated in vascular bifurcations and branching points along the arterial tree. It is often confined to a single ring of endothelial cells functioning as sphincters and hence experiencing the steepest gradient in shear stress. In aortic valves, tie1 is asymmetrically induced only in endothelial cells encountering changes in flow direction. Disturbance of laminar flow by a surgical interposition of a vein into an artery led to induction of tie1, specifically in the region where the differently sized vessels adjoin. In pathological settings, tie1 expression is specifically induced in areas of disturbed flow because of the emergence of aneurysms and, importantly, in endothelial cells precisely overlying atherosclerotic plaques. Hemodynamic features of atherosclerotic lesion-prone regions, recreated in vitro with the aid of a flow chamber with a built-in step, corroborated an upregulated tie1 promoter activity only in cells residing where flow separation and recirculation take place. These defined promoter elements might be harnessed for targeting gene expression to atherosclerotic lesions.

Loss of neural cell adhesion molecule induces tumor metastasis by up-regulating lymphangiogenesis.

Abstract: Reduced expression of neural cell adhesion molecule (NCAM) has been implicated in the progression to tumor malignancy in cancer patients. Previously, we have shown that the loss of NCAM function causes the formation of lymph node metastasis in a transgenic mouse model of pancreatic beta cell carcinogenesis (Rip1Tag2). Here we show that tumors of NCAM-deficient Rip1Tag2 transgenic mice exhibit up-regulated expression of the lymphangiogenic factors vascular endothelial growth factor (VEGF)-C and -D (17% in wild-type versus 60% in NCAM-deficient Rip1Tag2 mice) and, with it, increased lymphangiogenesis (0% in wild-type versus 19% in NCAM-deficient Rip1Tag2 mice). Repression of VEGF-C and -D function by adenoviral expression of a soluble form of their cognate receptor, VEGF receptor-3, results in reduced tumor lymphangiogenesis (56% versus 28% in control versus treated mice) and lymph node metastasis (36% versus 8% in control versus treated mice). The results indicate that the loss of NCAM function causes lymph node metastasis via VEGF-C- and VEGF-D-mediated lymphangiogenesis. These results also establish Rip1Tag2;NCAM-deficient mice as a unique model for stochastic, endogenous tumor lymphangiogenesis and lymph node metastasis in immunocompetent mice.

Lymphangiogenesis and cancer: meeting report

Abstract: The presence of tumor cells in lymph nodes (LNs) is a sign that metastatic spread of cancer has occurred, and it also signals poor prognosis for patients with cancer. However, it is uncertain whether the lymphatic vasculature is a route for the further dissemination of tumor cells to distant organs

Gene transfer into rabbit arteries with adeno-associated virus and adenovirus vectors.

Abstract: Background Gene transfer offers considerable potential for altering vessel wall physiology and intervention in vascular disease. Therefore, there is great interest in developing optimal strategies and vectors for efficient, targeted gene delivery into a vessel wall. Methods We studied adeno-associated viruses (AAV; 9 × 108 to 4 × 109 TU/ml) for their usefulness to transduce rabbit arteries in vivo in comparison with adenoviruses (Adv; 1 × 109 to 1 × 1010 pfu/ml). 100 µl of viruses or placebo solution were injected intraluminally into transiently isolated carotid segments. Results In normal arteries AAV transduced mainly medial smooth muscle cells (SMC) while Adv transduced exclusively endothelial cells (EC). Mechanical injury to EC layer and internal elastic lamina enabled Adv to penetrate and transduce medial SMC. Transgene expression in EC after the AAV-mediated gene transfer was very low. The use of the EC-specific Tie-1 promoter did not lead to specific transgene expression in EC. Transgene expression in SMC persisted for at least 100 days after the AAV treatment whereas the Adv-mediated effect diminished in 14 days. AAV caused only a modest increase in EC VCAM-1 expression and proliferation rate of vascular cells as compared with the mock-treated arteries while Adv caused an extensive inflammatory cell infiltration, VCAM-1 expression, vascular cell proliferation and morphological damages. Conclusions Significant differences were observed between the AAV and the Adv vectors in their patterns of arterial transduction and consequent inflammatory responses. These distinct properties may be utilized for different applications in vascular biology research and gene therapy for cardiovascular diseases. Copyright © 2004 John Wiley & Sons, Ltd.

Oral Imatinib Mesylate (STI571/Gleevec) Improves the Efficacy of Local Intravascular Vascular Endothelial Growth Factor-C Gene Transfer in Reducing Neointimal Growth in Hypercholesterolemic Rabbits

Abstract: Background— Platelet-derived growth factor (PDGF) antagonists have demonstrated beneficial effects on neointima formation, but in studies using PDGF inhibitors and extended follow-up, the lesions reoccur. These findings implicate a need to combine targeting of PDGF with other strategies. Stimulation of reendothelialization by treatment with endothelial cell mitogens of the vascular endothelial growth factor (VEGF) family counteracts restenosis, but there are also concerns regarding the durability of the effect with this approach. Methods and Results— To explore whether a combined use of PDGF antagonist and stimulation of reendothelialization confers better results than each therapy alone, we combined systemic administration of imatinib mesylate (STI571/Gleevec, 10 mg/kg−1 per d−1), a tyrosine kinase inhibitor with activity against PDGF receptors, with local intravascular adenovirus-mediated VEGF-C gene transfer (1.15×1010 pfu) in cholesterol-fed, balloon-injured rabbits. Throughout the course of the STI57...

Immunodetection and quantification of vascular endothelial growth factor receptor-3 in human malignant tumor tissues

Abstract: Vascular endothelial growth factor receptor-3 (VEGFR-3) and its ligands, vascular endothelial growth factor-C (VEGF-C) and -D (VEGF-D), are the major molecules involved in developmental and pathological lymphangiogenesis. Here we describe for the first time the development of a specific indirect enzyme-linked immunosorbent assay (ELISA) for the quantification of VEGFR-3 in different human cell and tissue lysates. A combination of the goat polyclonal anti-VEGFR-3 antibody and the mouse monoclonal anti-human VEGFR-3 antibody was used. The assay was highly sensitive and reproducible with a detection range of 0.2-25 ng/ml. The assay was specific for VEGFR-3, with no cross-reactivity to VEGFR-1 or VEGFR-2. Complex formation with VEGF-C and VEGF-D had no effect on the sensitivity of the assay. The VEGFR-3 concentration in the lysates of cultured human dermal microvascular endothelial cells was 14-fold higher than in the lysates from human umbilical vein endothelial cells. In human kidney, breast, colon, gastric and lung cancer tissues the protein levels of VEGFR-3 were in the range of 0.6-16.7 ng/mg protein. Importantly, the level of VEGFR-3 protein detected in the ELISA correlated significantly with the number of VEGFR-3 positive vessels observed in histochemical sections, suggesting that the ELISA assay may be a reliable surrogate of measuring VEGFR-3-positive vessel density. The protein levels of VEGFR-3 in 27 renal cell carcinoma samples had a significant correlation with the levels of VEGF-C (p<0.001), or biological active, free VEGF-A (p<0.0001), but not with VEGFR-1 or total VEGF-A. This assay provides a useful tool for the investigations of the expression levels of VEGFR-3 in physiological and pathological processes, particular in cancer and in lymphangiogenesis-related disease.

Vascular endothelial growth factor receptor-3 in hypoxia-induced vascular development

Abstract: Reduced tissue oxygen tension (hypoxia) is appreciated as an efficient stimulus for neovascularization. The effect of hypoxia on the very first stages of vascular development is, however, less well characterized. Here we show that hypoxic conditions (1% O2) potently stimulated formation of an extensive vascular network during a discrete stage of mouse embryonal stem cell differentiation. The morphological changes correlated with an expanding pool of endothelial cells and with activation of the vascular endothelial growth factor-d (Vegf-d) and Vegf receptor-3 genes. VEGF receptor-3 expression was confined to vascular endothelial cells and analysis of the lymphatic marker Prox-1 revealed no expansion of lymphatic endothelial cells. Administration of neutralizing antibodies against either VEGF receptor-3 or VEGF receptor-2 impaired vascular network formation, whereas neutralizing antibodies against VEGF receptor-1 potentiated development of immature vascular structures. In addition, sequestering of VEGF rece...

Bmx tyrosine kinase transgene induces skin hyperplasia, inflammatory angiogenesis, and accelerated wound healing.

Abstract: The Bmx gene, a member of the Tec family of nonreceptor protein tyrosine kinases, is expressed in arterial endothelium and in certain hematopoietic and epithelial cells Previous in vitro studies have implicated Bmx signaling in cell migration and survival and suggested that it contributes to the progression of prostate carcinomas However, the function of Bmx in normal tissues in vivo is unknown We show here that Bmx expression is induced in skin keratinocytes during wound healing To analyze the role of Bmx in epidermal keratinocytes in vivo, we generated transgenic mice overexpressing Bmx in the skin We show that Bmx overexpression accelerates keratinocyte proliferation and wound reepithelialization Bmx expression also induces chronic inflammation and angiogenesis in the skin, and gene expression profiling suggests that this occurs via cytokine-mediated recruitment of inflammatory cells Our studies provide the first data on Bmx function in vivo and form the basis of evaluation of its role in epithelial neoplasia

Antibodies to VEGF-C

Abstract: Provided are purified and isolated VEGF-C polypeptides capable of binding to at least one of KDR receptor tyrosine kinase (VEGFR-2) and Flt4 receptor tyrosine kinase (VEGFR-3); analogs of such peptides that have VEGF-C-like or VEGF-like biological activities or that are VEGF or VEGF-C inhibitors; polynucleotides encoding the polypeptides; vectors and host cells that embody the polynucleotides; pharmaceutical compositions and diagnostic reagents comprising the polypeptides; and methods of making and using the polypeptides.

Heparin binding veger-3 ligands

Abstract: The present invention is directed to methods and compositions for making and using chimeric polypeptides that comprise a VEGFR-3 ligand and a heparin binding domain. The chimeric molecules of the present invention retain VEGFR-3 binding activity and an enhanced heparin binding activity as compared to native VEGF-C and/or VEFG-D.

Processed vascular endothelial growth factor-B polypeptides

Abstract: VEGF-B polypeptides from the PDGF family of growth factors having the property of promoting mitosis and proliferation of vascular endothelial cells, DNA sequences encoding these polypeptides, pharmaceutical compositions containing them and antibodies which react with them. The VEGF-B polypeptides are useful in stimulating angiogenesis as well as in diagnostic applications.

Plasmin activates VEGF-C and VEGF-D

Abstract: Angiogenesis and lymphangiogenesis (growth of lymphatic vessels) are guided by members of the vascular endothelial growth factor (VEGF) family of secreted glycoproteins. In particular, VEGF-C and VEGF-D promote lymphangiogenesis as demonstrated in transgenic animal models and gene delivery studies. VEGF-C and VEGF-D are secreted as full-length forms that require proteolytic activation for high affinity binding to VEGF receptor-2 (VEGFR-2) and VEGFR-3, cell surface receptor tyrosine kinases localised on the endothelial cells of blood vessels and lymphatics. The proteases that activate these lymphangiogenic growth factors are key regulators of lymphatic development but were previously unknown. Here we report identification of the serine protease plasmin as an enzyme capable of activating both VEGF-C and VEGF-D.

Detection, Isolation and Culture of Lymphatic Endothelial Cells

Abstract: 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.

Ligands de recepteur 3 du facteur de croissance des cellules endotheliales de liaison a l'heparine

Abstract: La presente invention a trait a des procedes et des compositions pour la production et l'utilisation de polypeptides chimeriques comportant un ligand du recepteur 3 du facteur de croissance des cellules endotheliales et un domaine de liaison a l'heparine. Les molecules chimeriques de la presente invention preservent l'activite de liaison du recepteur 3 du facteur de croissance des cellules endotheliales et une activite de liaison a l'heparine accrue par rapport au facteur C de croissance des cellules endotheliales et/ou au facteur D de croissance de cellules endotheliales.