Showing papers by "Kari Alitalo published in 2000"

Regulation of angiogenesis via vascular endothelial growth factor receptors.

Abstract: Endothelial cell signal transduction mechanisms involved in angiogenesis have come into focus in cancer research when it was realized that solid tumors are dependent on neovascularization [(1)][1] . Unlike normal human endothelial cells, which are quiescent except in the reproductive organs of

Missense mutations interfere with VEGFR-3 signalling in primary lymphoedema

Abstract: Primary lymphoedema is a rare, autosomal dominant disorder that leads to a disabling and disfiguring swelling of the extremities and, when untreated, tends to worsen with time. Here we link primary human lymphoedema to the FLT4 locus, encoding vascular endothelial growth factor receptor-3 (VEGFR-3), in several families. All disease-associated alleles analysed had missense mutations and encoded proteins with an inactive tyrosine kinase, preventing downstream gene activation. Our study establishes that VEGFR-3 is important for normal lymphatic vascular function and that mutations interfering with VEGFR-3 signal transduction are a cause of primary lymphoedema.

PDGF-C is a new protease-activated ligand for the PDGF alpha-receptor.

Abstract: Platelet-derived growth factors (PDGFs) are important in many types of mesenchymal cell. Here we identify a new PDGF, PDGF-C, which binds to and activates the PDGF alpha-receptor. PDGF-C is activated by proteolysis and induces proliferation of fibroblasts when overexpressed in transgenic mice. In situ hybridization analysis in the murine embryonic kidney shows preferential expression of PDGF-C messenger RNA in the metanephric mesenchyme during epithelial conversion. Analysis of kidneys lacking the PDGF alpha-receptor shows selective loss of mesenchymal cells adjacent to sites of expression of PDGF-C mRNA; this is not found in kidneys from animals lacking PDGF-A or both PDGF-A and PDGF-B, indicating that PDGF-C may have a unique function.

Absence of Functional Lymphatics within a Murine Sarcoma: A Molecular and Functional Evaluation

Abstract: Despite a clinically recognized association between the lymphatics and metastasis, the biology of tumor-lymphatic interaction is not clearly understood. We report here that functional lymphatic capillaries are absent from the interior of a solid tumor, despite the presence within the tumor of the lymphangiogenic molecule vascular endothelial growth factor (VEGF)-C and endothelial cells bearing its receptor, VEGF receptor 3. Functional lymphatics, enlarged and VEGF receptor 3 positive, were detected in some tumors only at the tumor periphery (within 100 microm of the interface with normal tissue). We conclude that although lymphangiogenic factors are present, formation of functional lymphatic vessels is prevented, possibly due to collapse by the solid stress exerted by growing cancer cells.

Congenital Hereditary Lymphedema Caused by a Mutation That Inactivates VEGFR3 Tyrosine Kinase

Abstract: Hereditary lymphedema is a chronic swelling of limbs due to dysfunction of lymphatic vessels. An autosomal dominant, congenital form of the disease, also known as "Milroy disease," has been mapped to the telomeric part of chromosome 5q, in the region 5q34-q35. This region contains a good candidate gene for the disease, VEGFR3 (FLT4), that encodes a receptor tyrosine kinase specific for lymphatic vessels. To clarify the role of VEGFR3 in the etiology of the disease, we have analyzed a family with hereditary lymphedema. We show linkage of the disease with markers in 5q34-q35, including a VEGFR3 intragenic polymorphism, and we describe an A-->G transition that cosegregates with the disease, corresponding to a histidine-to-arginine substitution in the catalytic loop of the protein. In addition, we show, by in vitro expression, that this mutation inhibits the autophosphorylation of the receptor. Thus, defective VEGFR3 signaling seems to be the cause of congenital hereditary lymphedema linked to 5q34-q35.

Vascular Endothelial Growth Factor Receptor-3 in Lymphangiogenesis in Wound Healing

Abstract: Vascular endothelial growth factor receptor-3 (VEGFR-3) is essential for embryonic cardiovascular development, but thereafter becomes confined to the lymphatic endothelium in adult tissues. We have here studied VEGFR-3 expression in experimental wounds of pigs and chronic inflammatory wounds of humans. In healing incisional and punch biopsy wounds made in the dorsal skin of pigs, angiogenic blood vessels, identified by use of the blood vascular endothelial markers vWF and PAL-E and the basal lamina protein laminin, developed into the granulation tissue stroma from day 4 onward, being most abundant on days 5 and 6 and regressing thereafter. VEGFR-3-positive vessels were observed in the granulation tissue from day 5 onward. These vessels were distinct from the PAL-E/laminin/vWF-positive vessels and fewer in number, and they appeared to sprout from pre-existing VEGFR-3-positive lymphatic vessels at the wound edge. Unlike the blood vessels, very few VEGFR-3-positive lymphatic vessels persisted on day 9 and none on day 14. In chronic wounds such as ulcers and decubitus wounds of the lower extremity of. humans, VEGFR-3 was also weakly expressed in the vascular endothelium. Our results suggest that transient lymphangiogenesis occurs in parallel with angiogenesis in healing wounds and that VEGFR-3 becomes up-regulated in blood vessel endothelium. in chronic inflammatory wounds.

VEGF-C and VEGF-D expression in neuroendocrine cells and their receptor, VEGFR-3, in fenestrated blood vessels in human tissues

Abstract: Recently, vascular endothelial growth factor receptor 3 (VEGFR-3) has been shown to provide a specific marker for lymphatic endothelia in certain human tissues. In this study, we have investigated the expression of VEGFR-3 and its ligands VEGF-C and VEGF-D in fetal and adult tissues. VEGFR-3 was consistently detected in the endothelium of lymphatic vessels such as the thoracic duct, but fenestrated capillaries of several organs including the bone marrow, splenic and hepatic sinusoids, kidney glomeruli and endocrine glands also expressed this receptor. VEGF-C and VEGF-D, which bind both VEGFR-2 and VEGFR-3 were expressed in vascular smooth muscle cells. In addition, intense cytoplasmic staining for VEGF-C was observed in neuroendocrine cells such as the α cells of the islets of Langerhans, prolactin secreting cells of the anterior pituitary, adrenal medullary cells, and dispersed neuroendocrine cells of the gastrointestinal tract. VEGF-D was observed in the innermost zone of the adrenal cortex and in certa...

Mechanisms of angiogenesis and their use in the inhibition of tumor growth and metastasis.

Abstract: There is a constant requirement for vascular supply in solid tumors. Tumor-associated neovascularization allows the tumor cells to express their critical growth advantage. Axillary lymph node status is the most important prognostic factor in operable breast cancer, and experimental and clinical evidence suggests that the process of metastasis is also angiogenesis-dependent. Various angiogenic growth factors and cytokines induce neovascularization in tumors, namely members of the vascular endothelial growth factor (VEGF) and angiopoietin (Ang) gene families. A strong correlation has been found between VEGF expression and increased tumor microvasculature, malignancy, and metastasis in breast cancer. Anti-angiogenic therapy approaches offer a new promising anti-cancer strategy and a remarkably diverse group of over 20 such drugs is currently undergoing evaluation in clinical trials.

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

Abstract: Background—Gene 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 Results—Aortas 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 0.5760.04. VEGF-C gene transfer reduced I/M to 0.3860.02 (P,0.05 versus lacZ group). I/M in VEGF-A‐treated animals was 0.4960.17 (P5NS). 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 0.7360.16, the VEGF-C group 0.4460.14, and the VEGF-A group 0.6360.21 (P5NS). 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. Conclusions—Adenovirus-mediated VEGF-C gene transfer may be useful for the treatment of postangioplasty restenosis and vessel wall thickening after vascular manipulations. (Circulation. 2000;102:2262-2268.)

Monoclonal antibodies to vascular endothelial growth factor‐D block its interactions with both VEGF receptor‐2 and VEGF receptor‐3

Abstract: Vascular endothelial growth factor-D (VEGF-D), the most recently discovered mammalian member of the VEGF family, is an angiogenic protein that activates VEGF receptor-2 (VEGFR-2/Flk1/KDR) and VEGFR-3 (Flt4). These receptor tyrosine kinases, localized on vascular and lymphatic endothelial cells, signal for angiogenesis and lymphangiogenesis. VEGF-D consists of a central receptor-binding VEGF homology domain (VHD) and N-terminal and C-terminal propeptides that are cleaved from the VHD to generate a mature, bioactive form consisting of dimers of the VHD. Here we report characterization of mAbs raised to the VHD of human VEGF-D in order to generate VEGF-D antagonists. The mAbs bind the fully processed VHD with high affinity and also bind unprocessed VEGF-D. We demonstrate, using bioassays for the binding and cross-linking of VEGFR-2 and VEGFR-3 and biosensor analysis with immobilized receptors, that one of the mAbs, designated VD1, is able to compete potently with mature VEGF-D for binding to both VEGFR-2 and VEGFR-3 for binding to mature VEGF-D. This indicates that the binding epitopes on VEGF-D for these two receptors may be in close proximity. Furthermore, VD1 blocks the mitogenic response of human microvascular endothelial cells to VEGF-D. The anti-(VEGF-D) mAbs raised to the bioactive region of this growth factor will be powerful tools for analysis of the biological functions of VEGF-D.

Developmental expression of pim kinases suggests functions also outside of the hematopoietic system.

Abstract: We have cloned a novel quail cDNA with strong homology to the pim family of proto-oncogenes. The deduced amino acid (aa) sequence of the cDNA, named qpim, is more closely related to Xenopus Pim and to the recently identified rat Pim-3 than to human or rodent Pim-1 or Pim-2. The protein encoded by the qpim cDNA can autophosphorylate itself and share substrates with murine Pim-1, suggesting functional redundancy to other Pim family serine/threonine kinases. We have compared the expression of qpim in avian embryos to mouse pim-1, -2 and -3 by in situ hybridization. qpim shows a highly dynamic expression pattern, particularly at early developmental stages. Surprisingly, its expression pattern is not identical to any of the murine pim genes, which show complementary and/or partially overlapping expression sites both in- and outside of the hematopoietic system. Altogether, our results suggest novel functions for Pim family kinases during embryonic development, in particular in epithelia and in the central nervous system.

Vascular Endothelial Growth Factor-C and Its Receptor VEGFR-3 in the Nasal Mucosa and in Nasopharyngeal Tumors

Abstract: Vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs) are important regulators of blood and lymphatic vessel growth and vascular permeability. Both blood and lymphatic vessels of the upper respiratory tract play important roles in pathological conditions, such as infections and tumors. Here we have studied the expression of VEGF-C and its receptor VEGFR-3 in the upper respiratory system by Northern blot analysis and immunohistochemistry of human tissues, and in situ mRNA hybridization of developing mouse embryos and β-galactosidase staining of mouse embryos having a LacZ marker gene in the VEGFR-3 gene locus. The results demonstrate expression of VEGF-C and VEGFR-3 in the developing and adult nasal respiratory epithelium and in the nasal vascular plexus, respectively. Unlike in most other tissues, in the nasal mucosa VEGFR-3 is expressed in both blood and lymphatic vessels. Expression of VEGF-C was also detected in nasal and nasopharyngeal tumor islands, which were surrounded by VEGFR-3-positive angiogenic blood vessels. These results suggest that VEGF-C and VEGFR-3 have a role in the development of the nasal submucosal vascular plexus and in its normal function and that they are associated with angiogenesis in nasal and nasopharyngeal tumors.

The Bmx tyrosine kinase is activated by IL-3 and G-CSF in a PI-3K dependent manner.

Abstract: Cytoplasmic protein tyrosine kinases play crucial roles in signaling via a variety of cell surface receptors. The Bmx tyrosine kinase, a member of the Tec family, is expressed in hematopoietic cells of the granulocytic and monocytic lineages. Here we show that Bmx is catalytically activated by interleukin-3 (IL-3) and granulocyte-colony stimulating factor (G-CSF) receptors. Activation of Bmx required phosphatidylinositol 3-kinase (PI-3K) as demonstrated by the ability of PI-3K inhibitors to block the activation signal. A green fluorescent protein (GFP) tagged Bmx was translocated to cellular membranes upon co-expression of a constitutively active form of PI-3K, further indicating a role for PI-3K in signaling upstream of Bmx. The expression of wild type Bmx in 32D myeloid progenitor cells resulted in apoptosis in the presence of G-CSF, while cells expressing a kinase dead mutant of Bmx differentiated into mature granulocytes. However, Bmx did not modulate IL-3-dependent proliferation of the cells. These results demonstrate distinct effects of Bmx in cytokine induced proliferation and differentiation of myeloid cells, and suggest that the stage specific expression of Bmx is critical for the differentiation of myeloid cells.

An avian model for studies of embryonic lymphangiogenesis.

Abstract: Embryonic development of lymphatics (lymphangiogenesis) in recent years has rarely beenstudied experimentally. Using an avian model, we showed that both intra- and extra-embryonicblood vessels of chick and quail embryos are accompanied by lymphatics. The lymphatics of thechorioallantoic membrane (CAM) are drained by lymphatic trunks of the umbilicus and areconnected to the posterior lymph hearts. Intra-embryonic lymphatics are drained via pairedthoracic ducts into the jugulo-subclavian junction. The lymphatic endothelial cells arecharacterized by the expression of Vascular Endothelial Growth Factor Receptors (VEGFR) -2and -3. Application of VEGF-C, the ligand of these two receptors, on the differentiated CAM,induces proliferation of lymphatic endothelial cells and formation of huge lymphatic sinuses.These lymphatics derive from pre-existing lymphatic endothelial cells, whereas, in early embryoslymphangioblasts are present in the mesenchyme. This phenomenon can be demonstrated byinterspecific grafting experiments between chick and quail embryos. Together with the earlylymph sacs, the lymphangioblasts form the embryonic lymphatic system. Our studies demonstratethe importance of lymphangioblasts and lymphangiogenic growth factors in embryoniclymphangiogenesis.

Stimulation of lymphatic growth with an FLT4 ligand

Abstract: Provided are methods of stimulating the growth of lymphatic endothelia using an Flt 4 ligand.

Growth Factors Regulating Lymphatic Vessels

Abstract: The lymphatic vessels penetrate tissues of the body as a dense network that controls the microcirculation by draining fluid from the interstitial spaces. This fluid, lymph, is filtered by lymph nodes and returns to the systemic circulation through the thoracic and lymphatic ducts and the lymphaticovenous anastomoses [1]. The lymphatic endothelium differs from the blood vascular endothelium in many morphological aspects. Lymphatics have an irregular and wide lumen, an attenuated endothelial wall and, in general, do not develop a continuous basement membrane [2]. Lymphatic endothelial cells lack tight junctions and possess anchoring filaments attaching them to the surrounding connective tissue [1]. Due to their highly permeable nature and poorly developed basement membranes, the lymphatics have been proposed as the main routes for tumor metastasis [3]. Abnormal function of the lymphatics is implicated in disease states such as lymphedema, inflammation, infectious and immune diseases, fibrosis and tumors such as Kaposi’s sarcoma and lymphangioma/lymphangiomatosis [4].

Platelet-derived growth factor/vascular endothelial growth factor-like growth factor h, and uses thereof

Abstract: A portion of the PDGF/VEGF-Like Growth Factor H, a new member of the VEGF family of growth factors, is described, as well as the nucleotide sequence encoding this portion of the growth factor, methods for producing it, antibodies and other antagonists to it, transfected and transformed host cells for expressing it, pharmaceutical compositions containing it, and uses thereof in medical and diagnostic applications.