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Biomedical subjects

M G Achen

Publications and source records attributed to M G Achen.

At least 19 recordsLinked to original sources
PubMed2006

Targeting lymphangiogenesis to prevent tumour metastasis.

Recent studies involving animal models of cancer and clinicopathological analyses of human tumours suggest that the growth of lymphatic vessels (lymphangiogenesis) in or nearby tumours is associated with the metastatic spread of cancer. The best validated molecular signalling system for tumour lymphangiogenesis involves the secreted proteins vascular endothelial growth factor-C (VEGF-C) and VEGF-D that induce growth of lymphatic vessels via activation of VEGF receptor-3 (VEGFR-3) localised on the surface of lymphatic endothelial cells. In this review, we discuss the evidence supporting a role for this signalling system in the spread of cancer and potential approaches for blocking this system to prevent tumour metastasis.

Animals↗
PubMed2006

Current strategies for modulating lymphangiogenesis signalling pathways in human disease.

The recent discovery that members of the vascular endothelial growth factor (VEGF) family of secreted glycoproteins can mediate lymphatic vessel growth (lymphangiogenesis) via cell surface receptor tyrosine kinases expressed on endothelial cells has opened the way for therapeutic intervention for pathologies involving dysregulated lymphatic vessel function. At least two members of this family, VEGF-C and VEGF-D, have been shown to induce lymphangiogenesis in vivo. Lymphatic vessels and their specific growth factors have been directly implicated in a number of significant human pathologies. In cancer, VEGF-C and VEGF-D appear to correlate with tumor metastasis and poor patient outcome in a range of prevalent human cancers. Experimental studies have demonstrated that expression of the lymphangiogenic growth factors in tumor models induces increased lymphangiogenesis and results in spread of tumor cells via the lymphatics. In contrast, conditions such as lymphedema, where lymphatic vessels fail to clear fluid from interstitial spaces, are opportunities for which the application of growth factors to generate new lymphatic vessels may be a viable therapeutic option. The list of molecules that control lymphangiogenesis is now expanding, allowing more opportunities for the development of drugs with which to manipulate the relevant signalling pathways. Modulating these pathways and other molecules with specificity to the lymphatic endothelium could offer alternative treatments for a number of important clinical conditions.

Disease↗
PubMed2005

Gene transfer using the mature form of VEGF-D reduces neointimal thickening through nitric oxide-dependent mechanism.

Gene transfer to the vessel wall using vascular endothelial growth factors (VEGFs) has shown therapeutic potential for the treatment of restenosis. In this study, we evaluated the effect of catheter-mediated adenoviral (Ad) gene transfer of the mature form of VEGF-D (VEGF-D(DeltaNDeltaC)) in balloon-denuded cholesterol-fed rabbit aorta. AdLacZ was used as a control. Transduced VEGF-D(DeltaNDeltaC) mRNA was detectable in the arterial wall with RT-PCR at 6, 14 and 28 days. Gene transfer efficiency as detected with X-gal staining 6 days after the AdLacZ transduction was 1.91 +/- 1.32% in intima. AdVEGF-D(DeltaNDeltaC) gene transfer led to 52% reduction in intima/media ratio (I/M) as compared to the AdLacZ controls at 14 days time point. At 6 days there were no differences in I/M, but the number of macrophages in the vessel wall was 85% lower in the AdVEGF-D(DeltaNDeltaC) group as compared to the controls. The therapeutic effect was no longer detectable 28 days after the gene transfer. The therapeutic effect of VEGF-D(DeltaNDeltaC) was nitric oxide (NO)-dependent as the feeding of NO synthase inhibitor, L-NAME, blocked the reduction in intimal thickening. It is concluded that AdVEGF-D(DeltaNDeltaC) gene transfer reduces intimal thickening and macrophage influx into the vessel wall in balloon-denuded rabbit aortas.

Adenoviridae↗
PubMed2004

Molecular targeting of lymphatics for therapy.

The dysfunction or proliferation of lymphatic vessels (lymphangiogenesis) is linked to a number of pathological conditions including lymphedema and cancer. The recent discovery and characterisation of the lymphangiogenic growth factors vascular endothelial growth factor-C (VEGF-C) and VEGF-D and of their receptor on lymphatic endothelial cells, VEGFR-3, has provided an understanding of the molecular mechanisms controlling the growth of lymphatic vessels. In addition, other genes and protein markers have been identified with specificity for lymphatic endothelium that have enhanced the characterization and isolation of lymphatic endothelial cells. Our growing understanding of the molecules that control lymphangiogenesis allows us to design more specific drugs with which to manipulate the relevant signalling pathways. Modulating these pathways and other molecules with specificity to the lymphatic system could offer alternative treatments for a number of important clinical conditions.

Animals↗
PubMed2001

Multiple forms of mouse vascular endothelial growth factor-D are generated by RNA splicing and proteolysis.

The secreted glycoprotein vascular endothelial growth factor-D (VEGF-D) is angiogenic, lymphangiogenic, and promotes metastatic spread of tumor cells via lymphatic vessels. VEGF-D consists of a receptor-binding domain (VEGF homology domain) and N- and C-terminal propeptides. Proteolytic processing produces numerous forms of human VEGF-D, including fully processed derivatives (containing only the VEGF homology domain), partially processed, and unprocessed derivatives. Proteolysis is essential to generate human VEGF-D that binds the angiogenic receptor VEGF receptor-2 (VEGFR-2) and the lymphangiogenic receptor VEGFR-3 with high affinity. Here, we report that alternative use of an RNA splice donor site in exon 6 of the mouse VEGF-D gene produces two different protein isoforms, VEGF-D(358) and VEGF-D(326), with distinct C termini. The two isoforms were both expressed in all adult mouse tissues and embryonic stages of development analyzed. Both isoforms are proteolytically processed in a similar fashion to human VEGF-D to generate a range of secreted derivatives and bind and cross-link VEGFR-3 with similar potency. The isoforms are differently glycosylated when expressed in vitro. This study demonstrates that RNA splicing, protein glycosylation, and proteolysis are mechanisms for generating structural diversity of mouse VEGF-D.

Amino Acid Sequence↗
PubMed2001

Isolated lymphatic endothelial cells transduce growth, survival and migratory signals via the VEGF-C/D receptor VEGFR-3.

Vascular endothelial growth factor receptor-3 (VEGFR-3/Flt4) binds two known members of the VEGF ligand family, VEGF-C and VEGF-D, and has a critical function in the remodelling of the primary capillary vasculature of midgestation embryos. Later during development, VEGFR-3 regulates the growth and maintenance of the lymphatic vessels. In the present study, we have isolated and cultured stable lineages of blood vascular and lymphatic endothelial cells from human primary microvascular endothelium by using antibodies against the extracellular domain of VEGFR-3. We show that VEGFR-3 stimulation alone protects the lymphatic endothelial cells from serum deprivation-induced apoptosis and induces their growth and migration. At least some of these signals are transduced via a protein kinase C-dependent activation of the p42/p44 MAPK signalling cascade and via a wortmannin-sensitive induction of Akt phosphorylation. These results define the critical role of VEGF-C/VEGFR-3 signalling in the growth and survival of lymphatic endothelial cells. The culture of isolated lymphatic endothelial cells should now allow further studies of the molecular properties of these cells.

Apoptosis↗
PubMed2001

Signalling via vascular endothelial growth factor receptor-3 is sufficient for lymphangiogenesis in transgenic mice.

Vascular endothelial growth factor receptor-3 (VEGFR-3) has an essential role in the development of embryonic blood vessels; however, after midgestation its expression becomes restricted mainly to the developing lymphatic vessels. The VEGFR-3 ligand VEGF-C stimulates lymphangiogenesis in transgenic mice and in chick chorioallantoic membrane. As VEGF-C also binds VEGFR-2, which is expressed in lymphatic endothelia, it is not clear which receptors are responsible for the lymphangiogenic effects of VEGF-C. VEGF-D, which binds to the same receptors, has been reported to induce angiogenesis, but its lymphangiogenic potential is not known. In order to define the lymphangiogenic signalling pathway we have created transgenic mice overexpressing a VEGFR-3-specific mutant of VEGF-C (VEGF-C156S) or VEGF-D in epidermal keratinocytes under the keratin 14 promoter. Both transgenes induced the growth of lymphatic vessels in the skin, whereas the blood vessel architecture was not affected. Evidence was also obtained that these growth factors act in a paracrine manner in vivo. These results demonstrate that stimulation of the VEGFR-3 signal transduction pathway is sufficient to induce specifically lymphangiogenesis in vivo.

Animals↗
PubMed2001

The specificity of receptor binding by vascular endothelial growth factor-d is different in mouse and man.

Human vascular endothelial growth factor-D (VEGF-D) binds and activates VEGFR-2 and VEGFR-3, receptors expressed on vascular and lymphatic endothelial cells. As VEGFR-2 signals for angiogenesis and VEGFR-3 is thought to signal for lymphangiogenesis, it was proposed that VEGF-D stimulates growth of blood vessels and lymphatic vessels into regions of embryos and tumors. Here we report the unexpected finding that mouse VEGF-D fails to bind mouse VEGFR-2 but binds and cross-links VEGFR-3 as demonstrated by biosensor analysis with immobilized receptor domains and bioassays of VEGFR-2 and VEGFR-3 cross-linking. Mutation of amino acids in mouse VEGF-D to those in the human homologue indicated that residues important for the VEGFR-2 interaction are clustered at, or are near, the predicted receptor-binding surface. Coordinated expression of VEGF-D and VEGFR-3 in mouse embryos was detected in the developing skin where the VEGF-D gene was expressed in a layer of cells beneath the developing epidermis and VEGFR-3 was localized on a network of vessels immediately beneath the VEGF-D-positive cells. This suggests that VEGF-D and VEGFR-3 may play a role in establishing vessels of the skin by a paracrine mechanism. Our study of receptor specificity suggests that VEGF-D may have different biological functions in mouse and man.

Amino Acid Sequence↗
PubMed2001

Localization of vascular endothelial growth factor-D in malignant melanoma suggests a role in tumour angiogenesis.

Expression of angiogenic and lymphangiogenic factors by tumours may influence the route of metastatic spread. Vascular endothelial growth factor (VEGF) is a regulator of tumour angiogenesis, but studies of the inhibition of solid tumour growth by neutralizing anti-VEGF antibodies indicated that other angiogenic factors may be involved. VEGF-D may be an alternative regulator because like VEGF it is angiogenic and it activates VEGF receptor-2 (VEGFR-2), an endothelial cell receptor which is a key signalling molecule in tumour angiogenesis. This study reports the generation of monoclonal antibodies to the receptor-binding domain of VEGF-D and the use of these antibodies to localize VEGF-D in malignant melanoma. VEGF-D was detected in tumour cells and in vessels adjacent to immunopositive tumour cells, but not in vessels distant from the tumours. These findings are consistent with a model in which VEGF-D, secreted by tumour cells, activates endothelial cell receptors and thereby contributes to the regulation of tumour angiogenesis and possibly lymphangiogenesis. In addition, VEGF-D was detected in the vascular smooth muscle, but not the endothelium, of vessels in adult colon. The endothelium of these vessels was negative for VEGFR-2 and VEGFR-3. As VEGF receptors can be up-regulated on endothelium in response to vessel damage and ischaemia, these findings of a specific localization of VEGF-D in smooth muscle of the blood vessels suggest that VEGF-D produced by vascular smooth muscle could play a role in vascular repair by stimulating the proliferation of endothelial cells.

Animals↗
PubMed2001

VEGF-D promotes the metastatic spread of tumor cells via the lymphatics.

Metastasis to local lymph nodes via the lymphatic vessels is a common step in the spread of solid tumors. To investigate the molecular mechanisms underlying the spread of cancer by the lymphatics, we examined the ability of vascular endothelial growth factor (VEGF)-D, a ligand for the lymphatic growth factor receptor VEGFR-3/Flt-4, to induce formation of lymphatics in a mouse tumor model. Staining with markers specific for lymphatic endothelium demonstrated that VEGF-D induced the formation of lymphatics within tumors. Moreover, expression of VEGF-D in tumor cells led to spread of the tumor to lymph nodes, whereas expression of VEGF, an angiogenic growth factor which activates VEGFR-2 but not VEGFR-3, did not. VEGF-D also promoted tumor angiogenesis and growth. Lymphatic spread induced by VEGF-D could be blocked with an antibody specific for VEGF-D. This study demonstrates that lymphatics can be established in solid tumors and implicates VEGF family members in determining the route of metastatic spread.

Animals↗
PubMed2001

VEGF-D is an X-linked/AP-1 regulated putative onco-angiogen in human glioblastoma multiforme.

BACKGROUND: Glioblastoma multiforme (GBM) is a hypervascularized and locally infiltrating brain tumor of astroglial origin with a very poor prognosis. An X-linked c-fos oncogene-inducible mitogenic, morphogenic, and angiogenic factor, endothelial growth factor-D (VEGF-D), is the newest mammalian member of VEGF family. We analyzed VEGF-D in GBM because of its high angiogenic potential and its linkage to the X chromosome. MATERIALS AND METHODS: Nonmalignant brain and GBM tissue sections as well as GBM cell lines were analyzed by immunofluorescence for the expression of VEGF-D, factor VIII (endothelial cell marker), glial-fibrillary acidic protein (GFAP) (astrocytic cell lineage cytoplasmic marker), and several Fos family transcription factors, including c-Fos and Fra-1. The proteins were also detected by Western blots. The differences between genotypes of normal brain and GBM cells were examined by cDNA microarrays. RESULTS AND CONCLUSIONS: GBM expressed ubiquitously VEGF-D, which colocalized with GFAP. Contrary to our expectations, low levels of c-Fos were detected in GBM cells. However, we identified another Fos family member, Fra-1, together with its transcriptional activation partner, c-Jun, as being stably up-regulated in GBM cells. Furthermore, we demonstrated that a fra-1 transgene induced VEGF-D expression in cultured cells and GBM cell stimulation evoked a sustained increase in both Fra-1 and VEGF-D levels. This study reveals that an up-regulation of AP-1 factors may be a hallmark of GBM. Because VEGF-D activates VEGF receptor 2 and 3, receptors important for tumor angiogenesis, it may represent an X-linked/AP-1-regulated onco-angiogen in human GBM. The VEGF-D system and AP-1 activity appear to be very attractive targets for new molecular diagnostics and rational molecular anti-cancer therapies.

Animals↗
PubMed2000

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

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.

Antibodies, Monoclonal↗
PubMed2000

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

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 alpha 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 certain dispersed neuroendocrine cells. These results suggest that VEGF-C and VEGF-D have a paracrine function and perhaps a role in peptide release from secretory granules of certain neuroendocrine cells to surrounding capillaries.

Adult↗
PubMed1999

A mutant form of vascular endothelial growth factor (VEGF) that lacks VEGF receptor-2 activation retains the ability to induce vascular permeability.

Vascular endothelial growth factor (VEGF) is a major mediator of vasculogenesis and angiogenesis both during development and in pathological conditions. VEGF has a variety of effects on vascular endothelium, including the ability to stimulate endothelial cell mitogenesis, and the potent induction of vascular permeability. These activities are at least in part mediated by binding to two high affinity receptors, VEGFR-1 and VEGFR-2. In this study we have made mutations of mouse VEGF in order to define the regions that are required for VEGFR-2-mediated functions. Development of a bioassay, which responds only to signals generated by cross-linking of VEGFR-2, has allowed evaluation of these mutants for their ability to activate VEGFR-2. One mutant (VEGF0), which had amino acids 83-89 of VEGF substituted with the analogous region of the related placenta growth factor, demonstrated significantly reduced VEGFR-2 binding compared with wild type VEGF, indicating that this region was required for VEGF-VEGFR-2 interaction. Intriguingly, when this mutant was evaluated in a Miles assay for its ability to induce vascular permeability, no difference was found when compared with wild type VEGF. In addition we have shown that the VEGF homology domain of the structurally related growth factor VEGF-D is capable of binding to and activating VEGFR-2 but has no vascular permeability activity, indicating that VEGFR-2 binding does not correlate with permeability activity for all VEGF family members. These data suggest different mechanisms for VEGF-mediated mitogenesis and vascular permeability and raise the possibility of an alternative receptor mediating vascular permeability.

Amino Acid Sequence↗
PubMed1999

Biosynthesis of vascular endothelial growth factor-D involves proteolytic processing which generates non-covalent homodimers.

Vascular endothelial growth factor-D (VEGF-D) binds and activates the endothelial cell tyrosine kinase receptors VEGF receptor-2 (VEGFR-2) and VEGF receptor-3 (VEGFR-3), is mitogenic for endothelial cells, and shares structural homology and receptor specificity with VEGF-C. The primary translation product of VEGF-D has long N- and C-terminal polypeptide extensions in addition to a central VEGF homology domain (VHD). The VHD of VEGF-D is sufficient to bind and activate VEGFR-2 and VEGFR-3. Here we report that VEGF-D is proteolytically processed to release the VHD. Studies in 293EBNA cells demonstrated that VEGF-D undergoes N- and C-terminal cleavage events to produce numerous secreted polypeptides including a fully processed form of M(r) approximately 21,000 consisting only of the VHD, which is predominantly a non-covalent dimer. Biosensor analysis demonstrated that the VHD has approximately 290- and approximately 40-fold greater affinity for VEGFR-2 and VEGFR-3, respectively, compared with unprocessed VEGF-D. In situ hybridization demonstrated that embryonic lung is a major site of expression of the VEGF-D gene. Processed forms of VEGF-D were detected in embryonic lung indicating that VEGF-D is proteolytically processed in vivo.

Cell Division↗
PubMed1998

Sampling the genomic pool of protein tyrosine kinase genes using the polymerase chain reaction with genomic DNA.

The polymerase chain reaction (PCR), with cDNA as template, has been widely used to identify members of protein families from many species. A major limitation of using cDNA in PCR is that detection of a family member is dependent on temporal and spatial patterns of gene expression. To circumvent this restriction, and in order to develop a technique that is broadly applicable we have tested the use of genomic DNA as PCR template to identify members of protein families in an expression-independent manner. This test involved amplification of DNA encoding protein tyrosine kinase (PTK) genes from the genomes of three animal species that are well known development models; namely, the mouse Mus musculus, the fruit fly Drosophila melanogaster, and the nematode worm Caenorhabditis elegans. Ten PTK genes were identified from the mouse, 13 from the fruit fly, and 13 from the nematode worm. Among these kinases were 13 members of the PTK family that had not been reported previously. Selected PTKs from this screen were shown to be expressed during development, demonstrating that the amplified fragments did not arise from pseudogenes. This approach will be useful for the identification of many novel members of gene families in organisms of agricultural, medical, developmental and evolutionary significance and for analysis of gene families from any species, or biological sample whose habitat precludes the isolation of mRNA. Furthermore, as a tool to hasten the discovery of members of gene families that are of particular interest, this method offers an opportunity to sample the genome for new members irrespective of their expression pattern.

Amino Acid Sequence↗