Ex vivo intracoronary gene transfer of adeno-associated virus 2 leads to superior transduction over serotypes 8 and 9 in rat heart transplants.

Heart transplant gene therapy requires vectors with long-lasting gene expression, high cardiotropism, and minimal pathological effects. Here, we examined transduction properties of ex vivo intracoronary delivery of adeno-associated virus (AAV) serotype 2, 8, and 9 in rat syngenic and allogenic heart transplants. Adult Dark Agouti (DA) rat hearts were intracoronarily perfused ex vivo with AAV2, AAV8, or AAV9 encoding firefly luciferase and transplanted heterotopically into the abdomen of syngenic DA or allogenic Wistar-Furth (WF) recipients.

Cardiomyocyte-targeted HIF-1alpha gene therapy inhibits cardiomyocyte apoptosis and cardiac allograft vasculopathy in the rat.

Background: Hypoxia-inducible factor-1 (HIF-1), a key transcription factor in hypoxia, affects a wide range of adaptive cell functions. We examined the kinetics of endogenous HIF-1alpha during acute and chronic rejection, and the effect of exogenous HIF-1alpha in chronically rejecting rat cardiac allografts.

Methods: Heterotopic cardiac transplantations were performed between major MHC-mismatched Dark Agouti and Wistar-Furth rats.

Differential sensitivity of the inner ear sensory cell populations to forced cell cycle re-entry and p53 induction.

Previous studies have shown that the maintenance of post-mitotic state is critical for the life-long survival of the inner ear mechanosensory cells, the hair cells. A general concept is that differentiated, post-mitotic cells rapidly die following cell cycle re-entry. Here we have compared the response of postnatal cochlear (auditory) and utricular (balance) hair cells to forced cell cycle reactivation and p53 up-regulation.

PDGF-A, -C, and -D but not PDGF-B increase TGF-beta1 and chronic rejection in rat cardiac allografts.

Objective: Chronic rejection is the main reason for the poor long-term survival of heart transplant recipients and is characterized by cardiac allograft inflammation, fibrosis, and arteriosclerosis. We examined the specific roles of different platelet-derived growth factor (PDGF) ligands (A-D)--potent mesenchymal cell mitogens--in rat cardiac allografts.

Methods And Results: PDGFR-alpha mRNA was upregulated in acutely-rejecting, and PDGF-A and PDGF-C mRNA in chronically-rejecting cardiac centhatn allografts.

Distinct architecture of lymphatic vessels induced by chimeric vascular endothelial growth factor-C/vascular endothelial growth factor heparin-binding domain fusion proteins.

Vascular endothelial growth factor (VEGF)-C and VEGF-D are composed of the receptor-binding VEGF homology domain and a carboxy-terminal silk homology domain that requires proteolytic cleavage for growth factor activation. Here, we explored whether the C-terminal heparin-binding domain of the VEGF(165) or VEGF(189) isoform also containing neuropilin-binding sequences could substitute for the silk homology domain of VEGF-C. Such VEGF-C/VEGF-heparin-binding domain chimeras were produced and shown to activate VEGF-C receptors, and, when expressed in tissues via adenovirus or adeno-associated virus vectors, stimulated lymphangiogenesis in vivo.

Common protective and diverse smooth muscle cell effects of AAV-mediated angiopoietin-1 and -2 expression in rat cardiac allograft vasculopathy.

Angiopoietin-1 (Ang1) and Ang2 regulate the maintenance of normal vasculature by direct endothelial and indirect smooth muscle cell (SMC) effects. Dysfunction of vascular wall cells is considered central in cardiac allograft vasculopathy (CAV), where inflammation and arterial injury initiate subsequent intimal SMC proliferation. In this study, we investigated the effect of exogenous Ang1 and Ang2 in chronically rejecting rat cardiac allografts by intracoronary adeno-associated virus (AAV)-mediated gene transfer.

Stabilized HIF-1alpha is superior to VEGF for angiogenesis in skeletal muscle via adeno-associated virus gene transfer.

Therapeutic angiogenesis provides a potential alternative for the treatment of cardiovascular ischemic diseases. Vascular endothelial growth factor (VEGF) is an important component of the angiogenic response to ischemia. Here we used adeno-associated virus (AAV) gene delivery to skeletal muscle to examine the effects of VEGF vs.

Vascular endothelial cell growth factor receptor 3-mediated activation of lymphatic endothelium is crucial for tumor cell entry and spread via lymphatic vessels.

Lymphangiogenic growth factors vascular endothelial growth factor (VEGF)-C and VEGF-D have been shown to promote lymphatic metastasis by inducing tumor-associated lymphangiogenesis. In this study, we have investigated how tumor cells gain access into lymphatic vessels and at what stage tumor cells initiate metastasis. We show that VEGF-C produced by tumor cells induced extensive lymphatic sprouting towards the tumor cells as well as dilation of the draining lymphatic vessels, suggesting an active role of lymphatic endothelial cells in lymphatic metastasis.

Angiopoietin-1 promotes lymphatic sprouting and hyperplasia.

Angiopoietin 1 (Ang1), a ligand for the receptor tyrosine kinase Tie2, regulates the formation and stabilization of the blood vessel network during embryogenesis. In adults, Ang1 is associated with blood vessel stabilization and recruitment of perivascular cells, whereas Ang2 acts to counter these actions. Recent results from gene-targeted mice have shown that Ang2 is also essential for the proper patterning of lymphatic vessels and that Ang1 can be substituted for this function.

PDGF-D induces macrophage recruitment, increased interstitial pressure, and blood vessel maturation during angiogenesis.

Platelet-derived growth factor-D (PDGF-D) is a recently characterized member of the PDGF family with unknown in vivo functions. We investigated the effects of PDGF-D in transgenic mice by expressing it in basal epidermal cells and then analyzed skin histology, interstitial fluid pressure, and wound healing. When compared with control mice, PDGF-D transgenic mice displayed increased numbers of macrophages and elevated interstitial fluid pressure in the dermis.

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

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 x 10(8) to 4 x 10(9) TU/ml) for their usefulness to transduce rabbit arteries in vivo in comparison with adenoviruses (Adv; 1 x 10(9) to 1 x 10(10) pfu/ml).

Adeno-associated virus-mediated gene transfer of a secreted decoy human macrophage scavenger receptor reduces atherosclerotic lesion formation in LDL receptor knockout mice.

Macrophage scavenger receptors (MSR) promote atherosclerotic lesion formation, and modulation of MSR activity has been shown to influence atherosclerosis. Soluble receptors are effective in inhibiting receptor-mediated functions in various diseases. We have generated a secreted macrophage scavenger receptor (sMSR) that consists of the bovine growth hormone signal sequence and the human MSR A I extracellular domains.

Cell-type-specific characteristics modulate the transduction efficiency of adeno-associated virus type 2 and restrain infection of endothelial cells.

Adeno-associated viruses (AAVs) are promising vectors for various gene therapy applications due to their long-lasting transgene expression and wide spectrum of target cells. Recently, however, it has become apparent that there are considerable differences in the efficiencies of transduction of different cell types by AAVs. Here, we analyzed the efficiencies of transduction and the transport mechanisms of AAV type 2 (AAV-2) in different cell types, emphasizing endothelial cells.

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.

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.

A model for gene therapy of human hereditary lymphedema.

Primary human lymphedema (Milroy's disease), characterized by a chronic and disfiguring swelling of the extremities, is associated with heterozygous inactivating missense mutations of the gene encoding vascular endothelial growth factor C/D receptor (VEGFR-3). Here, we describe a mouse model and a possible treatment for primary lymphedema. Like the human patients, the lymphedema (Chy) mice have an inactivating Vegfr3 mutation in their germ line, and swelling of the limbs because of hypoplastic cutaneous, but not visceral, lymphatic vessels.

VEGFR3 gene structure, regulatory region, and sequence polymorphisms.

Vascular endothelial growth factor receptor 3 (VEGFR-3) is required for cardiovascular development during embryogenesis. In adults, this receptor is expressed in lymphatic endothelial cells, and mutant VEGFR3 alleles have been implicated in human hereditary lymphedema. To better understand the basis of its specific endothelial lineage-restricted expression, we have characterized the VEGFR3 gene and its regulatory 5' flanking region.

Cardiovascular failure in mouse embryos deficient in VEGF receptor-3.

Vascular endothelial growth factor (VEGF) is a key regulator of blood vessel development in embryos and angiogenesis in adult tissues. Unlike VEGF, the related VEGF-C stimulates the growth of lymphatic vessels through its specific lymphatic endothelial receptor VEGFR-3. Here it is shown that targeted inactivation of the gene encoding VEGFR-3 resulted in defective blood vessel development in early mouse embryos.

Genomic organization of the mouse and human genes for vascular endothelial growth factor B (VEGF-B) and characterization of a second splice isoform.

A second isoform and the genomic structures of mouse and human vascular endothelial growth factor B are described. Both genes consist of seven coding exons and span about 4 kilobases of DNA. The two identified isoforms of vascular endothelial growth factor B are generated by alternative splicing where different splice acceptor sites in exon 6 introduce a frameshift and a partial use of different but overlapping reading frames.

Vascular endothelial growth factor B, a novel growth factor for endothelial cells.

We have isolated and characterized a novel growth factor for endothelial cells, vascular endothelial growth factor B (VEGF-B), with structural similarities to vascular endothelial growth factor (VEGF) and placenta growth factor. VEGF-B was particularly abundant in heart and skeletal muscle and was coexpressed with VEGF in these and other tissues. VEGF-B formed cell-surface-associated disulfide-linked homodimers and heterodimerized with VEGF when coexpressed.

Novel human vascular endothelial growth factor genes VEGF-B and VEGF-C localize to chromosomes 11q13 and 4q34, respectively.

Background: Vascular endothelial growth factor (VEGF) is an important regulator of endothelial cell proliferation, migration, and permeability during embryonic vasculogenesis as well as in physiological and pathological angiogenesis. The recently isolated VEGF-B and VEGF-C cDNAs encode novel growth factor genes of the VEGF family.

Methods And Results: Southern blotting and polymerase chain reaction analysis of somatic cell hybrids and fluorescence in situ hybridization (FISH) of metaphase chromosomes were used to assess the chromosomal localization of VEGF-B and VEGF-C genes.

A novel vascular endothelial growth factor, VEGF-C, is a ligand for the Flt4 (VEGFR-3) and KDR (VEGFR-2) receptor tyrosine kinases.

Angiogenesis, the sprouting of new blood vessels from pre-existing ones, and the permeability of blood vessels are regulated by vascular endothelial growth factor (VEGF) via its two known receptors Flt1 (VEGFR-1) and KDR/Flk-1 (VEGFR-2). The Flt4 receptor tyrosine kinase is related to the VEGF receptors, but does not bind VEGF and its expression becomes restricted mainly to lymphatic endothelia during development. In this study, we have purified the Flt4 ligand, VEGF-C, and cloned its cDNA from human prostatic carcinoma cells.

Signalling properties of FLT4, a proteolytically processed receptor tyrosine kinase related to two VEGF receptors.

The FLT4, FLT1 and KDR/FLK1 genes encode structurally similar endothelial cell receptor tyrosine kinases. Recently it has been shown that the FLT1 and KDR/FLK-1 proteins function as high-affinity receptors for vascular endothelial growth factor (VEGF). Here we show that FLT4 does not act as a receptor for VEGF, as VEGF did not show specific binding to the FLT4 tyrosine kinase or induce its autophosphorylation.