Increased vascularization in mice overexpressing angiopoietin-1.

The angiopoietins and members of the vascular endothelial growth factor (VEGF) family are the only growth factors thought to be largely specific for vascular endothelial cells. Targeted gene inactivation studies in mice have shown that VEGF is necessary for the early stages of vascular development and that angiopoietin-1 is required for the later stages of vascular remodeling. Here it is shown that transgenic overexpression of angiopoietin-1 in the skin of mice produces larger, more numerous, and more highly branched vessels. These results raise the possibility that angiopoietins can be used, alone or in combination with VEGF, to promote therapeutic angiogenesis.

Leakage-resistant blood vessels in mice transgenically overexpressing angiopoietin-1.

Angiopoietin-1 (Ang1) and vascular endothelial growth factor (VEGF) are endothelial cell-specific growth factors. Direct comparison of transgenic mice overexpressing these factors in the skin revealed that the VEGF-induced blood vessels were leaky, whereas those induced by Ang1 were nonleaky. Moreover, vessels in Ang1-overexpressing mice were resistant to leaks caused by inflammatory agents. Coexpression of Ang1 and VEGF had an additive effect on angiogenesis but resulted in leakage-resistant vessels typical of Ang1. Ang1 therefore may be useful for reducing microvascular leakage in diseases in which the leakage results from chronic inflammation or elevated VEGF and, in combination with VEGF, for promoting growth of nonleaky vessels.

Angiopoietin-2, a natural antagonist for Tie2 that disrupts in vivo angiogenesis.

Angiogenesis is thought to depend on a precise balance of positive and negative regulation. Angiopoietin-1 (Ang1) is an angiogenic factor that signals through the endothelial cell-specific Tie2 receptor tyrosine kinase. Like vascular endothelial growth factor, Ang1 is essential for normal vascular development in the mouse. An Ang1 relative, termed angiopoietin-2 (Ang2), was identified by homology screening and shown to be a naturally occurring antagonist for Ang1 and Tie2. Transgenic overexpression of Ang2 disrupts blood vessel formation in the mouse embryo. In adult mice and humans, Ang2 is expressed only at sites of vascular remodeling. Natural antagonists for vertebrate receptor tyrosine kinases are atypical; thus, the discovery of a negative regulator acting on Tie2 emphasizes the need for exquisite regulation of this angiogenic receptor system.

Persistence of platelet thrombus formation in arterioles of mice lacking both von Willebrand factor and fibrinogen.

We used intravital microscopy to observe the formation of platelet plugs in ferric chloride-injured arterioles of live mice. With this model, we evaluated thrombus growth in mice lacking von Willebrand factor (vWF) and fibrinogen (Fg), the two key ligands known to mediate platelet adhesion and aggregation. In vWF(-/-) mice, despite the presence of arterial shear, delayed platelet adhesion occurred and stable thrombi formed. In many mice, a persisting high-shear channel never occluded. Abundant thrombi formed in Fg(-/-) mice, but they detached from the subendothelium, which ultimately caused downstream occlusion in all cases. Surprisingly, mice deficient in both vWF and Fg successfully formed thrombi with properties characteristic of both mutations, leading to vessel occlusion in the majority of vessels. Platelets of these doubly deficient mice specifically accumulated fibronectin in their alpha-granules, suggesting that fibronectin could be the ligand supporting the platelet aggregation.

Tie1, a receptor tyrosine kinase essential for vascular endothelial cell integrity, is not critical for the development of hematopoietic cells.

The receptor tyrosine kinase Tie1 is expressed in both vascular endothelial cells and immature hematopoietic cells. Expression of a common signaling molecule in distinct cellular lineages may suggest common ancestry of these lineages in ontogeny and/or utilization of shared signaling pathways. Tie 1-deficient mice carrying a targeted insertional mutation in germ line show defects in endothelial cell integrity resulting in edema and hemorrhage. To analyse the potential role of this kinase in hematopoietic cells, we have now compared hematopoietic compartments in wildtype and Tiel-deficient mice. The results show: (1) Total cellularity is mildly reduced comparing Tie1-/- and wildtype fetal liver from day 15.5 of gestation. (2) In vitro colony assays and cell transfer experiments of fetal liver cells into lymphocyte-deficient recombination-activating-gene-2-/- mice reveal that Tie1-/- hematopoietic progenitor cells can generate myeloid lineages as well as T and B lymphocytes. (3) Tie1-/- fetal liver cells contain long-term (at least 4 months) bone marrow-reconstituting hematopoietic stem cells suggesting that this kinase is not critical for stem cell-engraftment nor self-renewal.

Angiopoietin and Tie signaling pathways in vascular development.

The angiopoietin ligands and Tie receptors belong to a novel class of ligand/receptor families, which play critical roles in blood vessel formation. They are considered to control numerous signaling pathways that are involved in diverse cellular processes, such as cell migration, proliferation and survival, and reorganization of the actin cytoskeleton. In this review, we summarize the important biochemical and biological properties of this interesting ligand/receptor family. Particular emphasis will be made on potential downstream targets and consequences of the endothelial cell behavior, due to regulation by the angiopoietin/Tie pathway.

Immediate upstream promoter regions required for neurospecific expression of SNAP-25.

The promoter structure and regulation of Snap, a gene encoding the presynaptic t-SNARE SNAP-25 implicated in synaptic vesicle docking and fusion, was studied. Transcription start-site analysis revealed two major start sites located 42 nucleotides apart. Nucleotide sequence of a promoter region 2073 nucleotides upstream of the first transcription site contains three AP-1, one CRE sequence, and three Sp1-like sites close to the TATA box. Further upstream of these sites two TG repeats were found. The ability of regions within the 5' upstream sequence to promote basal neural-specific expression in tissue culture cells was evaluated using a series of constructs containing both Snap gene start sites with progressively restricted 5' sequence linked to the chloramphenicol acetyl transferase (CAT) reporter gene. CAT expression was maximal in neuron-like undifferentiated ND7 and PC12 cells transfected with constructs containing Snap sequences up to 127 bp from the start site. In contrast, nonneuronal fibroblast cell lines did not express significant amounts of CAT, suggesting that this short 127-bp sequence is sufficient to drive neural specific expression of SNAP-25. Band shift analysis of oligonucleotides spanning from -127 to -41 bp of the Snap promoter revealed three distinct DNA-protein complexes generated by brain nuclear extracts and one by liver nuclear extracts, indicating that transcription factors that bind to this 86-bp sequence located just upstream of the TATA box are involved in regulation of basal neurospecific expression of this gene.

On the origin of species specificity of fibronectin. Immunological identification of a species-specific domain of human fibronectin.

We have investigated whether species-specific epitopes of human fibronectin are localized at a specific domain of fibronectin using rabbit polyclonal antibodies. Tryptic fragments of human fibronectin were tested for reactivity with anti-human fibronectin antibody, which had been previously absorbed with other animal fibronectin to establish species specificity. Human-specific epitopes were found to be present on 75,000, 65,000, and 42,000 dalton fragments. The 42,000-dalton fragment shares almost all the epitopes with the 75,000 and 65,000 dalton fragments. It does not promote BHK cell spreading, whereas the 75,000 and 65,000 dalton fragments do. The amino acid sequence from the amino terminus of the 42,000-dalton fragment is Asp/Gly-Gln/Val-?-Ile-Val-, which is almost identical to the sequence Asp-Gln-Cys-Ile-Val- located in the carboxyl terminal 1/3 of the collagen-binding domain of human fibronectin (Kornblihtt et al. (1985) EMBO J. 4, 1755-1759). These results suggest that human fibronectin bears human-specific epitopes mainly on the amino-terminal half of domain 4 (Hayashi & Yamada (1983) J. Biol. Chem. 258, 3332-3340) located between the collagen and cell binding domains almost at the center of the fibronectin polypeptide. The domain specific for human fibronectin may be a general species-specific domain of animal fibronectins.

Purification and characterization of bovine milk fibronectin.

Fibronectins were isolated from both bovine colostrum and fresh milk, and they were found to be very similar to plasma fibronectin. The concentration of fibronectin in milk decreased post partum: 30 micrograms/ml after 0 d and 2 micrograms/ml after 7-851 d. No fibronectin could be detected in any brands of ultra heat treated liquid milk on the market (less than or equal to 0.006 micrograms/ml).

A combinatorial role of angiopoietin-1 and orphan receptor TIE1 pathways in establishing vascular polarity during angiogenesis.

Vascular polarity is a fundamental feature of angiogenesis and left-right asymmetry of the vascular network. Contrary to this importance, the molecular basis of vascular polarity is completely unknown. In this report, we show that the combinatorial function of angiopoietin-1 and the orphan receptor TIE1 is critical specifically for the development of the right-hand side venous system but is dispensable for the left-hand side venous system. Furthermore, our current finding reveals the existence of a distinct genetic program for the establishment of the right-hand side and left-hand side vascular networks well before the network asymmetry becomes morphologically discernible.

The type I and type II gamma-aminobutyric acid/benzodiazepine receptor: 1. Purification and two-dimensional electrophoretic analysis of the receptor from cortex and cerebellum.

The gamma-aminobutyric acid/benzodiazepine receptor complex was purified from rat cortex and cerebellum by benzodiazepine affinity chromatography. Receptors purified from cortex and cerebellum showed different relative affinities for Cl 218872, a non-benzodiazepine ligand which discriminates type I and type II receptors. In contrast, no differences in subunit composition could be detected between these two purified receptor preparations when analyzed by two-dimensional gel electrophoresis.

Type I and type II gamma-aminobutyric acid/benzodiazepine receptors: purification and analysis of novel receptor complex from neonatal cortex.

The gamma-aminobutyric acid (GABA) type A receptor was purified several thousandfold by affinity chromatography from rat cerebellum, adult cortex, and neonatal cortex. Competition for the benzodiazepine binding site by CL 218872 indicated that cerebellar receptors were predominantly type I, adult cortical receptors were a mixture of subtypes, and neonatal cortex was enriched in type II receptor. The receptor purified from neonatal cortex contained predominantly a 54-kilodalton (kDa), beta-subunit-like protein, whereas receptors from cerebellum and adult cortex contained nearly equal amounts of a 50-kDa, alpha-subunit-like protein and a 54-kDa polypeptide. Peptide maps of trypsin-digested 54-kDa subunits from cerebellum, adult cortex, and neonatal cortex exhibited very similar profiles, a result indicating considerable homology between these proteins in the receptor subtypes. A 59-kDa subunit protein was detected in the receptor complex purified from neonatal cortex. Like the 50-kDa, alpha-subunit of the type I receptor, this protein was photolabeled with [3H]flunitrazepam. The photolabeled peptide fragments, produced by trypsin digestion of these alpha 50- and alpha 59-subunits, exhibited the same retention times on reverse-phase HPLC. A less highly purified GABAA receptor preparation from adult rat spinal cord possessed characteristics that were very similar to those of the receptors purified from neonatal cortex.

Immunological identification of multiple alpha-like subunits of the gamma-aminobutyric acidA receptor complex purified from neonatal rat cortex.

Antibodies were prepared against a synthetic peptide corresponding to amino acid sequences 174-203 of the bovine gamma-aminobutyric acidA (GABAA) receptor alpha 1-subunit. The antibodies recognized this synthetic alpha 1-peptide, but failed to react with the homologous peptide sequence, 170-199, of the bovine beta 1-subunit. On Western blots, anti-alpha 1-subunit antibody recognized a 50-kilodalton (kDa) protein in affinity-purified receptor preparations from adult rat cortex and cerebellum. In receptor purified from neonatal cortex, the anti-alpha 1-antibody reacted with 50-kDa, 53-54-kDa, and 59-kDa proteins. After digestion with endoglycosidase F, these three protein bands retained differing electrophoretic mobilities. The 50-kDa and 59-kDa subunits of affinity-purified neonatal receptor, which were photoaffinity-labeled with [3H]flunitrazepam, were immunoprecipitated to different extents by alpha-subunit antibody. These data suggest the existence in GABAA receptor from neonatal cortex of three proteins (50 kDa, 53 kDa, and 59 kDa) which have immunological homology to alpha 1-subunit of bovine GABAA receptor. The presence of an alpha- and a beta-like subunit with similar mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis may account for the relatively high concentration of protein in the 53-54-kDa band which has been observed in receptor purified from neonatal cortex. The presence of multiple alpha-like subunits may be related to the presence of a relatively high concentration of type II GABA receptor in this tissue.

Mouse multidrug resistance 1a/3 gene is the earliest known endothelial cell differentiation marker during blood-brain barrier development.

Molecular mechanisms of endothelial cell differentiation during blood-brain barrier (BBB) development is not well understood due to the lack of specific molecular markers. Here we describe that expression of the mouse multidrug resistance 1a/3 (mdr1a/3) gene can be detected specifically in subsets of vascular endothelial cells associated with neural tissues at as early as embryonic day 10.5 (E10.5). This onset of mdr1a/3 gene expression coincides with the previously described first appearance of morphologically distinct endothelial cells in neural tissues during BBB development. To our knowledge, the mdr1a/3 gene is the earliest endothelial cell differentiation marker gene during BBB development described thus far. In addition, we have found that neither the level nor pattern of mdr1a/3 gene expression in BBB endothelial cells is affected by aberrant cortical neuronal layers in mutant mouse reeler.

Vascular endothelial cell lineage-specific promoter in transgenic mice.

Vascular endothelial cells play essential roles in the function and development of the cardiovascular system. However, due to the lack of lineage-specific markers suitable for molecular and biochemical analyses, very little is known about the molecular mechanisms that regulate endothelial cell differentiation. We report the first vascular endothelial cell lineage-specific (including angioblastic precursor cells) 1.2 kb promoter in transgenic mice. Moreover, deletion analysis of this promoter region in transgenic embryos revealed multiple elements that are required for the maximum endothelial cell lineage-specific expression. This is a powerful molecular tool that will enable us to identify factors and cellular signals essential for the establishment of vascular endothelial cell lineage. It will also allow us to deliver genes specifically into this cell type in vivo to test specifically molecules that have been implicated in cardiovascular development. Furthermore, we have established embryonic stem (ES) cells from the blastocysts of the transgenic mouse that carry the 1.2 kb promoter-LacZ reporter transgene. These ES cells were able to differentiate in vitro to form cystic embryoid bodies (CEB) that contain endothelial cells determined by PECAM immunohistochemistry. However, these in vitro differentiated endothelial cells did not express the LacZ reporter gene. This indicates the lack of factors and/or cellular interactions which are required to induce the expression of the reporter gene mediated by this 1.2 kb promoter in this in vitro differentiation system. Thus this system will allow us to screen for the putative inducers that exist in vivo but not in vitro. These putative inducers are presumably important for in vivo differentiation of vascular endothelial cells.

Role of the Ets transcription factors in the regulation of the vascular-specific Tie2 gene.

The Tie2 gene encodes a vascular endothelium-specific receptor tyrosine kinase that is required for normal vascular development and is also upregulated during angiogenesis. The regulatory regions of the Tie2 gene that are required for endothelium-specific gene expression in vivo have been identified. However, the transcription factors required for Tie2 gene expression remain largely unknown. We have identified highly conserved binding sites for Ets transcription factors in the Tie2 promoter. Mutations in 2 particular binding sites lead to a 50% reduction in the endothelium-specific activity of the promoter. We have compared the ability of several members of the Ets family to transactivate the Tie2 promoter. Our results demonstrate that 1 of 3 distinct isoforms of the novel Ets transcription factor NERF, NERF2, is expressed in endothelial cells and can strongly transactivate the regulatory regions of the Tie2 gene in comparison to other Ets factors, which have little or no effect. NERF2 can bind to the Tie2 promoter Ets sites in electrophoretic mobility shift assays. These studies support a role for Ets factors in the regulation of vascular-specific gene expression and suggest that the novel Ets factor NERF2 may be a critical transcription factor in specifying the expression of the Tie2 gene in vascular endothelial cells.