Birgitta Olofsson

Comparison of VEGF, VEGF-B, VEGF-C and Ang-1 mRNA regulation by serum, growth factors, oncoproteins and hypoxia.

The vascular endothelial growth factor (VEGF) family has recently been expanded by the isolation of two additional growth factors, VEGF-B and VEGF-C. Here we compare the regulation of steady-state levels of VEGF, VEGF-B and VEGF-C mRNAs in cultured cells by a variety of stimuli implicated in angiogenesis and endothelial cell physiology. Hypoxia, Ras oncoprotein and mutant p53 tumor suppressor, which are potent inducers of VEGF mRNA did not increase VEGF-B or VEGF-C mRNA levels. Serum and its component growth factors, platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) as well as transforming growth factor-β (TGF-β) and the tumor promoter phorbol myristate 12,13-acetate (PMA) stimulated VEGF-C, but not VEGF-B mRNA expression. Interestingly, these growth factors and hypoxia simultaneously downregulated the mRNA of another endothelial cell specific ligand, angiopoietin-1. Serum induction of VEGF-C mRNA occurred independently of protein synthesis; with an increase of the mRNA half-life from 3.5 h to 5.5 – 6 h, whereas VEGF-B mRNA was very stable (T1/2>8 h). Our results reveal that the three VEGF genes are regulated in a strikingly different manner, suggesting that they serve distinct, although perhaps overlapping functions in vivo.

Differential Binding of Vascular Endothelial Growth Factor B Splice and Proteolytic Isoforms to Neuropilin-1

Vascular endothelial growth factor B (VEGF-B) is expressed in various tissues, especially strongly in the heart, and binds selectively to one of the VEGF receptors, VEGFR-1. The two splice isoforms, VEGF-B167 and VEGF-B186, have identical NH2-terminal cystine knot growth factor domains but differ in their COOH-terminal domains which give these forms their distinct biochemical properties. In this study, we show that both splice isoforms of VEGF-B bind specifically to Neuropilin-1 (NRP1), a receptor for collapsins/semaphorins and for the VEGF165isoform. The NRP1 binding of VEGF-B could be competed by an excess of VEGF165. The binding of VEGF-B167 was mediated by the heparin binding domain, whereas the binding of VEGF-B186 to NRP1 was regulated by exposure of a short COOH-terminal proline-rich peptide upon its proteolytic processing. In immunohistochemistry, NRP1 distribution was found to be overlapping or adjacent to known sites of VEGF-B expression in several tissues, in particular in the developing heart, suggesting the involvement of VEGF-B in NRP1-mediated signaling.

Expression of the vascular endothelial growth factor C receptor VEGFR-3 in lymphatic endothelium of the skin and in vascular tumors.

It is difficult to identify lymph vessels in tissue sections by histochemical staining, and thus a specific marker for lymphatic endothelial cells would be more practical in histopathological diagnostics. Here we have applied a specific antigenic marker for lymphatic endothelial cells in the human skin, the vascular endothelial growth factor receptor-3 (VEGFR-3), and show that it identifies a distinct vessel population both in fetal and adult skin, which has properties of lymphatic vessels. The expression of VEGFR-3 was studied in normal human skin by in situ hybridization, iodinated ligand binding, and immunohistochemistry. A subset of developing vessels expressed the VEGFR-3 mRNA in fetal skin as shown by in situ hybridization and radioiodinated vascular endothelial growth factor (VEGF)-C bound selectively to a subset of vessels in adult skin that had morphological characteristics of lymphatic vessels. Monoclonal antibodies against the extracellular domain of VEGFR-3 stained specifically endothelial cells of dermal lymph vessels, in contrast to PAL-E antibodies, which stained only blood vessel endothelia. In addition, staining for VEGFR-3 was strongly positive in the endothelium of cutaneous lymphangiomatosis, but staining of endothelial cells in cutaneous hemangiomas was weaker. These results establish the utility of anti-VEGFR-3 antibodies in the identification of lymphovascular channels in the skin and in the differential diagnosis of skin lesions involving lymphatic or blood vascular endothelium.

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. Consequently, the COOH-terminal domains in the two isoforms show no resemblance. Mouse and human cDNA clones for the novel isoform of vascular endothelial growth factor B encoded a secreted protein of 186 amino acid residues. Expression in transfected cells generated a protein of 25 kDa which upon secretion was modified by O-linked glycosylation and displayed a molecular mass of 32 kDa under reducing conditions. The protein was expressed as a disulfide-linked homodimer, and heterodimers were generated when coexpressed with vascular endothelial growth factor. The entirely different COOH-terminal domains in the two isoforms of vascular endothelial growth factor B imply that some functional properties of the two proteins are distinct.

Current Biology of VEGF-B and VEGF-C

Endothelial growth factors and their receptors may provide important therapeutic tools for the treatment of pathological conditions characterised by defective or aberrant angiogenesis. Vascular endothelial growth factor (VEGF) is pivotal for vasculogenesis and for angiogenesis in normal and pathological conditions. VEGF-B and VEGF-C provide this gene family with additional functions, for example, VEGF-C also regulates lymphangiogenesis.

Vascular endothelial growth factors VEGF‐B and VEGF‐C

Vascular endothelial growth factor, which was idenheart cDNA library, respectively, by using a serendipitously found partial mouse cDNA clone as a probe tified almost 10 years ago, has so far been considered as the only growth factor relatively specific for endothelial (20,24). Independently, another group found the same gene when attempting to identify candidate genes for cells. VEGF is an important regulator of endothelial cell proliferation, migration, and permeability during multiple endocrine neoplasia type 1 (MEN1). The product of this alternatively spliced gene was designated as embryonic vasculogenesis and in physiological and pathological angiogenesis [reviewed in (1–3)]. The pivVRF (21). The two currently known isoforms of VEGF-B are otal role of VEGF in embryogenesis is emphasized by the unprecedented result that the inactivation of even a generated by alternative splicing of mRNA from the VEGF-B gene, spanning about 4 kb of DNA. The human single VEGF allele results in embryonic lethality (4,5). VEGF acts through its two known high-affinity recepand murine VEGF-B genes are composed of 7 exons, and their exon–intron organization resembles that of tors Flt1, vascular endothelial growth factor receptor 1 (VEGFR-1) and KDR/Flk1, or VEGFR-2 (6–10). A VEGF and PlGF genes (21,24,25). The mature VEGFB proteins (devoid of signal sequence) have 167 (VEGFthird receptor tyrosine kinase homologous with VEGFR-1 and VEGFR-2, designated Flt4, was cloned B167) and 186 (VEGF-B186) amino acid residues, respectively. VEGF-B186 is generated by using an alternative as an orphan receptor by two research groups and was shown not to bind VEGF (11–14). Additional VEGF splice acceptor site in exon 6, resulting in an insertion of 101 bp between nucleotides 410 and 411 in the coding receptors of unknown nature also exist on endothelial and tumor cells (15,16). sequence of VEGF-B167. This insertion introduces a frame shift and a stop codon at the position correspondThe second member of the VEGF family of growth factors, placenta growth factor (PlGF), is 53% identical ing to nucleotides 521–523 of the coding region of VEGF-B167 cDNA (Fig. 1). Thus, the two VEGF-B isowith VEGF within its platelet-derived growth factorlike region and binds only VEGFR-1 (17–19). Both forms have an identical NH2-terminal domain of 115 aa and different COOH-terminal domains. Although VEGF and PlGF are dimeric glycoproteins related in structure to the platelet-derived growth factors A and the C-terminus of VEGF-B167 is highly basic, that of VEGF-B186 is rich in alanine, proline, serine, and threoB (PDGF-A and PDGF-B). This relation is based on the presence of several conserved amino acid residues nine amino acid residues and has no significant similarity with amino acid sequences of known proteins including 8 equally spaced cysteines. Compared with VEGF, the mitogenic or permeability-enhancing activi(21,24). Unlike other growth factors of the VEGF-family, both isoforms of human and mouse VEGF-B lack ties of PlGF are weak; however, PlGF is able to potentiate the action of VEGF in vivo and in vitro (19). PlGF– the consensus sequence for N-linked glycosylation (NXT/S); instead, VEGF186 is O-glycosylated (24). VEGF heterodimers occur in vivo and have intermediate potency in mitogenic stimulation of endothelial VEGF-B167 remains cell associated with secretion, but it is released into the culture medium with treatcells (35). Two novel endothelial cell-specific growth factors, ment of the producing cells with heparin or high salt. The cell (or matrix) association of VEGF-B167 likely ocstructurally related to VEGF and PlGF, were recently discovered. These factors, designated as vascular endocurs via its basic region, as observed for the highly basic splice variants of VEGF. This notion is supported thelial growth factor B (VEGF-B) or VEGF-related factor (20,21) and vascular endothelial growth factor C by the fact that VEGF-B186 , lacking the highly basic region, is freely secreted from cells and is not bound to (VEGF-C) or VEGF-related protein (22,23) expand the known VEGF family and demonstrate the complexity cell-surface or pericellular heparan sulfate proteoglycans (20,24). of regulation of endothelial functions. This review summarizes the initial studies on VEGF-B and VEGF-C. The apparent molecular masses of the secreted VEGF-B167 and VEGF-B186 polypeptides are 21 kDa VEGF-B/VRF

The cellular retinoic acid binding proteins

The two cellular retinoic acid binding proteins, CRABP I and CRABP II, belong to a family of small cytosolic lipid binding proteins and are highly conserved during evolution. Both proteins are expressed during embryogenesis, particularly in the developing nervous system, craniofacial region and limb bud. CRABP I is also expressed in several adult tissues, however, in contrast, CRABP II expression appears to be limited to the skin. It is likely that these proteins serve as regulators in the transport and metabolism of retinoic acid in the developing embryo and throughout adult life. It has been proposed that CRABP I sequesters retinoic acid in the cytoplasm and prevents nuclear uptake of retinoic acid. A role in catabolism of retinoic acid has also been proposed. Recent gene targeting experiments have shown that neither of the two CRABPs are essential for normal embryonic development or adult life. Examination of CRABP I expression at subcellular resolution reveals a differential cytoplasmic and/or nuclear localization of the protein. A regulated nuclear uptake of CRABP I implies a role for this protein in the intracellular transport of retinoic acid. A protein mediated mechanism which controls the nuclear uptake of retinoic acid may play an important role in the transactivation of the nuclear retinoic acid receptors.

Localization of VEGF‐B in the mouse embryo suggests a paracrine role of the growth factor in the developing vasculature

Vascular endothelial growth factor B (VEGF‐B) is structurally closely related to VEGF and binds one of its receptors, VEGFR‐1. In situ hybridization and immunohistochemistry were used to localize VEGF‐B mRNA and protein in embryonic mouse tissues. In 8.5–17.5 day embryos, VEGF‐B was most prominently expressed in the developing myocardium, but not in the cardiac cushion tissue. The strong expression in the heart persisted at later developmental stages, while weaker signals were obtained from several other tissues, including developing muscle, bone, pancreas, adrenal gland, and from the smooth muscle cell layer of several larger vessels, but not from endothelial cells. VEGF‐B is likely to act in a paracrine fashion, as its receptor is almost exclusively present in endothelial cells. VEGF‐B may have a role in vascularization of the heart, skeletal muscles and developing bones, and in paracrine interactions between endothelial and surrounding muscle cells. Dev Dyn 1999;215:12–25.

Vascular Endothelial Growth Factor (VEGF) and VEGF-C Show Overlapping Binding Sites in Embryonic Endothelia and Distinct Sites in Differentiated Adult Endothelia

Vascular endothelial growth factor (VEGF) is a key modulator of angiogenesis during development and in adult tissues, whereas the related VEGF-C has been shown to induce both lymphangiogenesis and angiogenesis. To better understand the specific functions of these growth factors, we have here analyzed their binding to sections of mouse embryonic and adult tissues and compared the distribution of the bound growth factors with the expression patterns of the 3 known members of the VEGF receptor family as well as with neuropilin-1, a coreceptor for VEGF(165). Partially overlapping patterns of VEGF and VEGF-C binding were obtained in embryonic tissues, consistent with the expression of all known VEGF receptors by vascular endothelial cells. However, the most striking differences of binding were observed in the developing and adult heart, in which VEGF decorated all vessels, whereas strong VEGF-C signals were obtained only from epicardial vessels. In the lymph nodes, VEGF and VEGF-C showed distinct binding patterns in agreement with the differential location of their specific receptors. These results show that both VEGF-C and VEGF target embryonic blood vessels, whereas a more selective binding of VEGF-C occurs to its lymphatic vascular receptor in certain adult tissues. Our results suggest that VEGF and VEGF-C have both overlapping and distinct activities via their endothelial receptors.

RhoGDI3 and RhoG

RhoGDIs are negative regulators of small GTP-binding proteins of the Rho family, which have essential cellular functions in most aspects of actin-based morphology and motility processes. They extract Rho proteins from membranes, keep them in inactive rhoGDI/Rho complexes, and eventually deliver them again to specific membranes in response to cellular signals. RhoGDI3, the most divergent member of the rhoGDI family, is well suited to document the underlying molecular mechanisms, since the active and inactive forms of its cellular target, RhoG, have well-separated subcellular localizations. In this study, we investigate trafficking structures and molecular interactions involved in rhoGDI3-mediated shuttling of RhoG between the Golgi and the plasma membrane. Bimolecular fluorescence complementation and acceptor-photobleaching FRET experiments suggest that rhoGDI3 and RhoG form complexes on Golgi and vesicular structures in mammalian cells. 4D-videomicroscopy confirms this localization, and show that RhoG/rhoGDI3-labelled structures are less dynamic than RhoG and rhoGDI3-labeled vesicles, consistent with the inhibitory function of rhoGDI3. Next, we identify the Exocyst subunit Sec3 as a candidate rhoGDI3 partner in cells. RhoGDI3 relocates a subcomplex of the Exocyst (Sec3 and Sec8) from the cytoplasm to the Golgi, while Sec6 is unaffected. Remarkably, Sec3 increases the level of GTP-bound endogenous RhoG, the RhoG-dependent induction of membrane ruffles, and the formation of intercellular tunneling nanotube-like protrusions. Altogether, our study identifies a novel link between vesicular traffic and the regulation of Rho proteins by rhoGDIs. It also suggests that components of the Exocyst machinery may be involved in RhoG functions, possibly regulated by rhoGDI3.