Interaction of a multi-domain adaptor protein, vinexin, with a Rho-effector, Rhotekin.

Among various effector proteins for the Rho small GTPase, the function(s) of Rhotekin is almost unknown. We have identified a multi-domain adaptor protein, vinexin, as a binding partner for Rhotekin, using yeast two-hybrid screening of a human heart library. Rhotekin was found to associate with vinexin in vitro, in COS7 cells, and in brain tissues. The C-terminal Pro-rich motif of Rhotekin exhibited binding to the third SH3 domain of vinexin. The binding was little affected by Rho but was inhibited by activated Cdc42 in COS7 cells. Immunofluorescence analyses revealed partial colocalization of vinexin-alpha with Rhotekin at focal adhesions in REF52 fibroblast cells. These results suggest that Rhotekin forms a complex with vinexin and may play a role at focal adhesions.

Sept8 controls the binding of vesicle-associated membrane protein 2 to synaptophysin.

Septins, a conserved family of GTP/GDP-binding proteins, are present in organisms as diverse as yeast and mammals. We analyzed the distribution of five septins, Sept6, Sept7, Sept8, Sept9 and Sept11, in various rat tissues by western blot analyses and found all septins to be expressed in brain. We also examined the developmental changes of expression of these septins in the rat brain and found that the level of Sept8 increased during post-natal development. Morphological analyses revealed that Sept8 is enriched at pre-synapses. Using yeast two-hybrid screening, we identified vesicle-associated membrane protein 2 (VAMP2), a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE), as an interacting protein for Sept8. Synaptophysin is reported to associate with and recruit VAMP2 to synaptic vesicles and dissociate prior to forming the SNARE complex consisting of VAMP2, syntaxin and synaptosome-associated protein of 25 kDa. We showed that Sept8 suppresses the interaction between VAMP2 and synaptophysin through binding to VAMP2. In addition, we found that Sept8 forms a complex with syntaxin1A, and the Sept8-VAMP2 interaction is disrupted by synaptosome-associated protein of 25 kDa. These results suggest that Sept8 may participate in the process of the SNARE complex formation and subsequent neurotransmitter release.

Characterization of a multidomain adaptor protein, p140Cap, as part of a pre-synaptic complex.

p140Cap (Cas-associated protein) is an adaptor protein considered to play pivotal roles in cell adhesion, growth and Src tyrosine kinase-related signaling in non-neuronal cells. It is also reported to interact with a pre-synaptic membrane protein, synaptosome-associated protein of 25 kDa, and may participate in neuronal secretion. However, properties and precise functions of p140Cap in neuronal cells are almost unknown. Here we show, using biochemical analyses, that p140Cap is expressed in rat brain in a developmental stage-dependent manner, and is relatively abundant in the synaptic plasma membrane fraction in adults. Immunohistochemistry showed localization of p140Cap in the neuropil in rat brain and immunofluorescent analyses detected p140Cap at synapses of primary cultured rat hippocampal neurons. Electron microscopy further revealed localization at pre- and post-synapses. Screening of p140Cap-binding proteins identified a multidomain adaptor protein, vinexin, whose third Src-homology 3 domain interacts with the C-terminal Pro-rich motif of p140Cap. Immunocomplexes between the two proteins were confirmed in COS7 and rat brain. We also clarified that a pre-synaptic protein, synaptophysin, interacts with p140Cap. These results suggest that p140Cap is involved in neurotransmitter release, synapse formation/maintenance, and signaling.

Heterotrimeric G protein betagamma subunits stimulate FLJ00018, a guanine nucleotide exchange factor for Rac1 and Cdc42.

We previously reported that Gbetagamma signaling regulates cell spreading or cell shape change through activation of a Rho family small GTPase, suggesting the existence of a Gbetagamma-regulated Rho guanine-nucleotide exchange factor (RhoGEF). In this study we examined various RhoGEF clones, found FLJ00018 to beaGbetagamma-activated RhoGEF, and investigated the molecular mechanism of Gbetagamma-induced activation of Rho family GTPases. Co-expression of the genes for FLJ00018 and Gbetagamma enhanced serum response element-mediated gene transcription in HEK-293 cells. Combined expression of Gbetagamma and FLJ00018 significantly induced activation of Rac and Cdc42 but not RhoA. FLJ00018 also enhanced gene transcription induced by carbachol-stimulated m2 muscarinic acetylcholine receptor, and this enhancement was blocked by pertussis toxin. Furthermore, we demonstrated Gbetagamma to interact directly with the N-terminal region of FLJ00018 and the N-terminal fragment of this molecule to inhibit serum response element-dependent transcription induced by Gbetagamma/FLJ00018 and carbachol. In NIH3T3 cells, FLJ00018 enhanced lysophosphatidic acid-induced cell spreading, which was also blocked by the N-terminal fragment of FLJ00018. These results provide evidence for a signaling pathway by which G(i)-coupled receptor specifically induces Rac and Cdc42 activation through direct interaction of Gbetagamma with FLJ00018.

Involvement of Gq/11 in both integrin signal-dependent and -independent pathways regulating endothelin-induced neural progenitor proliferation.

We have previously shown that endothelin-B receptor stimulation increases neural progenitor proliferation, partly in G(i) and extracellular matrix molecule-dependent manner. In the present study, we investigated whether G(q/11) is also involved in this response and how G(i) and G(q/11) might regulate the extracellular signal-regulated kinase (ERK) pathway and integrin signaling. Endothelin-induced ERK phosphorylation was independent of integrin ligands, and an inhibitor of G(q/11), YM-254890, as well as pertussis toxin, partially inhibited endothelin-stimulated phosphorylation of Raf-1 and ERK. Endothelin-stimulated protein kinase C (PKC) was partially inhibited by both YM-254890 and pertussis toxin, while only pertussis toxin attenuated endothelin-induced Ras activation. In contrast, endothelin increased tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin in an integrin ligand-dependent manner. Both YM-254890 and pertussis toxin partially inhibited endothelin-stimulated phosphorylation of these proteins. A PKC inhibitor and down-regulation of PKC prevented endothelin-induced phosphorylation of paxillin and ERK. In addition, endothelin-induced proliferation and DNA synthesis were partially inhibited by YM-254890 and pertussis toxin. Taken together, the results indicate that endothelin activates PKC via G(q/11) and G(i), and consequently stimulates the ERK cascade in cooperation with Ras signaling stimulated by G(i). PKC appears to increase tyrosine phosphorylation of paxillin to enhance integrin signaling, which further increases DNA synthesis and proliferation.

SEPT9 sequence alternations causing hereditary neuralgic amyotrophy are associated with altered interactions with SEPT4/SEPT11 and resistance to Rho/Rhotekin-signaling.

SEPT9 is a member of the cytoskeleton-related septin family, which is highly expressed in glia cells in neuronal tissues. Sequence alterations in SEPT9 are known to cause hereditary neuralgic amyotrophy (HNA) but precise cellular consequences have yet to be determined. Since SEPT9 is thought to function through interaction with other septins and small GTPase Rho-mediated signaling, we analyzed the properties of HNA-associated SEPT9 missense variants, SEPT9F (c.278C>T/p.Ser93Phe in SEPT9_v3; NM_006640.3) and SEPT9W (c.262C>T/p.Arg88Trp in SEPT9_v3). We found both sequence variants, but not the wild type, to form filaments with SEPT4 along stress fibers in mesenchymal mouse mammary gland NMuMG cells. In the epithelial cells, the variants, but not the wild type, were colocalized with SEPT11 at cell-cell junctions. In addition, although septin filaments containing SEPT9_v3 were disrupted by Rho/Rhotekin signaling, this was not the case with SEPT9F and SEPT9W. Sequence variations in SEPT9 causing HNA are thus likely to alter modes of interaction with partner molecules in cells, and consequently contribute to the pathogenesis of HNA.

Phosphorylation by extracellular signal-regulated kinase of a multidomain adaptor protein, vinexin, at synapses.

Vinexin is an adaptor protein that is supposed to play pivotal roles in cell adhesion, cytoskeletal organization and signaling. At least three splice variants, vinexinalpha, beta and gamma, have so far been reported. In spite of the possible importance of vinexin, the properties and functions of vinexin in neuronal cells are almost unknown. Here we show that vinexin isoforms are expressed in rat brain in a developmental stage-dependent manner, and that vinexinalpha is relatively abundant in the telencephalon regions of the adult rat brain. An immunohistochemical study showed the localization of vinexinalpha in neurons and glia in the rat brain. In primary cultured rat hippocampal neurons, vinexin was found to be present at synapses and filopodia in growth cones by immunofluorescent analyses. Biochemical fractionation revealed the distribution of vinexin in synaptosomes. Nerve terminal localization of vinexin was confirmed by electron microscopy. Vinexinbeta is reported to be phosphorylated by extracellular signal-regulated kinase (ERK) at Ser189, which is equivalent to Ser593 of vinexinalpha. We thus constructed a site- and phosphorylation state-specific antibody to monitor the ERK-mediated phosphorylation of vinexin. In immunofluorescent analyses, the phosphorylation was observed at synapses formed among cultured rat hippocampal neurons and it was reduced by treatment of the cells with PD98059. In an immunoelectron microscopic examination, the phosphorylation signal was mainly detected on the postsynaptic side of synapses in the rat hippocampal neurons. As active ERK was co-localized with vinexin in synapses, the ERK signal is likely to be involved in the regulation of vinexin-dependent cellular processes in synapses. On the other hand, the phosphorylation was hardly detected in neurons cultured for 3 days, suggesting the presence of a yet unidentified regulatory mechanism of vinexin at the growth cone.

Expression of smooth muscle cell-specific proteins in neural progenitor cells induced by agonists of G protein-coupled receptors and transforming growth factor-beta.

Neural progenitor cells isolated from the embryonic cerebral cortex are well known to differentiate into neurons and glial cells, but recent reports have demonstrated differentiation into smooth muscle cells (SMCs) under the influence of fetal bovine serum. In this study, we report that agonists for G protein-coupled receptors (GPCRs), including endothelin, lysophosphatidic acid and carbachol, effectively promote the expression of SMC-specific proteins in the presence of transforming growth factor-beta (TGF-beta). Incubation of neural progenitor cells with agonists for GPCRs or TGF-beta alone induced the expression of an SMC-specific protein, alpha-smooth muscle actin (SMA), and their combination resulted in incremental increase. Stimulation with combinations of each GPCR agonist and TGF-beta increased the numbers of large, flat cells with thick actin fibers and also caused expression of other SMC marker proteins. Endothelin and TGF-beta enhanced SMA promoter-luciferase reporter activity at different times after stimulation. The mutation of TGF-beta control element of SMA promoter constructs decreased TGF-beta-enhanced luciferase activity but not endothelin-stimulated activity. Transfection of active forms of RhoA and its effector, mDia, strongly enhanced SMA promoter activity, and a dominant negative form of RhoA inhibited endothelin-stimulated promoter activity but not TGF-beta-stimulated activity. Whereas endothelin consistently activated RhoA, TGF-beta did not, and a specific inhibitor of TGF-beta type I receptor blocked TGF-beta-enhanced SMA promoter activity, suggesting involvement of Smad phosphorylation. These results suggest that separate signaling pathways of G protein and TGF-beta cooperatively promote the expression of SMC-specific proteins in neural progenitor cells.

Identification of a cell polarity-related protein, Lin-7B, as a binding partner for a Rho effector, Rhotekin, and their possible interaction in neurons.

Rhotekin, an effector of Rho, is highly expressed in the brain but its function(s) in neurons is almost unknown. In an attempt to define the properties of Rhotekin in neuronal cells, we focused on its interaction with polarity-related molecules. In the present study, we identified a PDZ protein, Lin-7B, as a binding partner for Rhotekin by yeast two-hybrid screening of human brain cDNA library. We then found that Rhotekin interacts with Lin-7B in in vitro pull-down assays, and forms an immunocomplex in COS7 cells and the rat brain. The C-terminal three amino acids of Rhotekin were essential for the interaction with Lin-7B. Their binding affinity became increased in the presence of active RhoA in the COS7 cell expression system. In addition, immunohistochemical analyses demonstrated that Lin-7 as well as Rhotekin is enriched in neurons. These results suggest that Lin-7 plays some role in neuronal functions in concert with Rho/Rhotekin signals.

Possible interaction of a Rho effector, Rhotekin, with a PDZ-protein, PIST, at synapses of hippocampal neurons.

Rhotekin, an effector of Rho, is highly expressed in the brain but its function is almost unknown. In an attempt to define the properties of Rhotekin in neuronal cells, we focused on its interaction with polarity-related molecules. In the present study, we raised the possibility that Rhotekin interacts with a PDZ-protein, PIST (PDZ domain protein interacting specifically with TC10) in vitro, and found that these proteins form complex in the rat brain tissues. We then demonstrated that Rhotekin and PIST are expressed in developmental stage-specific manners in the rat brain. In immunofluorescence analyses, Rhotekin and PIST were suggested to co-localize at synapses in rat primary cultured hippocampal neurons. On the other hand, PIST was found to form immunocomplex with another PDZ-binding protein, beta-catenin, in HEK293 cells and in the rat brain, and co-localized with this protein at dendritic filopods. The interaction of PIST with beta-catenin was inhibited by the presence of Rhotekin. These results suggest a possible yet unidentified role of Rhotekin, in harmony with PIST and beta-catenin, in neuronal cells.

Identification of a PDZ protein, PIST, as a binding partner for Rho effector Rhotekin: biochemical and cell-biological characterization of Rhotekin-PIST interaction.

Among various effector proteins for the small GTPase Rho, the function(s) of Rhotekin is (are) almost unknown. We have identified PIST [PDZ (PSD-95, Discs-large and ZO-1) domain protein interacting specifically with TC10 (a Rho-family small GTPase)] as a binding partner for Rhotekin, using yeast two-hybrid screening. Rhotekin was found to associate with PIST in vitro and in both polarized and non-polarized MDCK (Madin-Darby canine kidney) cells. The C-terminal SPV (Ser-Pro-Val) motif of Rhotekin exhibited binding to the PDZ domain of PIST. The binding was markedly inhibited by an activated version of Rho and partially by that of Rac or Cdc42 in COS7 cells. In contrast, TC10 had no effects on the binding. Immunofluorescence analyses revealed the co-localization of PIST and Rhotekin at the Golgi apparatus in non-polarized fibroblast-like MDCK cells and AJs (adherens junctions) in the fully polarized cells. PIST and Rhotekin are recruited from the cytosol to AJs as the cell becomes polarized. Expression of constitutively active Rho or prevention of Rhotekin-PIST interaction induced diffuse cytoplasmic distribution of Rhotekin in polarized MDCK cells. These results suggest that there is (1) Rho-dependent regulation of Rhotekin-PIST interaction, (2) involvement of PIST in the recruitment of Rhotekin to AJs and (3) a possible role(s) for these two proteins in cell-polarity development and/or maintenance.

Possible role of Rho/Rhotekin signaling in mammalian septin organization.

There is growing evidence for crosstalk between septin filaments and actin cytoskeleton which is regulated by Rho family of GTPases. Here we show that active Rho disrupts septin filament structures in rat embryonic fibroblast REF52 cells. Among Rho effector molecules tested, Rhotekin induced morphological changes of septin filaments similar to those by activated Rho. The center region of Rhotekin was sufficient for the septin reorganization in the cells, and likely to interact indirectly with the C-terminal half of a septin Sept9b, where a GTPase domain is located. Rhotekin and Sept9b are colocalized mainly in perinuclear regions in serum-starved REF52 cells. Upon stimulation with lysophosphatidic acid, they translocated to actin stress fibers in 10 min and then redistributed again to cytoplasm after 90 min treatment. In neuroblastoma Neuro2a cells, Rhotekin and Sept9b were enriched in the tip of neurites, a location where cortical actin reorganization is induced upon stimulation with lysophosphatidic acid. Taken together, we propose that Rhotekin is a novel regulator organizing mammalian septin structures and provide a new link between the septin and Rho-signaling.

Endoplasmic reticulum stress induces the phosphorylation of small heat shock protein, Hsp27.

There are several reports describing participation of small heat shock proteins (sHsps) in cellular protein quality control. In this study, we estimated the endoplasmic reticulum (ER) stress-induced response of Hsp27 and alphaB-crystallin in mammalian cells. Treatment targeting the ER with tunicamycin or thapsigargin induced the phosphorylation of Hsp27 but not of alphaB-crystallin in U373 MG cells, increase being observed after 2-10 h and decline at 24 h. Similar phosphorylation of Hsp27 by ER stress was also observed with U251 MG and HeLa but not in COS cells and could be blocked using SB203580, an inhibitor of p38 MAP kinase. Other protein kinase inhibitors, like Gö6983, PD98059, and SP600125, inhibitors of protein kinase C (PKC), p44/42 MAP kinase, and JNK, respectively, were without major influence. Prolonged treatment with tunicamycin but not thapsigargin for 48 h caused the second induction of the phosphorylation of Hsp27 in U251 MG cells. Under these conditions, the intense perinuclear staining of Hsp27, with some features of aggresomes, was observed in 10%-20% of the cells.

Nordihydroguaiaretic acid (NDGA) blocks the differentiation of C2C12 myoblast cells.

Addition of nordihydroguaiaretic acid (NDGA) to the differentiation medium of C2C12 mouse myoblast cells caused severe inhibition of the formation of myotubes and suppressed differentiation-dependent elevation in the levels of the creatine kinase M isozyme (CKM). Under these conditions, NDGA did not cause significant increase of damaged cells, as detected by annexin-V-FITC assay, or induction of heat shock proteins, known to be a response against extracellular stress. The results suggest that NDGA itself is not toxic but can effectively blocks the differentiation-dependent increase of CKM during C2C12 differentiation. The levels of muscle specific bHLH proteins MyoD, Myf5, and myogenin were also decreased by addition of NDGA, indicating a block of the initial step of the myogenesis through downregulation of muscle specific genes. NDGA is known to be a lipoxygenase inhibitor but other examples, like MK-886 and CDC, did not exert the same effects on differentiation of muscle cells, indicating that mechanisms of NDGA action are independent of its influence on lipoxygenase.

Biochemical and cell biological analyses of a mammalian septin complex, Sept7/9b/11.

Septins are members of a conserved family of cytoskeletal GTPases present in organisms as diverse as yeast and mammals. Unlike lower eukaryotic cells, the physiological significance of mammalian septin complexes is largely unknown. Using specific antibodies, we found at least five septins, Sept2, Sept7, Sept8, Sept9b, and Sept11, in septin complexes affinity-purified with anti-Sept7 antibody-conjugated column from rat embryonic fibroblast REF52 cells. Immunofluorescence studies revealed co-localization of Sept7, Sept9b, and Sept11 along stress fibers in REF52 cells. Biochemical and immunoprecipitation analyses revealed that the three septins directly bind with each other through their N- or C-terminal divergent regions. These septins per se formed distinct and characteristic filament structures when transiently expressed in COS7 cells. When two of the three septins were co-expressed in COS7 cells, combination-dependent filament elongation, bundling, or disruption was observed. Taken together, our results suggest that septin filament structures may be affected by interactions with other septins included in the complex.

Gi2 signaling enhances proliferation of neural progenitor cells in the developing brain.

Our previous study showed that the pertussis toxin-sensitive G protein, Gi2, is selectively localized in the ventricular zone of embryonic brains, where the neuroepithelial cells undergo active proliferation. In order to clarify the role of Gi2 in this site, we first administered pertussis toxin by an exo-utero manipulation method into the lateral ventricle of mouse brain at embryonic day 14.5. Examination at embryonic day 18.5 revealed that pertussis toxin-injected embryos had brains with thinner cerebral cortices, made up of fewer constituent cells. Bromodeoxyuridine labeling revealed fewer numbers of bromodeoxyuridine-positive cells in the cerebral cortices of pertussis toxin-injected embryos, suggesting impaired proliferation of neuroepithelial cells. Next we cultured neural progenitor cells from rat embryonic brains and evaluated the mitogenic effects of agonists for several Gi-coupled receptors that are known to be expressed in the ventricular zone. Among agonists tested, endothelin most effectively stimulated the incorporation of [3H]thymidine in the presence of fibronectin, via the endothelin-B receptor. This was associated with phosphorylation of extracellular signal-regulated kinase, and pertussis toxin partially inhibited both endothelin-stimulated DNA synthesis and phosphorylation of extracellular signal-regulated kinase. Injection of endothelin-3 into the ventricle of embryonic brains increased numbers of bromodeoxyuridine-positive cells in the cerebral cortex, whereas injection of an endothelin-B receptor antagonist decreased them. These findings indicate that Gi2 mediates signaling from receptors such as the endothelin-B receptor to maintain mitogenic activity in the neural progenitor cells of developing brain.

Galphaq/11 signaling induces apoptosis through two pathways involving reduction of Akt phosphorylation and activation of RhoA in HeLa cells.

We have previously reported that expression of the constitutively active mutant of Galpha11 or stimulation of m1 muscarinic acetylcholine receptor induced proteolytic activation of Rho-associated kinase (ROCK-I) by caspase and apoptosis in HeLa cells. In this study, we investigate the molecular mechanisms of Galphaq/11-induced apoptosis in m1 muscarinic acetylcholine receptor-expressing HeLa cells. Overexpression of Bcl-2 inhibited carbachol-induced ROCK-I cleavage, indicating a mitochondrial apoptotic pathway. Overexpression of the constitutively active mutant of Akt that delivers an anti-apoptotic survival signal had a similar influence. Insulin, a major survival factor in many cells, strongly increased phosphorylation of Akt, which was completely blocked by carbachol. This latter effect was partially inhibited by treatment with the tyrosine phosphatase inhibitors, orthovanadate and pervanadate. In parallel with these observations, carbachol attenuated insulin-stimulated tyrosine phosphorylation of insulin receptor substrate-1, an effect eliminated by orthovanadate. On the other hand, carbachol induced rapid stimulation of endogenous RhoA, and expression of a constitutively active mutant of RhoA increased ROCK-I cleavage. Orthovanadate and the dominant negative mutant of RhoA partially, and their combination completely, inhibited carbachol-induced ROCK-I cleavage and apoptosis. These results demonstrate that Gq/11 signaling induces apoptosis by reducing insulin-stimulated Akt phosphorylation through tyrosine dephosphorylation and activating RhoA in HeLa cells.

Riluzole enhances expression of brain-derived neurotrophic factor with consequent proliferation of granule precursor cells in the rat hippocampus.

The dentate gyrus of the hippocampus, generating new cells throughout life, is essential for normal recognition memory performance. Reduction of brain-derived neurotrophic factor (BDNF) in this structure impairs its functions. To elucidate the association between BDNF levels and hippocampal neurogenesis, we first conducted a search for compounds that stimulate endogenous BDNF production in hippocampal granule neurons. Among ion channel modulators tested, riluzole, a neuroprotective agent with anticonvulsant properties that is approved for treatment of amyotrophic lateral sclerosis, was highly effective as a single dose by an intraperitoneal injection, causing a rise in BDNF localized in dentate granule neurons, the hilus, and the stratum radiatum of the CA3 region. Repeated, but not single, injections resulted in prolonged elevation of hippocampal BDNF and were associated with increased numbers of newly generated cells in the granule cell layer. This appeared due to promoted proliferation rather than survival of precursor cells, many of which differentiated into neurons. Intraventricular administration of BDNF-specific antibodies blocked such riluzole effects, suggesting that BDNF increase is necessary for the promotion of precursor proliferation. Our results suggest the basis for a new strategy for treatment of memory dysfunction.

Goniodomin A, an antifungal polyether macrolide, exhibits antiangiogenic activities via inhibition of actin reorganization in endothelial cells.

Goniodomin A (GDA) is an antifungal polyether macrolide isolated from the dinoflagellate Goniodoma pseudogoniaulax. Previous studies revealed that GDA profoundly affected cytoskeletal reorganization. We examined the effect of GDA on the angiogenic properties of vascular endothelial cells. GDA itself did not affect proliferation of, migration of, and tube formation in type I collagen gels by, bovine aortic endothelial cells (BAECs). Proliferation of BAECs stimulated by bFGF was not affected by GDA at concentrations of up to 10 nM. However, at similar concentrations, GDA significantly inhibited bFGF-induced migration and tube formation in type I collagen gels by BAECs. Actin reorganization is required for cell migration. GDA caused the perinuclear aggregation of filamentous actin and inhibited stress fiber formation in bFGF- or VEGF-stimulated BAECs and lysophosphatidic acid-stimulated HeLa cells. However, GDA did not affect stress fiber structures already formed through Gbetagamma expression or in constitutively active RhoA mutant HeLa cells. Finally, GDA inhibited forming of vasucular system in a chorioallantoic membrane. Our results indicated that GDA suppressed angiogenic properties of ECs at least in part through the inhibition of actin reorganization and inhibited angiogenesis in vivo.