Abstract only β1-integrins are essential for angiogenesis. However, the mechanisms regulating integrin function in endothelial cells (EC) and their contribution to angiogenesis are unclear. Brag2 is ...a guanine nucleotide exchange factor for the small GTPase ARF6, which is involved in the traffic of receptors and in the regulation of actin cytoskeleton. Here, we demonstrate that siRNA-mediated silencing of Brag2 reduced basal and VEGF-induced activation of Arf6 and significantly blocked basal and VEGF-induced angiogenic sprouting (by 40 ± 7 %), tube formation and 3-dimensional migration of EC. Transfection with Brag2 siRNA increased focal adhesions and enhanced adhesion of EC on β1-integrin ligands, such as fibronectin and collagen. In line with these results, silencing of Brag2 significantly increased the surface expression of the α5β1-integrin. Moreover, silencing of Brag2 significantly reduced the endocytosis of β1-integrins and specifically of the active (matrix bound) α5β1-integrin present in focal adhesions, suggesting that Brag2 may contribute to the disassembly of focal adhesions during migration via β1-integrin-endocytosis. Strikingly, in vivo silencing of the Brag2 orthologues in zebrafish embryos with morpholinos perturbed vascular development by leading to defects of the parachordal vessels, the dorsal longitudinal anastomotic vessel and the intersomitic vessels. Beyond developmental angiogenesis, in vivo silencing of Brag2 with intravitreal injection of shRNA significantly reduced pathological retinal neovascularization in the model of retinopathy of prematurity (32 % inhibition) and pathological choroidal neovascularization in a model of laser-induced choroidal neovascularization. These data reveal that the Arf6 GEF, Brag2 is essential for developmental and pathological angiogenesis probably by mediating EC migration and angiogenic sprouting through regulation of adhesion by promoting integrin internalization.
beta1-Integrins are essential for angiogenesis. The mechanisms regulating integrin function in endothelial cells (EC) and their contribution to angiogenesis remain elusive. Brag2 is a guanine ...nucleotide exchange factor for the small Arf-GTPases Arf5 and Arf6. The role of Brag2 in EC and angiogenesis and the underlying molecular mechanisms remain unclear. siRNA-mediated Brag2-silencing reduced EC angiogenic sprouting and migration. Brag2-siRNA transfection differentially affected alpha5beta1- and alphaVbeta3-integrin function: specifically, Brag2-silencing increased focal/fibrillar adhesions and adhesion on beta1-integrin ligands (fibronectin and collagen), while reducing the adhesion on the alphaVbeta3-integrin ligand, vitronectin. Consistent with these results, Brag2-silencing enhanced surface expression of alpha5beta1-integrin, while reducing surface expression of alphaVbeta3-integrin. Mechanistically, Brag2-mediated alphaVbeta3-integrin-recycling and beta1-integrin endocytosis and specifically of the active/matrix-bound alpha5beta1-integrin present in fibrillar/focal adhesions (FA), suggesting that Brag2 contributes to the disassembly of FA via beta1-integrin endocytosis. Arf5 and Arf6 are promoting downstream of Brag2 angiogenic sprouting, beta1-integrin endocytosis and the regulation of FA. In vivo silencing of the Brag2-orthologues in zebrafish embryos using morpholinos perturbed vascular development. Furthermore, in vivo intravitreal injection of plasmids containing Brag2-shRNA reduced pathological ischemia-induced retinal and choroidal neovascularization. These data reveal that Brag2 is essential for developmental and pathological angiogenesis by promoting EC sprouting through regulation of adhesion by mediating beta1-integrin internalization and link for the first time the process of beta1-integrin endocytosis with angiogenesis.PUBLICATION ABSTRACT
Apomorphine, the catechol‐derived dopamine D1/D2 receptor agonist, is currently in use as an antiparkinsonian drug. It has previously been reported that apomorphine was able to elicit expression of ...the enzyme tyrosine hydroxylase, a marker for DA neurons, in the fetal rat cerebrocortical cultures whilst in the presence of brain‐derived neurotrophic factor. The present study demonstrated that treatment of fetal rat ventral mesencephalic cultures with apomorphine caused a marked increase in the number of dopaminergic neurons. The action of apomorphine can be mimicked by dopamine receptor (D1 and D2) agonists or blocked by preincubation with D1/D2 receptor antagonists. Incubation of recipient mesencephalic cultures with the conditioned medium derived from apomorphine‐stimulated donor mesencephalic cultures elicited a 3.72‐fold increase in the number of TH‐positive neurons. Increased mRNA expression levels of brain‐derived neurotrophic factor and glial cell line‐derived neurotrophic factor were also found in the apomorphine‐treated mesencephalic cells along with concomitant protein expression increases in the conditioned medium. Moreover, the trophic activity observed could be partially neutralized by antibodies against either brain‐derived neurotrophic factor or glial cell line‐derived neurotrophic factor. Cultured fetal striatal cells, but not hippocampal cells, also responded to apomorphine treatment. The membrane filtration studies revealed that both <30 kDa and >50 kDa fractions contained trophic activities. The latter characterization distinguishes them from most known neurotrophic factors. These results suggest that the apomorphine‐modulated development of dopaminergic neurons may be mediated by activation of the dopamine receptor subtypes D1and D2 thereby increasing the production of multiple growth factors.
Platelet-derived growth factor-DD (PDGF-DD) is a recently discovered member of the PDGF family. The role of PDGF-DD in pathological angiogenesis and the underlying cellular and molecular mechanisms ...remain largely unexplored. In this study, using different animal models, we showed that PDGF-DD expression was up-regulated during pathological angiogenesis, and inhibition of PDGF-DD suppressed both choroidal and retinal neovascularization. We also demonstrated a novel mechanism mediating the function of PDGF-DD. PDGF-DD induced glycogen synthase kinase-3beta (GSK3beta) Ser(9) phosphorylation and Tyr(216) dephosphorylation in vitro and in vivo, leading to increased cell survival. Consistently, GSK3beta activity was required for the antiangiogenic effect of PDGF-DD targeting. Moreover, PDGF-DD regulated the expression of GSK3beta and many other genes important for angiogenesis and apoptosis. Thus, we identified PDGF-DD as an important target gene for antiangiogenic therapy due to its pleiotropic effects on vascular and non-vascular cells. PDGF-DD inhibition may offer new therapeutic options to treat neovascular diseases.
During neurodegenerative processes, cascades of degeneration and subsequent regeneration are triggered. However, the molecular nature of the factors involved in the neurodegeneration of the CNS ...remains largely unknown. In this study, the variations of protein expression in the striatum of adult Sprague–Dawley rats following 6‐hydroxydopamine lesions were investigated, in order to better understand the molecular events occurring in the denervated target tissue. The rat striatum, ipsilateral to the lesion was analysed by two‐dimensional gel electrophoresis followed by matrix assisted laser desorption/ionization‐time of flight mass spectrometry. Seven proteins were up‐regulated (188.1–750% compared to control) in response to the lesion: amyloid precursor‐like protein 2 (APLP2), kininogen, glucokinase, tropomyosin α chain, type brain‐1 and calpactin I light chain; whilst four proteins, neural epidermal growth factor‐like 2, minichromosome maintenance 6, and thyroid hormone receptor β‐2, were down‐regulated (to between 36% and 59% of levels in sham‐operated controls). Three proteins that did not match with available data in the SWISS‐PROT protein database were also determined. Immunohistochemical analysis demonstrated colocalization of APLP2 and tyrosine hydroxylase in the nigral neurons. Moreover, reduction of APLP2‐positive neurons in the substantia nigra pars compacta as well as the increases in the substantia nigra pars reticulata and in the striatum were observed. Furthermore, the conditioned medium of the Chinese hamster ovary cells over‐expressing APLP2‐751 (chondroitin sulphate‐modified), but not APLP2‐763 (nonchondroitin sulphate‐modified), was able to increase the number of the tyrosine hydroxylase‐positive neurons in fetal mesencephalic cultures. These results suggest that the expression of APLP2, a protein that has been thought to be associated with Alzheimer's disease, is up‐regulated in the striatum following dopaminergic denervation. They also support the view that chondroitin sulphate‐modified APLP2 protein may play an important role in the dopaminergic nigrostriatal system.
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