1,721,010 research outputs found
Involvement of α(v)β (3) integrin in gremlin-induced angiogenesis.
No full textα(v)β(3) integrin modulates pro-angiogenic endothelial cell (EC) responses following vascular endothelial growth factor receptor-2 (VEGFR2) engagement. The bone morphogenic protein antagonist gremlin is a novel non-canonical VEGFR2 ligand that promotes the acquisition of a pro-angiogenic phenotype in ECs. Here we investigated the role of α(v)β(3) and extracellular matrix components on EC activation induced by gremlin. Gremlin triggers VEGFR2 phosphorylation and cell motility in ECs adherent to the α(v)β(3) ligand fibrinogen but not in ECs adherent to type-I collagen or fibronectin. Also, gremlin and VEGF-A stimulate the formation of VEGFR2/α(v)β(3) integrin complexes as shown by co-immunoprecipitation experiments and fluorescence resonance energy transfer analysis of β(3)-ECFP/VEGFR2-EYFP co-transfected ECs. Accordingly, anti-β(3) antibodies block the angiogenic activity exerted by gremlin or VEGF-A in vitro, ex vivo and in vivo. The results demonstrate a non-redundant role for α(v)β(3) in gremlin-induced angiogenesis and emphasize its contribution to the formation of functional multi-molecular VEGFR2 complexes responsible for the neovascularization events triggered by canonical and non-canonical pro-angiogenic VEGFR2 ligands
Role of VEGFR2 co-receptors in the modulation of the pro-angiogenic activity of gremlin.
No full textMutation in the Kinase Domain Alters the VEGFR2 Membrane Dynamics
Full text linkBackground: Recently, the substitution R1051Q in VEGFR2 has been described as a cancer-associated "gain of function" mutation. VEGFR2R1051Q phosphorylation is ligand-independent and enhances the activation of intracellular pathways and cell growth both in vitro and in vivo. In cancer, this mutation is found in heterozygosity, suggesting that an interaction between VEGFR2R1051Q and VEGFR2WT may occur and could explain, at least in part, how VEGFR2R1051Q acts to promote VEGFR2 signaling. Despite this, the biochemical/biophysical mechanism of the activation of VEGFR2R1051Q remains poorly understood. On these bases, the aim of our study is to address how VEGFR2R1051Q influences the biophysical behavior (dimerization and membrane dynamics) of the co-expressed VEGFR2WT. Methods: We employed quantitative FLIM/FRET and FRAP imaging techniques using CHO cells co-transfected with the two forms of VEGFR2 to mimic heterozygosity. Results: Membrane protein biotinylation reveals that VEGFR2WT is more exposed on the cell membrane with respect to VEGFR2R1051Q. The imaging analyses show the ability of VEGFR2WT to form heterodimers with VEGFR2R1051Q and this interaction alters its membrane dynamics. Indeed, when the co-expression of VEGFR2WT/VEGFR2R1051Q occurs, VEGFR2WT shows reduced lateral motility and a minor pool of mobile fraction. Conclusions: This study demonstrates that active VEGFR2R1051Q can affect the membrane behavior of the VEGFR2WT
Alternative method to visualize receptor dynamics in cell membranes
Full text linkThere is a close relation between membrane receptor dynamics and their behavior. Several microscopy techniques have been developed to study protein dynamics in live cells such as the Fluorescence Recovery After Photobleaching (FRAP) or the Single Particle Tracking (SPT). These methodologies require expensive instruments, are time consuming, allow the analysis of a small portion of the cell or an extremely small number of receptors at a time. Here we propose a time-saving approach that allows to visualize the entire receptor pool and its localization in time. This protocol requires an epifluorescence microscope equipped for structured illuminated sectioning and for live cell imaging. It can be applied to characterize membrane receptor and multi-protein complex and their response to activators or inhibitors. Image acquisition and analysis can be performed in two days, while cells and substratum preparation require a few minutes a day for three days
Heparin-mimicking sulfonic acid polymers as multitarget inhibitors of HIV-1 Tat and gp120 proteins
No full textHuman immunodeficiency virus (HIV) Tat and gp120 intriguingly share the feature of being basic peptides
that, once released by HIV cells, bind to polyanionic heparan sulfate proteoglycans (HSPGs) on target
uninfected cells, contributing to the onset of AIDS-associated pathologies. To identify multitarget anti-HIV
prodrugs, we investigated the gp120 and Tat antagonist potentials of a series of polyanionic synthetic sulfonic
acid polymers (SSAPs). Surface plasmon resonance revealed that SSAPs inhibit with a competitive mechanism
of action the binding of Tat and gp120 to surface-immobilized heparin, an experimental condition that
resembles binding to cellular HSPGs. Accordingly, SSAPs inhibited HSPG-dependent cell internalization and
the transactivating activity of Tat. Little is known about the binding of free gp120 to target cells. Here, we
identified two classes of gp120 receptors expressed on endothelial cells, one of which was consistent with an
HSPG-binding, low-affinity/high-capacity receptor that is inhibited by free heparin. SSAPs inhibited the
binding of free gp120 to endothelial cells, as well as its capacity to induce apoptosis in the same cells. In all
the assays, poly(4-styrenesulfonic acid) (PSS) proved to be the most potent antagonist of Tat and gp120.
Accordingly, PSS bound both proteins with high affinity. In conclusion, SSAPs represent an interesting class
of compounds that bind both gp120 and Tat and inhibit their HSPG-dependent cell surface binding and
pathological effects. As these activities contribute to both AIDS progression and associated pathologies, SSAPs
can be considered prototypic molecules for the development of multitarget drugs for the treatment of HIV
infection and AIDS-associated pathologies
VEGFR2 and v3 recruitment and cross-talk at the basal aspect of HIV-1 Tat-adherent endothelial cells drives pp60src/ERK1/2 activation, cytoskeleton organization and pro-angiogenic activation
No full textFlk-1/KDR and v3 recruitment and cross-talk at the basal aspect of HIV-1 Tat-adherent endothelial cells drives pp60src/ERK1/2 activation, cytoskeleton organization and pro-angiogenic activation
No full text- …
