HIV TAT Basic Peptide Is Not a High-Affinity Ligand for VEGF Receptor 2

2003 ◽  
Vol 384 (10-11) ◽  
pp. 1435-1441 ◽  
Author(s):  
A. Rubio Demirovic ◽  
J. Canadi ◽  
W. Weiglhofer ◽  
P. Scheidegger ◽  
R. Jaussi ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Peptide Sequence ◽  
Vegf Receptor ◽  
Binding Studies ◽  
Basic Peptide ◽  
Signalling Molecules ◽  
High Affinity ◽  
Affinity Ligand ◽  
Domain 3

AbstractThe 'transactivator of transcription' (TAT) protein of human immunodeficiency virus transforms cells in culture and promotes the development of tumors, socalled Kaposi's sarcoma, in AIDS patients. TAT induces growth and differentiation of blood vessels and has been suggested to directly activate VEGF receptor 2 expressed on endothelial cells through a peptide sequence located between amino acids 46 and 64, the so-called basic domain. This peptide mimics many aspects of TAT function when added to endothelial cells, even when expressed in the context of recombinant chimeric proteins. To define the exact sites of interaction between this peptide and VEGF receptor 2 we performed binding studies with recombinant proteins derived from the extracellular ligand binding domain of VEGF receptor 2. These in vitro binding studies showed that the TAT peptide binds with only low specificity to Iglike domain 3 of the receptor, while VEGF interacts with receptor-derived proteins encompassing at least extracellular domains 1 through 3. The original concept that the angiogenic properties of TAT basic peptide result from specific, high-affinity interaction with VEGF receptor 2 must therefore be revised. Apparently this peptide interacts with cells in multiple ways: by directly activating acidic cell surface-exposed receptors, by releasing extracellular matrix-bound growth factors such as bFGF and VEGF which then bind to their cognate receptors, and by activating intracellular signalling molecules with which basic peptide interacts upon translocation into cells.

Neuropilin-1 on hematopoietic cells as a source of vascular development

Blood ◽  
2003 ◽  
Vol 101 (5) ◽  
pp. 1801-1809 ◽  
Author(s):  
Yoshihiro Yamada ◽  
Yuichi Oike ◽  
Hisao Ogawa ◽  
Yasuhiro Ito ◽  
Hajime Fujisawa ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Fetal Liver ◽  
Vascular Development ◽  
Hematopoietic Cells ◽  
Vegf Receptor ◽  
Knock Out ◽  
Neuropilin 1 ◽  
Vasculogenesis And Angiogenesis

Neuropilin-1 (NP-1) is a receptor for vascular endothelial growth factor-165 (VEGF165) and acts as a coreceptor that enhances the function of VEGF165 through VEGF receptor-2 (VEGFR-2). Studies using transgenic and knock-out mice of NP-1 indicated that this molecule is important for vascular development as well as neuronal development. We recently reported that clustered soluble NP-1 phosphorylates VEGFR-2 on endothelial cells with a low dose of VEGF165 and rescues the defective vascularity of the NP-1−/− embryo in vitro and in vivo. Here we show that NP-1 is expressed by CD45+ hematopoietic cells in the fetal liver, can bind VEGF165, and phosphorylates VEGFR-2 on endothelial cells. CD45+NP-1+ cells rescued the defective vasculogenesis and angiogenesis in the NP-1−/− P-Sp (para-aortic splanchnopleural mesodermal region) culture, although CD45+NP-1− cells did not. Moreover, CD45+NP-1+ cells together with VEGF165 induced angiogenesis in an in vivo Matrigel assay and cornea neovascularization assay. The extracellular domain of NP-1 consists of “a,” “b,” and “c” domains, and it is known that the “a” and “c” domains are necessary for dimerization of NP-1. We found that both the “a” and “c” domains are essential for such rescue of defective vascularities in the NP-1 mutant. These results suggest that NP-1 enhances vasculogenesis and angiogenesis exogenously and that dimerization of NP-1 is important for enhancing vascular development. In NP-1−/− embryos, vascular sprouting is impaired at the central nervous system (CNS) and pericardium where VEGF is not abundant, indicating that NP-1–expressing cells are required for normal vascular development.

scholarly journals Acute ethanol exposure disrupts VEGF receptor cell signaling in endothelial cells

2008 ◽  
Vol 295 (1) ◽  
pp. H174-H184 ◽  
Author(s):  
Katherine A. Radek ◽  
Elizabeth J. Kovacs ◽  
Richard L. Gallo ◽  
Luisa A. DiPietro
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Cell Signaling ◽  
Network Formation ◽  
Ethanol Exposure ◽  
Capillary Network ◽  
Ethanol Metabolism ◽  
Vegf Receptor ◽  
Acute Ethanol

Physiological angiogenesis is regulated by various factors, including signaling through vascular endothelial growth factor (VEGF) receptors. We previously reported that a single dose of ethanol (1.4 g/kg), yielding a blood alcohol concentration of 100 mg/dl, significantly impairs angiogenesis in murine wounds, despite adequate levels of VEGF, suggesting direct effects of ethanol on endothelial cell signaling (40). To examine the mechanism by which ethanol influences angiogenesis in wounds, we employed two different in vitro angiogenesis assays to determine whether acute ethanol exposure (100 mg/dl) would have long-lasting effects on VEGF-induced capillary network formation. Ethanol exposure resulted in reduced VEGF-induced cord formation on collagen and reduced capillary network structure on Matrigel in vitro. In addition, ethanol exposure decreased expression of endothelial VEGF receptor-2, as well as VEGF receptor-2 phosphorylation in vitro. Inhibition of ethanol metabolism by 4-methylpyrazole partially abrogated the effect of ethanol on endothelial cell cord formation. However, mice treated with t-butanol, an alcohol not metabolized by alcohol dehydrogenase, exhibited no change in wound vascularity. These results suggest that products of ethanol metabolism are important factors in the development of ethanol-induced changes in endothelial cell responsiveness to VEGF. In vivo, ethanol exposure caused both decreased angiogenesis and increased hypoxia in wounds. Moreover, in vitro experiments demonstrated a direct effect of ethanol on the response to hypoxia in endothelial cells, as ethanol diminished nuclear hypoxia-inducible factor-1α protein levels. Together, the data establish that acute ethanol exposure significantly impairs angiogenesis and suggest that this effect is mediated by changes in endothelial cell responsiveness to both VEGF and hypoxia.

scholarly journals Islet vascularization is regulated by primary endothelial cilia via VEGF-A-dependent signaling

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Yan Xiong ◽  
M Julia Scerbo ◽  
Anett Seelig ◽  
Francesco Volta ◽  
Nils O'Brien ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Primary Cilia ◽  
Capillary Density ◽  
Vegf Receptor ◽  
Vascular Endothelial ◽  
Downstream Signaling ◽  
Vegfr2 Signaling ◽  
And Migration ◽  
Endothelial Growth Factor

Islet vascularization is essential for intact islet function and glucose homeostasis. We have previously shown that primary cilia directly regulate insulin secretion. However, it remains unclear whether they are also implicated in islet vascularization. At eight weeks, murine Bbs4-/-islets show significantly lower intra-islet capillary density with enlarged diameters. Transplanted Bbs4-/- islets exhibit delayed re-vascularization and reduced vascular fenestration after engraftment, partially impairing vascular permeability and glucose delivery to β-cells. We identified primary cilia on endothelial cells as the underlying cause of this regulation, via the vascular endothelial growth factor-A (VEGF-A)/VEGF receptor 2 (VEGFR2) pathway. In vitro silencing of ciliary genes in endothelial cells disrupts VEGF-A/VEGFR2 internalization and downstream signaling. Consequently, key features of angiogenesis including proliferation and migration are attenuated in human BBS4 silenced endothelial cells. We conclude that endothelial cell primary cilia regulate islet vascularization and vascular barrier function via the VEGF-A/VEGFR2 signaling pathway.

scholarly journals Two peptides targeting endothelial receptors are internalized into murine brain endothelial cells

PLoS ONE ◽  
2021 ◽  
Vol 16 (4) ◽  
pp. e0249686
Author(s):  
Diána Hudecz ◽  
Sara Björk Sigurdardóttir ◽  
Sarah Christine Christensen ◽  
Casper Hempel ◽  
Andrew J. Urquhart ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Transferrin Receptor ◽  
Vesicular Transport ◽  
Brain Diseases ◽  
Brain Endothelial Cells ◽  
Binding Studies ◽  
Wide Range ◽  
Murine Brain ◽  
The Brain

The blood-brain barrier (BBB) is one of the main obstacles for therapies targeting brain diseases. Most macromolecules fail to pass the tight BBB, formed by brain endothelial cells interlinked by tight junctions. A wide range of small, lipid-soluble molecules can enter the brain parenchyma via diffusion, whereas macromolecules have to transcytose via vesicular transport. Vesicular transport can thus be utilized as a strategy to deliver brain therapies. By conjugating BBB targeting antibodies and peptides to therapeutic molecules or nanoparticles, it is possible to increase uptake into the brain. Previously, the synthetic peptide GYR and a peptide derived from melanotransferrin (MTfp) have been suggested as candidates for mediating transcytosis in brain endothelial cells (BECs). Here we study uptake, intracellular trafficking, and translocation of these two peptides in BECs. The peptides were synthesized, and binding studies to purified endocytic receptors were performed using surface plasmon resonance. Furthermore, the peptides were conjugated to a fluorophore allowing for live-cell imaging studies of their uptake into murine brain endothelial cells. Both peptides bound to low-density lipoprotein receptor-related protein 1 (LRP-1) and the human transferrin receptor, while lower affinity was observed against the murine transferrin receptor. The MTfp showed a higher binding affinity to all receptors when compared to the GYR peptide. The peptides were internalized by the bEnd.3 mouse endothelial cells within 30 min of incubation and frequently co-localized with endo-lysosomal vesicles. Moreover, our in vitro Transwell translocation experiments confirmed that GYR was able to cross the murine barrier and indicated the successful translocation of MTfp. Thus, despite binding to endocytic receptors with different affinities, both peptides are able to transcytose across the murine BECs.

Vascular endothelial growth factor (VEGF) in cartilage neovascularization and chondrocyte differentiation: auto-paracrine role during endochondral bone formation

2000 ◽  
Vol 113 (1) ◽  
pp. 59-69 ◽  
Author(s):  
M.F. Carlevaro ◽  
S. Cermelli ◽  
R. Cancedda ◽  
F. Descalzi Cancedda
Keyword(s):  
Endothelial Cells ◽  
Cell Migration ◽  
Endothelial Cell ◽  
Hypertrophic Chondrocytes ◽  
Endothelial Cell Migration ◽  
Vegf Receptor ◽  
Hypertrophic Cartilage ◽  
Endothelial Growth Factor

Vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) induces endothelial cell migration and proliferation in culture and is strongly angiogenic in vivo. VEGF synthesis has been shown to occur in both normal and transformed cells. The receptors for the factor have been shown to be localized mainly in endothelial cells, however, the presence of VEGF synthesis and the VEGF receptor in cells other than endothelial cells has been demonstrated. Neoangiogenesis in cartilage growth plate plays a fundamental role in endochondral ossification. We have shown that, in an avian in vitro system for chondrocyte differentiation, VEGF was produced and localized in cell clusters totally resembling in vivo cartilage. The factor was synthesized by hypertrophic chondrocytes and was released into their conditioned medium, which is highly chemotactic for endothelial cells. Antibodies against VEGF inhibited endothelial cell migration induced by chondrocyte conditioned media. Similarly, endothelial cell migration was inhibited also by antibodies directed against the VEGF receptor 2/Flk1 (VEGFR2). In avian and mammalian embryo long bones, immediately before vascular invasion, VEGF was distinctly localized in growth plate hypertrophic chondrocytes. In contrast, VEGF was not observed in quiescent and proliferating chondrocytes earlier in development. VEGF receptor 2 colocalized with the factor both in hypertrophic cartilage in vivo and hypertrophic cartilage engineered in vitro, suggesting an autocrine loop in chondrocytes at the time of their maturation to hypertrophic cells and of cartilage erosion. Regardless of cell exposure to exogenous VEGF, VEGFR-2 phosphorylation was recognized in cultured hypertrophic chondrocytes, supporting the idea of an autocrine functional activation of signal transduction in this non-endothelial cell type as a consequence of the endogenous VEGF production. In summary we propose that VEGF is actively responsible for hypertrophic cartilage neovascularization through a paracrine release by chondrocytes, with invading endothelial cells as a target. Furthermore, VEGF receptor localization and signal transduction in chondrocytes strongly support the hypothesis of a VEGF autocrine activity also in morphogenesis and differentiation of a mesoderm derived cell.

scholarly journals Characterization of a new monoclonal antibody (PG-M3) directed against the aminoterminal portion of the PML gene product: immunocytochemical evidence for high expression of PML proteins on activated macrophages, endothelial cells, and epithelia

Blood ◽  
1995 ◽  
Vol 85 (7) ◽  
pp. 1871-1880 ◽  
Author(s):  
L Flenghi ◽  
M Fagioli ◽  
L Tomassoni ◽  
S Pileri ◽  
M Gambacorta ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Endothelial Cells ◽  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Retinoic Acid Receptor ◽  
Chromosomal Translocation ◽  
Peptide Sequence ◽  
Nuclear Staining

PG-M3 is a new monoclonal antibody (MoAb) specifically directed against a peptide sequence located in the aminoterminal region of the human PML protein. PML gene fuses with the retinoic acid receptor alpha (RAR alpha) gene during the t(15; 17) chromosomal translocation of acute promyelocytic leukemia (APL). The epitope recognized by PG-M3 is species-specific and fixative-resistant and is shared by most PML isoforms and PML/RAR alpha fusion proteins. PML is consistently located within the nucleus, although a minority of cells (about 20%), both in vitro and in vivo, show positivity for PML also in the cytoplasm. The nuclear staining pattern of PG-M3 varies from speckled (cells other than APL) to micropunctate (APL cells). Although two physiologically expressed PML isoforms are detectable by immunocytochemistry only or predominantly in the cytoplasm of transfected cells, the cytoplasmic localization of PML is a property also shared by the PML isoforms that predominantly localize to the nuclei. Immunohistologic analysis of normal human tissues with the PG-M3 MoAb showed variable PML expression, with the highest levels of the protein in postmitotic, differentiated cell types, such as endothelial cells, epithelia, and tissue macrophages, especially activated ones. In keeping with this in vivo finding, PML appears strongly upregulated in the U937 promonocyte cell line after exposure to agents that induce monocyte/macrophage activation (interferon gamma) or maturation (vitamin D3 and transforming growth factor beta 1).

scholarly journals Inhibiting the NLRP3 inflammasome with MCC950 ameliorates retinal neovascularization and leakage by reversing the IL-1β/IL-18 activation pattern in an oxygen-induced ischemic retinopathy mouse model

2020 ◽  
Vol 11 (10) ◽  
Author(s):  
Ailing Sui ◽  
Xiuping Chen ◽  
Jikui Shen ◽  
Anna M. Demetriades ◽  
Yiyun Yao ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Growth Factor ◽  
Nlrp3 Inflammasome ◽  
Retinal Neovascularization ◽  
Platelet Derived Growth Factor ◽  
Vegf Receptor ◽  
Activation Pattern ◽  
Ischemic Retinopathy

Abstract Activation of the nucleotide-binding domain leucine-rich repeat and pyrin domain containing receptor 3 (NLRP3) inflammasome plays an important role in ocular neovascularization. In our study, we found that the expression and activation levels of NLRP3 inflammasome components, including NLRP3, an apoptosis-associated speck-like protein (ASC) containing caspase activation and recruitment domain (CARD) and caspase-1 (CAS1), were significantly upregulated. In addition, we found interleukin (IL)-1β activity increased while IL-18 activity decreased in the retinas of oxygen-induced ischemic retinopathy (OIR) mice. MCC950, an inhibitor of NLRP3, reversed the IL-1β/IL-18 activation pattern, inhibited the formation of retinal neovascularization (RNV), decreased the number of acellular capillaries and reduced leakage of retinal vessels. Moreover, MCC950 could regulate the expression of endothelial cell- and pericyte function-associated molecules, such as vascular endothelial growth factor (VEGF), VEGF receptor (VEGFR)1, VEGFR2, matrix metalloproteinase (MMP)2, MMP9, tissue inhibitor of metalloproteinases (TIMP)1, TIMP2, platelet-derived growth factor receptor-β (PDGFR-β), platelet-derived growth factor-B (PDGF-B), and angiopoietin2 (Ang2). In vitro, recombinant human (r)IL-18 and rIL-1β regulated the expression of endothelial cell- and pericyte function-associated molecules and the proliferation and migration of endothelial cells and pericytes. We therefore determined that inhibiting the NLRP3 inflammasome with MCC950 can regulate the function of endothelial cells and pericytes by reversing the IL-1β/IL-18 activation pattern to ameliorate RNV and leakage; thereby opening new avenues to treat RNV-associated ocular diseases.

Exogenous clustered neuropilin 1 enhances vasculogenesis and angiogenesis

Blood ◽  
2001 ◽  
Vol 97 (6) ◽  
pp. 1671-1678 ◽  
Author(s):  
Yoshihiro Yamada ◽  
Nobuyuki Takakura ◽  
Hirofumi Yasue ◽  
Hisao Ogawa ◽  
Hajime Fujisawa ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Low Dose ◽  
Vascular Development ◽  
Vegf Receptor ◽  
Wild Type ◽  
Flag Epitope ◽  
Neuropilin 1 ◽  
Vasculogenesis And Angiogenesis

Neuropilin 1 (NP-1) is a receptor for vascular endothelial growth factor (VEGF) 165 (VEGF165) and acts as a coreceptor that enhances VEGF165 function through tyrosine kinase VEGF receptor 2 (VEGFR-2). Transgenic overexpression of np-1results in an excess of capillaries and blood vessels and a malformed heart. Thus, NP-1 may have a key role in vascular development. However, how NP-1 regulates vascular development is not well understood. This study demonstrates how NP-1 can regulate vasculogenesis and angiogenesis in vitro and in vivo. In homozygous np-1mutant (np-1−/−) murine embryos, vascular sprouting was impaired in the central nervous system and pericardium. Para-aortic splanchnopleural mesoderm (P-Sp) explants fromnp-1−/− mice also had vascular defects in vitro. A monomer of soluble NP-1 (NP-1 tagged with Flag epitope) inhibited vascular development in cultured wild-type P-Sp explants by sequestering VEGF165. In contrast, a dimer of soluble NP-1 (NP-1 fused with the Fc part of human IgG) enhanced vascular development in cultured wild-type P-Sp explants. Moreover, the NP-1–Fc rescued the defective vascular development in culturednp-1−/− P-Sp explants. A low dose of VEGF alone did not promote phosphorylation of VEGFR-2 on endothelial cells from np-1−/− embryos, but simultaneous addition of a low dose of VEGF and NP-1–Fc phosphorylated VEGFR-2 significantly. Moreover, NP-1–Fc rescued the defective vascularity of np-1−/− embryos in vivo. These results suggest that a dimer form of soluble NP-1 delivers VEGF165 to VEGFR-2–positive endothelial cells and promotes angiogenesis.

Platelet-Derived Microparticles Induce Angiogenesis and Stimulate Post-Ischemic Revascularization.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3916-3916
Author(s):  
Olga Dashevsky ◽  
Alexander Brill ◽  
Julia Rivo ◽  
David Varon
Keyword(s):  
Endothelial Cells ◽  
Aortic Ring ◽  
In Vivo Model ◽  
Preliminary Evaluation ◽  
Vegf Receptor ◽  
In Vivo Models ◽  
Ischemic Region

Abstract Platelet attachment to the subcellular matrix at injured sites of the vasculature is followed by their activation and release of microparticles. Platelet-derived microparticles (PMP) have been shown to be involved in the regulation of hemostasis. However, little is known about the role of PMP in the regulation of angiogenesis and related clinical conditions. We have recently demonstrated that platelets as a cellular system induce angiogenic responses both in vitro and in vivo. In the present study, we investigated the potential role of PMP in angiogenesis. A strong dose-dependent pro-angiogenic effect of PMP in the rat aortic ring model (5.3±2.1 mm2 surface covered with sprouting vessels versus 0.24±0.2 mm2 in the control, p<0.001) was observed. This effect was reversed by selective inhibition of VEGF, bFGF and PDGF (surface covered with vessels 0.7±0.5 mm2, 1.7±1.5 mm2, and 2.4±1.2 mm2, respectively, p<0.02 versus control), but not by inhibition of heparanase (5.1±0.8 mm2, p>0.5 versus control). PMP exert their stimulatory effect via PI3-kinase, Src kinase and ERK, whereas protein kinase C seems not to be involved, as judged by the aortic ring sprouting model. Using confocal and electron microscopy, we also demonstrate that PMP bind to non-activated endothelial cells. In addition, PMP markedly increased invasion of human endothelial cells through a layer of matrigel. This effect was abolished by an inhibitor of VEGF receptor tyrosine phosphorylation or laminaran sulfate (heparanase inhibitor). It was also partially reduced by PDGF blocking mAb, whereas blocking of bFGF had no effect. Furthermore, we have demonstrated that PMP induce angiogenesis in an in vivo model, in which beads (30 μl) of 4% agarose gel containing the substances under study were transplanted subcutaneously into mice. Image analysis of the capillary area revealed the following: control beads − 0.2±0.05 mm2, VEGF + bFGF containing beads − 4.8±1.1 mm2, PMP (100 μg/ml) containing beads − 5.1±1.3 mm2, p<0.001 versus control. The latter finding was further supported by immunohistochemical staining of the skin in the vicinity of the beads for von Willebrand factor, a marker of endothelial cells (control − 4.0±3.2, VEGF+bFGF − 12±4.4, PMP − 17±6.5 capillaries per view field, p<0.05 versus control). Finally, we explored the potential effect of PMP in a rat myocardial infarction model. Ischemia was induced by LAD ligation followed by injection of either PMP or PBS into the ischemic region. Preliminary evaluation of the LAD myocardial territory in sham-operated animals revealed 157±42.0 capillaries per view field. In contrast, number of capillaries observed 3 weeks after induction of ischemia was reduced to 34±21.5. When PMP were injected into the ischemic region, there was an increase in capillary number up to 97±27.3. In conclusion, PMP induce angiogenesis in both in vitro and in vivo models. Local injection of PMP into the ischemic myocardium may improve revascularization.

scholarly journals Analysis of pre- and postsynaptic factors of the serotonin system in rabbit retina.

1985 ◽  
Vol 100 (1) ◽  
pp. 64-73 ◽  
Author(s):  
C K Mitchell ◽  
D A Redburn
Keyword(s):  
Serotonin Receptor ◽  
Amacrine Cells ◽  
Uptake System ◽  
Rabbit Retina ◽  
Binding Studies ◽  
High Affinity ◽  
Serotonin System ◽  
Significant Difference

[3H]Serotonin is accumulated by a specific set of amacrine cells in the rabbit retina. These cells also accumulate the neurotoxin, 5,7-dihydroxytryptamine, and show signs of necrosis within 4 h of in vivo exposure to the drug. Biochemical analysis of [3H]serotonin uptake reveal a sodium- and temperature-dependent, high affinity uptake system with a Km of 0.94 microM and Vmax of 1.08 pmol/mg protein/min. [3H]Tryptophan is also accumulated in rabbit retinal homogenates by a high affinity process. Accumulated [3H]serotonin is released in response to potassium-induced depolarization of intact, isolated retinas. In vitro binding studies of rabbit retinal homogenate membranes demonstrate specific sets of binding sites with characteristics of the postsynaptic serotonin receptor. These data strongly suggest that rabbit retina contains virtually all of the molecular components required for a functional serotonergic neurotransmitter system. The only significant difference between the serotonin system in rabbit retina and that in the well-established serotonin transmitter systems in nonmammalin retinas and in brains of most species is the relatively low concentration of endogenous serotonin in rabbit retinas, as demonstrated by high-performance liquid chromatography, histofluorescence, or immunocytochemistry.

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