Temporal Dynamics of VEGFA-Induced VEGFR2/FAK Co-Localization Depend on SHB

Focal adhesion kinase (FAK) is essential for vascular endothelial growth factor-A (VEGFA)/VEGF receptor-2 (VEGFR2)-stimulated angiogenesis and vascular permeability. We have previously noted that presence of the Src homology-2 domain adapter protein B (SHB) is of relevance for VEGFA-stimulated angiogenesis in a FAK-dependent manner. The current study was conducted in order address the temporal dynamics of co-localization between these components in HEK293 and primary lung endothelial cells (EC) by total internal reflection fluorescence microscopy (TIRF). An early (<2.5 min) VEGFA-induced increase in VEGFR2 co-localization with SHB was dependent on tyrosine 1175 in VEGFR2. VEGFA also enhanced SHB co-localization with FAK. FAK co-localization with VEGFR2 was dependent on SHB since it was significantly lower in SHB deficient EC after VEGFA addition. Absence of SHB also resulted in a gradual decline of VEGFR2 co-localization with FAK under basal (prior to VEGFA addition) conditions. A similar basal response was observed with expression of the Y1175F-VEGFR2 mutant in wild type EC. The distribution of focal adhesions in SHB-deficient EC was altered with a primarily perinuclear location. These live cell data implicate SHB as a key component regulating FAK activity in response to VEGFA/VEGFR2.

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Mathematical models of the VEGF receptor and its role in cancer therapy

Journal of The Royal Society Interface ◽

10.1098/rsif.2006.0170 ◽

2006 ◽

Vol 4 (13) ◽

pp. 283-304 ◽

Cited By ~ 19

Author(s):

Tomás Alarcón ◽

Karen M Page

Keyword(s):

Vascular Endothelial Growth Factor ◽

Receptor Internalization ◽

Vegf Receptor ◽

Vascular Endothelial ◽

Src Homology 2 ◽

Low Concentrations ◽

Increased Sensitivity ◽

Src Homology ◽

Src Homology 2 Domain ◽

Endothelial Growth Factor

We present an analysis of a stochastic model of the vascular endothelial growth factor (VEGF) receptor. This analysis addresses the contribution of ligand-binding-induced oligomerization, activation of src-homology 2 domain-carrying kinases and receptor internalization in the overall behaviour of the VEGF/VEGF receptor (VEGFR) system. The analysis is based upon a generalization of a Wentzel–Kramers–Brillouin (WKB) approximation of the solution of the corresponding master equation. We predict that tumour-mediated overexpression of VEGFRs in the endothelial cells (ECs) of tumour-engulfed vessels leads to an increased sensitivity of the ECs to low concentrations of VEGF, thus endowing the tumour with increased resistance to anti-angiogenic treatment.

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VEGF Promotes Proliferation of Human Glioblastoma Multiforme Stem-Like Cells through VEGF Receptor 2

The Scientific World JOURNAL ◽

10.1155/2013/417413 ◽

2013 ◽

Vol 2013 ◽

pp. 1-8 ◽

Cited By ~ 18

Author(s):

Chengshi Xu ◽

Xing Wu ◽

Jianhong Zhu

Keyword(s):

Glioblastoma Multiforme ◽

Vegf Receptor ◽

Dependent Manner ◽

Vascular Endothelial ◽

Tumor Initiating Cells ◽

Atp Assay ◽

Human Glioblastoma Multiforme ◽

Human Gbm ◽

Endothelial Growth Factor ◽

Dose Dependent

Cancer stem-like cells, which have been described as tumor-initiating cells or tumor-propagating cells, play a crucial role in our fundamental understanding of glioblastoma multiforme (GBM) and its recurrence. GBM is a lethal cancer, characterized by florid vascularization and aberrantly elevated vascular endothelial growth factor (VEGF). VEGF promotes tumorigenesis and angiogenesis of human GBM stem-like cells (GBSCs). However, whether and how VEGF contributes to GBSCs proliferation remain largely uncertain. In this study, human GBSCs were isolated from surgical specimens of glioblastoma and cultured in medium favored for stem cell growth. Neural Colony-Forming Cell Assay and ATP assay were performed to measure GBSC proliferation under normoxia (20% O2) and hypoxia (1% O2). Our observations demonstrate that exogenous VEGF stimulates GBSC proliferation in a dose-dependent manner via VEGF Receptor 2 (VEGFR2); while VEGF Receptor 1 (VEGFR1) has a negative feedback effect on VEGFR2 when cells were exposed to higher concentration of VEGF. These results suggest that suppressing VEGFR2-dependent GBSC proliferation is a potentially therapeutic strategy in GBM.

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Lyn and PECAM-1 function as interdependent inhibitors of platelet aggregation

Blood ◽

10.1182/blood-2010-09-304816 ◽

2011 ◽

Vol 117 (14) ◽

pp. 3903-3906 ◽

Cited By ~ 33

Author(s):

Zhangyin Ming ◽

Yu Hu ◽

Jizhou Xiang ◽

Peter Polewski ◽

Peter J. Newman ◽

...

Keyword(s):

Thrombus Formation ◽

Dependent Manner ◽

Glycoprotein Vi ◽

Src Homology 2 ◽

Platelet Endothelial Cell Adhesion ◽

Src Homology ◽

Src Homology 2 Domain ◽

Cell Adhesion Molecule 1 ◽

Collagen Receptor ◽

Deficient Mice

Abstract Inhibition of platelet responsiveness is important to control pathologic thrombus formation. Platelet–endothelial cell adhesion molecule-1 (PECAM-1) and the Src family kinase Lyn inhibit platelet activation by the glycoprotein VI (GPVI) collagen receptor; however, it is not known whether PECAM-1 and Lyn function in the same or different inhibitory pathways. In these studies, we found that, relative to wild-type platelets, platelets derived from PECAM-1–deficient, Lyn-deficient, or PECAM-1/Lyn double-deficient mice were equally hyperresponsive to stimulation with a GPVI-specific agonist, indicating that PECAM-1 and Lyn participate in the same inhibitory pathway. Lyn was required for PECAM-1 tyrosine phosphorylation and subsequent binding of the Src homology 2 domain–containing phosphatase-2, SHP-2. These results support a model in which PECAM-1/SHP-2 complexes, formed in a Lyn-dependent manner, suppress GPVI signaling.

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Regulation of the vascular endothelial growth factor (VEGF) receptor Flk-1/KDR by estradiol through VEGF in uterus

Journal of Endocrinology ◽

10.1677/joe.1.06184 ◽

2006 ◽

Vol 188 (1) ◽

pp. 91-99 ◽

Cited By ~ 59

Author(s):

M A J Hervé ◽

G Meduri ◽

F G Petit ◽

T S Domet ◽

G Lazennec ◽

...

Keyword(s):

Vascular Endothelial Growth Factor ◽

Endothelial Cells ◽

Growth Factor ◽

Receptor Expression ◽

Maximal Response ◽

Target Cells ◽

Vegf Receptor ◽

Dependent Manner ◽

Vascular Endothelial ◽

Endothelial Growth Factor

The induction of vascular endothelial growth factor (VEGF) expression by 17β-estradiol (E2) in many target cells, including epithelial cells, fibroblasts and smooth muscle cells, suggests a role for this hormone in the modulation of angiogenesis and vascular permeability. We have already described a cyclic increase in Flk-1/KDR-expressing capillaries in the human endometrium during the proliferative and mid-secretory phases, strongly suggestive of an E2 effect on Flk-1/KDR expression in the endometrial capillaries. However, it is unclear whether these processes are due to a direct effect of E2 on endothelial cells. Using immunohistochemistry, we report an increase in Flk-1/KDR expression in endometrial capillaries of ovariectomized mice treated with E2, or both E2 and progesterone. This process is mediated through estrogen receptor (ER) activation. In vitro experiments using quantitative RT-PCR analysis demonstrate that Flk-1/KDR expression was not regulated by E2 in human endothelial cells from the microcirculation (HMEC-1) or macrocirculation (HUVEC), even in endothelial cells overexpressing ERα or ERβ after ER-mediated adenovirus infection. In contrast, Flk-1/KDR expression was up-regulated by VEGF itself, in a time- and dose-dependent manner, with the maximal response at 10 ng/ml. Thus, we suggest that E2 up-regulates Flk-1/KDR expression in vivo in endothelial cells mainly through the modulation of VEGF by a paracrine mechanism. It is currently unknown whether or not the endothelial origin might account for differences in the E2-modulation of VEGF receptor expression, particularly in relation to the vascular bed of sex steroid-responsive tissues.

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Rap1 promotes VEGFR2 activation and angiogenesis by a mechanism involving integrin αvβ3

Blood ◽

10.1182/blood-2011-04-349282 ◽

2011 ◽

Vol 118 (7) ◽

pp. 2015-2026 ◽

Cited By ~ 60

Author(s):

Sribalaji Lakshmikanthan ◽

Magdalena Sobczak ◽

Changzoon Chun ◽

Angela Henschel ◽

Jillian Dargatz ◽

...

Keyword(s):

Integrin Αvβ3 ◽

In Vivo Model ◽

Vegf Receptor ◽

Dependent Manner ◽

Vascular Endothelial ◽

Kinase Activation ◽

Vegf Signaling ◽

Endothelial Growth Factor

Abstract Vascular endothelial growth factor (VEGF) acting through VEGF receptor 2 (VEGFR2) on endothelial cells (ECs) is a key regulator of angiogenesis, a process essential for wound healing and tumor metastasis. Rap1a and Rap1b, 2 highly homologous small G proteins, are both required for angiogenesis in vivo and for normal EC responses to VEGF. Here we sought to determine the mechanism through which Rap1 promotes VEGF-mediated angiogenesis. Using lineage-restricted Rap1-knockout mice we show that Rap1-deficiency in endothelium leads to defective angiogenesis in vivo, in a dose-dependent manner. Using ECs obtained from Rap1-deficient mice we demonstrate that Rap1b promotes VEGF-VEGFR2 kinase activation and regulates integrin activation. Importantly, the Rap1b-dependent VEGF-VEGFR2 activation is in part mediated via integrin αvβ3. Furthermore, in an in vivo model of zebrafish angiogenesis, we demonstrate that Rap1b is essential for the sprouting of intersomitic vessels, a process known to be dependent on VEGF signaling. Using 2 distinct pharmacologic VEGFR2 inhibitors we show that Rap1b and VEGFR2 act additively to control angiogenesis in vivo. We conclude that Rap1b promotes VEGF-mediated angiogenesis by promoting VEGFR2 activation in ECs via integrin αvβ3. These results provide a novel insight into the role of Rap1 in VEGF signaling in ECs.

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Suppression of Development of Ankylosing Spondylitis Through Soluble Flt-1

Cellular Physiology and Biochemistry ◽

10.1159/000438571 ◽

2015 ◽

Vol 37 (6) ◽

pp. 2135-2142 ◽

Cited By ~ 2

Author(s):

Zhongxiang Yu ◽

Yuting Zhang ◽

Ningyang Gao ◽

Kuang Yong

Keyword(s):

Endothelial Cells ◽

Ankylosing Spondylitis ◽

Vegf Receptor ◽

Dependent Manner ◽

Vascular Endothelial ◽

Treatment Results ◽

Vegf Signaling ◽

Before And After ◽

Endothelial Growth Factor ◽

Pro Inflammatory Cytokines

Background/Aims: Circulating monocytes/macrophages are origins of osteoclasts that mediate the development of ankylosing spondylitis (AS). Moreover, infiltrated macrophages facilitate the AS progression through production and secretion of pro-inflammatory cytokines. Thus, suppression of the recruitment of circulating monocytes/macrophages may be an effective AS treatment, which is, however, not available so far in clinic. Soluble fms-like tyrosine kinase-1 (sFlt-1) is a decoy receptor for vascular endothelial growth factor (VEGF) to compete with VEGF receptor (VEGFR2) for VEGF binding in endothelial cells, while its application in treating AS and effects on the recruitment of circulating monocytes/macrophages has not been reported before. Methods: We used a proteoglycan-induced arthritis (PGIA) mouse model for human AS. We injected sFlt-1 into the articular cavity and evaluated its effects on PGIA by incidence of arthritis, and clinical and pathological arthritis severity. We isolated and analyzed macrophages and endothelial cells in the articular cavity before and after treatment. Results: Injection of sFlt-1 significantly decreased the incidence and severity of PGIA in mice, and significantly reduced the number of infiltrated macrophages, possibly through reduction of vessel permeability, in a VEGFR2-dependent manner. Conclusion: Our data suggest that sFlt-1 may have a therapeutic effect on AS, resulting from suppression of VEGF signaling-mediated recruitment of circulating monocytes/macrophages.

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Leishmania Infection Induces Macrophage Vascular Endothelial Growth Factor A Production in an ARNT/HIF-Dependent Manner

Infection and Immunity ◽

10.1128/iai.00088-19 ◽

2019 ◽

Vol 87 (11) ◽

Author(s):

Tiffany Weinkopff ◽

Hayden Roys ◽

Anne Bowlin ◽

Phillip Scott

Keyword(s):

Vascular Endothelial Growth Factor ◽

Growth Factor ◽

Leishmania Major ◽

Leishmania Infection ◽

Vegf Receptor ◽

Dependent Manner ◽

Vascular Endothelial ◽

Cell Type ◽

Content Type ◽

Endothelial Growth Factor

ABSTRACT Cutaneous leishmaniasis is characterized by vascular remodeling. Following infection with Leishmania parasites, the vascular endothelial growth factor A (VEGF-A)/VEGF receptor 2 (VEGFR-2) signaling pathway mediates lymphangiogenesis, which is critical for lesion resolution. Therefore, we investigated the cellular and molecular mediators involved in VEGF-A/VEGFR-2 signaling using a murine model of infection. We found that macrophages are the predominant cell type expressing VEGF-A during Leishmania major infection. Given that Leishmania parasites activate hypoxia-inducible factor 1α (HIF-1α) and this transcription factor can drive VEGF-A expression, we analyzed the expression of HIF-1α during infection. We showed that macrophages were also the major cell type expressing HIF-1α during infection and that infection-induced VEGF-A production is mediated by ARNT/HIF activation. Furthermore, mice deficient in myeloid ARNT/HIF signaling exhibited larger lesions without differences in parasite numbers. These data show that L. major infection induces macrophage VEGF-A production in an ARNT/HIF-dependent manner and suggest that ARNT/HIF signaling may limit inflammation by promoting VEGF-A production and, thus, lymphangiogenesis during infection.

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p56lck, LFA-1 and PI3K but not SHP-2 interact with GM1- or GM3-enriched microdomains in a CD4–p56lck association-dependent manner

Biochemical Journal ◽

10.1042/bj20061061 ◽

2007 ◽

Vol 402 (3) ◽

pp. 471-481 ◽

Cited By ~ 16

Author(s):

Christiane Barbat ◽

Maylis Trucy ◽

Maurizio Sorice ◽

Tina Garofalo ◽

Valeria Manganelli ◽

...

Keyword(s):

Tyrosine Phosphatase ◽

Structural Diversity ◽

Mutant Form ◽

Lymphocyte Function ◽

Dependent Manner ◽

Down Regulation ◽

Src Homology 2 ◽

Src Homology ◽

Src Homology 2 Domain ◽

Phosphoinositide 3 Kinase

We previously showed that the association of CD4 and GM3 ganglioside induced by CD4 ligand binding was required for the down-regulation of adhesion and that aggregation of ganglioside-enriched domains was accompanied by transient co-localization of LFA-1 (lymphocyte function-associated antigen-1), PI3K (phosphoinositide 3-kinase) and CD4. We also showed that these proteins co-localized with the GM1 ganglioside that partially co-localized with GM3 in these domains. In the present study, we show that CD4–p56lck association in CD4 signalling is required for the redistribution of p56lck, PI3K and LFA-1 in ganglioside-enriched domains, since ganglioside aggregation and recruitment of these proteins were not observed in a T-cell line (A201) expressing the mutant form of CD4 that does not bind p56lck. In addition, we show that although these proteins associated in different ways with GM1 and GM3, all of the associations were dependent on CD4–p56lck association. Gangliosides could associate with these proteins that differ in affinity binding and could be modified following CD4 signalling. Our results suggest that through these associations, gangliosides transiently sequestrate these proteins and consequently inhibit LFA-1-dependent adhesion. Furthermore, while structural diversity of gangliosides may allow association with distinct proteins, we show that the tyrosine phosphatase SHP-2 (Src homology 2 domain-containing protein tyrosine phosphatase 2), also required for the down-regulation of LFA-1-dependent adhesion, transiently and partially co-localized with PI3K and p56lck in detergent-insoluble membranes without association with GM1 or GM3. We propose that CD4 ligation and binding with p56lck and their interaction with GM3 and/or GM1 gangliosides induce recruitment of distinct proteins important for CD4 signalling to form a multimolecular signalling complex.

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