Modulation of VEGFR-2–mediated endothelial-cell activity by VEGF-C/VEGFR-3

Vascular endothelial growth factor (VEGF) receptor 3 (VEGFR-3), a receptor for VEGF-C, was shown to be essential for angiogenesis as well as for lymphangiogenesis. Targeted disruption of theVEGFR-3 gene in mice and our previous study using an antagonistic monoclonal antibody (MoAb) for VEGFR-3 suggested that VEGF-C/VEGFR-3 signals might be involved in the maintenance of vascular integrity. In this study we used an in vitro embryonic stem (ES) cell culture system to maintain the VEGFR-3+ endothelial cell (EC) and investigated the role of VEGFR-3 signals at the cellular level. In this system packed clusters of ECs were formed. Whereas addition of exogenous VEGF-A induced EC dispersion, VEGF-C, which can also stimulate VEGFR-2, promoted EC growth without disturbing the EC clusters. Moreover, addition of AFL4, an antagonistic MoAb for VEGFR-3, resulted in EC dispersion. Cytological analysis showed that VEGF-A– and AFL4-treated ECs were indistinguishable in many aspects but were distinct from the cytological profile induced by antagonistic MoAb for VE-cadherin (VECD-1). As AFL4- induced EC dispersion requires VEGF-A stimulation, it is likely that VEGFR-3 signals negatively modulate VEGFR-2. This result provides new insights into the involvement of VEGFR-3 signals in the maintenance of vascular integrity through modulation of VEGFR-2 signals. Moreover, our findings suggest that the mechanisms underlying AFL4-induced EC dispersion are distinct from those underlying VECD-1–induced dispersion for maintenance of EC integrity.

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The Role of the Vascular Endothelial Growth Factor–Delta-like 4 Ligand/Notch4-Ephrin B2 Cascade in Tumor Vessel Remodeling and Endothelial Cell Functions

Cancer Research ◽

10.1158/0008-5472.can-05-4226 ◽

2006 ◽

Vol 66 (17) ◽

pp. 8501-8510 ◽

Cited By ~ 120

Author(s):

Patricia Hainaud ◽

Jean-Olivier Contrerès ◽

Aude Villemain ◽

Lang-Xia Liu ◽

Jean Plouët ◽

...

Keyword(s):

Vascular Endothelial Growth Factor ◽

Endothelial Cell ◽

Growth Factor ◽

Vascular Endothelial ◽

Tumor Vessel ◽

Cell Functions ◽

Vessel Remodeling ◽

Endothelial Growth Factor

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Enhanced Vascular Endothelial Cell Function on Nanostructured Titanium Surface Features: The Role of Nano to Submicron Roughness

MRS Proceedings ◽

10.1557/proc-1136-dd04-04 ◽

2008 ◽

Vol 1136 ◽

Cited By ~ 2

Author(s):

Jing Lu ◽

Dongwoo Khang ◽

Thomas J. Webster

Keyword(s):

Cell Adhesion ◽

Endothelial Cell ◽

Cell Function ◽

Vascular Endothelial Cell ◽

Vascular Endothelial ◽

Endothelial Cell Function ◽

Titanium Surfaces ◽

Endothelial Cell Adhesion

ABSTRACTTo study the contribution of different surface feature properties in improving vascular endothelial cell adhesion, rationally designed nano/sub-micron patterns with various dimensions were created on titanium surfaces in this study. In vitro results indicated that endothelial cell adhesion was improved when the titanium pattern dimensions decreased into the nano-scale. Specifically, endothelial cells preferred to adhere on sub-micron and nano rough titanium substrates compared to flat titanium. Moreover, titanium with nano and sub-micron roughness and with the same chemistry as compared to flat titanium, had significantly greater surface energy. Thus, the present study indicated the strong potential of surface nanotopography and nano/sub-micron roughness for improving current vascular stent design.

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Maturation of Embryonic Stem Cells Into Endothelial Cells in an In Vitro Model of Vasculogenesis

Blood ◽

10.1182/blood.v93.4.1253.404k31_1253_1263 ◽

1999 ◽

Vol 93 (4) ◽

pp. 1253-1263 ◽

Cited By ~ 5

Author(s):

Masanori Hirashima ◽

Hiroshi Kataoka ◽

Satomi Nishikawa ◽

Norihisa Matsuyoshi ◽

Shin-Ichi Nishikawa

Keyword(s):

Cell Adhesion ◽

Endothelial Cell ◽

Growth Factor ◽

Culture System ◽

Molecular Mechanisms ◽

Es Cells ◽

Embryonic Stem ◽

Vascular Endothelial ◽

Cell Cell

A primitive vascular plexus is formed through coordinated regulation of differentiation, proliferation, migration, and cell-cell adhesion of endothelial cell (EC) progenitors. In this study, a culture system was devised to investigate the behavior of purified EC progenitors in vitro. Because Flk-1+ cells derived from ES cells did not initially express other EC markers, they were sorted and used as EC progenitors. Their in vitro differentiation into ECs, via vascular endothelial-cadherin (VE-cadherin)+ platelet-endothelial cell adhesion molecule-1 (PECAM-1)+ CD34−to VE-cadherin+ PECAM-1+CD34+ stage, occurred without exogenous factors, whereas their proliferation, particularly at low cell density, required OP9 feeder cells. On OP9 feeder layer, EC progenitors gave rise to sheet-like clusters of Flk-1+ cells, with VE-cadherin concentrated at the cell-cell junction. The growth was suppressed by Flt-1-IgG1 chimeric protein and dependent on vascular endothelial growth factor (VEGF) but not placenta growth factor (PIGF). Further addition of VEGF resulted in cell dispersion, indicating the role of VEGF in the migration of ECs as well as their proliferation. Cell-cell adhesion of ECs in this culture system was mediated by VE-cadherin. Thus, the culture system described here is useful in dissecting the cellular events of EC progenitors that occur during vasculogenesis and in investigating the molecular mechanisms underlying these processes.

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

eLife ◽

10.7554/elife.56914 ◽

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.

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

Journal of Cell Science ◽

10.1242/jcs.113.1.59 ◽

2000 ◽

Vol 113 (1) ◽

pp. 59-69 ◽

Cited By ~ 8

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.

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Vascular endothelial growth factor synergistically enhances bone morphogenetic protein-4-dependent lymphohematopoietic cell generation from embryonic stem cells in vitro

Blood ◽

10.1182/blood.v95.7.2275 ◽

2000 ◽

Vol 95 (7) ◽

pp. 2275-2283 ◽

Cited By ~ 65

Author(s):

Naoki Nakayama ◽

Jae Lee ◽

Laura Chiu

Keyword(s):

Vascular Endothelial Growth Factor ◽

Growth Factor ◽

Bone Morphogenetic Protein ◽

Es Cells ◽

Embryonic Stem ◽

Dependent Manner ◽

Vascular Endothelial ◽

Morphogenetic Protein ◽

Endothelial Growth Factor

Abstract The totipotent mouse embryonic stem (ES) cell is known to differentiate into cells expressing the β-globin gene when stimulated with bone morphogenetic protein (BMP)-4. Here, we demonstrate that BMP-4 is essential for generating both erythro-myeloid colony-forming cells (CFCs) and lymphoid (B and NK) progenitor cells from ES cells and that vascular endothelial growth factor (VEGF) synergizes with BMP-4. The CD45+ myelomonocytic progenitors and Ter119+ erythroid cells began to be detected with 0.5 ng/mL BMP-4, and their levels plateaued at approximately 2 ng/mL. VEGF alone weakly elevated the CD34+ cell population though no lymphohematopoietic progenitors were induced. However, when combined with BMP-4, 2 to 20 ng/mL VEGF synergistically augmented the BMP-4-dependent generation of erythro-myeloid CFCs and lymphoid progenitors from ES cells, which were enriched in CD34+ CD31lo and CD34+CD45− cell populations, respectively, in a dose-dependent manner. Furthermore, during the 7 days of in vitro differentiation, BMP-4 was required within the first 4 days, whereas VEGF was functional after the action of BMP-4 (in the last 3 days). Thus, VEGF is a synergistic enhancer for the BMP-4-dependent differentiation processes, and it seems to be achieved by the ordered action of the 2 factors.

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Role of kinase-independent and -dependent functions of FAK in endothelial cell survival and barrier function during embryonic development

The Journal of Cell Biology ◽

10.1083/jcb.200912094 ◽

2010 ◽

Vol 189 (6) ◽

pp. 955-965 ◽

Cited By ~ 76

Author(s):

Xiaofeng Zhao ◽

Xu Peng ◽

Shaogang Sun ◽

Ann Y.J. Park ◽

Jun-Lin Guan

Keyword(s):

Endothelial Cell ◽

Normal Development ◽

Vascular Development ◽

Vascular Endothelial ◽

Vascular Endothelial Cadherin ◽

Abnormal Distribution ◽

Endothelial Cell Survival ◽

Independent Functions

Focal adhesion kinase (FAK) is essential for vascular development as endothelial cell (EC)–specific knockout of FAK (conditional FAK knockout [CFKO] mice) leads to embryonic lethality. In this study, we report the differential kinase-independent and -dependent functions of FAK in vascular development by creating and analyzing an EC-specific FAK kinase-defective (KD) mutant knockin (conditional FAK knockin [CFKI]) mouse model. CFKI embryos showed apparently normal development through embryonic day (E) 13.5, whereas the majority of CFKO embryos died at the same stage. Expression of KD FAK reversed increased EC apoptosis observed with FAK deletion in embryos and in vitro through suppression of up-regulated p21. However, vessel dilation and defective angiogenesis of CFKO embryos were not rescued in CFKI embryos. ECs without FAK or expressing KD FAK showed increased permeability, abnormal distribution of vascular endothelial cadherin (VE-cadherin), and reduced VE-cadherin Y658 phosphorylation. Together, our data suggest that kinase-independent functions of FAK can support EC survival in vascular development through E13.5 but are insufficient for maintaining EC function to allow for completion of embryogenesis.

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Three-Dimensional Simulation of In Vitro Angiogenesis: Effects of Extracellular Matrix Structure and Density

ASME 2010 Summer Bioengineering Conference, Parts A and B ◽

10.1115/sbc2010-19502 ◽

2010 ◽

Author(s):

Lowell Taylor Edgar ◽

James E. Guilkey ◽

Clayton J. Underwood ◽

Brenda Baggett ◽

Urs Utzinger ◽

...

Keyword(s):

Extracellular Matrix ◽

Endothelial Cells ◽

Three Dimensional ◽

Vascular Endothelial ◽

Chemical Factors ◽

Dimensional Simulation ◽

Endothelial Growth Factor ◽

In Vitro Angiogenesis

The process of angiogenesis is regulated by both chemical and mechanical signaling. While the role of chemical factors such as vascular endothelial growth factor (VEGF) during angiogenesis has been extensively studied, the influence of the mechanostructural environment on new vessel generation has received significantly less attention. During angiogenesis, endothelial cells in the existing vasculature detach and migrate out into the surrounding extracellular matrix (ECM), forming tubular structures that eventually mature into new blood vessels. This process is modulated by the structure and composition of the ECM [1]. The ECM is then remodeled by endothelial cells in the elongating neovessel tip, resulting in matrix condensation and changes in fiber orientation [2]. The mechanism as to how angiogenic vasculature and the ECM influence each other is poorly understood.

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Natural Killer and B-Lymphoid Potential in CD34+ Cells Derived From Embryonic Stem Cells Differentiated in the Presence of Vascular Endothelial Growth Factor

Blood ◽

10.1182/blood.v91.7.2283 ◽

1998 ◽

Vol 91 (7) ◽

pp. 2283-2295 ◽

Cited By ~ 42

Author(s):

Naoki Nakayama ◽

Inghwa Fang ◽

Gary Elliott

Keyword(s):

Vascular Endothelial Growth Factor ◽

Growth Factor ◽

Natural Killer ◽

Es Cells ◽

Embryonic Stem ◽

Cytotoxic Lymphocytes ◽

Vascular Endothelial ◽

Cd34 Cells ◽

Endothelial Growth Factor

Abstract Differentiation of totipotent mouse embryonic stem (ES) cells to various lymphohematopoietic cells is an in vitro model of the hematopoietic cell development during embryogenesis. To understand this process at cellular levels, differentiation intermediates were investigated. ES cells generated progeny expressing CD34, which was significantly enhanced by vascular endothelial growth factor (VEGF). The isolated CD34+ cells were enriched for myeloid colony-forming cells but not significantly for erythroid colony-forming cells. When cultured on OP9 stroma cells in the presence of interleukin-2 and interleukin-7, the CD34+ cells developed two types of B220+ CD34−lymphocytes: CD3− cytotoxic lymphocytes and CD19+ pre-B cells, and such lymphoid potential was highly enriched in the CD34+ population. Interestingly, the cytotoxic cells expressed the natural killer (NK) cell markers, such as NKR-P1, perforin, and granzymes, classified into two types, one of which showed target specificity of NK cells. Thus, ES cells have potential to generate NK-type cytotoxic lymphocytes in vitro in addition to erythro-myeloid cells and pre-B cells, and both myeloid and lymphoid cells seem to be derived from the CD34+intermediate, on which VEGF may play an important role.

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