Acute ethanol exposure disrupts VEGF receptor cell signaling in endothelial cells

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.

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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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Serum IL-6 and sIL-6R in type 2 diabetes contribute to impaired capillary-like network formation

Journal of Applied Physiology ◽

10.1152/japplphysiol.00002.2019 ◽

2019 ◽

Vol 127 (2) ◽

pp. 385-392

Author(s):

Rian Q. Landers-Ramos ◽

Jacob B. Blumenthal ◽

Steven J. Prior

Keyword(s):

Type 2 Diabetes ◽

Endothelial Cells ◽

Endothelial Cell ◽

Glucose Tolerance ◽

Interleukin 6 ◽

Network Formation ◽

Normal Glucose Tolerance ◽

Normal Glucose

We hypothesized that the serum from individuals with type 2 diabetes mellitus (T2DM) and impaired glucose tolerance (IGT) would reduce in vitro capillary-like network formation compared with normal glucose tolerance (NGT) serum and that this would occur along with higher serum concentrations of inflammatory cytokines and lower concentrations of angiogenic growth factors. Subjects were sedentary, older (55–65 yr) adults with NGT, IGT, or T2DM ( n = 10/group) matched for body mass index. Human retroviral telomerized endothelial cells (HRVT-ECs) or coronary artery endothelial cells (CECs) were used in a capillary-like network formation assay using endothelial basal medium supplemented with 7.5% serum. Quantification of HRVT-EC network length indicated that serum from the T2DM group resulted in 32 and 35% lower network formation than when using serum from the NGT and IGT groups, respectively ( P < 0.05). Serum from T2DM subjects resulted in CEC network formation that was 11 and 8% lower than when using serum from NGT and IGT subjects, respectively ( P < 0.05). Analysis of serum cytokines indicated that IL-6 was 41% and 49% higher in the IGT and T2DM groups, respectively, compared with the NGT group ( P < 0.05) and there was a trend for higher soluble interleukin-6 receptor (sIL-6R; P = 0.06) and IL-8 ( P = 0.08) in the T2DM serum compared with NGT. The use of recombinant IL-6 and sIL-6R at concentrations detected in the T2DM serum also reduced capillary network formation compared with NGT concentrations ( P < 0.05). These results suggest that IL-6 and sIL-6R present in the serum of T2DM individuals impair in vitro endothelial cell function across different cell lines. Our findings may have implications for the microvascular complications associated with T2DM. NEW & NOTEWORTHY Higher concentrations of serum factors, specifically Interleukin-6 and its soluble receptor found in individuals with type 2 diabetes (T2DM) appear to impair endothelial cell capillary-like network formation compared with those present in serum from individuals with impaired glucose tolerance and normal glucose tolerance. This may have implications for the vascular complications associated with T2DM.

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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

Cell Death and Disease ◽

10.1038/s41419-020-03076-7 ◽

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.

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Type 5 phosphodiesterase expression is a critical determinant of the endothelial cell angiogenic phenotype

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.90474.2008 ◽

2009 ◽

Vol 296 (2) ◽

pp. L220-L228 ◽

Cited By ~ 19

Author(s):

Bing Zhu ◽

Li Zhang ◽

Mikhail Alexeyev ◽

Diego F. Alvarez ◽

Samuel J. Strada ◽

...

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Network Formation ◽

Microvascular Endothelial Cells ◽

Critical Determinant ◽

Angiogenic Potential ◽

Matrigel Plug ◽

Microvascular Endothelial

Type 5 phosphodiesterase (PDE5) inhibitors increase endothelial cell cGMP and promote angiogenesis. However, not all endothelial cell phenotypes express PDE5. Indeed, whereas conduit endothelial cells express PDE5, microvascular endothelial cells do not express this enzyme, and they are rapidly angiogenic. These findings bring into question whether PDE5 activity is a critical determinant of the endothelial cell angiogenic potential. To address this question, human full-length PDE5A1 was stably expressed in pulmonary microvascular endothelial cells. hPDE5A1 expression reduced the basal and atrial natriuretic peptide (ANP)-stimulated cGMP concentrations in these cells. hPDE5A1-expressing cells displayed attenuated network formation on Matrigel in vitro and also produced fewer blood vessels in Matrigel plug assays in vivo; the inhibitory actions of hPDE5A1 were reversed using sildenafil. To examine whether endogenous PDE5 activity suppresses endothelial cell angiogenic potential, small interfering RNA (siRNA) constructs were stably expressed in pulmonary artery endothelial cells. siRNA selectively decreased PDE5 expression and increased basal and ANP-stimulated cGMP concentrations in these conduit cells. PDE5 downregulation increased network formation on Matrigel in vitro and increased blood vessel formation in Matrigel plug assays in vivo. Collectively, our results indicate that PDE5 activity is an essential determinant of angiogenesis and suggest that PDE5 downregulation in microvascular endothelium imparts a stable, enhanced angiogenic potential to this cell type.

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Fibroblast growth factor 2 is a key determinant of vascular sprouting during bovine luteal angiogenesis

Reproduction ◽

10.1530/rep-11-0277 ◽

2012 ◽

Vol 143 (1) ◽

pp. 35-43 ◽

Cited By ~ 28

Author(s):

Kathryn J Woad ◽

Morag G Hunter ◽

George E Mann ◽

Mhairi Laird ◽

Amanda J Hammond ◽

...

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Growth Factor ◽

Fibroblast Growth Factor ◽

Network Formation ◽

Corpora Lutea ◽

Vegf Receptor ◽

Fibroblast Growth ◽

Branch Points ◽

Mixed Cell

Fibroblast growth factor (FGF) 2 and vascular endothelial growth factor (VEGF) A are thought to be key controllers of luteal angiogenesis; however, their precise roles in the regulation and coordination of this complex process remain unknown. Thus, the temporal and spatial patterns of endothelial network formation were determined by culturing mixed cell types from early bovine corpora lutea on fibronectin in the presence of FGF2 and VEGFA (6 h to 9 days). Endothelial cells, as determined by von Willebrand factor immunohistochemistry, initially grew in cell islands (days 0–3), before undergoing a period of vascular sprouting to display a more tubule-like appearance (days 3–6), and after 9 days in culture had formed extensive intricate networks. Mixed populations of luteal cells were treated with SU1498 (VEGF receptor 2 inhibitor) or SU5402 (FGF receptor 1 inhibitor) or control on days 0–3, 3–6 or 6–9 to determine the role of FGF2 and VEGFA during these specific windows. The total area of endothelial cells was unaffected by SU1498 treatment during any window. In contrast, SU5402 treatment caused maximal reduction in the total area of endothelial cell networks on days 3–6 vs controls (mean reduction 81%;P<0.001) during the period of tubule initiation. Moreover, SU5402 treatment on days 3–6 dramatically reduced the total number of branch points (P<0.001) and degree of branching per endothelial cell island (P<0.05) in the absence of changes in mean island area. This suggests that FGF2 is a key determinant of vascular sprouting and hence critical to luteal development.

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A Quantitative Study of the Effects of Ionizing Radiation on Endothelial Cells and Capillary-like Network Formation

Technology in Cancer Research & Treatment ◽

10.1177/153303460600500206 ◽

2006 ◽

Vol 5 (2) ◽

pp. 127-134 ◽

Cited By ~ 20

Author(s):

Xiao Wen Mao

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Laser Scanning ◽

Cell Activation ◽

Network Formation ◽

Cell Damage ◽

Dna Breaks ◽

Endothelial Cell Activation ◽

Radiation Induced

The initial events of angiogenesis comprise endothelial cell activation, migration, and proliferation. The characteristics of retinal endothelial cells and capillaries are significantly altered in a number of diseases including cancer. Since radiation has been shown as a useful tool in radiotherapy by altering the proliferative changes, it is important to evaluate the responses of the endothelial cells and the capillary network to radiation. We quantified functional and kinetic responses of endothelial cells and capillaries to radiation in an in vitro model. An in vitro angiogenesis model was introduced in our study with endothelial cells cultured on an extracellular matrix gel in which hollow tube-like structures could be rapidly formed. Vessel formation was quantified using stereological techniques. The cell cycle kinetics of endothelial cells and accumulation of DNA damage after radiation were measured using laser scanning cytometry. To study the response of proliferative capillary-like structures to radiation, the vessel network was irradiated with 2 gray (Gy). To evaluate functional and kinetic responses and differentiation of endothelial cells to radiation, cells were irradiated with 2 and 6 Gy. Progressive time- and dose-dependent loss of endothelial cells occurred starting 24 hours after radiation. Vessel growth was significantly retarded at the higher dose. A significant percentage of DNA breaks were detected dose-dependently. A large percentage of G1 cells were measured in the irradiated endothelial cell population when compared to the respective sham-treated control population. These results indicate that radiation-induced endothelial cell injuries destroy the integrity of vascular structure. We postulated that apoptosis may represent a biologically relevant mechanism of radiation-induced endothelial cell damage.

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Wnt5a Signaling Induces Proliferation and Survival of Endothelial Cells In Vitro and Expression of MMP-1 and Tie-2

Molecular Biology of the Cell ◽

10.1091/mbc.e06-04-0320 ◽

2006 ◽

Vol 17 (12) ◽

pp. 5163-5172 ◽

Cited By ~ 140

Author(s):

T. Néstor H. Masckauchán ◽

Dritan Agalliu ◽

Marina Vorontchikhina ◽

Audrey Ahn ◽

Nancy L. Parmalee ◽

...

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Wnt Signaling ◽

Network Formation ◽

Endothelial Cell Migration ◽

Endothelial Cell Proliferation ◽

Wnt5a Expression ◽

Matrix Metalloproteinase 1 ◽

Canonical Wnt

Wnts are lipid-modified secreted glycoproteins that regulate diverse biological processes. We report that Wnt5a, which functions in noncanonical Wnt signaling, has activity on endothelial cells. Wnt5a is endogenously expressed in human primary endothelial cells and is expressed in murine vasculature at several sites in mouse embryos and tissues. Expression of exogenous Wnt5a in human endothelial cells promoted angiogenesis. Wnt5a induced noncanonical Wnt signaling in endothelial cells, as measured by Dishevelled and ERK1/2 phosphorylation, and inhibition of canonical Wnt signaling, a known property of Wnt5a. Wnt5a induced endothelial cell proliferation and enhanced cell survival under serum-deprived conditions. The Wnt5a-mediated proliferation was blocked by Frizzled-4 extracellular domain. Wnt5a expression enhanced capillary-like network formation, whereas reduction of Wnt5a expression decreased network formation. Reduced Wnt5a expression inhibited endothelial cell migration. Screening for Wnt5a-regulated genes in cultured endothelial cells identified several encoding angiogenic regulators, including matrix metalloproteinase-1, an interstitial collagenase, and Tie-2, a receptor for angiopoietins. Thus, Wnt5a acts through noncanonical Wnt signaling to promote angiogenesis.

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

Blood ◽

10.1182/blood-2002-01-0119 ◽

2003 ◽

Vol 101 (5) ◽

pp. 1801-1809 ◽

Cited By ~ 35

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.

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Transforming Growth Factor β1 (TGF-β1) Promotes Endothelial Cell Survival during In Vitro Angiogenesis via an Autocrine Mechanism Implicating TGF-α Signaling

Molecular and Cellular Biology ◽

10.1128/mcb.21.21.7218-7230.2001 ◽

2001 ◽

Vol 21 (21) ◽

pp. 7218-7230 ◽

Cited By ~ 145

Author(s):

Francesc Viñals ◽

Jacques Pouysségur

Keyword(s):

Endothelial Cells ◽

Growth Factor ◽

Cell Survival ◽

Network Formation ◽

Egf Receptor ◽

Transforming Growth Factor ◽

Transforming Growth Factor Β1 ◽

In Vitro Angiogenesis ◽

Tgf Β1

ABSTRACT Mouse capillary endothelial cells (1G11 cell line) embedded in type I collagen gels undergo in vitro angiogenesis. Cells rapidly reorganize and form capillary-like structures when stimulated with serum. Transforming growth factor β1 (TGF-β1) alone can substitute for serum and induce cell survival and tubular network formation. This TGF-β1-mediated angiogenic activity depends on phosphatidylinositol 3-kinase (PI3K) and p42/p44 mitogen-activated protein kinase (MAPK) signaling. We showed that specific inhibitors of either pathway (wortmannin, LY-294002, and PD-98059) all suppressed TGF-β1-induced angiogenesis mainly by compromising cell survival. We established that TGF-β1 stimulated the expression of TGF-α mRNA and protein, the tyrosine phosphorylation of a 170-kDa membrane protein representing the epidermal growth factor (EGF) receptor, and the delayed activation of PI3K/Akt and p42/p44 MAPK. Moreover, we showed that all these TGF-β1-mediated signaling events, including tubular network formation, were suppressed by incubating TGF-β1-stimulated endothelial cells with a soluble form of an EGF receptor (ErbB-1) or tyrphostin AG1478, a specific blocker of EGF receptor tyrosine kinase. Finally, addition of TGF-α alone poorly stimulated angiogenesis; however, by reducing cell death, it strongly potentiated the action of TGF-β1. We therefore propose that TGF-β1 promotes angiogenesis at least in part via the autocrine secretion of TGF-α, a cell survival growth factor, activating PI3K/Akt and p42/p44 MAPK.

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Structure and Function of a Vimentin-associated Matrix Adhesion in Endothelial Cells

Molecular Biology of the Cell ◽

10.1091/mbc.12.1.85 ◽

2001 ◽

Vol 12 (1) ◽

pp. 85-100 ◽

Cited By ~ 111

Author(s):

Meredith Gonzales ◽

Babette Weksler ◽

Daisuke Tsuruta ◽

Robert D. Goldman ◽

Kristine J. Yoon ◽

...

Keyword(s):

Endothelial Cells ◽

Growth Factors ◽

Endothelial Cell ◽

Branching Morphogenesis ◽

Basement Membranes ◽

Αvβ3 Integrin ◽

Dependent Manner ◽

Matrix Adhesion ◽

Focal Contacts

The α4 laminin subunit is a component of endothelial cell basement membranes. An antibody (2A3) against the α4 laminin G domain stains focal contact-like structures in transformed and primary microvascular endothelial cells (TrHBMECs and HMVECs, respectively), provided the latter cells are activated with growth factors. The 2A3 antibody staining colocalizes with that generated by αv and β3 integrin antibodies and, consistent with this localization, TrHBMECs and HMVECs adhere to the α4 laminin subunit G domain in an αvβ3-integrin–dependent manner. The αvβ3 integrin/2A3 antibody positively stained focal contacts are recognized by vinculin antibodies as well as by antibodies against plectin. Unusually, vimentin intermediate filaments, in addition to microfilament bundles, interact with many of the αvβ3 integrin-positive focal contacts. We have investigated the function of α4-laminin and αvβ3-integrin, which are at the core of these focal contacts, in cultured endothelial cells. Antibodies against these proteins inhibit branching morphogenesis of TrHBMECs and HMVECs in vitro, as well as their ability to repopulate in vitro wounds. Thus, we have characterized an endothelial cell matrix adhesion, which shows complex cytoskeletal interactions and whose assembly is regulated by growth factors. Our data indicate that this adhesion structure may play a role in angiogenesis.

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