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

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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Acute ethanol exposure disrupts VEGF receptor cell signaling in endothelial cells

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00699.2007 ◽

2008 ◽

Vol 295 (1) ◽

pp. H174-H184 ◽

Cited By ~ 36

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.

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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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Endothelial cell integrin alpha5beta1 expression is modulated by cytokines and during migration in vitro

Journal of Cell Science ◽

10.1242/jcs.112.4.569 ◽

1999 ◽

Vol 112 (4) ◽

pp. 569-578 ◽

Cited By ~ 1

Author(s):

G. Collo ◽

M.S. Pepper

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Growth Factor ◽

Transforming Growth Factor ◽

Integrin Subunit ◽

Subunit Expression ◽

Synergistic Induction ◽

Co Addition ◽

Extracellular Matrix Interactions

Alterations in endothelial cell-extracellular matrix interactions are central to the process of angiogenesis. We have investigated the effect of wound-induced two-dimensional migration, basic fibroblast growth factor (bFGF), transforming growth factor-beta1 (TGF-beta1) and leukemia inhibitory factor (LIF) on expression of the alpha5beta1 integrin in endothelial cells. In multiple-wounded monolayers of bovine microvascular endothelial (BME) cells, an increase in mRNA and total protein for both alpha5 and beta1 subunits was observed, and this could be correlated with a reduction in cell density but not proliferation, both of which are induced following wounding. Although as previously reported, the alpha5 subunit was increased when cells were exposed to TGF-beta1 alone, co-addition of bFGF and TGF-beta1 resulted in a striking synergistic induction of alpha5, with no significant changes in the expression of beta1. In contrast, the alpha5 subunit was decreased by LIF in bovine aortic endothelial but not in BME cells. These findings suggest that quantitative alterations in alpha5 and beta1 integrin subunit expression modulate the adhesive and migratory properties of endothelial cells during angiogenesis.

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Neuropilin-1 regulates attachment in human endothelial cells independently of vascular endothelial growth factor receptor-2

Blood ◽

10.1182/blood-2004-07-2598 ◽

2005 ◽

Vol 105 (5) ◽

pp. 1992-1999 ◽

Cited By ~ 83

Author(s):

Matilde Murga ◽

Oscar Fernandez-Capetillo ◽

Giovanna Tosato

Keyword(s):

Vascular Endothelial Growth Factor ◽

Endothelial Cells ◽

Endothelial Cell ◽

Growth Factor ◽

Critical Role ◽

Vegf Receptor ◽

Vascular Endothelial ◽

Human Endothelial Cells ◽

Neuropilin 1 ◽

Endothelial Growth Factor

AbstractNeuropilin-1 (NRP-1) is a type 1 membrane protein that binds the axon guidance factors belonging to the class-3 semaforin family. In endothelial cells, NRP-1 serves as a co-receptor for vascular endothelial growth factor (VEGF) and regulates VEGF receptor 2 (VEGFR-2)–dependent angiogenesis. Although gene-targeting studies documenting embryonic lethality in NRP-1 null mice have demonstrated a critical role for NRP-1 in vascular development, the activities of NRP-1 in mature endothelial cells have been incompletely defined. Using RNA interference-mediated silencing of NRP-1 or VEGFR-2 in primary human endothelial cells, we confirm that NRP-1 modulates VEGFR-2 signaling-dependent mitogenic functions of VEGF. Importantly, we now show that NRP-1 regulates endothelial cell adhesion to extracellular matrix proteins independently of VEGFR-2. Based on its dual role as an enhancer of VEGF activity and a mediator of endothelial cell adhesiveness described here, NRP-1 emerges as a promising molecular target for the development of antiangiogenic drugs.

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An endothelial cell-derived growth factor.

The Journal of Cell Biology ◽

10.1083/jcb.85.2.467 ◽

1980 ◽

Vol 85 (2) ◽

pp. 467-472 ◽

Cited By ~ 235

Author(s):

C Gajdusek ◽

P DiCorleto ◽

R Ross ◽

S M Schwartz

Keyword(s):

Endothelial Cells ◽

Smooth Muscle ◽

Endothelial Cell ◽

Growth Factor ◽

Smooth Muscle Cells ◽

Muscle Cells ◽

Platelet Derived Growth Factor ◽

3T3 Cells ◽

Free Plasma ◽

Heat Instability

Cell-free plasma-derived serum (PDS) is deficient in the platelet-derived growth factor and will not support the growth of 3T3 cells, fibroblasts, or smooth muscle cells. However, when PDS-containing medium is preincubated with endothelial cells, the medium becomes modified so that it will support growth. The activity produced by the endothelial cells results from a polypeptide of 10,000 to 30,000 daltons which has several features that differ from those of the platelet-derived growth factor, including heat instability and lack of adsorption to CM Sephadex.

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Model of competitive binding of vascular endothelial growth factor and placental growth factor to VEGF receptors on endothelial cells

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00254.2003 ◽

2004 ◽

Vol 286 (1) ◽

pp. H153-H164 ◽

Cited By ~ 86

Author(s):

Feilim Mac Gabhann ◽

Aleksander S. Popel

Keyword(s):

Vascular Endothelial Growth Factor ◽

Endothelial Cells ◽

Growth Factor ◽

Synergistic Effects ◽

Placental Growth Factor ◽

Vegf Receptor ◽

Vascular Endothelial ◽

Relative Contribution ◽

Endothelial Growth Factor

Placental growth factor (PlGF) competes with vascular endothelial growth factor (VEGF) for binding to VEGF receptor (VEGFR)-1 but does not bind VEGFR2. Experiments show that PlGF can augment the response to VEGF in pathological angiogenesis and in models of endothelial cell survival, migration, and proliferation. This synergy has been hypothesized to be due to a combination of the following: signaling by PlGF through VEGFR1 and displacement of VEGF from VEGFR1 to VEGFR2 by PlGF, causing increased signaling through VEGFR2. In this study, the relative contribution of PlGF-induced VEGF displacement to the synergy is quantified using a mathematical model of ligand-receptor binding to examine the effect on ligand-receptor complex formation of VEGF and PlGF acting together. Parameters specific to the VEGF-PlGF system are used based on existing data. The model is used to simulate in silico a specific in vitro experiment in which VEGF-PlGF synergy is observed. We show that, whereas a significant change in the formation of endothelial surface growth factor-VEGFR1 complexes is predicted in the presence of PlGF, the increase in the number of VEGFR2-containing signaling complexes is less significant; these results were shown to be robust to significant variation in the kinetic parameters of the model. Synergistic effects observed in that experiment thus appear unlikely to be due to VEGF displacement but to a shift from VEGF-VEGFR1 to PlGF-VEGFR1 complexes and an increase in total VEGFR1 complexes. These results suggest that VEGFR1 signaling can be functional in adult-derived endothelial cells.

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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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Hepatocyte growth factor is a potent angiogenic factor which stimulates endothelial cell motility and growth.

The Journal of Cell Biology ◽

10.1083/jcb.119.3.629 ◽

1992 ◽

Vol 119 (3) ◽

pp. 629-641 ◽

Cited By ~ 940

Author(s):

F Bussolino ◽

M F Di Renzo ◽

M Ziche ◽

E Bocchietto ◽

M Olivero ◽

...

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Growth Factor ◽

Hepatocyte Growth Factor ◽

Cell Monolayer ◽

Angiogenic Factor ◽

Tyrosine Kinase Receptor ◽

Collagen Gels ◽

Hepatocyte Growth

Hepatocyte Growth Factor (HGF, also known as Scatter Factor) is a powerful mitogen or motility factor in different cells, acting through the tyrosine kinase receptor encoded by the MET protooncogene. Endothelial cells express the MET gene and expose at the cell surface the mature protein (p190MET) made of a 50 kD (alpha) subunit disulfide linked to a 145-kD (beta) subunit. HGF binding to endothelial cells identifies two sites with different affinities. The higher affinity binding site (Kd = 0.35 nM) corresponds to the p190MET receptor. Sub-nanomolar concentrations of HGF, but not of a recombinant inactive precursor, stimulate the receptor kinase activity, cell proliferation and motility. HGF induces repairs of a wound in endothelial cell monolayer. HGF stimulates the scatter of endothelial cells grown on three-dimensional collagen gels, inducing an elongated phenotype. In the rabbit cornea, highly purified HGF promotes neovascularization at sub-nanomolar concentrations. HGF lacks activities related to hemostasis-thrombosis, inflammation and endothelial cells accessory functions. These data show that HGF is an in vivo potent angiogenic factor and in vitro induces endothelial cells to proliferate and migrate.

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Inhibition of vascular endothelial growth factor receptor 2–mediated endothelial cell activation by Axl tyrosine kinase receptor

Blood ◽

10.1182/blood-2004-04-1469 ◽

2005 ◽

Vol 105 (5) ◽

pp. 1970-1976 ◽

Cited By ~ 67

Author(s):

Margherita Gallicchio ◽

Stefania Mitola ◽

Donatella Valdembri ◽

Roberto Fantozzi ◽

Brian Varnum ◽

...

Keyword(s):

Vascular Endothelial Growth Factor ◽

Endothelial Cells ◽

Endothelial Cell ◽

Growth Factor ◽

Tyrosine Kinase ◽

Tyrosine Phosphatase ◽

Tyrosine Kinase Receptor ◽

Vegf Receptor ◽

Vascular Endothelial ◽

Endothelial Growth Factor

AbstractGAS6, the product of a growth arrest specific (GAS) gene, is the ligand of the tyrosine kinase receptor Axl. GAS6 and Axl are both expressed in endothelial cells, where they are involved in many processes such as leukocyte transmigration through capillaries and neointima formation in injured vessels. Here, we show that Axl stimulation by GAS6 results in inhibition of the ligand-dependent activation of vascular endothelial growth factor (VEGF) receptor 2 and the consequent activation of an angiogenic program in vascular endothelial cells. GAS6 inhibits chemotaxis of endothelial cells stimulated by VEGF-A isoforms, but not that triggered by fibroblast growth factor-2 or hepatocyte growth factor. Furthermore, it inhibits endothelial cell morphogenesis on Matrigel and VEGF-A–dependent vascularization of chick chorion allantoid membrane. GAS6 activates the tyrosine phosphatase SHP-2 (SH2 domain-containing tyrosine phosphatase 2), which is instrumental in the negative feedback exerted by Axl on VEGF-A activities. A dominant-negative SHP-2 mutant, in which Cys 459 is substituted by Ser, reverted the effect of GAS6 on stimulation of VEGF receptor 2 and endothelial chemotaxis triggered by VEGF-A. These studies provide the first demonstration of a cross talk between Axl and VEGF receptor 2 and add new information on the regulation of VEGF-A activities during tissue vascularization.

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Selective recognition of fibroblast growth factor-2 by the long pentraxin PTX3 inhibits angiogenesis

Blood ◽

10.1182/blood-2003-10-3433 ◽

2004 ◽

Vol 104 (1) ◽

pp. 92-99 ◽

Cited By ~ 117

Author(s):

Marco Rusnati ◽

Maura Camozzi ◽

Emanuela Moroni ◽

Barbara Bottazzi ◽

Giuseppe Peri ◽

...

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Growth Factor ◽

Fibroblast Growth Factor ◽

Solid Phase ◽

Cell Monolayer ◽

Vascular Lesions ◽

Fibroblast Growth Factor 2 ◽

Fibroblast Growth

Abstract The long pentraxin PTX3 is a soluble pattern recognition receptor produced by monocytes and endothelial cells that plays a nonredundant role in inflammation. Several pathologic conditions are characterized by local production of both PTX3 and the angiogenic fibroblast growth factor-2 (FGF2). Here, solid-phase binding assays demonstrated that PTX3 binds with high affinity to FGF2 but not to a panel of cytokines and growth factors, including FGF1, FGF4, and FGF8. Accordingly, PTX3 prevented 125I-FGF2 binding to endothelial cell receptors, leading to specific inhibition of FGF2-induced proliferation. PTX3 hampered also the motogenic activity exerted by endogenous FGF2 on a wounded endothelial cell monolayer. Moreover, PTX3 cDNA transduction in FGF2-transformed endothelial cells inhibited their autocrine FGF2-dependent proliferation and morphogenesis in vitro and their capacity to generate vascular lesions when injected in nude mice. Finally, PTX3 suppressed neovascularization triggered by FGF2 in the chick embryo chorioallantoic membrane with no effect on physiologic angiogenesis. In contrast, the short pentraxin C-reactive protein was a poor FGF2 ligand/antagonist. These results establish the selective binding of a member of the pentraxin superfamily to a growth factor. PTX3/FGF2 interaction may modulate angiogenesis in various physiopathologic conditions driven by inflammation, innate immunity, and/or neoplastic transformation.

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