IQGAP1-Dependent Signaling Pathway Regulates Endothelial Cell Proliferation and Angiogenesis

Background

Vascular endothelial growth factor receptor-2 (VEGFR-2) signaling is an obligate requirement for normal development and pathological angiogenesis such as cancer and age-related macular degeneration. Although autophosphorylation of tyrosine 1173 (Y1173) of VEGFR-2 is considered a focal point for its angiogenic signal relay, however, the mechanism of phosphorylation of Y1173, signaling proteins that are recruited to this residue and their role in angiogenesis is not fully understood.

Methodology/Principal Findings

In this study we demonstrate that c-Src kinase directly through its Src homology 2 (SH2) domain and indirectly via c-Cbl binds to phospho-Y1057 of VEGFR-2. Activation of c-Src kinase by a positive feedback mechanism phosphorylates VEGFR-2 at multi-docking site, Y1173. c-Src also catalyzes tyrosine phosphorylation of IQGAP1 and acts as an adaptor to bridge IQGAP1 to VEGFR-2. In turn, IQGAP1 activates b-Raf and mediates proliferation of endothelial cells. Silencing expression of IQGAP1 and b-Raf revealed that their activity is essential for VEGF to stimulate angiogenesis in an in vivo angiogenesis model of chicken chorioallantoic membrane (CAM).

Conclusions/Significance

Angiogenesis contributes to the pathology of numerous human diseases ranging from cancer to age-related macular degeneration. Determining molecular mechanism of tyrosine phosphorylation of VEGFR-2 and identification of molecules that are relaying its angiogenic signaling may identify novel targets for therapeutic intervention against angiogenesis-associated diseases. Our study shows that recruitment and activation of c-Src by VEGFR-2 plays a pivotal role in relaying angiogenic signaling of VEGFR-2; it phosphorylates VEGFR-2 at Y1173, facilitates association and activation of IQGAP1 and other signaling proteins to VEGFR-2. IQGAP1-dependent signaling, in part, is critically required for endothelial cell proliferation, a key step in angiogenesis. Thus, Y1057 of VEGFR-2 serves to regulate VEGFR-2 function in a combinatorial manner by supporting both diversity of recruitment of angiogenic signaling proteins to VEGFR-2, and its ability to promote angiogenesis.     

MicroRNA-147b Regulates Vascular Endothelial Barrier Function by Targeting ADAM15 Expression

A disintegrin and metalloproteinase15 (ADAM15) has been shown to be upregulated and mediate endothelial hyperpermeability during inflammation and sepsis. This molecule contains multiple functional domains with the ability to modulate diverse cellular processes including cell adhesion, extracellular matrix degradation, and ectodomain shedding of transmembrane proteins. These characteristics make ADAM15 an attractive therapeutic target in various diseases. The lack of pharmacological inhibitors specific to ADAM15 prompted our efforts to identify biological or molecular tools to alter its expression for further studying its function and therapeutic implications. The goal of this study was to determine if ADAM15-targeting microRNAs altered ADAM15-induced endothelial barrier dysfunction during septic challenge by bacterial lipopolysaccharide (LPS). An in silico analysis followed by luciferase reporter assay in human vascular endothelial cells identified miR-147b with the ability to target the 3′ UTR of ADAM15. Transfection with a miR-147b mimic led to decreased total, as well as cell surface expression of ADAM15 in endothelial cells, while miR-147b antagomir produced an opposite effect. Functionally, LPS-induced endothelial barrier dysfunction, evidenced by a reduction in transendothelial electric resistance and increase in albumin flux across endothelial monolayers, was attenuated in cells treated with miR-147b mimics. In contrast, miR-147b antagomir exerted a permeability-increasing effect in vascular endothelial cells similar to that caused by LPS. Taken together, these data suggest the potential role of miR147b in regulating endothelial barrier function by targeting ADAM15 expression.     

Cross Talk with Hematopoietic Cells Regulates the Endothelial Progenitor Cell Differentiation of CD34 Positive Cells

Introduction

Despite the crucial role of endothelial progenitor cells (EPCs) in vascular regeneration, the specific interactions between EPCs and hematopoietic cells remain unclear.

Methods

In EPC colony forming assays, we first demonstrated that the formation of EPC colonies was drastically increased in the coculture of CD34⁺ and CD34⁻ cells, and determined the optimal concentrations of CD34⁺ cells and CD34⁻ cells for spindle-shaped EPC differentiation.

Results

Functionally, the coculture of CD34⁺ and CD34⁻ cells resulted in a significant enhancement of adhesion, tube formation, and migration capacity compared with culture of CD34⁺ cells alone. Furthermore, blood flow recovery and capillary formation were remarkably increased by the coculture of CD34⁺ and CD34⁻ cells in a murine hind-limb ischemia model. To elucidate further the role of hematopoietic cells in EPC differentiation, we isolated different populations of hematopoietic cells. T lymphocytes (CD3⁺) markedly accelerated the early EPC status of CD34⁺ cells, while macrophages (CD11b⁺) or megakaryocytes (CD41⁺) specifically promoted large EPC colonies.

Conclusion

Our results suggest that specific populations of hematopoietic cells play a role in the EPC differentiation of CD34⁺ cells, a finding that may aid in the development of a novel cell therapy strategy to overcome the quantitative and qualitative limitations of EPC therapy.     

EB1 Directly Regulates APC-Mediated Actin Nucleation

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Heat Stress-Induced Disruption of Endothelial Barrier Function Is via PAR1 Signaling and Suppressed by Xuebijing Injection

Increased vascular permeability leading to acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) is central to the pathogenesis of heatstroke. Protease-activated receptor 1 (PAR1), the receptor for thrombin, plays a key role in disruption of endothelial barrier function in response to extracellular stimuli. However, the role of PAR1 in heat stress-induced endothelial hyper-permeability is unknown. In this study, we measured PAR1 protein expression in heat-stressed human umbilical venous endothelial cells (HUVECs), investigated the influences of PAR1 on endothelial permeability, F-actin rearrangement, and moesin phosphorylation by inhibiting PAR1 with its siRNA, neutralizing antibody (anti-PAR1), specific inhibitor(RWJ56110), and Xuebijing injection (XBJ), a traditional Chinese medicine used for sepsis treatment, and evaluated the role of PAR1 in heatstroke-related ALI/ARDS in mice by suppressing PAR1 with RWJ56110, anti-PAR1and XBJ. We found that heat stress induced PAR1 protein expression 2h after heat stress in endothelial cells, caused the release of endothelial matrix metalloprotease 1, an activator of PAR1, after 60 or 120 min of heat stimulation, as well as promoted endothelial hyper-permeability and F-actin rearrangement, which were inhibited by suppressing PAR1 with RWJ56110, anti-PAR1 and siRNA. PAR1 mediated moesin phosphorylation, which caused F-actin rearrangement and disruption of endothelial barrier function. To corroborate findings from in vitro experiments, we found that RWJ56110 and the anti-PAR1 significantly decreased lung edema, pulmonary microvascular permeability, protein exudation, and leukocytes infiltrations in heatstroke mice. Additionally, XBJ was found to suppress PAR1-moesin signal pathway and confer protective effects on maintaining endothelial barrier function both in vitro and in vivo heat-stressed model, similar to those observed above with the inhibition of PAR1. These results suggest that PAR1 is a potential therapeutic target in heatstroke.     

IQGAP1 Negatively Regulates HIV-1 Gag Trafficking and Virion Production

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Involvement of Local Lamellipodia in Endothelial Barrier Function

Recently we observed that endothelial cells cultured in tightly confluent monolayers display frequent local lamellipodia, and that thrombin, an agent that increases endothelial permeability, reduces lamellipodia protrusions. This led us to test the hypothesis that local lamellipodia contribute to endothelial barrier function. Movements of subcellular structures containing GFP-actin or VE-cadherin-GFP expressed in endothelial cells were recorded using time-lapse microscopy. Transendothelial electrical resistance (TER) served as an index of endothelial barrier function. Changes in both lamellipodia dynamics and TER were assessed during baseline and after cells were treated with either the barrier-disrupting agent thrombin, or the barrier-stabilizing agent sphingosine-1-phosphate (S1P). The myosin II inhibitor blebbistatin was used to selectively block lamellipodia formation, and was used to test their role in the barrier function of endothelial cell monolayers and isolated, perfused rat mesenteric venules. Myosin light chain (MLC) phosphorylation was assessed by immunofluorescence microscopy. Rac1 and RhoA activation were evaluated using G-LISA assays. The role of Rac1 was tested with the specific inhibitor NSC23766 or by expressing wild-type or dominant negative GFP-Rac1. The results show that thrombin rapidly decreased both TER and the lamellipodia protrusion frequency. S1P rapidly increased TER in association with increased protrusion frequency. Blebbistatin nearly abolished local lamellipodia protrusions while cortical actin fibers and stress fibers remained intact. Blebbistatin also significantly decreased TER of cultured endothelial cells and increased permeability of isolated rat mesenteric venules. Both thrombin and S1P increased MLC phosphorylation and activation of RhoA. However, thrombin and S1P had differential impacts on Rac1, correlating with the changes in TER and lamellipodia protrusion frequency. Overexpression of Rac1 elevated, while NSC23766 and dominant negative Rac1 reduced barrier function and lamellipodia activity. Combined, these data suggest that local lamellipodia, driven by myosin II and Rac1, are important for dynamic changes in endothelial barrier integrity.     

Caveolin-1 Regulates Endothelial Adhesion of Lung Cancer Cells via Reactive Oxygen Species-Dependent Mechanism

The knowledge regarding the role of caveolin-1 (Cav-1) protein on endothelium adhesion of cancer cells is unclear. The present study revealed that Cav-1 plays a negative regulatory role on cancer-endothelium interaction. Endogenous Cav-1 was shown to down-regulate during cell detachment and the level of such a protein was conversely associated with tumor-endothelial adhesion. Furthermore, the ectopic overexpression of Cav-1 attenuated the ability of the cancer cells to adhere to endothelium while shRNA-mediated Cav-1 knock-down exhibited the opposite effect. We found that cell detachment increased cellular hydrogen peroxide and hydroxyl radical generation and such reactive oxygen species (ROS) were responsible for the increasing interaction between cancer cells and endothelial cells through vascular endothelial cell adhesion molecule-1 (VCAM-1). Importantly, Cav-1 was shown to suppress hydrogen peroxide and hydroxyl radical formation by sustaining the level of activated Akt which was critical for the role of Cav-1 in attenuating the cell adhesion. Together, the present study revealed the novel role of Cav-1 and underlying mechanism on tumor adhesion which explain and highlight an important role of Cav-1 on lung cancer cell metastasis.     

PKC-Dependent Phosphorylation of eNOS at T495 Regulates eNOS Coupling and Endothelial Barrier Function in Response to G+ -Toxins

Gram positive (G⁺) infections make up ∼50% of all acute lung injury cases which are characterized by extensive permeability edema secondary to disruption of endothelial cell (EC) barrier integrity. A primary cause of increased permeability are cholesterol-dependent cytolysins (CDCs) of G⁺-bacteria, such as pneumolysin (PLY) and listeriolysin-O (LLO) which create plasma membrane pores, promoting Ca²⁺-influx and activation of PKCα. In human lung microvascular endothelial cells (HLMVEC), pretreatment with the nitric oxide synthase (NOS) inhibitor, ETU reduced the ability of LLO to increase microvascular cell permeability suggesting an endothelial nitric oxide synthase (eNOS)-dependent mechanism. LLO stimulated superoxide production from HLMVEC and this was prevented by silencing PKCα or NOS inhibition suggesting a link between these pathways. Both LLO and PLY stimulated eNOS T495 phosphorylation in a PKC-dependent manner. Expression of a phosphomimetic T495D eNOS (human isoform) resulted in increased superoxide and diminished nitric oxide (NO) production. Transduction of HLMVEC with an active form of PKCα resulted in the robust phosphorylation of T495 and increased peroxynitrite production, indicative of eNOS uncoupling. To determine the mechanisms underlying eNOS uncoupling, HLMVEC were stimulated with LLO and the amount of hsp90 and caveolin-1 bound to eNOS determined. LLO stimulated the dissociation of hsp90, and in particular, caveolin-1 from eNOS. Both hsp90 and caveolin-1 have been shown to influence eNOS uncoupling and a peptide mimicking the scaffolding domain of caveolin-1 blocked the ability of PKCα to stimulate eNOS-derived superoxide. Collectively, these results suggest that the G⁺ pore-forming toxins promote increased EC permeability via activation of PKCα, phosphorylation of eNOS-T495, loss of hsp90 and caveolin-1 binding which collectively promote eNOS uncoupling and the production of barrier disruptive superoxide.     

Regulation of Endothelial Cell Barrier Function by Antibody-driven Affinity Modulation of Platelet Endothelial Cell Adhesion Molecule-1 (PECAM-1)

PECAM-1 is a 130-kDa member of the immunoglobulin (Ig) superfamily that is expressed on the surface of platelets and leukocytes, and at the intracellular junctions of confluent endothelial cell monolayers. Previous studies have shown that PECAM-1/PECAM-1 homophilic interactions play a key role in leukocyte transendothelial migration, in allowing PECAM-1 to serve as a mechanosensory complex in endothelial cells, in its ability to confer cytoprotection to proapoptotic stimuli, and in maintaining endothelial cell junctional integrity. To examine the adhesive properties of full-length PECAM-1 in a native lipid environment, we purified it from platelets and assembled it into phospholipid nanodiscs. PECAM-1-containing nanodiscs retained not only their ability to bind homophilically to PECAM-1-expressing cells, but exhibited regulatable adhesive interactions that could be modulated by ligands that bind membrane-proximal Ig Domain 6. This property was exploited to enhance the rate of barrier restoration in endothelial cell monolayers subjected to inflammatory challenge. The finding that the adhesive properties of PECAM-1 are regulatable suggests novel approaches for controlling endothelial cell migration and barrier function in a variety of vascular permeability disorders.     

RhoC Regulates the Proliferation of Gastric Cancer Cells through Interaction with IQGAP1

Our previous research results showed that both Ras homolog family member C (RhoC) and IQ-domain GTPase-activating protein 1 (IQGAP1) were over-expressed in gastric cancer tissues and cells, but their role in tumorigenensis has not been addressed clearly. Herein we reported the proliferation stimulating effect of RhoC and IQGAP1 on gastric cancer cells and the interaction between two proteins in regulating the proliferation of gastric cancer cells. Plasmids and viral constructs encoding target siRNA and DNA were used to alter the expression of RhoC and IQGAP1. MTT method and BrdU incorporation assay were used for analyzing the effect of RhoC and different structures of IQGAP1 on proliferation. Protein levels of IQGAP1 and RhoC in cell lines were detected by Western blotting. Immunofluorescence and Co-Immunoprecipitation assays were applied to investigate the localization and binding between RhoC and IQGAP1. The results showed that RhoC, IQGAP1 and the C-terminal fragment of IQGAP1 significantly stimulated the proliferation of gastric cancer cells, and enhanced the expression of cyclin E and cyclin D1. By contrast, reduction of endogenous IQGAP1 or RhoC by siRNA attenuated cell proliferation. The depletion of IQGAP1 expression by siRNA significantly blocked the proliferative activity of constitutively active RhoC, while RhoC silencing by siRNA had no effect on IQGAP1-induced proliferation in gastric cancer cells. Co-immunoprecipitation and Immunofluorescence assays showed that RhoC and IQGAP1 bound each other. In conclusion, our results suggest that RhoC stimulates the proliferation of gastric cancer cells through recruiting IQGAP1 as an effector.     

Integration of Biochemical and Biomechanical Signals Regulating Endothelial Barrier Function

Endothelial barrier function is critical for tissue homeostasis throughout the body. Disruption of the endothelial monolayer leads to edema, vascular diseases and even cancer metastasis among other pathological conditions. Breakdown of the endothelial barrier integrity triggered by cytokines (e.g.IL-8,IL-1β) and growth factors (e.g.VEGF) is well documented. However, endothelial cells are subject to major biomechanical forces that affect their behavior. Due to their unique location at the interface between circulating blood and surrounding tissues, endothelial cells experience shear stress, strain and contraction forces. More than three decades ago, it was already appreciated that shear flow caused endothelial cells alignment in the direction of the flow. After that observation, it took around 20 years to begin to uncover some of the mechanisms used by the cells for mechanotransduction. In this review, we describe mechanosensors on the endothelium identified to date and the associated signaling pathways that integrate biochemical and biomechanical inputs into biological responses and how they modulate the integrity of the endothelial barrier.     

Adiponectin Regulates Vascular Endothelial Growth Factor-C Expression in Macrophages via Syk-ERK Pathway

Adiponectin is exclusively expressed in adipose tissues and exhibits protective effects against cardiovascular and metabolic diseases. It enhances AMP-activated kinase (AMPK) and peroxisome proliferator-activated receptor α (PPARα) signaling in the liver and skeletal muscles, however, its signaling pathways in macrophages remain to be elucidated. Here, we show that adiponectin upregulated the expression of vascular endothelial growth factor (VEGF)-C, and induced phosphorylation of extracellular signal-regulated kinase (ERK) in macrophages. Inhibition of Syk abrogated adiponectin-induced VEGF-C expression and ERK phosphorylation. Furthermore, inhibition of ERK blocked the induction of VEGF-C gene. Inhibition of Syk, but not that of ERK, abrogated adiponectin-induced expression of cyclooxygenase (COX)-2, tissue inhibitor of metalloproteinase (TIMP)-1, and interleukin (IL)-6. These results indicate that adiponectin regulates VEGF-C expression via Syk-ERK pathway in macrophages.     

Basement Membrane Proteins Influence Brain Capillary Endothelial Barrier Function In Vitro

Abstract: The influence of basement membrane proteins on cellular barrier properties of primary cultures of porcine brain capillary endothelial cells grown on permeable filter inserts has been investigated. Measurements of transcellular electrical resistance (TER) by impedance spectroscopy were performed with cells cultured on type IV collagen, fibronectin, laminin, and one-to-one mixtures of these proteins. Moreover, a one-to-one combination of type IV collagen and SPARC (secreted protein acidic and rich in cysteine) has been studied. Rat tail collagen has been used as a reference substratum. If TERs of cells from a given preparation were low (∼350 Ω× cm²) on the reference substratum, type IV collagen, fibronectin, and laminin as well as one-to-one combinations of these proteins elevated transcellular resistances significantly (2.3- to 2.9-fold) compared with rat tail collagen. TER of cells exhibiting a high reference level (∼1,000 Ω× cm²) could, by contrast, be increased only 1.1- to 1.2-fold. The type IV collagen/SPARC mixture did not elevate TER. Our findings suggest that type IV collagen, fibronectin, and laminin are involved in tight junction formation between cerebral capillary endothelial cells. The differential effects observed for individual preparations probably reflect more or less dedifferentiated states of the endothelium, in which basement membrane proteins can influence cellular differentiation more or less strongly. However, our results indicate that type IV collagen, fibronectin, and laminin enhance the reliability and suitability of primary microvascular endothelial cell cultures as an in vitro model of the blood-brain barrier.     

GTSE1 Is a Microtubule Plus-End Tracking Protein That Regulates EB1-Dependent Cell Migration

The regulation of cell migration is a highly complex process that is often compromised when cancer cells become metastatic. The microtubule cytoskeleton is necessary for cell migration, but how microtubules and microtubule-associated proteins regulate multiple pathways promoting cell migration remains unclear. Microtubule plus-end binding proteins (+TIPs) are emerging as important players in many cellular functions, including cell migration. Here we identify a +TIP, GTSE1, that promotes cell migration. GTSE1 accumulates at growing microtubule plus ends through interaction with the EB1+TIP. The EB1-dependent +TIP activity of GTSE1 is required for cell migration, as well as for microtubule-dependent disassembly of focal adhesions. GTSE1 protein levels determine the migratory capacity of both nontransformed and breast cancer cell lines. In breast cancers, increased GTSE1 expression correlates with invasive potential, tumor stage, and time to distant metastasis, suggesting that misregulation of GTSE1 expression could be associated with increased invasive potential.