Regulation Of Integrin Function Research Articles

Critical role of thrombospondin-1 in promoting intestinal mucosal wound repair.

Thrombospondin-1 (TSP1) is a matricellular protein associated with the regulation of cell migration through direct binding interactions with integrin proteins and by associating with other receptors known to regulate integrin function, including CD47 and CD36. We previously demonstrated that deletion of an epithelial TSP1 receptor CD47 attenuates epithelial wound repair following intestinal mucosal injury. However, the mechanisms by which TSP1 contributes to intestinal mucosal repair remains poorly understood. Our results show upregulated TSP1 expression in colonic mucosal wounds and impaired intestinal mucosal wound healing in vivo upon intestinal epithelial specific loss of TSP1 (VillinCre/+Thbs1f/f or Thbs1ΔIEC). We report that exposure to exogenous TSP1 enhanced migration of IECs in a CD47- and TGFβ1-dependent manner, and that deficiency of TSP1 in primary murine colonic epithelial cells resulted in impaired wound healing. Mechanistically, TSP1 modulated epithelial actin cytoskeletal dynamics by suppression of RhoA activity, activation of Rac1, and changes in F-actin bundling. Overall, TSP1 was found to regulate intestinal mucosal wound healing via CD47 and TGFβ1, coordinate integrin-containing cell-matrix adhesion dynamics and remodel the actin cytoskeleton in migrating epithelial cells to enhance cell motility and promote wound repair.

Endoplasmic Reticulum Protein 72 Regulates Integrin Mac-1 Activity to Influence Neutrophil Recruitment.

Integrins mediate the adhesion, crawling, and migration of neutrophils during vascular inflammation. Thiol exchange is important in the regulation of integrin functions. ERp72 (endoplasmic reticulum-resident protein 72) is a member of the thiol isomerase family responsible for the catalysis of disulfide rearrangement. However, the role of ERp72 in the regulation of Mac-1 (integrin αMβ2) on neutrophils remains elusive. Intravital microscopy of the cremaster microcirculation was performed to determine in vivo neutrophil movement. Static adhesion, flow chamber, and flow cytometry were used to evaluate in vitro integrin functions. Confocal fluorescent microscopy and coimmunoprecipitation were utilized to characterize the interactions between ERp72 and Mac-1 on neutrophil surface. Cell-impermeable probes and mass spectrometry were used to label reactive thiols and identify target disulfide bonds during redox exchange. Biomembrane force probe was performed to quantitatively measure the binding affinity of Mac-1. A murine model of acute lung injury induced by lipopolysaccharide was utilized to evaluate neutrophil-associated vasculopathy. ERp72-deficient neutrophils exhibited increased rolling but decreased adhesion/crawling on inflamed venules in vivo and defective static adhesion in vitro. The defect was due to defective activation of integrin Mac-1 but not LFA-1 (lymphocyte function-associated antigen-1) using blocking or epitope-specific antibodies. ERp72 interacted with Mac-1 in lipid rafts on neutrophil surface leading to the reduction of the C654-C711 disulfide bond in the αM subunit that is critical for Mac-1 activation. Recombinant ERp72, via its catalytic motifs, increased the binding affinity of Mac-1 with ICAM-1 (intercellular adhesion molecule-1) and rescued the defective adhesion of ERp72-deficient neutrophils both in vitro and in vivo. Deletion of ERp72 in the bone marrow inhibited neutrophil infiltration, ameliorated tissue damage, and increased survival during murine acute lung injury. Extracellular ERp72 regulates integrin Mac-1 activity by catalyzing disulfide rearrangement on the αM subunit and may be a novel target for the treatment of neutrophil-associated vasculopathy.

Heterotropic roles of divalent cations in the establishment of allostery and affinity maturation of integrin αXβ2.

SUMMARYAllosteric activation and silencing of leukocyte β2-integrins transpire through cation-dependent structural changes, which mediate integrin biosynthesis and recycling, and are essential to designing leukocyte-specific drugs. Stepwise addition of Mg2+ reveals two mutually coupled events for the αXβ2 ligand-binding domain—the αX I-domain—corresponding to allostery establishment and affinity maturation. Electrostatic alterations in the Mg2+-binding site establish long-range couplings, leading to both pH− and Mg2+-occupancy-dependent biphasic stability change in the αX I-domain fold. The ligand-binding sensorgrams show composite affinity events for the αX I-domain accounting for the multiplicity of the αX I-domain conformational states existing in the solution. On cell surfaces, increasing Mg2+ concentration enhanced adhesiveness of αXβ2. This work highlights how intrinsically flexible pH− and cation-sensitive architecture endows a unique dynamic continuum to the αI-domain structure on the intact integrin, thereby revealing the importance of allostery establishment and affinity maturation in both extracellular and intracellular integrin events.

Essential Functions of Glycans in Human Epithelia Dissected by a CRISPR-Cas9-Engineered Human Organotypic Skin Model.

SummaryThe glycome undergoes characteristic changes during histogenesis and organogenesis, but our understanding of the importance of select glycan structures for tissue formation and homeostasis is incomplete. Here, we present a human organotypic platform that allows genetic dissection of cellular glycosylation capacities and systematic interrogation of the roles of distinct glycan types in tissue formation. We used CRISPR-Cas9 gene targeting to generate a library of 3D organotypic skin tissues that selectively differ in their capacity to produce glycan structures on the main types of N- and O-linked glycoproteins and glycolipids. This tissue library revealed distinct changes in skin formation associated with a loss of features for all tested glycoconjugates. The organotypic skin model provides phenotypic cues for the distinct functions of glycoconjugates and serves as a unique resource for further genetic dissection and identification of the specific structural features involved. The strategy is also applicable to other organotypic tissue models.

Cell Adhesion Molecules and Their Roles and Regulation in the Immune and Tumor Microenvironment.

The immune system and cancer have a complex relationship with the immune system playing a dual role in tumor development. The effector cells of the immune system can recognize and kill malignant cells while immune system-mediated inflammation can also promote tumor growth and regulatory cells suppress the anti-tumor responses. In the center of all anti-tumor responses is the ability of the immune cells to migrate to the tumor site and to interact with each other and with the malignant cells. Cell adhesion molecules including receptors of the immunoglobulin superfamily and integrins are of crucial importance in mediating these processes. Particularly integrins play a vital role in regulating all aspects of immune cell function including immune cell trafficking into tissues, effector cell activation and proliferation and the formation of the immunological synapse between immune cells or between immune cell and the target cell both during homeostasis and during inflammation and cancer. In this review we discuss the molecular mechanisms regulating integrin function and the role of integrins and other cell adhesion molecules in immune responses and in the tumor microenvironment. We also describe how malignant cells can utilize cell adhesion molecules to promote tumor growth and metastases and how these molecules could be targeted in cancer immunotherapy.

TGFβ1-induced cell motility but not cell proliferation is mediated through Cten in colorectal cancer.

Cten (C-terminal tensin-like) is a member of the tensin protein family found in complex with integrins at focal adhesions. It promotes epithelial-mesenchymal transition (EMT) and cell motility. The precise mechanisms regulating Cten are unknown, although we and others have shown that Cten could be under the regulation of several cytokines and growth factors. Since transforming growth factor beta 1 (TGF-β1) regulates integrin function and promotes EMT/cell motility, we were prompted to investigate whether TGF-β1 induces EMT and cell motility through Cten signalling in colorectal cancer. TGF-β1 signalling was modulated by either stimulation with TGF-β1 or knockdown of TGF-β1 in the CRC cell lines SW620 and HCT116. The effect of this modulation on expression of Cten, EMT markers and on cellular function was tested. The role of Cten as a direct mediator of TGF-β1 signalling was investigated in a CRC cell line in which the Cten gene had been deleted (SW620ΔCten ). When TGF-β1 was stimulated or inhibited, this resulted in, respectively, upregulation and downregulation of Cten expression and EMT markers (Snail, Rock, N-cadherin, Src). Cell migration and cell invasion were significantly increased following TGF-β1 stimulation and lost by TGF-β1 knockdown. TGF-β1 stimulation of the SW620ΔCten cell line resulted in selective loss of the effect of TGF-β1 signalling pathway on EMT and cell motility while the stimulatory effect on cell proliferation was retained. These data suggested Cten may play an essential role in mediating TGF-β1-induced EMT and cell motility and may therefore play a role in metastasis in CRC.

Factors Regulating Human Extravillous Trophoblast Invasion: Chemokine-peptidase and CD9-integrin Systems.

The invasion of an extravillous trophoblast (EVT) into maternal decidual tissues, especially towards maternal spiral arteries, is an essential process in the human placental formation and subsequent normal fetal development. However, the precise regulatory mechanisms to induce EVT invasion towards arteries and/or to protect EVT from further invasion are not well understood. We found that a chemokine receptor, CCR1, was specifically expressed on EVT migrating towards maternal arteries. Using EVT isolated from a primary villous explant culture, RANTES, which is one of the ligands for CCR1, was shown to enhance EVT invasion. Furthermore, we observed that the platelets were deposited among intravascular EVT and platelet-derived soluble factors, which contained RANTES, enhanced EVT invasion. On the one hand, dipeptidyl peptidase IV (DPPIV), which can metabolize RANTES on the cell surface, was expressed on non-invading EVT and was demonstrated to suppress EVT invasion. In contrast, laeverin/aminopeptidase Q, which is specifically expressed on EVT, was shown to induce EVT invasion. Also, CD9 which is a cell surface marker of platelets and a regulator of integrin function was expressed on EVT and gene knockdown of the CD9 molecule enhanced EVT invasion. These findings suggest that the chemokine-chemokine receptor, chemokine-peptidase, and CD9-integrin systems play important roles in the regulation of EVT invasion during early human placental formation.

Junctional Adhesion Molecules (JAMs): The JAM-Integrin Connection.

Junctional adhesion molecules (JAMs) are cell surface adhesion receptors of the immunoglobulin superfamily. JAMs are involved in a variety of biological processes both in the adult organism but also during development. These include processes such as inflammation, angiogenesis, hemostasis, or epithelial barrier formation, but also developmental processes such as hematopoiesis, germ cell development, and development of the nervous system. Several of these functions of JAMs depend on a physical and functional interaction with integrins. The JAM – integrin interactions in trans regulate cell-cell adhesion, their interactions in cis regulate signaling processes originating at the cell surface. The JAM – integrin interaction can regulate the function of the JAM as well as the function of the integrin. Beyond the physical interaction with integrins, JAMs can regulate integrin function through intracellular signaling indicating an additional level of JAM – integrin cross-talk. In this review, we describe the various levels of the functional interplay between JAMs and integrins and the role of this interplay during different physiological processes.

FERMT3 contributes to glioblastoma cell proliferation and chemoresistance to temozolomide through integrin mediated Wnt signaling.

FERMT3, also known as kindlin-3, is one of three kindlin family members expressed in mammals. Kindlins are cytosolic, adaptor proteins that are important activators and regulators of integrin function. They have also been shown to play critical roles in the development and progression of various cancers. In the present study, we hypothesized that FERMT3 would enhance glioblastoma multiforme (GBM) cell survival. Indeed, expression level analyses showed significant FERMT3 upregulation in human glioma tissues as compared to normal brain tissues. The effect was particularly pronounced in high-grade gliomas. We then demonstrated that FERMT3 knockdown suppresses glioma cell proliferation and chemoresistance to temozolomide (TMZ). To determine the mechanism by which FERMT3 enhances glioma cell proliferation and chemoresistance, we examined the effects of FERMT3 on integrin activation and Wnt/β-catenin signaling. Through the use of western blot assays and TOPflash and FOPflash plasmid transfection into glioma cells lines, we demonstrated that FERMT3 regulates glioma cell activity through integrin-mediated Wnt/β-catenin signaling. These results suggest that FERMT3 activates integrin activity in high-grade gliomas to enhance glioma cell survival and chemoresistance. The present study thus indicates a potential role for FERMT3 as a genetic target in the treatment of GBM.

Tensin links energy metabolism to extracellular matrix assembly.

The regulation of integrin function is key to fundamental cellular processes, including cell migration and extracellular matrix (ECM) assembly. In this issue, Georgiadou et al. (2017. J. Cell Biol. https://doi.org/10.1083/jcb.201609066) report that the metabolic sensor adenosine monophosphate-activated protein kinase influences tensin production to regulate α5β1-integrin and fibrillar adhesion assembly and thus reveal an important connection between energy metabolism and ECM assembly.

Circulation : Clinical Summaries

<i>Circulation</i> : Clinical Summaries

Abstract 405: Kindlin-2 regulates integrin function and sensitivity to docetaxel in prostate cancer cells

Abstract Over the past decade, many lines of evidence have emerged to implicate the three members of the kindlin family of FERM domain proteins as important regulators of integrin adhesion receptors. Specifically, kindlins appear to cooperate with talin to be essential regulators of the ligand binding function of integrins and thereby enhance the adhesive and migratory responses of cells. Kindlin-2 (FERMT2) is the most broadly distributed of the kindlins. To explore the relationship between integrin activation and kindlin-2, prostate cancer cell lines were screened and LNCaP prostate cancer cells were found to express relatively low levels of kindlin-2 and endogenous levels of beta-1 integrin on their surface. A portion of this integrin was expressed in an activated state as detected with an activation specific antibody, HUTS-4. These cells were transfected with vectors for kindlin-2, with or without talin-head domain, a combination that leads to activation of β3 integrins in model cells. Transfections with kindlin-2 suppressed β1 integrin activation on the surface of the LNCaP cells. This inhibitory effect was abolished by mutation of kindlin-2 that prevented its binding to integrin cytoplasmic tail. This inhibitory response to kindlin-2 on β1 integrin was in stark contract to the effects of kindlin-2 plus talin-head expression on β3 function, where co-expression of the same constructs enhanced integrin activation. A mutation in kindlin-2, deletion of its last 15 amino acids, that blocks activation of β3 integrins without preventing its binding to the integrin, also prevents its inhibition of β1 function. We next looked at the effects of kindlin-2 on docetaxel induced apoptosis as monitored with Annexin V staining and cell death as monitored by propidium iodide staining (both by flow cytometry). Compared to LNCaP cells expressing EGFP alone, cells overexpressing EGFP-kindlin-2 were protected against apoptosis and cell death. This effect was dependent upon binding of kindlin-2 to the cytoplasmic tail of integrins. Hence, the capacity of kindlin-2 to influence integrin function depends upon the integrin subfamily and can range from activation to suppression of function. Kindlin-2, which is often overexpressed in transformed cells, may desensitize cells towards chemotherapeutic agents. Citation Format: Edward F. Plow, Mitali Das, Jamila Hirbawi, Khalid Sossey-Alaoui. Kindlin-2 regulates integrin function and sensitivity to docetaxel in prostate cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 405. doi:10.1158/1538-7445.AM2015-405

CEACAM1 regulates integrin αIIbβ3-mediated functions in platelets

Previous studies have implicated that the Ig-ITIM superfamily member, CEACAM1 may regulate integrin function. While CEACAM1 has been demonstrated to play a role as an inhibitory co-receptor of ITAM-associated GPVI/FcR γ-chain signaling pathways in platelets, its physiologic role in integrin αIIbβ3-mediated platelet function is unclear. In this study, we investigate the functional importance of Ceacam1 in murine platelets. We show that CEACAM1 is constitutively associated with integrin αIIbβ3 in resting human and mouse platelets as demonstrated by co-immunoprecipitation studies. Using Ceacam1-deficient mice, we show that they have prolonged tail bleeding times and volume of blood lost that is corrected by reconstitution with platelet Ceacam1. Ceacam1−/− platelets have moderate integrin αIIbβ3 mediated functional defects with impaired kinetics of platelet spreading on fibrinogen and failure to retract fibrin clots in vitro. This functional integrin αIIbβ3 defect could not be attributed to altered integrin αIIbβ3 expression. Ceacam1−/− platelets displayed normal “inside–out” signaling properties as demonstrated by normal agonist-induced binding of soluble (fluorescein isothiocyanate) FITC-fibrinogen, JON/A antibody binding, and increases in cytosolic free calcium levels. This study provides direct evidence that Ceacam1 is essential for normal integrin αIIbβ3-mediated platelet function and that disruption of mouse Ceacam1 induced moderate integrin αIIbβ3-mediated functional defects.

PI3K/Akt in platelet integrin signaling and implications in thrombosis.

Blood platelets are anucleated circulating cells that play a critical role in hemostasis and are also implicated in arterial thrombosis, a major cause of death worldwide. The biological function of platelets strongly relies in their reactiveness to a variety of extracellular agonists that regulate their adhesion to extracellular matrix at the site of vascular injury and their ability to form rapidly growing cell aggregates. Among the membrane receptors expressed on the cell surface, integrins are crucial for both platelet activation, adhesion and aggregation. Integrin affinity for specific ligands is regulated by intracellular signaling pathways activated in stimulated platelets, and, once engaged, integrins themselves generate and propagate signals inside the cells to reinforce and consolidate platelet response and thrombus formation. Phosphatidylinositol 3-Kinases (PI3Ks) have emerged as crucial players in platelet activation, and they are directly implicated in the regulation of integrin function. This review will discuss the contribution of PI3Ks in platelet integrin signaling, focusing on the role of specific members of class I PI3Ks and their downstream effector Akt on both integrin inside-out and outside-in signaling. The contribution of the PI3K/Akt pathways stimulated by integrin engagement and platelet activation in thrombus formation and stabilization will also be discussed in order to highlight the possibility to target these enzymes in effective anti-thrombotic therapeutic strategies.

Role of EGFR expression levels in the regulation of integrin function by EGF.

Activation of β1 integrins in dormant tumor cells has been linked to metastatic progression, suggesting that therapies designed to maintain β1 integrins in an inactive state may be useful in the prevention of metastatic disease. Our earlier studies have demonstrated that EGF regulates the activation state of the α5β1 integrin in EGFR overexpressing tumor cells through an ERK/p90RSK signaling pathway. Activation of this pathway by EGF resulted in the filamin A dependent inactivation of the α5β1 integrin receptor for fibronectin. The current study was designed to address the role of EGFR overexpression in the regulation of α5β1 integrin activation state by EGF. Lentiviral knockdown of EGFR coupled with limited dilution cloning was used to develop A431 squamous carcinoma cell lines expressing high, moderate, and low levels of EGFR. Inactivation of α5β1 integrin by EGF was shown to correlate with both the level of EGFR expression and the extent of p90RSK phosphorylation, but not with the level of ERK phosphorylation, suggesting that high levels of EGFR promote α5β1 integrin inactivation through sustained activation of p90RSK. Treatment of cells with EGFR kinase inhibitor resulted in a reactivation of the integrin which could be reversed with the phosphatase inhibitor, menadione. Taken together, these findings indicate that p90RSK may function to maintain dormancy in tumor cells expressing high levels of EGFR. © 2015 Wiley Periodicals, Inc.

Peptides derived from central turn motifs within integrin αIIb and αV cytoplasmic tails inhibit integrin activation

We previously found that peptides derived from the full length of integrin αIIb and αV cytoplasmic tails inhibited their parent integrin activation, respectively. Here we showed that the cell-permeable peptides corresponding to the conserved central turn motif within αIIb and αV cytoplasmic tails, myr-KRNRPPLEED (αIIb peptide) and myr-KRVRPPQEEQ (αV peptide), similarly inhibited both αIIb and αV integrin activation. Pre-treatment with αIIb or αV peptides inhibited Mn2+-activated αIIbβ3 binding to soluble fibrinogen as well as the binding of αIIbβ3-expressing Chinese Hamster Ovary cells to immobilized fibrinogen. Our turn peptides also inhibited adhesion of two breast cancer cell lines (MDA-MB-435 and MCF7) to αV ligand vitronectin. These results suggest that αIIb and αV peptides share a same mechanism in regulating integrin function. Using αIIb peptide as a model, we found that replacement of RPP with AAA significantly attenuated the inhibitory activity of αIIb peptide. Furthermore, we found that αIIb peptide specifically bound to β-tubulin in cells. Our work suggests that the central motif of α tails is an anchoring point for cytoskeletons during integrin activation and integrin-mediated cell adhesion, and its function depends on the turn structure at RPP. However, post-treatment of peptides derived from the full-length tail or from the turn motif did not reverse αIIb and αV integrin activation.

Abstract 2088: Kindlin-3 enhances breast cancer metastasis through upregulation of Twist-mediated tumor angiogenesis

Abstract The FERM domain containing protein, Kindlin-3, has been recognized as a major regulator of integrin function in hematopoietic cells but its role in neoplasia is totally unknown. We have examined the relationship between Kindlin-3 and breast cancer in mouse models and human tissues. Human breast tumors showed a ∼7-fold elevation in kindlin-3 mRNA compared to non-neoplastic tissue by quantitative polymerize chain reaction. At the protein level, Kindlin-3 was found to be expressed abundantly in breast cancer tissue, and its expression increases with the advancing stage of tumor development. Mining of the Oncomine database (www.oncomine.com) confirmed this association: Kindlin-3 was in the top 3 % of gene products elevated in breast cancer (p&amp;lt; 10-12). When Kindlin-3 was over-expressed in a breast cancer cell line, primary tumor growth and lung metastasis increased by 2.5- and 3-fold, respectively when implanted into mice compared to cells expressing vector alone. Mechanistically, the Kindlin-cells displayed a 2.2-fold increase in Vascular Endothelial Growth Factor (VEGF) secretion and B1 integrin activation. Increased VEGF secretion resulted from enhanced production of Twist, a transcription factor which promotes tumor angiogenesis. Knockdown of Twist diminished VEGF production and knockdown of B1 integrins diminished Twist and VEGF production by Kindlin-3 cells while non-targeting si-RNA had no effect on expression of these gene products. The activation of this signaling cascade resulted in the enhancement of breast cancer cell invasion, and tumor angiogenesis and metastasis. Hence, our studies established the pathway in which cross-talk between Kindlin-3 and B1 integrins activates the nuclear translocation of TWIST, which leads to enhanced VEGF-A production and secretion, macrophage infiltration as well as activation of the Epithelial-to-mesenchymal-transition (EMT) program. The activation of this signaling cascade culminates in enhanced breast cancer cell migration, invasion and tumor angiogenesis, all of which are required for tumor progression and metastasis. In summary, by analysis of human tissue samples and in vitro and in vivo studies, we have identified a novel role for Kindlin-3 in the progression of breast cancer. To our knowledge, this is the first demonstration of the presence and implication of Kindlin-3 in breast cancer or any cancer. Citation Format: Khalid Sossey-Alaoui, Elzbieta Pluskota, Gangarao Davuluri, Katarzyna Bialkowska, Mitali Das, Daniel Lindner, Edward F. Plow. Kindlin-3 enhances breast cancer metastasis through upregulation of Twist-mediated tumor angiogenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2088. doi:10.1158/1538-7445.AM2014-2088

Membrane tension drives ligand-independent integrin signaling.

Integrins anchor cells to the extracellular matrix (ECM) and control a multitude of essential cellular functions by activating a variety of signaling pathways. In this issue of The EMBO Journal, a study conducted by Ferraris and colleagues report that binding of urokinase-type plasminogen activator receptor (uPAR) to vitronectin is sufficient to trigger ligand-independent β1 and β3 integrin signaling. The coupling of uPAR and integrins occurs independent of lateral interaction between the two receptors, but relies on membrane tension (Ferraris et al, 2014).

Brag2 differentially regulates β1- and β3-integrin-dependent adhesion in endothelial cells and is involved in developmental and pathological angiogenesis

β1-Integrins are essential for angiogenesis. The mechanisms regulating integrin function in endothelial cells (EC) and their contribution to angiogenesis remain elusive. Brag2 is a guanine nucleotide exchange factor for the small Arf-GTPases Arf5 and Arf6. The role of Brag2 in EC and angiogenesis and the underlying molecular mechanisms remain unclear. siRNA-mediated Brag2-silencing reduced EC angiogenic sprouting and migration. Brag2-siRNA transfection differentially affected α5β1- and αVβ3-integrin function: specifically, Brag2-silencing increased focal/fibrillar adhesions and adhesion on β1-integrin ligands (fibronectin and collagen), while reducing the adhesion on the αVβ3-integrin ligand, vitronectin. Consistent with these results, Brag2-silencing enhanced surface expression of α5β1-integrin, while reducing surface expression of αVβ3-integrin. Mechanistically, Brag2-mediated αVβ3-integrin-recycling and β1-integrin endocytosis and specifically of the active/matrix-bound α5β1-integrin present in fibrillar/focal adhesions (FA), suggesting that Brag2 contributes to the disassembly of FA via β1-integrin endocytosis. Arf5 and Arf6 are promoting downstream of Brag2 angiogenic sprouting, β1-integrin endocytosis and the regulation of FA. In vivo silencing of the Brag2-orthologues in zebrafish embryos using morpholinos perturbed vascular development. Furthermore, in vivo intravitreal injection of plasmids containing Brag2-shRNA reduced pathological ischemia-induced retinal and choroidal neovascularization. These data reveal that Brag2 is essential for developmental and pathological angiogenesis by promoting EC sprouting through regulation of adhesion by mediating β1-integrin internalization and link for the first time the process of β1-integrin endocytosis with angiogenesis.

Abstract 18516: Brag2, an Arf-GEF, Regulates Integrin-Dependent Endothelial Adhesion and is Involved in Developmental and Pathological Angiogenesis

β1-integrins are essential for angiogenesis. The mechanisms regulating integrin function in endothelial cells (EC) and their contribution to angiogenesis remain elusive. Brag2 is a guanine nucleotide exchange factor (GEF) for the small Arf-GTPases Arf5 and Arf6. The role of Brag2 in EC and angiogenesis and the underlying molecular mechanisms remain unclear. siRNA-mediated silencing of Brag2 significantly blocked basal and VEGF-induced angiogenic sprouting (by 40 ± 7 %), tube formation and migration of HUVEC (49 % inhibition). Transfection with Brag2-siRNA differentially affected α5β1- and αVβ3-integrin function: specifically, Brag2-silencing increased focal/fibrillar adhesions and EC adhesion on β1-integrin-ligands (fibronectin and collagen), while reducing the adhesion on the αVβ3-integrin-ligand, vitronectin. Consistent with these results, Brag2-silencing enhanced surface expression of α5β1-integrin, while reducing surface expression of αVβ3-integrin. Mechanistically, Brag2 mediated recycling of αVβ3-integrins and endocytosis of β1-integrins and specifically of the active/matrix bound α5β1-integrin present in fibrillar/focal adhesions (FA), suggesting that Brag2 contributes to the disassembly of FA via β1-integrin-endocytosis. Furthermore, we found that Arf5 and Arf6 are promoting downstream of Brag2 angiogenic sprouting, β1-integrin-endocytosis and the regulation of FA. In vivo silencing of the Brag2-orthologues in zebrafish embryos using morpholinos perturbed vascular development. Moreover, in vivo intravitreal injection of plasmids containing Brag2-shRNA reduced pathological ischemia-induced retinal and choroidal neovascularization. These data reveal that Brag2 is essential for developmental and pathological angiogenesis by promoting EC sprouting through regulation of adhesion by mediating β1-integrin internalization and link for the first time the process of β1-integrin endocytosis with angiogenesis.