Low-Level Nanog Expression in the Regulation of Quiescent Endothelium.

OBJECTIVE: Nanog is expressed in adult endothelial cells (ECs) at a low-level, however, its functional significance is not known. The goal of our study was to elucidate the role of Nanog in adult ECs using a genetically engineered mouse model system. Approach and Results: Biochemical analyses showed that Nanog is expressed in both adult human and mouse tissues. Primary ECs isolated from adult mice showed detectable levels of Nanog, Tert (telomerase reverse transcriptase), and eNos (endothelial nitric oxide synthase). Wnt3a (Wnt family member 3A) increased the expression of Nanog and hTERT (human telomerase reverse transcriptase) in ECs and increased telomerase activity in these cells. In a chromatin immunoprecipitation experiment, Nanog directly bound to the hTERT and eNOS promoter/enhancer DNA elements, thereby regulating their transcription. Administration of low-dose tamoxifen to ROSAmT/mG::Nanogfl/+::Cdh5CreERT2 mice induced deletion of a single Nanog allele, simultaneously labeling ECs with green fluorescent protein and resulting in decreased Tert and eNos levels. Histological and morphometric analyses of heart tissue sections prepared from these mice revealed cell death, microvascular rarefaction, and increased fibrosis in cardiac vessels. Accordingly, EC-specific Nanog-haploinsufficiency resulted in impaired EC homeostasis and angiogenesis. Conversely, re-expression of cDNA encoding the hTERT in Nanog-depleted ECs, in part, restored the effect of loss of Nanog. CONCLUSIONS: We showed that low-level Nanog expression is required for normal EC homeostasis and angiogenesis in adulthood.

LDL induces cholesterol loading and inhibits endothelial proliferation and angiogenesis in Matrigels: correlation with impaired angiogenesis during wound healing.

Hypercholesterolemia is a major risk factor for adverse cardiovascular outcomes, but its effect on angiogenesis and wound healing is not well understood. In this study, using a combination of mass spectrometry and laurdan two-photon imaging, we show that elevated levels of low-density lipoprotein (LDL), like those seen in hypercholesterolemic patients, lead to an increase in both free cholesterol and cholesterol esters, as well as increase in lipid order of endothelial cell membranes. Notably, these effects are distinct and opposite to the lack of cholesterol loading and the disruption of lipid order observed in our earlier studies in response to oxidized LDL (oxLDL). The same pathological level of LDL leads to a significant inhibition of endothelial proliferation and cell cycle arrest in G2/M phase, whereas oxLDL enhances endothelial proliferation in S phase of the cycle. LDL but not oxLDL suppresses the expression of vascular endothelial growth factor receptor-2 while enhancing the expression of vascular endothelial growth factor (VEGF). Furthermore, we show that aged (8-10 mo) hypercholesterolemic apolipoprotein E-deficient (ApoE-/-) mice display delayed wound closure compared with age-matched C57/BL6 wild-type controls following a skin punch biopsy. The delay in wound healing is associated with a decreased expression of cluster of differentiation 31 platelet endothelial cell adhesion molecule endothelial marker and decreased angiogenesis within the wound bed. Furthermore, decreased endothelial responsiveness to the growth factors VEGF and basic fibroblast growth factor is observed in ApoE-/- mice in Matrigel plugs and in Matrigels with high levels of LDL in wild-type mice. We propose that plasma hypercholesterolemia is antiangiogenic due to elevated levels of LDL.

Tumor-derived exosomes in the regulation of macrophage polarization.

BACKGROUND: This review focuses on exosomes derived from various cancer cells. The review discusses the possibility of differentiating macrophages in alternatively activated anti-inflammatory pro-tumorigenic M2 macrophage phenotypes and classically activated pro-inflammatory, anti-tumorigenic M1 macrophage phenotypes in the tumor microenvironment (TME). The review is divided into two main parts, as follows: (1) role of exosomes in alternatively activating M2-like macrophages-breast cancer-derived exosomes, hepatocellular carcinoma (HCC) cell-derived exosomes, lung cancer-derived exosomes, prostate cancer-derived exosomes, Oral squamous cell carcinoma (OSCC)-derived exosomes, epithelial ovarian cancer (EOC)-derived exosomes, Glioblastoma (GBM) cell-derived exosomes, and colorectal cancer-derived exosomes, (2) role of exosomes in classically activating M1-like macrophages, oral squamous cell carcinoma-derived exosomes, breast cancer-derived exosomes, Pancreatic-cancer derived modified exosomes, and colorectal cancer-derived exosomes, and (3) exosomes and antibody-dependent cellular cytotoxicity (ADCC). This review addresses the following subjects: (1) crosstalk between cancer-derived exosomes and recipient macrophages, (2) the role of cancer-derived exosome payload(s) in modulating macrophage fate of differentiation, and (3) intracellular signaling mechanisms in macrophages regarding the exosome's payload(s) upon its uptake and regulation of the TME. EVIDENCE: Under the electron microscope, nanoscale exosomes appear as specialized membranous vesicles that emerge from the endocytic cellular compartments. Exosomes harbor proteins, growth factors, cytokines, lipids, miRNA, mRNA, and DNAs. Exosomes are released by many cell types, including reticulocytes, dendritic cells, B-lymphocytes, platelets, mast cells, and tumor cells. It is becoming clear that exosomes can impinge upon signal transduction pathways, serve as a mediator of signaling crosstalk, thereby regulating cell-to-cell wireless communications. CONCLUSION: Based on the vesicular cargo, the molecular constituents, the exosomes have the potential to change the fate of macrophage phenotypes, either M1, classically activated macrophages, or M2, alternatively activated macrophages. In this review, we discuss and describe the ability of tumor-derived exosomes in the mechanism of macrophage activation and polarization.

The allosteric glycogen synthase kinase-3 inhibitor NP12 limits myocardial remodeling and promotes angiogenesis in an acute myocardial infarction model.

A key feature of acute myocardial infarction (AMI) is an alteration in cardiac architecture. Signaling events that result in the inhibition of glycogen synthase kinase-3 (GSK-3)ß represent an adaptive response that might limit the extent of adverse remodeling in the aftermath of AMI. Here, we report that an allosteric inhibitor of GSK-3ß, 4-benzyl-2-(naphthalene-1-yl)-1,2,4-thiadiazolidine-3,5-dione (NP12), lessens the magnitude of adverse myocardial remodeling and promotes angiogenesis. Male and female mice 8-10 weeks old were grouped (six animals in each group) into sham surgery (sham group), left anterior descending (LAD) ligation of the coronary artery followed by intramyocardial PBS injections (control group), and LAD ligation followed by NP12 administration (NP12 group). After 7 and 14 days, the extents of fibrosis and integrity of blood vessels were determined. Intramyocardial administration of NP12 increased phosphorylation of GSK-3ß, reduced fibrosis, and restored diastolic function in the mice that had experienced an AMI. Morphometric analyses revealed increased CD31+ and Ki67+ vascular structures and decreased apoptosis in these mice. NP12 administration mediated proliferation of reparative cells in the AMI hearts. In a time-course analysis, Wnt3a and NP12 stabilized ß-catenin and increased expression of both Nanog and VEGFR2. Moreover, NP12 increased the expression of ß-catenin and Nanog in myocardium from AMI mice. Finally, loss- and gain-of-function experiments indicated that the NP12-mediated benefit is, in part, Nanog-specific. These findings indicate that NP12 reduces fibrosis, reestablishes coronary blood flow, and improves ventricular function following an AMI. We conclude that NP12 might be useful for limiting ventricular remodeling after an AMI.

oxLDL induces endothelial cell proliferation via Rho/ROCK/Akt/p27kip1 signaling: opposite effects of oxLDL and cholesterol loading.

Oxidized modifications of LDL (oxLDL) play a key role in the development of endothelial dysfunction and atherosclerosis. However, the underlying mechanisms of oxLDL-mediated cellular behavior are not completely understood. Here, we compared the effects of two major types of oxLDL, copper-oxidized LDL (Cu2+-oxLDL) and lipoxygenase-oxidized LDL (LPO-oxLDL), on proliferation of human aortic endothelial cells (HAECs). Cu2+-oxLDL enhanced HAECs' proliferation in a dose- and degree of oxidation-dependent manner. Similarly, LPO-oxLDL also enhanced HAEC proliferation. Mechanistically, both Cu2+-oxLDL and LPO-oxLDL enhance HAEC proliferation via activation of Rho, Akt phosphorylation, and a decrease in the expression of cyclin-dependent kinase inhibitor 1B (p27kip1). Both Cu2+-oxLDL or LPO-oxLDL significantly increased Akt phosphorylation, whereas an Akt inhibitor, MK2206, blocked oxLDL-induced increase in HAEC proliferation. Blocking Rho with C3 or its downstream target ROCK with Y27632 significantly inhibited oxLDL-induced Akt phosphorylation and proliferation mediated by both Cu2+- and LPO-oxLDL. Activation of RhoA was blocked by Rho-GDI-1, which also abrogated oxLDL-induced Akt phosphorylation and HAEC proliferation. In contrast, blocking Rac1 in these cells had no effect on oxLDL-induced Akt phosphorylation or cell proliferation. Moreover, oxLDL-induced Rho/Akt signaling downregulated cell cycle inhibitor p27kip1 Preloading these cells with cholesterol, however, prevented oxLDL-induced Akt phosphorylation and HAEC proliferation. These findings provide a new understanding of the effects of oxLDL on endothelial proliferation, which is essential for developing new treatments against neovascularization and progression of atherosclerosis.

Oxidized LDL signals through Rho-GTPase to induce endothelial cell stiffening and promote capillary formation.

Endothelial biomechanics is emerging as a key factor in endothelial function. Here, we address the mechanisms of endothelial stiffening induced by oxidized LDL (oxLDL) and investigate the role of oxLDL in lumen formation. We show that oxLDL-induced endothelial stiffening is mediated by CD36-dependent activation of RhoA and its downstream target, Rho kinase (ROCK), via inhibition of myosin light-chain phosphatase (MLCP) and myosin light-chain (MLC)2 phosphorylation. The LC-MS/MS analysis identifies 7-ketocholesterol (7KC) as the major oxysterol in oxLDL. Similarly to oxLDL, 7KC induces RhoA activation, MLCP inhibition, and MLC2 phosphorylation resulting in endothelial stiffening. OxLDL also facilitates formation of endothelial branching networks in 3D collagen gels in vitro and induces increased formation of functional blood vessels in a Matrigel plug assay in vivo. Both effects are RhoA and ROCK dependent. An increase in lumen formation was also observed in response to pre-exposing the cells to 7KC, an oxysterol that induces endothelial stiffening, but not to 5α,6α epoxide that does not affect endothelial stiffness. Importantly, loading cells with cholesterol prevented oxLDL-induced RhoA activation and the downstream signaling cascade, and reversed oxLDL-induced lumen formation. In summary, we show that oxLDL-induced endothelial stiffening is mediated by the CD36/RhoA/ROCK/MLCP/MLC2 pathway and is associated with increased endothelial angiogenic activity.

KIF13B regulates angiogenesis through Golgi to plasma membrane trafficking of VEGFR2.

Although the trafficking of newly synthesized VEGFR2 to the plasma membrane is a key determinant of angiogenesis, the molecular mechanisms of Golgi to plasma membrane trafficking are unknown. Here, we have identified a key role of the kinesin family plus-end molecular motor KIF13B in delivering VEGFR2 cargo from the Golgi to the endothelial cell surface. KIF13B is shown to interact directly with VEGFR2 on microtubules. We also observed that overexpression of truncated versions of KIF13B containing the binding domains that interact with VEGFR2 inhibited VEGF-induced capillary tube formation. KIF13B depletion prevented VEGF-mediated endothelial migration, capillary tube formation and neo-vascularization in mice. Impairment in trafficking induced by knockdown of KIF13B shunted VEGFR2 towards the lysosomal degradation pathway. Thus, KIF13B is an essential molecular motor required for the trafficking of VEGFR2 from the Golgi, and its delivery to the endothelial cell surface mediates angiogenesis.

Integrin α6ß1 Expressed in ESCs Instructs the Differentiation to Endothelial Cells.

Adhesion of embryonic stem cells (ESCs) to the extracellular matrix may influence differentiation potential and cell fate decisions. Here, we investigated the inductive role of binding of integrin α6ß1 expressed in mouse (m)ESCs to laminin-1 (LN1) in mediating the differentiation of ESCs to endothelial cells (ECs). We observed that α6ß1 binding to LN1 was required for differentiation to ECs. α6ß1 functioned by recruiting the adaptor tetraspanin protein CD151, which activated FAK and Akt signaling and mediated the EC lineage-specifying transcription factor Er71. In contrast, association of the ESC-expressed α3ß1, another highly expressed LN1 binding integrin, with CD151, prevented α6ß1-mediated differentiation. CD151 thus functioned as a bifurcation router to direct ESCs toward ECs when α6ß1 associated with CD151, or prevented transition to ECs when α3ß1 associated with CD151. These observations were recapitulated in mice in which α6 integrin or CD151 knockdown reduced the expression of Er71-regulated angiogenesis genes and development of blood vessels. Thus, interaction of α6ß1 in ESCs with LN1 activates α6ß1/CD151 signaling which programs ESCs toward the EC lineage fate.

Role of c-Met/phosphatidylinositol 3-kinase (PI3k)/Akt signaling in hepatocyte growth factor (HGF)-mediated lamellipodia formation, reactive oxygen species (ROS) generation, and motility of lung endothelial cells.

Hepatocyte growth factor (HGF) mediated signaling promotes cell proliferation and migration in a variety of cell types and plays a key role in tumorigenesis. As cell migration is important to angiogenesis, we characterized HGF-mediated effects on the formation of lamellipodia, a pre-requisite for migration using human lung microvascular endothelial cells (HLMVECs). HGF, in a dose-dependent manner, induced c-Met phosphorylation (Tyr-1234/1235, Tyr-1349, Ser-985, Tyr-1003, and Tyr-1313), activation of PI3k (phospho-Yp85) and Akt (phospho-Thr-308 and phospho-Ser-473) and potentiated lamellipodia formation and HLMVEC migration. Inhibition of c-Met kinase by SU11274 significantly attenuated c-Met, PI3k, and Akt phosphorylation, suppressed lamellipodia formation and endothelial cell migration. LY294002, an inhibitor of PI3k, abolished HGF-induced PI3k (Tyr-458), and Akt (Thr-308 and Ser-473) phosphorylation and suppressed lamellipodia formation. Furthermore, HGF stimulated p47(phox)/Cortactin/Rac1 translocation to lamellipodia and ROS generation. Moreover, inhibition of c-Met/PI3k/Akt signaling axis and NADPH oxidase attenuated HGF- induced lamellipodia formation, ROS generation and cell migration. Ex vivo experiments with mouse aortic rings revealed a role for c-Met signaling in HGF-induced sprouting and lamellipodia formation. Taken together, these data provide evidence in support of a significant role for HGF-induced c-Met/PI3k/Akt signaling and NADPH oxidase activation in lamellipodia formation and motility of lung endothelial cells.

Low-dose 6-bromoindirubin-3'-oxime induces partial dedifferentiation of endothelial cells to promote increased neovascularization.

Endothelial cell (EC) dedifferentiation in relation to neovascularization is a poorly understood process. In this report, we addressed the role of Wnt signaling in the mechanisms of neovascularization in adult tissues. Here, we show that a low-dose of 6-bromoindirubin-3'-oxime (BIO), a competitive inhibitor of glycogen synthase kinase-3ß, induced the stabilization of ß-catenin and its subsequent direct interaction with the transcription factor NANOG in the nucleus of ECs. This event induced loss of VE-cadherin from the adherens junctions, increased EC proliferation accompanied by asymmetric cell division (ACD), and formed cellular aggregates in hanging drop assays indicating the acquisition of a dedifferentiated state. In a chromatin immunoprecipitation assay, nuclear NANOG protein bound to the NANOG- and VEGFR2-promoters in ECs, and the addition of BIO activated the NANOG-promoter-luciferase reporter system in a cell-based assay. Consequently, NANOG-knockdown decreased BIO-induced NOTCH-1 expression, thereby decreasing cell proliferation, ACD, and neovascularization. In a Matrigel plug assay, BIO induced increased neovascularization, secondary to the presence of vascular endothelial growth factor (VEGF). Moreover, in a mouse model of hind limb ischemia, BIO augmented neovascularization that was coupled with increased expression of NOTCH-1 in ECs and increased smooth muscle α-actin(+) cell recruitment around the neovessels. Thus, these results demonstrate the ability of a low-dose of BIO to augment neovascularization secondary to VEGF, a process that was accompanied by a partial dedifferentiation of ECs via ß-catenin and the NANOG signaling pathway.

NANOG induction of fetal liver kinase-1 (FLK1) transcription regulates endothelial cell proliferation and angiogenesis.

NANOG is a master transcription factor associated with the maintenance of stem cell pluripotency. Here, we demonstrate that transcription factor NANOG is expressed in cultured endothelial cells (ECs) and in a subset of tumor cell lines. Importantly, we provide evidence that WNT3A stimulation of ECs induces the transcription of NANOG which mediates the expression of vascular endothelial growth factor receptor-2, also known as fetal liver kinase-1 (FLK1). We defined ATTA as a minimal binding site for NANOG. Accordingly, a luciferase reporter assay showed that NANOG binds to and activates 4 ATTA binding sites identified in the FLK1 promoter after WNT3A stimulation. Consistent with this data, we found that, under basal conditions and in response to WNT3A stimulation, NANOG binding to these ATTA sequences markedly induced the expression of FLK1. Thus, our data indicate an essential role in angiogenesis for NANOG binding to these 4 ATTA sites. Surprisingly, NANOG depletion not only decreased FLK1 expression but also reduced cell proliferation and angiogenesis. These findings show the necessary and sufficient role of NANOG in inducing the transcription of FLK1 to regulate the angiogenic phenotypes of ECs.

Focal adhesion kinase regulation of neovascularization.

In this review, we discuss the role of focal adhesion kinase (FAK), an intracellular tyrosine kinase, in endothelial cells in relation to neovascularization. Genetic and in vitro studies have identified critical factors, receptor systems, and their intracellular signaling components that regulate the neovasculogenic phenotypes of endothelial cells. Among these factors, FAK appears to regulate several aspects of endothelial cellular behavior, including migration, survival, cytoskeletal organization, as well as cell proliferation. Upon adhesion of endothelial cells to extracellular matrix (ECM) ligands, integrins cluster on the plane of plasma-membrane, while cytoplasmic domains of integrins interact with cytoskeletal proteins and signaling molecules including FAK. However, FAK not only serves as a critical component of integrin signaling, but is also a downstream element of the VEGF/VEGF-receptor and other ligand-receptor systems that regulate neovascularization. A complete understanding of FAK-mediated neovascularization, therefore, should address the molecular and cellular mechanisms that regulate the biology of FAK. Continued research on FAK may, therefore, yield novel therapies to improve treatment modalities for the pathological neovascularization associated with diseases.

Kruppel-like factor-4 transcriptionally regulates VE-cadherin expression and endothelial barrier function.

RATIONALE: Vascular endothelial (VE)-cadherin localized at adherens junctions (AJs) regulates endothelial barrier function. Because WNT (wingless) signaling-induced activation of the transcription factor Krüppel-like factor (KLF)4 may have an important role in mediating the expression of VE-cadherin and AJ integrity, we studied the function of KLF4 in regulating VE-cadherin expression and the control of endothelial barrier function. OBJECTIVE: The goal of this study was to determine the transcriptional role of KLF4 in regulating VE-cadherin expression and endothelial barrier function. METHODS AND RESULTS: Expression analysis, microscopy, chromatin immunoprecipitation, electrophoretic mobility shift assays, and VE-cadherin-luciferase reporter experiments demonstrated that KLF4 interacted with specific domains of VE-cadherin promoter and regulated the expression of VE-cadherin at AJs. KLF4 knockdown disrupted the endothelial barrier, indicating that KLF4 is required for normal barrier function. In vivo studies in mice showed augmented lipopolysaccharide-induced lung injury and pulmonary edema following Klf4 depletion. CONCLUSION: Our data show the key role of KLF4 in the regulation of VE-cadherin expression at the level of the AJs and in the acquisition of VE-cadherin-mediated endothelial barrier function. Thus, KLF4 maintains the integrity of AJs and prevents vascular leakage in response to inflammatory stimuli.

A requirement for Krüppel Like Factor-4 in the maintenance of endothelial cell quiescence.

Rationale and Goal: Endothelial cells (ECs) are quiescent and critical for maintaining homeostatic functions of the mature vascular system, while disruption of quiescence is at the heart of endothelial to mesenchymal transition (EndMT) and tumor angiogenesis. Here, we addressed the hypothesis that KLF4 maintains the EC quiescence. Methods and Results: In ECs, KLF4 bound to KLF2, and the KLF4-transctivation domain (TAD) interacted directly with KLF2. KLF4-depletion increased KLF2 expression, accompanied by phosphorylation of SMAD3, increased expression of alpha-smooth muscle actin (αSMA), VCAM-1, TGF-ß1, and ACE2, but decreased VE-cadherin expression. In the absence of Klf4, Klf2 bound to the Klf2-promoter/enhancer region and autoregulated its own expression. Loss of EC-Klf4 in Rosa mT/mG ::Klf4 fl/fl ::Cdh5 CreERT2 engineered mice, increased Klf2 levels and these cells underwent EndMT. Importantly, these mice harboring EndMT was also accompanied by lung inflammation, disruption of lung alveolar architecture, and pulmonary fibrosis. Conclusion: In quiescent ECs, KLF2 and KLF4 partnered to regulate a combinatorial mechanism. The loss of KLF4 disrupted this combinatorial mechanism, thereby upregulating KLF2 as an adaptive response. However, increased KLF2 expression overdrives for the loss of KLF4, giving rise to an EndMT phenotype.

Lipid phosphate phosphatase-3 regulates tumor growth via ß-catenin and CYCLIN-D1 signaling.

BACKGROUND: The acquisition of proliferative and invasive phenotypes is considered a hallmark of neoplastic transformation; however, the underlying mechanisms are less well known. Lipid phosphate phosphatase-3 (LPP3) not only catalyzes the dephosphorylation of the bioactive lipid sphingosine-1-phosphate (S1P) to generate sphingosine but also may regulate embryonic development and angiogenesis via the Wnt pathway. The goal of this study was to determine the role of LPP3 in tumor cells. RESULTS: We observed increased expression of LPP3 in glioblastoma primary tumors and in U87 and U118 glioblastoma cell lines. We demonstrate that LPP3-knockdown inhibited both U87 and U118 glioblastoma cell proliferation in culture and tumor growth in xenograft assays. Biochemical experiments provided evidence that LPP3-knockdown reduced ß-catenin, CYCLIN-D1, and CD133 expression, with a concomitant increase in phosphorylated ß-catenin. In a converse experiment, the forced expression of LPP3 in human colon tumor (SW480) cells potentiated tumor growth via increased ß-catenin stability and CYCLIN-D1 synthesis. In contrast, elevated expression of LPP3 had no tumorigenic effects on primary cells. CONCLUSIONS: These results demonstrate for the first time an unexpected role of LPP3 in regulating glioblastoma progression by amplifying ß-catenin and CYCLIN-D1 activities.

Bone marrow progenitor cells induce endothelial adherens junction integrity by sphingosine-1-phosphate-mediated Rac1 and Cdc42 signaling.

RATIONALE: Little is known about the contribution of bone marrow-derived progenitor cells (BMPCs) in the regulation endothelial barrier function as defined by microvascular permeability alterations at the level of adherens junctions (AJs). OBJECTIVE: We investigated the role of BMPCs in annealing AJs and thereby in preventing lung edema formation induced by endotoxin (LPS). METHODS AND RESULTS: We observed that BMPCs enhanced basal endothelial barrier function and prevented the increase in pulmonary microvascular permeability and edema formation in mice after LPS challenge. Coculture of BMPCs with endothelial cells induced Rac1 and Cdc42 activation and AJ assembly in endothelial cells. However, transplantation of BMPCs isolated from sphingosine kinase-1-null mice (SPHK1(-/-)), having impaired S1P production, failed to activate Rac1 and Cdc42 or protect the endothelial barrier. CONCLUSIONS: These results demonstrate that BMPCs have the ability to reanneal endothelial AJs by paracrine S1P release in the inflammatory milieu and the consequent activation of Rac-1 and Cdc42 in endothelial cells.

TIRAP in the Mechanism of Inflammation.

The Toll-interleukin-1 Receptor (TIR) domain-containing adaptor protein (TIRAP) represents a key intracellular signalling molecule regulating diverse immune responses. Its capacity to function as an adaptor molecule has been widely investigated in relation to Toll-like Receptor (TLR)-mediated innate immune signalling. Since the discovery of TIRAP in 2001, initial studies were mainly focused on its role as an adaptor protein that couples Myeloid differentiation factor 88 (MyD88) with TLRs, to activate MyD88-dependent TLRs signalling. Subsequent studies delineated TIRAP's role as a transducer of signalling events through its interaction with non-TLR signalling mediators. Indeed, the ability of TIRAP to interact with an array of intracellular signalling mediators suggests its central role in various immune responses. Therefore, continued studies that elucidate the molecular basis of various TIRAP-protein interactions and how they affect the signalling magnitude, should provide key information on the inflammatory disease mechanisms. This review summarizes the TIRAP recruitment to activated receptors and discusses the mechanism of interactions in relation to the signalling that precede acute and chronic inflammatory diseases. Furthermore, we highlighted the significance of TIRAP-TIR domain containing binding sites for several intracellular inflammatory signalling molecules. Collectively, we discuss the importance of the TIR domain in TIRAP as a key interface involved in protein interactions which could hence serve as a therapeutic target to dampen the extent of acute and chronic inflammatory conditions.

Requirement of alpha(4)beta(1) and alpha(5)beta(1) integrin expression in bone-marrow-derived progenitor cells in preventing endotoxin-induced lung vascular injury and edema in mice.

The goal of this study was to determine the role of integrin-mediated adhesion of bone-marrow-derived progenitor cells (BMPCs) as a requirement for the endothelial barrier protection in a lung injury model. C57BL mice were used as the source for BMPCs, which were characterized as CD34(+) and fetal liver kinase-1 (Flk1)(+) and also an expression of a repertoire of integrins. We used a mouse model of bacterial lipopolysaccharide (LPS)-induced lung vascular injury and edema formation to test the effects of BMPC integrin expression in preventing endothelial barrier injury. Adhesion of BMPCs to purified extracellular matrix proteins induced focal adhesion kinase (Fak) phosphorylation and formation of branching point structures in a alpha(4) and alpha(5) integrin-dependent manner. BMPCs expressing red fluorescent protein (RFP) were administered via the retro-orbital venous route in mice treated intraperitonially with LPS (7.5 mg/kg body weight). We observed increased retention of RFP-labeled Flk1(+) and CD34(+) BMPCs for up to 8 weeks in mice injured with LPS. BMPC transplantation increased survival by 50% (at 72-96 hours after LPS) and reduced lung vascular injury and extravascular water content induced by LPS. However, blocking with anti-alpha(4) or anti-alpha(5) integrin antibody or shRNA-mediated silencing of alpha(4) or alpha(5) integrins in donor BMPCs failed to prevent the vascular injury or edema formation and mortality. Thus, alpha(4) and alpha(5) integrin-dependent adhesion of BMPCs in lung tissue plays a critical role in preventing lung vascular injury and increasing survival in a mouse model of LPS-induced acute lung injury.

Exosomes in the Regulation of Vascular Endothelial Cell Regeneration.

Exosomes have been described as nanoscale membranous extracellular vesicles that emerge from a variety of cells and tissues and are enriched with biologically active genomic and non-genomic biomolecules capable of transducing cell to cell communication. Exosome release, and exosome mediated signaling and cross-talks have been reported in several pathophysiological states. Therefore, exosomes have the potential to become suitable for the diagnosis, prognosis and treatment of specific diseases, including endothelial cell (EC) dysfunction and regeneration. The role of EC-derived exosomes in the mechanisms of cardiovascular tissue regenerative processes represents currently an area of intense research activity. Recent studies have described the potential of exosomes to influence the pathophysiology of immune signaling, tumor metastasis, and angiogenesis. In this review, we briefly discuss progress made in our understanding of the composition and the roles of exosomes in relation to EC regeneration as well as revascularization of ischemic tissues.

Sphingosine-1-Phosphate Receptor 1 Activity Promotes Tumor Growth by Amplifying VEGF-VEGFR2 Angiogenic Signaling.

The vascular endothelial growth factor-A (VEGF-A)-VEGFR2 pathway drives tumor vascularization by activating proangiogenic signaling in endothelial cells (ECs). Here, we show that EC-sphingosine-1-phosphate receptor 1 (S1PR1) amplifies VEGFR2-mediated angiogenic signaling to enhance tumor growth. We show that cancer cells induce S1PR1 activity in ECs, and thereby, conditional deletion of S1PR1 in ECs (EC-S1pr1-/- mice) impairs tumor vascularization and growth. Mechanistically, we show that S1PR1 engages the heterotrimeric G-protein Gi, which amplifies VEGF-VEGFR2 signaling due to an increase in the activity of the tyrosine kinase c-Abl1. c-Abl1, by phosphorylating VEGFR2 at tyrosine-951, prolongs VEGFR2 retention on the plasmalemma to sustain Rac1 activity and EC migration. Thus, S1PR1 or VEGFR2 antagonists, alone or in combination, reverse the tumor growth in control mice to the level seen in EC-S1pr1-/- mice. Our findings suggest that blocking S1PR1 activity in ECs has the potential to suppress tumor growth by preventing amplification of VEGF-VEGFR2 signaling.