[Show abstract][Hide abstract] ABSTRACT: Posttranscriptional RNA regulation is important in determining the plasticity of cellular phenotypes. However, mechanisms of how RNA binding proteins (RBPs) influence cellular behavior are poorly understood. We show here that the RBP embryonic lethal abnormal vision like 1 (ELAVL1, also know as HuR) regulates the alternative splicing of eukaryotic translation initiation factor 4E nuclear import factor 1 (Eif4enif1), which encodes an eukaryotic translation initiation factor 4E transporter (4E-T) protein and suppresses the expression of capped mRNAs. In the absence of ELAVL1, skipping of exon 11 of Eif4enif1 forms the stable, short isoform, 4E-Ts. This alternative splicing event results in the formation of RNA processing bodies (PBs), enhanced turnover of angiogenic mRNAs, and suppressed sprouting behavior of vascular endothelial cells. Further, endothelial-specific Elavl1 knockout mice exhibited reduced revascularization after hind limb ischemia and tumor angiogenesis in oncogene-induced mammary cancer, resulting in attenuated blood flow and tumor growth, respectively. ELAVL1-regulated alternative splicing of Eif4enif1 leading to enhanced formation of PB and mRNA turnover constitutes a novel posttranscriptional mechanism critical for pathological angiogenesis.
Proceedings of the National Academy of Sciences of the United States of America. 11/2014;
[Show abstract][Hide abstract] ABSTRACT: Atherosclerotic plaque localization correlates with regions of disturbed flow in which endothelial cells (ECs) align poorly, whereas sustained laminar flow correlates with cell alignment in the direction of flow and resistance to atherosclerosis. We now report that in hypercholesterolemic mice, deletion of syndecan 4 (S4(-/-)) drastically increased atherosclerotic plaque burden with the appearance of plaque in normally resistant locations. Strikingly, ECs from the thoracic aortas of S4(-/-) mice were poorly aligned in the direction of the flow. Depletion of S4 in human umbilical vein endothelial cells (HUVECs) using shRNA also inhibited flow-induced alignment in vitro, which was rescued by re-expression of S4. This effect was highly specific, as flow activation of VEGF receptor 2 and NF-κB was normal. S4-depleted ECs aligned in cyclic stretch and even elongated under flow, although nondirectionally. EC alignment was previously found to have a causal role in modulating activation of inflammatory versus antiinflammatory pathways by flow. Consistent with these results, S4-depleted HUVECs in long-term laminar flow showed increased activation of proinflammatory NF-κB and decreased induction of antiinflammatory kruppel-like factor (KLF) 2 and KLF4. Thus, S4 plays a critical role in sensing flow direction to promote cell alignment and inhibit atherosclerosis.
Proceedings of the National Academy of Sciences of the United States of America. 11/2014;
[Show abstract][Hide abstract] ABSTRACT: Vascular remodeling is essential for tissue repair and is regulated by multiple factors including thrombospondin-2 (TSP2) and hypoxia/VEGF-induced activation of Akt. In contrast to TSP2 knockout (KO) mice, Akt1 KO mice have elevated TSP2 expression and delayed tissue repair. To investigate the contribution of increased TSP2 to Akt1 KO mice phenotypes, we generated Akt1/TSP2 double KO (DKO) mice. Full thickness excisional wounds in DKO mice healed at an accelerated rate when compared to Akt1 KO mice. Isolated dermal Akt1 KO fibroblasts expressed increased TSP2 and displayed altered morphology and defects in migration and adhesion. These defects were rescued in DKO fibroblasts or after TSP2 knockdown. Conversely, addition of exogenous TSP2 to WT
cells induced cell morphology and migration rates that were similar to Akt1 KO cells. Akt1 KO fibroblasts displayed reduced adhesion to fibronectin with manganese stimulation when compared to WT and DKO cells, revealing an Akt1-dependent role for TSP2 in regulating integrin-mediated adhesions, however, this effect was not due to changes in β1 integrin surface expression or activation. Consistent with these results, Akt1 KO fibroblasts displayed reduced Rac1 activation that was dependent upon expression of TSP2 and could be rescued by a constitutively active Rac mutant. Our observations show that repression of TSP2 expression is a critical aspect of Akt1 function in tissue repair.
Journal of Biological Chemistry 11/2014; · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The PI3K/Akt pathway is necessary for several key endothelial cell (EC) functions, including cell growth, migration, survival, and vascular tone. However, existing literature supports the idea that Akt can be either pro- or antiangiogenic, possibly due to compensation by multiple isoforms in the EC when a single isoform is deleted. Thus, biochemical, genetic, and proteomic studies were conducted to examine isoform-substrate specificity for Akt1 vs. Akt2. In vitro, Akt1 preferentially phosphorylates endothelial nitric oxide synthase (eNOS) and promotes NO release, whereas nonphysiological overexpression of Akt2 can bypass the loss of Akt1. Conditional deletion of Akt1 in the EC, in the absence or presence of Akt2, retards retinal angiogenesis, implying that Akt1 exerts a nonredundant function during physiological angiogenesis. Finally, proteomic analysis of Akt substrates isolated from Akt1- or Akt2-deficient ECs documents that phosphorylation of multiple Akt substrates regulating angiogenic signaling is reduced in Akt1-deficient, but not Akt2-deficient, ECs, including eNOS and Forkhead box proteins. Therefore, Akt1 promotes angiogenesis largely due to phosphorylation and regulation of important downstream effectors that promote aspects of angiogenic signaling
Proceedings of the National Academy of Sciences 09/2014; 111(35):12865-70. · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Myeloid cells are important contributors to arteriogenesis, but their key molecular triggers and cellular effectors are largely unknown. We report, in inflammatory monocytes, that the combination of chemokine receptor (CCR2) and adhesion receptor (β2 integrin) engagement leads to an interaction between activated Rac2 and Myosin 9 (Myh9), the heavy chain of Myosin IIA, resulting in augmented vascular endothelial growth factor A (VEGF-A) expression and induction of arteriogenesis. In human monocytes, CCL2 stimulation coupled to ICAM-1 adhesion led to rapid nuclear-to-cytosolic translocation of the RNA-binding protein HuR. This activation of HuR and its stabilization of VEGF-A mRNA were Rac2-dependent, and proteomic analysis for Rac2 interactors identified the 226 kD protein Myh9. The level of induced Rac2-Myh9 interaction strongly correlated with the degree of HuR translocation. CCL2-coupled ICAM-1 adhesion-driven HuR translocation and consequent VEGF-A mRNA stabilization were absent in Myh9(-/-) macrophages. Macrophage VEGF-A production, ischemic tissue VEGF-A levels, and flow recovery to hind limb ischemia were impaired in myeloid-specific Myh9(-/-) mice, despite preserved macrophage recruitment to the ischemic muscle. Micro-CT arteriography determined the impairment to be defective induced arteriogenesis, whereas developmental vasculogenesis was unaffected. These results place the macrophage at the center of ischemia-induced arteriogenesis, and they establish a novel role for Myosin IIA in signal transduction events modulating VEGF-A expression in tissue.
[Show abstract][Hide abstract] ABSTRACT: Lymphatic vessels are intimately involved in regulation of water and solute homeostasis by returning interstitial fluid back to the venous circulation and play an equally important role in immune responses by providing avenues for immune cell transport. Defects in the lymphatic vasculature result in a number of pathological conditions, including lymphedema and lymphangiectasia. Knowledge of molecular mechanisms underlying lymphatic development and maintenance is therefore critical for understanding, prevention and treatment of lymphatic circulation-related diseases. Research in the past two decades has uncovered several key transcriptional factors (Prox1, Sox18 and Coup-TFII) controlling lymphatic fate specification. Most recently, ERK signaling has emerged as a critical regulator of this transcriptional program. This review summarizes our current understanding of lymphatic fate determination and its transcriptional controls.
Microvascular Research 08/2014; · 2.93 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: -Regulation of vascular endothelial growth factor receptor-2 (VEGFR2) signaling is a control point that determines the extent of vascular tree formation. Recent studies demonstrated an important role played by VEGFR2 endothelial trafficking in control of its activity and suggested the involvement of a phosphotyrosine phosphatase 1b (PTP1b) in this process. This study was designed to define the role of PTP1b in endothelial VEGFR2 signaling and its role in regulation of angiogenesis and arteriogenesis.
[Show abstract][Hide abstract] ABSTRACT: Autonomic sympathetic nerves innervate peripheral resistance arteries, thereby regulating vascular tone and controlling blood supply to organs. Despite the fundamental importance of blood flow control, how sympathetic arterial innervation develops remains largely unknown. Here, we identified the axon guidance cue netrin-1 as an essential factor required for development of arterial innervation in mice. Netrin-1 was produced by arterial smooth muscle cells (SMCs) at the onset of innervation, and arterial innervation required the interaction of netrin-1 with its receptor, deleted in colorectal cancer (DCC), on sympathetic growth cones. Function-blocking approaches, including cell type-specific deletion of the genes encoding Ntn1 in SMCs and Dcc in sympathetic neurons, led to severe and selective reduction of sympathetic innervation and to defective vasoconstriction in resistance arteries. These findings indicate that netrin-1 and DCC are critical for the control of arterial innervation and blood flow regulation in peripheral organs.
[Show abstract][Hide abstract] ABSTRACT: Receptor tyrosine kinases are involved in regulation of key process in roles in endothelial biology, including proliferation, migration, and angiogenesis. It is now generally accepted that receptor tyrosine kinase signaling occurs intracellularly and on the plasma membrane, although many important details remain to be worked out. Endocytosis and subsequent intracellular trafficking spatiotemporally regulate receptor tyrosine kinase signaling, whereas signaling endosomes provide a platform for the compartmentalization of signaling events. This review summarizes recent advances in our understanding of endothelial receptor tyrosine kinase endocytosis and signaling using vascular endothelial growth factor receptor-2 as a paradigm.
Arteriosclerosis Thrombosis and Vascular Biology 06/2014; · 6.34 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Vascular endothelial growth factors (VEGFs) signal via their cognate receptor tyrosine kinases designated VEGFR1-3. We report that the docking protein fibroblast growth factor receptor substrate 2 (FRS2α) plays a critical role in cell signaling via these receptors. In vitro FRS2α regulates VEGF-A and VEGF-C-dependent activation of extracellular signal-regulated receptor kinase signaling and blood and lymphatic endothelial cells migration and proliferation. In vivo endothelial-specific deletion of FRS2α results in the profound impairment of postnatal vascular development and adult angiogenesis, lymphangiogenesis, and arteriogenesis. We conclude that FRS2α is a previously unidentified component of VEGF receptors signaling.
Proceedings of the National Academy of Sciences 04/2014; · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Angiogenic growth factors have recently been linked to tissue metabolism. We have used genetic gain- and loss-of function models to elucidate the effects and mechanisms of action of vascular endothelial growth factor-B (VEGF-B) in the heart. A cardiomyocyte-specific VEGF-B transgene induced an expanded coronary arterial tree and reprogramming of cardiomyocyte metabolism. This was associated with protection against myocardial infarction and preservation of mitochondrial complex I function upon ischemia-reperfusion. VEGF-B increased VEGF signals via VEGF receptor-2 to activate Erk1/2, which resulted in vascular growth. Akt and mTORC1 pathways were upregulated and AMPK downregulated, readjusting cardiomyocyte metabolic pathways to favor glucose oxidation and macromolecular biosynthesis. However, contrasting with a previous theory, there was no difference in fatty acid uptake by the heart between the VEGF-B transgenic, gene-targeted or wildtype rats. Importantly, we also show that VEGF-B expression is reduced in human heart disease. Our data indicate that VEGF-B could be used to increase the coronary vasculature and to reprogram myocardial metabolism to improve cardiac function in ischemic heart disease.
EMBO Molecular Medicine 01/2014; · 7.80 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cell-cell communications typically involve receptor-mediated signaling initiated by soluble or cell-bound ligands. Here, we report a unique mode of endocytosis: proteins originating from cell-cell junctions and cytosolic cellular components from the neighboring cell are internalized, leading to direct exchange of cellular components between two adjacent endothelial cells. VE-cadherins form transcellular bridges between two endothelial cells that are the basis of adherence junctions. At such adherens junction sites, we observed the movement of the entire VE-cadherin molecule from one endothelial cell into the other with junctional and cytoplasmic components. This phenomenon, here termed trans-endocytosis, requires the establishment of a VE-cadherin homodimer in trans with internalization proceeding in a Rac1-, and actomyosin-dependent manner. Importantly, the trans-endocytosis is not dependent on any known endocytic pathway including clathrin-dependent endocytosis, macropinocytosis or phagocytosis. This novel form of cell-cell communications, leading to a direct exchange of cellular components, was observed in 2D and 3D-cultured endothelial cells as well as in the developing zebrafish vasculature.
PLoS ONE 01/2014; 9(3):e90736. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Abnormal vascular homeostasis can lead to increased proliferation of smooth muscle cells and deposition of extracellular matrix, resulting in neointima formation, which contributes to vascular lumen narrowing, a pathology that underlies diseases including transplant vasculopathy, the recurrence of stenosis, and atherosclerosis. Growth of neointima is in part due to endothelial-to-mesenchymal transition (EndMT), a transforming growth factor-β (TGFβ)-driven process, which leads to increased numbers of smooth muscle cells and fibroblasts and deposition of extracellular matrix. We reported that endothelial cell-specific knockout of fibroblast growth factor receptor 1 (FGFR1) led to activation of TGFβ signaling and development of EndMT in vitro and in vivo. Furthermore, EndMT in human diseased vasculature correlated with decreased abundance of FGFR1. These findings identify FGFR1 as the key regulator of TGFβ signaling and EndMT development.
[Show abstract][Hide abstract] ABSTRACT: The role of blood flow in regulating signaling pathways and gene expression in the blood vasculature is well known. Recent studies have identified equally important roles of flow-mediated signaling in the lymphatic circulation including control of lymphatic vascular growth, remodeling, regeneration and maintenance of the lymphatic fate. In this review, we summarize these advances focusing on the role of fluid dynamics in control of lymphatic vasculature formation.
[Show abstract][Hide abstract] ABSTRACT: Angiopoietin-2 (Ang2) is an extracellular protein and one of the principal ligands of Tie2 receptor that is involved in the regulation of vascular integrity, quiescence and inflammation. The mode of secretion of Ang2 has never been established, however. Here we provide evidence that Ang2 is secreted from endothelial cells via exosomes and that this process is inhibited by the PI3K/Akt/eNOS signaling pathway while it is positively regulated by syndecan-4 (S4)/syntenin pathway. Vascular defects in Akt1 null mice arise, in part, because of excessive Ang2 secretion and can be rescued by the syndecan-4 knockout that reduces extracellular Ang2 level. This novel mechanism connects three critical signaling pathways: angiopoietin/Tie2, PI3K/Akt/eNOS and syndecan/syntenin that play important roles in vascular growth and stabilization.
Journal of Biological Chemistry 11/2013; · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Aberrant blood vessel formation contributes to a wide variety of pathologies, and factors that regulate angiogenesis are attractive therapeutic targets. Endothelial and smooth muscle cell-derived neuropilin-like protein (ESDN) is a neuropilin-related transmembrane protein expressed in ECs; however, its potential effect on VEGF responses remains undefined. Here, we generated global and EC-specific Esdn knockout mice and demonstrated that ESDN promotes VEGF-induced human and murine EC proliferation and migration. Deletion of Esdn in the mouse interfered with adult and developmental angiogenesis, and knockdown of the Esdn homolog (dcbld2) in zebrafish impaired normal vascular development. Loss of ESDN in ECs blunted VEGF responses in vivo and attenuated VEGF-induced VEGFR-2 signaling without altering VEGF receptor or neuropilin expression. Finally, we found that ESDN associates with VEGFR-2 and regulates its complex formation with negative regulators of VEGF signaling, protein tyrosine phosphatases PTP1B and TC-PTP, and VE-cadherin. These findings establish ESDN as a regulator of VEGF responses in ECs that acts through a mechanism distinct from neuropilins. As such, ESDN may serve as a therapeutic target for angiogenesis regulation.
The Journal of clinical investigation 11/2013; · 15.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Arteriovenous malformations occur when abnormalities of vascular patterning result in the flow of blood from arteries to veins without an intervening capillary bed. Recent work has revealed the importance of the Notch and TGF-β signaling pathways in vascular patterning. Specifically, Notch signaling has an increasingly apparent role in arterial specification and suppression of branching, whereas TGF-β is implicated in vascular smooth muscle development and remodeling under angiogenic stimuli. These physiologic roles, consequently, have implicated both pathways in the pathogenesis of arteriovenous malformation. In this review, we summarize the studies of endothelial signaling that contribute to arteriovenous malformation and the roles of genes implicated in their pathogenesis. We further discuss how endothelial signaling may contribute to vascular smooth muscle development and how knowledge of signaling pathways may provide us targets for medical therapy in these vascular lesions.
Cellular and Molecular Life Sciences CMLS 09/2013; · 5.62 Impact Factor