[Show abstract][Hide abstract] ABSTRACT: The contribution of endothelial-derived miR-17∼92 to ischemia-induced arteriogenesis has not been investigated in an in vivo model. In the present study, we demonstrate a critical role for the endothelial-derived miR-17∼92 cluster in shaping physiological and ischemia-triggered arteriogenesis. Endothelial-specific deletion of miR-17∼92 results in an increase in collateral density limbs and hearts and in ischemic limbs compared with control mice, and consequently improves blood flow recovery. Individual cluster components positively or negatively regulate endothelial cell (EC) functions in vitro, and, remarkably, ECs lacking the cluster spontaneously form cords in a manner rescued by miR-17a, -18a, and -19a. Using both in vitro and in vivo analyses, we identified FZD4 and LRP6 as targets of miR-19a/b. Both of these targets were up-regulated in 17∼92 KO ECs compared with control ECs, and both were shown to be targeted by miR-19 using luciferase assays. We demonstrate that miR-19a negatively regulates FZD4, its coreceptor LRP6, and WNT signaling, and that antagonism of miR-19a/b in aged mice improves blood flow recovery after ischemia and reduces repression of these targets. Collectively, these data provide insights into miRNA regulation of arterialization and highlight the importance of vascular WNT signaling in maintaining arterial blood flow.
Proceedings of the National Academy of Sciences 09/2015; DOI:10.1073/pnas.1507094112 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Formation of arterial vasculature, here termed arteriogenesis, is a central process in embryonic vascular development as well as in adult tissues. Although the process of capillary formation, angiogenesis, is relatively well understood, much remains to be learned about arteriogenesis. Recent discoveries point to the key role played by vascular endothelial growth factor receptor 2 in control of this process and to newly identified control circuits that dramatically influence its activity. The latter can present particularly attractive targets for a new class of therapeutic agents capable of activation of this signaling cascade in a ligand-independent manner, thereby promoting arteriogenesis in diseased tissues.
Circulation Research 05/2015; 116(10):1712-1724. DOI:10.1161/CIRCRESAHA.116.302953 · 11.02 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Blood vascular networks in vertebrates are essential to tissue survival. Establishment of a fully functional vasculature is complex and requires a number of steps including vasculogenesis and angiogenesis that are followed by differentiation into specialized vascular tissues (i.e., arteries, veins, and lymphatics) and organ-specific differentiation. However, an equally essential step in this process is the pruning of excessive blood vessels. Recent studies have shown that pruning is critical for the effective perfusion of blood into tissues. Despite its significance, vessel pruning is the least understood process in vascular differentiation and development. Two recently published PLOS Biology papers provide important new information about cellular dynamics of vascular regression.
[Show abstract][Hide abstract] ABSTRACT: All F1000Prime Reports articles are distributed under the terms of the Creative Commons Attribution-Non Commercial License (http://creativecommons.org/licenses/by-nc/3.0/legalcode), which permits non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The electronic version of this article is the complete one and can be found at: Abstract In development and disease, vascular endothelial growth factor (VEGF) regulates the expansion of the vascular tree. In response to hypoxia, VEGF promotes new capillary formation through the process of angiogenesis by inducing endothelial cell sprouting, proliferation, and migration. Wound healing, tissue regeneration, and tumor growth depend on angiogenesis for adequate nutrient and oxygen delivery. Under different conditions, VEGF promotes arterial growth, modulates lumen expansion, and induces collateral vessel formation, events collectively referred to as arteriogenesis. Induction of arteriogenesis after cardiac or cerebral arterial occlusion can reduce ischemia and improve disease outcome. Endothelial VEGF receptor 2 (VEGFR2) signaling governs both processes. However, modulation of downstream VEGF signaling effectors, such as extracellular-signal-regulated kinase (ERK) activation, differs in order to achieve angiogenic versus arteriogenic outcomes. Recent reports show that neuropilin 1 (NRP1), a VEGF receptor, can instill VEGF signaling outcomes that specifically regulate either angiogenesis or arteriogenesis. Here, we discuss how NRP1 functions as a VEGFR2 co-receptor in angiogenesis and a modulator of VEGFR2 trafficking in arteriogenesis. The unique role played by neuropilin in different endothelial processes makes it an exciting therapeutic target to specifically enhance angiogenesis or arteriogenesis in disease settings.
F1000 Prime Reports 03/2015; 7. DOI:10.12703/P7-26)
[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 11/2014; 111(51). DOI:10.1073/pnas.1412172111 · 9.67 Impact Factor
[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 11/2014; 111(48). DOI:10.1073/pnas.1413725111 · 9.67 Impact Factor
[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; DOI:10.1074/jbc.M114.618421 · 4.57 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 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. DOI:10.1073/pnas.1408472111 · 9.67 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.
The Journal of Cell Biology 09/2014; 211(10). DOI:10.1084/jem.20132130 · 9.83 Impact Factor
[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; 96. DOI:10.1016/j.mvr.2014.07.016 · 2.13 Impact Factor