Dauren Biyashev

miR-27b controls venous specification and tip cell fate

We discovered that miR-27b controls 2 critical vascular functions: it turns the angiogenic switch on by promoting endothelial tip cell fate and sprouting and it promotes venous differentiation. We have identified its targets, a Notch ligand Delta-like ligand 4 (Dll4) and Sprouty homologue 2 (Spry2). miR-27b knockdown in zebrafish and mouse tissues severely impaired vessel sprouting and filopodia formation. Moreover, miR-27b was necessary for the formation of the first embryonic vein in fish and controlled the expression of arterial and venous markers in human endothelium, including Ephrin B2 (EphB2), EphB4, FMS-related tyrosine kinase 1 (Flt1), and Flt4. In zebrafish, Dll4 inhibition caused increased sprouting and longer intersegmental vessels and exacerbated tip cell migration. Blocking Spry2 caused premature vessel branching. In contrast, Spry2 overexpression eliminated the tip cell branching in the intersegmental vessels. Blockade of Dll4 and Spry2 disrupted arterial specification and augmented the expression of venous markers. Blocking either Spry2 or Dll4 rescued the miR-27b knockdown phenotype in zebrafish and in mouse vascular explants, pointing to essential roles of these targets downstream of miR-27b. Our study identifies critical role of miR-27b in the control of endothelial tip cell fate, branching, and venous specification and determines Spry2 and Dll4 as its essential targets.

NF-κB balances vascular regression and angiogenesis via chromatin remodeling and NFAT displacement

Extracellular factors control the angiogenic switch in endothelial cells (ECs) via competing survival and apoptotic pathways. Previously, we showed that proangiogenic and antiangiogenic factors target the same signaling molecules, which thereby become pivots of angiogenic balance. Here we show that in remodeling endothelium (ECs and EC precursors) natural angiogenic inhibitors enhance nuclear factor-kappaB (NF-kappaB) DNA binding, which is critical for antiangiogenesis, and that blocking the NF-kappaB pathway abolishes multiple antiangiogenic events in vitro and in vivo. NF-kappaB induction by antiangiogenic molecules has a dual effect on transcription. NF-kappaB acts as an activator of proapoptotic FasL and as a repressor of prosurvival cFLIP. On the FasL promoter, NF-kappaB increases the recruitment of HAT p300 and acetylated histones H3 and H4. Conversely, on cFLIP promoter, NF-kappaB increases histone deacetylase 1 (HDAC1), decreases p300 and histone acetylation, and reduces the recruitment of NFAT, a transcription factor critical for cFLIP expression. Finally, we found a biphasic effect, when HDAC inhibitors (HDACi) were used to test the dependence of pigment epithelial-derived factor activity on histone acetylation. The cooperative effect seen at low doses switches to antagonistic as the concentrations increase. Our study defines an interactive transcriptional network underlying angiogenic balance and points to HDACi as tools to manipulate the angiogenic switch.

Therapeutic manipulation of angiogenesis with miR-27b

BackgroundMultiple studies demonstrated pro-angiogenic effects of microRNA (miR)-27b. Its targets include Notch ligand Dll4, Sprouty (Spry)-2, PPARγ and Semaphorin (SEMA) 6A. miR-27 effects in the heart are context-dependent: although it is necessary for ventricular maturation, targeted overexpression in cardiomyocytes causes hypertrophy and dysfunction during development. Despite significant recent advances, therapeutic potential of miR-27b in cardiovascular disease and its effects in adult heart remain unexplored. Here, we assessed the therapeutic potential of miR-27b mimics and inhibitors in rodent models of ischemic disease and cancer.MethodsWe have used a number of models to demonstrate the effects of miR-27b mimicry and inhibition in vivo, including subcutaneous Matrigel plug assay, mouse models of hind limb ischemia and myocardial infarction and subcutaneous Lewis Lung carcinoma.ResultsUsing mouse model of myocardial infarction due to the coronary artery ligation, we showed that miR-27b mimic had overall beneficial effects, including increased vascularization, decreased fibrosis and increased ejection fraction. In mouse model of critical limb ischemia, miR-27b mimic also improved tissue re-vascularization and perfusion. In both models, miR-27b mimic clearly decreased macrophage recruitment to the site of hypoxic injury. In contrast, miR-27b increased the recruitment of bone marrow derived cells to the neovasculature, as was shown using mice reconstituted with fluorescence-tagged bone marrow. These effects were due, at least in part, to the decreased expression of Dll4, PPARγ and IL10. In contrast, blocking miR-27b significantly decreased vascularization and reduced growth of subcutaneous tumors and decreased BMDCs recruitment to the tumor vasculature.ConclusionsOur study demonstrates the utility of manipulating miR-27b levels in the treatment of cardiovascular disease and cancer.

Natural Angiogenesis Inhibitor Signals through Erk5 Activation of Peroxisome Proliferator-activated Receptor γ (PPARγ)

Erk-5, a member of the MAPK superfamily, has a catalytic domain similar to Erk1/2 and a unique C-terminal domain enabling binding with transcription factors. Aberrant vascularization in the Erk5-null mice suggested a link to angiogenesis. Ectopic expression of constitutively active Erk5 blocks endothelial cell morphogenesis and causes HIF1-α destabilization/degradation. However the mechanisms by which endogenous Erk5 regulates angiogenesis remain unknown. We show that Erk5 and its activating kinase MEK5 are the upstream mediators of the anti-angiogenic signal by the natural angiogenesis inhibitor, pigment epithelial-derived factor (PEDF). We demonstrate that Erk5 phosphorylation allows activation of PPARγ transcription factor by displacement of SMRT co-repressor. PPARγ, in turn is critical for NFκB activation, PEDF-dependent apoptosis, and anti-angiogenesis. The dominant negative MEK5 mutant and Erk5 shRNA diminished PEDF-dependent apoptosis, inhibition of the endothelial cell chemotaxis, and angiogenesis. This is the first evidence of Erk5-dependent transduction of signals by endogenous angiogenesis inhibitors.

E2F1 suppresses cardiac neovascularization by down-regulating VEGF and PlGF expression

AIMS The E2F transcription factors are best characterized for their roles in cell-cycle regulation, cell growth, and cell death. Here we investigated the potential role of E2F1 in cardiac neovascularization. METHODS AND RESULTS We induced myocardial infarction (MI) by ligating the left anterior descending artery in wild-type (WT) and E2F1(-/-) mice. E2F1(-/-) mice demonstrated a significantly better cardiac function and smaller infarct sizes than WT mice. At infarct border zone, capillary density and endothelial cell (EC) proliferation were greater, apoptotic ECs were fewer, levels of VEGF and placental growth factor (PlGF) were higher, and p53 level was lower in E2F1(-/-) than in WT mice. Blockade of VEGF receptor 2 (VEGFR2) signalling with the selective inhibitor SU5416 or with the VEGFR2-blocking antibody DC101 abolished the differences between E2F1(-/-) mice and WT mice in cardiac function, infarct size, capillary density, EC proliferation, and EC apoptosis. In vitro, hypoxia-induced VEGF and PlGF up-regulation was significantly greater in E2F1(-/-) than in WT cardiac fibroblasts, and E2F1 overexpression suppressed PlGF up-regulation in both WT and p53(-/-) cells; however, VEGF up-regulation was suppressed only in WT cells. E2F1 interacted with and stabilized p53 under hypoxic conditions, and both E2F1 : p53 binding and the E2F1-induced suppression of VEGF promoter activity were absent in cells that expressed an N-terminally truncated E2F1 mutant. CONCLUSION E2F1 limits cardiac neovascularization and functional recovery after MI by suppressing VEGF and PlGF up-regulation through p53-dependent and -independent mechanisms, respectively.

Contrasting Roles of E2F2 and E2F3 in Cardiac Neovascularization

Insufficient neovascularization, characterized by poor endothelial cell (EC) growth, contributes to the pathogenesis of ischemic heart disease and limits cardiac tissue preservation and regeneration. The E2F family of transcription factors are critical regulators of the genes responsible for cell-cycle progression and growth; however, the specific roles of individual E2Fs in ECs are not well understood. Here we investigated the roles of E2F2 and E2F3 in EC growth, angiogenesis, and their functional impact on myocardial infarction (MI). An endothelial-specific E2F3-deficient mouse strain VE-Cre; E2F3fl/fl was generated, and MI was surgically induced in VE-Cre; E2F3fl/fl and E2F2-null (E2F2 KO) mice and their wild-type (WT) littermates, VE-Cre; E2F3+/+ and E2F2 WT, respectively. The cardiac function, infarct size, and vascular density were significantly better in E2F2 KO mice and significantly worse in VE-Cre; E2F3fl/fl mice than in their WT littermates. The loss of E2F2 expression was associated with an increase in the proliferation of ECs both in vivo and in vitro, while the loss of E2F3 expression led to declines in EC proliferation. Thus, E2F3 promotes while E2F2 suppresses ischemic cardiac repair through corresponding changes in EC proliferation; and differential targeting of specific E2F members may provide a novel strategy for therapeutic angiogenesis of ischemic heart disease.

Abstract 3275: miR-194 counterbalances transcriptional activation of the anti-angiogenic factor thrombospondin-1 by p53

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Thrombospondin-1 (TSP-1) is a key endogenous inhibitor of angiogenesis, negatively regulated by oncogenes (e.g., c-Myc) and transcriptionally activated by the key tumor suppressor p53 (1). Yet in some cancers, such as colon adenocarcinomas, tumor progression accompanied by the loss of p53 does not result in TSP-1 down-regulation (2, 3). This paradox could involve promoter-independent mechanisms, for example microRNA-mediated regulation. In order to test this hypothesis, we conducted a global screen for microRNAs that regulate TSP-1 levels. Gain-of-function experiments and microarray data showed that in addition to the previously identified mir-17-92 cluster members (4, 5), several other miRs, including let-7, mir-199a-3p, mir-218 and miR-194, downregulate TSP-1. mir-194 was of particular interest, since its expression is known to be largely colon-specific (6), maintained by p53 and thus frequently reduced in advanced cancers (7). This reduced miR-194 expression could account for unexpectedly high TSP-1 levels in p53-null tumors. Indeed, mir-194 inhibition with antisense 2’-O-methyl ribonucleotides in p53-sufficient colon cancer cell lines strongly restored TSP-1 levels. Conversely, miR-194 mimics reduced TSP-1 expression at both RNA and protein levels. Furthermore, dual luciferase sensor assay demonstrated that this regulation was mediated by the single mir-194 site in the 3’UTR of the thbs1 gene. We then set up a retroviral transduction-based system to test the involvement of this miR-194 binding site in TSP-1 regulation by p53. Viruses expressing the thrombospondin-1 gene with 3’UTR in either wild type or miR-194-mutated configurations were introduced into HCT116-p53 sufficient cells (miR-194 levels are high) and their p53-null derivatives (miR-194 levels are low). In parental HCT116 cells, the mutation in the mir-194 site sharply increased TSP-1 levels, resulting in reduced angiogenesis in Matrigel plugs and tumor xenografts. However, the effects of this mutation disappeared in the absence of p53, resulting in lower TSP-1 expression levels. These results are consistent with the idea that miR-194 counterbalances transcriptional activation of TSP-1 by p53 and underscore the complex relationship between promoter-dependent and microRNA-mediated effects of p53. Grant Support: R01 [CA122334][1] (ATT) and T32 [CA009140][2] (PS) 1. K. M. Dameron, O. V. Volpert, M. A. Tainsky, N. Bouck, Science 265, 1582 (1994). 2. S. Kaiser et al., Genome Biol 8, R131 (2007). 3. D. A. Notterman, U. Alon, A. J. Sierk, A. J. Levine, Cancer Res 61, 3124 (2001). 4. M. Dews et al., Nat Genet 38, 1060 (2006). 5. M. Dews et al., Cancer Res 70, 8233 (2010). 6. K. Hino et al., RNA. 14, 1433 (2008). 7. C. J. Braun et al., Cancer Res 68, 10094 (2008). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3275. doi:10.1158/1538-7445.AM2011-3275 [1]: /lookup/external-ref?link_type=GEN&access_num=CA122334&atom=%2Fcanres%2F71%2F8_Supplement%2F3275.atom [2]: /lookup/external-ref?link_type=GEN&access_num=CA009140&atom=%2Fcanres%2F71%2F8_Supplement%2F3275.atom