Jason E. Fish

miR-126 Regulates Angiogenic Signaling and Vascular Integrity

Precise regulation of the formation, maintenance, and remodeling of the vasculature is required for normal development, tissue response to injury, and tumor progression. How specific microRNAs intersect with and modulate angiogenic signaling cascades is unknown. Here, we identified microRNAs that were enriched in endothelial cells derived from mouse embryonic stem (ES) cells and in developing mouse embryos. We found that miR-126 regulated the response of endothelial cells to VEGF. Additionally, knockdown of miR-126 in zebrafish resulted in loss of vascular integrity and hemorrhage during embryonic development. miR-126 functioned in part by directly repressing negative regulators of the VEGF pathway, including the Sprouty-related protein SPRED1 and phosphoinositol-3 kinase regulatory subunit 2 (PIK3R2/p85-beta). Increased expression of Spred1 or inhibition of VEGF signaling in zebrafish resulted in defects similar to miR-126 knockdown. These findings illustrate that a single miRNA can regulate vascular integrity and angiogenesis, providing a new target for modulating vascular formation and function.

MicroRNA Regulation of Cell Lineages in Mouse and Human Embryonic Stem Cells

Cell fate decisions of pluripotent embryonic stem (ES) cells are dictated by activation and repression of lineage-specific genes. Numerous signaling and transcriptional networks progressively narrow and specify the potential of ES cells. Whether specific microRNAs help refine and limit gene expression and, thereby, could be used to manipulate ES cell differentiation has largely been unexplored. Here, we show that two serum response factor (SRF)-dependent muscle-specific microRNAs, miR-1 and miR-133, promote mesoderm formation from ES cells but have opposing functions during further differentiation into cardiac muscle progenitors. Furthermore, miR-1 and miR-133 were potent repressors of nonmuscle gene expression and cell fate during mouse and human ES cell differentiation. miR-1s effects were in part mediated by translational repression of the Notch ligand Delta-like 1 (Dll-1). Our findings indicate that muscle-specific miRNAs reinforce the silencing of nonmuscle genes during cell lineage commitment and suggest that miRNAs may have general utility in regulating cell-fate decisions from pluripotent ES cells.

VHL Promotes E2 Box-Dependent E-Cadherin Transcription by HIF-Mediated Regulation of SIP1 and Snail

ABSTRACT The product of the von Hippel-Lindau gene (VHL) acts as the substrate-recognition component of an E3 ubiquitin ligase complex that ubiquitylates the catalytic α subunit of hypoxia-inducible factor (HIF) for oxygen-dependent destruction. Although emerging evidence supports the notion that deregulated accumulation of HIF upon the loss of VHL is crucial for the development of clear-cell renal cell carcinoma (CC-RCC), the molecular events downstream of HIF governing renal oncogenesis remain unclear. Here, we show that the expression of a homophilic adhesion molecule, E-cadherin, a major constituent of epithelial cell junctions whose loss is associated with the progression of epithelial cancers, is significantly down-regulated in primary CC-RCC and CC-RCC cell lines devoid of VHL. Reintroduction of wild-type VHL in CC-RCC (VHL−/−) cells markedly reduced the expression of E2 box-dependent E-cadherin-specific transcriptional repressors Snail and SIP1 and concomitantly restored E-cadherin expression. RNA interference-mediated knockdown of HIFα in CC-RCC (VHL−/−) cells likewise increased E-cadherin expression, while functional hypoxia or expression of VHL mutants incapable of promoting HIFα degradation attenuated E-cadherin expression, correlating with the disengagement of RNA polymerase II from the endogenous E-cadherin promoter/gene. These findings reveal a critical HIF-dependent molecular pathway connecting VHL, an established “gatekeeper” of the renal epithelium, with a major epithelial tumor suppressor, E-cadherin.

Stromal Cell–Derived Factor-1α Is Cardioprotective After Myocardial Infarction

Background— Heart disease is a leading cause of mortality throughout the world. Tissue damage from vascular occlusive events results in the replacement of contractile myocardium by nonfunctional scar tissue. The potential of new technologies to regenerate damaged myocardium is significant, although cell-based therapies must overcome several technical barriers. One possible cell-independent alternative is the direct administration of small proteins to damaged myocardium. Methods and Results— Here we show that the secreted signaling protein stromal cell–derived factor-1α (SDF-1α), which activates the cell-survival factor protein kinase B (PKB/Akt) via the G protein–coupled receptor CXCR4, protected tissue after an acute ischemic event in mice and activated Akt within endothelial cells and myocytes of the heart. Significantly better cardiac function than in control mice was evident as early as 24 hours after infarction as well as at 3, 14, and 28 days after infarction. Prolonged survival of hypoxic myocardium was followed by an increase in levels of vascular endothelial growth factor protein and neoangiogenesis. Consistent with improved cardiac function, mice exposed to SDF-1α demonstrated significantly decreased scar formation than control mice. Conclusion— These findings suggest that SDF-1α may serve a tissue-protective and regenerative role for solid organs suffering a hypoxic insult.

MicroRNA-146 represses endothelial activation by inhibiting pro-inflammatory pathways

Activation of inflammatory pathways in the endothelium contributes to vascular diseases, including sepsis and atherosclerosis. We demonstrate that miR‐146a and miR‐146b are induced in endothelial cells upon exposure to pro‐inflammatory cytokines. Despite the rapid transcriptional induction of the miR‐146a/b loci, which is in part mediated by EGR‐3, miR‐146a/b induction is delayed and sustained compared to the expression of leukocyte adhesion molecules, and in fact coincides with the down‐regulation of inflammatory gene expression. We demonstrate that miR‐146 negatively regulates inflammation. Over‐expression of miR‐146a blunts endothelial activation, while knock‐down of miR‐146a/b in vitro or deletion of miR‐146a in mice has the opposite effect. MiR‐146 represses the pro‐inflammatory NF‐κB pathway as well as the MAP kinase pathway and downstream EGR transcription factors. Finally, we demonstrate that HuR, an RNA binding protein that promotes endothelial activation by suppressing expression of endothelial nitric oxide synthase (eNOS), is a novel miR‐146 target. Thus, we uncover an important negative feedback regulatory loop that controls pro‐inflammatory signalling in endothelial cells that may impact vascular inflammatory diseases.

MicroRNAs: Opening a New Vein in Angiogenesis Research

MicroRNAs regulate angiogenic signaling in endothelial cells. Activation of the angiogenic program in endothelial cells is vital for normal embryonic development and for physiological angiogenesis in the adult. In addition, angiogenesis is an important therapeutic target: Formation of new blood vessels is desirable for regenerative purposes, such as during tissue healing or transplantation, but can be pathological, as in diabetic retinopathy and cancer. The response of the vascular endothelium to angiogenic stimuli is modulated by noncoding RNAs called microRNAs. The endothelial cell–specific microRNA microRNA-126 (miR-126) promotes angiogenesis in response to angiogenic growth factors, such as vascular endothelial growth factor or basic fibroblast growth factor, by repressing negative regulators of signal transduction pathways. Additional microRNAs have been implicated in the regulation of various aspects of angiogenesis. Thus, targeting the expression of microRNAs may be a novel therapeutic approach for diseases involving excess or insufficient vasculature.

A Slit/miR-218/Robo regulatory loop is required during heart tube formation in zebrafish

Members of the Slit family of secreted ligands interact with Roundabout (Robo) receptors to provide guidance cues for many cell types. For example, Slit/Robo signaling elicits repulsion of axons during neural development, whereas in endothelial cells this pathway inhibits or promotes angiogenesis depending on the cellular context. Here, we show that miR-218 is intronically encoded in slit2 and slit3 and that it suppresses Robo1 and Robo2 expression. Our data indicate that miR-218 and multiple Slit/Robo signaling components are required for heart tube formation in zebrafish and that this network modulates the previously unappreciated function of Vegf signaling in this process. These findings suggest a new paradigm for microRNA-based control of ligand-receptor interactions and provide evidence for a novel signaling pathway regulating vertebrate heart tube assembly.

Epigenetic Basis for the Transcriptional Hyporesponsiveness of the Human Inducible Nitric Oxide Synthase Gene in Vascular Endothelial Cells

A marked difference exists in the inducibility of inducible NO synthase (iNOS) between humans and rodents. Although important cis and trans factors in the murine and human iNOS promoters have been characterized using episomal-based approaches, a compelling molecular explanation for why human iNOS is resistant to induction has not been reported. In this study we present evidence that the hyporesponsiveness of the human iNOS promoter is based in part on epigenetic silencing, specifically hypermethylation of CpG dinucleotides and histone H3 lysine 9 methylation. Using bisulfite sequencing, we demonstrated that the iNOS promoter was heavily methylated at CpG dinucleotides in a variety of primary human endothelial cells and vascular smooth muscle cells, all of which are notoriously resistant to iNOS induction. In contrast, in human cell types capable of iNOS induction (e.g., A549 pulmonary adenocarcinoma, DLD-1 colon adenocarcinoma, and primary hepatocytes), the iNOS promoter was relatively hypomethylated. Treatment of human cells, such as DLD-1, with a DNA methyltransferase inhibitor (5-azacytidine) induced global and iNOS promoter DNA hypomethylation. Importantly, 5-azacytidine enhanced the cytokine inducibility of iNOS. Using chromatin immunoprecipitation, we found that the human iNOS promoter was basally enriched with di- and trimethylation of H3 lysine 9 in endothelial cells, and this did not change with cytokine addition. This contrasted with the absence of lysine 9 methylation in inducible cell types. Importantly, chromatin immunoprecipitation demonstrated the selective presence of the methyl-CpG-binding transcriptional repressor MeCP2 at the iNOS promoter in endothelial cells. Collectively, our work defines a role for chromatin-based mechanisms in the control of human iNOS gene expression.

Post-transcriptional Regulation of Endothelial Nitric-oxide Synthase by an Overlapping Antisense mRNA Transcript

Endothelial nitric-oxide synthase (eNOS) mRNA levels are abnormal in diseases of the cardiovascular system, but changes in gene expression cannot be accounted for by transcription alone. We found evidence for the existence of an antisense mRNA (sONE) that is derived from a transcription unit (NOS3AS) on the opposite DNA strand from which the human eNOS (NOS3) mRNA is transcribed at human chromosome 7q36. The genes are oriented in a tail-to-tail configuration, and the mRNAs encoding sONE and eNOS are complementary for 662 nucleotides. The mRNA for sONE could be detected in a variety of cell types, both in vivo and in vitro, but not vascular endothelial cells. In contrast, expression of eNOS is highly restricted to vascular endothelium. Most surprisingly, interrogation of transcriptional events across NOS3/NOS3AS genomic regions, using single- and double-stranded probes for nuclear run-off analyses and chromatin immunoprecipitation-based assessments of RNA polymerase II distribution, indicated that NOS3 and NOS3AS gene transcription did not correlate with steady-state mRNA levels. We found strong evidence supporting a role for NOS3AS in the post-transcriptional regulation of NOS3 expression. RNA interference-mediated inhibition of sONE expression in vascular smooth muscle cells increased eNOS expression. Overexpression of sONE in endothelial cells blunted eNOS expression. Finally, the histone deacetylase inhibitor trichostatin A is known to regulate the expression of eNOS via a post-transcriptional mechanism. We found that trichostatin A treatment of vascular endothelial cells increased expression of sONE mRNA levels prior to the observed decrease in eNOS mRNA expression. Taken together, these results indicate that an antisense mRNA (sONE) participates in the post-transcriptional regulation of eNOS and provide a newer model for endothelial cell-specific gene expression.

Hypoxic Repression of Endothelial Nitric-oxide Synthase Transcription Is Coupled with Eviction of Promoter Histones

Hypoxia elicits endothelial dysfunction, in part, through reduced expression of endothelial nitric-oxide synthase (eNOS). Here we present evidence that hypoxia causes a rapid decrease in the transcription of the eNOS/NOS3 gene, accompanied by decreased acetylation and lysine 4 (histone H3) methylation of eNOS proximal promoter histones. Surprisingly, we demonstrate that histones are rapidly evicted from the eNOS proximal promoter during hypoxia. We also demonstrate endothelium-specific H2A.Z incorporation at the eNOS promoter and find that H2A.Z is also evicted by hypoxic stimulation. After longer durations of hypoxia, histones are reincorporated at the eNOS promoter, but these histones lack substantial histone acetylation. Additionally, we identify a key role for the chromatin remodeler, BRG1, in re-establishing eNOS expression following reoxygenation of hypoxic cells. We posit that post-translational histone modifications are required to maintain constitutive eNOS transcriptional activity and that histone eviction rapidly resets histone marks and is a proximal event in the hypoxic repression of eNOS. Although nucleosome eviction has been reported in models of transcriptional activation, the observation that eviction can also accompany transcriptional repression in hypoxic mammalian cells argues that eviction may be broadly relevant to both positive and negative changes in transcription.