New Insights Into miR-17–92 Cluster Regulation and Angiogenesis

HomeCirculation ResearchVol. 118, No. 1New Insights Into miR-17–92 Cluster Regulation and Angiogenesis Free AccessEditorialPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessEditorialPDF/EPUBNew Insights Into miR-17–92 Cluster Regulation and Angiogenesis Jan Fiedler and Thomas Thum Jan FiedlerJan Fiedler From the Institute of Molecular and Translational Therapeutic Strategies (IMTTS), Integrated Research and Treatment Center Transplantation (J.F., T.T.), and Excellence Cluster REBIRTH (J.F., T.T.), Hannover Medical School, Hannover, Germany; and National Heart and Lung Institute, Imperial College London, London, UK (T.T.). Search for more papers by this author and Thomas ThumThomas Thum From the Institute of Molecular and Translational Therapeutic Strategies (IMTTS), Integrated Research and Treatment Center Transplantation (J.F., T.T.), and Excellence Cluster REBIRTH (J.F., T.T.), Hannover Medical School, Hannover, Germany; and National Heart and Lung Institute, Imperial College London, London, UK (T.T.). Search for more papers by this author Originally published8 Jan 2016https://doi.org/10.1161/CIRCRESAHA.115.307935Circulation Research. 2016;118:9–11Several noncoding microRNA (miR)-based regulatory mechanisms for endothelial cell (EC) biology have been identified over the last years. Initial studies of the miR biogenesis enzymes Drosha and Dicer evidenced a crucial need for a balanced miR expression to sustain EC integrity and angiogenic properties.1–3 For instance, the EC-enriched miR-126 was identified as a crucial player in vascular biology based on elegant gene deletion (mouse) and endogenous inhibition (zebrafish) approaches.4,5 Further detailed studies also highlighted the concept of using oligonucleotide-based anti-miR therapies to improve EC function, for example, in settings of ischemic disease.6,7 Because a single miR can target multiple downstream targets at the molecular level, bioinformatics target predicition databases8 offer a useful tool to characterize downstream signaling for selected miRs. Strategies to define regulatory elements for miRs are more difficult. Only a minority of scientific reports have taken effort to investigate either transcriptional activators or repressors for miR genes. For instance, the oncogenic miR-17–92 cluster was shown to be under the control of the transcription factor Myc in tumor angiogenesis,9 thereby offering an interesting entry point for therapeutic purposes in disease setting.Article, see p 38In this issue of Circulation Research, Chamorro-Jorganes et al10 provide novel insight into the vascular endothelial growth factor (VEGF)–dependent transcriptional activation of the angiogenic miR-17–92 cluster. Both in vitro and in vivo state-of-the-art genetic approaches were used to delineate the transcriptional regulation of one of the key angiogenic miR clusters. Pharmacological VEGF supplementation in freshly isolated primary human umbilical vein endothelial cells showed a strong response at the transcription level for the primary miR-17–92 cluster transcript and in parallel all miR-17–92 cluster members (miR-17, miR-17*, miR-18, miR-19a, miR-19b, miR-20, and miR-92a). Such VEGF-dependent transcriptional control was also detectable in mouse primary ECs, potentially indicating evolutionary conserved mechanisms. Detailed promoter analysis was subsequently performed to identify the relevant regulatory units within the miR-17–92 gene at chromosome 13. Using a luciferase-based reporter and sequential nucleotide deletion strategies, the crucial portion of the promotor was deciphered to be at positions −975 and −805 bp comprising cis-acting binding elements. Bioinformatic binding prediction tools identified the transcriptional activator Elk-1 (member of ETS oncogene family) as potentially binding to the miR-17–92 cluster promoter sequence which in turn was positively regulated by growth factor signaling via mitogen-activated protein kinase (MAPK). In line with this, VEGF stimulation of primary ECs triggered activation of MAPK and the phosphorylation of Elk-1. These effects were absent in experiments applying a MAPK-specific inhibitor, suggesting direct targeting of MAPK towards Elk-1 and the miR-17–92 cluster.Further evidence for participation of the VEGF-MAPK-Elk-1 axis was gained in approaches mutating the relevant base positions within the promoter construct for miR-17–92. In mutated reporter constructs, VEGF treatment or Elk-1 overexpression had no stimulatory effects on miR cluster expression. Similar results were obtained in experimental conditions with endogenous silencing of Elk-1, where VEGF stimulation was not potent enough to induce miR-17–92 cluster expression. More importantly, the direct bona fide binding of Elk-1 to the specified DNA sequence adjacent to gene cluster miR-17–92 was proved in a chromatin immunoprecipitation assay. Of interest, aforementioned findings on the regulatory role of the VEGF-MAPK-Elk-1 axis were not present in ECs of arterial origin, suggesting other modus operandi in pro-proliferative VEGF-stimulated conditions in arterial endothelial cells. Next to the detailed investigations at the miR-17–92 promoter level, several studies were performed to functionally characterize endogenous inhibition of miR cluster members in vitro. Synthetic deletion of miR cluster members triggered decreased EC proliferation rates independent from apoptosis events. Deteriorating effects via miR-17–92 cluster inhibition were also observed in a capillary sprouting assay, indicating main angiogenic characteristics that were lost after endothelial miR inhibition. Collectively, in vitro assays, therefore, confirmed intrinsic pro-angiogenic features of the miR-17–92 cluster.A substantial effort was undertaken to translate findings into the mouse, applying a pioneering genetic study using a postnatal inducible endothelial miR-17–92 cluster knockout mouse. Appropriate functional readouts for angiogenesis were initially conducted in the retina. Overall, endogenous deletion of the complete endothelial miR-17–92 cluster had detrimental effects on mature vessel biology. Loss of miRs induced constitutive vessel regression that ultimately led to lowered vascularization in the adult retina, highlighting first evidence for genetic participation of miR-17–92 during adulthood. Next to retina vascularization, VEGF-induced ear angiogenesis was analyzed by the application of a VEGF-encoding adenovirus that was injected locally. In line with previous findings, miR-17–92 knockout animals demonstrated an impaired response during VEGF-mediated ear angiogenesis. Analogous findings were also made in a cancer model, suggesting that interference with EC miR-17–92 cluster members is beneficial to counteract the proangiogenic tumor environment. Indeed, the herein presented in vivo findings are unique and, therefore, reveal valuable insight to the understanding of peripheral angiogenesis. To further decipher downstream effectors of miR-17–92 cluster members, bioinformatic target predictions were performed to identify target mRNAs relevant to angiogenic signaling. Using this approach, the conserved cluster target phosphatase and tensing homolog was identified and validated in the different settings of miR modulation either in vivo or in vitro. Phosphatase and tensing homolog expression was found to be upregulated in conditions where miR-17–92 was silenced, indeed suggesting repressive effect towards this specified miR target. However, anti-angiogenic effects for loss of miR-17–92 were not completely reverted or rescued after additional siRNA-mediated silencing of phosphatase and tensing homolog. This observation implies the participation of other mediators which was addressed in global expression analysis at the mRNA level. In addition to phosphatase and tensing homolog, the potent angiogenesis inhibitor thrombospondin-1 was attributed to contribute in the miR-17–92-dependent angiogenic signaling.Conclusively, Chamorro-Jorganes et al report the identification of several factors within the complex regulation of miR-17–92 and its downstream effects on angiogenic gene programmes (Figure 1). Despite the enormous effort to decipher miR cluster function, several open issues and limitations remain to be answered in future investigations. First, as therapeutic antagonism of the miR-17–92 cluster member miR-92a has been demonstrated to improve vascularization in ischemic disease both in mouse and large animal models,6,11 the here presented inducible EC knockout model should be tested in further ischemic disease models to clarify EC-specific function of the miR cluster in total. Indeed, that same group very recently presented data that under ischemic conditions, the endothelial-specific deletion of the miR-17–92 cluster results in an increase in collateral density of limbs and hearts and overall improves blood flow recovery.12 Previous individual miR-92a cluster member inhibition was performed by giving systemic applied miR inhibitors.6 The potential role of other additional cell types with importance for an angiogenic response, such as pericytes, inflammatory cells, smooth muscle cells, and so on, should additionally be investigated because this may explain certain differences between those studies. In addition, pro- and anti-angiogenic features of individual miR-17/92 cluster members may have a delicate balance only presenting a net effect when the complete cluster is silenced. Contribution of non-EC-derived miR-17–92 cluster members toward EC even in the herein applied knockout model, potentially via exosome-mediated cross talk as reported before in cardiac disease and atherosclerosis,13,14 add another layer of complexity to the observed angiogenic biological effects. In addition, the regulatory role of Elk-1 must be supported by one or several so far unknown factors which have to be identified. Next to VEGF-dependent transcriptional control for miR-17–92, other reports have shown, for example, HDAC9 to be important for cluster transcription,15 and other epigenetic modifiers altering methylation patterns could also may enter stage in the promoter region. Further, the understanding of the different outcomes in venous versus arterial ECs could be addressed in future research focusing on post-transcriptional mechanisms, such as the pri-miR processing machinery. Looking at the most downstream layer of miR action, the miR targetome, more information has also recently been investigated by Landskroner-Eiger et al identifying the miR-19a/b targets FZD4 and LRP6 directly involved to angiogenic signaling.12 Participation of both targets in the current setting for peripheral angiogenesis can also be assumed.Download figureDownload PowerPointFigure. Summary of vascular endothelial growth factor (VEGF)-dependent signaling in endothelial biology to enhance vascularization via microRNA (miR)-17–92 cluster induction. Proangiogenic VEGF triggers a cascade of signaling events, including mitogen activated protein kinase (MAPK) activation and Elk-1 phosphorylation. This ultimately increases expression of miR-17–92 cluster members and represses phosphatase and tensing homolog (PTEN), thereby supporting endothelial cell (EC) proliferation. In case of endothelial genetic inactivation for miR-17–92, loss of endothelial miRs leads to peripheral vascular impairment in vivo.The understanding of miR biology in health and disease is complex, and still we can await many more novel discoveries. Taken together, Chamorro-Jorganes et al report a sophisticated approach to delineate VEGF-dependent miR-17–92 cluster regulation and emphasize the pro-angiogenic role of many cluster members. This, however, may be different under stress conditions. The findings can contribute to a better understanding of miRs that may be used for therapeutic purpose to correct vascular defects, for example, in ischemic diseases or vessel-dependent tumor growth.Sources of FundingThe authors had financial support from the IFB-Tx (BMBF 01EO0802; T. Thum), DFG (TH 903/11-1; T. Thum), and Fondation Leducq (T. Thum).DisclosuresDr Fiedler and Dr Thum have filed patents for miR-based therapeutics in cardiac disease.FootnotesThe opinions expressed in this commentary are not necessarily those of the editors or of the American Heart Association.Correspondence to Thomas Thum, MD, PhD, Institute of Molecular and Translational Therapeutic Strategies (IMTTS), Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany. E-mail [email protected]References1. Kuehbacher A, Urbich C, Zeiher AM, Dimmeler S. Role of Dicer and Drosha for endothelial microRNA expression and angiogenesis.Circ Res. 2007; 101:59–68. doi: 10.1161/CIRCRESAHA.107.153916.LinkGoogle Scholar2. Suárez Y, Fernández-Hernando C, Pober JS, Sessa WC. Dicer dependent microRNAs regulate gene expression and functions in human endothelial cells.Circ Res. 2007; 100:1164–1173. doi: 10.1161/01.RES.0000265065.26744.17.LinkGoogle Scholar3. 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