Current Opinion in Hematology:
VASCULAR BIOLOGY: Edited by Thomas F. Deuel
Post-transcriptional gene regulation by HuR and microRNAs in angiogenesis
Chang, Sung-Hee; Hla, Timothy
Department of Pathology and Laboratory Medicine, Center for Vascular Biology, Weill Cornell Medical College, Cornell University, New York, New York, USA
Correspondence to Timothy Hla, Center for Vascular Biology, Department of Pathology and Laboratory Medicine, Room A607E, Weill Cornell Medical College, Cornell University, 1300 York Avenue, New York, New York 10021, USA. Tel: +1 212 746 9953; e-mail: email@example.com
Purpose of review
This review summarizes recent findings in the area of post-transcriptional regulation of gene expression during angiogenesis, also known as new blood vessel formation. Specifically, we focus on gene regulation by HuR, an RNA-binding protein (RBP), and microRNAs (miRNAs) and their interplay, which ultimately influences cellular phenotypes of cells involved in angiogenesis.
Recently, RBPs and miRNAs have emerged as key regulators of angiogenesis. We and others have demonstrated that the RBP HuR (a.k.a. Elavl1) stabilizes vascular endothelial growth factor-A mRNA, a potent angiogenic factor in the settings of tumor development and inflammation. However, several miRNAs were shown to modulate gene expression during developmental (miR-126), physiological (miR-126, miR-92a), and pathological angiogenesis (miR-200b, miR-132). Moreover, the interplay of HuR and miRNAs in the regulation of genes involved in angiogenesis was described. In addition, recent work suggests a new role of circulating miRNAs as paracrine mediators in angiogenesis.
The elucidation of novel posttranscriptional gene regulatory mechanisms has expanded our understanding of angiogenesis in physiological and pathological conditions. We anticipate that this knowledge will ultimately lead to new insights for discovering novel therapeutic strategies to control pathological angiogenesis.
Angiogenesis, the process by which new blood vessels develop from a preexisting vasculature, is a fundamental process required for embryonic development, postnatal physiological processes such as reproduction, wound healing, as well as pathological processes such as chronic inflammation, tissue responses to ischemia, and cancer [1,2]. During embryonic development, endothelial cell progenitors generate a primitive vascular network by a process called vasculogenesis, and subsequent sprouting angiogenesis is responsible for the formation of the embryonic vascular networks. It is also the latter process of sprouting angiogenesis that is responsible for most postnatal neo-vessel growth, which occurs in response to physiological demands as well as during pathological conditions such as cancer and chronic inflammatory diseases. Once the new vasculature is established, the endothelium returns to quiescence and thereby maintains physiological homeostasis. During physiological angiogenesis, stimuli such as hypoxia trigger a highly orchestrated process of angiogenesis, which is described below. Activated endothelium undergoes vessel sprouting, endothelial cell proliferation, and directed migration, leading to the formation of vascular sprouts, which ultimately fuse to form vascular networks. Nascent vessels, which become stabilized by blood flow and pericyte coverage, allow efficient blood flow and therefore tissue perfusion. Further maturation includes remodeling of the primitive vascular network and arterial/venous differentiation. However, during pathological conditions, the angiogenic process is dysregulated and leads to non-productive neovessel formation. Vessels in pathological conditions are abnormally organized and fail to function properly in their transport, barrier, and other functions. Thus, the tight control and coordination of endothelial cell behavior is essential for maintaining physiological homeostasis and tissue integrity.
During angiogenesis, endothelial cells receive paracrine signals, which are transmitted intracellularly, leading to proper gene expression programs. Regulation is exerted at multiple levels – such as transcriptional, post-transcriptional, and translational levels. Transcriptional regulation in endothelial cells is reviewed in this issue by Morini and Dejana (pp. 229–234). We will review current research in the area of post-transcriptional gene regulation in angiogenesis. Post-transcriptional regulation controls the fate of mRNAs at the steps of splicing, export, stabilization, subcellular localization, and translation. These processes are regulated by the interaction of specific cis-regulatory elements on transcripts and trans-acting elements such as RNA-binding proteins (RBPs) and microRNAs (miRNAs) [3,4]. This review will specifically focus on HuR, a ubiquitous RBP, and miRNAs during angiogenic processes.
THE ROLE OF HuR IN ANGIOGENESIS
HuR (a.k.a.Elavl1) modulates mRNA stability and/or translation by binding U-rich (URE) and AU-rich elements (ARE), which are regulatory sequence motifs generally located in the 3’ untranslated region (UTR) of transcripts . mRNAs bearing ARE sequences constitute about 8% of the transcribed genome and about 4700 transcripts have been identified to associate with HuR in the human genome by several cross-linking immunoprecipitation analyses [6–9]. HuR-associated mRNAs are shown to be involved in numerous cellular processes including inflammation, cell cycle, tumorigenesis, cell survival, and apoptosis, suggesting the role of HuR as a multifunctional post-transcriptional regulator. Indeed, in-vivo studies revealed that Elavl1 knockout mice were embryonic lethal because of defects in placentation and postnatal deletion of Elavl1 led to rapid lethality because of apoptosis of progenitor cells in the intestinal system [10,11]. Recently, we and others demonstrated that the loss of Elavl1 in the myeloid cells impaired tumor angiogenesis and attenuated ischemic muscle neovascularization after femoral artery ligation in mice [12▪,13▪]. These studies suggest that HuR-mediated posttranscriptional gene regulatory mechanisms are critical in the neovascular responses to stimuli provided by tumor microenvironment and tissue ischemia.
Several recent studies demonstrated that HuR stabilizes mRNAs that encode angiogenic factors. Although vascular endothelial growth factor-A (Vegfa) transcript is strongly induced by hypoxia at the transcriptional level, several studies demonstrated that Vegfa mRNA is stabilized by HuR. In response to hypoxia, HuR binds and stabilizes VEGFA mRNA in human embryonic kidney 293T cells . In tumor endothelial cells, HuR binds and stabilizes VEGFA and COX-2 mRNAs, thus supporting angiogenesis and tumor progression . A recent study demonstrated that macrophage β2 integrin engagement results in HuR-dependent stabilization of Vegfa and Mmp9 mRNAs and this event contributes to angiogenesis in response to tissue ischemia [13▪]. Our recent work showed that HuR in myeloid cells stabilizes Vegfa mRNA, resulting in the stimulation of angiogenesis in a paracrine manner [12▪]. These studies suggest that HuR-dependent stabilization of Vegfa mRNA is an important mechanism regulating the angiogenic response to various stimuli. It is likely that many angiogenic factors encoded by ARE and URE-bearing transcripts would also be regulated similarly.
THE ROLE OF microRNAs IN ANGIOGENESIS
Another mechanism of posttranscriptional regulation in angiogenesis appears to be through miRNAs, which are small noncoding RNAs that primarily repress gene expression by altering mRNA stability and/or translation [3,4,16,17]. The RNA-induced silencing complex, containing miRNAs and Argonaute (Ago) proteins, targets the partially complementary sequences in the 3’UTR of mRNAs. Several miRNAs have been shown to modulate gene expression during developmental (miR-126), physiological (miR-126, miR-92a), and pathological angiogenesis (miR-200b, miR-132). For example, miR-126, which is highly expressed in endothelial cells, is essential in normal vascular development and maintenance of vascular integrity in vivo[18,19]. Knockout of endothelial cell-specific miR-126 in mice causes partial embryonic lethality because of loss of vascular integrity. In addition, zebrafish mir-126 is shown as a mediator of integrating the physiological flow signal with VEGF signaling in endothelial cells to guide angiogenesis . In response to flow, which activates the zinc finger transcription factor Kruppel-like factor 2 (KLF2), the expression of mir-126 is induced. However, endothelial-miR-92a serves as an endogenous repressor of angiogenesis . Inhibition of miR-92a led to enhanced blood vessel growth and functional recovery of ischemic hind limb and heart muscle in mice, suggesting the important role of miR-92a for the maintenance of physiological homeostasis and tissue integrity. miR-132 is suggested as a marker of hyperproliferative/activated endothelium . Its expression in endothelial cells is up-regulated in human breast tumors but not detectable in normal breast tissue, suggesting a critical role in tumor angiogenesis. Recently, we demonstrated that miR-200b represses Vegfa gene expression in bone marrow-derived macrophages, leading to the inhibition of angiogenesis [12▪]. Others revealed that systemic delivery of miR-200a or 200b into tumor endothelium inhibited tumor angiogenesis and induced vascular normalization, implicating miR-200 members as potential antiangiogenic miRNAs [23▪▪]. These studies suggest that miRNA-mediated gene expression is an important gene regulatory mechanism in angiogenesis.
Another emerging concept is that the activity of miRNAs can be modulated by HuR function. We identified that the binding site of miR-200b in the Vegfa 3’UTR overlaps with the binding site of HuR [12▪]. HuR antagonized the suppressive effect of miR-200b, allowing Vegfa expression in bone marrow derived macrophages, thus supporting angiogenesis. This finding indicates that the role of miRNAs and HuR in post-transcriptional gene regulation is functionally interconnected [4,24]. Such collaborative interactions between miRNAs and RBPs exemplified by HuR are thought to achieve the specificity, precision, and robustness of regulation of gene expression in complex processes such as angiogenesis. It will be of interest to identify miRNAs that collaborate with HuR to regulate gene expression in endothelial cells as well as perivascular cells during developmental, physiological, and pathological angiogenesis.
CIRCULATING microRNAs AS PARACRINE MEDIATORS OF ANGIOGENESIS
Recently, several studies revealed that miRNAs are associated with extracellular microvesicles and interact with recipient cells leading to functional consequences. This new role of circulating miRNAs in mediating the transfer of genetic information is actively being investigated. The content of microvesicles and their effects depend on the origin and state of the donor and recipient cells. For example, miR-143/145 are enriched in extracellular vesicles secreted by shear stress-stimulated human endothelial cells, and act on vascular smooth muscle cells to induce an atheroprotective phenotype (Fig. 1) [25▪▪]. Specifically, the transfer of miR-143/145 into vascular smooth muscle cells leads to repression of miR-143/145 target genes and prevents smooth muscle cell de-differentiation. In another example, miR-126 is enriched in endothelial cell-derived apoptotic bodies that are generated during atherosclerosis to convey paracrine survival signals to neighboring endothelial cells (Fig. 1) . This transfer triggers the production of CXCL12 in recipient cells, which acts as an antiapoptotic factor and provides atheroprotective effects in vivo. Moreover, glioblastoma cell-derived exosomes containing miRNA, mRNA, and angiogenic regulatory proteins can enter human brain microvascular endothelial cells and induce angiogenesis . In addition, exosomes from T cells or dendritic cells release miRNAs into the target cells and modulate immunostimulatory and antitumor effects in vivo[28,29▪]. Furthermore, mast cell-derived exosomes also contain miRNA and modulate the function of the recipient cells . Taken together, these studies suggest that miRNAs can transfer gene regulatory information between cells via extracellular vesicles.
miRNA-mediated communication between cells occurs in both paracrine and endocrine modes. Thus, understanding how extracellular vesicles containing miRNAs are generated and transported through the circulation is an important area of research. miRNAs are found to be associated with several types of extracellular vesicles such as microvesicles, exosomes, or apoptotic bodies, as well as other carriers such as high-density lipoprotein (HDL) or Ago-2-associated ribonucleoprotein (RNP) particles (Fig. 2) [31▪]. Microvesicles are a heterogeneous population, differing in size from 100 to 1000 nm. They are released by budding of small cytoplasmic protrusions followed by their detachment from the plasma membrane. Exosomes, one of many sub-populations of microvesicles, are more homogeneous in size ranging from 30 to 100 nm. They are stored within multivesicular bodies (MVBs) of the late endosome system and are released when these MVBs fuse with the plasma membrane (Fig. 2). Exosome membranes are enriched in ceramide, sphingomyelin, as well as cholesterol . Ceramide, a product of hydrolysis of sphingomyelin by sphingomyelinase (SMase), triggers the secretion of exosomes . The inhibition of neutral SMase (nSMase) with GW4869 reduced the release of exosomes. Accordingly, inhibition of nSMase-2 reduced secretion of miRNA, whereas overexpression of nSMase-2 increased extracellular release of miRNAs . In addition, nSMase2-dependent exosomal miRNAs secretion induced angiogenesis in the tumor microenvironment [34▪]. These studies suggest that ceramide signalling pathway in cancer cells regulates the export of angiogenic miRNAs through the exosomal pathway, which contributes to cancer cell metastasis. A recent study extended the role of ceramide signaling in the regulation of multivesicular endosomes (MVEs). Sphingosine 1 phosphate (S1P), one of the metabolites of ceramide, acted on its inhibitory G protein (Gi)-coupled S1P receptors on MVEs and this activation mediated maturation of MVEs and exosome release [35▪▪]. Collectively, these data suggest that sphingolipid metabolites are critically involved in the secretory pathway of exosomes.
Apoptotic bodies, released as ‘blebs’ from apoptotic cells, have been identified as yet another form of extracellular vesicles containing miRNAs. miR-126 is found to be enriched in endothelial-derived apoptotic bodies and the transfer of miR-126 enriched apoptotic bodies to neighboring endothelial cells provides survival signals . Independent of the microvesicle-associated miRNAs, vesicle-free circulating miRNAs are also present in human plasma [25▪▪,36]. A recent study showed that HDL transports miR-143/145 and the delivery of HDL-miR143/145 complex to recipient cells is dependent on scavenger receptor class B type I [25▪▪]. In addition, Ago-2, the key effector protein of miRNA-mediated silencing, can form complexes with miRNAs and circulate in body fluids as RNPs .
Circulating miRNAs are relatively stable in blood and are now recognized as a new class of biomarkers for pathological conditions. Although the evidence for the contribution of extracellular vesicles containing miRNAs in pathological diseases has been accumulating, many questions still remain. What are the stimuli that trigger extracellular vesicles and how is the release of the vesicles regulated during angiogenesis? How are the miRNAs taken up by recipient vascular cells while avoiding intracellular degradation and remain biologically active? What are the selective miRNA species enriched in the vesicles and how is the selective enrichment of miRNA into vesicles regulated? Understanding these mechanisms and molecular pathways will likely accelerate novel therapeutic strategies to control angiogenesis both positively and negatively in diseases such as ischemic vascular insufficiency and cancer.
Accumulating evidence indicates that RBPs (i.e. HuR) and/or miRNAs regulate the expression of genes involved in angiogenesis. The global view of the angiogenic gene regulatory network regulated by HuR and/or miRNAs needs to be further elucidated. For example, efforts to identify the target genes of HuR and/or miRNAs in endothelial cells at the genome-wide scale need to be conducted. Understanding the functional interconnections between HuR and miRNAs in the angiogenic gene regulatory network will lead to deeper understanding of the process of angiogenesis and likely accelerate the design of novel therapies to treat pathological angiogenesis.
This work is supported by NIH grant H49094 to T.H.
Conflicts of interest
There are no conflicts of interest.
REFERENCES AND RECOMMENDED READING
Papers of particular interest, published within the annual period of review, have been highlighted as:
- ▪ of special interest
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angiogenesis; Elavl1; endothelial cells; HuR; microRNA
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