Connie Wu

Molecular basis for antagonism between PDGF and the TGFβ family of signalling pathways by control of miR-24 expression

Modulation of the vascular smooth‐muscle‐cell (vSMC) phenotype from a quiescent ‘contractile’ phenotype to a proliferative ‘synthetic’ phenotype has been implicated in vascular injury repair, as well as pathogenesis of vascular proliferative diseases. Both bone morphogenetic protein (BMP) and transforming growth factor‐β (TGFβ)‐signalling pathways promote a contractile phenotype, while the platelet‐derived growth factor‐BB (PDGF‐BB)‐signalling pathway promotes a switch to the synthetic phenotype. Here we show that PDGF‐BB induces microRNA‐24 (miR‐24), which in turn leads to downregulation of Tribbles‐like protein‐3 (Trb3). Repression of Trb3 coincides with reduced expression of Smad proteins and decrease in BMP and TGFβ signalling, promoting a synthetic phenotype in vSMCs. Inhibition of miR‐24 by antisense oligonuclotides abrogates the downregulation of Trb3 as well as pro‐synthetic activity of the PDGF‐signalling pathway. Thus, this study provides a molecular basis for the antagonism between the PDGF and TGFβ pathways, and its effect on the control of the vSMC phenotype.

Micromanaging Vascular Smooth Muscle Cell Differentiation and Phenotypic Modulation

The phenotype of vascular smooth muscle cells (VSMCs) is dynamically regulated in response to various stimuli. In a cellular process known as phenotype switching, VSMCs alternate between a contractile and synthetic phenotype state. Deregulation of phenotype switching is associated with vascular disorders such as atherosclerosis, restenosis after angioplasty, and pulmonary hypertension. An important role for microRNAs (miRNAs) in VSMC development and phenotype switching has recently been uncovered. Individual miRNAs are involved in promoting both contractile and synthetic VSMC phenotype. In this review, we summarize recent advances in the understanding of miRNA function in the regulation of VSMC phenotype regulation.

Hypoxia Potentiates MicroRNA-Mediated Gene Silencing through Posttranslational Modification of Argonaute2

ABSTRACT Hypoxia contributes to the pathogenesis of various human diseases, including pulmonary artery hypertension (PAH), stroke, myocardial or cerebral infarction, and cancer. For example, acute hypoxia causes selective pulmonary artery (PA) constriction and elevation of pulmonary artery pressure. Chronic hypoxia induces structural and functional changes to the pulmonary vasculature, which resembles the phenotype of human PAH and is commonly used as an animal model of this disease. The mechanisms that lead to hypoxia-induced phenotypic changes have not been fully elucidated. Here, we show that hypoxia increases type I collagen prolyl-4-hydroxylase [C-P4H(I)], which leads to prolyl-hydroxylation and accumulation of Argonaute2 (Ago2), a critical component of the RNA-induced silencing complex (RISC). Hydroxylation of Ago2 is required for the association of Ago2 with heat shock protein 90 (Hsp90), which is necessary for the loading of microRNAs (miRNAs) into the RISC, and translocation to stress granules (SGs). We demonstrate that hydroxylation of Ago2 increases the level of miRNAs and increases the endonuclease activity of Ago2. In summary, this study identifies hypoxia as a mediator of the miRNA-dependent gene silencing pathway through posttranslational modification of Ago2, which might be responsible for cell survival or pathological responses under low oxygen stress.

Weight Loss, Saline Loading, and the Natriuretic Peptide System

Background In epidemiologic studies, obesity has been associated with reduced natriuretic peptide (NP) concentrations. Reduced NP production could impair the ability of obese individuals to respond to salt loads, increasing the risk of hypertension and other disorders. We hypothesized that weight loss enhances NP production before and after salt loading. Methods and Results We enrolled 15 obese individuals (mean BMI 45±5.4 kg/m2) undergoing gastric bypass surgery. Before and 6 months after surgery, subjects were admitted to the clinical research center and administered a large‐volume intravenous saline challenge. Echocardiography and serial blood sampling were performed. From the pre‐operative visit to 6 months after surgery, subjects had a mean BMI decrease of 27%. At the 6‐month visit, N‐terminal pro‐atrial NP (Nt‐proANP) levels were 40% higher before, during, and after the saline infusion, compared with levels measured at the same time points during the pre‐operative visit (P<0.001). The rise in Nt‐pro‐ANP induced by the saline infusion (≈50%) was similar both before and after surgery (saline, P<0.001; interaction, P=0.2). Similar results were obtained for BNP and Nt‐proBNP; resting concentrations increased by 50% and 31%, respectively, after gastric bypass surgery. The increase in NP concentrations after surgery was accompanied by significant decreases in mean arterial pressure (P=0.004) and heart rate (P<0.001), and an increase in mitral annular diastolic velocity (P=0.02). Conclusion In obese individuals, weight loss is associated with a substantial increase in the “setpoint” of circulating NP concentrations. Higher NP concentrations could contribute to an enhanced ability to handle salt loads after weight loss.

Long Noncoding Mhrt RNA: Molecular Crowbar Unravel Insights into Heart Failure Treatment

Pathological cardiac hypertrophy and heart failure are known to involve changes in cardiac gene expression, particularly downregulation of Myh6 expression and upregulation of Myh7 expression.1 Previous studies have shown that the Brahma Related Gene 1 (Brg1)–histone deacetylase (Hdac)–poly (ADP ribose) polymerase (Parp) chromatin remodeling complex regulates the change in the ratio of Myh6 to Myh7 expression during cardiac stress.2 In the current study, Han et al3 add another layer to this mechanism by presenting evidence that a lncRNA interferes with the ability of the Brg1 complex to interact with its genomic DNA targets, including Myh6 and Myh7 , preventing the aberrant reinduction of fetal-gene expression seen in cardiac hypertrophy and heart failure. The authors3 performed rapid amplification of complementary DNA ends on RNA isolated from murine adult ventricles to identify a cluster of lncRNAs, which the authors named myosin heavy-chain-associated RNA transcripts ( Myheart , or Mhrt ), from the Myh7 loci. By using quantitative polymerase chain reaction with reverse transcription (RT-qPCR), the authors examined the expression levels of Mhrt in various murine tissues and compared the expression levels of Mhrt to the mRNA levels of Myh6 and Myh7 in murine hearts at different ages. The authors also performed RNA in situ hybridization to determine the cellular distribution of Mhrt in adult murine heart tissues. To study the role of Mhrt in cardiac hypertrophy and heart failure, the authors induced pressure overload by subjecting mice to transaortic constriction (TAC) and used RT-qPCR to measure the expression levels of Mhrt at different time points after TAC. The authors then focused on the most abundant Mhrt in murine adult ventricles ( Mhrt779 ) and generated transgenic mice overexpressing Mhrt779 (Tg779 mice) in a doxycycline-dependent and cardiomyocyte-specific manner. The authors subjected Tg779 mice to TAC to see whether overexpression of Mhrt779 could protect …

LMKB/MARF1 Localizes to mRNA Processing Bodies, Interacts with Ge-1, and Regulates IFI44L Gene Expression

The mRNA processing body (P-body) is a cellular structure that regulates the stability of cytoplasmic mRNA. MARF1 is a murine oocyte RNA-binding protein that is associated with maintenance of mRNA homeostasis and genomic stability. In this study, autoantibodies were used to identify Limkain B (LMKB), the human orthologue of MARF1, as a P-body component. Indirect immunofluorescence demonstrated that Ge-1 (a central component of the mammalian core-decapping complex) co-localized with LMKB in P-bodies. Two-hybrid and co-immunoprecipitation assays were used to demonstrate interaction between Ge-1 and LMKB. The C-terminal 120 amino acids of LMKB mediated interaction with Ge-1 and the N-terminal 1094 amino acids of Ge-1 were required for interaction with LMKB. LMKB is the first protein identified to date that interacts with this portion of Ge-1. LMKB was expressed in human B and T lymphocyte cell lines; depletion of LMKB increased expression of IFI44L, a gene that has been implicated in the cellular response to Type I interferons. The interaction between LMKB/MARF1, a protein that contains RNA-binding domains, and Ge-1, which interacts with core-decapping proteins, suggests that LMKB has a role in the regulation of mRNA stability. LMKB appears to have different functions in different cell types: maintenance of genomic stability in developing oocytes and possible dampening of the inflammatory response in B and T cells.

Novel microRNA regulators of atrial natriuretic peptide production

ABSTRACT Atrial natriuretic peptide (ANP) has a central role in regulating blood pressure in humans. Recently, microRNA 425 (miR-425) was found to regulate ANP production by binding to the mRNA of NPPA, the gene encoding ANP. mRNAs typically contain multiple predicted microRNA (miRNA)-binding sites, and binding of different miRNAs may independently or coordinately regulate the expression of any given mRNA. We used a multifaceted screening strategy that integrates bioinformatics, next-generation sequencing data, human genetic association data, and cellular models to identify additional functional NPPA-targeting miRNAs. Two novel miRNAs, miR-155 and miR-105, were found to modulate ANP production in human cardiomyocytes and target genetic variants whose minor alleles are associated with higher human plasma ANP levels. Both miR-155 and miR-105 repressed NPPA mRNA in an allele-specific manner, with the minor allele of each respective variant conferring resistance to the miRNA either by disruption of miRNA base pairing or by creation of wobble base pairing. Moreover, miR-155 enhanced the repressive effects of miR-425 on ANP production in human cardiomyocytes. Our study combines computational, genomic, and cellular tools to identify novel miRNA regulators of ANP production that could be targeted to raise ANP levels, which may have applications for the treatment of hypertension or heart failure.

MicroRNA Passenger Strand: Orchestral Symphony of Paracrine Signaling

During the past few years, increasing studies have identified microRNAs to be present in the circulation, either encapsulated in microvesicles or exosomes or associated with RNA-binding proteins or lipoproteins.1 Recent studies have suggested that circulating microRNAs may function in cell–cell communication, being transported from one cell type to another and regulating target gene expression in recipient cells.2,3 Although it has been generally thought that the passenger strand of the microRNA duplex is degraded during microRNA biogenesis and only the guide strand of the microRNA duplex is selected to become the mature functional microRNA, there is mounting evidence that passenger strand microRNAs can also target mRNAs and have biological functions in pathologies such as cancer.4,5 In the current study, Bang et al6 present evidence that exosomes produced by cardiac fibroblasts contain passenger strand microRNAs, which are transferred to cardiomyocytes and play a role in the development of fibroblast-derived cardiomyocyte hypertrophy, revealing a novel method of paracrine communication between cardiac fibroblasts and cardiomyocytes. The authors6 used electron microscopy to demonstrate the ability of neonatal rat cardiac fibroblasts to produce and secrete exosomes (fibroblast-derived exosomes). They further confirmed the identity of the exosomes by performing Western blotting and fluorescence-activated cell sorting analyses for the presence of an exosomal marker protein. To assess the microRNA content of fibroblast-derived exosomes, the authors used a microRNA …

Noncoding Genome-Wide Association Studies Variant for Obesity: Inroads Into Mechanism: An Overview From the AHA's Council on Functional Genomics and Translational Biology.

For the past decade in human genetics, genome‐wide association studies (GWAS) have been the backbone for uncovering novel loci that could underlie a disease or phenotype of interest. However, the rapid growth in the field of human genetics has been plagued by a bottleneck in translating these GWAS

Calcification of Vascular Smooth Muscle Cells and Imaging of Aortic Calcification and Inflammation.

Cardiovascular disease is the leading cause of morbidity and mortality in the world. Atherosclerotic plaques, consisting of lipid-laden macrophages and calcification, develop in the coronary arteries, aortic valve, aorta, and peripheral conduit arteries and are the hallmark of cardiovascular disease. In humans, imaging with computed tomography allows for the quantification of vascular calcification; the presence of vascular calcification is a strong predictor of future cardiovascular events. Development of novel therapies in cardiovascular disease relies critically on improving our understanding of the underlying molecular mechanisms of atherosclerosis. Advancing our knowledge of atherosclerotic mechanisms relies on murine and cell-based models. Here, a method for imaging aortic calcification and macrophage infiltration using two spectrally distinct near-infrared fluorescent imaging probes is detailed. Near-infrared fluorescent imaging allows for the ex vivo quantification of calcification and macrophage accumulation in the entire aorta and can be used to further our understanding of the mechanistic relationship between inflammation and calcification in atherosclerosis. Additionally, a method for isolating and culturing animal aortic vascular smooth muscle cells and a protocol for inducing calcification in cultured smooth muscle cells from either murine aortas or from human coronary arteries is described. This in vitro method of modeling vascular calcification can be used to identify and characterize the signaling pathways likely important for the development of vascular disease, in the hopes of discovering novel targets for therapy.