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DNA topoisomerase II α (TOP2 α ; 170 kDa, TOP2 α /170) is an essential enzyme for proper chromosome dysjunction by producing transient DNA double-stranded breaks and is an important target for DNA damage-stabilizing anticancer agents, such as etoposide. Therapeutic effects of TOP2 α poisons can be limited due to acquired drug resistance. We previously demonstrated decreased TOP2 α /170 levels in an etoposide-resistant human leukemia K562 subline, designated K/VP.5, accompanied by increased expression of a C-terminal truncated TOP2 α isoform (90 kDa; TOP2 α /90), which heterodimerized with TOP2 α /170 and was a determinant of resistance by exhibiting dominant-negative effects against etoposide activity. Based on 3'-rapid amplification of cDNA ends, we confirmed TOP2 α /90 as the translation product of a TOP2 α mRNA in which a cryptic polyadenylation site (PAS) harbored in intron 19 (I19) was used. In this report, we investigated whether the resultant intronic polyadenylation (IPA) would be attenuated by blocking or mutating the I19 PAS, thereby circumventing acquired drug resistance. An antisense morpholino oligonucleotide was used to hybridize/block the PAS in TOP2 α pre-mRNA in K/VP.5 cells, resulting in decreased TOP2 α /90 mRNA/protein levels in K/VP.5 cells and partially circumventing drug resistance. Subsequently, CRISPR/CRISPR-associated protein 9 with homology-directed repair was used to mutate the cryptic I19 PAS (A ATA AA→A CCC AA) to prevent IPA. Gene-edited clones exhibited increased TOP2 α /170 and decreased TOP2 α /90 mRNA/protein and demonstrated restored sensitivity to etoposide and other TOP2 α -targeted drugs. Together, results indicated that blocking/mutating a cryptic I19 PAS in K/VP.5 cells reduced IPA and restored sensitivity to TOP2 α -targeting drugs. SIGNIFICANCE STATEMENT: The results presented in this study indicate that CRISPR/CRISPR-associated protein 9 gene editing of a cryptic polyadenylation site (PAS) within I19 of the TOP2α gene results in the reversal of acquired resistance to etoposide and other TOP2-targeted drugs. An antisense morpholino oligonucleotide targeting the PAS also partially circumvented resistance., (Copyright © 2024 by The American Society for Pharmacology and Experimental Therapeutics.)

DNA topoisomerase II β (TOP2 β /180; 180 kDa) is a nuclear enzyme that regulates DNA topology by generation of short-lived DNA double-strand breaks, primarily during transcription. TOP2 β /180 can be a target for DNA damage-stabilizing anticancer drugs, whose efficacy is often limited by chemoresistance. Our laboratory previously demonstrated reduced levels of TOP2 β /180 (and the paralog TOP2 α /170) in an acquired etoposide-resistant human leukemia (K562) clonal cell line, K/VP.5, in part due to overexpression of microRNA-9-3p/5p impacting post-transcriptional events. To evaluate the effect on drug sensitivity upon reduction/elimination of TOP2 β /180, a premature stop codon was generated at the TOP2 β /180 gene exon 19/intron 19 boundary (A G A A //GTAA→A T A G //GTAA) in parental K562 cells (which contain four TOP2 β /180 alleles) by CRISPR/Cas9 editing with homology-directed repair to disrupt production of full-length TOP2 β /180. Gene-edited clones were identified and verified by quantitative polymerase chain reaction and Sanger sequencing, respectively. Characterization of TOP2 β /180 gene-edited clones, with one or all four TOP2 β /180 alleles mutated, revealed partial or complete loss of TOP2 β mRNA/protein, respectively. The loss of TOP2 β /180 protein correlated with decreased (2-{4-[(7-chloro-2-quinoxalinyl)oxy]phenoxy}propionic acid)-induced DNA damage and partial resistance in growth inhibition assays. Partial resistance to mitoxantrone was also noted in the gene-edited clone with all four TOP2 β /180 alleles modified. No cross-resistance to etoposide or mAMSA was noted in the gene-edited clones. Results demonstrated the role of TOP2 β /180 in drug sensitivity/resistance in K562 cells and revealed differential paralog activity of TOP2-targeted agents. SIGNIFICANCE STATEMENT: Data indicated that CRISPR/Cas9 editing of the exon 19/intron 19 boundary in the TOP2β/180 gene to introduce a premature stop codon resulted in partial to complete disruption of TOP2β/180 expression in human leukemia (K562) cells depending on the number of edited alleles. Edited clones were partially resistant to mitoxantrone and XK469, while lacking resistance to etoposide and mAMSA. Results demonstrated the import of TOP2β/180 in drug sensitivity/resistance in K562 cells and revealed differential paralog activity of TOP2-targeted agents., (Copyright © 2024 by The American Society for Pharmacology and Experimental Therapeutics.)

DNA topoisomerase II α (TOP2 α /170; 170 kDa) and topoisomerase II β (TOP2 β /180; 180 kDa) are targets for a number of anticancer drugs, whose clinical efficacy is attenuated by chemoresistance. Our laboratory selected for an etoposide-resistant K562 clonal subline designated K/VP.5. These cells exhibited decreased TOP2 α /170 and TOP2 β /180 expression. We previously demonstrated that a microRNA-9 (miR-9)-mediated posttranscriptional mechanism plays a role in drug resistance via reduced TOP2 α /170 protein in K/VP.5 cells. Here, it is hypothesized that a similar miR-9 mechanism is responsible for decreased TOP2 β /180 levels in K/VP.5 cells. Both miR-9-3p and miR-9-5p are overexpressed in K/VP.5 compared with K562 cells, demonstrated by microRNA (miRNA) sequencing and quantitative polymerase chain reaction. The 3'-untranslated region (3'-UTR) of TOP2 β /180 contains miRNA recognition elements (MRE) for both miRNAs. Cotransfection of K562 cells with a luciferase reporter plasmid harboring TOP2 β /180 3'-UTR plus miR-9-3p or miR-9-5p mimics resulted in statistically significant decreased luciferase expression. miR-9-3p and miR-9-5p MRE mutations prevented this decrease, validating direct interaction between these miRNAs and TOP2 β /180 mRNA. Transfection of K562 cells with miR-9-3p/5p mimics led to decreased TOP2 β protein levels without a change in TOP2 β /180 mRNA and resulted in reduced TOP2 β -specific XK469-induced DNA damage. Conversely, K/VP.5 cells transfected with miR-9-3p/5p inhibitors led to increased TOP2 β /180 protein without a change in TOP2 β /180 mRNA and resulted in enhancement of XK469-induced DNA damage. Taken together, these results strongly suggest that TOP2 β /180 mRNA is translationally repressed by miR-9-3p/5p, that these miRNAs play a role in acquired resistance to etoposide, and that they are potential targets for circumvention of resistance to TOP2-targeted agents. SIGNIFICANCE STATEMENT: Results presented here indicate that miR-9-3p and miR-9-5p play a role in acquired resistance to etoposide via decreased DNA topoisomerase II β 180 kDa protein levels. These findings contribute further information about and potential strategies for circumvention of drug resistance by modulation of microRNA levels. In addition, miR-9-3p and miR-9-5p overexpression in cancer chemoresistance may lead to future validation as biomarkers of responsiveness to DNA topoisomerase II-targeted therapy., (Copyright © 2023 by The American Society for Pharmacology and Experimental Therapeutics.)

Intronic polyadenylation (IPA) plays a critical role in malignant transformation, development, progression, and cancer chemoresistance by contributing to transcriptome/proteome alterations. DNA topoisomerase IIα (170 kDa, TOP2α/170) is an established clinical target for anticancer agents whose efficacy is compromised by drug resistance often associated with a reduction of nuclear TOP2α/170 levels. In leukemia cell lines with acquired resistance to TOP2α-targeted drugs and reduced TOP2α/170 expression, variant TOP2α mRNA transcripts have been reported due to IPA that resulted in the translation of C-terminal truncated isoforms with altered nuclear-cytoplasmic distribution or heterodimerization with wild-type TOP2α/170. This review provides an overview of the various mechanisms regulating pre-mRNA processing and alternative polyadenylation, as well as the utilization of CRISPR/Cas9 specific gene editing through homology directed repair (HDR) to decrease IPA when splice sites are intrinsically weak or potentially mutated. The specific case of TOP2α exon 19/intron 19 splice site editing is discussed in etoposide-resistant human leukemia K562 cells as a tractable strategy to circumvent acquired TOP2α-mediated drug resistance. This example supports the importance of aberrant IPA in acquired drug resistance to TOP2α-targeted drugs. In addition, these results demonstrate the therapeutic potential of CRISPR/Cas9/HDR to impact drug resistance associated with aberrant splicing/polyadenylation.

DNA Topoisomerase IIα (TOP2α/170) is an enzyme essential for proliferating cells. For rapidly multiplying malignancies, this has made TOP2α/170 an important target for etoposide and other clinically active anticancer drugs. Efficacy of these agents is often limited by chemoresistance related to alterations in TOP2α/170 expression levels. Our laboratory recently demonstrated reduced levels of TOP2α/170 and overexpression of a C-terminal truncated 90-kDa isoform, TOP2α/90, due to intronic polyadenylation (IPA; within intron 19) in an acquired etoposide-resistant K562 clonal cell line, K/VP.5. We previously reported that this isoform heterodimerized with TOP2α/170 and was a determinant of acquired resistance to etoposide. Optimization of the weak TOP2α exon 19/intron 19 5' splice site in drug-resistant K/VP.5 cells by gene-editing restored TOP2α/170 levels, diminished TOP2α/90 expression, and circumvented drug resistance. Conversely, in the present study, silencing of the exon 19/intron 19 5' splice site in parental K562 cells by CRISPR/Cas9 with homology-directed repair (HDR), and thereby forcing intron 19 retention, was used to induce resistance by disrupting normal RNA processing (i.e., gene knockout), and to further evaluate the role of TOP2α/170 and TOP2α/90 isoforms as resistance determinants. Gene-edited clones were identified by quantitative polymerase chain reaction (qPCR) and verified by Sanger sequencing. TOP2α/170 mRNA/protein expression levels were attenuated in the TOP2α gene-edited clones which resulted in resistance to etoposide as assessed by reduced etoposide-induced DNA damage (γH2AX, Comet assays) and growth inhibition. RNA-seq and qPCR studies suggested that intron 19 retention leads to decreased TOP2α/170 expression by degradation of the TOP2α edited mRNA transcripts. Forced expression of TOP2α/90 in the gene-edited K562 cells further decreased etoposide-induced DNA damage in support of a dominant negative role for this truncated isoform. Together results support the important role of both TOP2α/170 and TOP2α/90 as determinants of sensitivity/resistance to TOP2α-targeting agents., Competing Interests: The authors have declared that no competing interests exist.

An essential function of DNA topoisomerase II α (TOP2 α ; 170 kDa, TOP2 α /170) is to resolve DNA topologic entanglements during chromosome disjunction by introducing transient DNA double-stranded breaks. TOP2 α /170 is an important target for DNA damage-stabilizing anticancer drugs, whose clinical efficacy is compromised by drug resistance often associated with decreased TOP2 α /170 expression. We recently demonstrated that an etoposide-resistant K562 clonal subline, K/VP.5, with reduced levels of TOP2 α /170, expresses high levels of a novel C-terminal truncated TOP2 α isoform (90 kDa, TOP2 α /90). TOP2 α /90, the translation product of a TOP2 α mRNA that retains a processed intron 19 (I19), heterodimerizes with TOP2 α /170 and is a resistance determinant through a dominant-negative effect on drug activity. We hypothesized that genome editing to enhance I19 removal would provide a tractable strategy to circumvent acquired TOP2 α -mediated drug resistance. To enhance I19 removal in K/VP.5 cells, CRISPR/Cas9 was used to make changes (GAG//GTAA AC →GAG//GTAA GT ) in the TOP2 α gene's suboptimal exon 19/intron 19 5' splice site (E19/I19 5' SS). Gene-edited clones were identified by quantitative polymerase chain reaction and verified by sequencing. Characterization of a clone with all TOP2 α alleles edited revealed improved I19 removal, decreased TOP2 α /90 mRNA/protein, and increased TOP2 α /170 mRNA/protein. Sensitivity to etoposide-induced DNA damage ( γ H2AX, Comet assays) and growth inhibition was restored to levels comparable to those in parental K562 cells. Together, the results indicate that our gene-editing strategy for optimizing the TOP2 α E19/I19 5' SS in K/VP.5 cells circumvents resistance to etoposide and other TOP2 α -targeted drugs. SIGNIFICANCE STATEMENT: Results presented here indicate that CRISPR/Cas9 gene editing of a suboptimal exon 19/intron 19 5' splice site in the DNA topoisomerase II α (TOP2 α ) gene results in circumvention of acquired drug resistance to etoposide and other TOP2 α -targeted drugs in a clonal K562 cell line by enhancing removal of intron 19 and thereby decreasing formation of a truncated TOP2 α 90 kDa isoform and increasing expression of full-length TOP2 α 170 kDa in these resistant cells. Results demonstrate the importance of RNA processing in acquired drug resistance to TOP2 α -targeted drugs., (Copyright © 2021 by The American Society for Pharmacology and Experimental Therapeutics.)

DNA topoisomerase II α protein (TOP2 α ) 170 kDa (TOP2 α /170) is an important target for anticancer agents whose efficacy is often attenuated by chemoresistance. Our laboratory has characterized acquired resistance to etoposide in human leukemia K562 cells. The clonal resistant subline K/VP.5 contains reduced TOP2 α /170 mRNA and protein levels compared with parental K562 cells. The aim of this study was to determine whether microRNA (miRNA)-mediated mechanisms play a role in drug resistance via decreased expression of TOP2 α /170. miRNA-sequencing revealed that human miR-9-3p and miR-9-5p were among the top six of those overexpressed in K/VP.5 compared with K562 cells; validation by quantitative polymerase chain reaction demonstrated overexpression of both miRNAs. miRNA recognition elements (MREs) for both miRNAs are present in the 3'-untranslated region (UTR) of TOP2 α /170. Transfecting K562 cells with a reporter plasmid harboring the TOP2 α /170 3'-UTR together with either miR-9-3p or miR-9-5p mimics resulted in a statistically significant decrease in luciferase expression. Mutating the miR-9-3p or miR-9-5p MREs prevented this decrease, demonstrating direct interaction between these miRNAs and TOP2 α /170 mRNA. Transfection of K562 cells with miR-9-3p or miR-9-5p mimics led to decreased TOP2 α /170 protein levels without a change in TOP2 α /170 mRNA and resulted in attenuated etoposide-induced DNA damage (gain-of-miRNA-inhibitory function). Conversely, transfection of miR-9-3p or miR-9-5p inhibitors in K/VP.5 cells (overexpressed miR-9 and low TOP2 α /170) led to increased TOP2 α /170 protein expression without a change in TOP2 α /170 mRNA levels and resulted in enhancement of etoposide-induced DNA damage (loss-of-miRNA-inhibitory function). Taken together, these results strongly suggest that these miRNAs play a role in and are potential targets for circumvention of acquired resistance to etoposide. SIGNIFICANCE STATEMENT: Results presented here indicate that miR-9-3p and miR-9-5p decrease DNA topoisomerase II α protein 170 kDa expression levels in acquired resistance to etoposide. These findings contribute new information about and potential strategies for circumvention of drug resistance by modulation of microRNA levels. Furthermore, increased expression of miR-9-3p and miR-9-5p in chemoresistant cancer cells may support their validation as biomarkers of responsiveness to DNA topoisomerase II-targeted therapy., (Copyright © 2020 by The American Society for Pharmacology and Experimental Therapeutics.)

DNA topoisomerase IIα (170 kDa, TOP2α/170) induces transient DNA double-strand breaks in proliferating cells to resolve DNA topological entanglements during chromosome condensation, replication, and segregation. Therefore, TOP2α/170 is a prominent target for anticancer drugs whose clinical efficacy is often compromised due to chemoresistance. Although many resistance mechanisms have been defined, acquired resistance of human cancer cell lines to TOP2α interfacial inhibitors/poisons is frequently associated with a reduction of Top2α/170 expression levels. Recent studies by our laboratory, in conjunction with earlier findings by other investigators, support the hypothesis that a major mechanism of acquired resistance to TOP2α-targeted drugs is due to alternative RNA processing/splicing. Specifically, several TOP2α mRNA splice variants have been reported which retain introns and are translated into truncated TOP2α isoforms lacking nuclear localization sequences and subsequent dysregulated nuclear-cytoplasmic disposition. In addition, intron retention can lead to truncated isoforms that lack both nuclear localization sequences and the active site tyrosine (Tyr805) necessary for forming enzyme-DNA covalent complexes and inducing DNA damage in the presence of TOP2α-targeted drugs. Ultimately, these truncated TOP2α isoforms result in decreased drug activity against TOP2α in the nucleus and manifest drug resistance. Therefore, the complete characterization of the mechanism(s) regulating the alternative RNA processing of TOP2α pre-mRNA may result in new strategies to circumvent acquired drug resistance. Additionally, novel TOP2α splice variants and truncated TOP2α isoforms may be useful as biomarkers for drug resistance, prognosis, and/or direct future TOP2α-targeted therapies., Competing Interests: Conflicts of interest All authors declared that there are no conflicts of interest.

DNA topoisomerase II α (170 kDa, TOP2 α /170) is essential in proliferating cells by resolving DNA topological entanglements during chromosome condensation, replication, and segregation. We previously characterized a C-terminally truncated isoform (TOP2 α /90), detectable in human leukemia K562 cells but more abundantly expressed in a clonal subline, K/VP.5, with acquired resistance to the anticancer agent etoposide. TOP2 α /90 (786 aa) is the translation product of a TOP2 α mRNA that retains a processed intron 19. TOP2 α /90 lacks the active-site tyrosine-805 required to generate double-strand DNA breaks as well as nuclear localization signals present in the TOP2 α /170 isoform (1531 aa). Here, we found that TOP2 α /90, like TOP2 α /170, was detectable in the nucleus and cytoplasm of K562 and K/VP.5 cells. Coimmunoprecipitation of endogenous TOP2 α /90 and TOP2 α /170 demonstrated heterodimerization of these isoforms. Forced expression of TOP2 α /90 in K562 cells suppressed, whereas siRNA-mediated knockdown of TOP2 α /90 in K/VP.5 cells enhanced, etoposide-mediated DNA strand breaks compared with similarly treated cells transfected with empty vector or control siRNAs, respectively. In addition, forced expression of TOP2 α /90 in K562 cells inhibited etoposide cytotoxicity assessed by clonogenic assays. qPCR and immunoassays demonstrated TOP2 α /90 mRNA and protein expression in normal human tissues/cells and in leukemia cells from patients. Together, results strongly suggest that TOP2 α /90 expression decreases drug-induced TOP2 α -DNA covalent complexes and is a determinant of chemoresistance through a dominant-negative effect related to heterodimerization with TOP2 α /170. Alternative processing of TOP2 α pre-mRNA, and subsequent synthesis of TOP2 α /90, may be an important mechanism regulating the formation and/or stability of cytotoxic TOP2 α /170-DNA covalent complexes in response to TOP2 α -targeting agents., (Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.)

DNA topoisomerase IIα (TOP2α) is a prominent target for anticancer drugs whose clinical efficacy is often limited by chemoresistance. Using antibody specific for the N-terminal of TOP2α, immunoassays indicated the existence of two TOP2α isoforms, 170 and 90 kDa, present in K562 leukemia cells and in an acquired etoposide (VP-16)-resistant clone (K/VP.5). TOP2α/90 expression was dramatically increased in etoposide-resistant K/VP.5 compared with parental K562 cells. We hypothesized that TOP2α/90 was the translation product of novel alternatively processed pre-mRNA, confirmed by 3'-rapid amplification of cDNA ends, polymerase chain reaction, and sequencing. TOP2α/90 mRNA includes retained intron 19, which harbors an in-frame stop codon, and two consensus poly(A) sites. The processed transcript is polyadenylated. TOP2α/90 mRNA encodes a 90,076-Da translation product missing the C-terminal 770 amino acids of TOP2α/170, replaced by 25 unique amino acids through translation of the exon 19/intron 19 read-through. Immunoassays, utilizing antisera raised against these unique amino acids, confirmed that TOP2α/90 is expressed in both cell types, with overexpression in K/VP.5 cells. Immunodetection of complex of enzyme-to-DNA and single-cell gel electrophoresis (Comet) assays demonstrated that K562 cells transfected with a TOP2α/90 expression plasmid exhibited reduced etoposide-mediated TOP2α-DNA covalent complexes and decreased etoposide-induced DNA damage, respectively, compared with similarly treated K562 cells transfected with empty vector. Because TOP2α/90 lacks the active site tyrosine (Tyr 805 ) of full-length TOP2α, these results strongly suggest that TOP2α/90 exhibits dominant-negative properties. Further studies are underway to characterize the mechanism(s) by which TOP2α/90 plays a role in acquired resistance to etoposide and other TOP2α targeting agents., (Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.)

Functionally matured microRNAs (miRNAs) are small single-stranded non-coding RNA molecules which are emerging as important post-transcriptional regulators of gene expression and consequently are central players in many physiological and pathological processes. Since the biological roles of individual miRNAs will be dictated by the mRNAs that they regulate, the identification and validation of miRNA/mRNA target interactions is critical for our understanding of the regulatory networks governing biological processes. We promulgate the combined use of prediction algorithms, the examination of curated databases of experimentally supported miRNA/mRNA interactions, manual sequence inspection of cataloged miRNA binding sites in specific target mRNAs, and review of the published literature as a reliable practice for identifying and prioritizing biologically important miRNA/mRNA target pairs. Once a preferred miRNA/mRNA target pair has been selected, we propose that the authenticity of a functional miRNA/mRNA target pair be validated by fulfilling four well-defined experimental criteria. This review summarizes our current knowledge of miRNA biology, miRNA/mRNA target prediction algorithms, validated miRNA/mRNA target data bases, and outlines several experimental methods by which miRNA/mRNA targets can be authenticated. In addition, a case study of human endoglin is presented as an example of the utilization of these methodologies.

MicroRNAs (miRNAs), a family of small nonprotein-coding RNAs, play a critical role in posttranscriptional gene regulation by acting as adaptors for the miRNA-induced silencing complex to inhibit gene expression by targeting mRNAs for translational repression and/or cleavage. miR-155-5p and miR-155-3p are processed from the B-cell Integration Cluster (BIC) gene (now designated, MIR155 host gene or MIR155HG). MiR-155-5p is highly expressed in both activated B- and T-cells and in monocytes/macrophages. MiR-155-5p is one of the best characterized miRNAs and recent data indicate that miR-155-5p plays a critical role in various physiological and pathological processes such as hematopoietic lineage differentiation, immunity, inflammation, viral infections, cancer, cardiovascular disease, and Down syndrome. In this review we summarize the mechanisms by which MIR155HG expression can be regulated. Given that the pathologies mediated by miR-155-5p result from the over-expression of this miRNA it may be possible to therapeutically attenuate miR-155-5p levels in the treatment of several pathological processes., (© 2013 Elsevier B.V. All rights reserved.)

MicroRNA-29b (miR-29b) expression has been shown to be reduced in non-small-cell lung cancer (NSCLC) tissues. Here, we have identified the oncogene cyclin-dependent protein kinase 6 (CDK6) as a direct target of miR-29b in lung cancer. We hypothesized that in vivo restoration of miR-29b and thus targeting of genes important to tumor initiation and progression may represent an option for lung cancer treatment. We developed a cationic lipoplexes (LPs)-based carrier that efficiently delivered miR-29b both in vitro and in vivo. LPs containing miR-29b (LP-miR-29b) efficiently delivered miR-29b to NSCLC A549 cells, reduced the expression of key targets CDK6, DNMT3B, and myeloid cell leukemia sequence 1 (MCL1), as well as cell growth and clonogenicity of A549 cells. In addition, the IC50 for cisplatin in the miR-29b-treated cells was effectively reduced. In a xenograft murine model, LPs efficiently accumulated at tumor sites. Systemic delivery of LP-miR-29b increased the tumor miR-29b expression by approximately fivefold, downregulated the tumor mRNA expression of CDK6, DNMT3B, and MCL1 by ~57.4, ~40.5, and ~52.4%, respectively, and significantly inhibited tumor growth by ~60% compared with LP-miR-NC (negative control). Our results demonstrate that cationic LPs represent an efficient delivery system that holds great potential in the development of miRNA-based therapeutics for lung cancer treatment.Molecular Therapy-Nucleic Acids (2013) 2, e84; doi:10.1038/mtna.2013.14; published online 16 April 2013.

Acute coronary syndrome refers to any group of clinical symptoms compatible with acute myocardial infarction (AMI). AMI is a major cause of death and disability worldwide with the greatest risk of death within the first hours of AMI onset. Therefore, delays in 'ruling in' AMI may increase morbidity and mortality due to the time lag in initiating therapy. Likewise, since the majority of patients presenting with acute chest pain do not have AMI, the rapid 'ruling out' of AMI in those patients would increase emergency department triage efficiency, decrease medical costs, and reduce morbidity and mortality. Thus, the identification of novel biomarkers that improve current strategies and/or accurately identify subjects who are at risk of developing acute and chronic manifestations of cardiovascular disease are desperately needed. This article discusses the potential of peripheral blood microRNAs as clinical biomarkers for the diagnosis and prognosis of cardiovascular diseases such as AMI.

Lung vascular alterations and pulmonary hypertension associated with oxidative stress have been reported to be involved in idiopathic lung fibrosis (ILF). Therefore, here, we hypothesize that the widely used lung fibrosis inducer, bleomycin, would cause cytoskeletal rearrangement through thiol-redox alterations in the cultured lung vascular endothelial cell (EC) monolayers. We exposed the monolayers of primary bovine pulmonary artery ECs to bleomycin (10 µg) and studied the cytotoxicity, cytoskeletal rearrangements, and the macromolecule (fluorescein isothiocyanate-dextran, 70,000 mol. wt.) paracellular transport in the absence and presence of two thiol-redox protectants, the classic water-soluble N-acetyl-L-cysteine (NAC) and the novel hydrophobic N,N'-bis-2-mercaptoethyl isophthalamide (NBMI). Our results revealed that bleomycin induced cytotoxicity (lactate dehydrogenase leak), morphological alterations (rounding of cells and filipodia formation), and cytoskeletal rearrangement (actin stress fiber formation and alterations of tight junction proteins, ZO-1 and occludin) in a dose-dependent fashion. Furthermore, our study demonstrated the formation of reactive oxygen species, loss of thiols (glutathione, GSH), EC barrier dysfunction (decrease of transendothelial electrical resistance), and enhanced paracellular transport (leak) of macromolecules. The observed bleomycin-induced EC alterations were attenuated by both NAC and NBMI, revealing that the novel hydrophobic thiol-protectant, NBMI, was more effective at µM concentrations as compared to the water-soluble NAC that was effective at mM concentrations in offering protection against the bleomycin-induced EC alterations. Overall, the results of the current study suggested the central role of thiol-redox in vascular EC dysfunction associated with ILF.

Intronic microRNA (miRNAs) suppressed the expression of endothelial nitric oxide synthase (eNOS) gene in endothelial cells (ECs). This study was to investigate the role of signal transducer and activator of transcription 3 (STAT3) in the regulation of eNOS expression and vascular EC proliferation by the intronic 27-nucleotide (nt) miRNA derived from the 27-base pair repeats in intron 4 of eNOS gene. A detectable level of the 27-nt miRNA was present in the control ECs. Overexpression of the 27-nt miRNA dramatically suppressed the expression of eNOS and STAT3 at both transcription and translation levels in ECs in association with significant inhibition of EC proliferation. Mutation of the 27-nt miRNA at the 3'-terminal region resulted in substantial reduction of the inhibitory effect of miRNA on eNOS and STAT3 expression, and EC proliferation. Overexpression of active STAT3 significantly reversed the inhibitory effect of the 27-nt miRNA on eNOS expression and EC proliferation. In summary, we demonstrated that the 27-nt intronic miRNA functioned as a negative regulator for the expression of its host gene eNOS and cell proliferation in ECs. The sequence in 3'-terminal region played a key role in the function of the 27-nt miRNA. The regulatory effect of the intronic miRNA on eNOS gene expression was associated with miRNA polymorphisms, and mediated through inhibition of STAT3 signaling in ECs.

Aims: Heart failure is a common antecedent to atrial fibrillation; both heart failure and atrial fibrillation are associated with increased myocardial oxidative stress. Chronic canine heart failure reduces atrial action potential duration and atrial refractoriness. We hypothesized that inducible nitric oxide synthase 2 (NOS2) contributes to atrial oxidative stress and electrophysiologic alterations., Methods and Results: A 16-week canine tachypacing model of heart failure was used (n= 21). At 10 weeks, dogs were randomized to either placebo (n = 12) or active treatment (n = 9) with NOS cofactor, tetrahydrobiopterin (BH(4), 50 mg), and NOS substrate (L-arginine, 3 g) twice daily for 6 weeks. A group of matched controls (n = 7) was used for comparison. Heart failure increased atrial NOS2 and reduced atrial BH(4), while L-arginine was unchanged. Treatment reduced inducible atrial fibrillation and normalized the heart failure-induced shortening of the left atrial myocyte action potential duration. Treatment increased atrial [BH(4)] while [L-arginine] was unchanged. Treatment did not improve left ventricular function or dimensions. Heart failure-induced reductions in atrial [BH(4)] resulted in NOS uncoupling, as measured by NO and superoxide anion (O(2)(·-)) production, while BH(4) and L-arginine treatment normalized NO and O(2)(·-). Heart failure resulted in left atrial oxidative stress, which was attenuated by BH(4) and L-arginine treatment., Conclusion: Chronic non-ischaemic heart failure results in atrial oxidative stress and electrophysiologic abnormalities by depletion of BH(4) and uncoupling of NOS2. Modulation of NOS2 activity by repletion of BH(4) may be a safe and effective approach to reduce the frequency of atrial arrhythmias during heart failure.

Despite significant advances in treatments, cardiovascular disease (CVD) remains the leading cause of human morbidity and mortality in developed countries. The development of novel and efficient treatment strategies requires an understanding of the basic molecular mechanisms underlying cardiac function. MicroRNAs (miRNAs) are a family of small nonprotein-coding RNAs that have emerged as important regulators in cardiac and vascular developmental and pathological processes, including cardiac arrhythmia, fibrosis, hypertrophy and ischemia, heart failure and vascular atherosclerosis. The miRNA acts as an adaptor for the miRNA-induced silencing complex (miRISC) to specifically recognize and regulate particular mRNAs. Mature miRNAs recognize their target mRNAs by base-pairing interactions between nucleotides 2 and 8 of the miRNA (the seed region) and complementary nucleotides in the 3'-untranslated region (3'-UTR) of mRNAs and miRISCs subsequently inhibit gene expression by targeting mRNAs for translational repression or cleavage. In this review we summarize the basic mechanisms of action of miRNAs as they are related to cardiac arrhythmia and address the potential for miRNAs to be therapeutically manipulated in the treatment of arrhythmias., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Rapid and accurate diagnosis of heart attacks-and the assessment of damage-are critical for improving coronary care. Mature microRNAs (miRNAs) are abundant, easily measured, and relatively stable in blood plasma. If they prove indicative of disease states, miRNAs measured from peripheral blood may be a particularly attractive source for routine clinical assessments.

In heart failure (HF), arrhythmogenic spontaneous sarcoplasmic reticulum (SR) Ca(2+) release and afterdepolarizations in cardiac myocytes have been linked to abnormally high activity of ryanodine receptors (RyR2s) associated with enhanced phosphorylation of the channel. However, the specific molecular mechanisms underlying RyR2 hyperphosphorylation in HF remain poorly understood. The objective of the current study was to test the hypothesis that the enhanced expression of muscle-specific microRNAs (miRNAs) underlies the HF-related alterations in RyR2 phosphorylation in ventricular myocytes by targeting phosphatase activity localized to the RyR2. We studied hearts isolated from canines with chronic HF exhibiting increased left ventricular (LV) dimensions and decreased LV contractility. qRT-PCR revealed that the levels of miR-1 and miR-133, the most abundant muscle-specific miRNAs, were significantly increased in HF myocytes compared with controls (2- and 1.6-fold, respectively). Western blot analyses demonstrated that expression levels of the protein phosphatase 2A (PP2A) catalytic and regulatory subunits, which are putative targets of miR-133 and miR-1, were decreased in HF cells. PP2A catalytic subunit mRNAs were validated as targets of miR-133 by using luciferase reporter assays. Pharmacological inhibition of phosphatase activity increased the frequency of diastolic Ca(2+) waves and afterdepolarizations in control myocytes. The decreased PP2A activity observed in HF was accompanied by enhanced Ca(2+)/calmodulin-dependent protein kinase (CaMKII)-mediated phosphorylation of RyR2 at sites Ser-2814 and Ser-2030 and increased frequency of diastolic Ca(2+) waves and afterdepolarizations in HF myocytes compared with controls. In HF myocytes, CaMKII inhibitory peptide normalized the frequency of pro-arrhythmic spontaneous diastolic Ca(2+) waves. These findings suggest that altered levels of major muscle-specific miRNAs contribute to abnormal RyR2 function in HF by depressing phosphatase activity localized to the channel, which in turn, leads to the excessive phosphorylation of RyR2s, abnormal Ca(2+) cycling, and increased propensity to arrhythmogenesis.

Down syndrome (DS) or Trisomy 21 (Ts21) is caused by the presence of an extra copy of all or part of human chromosome 21 (Hsa21) and is the most frequent survivable congenital chromosomal abnormality. Bioinformatic annotation has established that Hsa21 harbors more than 400 genes, including five microRNA (miRNA) genes (miR-99a, let-7c, miR-125b-2, miR-155, and miR-802). MiRNAs are endogenous, single-stranded, small non-coding RNA molecules that regulate gene expression by interacting with specific recognition elements harbored within the 3'-untranslated region (3'-UTR) of mRNAs and subsequently target these mRNAs for translational repression or destabilization. MiRNA expression profiling, miRNA RT-PCR, and miRNA in situ hybridization experiments have demonstrated that Hsa21-derived miRNAs were over-expressed in fetal brain and heart specimens isolated from individuals with DS. We now propose that Ts21 gene dosage over-expression of Hsa21-derived miRNAs in DS individuals result in the decreased expression of specific target proteins (i.e. haploinsufficiency) that contribute, in part, to the DS phenotype.

Studies have demonstrated that angiotensin II (Ang II) can regulate intestinal fluid and electrolyte transport and control intestinal wall muscular activity. Ang II is also a proinflammatory mediator that participates in inflammatory responses such as apoptosis, angiogenesis, and vascular remodeling; accumulating evidence suggests that this hormone may be involved in gastrointestinal (GI) inflammation and carcinogenesis. Ang II binds to two distinct G protein-coupled receptor subtypes, the AT(1)R and AT(2)R, which are widely expressed in the GI system. Together these studies suggest that Ang II-AT(1)R/-AT(2)R actions may play an important role in GI tract physiology and pathophysiology. Currently it is not known whether miRNAs can regulate the expression of the human AT(1)R (hAT(1)R) in the GI system. PCR and in situ hybridization experiments demonstrated that miR-802 was abundantly expressed in human colon and intestine. Luciferase reporter assays demonstrated that miR-802 could directly interact with the bioinformatics-predicted target site harbored within the 3'-untranslated region of the hAT(1)R mRNA. To validate that the levels of miR-802 were physiologically relevant in the GI system, we demonstrated that miR-802 "loss-of-function" experiments resulted in augmented hAT(1)R levels and enhanced Ang II-induced signaling in a human intestinal epithelial cell line. These results suggest that miR-802 can modulate the expression of the hAT(1)R in the GI tract and ultimately play a role in regulating the biological efficacy of Ang II in this system.

Essential hypertension is a complex disorder, caused by the interplay between many genetic variants, gene-gene interactions, and environmental factors. Given that the renin-angiotensin system (RAS) plays an important role in blood pressure (BP) control, cardiovascular regulation, and cardiovascular remodeling, special attention has been devoted to the investigation of single-nucleotide polymorphisms (SNP) harbored in RAS genes that may be associated with hypertension and cardiovascular disease. MicroRNAs (miRNAs) are a family of small, ∼21-nucleotide long, and nonprotein-coding RNAs that recognize target mRNAs through partial complementary elements in the 3'-untranslated region (3'-UTR) of mRNAs and inhibit gene expression by targeting mRNAs for translational repression or destabilization. Since miRNA SNPs (miRSNPs) can create, destroy, or modify miRNA binding sites, this review focuses on the hypothesis that transcribed target SNPs harbored in RAS mRNAs, that alter miRNA gene regulation and consequently protein expression, may contribute to cardiovascular disease susceptibility.

Down syndrome (DS), or Trisomy 21, is the most common genetic cause of cognitive impairment and congenital heart defects in the human population. Bioinformatic annotation has established that human chromosome 21 (Hsa21) harbors five microRNA (miRNAs) genes: miR-99a, let-7c, miR-125b-2, miR-155, and miR-802. Our laboratory recently demonstrated that Hsa21-derived miRNAs are overexpressed in DS brain and heart specimens. The aim of this study was to identify important Hsa21-derived miRNA/mRNA target pairs that may play a role, in part, in mediating the DS phenotype. We demonstrate by luciferase/target mRNA 3'-untranslated region reporter assays, and gain- and loss-of-function experiments that miR-155 and -802 can regulate the expression of the predicted mRNA target, the methyl-CpG-binding protein (MeCP2). We also demonstrate that MeCP2 is underexpressed in DS brain specimens isolated from either humans or mice. We further demonstrate that, as a consequence of attenuated MeCP2 expression, transcriptionally activated and silenced MeCP2 target genes, CREB1/Creb1 and MEF2C/Mef2c, are also aberrantly expressed in these DS brain specimens. Finally, in vivo silencing of endogenous miR-155 or -802, by antagomir intra-ventricular injection, resulted in the normalization of MeCP2 and MeCP2 target gene expression. Taken together, these results suggest that improper repression of MeCP2, secondary to trisomic overexpression of Hsa21-derived miRNAs, may contribute, in part, to the abnormalities in the neurochemistry observed in the brains of DS individuals. Finally these results suggest that selective inactivation of Hsa21-derived miRNAs may provide a novel therapeutic tool in the treatment of DS.

The intracellular gram-negative bacterium Francisella tularensis causes the disease tularemia and is known for its ability to subvert host immune responses. Previous work from our laboratory identified the PI3K/Akt pathway and SHIP as critical modulators of host resistance to Francisella. Here, we show that SHIP expression is strongly down-regulated in monocytes and macrophages following infection with F. tularensis novicida (F.n.). To account for this negative regulation we explored the possibility that microRNAs (miRs) that target SHIP may be induced during infection. There is one miR that is predicted to target SHIP, miR-155. We tested for induction and found that F.n. induced miR-155 both in primary monocytes/macrophages and in vivo. Using luciferase reporter assays we confirmed that miR-155 led to down-regulation of SHIP, showing that it specifically targets the SHIP 3'UTR. Further experiments showed that miR-155 and BIC, the gene that encodes miR-155, were induced as early as four hours post-infection in primary human monocytes. This expression was dependent on TLR2/MyD88 and did not require inflammasome activation. Importantly, miR-155 positively regulated pro-inflammatory cytokine release in human monocytes infected with Francisella. In sharp contrast, we found that the highly virulent type A SCHU S4 strain of Francisella tularensis (F.t.) led to a significantly lower miR-155 response than the less virulent F.n. Hence, F.n. induces miR-155 expression and leads to down-regulation of SHIP, resulting in enhanced pro-inflammatory responses. However, impaired miR-155 induction by SCHU S4 may help explain the lack of both SHIP down-regulation and pro-inflammatory response and may account for the virulence of Type A Francisella.

MicroRNAs are small endogenous noncoding RNAs that regulate protein expression by hybridization to imprecise complementary sequences of target mRNAs. Changes in abundance of muscle-specific microRNA, miR-1, have been implicated in cardiac disease, including arrhythmia and heart failure. However, the specific molecular targets and cellular mechanisms involved in the action of miR-1 in the heart are only beginning to emerge. In this study we investigated the effects of increased expression of miR-1 on excitation-contraction coupling and Ca(2+) cycling in rat ventricular myocytes using methods of electrophysiology, Ca(2+) imaging and quantitative immunoblotting. Adenoviral-mediated overexpression of miR-1 in myocytes resulted in a marked increase in the amplitude of the inward Ca(2+) current, flattening of Ca(2+) transients voltage dependence, and enhanced frequency of spontaneous Ca(2+) sparks while reducing the sarcoplasmic reticulum Ca(2+) content as compared with control. In the presence of isoproterenol, rhythmically paced, miR-1-overexpressing myocytes exhibited spontaneous arrhythmogenic oscillations of intracellular Ca(2+), events that occurred rarely in control myocytes under the same conditions. The effects of miR-1 were completely reversed by the CaMKII inhibitor KN93. Although phosphorylation of phospholamban was not altered, miR-1 overexpression increased phosphorylation of the ryanodine receptor (RyR2) at S2814 (Ca(2+)/calmodulin-dependent protein kinase) but not at S2808 (protein kinase A). Overexpression of miR-1 was accompanied by a selective decrease in expression of the protein phosphatase PP2A regulatory subunit B56alpha involved in PP2A targeting to specialized subcellular domains. We conclude that miR-1 enhances cardiac excitation-contraction coupling by selectively increasing phosphorylation of the L-type and RyR2 channels via disrupting localization of PP2A activity to these channels.

The assembly of collagen fibers, the major component of the extracellular matrix (ECM), governs a variety of physiological processes. Collagen fibrillogenesis is a tightly controlled process in which several factors, including collagen binding proteins, have a crucial role. Discoidin domain receptors (DDR1 and DDR2) are receptor tyrosine kinases that bind to and are phosphorylated upon collagen binding. The phosphorylation of DDRs is known to activate matrix metalloproteases, which in turn cleave the ECM. In our earlier studies, we established a novel mechanism of collagen regulation by DDRs; that is, the extracellular domain (ECD) of DDR2, when used as a purified, soluble protein, inhibits collagen fibrillogenesis in-vitro. To extend this novel observation, the current study investigates how the DDR2-ECD, when expressed as a membrane-anchored, cell-surface protein, affects collagen fibrillogenesis by cells. We generated a mouse osteoblast cell line that stably expresses a kinase-deficient form of DDR2, termed DDR2/-KD, on its cell surface. Transmission electron microscopy, fluorescence microscopy, and hydroxyproline assays demonstrated that the expression of DDR2/-KD reduced the rate and abundance of collagen deposition and induced significant morphological changes in the resulting fibers. Taken together, our observations extend the functional roles that DDR2 and possibly other membrane-anchored, collagen-binding proteins can play in the regulation of cell adhesion, migration, proliferation and in the remodeling of the extracellular matrix.

Activation of discoidin domain receptor (DDR) 1 by collagen is reported to regulate cell migration and survival processes. While the oligomeric state of DDR1 is reported to play a significant role in collagen binding, not much is known about the effect of collagen binding on DDR1 oligomerization and cellular distribution. Using fluorescence resonance energy transfer (FRET) microscopy, we monitored the interaction between DDR1 tagged with cyan fluorescent protein and DDR1 tagged with yellow fluorescent protein in live cells. Significant FRET signal indicative of receptor dimerization was found even in the absence of collagen stimulation. Collagen stimulation induced aggregation of DDR1, followed by a sharp increase in FRET signal, localized in the regions of aggregated receptor. Further analysis of DDR1 aggregation revealed that DDR1 undergoes cytoplasmic internalization and incorporation into the early endosome. We found the kinetics of DDR1 internalization to be fast, with a significant percentage of the receptor population being internalized in the first few minutes of collagen stimulation. Our results indicate that collagen stimulation induces the aggregation and internalization of DDR1 dimers at timescales much before receptor activation. These findings provide new insights into the cellular redistribution of DDR1 following its interaction with collagen type I.

HIV replication occurs throughout the natural course of infection in secondary lymphoid tissues and in particular within the germinal centers (GCs), where follicular dendritic cells (FDCs) are adjacent to CD4(+) T cells. Because FDCs provide signaling that increases lymphocyte activation, we postulated that FDCs could increase human immunodeficiency virus (HIV) replication. We cultured HIV-infected CD4(+) T cells alone or with FDCs and measured subsequent virus expression using HIV-p24 production and reverse transcription-PCR analyses. When cultured with FDCs, infected CD4(+) T cells produced almost fourfold more HIV than when cultured alone, and the rate of virus transcription was doubled. Both FDCs and their supernatant increased HIV transcription and resulted in nuclear translocation of NF-kappaB and phosphorylated c-Jun in infected cells. FDCs produced soluble tumor necrosis factor alpha (TNF-alpha) ex vivo, and the addition of a blocking soluble TNF receptor ablated FDC-mediated HIV transcription. Furthermore, TNF-alpha was found highly expressed within GCs, and ex vivo GC CD4(+) T cells supported greater levels of HIV-1 replication than other CD4(+) T cells. These data indicated that FDCs increase HIV transcription and production by a soluble TNF-alpha-mediated mechanism. This FDC-mediated effect may account, at least in part, for the presence of persistent HIV replication in GCs. Therefore, in addition to providing an important reservoir of infectious virus, FDCs increase HIV production, contributing to a tissue microenvironment that is highly conducive to HIV transmission and expression.

There are relatively few protocols described for the in situ detection of microRNA (miRNA) and they often use cryostat sections, signal amplification and hybridization or washes of 50-60 degrees C. This protocol describes in situ miRNA detection that can be done in paraffin-embedded, formalin-fixed tissue. Detection of the miRNA precursors can be done by RT in situ PCR, which can theoretically detect one copy per cell. The key variable for the RT in situ PCR protocol is optimal protease digestion, which is then followed by overnight DNase digestion and target specific incorporation of the reported nucleotide into the amplified cDNA. Detection of mature miRNAs is achieved by in situ hybridization with locked nucleic acid probes. This part of the protocol involves a brief protease digestion, followed by an overnight hybridization, short low stringency wash and detection of the labeled probe. The key variables for this method include probe concentration and stringency conditions. Each miRNA in situ method takes 1 d. The final step of the protocol involves colabeling by immunohistochemistry for the putative target of the miRNA, which is done after the in situ hybridization step and takes a few hours.

Vascular ATP-sensitive K(+) (K(ATP)) channels are critical regulators of arterial tone and, thus, blood flow in response to local metabolic needs. They are important targets for clinically used drugs to treat hypertensive emergency and angina. It is known that protein kinase C (PKC) activation inhibits K(ATP) channels in vascular smooth muscles. However, the mechanism by which PKC inhibits the channel remains unknown. Here we report that caveolin-dependent internalization is involved in PKC-epsilon-mediated inhibition of vascular K(ATP) channels (Kir6.1 and SUR2B) by phorbol 12-myristate 13-acetate or angiotensin II in human embryonic kidney 293 cells and human dermal vascular smooth muscle cells. We showed that Kir6.1 substantially overlapped with caveolin-1 at the cell surface. Cholesterol depletion with methyl-beta-cyclodextrin significantly reduced, whereas overexpression of caveolin-1 largely enhanced, PKC-induced inhibition of Kir6.1/SUR2B currents. Importantly, we demonstrated that activation of PKC-epsilon caused internalization of K(ATP) channels, the effect that was blocked by depletion of cholesterol with methyl-beta-cyclodextrin, expression of dominant-negative dynamin mutant K44E, or knockdown of caveolin-1 with small interfering RNA. Moreover, patch-clamp studies revealed that PKC-epsilon-mediated inhibition of the K(ATP) current induced by PMA or angiotensin II was reduced by a dynamin mutant, as well as small interfering RNA targeting caveolin-1. The reduction in the number of plasma membrane K(ATP) channels by PKC activation was further confirmed by cell surface biotinylation. These studies identify a novel mechanism by which the levels of vascular K(ATP) channels could be rapidly downregulated by internalization. This finding provides a novel mechanistic insight into how K(ATP) channels are regulated in vascular smooth muscle cells.

Down syndrome (DS), or Trisomy 21, is the most common genetic cause of cognitive impairment and congenital heart defects in the human population. To date, the contribution of microRNAs (miRNAs) in DS has not been investigated. Bioinformatic analyses demonstrate that human chromosome 21 (Hsa21) harbors five miRNA genes; miR-99a, let-7c, miR-125b-2, miR-155, and miR-802. MiRNA expression profiling, miRNA RT-PCR, and miRNA in situ hybridization experiments demonstrate that these miRNAs are overexpressed in fetal brain and heart specimens from individuals with DS when compared with age- and sex-matched controls. We hypothesize that trisomic 21 gene dosage overexpression of Hsa21-derived miRNAs results in the decreased expression of specific target proteins and contribute, in part, to features of the neuronal and cardiac DS phenotype. Importantly, Hsa21-derived miRNAs may provide novel therapeutic targets in the treatment of individuals with DS.

Acute decompensated heart failure (ADHF) is responsible for more than 1 million hospital admissions each year in the US. Clinicians and scientists have developed therapeutic strategies that reduce mortality in patients with chronic heart failure (HF). Despite the widely appreciated magnitude of the ADHF problem, there is still a critical gap in our understanding of the cellular mechanisms involved and effective treatment strategies for hospitalized patients. Irrespective of the etiology, patients with ADHF present with similar symptoms (e.g. edema, altered hemodynamics and congestion) as multiple signaling pathways converge in a common phenotypic presentation. Investigations have shown that patients with ADHF have increased catecholamine levels, which cause chronic stimulation of beta-adrenergic receptors. This overstimulation leads to chronic G-protein activation and perturbations in myocyte signaling, as the patient's heart attempts to adapt to progressive HF. Over time, these compensatory signaling mechanisms ultimately fail, and maladaptive signaling prevails with progressive worsening of symptoms. This Review summarizes some of the changes that occur during chronic adrenergic stimulation, and examines how downstream contractile dysfunction and myocyte death can alter the prognosis of patients with HF hospitalized for acute events.

MicroRNAs are natural, single-stranded, small RNA molecules that regulate gene expression by binding to target mRNAs and suppress its translation or initiate its degradation. In contrast to the identification and validation of many miRNA genes is the lack of experimental evidence identifying their corresponding mRNA targets. The most fundamental challenge in miRNA biology is to define the rules of miRNA target recognition. This is critical since the biological role of individual miRNAs will be dictated by the mRNAs that they regulate. Therefore, only as target mRNAs are validated will it be possible to establish commonalities that will enable more precise predictions of miRNA/mRNA interactions. Currently there is no clear agreement as to what experimental procedures should be followed to demonstrate that a given mRNA is a target of a specific miRNA. Therefore, this review outlines several methods by which to validate miRNA targets. Additionally, we propose that multiple criteria should be met before miRNA target validation should be considered "confirmed."

microRNAs (miRNAs) are challenging molecules to amplify by PCR because the miRNA precursor consists of a stable hairpin and the mature miRNA is roughly the size of a standard PCR primer. Despite these difficulties, successful real-time RT-PCR technologies have been developed to amplify and quantify both the precursor and mature microRNA. An overview of real-time PCR technologies developed by us to detect precursor and mature microRNAs is presented here. Protocols describe presentation of the data using relative (comparative C(T)) and absolute (standard curve) quantification. Real-time PCR assays were used to measure the time course of precursor and mature miR-155 expression in monocytes stimulated by lipopolysaccharide. Protocols are provided to configure the assays as low density PCR arrays for high throughput gene expression profiling. By profiling over 200 precursor and mature miRNAs in HL60 cells induced to differentiate with 12-O-tetradecanoylphorbol-13-acetate, it was possible to identify miRNAs who's processing is regulated during differentiation. Real-time PCR has become the gold standard of nucleic acid quantification due to the specificity and sensitivity of the PCR. Technological advancements have allowed for quantification of miRNA that is of comparable quality to more traditional RNAs.

Both angiotensin II (ANG II) and transforming growth factor-beta1 (TGF-beta1) are thought to be involved in mediating pulmonary fibrosis. Interactions between the renin-angiotensin system (RAS) and TGF-beta1 have been well documented, with most studies describing the effect of ANG II on TGF-beta1 expression. However, recent gene expression profiling experiments demonstrated that the angiotensin II type 1 receptor (AT(1)R) gene was a novel TGF-beta1 target in human adult lung fibroblasts. In this report, we show that TGF-beta1 augments human AT(1)R (hAT(1)R) steady-state mRNA and protein levels in a dose- and time-dependent manner in primary human fetal pulmonary fibroblasts (hPFBs). Nuclear run-on experiments demonstrate that TGF-beta1 transcriptionally activates the hAT(1)R gene and does not influence hAT(1)R mRNA stability. Pharmacological inhibitors and specific siRNA knockdown experiments demonstrate that the TGF-beta1 type 1 receptor (TbetaRI/ALK5), Smad2/3, and Smad4 are essential for TGF-beta1-stimulated hAT(1)R expression. Additional pharmacological inhibitor and small interference RNA experiments also demonstrated that p38 MAPK, JNK, and phosphatidylinositol 3-kinase (PI3K) signaling pathways are also involved in the TGF-beta1-stimulated increase in hAT(1)R density. Together, our results suggest an important role for cross talk among Smad, p38 MAPK, JNK, and PI3K pathways in mediating the augmented expression of hAT(1)R following TGF-beta1 treatment in hPFB. This study supports the hypothesis that a self-potentiating loop exists between the RAS and the TGF-beta1 signaling pathways and suggests that ANG II and TGF-beta1 may cooperate in the pathogenesis of pulmonary fibrosis. The synergy between these systems may require that both pathways be simultaneously inhibited to treat fibrotic lung disease.

The adverse effects of angiotensin II (Ang II) are primarily mediated through the Ang II type 1 receptor (AT1R). A silent polymorphism (+1166 A/C) in the human AT1R gene has been associated with cardiovascular disease, possibly as a result of enhanced AT(1)R activity. Because this polymorphism occurs in the 3'-untranslated region of the human AT1R gene, the biological importance of this mutation has always been questionable. Computer alignment demonstrated that the +1166 A/C polymorphism occurred in a cis-regulatory site, which is recognized by a specific microRNA (miRNA), miR-155. miRNAs are noncoding RNAs that silence gene expression by base-pairing with complementary sequences in the 3'-untranslated region of target RNAs. When the +1166 C-allele is present, base-pairing complementarity is interrupted, and the ability of miR-155 to interact with the cis-regulatory site is decreased. As a result, miR-155 no longer attenuates translation as efficiently as demonstrated by luciferase reporter and Ang II radioreceptor binding assays. In situ hybridization experiments demonstrated that mature miR-155 is abundantly expressed in the same cell types as the AT1R (e.g. endothelial and vascular smooth muscle). Finally, when human primary vascular smooth muscle cells were transfected with an antisense miR-155 inhibitor, endogenous human AT1R expression and Ang II-induced ERK1/2 activation were significantly increased. Taken together, our study demonstrates that the AT1R and miR-155 are co-expressed and that miR-155 translationally represses the expression of AT1R in vivo. Therefore, our study provides the first feasible biochemical mechanism by which the +1166 A/C polymorphism can lead to increased AT1R densities and possibly cardiovascular disease.

Animal microRNAs (miRNAs) regulate gene expression through base pairing to their targets within the 3' untranslated region (UTR) of protein-coding genes. Single-nucleotide polymorphisms (SNPs) located within such target sites can affect miRNA regulation. We mapped annotated SNPs onto a collection of experimentally supported human miRNA targets. Of the 143 experimentally supported human target sites, 9 contain 12 SNPs. We further experimentally investigated one of these target sites for hsa-miR-155, within the 3' UTR of the human AGTR1 gene that contains SNP rs5186. Using reporter silencing assays, we show that hsa-miR-155 down-regulates the expression of only the 1166A, and not the 1166C, allele of rs5186. Remarkably, the 1166C allele has been associated with hypertension in many studies. Thus, the 1166C allele may be functionally associated with hypertension by abrogating regulation by hsa-miR-155, thereby elevating AGTR1 levels. Since hsa-miR-155 is on chromosome 21, we hypothesize that the observed lower blood pressure in trisomy 21 is partially caused by the overexpression of hsa-miR-155 leading to allele-specific underexpression of AGTR1. Indeed, we have shown in fibroblasts from monozygotic twins discordant for trisomy 21 that levels of AGTR1 protein are lower in trisomy 21.

Hypertension is a common cause of heart failure, and ventricular arrhythmias are a major cause of death in heart failure. The spontaneous hypertension heart failure (SHHF) rat model was used to study altered ventricular electrophysiology in hypertension and heart failure. We hypothesized that a reduction in the inward rectifier K(+) current (I(K1)) and expression of pacemaker current (I(f)) would favor abnormal automaticity in the SHHF ventricle. SHHF ventricular myocytes were isolated at 2 and 8 mo of age and during end-stage heart failure (>/=17 mo); myocytes from age-matched rats served as controls. Inward I(K1) was significantly reduced at both 8 and >/=17 mo in SHHF rats compared with controls. There was a reduction in inward I(K1) due to aging in the controls only at >/=17 mo. We found a significant increase in I(f) at all ages in the SHHF rats, compared with young controls. In controls, there was an age-dependent increase in I(f). Action potential recordings in the SHHF rats demonstrated abnormal automaticity, which was abolished by the addition of an I(f) blocker (10 muM zatebradine). Increased I(f) during hypertension alone or combined increases in I(f) with reduced I(K1) during the progression to hypertensive heart failure contribute to a substrate for arrhythmogenesis.

Discoidin domain receptors (DDR1 and DDR2) are widely expressed cell-surface receptors, which bind to and are activated by collagens, including collagen type 1. Activation of DDRs and the resulting downstream signaling is known to regulate the extracellular matrix. However, little is known about how DDRs interact with collagen and its direct impact on collagen regulation. Here, we have established that by binding to collagen, the extracellular domain (ECD) of DDR2 inhibits collagen fibrillogenesis and alters the morphology of collagen type 1 fibers. Our in vitro assays utilized DDR2-Fc fusion proteins, which contain only the ECD of DDR2. Using surface plasmon resonance, we confirmed that further oligomerization of DDR2-Fc (by means of anti-Fc antibody) greatly enhances its binding to immobilized collagen type 1. Collagen turbidity measurements and biochemical assays indicated that DDR2 delays the formation of collagen fibrils. Atomic force microscopy of soluble collagen revealed that a predominately monomeric state of collagen was present with DDR2, while control solutions had an abundance of polymeric collagen. Transmission electron microscopy of collagen fibers, showed that the native periodic banded structure of collagen fibers was weakened and nearly absent in the presence of DDR2. Further, using a cell-based assay we demonstrate that overexpression of full length DDR2 inhibits fibrillogenesis of collagen type 1. Our results demonstrate a novel and important functional role of the DDR2 ECD that may contribute to collagen regulation via modulation of fibrillogenesis.

A large number of studies have demonstrated that the expression of the angiotensin II type 1 receptor (AT(1)R) is regulated predominantly by post-transcriptional mechanisms. Recently, it has been suggested that 10% of human genes may be regulated, in part, by a novel post-transcriptional mechanism involving microRNAs (miRNAs). miRNAs are small RNAs that regulate gene expression primarily through translational repression. The aim of this study was to determine whether miRNAs could regulate human AT(1)R expression. Luciferase reporter assays demonstrated that miR-155 could directly interact with the 3'-untranslated region of the hAT(1)R mRNA. Functional studies demonstrated that transfection of miR-155 into human primary lung fibroblasts (hPFBs) reduced the endogenous expression of the hAT(1)R compared with non-transfected cells. Additionally, miR-155 transfected cells showed a significant reduction in angiotensin II-induced extracellular signal-related kinase 1/2 (ERK1/2) activation. Furthermore, when hPFBs were transfected with an antisense miR-155 inhibitor, anti-miR-155, endogenous hAT(1)R expression and angiotensin II-induced ERK1/2 activation were significantly increased. Finally, transforming growth factor-beta(1) treatment of hPFBs resulted in the decreased expression of miR-155 and the increased expression of the hAT(1)R. In summary, our studies suggest that miR-155 can bind to the 3'-untranslated region (UTR) of hAT(1)R mRNAs and translationally repress the expression of this protein in vivo. Importantly, the translational repression mediated by miR-155 can be regulated by physiological stimuli.

At least four alternatively spliced mRNAs can be synthesized from the human AT(1)R (hAT(1)R) gene that differ only in the inclusion or exclusion of exon 2 and/or 3. RT-PCR experiments demonstrate that splice variants harboring exon 2 accounts for at least 30% of all the hAT(1)R mRNA transcripts expressed in the human tissues investigated. Since exon 2 contains two upstream AUGs or open reading frames (uORFs), we hypothesized that these AUGs would inhibit the translation of the downstream hAT(1)R protein ORF harbored in exon 4. This study demonstrates that the inclusion of exon 2 in hAT(1)R mRNA transcripts dramatically reduces hAT(1)R protein levels (nine-fold) and significantly attenuates Ang II responsiveness ( approximately four-fold). Interestingly, only when both AUGs were mutated in combination were the hAT(1)R density and Ang II signaling levels comparable with those values obtained using mRNA splice variants that did not include exon 2. This observation is consistent with a model where the majority of the ribosomes likely translate uORF#1 and are then unable to reinitiate at the downstream hAT(1)R ORF, in part due to the presence of AUG#2 and to the short intercistronic spacing. Importantly, TGF-beta(1) treatment (4ng/ml for 4h) of fibroblasts up-regulated hAT(1)R mRNA splice variants, which harbored exon 2, six-fold. Since AT(1)R activation is closely associated with cardiovascular disease, the inclusion of exon 2 by alternative splicing represents a novel mechanism to reduce the overall production of the hAT(1)R protein and possibly limit the potential pathological effects of AT(1)R activation.

Sarcosine1, glycine8 angiotensin II (SG Ang II) displayed unusual characteristics in early pharmacological studies. It was a potent antagonist of the dipsogenic actions of intracerebroventricularly administered Ang II in the rat, but showed low affinity for bovine cerebellar Ang II receptors. It has recently been shown that SG Ang II binds preferentially to the AT1 receptor. To determine if SG Ang II is a functional antagonist of the AT1 receptor-mediated calcium signaling, CHO cells stably transfected with AT1 receptors were exposed to Ang II in the presence and absence of SG Ang II. At concentrations of 10-100 nM, SG Ang II completely inhibited Ang II-stimulated intracellular Ca2+ mobilization in AT1A and AT1B receptor-transfected cells. SG Ang II and 125I- SG Ang II bound to AT1A and AT1B receptor-transfected cells with KD and KI values of 2-4 nM. In contrast, SG Ang II bound to AT2 transfected cells with a KI value of 7.86 microM. These results demonstrate that SG Ang II is a selective and functional peptide antagonist of the AT1 angiotensin II receptor subtype.

Studies investigating the mechanisms that govern the expression of the human angiotensin II (Ang II) type 1 receptor (hAT1R) gene have progressed slowly due to the lack of human cell lines that express the AT1R. Recently, however, an immortalized human trophoblast cell line (HTR-8/SVNeo) was demonstrated to respond to Ang II. Therefore, we utilized this cell line to characterize the AT1R expressed on the cell surface and to investigate the mechanisms by which the hAT1R gene is regulated in these cells. HTR-8/SVNeo cells were shown to express functional high affinity AT1Rs having a Bmax value of 114+/-11 fmol/mg protein and a Kd value of 0.14+/-0.1 nM. Additionally, Ang II-induced IP3 production was mediated via the AT1R. Deletional analysis of the hAT1R promoter localized a major basal regulatory sequence within the -105 to -79 bp region, relative to the transcription start site, in HTR-8/SVNeo cells. Electrophoretic mobility shift assay (EMSA) and Chromatin Immunoprecipitation (ChIP) assay demonstrated that the transcription factors, Sp1 and Sp3, interact with this region of the hAT1R promoter in vitro and in vivo. Taken together, our data demonstrate that HTR-8/SVNeo cells express functional AT1Rs and that basal level expression of this gene is regulated, in part, by Sp1 and Sp3 in this cell line.

Activation of the angiotensin II type 1 receptor (AT1R) is closely involved in the pathogenesis of cardiovascular disease. The human AT1R (hAT1R) mRNA splice variants have long 5'-untranslated regions (5'-UTRs) ranging from 272 to 414 bp that have the potential to form stable secondary structures. In this study, we show that the 5'-UTR of hAT(1)R mRNAs contains an internal ribosome entry site (IRES) located within the first 40 bp of the proximal end of exon 1. Experiments utilizing the hAT1R 5'-UTR as a molecular decoy demonstrate a reduction in IRES activity of approximately 50%. This inhibition is most efficient for the hAT1R IRES suggesting that a defined set of trans-factors are required to initiate translation through this cis-element. Translation initiation from the hAT1R IRES appears to be physiologically relevant since IRES activity was maintained during serum starvation, a cellular stress known to inhibit cap-dependent translation. These results suggest that cap-independent translation initiation by internal ribosome entry may represent an important mechanism for the regulation of hAT1R expression.