Tropoelastin Improves Post-Infarct Cardiac Function.

BACKGROUND: Myocardial infarction (MI) is among the leading causes of death worldwide. Following MI, necrotic cardiomyocytes are replaced by a stiff collagen-rich scar. Compared to collagen, the extracellular matrix protein elastin has high elasticity and may have more favorable properties within the cardiac scar. We sought to improve post-MI healing by introducing tropoelastin, the soluble subunit of elastin, to alter scar mechanics early after MI. METHODS AND RESULTS: We developed an ultrasound-guided direct intramyocardial injection method to administer tropoelastin directly into the left ventricular anterior wall of rats subjected to induced MI. Experimental groups included shams and infarcted rats injected with either PBS vehicle control or tropoelastin. Compared to vehicle treated controls, echocardiography assessments showed tropoelastin significantly improved left ventricular ejection fraction (64.7±4.4% versus 46.0±3.1% control) and reduced left ventricular dyssynchrony (11.4±3.5 ms versus 31.1±5.8 ms control) 28 days post-MI. Additionally, tropoelastin reduced post-MI scar size (8.9±1.5% versus 20.9±2.7% control) and increased scar elastin (22±5.8% versus 6.2±1.5% control) as determined by histological assessments. RNA sequencing (RNAseq) analyses of rat infarcts showed that tropoelastin injection increased genes associated with elastic fiber formation 7 days post-MI and reduced genes associated with immune response 11 days post-MI. To show translational relevance, we performed immunohistochemical analyses on human ischemic heart disease cardiac samples and showed an increase in tropoelastin within fibrotic areas. Using RNA-seq we also demonstrated the tropoelastin gene ELN is upregulated in human ischemic heart disease and during human cardiac fibroblast-myofibroblast differentiation. Furthermore, we showed by immunocytochemistry that human cardiac fibroblast synthesize increased elastin in direct response to tropoelastin treatment. CONCLUSIONS: We demonstrate for the first time that purified human tropoelastin can significantly repair the infarcted heart in a rodent model of MI and that human cardiac fibroblast synthesize elastin. Since human cardiac fibroblasts are primarily responsible for post-MI scar synthesis, our findings suggest exciting future clinical translation options designed to therapeutically manipulate this synthesis.

Impact of the EndoPredict genomic assay on treatment decisions for oestrogen receptor-positive early breast cancer patients: benefits of physician selective testing.

PURPOSE: Genomic tests improve accuracy of risk prediction for early breast cancers but these are expensive. This study evaluated the clinical utility of EndoPredict®, in terms of impact on adjuvant therapy recommendations and identification of parameters to guide selective application. METHODS: Patients with ER-positive, HER2-negative, and early-stage invasive breast cancer were tested with EndoPredict®. Two cohorts were recruited: one consecutively and another at clinical team discretion. Systemic treatment recommendations were recorded before and after EndoPredict® results were revealed to the multidisciplinary team. RESULTS: 233 patients were recruited across five sites: 123 consecutive and 110 at clinical team discretion. In the consecutive cohort 50.6% (62/123) cases were classified high risk of recurrence by EndoPredict®, compared with 62.7% (69/110) in the selective cohort. A change in treatment recommendation was significantly more likely (p < 0.0001) in the selective cohort (43/110, 39.1%) compared to the consecutive group (11/123, 8.9%). The strongest driver of selective recruitment was intermediate grade histology, whilst logistic regression modelling demonstrated that nodal status (p < 0.001), proliferative rate (p = 0.001), and progesterone receptor positivity (p < 0.001) were the strongest discriminators of risk. CONCLUSION: Whilst molecular risk can be predicted by traditional variables in a high proportion of cases, EndoPredict® had a greater impact on treatment decisions in those cases selected for testing at team discretion. This is indicative of the robust ability of the clinical team to identify cases most likely to benefit from testing, underscoring the value of genomic tests in the oncologists' tool kit.

A tumour suppressive relationship between mineralocorticoid and retinoic acid receptors activates a transcriptional program consistent with a reverse Warburg effect in breast cancer.

BACKGROUND: The role of nuclear receptors in both the aetiology and treatment of breast cancer is exemplified by the use of the oestrogen receptor (ER) as a prognostic marker and treatment target. Treatments targeting the oestrogen signalling pathway are initially highly effective for most patients. However, for the breast cancers that fail to respond, or become resistant, to current endocrine treatments, the long-term outlook is poor. ER is a member of the nuclear receptor superfamily, comprising 48 members in the human, many of which are expressed in the breast and could be used as alternative targets in cases where current treatments are ineffective. METHODS: We used sparse canonical correlation analysis to interrogate potential novel nuclear receptor expression relationships in normal breast and breast cancer. These were further explored using whole transcriptome profiling in breast cancer cells after combinations of ligand treatments. RESULTS: Using this approach, we discovered a tumour suppressive relationship between the mineralocorticoid receptor (MR) and retinoic acid receptors (RAR), in particular RARß. Expression profiling of MR expressing breast cancer cells revealed that mineralocorticoid and retinoid co-treatment activated an expression program consistent with a reverse Warburg effect and growth inhibition, which was not observed with either ligand alone. Moreover, high expression of both MR and RARB was associated with improved breast cancer-specific survival. CONCLUSION: Our study reveals a previously unknown relationship between MR and RAR in the breast, which is dependent on menopausal state and altered in malignancy. This finding identifies potential new targets for the treatment of breast cancers that are refractory to existing therapeutic options.

Analysis of steric effects in DamID profiling of transcription factor target genes.

DNA adenine methyltransferase identification (DamID) is an enzymatic technology for detecting DNA regions targeted by chromatin-associated proteins. Proteins are fused to bacterial DNA adenine methyltransferase (Dam) and expressed in cultured cells or whole organisms. Here, we used DamID to detect DNA regions bound by the cardiac-restricted transcription factors (TFs) NKX2-5 and SRF, and ubiquitously-expressed co-factors ELK1 and ELK4. We compared targets bound by these TFs as N- and C-terminal fusions with Dam, for both wild type (WT) NKX2-5 and mutant proteins mimicking those found in congenital heart disease. Overall, DamID is highly robust: while the orientation of WT Dam fusions can affect the size of the target sets, their signatures remained largely reproducible. Furthermore, a severe NKX2-5 mutant lacking the homeodomain showed strong steric effects negatively impacting target discovery. The extent of steric effect is likely to be dependent on the protein in question and the orientation of Dam fusion.

The multifaceted role of the mineralocorticoid receptor in cancers.

The mineralocorticoid receptor (MR/NR3C2) is a member of the family of steroid receptors (SR) which also includes the estrogen receptor (ER), progesterone receptor (PR), androgen receptor (AR) and glucocorticoid receptor (GR). They function primarily as nuclear receptors to regulate gene expression. While the other steroid hormone receptors are known to play important roles in the pathogenesis and progression of many cancers, relatively little is understood about the role of MR in cancer biology. This review focuses on examining new insights into the potential roles and mechanisms of action of MR in cancers.

PDGF-AB Reduces Myofibroblast Differentiation Without Increasing Proliferation After Myocardial Infarction.

After myocardial infarction (MI), fibroblasts progress from proliferative to myofibroblast states, resulting in fibrosis. Platelet-derived growth factors (PDGFs) are reported to induce fibroblast proliferation, myofibroblast differentiation, and fibrosis. However, we have previously shown that PDGFs improve heart function post-MI without increasing fibrosis. We treated human cardiac fibroblasts with PDGF isoforms then performed RNA sequencing to show that PDGFs reduced cardiac fibroblasts myofibroblast differentiation and downregulated cell cycle pathways. Using mouse/pig MI models, we reveal that PDGF-AB infusion increases cell-cell interactions, reduces myofibroblast differentiation, does not affect proliferation, and accelerates scar formation. RNA sequencing of pig hearts after MI showed that PDGF-AB reduces inflammatory cytokines and alters both transcript variants and long noncoding RNA expression in cell cycle pathways. We propose that PDGF-AB could be used therapeutically to manipulate post-MI scar maturation with subsequent beneficial effects on cardiac function.

Quantitative trait and transcriptome analysis of genetic complexity underpinning cardiac interatrial septation in mice using an advanced intercross line.

Unlike single-gene mutations leading to Mendelian conditions, common human diseases are likely to be emergent phenomena arising from multilayer, multiscale, and highly interconnected interactions. Atrial and ventricular septal defects are the most common forms of cardiac congenital anomalies in humans. Atrial septal defects (ASD) show an open communication between the left and right atria postnatally, potentially resulting in serious hemodynamic consequences if untreated. A milder form of atrial septal defect, patent foramen ovale (PFO), exists in about one-quarter of the human population, strongly associated with ischaemic stroke and migraine. The anatomic liabilities and genetic and molecular basis of atrial septal defects remain unclear. Here, we advance our previous analysis of atrial septal variation through quantitative trait locus (QTL) mapping of an advanced intercross line (AIL) established between the inbred QSi5 and 129T2/SvEms mouse strains, that show extremes of septal phenotypes. Analysis resolved 37 unique septal QTL with high overlap between QTL for distinct septal traits and PFO as a binary trait. Whole genome sequencing of parental strains and filtering identified predicted functional variants, including in known human congenital heart disease genes. Transcriptome analysis of developing septa revealed downregulation of networks involving ribosome, nucleosome, mitochondrial, and extracellular matrix biosynthesis in the 129T2/SvEms strain, potentially reflecting an essential role for growth and cellular maturation in septal development. Analysis of variant architecture across different gene features, including enhancers and promoters, provided evidence for the involvement of non-coding as well as protein-coding variants. Our study provides the first high-resolution picture of genetic complexity and network liability underlying common congenital heart disease, with relevance to human ASD and PFO.

Inhibition of Melanosis in Whiteleg Shrimp (Litopenaeus vannamei) during Refrigerated Storage Using Extracts of Different Avocado (Persea americana Mill.) By-Products.

Melanosis in shrimp usually leads to reduction in its shelf life and quality, which causes a significant loss in economic value of shrimp products. This study reports potential applications of nine ethanolic extracts of by-products, i.e., peel and/or seed from three Vietnamese avocado varieties as effective inhibitors of melanosis in whiteleg shrimp. Six out of nine shrimp samples treated with the prepared extracts (0.025%, w/v) reduced melanosis and lipid oxidation more significantly as compared to those treated with sodium metabisulfite (SMS, 1.25%, w/v) and control groups (treated with water) during 8-day storage at 4°C (P<0.05). These six extracts had mean gray values ranging from 47.0±0.7 to 57.3±0.4% were lower than those treated with SMS (mean gray of 39.8±0.4%). The inhibition of melanosis and lipid oxidation in shrimp for these extracts could be attributed to their high content of polyphenols [total phenolic content (TPC) from 44.5±1.1 to 144.7±1.9 mg gallic acid equivalents/g dried weight] and strong antioxidant activities [including 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, ferric reducing antioxidant power (FRAP), and tyrosinase enzyme inhibition]. Pearson statistical analysis showed strong correlation for melanosis inhibition to TPC and DPPH scavenging (r>0.80) followed by tyrosinase inhibition and FRAP (r>0.50). The findings obtained from this study suggest potential utilization of avocado by-product extracts as safe and cheap natural alternatives to traditional sulfites for anti-melanosis and shelf life extension of whiteleg shrimp.

MNAzymes, a versatile new class of nucleic acid enzymes that can function as biosensors and molecular switches.

To increase the versatility and utility of nucleic acid enzymes, we developed multicomponent complexes, known as MNAzymes, which produce amplified "output" signals in response to specific "input" signals. Multiple oligonucleotide partzymes assemble into active MNAzymes only in the presence of an input assembly facilitator such as a target nucleic acid. Once formed, MNAzymes catalytically modify a generic substrate, generating an amplified output signal that heralds the presence of the target while leaving the target intact. We demonstrated several applications including sensitive, isothermal target detection; discrimination of polymorphisms; and highly specific monitoring of real-time polymerase chain reaction (PCR). Furthermore, we showed their capacity to function as molecular switches and to work in series to create a molecular cascade. The modular nature of MNAzymes, together with the separation of input and output functionalities, provides potential for their integration into diverse devices such as diagnostic biosensors, molecular computers, and/or nanoscale machines.

Sequence determinants of innate immune activation by short interfering RNAs.

BACKGROUND: Short interfering RNAs (siRNAs) have been shown to induce immune stimulation through a number of different receptors in a range of cell types. In primary cells, both TLR7 and TLR8 have been shown to recognise siRNAs however, despite the identification of a number of TLR7/8 stimulatory RNA motifs, the complete and definitive sequence determinants of TLR7 and TLR8 are yet to be elucidated. RESULTS: A total of 207 siRNA sequences were screened for TLR7/8 stimulation in human PBMCs. There was a significant correlation between the U count of the U-rich strand and the immunostimulatory activity of the duplex. Using siRNAs specifically designed to analyse the effect of base substitutions and hybridisation of the two strands, we found that sequence motifs and the thermodynamic properties of the duplexes appeared to be the major determinants of siRNA immunogenicity and that the strength of the hybridisation interaction between the two strands correlated negatively with immunostimulatory activity. CONCLUSION: The data presented favour a model of TLR7/8 activation by siRNAs, in which the two strands are denatured in the endosome, and single-stranded, U-rich RNA species activate TLR7/8. These findings have relevance to the design of siRNAs, particularly for in vivo or clinical applications.

Emerging functional roles of nuclear receptors in breast cancer.

Nuclear receptors (NRs) have been targets of intensive drug development for decades due to their roles as key regulators of multiple developmental, physiological and disease processes. In breast cancer, expression of the estrogen and progesterone receptor remains clinically important in predicting prognosis and determining therapeutic strategies. More recently, there is growing evidence supporting the involvement of multiple nuclear receptors other than the estrogen and progesterone receptors, in the regulation of various processes important to the initiation and progression of breast cancer. We review new insights into the mechanisms of action of NRs made possible by recent advances in genomic technologies and focus on the emerging functional roles of NRs in breast cancer biology, including their involvement in circadian regulation, metabolic reprogramming and breast cancer migration and metastasis.

NKX2-5 mutations causative for congenital heart disease retain functionality and are directed to hundreds of targets.

We take a functional genomics approach to congenital heart disease mechanism. We used DamID to establish a robust set of target genes for NKX2-5 wild type and disease associated NKX2-5 mutations to model loss-of-function in gene regulatory networks. NKX2-5 mutants, including those with a crippled homeodomain, bound hundreds of targets including NKX2-5 wild type targets and a unique set of "off-targets", and retained partial functionality. NKXΔHD, which lacks the homeodomain completely, could heterodimerize with NKX2-5 wild type and its cofactors, including E26 transformation-specific (ETS) family members, through a tyrosine-rich homophilic interaction domain (YRD). Off-targets of NKX2-5 mutants, but not those of an NKX2-5 YRD mutant, showed overrepresentation of ETS binding sites and were occupied by ETS proteins, as determined by DamID. Analysis of kernel transcription factor and ETS targets show that ETS proteins are highly embedded within the cardiac gene regulatory network. Our study reveals binding and activities of NKX2-5 mutations on WT target and off-targets, guided by interactions with their normal cardiac and general cofactors, and suggest a novel type of gain-of-function in congenital heart disease.

Breast cancer prognosis predicted by nuclear receptor-coregulator networks.

Although molecular signatures based on transcript expression in breast cancer samples have provided new insights into breast cancer classification and prognosis, there are acknowledged limitations in current signatures. To provide rational, pathway-based signatures of disrupted physiology in cancer tissues that may be relevant to prognosis, this study has directly quantitated changed gene expression, between normal breast and cancer tissue, as a basis for signature development. The nuclear receptor (NR) family of transcription factors, and their coregulators, are fundamental regulators of every aspect of metazoan life, and were rigorously quantified in normal breast tissues and ERα positive and ERα negative breast cancers. Coregulator expression was highly correlated with that of selected NR in normal breast, particularly from postmenopausal women. These associations were markedly decreased in breast cancer, and the expression of the majority of coregulators was down-regulated in cancer tissues compared with normal. While in cancer the loss of NR-coregulator associations observed in normal breast was common, a small number of NR (Rev-ERBß, GR, NOR1, LRH-1 and PGR) acquired new associations with coregulators in cancer tissues. Elevated expression of these NR in cancers was associated with poorer outcome in large clinical cohorts, as well as suggesting the activation of ERα -related, but ERα-independent, pathways in ERα negative cancers. In addition, the combined expression of small numbers of NR and coregulators in breast cancer was identified as a signature predicting outcome in ERα negative breast cancer patients, not linked to proliferation and with predictive power superior to existing signatures containing many more genes. These findings highlight the power of predictive signatures derived from the quantitative determination of altered gene expression between normal breast and breast cancers. Taken together, the findings of this study identify networks of NR-coregulator associations active in normal breast but disrupted in breast cancer, and moreover provide evidence that signatures based on NR networks disrupted in cancer can provide important prognostic information in breast cancer patients.

Acquired convergence of hormone signaling in breast cancer: ER and PR transition from functionally distinct in normal breast to predictors of metastatic disease.

Cumulative exposure to estrogen (E) and progesterone (P) over the menstrual cycle significantly influences the risk of developing breast cancer. Despite the dogma that PR in the breast merely serves as a marker of an active estrogen receptor (ER), and as an inhibitor of the proliferative actions of E, it is now clear that in the breast P increases proliferation independently of E action. We show here that the progesterone receptor (PR) and ER are expressed in different epithelial populations, and target non-overlapping pathways in the normal human breast. In breast cancer, PR becomes highly correlated with ER, and this convergence is associated with signaling pathways predictive of disease metastasis. These data challenge the established paradigm that ER and PR function co-operatively in normal breast, and have significant implications not only for our understanding of normal breast biology, but also for diagnosis, prognosis and/or treatment options in breast cancer patients.

Comprehensive transcriptome and immunophenotype analysis of renal and cardiac MSC-like populations supports strong congruence with bone marrow MSC despite maintenance of distinct identities.

Cells resembling bone marrow mesenchymal stem cells (MSC) have been isolated from many organs but their functional relationships have not been thoroughly examined. Here we compared the immunophenotype, gene expression, multipotency and immunosuppressive potential of MSC-like colony-forming cells from adult murine bone marrow (bmMSC), kidney (kCFU-F) and heart (cCFU-F), cultured under uniform conditions. All populations showed classic MSC morphology and in vitro mesodermal multipotency. Of the two solid organ-specific CFU-F, only kCFU-F displayed suppression of T-cell alloreactivity in vitro, albeit to a lesser extent than bmMSC. Quantitative immunophenotyping using 81 phycoerythrin-conjugated CD antibodies demonstrated that all populations contained high percentages of cells expressing diagnostic MSC surface markers (Sca1, CD90.2, CD29, CD44), as well as others noted previously on murine MSC (CD24, CD49e, CD51, CD80, CD81, CD105). Illumina microarray expression profiling and bioinformatic analysis indicated a correlation of gene expression of 0.88-0.92 between pairwise comparisons. All populations expressed approximately 66% of genes in the pluripotency network (Plurinet), presumably reflecting their stem-like character. Furthermore, all populations expressed genes involved in immunomodulation, homing and tissue repair, suggesting these as conserved functions for MSC-like cells in solid organs. Despite this molecular congruence, strong biases in gene and protein expression and pathway activity were seen, suggesting organ-specific functions. Hence, tissue-derived MSC may also retain unique properties potentially rendering them more appropriate as cellular therapeutic agents for their organ of origin.