Advanced detection strategies for cardiotropic virus infection in a cohort study of heart failure patients.

The prevalence and contribution of cardiotropic viruses to various expressions of heart failure are increasing, yet primarily underappreciated and underreported due to variable clinical syndromes, a lack of consensus diagnostic standards and insufficient clinical laboratory tools. In this study, we developed an advanced methodology for identifying viruses across a spectrum of heart failure patients. We designed a custom tissue microarray from 78 patients with conditions commonly associated with virus-related heart failure, conditions where viral contribution is typically uncertain, or conditions for which the etiological agent remains suspect but elusive. Subsequently, we employed advanced, highly sensitive in situ hybridization to probe for common cardiotropic viruses: adenovirus 2, coxsackievirus B3, cytomegalovirus, Epstein-Barr virus, hepatitis C and E, influenza B and parvovirus B19. Viral RNA was detected in 46.4% (32/69) of heart failure patients, with 50% of virus-positive samples containing more than one virus. Adenovirus 2 was the most prevalent, detected in 27.5% (19/69) of heart failure patients, while in contrast to previous reports, parvovirus B19 was detected in only 4.3% (3/69). As anticipated, viruses were detected in 77.8% (7/9) of patients with viral myocarditis and 37.5% (6/16) with dilated cardiomyopathy. Additionally, viruses were detected in 50% of patients with coronary artery disease (3/6) and hypertrophic cardiomyopathy (2/4) and in 28.6% (2/7) of transplant rejection cases. We also report for the first time viral detection within a granulomatous lesion of cardiac sarcoidosis and in giant cell myocarditis, conditions for which etiological agents remain unknown. Our study has revealed a higher than anticipated prevalence of cardiotropic viruses within cardiac muscle tissue in a spectrum of heart failure conditions, including those not previously associated with a viral trigger or exacerbating role. Our work forges a path towards a deeper understanding of viruses in heart failure pathogenesis and opens possibilities for personalized patient therapeutic approaches.

Overlapping and distinct biological effects of IL-6 classic and trans-signaling in vascular endothelial cells.

IL-6 affects tissue protective/reparative and inflammatory properties of vascular endothelial cells (ECs). This cytokine can signal to cells through classic and trans-signaling mechanisms, which are differentiated based on the expression of IL-6 receptor (IL-6R) on the surface of target cells. The biological effects of these IL-6-signaling mechanisms are distinct and have implications for vascular pathologies. We have directly compared IL-6 classic and trans-signaling in ECs. Human ECs expressed IL-6R in culture and in situ in coronary arteries from heart transplants. Stimulation of human ECs with IL-6, to model classic signaling, triggered the activation of phosphatidylinositol 3-kinase (PI3K)-Akt and ERK1/2 signaling pathways, whereas stimulation with IL-6 + sIL-6R, to model trans-signaling, triggered activation of STAT3, PI3K-Akt, and ERK1/2 pathways. IL-6 classic signaling reduced persistent injury of ECs in an allograft model of vascular rejection and inhibited cell death induced by growth factor withdrawal. When inflammatory effects were examined, IL-6 classic signaling did not induce ICAM or CCL2 expression but was sufficient to induce secretion of CXCL8 and support transmigration of neutrophil-like cells. IL-6 trans-signaling induced all inflammatory effects studied. Our findings show that IL-6 classic and trans-signaling have overlapping but distinct properties in controlling EC survival and inflammatory activation. This has implications for understanding the effects of IL-6 receptor-blocking therapies as well as for vascular responses in inflammatory and immune conditions.

HEARTBiT: A Transcriptomic Signature for Excluding Acute Cellular Rejection in Adult Heart Allograft Patients.

BACKGROUND: Nine mRNA transcripts associated with acute cellular rejection (ACR) in previous microarray studies were ported to the clinically amenable NanoString nCounter platform. Here we report the diagnostic performance of the resulting blood test to exclude ACR in heart allograft recipients: HEARTBiT. METHODS: Blood samples for transcriptomic profiling were collected during routine post-transplantation monitoring in 8 Canadian transplant centres participating in the Biomarkers in Transplantation initiative, a large (n = 1622) prospective observational study conducted between 2009 and 2014. All adult cardiac transplant patients were invited to participate (median age = 56 [17 to 71]). The reference standard for rejection status was histopathology grading of tissue from endomyocardial biopsy (EMB). All locally graded ISHLT ≥ 2R rejection samples were selected for analysis (n = 36). ISHLT 1R (n = 38) and 0R (n = 86) samples were randomly selected to create a cohort approximately matched for site, age, sex, and days post-transplantation, with a focus on early time points (median days post-transplant = 42 [7 to 506]). RESULTS: ISHLT ≥ 2R rejection was confirmed by EMB in 18 and excluded in 92 samples in the test set. HEARTBiT achieved 47% specificity (95% confidence interval [CI], 36%-57%) given ≥ 90% sensitivity, with a corresponding area under the receiver operating characteristic curve of 0.69 (95% CI, 0.56-0.81). CONCLUSIONS: HEARTBiT's diagnostic performance compares favourably to the only currently approved minimally invasive diagnostic test to rule out ACR, AlloMap (CareDx, Brisbane, CA) and may be used to inform care decisions in the first 2 months post-transplantation, when AlloMap is not approved, and most ACR episodes occur.

Deficiency of Circulating Monocytes Ameliorates the Progression of Myxomatous Valve Degeneration in Marfan Syndrome.

BACKGROUND: Myxomatous valve degeneration (MVD) involves the progressive thickening and degeneration of the heart valves, leading to valve prolapse, regurgitant blood flow, and impaired cardiac function. Leukocytes composed primarily of macrophages have recently been detected in myxomatous valves, but the timing of the presence and the contributions of these cells in MVD progression are not known. METHODS: We examined MVD progression, macrophages, and the valve microenvironment in the context of Marfan syndrome (MFS) using mitral valves from MFS mice (Fbn1C1039G/+), gene-edited MFS pigs (FBN1Glu433AsnfsX98/+), and patients with MFS. Additional histological and transcriptomic evaluation was performed by using nonsyndromic human and canine myxomatous valves, respectively. Macrophage ontogeny was determined using MFS mice transplanted with mTomato+ bone marrow or MFS mice harboring RFP (red fluorescent protein)-tagged C-C chemokine receptor type 2 (CCR2) monocytes. Mice deficient in recruited macrophages (Fbn1C1039G/+;Ccr2RFP/RFP) were generated to determine the requirements of recruited macrophages to MVD progression. RESULTS: MFS mice recapitulated histopathological features of myxomatous valve disease by 2 months of age, including mitral valve thickening, increased leaflet cellularity, and extracellular matrix abnormalities characterized by proteoglycan accumulation and collagen fragmentation. Diseased mitral valves of MFS mice concurrently exhibited a marked increase of infiltrating (MHCII+, CCR2+) and resident macrophages (CD206+, CCR2-), along with increased chemokine activity and inflammatory extracellular matrix modification. Likewise, mitral valve specimens obtained from gene-edited MFS pigs and human patients with MFS exhibited increased monocytes and macrophages (CD14+, CD64+, CD68+, CD163+) detected by immunofluorescence. In addition, comparative transcriptomic evaluation of both genetic (MFS mice) and acquired forms of MVD (humans and dogs) unveiled a shared upregulated inflammatory response in diseased valves. Remarkably, the deficiency of monocytes was protective against MVD progression, resulting in a significant reduction of MHCII macrophages, minimal leaflet thickening, and preserved mitral valve integrity. CONCLUSIONS: All together, our results suggest sterile inflammation as a novel paradigm to disease progression, and we identify, for the first time, monocytes as a viable candidate for targeted therapy in MVD.

Blood biomarkers to predict short-term pulmonary exacerbation risk in children and adolescents with CF: A pilot study.

In CF, pulmonary exacerbations (PEx) can lead to permanent loss in lung function and thus should be prevented. Previously, we identified a blood protein biosignature consisting of 6 proteins capable of predicting short-term PEx events in CF adults. In this study, we utilized blood samples from the placebo arm of a randomized controlled trial to assess whether this candidate protein biosignature was also capable of predicting short-term PEx events in CF children and adolescents. This pilot study provides preliminary evidence that blood inflammation can be monitored to predict short-term PEx risk in CF children and adolescents.

Cleavage and Sub-Cellular Redistribution of Nuclear Pore Protein 98 by Coxsackievirus B3 Protease 2A Impairs Cardioprotection.

Myocarditis, inflammation of the heart muscle, affects all demographics and is a major cause of sudden and unexpected death in young people. It is most commonly caused by viral infections of the heart, with coxsackievirus B3 (CVB3) being among the most prevalent pathogens. To understand the molecular pathogenesis of CVB3 infection and provide strategies for developing treatments, we examined the role of a key nuclear pore protein 98 (NUP98) in the setting of viral myocarditis. NUP98 was cleaved as early as 2 h post-CVB3 infection. This cleavage was further verified through both the ectopic expression of viral proteases and in vitro using purified recombinant CVB3 proteases (2A and 3C), which demonstrated that CVB3 2A but not 3C is responsible for this cleavage. By immunostaining and confocal imaging, we observed that cleavage resulted in the redistribution of NUP98 to punctate structures in the cytoplasm. Targeted siRNA knockdown of NUP98 during infection further increased viral protein expression and viral titer, and reduced cell viability, suggesting a potential antiviral role of NUP98. Moreover, we discovered that expression levels of neuregulin-1 (NRG1), a cardioprotective gene, and presenilin-1 (PSEN1), a cellular protease processing the tyrosine kinase receptor ERBB4 of NRG1, were reliant upon NUP98 and were downregulated during CVB3 infection. In addition, expression of these NUP98 target genes in myocardium tissue not only occurred at an earlier phase of infection, but also appeared in areas away from the initial inflammatory regions. Collectively, CVB3-induced cleavage of NUP98 and subsequent impairment of the cardioprotective NRG1-ERBB4/PSEN1 signaling cascade may contribute to increased myocardial damage in the context of CVB3-induced myocarditis. To our knowledge, this is the first study to demonstrate the link between NUP98 and the NRG1 signaling pathway in viral myocarditis.

Multiplexed LC-ESI-MRM-MS-based Assay for Identification of Coronary Artery Disease Biomarkers in Human Plasma.

PURPOSE: A highly-multiplexed LC-ESI-multiple reaction monitoring-MS-based assay is developed for the identification of coronary artery disease (CAD) biomarkers in human plasma. EXPERIMENTAL DESIGN: The assay is used to measure 107 stable isotope labeled peptide standards and native peptides from 64 putative biomarkers of cardiovascular diseases in tryptic digests of plasma from subjects with (n = 70) and without (n = 45) angiographic evidence of CAD and no subsequent cardiovascular mortality during follow-up. RESULTS: Extensive computational and statistical analysis reveals six plasma proteins associated with CAD, namely apolipoprotein CII, C reactive protein, CD5 antigen-like, fibronectin, inter alpha trypsin inhibitor heavy chain H1, and protein S. The identified proteins are combined into a LASSO-logistic score with high classification performance (cross-validated area under the curve = 0.74). When combined with a separate score computed from markers currently used in the clinic with similar performance, the area under the receiver operating curve increases to 0.84. Similar results are observed in an independent set of subjects (n = 87). CONCLUSIONS AND CLINICAL RELEVANCE: If externally validated, the assay and identified biomarkers can improve CAD risk stratification.

Inhibition of vascular endothelial growth factor reduces cardiac allograft vasculopathy.

BACKGROUND: Cardiac allograft vasculopathy (CAV) is a leading expression of chronic organ rejection at and beyond 1 year post-transplantation. Host bone marrow (BM)-derived cell migration to the allograft has been demonstrated in earlier work. Vascular endothelial growth factor (VEGF) is endogenously overexpressed within allografts. Graft neo-angiogenesis has been proposed as a mechanism by which VEGF may contribute to CAV. Herein we assess the therapeutic effect of inhibition of VEGF expression in CAV. METHODS: In 129J mice, female donor hearts were heterotopically transplanted into C57/B16 males and treated with soluble VEGF receptor 1 (sVEGFR1) or vehicle control. The effect of VEGF inhibition on BM-mediated microvascular outgrowth and endothelial cell migration and proliferation were assessed using in vitro assays of aortic ring angiogenesis, wound healing and proliferation, respectively. RESULTS: At 21 days post-transplantation, treatment with sVEGFR1 significantly reduced both percent luminal narrowing (p < 0.05) and percent of vessels affected (p < 0.005). sVEGFR1 significantly reduced average wet heart weight (p < 0.05), whereas mean ventricular cross-sectional area remained similar. Treatment of aortic rings with both sVEGFR1 and VEGFR2 tyrosine phosphorylation inhibitor (Ki 8751) significantly reduced BM-mediated microvascular outgrowth length (p < 0.05) and area (p < 0.05). Treatment of human coronary artery endothelial cells with sVEGFR1 and Ki 8751 significantly reduced BM-mediated endothelial cell migration (p < 0.005) and proliferation (p < 0.05). CONCLUSIONS: VEGF inhibition reduces the severity and incidence of CAV in mouse models of cardiac transplantation, while attenuating myocardial edema and neo-angiogenesis. Using this model, we provide in vitro evidence of the role of VEGF signaling in BM-mediated microvascular outgrowth and endothelial cell migration and proliferation. VEGF inhibition may represent a novel approach to CAV treatment and prevention.

Gender-specific plasma proteomic biomarkers in patients with Anderson-Fabry disease.

AIMS: Anderson-Fabry disease (AFD) is an important X-linked metabolic disease resulting in progressive end-organ involvement, with cardiac disease being the dominant determinant of survival in a gender-dependent manner. Recent epidemiological screening for AFD suggests the prevalence is much higher than previously recognized, with estimates of 1:3000. Our aim was to discover novel plasma biomarker signatures in adult patients with AFD. METHODS AND RESULTS: We used an unbiased proteomic screening approach to discover novel plasma biomarker signatures. In the discovery cohort of 46 subjects, 14 healthy controls and 32 patients with AFD, we used a mass spectrometry iTRAQ proteomic approach followed by multiple reaction monitoring (MRM) assays to identify biomarkers. Of the 38 protein groups discovered by iTRAQ, 18 already had existing MRM assays. Based on MRM, we identified an eight-protein biomarker panel (22 kDa protein, afamin, α1 antichymotrypsin, apolipoprotein E, ß-Ala His dipeptidase, haemoglobin α-2, isoform 1 of sex hormone-binding globulin, and peroxiredoxin 2) that was very specific and sensitive for male AFD patients. In female AFD patients, we identified a nine-marker panel of proteins with only three proteins, apolipoprotein E, haemoglobin α-2, and peroxiredoxin 2, common to both genders, suggesting a gender-specific alteration in plasma biomarkers in patients with AFD. The biomarkers were validated in plasma samples from 48 subjects using MRM, and they performed inferiorly in patients with heart failure. CONCLUSIONS: We have identified gender-specific plasma protein biomarker panels that are specific and sensitive for the AFD phenotype. The gender-specific panels offer important insight into potential differences in pathophysiology and prognosis between males and females with AFD.

A role for versican in the development of leiomyosarcoma.

Leiomyosarcoma (LMS) is a mesenchymal cancer that occurs throughout the body. Although LMS is easily recognized histopathologically, the cause of the disease remains unknown. Versican, an extracellular matrix proteoglycan, increases in LMS. Microarray analyses of 80 LMSs and 24 leiomyomas showed a significant elevated expression of versican in human LMS versus benign leiomyomas. To explore the importance of versican in this smooth muscle cell tumor, we used versican-directed siRNA to knock down versican expression in a LMS human cell line, SK-LMS-1. Decreased versican expression was accompanied by slower rates of LMS cell proliferation and migration, increased adhesion, and decreased accumulation of the extracellular matrix macromolecule hyaluronan. Addition of purified versican to cells expressing versican siRNA restored cell proliferation to the level of LMS controls, increased the pericellular coat and the retention of hyaluronan, and decreased cell adhesion in a dose-dependent manner. The presence of versican was not only synergistic with hyaluronan in increasing cell proliferation, but the depletion of versican decreased hyaluronan synthase expression and decreased the retention of hyaluronan. When LMS cells stably expressing versican siRNA were injected into nude mice, the resulting tumors displayed significantly less versican and hyaluronan staining, had lower volumes, and had reduced levels of mitosis as compared with controls. Collectively, these results suggest a role for using versican as a point of control in the management and treatment of LMS.

A new transcriptional role for matrix metalloproteinase-12 in antiviral immunity.

Interferon-α (IFN-α) is essential for antiviral immunity, but in the absence of matrix metalloproteinase-12 (MMP-12) or IκBα (encoded by NFKBIA) we show that IFN-α is retained in the cytosol of virus-infected cells and is not secreted. Our findings suggest that activated IκBα mediates the export of IFN-α from virus-infected cells and that the inability of cells in Mmp12(-/-) but not wild-type mice to express IκBα and thus export IFN-α makes coxsackievirus type B3 infection lethal and renders respiratory syncytial virus more pathogenic. We show here that after macrophage secretion, MMP-12 is transported into virus-infected cells. In HeLa cells MMP-12 is also translocated to the nucleus, where it binds to the NFKBIA promoter, driving transcription. We also identified dual-regulated substrates that are repressed both by MMP-12 binding to the substrate's gene exons and by MMP-12-mediated cleavage of the substrate protein itself. Whereas intracellular MMP-12 mediates NFKBIA transcription, leading to IFN-α secretion and host protection, extracellular MMP-12 cleaves off the IFN-α receptor 2 binding site of systemic IFN-α, preventing an unchecked immune response. Consistent with an unexpected role for MMP-12 in clearing systemic IFN-α, treatment of coxsackievirus type B3-infected wild-type mice with a membrane-impermeable MMP-12 inhibitor elevates systemic IFN-α levels and reduces viral replication in pancreas while sparing intracellular MMP-12. These findings suggest that inhibiting extracellular MMP-12 could be a new avenue for the development of antiviral treatments.

Longitudinal analysis of whole blood transcriptomes to explore molecular signatures associated with acute renal allograft rejection.

In this study, we explored a time course of peripheral whole blood transcriptomes from kidney transplantation patients who either experienced an acute rejection episode or did not in order to better delineate the immunological and biological processes measureable in blood leukocytes that are associated with acute renal allograft rejection. Using microarrays, we generated gene expression data from 24 acute rejectors and 24 nonrejectors. We filtered the data to obtain the most unambiguous and robustly expressing probe sets and selected a subset of patients with the clearest phenotype. We then performed a data-driven exploratory analysis using data reduction and differential gene expression analysis tools in order to reveal gene expression signatures associated with acute allograft rejection. Using a template-matching algorithm, we then expanded our analysis to include time course data, identifying genes whose expression is modulated leading up to acute rejection. We have identified molecular phenotypes associated with acute renal allograft rejection, including a significantly upregulated signature of neutrophil activation and accumulation following transplant surgery that is common to both acute rejectors and nonrejectors. Our analysis shows that this expression signature appears to stabilize over time in nonrejectors but persists in patients who go on to reject the transplanted organ. In addition, we describe an expression signature characteristic of lymphocyte activity and proliferation. This lymphocyte signature is significantly downregulated in both acute rejectors and nonrejectors following surgery; however, patients who go on to reject the organ show a persistent downregulation of this signature relative to the neutrophil signature.

Contribution of intimal smooth muscle cells to cholesterol accumulation and macrophage-like cells in human atherosclerosis.

BACKGROUND: Intimal smooth muscle cells (SMCs) contribute to the foam cell population in arterial plaque, and express lower levels of the cholesterol exporter ATP-binding cassette transporter A1 (ABCA1) in comparison with medial arterial SMCs. The relative contribution of SMCs to the total foam cell population and their expression of ABCA1 in comparison with intimal monocyte-derived macrophages, however, are unknown. Although the expression of macrophage markers by SMCs following lipid loading has been described, the relevance of this phenotypic switch by SMCs in human coronary atherosclerosis has not been determined. METHODS AND RESULTS: Human coronary artery sections from hearts explanted at the time of transplantation were processed to clearly delineate intracellular and extracellular lipids and allow costaining for cell-specific markers. Costaining for oil red O and the SMC-specific marker SM α-actin of foam cell-rich lesions revealed that 50±7% (average±standard error of the mean, n=14 subjects) of total foam cells were SMC derived. ABCA1 expression by intimal SMCs was significantly reduced between early and advanced atherosclerotic lesions, with no loss in ABCA1 expression by myeloid lineage cells. Costaining with the macrophage marker CD68 and SM α-actin revealed that 40±6% (n=15) of CD68-positive cells originated as SMCs in advanced human coronary atherosclerosis. CONCLUSIONS: These findings suggest SMCs contain a much larger burden of the excess cholesterol in human coronary atherosclerosis than previously known, in part, because of their relative inability to release excess cholesterol via ABCA1 in comparison with myeloid lineage cells. Our results also indicate that many cells identified as monocyte-derived macrophages in human atherosclerosis are in fact SMC derived.

Clinical, biochemical, and genetic predictors of coronary artery bypass graft failure.

OBJECTIVES: To identify novel predictors for coronary artery bypass grafting failure, we probed for associations with known clinical and biochemical risk factors for atherosclerosis. We also used microarray analysis to identify novel single nucleotide polymorphisms to better understand the genetics and pathogenesis of graft occlusion. METHODS: The present study was a nested case-control substudy of the Radial Artery Patency Study 5-year follow-up data. From 1996 to 2001, 87 patients underwent coronary artery bypass grafting. Of these, 26 patients (29.9%) had an occluded study graft (saphenous vein or radial artery) at 8.0 ± 1.1 years. The clinical parameters, late angiography, blood biomarker levels, and surgical outcomes data were included in a multivariate analysis to determine the independent predictors of graft failure. RESULTS: The risk factors of graft failure were fibrinogen (odds ratio [OR], 3.94; 95% confidence interval [CI], 1.33-11.63; P = .01), creatinine (OR, 1.06; 95% CI, 1.02-1.10; P = .006), and diabetes mellitus (OR, 5.15; 95% CI, 1.08-24.59; P = .04). High-density lipoprotein (OR, 0.74; 95% CI, 0.53-1.02; P = .06) was weakly protective; however, low-density lipoprotein and total cholesterol were not predictors. We then identified the association of several human single nucleotide polymorphisms with graft failure, including mutations in glutathione-S-transferase α3. Human coronary arteries and bypass grafts demonstrated increased protein expression of glutathione-S-transferase α3, a known cardioprotective factor, in the atherosclerotic regions and surrounding adventitial tissues. CONCLUSIONS: We identified diabetes as a potential clinical predictor and plasma fibrinogen, creatinine, and high-density lipoprotein as potential novel biomarkers. These might help risk stratify patients for the development of graft failure. We also demonstrated a novel association between glutathione-S-transferase α3 and graft failure.

Alteration of human blood cell transcriptome in uremia.

BACKGROUND: End-stage renal failure is associated with profound changes in physiology and health, but the molecular causation of these pleomorphic effects termed "uremia" is poorly understood. The genomic changes of uremia were explored in a whole genome microarray case-control comparison of 95 subjects with end-stage renal failure (n = 75) or healthy controls (n = 20). METHODS: RNA was separated from blood drawn in PAXgene tubes and gene expression analyzed using Affymetrix Human Genome U133 Plus 2.0 arrays. Quality control and normalization was performed, and statistical significance determined with multiple test corrections (qFDR). Biological interpretation was aided by knowledge mining using NIH DAVID, MetaCore and PubGene RESULTS: Over 9,000 genes were differentially expressed in uremic subjects compared to normal controls (fold change: -5.3 to +6.8), and more than 65% were lower in uremia. Changes appeared to be regulated through key gene networks involving cMYC, SP1, P53, AP1, NFkB, HNF4 alpha, HIF1A, c-Jun, STAT1, STAT3 and CREB1. Gene set enrichment analysis showed that mRNA processing and transport, protein transport, chaperone functions, the unfolded protein response and genes involved in tumor genesis were prominently lower in uremia, while insulin-like growth factor activity, neuroactive receptor interaction, the complement system, lipoprotein metabolism and lipid transport were higher in uremia. Pathways involving cytoskeletal remodeling, the clathrin-coated endosomal pathway, T-cell receptor signaling and CD28 pathways, and many immune and biological mechanisms were significantly down-regulated, while the ubiquitin pathway and certain others were up-regulated. CONCLUSIONS: End-stage renal failure is associated with profound changes in human gene expression which appears to be mediated through key transcription factors. Dialysis and primary kidney disease had minor effects on gene regulation, but uremia was the dominant influence in the changes observed. This data provides important insight into the changes in cellular biology and function, opportunities for biomarkers of disease progression and therapy, and potential targets for intervention in uremia.

MicroRNA-203 enhances coxsackievirus B3 replication through targeting zinc finger protein-148.

Coxsackievirus B3 (CVB3) is the primary causal agent of viral myocarditis. During infection, it hijacks host genes to favour its own replication. However, the underlying mechanism is still unclear. Although the viral receptor is an important factor for viral infectivity, other factors such as microRNAs (miRNA) may also play an essential role in its replication after host cell entry. miRNAs are post-transcriptional gene regulators involved in various fundamental biological processes as well as in diseases. To identify miRNAs involved in CVB3 pathogenesis, we performed microarray analysis of miRNAs using CVB3-infected murine hearts and identified miR-203 as one of the most upregulated candidates. We found that miR-203 upregulation is through the activation of protein kinase C/transcription factor AP-1 pathway. We further identified zinc finger protein-148 (ZFP-148), a transcription factor, as a novel target of miR-203. Ectopic expression of miR-203 downregulated ZFP-148 translation, increased cell viability and subsequently enhanced CVB3 replication. Silencing of ZFP-148 by siRNA showed similar effects on CVB3 replication. Finally, analyses of the signalling cascade downstream of ZFP-148 revealed that miR-203-induced suppression of ZFP-148 differentially regulated the expression of prosurvival and proapoptotic genes of the Bcl-2 family proteins as well as the cell cycle regulators. This altered gene expression promoted cell survival and growth, which provided a favourable environment for CVB3 replication, contributing to the further damage of the infected cells. Taken together, this study identified a novel target of miR-203 and revealed, for the first time, the molecular link between miR-203/ZFP-148 and the pathogenesis of CVB3.

Predicting acute cardiac rejection from donor heart and pre-transplant recipient blood gene expression.

BACKGROUND: Acute rejection in cardiac transplant patients remains a contributory factor to limited survival of implanted hearts. Currently, there are no biomarkers in clinical use that can predict, at the time of transplantation, the likelihood of post-transplant acute cellular rejection. Such a development would be of great value in personalizing immunosuppressive treatment. METHODS: Recipient age, donor age, cold ischemic time, warm ischemic time, panel-reactive antibody, gender mismatch, blood type mismatch and human leukocyte antigens (HLA-A, -B and -DR) mismatch between recipients and donors were tested in 53 heart transplant patients for their power to predict post-transplant acute cellular rejection. Donor transplant biopsy and recipient pre-transplant blood were also examined for the presence of genomic biomarkers in 7 rejection and 11 non-rejection patients, using non-targeted data mining techniques. RESULTS: The biomarker based on the 8 clinical variables had an area under the receiver operating characteristic curve (AUC) of 0.53. The pre-transplant recipient blood gene-based panel did not yield better performance, but the donor heart tissue gene-based panel had an AUC = 0.78. A combination of 25 probe sets from the transplant donor biopsy and 18 probe sets from the pre-transplant recipient whole blood had an AUC = 0.90. Biologic pathways implicated include VEGF- and EGFR-signaling, and MAPK. CONCLUSIONS: Based on this study, the best predictive biomarker panel contains genes from recipient whole blood and donor myocardial tissue. This panel provides clinically relevant prediction power and, if validated, may personalize immunosuppressive treatment and rejection monitoring.