Michael P. Vallely

Haemolysis during Sample Preparation Alters microRNA Content of Plasma

The presence of cell-free microRNAs (miRNAs) has been detected in a range of body fluids. The miRNA content of plasma/serum in particular has been proposed as a potential source of novel biomarkers for a number of diseases. Nevertheless, the quantification of miRNAs from plasma or serum is made difficult due to inefficient isolation and lack of consensus regarding the optimal reference miRNA. The effect of haemolysis on the quantification and normalisation of miRNAs in plasma has not been investigated in great detail. We found that levels of miR-16, a commonly used reference gene, showed little variation when measured in plasma samples from healthy volunteers or patients with malignant mesothelioma or coronary artery disease. Including samples with evidence of haemolysis led to variation in miR-16 levels and consequently decreased its ability to serve as a reference. The levels of miR-16 and miR-451, both present in significant levels in red blood cells, were proportional to the degree of haemolysis. Measurements of the level of these miRNAs in whole blood, plasma, red blood cells and peripheral blood mononuclear cells revealed that the miRNA content of red blood cells represents the major source of variation in miR-16 and miR-451 levels measured in plasma. Adding lysed red blood cells to non-haemolysed plasma allowed a cut-off level of free haemoglobin to be determined, below which miR-16 and miR-451 levels displayed little variation between individuals. In conclusion, increases in plasma miR-16 and miR-451 are caused by haemolysis. In the absence of haemolysis the levels of both miR-16 and miR-451 are sufficiently constant to serve as normalisers.

The Impact of Hemolysis on Cell-Free microRNA Biomarkers

Cell-free microRNAs in plasma and serum have become a promising source of biomarkers for various diseases. Despite rapid progress in this field, there remains a lack of consensus regarding optimal quantification methods, reference genes, and quality control of samples. Recent studies have shown that hemolysis occurring during blood collection has substantial impact on the microRNA content in plasma/serum. To date, the impact of hemolysis has only been investigated for a limited number of microRNAs, mainly the red blood cell (RBC)-enriched miRs-16 and -451. In contrast, the effect of hemolysis on other microRNAs – in particular those proposed as biomarkers – has not been addressed. In this study we profiled the microRNA content of hemolyzed and non-hemolyzed plasma as well as RBCs to obtain a profile of microRNAs in the circulation affected or unaffected by hemolysis. Profiling by TaqMan Array Microfluidic Cards was used to compare three pairs of hemolyzed and non-hemolyzed plasma (with varying degrees of hemolysis) and one RBC sample. A total of 136 microRNAs were detectable in at least two of the samples, and of those 15 were at least twofold elevated in all three hemolyzed samples. This number increased to 88 microRNAs for the sample with the highest level of hemolysis, with all of these also detected in the RBC profile. Thus these microRNAs represent a large proportion of detectable microRNAs and those most likely to be affected by hemolysis. Several of the hemolysis-susceptible microRNAs (e.g., miRs-21, -106a, -92a, -17, -16) have also been previously proposed as plasma/serum biomarkers of disease, highlighting the importance of rigorous quality control of plasma/serum samples used for measurement of circulating microRNAs. As low-level hemolysis is a frequent occurrence during plasma/serum collection it is critical that this is taken into account in the measurement of any candidate circulating microRNA.

Transcatheter aortic valve implantation for high-risk patients with severe aortic stenosis: A systematic review.

OBJECTIVES The present systematic review objectively assessed the safety and clinical effectiveness of transcatheter aortic valve implantation for patients at high surgical risk with severe aortic stenosis. METHODS Electronic searches were performed in 6 databases from January 2000 to March 2009. The end points included feasibility, safety, efficacy, and durability. Clinical effectiveness was synthesized through a narrative review with full tabulation of results of all included studies. RESULTS The current evidence on transcatheter aortic valve implantation for aortic stenosis is limited to short-term observational studies. The overall procedural success rates ranged from 74% to 100%. The incidence of major adverse events included 30-day mortality (0%-25%), major ventricular tachyarrhythmia (0%-4%), myocardial infarction (0%-15%), cardiac tamponade (2%-10%), stroke (0%-10%), conversion to surgery (0%-8%), moderate to major paravalvular leak (4%-35%), vascular complication (8%-17%), valve-in-valve procedure (2%-12%), and aortic dissection/perforation (0%-4%). The overall 30-day major adverse cardiovascular and cerebral events ranged from 3% to 35%. The mean aortic valve area ranged from 0.5 to 0.8 cm(2) before and 1.3 to 2.0 cm(2) after transcatheter aortic valve implantation. The mean pressure gradient ranged from 34 to 58 mm Hg before and 3 to 12 mm Hg after transcatheter aortic valve implantation. There was no significant deterioration in echocardiography measurements during the assessment period. Death rate at 6 months postprocedure ranged from 18% to 48%. No studies had adequate follow-up to reliably evaluate long-term outcomes. CONCLUSIONS The procedure has a potential for serious complications. Although short-term efficacy based on echocardiography measurements is good, there is little evidence on long-term outcomes. The use of transcatheter aortic valve implantation should be considered only within the boundaries of clinical trials.

Restoring expression of miR-16: a novel approach to therapy for malignant pleural mesothelioma

BACKGROUND Malignant pleural mesothelioma (MPM) is recalcitrant to treatment and new approaches to therapy are needed. Reduced expression of miR-15/16 in a range of cancer types has suggested a tumour suppressor function for these microRNAs, and re-expression has been shown to inhibit tumour cell proliferation. The miR-15/16 status in MPM is largely unknown. MATERIALS AND METHODS MicroRNA expression was analysed by TaqMan-based RT-qPCR in MPM tumour specimens and cell lines. MicroRNA expression was restored in vitro using microRNA mimics, and effects on proliferation, drug sensitivity and target gene expression were assessed. Xenograft-bearing mice were treated with miR-16 mimic packaged in minicells targeted with epidermal growth factor receptor (EGFR)-specific antibodies. RESULTS Expression of the miR-15 family was consistently downregulated in MPM tumour specimens and cell lines. A decrease of 4- to 22-fold was found when tumour specimens were compared with normal pleura. When MPM cell lines were compared with the normal mesothelial cell line MeT-5A, the downregulation of miR-15/16 was 2- to 10-fold. Using synthetic mimics to restore miR-15/16 expression led to growth inhibition in MPM cell lines but not in MeT-5A cells. Growth inhibition caused by miR-16 correlated with downregulation of target genes including Bcl-2 and CCND1, and miR-16 re-expression sensitised MPM cells to pemetrexed and gemcitabine. In xenograft-bearing nude mice, intravenous administration of miR-16 mimics packaged in minicells led to consistent and dose-dependent inhibition of MPM tumour growth. CONCLUSIONS The miR-15/16 family is downregulated and has tumour suppressor function in MPM. Restoring miR-16 expression represents a novel therapeutic approach for MPM.

Increased Circulating miR-625-3p: A Potential Biomarker for Patients With Malignant Pleural Mesothelioma

Introduction: We investigated the ability of cell-free microRNAs (miRNAs) in plasma and serum to serve as a biomarker for malignant mesothelioma (MM). Methods: Using miRNA microarrays, we profiled plasma samples from MM patients and healthy controls. miRNAs with significantly different abundance between cases and controls were validated in a larger series of MM patients and in an independent series of MM patients using quantitative real-time polymerase chain reaction. Levels of candidate miRNAs were also quantified in MM tumor samples. Results: We compared cell-free miRNA profiles in plasma from MM patients with healthy controls. Reviewing 90 miRNAs previously reported to be associated with MM, we found that the levels of two miRNAs, miR-29c* and miR-92a, were elevated in plasma samples from MM patients. In addition, we identified 15 novel miRNAs present at significantly higher levels in the plasma of MM patients. Further analysis of candidate miRNAs by real time-quantitative polymerase chain reaction confirmed that one of them, miR-625-3p, was present in significantly higher concentration in plasma/serum from MM patients and was able to discriminate between cases and controls, in both the original and the independent series of patients. MiR-625-3p was also found to be up-regulated in tumor specimens from a group of 18 MM patients, who underwent extrapleural pneumonectomy. Conclusion: Our data confirm the potential of miR-29c* and miR-92a as candidate tumor markers and reveal that miR-625-3p is a promising novel diagnostic marker for MM.

Coronary Artery Bypass Grafting With and Without Manipulation of the Ascending Aorta – A Meta-Analysis

BACKGROUND The main criticism of surgery in the SYNTAX trial was increased rate of stroke when compared to percutaneous coronary intervention. We aimed to determine whether avoiding aortic manipulation would decrease the rate of stroke. METHOD We performed a meta-analysis of seven studies comparing coronary artery bypass grafting (CABG) with and without manipulation of the ascending aorta. RESULTS When anaortic off-pump coronary artery bypass grafting (OPCAB) was compared with conventional CABG, the rate of stroke was 0.38% vs. 1.87% (p<0.0001). When anaortic OPCAB was compared with OPCAB using a side-clamp or proximal graft anastomosis device the rate of stroke was 0.31% vs. 1.35% (p=0.003). CONCLUSION Avoiding aortic manipulation during CABG may decrease the rate of peri-operative stroke.

Management of the Ascending Aorta in Patients with Bicuspid Aortic Valve Disease

Bicuspid aortic valve (BAV) disease is the most common form of congenital heart disease, affecting 1-2% of the population. Only 20% of patients will maintain normal valve function throughout their life and more than 30% of patients will develop serious morbidity. It is a highly heritable condition, with transmission likely to be autosomal dominant. Patients with BAV have a 10-fold risk of aortic dissection when compared to the normal population. Management of BAV associated aortopathy represents a significant clinical challenge.

The genetic and molecular basis of bicuspid aortic valve associated thoracic aortopathy: a link to phenotype heterogeneity

Bicuspid aortic valve (BAV) is the most common congenital cardiac anomaly, affecting 0.5-1.4% of the general population (1-4). BAV is a clinically heterogeneous disorder with a high rate of surgically relevant aortic valve and ascending aortic complications, which occur in over 35% of those affected (5). As such, BAV confers a greater burden of disease than all other congenital heart diseases combined (3,6). Familial clustering and genetic studies have established that BAV is a heritable trait, with approximately 9% prevalence amongst firstdegree relatives, and up to 24% in families with more than one affected family member (3,7,8). Despite its importance, the etiology of BAV is largely undetermined, although it is likely to involve genetic heterogeneity, abnormal signaling pathways and aberrant neural crest cell migration (3). BAV is not only a disorder of valvular development, but also represents a complex coexistent genetic disease of the aorta and cardiac development (3,7). Thoracic aortic enlargement is common in BAV, reported in up to 50-60% of affected individuals (6,9,10). Significant phenotype heterogeneity, which may occur independently of the underlying valvular morphology and function (6,9-11), has been described, most commonly manifesting as asymmetric ascending aortic dilatation beyond the sinotubular junction with variable arch involvement and/or varying degrees of annuloaortic ectasia, predisposing to aortic dissection/ rupture, a feared complication. Indeed, BAV conveys an 8-fold increased risk of aortic dissection and over a 25-year period, the risk for aneurysm formation is 26% and for aortic surgery is 25% (2), further highlighting its health burden. Presently, ongoing controversies exist in the literature regarding the underlying pathogenesis of BAV-associated thoracic aortopathy (BAV-TA), specifically whether it is genetic or hemodynamic in origin, which have important implications for planning intervention. Intense work is currently ongoing to address this question, which has shed light on the pathogenesis of BAV-TA. Therefore, it is timely to review the current state of knowledge in this common association of BAV, focusing primarily on the interaction between genetics, molecular pathway and hemodynamic factors in influencing the heterogeneous manifestation of aortopathy seen in BAV patients.

Percutaneous Tricuspid Valve Replacement for a Stenosed Bioprosthesis

Percutaneous replacement of the aortic and pulmonic valves has rapidly gained acceptance in clinical practice as a feasible alternative to open valve surgery in patients deemed to be at high operative risk.1,2 Total percutaneous tricuspid valve replacement (TVR), on the other hand, has not yet been documented in humans, although this approach has been described in experimental animals.3 Webb et al4 have recently reported 1 case of TVR, using a valve designed for percutaneous use but inserted through a thoracotomy with direct puncture of the right atrium. We describe a …

Quantitative and temporal differences in coagulation, fibrinolysis and platelet activation after on-pump and off-pump coronary artery bypass surgery.

BACKGROUND With the increasing use of OPCAB, potentially devastating thromboembolic events, including graft thrombosis may become increasingly evident. We present a study of the quantitative and temporal differences of the coagulation system, fibrinolysis and platelet activation after coronary artery surgery with or without cardiopulmonary bypass. METHODS Patients undergoing on-pump CABG (n=10) or OPCAB (n=10) had six blood samples taken before surgery and up to 24h post-operatively. Activation of the coagulation cascade (tissue factor pathway-factor VIIa), endothelial injury (von Willebrand Factor antigen), thrombin generation (prothrombin fragments FI+II), fibrinolysis (decreased plasminogen levels), fibrin degradation (D-Dimer), platelet counts and platelet activation (soluble P-selectin) were quantified. RESULTS CABG caused earlier and more significant generation of thrombin, however OPCAB caused a late and sustained generation of thrombin. CABG caused intraoperative activation of fibrinolysis and fibrin degradation, however, at 24h these parameters were equally elevated in both groups. Platelet activation was significant in the CABG group, but did not occur in the OPCAB group. CONCLUSIONS Late thrombin generation and reduced fibrinolysis in the presence of intact, functioning platelets may contribute to adverse thromboembolic events after OPCAB surgery. Thromboembolic prophylaxis and anti-platelet therapy may need to be more aggressive after OPCAB surgery.