Jatinderpal K. Sandhu

Vitamin D and prognosis in acute myocardial infarction

BACKGROUND Vitamin D status (VDS) has been linked to mortality and incident acute myocardial infarction (AMI) in healthy cohorts. Associations with recurrent adverse cardiovascular events in those with cardiovascular disease are less clear. Our objective was to assess the prevalence and prognostic impact of VDS on patients presenting with AMI. METHODS We measured plasma 25-(OH)D3 and 25-(OH)D2 using isotope dilution tandem mass spectrometry, in 1259 AMI patients (908 men, mean age 65.7 ± 12.8 years). The primary endpoint was major adverse events (MACE), a composite of death (n=141), heart failure hospitalisation (n=111) and recurrent AMI (n=147) over median follow-up of 550 days (range 131-1095). Secondary endpoints were fatal and non-fatal MACE. RESULTS Almost 74% of the patients were vitamin D deficient (<20 ng/ml 25-(OH)D). Plasma 25-(OH)D existed mainly as 25-(OH)D3 which varied with month of recruitment. Multivariable survival Cox regression models stratified by recruitment month (adjusted for age, gender, past history of AMI/angina, hypertension, diabetes, hypercholesterolaemia, ECG ST change, Killip class, eGFR, smoking, plasma NTproBNP), showed 25-(OH)D3 quartile as an independent predictor of MACE(P<0.001) and non-fatal MACE(P<0.01), but not death. Using the lowest 25-(OH)D3 quartile(<7.3 ng/ml) as reference for MACE prediction, the 2nd, 3rd and 4th quartiles showed significantly lower hazard ratios (HR 0.59(P<0.002), 0.58(P<0.001), and 0.59(P<0.003) respectively). For non-fatal MACE prediction, the 2nd, 3rd and 4th 25-(OH)D3 quartiles were all significantly different from the lowest reference quartile (HR 0.69(P<0.05), 0.54(P<0.003) and 0.59(P<0.014) respectively). CONCLUSIONS VDS is prognostic for MACE (predominantly non-fatal MACE) post-AMI, with approximate 40% risk reduction for 25-(OH)D3 levels above 7.3 ng/ml.

Pleiotropic effects of statins in hypercholesterolaemia: a prospective observational study using a lipoproteomic based approach

BACKGROUND The benefit of statins in the prevention of cardiovascular disease is well founded, derived from their lipid lowering and pleiotropic effects. The concept of lipoproteins as lipid transporters has evolved to encompass functions in coagulation, inflammation, and redox reactions due to their unique protein cargo. The aim of this study was to determine the effect of statin therapy on lipoproteins and their protein cargo by use of an unbiased bottom-up proteomics approach in people with hypercholesterolaemia. METHODS 11 people fulfilling the inclusion criteria were recruited into this UK-based single centre prospective observational study. They were started on statins for primary prevention. Blood was withdrawn at baseline and after a minimum of 2 months of statin therapy. Plasma was co-incubated with a lipoaffinity resin. Isolated proteins were digested and analysed with label-free two-dimensional liquid chromatography coupled to electrospray high-definition ion mobility tandem mass spectrometry. FINDINGS 218 proteins were identified with Progenesis QI software, with 33 proteins demonstrating significant differential expression between the pre-statin and the on-statin samples (each p<0·05). 17 proteins were upregulated by statin therapy, including proteins concerned with cytoskeletal organisation (vinculin p<0·0001, tropomyosin α4 p=0·0108), antioxidative (peroxiredoxin 2 p=0·0092), and anti-inflammatory effects (transgelin-2 p=0·0071). Apolipoprotein B100 was downregulated by statin therapy, consistent with it mechanism of action (p=0·0006). Statin therapy downregulated novel proteins concerned with the modulation of pancreatic β-cell function (adipsin p=0·0056) and haemopoietic precursor proliferation (stem cell growth factor p<0·0001). INTERPRETATION Our findings show that statins remodel the cytoskeletal architecture and mediate various anti-inflammatory, antioxidant, and antiproliferative effects that might limit endothelial dysfunction. The downregulation of adipsin, a novel adipokine that stimulates insulin secretion, could explain the controversial link between statin use and the development of diabetes. This study extends our understanding of the beneficial and harmful pleiotropic effects of statin therapy. FUNDING British Heart Foundation.

Identification of novel biomarkers in plasma for prediction of treatment response in patients with heart failure.

BACKGROUND Heart failure is a complex clinical syndrome that occurs at the end stage of heart disease. Despite advances in therapy for heart failure, improvement of clinical outcomes remains a challenge for physicians. The identification of treatment response early in the course of disease would be useful to improve management of these patients. The aim of this study was to identify novel biomarkers in plasma that could predict treatment response in patients with heart failure. METHODS Patients with heart failure who met inclusion and exclusion criteria according to the guidelines of the European Society of Cardiology were recruited. Uptitration of angiotensin-converting enzyme inhibitors and β blockers was performed over 6 months. Patients were followed up for clinical events within the next 24 months. Plasma proteins in patients who responded to standard treatment (responders) were compared with patients who died or were re-admitted for heart failure (non-responders). Plasma samples were depleted of 14 high abundance proteins with a multiple affinity removal system column (MARS). Then plasma samples were analysed on two-dimensional liquid chromatography coupled to a tandem mass spectrometry (2D LC-ESI-MS/MS) in high definition mode (HDMS(E)) to identify and quantify the different expression of proteins in plasma. Finally, ELISA was used to verify candidate biomarkers. FINDINGS Participants were 100 patients with heart failure matched for sex and age (50 responders [25 women], 50 non-responders [25 women], mean age 76·6 years [SD 8·1]). Of the non-responders, 18 died and 32 were re-admitted to hospital. 2D LC-ESI-MS/MS showed that the expression of neurotrimin (NTM) was highly upregulated, by 26·5 times (p<0·0001), in the responder group compared with the non-responder group. ELISA in the verification phase showed that the concentrations of NTM in plasma were significantly higher in the responders and lower in the non-responders (mean 4·73 log10 relative light units [SD 0·07] vs 4·70 [0·08], p=0·036). When ANOVA with Bonferroni post-hoc comparisons was used in three outcome subgroups (responders, patients re-admitted to hospital, and deaths), NTM concentrations were significantly different between death and the other groups (higher in responder vs death group, p<0·0001; higher in re-admission vs death group, p=0·001). INTERPRETATION Our findings suggest that NTM as a novel biomarker in heart failure will not only add information to understand the pathophysiological mechanisms of heart failure better, but also might provide a more accurate prediction of treatment response to guide medical therapy. In addition, a novel therapeutic target could be identified for design of drugs to improve outcomes. Futher work is required in larger populations to confirm this biomarker. FUNDING European Unions Seventh Framework Programme (BIOSTAT-CHF), John and Lucille van Geest Foundation.

DNMTs are required for delayed genome instability caused by radiation

The ability of ionizing radiation to initiate genomic instability has been harnessed in the clinic where the localized delivery of controlled doses of radiation is used to induce cell death in tumor cells. Though very effective as a therapy, tumor relapse can occur in vivo and its appearance has been attributed to the radio-resistance of cells with stem cell-like features. The molecular mechanisms underlying these phenomena are unclear but there is evidence suggesting an inverse correlation between radiation-induced genomic instability and global hypomethylation. To further investigate the relationship between DNA hypomethylation, radiosensitivity and genomic stability in stem-like cells we have studied mouse embryonic stem cells containing differing levels of DNA methylation due to the presence or absence of DNA methyltransferases. Unexpectedly, we found that global levels of methylation do not determine radiosensitivity. In particular, radiation-induced delayed genomic instability was observed at the Hprt gene locus only in wild-type cells. Furthermore, absence of Dnmt1 resulted in a 10-fold increase in de novo Hprt mutation rate, which was unaltered by radiation. Our data indicate that functional DNMTs are required for radiation-induced genomic instability, and that individual DNMTs play distinct roles in genome stability. We propose that DNMTS may contribute to the acquirement of radio-resistance in stem-like cells.

Pro-substance p for evaluation of risk in acute myocardial infarction.

BACKGROUND Pro-substance P (ProSP) is a stable surrogate marker for labile substance P, which has pro-inflammatory effects, increases platelet aggregation and clot strength, and reduces fibrinolysis. OBJECTIVES This study assessed whether ProSP was associated with poor prognosis after acute myocardial infarction (AMI) to identify novel pathophysiological mechanisms. METHODS ProSP was measured in 1,148 AMI patients (825 men, mean age 66.2 ± 12.8 years). Endpoints were major adverse cardiac events (composite of death, reinfarction, and heart failure [HF] hospitalization), death/reinfarction, and death/HF. GRACE (Global Registry of Acute Coronary Events) scores were compared with ProSP for death and/or reinfarction at 6 months. RESULTS During 2-year follow-up, there were 140 deaths, 112 HF hospitalizations, and 149 re-AMI. ProSP levels were highest on the first 2 days after admission and related to estimated glomerular filtration rate, age, history of diabetes, ischemic heart disease or hypertension, Killip class, left ventricular wall motion index, and sex. Multivariate Cox regression models showed ProSP level was a predictor of major adverse events (hazard ratio [HR]: 1.30; 95% confidence interval [CI]: 1.10 to 1.54; p < 0.002), death and/or AMI (HR: 1.42; 95% CI: 1.20 to 1.68; p < 0.0005), death and/or HF (HR: 1.38; 95% CI: 1.14 to 1.67; p < 0.001). ProSP levels with GRACE scores were independent predictors of 6-month death and/or reinfarction (p < 0.0005 for both). ProSP-adjusted GRACE scores reclassified patients significantly (overall category-free net reclassification improvement of 31.6 (95% CI: 14.3 to 49.0; p < 0.0005) mainly by down-classifying those without endpoints. CONCLUSIONS ProSP levels post-AMI are prognostic for death, recurrent AMI, or HF, and they improve risk prediction of GRACE scores, predominantly by down-classifying risk in those without events.

Improved analysis of vitamin D metabolites in plasma using liquid chromatography tandem mass spectrometry, and its application to cardiovascular research

The accurate and specific measurement of vitamin D is increasingly important for determining the role of vitamin D in the pathogenesis of disease. Liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS) has increasingly become the analytical modality of choice for the analysis of vitamin D. There are many advantages to using LC/MS/MS, such as high specificity and sensitivity to help distinguish the isomers of vitamin D. This rapid method, modified from a Waters Corporation application note, consists of minimal sample manipulation using liquid-liquid extraction and incorporates an internal standard. The supernatant is dried down and injected onto an ultra-high-performance liquid chromatography/electrospray ionization tandem mass spectrometer. The total analysis time is 10 min per injection, enabling high throughput of samples. This method also incorporates two commercial quality control standards to provide a robust system with acceptable coefficient of variation. The analysis of control and heart failure plasma samples showed significant differences in the levels of vitamin D3 between these two groups; however, in the control group, there were individuals who were vitamin D deficient. Overall, the vitamin D3 levels were higher in control samples than in heart failure individuals. As expected, vitamin D2 levels were not observed in many of the samples analysed. This modified method is quick and incorporates an internal standard to allow for any loss in the extraction procedure. The method also includes quality control samples to enable assay standardization. The assay involves inexpensive pre-sample clean-up, aiding high throughput, which is important in many laboratories.

Development of a novel site-specific mutagenesis assay using MALDI-ToF MS (SSMA-MS)

We have developed and validated a novel site-specific mutagenesis assay, termed SSMA-MS, which incorporates MALDI-ToF mass spectrometry (MALDI-MS) analysis as a means of determining the mutations induced by a single DNA adduct. The assay involves ligating an adducted deoxyoligonucleotide into supF containing pSP189 plasmid. The plasmid is transfected into human Ad293 kidney cells allowing replication and therefore repair or a mutagenic event to occur. Escherichia coli indicator bacteria are transformed with recovered plasmid and plasmids containing the insert are identified colormetrically, as they behave as frameshift mutations. The plasmid is then amplified and digested using a restriction cocktail of Mbo11 and Mnl1 to yield 12 bp deoxyoligonucleotides, which are characterized by MALDI-MS. MALDI-MS takes advantage of the difference in molecular weight between bases to identify any induced mutations. This analysis method therefore provides qualitative and quantitative information regarding the type and frequency of mutations induced. This assay was developed and validated using an O6-methyl-2′-deoxyguanosine adduct, which induced the expected GC→AT substitutions, when replicated in human or bacterial cells. This approach can be applied to the study of any DNA adduct in any biologically relevant gene sequence (e.g. p53) in human cells and would be particularly amenable to high-throughput analysis.

Novel biomarkers for prediction of poor treatment response in heart failure to guide therapy

Abstract Background Heart failure is a complex clinical syndrome that occurs at the end stage of heart disease. It is a major health problem in western countries with an overall population prevalence of 2–3% which rises sharply to 10–20% at 75 years of age. Despite advances in therapy for heart failure, improvment of clinical outcomes remains a challenge for physicians: half of patients die within 4 years of diagnosis and 40% of patients, who have been admitted to hospital are dead or re-admitted to hospital within 1 year. The aim of this study was to find plasma proteins that predict differences in clinical response from standard therapy in patients with heart failure. Methods Patients with heart failure who met inclusion and exclusion criteria were recruited. Uptitration of angiotensin-converting-enzyme-inhibitors and β blockers was done over 6 months. Patients were followed up for clinical events such as death and admission to hospital for heart failure within the next 24 months. We compared plasma proteins in 50 patients who responded to standard treatment (responders) with 50 patients who died or were re-admitted to hospital (non-responders). Plasma samples were pooled and depleted of 14 high abundance proteins and then reduced and alkylated, before digestion with trypsin to peptides. Peptides were analysed on two-dimensional liquid chromatography coupled to a tandem mass spectrometry in high definition mode which used a high pH reverse phase liquid chromatography in the first step before separation with conventional low pH reverse phase columns in the second step. Findings 434 proteins were identified in the plasma of patients with heart failure. 137 proteins in both responders and non-responders were significantly overexpressed or underexpressed (p Interpretation The discovery of novel biomarkers in this study will not only help to understand the pathophysiology of heart failure better, but also lead to the development of a more personalised approach in predicting treatment response to guide medical therapy. For example, in patients with heart failure who have abnormally increased concentrations of retinol-binding protein 4, a marker for matrix and cellular remodelling, a drug for reducing collagen deposition could be useful. In this way, unnecessary treatment in non-responders could be avoided (with implications for cost and adverse effects) and novel therapeutic targets could be identified for design of therapies to improve outcomes. Funding European Union FP7 Project.

20 Proteomics of human plasma in diastolic heart failure (DHF) using novel chemical affinity, mixed mode matrix (M3)

Background The understanding of diastolic heart failure (DHF) has developed in recent years, a condition that was often misdiagnosed for systolic heart failure (SHF). Although there are constant advances in diagnostic technologies, treatment and diagnosis for DHF still remain a challenge prompting a need for biomarker discovery within human plasma. Prospecting for new protein biomarkers can be hindered by the presence of high abundant proteins (HAPs), masking the appearance of potentially diagnostic low abundance proteins (LAPs) due to their dominance in the sample matrix. A novel method, termed Mixed Mode Matrix (M3), is described to reduce the complexity and improve the dynamic range of plasma proteomics to investigate biomarkers in DHF. A pilot study involving controls (n = 10) and disease (DHF n = 10, SHF n = 10) plasma was carried out. Method HAPs were depleted using MARS14 column, bound to M3, eluted into fractions using increasing concentrations of NaH2PO4 and purified by solid-phase-extraction. A BCA protein assay was used to determine protein concentration prior to digestion. Peptides were separated using high-resolution liquid-chromatography (nano-UPLC) and high-definition-MSE analysis. Results A method for LAPs enrichment was developed. Over 300 LAPs were identified with 1% false discovery rate and contained at least 2 identified peptides. In depleting and enriching plasma with M3, we are able to expand the dynamic range of plasma thus enabling us to identify the LAPs. Conclusion M3 has the potential to improve efficiency and cost-effectiveness of LAPs enrichment, leading to plasma protein biomarker identification for diagnosis and treatment of a multitude of disease states.

Growth hormone for risk stratification and effects of therapy in acute myocardial infarction

Abstract Context: Excess growth hormone (GH) is associated with early mortality. Objectives: We assessed the association of GH with prognosis after acute myocardial infarction (AMI), and the effects of secondary prevention therapies. Methods: GH was measured using a high-sensitivity assay in 953 AMI patients (687 males, mean age 66.1 ± 12.8 years). Results: During 2 years follow-up, there were 281 major adverse cardiac events (MACE). Patients with MACE had higher GH levels (median [range], 0.91 [0.04–26.28] μg/L) compared to event-free survivors (0.59 [0.02–21.6], p < 0.0005). In multivariate Cox survival analysis, GH was a significant predictor of MACE (hazard ratios 1.43, p = 0.026 and 1.49, p = 0.01, respectively) with significant interactions with beta blocker therapy (p = 0.047) and angiotensin converting enzyme inhibitor or angiotensin receptor blocker (ACE/ARB) therapy (p = 0.016). Conclusions: GH levels post-AMI are prognostic for MACE and may indicate those patients who benefit from beta blocker and ACE/ARB therapy.