Mark D. Benson

Aptamer-Based Proteomic Profiling Reveals Novel Candidate Biomarkers and Pathways in Cardiovascular Disease.

Background: Single-stranded DNA aptamers are oligonucleotides of ≈50 base pairs in length selected for their ability to bind proteins with high specificity and affinity. Emerging DNA aptamer-based technologies may address limitations of existing proteomic techniques, including low sample throughput, which have hindered proteomic analyses of large cohorts. Methods: To identify early biomarkers of myocardial injury, we applied an aptamer-based proteomic platform that measures 1129 proteins to a clinically relevant perturbational model of planned myocardial infarction (PMI), patients undergoing septal ablation for hypertrophic cardiomyopathy. Blood samples were obtained before and at 10 and 60 minutes after PMI, and protein changes were assessed by repeated-measures analysis of variance. The generalizability of our PMI findings was evaluated in a spontaneous myocardial infarction cohort (Wilcoxon rank-sum). We then tested the platform’s ability to detect associations between proteins and Framingham Risk Score components in the Framingham Heart Study, performing regression analyses for each protein versus each clinical trait. Results: We found 217 proteins that significantly changed in the peripheral vein blood after PMI in a derivation cohort (n=15; P<5.70E-5). Seventy-nine of these proteins were validated in an independent PMI cohort (n=15; P<2.30E-4); >85% were directionally consistent and reached nominal significance. We detected many protein changes that are novel in the context of myocardial injury, including Dickkopf-related protein 4, a WNT pathway inhibitor (peak increase 124%, P=1.29E-15) and cripto, a growth factor important in cardiac development (peak increase 64%, P=1.74E-4). Among the 40 validated proteins that increased within 1 hour after PMI, 23 were also elevated in patients with spontaneous myocardial infarction (n=46; P<0.05). Framingham Heart Study analyses revealed 156 significant protein associations with the Framingham Risk Score (n=899), including aminoacylase 1 (&bgr;=0.3386, P=2.54E-22) and trigger factor 2 (&bgr;=0.2846, P=5.71E-17). Furthermore, we developed a novel workflow integrating DNA-based immunoaffinity with mass spectrometry to analytically validate aptamer specificity. Conclusions: Our results highlight an emerging proteomics tool capable of profiling >1000 low-abundance analytes with high sensitivity and high precision, applicable both to well-phenotyped perturbational studies and large human cohorts, as well.

Genetic Architecture of the Cardiovascular Risk Proteome

Background: We recently identified 156 proteins in human plasma that were each associated with the net Framingham Cardiovascular Disease Risk Score using an aptamer-based proteomic platform in Framingham Heart Study Offspring participants. Here we hypothesized that performing genome-wide association studies and exome array analyses on the levels of each of these 156 proteins might identify genetic determinants of risk-associated circulating factors and provide insights into early cardiovascular pathophysiology. Methods: We studied the association of genetic variants with the plasma levels of each of the 156 Framingham Cardiovascular Disease Risk Score–associated proteins using linear mixed-effects models in 2 population-based cohorts. We performed discovery analyses on plasma samples from 759 participants of the Framingham Heart Study Offspring cohort, an observational study of the offspring of the original Framingham Heart Study and their spouses, and validated these findings in plasma samples from 1421 participants of the MDCS (Malmö Diet and Cancer Study). To evaluate the utility of this strategy in identifying new biological pathways relevant to cardiovascular disease pathophysiology, we performed studies in a cell-model system to experimentally validate the functional significance of an especially novel genetic association with circulating apolipoprotein E levels. Results: We identified 120 locus-protein associations in genome-wide analyses and 41 associations in exome array analyses, the majority of which have not been described previously. These loci explained up to 66% of interindividual plasma protein-level variation and, on average, accounted for 3 times the amount of variation explained by common clinical factors, such as age, sex, and diabetes mellitus status. We described overlap among many of these loci and cardiovascular disease genetic risk variants. Finally, we experimentally validated a novel association between circulating apolipoprotein E levels and the transcription factor phosphatase 1G. Knockdown of phosphatase 1G in a human liver cell model resulted in decreased apolipoprotein E transcription and apolipoprotein E protein levels in cultured supernatants. Conclusions: We identified dozens of novel genetic determinants of proteins associated with the Framingham Cardiovascular Disease Risk Score and experimentally validated a new role for phosphatase 1G in lipoprotein biology. Further, genome-wide and exome array data for each protein have been made publicly available as a resource for cardiovascular disease research.

Application of Large-Scale Aptamer-Based Proteomic Profiling to Planned Myocardial Infarctions

Background: Emerging proteomic technologies using novel affinity-based reagents allow for efficient multiplexing with high-sample throughput. To identify early biomarkers of myocardial injury, we recently applied an aptamer-based proteomic profiling platform that measures 1129 proteins to samples from patients undergoing septal alcohol ablation for hypertrophic cardiomyopathy, a human model of planned myocardial injury. Here, we examined the scalability of this approach using a markedly expanded platform to study a far broader range of human proteins in the context of myocardial injury. Methods: We applied a highly multiplexed, expanded proteomic technique that uses single-stranded DNA aptamers to assay 4783 human proteins (4137 distinct human gene targets) to derivation and validation cohorts of planned myocardial injury, individuals with spontaneous myocardial infarction, and at-risk controls. Results: We found 376 target proteins that significantly changed in the blood after planned myocardial injury in a derivation cohort (n=20; P<1.05E-05, 1-way repeated measures analysis of variance, Bonferroni threshold). Two hundred forty-seven of these proteins were validated in an independent planned myocardial injury cohort (n=15; P<1.33E-04, 1-way repeated measures analysis of variance); >90% were directionally consistent and reached nominal significance in the validation cohort. Among the validated proteins that were increased within 1 hour after planned myocardial injury, 29 were also elevated in patients with spontaneous myocardial infarction (n=63; P<6.17E-04). Many of the novel markers identified in our study are intracellular proteins not previously identified in the peripheral circulation or have functional roles relevant to myocardial injury. For example, the cardiac LIM protein, cysteine- and glycine-rich protein 3, is thought to mediate cardiac mechanotransduction and stress responses, whereas the mitochondrial ATP synthase F0 subunit component is a vasoactive peptide on its release from cells. Last, we performed aptamer-affinity enrichment coupled with mass spectrometry to technically verify aptamer specificity for a subset of the new biomarkers. Conclusions: Our results demonstrate the feasibility of large-scale aptamer multiplexing at a level that has not previously been reported and with sample throughput that greatly exceeds other existing proteomic methods. The expanded aptamer-based proteomic platform provides a unique opportunity for biomarker and pathway discovery after myocardial injury.

Bulging at the Root: An Inflammatory Tale

Information about a real patient is presented in stages (boldface type) to an expert clinician (Dr Bhatt), who responds to the information, sharing his reasoning with the reader (regular type). A discussion by the authors follows. Patient presentation: A 54-year-old man was transferred from an outside institution after he presented to the emergency department following the sudden onset of nonradiating, substernal chest pressure, dyspnea, diaphoresis, and nausea while brushing his teeth. Before transfer, the patient received a full dose of aspirin and was started on unfractionated heparin with biomarker and electrocardiographic evidence of a non–ST-segment–elevation myocardial infarction. His medical history included hypertension, dyslipidemia, long-standing severe atopic dermatitis, and remote large-cell non-Hodgkin lymphoma that had remained in remission for the past 9 years after treatment with rituximab, cyclophosphamide, doxorubicin hydrochloride, vincristine, and prednisolone. Over the preceding year, the patient had suffered recurrent admissions for acutely decompensated heart failure with a newly appreciated depressed left ventricular (LV) ejection fraction of 20% and evidence of global hypokinesis by echocardiography. Regadenoson radionuclide myocardial perfusion imaging had demonstrated a moderate-sized scar in the midanterior wall with no evidence of ischemia. During the same time period, the patient had developed rapidly progressive end-stage renal disease of unknown origin requiring the initiation of hemodialysis. The patient had not yet undergone a renal biopsy. Renal ultrasound had incidentally demonstrated a 4.3-cm infrarenal abdominal aortic aneurysm (AAA) and bilateral common iliac artery aneurysms (right, 2.6 cm; left, 1.7 cm). A computerized tomography (CT) scan had additionally detected a 1.7-cm superior mesenteric artery aneurysm with a dissection. The patient’s medications included daily aspirin 81 mg, atorvastatin 80 mg, lisinopril 2.5 mg, metoprolol succinate 25 mg, furosemide 20 mg, a multivitamin, and sublingual nitroglycerin as needed. He had no allergies to medicines and no history of smoking, alcohol use, or drug …