Identification of cis-regulatory variation influencing protein abundance levels in human plasma.

Proteins are central to almost all cellular processes, and dysregulation of expression and function is associated with a range of disorders. A number of studies in human have recently shown that genetic factors significantly contribute gene expression variation. In contrast, very little is known about the genetic basis of variation in protein abundance in man. Here, we assayed the abundance levels of proteins in plasma from 96 elderly Europeans using a new aptamer-based proteomic technology and performed genome-wide local (cis-) regulatory association analysis to identify protein quantitative trait loci (pQTL). We detected robust cis-associations for 60 proteins at a false discovery rate of 5%. The most highly significant single nucleotide polymorphism detected was rs7021589 (false discovery rate, 2.5 × 10(-12)), mapped within the gene coding sequence of Tenascin C (TNC). Importantly, we identified evidence of cis-regulatory variation for 20 previously disease-associated genes encoding protein, including variants with strong evidence of disease association show significant association with protein abundance levels. These results demonstrate that common genetic variants contribute to the differences in protein abundance levels in human plasma. Identification of pQTLs will significantly enhance our ability to discover and comprehend the biological and functional consequences of loci identified from genome-wide association study of complex traits. This is the first large-scale genetic association study of proteins in plasma measured using a novel, highly multiplexed slow off-rate modified aptamer (SOMAmer) proteomic platform.

Age-dependent changes in the cerebrospinal fluid proteome by slow off-rate modified aptamer array.

An important precondition for the successful development of diagnostic assays of cerebrospinal fluid (CSF) biomarkers of age-related neurodegenerative diseases is an understanding of the dynamic nature of the CSF proteome during the normal aging process. In this study, a novel proteomic technology was used to quantify hundreds of proteins simultaneously in the CSF from 90 cognitively normal adults 21 to 85 years of age. SomaLogic's highly multiplexed proteomic platform can measure more than 800 proteins simultaneously from small volumes of biological fluids using novel slow off-rate modified aptamer (SOMAmer) protein affinity reagents with sensitivity, specificity, and dynamic ranges that meet or exceed those of enzyme-linked immunosorbent assays. In the first application of this technology to CSF, we detected 248 proteins that possessed signals greater than twofold over background. Several novel correlations between detected protein concentrations and age were discovered that indicate that both inflammation and response to injury in the central nervous system may increase with age. Applying this powerful proteomic approach to CSF provides potential new insight into the aging of the human central nervous system that may have utility in discovering new disease-related changes in the CSF proteome.

The induction of human superoxide dismutase and catalase in vivo: a fundamentally new approach to antioxidant therapy.

A composition consisting of extracts of five widely studied medicinal plants (Protandim) was administered to healthy human subjects ranging in age from 20 to 78 years. Individual ingredients were selected on the basis of published findings of induction of superoxide dismutase (SOD) and/or catalase in rodents in vivo, combined with evidence of decreasing lipid peroxidation. Each ingredient was present at a dosage sufficiently low to avoid any accompanying unwanted pharmacological effects. Blood was analyzed before supplementation and after 30 and 120 days of supplementation (675 mg/day). Erythrocytes were assayed for SOD and catalase, and plasma was assayed for lipid peroxidation products as thiobarbituric acid-reacting substances (TBARS), as well as uric acid, C-reactive protein, and cholesterol (total, LDL, and HDL). Before supplementation, TBARS showed a strong age-dependent increase. After 30 days of supplementation, TBARS declined by an average of 40% (p = 0.0001) and the age-dependent increase was eliminated. By 120 days, erythrocyte SOD increased by 30% (p < 0.01) and catalase by 54% (p < 0.002). We conclude that modest induction of the catalytic antioxidants SOD and catalase may be a much more effective approach than supplementation with antioxidants (such as vitamins C and E) that can, at best, stoichiometrically scavenge a very small fraction of total oxidant production.

Oxidative stress in organ preservation: a multifaceted approach to cardioplegia.

Every transplant is a reperfused organ and, therefore, undergoes some degree of oxidative damage. Postischemic reperfusion injury results in non-specific free radical-mediated acute endothelial damage, cell death and organ failure. The endothelium is a key site of injury from reactive oxygen species (ROS), and the endothelial cell dysfunction is central to the pathogenesis of arteriosclerosis. Accelerated arteriosclerosis, secondary to chronic allograft rejection, is a major long-term complication of heart transplantation. Therefore, preservation methods that would decrease injury during reperfusion are very important. We have developed a unique preservation solution, with a multifaceted approach, which best preserves the organ from ROS for an extended period of time before transplantation. The advantages of extending this period of preservation include an expansion of the donor pool, by permitting more distant procurement, the ability to perform detailed tissue typing, therefore, improves histocompatibility match and a reduction in emergency surgery as a result of graft rejection.

A novel heparin-binding, human chimeric, superoxide dismutase improves myocardial preservation and protects from ischemia-reperfusion injury.

BACKGROUND: The plasma membranes of endothelial cells are sites of physiologic injury caused by superoxide attack, whether the radicals are generated within the cell (i.e., from enzymatic sources such as xanthine oxidase or from ischemically injured mitochondria) or are generated within the interstitial spaces by activated neutrophils or macrophages. An extracellular superoxide dismutase (ECSOD) electrostatically bound to endothelial surfaces partially protects against this oxidative attack. To provide a therapeutic equivalent of this ECSOD activity, we evaluated the product of a fusion gene encoding a chimeric manganese SOD (chimeric-SOD) and the carboxyl-terminal 26-amino acid basic "tail" from ECSOD with high affinity for heparin-like proteoglycans on cell surfaces. METHODS: We tested the chimeric-SOD in isolated rabbit hearts during warm and cold ischemia. RESULTS: When perfused through an isolated rabbit heart, chimeric-SOD bound to endothelial surfaces and was displaced by a bolus dose of heparin. In an established model of no-flow ischemia followed by reperfusion of the isolated rabbit heart, the chimeric-SOD was as protective as native Mn-SOD or Cu,Zn-SOD, but at doses nearly 2 orders of magnitude lower. In a rabbit-heart preservation model, the chimeric-SOD provided better recovery of function after 4 hours of cold ischemia than did University of Wisconsin cardioplegia solution. CONCLUSION: This chimeric-SOD can bind to cell surfaces and may aid in preventing superoxide-mediated endothelial damage and may function as a rational therapeutic agent for treating free-radical-mediated diseases.

Candidate blood proteome markers of Alzheimer's disease onset and progression: a systematic review and replication study.

A blood-based protein biomarker, or set of protein biomarkers, that could predict onset and progression of Alzheimer's disease (AD) would have great utility; potentially clinically, but also for clinical trials and especially in the selection of subjects for preventative trials. We reviewed a comprehensive list of 21 published discovery or panel-based (> 100 proteins) blood proteomics studies of AD, which had identified a total of 163 candidate biomarkers. Few putative blood-based protein biomarkers replicate in independent studies but we found that some proteins do appear in multiple studies; for example, four candidate biomarkers are found to associate with AD-related phenotypes in five independent research cohorts in these 21 studies: α-1-antitrypsin, α-2-macroglobulin, apolipoprotein E, and complement C3. Using SomaLogic's SOMAscan proteomics technology, we were able to conduct a large-scale replication study for 94 of the 163 candidate biomarkers from these 21 published studies in plasma samples from 677 subjects from the AddNeuroMed (ANM) and the Alzheimer's Research UK/Maudsley BRC Dementia Case Registry at King's Health Partners (ARUK/DCR) research cohorts. Nine of the 94 previously reported candidates were found to associate with AD-related phenotypes (False Discovery Rate (FDR) q-value < 0.1). These proteins show sufficient replication to be considered for further investigation as a biomarker set. Overall, we show that there are some signs of a replicable signal in the range of proteins identified in previous studies and we are able to further replicate some of these. This suggests that AD pathology does affect the blood proteome with some consistency.

Aptamer-based multiplexed proteomic technology for biomarker discovery.

BACKGROUND: The interrogation of proteomes ("proteomics") in a highly multiplexed and efficient manner remains a coveted and challenging goal in biology and medicine. METHODOLOGY/PRINCIPAL FINDINGS: We present a new aptamer-based proteomic technology for biomarker discovery capable of simultaneously measuring thousands of proteins from small sample volumes (15 µL of serum or plasma). Our current assay measures 813 proteins with low limits of detection (1 pM median), 7 logs of overall dynamic range (~100 fM-1 µM), and 5% median coefficient of variation. This technology is enabled by a new generation of aptamers that contain chemically modified nucleotides, which greatly expand the physicochemical diversity of the large randomized nucleic acid libraries from which the aptamers are selected. Proteins in complex matrices such as plasma are measured with a process that transforms a signature of protein concentrations into a corresponding signature of DNA aptamer concentrations, which is quantified on a DNA microarray. Our assay takes advantage of the dual nature of aptamers as both folded protein-binding entities with defined shapes and unique nucleotide sequences recognizable by specific hybridization probes. To demonstrate the utility of our proteomics biomarker discovery technology, we applied it to a clinical study of chronic kidney disease (CKD). We identified two well known CKD biomarkers as well as an additional 58 potential CKD biomarkers. These results demonstrate the potential utility of our technology to rapidly discover unique protein signatures characteristic of various disease states. CONCLUSIONS/SIGNIFICANCE: We describe a versatile and powerful tool that allows large-scale comparison of proteome profiles among discrete populations. This unbiased and highly multiplexed search engine will enable the discovery of novel biomarkers in a manner that is unencumbered by our incomplete knowledge of biology, thereby helping to advance the next generation of evidence-based medicine.

Bucillamine, a thiol antioxidant, prevents transplantation-associated reperfusion injury.

Ischemia/reperfusion (I/R) injury is a serious potential threat to outcomes in organ transplantation and other clinical arenas in which there is temporary interruption of blood flow. I/R is a frequent cause of primary failure in organ transplantation. We hypothesized that the antioxidant bucillamine, a potent sulfhydryl donor, would protect against I/R injury in high-risk organ transplants. Because livers subjected to prolonged ischemia and very fatty livers are highly susceptible to severe I/R injury, we studied the effect of bucillamine in three animal models of liver transplantation: two ex vivo models of isolated perfused livers, either normal or fatty rat livers, and an in vivo model of syngenic orthotopic liver transplants in rats. In all models, livers were deprived of oxygen for 24 h before either ex vivo reperfusion or transplantation. In the ex vivo models, bucillamine treatment significantly improved portal vein blood flow and bile production, preserved normal liver architecture, and significantly reduced liver enzyme release and indices of oxidative stress. Moreover, bucillamine treatment significantly increased levels of reduced glutathione in the liver and lowered levels of oxidized glutathione in both liver and blood. In rats subjected to liver transplants, bucillamine significantly enhanced survival and protected against hepatic injury. Possible mechanisms of this protection include prevention of excessive accumulation of toxic oxygen species, interruption of redox signaling in hepatocytes, and inhibition of macrophage activation. This study demonstrates the potential utility of bucillamine or other cysteine-derived thiol donors for improving outcomes in organ transplantation and other clinical settings involving I/R injury.

Thiol antioxidants inhibit the adjuvant effects of aerosolized diesel exhaust particles in a murine model for ovalbumin sensitization.

Although several epidemiological studies indicate a correlation between exposure to ambient particulate matter and adverse health effects in humans, there is still a fundamental lack of understanding of the mechanisms involved. We set out to test the hypothesis that reactive oxygen species are involved in the adjuvant effects of diesel exhaust particles (DEP) in a murine OVA sensitization model. First, we tested six different antioxidants, N-acetylcysteine (NAC), bucillamine (BUC), silibinin, luteolin, trolox (vitamin E), and ascorbic acid, for their ability to interfere in DEP-mediated oxidative stress in vitro. Of the six agents tested, only the thiol antioxidants, BUC and NAC, were effective at preventing a decrease in intracellular reduced glutathione:glutathione disulfide ratios, protecting cells from protein and lipid oxidation, and preventing heme oxygenase 1 expression. Therefore, we selected the thiol antioxidants for testing in the murine OVA inhalation sensitization model. Our data demonstrate that NAC and BUC effectively inhibited the adjuvant effects of DEP in the induction of OVA-specific IgE and IgG1 production. Furthermore, NAC and BUC prevented the generation of lipid peroxidation and protein oxidation in the lungs of OVA- plus DEP-exposed animals. These findings indicate that NAC and BUC are capable of preventing the adjuvant effects of inhaled DEP and suggest that oxidative stress is a key mechanistic component in the adjuvant effect of DEP. Antioxidant treatment strategies may therefore serve to alleviate allergic inflammation and may provide a rational basis for treating the contribution of particulate matter to asthmatic disease.