Scott M. Gordon

Proteomic Characterization of Human Plasma High Density Lipoprotein Fractionated by Gel Filtration Chromatography

Plasma levels of high density lipoprotein cholesterol (HDL-C) are inversely proportional to the incidence of cardiovascular disease. Recent applications of modern proteomic technologies have identified upward of 50 distinct proteins associated with HDL particles with many of these newly discovered proteins implicating HDL in nonlipid transport processes including complement activation, acute phase response and innate immunity. However, almost all MS-based proteomic studies on HDL to date have utilized density gradient ultracentrifugation techniques for HDL isolation prior to analysis. These involve high shear forces and salt concentrations that can disrupt HDL protein interactions and alter particle function. Here, we used high-resolution size exclusion chromatography to fractionate normal human plasma to 17 phospholipid-containing subfractions. Then, using a phospholipid binding resin, we identified proteins that associate with lipoproteins of various sizes by electrospray ionization mass spectrometry. We identified 14 new phospholipid-associated proteins that migrate with traditionally defined HDL, several of which further support roles for HDL in complement regulation and protease inhibition. The increased fractionation inherent to this method allowed us to visualize HDL protein distribution across particle size with unprecedented resolution. The observed heterogeneity across subfractions suggests the presence of HDL particle subpopulations each with distinct protein components that may prove to impart distinct physiological functions.

High density lipoprotein: it's not just about lipid transport anymore

Plasma levels of high density lipoprotein cholesterol (HDL-C) have long been associated with protection against cardiovascular disease (CVD) in large populations. However, HDL-C has been significantly less useful for predicting CVD risk in individual patients. This has ignited a new debate on the merits of measuring HDL quantity versus quality in terms of protective potential. In addition, numerous recent studies have begun to uncover HDL functions that vary surprisingly from traditional lipid transport roles. In this paper, we review recent findings that point to important functions for HDL that go well beyond lipid transport. These discoveries suggest that HDL might be a platform that mediates protection from a host of disease states ranging from CVD to diabetes to infectious disease.

The Effects of Type 2 Diabetes on Lipoprotein Composition and Arterial Stiffness in Male Youth

Recent studies suggest HDL exists as numerous subpopulations with distinct protein/lipid compositions that are not reflected in the HDL cholesterol (HDL-C) number. In this study, we sought to evaluate HDL subpopulations in adolescents with type 2 diabetes (T2D) to determine if changes in HDL composition are associated with early vascular disease. T2D (n = 10), lean (n = 9), and obese (n = 11) youth were recruited. Plasma was fractionated using gel-filtration chromatography, and lipid-associated proteins were identified using mass spectrometry. Concurrently, vascular stiffness was assessed using pulse wave velocity (PWV). We found youth with T2D exhibited decreased phospholipid content in fractions containing large HDL particles that was inversely associated with PWV (P < 0.001). No association was noted between HDL-C and PWV. Proteomic analysis revealed changes in 7 of 45 identified proteins in the T2D group, including apolipoprotein (apo) A-II, apoE, and paraoxonase-1 (P < 0.05). Our data demonstrate early changes in the lipid and protein compositions of specific HDL subspecies in adolescents with T2D that are related to early markers of arterial disease. These findings suggest that analyzing the composition of HDL, rather than HDL-C, may be useful in assessing cardiovascular risk in this population.

High-Density Lipoprotein Maintains Skeletal Muscle Function by Modulating Cellular Respiration in Mice

Background— Abnormal glucose metabolism is a central feature of disorders with increased rates of cardiovascular disease. Low levels of high-density lipoprotein (HDL) are a key predictor for cardiovascular disease. We used genetic mouse models with increased HDL levels (apolipoprotein A-I transgenic [apoA-I tg]) and reduced HDL levels (apoA-I–deficient [apoA-I ko]) to investigate whether HDL modulates mitochondrial bioenergetics in skeletal muscle. Methods and Results— ApoA-I ko mice exhibited fasting hyperglycemia and impaired glucose tolerance test compared with wild-type mice. Mitochondria isolated from gastrocnemius muscle of apoA-I ko mice displayed markedly blunted ATP synthesis. Endurance capacity during exercise exhaustion test was impaired in apoA-I ko mice. HDL directly enhanced glucose oxidation by increasing glycolysis and mitochondrial respiration rate in C2C12 muscle cells. ApoA-I tg mice exhibited lower fasting glucose levels, improved glucose tolerance test, increased lactate levels, reduced fat mass, associated with protection against age-induced decline of endurance capacity compared with wild-type mice. Circulating levels of fibroblast growth factor 21, a novel biomarker for mitochondrial respiratory chain deficiencies and inhibitor of white adipose lipolysis, were significantly reduced in apoA-I tg mice. Consistent with an increase in glucose utilization of skeletal muscle, genetically increased HDL and apoA-I levels in mice prevented high-fat diet–induced impairment of glucose homeostasis. Conclusions— In view of impaired mitochondrial function and decreased HDL levels in type 2 diabetes mellitus, our findings indicate that HDL-raising therapies may preserve muscle mitochondrial function and address key aspects of type 2 diabetes mellitus beyond cardiovascular disease.

Multi-dimensional Co-separation Analysis Reveals Protein–Protein Interactions Defining Plasma Lipoprotein Subspecies

The distribution of circulating lipoprotein particles affects the risk for cardiovascular disease (CVD) in humans. Lipoproteins are historically defined by their density, with low-density lipoproteins positively and high-density lipoproteins (HDLs) negatively associated with CVD risk in large populations. However, these broad definitions tend to obscure the remarkable heterogeneity within each class. Evidence indicates that each class is composed of physically (size, density, charge) and compositionally (protein and lipid) distinct subclasses exhibiting unique functionalities and differing effects on disease. HDLs in particular contain upward of 85 proteins of widely varying function that are differentially distributed across a broad range of particle diameters. We hypothesized that the plasma lipoproteins, particularly HDL, represent a continuum of phospholipid platforms that facilitate specific protein–protein interactions. To test this idea, we separated normal human plasma using three techniques that exploit different lipoprotein physicochemical properties (gel filtration chromatography, ionic exchange chromatography, and preparative isoelectric focusing). We then tracked the co-separation of 76 lipid-associated proteins via mass spectrometry and applied a summed correlation analysis to identify protein pairs that may co-reside on individual lipoproteins. The analysis produced 2701 pairing scores, with the top hits representing previously known protein–protein interactions as well as numerous unknown pairings. A network analysis revealed clusters of proteins with related functions, particularly lipid transport and complement regulation. The specific co-separation of protein pairs or clusters suggests the existence of stable lipoprotein subspecies that may carry out distinct functions. Further characterization of the composition and function of these subspecies may point to better targeted therapeutics aimed at CVD or other diseases.

Apolipoprotein A-I mimetics and high-density lipoprotein function.

Purpose of reviewTo review recently published advances in the development of apolipoprotein A-I (apoA-I) mimetic peptides as a potential treatment for cardiovascular diseases. Recent findingsVarious apoA-I mimetic peptides are currently in development and these display potent cardioprotective features that can rival or even surpass those of full length apoA-I and high-density lipoproteins (HDLs). These features include the ability to efflux cholesterol from various cell types as well as anti-inflammatory and antioxidative properties. Recent work has been aimed at identifying the structural features of these peptides that are responsible for these various functions and also for determining the operational mechanisms. There is also interesting new data suggesting that the intestine may be playing an important role in the action of these peptides. SummaryIn the last year, there have been many important advances in the relatively new field of apoA-I mimetic therapy. These findings support a strong potential for their development as treatment for not only cardiovascular disease but other disease states involving chronic inflammation and oxidation as well.

Rosuvastatin Alters the Proteome of High Density Lipoproteins: Generation of alpha-1-antitrypsin Enriched Particles with Anti-inflammatory Properties

Statins lower plasma cholesterol by as much as 50%, thus reducing future cardiovascular events. However, the physiological effects of statins are diverse and not all are related to low density lipoprotein cholesterol (LDL-C) lowering. We performed a small clinical pilot study to assess the impact of statins on lipoprotein-associated proteins in healthy individuals (n = 10) with normal LDL-C (<130 mg/dL), who were treated with rosuvastatin (20 mg/day) for 28 days. Proteomic analysis of size-exclusion chromatography isolated LDL, large high density lipoprotein (HDL-L), and small HDL (HDL-S) fractions and spectral counting was used to compare relative protein detection before and after statin therapy. Significant protein changes were found in each lipoprotein pool and included both increases and decreases in several proteins involved in lipoprotein metabolism, complement regulation and acute phase response. The most dramatic effect of the rosuvastatin treatment was an increase in α-1-antirypsin (A1AT) spectral counts associated with HDL-L particles. Quantitative measurement by ELISA confirmed an average 5.7-fold increase in HDL-L associated A1AT. Molecular modeling predictions indicated that the hydrophobic reactive center loop of A1AT, the functional domain responsible for its protease inhibitor activity, is likely involved in lipid binding and association with HDL was found to protect A1AT against oxidative inactivation. Cell culture experiments, using J774 macrophages, demonstrated that the association of A1AT with HDL enhances its antiprotease activity, preventing elastase induced production of tumor necrosis factor α. In conclusion, we show that statins can significantly alter the protein composition of both LDL and HDL and our studies reveal a novel functional relationship between A1AT and HDL. The up-regulation of A1AT on HDL enhances its anti-inflammatory functionality, which may contribute to the non-lipid lowering beneficial effects of statins.

A Comparison of the Mouse and Human Lipoproteome: Suitability of the Mouse Model for Studies of Human Lipoproteins

Plasma levels of low density lipoproteins (LDL) and high density lipoproteins (HDL) exhibit opposing associations with cardiovascular disease in human populations and mouse models have been heavily used to derive a mechanistic understanding of these relationships. In humans, recent mass spectrometry studies have revealed that the plasma lipoproteome is significantly more complex than originally appreciated. This is particularly true for HDL which contains some 90 distinct proteins, a majority of which play functional roles that go beyond those expected for simple lipid transport. Unfortunately, the mouse lipoproteome remains largely uncharacterized-a significant gap given the heavy reliance on the model. Using a gel filtration chromatography and mass spectrometry analysis that targets phospholipid-bound plasma proteins, we compared the mouse lipoproteome and its size distribution to a previous, identical human analysis. We identified 113 lipid associated proteins in the mouse. In general, the protein diversity in the LDL and HDL size ranges was similar in mice versus humans, though some distinct differences were noted. For the majority of proteins, the size distributions, that is, whether a given protein was associated with large versus small HDL particles, for example, were also similar between species. Again, however, there were clear differences exhibited by a minority of proteins that may reflect metabolic differences between species. Finally, by correlating the lipid and protein size profiles, we identified five proteins that closely track with the major HDL protein, apolipoprotein A-I across both species. Thus, mice have most of the minor proteins identified in human lipoproteins that play key roles in inflammation, innate immunity, proteolysis and its inhibition, and vitamin transport. This provides support for the continued use of the mouse as a model for many aspects of human lipoprotein metabolism.

Creation of Apolipoprotein C-II (ApoC-II) Mutant Mice and Correction of Their Hypertriglyceridemia with an ApoC-II Mimetic Peptide

Apolipoprotein C-II (apoC-II) is a cofactor for lipoprotein lipase, a plasma enzyme that hydrolyzes triglycerides (TGs). ApoC-II deficiency in humans results in hypertriglyceridemia. We used zinc finger nucleases to create Apoc2 mutant mice to investigate the use of C-II-a, a short apoC-II mimetic peptide, as a therapy for apoC-II deficiency. Mutant mice produced a form of apoC-II with an uncleaved signal peptide that preferentially binds high-density lipoproteins (HDLs) due to a 3-amino acid deletion at the signal peptide cleavage site. Homozygous Apoc2 mutant mice had increased plasma TG (757.5 ± 281.2 mg/dl) and low HDL cholesterol (31.4 ± 14.7 mg/dl) compared with wild-type mice (TG, 55.9 ± 13.3 mg/dl; HDL cholesterol, 55.9 ± 14.3 mg/dl). TGs were found in light (density < 1.063 g/ml) lipoproteins in the size range of very-low-density lipoprotein and chylomicron remnants (40–200 nm). Intravenous injection of C-II-a (0.2, 1, and 5 μmol/kg) reduced plasma TG in a dose-dependent manner, with a maximum decrease of 90% occurring 30 minutes after the high dose. Plasma TG did not return to baseline until 48 hours later. Similar results were found with subcutaneous or intramuscular injections. Plasma half-life of C-II-a is 1.33 ± 0.72 hours, indicating that C-II-a only acutely activates lipolysis, and the sustained TG reduction is due to the relatively slow rate of new TG-rich lipoprotein synthesis. In summary, we describe a novel mouse model of apoC-II deficiency and show that an apoC-II mimetic peptide can reverse the hypertriglyceridemia in these mice, and thus could be a potential new therapy for apoC-II deficiency.

Kit signaling is required for development of coordinated motility patterns in zebrafish gastrointestinal tract

Interstitial cells of Cajal (ICC) provide a pacemaker signal for coordinated motility patterns in the mammalian gastrointestinal (GI) tract. Kit signaling is required for development and maintenance of ICC, and these cells can be identified by Kit-like immunoreactivity. The zebrafish GI tract has two distinct ICC networks similar to mammals, suggesting a similar role in the generation of GI motility; however, a functional role for Kit-positive cells in zebrafish has not been determined. Analysis of GI motility in intact zebrafish larvae was performed during development and after disruption of Kit signaling. Development of coordinated motility patterns occurred after 5 days post-fertilization (dpf) and correlated with appearance of Kit-positive cells. Disruptions of Kit signaling using the Kit antagonist imatinib mesylate, and in Sparse, a null kita mutant, also disrupted development of coordinated motility patterns. These data suggest that Kit signaling is necessary for development of coordinated motility patterns and that Kit-positive cells in zebrafish are necessary for coordinated motility patterns.