High-density Lipoprotein Proteome Research Articles

Abstract 3040: Quantitative Proteomic Analysis Of HDL Shows Apolipoprotein M (ApoM) As A Possible Marker For COVID-19 Mortality

Introduction: High-density lipoprotein (HDL) comprises a heterogeneous group of particles that differ in many aspects, such as density, size, composition and function. The use of mass spectrometry-based proteomics to investigate HDL protein composition has brought some new insights into the field. The qualitative and quantitative HDL proteome is remodeled in different disease states, such as diabetes mellitus, hypercholesterolemia and kidney dysfunction. Beyond its established role in inflammatory and cardiovascular diseases, HDL's involvement in infectious diseases, such as COVID-19, has recently come to light. Here, we investigate the remodeling of HDL proteome in hospitalized and non-hospitalized COVID-19 patients. Hypothesis: We hypothesize HDL proteome is remodeled in infectious diseases and may be a marker and perhaps a predictor of a poor prognosis. Methods: We implemented a robust, global label-free quantification proteomic approach to unravel relative changes in HDL protein composition. We analyzed the proteome of COVID-19 subjects from Brazil categorized in two groups, hospitalized (n=30) and non-hospitalized (n=11). Results: Comparative analysis between hospitalized and non-hospitalized patients revealed a more than 50% increase in relative levels of certain proteins in hospitalized patients, including the inflammatory proteins serum amyloid A1 and A2, as well as pulmonary surfactant-associated protein B. We also observed reductions in the lipid-metabolism related apolipoprotein A2 and phospholipid transfer protein. These findings, independent of sex, triglyceride and HDL-cholesterol, suggest an inflammatory remodeling of the HDL particles. Further analysis of surviving (n=35) and deceased patients (n=6) indicated a negative association between ApoM levels and odds of death due to COVID-19 complications (odds ratio per 1-SD increase in ApoM was 0.27 [0.07 to 0.72], P=0.007). Conclusions: Our results point out HDL remodeling is a marker of COVID-19 severity and ApoM-carried sphingosine-1-phosphate signaling pathway may play a pivotal role in the disease outcome.

Targeting the High-Density Lipoprotein Proteome for the Treatment of Post-Acute Sequelae of SARS-CoV-2.

Here, we target the high-density lipoprotein (HDL) proteome in a case series of 16 patients with post-COVID-19 symptoms treated with HMG-Co-A reductase inhibitors (statin) plus angiotensin II type 1 receptor blockers (ARBs) for 6 weeks. Patients suffering from persistent symptoms (post-acute sequelae) after serologically confirmed SARS-CoV-2 infection (post-COVID-19 syndrome, PCS, n = 8) or following SARS-CoV-2 vaccination (PVS, n = 8) were included. Asymptomatic subjects with corresponding serological findings served as healthy controls (n = 8/8). HDL was isolated using dextran sulfate precipitation and the HDL proteome of all study participants was analyzed quantitatively by mass spectrometry. Clinical symptoms were assessed using questionnaires before and after therapy. The inflammatory potential of the patients' HDL proteome was addressed in human endothelial cells. The HDL proteome of patients with PCS and PVS showed no significant differences; however, compared to controls, the HDL from PVS/PCS patients displayed significant alterations involving hemoglobin, cytoskeletal proteins (MYL6, TLN1, PARVB, TPM4, FLNA), and amyloid precursor protein. Gene Ontology Biological Process (GOBP) enrichment analysis identified hemostasis, peptidase, and lipoprotein regulation pathways to be involved. Treatment of PVS/PCS patients with statins plus ARBs improved the patients' clinical symptoms. After therapy, three proteins were significantly increased (FAM3C, AT6AP2, ADAM10; FDR < 0.05) in the HDL proteome from patients with PVS/PCS. Exposure of human endothelial cells with the HDL proteome from treated PVS/PCS patients revealed reduced inflammatory cytokine and adhesion molecule expression. Thus, HDL proteome analysis from PVS/PCS patients enables a deeper insight into the underlying disease mechanisms, pointing to significant involvement in metabolic and signaling disturbances. Treatment with statins plus ARBs improved clinical symptoms and reduced the inflammatory potential of the HDL proteome. These observations may guide future therapeutic strategies for PVS/PCS patients.

Bridging the Gap Between the Bench and Bedside: Clinical Applications of High-density Lipoprotein Function.

Decades of research have reshaped our understanding of high-density lipoprotein (HDL) , shifting our focus from cholesterol (C) levels to multifaceted functionalities. Epidemiological studies initially suggested an association between HDL-C levels and cardiovascular disease (CVD) risk; however, such a simple association has not been indicated by recent studies. Notably, genome-wide studies have highlighted discrepancies between HDL-C levels and CVD outcomes, urging a deeper exploration of the role of HDL. The key to this shift lies in elucidating the role of HDL in reverse cholesterol transport (RCT), which is a fundamental anti-atherosclerotic mechanism. Understanding RCT has led to the identification of therapeutic targets and novel interventions for atherosclerosis. However, clinical trials have underscored the limitations of HDL-C as a therapeutic target, prompting the re-evaluation of the role of HDL in disease prevention. Further investigations have revealed the involvement of HDL composition in various diseases other than CVD, including chronic kidney disease, Alzheimer's disease, and autoimmune diseases. The anti-inflammatory, antioxidative, and anti-infectious properties of HDL have emerged as crucial aspects of its protective function, opening new avenues for novel biomarkers and therapeutic targets. Omics technologies have provided insights into the diverse composition of HDL, revealing disease-specific alterations in the HDL proteome and lipidome. In addition, combining cell-based and cell-free assays has facilitated the evaluation of the HDL functionality across diverse populations, offering the potential for personalized medicine. Overall, a comprehensive understanding of HDL multifunctionality leads to promising prospects for future clinical applications and therapeutic developments, extending beyond cardiovascular health.

Understanding HDL Metabolism and Biology Through In Vivo Tracer Kinetics.

HDL (high-density lipoprotein), owing to its high protein content and small size, is the densest circulating lipoprotein. In contrast to lipid-laden VLDL (very-low-density lipoprotein) and LDL (low-density lipoprotein) that promote atherosclerosis, HDL is hypothesized to mitigate atherosclerosis via reverse cholesterol transport, a process that entails the uptake and clearance of excess cholesterol from peripheral tissues. This process is mediated by APOA1 (apolipoprotein A-I), the primary structural protein of HDL, as well as by the activities of additional HDL proteins. Tracer-dependent kinetic studies are an invaluable tool to study HDL-mediated reverse cholesterol transport and overall HDL metabolism in humans when a cardiovascular disease therapy is investigated. Unfortunately, HDL cholesterol-raising therapies have not been successful at reducing cardiovascular events suggesting an incomplete picture of HDL biology. However, as HDL tracer studies have evolved from radioactive isotope- to stable isotope-based strategies that in turn are reliant on mass spectrometry technologies, the complexity of the HDL proteome and its metabolism can be more readily addressed. In this review, we outline the motivations, timelines, advantages, and disadvantages of the various tracer kinetics strategies. We also feature the metabolic properties of select HDL proteins known to regulate reverse cholesterol transport, which in turn underscore that HDL lipoproteins comprise a heterogeneous particle population whose distinct protein constituents and kinetics likely determine its function and potential contribution to cholesterol clearance.

Associations between neighborhood socioeconomic deprivation, IFNγ, and high-density lipoprotein particle size: Data from the Washington, D.C. cardiovascular health and needs assessment

IntroductionNeighborhood socioeconomic deprivation is associated with increased cardiovascular risk factors, including inflammation. Inflammation plays an important role in modifying the cardioprotective function of high-density lipoprotein (HDL). Moreover, recent studies suggest that very high HDL is associated with adverse cardiovascular disease (CVD) outcomes. Thus, we sought to explore the relationships between neighborhood socioeconomic deprivation as a marker of chronic stress, inflammation, proprotein convertase subtilisin/kexin type 9 (PCSK9) (a core component of the HDL proteome), HDL characterisitcs, and biological aging as a predictor of CVD and all-cause mortality. MethodsSixty African American subjects were recruited to the NIH Clinical Center as part of a community-based participatory research-designed observational study. Neighborhood deprivation index (NDI), a marker of neighborhood socioeconomic deprivation, was measured using US Census data. HDL characteristics (cholesterol, particle number, size, subspecies) were determined from NMR lipoprotein profiling, and plasma cytokines (IL-1β, IL-6, IL-8, TNFα, IFNγ) were measured using an ELISA-based multiplex technique. Epigenetic clock biomarkers of aging were measured using DNA methylation data obtained from participants' buffy coat samples. We used linear regression modeling adjusted for atherosclerotic cardiovascular disease (ASCVD) risk score, body mass index (BMI), and lipid-lowering medication use to investigate relationships of interest. ResultsNDI directly associated with large HDL particle count (H7P) and IFNγ and trended toward significance with HDL-C and PCSK9. IFNγ and PCSK9 then directly associated with H7P. H7P also directly associated with higher DNA methylation phenotypic age (PhenoAge). ConclusionWe highlight associations between neighborhood socioeconomic deprivation, IFNγ, PCSK9, HDL subspecies, and epigenetic biomarkers of aging. Taken together, our findings suggest indirect pathways linking neighborhood deprivation-related stress and inflammation to HDL and immune epigenetic changes. Moreover, these results add to recent work showing the pathogenicity of high HDL levels and underscore the need to understand how chronic stress-related inflammation and lipoprotein subspecies relate to CVD risk across diverse populations.

ONS in high-density lipoprotein proteome and lipidome associated with the improvement in cholesterol efflux capacity in individuals with acute myocardial infarction: A longitudinal study

ONS in high-density lipoprotein proteome and lipidome associated with the improvement in cholesterol efflux capacity in individuals with acute myocardial infarction: A longitudinal study

HDL functionality is dependent on hepatocyte stress defense factors Nrf1 and Nrf2.

High density lipoproteins (HDL) promote homeostasis and counteract stressful tissue damage that underlie cardiovascular and other diseases by mediating reverse cholesterol transport, reducing inflammation, and abrogating oxidative damage. However, metabolically stressful conditions associated with atherosclerosis can impair these effects. Hepatocytes play a major role in the genesis and maturation of circulating HDL, and liver stress elicits marked regulatory changes to circulating HDL abundance and composition, which affect its functionality. The mechanisms linking liver stress to HDL function are incompletely understood. In this study, we sought to determine whether stress defending transcription factors nuclear factor erythroid 2 related factor-1 (Nrf1) and -2 (Nrf2) promote hepatocyte production of functional HDL. Using genetically engineered mice briefly fed a mild metabolically stressful diet, we investigated the effect of hepatocyte-specific deletion of Nrf1, Nrf2, or both on circulating HDL cholesterol, protein composition, and function. Combined deletion, but not single gene deletion, reduced HDL cholesterol and apolipoprotein A1 levels as well as the capacity of HDL to accept cholesterol undergoing efflux from cultured macrophages and to counteract tumor necrosis factor α-induced inflammatory effect on cultured endothelial cells. This coincided with substantial alteration to the HDL proteome, which correlated with liver gene expression profiles of corresponding proteins. Thus, our findings show complementary actions by hepatocyte Nrf1 and Nrf2 play a role in shaping HDL abundance and composition to promote production of functionally viable HDL. Consequently, our study illuminates the possibility that enhancing stress defense programming in the liver may improve atheroprotective and perhaps other health promoting actions of HDL.

Strategies for consistent and automated quantification of HDL proteome using data-independent acquisition

The introduction of mass spectrometry-based proteomics has revolutionized the high-density lipoprotein (HDL) field, with the description, characterization, and implication of HDL-associated proteins in an array of pathologies. However, acquiring robust, reproducible data is still a challenge in the quantitative assessment of HDL proteome. Data-independent acquisition (DIA) is a mass spectrometry methodology that allows the acquisition of reproducible data, but data analysis remains a challenge in the field. To date, there is no consensus on how to process DIA-derived data for HDL proteomics. Here, we developed a pipeline aiming to standardize HDL proteome quantification. We optimized instrument parameters and compared the performance of four freely available, user-friendly software tools (DIA-NN, EncyclopeDIA, MaxDIA, and Skyline) in processing DIA data. Importantly, pooled samples were used as quality controls throughout our experimental setup. A careful evaluation of precision, linearity, and detection limits, first using E. coli background for HDL proteomics and second using HDL proteome and synthetic peptides, was undertaken. Finally, as a proof of concept, we employed our optimized and automated pipeline to quantify the proteome of HDL and apolipoprotein B–containing lipoproteins. Our results show that determination of precision is key to confidently and consistently quantifying HDL proteins. Taking this precaution, any of the available software tested here would be appropriate for quantification of HDL proteome, although their performance varied considerably.

Proteomic analysis of postprandial high-density lipoproteins in healthy subjects

The relationship between the functionality and composition of high-density lipoproteins (HDL) is yet not fully studied, and little is known about the influence of the diet in HDL proteome. Therefore, the aim of this research was to elucidate the HDL proteome associated to postprandial hyperlipidemia. Male volunteers were recruited for an interventional study with high fatty acid-based meals. Blood samples were collected before the intake (baseline), and 2–3 (postprandial peak) and 5–6 (postprandial post peak) hours later. HDL were purified and the protein composition was quantified by LC-MS/MS. Statistical analysis was performed by lineal models (amica) and by ANOVA and multi-t-test of the different conditions (MetaboAnalyst). Additionally, a clustering of the expression profiles of each protein was done with coseq R package (RStudio). Initially, 320 proteins were identified but only 119 remained after the filtering. APOM, APOE, APOB, and APOA2, proteins previously identified in the HDL proteome, were the only proteins with a statistically significant altered expression in postprandial hyperlipidemia when compared to baseline (p values <0.05 and logFC >1). In conclusion, we have been able to describe several behaviors of the whole HDL proteome during the postprandial hyperlipidemic metabolism.

Metabolic labeling unveils alterations in the turnover of HDL-associated proteins during diabetes progression in mice.

Several aspects of diabetes pathophysiology and complications result from hyperglycemia-induced alterations in the structure and function of plasma proteins. Furthermore, insulin has a significant influence on protein metabolism by affecting both the synthesis and degradation of proteins in various tissues. To understand the role of progressive hyperglycemia on plasma proteins, in this study, we measured the turnover rates of high-density lipoprotein (HDL)-associated proteins in control (chow diet), prediabetic [a high-fat diet (HFD) for 8 wk] or diabetic [HFD for 8 wk with low-dose streptozotocin (HFD + STZ) in weeks 5-8 of HFD] C57BL/6J mice using heavy water (2H2O)-based metabolic labeling approach. Compared with control mice, HFD and HFD + STZ mice showed elevations of fasting plasma glucose levels in the prediabetic and diabetic range, respectively. Furthermore, the HFD and HFD + STZ mice showed increased hepatic triglyceride (TG) levels, total plasma cholesterol, and plasma TGs. The kinetics of 40 proteins were quantified using the proteome dynamics method, which revealed an increase in the fractional synthesis rate (FSR) of HDL-associated proteins in the prediabetic mice compared with control mice, and a decrease in FSR in the diabetic mice. The pathway analysis revealed that proteins with altered turnover rates were involved in acute-phase response, lipid metabolism, and coagulation. In conclusion, prediabetes and diabetes have distinct effects on the turnover rates of HDL proteins. These findings suggest that an early dysregulation of the HDL proteome dynamics can provide mechanistic insights into the changes in protein levels in these conditions.NEW & NOTEWORTHY This study is the first to examine the role of gradual hyperglycemia during diabetes disease progression on HDL-associated protein dynamics in the prediabetes and diabetic mice. Our results show that the fractional synthesis rate of HDL-associated proteins increased in the prediabetic mice whereas it decreased in the diabetic mice compared with control mice. These kinetic changes can help to elucidate the mechanism of altered protein levels and HDL dysfunction during diabetes disease progression.

Abstract 197: Proteomic Characterization Of Human Brain Lipoproteins Speciated By Size Exclusion Chromatography

Like plasma, the cerebrospinal fluid (CSF) contains lipid-protein complexes called lipoproteins tasked with transporting lipids and protecting against cerebrovascular dysfunction. These brain lipoproteins (BLps) are generated de novo in the central nervous system (CNS), but little is known about them, because they exist in CSF at &lt;0.5% the concentration of lipoproteins in plasma making them difficult to study. BLps are thought to resemble plasma high-density lipoproteins (HDL), a heterogenous population of compositionally distinct particles important for transporting lipid, protecting against oxidative stress, and modulating immune and inflammatory responses in the periphery. Mass spectrometry (LC-MS/MS) studies have shown that 260 different proteins can interact with different HDLs in the periphery and specific combinations of these proteins cluster on the lipid surface to govern particle function. In the current study, we developed a lipoprotein profiling technology that overcomes the BLp abundance problem in CSF to capture BLp subspecies with unprecedented sensitivity and resolution. Using high-resolution size exclusion chromatography, we identified at least 10 distinctly sized BLp populations in human CSF with a majority of the particles eluting in size ranges between plasma low-density lipoproteins (LDL) and HDL. BLps were isolated from fractionated CSF using a lipid-binding resin. Proteomic analysis of the particles using LC-MS/MS identified 299 different lipid-associated proteins across fractions with 189 of those proteins not currently reported on the human HDL Proteome Watch. Co-migration analysis further revealed at least 27 unique protein communities across the fractions indicating that multiple distinct subspecies exist within the same sized subfractions. DAVID functional analysis shows that the communities are highly enriched in proteins important in the immune response and neurometabolic functions such as neuron generation and development. In summary, our study shows that similar to plasma HDL, BLps are a highly speciated population of compositionally distinct particles but are enriched in CNS-derived proteins that are essential for maintaining cognitive health.

Sphingosine-1-phosphate: metabolism, transport, atheroprotection and effect of statin treatment.

To better define the metabolism of sphingosine-1-phosphate (S1P), its transport in plasma and its interactions with S1P receptors on vascular cells, and to evaluate the effect of statin treatment on the subnormal plasma levels of high-density lipoprotein (HDL)-bound S1P characteristic of the atherogenic dyslipidemia of metabolic syndrome (MetS). Neither clinical intervention trials targeted to raising high-density lipoprotein-cholesterol (HDL-C) levels nor human genome-wide association studies (GWAS) studies have provided evidence to support an atheroprotective role of HDL. Recently however a large monogenic univariable Mendelian randomization on the N396S mutation in the gene encoding endothelial lipase revealed a causal protective effect of elevated HDL-C on coronary artery disease conferred by reduced enzyme activity. Given the complexity of the HDL lipidome and proteome, components of HDL other than cholesterol may in all likelihood contribute to such a protective effect. Among HDL lipids, S1P is a bioactive sphingolipid present in a small proportion of HDL particles (about 5%); indeed, S1P is preferentially enriched in small dense HDL3. As S1P is bound to apolipoprotein (apo) M in HDL, such enrichment is consistent with the elevated apoM concentration in HDL3. When HDL/apoM-bound S1P acts on S1P1 or S1P3 receptors in endothelial cells, potent antiatherogenic and vasculoprotective effects are exerted; those exerted by albumin-bound S1P at these receptors are typically weaker. When HDL/apoM-bound S1P binds to S1P2 receptors, proatherogenic effects may potentially be induced. Subnormal plasma levels of HDL-associated S1P are typical of dyslipidemic individuals at high cardiovascular risk and in patients with coronary heart disease. International Guidelines recommend statin treatment as first-line lipid lowering therapy in these groups. The cardiovascular benefits of statin therapy are derived primarily from reduction in low-density lipoprotein (LDL)-cholesterol, although minor contributions from pleiotropic actions cannot be excluded. Might statin treatment therefore normalize, directly or indirectly, the subnormal levels of S1P in dyslipidemic subjects at high cardiovascular risk? Our unpublished findings in the CAPITAIN study (ClinicalTrials.gov: NCT01595828), involving a cohort of obese, hypertriglyceridemic subjects (n = 12) exhibiting the MetS, showed that pitavastatin calcium (4 mg/day) treatment for 180days was without effect on either total plasma or HDL-associated S1P levels, suggesting that statin-mediated improvement of endothelial function is not due to normalization of HDL-bound S1P. Statins may however induce the expression of S1P1 receptors in endothelial cells, thereby potentiating increase in endothelial nitric oxide synthase response to HDL-bound S1P, with beneficial downstream vasculoprotective effects. Current evidence indicates that S1P in small dense HDL3 containing apoM exerts antiatherogenic effects and that statins exert vasculoprotective effects through activation of endothelial cell S1P1 receptors in response to HDL/apoM-bound S1P.

Sodium salicylate rewires hepatic metabolic pathways in obesity and attenuates IL-1β secretion from adipose tissue: The implications for obesity-impaired reverse cholesterol transport

IntroductionHigh-fat diet (HFD)-induced obesity impairs clearance of cholesterol through the Reverse Cholesterol Transport (RCT) pathway, with downregulation in hepatic expression of cholesterol and bile acid transporters, namely ABCG5/8 and ABCB11, and reduced high-density lipoprotein (HDL) cholesterol efflux capacity (CEC). In the current study, we hypothesized that the development of hepatosteatosis, secondary to adipose-tissue dysfunction, contributes to obesity-impaired RCT and that such effects could be mitigated using the anti-inflammatory drug sodium salicylate (NaS). Materials and methodsC57BL/6J mice, fed HFD ± NaS or low-fat diet (LFD) for 24 weeks, underwent glucose and insulin tolerance testing. The 3H-cholesterol movement from macrophage-to-feces was assessed in vivo. HDL-CEC was determined ex vivo. Cytokine secretion from adipose-derived stromal vascular fraction (SVF) cells was measured ex vivo. Liver and HDL proteins were determined by mass spectrometry and analyzed using Ingenuity Pathway Analysis. ResultsNaS delayed HFD-induced weight gain, abrogated priming of pro-IL-1β in SVFs, attenuated insulin resistance, and prevented steatohepatitis (ectopic fat accumulation in the liver). Prevention of hepatosteatosis coincided with increased expression of PPAR-alpha/beta-oxidation proteins with NaS and reduced expression of LXR/RXR-induced proteins including apolipoproteins. The latter effects were mirrored within the HDL proteome in circulation. Despite remarkable protection shown against steatosis, HFD-induced hypercholesterolemia and repression of the liver-to-bile cholesterol transporter, ABCG5/8, could not be rescued with NaS. Discussions and conclusionsThe cardiometabolic health benefits of NaS may be attributed to the reprogramming of hepatic metabolic pathways to increase fatty acid utilization in the settings of nutritional overabundance. Reduced hepatic cholesterol levels, coupled with reduced LXR/RXR-induced proteins, may underlie the lack of rescue of ABCG5/8 expression with NaS. This remarkable protection against HFD-induced hepatosteatosis did not translate to improvements in cholesterol homeostasis.

Abstract 11554: Measures of High-Density Lipoprotein Function and Composition in Men and Women with Severe Aortic Valve Stenosis

Introduction and Hypothesis: Aortic valve calcification (AVC), is the intrinsic mechanism of valvular obstruction in severe aortic stenosis (AS), sharing similarities with atherosclerosis. There are differences in AVC by sex, with women having a lower burden of calcification than men, but the mechanism for this is unclear. Improved HDL functionality has been associated with protection against coronary artery calcification, but its relationship to AVC is less studied. Methods: We evaluated measures of high-density lipoprotein (HDL) functionality in 46 patients with severe AS, n= 30 men, n=16 women. ATP-Binding Cassette A1 (ABCA1) mediated cholesterol efflux capacity (CEC) was measured from human cultured Tamm-Horsfall protein 1 (THP-1) macrophages to apoB-depleted patient plasma samples. Lipid profile, lecithin-cholesterol acyltransferase activity (LCAT), fast protein liquid chromatography (FPLC), HDL size, and small pre-β1 and α-HDL particles, were measured in apolipoprotein B-depleted patient plasma. Proteomic analysis was used to explore sex-based differences in the HDL proteome. Results: Women with severe AS had higher ABCA1-mediated CEC, as compared to men (n=46, p=0.04). We found differences in FPLC profiles between sexes in severe AS along with a reduction in preβ and α-particles in men vs women by spectral counts, (preβ-HDL, 15661.74±789.00 vs 20298.29±1076.15, p=0.002), and (α-HDL, 50447.00± 546.52 vs 63006.35±756.81, p=0.03). Conversion of free cholesterol into cholesteryl esters by LCAT was decreased in men vs. women (12.00±8.07 %/h vs 16.44±9.11 %/h, p=0.03). We identified 108 HDL-associated proteins in a representative man and woman, of which, 15 were found only in the female sample Conclusions: Sex-specific differences in measures of HDL functionality were found in patients with severe AS. Future studies should consider whether sex-based differences in HDL functionality might account for the decreased burden of calcification in women vs. men with severe AS.

HDL proteome remodeling associates with COVID-19 severity

BackgroundBesides the well-accepted role in lipid metabolism, high-density lipoprotein (HDL) also seems to participate in host immune response against infectious diseases.Objective: We used a quantitative proteomic approach to test the hypothesis that alterations in HDL proteome associate with severity of Coronavirus disease 2019 (COVID-19).MethodsBased on clinical criteria, subjects (n=41) diagnosed with COVID-19 were divided into two groups: a group of subjects presenting mild symptoms and a second group displaying severe symptoms and requiring hospitalization. Using a proteomic approach, we quantified the levels of 29 proteins in HDL particles derived from these subjects.ResultsWe showed that the levels of serum amyloid A 1 and 2 (SAA1 and SAA2, respectively), pulmonary surfactant-associated protein B (SFTPB), apolipoprotein F (APOF), and inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4) were increased by more than 50% in hospitalized patients, independently of sex, HDL-C or triglycerides when comparing with subjects presenting only mild symptoms. Altered HDL proteins were able to classify COVID-19 subjects according to the severity of the disease (error rate 4.9%). Moreover, apolipoprotein M (APOM) in HDL was inversely associated with odds of death due to COVID-19 complications (odds ratio [OR] per 1-SD increase in APOM was 0.27, with 95% confidence interval [CI] of 0.07 to 0.72, P=0.007).ConclusionOur results point to a profound inflammatory remodeling of HDL proteome tracking with severity of COVID-19 infection. They also raise the possibility that HDL particles could play an important role in infectious diseases.

Reproducible Determination of High-Density Lipoprotein Proteotypes.

High-density lipoprotein (HDL) is a heterogeneous mixture of blood-circulating multimolecular particles containing many different proteins, lipids, and RNAs. Recent advancements in mass spectrometry-based proteotype analysis show promise for the analysis of proteoforms across large patient cohorts. In order to create the required spectral libraries enabling these data-independent acquisition (DIA) strategies, HDL was isolated from the plasma of more than 300 patients with a multiplicity of physiological HDL states. HDL proteome spectral libraries consisting of 296 protein groups and more than 786 peptidoforms were established, and the performance of the DIA strategy was benchmarked for the detection of HDL proteotype differences between healthy individuals and a cohort of patients suffering from diabetes mellitus type 2 and/or coronary heart disease. Bioinformatic interrogation of the data using the generated spectral libraries showed that the DIA approach enabled robust HDL proteotype determination. HDL peptidoform analysis enabled by using spectral libraries allowed for the identification of post-translational modifications, such as in APOA1, which could affect HDL functionality. From a technical point of view, data analysis further shows that protein and peptide quantities are currently more discriminative between different HDL proteotypes than peptidoforms without further enrichment. Together, DIA-based HDL proteotyping enables the robust digitization of HDL proteotypes as a basis for the analysis of larger clinical cohorts.

PCSK9 Activity Is Potentiated Through HDL Binding.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) circulates in a free and lipoprotein-bound form, yet the functional consequence of the association between PCSK9 and high-density lipoprotein (HDL) remains unexplored. This study sought to interrogate the novel relationship between PCSK9 and HDL in humans. Comparing lipoprotein and apolipoprotein profiles by nuclear magnetic resonance and targeted mass spectrometry measurements with PCSK9 levels in the community-based Bruneck (n=656) study revealed a positive association of plasma PCSK9 with small HDL, alongside a highly significant positive correlation between plasma levels of PCSK9 and apolipoprotein-C3, an inhibitor of lipoprotein lipase. The latter association was replicated in an independent cohort, the SAPHIR study (n=270). Thus, PCSK9-HDL association was determined during the postprandial response in two dietary studies (n=20 participants each, 8 times points). Peak triglyceride levels coincided with an attenuation of the PCSK9-HDL association, a loss of apolipoprotein-C3 from HDL and lower levels of small HDL as measured by nuclear magnetic resonance. Crosslinking mass spectrometry (XLMS) upon isolated HDL identified PCSK9 as a potential HDL-binding partner. PCSK9 association with HDL was confirmed through size-exclusion chromatography and immuno-isolation. Quantitative proteomics upon HDL isolated from patients with coronary artery disease (n=172) returned PCSK9 as a core member of the HDL proteome. Combined interrogation of the HDL proteome and lipidome revealed a distinct cluster of PCSK9, phospholipid transfer protein, clusterin and apolipoprotein-E within the HDL proteome, that was altered by sex and positively correlated with sphingomyelin content. Mechanistically, HDL facilitated PCSK9-mediated low-density lipoprotein receptor degradation and reduced low-density lipoprotein uptake through the modulation of PCSK9 internalisation and multimerisation. This study reports HDL as a binder of PCSK9 and regulator of its function. The combination of -omic technologies revealed postprandial lipaemia as a driver of PCSK9 and apolipoprotein-C3 release from HDL.

The Problem of Abnormal Body Weight and Dyslipidemia as Risk Factors for Cardiovascular Diseases in Children and Adolescents with Type 1 Diabetes.

Diabetes is a disease that affects many people around the world. Its complications are the cause of cardiovascular diseases (CVD) and increased mortality. That is why the search for predictive biomarkers is so important. The aim of the study was to show the prevalence of the problem and risk factors in children and adolescents with type 1 diabetes. These patients are often overweight and obese, and the percentage of lipid disorders is particularly high. The discussed markers of CVD risk in type 1 diabetes include apolipoproteins (apo-B and apo-C3), modified forms of LDL, and the role of high-density lipoprotein (HDL). Recently, a new look at the vasoprotective effect of HDL has appeared, which due to its dysfunctional form in type 1 diabetes may not protect against cardiovascular risk. The HDL proteome in type 1 diabetes has an altered protein composition compared to the healthy population. Another direction of research is determining the importance of trace elements (mainly Mg) in the development of diabetes complications.

Maternal and Child Supplementation With Small-Quantity Lipid-Based Nutrient Supplements Increases Child HDL Cholesterol Efflux Capacity

ObjectivesThe purpose of this secondary outcome analysis is to investigate whether small-quantity lipid-based nutrient supplements (SQ-LNS) alters lipid, protein or glycan composition, or the cholesterol efflux capacity (CEC), of high-density lipoprotein (HDL) particles in children in the International Lipid-Based Nutrient Supplements (iLiNS) DYAD trial in Ghana. MethodsPlasma samples were obtained from a subcohort of 80 children at 18 months of age from the iLiNS-DYAD-Ghana trial. Mothers were randomized to either iron and folic acid (IFA) in pregnancy and 200 mg/d calcium for 6 months postpartum or SQ-LNS (pregnancy and 6 months postpartum). Children in the SQ-LNS group received SQ-LNS from 6 to 18 months while children in the IFA group did not receive supplements. HDL was isolated from plasma by sequential ultracentrifugation followed by size-exclusion chromatography. Assay of cholesterol efflux was performed in vitro, and glycoproteomic and lipidomic composition were analyzed by mass spectrometry. The primary analysis was a comparison of the effects of intervention groups on HDL lipidome, proteome, and CEC. In the exploratory analysis, we compared the enrichment of glycopeptides in measured HDL-associated proteins between groups. ResultsMean (±SD) HDL CEC was higher among children in the SQ-LNS vs. IFA group (20.9 ± 4.1% vs. 19.4 ± 3.3%; one-tailed p = 0.038). We found no differences in HDL lipidomic or proteomic composition between groups. ConclusionsPrenatal and postnatal SQ-LNS may improve the CEC of child HDL particles. These improvements may have a potential impact on child health outcomes. Funding SourcesSupported by Bill & Melinda Gates Foundation grant to the University of California, Davis.

Immature Circulating SP-B, Bound to HDL, Represents an Early Sign of Smoke-Induced Pathophysiological Alterations.

Cigarette smoking is a major independent risk factor for cardiovascular diseases (CVD). The underlying mechanisms, however, are not clearly understood. Lungs are the primary route of exposure to smoke, with pulmonary cells and surfactant being the first structures directly exposed, resulting in the leakage of the immature proteoform of surfactant protein B (proSP-B). Herein, we evaluated whether proSP-B joined the cargo of high-density lipoprotein (HDL) proteins in healthy young subjects (n = 106) without any CVD risk factor other than smoking, and if HDL-associated proSP-B (HDL-SPB) correlated with pulmonary function parameters, systemic inflammation, and oxidative stress. At univariable analysis, HDL-SPB resulted significantly higher in smokers (2.2-fold, p < 0.001) than in non-smokers. No significant differences have been detected between smokers and non-smokers for inflammation, oxidation variables, and alveolar-capillary diffusion markers. In a multivariable model, HDL-SPB was independently associated with smoking. In conclusion, HDL-SPB is not only a precocious and sensitive index of the acute effects of smoke, but it might be also a potential causal factor in the onset of the vascular damage induced by modified HDL. These findings contribute to the emerging concept that the quality of the HDL proteome, rather than the quantity of particles, plays a central role in CVD risk protection.