HDL Protein Research Articles

Abstract 4136813: Redox Serum Proteomics Of Left Ventricular Assist Device Unloaded Human Heart

Heart failure (HF) is a global epidemic claiming millions of lives worldwide. Unloading the heart of end-stage HF patients via left ventricular assist devices (LVADs) is used not only as a bridge to transplantation but also as a bridge to recovery in certain selected patients. To date, assessment of patients’ recovery from HF is based on clinical and symptomatic evaluation. The use of non-invasive biomarkers that monitor the remodeling/reverse remodeling associated with HF progression/recovery is still lacking. In this study we applied mass spectrometry-based quantitative redox proteomics to identify serum biomarkers to monitor both the progression of HF and its recovery after LVAD treatment. A fingerprint of reversed behavior at the protein and at the oxidized Cys peptide level has been discovered when comparing HF and LVAD patients. HDL proteins related to lipid metabolism were decreased in HF samples and increased in LVAD patients. In addition, a number of inflammatory proteins showed a decrease in LVAD samples and an increase in HF samples. In particular APOA4, C7, F2 or SAA2 discriminated between HF and LVAD patients. A pool of peptides containing specific oxidized Cys residues showed an inverse relationship in HF as opposed to LVAD samples. Oxidized Cys-containing peptides derived from Albumin, IGK and IGG1 discriminated HF from LVAD samples. Discriminatory sensitivity and specificity were enhanced by using a combination of the proteins APOA4, C7, F2 and the above-mentioned peptide derived from Albumin. Responders and non-responders could also be separated by proteins such as APOA4, and Cys-oxidized peptides, such as the ones derived from Albumin or IGK mentioned above. This is the first study to apply redox proteomics to explore the protein profile of the LVAD unloaded human heart. These data suggest that HDL plays an important role in LVAD recovery and that a specific group of peptides is associated with the recovery process; the mechanisms involved need further study. Our data could have important implications to the management of patients in the LVAD recovery program and could serve to identify new therapeutic targets.

Dismountable Protein Corona-Modified Virus-Like Manganese-Arsenic Nanomedicine Enables Safe and Targeted Delivery for Synergistic Arsenotherapy.

Arsenic agents have shown great potential in fighting leukemia, but are poorly known in treating solid tumors, mainly ascribing to the rapid clearance and low targeting ability. It is reported that morphology modulation can enhance the interaction between nanoparticles and cell membrane. Herein, a dismountable protein corona-modified virus-like manganese-arsenic nanomedicine (vMnAs@HR) is rationally proposed for realizing safe and targeted delivery and synergistic arsenotherapy. The virus-like manganese-arsenic nanoparticle (vMnAs) is constructed followed by modification of a temporary R848-loaded HDL (HR) protein corona. Upon intravenous injection, the HR protein corona is stable and actively targeted to tumor tissue by taking advantage of the interaction between HDL and its receptor SR-BI. Intriguingly, upon accumulated in the tumor, HR can be jettisoned and interacted with macrophages for proinflammatory phenotype modulation. The re-exposed vMnAs can efficiently enhance endocytosis by taking advantage of the rationally designed spiky morphology. Moreover, the released double-stranded DNA (dsDNA) and manganese ions during tumor cell apoptosis can cooperatively activate cyclic guanosine monophosphate adenosine monophosphate synthase (cGAS)-stimulator of interferon genes (STING) signaling pathway of DCs for systematic immune activation. It is anticipated that this morphology-transformable nanomedicine can realize safe and efficient arsenic delivery for synergistic arsenotherapy.

HDL cholesterol efflux capacity and protein content are associated with vasospasm after aneurysmal subarachnoid hemorrhage

HDL cholesterol efflux capacity and protein content are associated with vasospasm after aneurysmal subarachnoid hemorrhage

Abstract 38: Prediction of Coronary Artery Disease Subtypes With Polygenic Risk Scores

Background: Coronary Artery Disease (CAD) is a complex heterogeneous disease with multiple known etiological mechanisms. Consequently, CAD could be considered to have multiple subtypes, reflecting alternative driving etiologies that may warrant distinct therapeutic approaches. Motivated by this, we developed and optimized statistical models to predict CAD subtypes informed by genetic and clinical risk factors. Methods: We developed three models, each using logistic regression: (a) clinical risk factors only, (b) clinical risk factors plus genome-wide polygenic risk scores (PRS), and (c) clinical risk factors and pathway-based PRSs. We defined 5 different CAD subtypes on the basis of the literature: (1) stable vs. unstable, (2) occlusive vs non-occlusive, (3) high vs normal LDL, (4) high vs normal Lp(a), and (5) high vs normal ASCVD score. Clinical risk factors included: ASCVD score, ApoA, ApoB, LDL, HDL, triglycerides, and C-reactive protein. We calculated 4,402 pathway PRSs obtained from six genomic pathway databases (e.g. KEGG). We optimized the models using 80% of the samples as training, and then performed model evaluation (Bonferroni-corrected P-values) in an unseen 20% of the samples as validation. Results: Among 20,935 UK Biobank participants with CAD (mean age 63 years, 58% female), addition of pathway-based PRSs improved prediction of high LDL (P=1e-19) and high Lp(a) subtypes (P=1.8e-78). Using a lasso dimension reduction approach, the pathways most associated with the high Lp(a) subtype of CAD were: Fibronectin Binding (P=6xe-260), Apolipoprotein Binding (P=6.2e-252) and Serine-Type Endopeptidase Activity (P=1.7e-203). Conclusions: We demonstrate that prediction models distinguish CAD subtypes and that a novel pathway PRS approach can increase predictive power and provide insights into the genetic underpinnings of CAD subtypes. Pathway PRSs hold promise for the targeted therapeutic management for subgroups of CAD patients.

Single Nucleotide Variation in the Promoter Region of the APOA1 Gene as a Candidate Biomarker for Dyslipidemia

Dyslipidemia is a lipid profile abnormality that can escalate the risk of cardiovascular disease. The rate of cardiovascular events in Indonesia is very high. One of the causes of dyslipidemia is due to polymorphisms in genes associated with lipid metabolism. The APOA-1 gene encodes the APOA-1 protein which functions to regulate HDL protein synthesis. The objective of this study is to determine APOA1 gene polymorphisms in patients with dyslipidemia. Samples of healthy controls and dyslipidemia patients were used in this investigation. The lipid profiles of the patients and normal controls are determined at the onset of the study. Following DNA extraction, the APOA-1 gene was amplified and sequenced using the serum sample. MEGA X and BLAST were used to analyze the sequencing results. The study's findings demonstrated that the APOA-1 gene length in samples of dyslipidemic patients and normal controls was 433 bp. While the normal control samples have the same sequence as the database, the dyslipidemic patient samples have an APOA-1 gene polymorphism in the promoter region. APOA-1 gene polymorphism results in disturbances in lipid profiles, particularly HDL which is at risk of developing dyslipidemia. The APOA-1 gene has the potential to be developed as a biomarker for diagnosing dyslipidemia involving a larger number of samples.

Obesity, diabetes and their metabolic correlates in middle-aged adults with Down syndrome.

Obesity in adults without Down syndrome is associated with an adverse metabolic profile including high prevalence of pre-diabetes and diabetes, high levels of insulin, non-high-density lipoprotein (HDL) cholesterol, leptin and high-sensitivity C-reactive protein (hsCRP) and low levels of HDL and adiponectin. We examined whether obesity in middle-aged adults with Down syndrome is also related to an adverse metabolic profile. This cross-sectional study included 143 adults with Down syndrome, with a mean age of 55.7±5.7years and 52.5% women. Body mass index (BMI) was classified as underweight (BMI<18.5kg/m2 ), normal (BMI 18.5-24.9kg/m2 ), overweight (BMI 25-29.9kg/m2 ) and obese (BMI≥30kg/m2 ). Diabetes was ascertained by history or by haemoglobin A1c (HbA1c) as normal glucose tolerance (HbA1c<5.7%), pre-diabetes (HbA1c 5.7-6.4%) and diabetes (HbA1c≥6.5%). We measured non-fasting lipids, hsCRP, insulin, adiponectin and leptin. The majority of the sample had an overweight (46.9%) or obesity (27.3%) status. However, there was a relatively low prevalence of pre-diabetes (9.8%) and diabetes (6.9%). Overweight and obesity status were not associated with lower HDL and adiponectin and higher insulin, non-HDL cholesterol and hsCRP as expected in adults without Down syndrome. However, overweight and obesity were strongly associated with higher leptin (P<0.001). The only metabolic correlate of obesity in middle-aged adults with Down syndrome was high leptin levels. Our findings are limited by non-fasting laboratory tests but suggest that middle-aged adults with Down syndrome do not have the adverse metabolic profile related to obesity found in adults without Down syndrome.

Rosuvastatin effects on the HDL proteome in hyperlipidemic patients.

The advancements in proteomics have provided a better understanding of the functionality of apolipoproteins and lipoprotein-associated proteins, with the HDL lipoprotein fraction being the most studied. The focus of this study was to evaluate the HDL proteome in dyslipidemic subjects without an established cardiovascular disease, as well as to test whether rosuvastatin treatment alters the HDL proteome. Patients with primary hypercholesterolemia or mixed dyslipidemia were assigned to 20 mg/day rosuvastatin and blood samples were drawn at study entry and after 12 weeks of treatment. A label-free LC-MS/MS protein profiling was conducted, coupled with bioinformatics analysis. Sixty-nine HDL proteins were identified, belonging to four main biological function clusters: lipid transport and metabolism; platelet activation, degranulation, and aggregation, wound response and wound healing; immune response; inflammatory and acute phase response. Five HDL proteins showed statistically significant differences in the abundance (Anova ≤ 0.05), before and after rosuvastatin treatment. Platelet factor 4 variant (PF4V1), Pregnancy-specific beta-1-glycoprotein 2 (PSG2), Profilin-1 (PFN1) and Keratin type II cytoskeletal 2 epidermal (KRT2) showed decreased expressions, while Integrin alpha-IIb (ITGA2B) showed an increased expression after treatment with rosuvastatin. The ELISA validation of PFN1 segregated the subjects into responders and non-responders, as PFN1 levels after rosuvastatin were shown to mostly depend on the subjects' inflammatory phenotype. Findings from this study introduce novel insights into the HDL proteome and statin pleiotropism.

Apolipoprotein E-containing HDL decreases caspase-dependent apoptosis of memory regulatory T lymphocytes

Plasma levels of HDL cholesterol are inversely associated with CVD progression. It is becoming increasingly clear that HDL plays important roles in immunity that go beyond its traditionally understood roles in lipid transport. We previously reported that HDL interaction with regulatory T cells (Treg) protected them from apoptosis, which could be a mechanism underlying the broad anti-inflammatory effect of HDL. Herein, we extend our work to show that HDL interacts mainly with memory Treg, particularly with the highly suppressive effector memory Treg, by limiting caspase-dependent apoptosis in an Akt-dependent manner. Reconstitution experiments identified the protein component of HDL as the primary driver of the effect, though the most abundant HDL protein, apolipoprotein A-I (APOA1), was inactive. In contrast, APOE-depleted HDL failed to rescue effector memory Treg, suggesting the critical role of APOE proteins. HDL particles reconstituted with APOE, and synthetic phospholipids blunted Treg apoptosis at physiological concentrations. The APOE3 and APOE4 isoforms were the most efficient. Similar results were obtained when lipid-free recombinant APOEs were tested. Binding experiments showed that lipid-free APOE3 bound to memory Treg but not to naive Treg. Overall, our results show that APOE interaction with Treg results in blunted caspase-dependent apoptosis and increased survival. As dysregulation of HDL-APOE levels has been reported in CVD and obesity, our data bring new insight on how this defect may contribute to these diseases.

Metabolic disorders in patients with post-COVID-19 Tuberculosis: A Peruvian unicentric experience

Background: During the COVID-19 pandemic, the diagnosis, monitoring, and prevention of many significant pathologies began to be ignored, tuberculosis (TB) being one of these pathologies. The objective was to determine metabolic disorders and their association with TB stages in post-COVID-19 TB patients from the Félix Torrealva Gutiérrez Hospital in Ica, Peru. Methods: The research was observational, cross-sectional, and descriptive-correlational. The study was carried out on 80 patients diagnosed with post-COVID-19 tuberculosis. A questionnaire was used to collect the sociodemographic characteristics and the metabolites analyzed were glucose, lipid profile, and hepatic profile. Results: Among the 80 patients, a mean age of 56.6 years was observed and 52.5% were male. The presence of sensitive tuberculosis (56.3%), multiresistant tuberculosis (15%), and recurrent tuberculosis (28.7%) with 5.94 months of treatment was found. Alterations were found in the concentration of glucose (36.3%), total cholesterol (31.3%), triglycerides (52.5%), HDL (52.5%), LDL (53.8%), albumin (32.5%), total bilirubin (46.3%), direct bilirubin (5.0%), TGO (30.0%), TGP (56.3%), alkaline phosphatase (50.0%), GGTP (46.3%) and total protein (22.5%). TB stage was significantly associated with sex (p=0.011), treatment time (p˂0.001) and total cholesterol (p=0.021). Conclusions: There are post-COVID-19 metabolic disorders in patients with tuberculosis at the Félix Torrealva Gutiérrez Hospital in Ica, Peru.

Abnormal HDL lipid and protein composition following pediatric cancer treatment: an associative study

BackgroundLong-term childhood cancer survivors (CCS) are at high risk of having dyslipidemia including low high density lipoprotein cholesterol (HDL-C). However, little is known about the prevalence of low HDL-C and the impact of therapy exposure on HDL composition early after treatment is terminated.MethodsThis associative study included 50 children and adolescents who had completed their cancer treatments (< 4 years). Clinical characteristics (demographic, diagnosis, treatment, anthropometric parameters), fasting plasma lipids, apoliporoteins (Apo) A-I and composition of HDL fractions (HDL2 and HDL3) were assessed. Data were stratified according to the presence of dyslipidemia and median doses of therapeutic agents and compared using Fisher exact or Mann–Whitney tests. Univariate binary logistic regression analyses were carried out to evaluate the associations between the clinical and biochemical characteristics and having low HDL-C. Composition of HDL2 and HDL3 particles was assessed in a sub-group of 15 patients and compared to 15 age- and sex-matched healthy controls using Wilcoxon paired test.ResultsOf the 50 pediatric cancer patients included in this study (mean age: 11.30 ± 0.72 y; mean time since end of treatment: 1.47 ± 0.12 y; male: 38%), 8 had low HDL-C (16%), all of which were adolescent at diagnosis. Higher doses of doxorubicin were associated with lower HDL-C and Apo A-I levels. In hypertriglyceridemic patients and compared to normolipidemics, triglycerides (TG) content was greater in HDL2 and HDL3 fractions whereas esterified cholesterol (EC) content was lower in HDL2. Enrich TG content of HDL3 and lower EC of HDL2 was found in patients exposed to ≥ 90 mg/m2 doxorubicin. Factors positively associated with the risk of having low HDL-C were age, being overweight or obese and exposure to doxorubicin ≥ 90 mg/m2. Compared to healthy controls, a sub-group of 15 patients showed higher TG and free cholesterol (FC) content of HDL2 and HDL3 and lower EC content in HDL3.ConclusionsOverall, we found abnormalities in HDL-C and Apo A-I levels and in HDL composition early after pediatric cancer treatment that are influenced by age, overweight or obesity status and exposure to doxorubicin.

Assessment of the physicochemical and sensory characteristics of fermented camel milk fortified with Cordia myxa and its biological effects against oxidative stress and hyperlipidemia in rats.

Natural feed additives and their potential benefits in production of safe and highly nutritious food have gained the attention of many researchers the last decades. Cordia myxa is a nutrient-dense food with various health benefits. Despite this fact, very limited studied investigated the physicochemical and sensory impacts of incorporation of fermented camel milk with Cordia myxa and its biological effects. The current study aimed to assess the physical, chemical, and sensory characteristics of fermented camel milk (FCM) fortified with 5, 10, and 15% Cordia myxa pulp. The study demonstrated that fortification of camel milk efficiently enhanced protein, total solids, ash, fiber, phenolic substance, and antioxidant activity. When compared to other treatments, FCM supplemented with 10% Cordia myxa pulp had the best sensory features. In addition, FCM fortified with 10% Cordia myxa pulp was investigated as a potential inhibitor of hypercholesterolemia agents in obese rats. Thirty-two male Wistar rats were split into two main groups including normal pellet group (n = 8) served as negative control group (G1) and a group of hyperlipidemic animals (n = 24) were feed on a high-fat diet (HFD). Hyperlipidemic rats group (n = 24) were then divided into three subgroups (8 per each); second group or positive control (G2) which include hyperlipidemic rats received distilled water (1 mL/day), the third group (G3) involved hyperlipidemic rats feed on FCM (10 g/day) and the fourth group (G4) included hyperlipidemic animals feed on 10 g/day FCM fortified with 10% of Cordia myxa pulp by oral treatment via an intestinal tube for another 4 weeks. In contrast to the positive control group, G4 treated with Cordia myxa showed a substantial decrease in malondialdehyde, LDL, cholesterol, triglycerides, AST, ALT, creatinine, and urea levels, while a significant increase in HDL, albumin, and total protein concentrations. The number of large adipocytes decreased while the number of small adipocytes increased after consumption of fortified FCM. The results indicated that fermented milk fortified with Cordia myxa pulp improved the functions of the liver and kidney in hyperlipidemic rats. These results demonstrated the protective effects of camel milk and Cordia myxa pulp against hyperlipidemia in rats.

Abstract 167: Immunomodulatory Properties of HDL are Associated with Lipidomic and Proteomic Composition and are Modifiable by Diet

HDL is proposed to be an important immune regulator; however, the direct effects of HDL lipidomic and proteomic composition on immune activity, and the capacity to modulate immunomodulatory properties of HDL through dietary intervention, requires further study. We hypothesized that daily intake of different egg-based diets, which we have reported to induce HDL lipidome and proteome remodeling, would correspond to changes in the capacity of HDL to modulate macrophage and T cell inflammation. Healthy men and women (n = 26) participated in a 16-week randomized, crossover intervention trial in which they followed a 4-week egg-free diet, followed by random assignment to a 4-week diet period in which they consumed 3 whole eggs/day or 3 egg whites/day. Subjects then switched to the alternative egg treatment following a second 4-week egg-free washout period. Fasted blood samples were collected after each diet period to isolate HDL for compositional analysis and perform immune activation assays. We observed that subject-derived HDL increased lipopolysaccharide (LPS)-induced tumor necrosis factor α (TNFα) secretion from RAW 264.7 macrophages following the whole egg diet period, whereas there was a trend toward reduced LPS-induced interleukin 6 (IL-6) secretion and no effect on IL-10. Further, HDL dose-dependently increased Jurkat T cell counts and interleukin-2 (IL-2) secretion following activation, and these changes were inversely associated with lipid raft formation. HDL following the whole egg period increased IL-2 secretion from activated T cells, with greater increases in cell numbers observed in female subjects. Interestingly, changes in HDL-phosphatidylcholine, -phosphatidylethanolamine, and -ceramide species correlated with changes in macrophage TNFα between diet periods, whereas changes in macrophage IL-1β and T cell IL-2 were associated with changes in HDL-triacylglycerol and sphingomyelin species. Changes in macrophage and T cell cytokine production correlated with changes in 25 and 34 HDL proteins, respectively, but not HDL-cholesterol or cholesterol efflux capacity. HDL lipidomic and proteomic composition are determinants of its immunomodulatory properties, which can be modified through dietary intervention.

Ethanol Extracts of Eriobotrya japonica (Loquat) Seeds, Leaves, and Fruits Have Anti-obesity and Hypolipidemic Effects in Rats

Background: Obesity is a serious public health problem contributing to development of several diseases, including hyperlipidemia, hyperglycemia, and hypertension. The plant Eriobotrya japonica (loquat) has been used in traditional Chinese medicine to treat many ailments and traditional healers used it to reduce weight. Objectives: To examine the potential anti-obesity and hypolipidemic effects of ethanol extract of loquat in rats. Materials and Methods: Loquat leaves, fruits, or seeds were extracted with ethanol. About 96 Wistar male rats were fed either a normal rat diet (normal control group; group 1) or a high-fat diet (HFD) for 12 weeks (obese; groups 2−12). Obese rats were divided into11 groups as follows: (obese control: group 2) (obese positive control which received the hypolipidemic reference drug atorvastatin: group 3). Groups (4−6), (7−9), and (10−12) were given seed, leaf, or fruit extract, respectively, at 40,100, and 400 mg/kg. Body weight, serum glucose, lipid profile, creatinine, liver enzymes, albumin, and total protein were measured weekly. Results: HFD consumption significantly increased body weight and serum total cholesterol (TC), triglycerides (TGs), low-density lipoprotein (LDL), very-low density lipoprotein (VLDL), and glucose and decreased HDL compared to rats fed the normal diet. HFD also increased serum glutamic-oxaloacetic transaminase (GOT), gamma-glutamyl transferase (GGT), alkaline phosphatase (ALP) and creatinine but decreased serum albumin and total protein. Administration of E. japonica seed, leaf, or fruit extract significantly decreased body weight, TC, TGs, LDL, VLDL, glucose, liver, and renal enzymes but increased HDL, albumin, and total protein levels. Conclusion: E. japonica seed, leaf, and fruit ethanol extract regulates body weight gain, has hypolipidemic properties, and positively affects liver and kidney enzymes.

Abstract 14231: Egg Consumption Induces Sex-Specific Changes in High-Density Lipoprotein Composition and Function

Introduction: HDL composition is an important determinant of cardioprotective functions. Importantly, sex-specific differences in HDL composition and function have been observed. While diet represents a promising therapeutic strategy to optimize HDL composition and function, it is unclear whether the capacity to modulate HDL profiles through diet is sex-specific. Hypothesis: We hypothesized that daily consumption of different egg-based diets, which differentially impact HDL particle profiles and efflux capacity, would alter HDL composition and function in a sex-dependent manner. Methods: Healthy men and women (n = 26) participated in a 16-week randomized, crossover intervention trial (NCT03577223), in which they consumed a 4-week egg-free diet, followed by a 4-week diet containing either 3 whole eggs or 3 egg whites per day. Participants then followed a 4-week egg-free washout diet, before switching to the alternative whole egg or egg white diet treatment. Fasted blood samples were collected after each diet period to determine HDL particle profiles by NMR, serum paraoxonase 1 (PON1) lactonase activity, cholesterol efflux capacity, and HDL lipid and protein composition. Results: We observed sex-specific differences in HDL profiles following egg intake. In women, whole egg intake increased HDL-phosphatidylcholine and phosphatidylethanolamine content, whereas HDL-phosphatidylinositol and sphingomyelin were increased and decreased, respectively, in men. Similarly, egg diets differentially and sex-dependently altered HDL proteins implicated in cholesterol efflux capacity and cardiovascular disease risk. Whole egg intake increased HDL-apoAII, apoL1, apoM, and lowered serum amyloid P compared to the egg-free and egg white diets in women, whereas HDL-PON3 was increased by egg white intake in men. PON1 activity and cholesterol efflux capacity were increased following whole egg intake in women only. Independent of sex, changes in cholesterol efflux capacity between the egg white and whole egg periods were positively correlated with the number of total and small HDL particles. Conclusions: Modulation of HDL profiles by diet are sex-specific, with whole egg intake promoting more significant changes in HDL composition and function in women.

RF12 | PSUN125 Lymphoma in Congenital Leptin Deficiency: Comorbidity or Adverse Effect?

Abstract Background Congenital leptin deficiency (CLD) is a rare autosomal recessive form of monogenic obesity caused by loss-of-function mutations in the leptin gene. Targeted therapy is available in the form of recombinant human leptin (metreleptin). We previously reported baseline characteristics of one of the first cases of CLD diagnosed in the United States, a patient with a comorbid diagnosis of Hodgkin lymphoma prior to initiation of therapy. Metreleptin has a black box warning for risk of T-cell lymphoma, which has been reported in acquired generalized lipodystrophy patients, both treated and untreated with metreleptin. Clinical Case A 20 yr old female (Pt B) was referred for evaluation of obesity (BMI 45.0 kg/m2) with a history of hyperphagia and excessive weight gain by three months of life. Pt B is of Pakistani origin with a family history of consanguinity. She was treated with combination OCPs in adolescence due to primary amenorrhea and hypogonadotropic hypogonadism. At the time of her initial evaluation, serum leptin levels were undetectable. Pt B was homozygous for the pathogenic variant c398delG in exon 3 of the leptin gene (LEP), which causes a frameshift/premature stop codon. On MR elastography performed for hepatic steatosis, she had an incidental finding of axillary lymphadenopathy. Surgical biopsy and staging work-up confirmed diagnosis of nodular lymphocyte predominant Hodgkin lymphoma, Stage II disease. She was treated with 2 cycles of R-ABVD followed by adjuvant radiation (2000 cGy) to the right supraclavicular and axillary region, achieving clinical remission. Pt B was then treated with 3.75-5 mg metreleptin once daily in an observational treatment protocol for 18 months. With therapy, pt B lost 39.1 kg or 33% of initial body weight. Clinically significant improvements in related parameters were observed, including percent body fat (-18%), estimated visceral adipose tissue (-53%), HOMA2 IR (-80%), ALT (-91%), total cholesterol (-10%), triglycerides (-26%), HDL (+6%), LDL (-19%), and high-sensitivity C-reactive protein (-84%). In addition, prediabetes (with abnormal 2 hour OGTT) resolved, hepatic steatosis improved, bone mineral density increased (+0.6 SD), and pt B developed spontaneous regular menses. She tolerated metreleptin well without serious adverse effects and her lymphoma remained in remission throughout the study. Conclusion To our knowledge, this is the first report of lymphoma in a patient with CLD, occurring prior to treatment with metreleptin. Pt B achieved clinical remission prior to initiation of therapy with metreleptin, with no recurrence on 18 months of therapy. She experienced substantial weight loss and marked improvements in body composition, glucose metabolism, insulin sensitivity, lipid metabolism, liver and reproductive health. Untreated CLD has been associated with impaired lymphocyte production and function. The risk for lymphoma associated with metreleptin may relate to preexisting autoimmunity or immunologic abnormalities related to leptin deficiency rather than medication adverse effect. Presentation: Saturday, June 11, 2022 1:00 p.m. - 1:05 p.m., Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.

Abstract 193: Apolipoprotein A-I Rotamers Determine The Size And Cholesterol Efflux Capacity Of High-Density Lipoprotein

Cardiovascular disease (CVD) risk is strongly and inversely associated with plasma levels of high-density lipoprotein cholesterol (HDL-C). Recent clinical studies suggest that the association between HDL-C and CVD risk is indirect and that HDL’s cardioprotective function may be attributed to its ability to promote cholesterol efflux from macrophages. Apolipoprotein A-I (APOA1), the major protein in HDL, serves as a structural scaffold to facilitate protein binding and enzyme activation. Our previous work demonstrated that two or three molecules of APOA1 form antiparallel dimer structures (rotamers) that wrap around discoidal and spherical HDL. Human HDL contains both LL5/5 and LL5/4 rotamers. Because little is known about the impact of different rotamers on HDL function, we generated human APOA1 mutants with single cysteine mutations that produced reconstituted HDL (rHDL) locked in specific rotamer (K133C for LL5/5 and L122C for LL5/4). The LL5/5 rotamer of rHDL demonstrated four-fold higher LCAT activation efficiency than the LL5/4 rotamer. To investigate the roles of APOA1 rotamers in vivo , we used a liver-targeted adeno-associated virus (AAV) to express WT hAPOA1 and the two cysteine mutants in APOA1-deficient ( Apoa1 -/- ) mice. HDL particle concentration (HDL-P) in WT and LL5/5 mice were similar but were 250% higher (n=8; P&lt;0.0001) than those of LL5/4 mice. Importantly, mice expressing different APOA1 rotamers had markedly different HDL size patterns: HDL in LL5/4 hAPOA1 mice consisted mainly of extra-small HDL (7.8 nm in diameter), consistent with our finding that the LL5/4 rotamer had a low efficiency in activating LCAT. HDL in WT and LL5/5 mice had the same size distribution with one major peak at 10 nm. LC-MS/MS analysis identified 48 HDL proteins. One-third of the proteins (including APOA2, APOC1, APOC3 and LCAT) differed significantly in relative abundance in the different rotamers. Importantly, on a per particle basis, HDL from LL5/4 mice demonstrated three times higher ABCA1 cholesterol efflux capacity than HDL from WT and LL5/5 mice. Our observations strongly suggest that APOA1 rotamers play distinct roles in HDL metabolism, raising the possibility that differences in APOA1 rotamer distribution alter the cardioprotective functions of HDL.

Abstract 445: Pulmonary Surfactant Protein B Carried By HDL Is Associated With Incident Cardiovascular Disease In Patients With Type 1 Diabetes

Atherosclerotic cardiovascular disease (CVD) is the major cause of death in people with type 1 diabetes mellitus (T1DM). Alterations in the HDL proteome associate with prevalent CVD in T1DM. We therefore determined which proteins carried by HDL are associated with incident CVD in T1DM patients. We conducted a case-cohort study of incident CVD in the prospective Coronary Artery Calcification in Type 1 Diabetes (CACTI) study. We randomly sampled 145 CACTI participants without prevalent CVD, 11 of whom subsequently developed a CVD event as a cohort, plus all additional T1DM participants in CACTI (N=36) who developed an incident CVD event (a total of 47 CVD cases). Using targeted MS/MS, we quantified 51 proteins in HDL isolated from baseline plasma samples. We used Cox proportional regression analysis and a case-cohort design to test associations of HDL proteins with incident CVD (myocardial infarction, coronary artery bypass grafting, angioplasty, or death from coronary heart disease). Only one HDL protein—SFTPB (pulmonary surfactant protein B)—was associated with incident CVD in T1DM in all of the models tested. In a fully adjusted model that controlled for smoking status, lipids and other risk factors, the hazard ratio was 2.17 per SD increase of SFTPB (95% confidence interval, 1.12-4.21, P=0.022). Plasma fractionation and targeted MS/MS quantification demonstrated that nearly 95% of plasma SFTPB is quantitatively bound to HDL. Our results show that elevated levels of SFTPB in HDL are strongly associated with incident CVD in CACTI subjects with T1DM independently of smoking status and a wide range of other confounding factors. Because HDL is the major carrier of SFTPB in plasma, our observations support the proposal that SFTPB carried by HDL is a novel marker of CVD risk in patients with T1DM.

Abstract 198: Robust Quantification Of Hdl Proteome For Mechanistic And Clinical Studies

Introduction: Emerging studies have proposed novel roles of HDL in cardiovascular and noncardiovascular diseases. The understanding of how HDL participates in a pathophysiological process involves animal models, as well as clinical studies. A key issue involving HDL studies is the accurate quantification of its proteome. HDL proteome is complex, and its composition may be modulated by the subject’s health state. The main goal of this work is to establish a pipeline for accurate quantification of HDL proteome in clinical and animal studies. Hypothesis: The knowledge of the composition of HDL proteome in clinical and animal studies is critical to understand its biological functions. Methods: We first performed a deep proteomic analysis to identify proteins belonging to HDL of mice and humans. We then established a targeted, data independent acquisition (DIA) strategy that works for mouse and humans. Next, by using different software platforms, we developed a quantification methodology specific for each species. Results: Although mice and humans share many of their HDL proteins, the protein sequence is unique for each species, and thus, HDL proteome quantification methods must be species specific. Using recent advances in mass spectrometry, we employed a single acquisition method that worked for both, mice and human samples. Differentiation occurred after acquisition, using spectral libraries specifically built for each species. Using different software platforms, we compared four strategies of quantification for HDL proteome, showing that CVs depend on the strategy employed. Thus, using quality control samples (n=8), we showed that for the same protein, CVs may vary up to 50 %. The quantification strategy was thus optimized in order to provide the best quantification with the least variance, reaching CVs lower than 15 % for 90 % of the proteins. Conclusions: Using the same mass spectrometry acquisition method, it is possible to quantify HDL proteome of mice and humans in a consistent and accurate way. These advances may integrate animal models and clinical studies of HDL proteome.

Apolipoprotein E enhance survival of effector memory regulatory T lymphocytes by regulating caspase-dependent apoptosis and lipid metabolism.

Abstract Mechanisms underlying the pleiotropic anti-inflammatory effect of HDL remain incompletely understood. Regulatory T cells (Tregs) are essential for maintaining immune homeostasis in cardiovascular diseases (CVD) and metabolic disorders. We recently show Tregs, particularly effector memory Tregs (emTregs), bind/internalize HDL, which promotes their survival by limiting caspase-dependent apoptosis. As HDL contain a complex mix of proteins and lipids, we investigated which components of HDL mediate this process and found HDL proteins were the most critical. Using reconstituted HDL mimic vesicles containing recombinant HDL proteins and POPC, we found that APOE, but none of the APOA proteins, significantly decreased caspase expression in emTregs similarly to total HDL. Neither APOE nor HDL had any effect on naïve Tregs. APOE-depleted HDL did not rescue emTregs, confirming the critical role of APOE. Because Tregs have distinct metabolic requirements, we next investigated the effect of HDL, APOE3-POPC and APOE-depleted HDL on lipid metabolism in emTregs. APOE3-POPC and HDL significantly increased the concentration of long chain (14:0 20:0) and polyunsaturated fatty acids (FA, 18:3 22:6) in emTregs. Our data also suggested the involvement of de novo FA synthesis, as APOE3-POPC and HDL increased the concentration of palmitate (16:0), the earlier precursor of this pathway. Further, treatment of Tregs with the fatty acid synthase inhibitor C75 abolished HDL survival effect. Our results show that HDL, specifically APOE, promote emTregs survival by regulating de novo FA synthesis and caspase-dependent apoptosis. HDL-APOE levels seem to be dysregulated in CVD and obesity, suggesting this mechanism may contribute to HDL protective role

Abstract 237: Association Between The HDL-sized And Circulating Plasma Proteomes

Background: Mass spectrometry (MS) profiling has identified over 250 proteins associated with HDL that are thought to underlie the diverse atheroprotective properties of HDL particles and thus may be important biomarkers of cardiovascular disease (CVD) risk. Likewise, recent studies have identified circulating plasma proteins as biomarkers of CVD risk factors and health outcomes. However, few studies have compared the HDL proteome with the circulating plasma proteome. Purpose: The purpose of this analysis was to examine the relationship between the abundance of individual proteins measured in whole plasma and the HDL-sized plasma fraction. Methods: We examined the HDL-sized and circulating plasma proteomes in 156 Black (30%) and White men and women (61%) from the HERITAGE Family Study. HDL was isolated from plasma via gel filtration chromatography and untargeted MS analysis was performed via nano-HPLC-MS/MS. The whole plasma proteome was measured using a modified aptamer (SOMAscan) assay. The correlations between protein abundances in HDL and whole plasma were examined for 101 HDL-associated proteins present in at least 40% of the sample. Results: The abundance of 56 proteins in HDL-sized and whole plasma were significantly (5% FDR) correlated with the strongest correlation for Haptoglobin levels (r=0.83, p=1.1x10 -40 ) ( Table 1 ). The remaining significant correlations ranged from weak to moderate (r= 0.18-0.64) and were found among a mix of frequently and occasionally observed HDL proteins. Discussion: We found that protein abundance measured in HDL-sized and whole plasma were moderately to strongly correlated for several proteins, whereas 45% of protein levels showed no association between HDL-sized and whole plasma fractions. Given the inherit differences in measurement techniques and sources of proteins, it appears that the plasma HDL-sized proteome is mostly distinct from the circulating whole plasma proteome as measured by the SOMAscan assay.