Low concentrations of medium-sized HDL particles predict incident CVD in chronic kidney disease patients

Patients with chronic kidney disease (CKD) are at high risk for CVD. However, traditional CVD risk factors cannot completely explain the increased risk. Altered HDL proteome is linked with incident CVD in CKD patients, but it is unclear whether other HDL metrics are associated with incident CVD in this population. In the current study, we analyzed samples from two independent prospective case-control cohorts of CKD patients, the Clinical Phenotyping and Resource Biobank Core (CPROBE) and the Chronic Renal Insufficiency Cohort (CRIC). We measured HDL particle sizes and concentrations (HDL-P) by calibrated ion mobility analysis and HDL cholesterol efflux capacity (CEC) by cAMP-stimulated J774 macrophages in 92 subjects from the CPROBE cohort (46 CVD and 46 controls) and in 91 subjects from the CRIC cohort (34 CVD and 57 controls). We tested associations of HDL metrics with incident CVD using logistic regression analysis. No significant associations were found for HDL-C or HDL-CEC in either cohort. Total HDL-P was only negatively associated with incident CVD in the CRIC cohort in unadjusted analysis. Among the six sized HDL subspecies, only medium-sized HDL-P was significantly and negatively associated with incident CVD in both cohorts after adjusting for clinical confounders and lipid risk factors with odds ratios (per 1-SD) of 0.45 (0.22–0.93, P = 0.032) and 0.42 (0.20–0.87, P = 0.019) for CPROBE and CRIC cohorts, respectively. Our observations indicate that medium-sized HDL-P—but not other-sized HDL-P or total HDL-P, HDL-C, or HDL-CEC—may be a prognostic cardiovascular risk marker in CKD.     

HDL Isolated by Immunoaffinity,Ultracentrifugation, or Precipitation is Compositionally and Functionally Distinct

The HDL proteome has been widely recognized as an important mediator of HDL function. While a variety of HDL isolation methods exist, their impact on the HDL proteome and its associated function remain largely unknown. Here, we compared three of the most common methods for HDL isolation, namely immunoaffinity (IA), density gradient ultracentrifugation (UC), and dextran-sulfate precipitation (DS), in terms of their effects on the HDL proteome and associated functionalities. We used state-of-the-art mass spectrometry to identify 171 proteins across all three isolation methods. IA-HDL contained higher levels of paraoxonase 1, apoB, clusterin, vitronectin, and fibronectin, while UC-HDL had higher levels of apoA2, apoC3, and α-1-antytrypsin. DS-HDL was enriched with apoA4 and complement proteins, while the apoA2 content was very low. Importantly, size-exclusion chromatography analysis showed that IA-HDL isolates contained subspecies in the size range above 12 nm, which were entirely absent in UC-HDL and DS-HDL isolates. Analysis of these subspecies indicated that they primarily consisted of apoA1, IGκC, apoC1, and clusterin. Functional analysis revealed that paraoxonase 1 activity was almost completely lost in IA-HDL, despite high paraoxonase content. We observed that the elution conditions, using 3M thiocyanate, during IA resulted in an almost complete loss of paraoxonase 1 activity. Notably, the cholesterol efflux capacity of UC-HDL and DS-HDL was significantly higher compared to IA-HDL. Together, our data clearly demonstrate that the isolation procedure has a substantial impact on the composition, subclass distribution, and functionality of HDL. In summary, our data show that the isolation procedure has a significant impact on the composition, subclass distribution and functionality of HDL. Our data can be helpful in the comparison, replication and analysis of proteomic datasets of HDL.     

Cholesteryl hemiazelate identified in CVD patients causes in vitro and in vivo inflammation

Oxidation of PUFAs in LDLs trapped in the arterial intima plays a critical role in atherosclerosis. Though there have been many studies on the atherogenicity of oxidized derivatives of PUFA-esters of cholesterol, the effects of cholesteryl hemiesters (ChEs), the oxidation end products of these esters, have not been studied. Through lipidomics analyses, we identified and quantified two ChE types in the plasma of CVD patients and identified four ChE types in human endarterectomy specimens. Cholesteryl hemiazelate (ChA), the ChE of azelaic acid (n-nonane-1,9-dioic acid), was the most prevalent ChE identified in both cases. Importantly, human monocytes, monocyte-derived macrophages, and neutrophils exhibit inflammatory features when exposed to subtoxic concentrations of ChA in vitro. ChA increases the secretion of proinflammatory cytokines such as interleukin-1β and interleukin-6 and modulates the surface-marker profile of monocytes and monocyte-derived macrophage. In vivo, when zebrafish larvae were fed with a ChA-enriched diet, they exhibited neutrophil and macrophage accumulation in the vasculature in a caspase 1- and cathepsin B-dependent manner. ChA also triggered lipid accumulation at the bifurcation sites of the vasculature of the zebrafish larvae and negatively impacted their life expectancy. We conclude that ChA behaves as an endogenous damage-associated molecular pattern with inflammatory and proatherogenic properties.     

10,12-Conjugated linoleic acid supplementation improves HDL composition and function in mice

Obesity is associated with inflammation, insulin resistance, and type 2 diabetes, which are major risk factors for CVD. One dietary component of ruminant animal foods, 10,12-conjugated linoleic acid (10,12 CLA), has been shown to promote weight loss in humans. Previous work has shown that 10,12 CLA is atheroprotective in mice by a mechanism that may be distinct from its weight loss effects, but this exact mechanism is unclear. To investigate this, we evaluated HDL composition and function in obese LDL receptor (Ldlr^?/?) mice that were losing weight because of 10,12 CLA supplementation or caloric restriction (CR; weight-matched control group) and in an obese control group consuming a high-fat high-sucrose diet. We show that 10,12 CLA-HDL exerted a stronger anti-inflammatory effect than CR- or high-fat high-sucrose-HDL in cultured adipocytes. Furthermore, the 10,12 CLA-HDL particle (HDL-P) concentration was higher, attributed to more medium- and large-sized HDL-Ps. Passive cholesterol efflux capacity of 10,12 CLA-HDL was elevated, as was expression of HDL receptor scavenger receptor class B type 1 in the aortic arch. Murine macrophages treated with 10,12 CLA in vitro exhibited increased expression of cholesterol transporters Abca1 and Abcg1, suggesting increased cholesterol efflux potential of these cells. Finally, proteomics analysis revealed elevated Apoa1 content in 10,12 CLA-HDL-Ps, consistent with a higher particle concentration, and particles were also enriched with alpha-1-antitrypsin, an emerging anti-inflammatory and antiatherosclerotic HDL-associated protein. We conclude that 10,12 CLA may therefore exert its atheroprotective effects by increasing HDL-P concentration, HDL anti-inflammatory potential, and promoting beneficial effects on cholesterol efflux.     

Dalcetrapib and anacetrapib increase apolipoprotein E-containing HDL in rabbits and humans

The large HDL particles generated by administration of cholesteryl ester transfer protein inhibitors (CETPi) remain poorly characterized, despite their potential importance in the routing of cholesterol to the liver for excretion, which is the last step of the reverse cholesterol transport. Thus, the effects of the CETPi dalcetrapib and anacetrapib on HDL particle composition were studied in rabbits and humans. The association of rabbit HDL to the LDL receptor (LDLr) in vitro was also evaluated. New Zealand White rabbits receiving atorvastatin were treated with dalcetrapib or anacetrapib. A subset of patients from the dal-PLAQUE-2 study treated with dalcetrapib or placebo were also studied. In rabbits, dalcetrapib and anacetrapib increased HDL-C by more than 58% (P < 0.01) and in turn raised large apo E-containing HDL by 66% (P < 0.001) and 59% (P < 0.01), respectively. Additionally, HDL from CETPi-treated rabbits competed with human LDL for binding to the LDLr on HepG2 cells more than control HDL (P < 0.01). In humans, dalcetrapib increased concentrations of large HDL particles (+69%, P < 0.001) and apo B-depleted plasma apo E (+24%, P < 0.001), leading to the formation of apo E-containing HDL (+47%, P < 0.001) devoid of apo A-I. Overall, in rabbits and humans, CETPi increased large apo E-containing HDL particle concentration, which can interact with hepatic LDLr. The catabolism of these particles may depend on an adequate level of LDLr to contribute to reverse cholesterol transport.     

Coronin-1 is phosphorylated at Thr-412 by protein kinase Cα in human phagocytic cells

Coronin-1, a hematopoietic cell-specific actin-binding protein, is thought to be involved in the phagocytic process through its interaction with actin filaments. The dissociation of coronin-1 from phagosomes after its transient accumulation on the phagosome surface is associated with lysosomal fusion. We previously reported that 1) coronin-1 is phosphorylated by protein kinase C (PKC), 2) coronin-1 has two phosphorylation sites, Ser-2 and Thr-412, and 3) Thr-412 of coronin-1 is phosphorylated during phagocytosis. In this study, we examined which PKC isoform is responsible for the phosphorylation of coronin-1 at Thr-412 by using isotype-specific PKC inhibitors and small interfering RNAs (siRNAs). Thr-412 phosphorylation of coronin-1 was suppressed by Gö6976, an inhibitor of PKCα and PKCβI. This phosphorylation was attenuated by siRNA for PKCα, but not by siRNA for PKCβ. Furthermore, Thr-412 of coronin-1 was phosphorylated by recombinant PKCα in vitro, but not by recombinant PKCβ. We next examined the effects of Gö6976 on the intracellular distribution of coronin-1 in HL60 cells during phagocytosis. The confocal fluorescence microscopic observation showed that coronin-1 was not dissociated from phagosomes in Gö6976-treated cells. These results indicate that phosphorylation of coronin-1 at Thr-412 by PKCα regulates intracellular distribution during phagocytosis.     

Methylglyoxal attenuates isoproterenol-induced increase in uncoupling protein 1 expression through activation of JNK signaling pathway in beige adipocytes

Methylglyoxal (MG) is a metabolite derived from glycolysis whose levels in the blood and tissues of patients with diabetes are higher than those of healthy individuals, suggesting that MG is associated with the development of diabetic complications. However, it remains unknown whether high levels of MG are a cause or consequence of diabetes. Here, we show that MG negatively affects the expression of uncoupling protein 1 (UCP1), which is involved in thermogenesis and the regulation of systemic metabolism. Decreased Ucp1 expression is associated with obesity and type 2 diabetes. We found that MG attenuated the increase in Ucp1 expression following treatment with isoproterenol in beige adipocytes. However, MG did not affect protein kinase A signaling, the core coordinator of isoproterenol-induced Ucp1 expression. Instead, MG activated c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinases. We found that JNK inhibition, but not p38, recovered isoproterenol-stimulated Ucp1 expression under MG treatment. Altogether, these results suggest an inhibitory role of MG on the thermogenic function of beige adipocytes through the JNK signaling pathway.     

Cytochrome P450-soluble epoxide hydrolase derived linoleic acid oxylipins and cognitive performance in type 2 diabetes

Type 2 diabetes mellitus (T2DM) increases the risk of cognitive decline and dementia. Disruptions in the cytochrome P450-soluble epoxide hydrolase (CYP450-sEH) pathway have been reported in T2DM, obesity and cognitive impairment. We examine linoleic acid (LA)-derived CYP450-sEH oxylipins and cognition in T2DM and explore potential differences between obese and nonobese individuals. The study included 51 obese and 57 nonobese participants (mean age 63.0 ± 9.9, 49% women) with T2DM. Executive function was assessed using the Stroop Color-Word Interference Test, FAS-Verbal Fluency Test, Digit Symbol Substitution Test, and Trails Making Test-Part B. Verbal memory was assessed using the California Verbal Learning Test, second Edition. Four LA-derived oxylipins were analyzed by ultra-high-pressure–LC/MS, and the 12,13-dihydroxyoctadecamonoenoic acid (12,13-DiHOME) considered the main species of interest. Models controlled for age, sex, BMI, glycosylated hemoglobin A1c, diabetes duration, depression, hypertension, and education. The sEH-derived 12,13-DiHOME was associated with poorer executive function scores (F_1,98 = 7.513, P = 0.007). The CYP450-derived 12(13)-epoxyoctadecamonoenoic acid (12(13)-EpOME) was associated with poorer executive function and verbal memory scores (F_1,98 = 7.222, P = 0.008 and F_1,98 = 4.621, P = 0.034, respectively). There were interactions between obesity and the 12,13-DiHOME/12(13)-EpOME ratio (F_1,97 = 5.498, P = 0.021) and between obesity and 9(10)-epoxyoctadecamonoenoic acid (9(10)-EpOME) concentrations (F_1,97 = 4.126, P = 0.045), predicting executive function such that relationships were stronger in obese individuals. These findings suggest that the CYP450-sEH pathway as a potential therapeutic target for cognitive decline in T2DM. For some markers, relationships may be obesity dependent.     

Programmed cell death 1 positive lymphocytes at palate tonsils in the elder patients with chronic tonsillitis

Circulating lymphocytes infiltrate into local foci at the inflammatory phase of acute wound healing for activation of the immune system and express an immune checkpoint protein programmed cell death 1 (PD-1) at the resolution phase for inactivation of the immune system. Conversely, the PD-1 expression was still found even on circulating lymphocytes of the elder patients with chronic tonsillitis at the palliative stage. Recently, an adhesion G protein coupled receptor 56 (GPR56) was reported to at least work as a proliferation factor for infiltrated lymphocytes into local foci at the resolution phase of acute wound healing. To preliminary examine a similar role of PD-1 and GPR56 at local foci at chronic inflammation, palate tonsils were prepared from small amounts of patients with chronic tonsillitis and tonsillar hypertrophy. A positive relationship of RNA expression might be observed between PD-1 and GPR56 in the elder patients with chronic tonsillitis. In regard to immunohistopathological findings, there were huge and small amounts of PD-1 and GPR56 expression at the marginal zone of lymphoid follicles of palate tonsils with chronic tonsillitis. Moreover, the positive relationship of RNA expression between PD-1 and GPR56 confirmed in large numbers of the elder patients with chronic tonsillitis. Probably, GPR56 participates in a supplement of PD-1⁺ lymphocytes to circulating bloods of the elder patients with chronic tonsillitis through a lymphocyte cell maintenance system at the marginal zone of the lymphoid follicles of palate tonsils.     

Characteristics and Risk Factors of Pulmonary Hypertension in Patients With Hyperthyroidism

ObjectiveThis study aimed to comprehensively assess the characteristics and risk factors of hyperthyroidism with pulmonary hypertension (PH).MethodsThis was a retrospective cross-sectional analysis of 315 consecutive patients with hyperthyroidism admitted to the endocrinology department of Tongji Hospital from February 2016 to December 2017. PH was defined as a pulmonary arterial systolic pressure above 35 mm Hg measured by echocardiography.ResultsAmong the 315 patients, 208 were females, the median age was 42 (30-51) years, and the median disease duration was 12 (3-48) months. Thirty-five percent (111/315) of patients were identified with PH. Patients with hyperthyroidism and PH showed significantly higher serum concentrations of free thyroxine (FT4), free triiodothyronine, thyroid receptor antibodies, total bilirubin (TB), direct and indirect bilirubin, lower serum levels of hemoglobin and creatinine, and more severe cardiac load (P < .05 for each) compared with patients without PH. Levels of serum FT4, free triiodothyronine, thyroid receptor antibodies, and thyroid peroxidase antibody were different among groups of patients with different levels of pulmonary arterial systolic pressure (P < .05 for each). Multivariate logistic regression analysis indicated that serum FT4 (odds ratio, 1.02; 95% CI, 1.01-1.04; P = .004) and TB (OR, 1.03; 95% CI, 1.00-1.06; P = .030) were independent risk factors for PH in patients with hyperthyroidism.ConclusionElevated serum FT4 and TB levels may be independent risk factors for PH in patients with hyperthyroidism and valuable indicators for the identification and treatment of patients with PH and hyperthyroidism.     

First report of computational protein–ligand docking to evaluate susceptibility to HIV integrase inhibitors in HIV-infected Iranian patients

BackgroundIran has recently included integrase (INT) inhibitors (INTIs) in the first‐line treatment regimen in human immunodeficiency virus (HIV)-infected patients. However, there is no bioinformatics data to elaborate the impact of resistance-associated mutations (RAMs) and naturally occurring polymorphisms (NOPs) on INTIs treatment outcome in Iranian patients.MethodIn this cross-sectional survey, 850 HIV-1-infected patients enrolled; of them, 78 samples had successful sequencing results for INT gene. Several analyses were performed including docking screening, genotypic resistance, secondary/tertiary structures, post-translational modification (PTM), immune epitopes, etc.ResultThe average docking energy (E value) of different samples with elvitegravir (EVG) and raltegravir (RAL) was more than other INTIs. Phylogenetic tree analysis and Stanford HIV Subtyping program revealed HIV-1 CRF35-AD was the predominant subtype (94.9%) in our cases; in any event, online subtyping tools confirmed A1 as the most frequent subtype. For the first time, CRF-01B and BF were identified as new subtypes in Iran. Decreased CD4 count was associated with several factors: poor or unstable adherence, naïve treatment, and drug user status.ConclusionAs the first bioinformatic report on HIV-integrase from Iran, this study indicates that EVG and RAL are the optimal INTIs in first-line antiretroviral therapy (ART) in Iranian patients. Some conserved motifs and specific amino acids in INT-protein binding sites have characterized that mutation(s) in them may disrupt INT-drugs interaction and cause a significant loss in susceptibility to INTIs. Good adherence, treatment of naïve patients, and monitoring injection drug users are fundamental factors to control HIV infection in Iran effectively.     

Thyroid Immune-Related Adverse Events in Patients with Cancer Treated with anti-PD1/anti-CTLA4 Immune Checkpoint Inhibitor Combination: Clinical Course and Outcomes

ObjectiveThyroid immune-related adverse events (irAEs) have been reported to have prognostic significance among patients with cancer treated with anti-programmed cell death-1 (PD1) and anti-programmed death-ligand 1 monotherapies. We evaluated the clinical course and predictors of thyroid irAEs in relation to outcomes of patients with advanced cancer treated with combination anti-PD1/anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA4).MethodsWe conducted a regional study and identified patients with advanced cancer who received ≥1 cycle of combination anti-PD1/anti-CTLA4 between 2015 and 2019 in Hong Kong. Thyroid function tests (TFTs) were monitored every 3 weeks. Thyroid irAE was defined by ≥2 abnormal TFTs after initiation of combination anti-PD1/anti-CTLA4 in the absence of other causes.ResultsOne hundred and three patients were included (median age: 59 years; 71.8% men). About 45% had prior anti-PD1 exposure. Upon median follow-up of 6.8 months, 17 patients (16.5%) developed thyroid irAEs, where 6 initially presented with thyrotoxicosis (overt, n = 4; subclinical, n = 2) and 11 with hypothyroidism (overt, n = 2; subclinical, n = 9). Eventually, 10 patients (58.8%) required continuous thyroxine replacement. Systemic steroid was not required in all cases. Prior anti-PD1 exposure (odds ratio, 3.67; 95% CI, 1.19–11.4; P = .024) independently predicted thyroid irAEs. Multivariable Cox regression analysis revealed that occurrence of thyroid irAEs was independently associated with better overall survival (adjusted hazard ratio, 0.34; 95% CI, 0.17–0.71; P = .004).ConclusionThyroid irAEs are common in routine clinical practice among patients with advanced cancer treated with anti-PD1/anti-CTLA4 combination and might have potential prognostic significance. Regular TFT monitoring is advised for timely treatment of thyroid irAEs to prevent potential morbidities.     

Ebola virus replication is regulated by the phosphorylation of viral protein VP35

Ebola virus (EBOV) is a zoonotic pathogen, the infection often results in severe, potentially fatal, systematic disease in human and nonhuman primates. VP35, an essential viral RNA-dependent RNA polymerase cofactor, is indispensable for Ebola viral replication and host innate immune escape. In this study, VP35 was demonstrated to be phosphorylated at Serine/Threonine by immunoblotting, and the major phosphorylation sites was S187, S205, T206, S208 and S317 as revealed by LC-MS/MS. By an EBOV minigenomic system, EBOV minigenome replication was shown to be significantly inhibited by the phosphorylation-defective mutant, VP35 S187A, but was potentiated by the phosphorylation mimic mutant VP35 S187D. Together, our findings demonstrate that EBOV VP35 is phosphorylated on multiple residues in host cells, especially on S187, which may contribute to efficient viral genomic replication and viral proliferation.     

Ceramide-1-phosphate transfer protein promotes sphingolipid reorientation needed for binding during membrane interaction

Lipid transfer proteins acquire and release their lipid cargoes by interacting transiently with source and destination biomembranes. In the GlycoLipid Transfer Protein (GLTP) superfamily, the two-layer all-α-helical GLTP-fold defines proteins that specifically target sphingolipids (SLs) containing either sugar or phosphate headgroups via their conserved but evolutionarily-modified SL recognitions centers. Despite comprehensive structural insights provided by X-ray crystallography, the conformational dynamics associated with membrane interaction and SL uptake/release by GLTP superfamily members have remained unknown. Herein, we report insights gained from molecular dynamics (MD) simulations into the conformational dynamics that enable ceramide-1-phosphate transfer proteins (CPTPs) to acquire and deliver ceramide-1-phosphate (C1P) during interaction with 1-palmitoyl-2-oleoyl phosphatidylcholine bilayers. The focus on CPTP reflects this protein’s involvement in regulating pro-inflammatory eicosanoid production and autophagy-dependent inflammasome assembly that drives interleukin (IL-1β and IL-18) production and release by surveillance cells. We found that membrane penetration by CPTP involved α-6 helix and the α-2 helix N-terminal region, was confined to one bilayer leaflet, and was relatively shallow. Large-scale dynamic conformational changes were minimal for CPTP during membrane interaction or C1P uptake except for the α-3/α-4 helices connecting loop, which is located near the membrane interface and interacts with certain phosphoinositide headgroups. Apart from functioning as a shallow membrane-docking element, α-6 helix was found to adeptly reorient membrane lipids to help guide C1P hydrocarbon chain insertion into the interior hydrophobic pocket of the SL binding site.These findings support a proposed ‘hydrocarbon chain-first’ mechanism for C1P uptake, in contrast to the ‘lipid polar headgroup-first’ uptake used by most lipid-transfer proteins.     

PD-L1 Expression in Normal Endocrine Tissues Is Not Increased Despite High Incidence of PD-1 Inhibitor-Associated Endocrinopathies

ObjectiveTreatment with immune-checkpoint inhibitors often results in endocrine immune-related adverse events (irAEs), affecting the pituitary, thyroid, adrenal, and parathyroid glands and pancreas. The mechanism underlying the endocrine irAEs has not been fully elucidated, and it remains unclear why endocrine organs are so commonly affected. In the present study, we evaluated immunostaining patterns of programmed death-ligand 1 (PD-L1) in normal endocrine tissues to determine whether increased expression may explain the predilection of endocrinopathies in patients treated with programmed cell death-1 inhibitors.MethodsNormal formalin-fixed paraffin-embedded endocrine tissues (pituitary, thyroid, adrenal, pancreas, and parathyroid) were collected from our hospital’s pathology tissue archive. The tissues were assessed for membranous and cytoplasmic PD-L1 immunostaining using the Dako 22C3 pharmDx assay on an automated staining platform.ResultsWe examined 49 endocrine tissues, including 12 thyroid, 5 pancreatic, 17 adrenal, 5 parathyroid, and 10 pituitary samples. Samples with less than 1% membranous PD-L1–positive cells were considered negative, while those with more than 1% of PD-L1 membranous staining were considered positive. Immunostaining result of immune-related cells was also evaluated, considering the cytoplasmic PD-L1–positive cells with the same cutoff of 1%. None of the endocrine tissues demonstrated PD-L1 positivity higher than 1% in the relevant cells.ConclusionWhile our results do not suggest a role of PD-L1 expression in the pathogenesis of endocrine irAEs, they may serve as a basis for future studies further investigating the mechanisms of autoimmune, inflammatory, or malignant endocrine conditions.