Pulmonary surfactant protein B carried by HDL predicts incident CVD in patients with type 1 diabetes

Atherosclerotic CVD is the major cause of death in patients with type 1 diabetes mellitus (T1DM). Alterations in the HDL proteome have been shown to associate with prevalent CVD in T1DM. We therefore sought to determine which proteins carried by HDL might predict incident CVD in patients with T1DM. Using targeted MS/MS, we quantified 50 proteins in HDL from 181 T1DM subjects enrolled in the prospective Coronary Artery Calcification in Type 1 Diabetes study. 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). We found that only one HDL protein—SFTPB (pulmonary surfactant protein B)—predicted incident CVD in all the models tested. In a fully adjusted model that controlled for 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). In addition, plasma fractionation demonstrated that SFTPB is nearly entirely bound to HDL. Although previous studies have shown that high plasma levels of SFTPB associate with prevalent atherosclerosis only in smokers, we found that SFTPB predicted incident CVD in T1DM independently of smoking status and a wide range of confounding factors, including HDL-C, LDL-C, and triglyceride levels. Because SFTPB is almost entirely bound to plasma HDL, our observations support the proposal that SFTPB carried by HDL is a marker—and perhaps mediator—of CVD risk in patients with T1DM.     

Distinct roles in phagocytosis of the early and late increases of cell surface calreticulin induced by oxaliplatin

Calreticulin (CRT), a chaperone typically located in the endoplasmic reticulum (ER), is known to translocate to the cell surface in response to anticancer drugs. Cell surface CRT (ecto-CRT) on apoptotic or pre-apoptotic cells serves as an “eat me” signal that can promote phagocytosis. In this study, we observed the biphasic (early transient and late sustained) increase of ecto-CRT on HT-29 cells after treatment with oxaliplatin (L-OHP). To investigate the role of ecto-CRT that accumulates in the early and late phases as “eat me” signals, we examined the phagocytosis of HT-29 cells by macrophage-like cells and dendritic cell (DC) -like cells prepared from THP-1 cells. The results indicated that the early ecto-CRT-expressed cells were phagocytosed by immature DC-like cells, and the late ecto-CRT-expressed cells were phagocytosed primarily by macrophage-like cells, while mature DC-like cells did not respond to the either class of ecto-CRT-expressed cells. Both types of phagocytotic events were inhibited by CRT Blocking Peptide, suggesting that such events depended on the ecto-CRT. Our results suggested that the early increase of ecto-CRT is related to phagocytosis as part of immunogenic cell death (ICD), while the late increase of ecto-CRT is related to the removal of apoptotic cells by macrophages.     

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.     

Under time pressure,the exogenous modulation of saccade plans is ubiquitous,intricate, and lawful

The choice of where to look next is determined by both exogenous (bottom-up) and endogenous (top-down) factors, but details of their interaction and distinct contributions to target selection have remained elusive. Recent experiments with urgent choice tasks, in which stimuli are evaluated while motor plans are already advancing, have greatly clarified these contributions. Specifically, exogenous modulations associated with stimulus detection act rapidly and briefly (∼25 ms) to automatically halt and/or boost ongoing motor plans as per spatial congruence rules. These stereotypical modulations explain, in quantitative detail, characteristic features of many saccadic tasks (e.g. antisaccade, countermanding, saccadic-inhibition, gap, and double-step). Thus, the same low-level visuomotor interactions contribute to diverse oculomotor phenomena traditionally attributed to different neural mechanisms.     

Vascular lipid droplets formed in response to TNF,hypoxia, or OA: biochemical composition and prostacyclin generation

Biogenesis of lipid droplets (LDs) in various cells plays an important role in various physiological and pathological processes. However, the function of LDs in endothelial physiology and pathology is not well understood. In the present work, we investigated the formation of LDs and prostacyclin (PGI₂) generation in the vascular tissue of isolated murine aortas following activation by proinflammatory factors: tumor necrosis factor (TNF), lipopolysaccharides (LPS), angiotensin II (AngII), hypoxic conditions, or oleic acid (OA). The abundance, size, and biochemical composition of LDs were characterized based on Raman spectroscopy and fluorescence imaging. We found that blockade of lipolysis by the adipose triglyceride lipase (ATGL) delayed LDs degradation and simultaneously blunted PGI₂ generation in aorta treated with all tested proinflammatory stimuli. Furthermore, the analysis of Raman spectra of LDs in the isolated vessels stimulated by TNF, LPS, AngII, or hypoxia uncovered that these LDs were all rich in highly unsaturated lipids and had a negligible content of phospholipids and cholesterols. Additionally, by comparing the Raman signature of endothelial LDs under hypoxic or OA-overload conditions in the presence or absence of ATGL inhibitor, atglistatin (Atgl), we show that Atgl does not affect the biochemical composition of LDs. Altogether, independent of whether LDs were induced by pro-inflammatory stimuli, hypoxia, or OA and of whether they were composed of highly unsaturated or less unsaturated lipids, we observed LDs formation invariably associated with ATGL-dependent PGI₂ generation. In conclusion, vascular LDs formation and ATGL-dependent PGI₂ generation represent a universal response to vascular proinflammatory insult.     

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.     

Human apoA-I[Lys107del] mutation affects lipid surface behavior of apoA-I and its ability to form large nascent HDL

Population studies have found that a natural human apoA-I variant, apoA-I[K107del], is strongly associated with low HDL-C but normal plasma apoA-I levels. We aimed to reveal properties of this variant that contribute to its unusual phenotype associated with atherosclerosis. Our oil-drop tensiometry studies revealed that compared to WT, recombinant apoA-I[K107del] adsorbed to surfaces of POPC-coated triolein drops at faster rates, remodeled the surfaces to a greater extent, and was ejected from the surfaces at higher surface pressures on compression of the lipid drops. These properties may drive increased binding of apoA-I[K107del] to and its better retention on large triglyceride-rich lipoproteins, thereby increasing the variant’s content on these lipoproteins. While K107del did not affect apoA-I capacity to promote ABCA1-mediated cholesterol efflux from J774 cells, it impaired the biogenesis of large nascent HDL particles resulting in the formation of predominantly smaller nascent HDL. Size-exclusion chromatography of spontaneously reconstituted 1,2-dimyristoylphosphatidylcholine-apoA-I complexes showed that apoA-I[K107del] had a hampered ability to form larger complexes but formed efficiently smaller-sized complexes. CD analysis revealed a reduced ability of apoA-I[K107del] to increase α-helical structure on binding to 1,2-dimyristoylphosphatidylcholine or in the presence of trifluoroethanol. This property may hinder the formation of large apoA-I[K107del]-containing discoidal and spherical HDL but not smaller HDL. Both factors, the increased content of apoA-I[K107del] on triglyceride-rich lipoproteins and the impaired ability of the variant to stabilize large HDL particles resulting in reduced lipid:protein ratios in HDL, may contribute to normal plasma apoA-I levels along with low HDL-C and increased risk for CVD.     

The SARS-CoV2 envelope differs from host cells,exposes procoagulant lipids,and is disrupted in vivo by oral rinses

The lipid envelope of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an essential component of the virus; however, its molecular composition is undetermined. Addressing this knowledge gap could support the design of antiviral agents as well as further our understanding of viral-host protein interactions, infectivity, pathogenicity, and innate immune system clearance. Lipidomics revealed that the virus envelope comprised mainly phospholipids (PLs), with some cholesterol and sphingolipids, and with cholesterol/phospholipid ratio similar to lysosomes. Unlike cellular membranes, procoagulant amino-PLs were present on the external side of the viral envelope at levels exceeding those on activated platelets. Accordingly, virions directly promoted blood coagulation. To investigate whether these differences could enable selective targeting of the viral envelope in vivo, we tested whether oral rinses containing lipid-disrupting chemicals could reduce infectivity. Products containing PL-disrupting surfactants (such as cetylpyridinium chloride) met European virucidal standards in vitro; however, components that altered the critical micelle concentration reduced efficacy, and products containing essential oils, povidone-iodine, or chlorhexidine were ineffective. This result was recapitulated in vivo, where a 30-s oral rinse with cetylpyridinium chloride mouthwash eliminated live virus in the oral cavity of patients with coronavirus disease 19 for at least 1 h, whereas povidone-iodine and saline mouthwashes were ineffective. We conclude that the SARS-CoV-2 lipid envelope i) is distinct from the host plasma membrane, which may enable design of selective antiviral approaches; ii) contains exposed phosphatidylethanolamine and phosphatidylserine, which may influence thrombosis, pathogenicity, and inflammation; and iii) can be selectively targeted in vivo by specific oral rinses.     

Hormone-sensitive lipase is localized at synapses and is necessary for normal memory functioning in mice

Hormone-sensitive lipase (HSL) is mainly present in adipose tissue where it hydrolyzes diacylglycerol. Although expression of HSL has also been reported in the brain, its presence in different cellular compartments is uncertain, and its role in regulating brain lipid metabolism remains hitherto unexplored. We hypothesized that HSL might play a role in regulating the availability of bioactive lipids necessary for neuronal function and therefore investigated whether dampening HSL activity could lead to brain dysfunction. In mice, we found HSL protein and enzymatic activity throughout the brain, localized within neurons and enriched in synapses. HSL-null mice were then analyzed using a battery of behavioral tests. Relative to wild-type littermates, HSL-null mice showed impaired short-term and long-term memory, yet preserved exploratory behaviors. Molecular analysis of the cortex and hippocampus showed increased expression of genes involved in glucose utilization in the hippocampus, but not cortex, of HSL-null mice compared with controls. Furthermore, lipidomics analyses indicated an impact of HSL deletion on the profile of bioactive lipids, including a decrease in endocannabinoids and eicosanoids that are known to modulate neuronal activity, cerebral blood flow, and inflammation processes. Accordingly, mild increases in the expression of proinflammatory cytokines in HSL mice compared with littermates were suggestive of low-grade inflammation. We conclude that HSL has a homeostatic role in maintaining pools of lipids required for normal brain function. It remains to be tested, however, whether the recruitment of HSL for the synthesis of these lipids occurs during increased neuronal activity or whether HSL participates in neuroinflammatory responses.     

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.     

Lipoprotein size is a main determinant for the rate of hydrolysis by exogenous LPL in human plasma

LPL is a key player in plasma triglyceride metabolism. Consequently, LPL is regulated by several proteins during synthesis, folding, secretion, and transport to its site of action at the luminal side of capillaries, as well as during the catalytic reaction. Some proteins are well known, whereas others have been identified but are still not fully understood. We set out to study the effects of the natural variations in the plasma levels of all known LPL regulators on the activity of purified LPL added to samples of fasted plasma taken from 117 individuals. The enzymatic activity was measured at 25°C using isothermal titration calorimetry. This method allows quantification of the ability of an added fixed amount of exogenous LPL to hydrolyze triglyceride-rich lipoproteins in plasma samples by measuring the heat produced. Our results indicate that, under the conditions used, the normal variation in the endogenous levels of apolipoprotein C1, C2, and C3 or the levels of angiopoietin-like proteins 3, 4, and 8 in the fasted plasma samples had no significant effect on the recorded activity of the added LPL. Instead, the key determinant for the LPL activity was a lipid signature strongly correlated to the average size of the VLDL particles. The signature involved not only several lipoprotein and plasma lipid parameters but also apolipoprotein A5 levels. While the measurements cannot fully represent the action of LPL when attached to the capillary wall, our study provides knowledge on the interindividual variation of LPL lipolysis rates in human plasma.     

Membrane Binding and Homodimerization of Atg16 Via Two Distinct Protein Regions is Essential for Autophagy in Yeast

Macroautophagy is a bulk degradation mechanism in eukaryotic cells. Efficiency of an essential step of this process in yeast, Atg8 lipidation, relies on the presence of Atg16, a subunit of the Atg12–Atg5-Atg16 complex acting as the E3-like enzyme in the ubiquitination-like reaction. A current view on the functional structure of Atg16 in the yeast S. cerevisiae comes from the two crystal structures that reveal the Atg5-interacting α-helix linked via a flexible linker to another α-helix of Atg16, which then assembles into a homodimer. This view does not explain the results of previous in vitro studies revealing Atg16-dependent deformations of membranes and liposome-binding of the Atg12–Atg5 conjugate upon addition of Atg16. Here we show that Atg16 acts as both a homodimerizing and peripheral membrane-binding polypeptide. These two characteristics are imposed by the two distinct regions that are disordered in the nascent protein. Atg16 binds to membranes in vivo via the amphipathic α-helix (amino acid residues 113–131) that has a coiled-coil-like propensity and a strong hydrophobic face for insertion into the membrane. The other protein region (residues 64–99) possesses a coiled-coil propensity, but not amphipathicity, and is dispensable for membrane anchoring of Atg16. This region acts as a Leu-zipper essential for formation of the Atg16 homodimer. Mutagenic disruption in either of these two distinct domains renders Atg16 proteins that, in contrast to wild type, completely fail to rescue the autophagy-defective phenotype of atg16Δ cells. Together, the results of this study yield a model for the molecular mechanism of Atg16 function in macroautophagy.     

Mass Spectrometry and Machine Learning Reveal Determinants of Client Recognition by Antiamyloid Chaperones

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Inhibition of Cancer Cell Adhesion,Migration and Proliferation by a Bispecific Antibody that Targets two Distinct Epitopes on αv Integrins

Members of the αv family of integrins regulate activation of transforming growth factor beta (TGFβ) and are directly involved in pro-tumorigenic phenotypes. Thus, αv integrins may be therapeutic targets for fibrosis and cancer, yet the isolation of selective inhibitors is currently a challenge. We generated synthetic antibodies selective for αv integrins by phage display selections on cell lines that displayed integrin heterodimers. We identified antibodies that targeted two distinct epitopes on cell-surface αv integrins and partially inhibited cell adhesion mediated by interactions between integrins and the latency-associated peptide, part of the pro-form of TGFβ. Using the isolated antibody paratope sequences we engineered a bispecific antibody capable of binding to both epitopes simultaneously; this antibody potently and completely inhibited cell adhesion mediated by integrins αvβ1, αvβ3 and αvβ5. In addition, the bispecific antibody inhibited proliferation and migration of lung carcinoma lines, where the highest and lowest potencies observed correlated with integrin-αv cell surface expression levels. Taken together, our results demonstrate that phage display selections with live cells can yield high quality anti-integrin antibodies, which we used as biparatopic building blocks to construct a bispecific antibody that strongly inhibited integrin function and may be a therapeutic candidate for cancer and fibrosis.     

Nordic Walking Improves Cardiometabolic Parameters,Fitness Performance,and Quality of Life in Older Adults With Type 2 Diabetes

ObjectiveTo assess the effect of Nordic walking (NW) on cardiometabolic health, physical performance, and well-being in sedentary older adults with type 2 diabetes (T2D).MethodsFifteen subjects with T2D (female, 5; male, 10; age, 65 ± 6.2 years [mean ± standard deviation]; body mass index, 27.3 ± 4.9 kg/m² [mean ± standard deviation]) were enrolled in a 6-month NW training program. The fasting glucose and glycosylated hemoglobin levels, lipid profile (total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, and triglycerides), systolic blood pressure (SBP), and diastolic blood pressures were measured before and after the intervention. Participants’ quality of life (Short-Form Health Survey) and physical fitness (6-minute walking test) were also evaluated.ResultsCompared with baseline, NW significantly improved the fasting glucose level (103.5 ± 18.5 vs 168.7 ± 37.7 mg/dL, P = .01), SBP (121.8 ± 12.2 vs 133 ± 14.4 mm Hg, P = .02), physical fitness (759.88 ± 69 vs 615.5 ± 62.6 m, P < .001), and both mental health (54.5 ± 4.4 vs 45.7 ± 5.6, P < .01) and physical health (49.8 ± 4.7 vs 40.3 ± 5.9, P < .01). The levels of glycosylated hemoglobin (6.15% ± 0.8% vs 6.4% ± 1%, P = .46), total cholesterol (162.2 ± 31.2 vs 175.5 ± 28.8 mg/dL, P = .13), low-density lipoprotein cholesterol (95.2 ± 24.2 vs 106.3 ± 32.3 mg/dL, P = .43), and triglycerides (135.5 ± 60.8 vs 127.6 ± 57.4 mg/dL, P = 0.26) improved without reaching significance.ConclusionNW training improved the glycemic levels, SBP, physical fitness, and perception of quality of life in older adults with T2D. NW represents a suitable complementary strategy to improve the global health status in this population.