Effects of Recombinant Human Lecithin Cholesterol Acyltransferase on Lipoprotein Metabolism in Humans.

BACKGROUND: LCAT (lecithin cholesterol acyl transferase) catalyzes the conversion of unesterified, or free cholesterol, to cholesteryl ester, which moves from the surface of HDL (high-density lipoprotein) into the neutral lipid core. As this iterative process continues, nascent lipid-poor HDL is converted to a series of larger, spherical cholesteryl ester-enriched HDL particles that can be cleared by the liver in a process that has been termed reverse cholesterol transport. METHODS: We conducted a randomized, placebocontrolled, crossover study in 5 volunteers with atherosclerotic cardiovascular disease, to examine the effects of an acute increase of recombinant human (rh) LCAT via intravenous administration (300-mg loading dose followed by 150 mg at 48 hours) on the in vivo metabolism of HDL APO (apolipoprotein)A1 and APOA2, and the APOB100-lipoproteins, very low density, intermediate density, and low-density lipoproteins. RESULTS: As expected, recombinant human LCAT treatment significantly increased HDL-cholesterol (34.9 mg/dL; P≤0.001), and this was mostly due to the increase in cholesteryl ester content (33.0 mg/dL; P=0.014). This change did not affect the fractional clearance or production rates of HDL-APOA1 and HDL-APOA2. There were also no significant changes in the metabolism of APOB100-lipoproteins. CONCLUSIONS: Our results suggest that an acute increase in LCAT activity drives greater flux of cholesteryl ester through the reverse cholesterol transport pathway without significantly altering the clearance and production of the main HDL proteins and without affecting the metabolism of APOB100-lipoproteins. Long-term elevations of LCAT might, therefore, have beneficial effects on total body cholesterol balance and atherogenesis.

Mitochondrial control of hypoxia-induced pathological retinal angiogenesis.

OBJECTIVE: Pathological retinal neovascularization is vision-threatening. In mouse oxygen-induced retinopathy (OIR) we sought to define mitochondrial respiration changes longitudinally during hyperoxia-induced vessel loss and hypoxia-induced neovascularization, and to test interventions addressing those changes to prevent neovascularization. METHODS: OIR was induced in C57BL/6J mice and retinal vasculature was examined at maximum neovessel formation. We assessed total proteome changes and the ratio of mitochondrial to nuclear DNA copy numbers (mtDNA/nDNA) of OIR vs. control retinas, and mitochondrial oxygen consumption rates (OCR) in ex vivo OIR vs. control retinas (BaroFuse). Pyruvate vs. vehicle control was supplemented to OIR mice either prior to or during neovessel formation. RESULTS: In OIR vs. control retinas, global proteomics showed decreased retinal mitochondrial respiration at peak neovascularization. OCR and mtDNA/nDNA were also decreased at peak neovascularization suggesting impaired mitochondrial respiration. In vivo pyruvate administration during but not prior to neovessel formation (in line with mitochondrial activity time course) suppressed NV. CONCLUSIONS: Mitochondrial energetics were suppressed during retinal NV in OIR. Appropriately timed supplementation of pyruvate may be a novel approach in neovascular retinal diseases.

The role of proprotein convertase subtilisin/kexin 9 (PCSK9) in macrophage activation: a focus on its LDL receptor-independent mechanisms.

Recent clinical trials demonstrated that proprotein convertase subtilisin/kexin 9 (PCSK9) inhibitors reduce cardiovascular events without affecting systemic inflammation in the patients with coronary artery disease, as determined by high sensitivity C-reactive protein (CRP) levels. However, its pro-inflammatory effects in cardiovascular disease in humans and experimental animals beyond the traditional cholesterol receptor-dependent lipid metabolism have also called attention of the scientific community. PCSK9 may target receptors associated with inflammation other than the low-density lipoprotein receptor (LDLR) and members of the LDLR family. Accumulating evidence suggests that PCSK9 promotes macrophage activation not only via lipid-dependent mechanisms, but also lipid-independent and LDLR-dependent or -independent mechanisms. In addition to dyslipidemia, PCSK9 may thus be a potential therapeutic target for various pro-inflammatory diseases.

Carnitine O-octanoyltransferase (CROT) deficiency in mice leads to an increase of omega-3 fatty acids.

Introduction: Carnitine O-octanoyltransferase (CROT) is a well-established peroxisomal enzyme involved in liver fatty acid oxidation, but less is known about its recently discovered role in promoting vascular calcification, and whether CROT-dependent liver metabolism contributes to the latter. To date, CROT function in the context of calcification potential has been conducted in the dyslipidemic low-density lipoprotein receptor-deficient (Ldlr-/-) mice. Objectives: To differentiate peroxisome and CROT-dependent lipid biology from that of lipoprotein-mediated lipid biology, we therefore conducted a metabolomic analysis of the liver and plasma of normolipidemic CROT-deficient (Crot-/-) mice. Methods: We performed LC-MS-based metabolomics on liver and plasma derived from Crot-/- and Crot +/- mice and sibling Crot+/+ mice, using a dual-phase metabolite extraction protocol, and multiple LC-MS acquisition strategies. Results: We identified between 79 to 453 annotated metabolites from annotated metabolites from liver samples, and 117 to 424 annotated metabolites from plasma samples. Through differential abundance analysis, we determined that omega-3 fatty acids such as EPA, DPA, and DHA were higher in the liver of Crot-/- and Crot +/- mice than Crot+/+ mice. EPA were higher in plasma of Crot-/- mice than Crot+/+ mice. We also determined that the anti-inflammatory dicarboxylic acids, tetradecanedioic acid and azelaic acid, were higher in the plasma of CROT-deficient mice. Conclusion: Our study associated genetic CROT deletion with increased levels of anti-inflammatory molecules in mouse liver and plasma. These results suggest a potential mechanism for anti-calcification effects of CROT suppression and the potential use of omega-3 fatty acids as biomarkers for future CROT inhibition therapies.

Polycarbonate Ultracentrifuge Tube Re-use in Proteomic Analyses of Extracellular Vesicles.

Single-use laboratory plastics exacerbate the pollution crisis and contribute to consumable costs. In extracellular vesicle (EV) isolation, polycarbonate ultracentrifuge (UC) tubes are used to endure the associated high centrifugal forces. EV proteomics is an advancing field and validated re-use protocols for these tubes are lacking. Re-using consumables for low-yield protein isolation protocols and downstream proteomics requires reagent compatibility with mass spectroscopy acquisitions, such as the absence of centrifuge tube-derived synthetic polymer contamination, and sufficient removal of residual proteins. This protocol describes and validates a method for cleaning polycarbonate UC tubes for re-use in EV proteomics experiments. The cleaning process involves immediate submersion of UC tubes in H2O to prevent protein drying, washing in 0.1% sodium dodecyl sulfate (SDS) detergent, rinsing in hot tap water, demineralized water, and 70% ethanol. To validate the UC tube re-use protocol for downstream EV proteomics, used tubes were obtained following an experiment isolating EVs from cardiovascular tissue using differential UC and density gradient separation. Tubes were cleaned and the experimental process was repeated without EV samples comparing blank never-used UC tubes to cleaned UC tubes. The pseudo-EV pellets obtained from the isolation procedures were lysed and prepared for liquid chromatography-tandem mass spectrometry using a commercial protein sample preparation kit with modifications for low-abundance protein samples. Following cleaning, the number of identified proteins was reduced by 98% in the pseudo-pellet versus the previous EV isolation sample from the same tube. Comparing a cleaned tube against a blank tube, both samples contained a very small number of proteins (≤20) with 86% similarity. The absence of polymer peaks in the chromatograms of the cleaned tubes was confirmed. Ultimately, the validation of a UC tube cleaning protocol suitable for the enrichment of EVs will reduce the waste produced by EV laboratories and lower the experimental costs.

Timed topical dexamethasone eye drops improve mitochondrial function to prevent severe retinopathy of prematurity.

Pathological neovascularization in retinopathy of prematurity (ROP) can cause visual impairment in preterm infants. Current ROP treatments which are not preventative and only address late neovascular ROP, are costly and can lead to severe complications. We showed that topical 0.1% dexamethasone eye drops administered prior to peak neovessel formation prevented neovascularization in five extremely preterm infants at high risk for ROP and suppressed neovascularization by 30% in mouse oxygen-induced retinopathy (OIR) modeling ROP. In contrast, in OIR, topical dexamethasone treatment before any neovessel formation had limited efficacy in preventing later neovascularization, while treatment after peak neovessel formation had a non-statistically significant trend to exacerbating disease. Optimally timed topical dexamethasone suppression of neovascularization in OIR was associated with increased retinal mitochondrial gene expression and decreased inflammatory marker expression, predominantly found in immune cells. Blocking mitochondrial ATP synthetase reversed the inhibitory effect of dexamethasone on neovascularization in OIR. This study provides new insights into topical steroid effects in retinal neovascularization and into mitochondrial function in phase II ROP, and suggests a simple clinical approach to prevent severe ROP.

Intracellular proteomics and extracellular vesiculomics as a metric of disease recapitulation in 3D-bioprinted aortic valve arrays.

In calcific aortic valve disease (CAVD), mechanosensitive valvular cells respond to fibrosis- and calcification-induced tissue stiffening, further driving pathophysiology. No pharmacotherapeutics are available to treat CAVD because of the paucity of (i) appropriate experimental models that recapitulate this complex environment and (ii) benchmarking novel engineered aortic valve (AV)-model performance. We established a biomaterial-based CAVD model mimicking the biomechanics of the human AV disease-prone fibrosa layer, three-dimensional (3D)-bioprinted into 96-well arrays. Liquid chromatography-tandem mass spectrometry analyses probed the cellular proteome and vesiculome to compare the 3D-bioprinted model versus traditional 2D monoculture, against human CAVD tissue. The 3D-bioprinted model highly recapitulated the CAVD cellular proteome (94% versus 70% of 2D proteins). Integration of cellular and vesicular datasets identified known and unknown proteins ubiquitous to AV calcification. This study explores how 2D versus 3D-bioengineered systems recapitulate unique aspects of human disease, positions multiomics as a technique for the evaluation of high throughput-based bioengineered model systems, and potentiates future drug discovery.

PARP14 is pro- and anti-viral host factor that promotes IFN production and affects the replication of multiple viruses.

PARP14 is a 203 kDa multi-domain protein that is primarily known as an ADP-ribosyltransferase, and is involved in a variety of cellular functions including DNA damage, microglial activation, inflammation, and cancer progression. In addition, PARP14 is upregulated by interferon (IFN), indicating a role in the antiviral response. Furthermore, PARP14 has evolved under positive selection, again indicating that it is involved in host-pathogen conflict. We found that PARP14 is required for increased IFN-I production in response to coronavirus infection lacking ADP-ribosylhydrolase (ARH) activity and poly(I:C), however, whether it has direct antiviral function remains unclear. Here we demonstrate that the catalytic activity of PARP14 enhances IFN-I and IFN-III responses and restricts ARH-deficient murine hepatitis virus (MHV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication. To determine if PARP14's antiviral functions extended beyond CoVs, we tested the ability of herpes simplex virus 1 (HSV-1) and several negative-sense RNA viruses, including vesicular stomatitis virus (VSV), Ebola virus (EBOV), and Nipah virus (NiV), to infect A549 PARP14 knockout (KO) cells. HSV-1 had increased replication in PARP14 KO cells, indicating that PARP14 restricts HSV-1 replication. In contrast, PARP14 was critical for the efficient infection of VSV, EBOV, and NiV, with EBOV infectivity at less than 1% of WT cells. A PARP14 active site inhibitor had no impact on HSV-1 or EBOV infection, indicating that its effect on these viruses was independent of its catalytic activity. These data demonstrate that PARP14 promotes IFN production and has both pro- and anti-viral functions targeting multiple viruses.