Aims Lipoprotein (a) [Lp(a)] is a lipoprotein species causatively associated with atherosclerosis. Unlike statins, PCSK9 inhibitors (PCSK9i) reduce Lp(a), but this reduction is highly variable. Levels of Lp(a) are chiefly governed by the size of its signature protein, apolipoprotein (a) [apo(a)]. Whether this parameter determines some of the reduction in Lp(a) induced by PCSK9i remains unknown. We aimed to investigate if the Lp(a) lowering efficacy of PCSK9i is modulated by the size of apo(a), which is genetically determined by the variable number of KIV domains present on that protein. Methods and results The levels of Lp(a) and the size of apo(a) were assessed in plasma samples from 268 patients before and after treatment with PCSK9i. Patients were recruited at the Outpatient Lipid Clinic of the Charite Hospital (Berlin) between 2015 and 2020. They were hypercholesterolaemic at very high cardiovascular disease risk with low-density lipoprotein (LDL)-cholesterol levels above therapeutic targets despite maximally tolerated lipid-lowering therapy. Patients received either Alirocumab (75 or 150 mg) or Evolocumab (140 mg) every 2 weeks. Apo(a), apoB100, and apoE concentrations as well as apoE major isoforms were determined by liquid chromatography high-resolution mass spectrometry. Apo(a) isoforms sizes were determined by western blot. PCSK9i sharply reduced LDL-cholesterol (-57%), apoB100 (-47%), and Lp(a) (-36%). There was a positive correlation between the size of apo(a) and the relative reduction in Lp(a) induced by PCSK9i (r = 0.363, P = 0.0001). The strength of this association remained unaltered after adjustment for baseline Lp(a) levels and all other potential confounding factors. In patients with two detectable apo(a) isoforms, there was also a positive correlation between the size of apo(a) and the reduction in Lp(a), separately for the smaller (r = 0.350, P = 0.0001) and larger (r = 0.324, P = 0.0003) isoforms. The relative contribution of the larger isoform to the total concentration of apo(a) was reduced from 29% to 15% (P < 0.0001). Conclusions The size of apo(a) is an independent determinant of the response to PCSK9i. Each additional kringle domain is associated with a 3% additional reduction in Lp(a). This explains in part the variable efficacy of PCSK9i and allows to identify patients who will benefit most from these therapies in terms of Lp(a) lowering.

Introduction: The pleiotropic protective effects of high-density lipoproteins (HDLs) on cerebral ischemia have never been tested under acute hyperglycemic conditions. The aim of this study is to evaluate the potential neuroprotective effect of HDL intracarotid injection in a mouse model of middle cerebral artery occlusion (MCAO) under hyperglycemic conditions. Methods: Forty-two mice were randomized to receive either an intracarotid injection of HDLs or saline. Acute hyperglycemia was induced by an intraperitoneal injection of glucose (2.2 g/kg) 20 min before MCAO. Infarct size (2,3,5-triphenyltetrazolium chloride (TTC)-staining), blood–brain barrier leakage (IgG infiltration), and hemorrhagic changes (hemoglobin assay by ELISA and hemorrhagic transformation score) were analyzed 24 h post-stroke. Brain tissue inflammation (IL-6 by ELISA, neutrophil infiltration and myeloperoxidase by immunohisto-fluorescence) and apoptosis (caspase 3 activation) were also assessed. Results: Intraperitoneal D-glucose injection allowed HDL- and saline-treated groups to reach a blood glucose level of 300 mg/dl in the acute phase of cerebral ischemia. HDL injection did not significantly reduce mortality (19% versus 29% in the saline-injected group) or cerebral infarct size (p = 0.25). Hemorrhagic transformations and inflammation parameters were not different between the two groups. In addition, HDL did not inhibit apoptosis under acute hyperglycemic conditions. Conclusion: We observed a nonsignificant decrease in cerebral infarct size in the HDL group. The deleterious consequences of reperfusion such as hemorrhagic transformation or inflammation were not improved by HDL infusion. In acute hyperglycemia, HDLs are not potent enough to counteract the adverse effects of hyperglycemia. The addition of antioxidants to therapeutic HDLs could improve their neuroprotective capacity.

Background and Aims: PCSK9 inhibitors can reduce LDL cholesterol down to extremely low levels. Despite PCSK9 inhibitors undisputed cardiovascular benefits, a relationship between very low LDL-C and the risk of hemorrhagic transformation (HT) following ischemic stroke has been suggested. The aim of the present study was to assess HT in PCSK9 knockout mice.

HomeStrokeVol. 52, No. 9PCSK9 (Proprotein Convertase Subtilisin Kexin Type 9) Inhibition in Hyperglycemic Mice Increases the Risk of Hemorrhagic Transformation of Ischemic Stroke Free AccessLetterPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyRedditDiggEmail Jump toSupplementary MaterialsFree AccessLetterPDF/EPUBPCSK9 (Proprotein Convertase Subtilisin Kexin Type 9) Inhibition in Hyperglycemic Mice Increases the Risk of Hemorrhagic Transformation of Ischemic Stroke Brice Nativel, PhD Stéphane Ramin-Mangata, PhD David Couret, MD, PhD Cynthia Planesse, BSc Mathias Roche, BSc Antonio Gallo, MD, PhD Olivier Meilhac, PhD Gilles Lambert, PhD Steeve BouranePhD Brice NativelBrice Nativel https://orcid.org/0000-0003-3719-7903 Université de La Réunion, INSERM, UMR 1188 Diabète athérothrombose Réunion Océan Indien (DéTROI), Saint-Denis de La Réunion, France (B.N., S.R.-M., D.C., C.P., M.R., A.G., O.M., G.L., S.B.). , Stéphane Ramin-MangataStéphane Ramin-Mangata https://orcid.org/0000-0002-3865-9084 Université de La Réunion, INSERM, UMR 1188 Diabète athérothrombose Réunion Océan Indien (DéTROI), Saint-Denis de La Réunion, France (B.N., S.R.-M., D.C., C.P., M.R., A.G., O.M., G.L., S.B.). , David CouretDavid Couret https://orcid.org/0000-0003-0305-8681 Université de La Réunion, INSERM, UMR 1188 Diabète athérothrombose Réunion Océan Indien (DéTROI), Saint-Denis de La Réunion, France (B.N., S.R.-M., D.C., C.P., M.R., A.G., O.M., G.L., S.B.). CHU de La Réunion, Saint-Pierre de la Réunion, France (D.C., O.M.). , Cynthia PlanesseCynthia Planesse Université de La Réunion, INSERM, UMR 1188 Diabète athérothrombose Réunion Océan Indien (DéTROI), Saint-Denis de La Réunion, France (B.N., S.R.-M., D.C., C.P., M.R., A.G., O.M., G.L., S.B.). , Mathias RocheMathias Roche Université de La Réunion, INSERM, UMR 1188 Diabète athérothrombose Réunion Océan Indien (DéTROI), Saint-Denis de La Réunion, France (B.N., S.R.-M., D.C., C.P., M.R., A.G., O.M., G.L., S.B.). , Antonio GalloAntonio Gallo https://orcid.org/0000-0002-8887-994X Université de La Réunion, INSERM, UMR 1188 Diabète athérothrombose Réunion Océan Indien (DéTROI), Saint-Denis de La Réunion, France (B.N., S.R.-M., D.C., C.P., M.R., A.G., O.M., G.L., S.B.). , Olivier MeilhacOlivier Meilhac Université de La Réunion, INSERM, UMR 1188 Diabète athérothrombose Réunion Océan Indien (DéTROI), Saint-Denis de La Réunion, France (B.N., S.R.-M., D.C., C.P., M.R., A.G., O.M., G.L., S.B.). CHU de La Réunion, Saint-Pierre de la Réunion, France (D.C., O.M.). , Gilles LambertGilles Lambert Correspondence to: Gilles Lambert, PhD, Laboratoire Inserm UMR 1188 DéTROI, Plateforme CYROI, 2 Rue Maxime Rivière, 97490 Sainte Clotilde, France. Email E-mail Address: [email protected] https://orcid.org/0000-0001-5632-0685 Université de La Réunion, INSERM, UMR 1188 Diabète athérothrombose Réunion Océan Indien (DéTROI), Saint-Denis de La Réunion, France (B.N., S.R.-M., D.C., C.P., M.R., A.G., O.M., G.L., S.B.). and Steeve BouraneSteeve Bourane https://orcid.org/0000-0002-9805-2196 Université de La Réunion, INSERM, UMR 1188 Diabète athérothrombose Réunion Océan Indien (DéTROI), Saint-Denis de La Réunion, France (B.N., S.R.-M., D.C., C.P., M.R., A.G., O.M., G.L., S.B.). Originally published28 Jul 2021https://doi.org/10.1161/STROKEAHA.121.035677Stroke. 2021;52:e545–e547AbstractDownload figureDownload PowerPointThe relationship between very low levels of LDL-C (low-density lipoprotein cholesterol) and the risk of hemorrhagic transformations (HT) after ischemic stroke (IS) remains a matter of intense controversy.1 HT is a too frequent complication of IS and is predictive of the worst clinical outcomes. Acute preischemic hyperglycemic conditions aggravate HT and thereby worsen IS prognosis.2 Since pharmacological inhibition of PCSK9 (proprotein convertase subtilisin kexin type 9) drastically lowers LDL-C, we aimed to evaluate whether chronic PCSK9 deficiency increases the risk of HT in a mouse model of acute hyperglycemic IS.MethodsThe data that support the findings of this study are available from the corresponding author upon reasonable request. In vivo experiments were conducted in accordance with the French and European Community Guidelines for the Use of Animals in Research (86/609/EEC and 2010/63/EU) and approved by the Ethics Committee for animal experimentations (CYROI APAFIS#9040-201611282211297 v9). Eight-week-old wild-type and PCSK9 knockout mice were infused intraperitoneally with 2.2 g of glucose per kg of body weight.2 Cerebral ischemia/reperfusion was induced by a 90 minutes intraluminal middle cerebral artery occlusion by introducing a monofilament into the right common carotid.3 After removing the monofilament, reperfusion was allowed for the next 22 hours. Mice were euthanized, and intravascular washout was performed by intracardiac perfusion of saline. Brains were harvested and cut into 1-mm coronal slices for evaluation of infarct volumes and HT by macroscopic scoring and extravascular hemoglobin quantification (Methods in the Data Supplement).ResultsAs anticipated, PCSK9 knockouts had sharply reduced plasma cholesterol, LDL-C, and triglycerides (Table I in the Data Supplement). Despite similar baseline glucose levels, PCSK9 knockouts had significantly higher glycemia and delayed glucose clearance compared with wild types (Figure I in the Data Supplement). Brain infarct volumes were similar in PCSK9 knockout and control mice, at 43±13% and 41±9% of the contralateral hemisphere, respectively (Figure II in the Data Supplement). In contrast, PCSK9 knockouts exhibited more hemorrhages macroscopically than wild types (Figure III in the Data Supplement and Figure [A]). The hemoglobin content of brain homogenates was higher in PCSK9 knockouts than in wild types, with respective ipsilateral/contralateral ratios of 4.5±2.4 and 2.5±1.0 (P=0.039; Figure [B]), and correlated positively with macroscopic hemorrhagic scores (r=0.42; P=0.025). Given that MMP-9 (matrix metalloprotease-9) overexpression and activation induce the degradation of the extracellular matrix, in particular that of cerebral blood vessels, leading to vascular leakage,4 we assessed its expression in the brain sections of these animals. MMP-9 expression was restricted to cerebral blood vessels of the infarcted areas and sharply increased in PCSK9 knockouts versus controls with respective fluorescence intensities of 1.97±0.62 and 0.95±0.42 (P=0.012; Figure IV in the Data Supplement and Figure [C]).Download figureDownload PowerPointFigure. Hemorrhagic transformations in PCSK9 (proprotein convertase subtilisin kexin type 9) knockout and wild-type mice in acute hyperglycemic conditions. Representative example of hemorrhagic transformation (HT) in wild-type and PCSK9 knockout mice brain slices (A). Hemoglobin content of brain homogenates 22 h post-middle cerebral artery occlusion (MCAO; B). Representative immunohistological analyses of infarcted brain sections areas of wild-type and PCSK9 knockout mice stained for MMP-9 (matrix metalloprotease-9; red) and the blood vessel marker IB4 (green). Cell nuclei were stained with DAPI and appear in blue (C).DiscussionOur first observation is at odds with previous studies showing similar HT in PCSK9 knockout and wild-type mice.1 The major difference between these studies is that here animals were under acute hyperglycemia, a very common condition associated with IS and known to alter the integrity of the blood-brain barrier.2 The fact that PCSK9 knockout mice are intolerant to glucose compared with wild types5 may in itself suffice to explain the worsening of HT in this animal model under acute hyperglycemia. We also showed that MMP-9 expression was twice as high in the infarcted brains of PCSK9 knockouts versus controls. Since MMP-9 weakens tight junctions and is overexpressed and activated after stroke, we propose this as the underlying mechanism aggravating the cerebrovascular leakages in hyperglycemic PCSK9 knockout mice after stroke.4 It remains to be seen whether increased MMP-9 results from the lack of endogenous PCSK9 in brain cells or from the impaired glucose tolerance observed in these animals. Both options indicate that drugs such as monoclonal antibodies that specifically target circulating PCSK9 will likely not increase the risk of HT of IS in humans.Sources of FundingThe project grant ANR-20-CE14-0009 from the Agence Nationale de la Recherche.DisclosuresDr Lambert reports honoraria and research support from Sanofi-Regeneron, Amgen Inc, Affiris AG, Nyrada Inc, and Pfizer. Dr Gallo reports personal fees from Akcea Therapeutics, Amgen Inc, Mylan, MSD, and SANOFI. The other authors report no conflicts.Supplemental MaterialsExpanded Materials and MethodsOnline Table IOnline Figures I–IVNonstandard Abbreviations and AcronymsHThemorrhagic transformationISischemic strokeLDL-Clow-density lipoprotein cholesterolMMP-9matrix metalloprotease-9PCSK9proprotein convertase subtilisin kexin type 9Footnotes*B. Nativel, S. Ramin-Mangata, and D. Couret contributed equally.The Data Supplement is available with this article at https://www.ahajournals.org/doi/suppl/10.1161/STROKEAHA.121.035677.For Sources of Funding and Disclosures, see page e546.Correspondence to: Gilles Lambert, PhD, Laboratoire Inserm UMR 1188 DéTROI, Plateforme CYROI, 2 Rue Maxime Rivière, 97490 Sainte Clotilde, France. Email gilles.[email protected]frReferences1. Tran-Dinh A, Levoye A, Lambert G, Louedec L, Journé C, Meilhac O, Amarenco P. Low levels of low-density lipoprotein-C associated with proprotein convertase subtilisin kexin 9 inhibition do not increase the risk of hemorrhagic transformation.Stroke. 2014; 45:3086–3088. doi: 10.1161/STROKEAHA.114.005958LinkGoogle Scholar2. Couret D, Bourane S, Catan A, Nativel B, Planesse C, Dorsemans AC, Ait-Arsa I, Cournot M, Rondeau P, Patche J, Tran-Dinh A, Lambert G, Diotel N, Meilhac O. A hemorrhagic transformation model of mechanical stroke therapy with acute hyperglycemia in mice.J Comp Neurol. 2018; 526:1006–1016. doi: 10.1002/cne.24386Google Scholar3. Hata R, Mies G, Wiessner C, Fritze K, Hesselbarth D, Brinker G, Hossmann KA. A reproducible model of middle cerebral artery occlusion in mice: hemodynamic, biochemical, and magnetic resonance imaging.J Cereb Blood Flow Metab. 1998; 18:367–375. doi: 10.1097/00004647-199804000-00004CrossrefMedlineGoogle Scholar4. Turner RJ, Sharp FR. Implications of MMP9 for blood brain barrier disruption and hemorrhagic transformation following ischemic stroke.Front Cell Neurosci. 2016; 10:56. doi: 10.3389/fncel.2016.00056CrossrefMedlineGoogle Scholar5. Da Dalt L, Ruscica M, Bonacina F, Balzarotti G, Dhyani A, Di Cairano E, Baragetti A, Arnaboldi L, De Metrio S, Pellegatta F, et al.. PCSK9 deficiency reduces insulin secretion and promotes glucose intolerance: the role of the low-density lipoprotein receptor.Eur Heart J. 2019; 40:357–368. doi: 10.1093/eurheartj/ehy357Google Scholar Previous Back to top Next FiguresReferencesRelatedDetails September 2021Vol 52, Issue 9Article InformationMetrics Download: 187 © 2021 American Heart Association, Inc.https://doi.org/10.1161/STROKEAHA.121.035677PMID: 34315254 Originally publishedJuly 28, 2021 Keywordsischemic strokeprognosisglucosecholesterollipoproteinsPDF download SubjectsLipids and CholesterolAnimal Models of Human Disease

Background and aims: Proprotein Convertase Subtilisin Kexin Type 9 (PCSK9) is an endogenous inhibitor of the LDL receptor (LDLR). Mendelian randomization studies suggest that PCSK9 deficiency increases diabetes risk, but the underlying mechanisms remain unknown. The aim of our study was to investigate whether PCSK9 or its inhibition may modulate beta cell function. Methods: We assessed PCSK9 and insulin colocalization in human pancreatic sections by epifluorescent and confocal microscopy. We also investigated the expression and the function of PCSK9 in the human EndoC-8H1 beta cell line, by ELISA and flow cytometry, respectively. PCSK9 was inhibited with Alirocumab or siRNA. LDLR expression and LDL uptake were assessed by flow cytometry. Results: PCSK9 was expressed and secreted from beta cells isolated from human pancreas as well as from EndoC-8H1 cells. PCSK9 secretion was enhanced by statin treatment. Recombinant PCSK9 decreased LDLR abundance at the surface of these cells, an effect abrogated by Alirocumab. Alirocumab as well as PCSK9 silencing increased LDLR expression at the surface of EndoC-8H1 cells. Neither exogenous PCSK9, nor Alirocumab, nor PCSK9 silencing significantly altered glucose-stimulated insulin secretion (GSIS) from these cells. High-low density lipoproteins (LDL) concentrations decreased GSIS, but the addition of PCSK9 or its inhibition did not modulate this phenomenon. Conclusions: While PCSK9 regulates LDLR abundance in beta cells, inhibition of exogenous or endogenous PCSK9 does not appear to significantly impact insulin secretion. This is reassuring for the safety of PCSK9 inhibitors in terms of beta cell function.

BACKGROUND AND AIMS:It remains unclear whether serum PCSK9 levels can predict the severity of the disease and the risk of future events in patients with coronary artery disease (CAD). We aimed to evaluate the association between PCSK9 levels, metabolic parameters, severity of CAD on coronary angiography (SYNTAX score), and the risk of in-hospital events and at one-year follow-up.METHODS AND RESULTS:From September 2015 to December 2016, serum PCSK9 levels were measured on admission in patients not previously receiving statin therapy, and admitted for an acute myocardial infarction (MI), in an intensive care unit from a university hospital. In a total of 648 patients (mean age: 66 years, 67% male), median PCSK9 was 263 ng/ml, higher for females compared with males (270 vs 256 ng/ml, p = 0.009). Serum PCSK9 was associated with LDL cholesterol (r = 0.083, p = 0.036), total cholesterol (r = 0.136, p = 0.001) and triglycerides (r = 0.137, p = 0.001). A positive association was also observed in the subgroup of patients with CRP >10 mg/L (p < 0.001), but not with NT-proBNP, troponin and creatine kinase. PCSK9 levels were similar whatever the SYNTAX score or the number of significant coronary lesions. PCSK9 levels were not associated with in-hospital events (death, recurrent MI and stroke) and events (cardiovascular death, cardiovascular events, recurrent MI) at one-year follow-up.CONCLUSIONS:In this large cohort of patients hospitalized for acute MI and not previously receiving statin therapy, PCSK9 levels was not associated with the severity or the recurrence of cardiovascular events. The clinical utility of measuring PCSK9 levels for this category of patients therefore appears limited.

Proprotein convertase (PC) subtilisin kexin type 9 (PCSK9) inhibits the clearance of low density lipoprotein (LDL) cholesterol from plasma by directly interacting with the LDL receptor (LDLR). As the interaction promotes elevated plasma LDL cholesterol levels and a predisposition to cardiovascular disease (CVD), it has attracted much interest as a therapeutic target. While anti-PCSK9 monoclonal antibodies have been successful in the treatment of hypercholesteremia by decreasing CVD risk, their high cost and a requirement for injection have prohibited widespread use. The advent of an orally bioavailable small molecule inhibitor of the PCSK9-LDLR interaction is an attractive alternative, however efforts have been tempered as the binding interface is unfavourable for binding by small organic molecules. Despite its challenging nature, we report herein the discovery of compound 3f as a small molecule inhibitor of PCSK9. The kinase inhibitor nilotinib emerged from a computational screen that was applied to identify compounds that may bind to a cryptic groove within PCSK9 and proximal to the LDLR-binding interface. A subsequent in vitro PCSK9-LDLR binding assay established that nilotinib was a bona fide but modest inhibitor of the interaction (IC50 = 9.8 µM). Through multiple rounds of medicinal chemistry, 3f emerged as a lead-like molecule by demonstrating disruption of the PCSK9-LDLR interaction at nanomolar levels in vitro (IC50 = 537 nM) with no inhibitory activity (IC50 > 10 µM) against a small panel of kinases. Compound 3f restored LDL uptake by liver cells at sub-micromolar levels and demonstrated excellent bioavailability when delivered subcutaneously in mice. Most significantly, compound 3f lowered total cholesterol levels in the plasma of wild-type mice, thereby providing proof-of-concept that the notion of a small molecule inhibitor against PCSK9 is therapeutically viable.

Bacterial DNA contains CpG oligonucleotide (ODN) motifs to trigger innate immune responses through the endosomal receptor Toll-like receptor 9 (TLR9). One of the cell surface receptors to capture and deliver microbial DNA to intracellular TLR9 is the C-type lectin molecule DEC-205 through its N-terminal C-type lectin-like domain (CTLD). CD93 is a cell surface protein and member of the lectin group XIV with a CTLD. We hypothesized that CD93 could interact with CpG motifs, and possibly serve as a novel receptor to deliver bacterial DNA to endosomal TLR9. Using ELISA and tryptophan fluorescence binding studies we observed that the soluble histidine-tagged CD93-CTLD was specifically binding to CpG ODN and bacterial DNA. Moreover, we found that CpG ODN could bind to CD93-expressing IMR32 neuroblastoma cells and induced more robust interleukin-6 secretion when compared with mock-transfected IMR32 control cells. Our data argue for a possible contribution of CD93 to control cell responsiveness to bacterial DNA in a manner reminiscent of DEC-205. We postulate that CD93 may act as a receptor at plasma membrane for DNA or CpG ODN and to grant delivery to endosomal TLR9.

Heme oxygenase-1 (HO-1), a rate-limiting enzyme involved in the degradation of heme, is induced in response to a wide range of stress conditions. HO-1 exerts antiviral activity against a broad range of viruses, including the Hepatitis C virus, the human immunodeficiency virus, and the dengue virus by inhibiting viral growth. It has been reported that HO-1 displays antiviral activity against the Zika virus (ZIKV) but the mechanisms of viral inhibition remain largely unknown. Using a ZIKV RNA replicon with the Green Fluorescent Protein (GFP) as a reporter protein, we were able to show that HO-1 expression resulted in the inhibition of viral RNA replication. Conversely, we observed a decrease in HO-1 expression in cells replicating the ZIKV RNA replicon. The study of human cells infected with ZIKV showed that the HO-1 expression level was significantly lower once viral replication was established, thereby limiting the antiviral effect of HO-1. Our work highlights the capacity of ZIKV to thwart the anti-replicative activity of HO-1 in human cells. Therefore, the modulation of HO-1 as a novel therapeutic strategy against ZIKV infection may display limited effect.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a crucial protein governing the circulating levels of low density lipoprotein-cholesterol (LDL-C), by virtue of its pivotal role in the degradation of the LDL receptor (LDLR). In the last 15 years, in vitro and in vivo studies have allowed our understanding of the physiological role of PCSK9. In the current report, we review the key studies that have established the mode of action of PCSK9, leading to the development of PCSK9 inhibitors for clinical use. Data from clinical trials investigating these therapies clearly and unambiguously demonstrate the safety and efficacy of these new drugs that have the power to dramatically reduce LDL-C and associated cardiovascular diseases.

Therapeutic antibodies targeting proprotein convertase subtilisin kexin type 9 (PCSK9) (e.g. alirocumab) lower low-density lipoprotein cholesterol (LDL-C) and lipoprotein (a) [Lp(a)] levels in clinical trials. We recently showed that PCSK9 enhances apolipoprotein(a) [apo(a)] secretion from primary human hepatocytes but does not affect Lp(a) cellular uptake. Here, we aimed to determine how PCSK9 neutralization modulates Lp(a) levels in vivoSix nonhuman primates (NHP) were treated with alirocumab or a control antibody (IgG1) in a crossover protocol. After the lowering of lipids reached steady state, NHP received an intravenous injection of [2H3]-leucine, and blood samples were collected sequentially over 48 h. Enrichment of apolipoproteins in [2H3]-leucine was assessed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Kinetic parameters were calculated using numerical models with the SAAMII software. Compared with IgG1, alirocumab significantly reduced total cholesterol (TC) (-28%), LDL-C (-67%), Lp(a) (-56%), apolipoprotein B100 (apoB100) (-53%), and apo(a) (-53%). Alirocumab significantly increased the fractional catabolic rate of apoB100 (+29%) but not that of apo(a). Conversely, alirocumab sharply and significantly reduced the production rate (PR) of apo(a) (-42%), but not significantly that of apoB100, compared with IgG1, respectively.In line with the observations made in human hepatocytes, the present kinetic study establishes that PCSK9 neutralization with alirocumab efficiently reduces circulating apoB100 and apo(a) levels by distinct mechanisms: apoB primarily by enhancing its catabolism and apo(a) primarily by lowering its production.

Human apoE exhibits three major isoforms (apoE2, apoE3, and apoE4) corresponding to polymorphism in the APOE gene. Total plasma apoE concentrations are closely related to these isoforms, but the underlying mechanisms are unknown. We aimed to describe the kinetics of apoE individual isoforms to explore the mechanisms for variable total apoE plasma concentrations. We used LC-MS/MS to discriminate between isoforms by identifying specific peptide sequences in subjects (three E2/E3, three E3/E3, and three E3/E4 phenotypes) who received a primed constant infusion of 2H3-leucine for 14 h. apoE concentrations and leucine enrichments were measured hourly in plasma. Concentrations of apoE2 were higher than apoE3, and concentrations of apoE4 were lower than apoE3. There was no difference between apoE3 and apoE4 catabolic rates and between apoE2 and apoE3 production rates (PRs), but apoE2 catabolic rates and apoE4 PRs were lower. The mechanisms leading to the difference in total plasma apoE concentrations are therefore related to contrasted kinetics of the isoforms. Production or catabolic rates are differently affected according to the specific isoforms. On these grounds, studies on the regulation of the involved biochemical pathways and the impact of pathological environments are now warranted.

Porphyromonas gingivalis is a key bacterium in chronic periodontitis, which is associated with several chronic inflammatory diseases. Lipopolysaccharides from P. gingivalis (Pg LPS) can activate multiple cell types via the production of pro-inflammatory cytokines. The receptors for Pg LPS have initially been reported as TLR2, contrasting with the well-studied TLR4 receptor for E. coli LPS; this observation remains controversial since synthetic Pg lipid A activates TLR4 but not TLR2. Despite this observation, the dogma of Pg LPS-mediated TLR2 activation remains the basis of many hypotheses and result interpretations. In the present work, we aimed at determining whether TLR4 or TLR2, or both, mediate Pg LPS pro-inflammatory activity using Pg LPS with different grades of purity, instead of synthetic lipid A from Pg LPS. Here we show that Pg LPS 1) acts exclusively through TLR4, and 2) are differently recognized by mouse and human TLR4 both in vitro and in vivo. Taken together, our results suggest that Pg LPS activity is mediated exclusively through TLR4 and only weakly induces proinflammatory cytokine secretion in mouse models. Caution should be taken when extrapolating data from mouse systems exposed to Pg or Pg LPS to humans.

Available rapid, simple and accurate methods for detection and diagnosis of emerging viral diseases are required. Recently, there was an urgent need for specific antibodies against mosquito-borne Zika virus (ZIKV), which is an emerging zoonotic disease of medical concern in different regions of the world. Here, we showed that overexpression of ZIKV antigens in ClearColi BL21(DE3), a bacteria strain expressing a non-endotoxic form of LPS, is suitable for the production of specific ZIKV antisera. Two major ZIKV antigenic domains, the domain III from envelope E glycoprotein, which brings the virus-specific epitopes, and the N-terminal region of nonstructural NS1 glycoprotein, which is responsible for pathophysiological conditions, were overexpressed in ClearColi BL21(DE3). Immunization of adult rat with insoluble recombinant ZIKV antigens in inclusion bodies resulted in the production of specific antibodies in a few weeks. Anti-E and anti-NS1 antibodies are efficient as biological tools for ZIKV detection by indirect ELISA and immunoblot assay. This method could successfully be applied to any emerging viruses.

OBJECTIVE:Evolocumab, a PCSK9 (proprotein convertase subtilisin kexin type 9)-neutralizing antibody, lowers low-density lipoprotein cholesterol (LDL-C) in homozygous familial hypercholesterolemic (HoFH) patients with reduced LDLR (low-density lipoprotein receptor) function. However, their individual responses are highly variable, even among carriers of identical LDLR genetic defects. We aimed to elucidate why HoFH patients variably respond to PCSK9 inhibition.APPROACH AND RESULTS:Lymphocytes were isolated from 22 HoFH patients enrolled in the TAUSSIG trial (Trial Assessing Long Term Use of PCSK9 Inhibition in Subjects With Genetic LDL Disorders). Ten patients were true homozygotes (FH1/FH1) and 5 identical compound heterozygotes (FH1/FH2). Lymphocytes were plated with or without mevastatin, recombinant PCSK9 (rPCSK9), or a PCSK9-neutralizing antibody. Cell surface LDLR expression was analyzed by flow cytometry. All HoFH lymphocytes had reduced cell surface LDLR expression compared with non-FH lymphocytes, for each treatment modality. Lymphocytes from FH1/FH2 patients (LDLR defective/negative) displayed the lowest LDLR expression levels followed by lymphocytes from FH1/FH1 patients (defective/defective). Mevastatin increased, whereas rPCSK9 reduced LDLR expression. The PCSK9-neutralizing antibody restored LDLR expression. Lymphocytes displaying higher LDLR expression levels were those isolated from patients presenting with lowest levels of LDL-C and apolipoprotein B, before and after 24 weeks of evolocumab treatment. These negative correlations remained significant in FH1/FH1 patients and appeared more pronounced when patients with apolipoprotein E3/E3 genotypes were analyzed separately. Significant positive correlations were found between the levels of LDLR expression and the percentage reduction in LDL-C on evolocumab treatment.CONCLUSIONS:Residual LDLR expression in HoFH is a major determinant of LDL-C levels and seems to drive their individual response to evolocumab.

Clinical benefit for mechanical thrombectomy (MT) in stroke was recently demonstrated in multiple large prospective studies. Acute hyperglycemia (HG) is an important risk factor of poor outcome in stroke patients, including those that underwent MT. The aim of this therapy is to achieve a complete reperfusion in a short time, given that reperfusion damage is dependent on the duration of ischemia. Here, we investigated the effects of acute HG in a mouse model of ischemic stroke induced by middle cerebral artery occlusion (MCAO). Hyperglycemic (intraperitoneal [ip] injection of glucose) and control (ip saline injection) 10-week male C57BL6 mice were subjected to MCAO (30, 90, and 180 min) followed by reperfusion obtained by withdrawal of the monofilament. Infarct volume, hemorrhagic transformation (HT), neutrophil infiltration, and neurological scores were assessed at 24 hr by performing vital staining, ELISA immunofluorescence, and behavioral test, respectively. Glucose injection led to transient HG (blood glucose5250-390 mg/dL) that significantly increased infarct volume, HT, and worsened neurological outcome. In addition, we report that HG promoted blood-brain barrier disruption as shown by hemoglobin accumulation in the brain parenchyma and tended to increase neutrophil extravasation within the infarcted area. Acute HG increased neurovascular damage for all MCAO durations tested. HTs were observed as early as 90 min after ischemia under hyperglycemic conditions. This model mimics MT ischemia/reperfusion and allows the exploration of brain injury in hyperglycemic conditions.

Zika virus (ZIKV) is an emerging flavivirus since the first epidemics in South Pacific in 2007. The recent finding that ZIKV is now circulating in Western Hemisphere and can be associated to severe human diseases, warrants the need for its study. Here we evaluate the susceptibility of human lung epithelial A549 cells to South Pacific epidemic strain of ZIKV isolated in 2013. We showed that ZIKV growth in A549 cells is greatly efficient. ZIKV infection resulted in the secretion of IFN-β followed by the expression of pro-inflammatory cytokines such as IL-1β, and transcriptional activity of IFIT genes. At the maximum of virus progeny production, ZIKV triggers mitochondrial apoptosis through activation of caspases-3 and -9. Whereas at early infection times, the rapid release of IFN-β which exerts an antiviral effect against ZIKV might delay apoptosis in infected cells.

CD93 belongs to the group XIV C-type lectin like domain (CTLD) and is closely related to thrombomodulin (CD141). Although CD93 is known to be involved in the regulation of cell adhesion and phagocytosis, its role in innate immunity remains to be fully investigated. Critically, published data about CD141 suggest that CD93 CTLD could be involved in the control of inflammation. In order to address further functional and structural analyses, we expressed human CD93 CTLD with several disulfide bonds in an E. coli expression system. As the E. coli cytoplasm is a reducing compartment, production of disulfide-bond proteins remains a challenge. Hence, we decided to over express CD93 CTLD in commercially available strains of E. coli and co-expressed a sulfhydryl oxidase (Erv1p) and a disulfide isomerase (DsbC). This strategy led to high yield expression of a native form of CD93 CTLD. NMR studies revealed that Ca2+ was not able to bind to CD93 CTLD. We also showed that the recombinant protein could alter LPS pro-inflammatory activity on THP1. This work provides new tool for further functional and structural studies to decipher the functions associated to the CTLD of CD93. This approach may also be used for others members of the group XIV C-type lectin like domain (CD141, CD248 and CLec14A).

http://dx.doi.org/10.1016/j.cyto.2015.01.028 1043-4666/ 2015 Elsevier Ltd. All rights reserved. Abbreviations: APC, antigen presenting cells; CD14, cluster of differentiation 14; DAMP, damage-associated molecular pattern; exHSP60, extracellular 60 kDa-heat shock protein; GST, glutathion-S-transferase; HSP, heat shock proteins; HSP60, 60 kDa-heat shock protein; hTNF-a, human tumor necrosis factor a; LPS, lipopolysaccharide; MD2, myeloid differentiation 2; NFjB, nuclear factor kappa B; PMA, phorbol 12-myristate 13-acetate; PMB, polymixin B; rhHSP60, recombinant human 60 kDa-heat shock protein; RPMI medium, Roswell Park Memorial Institute medium; SEAP, secreted alkaline phosphatase; TLR2, toll-like receptor 2; TLR4, toll-like receptor 4. ⇑ Corresponding author at: Laboratoire GRI, Université de La Réunion and plateforme CYROI, 15, Avenue René Cassin, BP 7151, 97715 Saint Denis Messag. Cedex 9, Reunion. Tel.: +262 262 93 88 29. E-mail address: wildriss.viranaicken@univ-reunion.fr (W. Viranaïcken). 1 These two authors contribute equally to this work. 2 Co-authors seniorship. Cynthia Planesse , Brice Nativel , Thomas Iwema , Philippe Gasque , Christine Robert-Da Silva , Wildriss Viranaïcken b,2,⇑