PCSK9 ablation attenuates Aß pathology, neuroinflammation and cognitive dysfunctions in 5XFAD mice.

BACKGROUND: Increasing evidence highlights the importance of novel players in Alzheimer's disease (AD) pathophysiology, including alterations of lipid metabolism and neuroinflammation. Indeed, a potential involvement of Proprotein convertase subtilisin/kexin type 9 (PCSK9) in AD has been recently postulated. Here, we first investigated the effects of PCSK9 on neuroinflammation in vitro. Then, we examined the impact of a genetic ablation of PCSK9 on cognitive performance in a severe mouse model of AD. Finally, in the same animals we evaluated the effect of PCSK9 loss on Aß pathology, neuroinflammation, and brain lipids. METHODS: For in vitro studies, U373 human astrocytoma cells were treated with Aß fibrils and human recombinant PCSK9. mRNA expression of the proinflammatory cytokines and inflammasome-related genes were evaluated by q-PCR, while MCP-1 secretion was measured by ELISA. For in vivo studies, the cognitive performance of a newly generated mouse line - obtained by crossing 5XFADHet with PCSK9KO mice - was tested by the Morris water maze test. After sacrifice, immunohistochemical analyses were performed to evaluate Aß plaque deposition, distribution and composition, BACE1 immunoreactivity, as well as microglia and astrocyte reactivity. Cholesterol and hydroxysterols levels in mouse brains were quantified by fluorometric and LC-MS/MS analyses, respectively. Statistical comparisons were performed according to one- or two-way ANOVA, two-way repeated measure ANOVA or Chi-square test. RESULTS: In vitro, PCSK9 significantly increased IL6, IL1B and TNFΑ mRNA levels in Aß fibrils-treated U373 cells, without influencing inflammasome gene expression, except for an increase in NLRC4 mRNA levels. In vivo, PCSK9 ablation in 5XFAD mice significantly improved the performance at the Morris water maze test; these changes were accompanied by a reduced corticohippocampal Aß burden without affecting plaque spatial/regional distribution and composition or global BACE1 expression. Furthermore, PCSK9 loss in 5XFAD mice induced decreased microgliosis and astrocyte reactivity in several brain regions. Conversely, knocking out PCSK9 had minimal impact on brain cholesterol and hydroxysterol levels. CONCLUSIONS: In vitro studies showed a pro-inflammatory effect of PCSK9. Consistently, in vivo data indicated a protective role of PCSK9 ablation against cognitive impairments, associated with improved Aß pathology and attenuated neuroinflammation in a severe mouse model of AD. PCSK9 may thus be considered a novel pharmacological target for the treatment of AD.

Evaluation of the effects of natural isoquinoline alkaloids on low density lipoprotein receptor (LDLR) and proprotein convertase subtilisin/kexin type 9 (PCSK9) in hepatocytes, as new potential hypocholesterolemic agents.

Nine different isoquinoline alkaloids, berberine, govaniadine, stylopine, adlumine, adlumidine, bicuculline, sanguinarine, protopine and californidine have been evaluated for their effects on a cellular model of hepatocyte for their effect on low density lipoprotein receptor (LDLR) and proprotein convertase subtilisin/kexin type 9 (PCSK9) expression compared to simvastatin. Berberine, californidine and govaniadine induced LDLR with an effect similar to 2.5 µM simvastatin. Californidine and berberine at tested doses reduced the expression of PCSK9, with an opposite behaviour to simvastatin on this target. Govaniadine, on the other hand, showed a statin-like effect, although less potently, by increasing both LDLR and PCSK9 levels. Berberine californidine and govaniadine were then tested on the same cellular model to assess possible effect of reduction of total cholesterol, compared to simvastatin. All compounds were able to reduce total cholesterol level in the hepatocytes.

PCSK9 Affects Astrocyte Cholesterol Metabolism and Reduces Neuron Cholesterol Supplying In Vitro: Potential Implications in Alzheimer's Disease.

The Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) involvement in Alzheimer's disease (AD) is poorly investigated. We evaluated the in vitro PCSK9 modulation of astrocyte cholesterol metabolism and neuronal cholesterol supplying, which is fundamental for neuronal functions. Moreover, we investigated PCSK9 neurotoxic effects. In human astrocytoma cells, PCSK9 reduced cholesterol content (−20%; p < 0.05), with a greater effect in presence of beta amyloid peptide (Aß) (−37%; p < 0.01). PCSK9 increased cholesterol synthesis and reduced the uptake of apoE-HDL-derived cholesterol (−36%; p < 0.0001), as well as the LDL receptor (LDLR) and the apoE receptor 2 (ApoER2) expression (−66% and −31%, respectively; p < 0.01). PCSK9 did not modulate ABCA1- and ABCG1-cholesterol efflux, ABCA1 levels, or membrane cholesterol. Conversely, ABCA1 expression and activity, as well as membrane cholesterol, were reduced by Aß (p < 0.05). In human neuronal cells, PCSK9 reduced apoE-HDL-derived cholesterol uptake (−41%; p < 0.001) and LDLR/apoER2 expression (p < 0.05). Reduced cholesterol internalization occurred also in PCSK9-overexpressing neurons exposed to an astrocyte-conditioned medium (−39%; p < 0.001). PCSK9 reduced neuronal cholesterol content overall (−29%; p < 0.05) and increased the Aß-induced neurotoxicity (p < 0.0001). Our data revealed an interfering effect of PCSK9, in cooperation with Aß, on brain cholesterol metabolism leading to neuronal cholesterol reduction, a potentially deleterious effect. PCSK9 also exerted a neurotoxic effect, and thus represents a potential pharmacological target in AD.

NMR, LC-MS Characterization of Rydingia michauxii Extracts, Identification of Natural Products Acting as Modulators of LDLR and PCSK9.

Rydingia michauxii (Briq.) Scheen and V.A.Albert (Lamiaceae) is used in Iranian traditional medicine to treat malaria, diabetes, hyperlipidemia, rheumatism and cardiovascular diseases. NMR and LC-DAD-MSn analyses were used to establish extract composition and phenylethanoid, flavonoid glycosides, lignans, labdane diterpenes and iridoids were identified and quantified. The main constituents were isolated, and structures were elucidated based on NMR, polarimetric and MS measurements. A new natural compound, ent-labda-8(17),13-dien-18-glucopyranosyl ester-15,16-olide is described here. The effects of ent-labda-8(17),13-dien-18-oic acid-15,16-olide (1), ent-labda-8(17),13-dien-18-glucopyranosyl es-ter-15,16-olide (2), antirrhinoside (3), echinacoside (4), verbascoside (5), and apigenin 6,8-di-C-glucoside (6), on the low-density lipoprotein receptor (LDLR) and proprotein convertase subtilisin/kexin type 9 (PCSK9), were studied in the human hepatocarcinoma cell line Huh7. Among the six constituents, (3) showed the strongest induction of the LDLR (3.7 ± 2.2 fold vs. control) and PCSK9 (3.2 ± 1.5 fold vs. control) at a concentration of 50 µM. The in vitro observations indicated a potential lipid lowering activity of (3) with a statin-like mechanism of action.

Investigating the in vitro mode of action of okra (Abelmoschus esculentus) as hypocholesterolemic, anti-inflammatory, and antioxidant food.

Okra (Abelmoschus esculentus) have been introduced as food relatively recently in Europe. It is native to India and one of the most important vegetables in Nigeria. The leaves can be consumed but also the fruit is rich in nutrients and bioactive compounds (i.e., dietary fiber, vitamins, oils, polysaccharides, polyphenols) and several health promoting actions have been ascribed, including a lipid-lowering properties. In this work the effects of fruit and leaf extracts on expression of key mediators of cholesterol metabolism, i.e., the low-density lipoprotein receptor (LDLR) and proprotein convertase subtilisin/kexin type 9 (PCSK9), were investigated in human hepatoma cell line Huh7. Furthermore, effects on proinflammatory cytokines (IL-1ß, IL-6 and TNF-α) expressed by THP1-derived macrophages were studied to assess potential anti-inflammatory actions. Okra fruit extract significantly induced the mRNA and protein levels of the LDLR by 1.4 ± 0.3 and 4.8 ± 1.5-fold, respectively without any significant modification of PCSK9 expression. In addition, fruit extract showed a significant sequestering ability of cholic acid. Leaf butanol extract exerted similar action by inducing the expression of both the LDLR (+3.1 ± 1.6-fold vs control) and PCSK9 (+1.3 ± 0.4-fold vs control). The evaluation of the potential anti-inflammatory effect revealed a significant action of leaf butanol extract with reduced mRNA levels of IL-1ß (-28 ± 8 % vs control), IL-6 (-11 ± 1 % vs control) and TNF-α (-43 ± 8 % vs control), while fruit extract did not show any anti-inflammatory activity. Finally, leaf ethyl acetate extract showed a significant antioxidant capacity comparable to ascorbic acid. Taken together, we provided evidence that leaf butanol extract and, more effectively, fruit extract induced the LDLR expression, effect that may explain the previously reported hypocholesterolemic action of okra. In addition, okra's extracts reduced the expression of pro-inflammatory cytokines from THP1-derived macrophages, an effect that may suggest a vascular protective action of okra.

In Vitro and In Vivo Sucrosomial® Berberine Activity on Insulin Resistance.

Background: Berberine is a natural alkaloid with hypoglycemic properties. However, its therapeutic use is limited by a very low oral bioavailability. Here we developed a new oral formulation of berberine based on Sucrosomial® technology and tested its effect on insulin resistance. Methods: Sucrosomial® berberine was first tested in vitro in the hepatoma cell line Huh7 to assess its effect on proteins involved in glucose homeostasis and insulin resistance. The pharmacokinetics and efficacy on insulin resistance were then studied in C57BL/6 mice fed with standard (SD) and high-fat diet (HFD) for 16 weeks and treated daily during the last 8 weeks with oral gavage of Sucrosomial® berberine or berberine. Results: Sucrosomial® berberine did not affect Huh7 cell viability at concentrations up to 40 µM. Incubation of Huh7 with 20 µM of Sucrosomial® and control berberine induced glucokinase (GK) and the phosphorylation of 5'-adenosine monophosphate (AMP)-activated protein kinase (AMPK), both known targets for the control of insulin resistance. In vivo, we observed an 8-fold higher plasma concentration after 3 weeks of oral administration of 50 mg/kg/day of Sucrosomial® formulation compared to berberine. HFD, compared to SD, induced insulin resistance in mice as determined by oral glucose tolerance test (OGTT). The treatment with a 6.25 mg/kg/daily dose of Sucrosomial® berberine significantly reduced the area under the curve (AUC) of OGTT (73,103 ± 8645 vs. 58,830 ± 5597 mg/dL × min), while control berberine produced the same effects at 50 mg/Kg/day (51518 ± 1984 mg/dL × min). Under these conditions, the two formulations resulted in similar berberine plasma concentration in mice. Nevertheless, a different tissue distribution of metabolites was observed with a significant accumulation of reduced, demethylated and glucuronide berberine in the brain after the oral administration of the Sucrosomial® form. Glucuronide berberine plasma concentration was higher with Sucrosomial® berberine compared to normal berberine. Finally, we observed similar increases of AMPK phosphorylation in the liver in response to the treatment with Sucrosomial® berberine and berberine. Conclusions: The Sucrosomial® formulation is an innovative and effective technology to improve berberine gastrointestinal (GI) absorption with proven in vitro and in vivo activity on insulin resistance.

PCSK9 promotes arterial medial calcification.

BACKGROUND AND AIMS: A complex interplay among chronic kidney disease (CKD), lipid metabolism and aortic calcification has been recognized. Here we investigated the influence of kidney function on PCSK9 levels and its potential direct action on smooth muscle cells (SMCs) calcification. METHODS AND RESULTS: In a cohort of 594 subjects, a negative association between glomerular filtration rate and plasma PCSK9 was found. Atherosclerotic cardiovascular disease, as co-morbidity, further increased PCSK9 plasma levels. Diet-induced uremic condition in rats led to aortic calcification and increased total cholesterol and Pcsk9 levels in plasma, livers, and kidneys. Both human and rat SMCs overexpressing human PCSK9 (SMCsPCSK9), cultured in a pro-calcific environment (2.0 mM or 2.4 mM inorganic phosphate, Pi) showed a significantly higher extracellular calcium (Ca2+) deposition compared to control SMCs. The addition of recombinant human PCSK9 did not increase the extracellular calcification of SMCs, suggesting the involvement of intracellular PCSK9. Accordingly, the further challenge with evolocumab did not affect calcium deposition in hSMCsPCSK9. Under pro-calcific conditions, SMCsPCSK9 released a higher number of extracellular vesicles (EVs) positive for three tetraspanin molecules, such as CD63, CD9, and CD81. EVs derived from SMCsPCSK9 tended to be more enriched in calcium and alkaline phosphatase (ALP), compared to EVs from control SMCs. In addition, PCSK9 has been detected in SMCsPCSK9-derived EVs. Finally, SMCsPCSK9 showed an increase in pro-calcific markers, namely bone morphogenetic protein 2 and ALP, and a decrease in anti-calcific mediator matrix GLA protein and osteopontin. CONCLUSIONS: Our study reveals a direct role of PCSK9 on vascular calcification induced by higher inorganic phosphate levels associated with renal impairment. This effect appears to be mediated by a switch towards a pro-calcific phenotype of SMCs associated with the release of EVs containing Ca2+ and ALP.