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This study investigates the role of 25-hydroxycholesterol (25HC), a metabolite produced by cholesterol hydroxylase encoded by the Ch25h gene, in modulating microglial function and its potential implications in Alzheimer’s disease (AD) pathology. We demonstrated that 25HC impairs microglial surveillance, reduces phagocytic capacity, and increases the production of pro-inflammatory cytokines. In vivo two-photon microscopy revealed that 25HC administration diminishes microglial response to brain lesions, while flow cytometry confirmed reduced phagocytosis in both in vivo and in vitro models. Additionally, amyloid-beta (Aβ) was shown to upregulate Ch25h expression and elevate 25HC levels in microglia, exacerbating these functional impairments. Mechanistically, 25HC was found to enhance cholesterol esterification, disrupt cell membrane dynamics, and further reduce microglial mobility and phagocytosis. Treatment with Avasimibe, a cholesterol esterification inhibitor, restored membrane dynamics and microglial function, leading to attenuated AD pathology in a 5XFAD mouse model. These findings suggest that 25HC-induced changes in microglial function contribute to AD progression, and targeting cholesterol metabolism could offer therapeutic potential.

The cardioprotective effects of estrogen are mediated by receptors expressed in vascular cells. Here we show that 27-hydroxycholesterol (27HC), an abundant cholesterol metabolite that is elevated with hypercholesterolemia and found in atherosclerotic lesions, is a competitive antagonist of estrogen receptor action in the vasculature. 27HC inhibited both the transcription-mediated and the non-transcription-mediated estrogen-dependent production of nitric oxide by vascular cells, resulting in reduced estrogen-induced vasorelaxation of rat aorta. Furthermore, increasing 27HC levels in mice by diet-induced hypercholesterolemia, pharmacologic administration or genetic manipulation (by knocking out the gene encoding the catabolic enzyme CYP7B1) decreased estrogen-dependent expression of vascular nitric oxide synthase and repressed carotid artery reendothelialization. As well as antiestrogenic effects, there were proestrogenic actions of 27HC that were cell-type specific, indicating that 27HC functions as an endogenous selective estrogen receptor modulator (SERM). Taken together, these studies point to 27HC as a contributing factor in the loss of estrogen protection from vascular disease.

Acute lung injury (ALI) is mainly caused by uncontrolled inflammatory response, and it remains without effective therapeutic options. 25‐hydroxycholesterol (25HC) has been reported to be a potent regulator of inflammation. The aim of this study was to investigate the effects of 25HC on lipopolysaccharide (LPS)‐induced ALI. C57BL/6 mice were pretreated with 25HC intraperitoneally before intratracheal exposure to LPS. Our results showed that 25HC pretreatment improved survival rate, attenuated the pathological changes of the lung and decreased the release of inflammatory cytokines in mice. Consistently, 25HC reduced expression of Toll‐like receptor (TLR4)‐mediated inflammatory cytokines in vitro. These effects of 25HC were obtained by preventing LPS binding to TLR4 via interaction with myeloid differentiation protein 2 (MD‐2). Crystal structure analysis suggested that 25HC could bind MD‐2 with high affinity into its hydrophobic pocket. Furthermore, LPS‐induced activation of Akt/NF‐κB pathway was partially down‐regulated by 25HC pretreatment. In summary, this study demonstrates that 25HC could inhibit the overwhelming inflammatory response through MD‐2 interaction, which suppresses Akt/NF‐κB signalling pathway. These findings suggest 25HC may be a promising candidate for ALI prevention.

We investigated effects of 7-oxygenated cholesterol derivatives present in atherosclerotic lesions, 7α-hydroxycholesterol (7αOHChol), 7β-hydroxycholesterol (7βOHChol), and 7-ketocholesterol (7K), on IL-8 expression. Transcript levels of IL-8 and secretion of its corresponding gene product by monocytes/macrophages were enhanced by treatment with 7αOHChol and, to a lesser extent, 7K, but not by 7βOHChol. The 7-oxygenated cholesterol derivatives, however, did not change transcription of the IL-8 gene in vascular smooth muscle cells. 7αOHChol-induced IL-8 gene transcription was inhibited by cycloheximide and Akt1 downregulation, but not by OxPAPC. Expression of C5a receptor was upregulated after stimulation with 7αOHChol, but not with 7K and 7βOHChol, and a specific antagonist of C5a receptor inhibited 7αOHChol-induced IL-8 gene expression in a dose dependent manner. Pharmacological inhibitors of PI3K and MEK almost completely inhibited expression of both IL-8 and cell-surface C5a receptor induced by 7αOHChol. These results indicate that 7-oxygenated cholesterol derivatives have differential effects on monocyte/macrophage expression of IL-8 and C5a receptor and that C5a receptor is involved in 7αOHChol-induced IL-8 expression via PI3K and MEK.

This study explores the pharmacokinetics of 22- S -hydroxycholesterol (22SHC) in vivo in rats. We also carried out a metabolic study to explore whether the beneficial effects observed of 22SHC on glucose and lipid metabolism in vitro could be seen in vivo in rats. In the pharmacokinetic study, rats were given 50 mg/kg of [ 3 H]22- S -hydroxycholesterol before absorption, distribution and excretion were monitored. In the metabolic study, the effect of 22SHC (30 mg/kg/day for 3 weeks) in rats on body weight gain [chow and high-fat diet (HFD)], serum lipids triacylglycerol (TAG) content and gene expression in liver and skeletal muscle were examined. Results showed that 22SHC was well absorbed after oral administration and distributed to most organs and mainly excreted in feces. Rats receiving 22SHC gained less body weight than their controls regardless whether the animals received chow diet or HFD. Moreover, we observed that animals receiving HFD had elevated levels of serum TAG while this was not observed for animals on HFD supplemented with 22SHC. The amount of TAG in liver was reduced after 22SHC treatment in animals receiving either chow diet or HFD. Gene expression analysis revealed that two genes (carnitine palmitoyltransferase 2 and uncoupling protein 3) involved in fatty acid oxidation and energy dissipation were increased in liver. Ucp3 expression (both protein and mRNA level) was increased in skeletal muscle, but insulin-stimulated glucose uptake and TAG content were unchanged. In conclusion, 22SHC seems to be an interesting model substance in the search of treatments for disorders involving aberrations in lipid metabolism.

Oxysterols are structurally similar to cholesterol, but are characterized by one or more additional oxygen-containing functional groups. These compounds are implicated in inflammation given their ability to cause irreversible damage to vascular cells. The aim of this study was to study the alteration of some inflammatory biomarkers in Wistar rats in response to dietary oxysterols. Eighteen rats were randomly divided into three groups of six rats each. A standard diet supplemented with 1% ( w / w ) pure cholesterol (Chol group) or 1% ( w / w ) of an oxidized cholesterol mixture (COPs group) was fed for 8 weeks. Blood serum was separated; abdominal, pericardial, and epididymal adipose tissue was removed carefully. The COPs subjects exhibited significant increase in blood pressure and serum triacylgycerols as well as increased body fat index and pericardic, abdominal, and epididymal adipose tissue. These effects were accompanied by elevated circulating levels of plasma high-sensitivity C-reactive protein, tumor necrosis factor alpha, and resistin. We suggest that dietary oxysterols have an important pro-inflammatory effect.

Interleukin-1 beta (IL-1beta) is an important mediator in intestinal inflammation. IL-1beta promotes IL-8 production, which can be modulated by a number of factors, including oxidative stress. Interestingly, oxysterols, which are thought to contribute to inflammation in atherosclerotic plaques, are also produced by intestinal epithelial cells. Thus, we investigated the effect of oxysterols, including 25-hydroxycholesterol and 7beta-hydroxycholesterol, on IL-1beta-induced IL-8 production in Caco-2 cells (a human colon carcinoma cell line). Pre-treatment of Caco-2 cells with 25-hydroxycholesterol significantly enhanced IL-1beta-induced IL-8 expression at both mRNA and protein levels. However, 7beta-hydroxycholesterol showed very little effect on IL-8 production. Furthermore, pre-treatment with 25-hydroxycholesterol, followed by IL-1beta stimulation, enhanced IL-8 promoter activity beyond that observed with IL-1beta alone. These results suggest that 25-hydroxycholesterol enhances IL-1beta-induced IL-8 production, possibly by enhancing promoter activity.

Niemann-Pick disease, type C1 (NPC1) is a lysosomal storage disorder characterised by progressive neurodegeneration. In preclinical testing, 2-hydroxypropyl-β-cyclodextrins (HPβCD) significantly delayed cerebellar Purkinje cell loss, slowed progression of neurological manifestations, and increased lifespan in mouse and cat models of NPC1. The aim of this study was to assess the safety and efficacy of lumbar intrathecal HPβCD. In this open-label, dose-escalation phase 1–2a study, we gave monthly intrathecal HPβCD to participants with NPC1 with neurological manifestation at the National Institutes of Health (NIH), Bethesda, MD, USA. To explore the potential effect of 2-week dosing, three additional participants were enrolled in a parallel study at Rush University Medical Center (RUMC), Chicago, IL, USA. Participants from the NIH were non-randomly, sequentially assigned in cohorts of three to receive monthly initial intrathecal HPβCD at doses of 50, 200, 300, or 400 mg per month. A fifth cohort of two participants received initial doses of 900 mg. Participants from RUMC initially received 200 or 400 mg every 2 weeks. The dose was escalated based on tolerance or safety data from higher dose cohorts. Serum and CSF 24(S)-hydroxycholesterol (24[S]-HC), which serves as a biomarker of target engagement, and CSF protein biomarkers were evaluated. NPC Neurological Severity Scores (NNSS) were used to compare disease progression in HPβCD-treated participants relative to a historical comparison cohort of 21 NPC1 participants of similar age range. Between Sept 21, 2013, and Jan 19, 2015, 32 participants with NPC1 were assessed for eligibility at the National Institutes of Health. 18 patients were excluded due to inclusion criteria not met (six patients), declined to participate (three patients), pursued independent expanded access and obtained the drug outside of the study (three patients), enrolled in the RUMC cohort (one patient), or too late for the trial enrolment (five patients). 14 patients were enrolled and sequentially assigned to receive intrathecal HPβCD at a starting dose of 50 mg per month (three patients), 200 mg per month (three patients), 300 mg per month (three patients), 400 mg per month (three patients), or 900 mg per month (two patients). During the first year, two patients had treatment interrupted for one dose, based on grade 1 ototoxicity. All 14 patients were assessed at 12 months. Between 12 and 18 months, one participant had treatment interrupted at 17 months due to hepatocellular carcinoma, one patient had dose interruption for 2 doses based on caregiver hardship and one patient had treatment interrupted for 1 dose for mastoiditis. 11 patients were assessed at 18 months. Between Dec 11, 2013, and June 25, 2014, three participants were assessed for eligibility and enrolled at RUMC, and were assigned to receive intrathecal HPβCD at a starting dose of 200 mg every 2 weeks (two patients), or 400 mg every two weeks (one patient). There were no dropouts in this group and all 3 patients were assessed at 18 months. Biomarker studies were consistent with improved neuronal cholesterol homoeostasis and decreased neuronal pathology. Post-drug plasma 24(S)-HC area under the curve (AUC8-72) values, an indicator of neuronal cholesterol homoeostasis, were significantly higher than post-saline plasma 24(S)-HC AUC8-72 after doses of 900 mg (p=0·0063) and 1200 mg (p=0·0037). CSF 24(S)-HC concentrations in three participants given either 600 or 900 mg of HPβCD were increased about two fold (p=0·0032) after drug administration. No drug-related serious adverse events were observed. Mid-frequency to high-frequency hearing loss, an expected adverse event, was documented in all participants. When managed with hearing aids, this did not have an appreciable effect on daily communication. The NNSS for the 14 participants treated monthly increased at a rate of 1·22, SEM 0·34 points per year compared with 2·92, SEM 0·27 points per year (p=0·0002) for the 21 patient comparison group. Decreased progression was observed for NNSS domains of ambulation (p=0·0622), cognition (p=0·0040) and speech (p=0·0423). Patients with NPC1 treated with intrathecal HPβCD had slowed disease progression with an acceptable safety profile. These data support the initiation of a multinational, randomised, controlled trial of intrathecal HPβCD. National Institutes of Health, Dana's Angels Research Trust, Ara Parseghian Medical Research Foundation, Hope for Haley, Samantha's Search for the Cure Foundation, National Niemann-Pick Disease Foundation, Support of Accelerated Research for NPC Disease, Vtesse, Janssen Research and Development, a Johnson & Johnson company, and Johnson & Johnson.

PurposeFedratinib, an oral selective kinase inhibitor with activity against both wild type and mutationally activated Janus kinase 2, has been approved for the treatment of adult patients with intermediate-2 or high-risk myelofibrosis by the US Food and Drug Administration. In vitro studies indicated that fedratinib was an inhibitor of several cytochrome P450 (CYP) enzymes. The primary objective of this study was to evaluate the effects of repeated doses of fedratinib on the activity of CYP2D6, CYP2C19, and CYP3A4 in patients with solid tumors using a CYP probe cocktail.MethodsAn open-label, one-sequence, two-period, two-treatment crossover study was conducted. Patients were administered a single oral dose cocktail of metoprolol (100 mg), omeprazole (20 mg), and midazolam (2 mg) used as probe substrates for CYP2D6, CYP2C19, and CYP3A4 enzyme activities, respectively, without fedratinib on Day -1 or with fedratinib on Day 15.Results Coadministration of 500 mg once-daily doses of fedratinib for 15 days increased the mean area under the plasma concentration–time curve from time zero to infinity following a single-dose cocktail containing metoprolol (CYP2D6 substrate), omeprazole (CYP2C19 substrate), and midazolam (CYP3A4 substrate) by 1.77-fold (90% confidence interval [CI] 1.27–2.47) for metoprolol, 2.82-fold (90% CI 2.26–3.53) for omeprazole, and 3.84-fold (90% CI 2.62–5.63) for midazolam, respectively. The mean plasma Day 14/Day 1 ratio of 4β-hydroxycholesterol, an endogenous biomarker of CYP3A4 activity, was 0.59 (90% CI 0.54-0.66), suggesting a net inhibition of CYP3A4 by fedratinib.ConclusionFedratinib is a weak inhibitor of CYP2D6, and a moderate inhibitor of CYP2C19 and CYP3A4. These results serve as the basis for dose modifications of these CYP substrate drugs when co-administered with fedratinib.

Purpose27-hydroxycholesterol (27HC), an endogenous selective estrogen receptor modulator (SERM), drives the growth of estrogen receptor-positive (ER+) breast cancer. 1,25-dihydroxyvitamin D (1,25(OH)2D), the active metabolite of vitamin D, is known to inhibit expression of CYP27B1, which is very similar in structure and function to CYP27A1, the synthesizing enzyme of 27HC. Therefore, we hypothesized that 1,25(OH)2D may also inhibit expression of CYP27A1, thereby reducing 27HC concentrations in the blood and tissues that express CYP27A1, including breast cancer tissue.Methods27HC, 25-hydroxyvitamin D (25OHD), and 1,25(OH)2D were measured in sera from 29 breast cancer patients before and after supplementation with low-dose (400 IU/day) or high-dose (10,000 IU/day) vitamin D in the interval between biopsy and surgery.ResultsA significant increase (p = 4.3E−5) in 25OHD and a decrease (p = 1.7E−1) in 27HC was observed in high-dose versus low-dose vitamin D subjects. Excluding two statistical outliers, 25OHD and 27HC levels were inversely correlated (p = 7.0E−3).ConclusionsVitamin D supplementation can decrease circulating 27HC of breast cancer patients, likely by CYP27A1 inhibition. This suggests a new and additional modality by which vitamin D can inhibit ER+ breast cancer growth, though a larger study is needed for verification.