The BET inhibitor apabetalone decreases neuroendothelial proinflammatory activation in vitro and in a mouse model of systemic inflammation.

Brain vascular inflammation is characterized by endothelial activation and immune cell recruitment to the blood vessel wall, potentially causing a breach in the blood - brain barrier, brain parenchyma inflammation, and a decline of cognitive function. The clinical-stage small molecule, apabetalone, reduces circulating vascular endothelial inflammation markers and improves cognitive scores in elderly patients by targeting epigenetic regulators of gene transcription, bromodomain and extraterminal proteins. However, the effect of apabetalone on cytokine-activated brain vascular endothelial cells (BMVECs) is unknown. Here, we show that apabetalone treatment of BMVECs reduces hallmarks of in vitro endothelial activation, including monocyte chemoattractant protein-1 (MCP-1) and RANTES chemokine secretion, cell surface expression of endothelial cell adhesion molecule VCAM-1, as well as endothelial capture of THP-1 monocytes in static and shear stress conditions. Apabetalone pretreatment of THP-1 downregulates cell surface expression of chemokine receptors CCR1, CCR2, and CCR5, and of the VCAM-1 cognate receptor, integrin α4. Consequently, apabetalone reduces THP-1 chemoattraction towards soluble CCR ligands MCP-1 and RANTES, and THP-1 adhesion to activated BMVECs. In a mouse model of brain inflammation, apabetalone counters lipopolysaccharide-induced transcription of endothelial and myeloid cell markers, consistent with decreased neuroendothelial inflammation. In conclusion, apabetalone decreases proinflammatory activation of brain endothelial cells and monocytes in vitro and in the mouse brain during systemic inflammation.

Molecular and biophysical mechanisms regulating hypertrophic differentiation in chondrocytes and mesenchymal stem cells.

Chondrocyte hypertrophy is one of the key physiological processes involved in the longitudinal growth of long bones, yet the regulation of hypertrophy is also becoming increasingly relevant for clinical application of mesenchymal stem cells (MSCs) and screening for drugs to treat hypertrophic osteoarthritis. The extraordinary cell volume increase during hypertrophy is accompanied by an up-regulation of collagen X, matrix metalloproteinases (MMPs), and vascular endothelial growth factor (VEGF), all which are targets of the runt-related transcription factor 2 (Runx2). Many pathways, including parathyroid hormone-related protein (PTHrP)/Indian Hedgehog, Wingless/Int (Wnt)/ß-catenin, and transforming growth factor beta (TGF-ß)/Sma and Mad Related Family (Smad) pathways, can regulate hypertrophy, but factors as diverse as hypoxia, co-culture, epigenetics and biomaterial composition can also potently affect Runx2 expression. Control of hypertrophic differentiation can be exploited both for cartilage repair, where a stable phenotype is desired, but also in bone regeneration, where hypertrophic cartilage could act as a template for endochondral bone formation. We hope this review will motivate the design of novel engineered microenvironments for skeletal regeneration applications.

Linking Aortic Mechanical Properties, Gene Expression and Microstructure: A New Perspective on Regional Weakening in Abdominal Aortic Aneurysms.

Background: Current clinical practice for the assessment of abdominal aortic aneurysms (AAA) is based on vessel diameter and does not account for the multifactorial, heterogeneous remodeling that results in the regional weakening of the aortic wall leading to aortic growth and rupture. The present study was conducted to determine correlations between a novel non-invasive surrogate measure of regional aortic weakening and the results from invasive analyses performed on corresponding ex vivo aortic samples. Tissue samples were evaluated to classify local wall weakening and the likelihood of further degeneration based on non-invasive indices. Methods: A combined, image-based fluid dynamic and in-vivo strain analysis approach was used to estimate the Regional Aortic Weakness (RAW) index and assess individual aortas of AAA patients prior to elective surgery. Nine patients were treated with complete aortic resection allowing the systematic collection of tissue samples that were used to determine regional aortic mechanics, microstructure and gene expression by means of mechanical testing, microscopy and transcriptomic analyses. Results: The RAW index was significantly higher for samples exhibiting lower mechanical strength (p = 0.035) and samples classified as low elastin content (p = 0.020). Samples with higher RAW index had the greatest number of genes differentially expressed compared to any constitutive metric. High RAW samples showed a decrease in gene expression for elastin and a down-regulation of pathways responsible for cell movement, reorganization of cytoskeleton, and angiogenesis. Conclusions: This work describes the first AAA index free of assumptions for material properties and accounting for patient-specific mechanical behavior in relation to aneurysm strength. Use of the RAW index captured biomechanical changes linked to the weakening of the aorta and revealed changes in microstructure and gene expression. This approach has the potential to provide an improved tool to aid clinical decision-making in the management of aortic pathology.

Retrieval of a metabolite from cells with polyelectrolyte microcapsules.

To monitor cellular processes in individual cells, it is important to measure the concentrations of intracellular metabolites and to retrieve them for analysis. The use of functionalized polyelectrolyte microcapsules as intracellular sensors for in vivo reporting is persented. Capsules loaded with streptavidin-rhodamine, which was introduced into fibroblasts by electroporation, autonomously escaped from an endocytic compartment and efficiently recruited biotin-fluorescein from the cytosol. This work demonstrates the utility of polyelectrolyte microcapsules for intracellular capture of metabolites and eventually for drug delivery on an organismic level.

Apabetalone (RVX-208) reduces vascular inflammation in vitro and in CVD patients by a BET-dependent epigenetic mechanism.

BACKGROUND: Apabetalone (RVX-208) is a bromodomain and extraterminal protein inhibitor (BETi) that in phase II trials reduced the relative risk (RR) of major adverse cardiac events (MACE) in patients with cardiovascular disease (CVD) by 44% and in diabetic CVD patients by 57% on top of statins. A phase III trial, BETonMACE, is currently assessing apabetalone's ability to reduce MACE in statin-treated post-acute coronary syndrome type 2 diabetic CVD patients with low high-density lipoprotein C. The leading cause of MACE is atherosclerosis, driven by dysfunctional lipid metabolism and chronic vascular inflammation (VI). In vitro studies have implicated the BET protein BRD4 as an epigenetic driver of inflammation and atherogenesis, suggesting that BETi may be clinically effective in combating VI. Here, we assessed apabetalone's ability to regulate inflammation-driven gene expression and cell adhesion in vitro and investigated the mechanism by which apabetalone suppresses expression. The clinical impact of apabetalone on mediators of VI was assessed with proteomic analysis of phase II CVD patient plasma. RESULTS: In vitro, apabetalone prevented inflammatory (TNFα, LPS, or IL-1ß) induction of key factors that drive endothelial activation, monocyte recruitment, adhesion, and plaque destabilization. BRD4 abundance on inflammatory and adhesion gene promoters and enhancers was reduced by apabetalone. BRD2-4 degradation by MZ-1 also prevented TNFα-induced transcription of monocyte and endothelial cell adhesion molecules and inflammatory mediators, confirming BET-dependent regulation. Transcriptional regulation by apabetalone translated into a reduction in monocyte adhesion to an endothelial monolayer. In a phase II trial, apabetalone treatment reduced the abundance of multiple VI mediators in the plasma of CVD patients (SOMAscan® 1.3 k). These proteins correlate with CVD risk and include adhesion molecules, cytokines, and metalloproteinases. Ingenuity® Pathway Analysis (IPA®) predicted that apabetalone inhibits pro-atherogenic regulators and pathways and prevents disease states arising from leukocyte recruitment. CONCLUSIONS: Apabetalone suppressed gene expression of VI mediators in monocytes and endothelial cells by inhibiting BET-dependent transcription induced by multiple inflammatory stimuli. In CVD patients, apabetalone treatment reduced circulating levels of VI mediators, an outcome conducive with atherosclerotic plaque stabilization and MACE reduction. Inhibition of inflammatory and adhesion molecule gene expression by apabetalone is predicted to contribute to MACE reduction in the phase III BETonMACE trial.

Human chondroprogenitors in alginate-collagen hybrid scaffolds produce stable cartilage in vivo.

The goal of this study was to evaluate human epiphyseal chondroprogenitor cells (ECPs) as a potential new cell source for cartilage regeneration. ECPs were compared to human bone marrow stromal cells (MSCs) and human adult articular chondrocytes (ACs) for their chondrogenic potential and phenotypic stability in vitro and in vivo. The cells were seeded in Optimaix-3D scaffolds at 5 × 104 cells/mm3 and gene expression, matrix production and mechanical properties were analysed up to 6 weeks. In vitro, ECPs synthesized consistently high collagen 2 and low collagen 10. AC-seeded constructs exhibited high donor variability in GAG/DNA values as well as in collagen 2 staining, but showed low collagen 10 production. MSCs, on the other hand, expressed high levels of collagen 2 but also of collagens 1 and 10, and were therefore not considered further. In vivo, there was considerable loss of matrix proteins in ECPs compared to in vitro cultured samples. To overcome this, a second implantation study investigated the effect of mixing cells with alginate prior to seeding in the scaffold. ECPs in alginate maintained their cartilage matrix and resisted mineralization and vessel infiltration better 6 weeks after subcutaneous implantation, whereas ACs lost their chondrogenic matrix completely. This study shows the great potential of ECPs as an off-the-shelf, highly chondrogenic cell type that produces stable cartilage in vivo. Copyright © 2016 John Wiley & Sons, Ltd.

Ribosomal protein l13a as a reference gene for human bone marrow-derived mesenchymal stromal cells during expansion, adipo-, chondro-, and osteogenesis.

In the field of human mesenchymal stromal cell (MSC) research, quantitative real-time reverse transcription-polymerase chain reaction (qPCR) is the method of choice to study changes in gene expression patterns upon differentiation, application of stimuli, or of factors such as inhibitors or siRNAs. To reliably detect small changes, the use of a reference gene (RG) that is stably expressed under all conditions is essential. The large number of different RGs used in the field and the lack of validation of their suitability make the comparison between studies impossible. Therefore, this work aims to establish one single RG for mesodermal differentiation studies that use MSCs. Seven commonly used RGs (glyceraldehyde-3-phosphate dehydrogenase [GAPDH], ribosomal protein L13a [RPL13a], beta-actin [ACTB], tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta-polypeptide [YWHAZ], eukaryotic translational elongation factor 1 alpha [EF1α], ß2-microglobulin [B2M], and 18S ribosomal RNA [18S]) were investigated concerning their mRNA expression stability during expansion of bone marrow-derived MSCs up to four passages as well as during their adipo-, chondro-, and osteogenenic differentiation on days 9, 16, and 22 after induction. RPL13a was validated for qPCR studies of MSCs (bone marrow- and placenta-derived) and, additionally, for primary human bone cells (HBCs) and the osteosarcoma cell line MG-63. GAPDH and ACTB, the two most frequently used RGs, showed the highest expression variance. The superior performance of RPL13a should make it the RG of choice for all MSC studies addressing mesodermal differentiation.

Design and synthesis of 4-substituted-8-(2-phenyl-cyclohexyl)-2,8-diaza-spiro[4.5]decan-1-one as a novel class of GlyT1 inhibitors: achieving selectivity against the mu opioid and nociceptin/orphanin FQ peptide (NOP) receptors.

A novel class of 4-substituted-8-(2-phenyl-cyclohexyl)-2,8-diaza-spiro[4.5]decan-1-ones have been discovered and developed as potent and selective GlyT1 inhibitors. The molecules are devoid of activity at the GlyT2 isoform and display excellent selectivities against the mu opioid receptor as well as the nociceptin/orphanin FQ peptide (NOP) receptor. A novel, straightforward and efficient synthetic strategy for the assembly of the target molecules is also presented.