Chondrocyte hypertrophy is one of the key physiological processes involved in the longitudinal growth of long bones, yet regulation of hypertrophy is 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)/βcatenin, and transforming growth factor beta (TGF-β)/ Sma and Mad Related Family (Smad) pathways, can regulate hypertrophy, but factors as diverse as hypoxia, coculture, 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.

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-1β) 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. Electronic supplementary material The online version of this article (10.1186/s13148-019-0696-z) contains supplementary material, which is available to authorized users.

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], β2-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.