Data on gene and protein expression changes induced by apabetalone (RVX-208) in ex vivo treated human whole blood and primary hepatocytes

Apabetalone (RVX-208) inhibits the interaction between epigenetic regulators known as bromodomain and extraterminal (BET) proteins and acetyl-lysine marks on histone tails. Data presented here supports the manuscript published in Atherosclerosis “RVX-208, a BET-inhibitor for Treating Atherosclerotic Cardiovascular Disease, Raises ApoA-I/HDL and Represses Pathways that Contribute to Cardiovascular Disease” (Gilham et al., 2016) [1]. It shows that RVX-208 and a comparator BET inhibitor (BETi) JQ1 increase mRNA expression and production of apolipoprotein A-I (ApoA-I), the main protein component of high density lipoproteins, in primary human and African green monkey hepatocytes. In addition, reported here are gene expression changes from a microarray-based analysis of human whole blood and of primary human hepatocytes treated with RVX-208.


Subject area
Molecular biology More specific subject area

Atherosclerosis
Type of data Graphs and tables How data was acquired Real-time PCR using TaqMan assays; ELISA; Microarray analysis using Affymetrix Human Genome U133 Plus 2.0 and 2.4 Arrays.

Data format
Analyzed Experimental factors in vitro treatment of cultured primary cells with RVX-208, JQ1 or DMSO for up to 72 h. Experimental features mRNA and media were collected from cultured primary hepatocytes and analyzed by real-time PCR and ELISA, respectively. Human whole blood from healthy volunteers was treated ex vivo with BET inhibitors. Total RNA was extracted from treated whole blood and hepatocytes and analyzed using gene microarrays. Data source location

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Data accessibility Data is supplied with this article.

Value of the data
Data demonstrate suitability of human and African green monkey primary hepatocyte 3D culture systems for expression studies of the ApoA-I gene and protein.
Data demonstrate suitability of a recently developed anti-proApoA-I antibody to measure newly produced ApoA-I protein in human primary hepatocytes.
The gene expression data from human whole blood and primary hepatocytes reported here provide an RVX-208 transcriptional signature that can be compared to other compounds targeting BET proteins.

Data
Data presented here supports the manuscript published in Atherosclerosis "RVX-208, a BETinhibitor for Treating Atherosclerotic Cardiovascular Disease, Raises ApoA-I/HDL and Represses Pathways that Contribute to Cardiovascular Disease" (Gilham et al., 2016) [1]. Combine with previous sentence. Expression of apolipoprotein A-I (ApoA-I) mRNA in response to RVX-208 treatment was assessed in primary hepatocytes from African green monkey grown in a 3-D culture system (Fig. 1). In primary human hepatocytes, the effect of RVX-208 on newly synthesized ApoA-I protein was compared to that of JQ1, a BETi with a distinct chemical scaffold (Fig. 2). Changes in expression of genes involved in inflammation and atherosclerosis  were identified by microarrays from human whole blood and human primary hepatocytes treated in vitro with RVX-208 (Tables 1-5).

Experimental design, materials and methods
2.1. Detection of ApoA-I mRNA in primary hepatocytes from African green monkey Primary hepatocytes from African green monkey were supplied by RegeneMed Inc. (San Diego, CA). Stromal cells were grown concurrently on a nylon mesh scaffold with fresh liver parenchymal cells to create a three dimensional culture. Cells were treated with 0.1% DMSO or RVX-208 for 3 h, 24 h or 48 h, mRNA was purified with mRNA Catcher TM PLUS kits (Life Technologies) and mRNA expression analysis was performed by TaqMan s based real-time PCR as described previously [80].

Detection of ApoA-I mRNA in primary human hepatocytes
Primary human hepatocytes (CellzDirect/Life Technologies) were plated as recommended by the supplier. Cells were treated with 0.1% DMSO, 30 mM RVX-208 or 0.6 mM JQ1 for 48 h, mRNA was purified and mRNA expression analyzed as above.

Detection of ApoA-I and proApoA-I secreted by primary human hepatocytes
Primary human hepatocytes (CellzDirect/Life Technologies) were treated with 0.1% DMSO, 30 mM RVX-208 or 0.6 mM JQ1 for 72 h and media samples containing secreted proteins were analyzed by ELISA. A rabbit monoclonal anti-proApoA-I antibody was generated using a synthetic peptide RHFWQQ_DEPP [1]. The antibody was used to coat EIA/RIA high binding surface microplates (Corning) overnight. Plates were washed, and then blocked with 5% skim milk. Recombinant poly-histidine tagged human proApoA-I (Genscript, Piscataway, NJ) was used as a standard. Recombinant protein or media samples with were introduced onto plates, incubated with anti-human ApoA-I (Calbiochem # 178470), and then with HRP conjugated anti-mouse IgG (Calbiochem # 401253). Color was developed by treatment with tetramethylbenzidine, followed by sulfuric acid. Plates were read on a Thermo Scientific Multiskan GO Spectrophotometer at 450 nm. ApoA-I ELISA was performed in a similar fashion as proApoA-I, except using the mouse anti-human ApoA-I antibody (Calbiochem # 178470). The standard was purified ApoA-I (Calbiochem # 178452) and it was detected using a polyclonal rabbit anti-human ApoA-I antibody (Calbiochem # 178422), followed by HRP conjugated anti-rabbit IgG (Calbiochem # 401353).

Gene expression microarray from human whole blood
After obtaining informed consent, whole blood was collected from three healthy volunteers into BD Vacutainer Sodium Heparin tubes (# 367874 ) and samples were inverted 10 times. Blood samples (1 mL) were combined with 1 mL of RPMI containing 2 mM glutamine, 1% penicillin/streptomycin, 20% FBS and 20 mM RVX-208 or vehicle (0.1% DMSO), followed by a 3 h or 24 h incubation at 37°C in a tissue culture incubator (CO 2 at 5.5% concentration). Treated samples were transferred to PAXgene Table 1 The effect of RVX-208 on expression of pro-atherogenic genes in human whole blood treated ex vivo for 24 h. RNA tubes (PreAnalytix/Qiagen), inverted 5 times and frozen. RNA was isolated with the PAXgene RNA kit according to the manufacturer's instructions. Microarrays were performed by Asuragen Inc.

Table 3
The effect of RVX-208 on expression of anti-atherogenic genes in human whole blood treated ex vivo for 24 h.

Gene symbol
Fold change Effects on atherosclerotic processes in vitro and in vivo Ref. (Austin, TX) using the Affymetrix human U133 plus 2.4 Array. Gene expression changes were calculated as a fold change relative to DMSO treated samples. Genes with known roles in atherosclerosis, thrombosis or inflammation (based on published literature) and whose expression changed in response to RVX-208 treatment (p-value o 0.05, Student's t-test) were compiled into pro-atherogenic and anti-atherogenic categories.

Gene expression microarray from primary human hepatocytes
Primary human hepatocytes (CellzDirect/Life Technologies) were plated in 24 well format at 500,000 cells/well, then overlaid with Matrigel™ as recommended by the supplier. Cells were treated with 0.1% DMSO or 30 mM RVX-208 for 48 h. Total RNA was extracted with the mirVana™ kit (Ambion) and sent to Asuragen Inc. (Austin, TX) for microarray analysis using Affymetrix Human Genome U133 Plus 2.0 Array. Gene expression changes were calculated as a fold change relative to DMSO treated cells. Genes encoding acute phase response proteins associated with HDL (based on http://homepages.uc.edu/ $ davidswm/HDLproteome.html) and whose expression changed in response to RVX-208 treatment (p-value o0.05, Student's t-test) were compiled.

Transparency document. supporting material
Transparency data associated with this article can be found in the online version at: http://dx.doi. org/10.1016/j.dib.2016.07.047.