Microarray data and pathway analyses for human microvascular endothelial cells supplemented with low dose vitamin D or niacin during lipotoxicity

Low dose niacin and vitamin D can directly improve human microvascular endothelial cell angiogenic function under lipotoxic conditions Peters et al.,2019. Despite exerting similar benefits on in vitro angiogenic function, these vitamins are known to signal through independent receptors, raising the possibility that differential changes in gene expression may underlie these effects. Here we provide data collected using Affymetrix GeneChip microarrays to compare gene expression in human microvascular endothelial cells treated for 16 h with growth medium containing BSA alone, or BSA complexed with the saturated fatty acid palmitate, and supplemented with 10 μM niacin or 10 nM vitamin D (1,25-dihydroxyvitamin D3). Data sets of differential gene expression included many genes involved in cellular stress responses. Pathway analyses of genes specific to vitamin D treatment identified a robust overrepresentation of pathways related to the cell cycle and DNA replication and repair.


Data
Microarray analyses comparing mRNA isolated from human microvascular endothelial cells (HMVEC) following 16 h treatment in growth medium containing bovine serum albumin (BSA) or 0.5 mM palmitate supplemented with 10 mM niacin or 10 nM vitamin D revealed significantly (p < 0.05) altered expression of many cellular stress response genes. These included genes involved in ER stress, oxidative stress, and inflammation, at a fold change of greater than 2 [1] (Supplemental material). These data were consistent with subsequent Reactome pathway analyses (Figs. 1e3, and Supplemental material). Pathway analyses of differentially expressed genes specific to vitamin D treatment identified robust overrepresentation of genes involved in cell cycle regulation, DNA replication, DNA repair, and signal transduction (Fig. 2, and Supplemental material).

Endothelial cell culture and treatments
Primary HMVEC (Lonza, Walkerville, MD USA) were maintained in Medium 199 (Life Technologies, Carlsbad, CA, USA) supplemented with EGM-2MV SingleQuots (Lonza) and subcultured according to manufacturer's protocol. For experiments, cells from a single donor were used at subculture 5. For lipotoxic treatments, cells were exposed to media supplemented with 0.5 mM palmitic acid complexed to bovine serum albumin (BSA) at a molar ratio of 2:1. Cell monolayers at 80% confluence were treated for 16 h with media containing either BSA, palmitate, palmitate with 10 nM vitamin D (1,25dihydroxyvitamin D3; Sigma Aldrich), solubilized in dimethyl sulfoxide (DMSO), or palmitate with 10 mM niacin, solubilized in cell culture grade water. DMSO (2 ml/ml of media) was used as the vehicle control. A total of twelve samples (three BSA, three palmitate, three palmitate plus vitamin D, and three palmitate plus niacin) were generated for subsequent gene expression analyses.

RNA isolation, quality assessment, probe preparation and gene chip hybridization
Following treatment, cells were lysed and RNA was isolated following a standard QIAGEN protocol as previously described. RNA concentrations and purity were measured using a NanoDrop Specifications  spectrophotometer (Thermo Scientific). Samples were diluted to a concentration of 200 ng/mL in nuclease-free water and RNA was stored at À80 C prior to microarray analyses. All sample labeling and GeneChip processing were performed at the London Regional Genomics Centre (Robarts Research Institute). RNA quality was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies) and the RNA 6000 Nano kit (Caliper Life Sciences). End labeled single stranded complementary DNA was

Statistical analyses of changes in global gene expression
Microarray probe data were imported into Partek Genomics Suite v6.6 using the robust multi-array average (RMA) algorithm [2]. Partek was used to determine gene level ANOVA p-values and fold changes. Filtered gene lists were generated for expression changes of greater than 2.0-fold and having a p-value of less than 0.05 (Supplementary material).

Pathway analyses
Lists of genes exhibiting at least a 2.0-fold change in expression with a p-value less than 0.05 were used for gene ontology (GO) and KEGG pathway enrichment analyses (Supplementary material). Overrepresented transcripts were also imported into the open-source Reactome curated pathway data base (v58) (http://www.reactome.org/) for further analyses. Pathway overview schematics, indicating significantly represented (p < 0.05) nodes (pathways) and relationships between nodes were generated for each list of differentially expressed genes.