ArrayExpress Uploader0human3352arrayexpress_sid4We generated the transcriptional regulatory footprint of phthalimide neovascular factor 1 (PNF1)—a novel synthetic small molecule that...E-GEOD-17119/profile/8773/arrayexpressuploader014serumDec.12, 2014Falsetranscriptional-profiling-of-a-novel-pro-angiogeniE-GEOD-17119_A-AFFY-44Transcriptional profiling of a novel pro-angiogenic small molecule phthalimide neovascular factor 1 (PNF1)Sep.12, 2014We generated the transcriptional regulatory footprint of phthalimide neovascular factor 1 (PNF1)—a novel synthetic small molecule that exhibits significant in vitro endothelial potency and significant in vivo microvascular network expansion—by performing comparative microarray analysis on PNF1-stimulated (versus control) human microvascular endothelial cells (HMVEC) spanning 1-48 h post-supplementation. We subsequently applied network analysis tools (including substantial libraries of information regarding known associations among network components) to elucidate key signaling components and pathways involved in the PNF1 mechanism-of-action. We identified that PNF1 first induces function of the tumor necrosis factor-alpha (TNF-α) signaling pathway, which in turn affects transforming growth factor-beta (TGF-β) signaling. HMVEC (Cambrex, Walkersville, MD, USA) were cultured in endothelial growth medium 2-microvascular (bulletkit, BioWhittaker, Walkersville, MD, USA) supplemented as directed with 5% fetal bovine serum. The cells (passage 9) were plated at 2.5 x 104 cells/cm2 at 37 degrees Celsius in a humidified chamber with 5% carbon dioxide. They were grown to confluence. After confluence, medium was refreshed, and 30 µM PNF1 or 0.6% dimethyl sulfoxide (DMSO) vehicle control was added to the sample. Total RNA from the PNF1 (n=1 at each timepoint) and control (n=1 at each timepoint) samples was isolated 1, 2, 4, 8, 16, 24 and 48 h post-supplementation using an RNeasy kit (Qiagen, Inc., Valencia, CA, USA) according to the manufacturer's protocol.http://www.ebi.ac.uk/arrayexpress/experiments/E-GEOD-17119http://www.ebi.ac.uk/arrayexpress/experiments/E-GEOD-17119/samples/