Novel pathway compendium analysis elucidates mechanism of pro-angiogenic synthetic small molecule.
Ontology highlight
ABSTRACT: Motivation
Computational techniques have been applied to experimental datasets to identify drug mode-of-action. A shortcoming of existing approaches is the requirement of large reference databases of compound expression profiles. Here, we developed a new pathway-based compendium analysis that couples multi-timepoint, controlled microarray data for a single compound with systems-based network analysis to elucidate drug mechanism more efficiently.Results
We applied this approach to a transcriptional regulatory footprint of phthalimide neovascular factor 1 (PNF1)-a novel synthetic small molecule that exhibits significant in vitro endothelial potency-spanning 1-48 h post-supplementation in human micro-vascular endothelial cells (HMVEC) to comprehensively interrogate PNF1 effects. We concluded that PNF1 first induces tumor necrosis factor-alpha (TNF-alpha) signaling pathway function which in turn affects transforming growth factor-beta (TGF-beta) signaling. These results are consistent with our previous observations of PNF1-directed TGF-beta signaling at 24 h, including differential regulation of TGF-beta-induced matrix metalloproteinase 14 (MMP14/MT1-MMP) which is implicated in angiogenesis. Ultimately, we illustrate how our pathway-based compendium analysis more efficiently generates hypotheses for compound mechanism than existing techniques.
SUBMITTER: Wieghaus KA
PROVIDER: S-EPMC2562016 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:We 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.
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Project description:We 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.
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