ABSTRACT
PURPOSE: In vivo experiments indicate that gas-plasma-treated D,L-polylactide polymers expressing basic fibroblast growth factor (bFGF) exhibit enhanced angiogenesis. bFGF is not a single entity, but it is instead a family of isoforms. Consequently, we sought to determine which bFGF isoforms and levels initiate angiogenesis in nude mice peritoneums. METHODS: Cytoplasmic and nuclear bFGF were characterized for nude mice peritoneums incubated with nontreated scaffolds containing HAEC (CW), its respective polymer-only scaffolds (Cp) and gas-plasma treated scaffolds with HAEC (TW) and without cells (Tp). NuPAGE electrophoresis and WesternBreeze Chemiluminescent kits were used to analyze relative bFGF densities and molecular weights. VEGF was quantified using ImageJ. RESULTS: bFGF bands were located at molecular weights of 24, 48, 58, 72 and 80 kDa, depending on whether they were from cytoplasms or nuclei. At 12, 24 and 72 days, 58-kDa bFGF bands were observed from nuclei of TW and Tp, 80-kDa bFGF bands were only observed in cytoplasmic fractions < or = 24 days. Total cytoplasmic and nuclear bFGF intensities increased from 12 to 24 days, then declined by 72 days. CONCLUSIONS: (1) Gas-plasma treated scaffolds up-regulate bFGF isoforms. (2) bFGF was expressed in the nuclei; however, 80-kDa bFGF was seen only in cytoplasms.
Subject(s)
Biocompatible Materials/pharmacology , Endothelium, Vascular/drug effects , Fibroblast Growth Factor 2/genetics , Fibroblast Growth Factor 2/pharmacology , Gene Expression/drug effects , Gene Expression/genetics , Neovascularization, Physiologic/drug effects , Neovascularization, Physiologic/genetics , Peritoneum/drug effects , Polyesters/pharmacology , Protein Isoforms/genetics , Protein Isoforms/pharmacology , Animals , Dose-Response Relationship, Drug , Female , Fibroblast Growth Factor 2/administration & dosage , Mice , Mice, Nude , Nuclear Matrix-Associated Proteins/administration & dosage , Nuclear Matrix-Associated Proteins/genetics , Nuclear Matrix-Associated Proteins/pharmacology , Protein Isoforms/administration & dosageABSTRACT
PURPOSE: Vascular endothelial growth factor (VEGF) isoforms play different roles in the temporal sprouting of endothelial-lined vessels in a nude mouse peritoneal model as cells respond to nontreated control and gas-plasma-treated bioresorbable poly-D,L-lactide acid 3D scaffolds with human aortic endothelial cells (HAEC). METHODS AND MATERIALS: Nude mice peritoneums were incubated with HAEC (CW = control; TW = gas-plasma treated) or polymer scaffolds (Cp = control; Tp = treated) for 12, 24 and 72 days. Cytoplasmic and nuclear protein fractions were isolated using NER, electrophoresized using NuPAGE-MES and analyzed by WesternBreeze Chemiluminescent. RESULTS: Prominent VEGF bands included 28, 45 and 62 kDa; 52-kDa VEGF observed in cytoplasmic TW fractions contributed about 18.6% at 12 days, 20.0% at 24 days and 13.1% at 72 days of the total VEGF signal. Yet, it was only noted in CW at 72 days where it accounted for 6.9%. A unique 32-kDa band appeared in both Cp (24.6%) and Tp (18.3%). Significant differences between band densities occurred for cytoplasmic nuclear CW24- TW24 (P = .022), CW72-TW72 (P = .011) and, also, cytoplasmic Cp24-Tp24 (P = .038). CONCLUSIONS: The temporal and spatial organization of the TW isoforms results in more angiogenesis.
