Objective: To develop a model permitting the study of the mechanisms of endothelin (ET-1)-induced vascular injury, we generated an inducible and endothelial cell (EC)-restricted human ET-1 (EDN1) cDNA overexpressing transgenic mouse, which would accordingly be devoid of developmental effects.
Methods and Results: A transgene was engineered that expresses chloramphenicol acetyltransferase (cat) cDNA prior to Cre-mediated excision, and EDN1 cDNA after Cre-mediated excision, under the control of cytomegalovirus immediate early enhancer/chicken β-actin promoter (CAG). Co-transfection of Cre expression vector and pCAG-cat-EDN1 caused a 30-fold increase in ET-1 production evaluated by ELISA. A DNA fragment containing the CAG-cat-EDN1 transgene was used to generate transgenic mice. PCR genotyping demonstrated that 14/132 mice were transgenic. Two of 7 transgenic lines were selected based on cardiac cat expression level, which were twice higher in line C-134 than C-170. Inducible EC-restricted EDN1 mice were generated crossing CAG-cat-EDN1 mice with mice expressing Cre recombinase fused to a tamoxifen (TAM)-inducible modified estrogen receptor ligand binding domain (CreERT2) under control of EC-specific receptor tyrosine kinase (Tie2) promoter. To investigate the extent of CreERT2 activation by TAM and tissue specificity, Tie2-CreERT2 mice were crossed with ROSAmT-mG/mT-mG reporter mice expressing a membrane-targeted tandem dimer tomato (mT) before Cre-mediated excision, and membrane-targeted enhanced green fluorescent protein (mG) after excision. Tie2-CreERT2/cat-EDN1 and Tie2-CreERT2/ROSAmT-mG/+ mice were treated with 1 mg TAM/d SC for 5 d and sacrificed 14-16 d later. mG expression was detected in 22±3% of mesenteric artery EC of Tie2-CreERT2/ROSAmT-mG/+ mice. Plasma ET-1 levels were similar in vehicle-treated Tie2-CreERT2/cat-EDN1 (1.2±0.1 pg/mL, n=4) and wild-type mice (1.1±0.1 pg/mL, n=2), demonstrating no leaky expression. TAM induced 8-fold increase in plasma ET-1 levels in Tie2-CreERT2/cat-EDN1 mice (9.1 ± 0.3 pg/mL, n = 4).
Conclusion:Tie2-CreERT2/cat-EDN1-inducible EC-restricted EDN1 overexpressing mice allow the study of ET-1 vascular effects in adult independently of developmental effects.