Tissue for histological analysis was collected from mice perfused with phosphate buffer saline (PBS) followed by 4% paraformaldehyde, tissue for biochemical analysis was collected from mice perfused with PBS only and was snap frozen in liquid nitrogen and stored at −80°C until analysis. Primary endothelial cells were isolated from lungs by immunoselection with CD31 antibody (BD Biosciences, UK) coated magnetic beads as described previously.10 (link) Vascular smooth muscle cells (VSMCs) were isolated from the aorta by digestion after removal of the endothelial cell layer and the adventitia as described previously.11 (link)
Total RNA was extracted using the Ambion Pure Link kit animal studies and QIAamp DNA blood Midi kit (Qiagen, UK) for human studies. Quantitative real-time RT-PCR was performed with an iCycler IQ real-time detection system (BioRad Laboratories) for animal studies and a QuantStudio7 (ABI) for human studies using primers and probes from the TaqMan Gene Expression Assay system (Life Technologies). Gene expression data were normalized to an appropriate house keeper using the delta CT method.
Total RNA was extracted using the Ambion Pure Link kit animal studies and QIAamp DNA blood Midi kit (Qiagen, UK) for human studies. Quantitative real-time RT-PCR was performed with an iCycler IQ real-time detection system (BioRad Laboratories) for animal studies and a QuantStudio7 (ABI) for human studies using primers and probes from the TaqMan Gene Expression Assay system (Life Technologies). Gene expression data were normalized to an appropriate house keeper using the delta CT method.