Angiotensin II regulated gene expression in rat primary vascular smooth muscle cells (VSMC)
Angiotensin II regulated gene expression in rat primary vascular smooth muscle cells (VSMC)
Primary rat vascular smooth muscle cells were isolated from 40-50 days old male Wistar rats weighing 170-250 g. Animals were sacrificed by decapitation and fast bleeding. The thoracic aorta was excised and placed in modified Krebs-Ringer solution (120 mM NaCl (Duchefa Biochemie); 4.7 mM KCl (Duchefa Biochemie); 1.8 mM CaCl2 (Duchefa Biochemie); 0.7mM MgSO4 (Fluka; Sleez; Hanover; Germany); 10 mM glucose (Serva); 10 mM Na-HEPES (Serva). Following the removal of connective tissue and adherent fat, the aorta was cut to 1 mm sections, and aorta-rings were digested with collagenase (Collagenase type I., Worthington) for 25 minutes at 37°C. VSMCs were allowed to grow onto 10 cm cell culture plates from the explants for 7-14 days incubated at 5% CO2 and 37°C. VSMCs were cultured in Dulbecco's modified Eagle media (DMEM High Glucose W/ L-Glutamine W/Sodium Pyruvate; Biosera) containing 10% fetal bovine serum (FBS; Biosera), 1% penicillin-streptomycin (Sigma-Aldrich) and 1% GlutaMAX (Gibco).  The VSMCs were passaged with trypsin (EuroClone, Milan, Italy) and were used between passages 2-3.  Primary rat vascular smooth muscle cells were serum deprived overnight prior to hormone stimulus. Vascular smooth muscle cells (VMSCs) were stimulated with 100 nM Angiotensin II for 2 hours at 37°C. VSMCs were washed twice with cold sterile PBS (137 mM NaCl; 2.7 mM KCl 2.7; 10.1 mM Na2HPO4; 1.8 mM KH2PO4, pH 7.4). Cell lysis was carried out using Trizol reagent (ThermoFisher Scientific). Trizol extraction of total RNA was performed according to the manufacturer's instructions (ThermoFisher Scientific). Poly-A RNAs were captured by oligo-dT conjugated magnetic beads; then, the mRNAs were eluted and fragmented at 94°C. First strand cDNA was generated by random priming reverse transcription, and after the second strand synthesis step, double-stranded cDNA was generated. After repairing ends, A-tailing and adapter ligation steps, adapter ligated fragments were amplified in enrichment PCR and finally sequencing libraries were generated.