Rmvecs

SS-AT₁WT and SS-AT₁KO rat cardiac microvascular endothelial cells (RMVECs) (Cell Biologics) grown according to company protocol were brought to 70–90% confluency. Media was aspirated and cells were scraped in MPER buffer (Pierce 78501) containing Protease Inhibitor (Roche 11697498001. Cells were lysed with a 21-gauge needle and assayed for protein concentration with MicroBCA kit (Pierce 23235). Thirty-five micrograms of protein from each sample was loaded on a 10% TGX PAGE gel (Biorad) and transferred to nitrocellulose. Blot was blocked overnight in 5% NFDM (Biorad 1706404) and 1% BSA (Sigma A7906). Blot was incubated with Mas1 primary antibody (Santa Cruz sc-135063) at 1:1000 dilution overnight at 4 degrees C. Blot was rinsed and incubated with goat anti-rabbit HRP-conjugated secondary antibody (Biorad 1706515) at 1:5000 dilution for 1 hour. Blot was visualized with SuperSignal West Pico Chemiluminescence Substrate (Pierce 34080). Membrane was imaged on ImageQuant LAS 500 (GE Healthcare Life Sciences) and images were analyzed using ImageJ software (https://imagej.nih.gov/ij/).

Tube formation assay was performed as in previous studies[3 (link)], with the exception of the slide format used. SS-AT₁WT and SS-AT₁KO rat cardiac microvascular endothelial cells (RMVECs) (Cell Biologics) grown according to company protocol were brought to 70–90% confluency, washed twice with DPBS, and lifted using Enzymatic Free Cell Dissociation Buffer (Millipore) with gentle agitation for 30 minutes at 37°C. RMVECs were then centrifuged at 300 x g for 5 minutes, washed twice with DPBS, resuspended in 1 mL MCDB131 basal media plus 2% FBS, and counted using the cell Countess system (Invitrogen). RMVECs were diluted for the addition of 1,250 cells in 50uL of media per well of u-Angiogenesis slides (iBidi) coated in 11 uL of Geltrex (Thermo Fisher). Serum-starved and growth factor depleted conditions were utilized as a tool to stunt normal RMVEC tube formation stimulation to better decipher changes that would be observed through the addition of 100 nM Ang-(1–7). Treatment conditions included RMVECs plus vehicle and RMVECs plus 100 nM Ang-(1–7). At 24 and 48 hours 10X magnification images were taken using a TS100 Inverted Microscope (Nikon Corporation) for analysis of the mean tube length per field (μm) using open-access PipeLine tube formation analysis software[18 (link)]. The results were averaged across biological and technical replicates followed by 1-way ANOVA.