Primary Murine Vascular Smooth Muscle Cell (VSMCs) Isolation and Culture

CO₂ asphyxiation, followed by cervical dislocation, was performed to euthanize the animals. Bodyweight and non-fasting blood glucose measurements were taken at the time of euthanasia, followed by removing the thoracic aorta (Supplementary Table 1). The same animal provided tissue for both VSMC and AFB isolations as described below. The adventitial layer was separated from the medial layer, and the medial layer was placed in a collagenase-elastase digestion solution [1950 U/A of collagenase type 2 enzyme (Worthington Biochemical), 11.275 units of elastase enzyme activity (Worthington Biochemical), 0.004% Trypsin (Corning), and 10 mL of High-Glucose Dulbecco’s Modified Eagles Medium (HG-DMEM, 4.5 g/L glucose; Corning)] to isolate the cells of the medial and intimal aortic tissue. Tissue pieces and enzymatic digestion solution were maintained in a water-jacketed spinner flask under constant agitation for 45 min. A 100 μm cell strainer was used to filter the tissue-collagenase-elastase solution and 5 mL of HG-DMEM with 30% heat-inactivated Fetal Bovine Serum (FBS) (Innovative Research) was added to neutralize the enzymes. The cell mixture was centrifuged at 225 × g for 10 min, and then VSMCs were resuspended in HG-DMEM [14.3 mM NaHCO₃, 15 mM HEPES, 15% FBS, 2% L-glutamine (Corning), 2X Primocin^TM (Invivogen), and Clonetics ^® Smooth Muscle Growth Media-2 SingleQuots (Lonza)]. The resuspended cell solution was placed at 37°C at 5% CO₂ until completion of digestion. The remaining tissue was removed from the strainer and placed in a collagenase digestion solution [100 U/mL type 2 collagenase, 0.1% trypsin (Gibco), and HG-DMEM] in a water-jacketed spinner flask under constant agitation for 30 min. The collagenase digestion was filtered through a 100 μm cell strainer and combined with the previously centrifuged cell solution. All solutions were centrifuged at 225 × g for 10 min and then subsequently plated on 100 μg/mL PureCol^® collagen-coated (Advanced Biomatrix) 60 mm plates in VSMC HG-DMEM. Twenty-four hours after plating, the cells were washed with appropriate glucose media according to genotype [i.e., HG-DMEM for diabetic and Low Glucose-DMEM (LG-DMEM, 1 g/L glucose- euglycemic media; Corning) for non-diabetic]. VSMCs were maintained in LG-DMEM or HG-DMEM (14.3 mM NaHCO₃, 15 mM HEPES, 15% FBS, 2% L-glutamine, 2X Primocin^TM, and Clonetics ^® Smooth Muscle Growth Media-2 SingleQuots). All studies used cells at P1 for baseline characterization and P2 for signaling experiments. The purity of the cultures (>90–95%) was confirmed by positive staining for the VSMC-specific marker, α-Smooth Muscle Actin (Sigma-Aldrich, St. Louis, MO, United States, A2547). Aortas from 3 to 4 mice were used per isolation. Data from 6 to 9 separate isolations were collected per genotype. VSMCs cultures were found to be pure with no endothelial cell presence (Supplementary Figure 1).