Quantifying Collagen Composition in Aortic Tissue

Snap frozen aortic samples from control and RhoA cKO mice were pulverized and washed once with PBS for 30 min at 4 ˚C. An aliquot of the sample was transferred into a new tube without pepsin digestion to determine the total collagen content for evaluating collagen synthesis. After centrifugation at 16,000 × g for 30 min at 4 ˚C, the precipitated samples were digested in 0.5 mol/L of acetic acid containing 1 mg/mL of pepsin for 16 h at 4 ˚C with gentle rotation. The undigested samples were assembled by centrifugation at 16,000 × g for 30 min at 4 ˚C, and were collected as cross-linked collagen. The supernatant was collected as non-cross-linked collagen, and was subsequently precipitated in 2 mol/L of NaCl for 30 min, followed by centrifugation at 16,000 × g for 30 min at 4 ˚C. Total, cross-linked and non-cross-linked collagens were quantified by the hydroxyproline assay^{53 (link)}, which were performed using the kit (Sigma-Aldrich, St. Louis, MO, USA). Results are reported as micrograms of hydroxyproline per milligram of wet tissue weight.