Pressure Myography

Freshly isolated 3^rd or 4^th order rat or mouse mesenteric arteries (< 220 μm) were mounted in a pressure arteriograph [Danish MyoTechnology (DMT)] and equilibrated for 30 min at 80 mmHg and 37°C, as previously described²⁹. If treated with L-nitro-arginine methyl ester (L-NAME, 100 μM), L-NAME was added 10 minutes into the equilibration step. The lumen and bath solutions were then replaced with EV containing Krebs-HEPES solution while control vessels were perfused with Krebs-HEPES. 30 μL of the isolated EV solution (or liposomes) were placed into 3 mL buffer for the lumen solution and 70 μL of EVs were placed into the 10 mL circulating bath. The vessels were then equilibrated for an additional 10 min followed by pre-constriction with 10 μM phenylephrine (PE). To complete a full ACh dose response curve for each vessel, increasing concentrations of ACh were then added into the bath to examine the endothelial-dependent vasodilation of these vessels. Endothelial cell health was confirmed using NS309 and smooth muscle cell health was confirmed by PE. Maximal passive diameter of the vessels was then obtained as previously described²⁹. Internal diameter was measured at each step using the DMT MyoVIEW software. Data were replotted in multiple figures (such as control WKY arteries) because comparisons were done across all groups as all treatment groups underwent the same experimental design. The EC₅₀ of the ACh dilation and raw lumen diameters for resting diameter (after EV treatment), maximal constriction, maximum dilation for each treatment group were calculated in the online-only supplemental Table S1 and Table S2 (please see http://hyper.ahajournals.org).