Diminazene aceturate uses different pathways to induce relaxation in healthy and atherogenic blood vessels

Diminazene aceturate (DIZE), a putative angiotensin-converting enzyme 2 (ACE2) activator, elicits relaxation in various animal models. This study aimed to determine the relaxing mechanisms in internal iliac arteries utilised by DIZE in healthy and atherogenic rabbit models. Studies were conducted on internal iliac artery rings retrieved from male New Zealand White rabbits fed a 4-week healthy control (n = 24) or atherogenic diet (n = 20). To investigate pathways utilised by DIZE to promote arterial relaxation, a DIZE dose response [10^-9.0 M - 10^-5.0 M] was performed on pre-contracted rings incubated with pharmaceuticals that target: components of the renin-angiotensin system; endothelial- and vascular smooth muscle-dependent mechanisms; protein kinases; and potassium channels. ACE2 expression was quantified by immunohistochemistry analysis following a 2 hr or 4 hr DIZE incubation. DIZE significantly enhanced vessel relaxation in atherogenic rings at doses [10^-5.5 M] (p < 0.01) and [10^-5.0 M] (p < 0.0001), when compared to healthy controls. Comprehensive results from functional isometric studies determined that DIZE causes relaxation via different mechanisms depending on pathology. For the first time, we report that in healthy blood vessels DIZE exerts its direct relaxing effect through ACE2/AT₂R and NO/sGC pathways; however, in atherogenesis this switches to MasR, arachidonic acid pathway (i.e., COX1/2, EET and DHET), MCLP, Ca²⁺ activated voltage channels, AMPK and ERK1/2. Moreover, quantitative immunohistochemical analysis demonstrated that DIZE increases artery ACE2 expression in a time dependent manner. We provide a detailed investigation of DIZE's mechanisms and demonstrate for the first time that in healthy and atherogenic arteries DIZE provides beneficial effects through directly inducing relaxation, albeit via different pathways.