A new mouse model of peripheral artery disease: development, validation and assessment of a potential intervention and a therapeutic target

Abstract

Peripheral arterial disease (PAD) affects more than 200 million people worldwide and is caused by occlusions resulting from atherosclerosis in arteries supplying blood to the lower limbs. Treatments for limb ischaemia in PAD is an unmet medical need. Novel treatments are sought to improve limb blood supply and function in PAD patients unlikely to benefit from existing treatments such as surgical revascularisation, structured exercise programs and cilostozol. Currently, the main pre-clinical experimental model employed in PAD research is based on induction of acute hind limb ischemia (HLI) which results in rapid natural recovery of blood supply to ischaemic tissues. There are concerns regarding the ability to translate findings from this mouse model to PAD patients and a clinically relevant mouse model of HLI is lacking. Evidence suggests metformin may be a potential treatment for PAD and angiotensin converting enzyme 2 (ACE2) may play a role in limb ischaemia. This thesis aimed to develop a clinically relevant mouse model of PAD that involved a two-stage induction of HLI and examine the effect of exercise training, metformin and ACE2 deficiency in the two-stage model of HLI. Two-stage HLI was induced in male Apolipoprotein E (ApoE-/-) deficient mice by slow onset of severe ischemia over 14 days. This 2-stage HLI model was compared to the acute HLI model and sham controls. Limb blood supply was assessed by Laser Doppler Perfusion Imaging (LDI). Ambulatory ability was assessed using a treadmill test and established scoring scales. Running wheel exercise training was examined in the two-stage mouse model and limb function was assessed by a treadmill exercise test and blood supply was assessed by LDI. Next, the effect of metformin on limb ischaemia was assessed by LDI in the two-stage HLI mouse model. Lastly, the effect of ACE2 deficiency (ACE2-/y) on limb ischaemia was examined in the two-stage mouse model of HLI. HLI was significantly more severe in mice receiving the two-stage compared to the acute HLI induction procedure as assessed by LDI (p=0.013), and reflected a higher ischemic score (p=0.003) and lower average distance travelled on a treadmill exercise test (p=0.045). Mice with two-stage HLI receiving exercise training showed significantly greater improvement in their ambulatory ability on a treadmill test than the sedentary control group (p=0.003). However, limb blood supply was comparable between mice receiving exercise compared to controls (p=0.700). Mice with two-stage HLI administered metformin had greater blood supply than mice with two-stage HLI receiving vehicle control (p<0.001). The two-stage HLI mice receiving metformin had increased adenosine monophosphate kinase alpha (AMPKα) activity (p=0.041), endothelial nitric oxide synthase (e-NOS) activity (p=0.031), nitric oxide (NO) bioavailability (p=0.024), peroxisome proliferator activated receptor 1 alpha (PGC1-α) expression (p=0.026) and decreased thioredoxin interacting protein (TXNIP) expression (p=0.038) compared to mice with two-stage HLI receiving vehicle. ACE2-/y in mice with two-stage HLI had comparable limb ischaemia to ACE2 unmodified control mice with two-stage HLI (p=0.263). In conclusion, this thesis showed that the novel two-stage mouse model of HLI had severe ongoing HLI and functional impairment. Exercise training augmented treadmill walking capacity in the new mouse model which was independent of changes to limb blood supply and mirroring patient response to exercise therapy. Metformin administration improved limb blood supply in the two-stage mouse model of ongoing HLI. Improvement in blood supply was associated with the upregulation of AMPKα activity, increased activation of e-NOS in the ischaemic muscles, increased bioavailability of circulating NO, increased expression of PGC1α and reduced expression of TXNIP. This suggests metformin may have potential to be used as a treatment to improve limb blood supply in PAD patients. ACE2-/ did not worsen limb blood supply after two-stage HLI induction suggesting ACE2 may not play an important role in limb ischemia. The results do not encourage the pursuit of ACE2 for pharmacological management of PAD in clinical trials and ACE2 is unlikely to be an important target for improving limb blood supply or function in patients with PAD.