Abstract
Objective: Peripheral arterial disease (PAD) patients with diabetes mellitus suffer from impaired neovascularization after ischemia which results in poorer outcomes. MicroRNA (miR)-133a is excessively expressed in endothelial cells under diabetic conditions. Here, we test whether diabetes-induced miR-133a up-regulation is involved in the impaired capability of neovascularization in experimental PAD models. Methods and results: MiR-133a level was measured by quantitative RT-PCR and showed a higher expression level in the ischemic muscle from diabetic mice when compared with nondiabetic mice. Knockdown of miR-133a using antagomir improved perfusion recovery and angiogenesis in experimental PAD model with diabetes day 21 after HLI. On the other hand, overexpression of miR-133a impaired perfusion recovery. Ischemic muscle was harvested day 7 after experimental PAD for biochemical test, miR-133a antagonism resulted in reduced malondialdehyde, and it increased GTP cyclohydrolase 1 (GCH1), and cyclic guanine monophosphate (cGMP) levels. In cultured endothelial cells, miR-133a antagonism resulted in reduced reactive oxygen species level, and it increased tube formation, nitric oxide (NO), and cGMP level. Moreover, miR-133a antagonism-induced angiogenesis was abolished by GCH1 inhibitor. In contrary, miR-133a overexpression impairs angiogenesis and it reduces GCH1, NO, and cGMP levels in nondiabetic models. Conclusion: Diabetes mellitus-induced miR-133a up-regulation impairs angiogenesis in PAD by reducing NO synthesis in endothelial cells. MiR-133a antagonism improves postischemic angiogenesis.
Highlights
Peripheral arterial disease (PAD) affects approximately 20% of the population over 55 years old worldwide [1,2,3,4], diabetes mellitus is an important risk factor for the development of PAD and puts PAD patients in a higher risk of lower extremity amputation and even death, which is mainly attributed to hyperglycemia-induced neovascularization impairment [1,2,5]
By using quantitative RT-PCR (qPCR), we found miR-133a expression level in ischemic mouse hindlimbs was significantly higher from mice with diabetes when compared with nondiabetic mice day 7 after hindlimb ischemia (HLI) (Figure 1A)
We analyzed the expression of miR-133a in Human umbilical vein endothelial cells (HUVEC) exposed to normal glucose (5 mM) and hyperglycemia (25 mM) under hypoxia and serum starvation (HSS) conditions for 48 h, consistent with what we found in ischemic muscle, miR-133a expression in HUVECs cultured with hyperglycemic culture medium was higher than HUVECs under normal glucose culture (Figure 1B)
Summary
Peripheral arterial disease (PAD) affects approximately 20% of the population over 55 years old worldwide [1,2,3,4], diabetes mellitus is an important risk factor for the development of PAD and puts PAD patients in a higher risk of lower extremity amputation and even death, which is mainly attributed to hyperglycemia-induced neovascularization impairment [1,2,5]. Ischemia-induced neovascularization is the key point of perfusion recovery for PAD; there is no-known medication that is able to induce enough functional blood vessel growth and treat PAD; especially when complicated with diabetes [6,7]. Mouse hindlimb ischemia (HLI) has been widely used as a preclinical PAD model, the processes involved in vascular adaptation following vessel occlusion are similar to PAD patients.
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