Apelin prevents diabetes-induced poor collateral vessel formation and blood flow reperfusion in ischemic limb

Abstract

IntroductionPeripheral arterial disease (PAD) is a major risk factor for lower-extremity amputation in diabetic patients. Unfortunately, previous clinical studies investigating therapeutic angiogenesis using the vascular endothelial growth factor (VEGF) have shown disappointing results in diabetic patients, which evokes the necessity for novel therapeutic agents. The apelinergic system (APJ receptor/apelin) is highly upregulated under hypoxic condition and acts as an activator of angiogenesis. Apelin treatment improves revascularization in nondiabetic models of ischemia, however, its role on angiogenesis in diabetic conditions remains poorly investigated. This study explored the impact of Pyr-apelin-13 in endothelial cell function and diabetic mouse model of hindlimb ischemia.MethodsNondiabetic and diabetic mice underwent femoral artery ligation to induce limb ischemia. Diabetic mice were implanted subcutaneously with osmotic pumps delivering Pyr-apelin-13 for 28 days. Blood flow reperfusion was measured for 4 weeks post-surgery and exercise willingness was assessed with voluntary wheels. In vitro, bovine aortic endothelial cells (BAECs) were exposed to normal (NG) or high glucose (HG) levels and hypoxia. Cell migration, proliferation and tube formation assays were performed following either VEGF or Pyr-apelin-13 stimulation.Results and DiscussionFollowing limb ischemia, blood flow reperfusion, functional recovery of the limb and vascular density were improved in diabetic mice receiving Pyr-apelin-13 compared to untreated diabetic mice. In cultured BAECs, exposure to HG concentrations and hypoxia reduced VEGF proangiogenic actions, whereas apelin proangiogenic effects remained unaltered. Pyr-apelin-13 induced its proangiogenic actions through Akt/AMPK/eNOS and RhoA/ROCK signaling pathways under both NG or HG concentrations and hypoxia exposure. Our results identified the apelinergic system as a potential therapeutic target for angiogenic therapy in diabetic patients with PAD.

Endothelial SHP-1 regulates diabetes-induced abnormal collateral vessel formation and endothelial cell senescence

“…Proteins from the adductors muscles or endothelial cells were lysed, extracted and treated as previously described 16,27 . Protein content quantification was performed using computer-assisted densitometry with ImageLab imaging software (Chemidoc, BioRad)…”

“…Bovine aortic endothelial cells (ECs) were isolated from freshly harvested aortas that were obtained from a local slaughterhouse as previously described 19,27 . Cells were cultured in DMEM 2.5% FBS, 1% penicillin-streptomycin and exposed to DMEM 0.1% FBS containing normal glucose (NG; 5.6 mmol/L + 19.4 mmol/L mannitol to adjust osmotic pressure) or high glucose (HG; 25 mmol/L) levels up to 48 h. To induce hypoxic conditions, cells were placed into an incubator set at 1% O2 for the last 16 hours and then stimulated with VEGF-a (10 ng/mL) for 5 minutes.…”

“…EC were infected with ad-GFP or Ad-dnSHP-1 and then exposed to NG or HG concentrations for 48 h with or without VEGF 25 ng/ml for 24 h (proliferation and migration) or 4 h (lumen formation) and on 1% O 2 for the last 16 h. Proliferation, migration and lumen formation quantification were performed as previously described 27 .…”

The apelinergic system accelerates skin repair of full-thickness excisional wounds in a diabetic murine model

“…Dulbecco's Modified Eagle Medium (DMEM) low glucose (31600-034) was obtained from Invitrogen (Burlington, ON, CA). Pyr-apelin-13 ([Pyr 1 ]-apelin-13) and Pyr-ELABELA-32 ([Pyr 1 ]-ELABELA-32) were synthesized as previously described 11 and provided by Dr. Boudreault's laboratory from the Institut de Pharmacologie de Sherbrooke. D-mannitol (M-120), sodium citrate (BP327.1) and citric acid (A940-500) were purchased from Fisher Scientific (Hampton, NH, USA).…”

“…Two separate group of diabetic mice received daily topical solution of Pyr-apelin-13 or Pyr-ELABELA-32 to the wounds for 14 days. Pyr-apelin-13 was used at the same concentration as in our previous study (7.05 μg/μl in PBS) 11 and Pyr-ELABELA-32 was used at an equimolar concentration (18.707 μg/μl in PBS). Interestingly, treatment with Pyr-apelin-13 or Pyr-ELABELA-32 significantly increased global wound closure in diabetic mice to 87% (P=0.0019) and 92% (P<0.0001,) respectively, compared to PBS-treated diabetic mice (Fig.…”