Arterial Baroreflex Dysfunction Impairs Ischemia-Induced Angiogenesis

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Arterial Baroreflex Dysfunction Impairs Ischemia-Induced Angiogenesis by Changning Hao, Zhen-Hao Huang, Shu-Wei Song, Yi-Qin Shi, Xian Wu Cheng, Toyoaki Murohara, Wei Lu, Ding-Feng Su, and Jun-Li Duan J Am Heart Assoc Volume 3(3):e000804 June 19, 2014 © 2014 Changning Hao et al.

Study protocol and sampling time points. Study protocol and sampling time points. HLI indicates hindlimb ischemia; Pyr, pyridostigmine; SAD, sinoaortic denervation. Changning Hao et al. J Am Heart Assoc 2014;3:e000804 © 2014 Changning Hao et al.

Hindlimb ischemia-induced angiogenesis in vivo. Hindlimb ischemia-induced angiogenesis in vivo. At day 14 after induction of ischemia, (A and B) infrared spectrum imaging assay, (C and D) anti-CD31 staining, (E) ELISA to the content of vascular endothelial growth factor (VEGF), and (F) Western blotting assay to expression of hypoxia-inducible factor 1 alpha (HIF-1α) in the ischemic area were performed to evaluate angiogenic actions. Infrared spectrum imaging data were analyzed and illustrated as an ischemic/normal hindlimb temperature ratio. Data of Western blotting are represented as fold of control. n=6 in each group. *P<0.05, **P<0.01; N.S., no significant difference; Tukey's post-hoc test. Temp, temperature. Scale bar: 50 μm. HPF indicates high power field; Pyr, pyridostigmine; SAD, sinoaortic denervation. Changning Hao et al. J Am Heart Assoc 2014;3:e000804 © 2014 Changning Hao et al.

Acetylcholine (ACh)-related protein expression in vivo. Acetylcholine (ACh)-related protein expression in vivo. Protein expressions of (A) α7-nicotinic acetylcholine receptor (α7-nAChR), (B) choline acetyltransferase (ChAT), and (C) vesicular acetylcholine transporter (VAChT) in the ischemic area were determined among 3 experimental groups. Data of Western blotting are represented as fold of control. n=6 in each group. *P<0.05; N.S., no significant difference; Tukey's post-hoc test. Pyr indicates pyridostigmine; SAD, sinoaortic denervation. Changning Hao et al. J Am Heart Assoc 2014;3:e000804 © 2014 Changning Hao et al.

Donepezil-mediated eACh activity in vitro. Donepezil-mediated eACh activity in vitro. After being cultured in serum-free EBM-2, HUVECs were treated with donepezil at a concentration of 10 μmol/L for 24 hours. (A and B) Results of ChAT and VAChT gene expressions determined with real-time PCR. (C and D) Results of ChAT and VAChT protein expressions determined with Western blotting. Data of real-time PCR and Western blotting are represented as fold of control. n=4 in each group. *P<0.05; **P<0.01; Student t test. (E and F) Representative images (×400 magnification) of anti-ChAT and -VAChT immunofluorescence. Scale bar: 50 μm. ChAT indicates choline acetyltransferase; eACh, endothelium-derived acetylcholine; EBM-2, endothelial basal medium 2; HUVECs, human umbilical vein endothelial cells; PCR, polymerase chain reaction; VAChT, vesicular acetylcholine transporter. Changning Hao et al. J Am Heart Assoc 2014;3:e000804 © 2014 Changning Hao et al.

Donepezil-mediated angiogenic effect in vitro. Donepezil-mediated angiogenic effect in vitro. (A) Quantitative analysis of HIF-1α expression in lysates of HUVECs treated with donepezil (10 μmol/L) and LY294002 (1 and 10 μmol/L; Tukey's post-hoc test). (B) HIF-1α expression in lysates of HUVECs when treated with nontargeting siRNA and siRNA HIF-1α (100 nmol/L; Student t test). (C) ELISA for VEGF in the supernatant of HUVECs when treated with donepezil (10 μmol/L) and HIF-1α (100 nmol/L; Tukey's post-hoc test). (D and E) Quantitative analysis of stromal derived factor 1 (SDF-1) and basic fibroblastic growth factor (bFGF) contained in the supernatant of HUVECs (Student t test). n=4 in each group. Data of real-time PCR and Western blotting are represented as folds of control. **P<0.01. HIF-1α indicates hypoxia-inducible factor 1 alpha; HUVECs, human umbilical vein endothelial cells; PCR, polymerase chain reaction; VEGFR, vascular endothelial growth factor receptor. Changning Hao et al. J Am Heart Assoc 2014;3:e000804 © 2014 Changning Hao et al.

Effects of carbachol on HUVECs Effects of carbachol on HUVECs. HUVECs were exposed to different concentration gradients of carbachol (0 to 100 μmol/L) for 24 hours. Effects of carbachol on HUVECs. HUVECs were exposed to different concentration gradients of carbachol (0 to 100 μmol/L) for 24 hours. (A) Concentrations of VEGF contained in the supernatant were measured by ELISA. After HUVECs were stimulated by carbachol (10 μmol/L), (B) levels of phosphorylation of VEGFR2 (p-VEGFR2; Tyr 996) were measured by Western blotting (**P<0.01 versus corresponding control; Tukey's post-hoc test). HUVECs were pretreated by atropine or methyllycaconitine (MLA) for 1 hour before carbachol (10 μmol/L) was added. Then, protein levels of (C) VEGF in supernatant and (D) p-VEGFR2 in cell lysates were quantified (**P<0.01; Tukey's post-hoc test). HUVECs were then exposed to carbachol (10 μmol/L) for 24 hours, and expression of (E) HIF-1α and (F) phospho-Akt (p-Akt; Ser473) were measured by Western blotting (**P<0.01 versus corresponding control; Tukey's post-hoc test). Data of Western blotting are represented as folds of control. n=4 in each group. HIF-1α indicates hypoxia-inducible factor 1 alpha; HUVECs, human umbilical vein endothelial cells; p-VEGFR2, phosphorylated vascular endothelial growth factor receptor 2. Changning Hao et al. J Am Heart Assoc 2014;3:e000804 © 2014 Changning Hao et al.

Pyridostigmine-mediated inhibition of acetylcholinesterase on angiogenesis. Pyridostigmine-mediated inhibition of acetylcholinesterase on angiogenesis. Using wild-type (WT) and α7-nAChR KO mice, unilateral hindlimb ischemia was surgically induced and fed by either pyridostigmine or saline to evaluate (A and B) the ischemic/normal limb temperature ratio, (C and D) the capillary density, and (E) levels of VEGF in ischemic muscles. n=6 in each group. **P<0.01; N.S., no significant difference; Tukey's post-hoc test. α7-nAChR indicates α7-nicotinic acetylcholine receptor; HPF, high power field; KO, knockout; Pyr, pyridostigmine; VEGFR, vascular endothelial growth factor receptor. Changning Hao et al. J Am Heart Assoc 2014;3:e000804 © 2014 Changning Hao et al.

Possible mechanisms of acetylcholinesterase inhibitor-mediated angiogenesis in the present study. Possible mechanisms of acetylcholinesterase inhibitor-mediated angiogenesis in the present study. ACh indicates acetylcholine; AChEI, acetylcholinesterase inhibitor; ChAT, choline acetyltransferase; HIF-1α, hypoxia-inducible factor 1 alpha; α7-nAChR, α7-nicotinic acetylcholine receptor; p-Akt, phosphor-Akt; VAChT, vesicular acetylcholine transporter; VEGFR, vascular endothelial growth factor receptor. Changning Hao et al. J Am Heart Assoc 2014;3:e000804 © 2014 Changning Hao et al.