Botulinum toxin type A induces protective autophagy in human dermal microvascular endothelial cells exposed to an in vitro model of ischemia/reperfusion injury

Abstract

Botulinum toxin type A (BTXA) has been reported to increase the survival of ischemic skin flaps; however, the exact mechanism underlying this effect remains unclear and needs to be further established. The present study aimed to elucidate whether autophagy caused by BTXA functions as a protection mechanism and to identify the mechanisms of its regulation by BTXA in human dermal microvascular endothelial cells (HDMECs) subjected to hypoxia/reoxygenation (H/R)-induced injury. HDMECs were harvested from the upper eyelid tissues of female blepharoplasty patients. HDMECs were exposed to BTXA treatment for 12 h and then subjected to hypoxia for 8 h, followed by reoxygenation for 24 h. Chloroquine diphosphate salt (CQ) was used as an autophagy inhibitor. H/R led to extreme injury to the HDMECs as indicated by the rise in the apoptosis rate, which was significantly attenuated by BTXA pretreatment. The outcomes demonstrated that H/R caused autophagy, as evidenced by a higher type II/type I ratio of light chain 3 (LC3), increased expression of Beclin-1 and increased autophagosome formation. BTXA enhanced autophagy and attenuated apoptosis in a dose-dependent manner, whereas CQ attenuated the BTXA antiapoptotic effects and inhibited the formation of autophagolysosomes, which caused clustering of the LC3-II in cells. In conclusion, autophagy promoted by BTXA serves as a potential protective effect on ischemia/reperfusion injury.

Keywords: autophagy; botulinum toxin type A; human dermal microvascular endothelial cells; ischemia/reperfusion injury.

Figure 1.

BTXA reduced apoptosis of human dermal microvascular endothelial cells in a dose-dependent manner during H/R. Apoptotic rates were determined by flow cytometry. Values are presented as the mean ± standard deviation. *P<0.01 vs. the control group; ^#P<0.01 vs. the H/R group. BTXA, botulinum toxin type A; H/R, hypoxia/reoxygenation.

Figure 2.

CQ, an autophagy inhibitor, attenuated the BTXA-induced antiapoptotic effects. CQ blocked the anti-apoptotic effect of BTXA following induction by H/R, as demonstrated by flow cytometry. Values are presented as the mean ± standard deviation. *P<0.01. CQ, chloroquine diphosphate salt; BTXA, botulinum toxin type A; H/R, hypoxia/reoxygenation.

Figure 3.

BTXA promoted autophagy in human dermal microvascular endothelial cells, whereas CQ inhibited autophagy. (A) Expression levels of LC3-II/LC3-I and Beclin-1 determined by western blot analysis. (B) Immunofluorescence staining of LC3-II (depicted as punctate dots). Values are presented as the mean ± standard deviation. Magnification, ×400. ^#P<0.01 vs. control group; *P<0.01 vs. H/R group; ^&P<0.01 vs. BTXA group. BTXA, botulinum toxin type A; CQ, chloroquine diphosphate salt; H/R, hypoxia/reoxygenation; LC3, light chain 3.

Figure 4.

Effect of BTXA on autophagosome formation and ultrastructure in H/R-treated human dermal microvascular endothelial cells by transmission electron microscopy. BTXA promoted the formation of autophagosomes to maintain intracellular homeostasis, while CQ reduced this effect. Black arrows indicate the autophagosomes, yellow arrows indicate the swollen mitochondria, and blue arrows indicate the dilated endoplasmic reticulum. Magnification, ×10. BTXA, botulinum toxin type A; H/R, hypoxia/reoxygenation; CQ, chloroquine diphosphate salt.