[Activation of the CaMKKβ/AMPK/HIF-1α signaling pathway promotes angiogenesis in endometriosis by regulating mitophagy]

OBJECTIVES: To investigate whether the CaMKKβ/AMPK/HIF-1α signaling pathway participates in angiogenesis in endometriosis by regulating mitophagy. METHODS: Human endometrial stromal cells (hEM15A) isolated from endometriosis patients and normal endometrial stromal cells (HESCs) from non-endometriosis women were tested for proliferation, invasion, migration and tube formation abilities using CCK-8 assay, scratch assay and in vitro angiogenesis test. The protein and mRNA expression levels of CaMKKβ, AMPK, HIF-1α, BNIP3, LC3, VEGF and CD31 in the cells were detected by Western blotting and qPCR. MitoTracker Green, MitoSOX Red fluorescent probes and immunofluorescence staining were used to observe mitochondrial morphology, mtROS level and BNIP3/LC3 co-localization. CaMKKβ agonists and inhibitors were used to verify the function of the CaMKKβ/AMPK/HIF-1α pathway. RESULTS: Compared with HESCs, hEM15A cells showed stronger proliferation, invasion, migration and angiogenesis abilities with significantly up-regulated expressions of CaMKKβ, AMPK, HIF-1α, BNIP3, LC3, VEGF and CD31. hEM15A cells showed increased number of mitochondria with intact structure, but the localization signals of BNIP3 and LC3 and mtROS level increased significantly. Mechanistically, inhibition of CaMKKβ significantly reduced expressions of AMPK, BNIP3, LC3, HIF-1α, VEGF and CD31, while activation of CaMKKβ increased expressions these proteins, down-regulated mitochondrial autophagy and inhibited angiogenesis. CONCLUSIONS: CaMKKβ/AMPK is abnormally activated in endometriosis to result in excessive accumulation of HIF-1α, which in turn increases the expression of its downstream target gene BNIP3, thus promoting mitophagy to trigger abnormal angiogenesis, which can be inhibited by inhibiting abnormal activation of CaMKKβ.