Hypoxia Stimulates Glucose Metabolism Of Eutopic And Ectopic Endometrial Stromal Cells From Women With Endometriosis And Its Pathological Significance

Background and Objectives:Endometriosis(EMS)is a prevalent gynecological disorder characterized by ectopic establishment of endometrium outside of the uterus cavity.Chronic pelvic pain and subfertility are often observed in endometriotic women.Endometrial cells with endometriosis may share multiple features with cancer such as invasion and relapse,so EMS is a benign disease with a malignant behavior.Despite intensive research efforts,endometriosis is still a challenge for treatment.Although genetic,endocrine and inflammatory are recognized as etiological factors,the molecular mechanisms and pathways of endometriosis remain ambiguous.Sampson’s retrograde theory is accepted by many investigators to explain the endometriotic pathology.According to this theory,shed endometrial tissues travel to the peritoneal cavity at the time of menstruation,thereby survive and form endometriotic lesions.It is reported that cancer have a unique feature called “Warburg Effect”,also known as “aerobic glycolysis” by Warburg eighty years ago.“Warburg effect”,is characterized by a switch in cell metabolism from mitochondrial glucose oxidation to cytoplasmic glycolysis.The shift in the mode of energy production may offer certain advantage to cell survival under some circumstances.For example,certain glycolysis enzymes have a role in anti-apoptosis effect.Lactate is able to promote invasion,metastasis,angiogenesis and immune escape.Reduced mitochondrial activity may also limit the mitochondrial-dependent apoptosis.Additionally,several cross-talk signaling pathways such as PI3K、AMPK pathways and cancer-related mutations including p53、PTEN、MYC、HIF are involved in the energy mechanism.Young et al.and our group found that endometriosis lesions underwent a change in glucose metabolism,which was consistent with Warburg Effect reported in tumor tissues.We cultured endometrial Stromal Cells(ESCs)in vitro to compare energy metabolism characters among different ESCs.The result was that ectopic ESCs had higher glucose consumption and lactate production than normal controls and eutopic ESCs.Why and what is the meaning of this phenomenon?Recent studies have revealed that hypoxia and hypoxia-inducible factor-1(HIF-1)appear to play a pivotal role in the pathogenesis of endometriosis.HIF-1,composed of HIF-1α and HIF-1β,is an important mediator for cellular responses to low oxygen level.HIF-1α normally degrades under nomoxia,but is activated and stabilized in hypoxia condition.HIF-1β appears to remain stable in the cells.HIF-1α regulates apoptosis,proliferation and angiogenesis.HIF-1α induces the expression of several enzymes during glucose metabolism,such as hexokinase 2(HK2),and pyruvate dehydrogenase kinase(PDK).HIF-1α-induced PDK1 expression will block tricarboxylic acid(TCA)cycle.The increased glycolysis will produce more pyruvate which will be turned into lactate by HIF-1α-induced lactic dehydrogenase A(LDHA).It is found that HIF-1α accumulated and transferred into the nuclear to constitute a heterodimer with HIF-1β under hypoxia.HIF-1 would then combine with Hypoxia response element(HRE)in the target gene promoter sequences and lead to a target gene transcription eventually.In vivo studies demonstrated that exposure to low oxygen could stimulate the angiogenesis and the growth of endometriosis lesions.The level of HIF-1α protein is higher in the endometriosis lesion compared to the eutopic tissue.Therefore,we supposed that the expression of HIF-1 elevated in the ectopic lesions in the abdominal cavity,which caused aerobic glycolysis of ESCs and thus leading to the development of EMS by altering the biological behavior of ESCs.To prove the above research hypothesis and further study on the “aerobic glycolysis” of EMS,in vitro ectopic,eutopic and normal ESCs were cultured as a model to investigate the role of hypoxia in “aerobic glycolysis” and both of their functions in the development of EMS.Material and Methods:(1)An in vitro study was conducted in ESCs isolated from endometrium of women with(EU)or without endometriosis(NE)as well as from endometrioma tissues(EC).Glucose consumption and lactate generation were determined by glucose and lactate kit.Meanwhile,protein expression of glucose metabolism related enzymes among NE,EC and EU by western blot analysis at different oxygen environment.(2)Mitochondrial membrane potential(MMP),adenosine triphosphate(ATP)production and mitochondrial morphology were measured to assess mitochondrial function among NE,EU and EC at different oxygen environment.(3)Cell apoptosis by a flow cytometer and invasion activities by transwell invasion assay were examined among NE,EU and EC as biological behavior at different oxygen environment.(4)Statistical analysis: Data were analyzed using SPSS software(Version 20.0).Quantitative data were expressed as mean ± standard error(Mean ± SD).One-way ANOVA was applied to determine the difference of means among groups.The differences between two groups were compared based on the multiple comparison.P<0.05 were considered to be statistically significant.Results:(1)EC consumed and produced more glucose and lactate in nomoxia than NE and EU.But upon exposure to low oxygen,the glucose metabolism of EU was mobilized vigorously.(2)In normal oxygen condition,the expression of glycolysis-related enzymes including HIF1α,HK2,PDK1 and LDHA was higher in EC than NE and EU.However,in hypoxia,the expression of glycolysis related enzymes of NE and EU increased.(3)Compared to NE and EU,the MMP of EC was the lowest,when placed in low oxygen,however,MMP of the three types of stromal cells decreased quickly.(4)Compared with NE and EU,the mitochondrion of EC was smaller and increased in number,and the cristae became vague.Occasionally two nucleoli were observed in a single nucleus.Particularly,mitochondrial cristae became obscure in NE and EU stromal cells under hypoxia.(5)The generation of ATP of NE,EU and EC stromal cells was more in reduced-oxygen than normoxia.The increase of ATP in EC stromal cells significantly exceeded that in NE and EU stromal cells under hypoxia condition.(6)EU showed the highest invasion potential compared to NE and EC in normoxia,Moreover,invasive ability of the three types of cells was significantly enhanced in hypoxia.While compared to NE and EC,the apoptosis rate of EU stromal cells was the lowest under both normoxia and hypoxia conditions.Conclusion:(1)EC showed higher glucose consumption and lactate production than the other two groups,which proved the aberrant energy metabolism existed in EMS.(2)Upon exposure to low oxygen,the glucose metabolism of EU and EC increased,which indicated hypoxia and glucose metabolism may have a vital role in the pathogenesis of EMS.(3)EU appeared to exhibit the stronger invasion ability and lower apoptosis rate compared to NE,implying EU is better adapted to hypoxia.(4)Further investigation needed to be done to study the role of hypoxia and glucose metabolism in EMS.