|Year : 2019 | Volume
| Issue : 2 | Page : 69-76
Activated platelets induce hypoxia-inducible factor-1α expression likely through transforming growth factor-β1 in human endometrial stromal cells
Qiu-Ming Qi1, Sun-Wei Guo2, Xi-Shi Liu2
1 Department of Gynecology, Shanghai Obstetrics and Gynecology Hospital, Fudan University, Shanghai 200011, China
2 Department of Gynecology, Shanghai Obstetrics and Gynecology Hospital; Shanghai Key Laboratory of Female Reproductive Endocrine-Related Diseases, Fudan University, Shanghai 200011, China
|Date of Submission||08-Apr-2019|
|Date of Web Publication||9-Jul-2019|
Prof. Xi-Shi Liu
Department of Gynecology, Shanghai Obstetrics and Gynecology Hospital, Fudan University, 419 Fangxie Road, Shanghai 200011
Source of Support: None, Conflict of Interest: None
Objective: Endometriosis is a common gynecological disease with an enigmatic pathogenesis. Recent studies suggest that the behavior of normal endometrial stromal cells can dramatically change under hypoxic conditions, which effectively turns them into endometriotic stromal cells. Because menstrual debris is not only hypoxic but may also contain platelet aggregates, at present, we aimed to approve that activated platelets could induce hypoxia-inducible factor-1α (HIF-1α) expression in endometrial stromal cells, signaling the presence of hypoxia.
Methods: We evaluated the gene and protein expression levels of HIF-1α and its target gene erythropoietin (EPO) in both human endometriotic stromal cells (HESCs) and a human endometrial stromal cell line (ESCL) cocultured with or without activated platelets for 48 h.
Results: We found that the gene and protein expression levels of HIF-1α and EPO in both HESC and ESCL were significantly increased after coculture with activated platelets. We also found that neutralization of transforming growth factor-β1 completely abolishes this induction.
Conclusions: Platelets can induce a hypoxic state in endometrial and endometriotic stromal cells, resulting in increased angiogenesis, as well as enhanced survival and proliferation. In conjunction with other roles that platelets play in the development of endometriosis, our findings further highlight the important roles of platelets in the development and initiation of endometriosis, shedding new light into the etiology of endometriosis.
Keywords: Endometriosis; Hypoxia, Hypoxia-inducible Factor-1α; Platelet, Stromal Cell
|How to cite this article:|
Qi QM, Guo SW, Liu XS. Activated platelets induce hypoxia-inducible factor-1α expression likely through transforming growth factor-β1 in human endometrial stromal cells. Reprod Dev Med 2019;3:69-76
|How to cite this URL:|
Qi QM, Guo SW, Liu XS. Activated platelets induce hypoxia-inducible factor-1α expression likely through transforming growth factor-β1 in human endometrial stromal cells. Reprod Dev Med [serial online] 2019 [cited 2019 Jul 15];3:69-76. Available from: http://www.repdevmed.org/text.asp?2019/3/2/69/262390
| Introduction|| |
Endometriosis is defined as the presence of endometrial tissue outside of the uterine cavity and is a common gynecological disease that affects approximately 10% women of reproductive age and involves pelvic pain and infertility as its clinical manifestations. Despite extensive research, its pathogenesis remains elusive. One widely accepted theory is the retrograde menstruation theory proposed by Sampson, which stipulates that, when viable endometrial cells are regurgitated into the pelvic cavity through the Fallopian tube More Detailss, they somehow invade the peritoneum and establish the initial lesions. However, the ectopic and eutopic endometria are known to be transcriptionally different, and the difference is presumably attributed to their different microenvironments, the key factor that determines the difference is still unclear.
Recently, it has been shown that hypoxia plays a critical role in the survival and angiogenesis of ectopic endometrial cells regurgitated from the uterus. Remarkably, hypoxic conditions alone dramatically change the behavior of endometrial cells that are otherwise “normal.”
One hallmark of hypoxia is the overexpression of the transcription factor hypoxia-inducible factor-1α (HIF-1α), a key mediator of cellular adaptation to low oxygen levels and a potential target for many types of cancer, such as ovarian, prostate, and breast; HIF-1α expression reflects the degree of cell hypoxia; its increased expression has been reported in most neoplasms; and it is responsible for increased angiogenesis in cancers. Similar to that in cancer, HIF-1α has also been shown to be overexpressed in endometriosis, reflecting the increasing need for nutrients and oxygen as endometriotic lesions grow. Conceivably, the menstrual debris, once regurgitated from the uterus to the peritoneal cavity as depicted in Sampson's hypothesis, would experience hypoxia due to a loss of blood supply.
Under hypoxic conditions, HIF-1α is stabilized and binds to core hypoxia response elements (HREs). This results in the transcriptional activation of hypoxia-regulated genes, including erythropoietin (EPO), vascular endothelial growth factor (VEGF), and cyclooxygenase-2 (COX-2),, which are genes known to promote angiogenesis, cellular proliferation, and the production of pro-inflammatory cytokines/chemokines. Under hypoxic stress, several events such as steroidogenesis, angiogenesis, and epigenetic modulation take place and effectively turn normal endometrial stromal cells into endometriotic stroma-like cells mainly through the induction of HIF-1α., This may explain why endometrial debris invades and establish endometriotic foci in ectopic sites.
In the last few years, growing evidence has suggested that endometriotic lesions are essentially wounds undergoing repeated tissue injury and repair,,,, and many nonendometriotic cells in the lesional microenvironment, such as platelets,, macrophages,, natural killer cells,, and nerve fibers,, are involved in facilitating the development of endometriosis. Because platelets are the first responders to injury, menstrual debris could be shrouded with activated platelets and thus activate HIF-1α expression. Thus, we hypothesized that activated platelets might induce HIF-1α expression in endometrial stromal cells, signaling the presence of hypoxia.
| Methods|| |
Patients and specimens
Endometriotic tissue samples were obtained after informed consent from 16 premenopausal patients (mean age = 30.7 ± 5.3 years) with histologically diagnosed ovarian endometriomas admitted to the Shanghai Obstetrics and Gynecology Hospital, Fudan University, from May to September 2015. All endometriotic tissue samples were used for primary culture of ectopic endometrial stromal cells (HESCs). Among these endometriotic tissue samples, eight were used for real-time polymerase chain reaction (PCR) analysis and the remaining eight were used for Western blot analysis. This study was approved by the Institutional Ethics Review Board of the Shanghai Obstetrics and Gynecology Hospital (Kyy2015-34).
The human endometrial stromal cell line (ESCL), established by Dr. Krikun et al., was kindly provided by Dr. Asgi Fazleabas of Michigan State University, Michigan, USA. The cells were cultured in Dulbecco's modified Eagle's medium/Ham's F-12 medium (DMEM/F-12, Hyclone) supplemented with 5% fetal bovine serum (FBS) and 1% antibiotics (100 IU/mL penicillin G, 100 mg/mL streptomycin, and 2.5 μg/mL amphotericin B).
Endometriosis-derived primary ectopic endometrial stromal cells (HESCs) were isolated and cultured as reported previously. Briefly, after washing with DMEM/F-12 medium supplemented with 5% FBS and 1% antibiotics, the tissue samples were minced into small pieces ~1 mm3 in size. Then, the minced tissues were enzymatically digested with 0.2% collagenase II (Sigma, St Louis, MO, USA) in a shaking bed for 1.5 h at 37°C. After that, they were separated by filtration through a 149-μm and a 37-μm (pore size) nylon mesh. The stromal cells remaining in the filtrate were collected by centrifugation, resuspended in DMEM/F12 (reconstituted with 10% FBS and 1% antibiotics), seeded into 25-cm2 cell culture flasks, and incubated at 37°C in a humidified atmosphere of 5% CO2 in air. Antibodies against vimentin (Abcam, Cambridge, UK), cytokeratin 7 (CK-7, Abcam), and follicle-stimulating hormone receptor (FSHR, Abcam) were used for immunocytochemistry to verify the purity and homogeneity of the stromal cell preparation (≥98%) after 3–4 passages.
Preparation of platelets
Platelets were isolated by centrifugation at room temperature of whole blood samples donated by healthy male volunteers who provided informed consent and did not take any medications for at least 2 weeks prior to donation as reported previously. The blood was first centrifuged at 150 ×g for 10 min, the supernatant platelet-rich plasma (PRP) was harvested and centrifuged at 300 ×g for 5 min, and finally the supernatant PRP was harvested again and centrifuged at 1000 ×g for 14 min. Finally, the deposited platelets were suspended in DMEM/F12 culture medium for subsequent experiments. We obtained about 2 × 108 platelets from 20 mL of peripheral blood samples.
Treatment of human endometriotic stromal cell/endometrial stromal cell line
HESC and ESCL were added to the serum-free DMEM/F-12 medium at 80% confluence and were starved for 24 h. Then, they were cocultured with 3.5 mL of different treatment media as follows: control (standard DMEM/F-12 medium), platelets (standard medium containing ~107 human platelets), activated platelets (standard medium containing ~107 human platelets and human thrombin [1.49 NIH U, Sigma, St. Louis, MO, USA]), and thrombin alone (standard medium containing human thrombin 1.49 NIH U).
To see whether transforming growth factor-β (TGF-β) neutralization can abolish the overexpression of HIF-1α induced by activated platelets in HESC and ESCL, the cells were pretreated for 2 h with 1 μmol/L of A83-01 compound (Santa Cruz, CA, USA), which is a TGF-β receptor inhibitor, and then incubated with activated platelets plus 1 μmol/L A83-01 as described in. After coculture for 48 h, all cells were harvested for real-time PCR and Western blot analysis. To ensure that the Western blot results were obtained from stromal cells but not platelets, we washed the HESCs and ESCL cells cocultured with platelets with sterile phosphate-buffered saline (PBS) three times to remove the platelets as reported previously.
RNA isolation and real-time polymerase chain reaction
TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was used to isolate total RNA from HESC and ESCL, and a reverse transcription kit (Takara, Otsu, Japan) was used for cDNA synthesis. Gene expression levels were evaluated by real-time PCR using SYBR Premix Ex Taq (Takara, Otsu, Japan). The oligonucleotide primer sequences, synthesized by Sangon Corporation (Shanghai, China), were as follows: HIF-1α: 5′ GTC GAC ACA GCC TGG ATA TGA A 3′ (forward) and 5′ CAT ATC ATG ATG AGT TTT GGT CAG ATG 3′ (reverse). EPO:5′ CAC CAC GCC TCA TCT GTG AC 3′ (forward) and 5′ CAC AGC CCG TCG TGA TAT TCT 3′ (reverse). GAPDH: 5′ TGC ACC ACC AAC TGC TTA G 3′ (forward) and 5′ GAT GCA GGG ATG ATG TTC 3′ (reverse). Melting curves of the products were obtained after cycling by a stepwise increase of temperature from 55°C to 95°C. After 40 cycles, the reaction products were separated electrophoretically on an agarose gel and stained with ethidium bromide for visual confirmation of the PCR products. Expression values were normalized to the geometric mean of GAPDH measurements, and the quantification of mRNA abundance was made using the method described previously.
Western blot analysis
Total protein was harvested from HESCs and ESCL cells by scraping and extracting using the commercial RIPA buffer (Thermo, Waltham, MA, USA). The protein concentration, after coculture with platelets, activated platelets, or PBS for 48 h, was determined using BCA protein quantitative analysis kit (P0010S, Beyotime, Shanghai, China). All proteins mixed with SDS-PAGE loading buffer (P0015, Beyotime) were heated for 10 min at 95°C for denaturation. The protein samples were loaded on a 10% SDS-PAGE and subsequently electroblotted onto PVDF membranes (Bio-Rad, Hercules, California, USA). After blocking in Western blocking buffer (P0023B, Beyotime) for 1 h at room temperature, the membranes were subsequently incubated at 4°C overnight with the following primary antibodies: HIF-1α (1:1,000, Abcam), EPO (1:1,000, Abcam), GAPDH (1:1,000, Cell Signaling Technology, MO, USA), and β-actin (1:1,000, Cell Signaling Technology). After the membranes were incubated with HRP-labeled secondary antibodies for 1 h at room temperature, the signal was detected using ECL (Pierce, Thermo Scientific, Rockford, IL, USA) on Image Quant LAS 4000 mini (GE Healthcare). The amount of protein was quantified by Quantity One software (Bio-Rad) and normalized to GAPDH or β-actin levels, which served as loading controls.
To compare gene and protein expression levels between cells with different treatments, Wilcoxon's test was used. P ≤ 0.05 was considered statistically significant. All calculations were carried out using SPSS, version 16.0 (SPSS Inc., Chicago, IL, USA).
| Results|| |
Activated platelets elevate the gene and protein expression levels of HIF-1α and erythropoietin in human endometriotic stromal cell and endometrial stromal cell line
The gene expression levels of HIF-1α were significantly increased in HESCs cocultured with platelets [P = 0.05, [Figure 1]a and with activated platelets (P = 0.017) as compared with those in the cells cocultured with PBS. Consistently, the protein expression levels of HIF-1α in HESCs cocultured with platelets and activated platelets were similarly elevated as compared with those in the controls [P = 0.017 and P = 0.012, respectively, [Figure 1]b and [Figure 1]c.
|Figure 1: Activated platelets induce the gene and protein expression levels of HIF-1α and EPO in HESC. (1) Fold change of the mRNA (a) and protein expression levels (b) of HIF-1α in HESC cocultured with PBS, platelets, activated platelets, and thrombin alone. (c) Representative Western blot results for HIF-1α protein expression. (2) Fold change of the mRNA (d) and protein expression levels (e) of EPO in HESC cocultured with PBS, platelets, activated platelets, and thrombin alone. (f) Representative Western blot results for EPO protein expression. In all experiments, n= 8. “*” denotes that the P-value of the difference with the PBS group is <0.05. PLT: Platelets, APLT: Activated platelets, TBMB: Thrombin; HIF-1α: Hypoxia-inducible factor-1α; EPO: Erythropoietin; ESCL: Endometrial stromal cell line; HESC: Human endometriotic stromal cell.|
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Next, we evaluated the gene and protein expression levels of EPO, a HIF-1α target gene. Consistently, the gene expression levels of EPO were significantly increased in HESCs cocultured with platelets and activated platelets [Figure 1]d, both P = 0.012] as compared with those in the control group; the same results were observed with the protein expression levels [Figure 1]e and [Figure 1]f, both P = 0.012].
We also evaluated the gene and protein expression levels of HIF-1α and EPO in ESCL. We found that the gene expression levels of HIF-1α were significantly increased when cocultured with platelets and activated platelets [both P's = 0.012, [Figure 2]a as compared with those in the cells cocultured with PBS. Interestingly, the gene expression levels of EPO in ESCL were significantly increased after coculture with activated platelets [P = 0.043, [Figure 2]d, but not with platelets alone [P = 0.69]. The protein expression levels of HIF-1α [both P's = 0.012, [Figure 2]b and [Figure 2]c and EPO [both P's = 0.043, [Figure 2]e and [Figure 2]f were significantly increased after coculture with platelets and with activated platelets.
|Figure 2: Activated platelets increased the gene and protein expression levels of HIF-1α and EPO in ESCL cells. (1) Fold change of the mRNA (a) and protein expression levels (b) of HIF-1α in ESCL cocultured with PBS, platelets, activated platelets, and thrombin alone. (c) Representative Western blot results for HIF-1α protein expression. (2) Fold change of mRNA (d) and protein expression levels (e) of EPO in ESCL cocultured with PBS, platelets, activated platelets, and thrombin alone. (f) Representative Western blot results for EPO protein expression. In all experiments, n= 5. “*” denotes that the P-value of the difference with the PBS group is <0.05. PLT: Platelets; APLT: Activated platelets; TBMB: Thrombin; HIF-1α: Hypoxia-inducible factor-1α; EPO: Erythropoietin; ESCL: Endometrial stromal cell line; PBS: Phosphate buffer saline.|
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Neutralization of TGF-β1 signaling completely abolishes activated platelet-induced expression of HIF-1α and erythropoietin in both human endometriotic stromal cell and endometrial stromal cell line
Activated platelets have been shown to induce the TGF-β1/Smad3 signaling pathway in endometrial and endometriotic stromal cells. Thus, we wondered whether the induction of HIF-1α is through the TGF-β1/Smad3 signaling pathway.
To study this, HESCs were preincubated with A83-01, and the gene and protein expression levels of HIF-1α and EPO were evaluated by real-time PCR and Western blot after coculture with activated platelets. We found that, as previously reported, the gene expression levels of both HIF-1α and EPO were significantly elevated [Figure 3]a and [Figure 3]d, P = 0.012 and 0.017, respectively] after coculture with activated platelets, and that TGF-β1 neutralization by A83-01 completely abolished the elevation [P = 0.012 and 0.025, respectively]. Similarly, after coculture with activated platelets, the protein expression levels of both HIF-1α and EPO were significantly increased [Figure 3]b, [Figure 3]c, [Figure 3]e and [Figure 3]f, both P's = 0.012], but TGF-β1 neutralization abrogated the increase in protein expression levels (both P's = 0.012).
|Figure 3: Neutralization of TGF-β1 signaling completely abolishes the expression of HIF-1α and EPO induced by activated platelets in HESC. (1) Fold change of the mRNA (a) and protein expression (b) levels of HIF-1α in HESC cocultured with a vehicle, activated platelets, and activated platelets plus A83-01. (c) Representative Western blot results for HIF-1α protein expression. (2) Fold change of the mRNA (d) and protein (e) expression levels of EPO in HESC cocultured with a vehicle, activated platelets, and activated platelets plus A83-01. (f) Representative Western blot results for EPO protein expression. In all experiments, n = 8. “*” denotes that the P-value of the difference with the vehicle group is <0.05. APLT: Activated platelets; APLT + A83-01: Activated platelets plus A83-01; HIF-1α: Hypoxia-inducible factor-1α; EPO: Erythropoietin; ESCL: Endometrial stromal cell line; TGF-β: Transforming growth factor-β; HESC: Human endometriotic stromal cell.|
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As with HESCs, nearly identical results were obtained for ESCL cells: the elevated gene [Figure 4]a and [Figure 4]d, both P's = 0.043] and protein expression levels [Figure 4]b, [Figure 4]c, [Figure 4]e, and [Figure 4]f, both P's = 0.043] of HIF-1α and EPO induced by activated platelets were completely abolished by TGF-β1 neutralization.
|Figure 4: Neutralization of TGF-β1 signaling completely abolishes the expression of HIF-1α and EPO induced by activated platelets in ESCL. (1) Fold change of the mRNA (a) and protein expression (b) levels of HIF-1α in ESCL cocultured with a vehicle, activated platelets, and activated platelets plus A83-01. (c) Representative Western blot results for HIF-1α protein expression. (2) Fold change of the mRNA (d) and protein (e) expression levels of EPO in ESCL cocultured with a vehicle, activated platelets, and activated platelets plus A83-01. (f) Representative Western blot results for EPO protein expression. In all experiments, n= 5. “*” denotes that the P-value of the difference with the vehicle group is <0.05. APLT: Activated platelets; A.PLT + A83-01: Activated platelets plus A83-01; HIF-1α: Hypoxia-inducible factor-1α; EPO: Erythropoietin; ESCL: Endometrial stromal cell line; TGF-β: Transforming growth factor-β.|
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| Discussion|| |
Hypoxia is a critical mediator of endothelial growth factors and signaling proteins during periods of metabolic stress and vascular remodeling like angiogenesis or regression, most of which are mediated via transcriptional regulation by HIF. HIF consists of two subunits, α and β, which form a heterodimeric complex. HIF-1β is present under both normoxia and hypoxia, while HIF-1α is present only under hypoxic conditions. As a transcription factor, HIF-1α regulates the activities of its downstream genes by binding the HREs in their promoter regions. For example, COX-2 expression is regulated by HIF-1α transcriptional activity. HIF-1α-induced COX-2 expression leads to elevated prostaglandin E2(PGE2) levels, which can induce PGE2-mediated vascularization., VEGF, a major driver of angiogenesis in cancer and in endometriosis, is also transcriptionally regulated by HIF-1α under hypoxic conditions. HIF-1α binds to the HRE regions of the VEGF promoter and is positively correlated with VEGF expression in endometriosis. Tissue hypoxia is the main stimulus of EPO production. HIF-1α upregulates the expression of EPO, which mediates its effects by binding to the EPO receptor (EPOR). Hypoxia results in the increased production of EPO via the induction of the HIF-1 pathway. The EPO gene is under the direct control of hypoxia through HIF-1α, which then binds to a cis-acting DNA site in the HRE of the EPO gene promoter., EPO, a widely known growth factor in erythropoiesis, can also stimulate angiogenesis, as well as tumor cell proliferation and survival. The expression levels of EPO, VEGF, and COX-2 have all been reported to be elevated in endometriosis, resulting in angiogenesis, proliferation, and enhanced survival in lesional development., We have previously shown that activated platelets can activate VEGF and COX-2 expression in HESCs; in this study, we found that activated platelets also induce the activation of HIF-1α and the expression of its target gene EPO in HESCs and ESCL cells. Thus, the upregulation of VEGF and COX-2 induced by activated platelets in endometriotic stromal cells was probably via HIF-1α induction, which occurs through the TGF-β1/Smad3 signaling pathway.
In this study, we investigated the gene and protein expression levels of HIF-1α in primary endometriotic stromal cells cocultured with platelets and demonstrated that platelets could induce HIF-1α expression, signaling the state of hypoxia. The upregulation of some downstream genes of HIF-1α, such as COX-2 and VEGF, induced by activated platelets have been reported previously. Activated platelets was able to also induce HIF-1α, as well as its downstream target gene EPO, expression in ESCL, indicating that activated platelets can effectively induce a hypoxic state in both endometrial and endometriotic stromal cells. However, TGF-β1 neutralization by A83-01 abrogates HIF-1α and EPO overexpression induced by activated platelets, suggesting that the induction of HIF-1α by activated platelets in both endometrial and endometriotic stromal cells probably occurs through the TGF-β1 signaling pathway.
Hypoxia is an important factor that regulates numerous physiological and pathological processes. It has been reported that HIF-1α is overexpressed in endometriotic stromal cells, resulting in increased cellular proliferation in an autocrine fashion.,, Our data demonstrate that activated platelets could induce hypoxia in the microenvironment of endometriotic stromal cells through the elevated expression of HIF-1α, resulting in cellular proliferation, angiogenesis, and lesional development.
The increased TGF-β1 expression in endometriosis has been well documented.,,, In a gene profiling study on a mouse model, TGF-β1 has been identified to play important roles in the gene network involved in the pathogenesis of endometriosis. It has also been reported to induce a Warburg-like metabolic reprogramming in peritoneal mesothelial cells, potentially facilitating the development of peritoneal endometriosis. We have shown previously that activated platelets, through the release of TGF-β1 and the induction of the TGF-β1/Smad3 signaling pathway, promote lesional development, and that TGF-β1 blockade reverses the development.
It has been reported that TGF-β1 promoter activity is regulated by hypoxia, and that HIF-1α could directly regulate TGF-β1 expression through the HRE, which is located in the TGF-β1 proximal promoter. On the other hand, exposure to TGF-β1 could increase HIF-1α gene and protein expression in many cell types.,, In this study, we found that neutralization of TGF-β1 completely abolished the HIF-1α induction by activated platelets in both HESC and ESCL, highlighting the important role of platelets in the initiation and development of endometriotic lesions.
We note that HIF-1α has also been reported to promote epithelial–mesenchymal transition (EMT) in several types of cancer by modulating one or more EMT-associated genes. Moreover, in this study, we demonstrated that TGF-β1 neutralization completely abolishes platelet-induced HIF-1α expression in endometriotic cells. However, more research is warranted to elucidate how TGF-β1 signaling can modulate platelet-induced HIF-1α expression in endometriosis.
Our data have shown that activated platelets can also turn normal endometrial stromal cells into a hypoxic phenotype, similar to that in HESCs. The results are consistent with the finding that activation of HIF-1α and consequent hypoxia can dramatically change the behavior of human endometrial stromal cells, resulting in completely different phenotypes. This is consistent with the report that both endometriotic and endometrial stromal cells have elevated expression of myofibroblast markers after stimulation with TGF-β1. While it may seem logical that the TGF-β1/Smad3 pathway is a therapeutic target for endometriosis, it is worth noting that this pathway also plays many important physiological functions in the endometrium. In other words, targeting this pathway directly may cause unintended collateral damage to normal tissues/organs. As shown in this study and in others,, a better, alternative therapeutic approach may be to target the coagulation pathways.
One notable limitation of this study is due to the in vitro nature of this study, which evaluated the results of cell culture only. Future in vivo studies are needed to validate our findings. However, because endometriotic stromal cells and platelets used in this study were all derived from humans, and both platelet aggregation and hypoxia are now well documented in endometriosis,,,,,,, we believe that platelets must play a role in hypoxia in endometriosis.
In summary, we have shown here that platelets play a critical role in driving hypoxia through the upregulation of HIF-1α and its downstream genes, facilitating the development of endometriosis. In addition, neutralization of TGF-β1 completely abolishes the activated platelet-induced expression of HIF-1α and its target gene EPO. These findings highlight the importance of platelets in the development, and perhaps as well as the initiation, of endometriosis. These results, in conjunction with other reports on the roles of platelets in driving lesional development, underscore the possibility of using anticoagulation therapy in the nonhormonal treatment of endometriosis, as well as hold promise for the development of novel biomarkers for endometriosis.
Financial support and sponsorship
This research was supported in part by grants 81530040 (SWG), 81771553 (SWG), 81671436 (XSL), and 81871144 (XSL) from the National Science Foundation of China and a grant for Shanghai Medical Center for Female Reproductive Disease (2017ZZ01016) from the Science and Technology Commission of Shanghai Municipality.
Conflicts of interest
There are no conflicts of interest.
| References|| |
Giudice LC, Kao LC. Endometriosis. Lancet 2004;364:1789-99. doi: 10.1016/S0140-6736(04)17403-5.
Guo SW. An overview of the current status of clinical trials on endometriosis: Issues and concerns. Fertil Steril 2014;101:183-90.e4. doi: 10.1016/j.fertnstert.2013.08.050.
Sampson JA. Metastatic or embolic endometriosis, due to the menstrual dissemination of endometrial tissue into the venous circulation. Am J Pathol 1927;3:93-110.43. doi: 10.1007/BF01995231
Wu Y, Kajdacsy-Balla A, Strawn E, Basir Z, Halverson G, Jailwala P, et al.
Transcriptional characterizations of differences between eutopic and ectopic endometrium. Endocrinology 2006;147:232-46. doi: 10.1210/en.2005-0426.
Hsiao KY, Lin SC, Wu MH, Tsai SJ. Pathological functions of hypoxia in endometriosis. Front Biosci (Elite Ed) 2015;7:309-21. doi: 10.2741/e736.
Semenza GL. HIF-1 and tumor progression: Pathophysiology and therapeutics. Trends Mol Med 2002;8:S62-7. doi: 10.1016/S1471-4914(02)02317-1.
Badowska-Kozakiewicz AM, Budzik MP, Przybylski J. Hypoxia in breast cancer. Pol J Pathol 2015;66:337-46. doi: 10.5114/pjp.2015.57245.
Zhan L, Wang W, Zhang Y, Song E, Fan Y, Wei B. Hypoxia-inducible factor-1alpha: A promising therapeutic target in endometriosis. Biochimie 2016;123:130-7. doi: 10.1016/j.biochi.2016.01.006.
Wu MH, Hsiao KY, Tsai SJ. Hypoxia: The force of endometriosis. J Obstet Gynaecol Res 2019;45:532-41. doi: 10.1111/jog.13900.
Badowska-Kozakiewicz A, Sobol M, Patera J. Expression of hypoxia-inducible factor 1α in invasive breast cancer with metastasis to lymph nodes: Correlation with steroid receptors, HER2 and EPO-R. Adv Clin Exp Med 2016;25:741-50. doi: 10.17219/acem/63143.
Nagaraju GP, Bramhachari PV, Raghu G, El-Rayes BF. Hypoxia inducible factor-1α: Its role in colorectal carcinogenesis and metastasis. Cancer Lett 2015;366:11-8. doi: 10.1016/j.canlet.2015.06.005.
Lin SC, Lee HC, Hou PC, Fu JL, Wu MH, Tsai SJ. Targeting hypoxia-mediated YAP1 nuclear translocation ameliorates pathogenesis of endometriosis without compromising maternal fertility. J Pathol 2017;242:476-87. doi: 10.1002/path.4922.
Duan J, Liu X, Wang H, Guo SW. The M2a macrophage subset may be critically involved in the fibrogenesis of endometriosis in mice. Reprod Biomed Online 2018;37:254-68. doi: 10.1016/j.rbmo.2018.05.017.
Guo SW, Ding D, Shen M, Liu X. Dating endometriotic ovarian cysts based on the content of cyst fluid and its potential clinical implications. Reprod Sci 2015;22:873-83. doi: 10.1177/1933719115570907.
Zhang Q, Duan J, Liu X, Guo SW. Platelets drive smooth muscle metaplasia and fibrogenesis in endometriosis through epithelial-mesenchymal transition and fibroblast-to-myofibroblast transdifferentiation. Mol Cell Endocrinol 2016;428:1-6. doi: 10.1016/j.mce.2016.03.015.
Zhang Q, Duan J, Olson M, Fazleabas A, Guo SW. Cellular changes consistent with epithelial-mesenchymal transition and fibroblast-to-myofibroblast transdifferentiation in the progression of experimental endometriosis in Baboons. Reprod Sci 2016;23:1409-21. doi: 10.1177/1933719116641763.
Ding D, Liu X, Duan J, Guo SW. Platelets are an unindicted culprit in the development of endometriosis: Clinical and experimental evidence. Hum Reprod 2015;30:812-32. doi: 10.1093/humrep/dev025.
Bacci M, Capobianco A, Monno A, Cottone L, Di Puppo F, Camisa B, et al.
Macrophages are alternatively activated in patients with endometriosis and required for growth and vascularization of lesions in a mouse model of disease. Am J Pathol 2009;175:547-56. doi: 10.2353/ajpath.2009.081011.
Guo SW, Du Y, Liu X. Endometriosis-derived stromal cells secrete thrombin and thromboxane A2, inducing platelet activation. Reprod Sci 2016;23:1044-52. doi: 10.1177/1933719116630428.
Du Y, Liu X, Guo SW. Platelets impair natural killer cell reactivity and function in endometriosis through multiple mechanisms. Hum Reprod 2017;32:794-810. doi: 10.1093/humrep/dex014.
Liu X, Yan D, Guo SW. Sensory nerve-derived neuropeptides accelerate the development and fibrogenesis of endometriosis. Hum Reprod 2019;34:452-68. doi: 10.1093/humrep/dey392.
Yan D, Liu X, Guo SW. Neuropeptides substance P
and calcitonin gene related peptide accelerate the development and fibrogenesis of endometriosis. Sci Rep 2019;9:2698. doi: 10.1038/s41598-019-39170-w.
Krikun G, Mor G, Alvero A, Guller S, Schatz F, Sapi E, et al.
A novel immortalized human endometrial stromal cell line with normal progestational response. Endocrinology 2004;145:2291-6. doi: 10.1210/en.2003-1606.
Zhang Q, Ding D, Liu X, Guo SW. Activated platelets induce estrogen receptor β expression in endometriotic stromal cells. Gynecol Obstet Invest 2015;80:187-92. doi: 10.1159/000377629.
Guo SW, Ding D, Geng JG, Wang L, Liu X. P-selectin as a potential therapeutic target for endometriosis. Fertil Steril 2015;103:990-1000.e8. doi: 10.1016/j.fertnstert.2015.01.001.
Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-delta delta C(T)) method. Methods 2001;25:402-8. doi: 10.1006/meth.2001.1262.
Benderro GF, LaManna JC. HIF-1α/COX-2 expression and mouse brain capillary remodeling during prolonged moderate hypoxia and subsequent re-oxygenation. Brain Res 2014;1569:41-7. doi: 10.1016/j.brainres.2014.04.035.
Semenza GL. Hypoxia-inducible factor 1 (HIF-1) pathway. Sci STKE 2007;2007:cm8. doi: 10.1126/stke.4072007cm8.
Zhang W, Shi X, Peng Y, Wu M, Zhang P, Xie R, et al.
HIF-1α promotes epithelial-mesenchymal transition and metastasis through direct regulation of ZEB1 in colorectal cancer. PLoS One 2015;10:e0129603. doi: 10.1371/journal.pone.0129603.
Kaidi A, Qualtrough D, Williams AC, Paraskeva C. Direct transcriptional up-regulation of cyclooxygenase-2 by hypoxia-inducible factor (HIF)-1 promotes colorectal tumor cell survival and enhances HIF-1 transcriptional activity during hypoxia. Cancer Res 2006;66:6683-91. doi: 10.1158/0008-5472.CAN-06-0425.
Greenhough A, Smartt HJ, Moore AE, Roberts HR, Williams AC, Paraskeva C, et al.
The COX-2/PGE2 pathway: Key roles in the hallmarks of cancer and adaptation to the tumour microenvironment. Carcinogenesis 2009;30:377-86. doi: 10.1093/carcin/bgp014.
Tsujii M, Kawano S, Tsuji S, Sawaoka H, Hori M, DuBois RN. Cyclooxygenase regulates angiogenesis induced by colon cancer cells. Cell 1998;93:705-16. doi: 10.1016/S0092-8674(00)81433-6.
Forsythe JA, Jiang BH, Iyer NV, Agani F, Leung SW, Koos RD, et al.
Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1. Mol Cell Biol 1996;16:4604-13. doi: 10.1128/MCB.16.9.4604.
Ferns GA, Heikal L. Hypoxia in atherogenesis. Angiology 2017;68:472-93. doi: 10.1177/0003319716662423.
Lai SY, Grandis JR. Understanding the presence and function of erythropoietin receptors on cancer cells. J Clin Oncol 2006;24:4675-6. doi: 10.1200/JCO.2006.08.1190.
Masson N, Ratcliffe PJ. Hypoxia signaling pathways in cancer metabolism: The importance of co-selecting interconnected physiological pathways. Cancer Metab 2014;2:3. doi: 10.1186/2049-3002-2-3.
Semenza GL. Hypoxia-inducible factor 1: Master regulator of O2 homeostasis. Curr Opin Genet Dev 1998;8:588-94. doi: 10.1016/S0959-437X(98)80016-6.
Hardee ME, Arcasoy MO, Blackwell KL, Kirkpatrick JP, Dewhirst MW. Erythropoietin biology in cancer. Clin Cancer Res 2006;12:332-9. doi: 10.1158/1078-0432.CCR-05-1771.
Matsuzaki S, Canis M, Yokomizo R, Yaegashi N, Bruhat MA, Okamura K. Expression of erythropoietin and erythropoietin receptor in peritoneal endometriosis. Hum Reprod 2003;18:152-6. doi: 10.1093/humrep/deg007.
Fan DM, Qi PW, Gao SG, Chen YW, Cheng XL. TGF-β1 mediates estrogen receptor-induced epithelial-to-mesenchymal transition in some tumor lines. Tumour Biol 2014;35:11277-82. doi: 10.1007/s13277-014-2166-8.
Lin SC, Wang CC, Wu MH, Yang SH, Li YH, Tsai SJ. Hypoxia-induced microRNA-20a expression increases ERK phosphorylation and angiogenic gene expression in endometriotic stromal cells. J Clin Endocrinol Metab 2012;97:E1515-23. doi: 10.1210/jc.2012-1450.
Wu MH, Chen KF, Lin SC, Lgu CW, Tsai SJ. Aberrant expression of leptin in human endometriotic stromal cells is induced by elevated levels of hypoxia inducible factor-1alpha. Am J Pathol 2007;170:590-8. doi: 10.2353/ajpath.2007.060477.
Zhang F, Liu XL, Wang W, Dong HL, Xia YF, Ruan LP, et al.
Expression of MMIF, HIF-1α and VEGF in serum and endometrial tissues of patients with endometriosis. Curr Med Sci 2018;38:499-504. doi: 10.1007/s11596-018-1906-1.
Ren X, He YL, Pan SL, Peng DX. Expression of hypoxia-inducible factor-1alpha in endometriosis. Nan Fang Yi Ke Da Xue Xue Bao 2007;27:538-40. doi: 10.3321/j.issn:1673-4254.2007.04.029.
Oosterlynck DJ, Meuleman C, Waer M, Koninckx PR. Transforming growth factor-beta activity is increased in peritoneal fluid from women with endometriosis. Obstet Gynecol 1994;83:287-92. doi: 10.1016/0378-5122(94)90052-3.
Komiyama S, Aoki D, Komiyama M, Nozawa S. Local activation of TGF-beta1 at endometriosis sites. J Reprod Med 2007;52:306-12. doi: 10.1038/sj.jp.7211670.
Pizzo A, Salmeri FM, Ardita FV, Sofo V, Tripepi M, Marsico S. Behaviour of cytokine levels in serum and peritoneal fluid of women with endometriosis. Gynecol Obstet Invest 2002;54:82-7. doi: 10.1159/000067717.
Young VJ, Brown JK, Saunders PT, Duncan WC, Horne AW. The peritoneum is both a source and target of TGF-β in women with endometriosis. PLoS One 2014;9:e106773. doi: 10.1371/journal.pone.0106773.
Hull ML, Escareno CR, Godsland JM, Doig JR, Johnson CM, Phillips SC, et al.
Endometrial-peritoneal interactions during endometriotic lesion establishment. Am J Pathol 2008;173:700-15. doi: 10.2353/ajpath.2008.071128.
Young VJ, Brown JK, Maybin J, Saunders PT, Duncan WC, Horne AW. Transforming growth factor-β induced warburg-like metabolic reprogramming may underpin the development of peritoneal endometriosis. J Clin Endocrinol Metab 2014;99:3450-9. doi: 10.1210/jc.2014-1026.
Hung SP, Yang MH, Tseng KF, Lee OK. Hypoxia-induced secretion of TGF-β1 in mesenchymal stem cell promotes breast cancer cell progression. Cell Transplant 2013;22:1869-82. doi: 10.3727/096368912X657954.
Young VJ, Ahmad SF, Brown JK, Duncan WC, Horne AW. ID2 mediates the transforming growth factor-β1-induced warburg-like effect seen in the peritoneum of women with endometriosis. Mol Hum Reprod 2016;22:648-54. doi: 10.1093/molehr/gaw045.
Higgins DF, Kimura K, Bernhardt WM, Shrimanker N, Akai Y, Hohenstein B, et al.
Hypoxia promotes fibrogenesis in vivo
via HIF-1 stimulation of epithelial-to-mesenchymal transition. J Clin Invest 2007;117:3810-20. doi: 10.1172/JCI30487.
Matsuzaki S, Darcha C. Involvement of the Wnt/β-catenin signaling pathway in the cellular and molecular mechanisms of fibrosis in endometriosis. PLoS One 2013;8:e76808. doi: 10.1371/journal.pone.0076808.
Luo X, Xu J, Chegini N. The expression of smads in human endometrium and regulation and induction in endometrial epithelial and stromal cells by transforming growth factor-beta. J Clin Endocrinol Metab 2003;88:4967-76. doi: 10.1210/jc.2003-030276.
Ding D, Liu X, Guo SW. Further evidence for hypercoagulability in women with ovarian endometriomas. Reprod Sci 2018;25:1540-8. doi: 10.1177/1933719118799195.
Wu Q, Ding D, Liu X, Guo SW. Evidence for a hypercoagulable state in women with ovarian endometriomas. Reprod Sci 2015;22:1107-14. doi: 10.1177/1933719115572478.
Alpay Z, Saed GM, Diamond MP. Female infertility and free radicals: Potential role in adhesions and endometriosis. J Soc Gynecol Investig 2006;13:390-8. doi: 10.1016/j.jsgi.2006.05.002.
Wu MH, Lin SC, Hsiao KY, Tsai SJ. Hypoxia-inhibited dual-specificity phosphatase-2 expression in endometriotic cells regulates cyclooxygenase-2 expression. J Pathol 2011;225:390-400. doi: 10.1002/path.2963.
[Figure 1], [Figure 2], [Figure 3], [Figure 4]