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 Table of Contents  
ORIGINAL ARTICLE
Year : 2019  |  Volume : 3  |  Issue : 2  |  Page : 69-76

Activated platelets induce hypoxia-inducible factor-1α expression likely through transforming growth factor-β1 in human endometrial stromal cells


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 Submission08-Apr-2019
Date of Web Publication9-Jul-2019

Correspondence Address:
Prof. Xi-Shi Liu
Department of Gynecology, Shanghai Obstetrics and Gynecology Hospital, Fudan University, 419 Fangxie Road, Shanghai 200011
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2096-2924.262390

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  Abstract 


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 Top


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.[1] Despite extensive research, its pathogenesis remains elusive.[2] One widely accepted theory is the retrograde menstruation theory proposed by Sampson,[3] 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,[4] 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.[5] Remarkably, hypoxic conditions alone dramatically change the behavior of endometrial cells that are otherwise “normal.”[5]

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;[6] 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.[7] 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.[8] Conceivably, the menstrual debris, once regurgitated from the uterus to the peritoneal cavity as depicted in Sampson's hypothesis,[3] would experience hypoxia due to a loss of blood supply.[9]

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),[10],[11] 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.[9],[12] 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,[13],[14],[15],[16] and many nonendometriotic cells in the lesional microenvironment, such as platelets,[15],[17] macrophages,[13],[18] natural killer cells,[19],[20] and nerve fibers,[21],[22] 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 Top


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).

Cell culture

The human endometrial stromal cell line (ESCL), established by Dr. Krikun et al.,[23] 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.[24] 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.[25] 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.[24] 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.[19]

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.[26]

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.

Statistical analysis

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 Top


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.[15] 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 Top


Hypoxia is a critical mediator of endothelial growth factors and signaling proteins during periods of metabolic stress and vascular remodeling like angiogenesis or regression,[27] most of which are mediated via transcriptional regulation by HIF. HIF consists of two subunits, α and β, which form a heterodimeric complex.[28] HIF-1β is present under both normoxia and hypoxia, while HIF-1α is present only under hypoxic conditions.[28] As a transcription factor, HIF-1α regulates the activities of its downstream genes by binding the HREs in their promoter regions.[29] For example, COX-2 expression is regulated by HIF-1α transcriptional activity.[30] HIF-1α-induced COX-2 expression leads to elevated prostaglandin E2(PGE2) levels, which can induce PGE2-mediated vascularization.[31],[32] VEGF, a major driver of angiogenesis in cancer and in endometriosis, is also transcriptionally regulated by HIF-1α under hypoxic conditions.[33] HIF-1α binds to the HRE regions of the VEGF promoter and is positively correlated with VEGF expression in endometriosis.[33] Tissue hypoxia is the main stimulus of EPO production.[34] HIF-1α upregulates the expression of EPO, which mediates its effects by binding to the EPO receptor (EPOR).[35] 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.[36],[37] EPO, a widely known growth factor in erythropoiesis, can also stimulate angiogenesis,[38] as well as tumor cell proliferation and survival.[35] The expression levels of EPO,[39] VEGF, and COX-2[17] have all been reported to be elevated in endometriosis, resulting in angiogenesis, proliferation, and enhanced survival in lesional development.[35],[40] We have previously shown that activated platelets can activate VEGF and COX-2 expression in HESCs;[17] 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.[17] 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.[41] It has been reported that HIF-1α is overexpressed in endometriotic stromal cells, resulting in increased cellular proliferation in an autocrine fashion.[42],[43],[44] 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.[45],[46],[47],[48] 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.[49] It has also been reported to induce a Warburg-like metabolic reprogramming in peritoneal mesothelial cells, potentially facilitating the development of peritoneal endometriosis.[50] 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.[15]

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.[51] On the other hand, exposure to TGF-β1 could increase HIF-1α gene and protein expression in many cell types.[48],[50],[52] 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.[53] 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.[9] This is consistent with the report that both endometriotic and endometrial stromal cells have elevated expression of myofibroblast markers after stimulation with TGF-β1.[54] 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.[55] In other words, targeting this pathway directly may cause unintended collateral damage to normal tissues/organs. As shown in this study and in others,[17],[19] 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,[15],[17],[42],[56],[57],[58],[59] 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.



 
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