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148]

Original Article

Activated Platelets Induce Hypoxia-Inducible Factor-1α

Expression Likely through Transforming Growth Factor-β1 in
Human Endometrial Stromal Cells
Qiu-Ming Qi1, Sun-Wei Guo1,2, Xi-Shi Liu1,2
1
Department of Gynecology, Shanghai Obstetrics and Gynecology Hospital, Fudan University, Shanghai 200011, China
2
Shanghai Key Laboratory of Female Reproductive Endocrine-Related Diseases, Fudan University, Shanghai 200011, China

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.

Key words: Endometriosis; Hypoxia, Hypoxia-inducible Factor-1α; Platelet, Stromal Cell

Introduction regurgitated from the uterus.[5] Remarkably, hypoxic conditions

alone dramatically change the behavior of endometrial cells
Endometriosis is defined as the presence of endometrial tissue
that are otherwise “normal.”[5]
outside of the uterine cavity and is a common gynecological
disease that affects approximately 10% women of reproductive One hallmark of hypoxia is the overexpression of the transcription
age and involves pelvic pain and infertility as its clinical factor hypoxia-inducible factor-1α (HIF-1α), a key mediator of
manifestations.[1] Despite extensive research, its pathogenesis cellular adaptation to low oxygen levels and a potential target
remains elusive.[2] One widely accepted theory is the retrograde for many types of cancer, such as ovarian, prostate, and breast;[6]
menstruation theory proposed by Sampson,[3] which stipulates HIF-1α expression reflects the degree of cell hypoxia; its
that, when viable endometrial cells are regurgitated into the increased expression has been reported in most neoplasms; and it
pelvic cavity through the fallopian tubes, they somehow
invade the peritoneum and establish the initial lesions. Address for correspondence: Prof. Xi-Shi Liu,
However, the ectopic and eutopic endometria are known to be Department of Gynecology, Shanghai Obstetrics and Gynecology Hospital,
transcriptionally different,[4] and the difference is presumably Fudan University, 419 Fangxie Road, Shanghai 200011, China
E-Mail: lxsdoc@hotmail.com
attributed to their different microenvironments, the key factor
that determines the difference is still unclear.
This is an open access journal, and articles are distributed under the terms of the Creative
Recently, it has been shown that hypoxia plays a critical role Commons Attribution-NonCommercial-ShareAlike 4.0 License, which allows others to remix,
in the survival and angiogenesis of ectopic endometrial cells tweak, and build upon the work non-commercially, as long as appropriate credit is given and
the new creations are licensed under the identical terms.
Access this article online For reprints contact: reprints@medknow.com
Quick Response Code: © 2019 Reproductive and Developmental Medicine | Published by Wolters Kluwer ‑ Medknow
Website:
www.repdevmed.org
Received: 08-04-2019 Edited by: Yong-Qing Zhu
How to cite this article: Qi QM, Guo SW, Liu XS. Activated Platelets
DOI: Induce Hypoxia-Inducible Factor-1α Expression Likely through
10.4103/2096-2924.262390 Transforming Growth Factor-β1 in Human Endometrial Stromal Cells.
Reprod Dev Med 2019;3:69-76.

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is responsible for increased angiogenesis in cancers.[7] Similar to (100 IU/mL penicillin G, 100 mg/mL streptomycin, and
that in cancer, HIF-1α has also been shown to be overexpressed 2.5 μg/mL amphotericin B).
in endometriosis, reflecting the increasing need for nutrients
Endometriosis-derived primary ectopic endometrial stromal cells
and oxygen as endometriotic lesions grow.[8] Conceivably,
(HESCs) were isolated and cultured as reported previously.[24]
the menstrual debris, once regurgitated from the uterus to the
Briefly, after washing with DMEM/F-12 medium supplemented
peritoneal cavity as depicted in Sampson’s hypothesis,[3] would with 5% FBS and 1% antibiotics, the tissue samples were minced
experience hypoxia due to a loss of blood supply.[9] into small pieces ~1 mm3 in size. Then, the minced tissues
Under hypoxic conditions, HIF-1α is stabilized and binds were enzymatically digested with 0.2% collagenase II (Sigma,
to core hypoxia response elements (HREs). This results in St Louis, MO, USA) in a shaking bed for 1.5 h at 37°C. After
the transcriptional activation of hypoxia-regulated genes, that, they were separated by filtration through a 149-μm and
including erythropoietin (EPO), vascular endothelial growth a 37-μm (pore size) nylon mesh. The stromal cells remaining
factor (VEGF), and cyclooxygenase-2 (COX-2),[10,11] which are in the filtrate were collected by centrifugation, resuspended in
genes known to promote angiogenesis, cellular proliferation, DMEM/F12 (reconstituted with 10% FBS and 1% antibiotics),
and the production of pro-inflammatory cytokines/chemokines. seeded into 25-cm2 cell culture flasks, and incubated at 37°C in
Under hypoxic stress, several events such as steroidogenesis, a humidified atmosphere of 5% CO2 in air. Antibodies against
angiogenesis, and epigenetic modulation take place and vimentin (Abcam, Cambridge, UK), cytokeratin 7 (CK-7,
effectively turn normal endometrial stromal cells into Abcam), and follicle-stimulating hormone receptor (FSHR,
endometriotic stroma-like cells mainly through the induction Abcam) were used for immunocytochemistry to verify the
of HIF-1α.[9,12] This may explain why endometrial debris purity and homogeneity of the stromal cell preparation (≥98%)
invades and establish endometriotic foci in ectopic sites. after 3–4 passages.

In the last few years, growing evidence has suggested that Preparation of platelets
endometriotic lesions are essentially wounds undergoing Platelets were isolated by centrifugation at room temperature of
repeated tissue injury and repair,[13-16] and many nonendometriotic whole blood samples donated by healthy male volunteers who
cells in the lesional microenvironment, such as platelets,[15,17] provided informed consent and did not take any medications
macrophages,[13,18] natural killer cells,[19,20] and nerve fibers,[21,22] for at least 2 weeks prior to donation as reported previously.[25]
are involved in facilitating the development of endometriosis. The blood was first centrifuged at 150 ×g for 10 min, the
Because platelets are the first responders to injury, menstrual supernatant platelet-rich plasma (PRP) was harvested and
centrifuged at 300 ×g for 5 min, and finally the supernatant PRP
debris could be shrouded with activated platelets and
was harvested again and centrifuged at 1000 ×g for 14 min.
thus activate HIF-1α expression. Thus, we hypothesized
Finally, the deposited platelets were suspended in DMEM/
that activated platelets might induce HIF-1α expression in F12 culture medium for subsequent experiments. We obtained
endometrial stromal cells, signaling the presence of hypoxia. about 2 × 108 platelets from 20 mL of peripheral blood samples.

Methods Treatment of human endometriotic stromal cell/endometrial

stromal cell line
Patients and specimens HESC and ESCL were added to the serum-free DMEM/F-12
Endometriotic tissue samples were obtained after medium at 80% confluence and were starved for 24 h. Then,
informed consent from 16 premenopausal patients they were cocultured with 3.5 mL of different treatment
(mean age = 30.7 ± 5.3 years) with histologically diagnosed media as follows: control (standard DMEM/F-12 medium),
ovarian endometriomas admitted to the Shanghai Obstetrics platelets (standard medium containing ~107 human platelets),
and Gynecology Hospital, Fudan University, from May to activated platelets (standard medium containing ~107 human
September 2015. All endometriotic tissue samples were used for platelets and human thrombin [1.49 NIH U, Sigma, St. Louis,
primary culture of ectopic endometrial stromal cells (HESCs). MO, USA]), and thrombin alone (standard medium containing
Among these endometriotic tissue samples, eight were used for human thrombin 1.49 NIH U).
real-time polymerase chain reaction (PCR) analysis and the
remaining eight were used for Western blot analysis. This study To see whether transforming growth factor-β (TGF-β)
was approved by the Institutional Ethics Review Board of the neutralization can abolish the overexpression of HIF-1α
Shanghai Obstetrics and Gynecology Hospital (Kyy2015-34). induced by activated platelets in HESC and ESCL, the cells
were pretreated for 2 h with 1 μmol/L of A83-01 compound
Cell culture (Santa Cruz, CA, USA), which is a TGF-β receptor inhibitor,
The human endometrial stromal cell line (ESCL), established by and then incubated with activated platelets plus 1 μmol/L
Dr. Krikun et al.,[23] was kindly provided by Dr. Asgi Fazleabas A83-01 as described in.[24] After coculture for 48 h, all cells
of Michigan State University, Michigan, USA. The cells were were harvested for real-time PCR and Western blot analysis.
cultured in Dulbecco’s modified Eagle’s medium/Ham’s To ensure that the Western blot results were obtained from
F-12 medium (DMEM/F-12, Hyclone) supplemented stromal cells but not platelets, we washed the HESCs and
with 5% fetal bovine serum (FBS) and 1% antibiotics ESCL cells cocultured with platelets with sterile phosphate-

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buffered saline (PBS) three times to remove the platelets as Results

reported previously.[19]
Activated platelets elevate the gene and protein expression
RNA isolation and real-time polymerase chain reaction levels of HIF-1α and erythropoietin in human endometriotic
TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was used stromal cell and endometrial stromal cell line
to isolate total RNA from HESC and ESCL, and a reverse The gene expression levels of HIF-1α were significantly
transcription kit (Takara, Otsu, Japan) was used for cDNA increased in HESCs cocultured with platelets [P = 0.05, Figure 1a]
synthesis. Gene expression levels were evaluated by real-time and with activated platelets (P = 0.017) as compared with
PCR using SYBR Premix Ex Taq (Takara, Otsu, Japan). The those in the cells cocultured with PBS. Consistently, the
oligonucleotide primer sequences, synthesized by Sangon protein expression levels of HIF-1α in HESCs cocultured with
Corporation (Shanghai, China), were as follows: HIF-1α: 5′ platelets and activated platelets were similarly elevated as
GTC GAC ACA GCC TGG ATA TGA A 3′ (forward) and 5′ compared with those in the controls [P = 0.017 and P = 0.012,
CAT ATC ATG ATG AGT TTT GGT CAG ATG 3′ (reverse). respectively, Figure 1b and 1c].
EPO: 5′ CAC CAC GCC TCA TCT GTG AC 3′ (forward)
and 5′ CAC AGC CCG TCG TGA TAT TCT 3′ (reverse). Next, we evaluated the gene and protein expression levels of
GAPDH: 5′ TGC ACC ACC AAC TGC TTA G 3′ (forward) EPO, a HIF-1α target gene. Consistently, the gene expression
and 5′ GAT GCA GGG ATG ATG TTC 3′ (reverse). Melting levels of EPO were significantly increased in HESCs
curves of the products were obtained after cycling by a stepwise cocultured with platelets and activated platelets [Figure 1d,
increase of temperature from 55°C to 95°C. After 40 cycles, both P = 0.012] as compared with those in the control group;
the reaction products were separated electrophoretically on the same results were observed with the protein expression
an agarose gel and stained with ethidium bromide for visual levels [Figure 1e and 1f, both P = 0.012].
confirmation of the PCR products. Expression values were We also evaluated the gene and protein expression levels of
normalized to the geometric mean of GAPDH measurements, HIF-1α and EPO in ESCL. We found that the gene expression
and the quantification of mRNA abundance was made using levels of HIF-1α were significantly increased when cocultured
the method described previously.[26] with platelets and activated platelets [both P's = 0.012,
Figure 2a] as compared with those in the cells cocultured with
Western blot analysis PBS. Interestingly, the gene expression levels of EPO in ESCL
Total protein was harvested from HESCs and ESCL cells by
were significantly increased after coculture with activated
scraping and extracting using the commercial RIPA buffer
platelets [P = 0.043, Figure 2d], but not with platelets alone
(Thermo, Waltham, MA, USA). The protein concentration,
[P = 0.69]. The protein expression levels of HIF-1α [both
after coculture with platelets, activated platelets, or PBS for
P's = 0.012, Figure 2b and 2c] and EPO [both P's = 0.043,
48 h, was determined using BCA protein quantitative analysis
Figure 2e and 2f] were significantly increased after coculture
kit (P0010S, Beyotime, Shanghai, China). All proteins mixed with platelets and with activated platelets.
with SDS-PAGE loading buffer (P0015, Beyotime) were heated
for 10 min at 95°C for denaturation. The protein samples were Neutralization of TGF-β1 signaling completely abolishes
loaded on a 10% SDS-PAGE and subsequently electroblotted activated platelet-induced expression of HIF-1α and
onto PVDF membranes (Bio-Rad, Hercules, California, USA). erythropoietin in both human endometriotic stromal cell
After blocking in Western blocking buffer (P0023B, Beyotime) and endometrial stromal cell line
for 1 h at room temperature, the membranes were subsequently Activated platelets have been shown to induce the TGF-β1/Smad3
incubated at 4°C overnight with the following primary signaling pathway in endometrial and endometriotic stromal
antibodies: HIF-1α (1:1,000, Abcam), EPO (1:1,000, Abcam), cells.[15] Thus, we wondered whether the induction of HIF-1α
GAPDH (1:1,000, Cell Signaling Technology, MO, USA), is through the TGF-β1/Smad3 signaling pathway.
and β-actin (1:1,000, Cell Signaling Technology). After the
membranes were incubated with HRP-labeled secondary To study this, HESCs were preincubated with A83-01, and the
antibodies for 1 h at room temperature, the signal was detected gene and protein expression levels of HIF-1α and EPO were
using ECL (Pierce, Thermo Scientific, Rockford, IL, USA) on evaluated by real-time PCR and Western blot after coculture
Image Quant LAS 4000 mini (GE Healthcare). The amount of with activated platelets. We found that, as previously reported,
protein was quantified by Quantity One software (Bio-Rad) the gene expression levels of both HIF-1α and EPO were
and normalized to GAPDH or β-actin levels, which served as significantly elevated [Figure 3a and 3d, P = 0.012 and 0.017,
loading controls. respectively] after coculture with activated platelets, and
that TGF-β1 neutralization by A83-01 completely abolished
Statistical analysis the elevation [P = 0.012 and 0.025, respectively]. Similarly,
To compare gene and protein expression levels between after coculture with activated platelets, the protein expression
cells with different treatments, Wilcoxon’s test was used. levels of both HIF-1α and EPO were significantly increased
P ≤ 0.05 was considered statistically significant. All [Figure 3b, 3c, 3e and 3f, both P's = 0.012], but TGF-β1
calculations were carried out using SPSS, version 16.0 neutralization abrogated the increase in protein expression
(SPSS Inc., Chicago, IL, USA). levels (both P's = 0.012).

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a b c

d e f
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.

As with HESCs, nearly identical results were stimulus of EPO production.[34] HIF-1α upregulates the expression
obtained for ESCL cells: the elevated gene of EPO, which mediates its effects by binding to the EPO receptor
[Figure 4a and 4d, both P's = 0.043] and protein expression (EPOR).[35] Hypoxia results in the increased production of EPO
levels [Figure 4b, 4c, 4e, and 4f, both P's = 0.043] of HIF- via the induction of the HIF-1 pathway. The EPO gene is under
1α and EPO induced by activated platelets were completely the direct control of hypoxia through HIF-1α, which then binds to
abolished by TGF-β1 neutralization. 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
Discussion stimulate angiogenesis,[38] as well as tumor cell proliferation and
survival.[35] The expression levels of EPO,[39] VEGF, and COX-2[17]
Hypoxia is a critical mediator of endothelial growth factors and
have all been reported to be elevated in endometriosis, resulting
signaling proteins during periods of metabolic stress and vascular
in angiogenesis, proliferation, and enhanced survival in lesional
remodeling like angiogenesis or regression,[27] most of which are
development.[35,40] We have previously shown that activated
mediated via transcriptional regulation by HIF. HIF consists of
platelets can activate VEGF and COX-2 expression in HESCs;[17]
two subunits, α and β, which form a heterodimeric complex.[28]
in this study, we found that activated platelets also induce the
HIF-1β is present under both normoxia and hypoxia, while HIF-
activation of HIF-1α and the expression of its target gene EPO
1α is present only under hypoxic conditions.[28] As a transcription
in HESCs and ESCL cells. Thus, the upregulation of VEGF and
factor, HIF-1α regulates the activities of its downstream genes
COX-2 induced by activated platelets in endometriotic stromal
by binding the HREs in their promoter regions.[29] For example,
cells was probably via HIF-1α induction, which occurs through
COX-2 expression is regulated by HIF-1α transcriptional
the TGF-β1/Smad3 signaling pathway.
activity.[30] HIF-1α-induced COX-2 expression leads to elevated
prostaglandin E2 (PGE2) levels, which can induce PGE2-mediated In this study, we investigated the gene and protein expression
vascularization.[31,32] VEGF, a major driver of angiogenesis in levels of HIF-1α in primary endometriotic stromal cells
cancer and in endometriosis, is also transcriptionally regulated by cocultured with platelets and demonstrated that platelets could
HIF-1α under hypoxic conditions.[33] HIF-1α binds to the HRE induce HIF-1α expression, signaling the state of hypoxia. The
regions of the VEGF promoter and is positively correlated with upregulation of some downstream genes of HIF-1α, such as
VEGF expression in endometriosis.[33] Tissue hypoxia is the main COX-2 and VEGF, induced by activated platelets have been

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a b c

d e f
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.

reported previously.[17] Activated platelets was able to also induce We have shown previously that activated platelets, through
HIF-1α, as well as its downstream target gene EPO, expression in the release of TGF-β1 and the induction of the TGF-β1/Smad3
ESCL, indicating that activated platelets can effectively induce signaling pathway, promote lesional development, and that
a hypoxic state in both endometrial and endometriotic stromal TGF-β1 blockade reverses the development.[15]
cells. However, TGF-β1 neutralization by A83-01 abrogates It has been reported that TGF-β1 promoter activity is
HIF-1α and EPO overexpression induced by activated platelets, regulated by hypoxia, and that HIF-1α could directly regulate
suggesting that the induction of HIF-1α by activated platelets TGF-β1 expression through the HRE, which is located
in both endometrial and endometriotic stromal cells probably in the TGF-β1 proximal promoter.[51] On the other hand,
occurs through the TGF-β1 signaling pathway. exposure to TGF-β1 could increase HIF-1α gene and protein
Hypoxia is an important factor that regulates numerous expression in many cell types.[48,50,52] In this study, we found
physiological and pathological processes.[41] It has been reported that neutralization of TGF-β1 completely abolished the HIF-1α
that HIF-1α is overexpressed in endometriotic stromal cells, induction by activated platelets in both HESC and ESCL,
resulting in increased cellular proliferation in an autocrine highlighting the important role of platelets in the initiation
fashion.[42-44] Our data demonstrate that activated platelets could and development of endometriotic lesions.
induce hypoxia in the microenvironment of endometriotic We note that HIF-1α has also been reported to promote
stromal cells through the elevated expression of HIF-1α, resulting epithelial–mesenchymal transition (EMT) in several types
in cellular proliferation, angiogenesis, and lesional development. of cancer by modulating one or more EMT-associated
The increased TGF-β1 expression in endometriosis has been genes.[53] Moreover, in this study, we demonstrated that TGF-β1
well documented.[45-48] In a gene profiling study on a mouse neutralization completely abolishes platelet-induced HIF-1α
model, TGF-β1 has been identified to play important roles in the expression in endometriotic cells. However, more research is
warranted to elucidate how TGF-β1 signaling can modulate
gene network involved in the pathogenesis of endometriosis.[49]
platelet-induced HIF-1α expression in endometriosis.
It has also been reported to induce a Warburg-like metabolic
reprogramming in peritoneal mesothelial cells, potentially Our data have shown that activated platelets can also turn
facilitating the development of peritoneal endometriosis.[50] normal endometrial stromal cells into a hypoxic phenotype,

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a b c

d e f
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.

similar to that in HESCs. The results are consistent with In summary, we have shown here that platelets play a critical
the finding that activation of HIF-1α and consequent role in driving hypoxia through the upregulation of HIF-
hypoxia can dramatically change the behavior of human 1α and its downstream genes, facilitating the development
endometrial stromal cells, resulting in completely different of endometriosis. In addition, neutralization of TGF-β1
phenotypes.[9] This is consistent with the report that both completely abolishes the activated platelet-induced expression
endometriotic and endometrial stromal cells have elevated of HIF-1α and its target gene EPO. These findings highlight
expression of myofibroblast markers after stimulation with the importance of platelets in the development, and perhaps
TGF-β1.[54] While it may seem logical that the TGF-β1/ as well as the initiation, of endometriosis. These results, in
Smad3 pathway is a therapeutic target for endometriosis, it conjunction with other reports on the roles of platelets in
is worth noting that this pathway also plays many important driving lesional development, underscore the possibility of
physiological functions in the endometrium. [55] In other using anticoagulation therapy in the nonhormonal treatment
words, targeting this pathway directly may cause unintended of endometriosis, as well as hold promise for the development
collateral damage to normal tissues/organs. As shown in of novel biomarkers for endometriosis.
this study and in others,[17,19] a better, alternative therapeutic
approach may be to target the coagulation pathways. Financial support and sponsorship
This research was supported in part by grants 81530040
One notable limitation of this study is due to the in vitro nature (SWG), 81771553 (SWG), 81671436 (XSL), and 81871144
of this study, which evaluated the results of cell culture only. (XSL) from the National Science Foundation of China and a
Future in vivo studies are needed to validate our findings. grant for Shanghai Medical Center for Female Reproductive
However, because endometriotic stromal cells and platelets Disease (2017ZZ01016) from the Science and Technology
used in this study were all derived from humans, and both Commission of Shanghai Municipality.
platelet aggregation and hypoxia are now well documented in
endometriosis,[15,17,42,56-59] we believe that platelets must play a Conflicts of interest
role in hypoxia in endometriosis. There are no conflicts of interest.

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a b c

d e f
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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