Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Jun 1;32(6):1218-1229.
doi: 10.1093/humrep/dex069.

Enhancement of trophoblast differentiation and survival by low molecular weight heparin requires heparin-binding EGF-like growth factor

Alan D Bolnick  1 , Jay M Bolnick  1 , Hamid-Reza Kohan-Ghadr  1 , Brian A Kilburn  1 , Omar J Pasalodos  1 , Pankaj K Singhal  2 , Jing Dai  1 , Michael P Diamond  3 , D Randall Armant  1   4 , Sascha Drewlo  1
Affiliations

Enhancement of trophoblast differentiation and survival by low molecular weight heparin requires heparin-binding EGF-like growth factor

Alan D Bolnick et al. Hum Reprod. .

Abstract

Study question: Does low molecular weight heparin (LMWH) require heparin-binding epidermal growth factor (EGF)-like growth factor (HBEGF) signaling to induce extravillous trophoblast differentiation and decrease apoptosis during oxidative stress?

Summary answer: LMWH increased HBEGF expression and secretion, and HBEGF signaling was required to stimulate trophoblast extravillous differentiation, increase invasion in vitro and reduce trophoblast apoptosis during oxidative stress.

What is known already: Abnormal trophoblast differentiation and survival contribute to placental insufficiency syndromes, including preeclampsia and intrauterine growth restriction. Preeclampsia often manifests as a pro-thrombotic state, with unsuccessful transformation of the spiral arteries that reduces oxygen supply and can produce placental infarction. LMWH improves placental function by increasing blood flow. Recent data suggest that the actions of LMWH transcend its anti-coagulative properties, but the molecular mechanism is unknown. There is evidence that LMWH alters the expression of human HBEGF in trophoblast cells, which regulates human trophoblast pathophysiology. HBEGF, itself, is capable of increasing trophoblast survival and invasiveness.

Study design, size, duration: First-trimester placental explants and the HTR-8/SVneo cell line, established using extravillous trophoblast outgrowths from first-trimester villous explants, were treated in vitro with LMWH to examine the effects on HBEGF signaling and trophoblast function under normal physiological and pathological conditions. A highly specific antagonist of HBEGF and other inhibitors of HBEGF downstream signaling were used to determine the relationship between LMWH treatment and HBEGF.

Participants/materials, setting, methods: Placental tissues (n = 5) were obtained with IRB approval and patient consent from first-trimester terminations. Placental explants and HTR-8/SVneo cells were cultured on plastic or Matrigel™ and treated with a therapeutic dose of LMWH (Enoxaparin; 10 IU/ml), with or without CRM197, pan Erb-B2 Receptor Tyrosine Kinase (ERBB) inhibitor, anti-ERBB1 or ERBB4 blocking antibodies, or pretreatment of cells with heparitinase I. Extravillous differentiation was assessed by immunocytochemistry to determine the relative levels of integrins α6β4 and α1β1. Trophoblast invasiveness was assessed in villous explants by measuring outgrowth from villous tips cultured on Matrigel, and by invasion assays with HTR-8/SVneo cells cultured on Matrigel-coated transwell insert. Placental explants and HTR-8/SVneo cells were exposed to oxidative stress in a hypoxia-reoxygenation (H-R) model, measuring cell death by TUNEL assay, caspase 3 cleavage, and BCL-2α expression.

Main results and the role of chance: LMWH induced extravillous differentiation, according to trophoblast invasion assays and integrin (α6β4-α1β1) switching. Treatment with LMWH rescued cytotrophoblasts and HTR-8/SVneo cells from apoptosis during exposure to reoxygenation injury, based on TUNEL, caspase 3 cleavage and BCL-2α expression. Experiments using CRM197, ERBB1 and ERBB4 blocking antibodies, pan-ERBB inhibitor and removal of cell surface heparin demonstrated that the effects of LMWH on trophoblast invasion and survival were dependent upon HBEGF signaling.

Large scale data: N/A.

Limitations, reasons for caution: The primary limitation of this study was the use of only in vitro experiments. Patient demographics from elective terminations were not available.

Wider implications of the findings: These data provide new insights into the non-coagulation-related aspects of perinatal LMWH treatment in the management of placental insufficiency disorders.

Study funding/competing interest(s): This research was supported by grants from the National Institutes of Health (HD071408 and HL128628), the March of Dimes, and the W. K. Kellogg Foundation. There were no conflicts or competing interests.

Keywords: BCL-2α; HBEGFimplantation; LMWH; apoptosis; caspase; differentiation; integrins; placenta; trophoblast.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Effect of LMWH on cell death in first-trimester placental explants. Dissected villous explants were cultured at 8% O2, 2% O2 or during H/R, with or without addition of 10 IU LMWH, as indicated. Cell death was assessed using a TUNEL assay (A–I). Villi were double-labeled using fluorescent indicators to visualize DNA fragmentation (dUTP) by TUNEL (A–D) or nuclei with DAPI (E–H). TUNEL Index was quantified as a percentage by dividing the number of TUNEL-positive cells by the total cell number of DAPI-labeled nuclei (I). Western blot analysis of villous explant extracts was performed for BCL-2α expression, and densities of immuno-reactive bands were measured as arbitrary units by ImageJ software (J). Protein levels were normalized to β-actin. (K–Y) first-trimester villous explants were fluorescently labeled with antibodies against BCL-2α and cleaved caspase 3 (cCASP3). Immunofluorescence images are shown for BCL-2α (without treatment at ambient green, K–N), cCASP3 (red, P–S) and merged fields (U–Y), as indicated. Non-immune IgG negative controls are shown in O and T. Arrows indicate STB and CTB cells. Bars = 50 µM. Error bars denote SEM. *P < 0.05 compared to treatments without LMWH (I, J). LMWH, low molecular weight heparin; STB, syncytiotrophoblast; CBT, cytotrophoblast; H/R, hypoxia/reoxygenation.
Figure 2
Figure 2
HBEGF protein expression and LMWH. HTR-8/SVneo (HTR) cells were cultured untreated (0, Control), or their medium was supplemented with 0.1–25 IU/ml LMWH for 24 h (A). Afterward, HBEGF was measured by ELISA in the cell lysates and media. Secreted HBEGF was measured by ELISA in first-trimester villous explants after 24 h of culture with 0 (Control) or 10 IU/ml of LMWH (B). HBEGF expression was localized by immunofluorescence in HTR cells (C–D) and first-trimester villous explants (F–G) for control and LMWH treatments, as indicated. DAPI nuclear counterstain (blue) is included in the images. Villi and cells treated with LMWH were labeled with non-immune goat IgG, as indicated (E and H). Bars = 50 μM. *P < 0.05, compared with control. HBEGF, heparin-binding epidermal growth factor-like growth factor.
Figure 3
Figure 3
Induction of trophoblast invasion and outgrowth by LMWH. HTR-8/SVneo cells were cultured on Matrigel™-coated transwell inserts for 72 h to determine their invasive capacity (A–G). Cells that invaded through the Matrigel™ were trypsinized from the bottom of the insert, and fixed. Examples of cells that penetrated through the Matrigel™ are shown after culture with no treatment (Control, A), 10 IU/ml LMWH (B), or combinations of LMWH and either 10 μg/ml CRM197 (C), pan-ERBB inhibitor (D), ERBB1 blocking antibody (E) or ERBB4 blocking antibody (F). The number of cells entering the lower chambers after 72 h was quantified for the indicated treatments that did (solid bars) or did not (open bars) include LMWH (G). First-trimester villous explants were cultured on Matrigel™-coated transwell inserts for 72 h, and assessed for trophoblast outgrowth from their distal ends (H–L). Examples of outgrowths from villous explants cultured with no treatment (Control, H), 10 IU/ml LMWH (I), and a combination of LMWH with either CRM197 (J), or ERBB4 blocking antibody (K) are shown. Outgrowth length was measured, as indicated by red arrows, using Simple PCI image analysis software, and is depicted graphically in L for triplicate experiments in which 10 villous explants were measured for each indicated treatment that did (solid bars) or did not (open bars) include LMWH. Size bars = 100 μM. *P < 0.05 compared to no treatment (open bars). Error bars represent SEM. ERBB, Erb-B2 Receptor Tyrosine Kinase.
Figure 4
Figure 4
Induction of integrin switching by LMWH, and HBEGF dependence. HTR-8/SVneo cells were cultured for 24 h with no treatment (Control; A, G), 10 IU/ml LMWH (B, H), and combinations of LMWH with 10 μg/ml CRM197 (C, I), pan-ERBB inhibitor (D, J) or heparitinase (E, K). Cells were assessed for α1 (A–E) or α6 (G–K) integrin subunits by immunofluorescence. Nuclei were counterstained with DAPI. Cells treated with LMWH were labeled with non-immune goat IgG (F) as a negative control. Bars = 100 μM. Integrin α1 and α6 protein expression was quantified by image analysis (L). * or †, P < 0.05 compared to no treatment. § or ‡, P < 0.05 compared to LMWH treatment.
Figure 5
Figure 5
LMWH rescue from cell death requires HBEGF. HTR-8/SVneo cells were cultured without treatment at ambient (20%) O2, or exposed to H/R with no treatment (Control), supplementation with 10 IU/ml LMWH, or combinations of LMWH and either 10 μg/ml CRM197, pan-ERBB inhibitor, heparitinase I, ERBB1 blocking antibody or ERBB4 blocking antibody. HTR-8/SVneo cells were fluorescently double-labeled after treatment to visualize cell death by TUNEL (green fluorescence) and nuclei with DAPI (blue fluorescence). Representative fluorescent images are shown of TUNEL (A–D) or merged TUNEL/DAPI (E–H) in cells cultured at ambient O2 (A, E), with H/R (B, F), with H/R + LMWH (C, G) and with H/R + LMWH + CRM197 (D, H). Bars = 100 μM. TUNEL Index is shown graphically for the indicated culture conditions and treatments, which did (solid bars) or did not (open bars) include LMWH (I). Error bars denote SEM. Bars labeled with dissimilar letters indicate differences at P < 0.05.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Adiguzel C, Jeske WP, Hoppensteadt D, Walenga JM, Bansal V, Fareed J. Structural and functional characterization of low-molecular-weight heparins: impact on the development of guidelines for generic products. Clin Appl Thromb Hemost 2009;15:137–144. - PubMed
    1. Aghajanova L, Shen S, Rojas AM, Fisher SJ, Irwin JC, Giudice LC. Comparative transcriptome analysis of human trophectoderm and embryonic stem cell-derived trophoblasts reveal key participants in early implantation. Biol Reprod 2012;86:1–21. - PubMed
    1. Akhtar MA, Sur S, Raine-Fenning N, Jayaprakasan K, Thornton J, Quenby S, Marjoribanks J. Heparin for assisted reproduction: summary of a Cochrane review. Fertil Steril 2015;103:33–34. - PubMed
    1. Allaire AD, Ballenger KA, Wells SR, McMahon MJ, Lessey BA. Placental apoptosis in preeclampsia. Obstet Gynecol 2000;96:271–276. - PubMed
    1. Armant DR, Kilburn BA, Petkova A, Edwin SS, Duniec-Dmuchowski ZM, Edwards HJ, Romero R, Leach RE. Human trophoblast survival at low oxygen concentrations requires metalloproteinase-mediated shedding of heparin-binding EGF-like growth factor. Development 2006;133:751–759. - PMC - PubMed

MeSH terms