Long-Acting Progestin-Only Contraceptives Enhance Human Endometrial Stromal Cell Expressed Neuronal Pentraxin-1 and Reactive Oxygen Species to Promote Endothelial Cell Apoptosis

Abstract

Context:

Despite the absence of progesterone receptor protein in human endometrial endothelial cells (HEECs), endometria of women receiving long-acting progestin-only contraceptives (LAPCs) display reduced uterine blood flow, elevated reactive oxygen species generation, increased angiogenesis, and irregularly distributed, enlarged, fragile microvessels resulting in abnormal uterine bleeding.

Objective:

We propose that paracrine factors from LAPC-treated human endometrial stromal cells (HESCs) impair HEEC functions by shifting the balance between HEEC viability and death in favor of the latter.

Design and Setting:

Proliferation, apoptosis, and transcriptome analyses were performed in HEECs treated with conditioned medium supernatant (CMS) derived from HESCs treated with estradiol (E₂) ± medroxyprogesterone acetate or etonogestrel under normoxia or hypoxia. Mass spectrometry interrogated the CMS secretome while immunostaining for neuronal pentraxin-1 (NPTX1), cleaved caspase-3, and cytochrome c was performed in cultured HEECs and paired endometria from women using LAPCs.

Main Outcome:

HEEC apoptosis and its underlying mechanism.

Results:

HESC CMS from E₂ + medroxyprogesterone acetate or E₂ + etonogestrel incubations under hypoxia induced HEEC apoptosis (P < .05), whereas mass spectrometry of the CMS revealed increased NPTX1 secretion (P < .05). Endothelial cleaved caspase-3 and stromal NPTX1 immunoreactivity were significantly higher in LAPC-treated endometria (P < .001). Transcriptomics revealed AKT signaling inhibition and mitochondrial dysfunction in HEECs incubated with HESC CMS. In vitro analyses proved that CMS decreased HEEC AKT phosphorylation (P < .05) and that recombinant NPTX1 (P < .05) or NPTX1 + H₂O₂ (P < .001) increase HEEC apoptosis and cytosolic cytochrome c levels.

Conclusions:

LAPC-enhanced NPTX1 secretion and reactive oxygen species generation in HESCs impair HEEC survival resulting in a loss in vascular integrity, demonstrating a novel paracrine mechanism to explain LAPC-induced abnormal uterine bleeding.

By providing safe, discrete, and highly effective contraception, long-acting progestin-only contraceptives (LAPCs) are ideal for use in underdeveloped countries with limited access to trained medical personnel and by women in whom estrogen use is contraindicated (1). Currently available formulations include Depo-Provera, an injectable form of medroxyprogesterone acetate (MPA); Implanon, a subdermal rod releasing etonogestrel (ETO); and Mirena, an intrauterine device that releases levonorgestrel. According to World Contraceptive Use, 2005, specific LAPCs, Depo-Provera, or Implanon, were used by 0.7% of women in developed countries and 3.6% of women in developing countries, whereas Mirena was used by 7.6% of women in developed countries and 14.5% of women in developing countries (http://www.dialoguedynamics.com/contenu/learning-forum/seminars/the-contraception-abortion-nexus/ and http://www.un.org/esa/population/publications/contraceptive2005/WCU2005.htm). Adherence to LAPCs is diminished by abnormal uterine bleeding (AUB) (2, 3), which is a source of personal annoyance, discomfort, and/or religious and social taboo in specific societies (2–4). Unlike menstrual bleeding, which originates globally from spiral arterioles as an organized response to circulating progesterone (P₄) withdrawal, LAPC-associated AUB occurs intermittently and focally from irregularly distributed, superficial, abnormally enlarged, fragile capillaries and venules (5).

Laser-Doppler fluxmetry reveals that LAPC administration to women impairs endometrial microvascular perfusion to induce local hypoxia (HX)/reperfusion injury (6). Administration of LAPCs also induces vasoconstriction in guinea pig uterine arteries (7) and generates excess reactive oxygen species (ROS) such as 8-isoprostane, a marker of oxidative fatty acids; 8-hydroxy-2′-deoxyguanosine, a marker of oxidative DNA damage; and nitrotyrosine, a marker of oxidative protein damage (7, 8).

Maintenance of microvascular integrity depends on endothelial cell survival, which reflects the balance between proliferation and apoptosis (9), with impaired survival of human endometrial microvascular endothelial cells (HEECs) likely playing a critical role in LAPC-induced AUB. The endometria of women administered LAPCs contains microvessels enmeshed in a matrix of decidualized stromal cells. Binding of P₄ to P₄ receptors (PRs) induces human endometrial stromal cells (HESCs) to undergo in vivo and in vitro decidualization (10, 11), whereas PRs are not expressed in primary HEECs (12, 13). We hypothesize that progestins interact with HESC-expressed PRs to induce secretion of paracrine factors that impair survival of HEECs.

To test this hypothesis, proliferation and apoptosis analyses were performed on primary cultures of HEECs and human umbilical venous endothelial cells (HUVECs) treated with HESC-derived conditioned medium supernatant (CMS) after incubation in estradiol (E₂) ± P₄, or MPA, or ETO. To mimic the reduced uterine blood flow resulting in local HX in women administered LAPCs, HESCs were cultured in parallel under normoxia (NX) or HX. Proteomic analysis was performed on CMSs by mass spectrometry (MS) for the presence of potential mediators of apoptosis and proliferation. HEECs and HUVECs were incubated with the MS-identified compounds. In situ confirmation of the expression of these identified compounds and the apoptosis index were assessed on paired endometrial sections from women before and after Depo-Provera by immunostaining. Transcriptome and pathway analyses were carried out to determine whole genome changes and related intracellular signaling cascades in HEECs treated with HESC-derived CMS.

Materials and Methods

Tissues

Immunohistochemistry was performed on paired endometrial tissues obtained from women before vs after Depo-Provera therapy (n = 5) after receiving written informed consent at New York University (NYU), under Institutional Review Board (IRB) approval. Endometrial specimens were collected by Pipelle biopsy during either the proliferative or secretory phase before Depo-Provera and 3 months after Depo-Provera injection. HESCs were isolated from endometrial tissues obtained from reproductive-age women with regular menstrual cycles undergoing laparoscopy or hysterectomy for benign gynecological conditions. Frozen HESCs from previously banked samples (n = 3; two from late secretory phase and one from early proliferative phase, established from the women's menstrual history and confirmed by a histopathologist) under NYU IRB approval were thawed and cultured.

Immunohistochemistry

Paraffin sections were blocked in 5% normal goat serum and incubated overnight at 4°C with a rabbit anticleaved caspase-3 (CASP3) (Cell Signaling) or a rabbit anti-neuronal pentraxin-1 (NPTX1) antibody (LSBio). Sections were washed in Tris-buffered saline containing 0.1% Tween-20 (TBS-T) and incubated with the biotinylated antirabbit secondary antibody (Vector Laboratories) for 30 minutes. After rinsing in TBS-T, sections were incubated in streptavidin-peroxidase complex (Elite ABC Kit; Vector Laboratories). Immunoreactivity was developed by diaminobenzidine tetrahydrochloride dihydrate (Vector Laboratories). Hematoxylin was used for background staining. Nonspecific rabbit IgG was used as a negative control at the same concentrations as the primary antibodies. Slides were analyzed by histological scoring (HSCORE) as described (14).

HESC cultures

Cultured HESCs were isolated, characterized, and cultured in basal medium, a phenol red-free 1:1 vol/vol mix of Dulbecco's MEM/F12 (Gibco) as previously described (15). Confluent HESCs were incubated in parallel in basal medium with 10⁻⁸ m E₂ (Sigma-Aldrich) used as the control, or E₂ + 10⁻⁷ m P₄ (Sigma-Aldrich), or E₂ + 10⁻⁷ m ETO (Organon), or E₂ + 10⁻⁷ m MPA (Sigma-Aldrich). After 7 days, the cultures were switched to serum-free defined media as described (15), containing corresponding vehicle control or steroids for 24 hours. The cultures were incubated in defined media for 24 and 48 hours under NX (20% O₂) or HX (0.5% O₂). After centrifugation, CMSs were stored in −70°C at the end of experimental test periods for future use in endothelial cell assays.

HEEC and HUVEC cultures

HEECs were isolated and characterized as described (13, 16) and grown in EGM-2 MV Singlequot Medium (Cambrex Bio Science) with 5% stripped fetal calf serum. HUVECs were purchased from Life Technologies and grown in EGM-2 MV with 5% stripped fetal calf serum.

Confluent HEECs (n = 3 patients) and HUVECs seeded at 2 × 10⁴/well were incubated for 48 hours in 96-well plates with HESC-derived CMS obtained after 24 and 48 hours of treatment as described above. Total RNA and protein lysates for microarray and immunoblot analysis, respectively, were obtained from HEECs cultured in six-well plates treated with HESC-derived CMS. In parallel experiments, HEECs were also incubated with serum-free EGM-2 MV containing vehicle, or 1, 10, and 100 ng of recombinant human (rh) serpin peptidase inhibitor, clade E member 1 (SERPINE1, aka plasminogen activator inhibitor-1) or rhNPTX1 (both from R&D Systems) ± 5 and 50 μm H₂O₂, a major cellular ROS (17).

Apoptosis and cell proliferation assay

An ELISA-based cell death detection kit (Roche) quantified cytoplasmic histone-associated DNA fragments in HEECs and HUVECs incubated with HESC-derived CMS at the end of each treatment period. Cell proliferation of HEECs and HUVECs was detected by bromodeoxyuridine (BrdU) incorporation into proliferating cellular DNA using the BrdU Cell Proliferation Kit (Cell Signaling) according to the manufacturer's instructions.

Microarray analysis

HEECs were incubated with HESC-derived CMS under NX or HX. After 6 hours, total RNA was isolated from HEECs using miRNeasy mini kit and RNeasy MinElute cleanup kit (QIAGEN) according to the manufacturer's instructions. Extracted total RNA was subjected to microarray analysis at the Keck Biotechnology Resource Laboratory at Yale University, using a HumanHT-12 v4 Expression BeadChip Kit (Illumina). Sample labeling and hybridization were performed per the manufacturer's instructions. Raw data without normalization were analyzed by GeneSpring GX12.5 software (Agilent Technologies-Silicon Genetics). Gene readouts were normalized to the 75th percentile of the distribution of all measurements in each chip. Normalization for each gene across chips was performed using the median value of each gene throughout different chips in the same experimental condition. Normalized data were first filtered to eliminate genes absent in all experimental conditions and replicates, and then filtered on a volcano plot with moderated t test without multiple testing correction. Genes with a fold change of >1.25 and a P value of <.05 were considered differentially expressed. Molecular functions and biological networks related to differentially expressed genes were explored using Ingenuity Pathway Analysis (IPA) software (Ingenuity Systems) according to the manufacturer's instructions or using the Database for Annotation, Visualization and Integrated Discovery (DAVID) version 6.7 software (which is freely accessed at http://david.abcc.ncifcrf.gov/, a web-based online bioinformatics resource from the National Institutes of Health).

MS analysis

Serum-free CMSs obtained from HESCs treated with E₂, or E₂+P₄, or E₂+ETO, or E₂+MPA under NX or HX were collected after 24 hours, then centrifuged for 10 minutes at 3000 × g. Eighty microliters of CMS was combined with 16 μL of 100 mm NH₃HCO₃ and 300 ng of trypsin for overnight 37°C incubation, followed by speed-vac drying. Twenty microliters of 2% acetonitrile and 0.1% formic acid was added, and the samples were assessed at A280 using formic acid as a blank on a Nanodrop spectrophotometer. Subsequently, 1.5 μg of peptides were analyzed by sensitive liquid chromatography-electrospray ionization/multistage MS using an Easy-nLC II (Thermo Scientific) coupled to a hybrid Orbitrap Elite ETD (Thermo Scientific). A 90-minute gradient from 7 to 35% acetonitrile in 0.1% formic acid at a flow rate of 300 nL/min was used after in-line desalting. Protein matches were retained, based on a false discovery rate threshold of 5% and the presence of two unique peptide matches. Differences in peptide abundances were corrected by normalizing each sample to the geometric mean of the 50 most highly detected spectral counts. Student's t test used normalized counts to calculate statistical significance of pair-wise comparisons. Each calculation is performed in the R statistical computing and graphics environment (http://www.r-project.org).

Immunoblot analysis

Immunoblot analysis was performed on whole cell extracts obtained from HEECs incubated with HESC-derived CMS for 15 minutes as described previously (14). The membranes were incubated with anti-total-(T) AKT rabbit, anti-phosphorylated (P)-AKT rabbit, and anti-T ERK1/2 MAPK rabbit and anti-P-ERK1/2 MAPK rabbit antibodies (Cell Signaling) for primary antibody labeling. The membrane was then incubated with horseradish peroxidase-conjugated goat antirabbit IgG (Vector Laboratories) for secondary antibody labeling. Signals were developed using a chemiluminescence kit (GE Healthcare). The membranes were sequentially stripped and finally reprobed with anti-β-actin rabbit monoclonal antibody (Cell Signaling).

Statistical analysis

Results for proliferation and apoptosis assays, immunoblot analysis, and HSCORE were analyzed by one-way ANOVA, followed by analysis by the Student-Newman-Keuls method. Immunostaining-associated HSCOREs for cleaved CASP3 and NPTX1 were analyzed using a t test. Statistical calculations were performed using SigmaStat version 3.0 (Systat Software).

Results

Selective induction of apoptosis in HEECs by hypoxic CMS from LAPC-induced HESCs

To determine whether LAPCs stimulate HESCs to secrete factor(s) that affect HEEC survival, primary HESCs were incubated with E₂, as the control, or with E₂+P₄, or E₂+ETO, or E₂+MPA under NX or HX for 24 and 48 hours. The resultant HESC CMSs were then used to treat HEECs for 48 hours. Thus, CMS derived from 48-hour steroid-treated HESCs maintained either under NX or HX failed to affect proliferation indices in either HEECs or HUVECs (Figure 1, A and B) compared with control CMS derived from HESCs treated for 48 hours with E₂ under NX or HX. In contrast, a significant increase in the apoptotic index was detected in HEECs (Figure 1C) incubated with HESC-derived CMS from cultures maintained in E₂+ETO or E₂+MPA under HX for 48 hours (mean ± SEM in HEECs, 0.87 ± 0.10 and 0.93 ± 0.11, respectively) compared to control HESC CMS (48-h E₂ treatment under HX; mean ± SEM in HEEC, 0.63 ± 0.07). However, no increase in the apoptotic index was detected after such treatment in HUVECs (Figure 1D), nor did treatment of HEECs or HUVECs with CMS from E₂+P₄-treated cultures under HX increase apoptosis (Figure 1, C and D). Treatment of HEECs and HUVECs with CMS derived from HESCs incubated with any steroid combination under either NX or HX for 24 hours failed to alter proliferation or the apoptotic index (Supplemental Figure 1, A–D). To ascertain whether progestins directly induce endothelial apoptosis, cultured HEECs were incubated in parallel with 10⁻⁶ to 10⁻⁸ m P₄ or ETO or MPA. Over this concentration range, none of the progestins significantly affected the apoptotic index (Supplemental Figure 1E), which is consistent with the absence of demonstrable PR expression in HEECs (12, 13).

Figure 1.

Enhanced apoptosis in HEECs incubated with secreted factors from LAPC-induced HESCs, and in endometrial microvascular endothelium of post-Depo users. BrdU incorporation in HEECs (A) or HUVECs (B) after 48 hours incubating with HESC CMS derived from 48-hour incubations with E₂ alone, or E₂+P₄, or E₂+ETO, or E₂+MPA under NX or HX. Bar represents mean ± SEM. Graphs represent replicates of eight from two independent experiments. Apoptotic index in HEECs (C) and HUVECs (D) after 48-hour incubations with HESC CMS derived from 48-hour incubations with E₂ alone, or E₂+P₄, or E₂+ETO or E₂+MPA under NX or HX. #, vs E₂ alone; *, HX vs NX; P < .05. Bar represents mean ± SEM; graphs represent replicates of 12 from three independent experiments. E, Immunoreactive cleaved CASP3 in endothelial cells (arrows) of paired endometrial sections obtained from women before Depo-Provera (pre-Depo) and after (post-Depo) use. Stronger CASP3 immunoreactivity at endothelium (arrowhead) of BL vs NBL site. Scale bar, 50 μm. Graph represents HSCOREs for cleaved CASP3 immunoreactivity in endometrial microvascular endothelial cells in pre = and post-Depo users. Bars represents means ± SEM. *, P < .001, post-Depo vs pre-Depo, n = 5. E₂, 10⁻⁸ m E₂; P, 10⁻⁷ m P₄; ETO, 10⁻⁷ m ETO; MPA, 10⁻⁷ m MPA.

Depo-Provera administration increases cleaved CASP3 immunoreactivity in microvascular endometrial endothelial cells

Paraffin sections from banked paired endometrial tissues obtained from women before and after Depo-Provera administration were immunostained using an antibody specific for cleaved CASP3. Compared with specimens obtained before Depo-Provera treatment (pre-Depo), cleaved CASP3 immunoreactivity was significantly higher in endometrial microvascular endothelial cells in post-Depo users (Figure 1E; P < .001). Moreover, more pronounced cleaved CASP3 immunoreactivity was seen in endometrial microvascular endothelium at bleeding (BL) vs nonbleeding (NBL) sites among women administered Depo-Provera (Figure 1E).

MS secretome analysis identifies candidates that induce HEEC apoptosis

To identify paracrine factors present in HESC-derived CMS capable of inducing HEEC apoptosis, MS compared secretome profiles of CMS collected from HESCs after 48-hour incubation with E₂ ± progestins under NX vs HX. Among several differentially expressed proteins, only SERPINE1 and NPTX1 were found to be significantly and selectively increased in the CMS of E₂+MPA- or E₂+ETO-treated HESCs under NX vs E₂ alone and further increased under HX (Figure 2A; P < .05).

Figure 2.

Identification of candidate secreted factors from LAPC-induced HESCs and their role in endometrium. A, MS identified increased expression of SERPINE1 and NPTX1 as potential apoptotic inducers increased in CMSs derived from E₂+MPA- and E₂+ETO-treated HESCs under HX vs parallel incubation under NX. Y-axis displays peptide numbers of candidate proteins counted in HESC-derived CMS by MS; P < .05 for changes in NPTX1 levels. B, Enhanced apoptotic index in HEECs treated with rhNPTX1 in a dose-dependent manner when compared to control (P < .05). C, Enhanced apoptotic index of HEECs by rhNPTX1 and H₂O₂ alone or by their combination. *, P < .05 vs control; #, P < .001 vs the corresponding concentration of rhNPTX1 or H₂O₂ alone. Bars represent mean ± SEM. D, Immunoreactive NPTX1 expression in paired endometrial samples from pre- and post-Depo users, and post-Depo BL and NBL endometrial sites. Higher NPTX1 immunoreactivity in HESCs in paired post- vs pre-Depo endometria (*, P < .001 vs pre-Depo). Increased NPTX1 immunoreactivity localized to vascular endothelium in BL vs NBL sites of post-Depo endometrium. Original magnification, ×40. Scale bar, 50 μm.

Synergistic induction of HEEC apoptosis by NPTX1 and H₂O₂

To evaluate the apoptotic and proliferative effects of the candidate proteins identified by MS, cultured HEECs were incubated with 1 to 100 ng/ml of rhSERPINE1 or rhNPTX1 for 48 hours. Neither rhSERPINE1 nor rhNPTX1 affected proliferation of HEECs (Supplemental Figure 2, A and B). Moreover, rhSERPINE1 failed to affect HEEC apoptosis over the entire concentration range (Supplemental Figure 2C). By contrast, 1 to 100 ng/mL of rhNPTX1 significantly increased HEEC apoptosis (Figure 2B; P < .05). Our prior observations that LAPC reduced endometrial blood flow and induced local HX/reperfusion-mediated ROS generation in women (18), taken together with similar observations in the LAPC-treated guinea pig endometrium (7), prompted evaluation of the separate and combined effects of ROS (such as H₂O₂) and rhNPTX1 in HEECs. Both 5 and 50 μm H₂O₂ significantly induced apoptosis in HEECs vs control (Figure 2C; P < .05). Moreover, the combination of H₂O₂ and rhNPTX1 synergistically increased HEEC apoptosis, with 10 ng/mL rhNPTX1 plus 5 μm H₂O₂ generating maximal effect (Figure 2C; P < .001).

LAPCs enhance NPTX1 expression in human endometrium

To confirm in vitro observations (Figure 2, A–C), immunohistochemical staining for NPTX1 was performed on paired endometrial sections from women pre- and post-Depo and from specific BL and NBL sites in post-Depo endometria. Increased NPTX1 immunoreactivity confirmed by HSCORES was observed in HESCs in post-Depo vs paired pre-Depo endometria (Figure 2D; P < .001). Moreover, post-Depo endometria displayed greater NPTX1 immunoreactivity in perivascular BL vs NBL sites (Figure 2D).

NPTX1-induced HEEC apoptosis is associated with increased cytoplasmic cytochrome c release

Microarray analysis was used to detect global transcriptional changes in HEECs treated with HESC-derived CMS. Specifically, in incubations under HX, HEECs incubated with CMS from E₂+MPA- or E₂+ETO-treated HESCs were observed differentially expressed 113 and 176 genes, respectively, compared to HEECs incubated with CMS from corresponding E₂-treated HESCs. Among the 113 MPA-regulated genes, 10 (8.8%) were found to be up-regulated, and 103 (91.2%) down-regulated (Figure 3A). By comparison, among the 176 ETO-regulated genes, six (3.4%) were found to be up-regulated, and 170 (96.6%) were down-regulated (Figure 3B). Further evaluation of the differentially regulated genes in HEECs treated with CMS derived from HESCs incubated with E₂+ ETO or E₂+MPA under HX utilizing IPA of the microarray gene expression data revealed common upstream mediators of apoptosis including activation of CD437 (synthetic retinoid as a selective agonist of retinoic acid receptor-γ) (Figure 3C; P value = 8.56E⁻⁰⁶), as well as inhibition of the anti-apoptotic serine/threonine-specific protein kinase (AKT; P value = 3.62E⁻⁰²), and extracellular-signal-regulated kinases (ERK1/2; P value = 3.82E⁻⁰²) signaling pathways. The pivotal role played by CD437 in promoting apoptosis has been attributed to promotion of mitochondrial dysfunction via enhanced mitochondrial permeability (19). Previous studies indicating that CD437 inhibited both AKT (20) and ERK1/2 (21) signaling prompted immunoblotting of total and phosphorylated levels of both signaling pathways. The results singled out reduced levels of P-AKT (Figure 3D; P < .05), but not P-ERK1/2 (data not shown) by CMS derived from HESCs incubated with E₂+ETO or E₂+MPA under HX, but not NX. In contrast, no effects were observed from CMS derived from HESCs incubated with E₂+P₄ under either NX or HX (Figure 3D).

Figure 3.

Regulation of gene expression in HEECs by CMS derived from E₂+MPA- or E₂+ETO-treated HESC. A and B, Percentage of genes up- or down-regulated in HEECs incubated with HX derived CMS from E₂+MPA-treated (A) or E₂+ETO-treated (B) HESCs compared to that in HEECs incubated with NX derived CMS from HESCs treated with corresponding steroids. C, IPA of microarray data predicts that activation of CD437 signaling as the upstream regulators in HEECs incubated with CMS derived from HESCs treated with E₂+MPA or E₂+ETO under HX. D, Representative immunoblot bands showing total (T)-AKT and phosphorylated (P)-AKT compared with β-Actin in 15-minute incubation of HEECs with CMS derived from HESCs treated 48 hours with E₂, or E₂+P_4, or E₂+ETO, or E₂+MPA under NX or HX. Bars represent mean ± SEM. *, P < .05 (n = 3).

DAVID analyses of HEEC microarray data revealed that CMS from E₂+MPA- or E₂+ETO-treated HESCs under HX down-regulates several genes in HEECs that control mitochondrial homeostasis (Figure 4A). These genes are essential for functioning of the mitochondrial electron transport complex (Cx) I, III, IV, and V (Figure 4, A and B). To confirm that NPTX1 mediates mitochondrial dysfunction in HEECs, we assessed its direct effect on cytosolic cytochrome c release (22). Specifically, immunocytochemistry for cytochrome c expression was performed on cultured HEECs treated with rhNPTX1 ± H₂O₂. Very weak or absent immunoreactivity for cytochrome c expression was observed in control HEECs (Figure 5A), whereas higher immunoreactivity was observed after incubation with 10 or 100 ng/mL rhNPTX1 (Figure 5, B and C, respectively). Significantly higher cytochrome c immunoreactivity was observed in HEECs treated with rhNPTX+H₂O₂ (Figure 5, D and E), with the combination of 10 ng/mL of rhNPTX1 plus 5 μm of H₂O₂ inducing the highest immunoreactivity (Figure 5D).

Figure 4.

Mitochondrial dysfunction in HEECs treated with HX-CMS derived from E₂+MPA- or E₂+ETO-treated HESC. A, List shows the differentially regulated genes causing mitochondrial dysfunction by DAVID analysis in HEECs treated with HX-CMSs from E₂+ETO- or E₂+MPA-treated HESC compared to that in HEECs incubated with NX derived CMS from HESCs treated with corresponding steroids. B, Localization of these down-regulated genes in mitochondrial Complex I (Cx I), Cx III, Cx IV, and Cx V (red stars) and their relationship to induce apoptosis. Schema were obtained from DAVID analysis and modified from KEGG pathway maps (http://www.genome.jp/kegg-bin/show_pathway?map05012).

Figure 5.

NPTX1 increases cytochrome c release by HEECs. Cytochrome c immunoreactivity in cultured HEECs treated with control (A), or with 10 and 100 ng/mL rhNPTX1 (B and C, respectively) alone, or with 5 and 50 μm H₂O₂ (D and E, respectively) for 72 hours. F, Graph represents HSCORE for cytochrome c immunoreactivity. Bars represent mean ± SEM. *, P < .001 vs all treatment conditions. Original image, ×40. Scale bar, 25 μm.

Discussion

The endometria of women receiving LAPCs contain enlarged thin-walled fragile microvessels that are irregularly distributed across the superficial layer and display intermittent focal bleeding. We observed that LAPC administration results in reduced endometrial blood flow, which promotes local HX and ROS generation, damaging microvascular endothelial cells and enhancing production of proangiogenic factors by HESCs (8) (Figure 6). Specifically, the endometria of women receiving LAPCs display elevated level of vascular endothelial growth factor (VEGF), the primary mediator of angiogenesis (23, 24). Although physiological VEGF levels promote angiogenesis, overexpressed VEGF induces endothelial vascular “leakiness” and perivascular extracellular matrix dissolution that culminates in bleeding (25). Moreover, our studies using primary cultures of HESCs and HEECs demonstrate that progestins inhibit HESC-expressed angiopoietin (Ang)-1, a vessel-stabilizing factor, whereas HX enhances HEEC-expressed Ang-2, which promotes vessel branching and enhances endothelial cell permeability. These findings were confirmed in situ by immunohistochemical studies of LAPC exposed endometria (18). The current study postulates that LAPCs target PRs expressed by HESCs to induce secretion of factors that compromise survival of adjacent PR-negative HEECs. This question was approached by incubating primary cultured HESCs with P₄, MPA, or ETO under either NX or HX to mimic the effects of LAPC-induced HX/ROS (18). A significant time-dependent increase in the apoptotic index was detected in HEECs, but not in HUVECs, incubated with HESC-derived CMS under HX with either of the synthetic progestins, ETO or MPA, but not with native P₄. Additional experiments confirmed the requirement for HESC mediation because no change in the apoptotic index was observed in HEECs directly treated with these progestins. In situ confirmation of these in vitro results was obtained by demonstrating increased immunostaining for cleaved CASP3, a marker apoptosis, in the microvascular endothelium of endometrial sections obtained from women post- vs pre-Depo use. In the former, higher levels of cleaved CASP3 were observed in the microvascular endothelium of BL vs NBL sites, providing a direct correlation between HEEC apoptosis and AUB. The absence of a proapoptotic effect of P₄ vs the synthetic progestins may reflect rapid metabolism of P₄ in cultured HESCs (28) or documented glucocorticoid and/or mineralocorticoid effects of these synthetic progestins (29).

Figure 6.

Mechanisms mediating LAPC-induced AUB in women. LAPCs reduce endometrial blood flow, causing HX and ROS generation that directly damages vessels and initiates aberrant angiogenesis mediated by excess VEGF and Ang-2, whereas progestins inhibit vessel stabilizing Ang-1. In turn, the combined effects of HX and MPA or ETO further enhances NPTX1 expression in HESCs that acts in a paracrine fashion to trigger HEEC apoptosis by inhibiting AKT signaling, down-regulating several mitochondrial function-related genes, enhancing cytochrome c release and CASP3 cleavage. The resulting increased endothelial cell death and loss of vascular integrity promotes AUB. (Previous findings are shown in black, whereas the findings of the current study are shown in red).

Consistent with the growing use of MS to identify protein mediators in various pathological processes, the current study utilized secretomic analyses to search the CMS from progestin/HX treated HESCs for potential paracrine inducers of apoptosis in HEECs. Among several candidate proteins, NPTX1 and SERPINE1 alone were significantly increased during incubation with either E₂+MPA or E₂+ETO under NX with a further increase observed in parallel incubations under HX. Subsequently, exogenous NPTX1, but not SERPINE1, was observed to significantly increase the apoptotic index in cultured HEECs. The failure of SERPINE1 to induce apoptosis in HEECs is consistent with in vivo and in vitro identification of SERPINE1 as a product of decidualized stromal cells that maintains hemostasis during the luteal phase of the menstrual cycle (30). Therefore, we explored NPTX1-induced apoptosis in HEECs.

The NPTX1 molecule is a 47-kDa secretory glycoprotein with homology to the pentraxin family and is highly expressed in neurons of the cerebellum, hippocampus, and cerebral cortex (31). Increased expression of NPTX1 in response to HX induces apoptosis in several cell types (32, 33). The involvement of enhanced HESC-expressed NPTX1 as a paracrine inducer of apoptosis in HEECs is supported by the in situ demonstration of significantly elevated immunoreactive NPTX1 expression in stromal cells surrounding compromised microvessels at BL vs NBL sites of the post-Depo endometrium.

Microarray analysis identified dysregulated transcription of multiple genes in HEECs after incubation with CMS derived from HESCs induced by either E₂+MPA or E₂+ETO under HX. Subsequent IPA analysis of these dysregulated genes revealed activation of CD437 and inhibition of AKT and ERK1/2. By enhancing mitochondrial permeability, CD437 triggers mitochondrial dysfunction (34) and induces apoptosis by stimulating the release of cytochrome c and subsequent Apaf-1-dependent activation of caspases (35). Moreover, CD437 also inhibits AKT signaling (20), which regulates glucose uptake, energy metabolism, mitochondrial integrity, and cell survival (36). Inhibition of the AKT signaling pathway triggers mitochondrial dysfunction and subsequent cell death (37–39). In the current study, 1) immunoblotting found significantly reduced HEEC AKT phosphorylation, consistent with induction of the apoptosis by HESC CMS; and 2) DAVID analysis identified down-regulation of eight genes essential for functioning of mitochondrial electron transport. The observed increase in cytosolic levels of cytochrome c in incubation of HEECs with NPTX1 ± ROS is consistent with the documented association of mitochondrial dysfunction and the subsequent release of cytochrome c to the cytoplasm (19, 22, 40). In conclusion, the results of this study indicate that increased HEEC apoptosis resulting from elevated NPTX1 secretion interacting with enhanced ROS generation by LAPC-induced PR-expressing HESCs impairs vascular integrity, which provides a novel paracrine mechanism that initiates AUB.