Hum. Reprod. Advance Access originally published online on October 23, 2007
Human Reproduction 2007 22(12):3159-3169; doi:10.1093/humrep/dem266
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Differentially expressed genes in human endometrial endothelial cells derived from eutopic endometrium of patients with endometriosis compared with those from patients without endometriosis
1 Department of Obstetrics and Gynecology, Peking Union Medical College Hospital, Peking Union Medical College, Chinese Academy of Medical Sciences, Peking, People's Republic of China 2 Beijing Huada Gene Research Centre, Chinese Academy of Science, Peking, People's Republic of China 3 Capitalbio Corporation, National Engineer Research Center for Beijing Biochip Technology, Peking, People's Republic of China
4Correspondence address. Tel: +86-10-65296201; Fax: +86-10-65296212; E-mail: langjinghe{at}hotmail.com
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionConclusionsSupplementary dataReferences |
|---|
BACKGROUND: The pathogenesis of endometriosis remains poorly defined. The aberrant angiogenesis that occurs in eutopic endometrium may play a role in the lesion formation and survival. The difference in gene expression profile between human endometrial endothelial cells (HEECs) from eutopic endometria of patients with and without endometriosis would be a factor that affects the occurrence of endometriosis.
METHODS: To explore the difference, we performed in vitro culture and identified the endothelial origin, as well as observed growth features, of HEECs from the two different sources. We also identified their differences in gene expression profiles by combined suppression subtractive hybridization (SSH) with Genechip, and confirmed the results by quantitative reverse transcription–polymerase chain reaction.
RESULTS: The HEECs derived from endometriosis patients exhibited a potent survival ability in vitro compared with those from non-endometriosis patients. In the HEECs from EM patients, an altered secretion pattern of extracellular matrix (ECM) components and up-regulation of GREM1 were found. These findings may be used to interpret the remarkable change of phenotype in HEECs from endometriosis patients. The synergistic action of these differentially expressed genes is to promote cell proliferation and concomitantly to inhibit apoptosis. Among the up-regulated ECM genes, TSP2 was the only one which exhibits the capacity to suppress angiogenesis; it may therefore function as an antagonist to the aberrant angiogenesis and may confine its extent and severity.
CONCLUSION: It may be postulated that differential regulation of some of these genes in eutopic HEECs plays a facilitating role during the peritoneal vascularization of ectopic endometrial lesions by enhancing angiogenic activity via a paracrine effect.
Key words: endometriosis/angiogenesis/HEEC/gremlin 1/ECM
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionsSupplementary dataReferences |
|---|
Endometriosis (EM) is a common gynecological disease characterized by extrauterine implantation and ectopic growth of endometrium; it affects
10% of women during their reproductive years. However, the underlying mechanism has not been completely elucidated yet.
Angiogenesis plays a pivotal role in implantation of ectopic endometrium and lesion formation thereafter (Groothuis et al., 2005
). Active endometriotic explants have pronounced vascularization both within and around the tissue (Nisolle et al., 1993). Inhibitors of angiogenesis effectively interfere with the maintenance and growth of EM (Nap et al., 2004; Laschke et al., 2006). The peritoneal fluid from patients with EM contains significantly greater amounts of vascular endothelial growth factor (VEGF) compared with that from controls (Mclaren et al., 1996a), and the major source of this VEGF is peritoneal macrophages which are activated in EM, to a greater extent than in controls (Mclaren et al., 1996b).
The corresponding endothelium plays a central role during the process of angiogenesis in specific tissues. With use of immunomagnetic binding, human endometrial endothelial cells (HEECs) have been isolated in vitro (Iruela-Arispe et al., 1999; Nikitenko et al., 2000; Schatz et al., 2000; Koolwijk et al., 2001; Kayisli et al., 2004). HEECs exhibit a different growth response to progesterone compared to endometrial epithelial cells (Kayisli et al., 2004).
To date, several groups of authors have reported their results on differential expression profiles with microarrays between eutopic endometria of EM patients and normal controls, or between ectopic lesions and their eutopic counterparts. They have paid attention to epithelial and/or stromal compartments through laser capture microdissection or have used entire endometrial tissue without discriminating between the cellular compartments (Wu et al., 2006). We have selected, apparently for the first time, the eutopic endothelial compartment as the subject for exploring the differential expression profile between EM patients and normal controls. It is important to consider that (i) the endothelium is enmeshed in a complex tissue consisting of vessel wall components, stromal cells as well as epithelial cells; (ii) only a small fraction of the cells within these tissues are endothelial (Croix et al., 2000) as well as that (iii) endometrial cyclical angiogenesis has been remained largely unexplored such that some low abundance or novel transcripts involved in this complex and dramatically changed process remain unknown. As a result, it is impossible to obtain the differences in expression of these genes with the microarray technique alone because of the absence of the dots representing these genes on present commercially available microarrays. We therefore chose to address the differences in phenotype and expression profile between the eutopic endometrial endothelial cells derived from patients with and without EM through the combination of diverse technologies available at present.
Thus, we isolated HEECs from eutopic endometria of patients with and without EM and observed the vigorous viability of HEECs from patients with EM. Thereafter, we explored the differential gene profile between the two sources of HEECs with the methods of suppression subtractive hybridization (SSH) and Affymetrix genechip® microarray, in which the differences of relatively high abundance genes are always displayed (Cao et al., 2004). The results were validated through quantitative reverse transcription polymerase chain reaction (qRT–PCR).
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionsSupplementary dataReferences |
|---|
Materials
Penicillin, streptomycin, L-glutamine, EDTA and Dulbecco's modified Eagle medium (DMEM) with or without phenol red were obtained from GIBCO (Invitrogen Corporation, Grant Island, NY, USA). Defined fetal bovine serum (FBS) and charcoal dextran treated FBS from which the influence of some growth factor and hormones had been excluded were obtained from HyClone Corporation, Logan, UT, USA. Collagenase I, trypsin, endothelial cell growth supplement (ECGS), tumor necrosis factor-
(TNF-) were obtained from Sigma Corporation, St. Louis, MO, USA. Rabbit anti-human vWF monoclonal antibody were obtained from Sigma, mouse anti-human CD31, CD34 and ELAM-1 monoclonal antibody were purchased from R & D Corp., Mineapolis, MN, USA. Goat anti-rabbit and rat anti-mouse FITC-conjugated secondary antibody were obtained from Sigma. Acetate-DiI-Low density lipoprotein (Ac-DiI-LDL) were obtained from Molecular probe Corporation, Eugene, OR, USA.
TRIzol®and ElectroMAX DH5-E cells were from Invitrogen, Carisbad, CA. Oligotex mRNA Spin-Column kit, QIAquick PCR purification kit and TA cloning kit were from Qiagen, GmbH, Hilden, Germany. PCR-SELECT cDNA subtraction kit was manufactured by CLONTECH, Palo Alto, CA. MessageAmpTM II aRNA Amplification Kit was from Ambion, Austin, TX, USA.
Isolation and culture of HEECs
The isolation and culture of HEECs were according to a previously reported procedure with a little modification (Koolwijk et al., 2001). Here, we used endometria scraped from premenopausal uteri which were resected for reasons other than endometrial abnormalities or endometria that were scraped during the process of laparoscopy operation or conization. The patients selected for obtaining endometria had regular cycles and were not on hormonal medications for 6 months. The diagnosis of EM was confirmed or excluded by either laparoscopy or laparotomy. Informed consent was obtained from all patients and the use of human specimens for these experiments was reviewed and approved by the committee of medical ethics, Peking Union Medical Collage Hospital, Beijing, China. The detailed information on case histories is listed on Table 1, and stages of the cycles were confirmed by histological examination and last menstruation period prior to the operations.
|
For cell isolation, the endometrium was minced and incubated in 0.5% collagenase I at 37°C for 20–30 min, followed by filtration through a metal mesh (70 µM) screen for removal of the undigested tissue. The primary cultures were performed in endothelium culture medium (DMEM supplemented with 20 mM HEPES, 100 U/ml penicillin, 100 µg/ml streptomycin, 20% FBS, 100 µg/ml ECGS, 40 U/ml heparin and 40 µU/ml Insulin) at 37°C in 5% CO2.
The primary heterogeneous cell populations were allowed to grow to near confluence before HEECs isolation. The cells were incubated in DMEM containing 0.1% bovine serum albumin (BSA) with mouse anti-human CD34 monoclonal antibody-coated beads (20 beads/target cell). A 15–30-min end-over-end rotation was performed at 4°C before the cells bound to beads were separated using a magnet (Dynal). The positively isolated cells were cultured in endothelium culture medium at a density of 500–1000 cells per cm2.
The homogenous endothelial cell clones were observed after several days. Occasionally, some scattered or clustered stroma-like cells around the endothelial clones appeared. According to different sensitivity to trypsin/EDTA, the stroma-like cells could be eliminated by transient treatment of trypsin/EDTA. The remaining cells were incubated and fed with fresh medium every 3 days until reaching confluence after 15–20 days. For subcultures, confluent HEECs were split 1:3 and maintained for 6–15 passages.
Characterization of isolated HEECs
Cells were plated on coverslips for subsequent manipulation. For examining the expression of vWF, CD31 and CD34, the cells were blocked with PBS containing 0.1% BSA, incubated with rabbit anti-human vWF monoclonal antibody or mouse anti-human CD31 or CD34, respectively, at room temperature for 45 min. After completely washing with PBS, incubation with FITC-labeled rat anti-rabbit or anti-mouse monoclonal antibody was performed for 30 min. The purity of cultured HEECs was examined by flow cytometry. Parallel procedures, except for the primary antibodies being substituted with related antibody derived from the same source, were performed as negative controls.
The medium of cells grown on coverslips was changed to DMEM containing 1 µg/100 µl 1,1'dioctadecyl-3,3,3',3'- tetramethyl-indocarbocyanine perchlorate acetylated low density lipoprotein (DiI-Ac-LDL). After incubation at 37°C for 4 h, the coverslips were fixed and observed.
After treatment with 30 ng/ml TNF- for 6 h, HEECs were incubated with mouse anti-human ELAM-1 monoclonal antibody for 30 min at 4°C. Rat anti-mouse monoclonal antibody was used as secondary antibody. The percentage of positive cells was examined by flow cytometry. For negative controls, HEECs were incubated in the absence of TNF- or with a related antibody derived from mouse as a substitute for the primary antibody.
Isolation of total RNA and polyadenlyated RNA
The HEECs used for total RNA isolation were passaged two times. The isolation of total RNA with TRIzol® was performed according to the manufacturer's instruction. The purity and yield of RNA were determined by the ratio of OD260/280 and OD 260. RNA integrity was examined by electrophoresis on a 1% formaldehyde denaturing gel. The samples with bright bands corresponding to ribosomal 28S and 18S RNA with a ratio of intensities of 1.5–2.5:1 were used in SSH, microarray assay and qRT–PCR. The polyadenlyated RNA was isolated from total RNA with the Qiagen Oligotex mRNA Spin-Column Kit.
SSH procedure
SSH was performed with the PCR-SELECT cDNA subtraction kit. Starting material consisted of 2 µg of mRNA from HEECs derived from eutopic endometrium of patient 5 and 2 µg of mRNA from HEECs derived from eutopic endometrium of control 5. In the forward subtraction (FS), mRNA from EM was termed as ‘tester', and that from non-EM as ‘driver'; in the reverse subtraction (RS), mRNA from EM was termed as ‘driver' and that from non-EM as ‘tester'. The procedure was operated strictly according to manufacturer's direction, and 27 primary PCR cycles and 12 secondary PCR cycles were performed. The PCR products were purified with QIAquick PCR purification kit.
PCR products generated by SSH were subcloned into pDriver vector using a TA cloning kit. We electroporated ElectroMAX DH5-E Cells with ligated vector DNA and applied the germ liquid to an agar plate containing X-gal, IPTG, ampicillin and kanamycin. The length of inserted fragments was examined by colony PCR amplification. Plasmid DNA was extracted and sequenced using an ABI 3730 sequencer. Base-calling was performed with phenol red and use of the Q20 standard to trim low-quality bases. Genes were identified by sequence similarity comparison against the COG (cluster of orthologous groups) (http://www.ncbi.nlm.nih.gov/COG/) by using BLASTX with E values <1x–10 and alignment percentage 
20%.
Microarray analysis
We collected 10 HEEC samples, of which 5 were derived from EM pateients and 5 were derived from patients without EM, to screen the differentially expressed genes between the two group samples. A aliquot of 2 µg of total HEECs RNA was used to synthesize double-stranded cDNA, then produce biotin-tagged cRNA using the MessageAmpTM II aRNA Amplification Kit. The resulting bio-tagged cRNA were fragmented to strands of 35–200 bases in length according to the protocols from Affymetrix. The fragmented cRNA was hybridized to Affymetrix human Genome U133 Plus 2.0 Array containing 47 000 transcripts. Hybridization was performed at 45°C with rotation for 16 h (Affymetrix GeneChip Hybridization Oven 640). The GeneChip arrays were washed and then stained (streptavidin–phycoerythrin) on an Affymetrix Fluidics Station 450 followed by scanning on a GeneChip Scanner 3000.
The hybridization data were analyzed using GeneChip Operating software (GCOS 1.4). The scanned images were first assessed by visual inspection then analyzed to generate raw data files saved as CEL files using the default setting of GCOS 1.4. A global scaling procedure was performed to normalize the different arrays using dChip software. In a comparison analysis, we applied a two class unpaired method in the Significant Analysis of Microarray software (SAM) to identify significantly differentially expressed genes between EM and non-EM groups.
Quantitative real-time PCR confirmation
We used the same RNAs from 10 HEEC samples to confirm the SSH and microarray data. Total RNAs from each of the 10 patients were subjected to genomic DNA digestion using DNase I (Taraka, Dalian, China). First-strand cDNAs were synthesized with Oligo-dT primer and gene-specific real-time PCR primers were added (Table 2). Parallel reactions using 
-actin were performed to normalize the amount of template cDNA. The protocol of real-time PCR was as follows: initiation with a 10 min denaturation at 95°C, followed by 40 cycles of amplification with 15 s of denaturation at 95°C, 5 s of annealing at 55°C, 15 s of extension at 72°C and reading the plate for fluorescence data collection at 76°C. A melting curve was performed from 75 to 95°C to check the specificity of the amplified product. Each of the amplifications was duplicated, then the mean value was calculated. The student's t-test of independent data was used to examine the statistical significance of the differential expression level of each gene or transcript within the 10 samples, for each group of 5 samples.
|
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionsSupplementary dataReferences |
|---|
Characterization of isolated HEECs
After magnetic selection and digestion using trypsin/EDTA, the remaining cells formed discrete clones which consisted of purified HEECs. HEEC cultures presented flattened monolayers, with typical cobblestone morphology and contact-inhibition (Fig. 1A). The yield of purified HEECs was always larger from secretory than proliferative endometrium. Purified HEECs expressed vWF, CD31 and CD34 as shown by indirect immunofluorescence (Fig. 1B–D) examination. In addition, no signal was detected after treatment with antibodies against the epithelial cell marker cytokeratin or stromal cell marker vimentin (data not shown). After incubation with Ac-DiI-LDL, we found that 100% of the cells demonstrated uptake of Ac-DiI-LDL (Fig. 1E). The up-regulation of ELAM-1 was also demonstrated, in response to 30 ng/ml TNF-
stimulus for 6 h, by flow cytometry (data not shown).
|
Differences in growth features between HEECs from EM and non-EM
An obviously vigorous viability could be observed in HEECs from EM. With the method described above, the HEECs from EM could be passaged at least 13 times and the growth status could be kept very well, whereas the HEECs from non-EM could be passaged no more than eight times. The passage numbers were compared for 24 separate isolation procedures and the difference was statistically significant when performing an unpaired student's t-test (P < 0.05). After 2–3 passages, some specific cell populations with high density could always be found in the culture of HEECs from EM; these cells partially lose the characteristic of contact-inhibition and exhibit a larger ratio of nuclear to cytoplasm as well as a heterogeneous cellular size, although a few items of characterization confirmed their endothelial origin. Smaller cells were predominant in these populations (Fig. 2). The changes in morphology of HEECs from EM were reversible through passage.
|
The differential expression profile of HEECs detected by SSH
After plating the library, bacterial colonies were randomly picked and a total of 6480 ESTs were sequenced: 3629 ESTs were generated from a FS library and 2851 ESTs were from a RS library. The 6480 sequences were trimmed of vector and low-quality sequence and filtered for minimum length (100 bp), allowing the identification of 6165 high-quality ESTs with an average length of 430 bp. PHRAP assembly program was used to identify those that represent redundant transcripts. The 6165 high-quality ESTs were assembled into 889 contigs and 2991 singlets, giving 3880 assembled sequences. From the 3880 assembled sequences, 41 were assembled by ESTs expressed in two libraries, 2051 were only expressed in a FS library and 1788 were only expressed in a RS library. Table 3 lists several items of differentially expressed genes or transcripts. The transcript for an unknown protein that was cloned from uteri leiomyosarcoma was the most predominant among those that were up-regulated. Among the genes of known function, the genes for gremlin1 (GREM1), proliferation-inducing gene 2 (PIG2), fibronectin (FN) were noticeably up-regulated. Moreover, some regions within which no transcript had been reported were found to hide transcripts that were up- or down-regulated in HEECs from EM compared with that of non-EM counterparts. Detailed information on the differential expression profile can be seen in Supplementary data 1 and 2.
|
The differential expression profile of HEECs detected by microarray
After SAM analysis of microarray data from the 10 subjects consisting of 5 with EM and 5 without EM, 288 genes were determined to be significantly differentially expressed with a selection threshold of false discovery rate, FDR = 8.06% and fold change >2.0 in the SAM output result (see Supplementary data 3). The SAM result was visualized in Fig. 3 using SAM software and visualized with TreeView tools after unsupervised hierarchical clustering. The data discussed here have been deposited in NCBIs Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/info/linking.html) and are accessible through GEO Series accession number GSE7846 [NCBI GEO] .
|
All 288 differentially expressed genes were analyzed using a free web-based Molecular Annotation System 2.0 (MAS 2.0, www.capitalbio.com) which integrates three different open source pathway resources—KEGG, BioCarta and GenMAPP. In the MAS 2.0 tool, the pathways are ranked with statistical significance by calculating their P-values based on hypergeometric distribution (Mao et al., 2005
). Here, we list the significant pathways associated with cell survival, after interrogating the 288 differentially expressed genes (Table 4). Among these, the pathways of extracellular matrix (ECM)–receptor interaction and focal adhesion were the most predominant.
|
Confirmation with qRT–PCR
We selected six differential genes or transcripts to confirm their expression differences with qRT–PCR. The results are illustrated in Fig. 4. The qRT–PCR data confirmed the up-regulation of GREM1, PIG2 and FN in HEECs from EM compared with that from non-EM.
|
In addition, SSH detected two up-regulated sequences AC090877 [GenBank] and AC098935 [GenBank] , which represent large fragments on chromosome 15 and 1, respectively, and one down-regulated sequence AC111194 [GenBank] , which represents a large fragment on BAC clone RP11-463H12 from chromosome 4. There has not been transcripts reported within these three fragments on genomic DNA until now. With their specific primers designed for the sequences within these three large fragments on genomic DNA, we amplified the target sequences and confirmed their expression changes in HEECs from EM (Fig. 4).
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionsSupplementary dataReferences |
|---|
Analysis of pathways involving genes with known function: characteristics of HEEC from EM compared with those from non-EM
ECM–receptor interaction and focal adhesion
ECM–cellular interaction and focal adhesion were the most predominantly changed pathways in HEECs from EM compared with controls, with the P-values of nearly zero. The binding of ECM including FN, collagen, laminin and thrombospondin (THBS or TSP) to their transmembrane receptor integrin family, plays an essential role in endothelial functional modulation, such as adhesion, migration and angiogenesis. Compared with HEECs from controls, the remarkable up-regulation of FN, COL6A1, COL6A2 and laminin
4 chain (LAMA4) may be used to explain in part the increased viability of HEECs from EM (Fig. 5).
|
Adhesion to FN through the integrin
51 enables human umbilical vein endothelial cells (HUVECs) to proliferate due to the activation of ERK one-half (Kim et al., 2000) and Rac in response to growth factors (Mettouchi et al., 2001) as well as NF-kappa B through a signaling pathway requiring Ras, PI3-kinase and Rho family protein (Klein et al., 2002). The myofiber of Col6a1–/– mice exhibit increased apoptosis due to mitochondrial dysfunction, whereas this was reversed by plating Col6a1–/– myofibers on collagen VI (Irwin et al., 2003). The LAMA4, a component of laminin-8 and -9, is expressed in endothelial cell basement membranes. The 31 and V1 can bind the G domain of laminin 4 subunit with high affinity (Gonzalez et al., 2002). An anti-laminin 4 antibody, directed against the G domain of the 4 laminin subunit, inhibits proliferation and enhances apoptosis of endothelial cells when cells are maintained in vitro (DeHahn et al., 2004).
Syndecan-2 (SDC2) is a member of cell surface heparan sulfate proteoglycan (HSPG), and has been shown to be expressed in a diversity of cell types, including human endothelial cells. The mRNA for SDC2 was up-regulated significantly in HEECs from EM compared with that from non-EM. Via HS-GAG chains, HSPGs may interact with a wide variety of extracellular ligands such as chemokines, adhesion molecules and growth factors, and acts as transmembrane co-factors with their cognate receptors. Studies with human endothelium from diverse sources have indicated that SDC2 has a modulatory role in transendothelial migration of monocytes (Floris et al., 2003) and IL-8 binds to SDC2 on HUVEC which are up-regulated concomitantly in response to TNF- (Halden et al., 2004). THBS or TSP2 is a secreted matricellular glycoprotein. Compared with its counterparts from non-EM, the HEECs from EM expressed a high level of mRNA for TSP2 with a fold change of 2.59. TSP2 is up-regulated in the chronic inflammatory lesions of rheumatoid arthritis (Park et al., 2004) as well as in some carcinomas. Overexpression of TSP2 leads to not only prompt inhibition of lesional vascularizaiton but also to suppression of the production of a proinflammatroy mediator (Park et al., 2004) and its down-regulation elicits increased and prolonged inflammation and angiogenesis in delayed-type hypersensitivity reactions in the skin of TSP2-deficient mice (Lange-Asschenfeldt et al., 2002).
Focal adhesion is a molecular bridge between the intracellular and extracellular spaces that integrate a variety of environmental stimuli and mediate two-way crosstalk between the ECM and the cytoskeleton (Romer et al., 2006). Src and FAK, with which focal adhesion is closely associated, exert their wide-ranging impact on vascular biology, for instance apoptosis and vasculogenesis. The downstream signaling is dependent upon the sequential phosphorylation of related proteins.
GREM1 and TGF- signaling pathway
The mRNA for GREM1, a member of the DAN family proteins, was significantly up-regulated in HEECs from EM compared with those from non-EM with a fold change of 3.06. GREM1 is a highly conserved, secreted protein that fulfils a pivotal function in diverse processes of growth, differentiation and development, by antagonizing the activity of bone morphogenetic proteins (BMPs) through being heterodimerized with specific BMPs and preventing their interactions with TGF- receptors (Merino et al., 1999; Khokha et al., 2003; Michos et al., 2004). In non-tumor cells, TGF- inhibits cell cycle progression and maintains G1 arrest, through up-regulation of gene expression of the P21Waf1/Cip1 (Pardali et al., 2000) and P15Ink4B (Feng et al., 2000). GREM1 is expressed by stromal cells in many carcinomas but not in normal tissue, and could provide a favorable microenvironment for cancer cell survival (Sneddon et al., 2006). GREM1 and FGF2 are major components secreted by human fetal skin feeder that has the ability of supporting human embryonic stem cells (Kueh et al., 2006). GREM1 exhibits endothelium mitogenic activity in mouse and bovine aortic endothelial cells. The potent angiogenic response was observed in chick embryo CAMs implanted with transfectants with GREM1 cDNA (Stabile et al., 2007).
In addition, GREM1 is up-regulated in diabetic nephropathy (Murphy et al., 1999) and diabetic retinopathy (Kane et al., 2005). It is known that diabetic nephropathy and diabetic retinopathy are characterized by microangiopathies, including proliferative capillary, microaneurysm, basement membrane thickening and neovascularisation. Notably, PIG2 (the second most abundant gene in FS cDNA library) exhibits a close similarity to the cDNA of GREM1.
TGF- is the major secretory product of macrophage, both of which were increased in quantity, while the capacity of phagocytosis is reduced in peritoneal fluid of EM patients. Macrophages in endometriotic tissue are a major source of TGF- (Tamura et al., 1999). Inhibition of TGF-1 alters the growth, anchor-dependent cell aggregation and integrin mRNA expression in U937 cells (a promonocytic human cell line) (Dou et al., 1997). Moreover, TGF- is involved in progesterone-induced inhibition of matrix metalloproteinase (MMP) production in normal secretory endometrium (Bruner-Tran et al., 2002). Thus, further studies need to explore if the inhibition of the TGF- pathway as a result of up-regulation of GREM1 from HEEC in EM patients involves in the altered function of macrophage and stromal cells with paracrine action.
Consistency with previously published studies
When comparing our results with other author's, the up-regulated expression of u-PA (Koolwijk et al., 2001), IL-8 (Luk et al., 2005), VEGF (Schatz et al., 2000) and fibronectin in HEEC (Schatz et al., 2000) have been reported previously. However, it has been reported that the expression of MMP-3 in endometrial glandular or luminal epithelial cell from EM is higher than that from non-EM (Ramon et al., 2005).
The interaction of HEEC with other cell types
Although cyclical endometrial regeneration is under the strict control of ovarian sex steroid hormones and HEECs express estrogen receptor- and a low level of progesterone receptor, no change has been observed in classic angiogenic genes under the influence of the steroids (Krikun et al., 2005). Krikun et al. postulated that steroids may act synergistically with paracrine mediators to mediate downstream angiogenic pathways, which is similar to the way in which estradiol plays a role in epithelial stimulation (Cooke et al., 1997). Thus, the stromal cell or the HEEC-stroma interaction may play an important role in the changed angiogenesis within the endometrium from EM. Endometrial stromal cell exhibits a dose-dependent increase of adhesion to FN with IL-8 treatment. IL-8 production is elevated when endometrial stromal cells are plated on ECM components (Garcia-Velasco et al., 1999). IL-8, which was also up-regulated in our data, is a chemoattractor and activator of neutrophils and is a potent angiogenic agent (Oral et al., 1996). In addition, the changes in production of a broad array of ECM could influence the adhesion, activation and transendothelial migration of macrophages, NK cells, neutrophils, T cells, etc. The interaction of HEECs with these cells deserves further investigation and the result will help to elucidate the pathogenesis of EM.
Angiogenesis is crucial for the establishment and survival of endometriotic lesions. A large number of descriptive studies indicate that there is increased angiogenic capacity in ectopic endometria or peritoneal fluid of patients with EM due to elevated levels of influencing factors such as adhesion molecules, ECM degradation proteolytic enzyme as well as angiogenic factors. Moreover, the local estrogen production within ectopic endometrium is increased due to the high level of aromatase (Zeitoun et al., 1999) and suppressed level of 17-hydroxysteroid dehydrogenase type 2 (Attia et al., 2000). The role of estrogen in the pathogenesis of EM is generally accepted.
However, endothelial cell play a central role in procedure of angiogenesis. Groothuis et al. (2005) believes that it is the endothelium from peritonium of human or murine host that form new vasculature to provide oxygen and nutrients to explanted endometriotic grafts. Grümmer et al. (2001) and Hull et al. (2003) demonstrated in a nude mouse model that human endometrial tissue attract murine vessels from the immediate environment. Other observations noted that extrapelvic EM was found in well-vascularized sites, e.g. lung, skin and muscle (Mclaren, 2000). It would be important for us to note that these mouse models may not accurately reflect the human disease process. The increased ability of adhesion could make the attachment of endometriotic endometrial fragments to peritoneum more easy than that for normal endometrium. It may be postulated that the abnormal survival of HEECs produce a changed ECM pattern of which endothelium from peritoneum take advantage to make up its own vasculature and induce a inflammatory reaction through chemotaxis and activation of leukocytes.
Some novel transcripts
In addition to the genes of known function mentioned above, we also obtained some changed genes which have been found to be transcribed in placenta or other organs previously, or their existences however have not been predicted by bioinformatics algorithms, although their functions are still unknown. Our study may provide some hints to their roles in the pathogenesis of EM.
Using one sample and one control (rather than pooled) for the SSH is a weakness, but the use of 5 paired samples in the microarray and qRT–PCR strengthened reliability of the data. Further, the first step of our next works is to examine the exact differences of selected transcripts, for instance GREM1 and BC073984 [GenBank] with enlarged sample sizes of eutopic endometria from patients and normal controls by in situ hybridization and/or RT–PCR.
|
Conclusions |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionsSupplementary dataReferences |
|---|
In vitro, HEECs from patients with EM display a more vigorous viability than those from normal controls. The altered ECM-secreting pattern and up-regulation of GREM1 may be used to interpret the remarkable change of phenotype in HEECs from EM. The increment of secretion of ECM components and increased production of GREM1, which have assured proangiogenic activity, may contribute synergistically to the promotion of proliferation and the inhibition of apopotosis. Among the up-regulated ECMs, TSP2 is the only one which exhibits the capacity to suppress angiogenesis; it maybe functions as the antagonist for the aberrant angiogenesis and confines its extent and severity. Additionally, GREM1 may exert its effect on HEECs through inhibition of TGF-
pathway, by which proliferation may also be promoted. Taken together, it may be postulated that eutopic HEECs play a facilitating role during the peritoneal vascularization of ectopic endometrial lesions by enhancing angiogenic activity via a paracrine effect. In addition, with SSH, we found some novel sequences of unknown function. Further investigation is required to determine how all of these differentially expressed genes play their roles in the formation and maintenance of an ectopic lesion. |
Supplementary data |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionsSupplementary dataReferences |
|---|
Supplementary data is available at at http://humrep.oxfordjournals.org/
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionsSupplementary dataReferences |
|---|
Attia GR, Zeitoun K, Edwards D, Johns A, Carr BR, Bulun SE. Progesterone receptor isoform A but not B is expressed in endometriosis. J Clin Endocrinol Metab (2000) 85:2897–2902.
Bruner-Tran KL, Eisenberg E, Yeaman GR, Anderson TA, McBean J, Osteen KG. Steroid and cytokine regulation of matrix metalloproteinase expression in endometriosis and the establishment of experimental endometriosis in nude mice. J Clin Endocrinol Metab (2002) 87:4782–4791.
Cao W, Epstein C, Liu H, DeLoughery C, Ge N, Lin J, Diao R, Cao H, Long F, Zilberstein A, et al. Comparing gene discovery from Affymetrix Genechip microarrys and Clontech PCR-select cDNA subtraction: a case study. BMC Genomics (2004) 5:26–35.[CrossRef][Medline]
Cooke PS, Buchanan DL, Young P, Setiawan T, Brody J, Korach KS, Taylor J, Lubahn DB, Cuhna GR. Stromal estrogen receptors mediate mitogenic effects of estradiol on uterine epithelium. PNAS USA (1997) 94:6535–6540.
Croix B, Rago C, Velculescu V, Traverso G, Romans KE, Montgomery E, Lal A, Riggins GJ, Lengauer C, Kinzler KW, et al. Genes expressed in human tumor endothelium. Science (2000) 289:1197–1202.
DeHahn KC, Gonzales M, Gonzalez AM, Hopkinson SB, Chandel NS, Brunelle JK, Jones JC, et al. The alpha4 laminin subunit regulates endothelial cell survival. Exp Cell Res (2004) 294:281–289.[CrossRef][Web of Science][Medline]
Dou Q, Williams RS, Chegini N. Inhibition of transforming growth factor-beta 1 alters the growth, anchor-dependent cell aggregation and integrin mRNA expression in human promonocytes: implications for endometriosis and peritoneal adhesion formation. Mol Hum Reprod (1997) 3:383–391.
Feng X, Lin X, Derynck R. Smad2, Smad3 and Smad4 cooperate with Sp1 to induce P15Ink4B transcription in response to TGF-. EMBO J (2000) 19:5178–5193.[CrossRef][Web of Science][Medline]
Floris S, van den Born J, van der Pol SM, Dijkstra CD, De Vries HE. Heparan sulfate proteoglycans modulate monocyte migration across cerebral endothelium. J Neuropathol Exp Neurol (2003) 62:780–790.[Web of Science][Medline]
Garcia-Velasco JA, Arici A. Interleukin-8 expression in endometrial stromal cells is regulated by intergrin-dependent cell adhesion. Mol Hum Reprod (1999) 5:1135–1140.
Gonzalez AM, Gonzales M, Herron GS, Nagavarapu U, Hopkinson SB, Tsuruta D, Jones JCR. Complex interactions between the laminin alpha 4 subunit and intergrins regulate endothelial cell behavior in vitro and angiogenesis in vivo. PNAS USA (2002) 99:16075–16080.
Groothuis PG, Nap AW, Winterhager E, Grümmer R. Vascular development in endometriosis. Angiogenesis (2005) 8:147–156.[CrossRef][Medline]
Grümmer R, Schwarzer F, Bainczyk K, Hess-Stumpp H, Regidor PA, Schindler AE, Winterhager E. Peritoneal endometriosis:Validation of an in-vivo model. Hum Reprod (2001) 16:1736–1743.
Halden Y, Rek A, Atzenhofer W, Szilak L, Wabnig A, Kungl AJ. Interleukin-8 binds to syndecan-2 on human endothelial cells. Biochem J (2004) 377:533–538.[CrossRef][Web of Science][Medline]
Hull ML, Charnock-Jones DS, Chan CL, Bruner-Tran KL, Osteen KG, Tom BD, Fan TP, Smith SK. Antiangiogenic agents are effective inhibitors of endometriosis. J Clin Endocrinol Metab (2003) 88:2889–2899.
Iruela-Arispe ML, Rodriguez-Manzaneque JC, Abu-Jawdeh G. Endometrial endothelial cells express estrogen and P receptors and exhibit a tissue specific response to angiogenic growth factors. Microcirculation (1999) 6:127–140.[CrossRef][Web of Science][Medline]
Irwin WA, Bergamin N, Sabatelli P, Reggiani C, Megighian A, Merlini L, Braghetta P, Columbaro M, Volpin D, Bonaldo P, et al. Mitochondrial dysfunction and apoptosis in myopathic mice with collagen VI deficiency. Nat Genet (2003) 35:367–371.[CrossRef][Web of Science][Medline]
Khokha MK, Hsu D, Brunet LJ, Dionne MS, Harland RM. Gremlin is the BMP antagonist required for maintenance of Shh and Fgf signals during limb patterning. Nat Genet (2003) 34:303–307.[CrossRef][Web of Science][Medline]
Kane R, Stevenson L, Godson C, Stitt AW, O'Brien C. Gremlin gene expression in bovine retinal pericytes exposed to elevated glucose. Br J Ophthalmol (2005) 89:1638–1642.
Kayisli UA, Luk J, Guzeloglu-Kayisli O, Seval Y, Demir R, Arici A. Regulation of angiogeic activity of human endometrial endothelial cells in culture by ovarian steroids. J Clin Endocrinol Metab (2004) 89:5794–5802.
Kim F, Corson MA. Adhesion to fibronectin enhances MKP-1 activation in human endothelial cell. Biochem Biophys Res Commun (2000) 273:539–545.[CrossRef][Web of Science][Medline]
Klein S, de Fougerolles AR, Blaikie P, Khan L, Pepe A, Green CD, Koteliansky V, Giancotti FG. Alpha5beta1 integrin activates an NF-kappa B-dependant program of gene expression important for angiogenesis and inflammation. Mol Cell Biol (2002) 22:5912–5922.
Koolwijk P, Kapiteijn K, Molenaar B, van Spronsen E, van der Vecht B, Helmerhorst FM, van Hinsbergh VW. Enhanced angiogenic capacity and urokinase-type plasminogen activator expression by endothelial cells isolated from human endometrium. J Clin Endocrinol Metab (2001) 86:3359–3367.
Krikun G, Schatz F, Taylor R, Critchley HO, Rogers PA, Huang J, Lockwood CJ. Endometrial endothelial cell steroid receptor expression and steroid effects on gene expression. J Clin Endocrinol Metab (2005) 90:1812–1818.
Kueh J, Richards M, Ng SW, Chan WK, Bongso A. The search for factors in human feeders that support the derivation and propagation of human embryonic stem cells: preliminary studies using transcriptome profiling by serial analysis of gene expression. Fertil Steril (2006) 85:1843–1846.[CrossRef][Web of Science][Medline]
Lange-Asschenfeldt B, Weninger W, Velasco P, Kyriakides TR, von Andrian UH, Bornstein P, Detmar M, et al. Increased and prolonged inflammation and angiogenesis in delayed-type hypersensitivity reactions elicited in the skin of thrombospondin- 2-deficient mice. Blood (2002) 99:538–545.
Laschke MW, Elitzsch A, Scheuer C, Holstein JH, Vollmar B, Menger MD. Rapamycin induces regression of endometriotic lesions by inhibiting neovascularization and cell proliferation. Br J Pharmacol (2006) 149:137–144.[CrossRef][Web of Science][Medline]
Luk J, Seval Y, Kayisli UA, Ulukus M, Ulukus CE, Arici A. Regulation of interleukin-8 expression in human endometrial endothelial cells: A potential mechanism for the pathogenesis of Endometriosis. J Clin Endocri Metab (2005) 90:1805–1811.
Mao X, Cai T, Olyarchuk JG, Wei L. Automated genome annotation and pathway identification using the KEGG Orthology (KO) as a controlled vocabulary. Bioinformatics (2005) 21:3787–3793.
Mclaren J. Vascular endothelial growth factor and endometriotic angiogenesis. Hum Reprod Update (2000) 6:45–55.
McLaren J, Prentice A, Charnock-Jones DS, Smith SK. Vascular endothelial growth factor (VEGF) concentrations are elevated in peritoneal fluid of women with endometriosis. Hum Reprod (1996) 11, a. 220–223.
McLaren J, Prentice A, Charnock-Jones DS, Millican SA, Muller KH, Sharkey AM, Smith SK. Vascular endothelial growth factor is produced by peritoneal fluid macrophages in endometriosis and is regulated by ovarian steroids. J Clin Invest (1996) 98, b. 482–489.[Web of Science][Medline]
Mettouchi A, Klein S, Guo W, Lopez-Lago M, Lemichez E, Westwich JK, Giancotti FG, et al. Integrin-specific activation of Rac controls progression through the G(1) phase of the cell cycle. Mol cell (2001) 8:115–127.[CrossRef][Web of Science][Medline]
Merino R, Rodriguez-Leon J, Macias D, Ganan Y, Economides AN, Hurle JM. The BMP antagonist Gremlin regulates outgrowth, chondrogenesis and programmed cell death in the developing limb. Development (1999) 26:5515–5522.
Michos O, Panman L, Vintersten K, Beier K, Zeller R, Zuniga A. Gremlin-mediated BMP antagonism induces the epithelial-mesenchymal feedback signaling controlling metanephic kidney and limb organogenesis. Development (2004) 131:3401–3410.
Murphy M, Godson C, Cannon S, Kato S, Mackenzie HS, Martin F, Brady HR. Suppression subtractive hybridization identifies high glucose levels as a stimulus for expression of connective tissue growth factor and other genes in human mesangial cells. J Biol Chem (1999) 274:5830–5834.
Nap AW, Griffioen AW, Dunselman GA, Bouma-Ter Steege JC, Thijssen VL, Evers JL, Groothuis PG. Antiangiogenesis therapy for endometriosis. J Clin Endocrin Metab (2004) 89:1089–1095.
Nikitenko LL, MacKenzie IZ, Rees MCP, Bichnell R. Adrenomedullin is an autocrine regulator of endothelial growth in human endometrium. Mol Hum Reprod (2000) 6:811–819.
Nisolle M, Casanas-Roux F, Anaf V, Mine JM, Donnez J. Morphometric study of the stromal vascularisation in peritoneal endometriosis. Fertil Steril (1993) 59:681–684.[Web of Science][Medline]
Oral E, Arici A. Peritoneal growth factors and endometriosis. Semin Reprod Endocrinol (1996) 14:257–267.[Web of Science][Medline]
Pardali K, Kurisaki A, Moren A, ten Dijke P, Kardassis D, Moustakas A. Role of Smad proteins and transcription factor Sp1 in P21Waf1/Cip1 regulation by transforming growth factor-. J Biol Chem (2000) 275:29244–29256.
Park YW, Kang YM, Butterfield J, Detmar M, Goronzy JJ, Weyand CM. Thrombospondin 2 functions as an endogenous regulator of angiogenesis and inflammation in rheumatoid arthritis. Am J Pathol (2004) 165:2087–2098.
Ramon L, Gilabert-Estelles J, Castello R, Gilabert J, Espana F, Romeu A, Chirivella M, Aznar J, Estelles A. mRNA analysis of several components of the plasminogen activator and matrix metalloproteinase system s in endometriosis using a real-time quantitative RT-PCR assay. Huma Reprod (2005) 20:272–278.
Romer LH, Birukov KG, Garcia JG. Focal adhesions: paradigm for a signaling nexus. Circ Res (2006) 98:606–616.
Schatz F, Soderland C, Hendricks-Munoz KD, Gerrets RP, Lockwood CJ. Human endometrial endothelial cells: isolation, characterization, and inflammatory-mediated expression of tissue factor and type 1 plasminogen activator inhibitor. Biol Reprod (2000) 62:691–697.
Sneddon JB, Zhen H, Montgomery K, van de Rijn M, Tward AD, West R, Gladstone H, Chang HY, Morganroth GS, Brown P. Bone morphogenetic protein antagonists gremlin 1 is widely expressed by cancer-associated stromal cells and can promote tumor cell proliferation. PNAS USA (2006) 103:14842–14847.
Stabile H, Mitola S, Moroni E, Belleri M, Nicoli S, Coltrini D, Peri F, Orastti L, Presta M. Bone morphogenic protein antagonist Drm/gremlin is a novel proangiogenic factor. Blood (2007) 109:1834–1840.
Tamura M, Fukaya T, Enomoto A, Murakami T, Uehara S, Yajima A. Transforming growth factor-beta isoforms receptors in endometriotic cysts of the human ovary. Am J Reprod Immunol (1999) 42:160–167.[Medline]
Wu Y, Kajdacsy-Balla A, Strawn E, Basir Z, Halverson G, Jailwala P, Wang Y, Wang X, Ghosh S, Guo SW. Transcriptional characterizations of differences between eutopic and ectopic endometrium. Endocrinology (2006) 147:232–246.
Zeitoun KM, Bulun SE. Aromatase: A key molecule in the pathophysiology of endometriosis and a therapeutic target. Fertil Steril (1999) 72:961–969.[CrossRef][Web of Science][Medline]
Submitted on June 6, 2007; resubmitted on July 14, 2007; accepted on July 25, 2007.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  O. Guzeloglu-Kayisli, G. Halis, S. Taskiran, U. A. Kayisli, and A. Arici DNA-Binding Ability of NF-{kappa}B is Affected Differently by ER{alpha} and ER{beta} and Its Activation Results in Inhibition of Estrogen Responsiveness Reproductive Sciences, May 1, 2008; 15(5): 493 - 505. [Abstract] [PDF]
|
|
|
|
 |
|
 A. Van Langendonckt, J. Donnez, S. Defrere, G. A.J. Dunselman, and P. G. Groothuis Antiangiogenic and vascular-disrupting agents in endometriosis: pitfalls and promises Mol. Hum. Reprod., May 1, 2008; 14(5): 259 - 268. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||