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Department of Obstetrics and Gynecology (K.Ko., Y.O., O.T., T.Y., O.Y., Ya.T., H.M., H.H., K.Ku., M.M., T.F., Yu.T.), University of Tokyo, Tokyo 113-8655, Japan; and CREST, Japan Science and Technology (O.T.), Kawaguchi 332-0012, Japan
Address all correspondence and requests for reprints to: Dr. Yuji Taketani, Department of Obstetrics and Gynecology, University of Tokyo, Tokyo 113-8655, Japan.
Abstract
Angiogenesis is thought to be crucial for normal physiology of the endometrium, where dynamic vascular remodeling occurs during the menstrual cycle and pregnancy. We investigated the presence of angiogenin, a potent inducer of angiogenesis, and the regulatory mechanisms of its production in the human endometrium. Western blot analysis demonstrated that angiogenin protein expression increased by 3- to 4-fold in the endometrium in the mid and late secretory phases and in early gestation relative to that during the proliferative phase. Quantitative mRNA analysis showed the similar tendency in the expression of angiogenin mRNA in the endometrium, with the highest levels observed in the mid and late secretory phases and early gestation. An immunohistochemical study showed that angiogenin was expressed in both stromal cells and epithelial cells, with indistinguishable intensity between these cells regardless of phases of the menstrual cycle. In support of the Western blot analysis, the intensity of staining appeared to be highest in the mid to late secretory phases relative to other phases.
Consistent with these in vivo results, decidualized cultured stromal cells, after treatment with progesterone or progesterone plus E2, exhibited the capacity to secrete significantly increased amounts of angiogenin compared with untreated or E2 alone-treated control group. Both the treatment with (Bu)2cAMP and hypoxic conditions stimulated angiogenin secretion by stromal cells. For isolated epithelial cells, hypoxia stimulated angiogenin secretion, whereas (Bu)2cAMP had no appreciable effect. In summary, we demonstrated the presence of angiogenin in human endometrium and its possible local regulatory factors, such as progesterone, cAMP, and hypoxia. These findings along with its enhanced expression in the endometrium in the secretory phase and in decidual tissues raise the possibility that angiogenin may play a role in establishing pregnancy.
ANGIOGENIN, A heparin-binding 14.1-kDa single chain polypeptide, was initially isolated from supernatants of colon carcinoma cells and was found to be a member of the pancreatic ribonuclease superfamily (1). Angiogenin is shown to be present in various body fluids, including serum of human (2) and other mammals (3), human follicular fluid (4), and bovine milk (5). There is persuasive evidence that angiogenin is a potent inducer of angiogenesis, thus playing a role in a variety of physiological and pathological states (6, 7, 8, 9).
Angiogenesis, the development of new capillaries, is thought to be crucial for normal physiology of the endometrium, where dynamic vascular remodeling occurs during the menstrual cycle. In the proliferative phase, spiral vessels extend and eventually form a loose capillary network. After ovulation, they further grow and exhibit intensified coiling.
In view of the complexity of vascular remodeling that occurs in the endometrium, it appears that a range of angiogenic factors pertains to endometrial angiogenesis in tandem during the menstrual cycle and pregnancy. At present, several angiogenic factors, such as vascular endothelial growth factor (VEGF) (10) and basic fibroblast growth factor (FGF) (11), have been demonstrated in human endometrium.
Prompted by the recent studies demonstrating a significant difference in serum levels of angiogenin between the proliferative and secretory phases (12) together with increased serum levels in women with endometrial cancer (9), we sought to examine the presence of angiogenin in human endometrium and regulators of its production.
Subjects and Methods
Patients and samples
Endometrial tissues were obtained from 40 patients undergoing hysterectomy for benign gynecological conditions. All patients had regular menstrual cycles, and none had received hormonal treatment at least 6 months before surgery. The specimens were dated according to the criteria of Noyes et al. (13) and were classified as early proliferative, mid proliferative, late proliferative, early secretory, mid secretory, and late secretory phases. Moreover, decidual tissues without contamination of trophoblasts were obtained from 3 women by dilatation and curettage at operation for ectopic pregnancy. Twenty-two endometrial tissues were collected under sterile conditions and processed for primary cell cultures. Eighteen endometrial tissues (n = 3 in each menstrual phase) and 3 decidual tissues were used for immunohistochemical analysis and extractions of protein and mRNA. The tissues for immunohistochemical analysis were fixed overnight in 4% paraformaldehyde at 4 C, immersed in OCT compound (Tissue Tek, Elkhart, IN), and snap-frozen in acetone precooled with dry ice. The tissues for protein and mRNA extractions were snap-frozen in liquid nitrogen and stored at -80 C. The experimental procedures were approved by the institutional review board of University of Tokyo, and signed informed consent for use of the endometrium and the decidua was obtained from each woman.
Western blotting
Endometrial (n = 3 in each phase) and decidual (n = 2) tissues were homogenized in lysis buffer containing 50 mM Tris-HCl (pH7.4), 0.1% SDS, 1 mM EDTA, 0.5% Igepal, and 50 mM dithiothreitol and diluted to 1 mg total protein/ml. Samples were resolved by 10% SDS-PAGE in parallel lanes with recombinant human angiogenin (Genzyme/Techne, Minneapolis, MN). Proteins were blotted onto a nitrocellulose membrane and incubated with an antihuman angiogenin goat antibody (1:500; Genzyme/Techne) as a primary antibody and an antigoat horseradish peroxidase antibody (1:1000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) as a secondary antibody. Immune complexes were visualized using the ECL Western blotting system (Amersham Pharmacia Biotech, Little Chalfont, UK). Densitometric analysis of bands on developed x-ray films was performed using NIH Image.
RT and real-time quantitative PCR
RT and real-time quantitative PCR were performed as we have reported previously (4). Total RNA was extracted from 18 endometrial (n = 3 in each phase) and 3 decidual tissues using the RNeasy Mini Kit (QIAGEN, Hilden, Germany). RT was performed using the TaKaRa RNA PCR Kit (Takara Shuzo, Tokyo, Japan), and real-time quantitative PCR and data analysis were carried out using a LightCycler (Roche, Mannheim, Germany) according to the manufacturer’s instructions. One microgram of total RNA was reverse-transcribed in a 20-µl volume. PCR primers for the angiogenin were 5'-CCTGGGCGTTTTGTTGTTGG-3' (sense primer corresponding to nucleotides 1820–1839 of the published sequence) (14) and 5'-TGTGGCTCGGTACTGGCATG-3' (antisense primer corresponding to nucleotides 2152–2171). Amplification was performed in a total volume of 20 µl mixture including 1 µl of each RT reaction, 2 µl LightCycler-FastStart Reaction Mix SYBR Green 1 (Roche), 0.5 µmol/liter of each primer, and 3 mmol/liter MgCl2, with 40 cycles of denaturing (95 C, 15 sec), annealing (60 C, 10 sec), and extension (72C, 14 sec), followed by melting curve analysis.
Immunohistochemistry
Cryostat sections (6 µm) cut from frozen tissues were mounted on poly-L-lysine-treated slides. Sections were treated with 0.3% hydrogen peroxide for 30 min to eliminate endogenous peroxidase. After blocking with 1.5% horse serum, the sections were incubated with antihuman angiogenin mouse antibody (1:25; Biogenesis, Poole, UK) for 30 min at room temperature. Control slides were incubated with nonimmune mouse IgG, the concentration of which was adjusted to that of the primary antibody. The sections were then incubated with biotinylated horse antimouse IgG, followed by avidin peroxidase using the Vectastain Elite ABC kit (Vector Laboratories, Inc., Burlingame, CA). The chromogenic reaction was carried out with diaminobenzidine (Vector Laboratories, Inc.). All sections were counterstained with hematoxylin. Assessments of immunostaining were based on agreement among three independent observers who were blind to the phases of the menstrual cycle at which specimens were collected.
Isolation and culture of human endometrial stromal and epithelial cells
Fresh endometrial biopsy specimens collected in sterile medium were rinsed to remove blood cells. The tissues were minced into small pieces and incubated in DMEM/Ham’s F-12 containing type I collagenase (0.25%; Sigma, St. Louis, MO) and deoxyribonuclease I (15 U/ml; Takara Shuzo) for 60 min at 37 C. The resultant dispersed endometrial cells were separated by filtration through a 40-µm nylon cell strainer (Becton Dickinson and Co., Franklin Lakes, NJ). Endometrial epithelial glands that remained intact were retained by the strainer, whereas dispersed stromal cells passed through the strainer into the filtrate.
Stromal cells in the filtrate were collected by centrifugation and resuspended in phenol-red free DMEM/Ham’s F-12 containing 10% charcoal-stripped FBS, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 0.25 µg/ml amphotericin. The stromal cells were plated in a 100-mm culture plate and kept at 37 C in a humidified 5% CO2/95% air atmosphere. At the first passage, the cells were plated at a density of 2 x 105 cells/ml into 60-mm or 24-well culture plates.
Epithelial cells were collected by backwashing the strainer with DMEM/Ham’s F-12, plated in a 100-mm plate, and incubated at 37 C for 30 min to allow contaminated stromal cells to attach to the plate wall. The nonattached epithelial cells were recovered and cultured in the culture medium as described above at a density of 2 x 105 cells/ml into 24-well culture plates (PRIMALIA, Becton Dickinson and Co.). The purity of both the stromal and epithelial cell preparations was more than 95%, as judged by positive cellular staining for vimentin or cytokeratin, respectively.
In vitro decidualization
Stromal cells were obtained from four patients. In vitro decidualization was achieved as described previously (15). Briefly, after 80% confluence in a 60-mm plate, the cells were rinsed and treated with 2.5% charcoal-stripped FBS in the presence of E2 (10 ng/ml), progesterone (100 ng/mL), E2 plus progesterone, or vehicle (0.1% ethanol). Culture media were collected and replenished every 3 or 4 d. The media were centrifuged and stored at -80 C for analysis of angiogenin levels by an ELISA. Decidualization was assessed by measurement of PRL in the culture medium using an SR1 analyzer (Stat Profile Ultra, Nova Biomedical, Boston, MA). At the end of each experiment, the cells were harvested for total protein measurements.
Treatment with (Bu)2cAMP and hypoxia
Experiments were conducted with both stromal and epithelial cells in normoxia or hypoxia in the presence or absence of (Bu)2cAMP. When stromal (n = 13) and epithelial (n = 9) cells reached confluence in 24-well plates, the culture media were replenished with serum-free medium with or without (Bu)2cAMP (1 mM; Sigma). Then, the cells were placed either under hypoxic conditions (1% O2) using an incubator (water-jacketed mini multi gas incubator, Juji field, Tokyo, Japan) or normoxic conditions (20% O2). Twenty-four hours later, conditioned media were collected, centrifuged, and stored at -80 C for analysis of angiogenin concentrations.
Measurement of angiogenin
Angiogenin concentrations in conditioned media were assayed by ELISA (Quantikine Human Angiogenin Immunoassay, Gemzyme/Techne). The limit of sensitivity of the ELISA was 6 pg/ml. The intra- and interassay coefficients of variation were 4.3% and 8.7%, respectively.
Statistical analysis
Data were evaluated using ANOVA with post-hoc analysis (Fisher’s protected least significance) for multiple comparisons.
Results
Expression of angiogenin protein and its mRNA in endometrial and decidual tissues
The expressions of angiogenin protein in endometrial and decidual
tissues were detected by Western blot analysis as a single band with
the same molecular size as recombinant angiogenin, i.e.14.3
kDa. Based on densitometric analysis, the magnitude of expression was
low in the early proliferative phase and increased stepwise, reaching a
plateau in the mid and late secretory phases, the peak level being 3-
to 4-fold higher than that in the proliferative phase (Fig. 1
). The amount of angiogenin expression
in decidual tissues was essentially the same as that found in the mid
and late secretory phases. Quantitative mRNA analysis for angiogenin
showed a similar tendency, with the highest levels observed in the mid
and late secretory phases, which were 3- to 5- fold higher than those
in the proliferative phase (Fig. 2). Likewise, decidual tissues had
amounts of mRNA comparable to those in the mid and late secretory
phases.
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|
, the presence of
immunoreactive angiogenin in human endometrium was demonstrated in both
proliferative and secretory phases. Both stromal and epithelial cells
were stained. The intensities of staining in both cell populations were
indistinguishable in the same section, regardless of the phase of the
menstrual cycle. The intensity of the staining was minimal during the
proliferative and early secretory phases and was enhanced during the
mid to late secretory phase. No staining was seen when the primary
antibody was replaced with nonimmune mouse IgG.
|
As shown in Fig. 4, the
concentrations of angiogenin in culture media of endometrial stromal
cells in the absence of hormones or during treatment with E2 were
virtually the same and remained constant during the period from 4–18
d. No significant differences were seen in angiogenin concentrations on
d 4 and 8 in the presence of progesterone or E2 plus progesterone
compared with those in the untreated control. A significant increase in
angiogenin concentrations in the presence of progesterone was observed
on d 14 and 18 compared with d 4. The concentrations of angiogenin in
the presence of E2 plus progesterone on d 14 and 18 were further
increased, being 3- and 5.5-fold higher than those on d 4,
respectively. As expected, the concentrations of PRL in the media were
significantly increased by progesterone with or without E2 on d 14 and
18 compared with those on d 4.
|
We examined the effects of (Bu)2cAMP and
hypoxia on the production of angiogenin by both stromal and epithelial
cells. Under the present experimental conditions, the amounts of
angiogenin released were essentially the same regardless of whether
stromal/epithelial cells were collected from the proliferative or
secretory endometrium. The same was true for the production of
angiogenin in response to (Bu)2cAMP and hypoxia.
Thus, we analyzed data on the production of angiogenin by
stromal/epithelial cells from the proliferative or secretory
endometrium collectively. Figure 5A
illustrates the production of angiogenin by stromal cells in response
to (Bu)2cAMP and hypoxia. Treatment with
(Bu)2cAMP and a hypoxic milieu stimulated the
production of angiogenin significantly; the combination of both further
augmented the production.
|
).
Interestingly, treatment with (Bu)2cAMP alone or
in combination with hypoxia resulted in no appreciable effect on the
production of angiogenin. Discussion
In the present study we present evidence for the presence of angiogenin in human endometrium and its localization in both epithelial and stromal cell subpopulations. The presence of angiogenic factors in the endometrium was suggested in a study by Markee (16), who observed regeneration of the vascular bed in intraocular endometrial transplants in the rhesus monkey. With the advent of molecular biology, the presence of various angiogenic factors has been identified in the endometrium, including VEGF (10), basic FGF (11), and platelet-derived endothelial cell growth factor (15). This study added angiogenin to the list of angiogenic factors present in the endometrium.
The proliferation of the vascular endothelium is not restricted to a particular stage of the menstrual cycle, suggesting an intricate interplay between ovarian steroid hormones and multiple angiogenic factors. In this regard various angiogenic factors detected in the endometrium exhibit differential patterns of expression during the menstrual cycle. For example, the expression of FGF is stable throughout the menstrual cycle (11), whereas the highest level of VEGF is expressed in the early proliferative phase (17). These angiogenic factors may stimulate angiogenesis in a temporo-spatially different fashion in the endometrium.
The expression of angiogenin is characterized by a significant increase in the endometrium during the mid to late secretory phase and in the decidua. This expression pattern may imply that angiogenin could participate in the process of convolution and thickening of the arterioles, which are the most pronounced events during the secretory phase. In this context, an interesting finding has been reported that VSMC around the spiral arterioles show an increased proliferative activity in the mid to late secretory phases (18). Besides, differentiation of VSMC is thought to proceed in parallel with decidualization (4). In light of the effect of angiogenin to stimulate the proliferation of endothelial cells and VSMC (19, 20, 21) along with an observed characteristic expression of angiogenin, it is intriguing to speculate that it might regulate the proliferation and functional differentiation of VSMC in the secretory endometrium and the decidua.
The increased expression of angiogenin in the endometrium at the secretory phase and decidua seems to suggest a regulatory role for progesterone. Furthermore, in vitro decidualized stromal cells in the presence of progesterone exhibited an increased ability to produce angiogenin. It is of note that the stimulatory effect of progesterone on the production of angiogenin was seen after exposure to progesterone for more than 8 d, the time frame of which is similar to progesterone-stimulated production of PRL, the best known marker for decidualization. Thus, it is conceivable that angiogenin might play a role in implantation and sustenance of pregnancy.
The production of angiogenin by endometrial stromal cells was augmented by the addition of (Bu)2cAMP, whereas (Bu)2cAMP did not affect angiogenin production from epithelial cells. Although the reason for this is far from clear at present, differential regulatory mechanisms between stromal and epithelial cells might be working for angiogenin production in the endometrium. A pivotal role of cAMP in the process of decidualization of endometrial stromal cells has been reported (22, 23), and PG- or forskolin-stimulated adenylate cyclase activity is enhanced in the endometrium in the secretory phase (24). Given these findings together with an enhanced expression of angiogenin in the decidualized endometrium, one may infer that cAMP might play a role as a physiological mediator of angiogenin production in the process of decidualization.
Hypoxia-induced up-regulation of angiogenin expression may have a teleological sense, in that it could support endometrial regeneration in the perimenstrual period, during which a marked decrease in uterine blood flow and intense vasoconstriction induce distal ischemia (16). The expression of other angiogenic factors, such as VEGF and FGF, is also shown to be stimulated under a hypoxic milieu (25). Thus, it may be that up-regulation by hypoxia is a common property of angiogenic factors. Recent emerging evidence is an elevation of serum VEGF levels in patients with eclampsia, a pathological condition characterized by diminished uterine blood flow and endothelial damage in the placental bed (26, 27). Taken in the context of increased expression of angiogenin in decidual tissues, it would be interesting to learn whether angiogenin, like VEGF, is involved in the pathogenesis of preeclampsia and other pregnancy-associated complications accompanied with placental ischemia.
In summary, the present study demonstrated the presence of angiogenin in human endometrium, with various factors, such as progesterone, cAMP, and hypoxia, up-regulating its production. Taken together with its characteristic expression during the menstrual cycle and in the process of decidualization, it appears that angiogenin may play a role in establishing pregnancy.
Acknowledgments
We thank Yuko Kai and Keiko Tomita for technical assistance. We also thank our medical colleagues for their assistance with tissue collection.
Footnotes
Abbreviations: FGF, Fibroblast growth factor; VEGF, vascular endothelial growth factor.
Received October 26, 2000.
Accepted July 25, 2001.
References
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|
|
|
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|
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