Akemi Nishigaki

Hypoxic stress simultaneously stimulates vascular endothelial growth factor via hypoxia-inducible factor-1α and inhibits stromal cell-derived factor-1 in human endometrial stromal cells

BACKGROUND Hypoxia of the human endometrium is a physiologic event occurring during the perimenstrual period and the local stimulus for angiogenesis. The aim of this study was to investigate the effects of hypoxic stress on the regulation of vascular endothelial growth factor (VEGF) and stromal cell-derived factor-1 (SDF-1/CXCL12), and the potential role of hypoxia-inducible factor-1α (HIF-1α) in the endometrium. METHODS Human endometrial stromal cells (ESCs, n= 22 samples) were studied in vitro. ESCs were cultured under hypoxic and normoxic conditions and treated with cobalt chloride (CoCl₂; a hypoxia-mimicking agent) and/or echinomycin, a small-molecule inhibitor of HIF-1α activity. The mRNA levels and production of VEGF and SDF-1 were assessed by real-time PCR and ELISA, respectively. The HIF-1α protein levels were measured using western blot analysis. RESULTS Hypoxia simultaneously induced the expression of mRNA and production of VEGF and attenuated the expression and production of SDF-1 from ESCs in a time-dependent manner. Similar changes were observed in the ESCs after stimulation with CoCl₂ in a dose-dependent manner. CoCl₂ significantly induced the expression of HIF-1α protein, and its highest expression was observed at 6 h. Echinomycin inhibited hypoxia-induced VEGF production without affecting the HIF-1α protein level and cell toxicity and had no effect on SDF-1 secretion (P < 0.01). CONCLUSIONS Hypoxia simultaneously acts to increase VEGF via HIF-1α and to decrease SDF-1 in a HIF-1α-independent manner in ESCs. These results indicate a potential mechanism for the action of hypoxic conditions that could influence angiogenesis in the human endometrium.

Regulation of decidualization and angiogenesis in the human endometrium: Mini review

The human endometrium is a dynamic tissue that undergoes regular cycles of menstruation, menstrual repair, proliferation and secretory differentiation in response to hypoxia and the female sex hormones.

Concentrations of stromal cell-derived factor-1 and vascular endothelial growth factor in relation to the diameter of human follicles

OBJECTIVE To determine the concentrations of stromal cell-derived factor-1 (SDF-1/CXCL12) and vascular endothelial growth factor (VEGF) in individual human preovulatory follicles in relation to their diameter or volume for clarifying the role of these molecules in folliculogenesis. DESIGN Prospective study. SETTING Research laboratory at Kansai Medical University. PATIENT(S) Twenty-seven women undergoing IVF. INTERVENTION(S) Follicular fluid (FF) was collected from individual follicles. A total of 373 follicles were analyzed. MAIN OUTCOME MEASURE(S) The concentrations of SDF-1 and VEGF in FF and oocyte recovery rates. RESULT(S) The concentrations of SDF-1 and VEGF in follicles with a diameter ≤ 14 mm were significantly lower than those in follicles with a diameter ≥ 15 mm. The concentrations of SDF-1 and VEGF in FF increased with follicular diameters or volume, with concentrations peaking in follicles with a diameter of 18-20 mm or a volume of 3.6-5.0 mL. Furthermore, we found that there exists a positive correlation between the concentrations of SDF-1 and VEGF in FF from follicles ≤ 20 mm in diameter. The oocyte recovery rates increased with concentrations of SDF-1 and VEGF in FF. CONCLUSION(S) Our data suggest that SDF-1, as well as VEGF, may play an important role in follicular growth and development.

Angiopoietin 1 and angiopoietin 2 in follicular fluid of women undergoing a long protocol.

OBJECTIVE To determine the concentrations of angiopoietin 1 (ANGPT1) and ANGPT2 in individual human preovulatory follicles in relation to their diameter or volume to clarify the role of these molecules in folliculogenesis. DESIGN Prospective study. SETTING Research laboratory at Kansai Medical University, Osaka, Japan. PATIENT(S) Twenty-three women undergoing IVF. INTERVENTION(S) On the day of oocyte retrieval, serum samples and follicular fluid (FF) from individual follicles were collected. We analyzed 348 follicles. MAIN OUTCOME MEASURE(S) ANGPT1 and ANGPT2 concentrations in FF and serum and oocyte recovery rates. RESULT(S) On average, ANGPT1 concentrations in FF were 150 times lower than those in serum, whereas ANGPT2 concentrations in FF were 8 times higher than those in serum. The concentrations of ANGPT1 in follicles with a diameter ≤17 mm were significantly higher than those in follicles with a diameter ≥18 mm. On the other hand, the concentrations of ANGPT2 in follicles with a diameter ≤17 mm were significantly lower than those in follicles with a diameter ≥18 mm. The ANGPT2/ANGPT1 ratio increased with enlargement of follicular diameter. ANGPT1 concentrations in FF decreased with follicular volume. ANGPT2 concentrations and the ANGPT2/ANGPT1 ratio in FF rose with follicular volume. The ANGPT2/ANGPT1 ratio in FF from the oocyte recovery group was significantly higher than that from the nonrecovery group. CONCLUSION(S) Our data suggested that the change in ANGPT1 and ANGPT2 levels may be associated with follicular growth and angiogenesis during the preovulatory period.

The concentration of human follicular fluid stromal cell-derived factor-1 is correlated with luteinization in follicles

Stromal cell-derived factor-1 (SDF-1/CXCL12) and vascular endothelial growth factor (VEGF) are angiogenic factors that have possible roles in ovarian function. The objectives of this study were to investigate the association between the individual concentrations of SDF-1 and VEGF and sex steroid hormones in human preovulatory follicles and to verify the SDF-1 expression in ovarian follicles. Follicular fluid (FF) and luteinizing granulosa cells (LGCs) were collected from follicles at the time of oocyte retrieval. The concentrations of SDF-1, VEGF, estradiol (E2) and progesterone (P4) were determined by biochemical assay. The expression levels of SDF-1 mRNA and protein were analyzed by RT-PCR and immunohistochemical analysis, respectively. A total of 177 follicles were analyzed. The FF concentrations of SDF-1 and VEGF positively correlated with P4 concentrations (r = 0.457 and p < 0.01, r = 0.698 and p < 0.01, respectively), but did not correlate with E2 concentrations in FF. Furthermore, we confirmed that SDF-1 mRNA was expressed in LGCs and SDF-1 protein is present in the granulosa cells of the human ovary. Our findings suggest that SDF-1, as well as VEGF, may play important modulatory roles in early luteinization of human preovulatory follicles.

Requirement of heart and neural crest derivatives–expressed transcript 2 during decidualization of human endometrial stromal cells in vitro

OBJECTIVE To investigate the role of heart and neural crest derivatives-expressed transcript 2 (HAND2) during decidualization of human endometrial stromal cells (ESCs). DESIGN In vitro experiment. SETTING Research laboratory. PATIENT(S) Twenty-six patients undergoing hysterectomy for benign reasons. INTERVENTION(S) ESCs were cultured for 12 days with HAND2 small interfering RNA (siRNA) or nonsilencing RNA during decidualization by medroxyprogesterone acetate (MPA) and E2. MAIN OUTCOME MEASURE(S) Decidualization was monitored by changes in cellular morphology and the expression of several decidual-specific genes. RESULT(S) HAND2 siRNA effectively suppressed HAND2 levels in ESCs after 12 days of E2 + MPA treatment. ESCs cultured with HAND2 siRNA retained a long fibroblast-like shape, whereas the cells cultured with control siRNA transformed into enlarged polygonal cells. Silencing of HAND2 expression significantly reduced connexin-43 involved in the morphologic changes. HAND2 silencing significantly reduced the mRNA levels of fibulin-1, prolactin, tissue inhibitor of metalloproteinase 3, interleukin-15, and forkhead box O1A (FOXO1A), but had no effect on the mRNA levels of dickkopf-1, serum glucocorticoid kinase 1, and insulin-like growth factor-binding protein 5. HAND2 siRNA effectively suppressed the levels of nuclear FOXO1A protein as a regulator of decidualization. CONCLUSION(S) These results suggest that HAND2 plays a key role in the regulation of progestin-induced decidualization of human ESCs.

Effects of the hypoxia-inducible factor-1 inhibitor echinomycin on vascular endothelial growth factor production and apoptosis in human ectopic endometriotic stromal cells.

Abstract Recent evidence points to a possible role for hypoxia-inducible factor (HIF)-1 in the pathogenesis and development of endometriosis. The objectives of this study were to investigate the critical role of HIF-1 in endometriosis and the effect of the HIF-1 inhibitor echinomycin on human ectopic endometriotic stromal cells (eESCs). Ectopic endometriotic tissues were obtained from 20 patients, who received an operation for ovarian endometriomas. We examined vascular endothelial growth factor (VEGF) and stromal cell-derived factor-1 (SDF-1) production, HIF-1 expression, cell proliferation and apoptosis of eESCs. Cobalt chloride (CoCl2) significantly induced expression of HIF-1α protein and VEGF production in a time-dependent manner in eESCs, but reduced SDF-1 production. VEGF production was significantly suppressed by treatment of 100 nM echinomycin without causing cell toxicity, but 0.1–10 nM echinomycin or 100 nM progestin had no significant effect. SDF-1 production was not affected by echinomycin treatment at any dose. Echinomycin inhibited cell proliferation and induced apoptotic cell death of the eESCs, and significantly inhibited expression of the anti-apoptotic proteins Bcl-2 and Bcl-xL. Echinomycin inhibits VEGF production and induces apoptosis of eESCs by suppression of Bcl-2 and Bcl-xL. These findings suggest the unique therapeutic potential for echinomycin as an inhibitor of HIF-1 activation for endometriosis treatment.

Effects of ovarian hormone treatment on the gene expression of muscarinic acetylcholine receptors in the ovariectomized rat myometrium.

We investigate the effects of ovarian hormone on the gene expression of muscarinic acetylcholine receptors (M1-M5) in the myometrium using real-time PCR and evaluate the relationships between their expression and that of ovarian hormone receptors (ERα, ERβ, and PgR). Wistar rats were sham operated (SO) or ovariectomized (OVX) and treated with vehicle, estradiol (E2), progesterone (P4), or both E2 and P4 for 2 days beginning on postoperative day 33. M1 and M4 mRNA expressions were not detected in the myometrium. M2 mRNA expression did not change significantly in the OVX and OVX+P4 groups compared to the SO group, but increased significantly in the OVX+E2 group and was normalized in the OVX+E2P4 group. M3 mRNA expression increased significantly in the OVX and OVX+P4 groups compared to the SO group, but was normalized in the OVX+E2 and OVX+E2P4 groups. M5 mRNA expression did not change significantly in all experimental groups. ERα mRNA expression increased significantly in the OVX, OVX+E2, and OVX+P4 groups compared to the SO group, but was normalized in the OVX+E2P4 group. The changes in ERβ mRNA expression were similar to those of M3 mRNA expression in all experimental groups. In contrast, the changes in PgR mRNA expression did not correspond with that of M2, M3, or M5 mRNA expression in any of the experimental groups. Additionally, we evaluated the relationship between the expression of muscarinic acetylcholine receptors and ovarian hormone receptors in estrus cycle. M2 mRNA expression increased significantly in diestus and metaestrus compared in proestrus and estrus. M3 mRNA expression increased significantly in only diestrus compared in the other stages. In contrast, M5 mRNA expression did not change in estrus cycle. The changes in ERα mRNA expression appeared to be similar to those of M2 in estrus cycle, but no significant difference was found. The changes in ERβ mRNA expression were similar to those of M3 mRNA expression. The change in PgR mRNA expression increased significantly in diestrus compared in metaestrus, but did not correspond with that of M2, M3, or M5 mRNA expression in estrus cycle. When acetylcholine sensitivity in the myometrium was compared between diestrus and estrus, the sensitivity is significantly lower in estrus than in diestrus. These results suggest that ovarian hormones influence the expression of M2 and M3 in the myometrium by regulating the expression of hormone receptors. E2 may upregulate M2 via ERα, but P4 may downregulate M2 by inhibiting ERα via PgR. E2 may downregulate M3 by inhibiting ERβ, but P4 may not regulate the expression of M3 and ERβ. M5 may be a constitutive muscarinic receptor in the myometrium because neither E2 nor P4 influence the expression of M5. The combination of E2 and P4 may contribute the reproduction by quieting down the acetylcholine-induced myometrial contraction.

Lipid-soluble fraction of Shakuyaku-kanzo-to inhibits myometrial contraction in pregnant women

Shakuyaku‐kanzo‐to, a Kampo medicine composed equally of shakuyaku and kanzo, is an antispasmodic drug that can inhibit contraction of uterine smooth muscles in pregnant women and rats. We aimed to test the inhibitory effects of water‐ and lipid‐soluble extracts of shakuyaku‐kanzo‐to, shakuyaku, and kanzo in order to identify the fraction responsible for inhibiting uterine smooth muscle contraction in pregnancy.