Keiko Takata

Fasudil inhibits vascular endothelial growth factor–induced angiogenesis in vitro and in vivo

Vascular endothelial growth factor (VEGF)–induced endothelial cell migration is an important component of tumor angiogenesis. Rho and Rho-associated kinase (ROCK) are key regulators of focal adhesion, stress fiber formation, and thus cell motility. Inhibitors of this pathway have been shown to inhibit endothelial cell motility and angiogenesis. In this study, we investigated the antiangiogenic effect of fasudil, one of the ROCK inhibitors. Fasudil inhibited VEGF-induced endothelial cell migration, viability, and tube formation in vitro in human umbilical vein endothelial cells. VEGF-induced endothelial cell migration was reduced by fasudil associated with loss of stress fiber formation, focal adhesion assembly, and with the suppression of tyrosine phosphorylation of focal adhesion proteins. Furthermore, fasudil inhibited VEGF-induced phosphorylation of myosin light chain, which is one of the main substrates of ROCK. Therefore, the effect of fasudil was suggested to be ROCK dependent. Fasudil not only inhibited VEGF-induced cell proliferation but also reversed the protective effect of VEGF on apoptosis, which resulted in the decrease of cell viability. Moreover, fasudil inhibited VEGF-induced angiogenesis in a directed in vivo angiogenesis assay. These data are the first demonstration that fasudil has antiangiogenic properties. Therefore, fasudil might be useful for the treatment of angiogenesis-related diseases, especially cancer. [Mol Cancer Ther 2007;6(5):1517–25]

Fasudil-induced hypoxia-inducible factor-1α degradation disrupts a hypoxia-driven vascular endothelial growth factor autocrine mechanism in endothelial cells

Hypoxic response of endothelial cells (EC) is an important component of tumor angiogenesis. Especially, hypoxia-inducible factor-1 (HIF-1)–dependent EC-specific mechanism is an essential component of tumor angiogenesis. Recently, the Rho/Rho-associated kinase (ROCK) signaling has been shown to play a key role in HIF-1α induction in renal cell carcinoma and trophoblast. The present study was designed to investigate whether low oxygen conditions might modulate HIF-1α expression through the Rho/ROCK signaling in human umbilical vascular ECs (HUVEC). Pull-down assay showed that hypoxia stimulated RhoA activity. Under hypoxic conditions, HUVECs transfected with small interfering RNA of RhoA and ROCK2 exhibited decreased levels of HIF-1α protein compared with nontargeted small interfering RNA transfectants, whereas HIF-1α mRNA levels were not altered. One of ROCK inhibitors, fasudil, inhibited hypoxia-induced HIF-1α expression without altering HIF-1α mRNA expression. Furthermore, proteasome inhibitor prevented the effect of fasudil on HIF-1α expression, and polyubiquitination was enhanced by fasudil. These results suggested that hypoxia-induced HIF-1α expression is through preventing HIF-1α degradation by activating the Rho/ROCK signaling in ECs. Furthermore, hypoxia induced both vascular endothelial growth factor (VEGF) and VEGF receptor-2 expression through the Rho/ROCK/HIF-1α signaling in HUVECs. Thus, augmented VEGF/VEGF receptor-2 autocrine mechanism stimulated HUVEC migration under hypoxic conditions. In summary, the Rho/ROCK/HIF-1α signaling is an essential mechanism for hypoxia-driven, VEGF-mediated autocrine loop in ECs. Therefore, fasudil might have the antimigratory effect against ECs in tumor angiogenesis. [Mol Cancer Ther 2008;7(6):1551–61]

Impact of surgical menopause on lipid and bone metabolism

Objective To clarify the effects of ovariectomy on lipid and bone metabolism. Methods This study was a prospective study with a longitudinal design within 1 year after surgery. Sixty-two premenopausal women were recruited and divided into two groups: group M (preservation of ovary, n = 27) and group BSO (bilateral ovariectomy, n = 35). Serum lipid levels, urinary N-telopeptide of type I collagen (NTx) and bone mineral density (BMD) were measured. We also examined the number of postoperative episodes requiring pharmacological intervention. Results There was a significant elevation in the level of low density lipoprotein cholesterol in group BSO from 6 to 12 months compared with the baseline level; the level did not change in group M. The NTx level significantly increased from 6 to 12 months, and the BMD was significantly decreased by as much as 6.7% at 12 months in group BSO; these variables did not change in group M. The effect of lipid and bone metabolism in group BSO was observed when the ages of the two groups were matched. Carbohydrate metabolism and arterial stiffness, measured by pulse wave velocity, did not change throughout the study period in either group. No subjects in group M required medication expect for two patients whose ovarian function was diminished by postoperative radiation and by natural menopause. Eleven women received medication in group BSO: nine for climacteric disorders using hormone therapy (25.7%), and two for dyslipidemia using statins (5.7%). Conclusions Bilateral ovariectomy seems to cause dyslipidemia and serious loss of bone mineral density within only 1 year, and patients who lose ovarian function may require careful medical care.

Differential regulation of the inducible nitric oxide synthase gene by estrogen receptors 1 and 2.

Estrogen has both rapid and longer term direct effects on cardiovascular tissues mediated by the two estrogen receptors, ESR1 and ESR2. Previous work identified that estrogen regulates the expression of inducible nitric oxide synthase (NOS2A) in vascular smooth muscle cells (VSMC). ESR2 knockout mice have vascular dysfunction due to dysregulation of NOS2A expression and these mice are hypertensive (Zhu et al. Science 2002 295 505-508). Here, we report studies to examine the differential regulation of NOS2A gene expression by ESR1 and 2. Immunoblotting and RT-PCR studies revealed that different VSMC lines expressed different levels of ESR1 and ESR2 protein and mRNA. VSMC from different vascular beds were studied, including aortic VSMC expressing ESR1 and radial (Rad) VSMC expressing ESR2. E(2) inhibited NO production and NOS2A protein expression in aortic VSMC. Human NOS2A promoter-reporter studies revealed suppression of NOS2A reporter activity by E(2) in aortic VSMC, and stimulation of NOS2A reporter activity by E(2) in Rad arterial VSMC. In heterologous expression studies of COS-7 cells lacking endogenous ER, E(2) treatment of COS-7 cells did not alter NOS2A reporter activity in the presence of ESR1, while reporter activity increased 2.3-fold in the presence of ESR2. Similar experiments in COS-7 cells using the selective estrogen receptor modulator raloxifene showed that raloxifene caused a reduction in NOS2A reporter activity with ESR1 coexpression and an increase with ESR2 coexpression. Rat VSMC expressing ESR2 but not ESR1 also showed increased NOS2A reporter activity with E(2) treatment, an effect lost when ESR1 was introduced into the cells. Taken together, these data support that hNOS2A transcription is regulated positively by ESR2 and negatively by ESR1 in VSMC, supporting differential actions of these two estrogen receptors on a physiologically relevant gene in VSMC.

Impact of Menopause on the Augmentation of Arterial Stiffness with Aging

Background/Aims: Aortic stiffness, determined by pulse wave velocity (PWV), is an independent marker of cardiovascular risk. PWV is mainly influenced by age-associated alterations in arterial wall structure and blood pressure. The present study was conducted to assess the impact of menopause on the brachial-ankle PWV (baPWV) in healthy women. Methods: Fifty premenopausal women aged 22–54 years and 40 postmenopausal women aged 40–73 years were recruited for this study. Subjects with hypertension, diabetes, and hyperlipidemia were strictly excluded. The results of baPWV were analyzed chronologically by 10- or 5-year age intervals. Results: There was no significant difference in baPWV between premenopausal and postmenopausal women in their 40s and 50s. The baPWV of postmenopausal women aged over 60 years was significantly higher than that of postmenopausal women in their 50s. To clarify the age-dependent elevation in baPWV in detail, women their 50s and 60s were divided into subgroups by 5-year age intervals. There was no significant difference in baPWV among the 50–54-, 55–59- and 60–64-year subgroups. baPWV significantly increased in the 65–69- year subgroup (p< 0.05). There was a significant relationship between baPWV and age in premenopausal (r = 0.452, p = 0.001) and postmenopausal (r = 0.581, p < 0.0001) women. The slope of the regression line for baPWV plotted against age was steeper in postmenopausal than in premenopausal women. Conclusions: This study produces suggestive evidence that menopause amplifies the age-dependent increase in arterial stiffness.

Molecular Mechanism of the Inhibition of Estradiol-Induced Endometrial Epithelial Cell Proliferation by Clomiphene Citrate

We examined the molecular mechanisms of the antiestrogenic effects of clomiphene citrate (CC) in the endometrium using two types of cell lines, Ishikawa and EM-E6/E7/hTERT cells. CC or ICI182780 inhibited 17beta-estradiol (E2)-induced endometrial cell proliferation and transcriptional activation of the estrogen response element (ERE) gene. We directly visualized the ligand-estrogen receptor (ER)alpha interaction using green fluorescent protein (GFP)-tagged ER alpha in a single living cell. Whereas E2 changed the nuclear localization of GFP-ER alpha to a punctate distribution within 5 min, CC or ICI182780 changed the slower and less mobilization of GFP-ER alpha compared with E2. Pretreatment with CC or ICI182780 partly prevented the E2-induced nuclear redistribution of GFP-ER alpha. Fluorescence recovery after photobleaching revealed that GFP-ER alpha mobility treated with E2 was more rapid than that treated by CC or ICI182780. As coactivator recruitment to the ER is essential for ER-dependent transcription, we examined the interaction between ER alpha and steroid receptor coactivator-1 (SRC-1). The complex formation between ER alpha and SRC-1 was significantly increased by E2 but was prevented in the presence of CC or ICI182780 by coimmunoprecipitation. Moreover, the E2-induced colocalization of GFP-ER alpha and SRC-1 was prevented in the presence of CC or ICI182780 according to an immunofluorescence assay. We also observed that the reduction of SRC-1 using small interfering RNA for SRC-1 resulted in the inhibition of E2-induced cell proliferation and transcriptional activation of the ERE gene. Collectively, these results suggest that CC may inhibit E2-induced endometrial epithelial cell proliferation and ERE transactivation by inhibiting the recruitment of SRC-1 to ER alpha.

Association of estrogen with glucocorticoid levels in visceral fat in postmenopausal women.

ObjectiveThe aim of this study is to clarify the association between estrogen and glucocorticoid levels in adipose tissues of premenopausal and postmenopausal women. MethodsForty (15 premenopausal and 25 postmenopausal) women aged 22 to 79 years were recruited through elective gynecological surgical operation, and both subcutaneous and visceral fat were collected during the surgical operation. Messenger RNA (mRNA) expressions of 11&bgr; hydroxysteroid dehydrogenase type 1 (HSD1), which catalyzes cortisone to cortisol, and 17&bgr;-HSD1 and 17&bgr; hydroxysteroid dehydrogenase type 2 (HSD2), which catalyze the interconversion of estrone (E1) and estradiol (E2), were measured by real-time reverse transcription polymerase chain reaction. The levels of E1, E2, cortisol, and cortisone in adipose tissues were measured by liquid chromatography tandem mass spectrometry. ResultsThe visceral fat area was significantly increased (P < 0.01) in postmenopausal women compared with that in premenopausal women. The cortisol/cortisone ratio (P < 0.01) and the expression of 11&bgr;-HSD1 mRNA (P < 0.001) in visceral fat, but not in subcutaneous fat, in postmenopausal women were significantly higher than those in premenopausal women. There was a significant correlation (r2 = 0.69, P < 0.05) between the E1/E2 ratio of visceral fat and body mass index in postmenopausal women. A significant correlation (r2 = 0.54, P < 0.05) between the cortisol/cortisone ratio and the E1/E2 ratio of visceral fat was observed in postmenopausal women. ConclusionsThe expression of 11&bgr;-HSD1 mRNA in visceral fat increases in postmenopausal women, and the E1/E2 ratio in visceral fat may be associated with local glucocorticoid levels after menopause.

Estrogen induces neurite outgrowth via Rho family GTPases in neuroblastoma cells.

Estrogen (E2) has direct in vivo and in vitro effects, such as inducing neurite outgrowth, on neurons. We investigated the morphological changes and intracellular signaling pathway induced by E2 in neuroblastoma (SH-SY5Y) cells. The effect of medroxyprogesterone acetate (MPA) or progesterone (P4) on the E2-induced neurite outgrowth was also examined using SH-SY5Y cells. Neurite outgrowth was induced by E2 in association with the phosphorylation of Akt, and these effects of E2 were abolished by MPA but not by P4. Progesterone receptor antagonist RU486 blocked the inhibitory effects of MPA. Estrogen receptor antagonist ICI 182,780 and phosphatidylinositol 3-kinase inhibitor LY294002 inhibited the E2-induced neurite outgrowth. Because the Rho family of small GTPases has been shown to be involved in the regulation of neurite outgrowth, we examined the cross-talk among Rac1, Cdc42 and RhoA in the E2-induced neurite outgrowth. E2 immediately increased the Rac1 and Cdc42 activity and decreased the RhoA activity. E2-induced neurite outgrowth was attenuated in cells expressing dominant-negative mutants for Rac1 or Cdc42. These results suggest that regulation of Rho family GTPase activity by E2 is important for the neurite outgrowth in neuroblastoma cells, and that MPA may have an antagonistic effect against E2.

Raloxifene improves the ovariectomy-induced impairment in endothelium-dependent vasodilation.

Objective:To examine the effect of raloxifene on the endothelial dysfunction caused by surgical menopause. Design:Ten premenopausal women who underwent gynecological surgery with ovariectomy were divided into two groups. Five participants used raloxifene (60 mg/d) for 7 days staring 1 week after the surgery, and the other five participants did not use raloxifene. We examined the changes in flow-mediated dilatation (FMD) of the brachial artery using ultrasonography. Vasodilation in response to nitroglycerin was also studied. We also measured the brachial-ankle pulse wave velocity to examine the change in arterial stiffness in these participants before and after surgical menopause. Results:In both the raloxifene and control groups, a significant decrease in FMD was observed 1 week after the surgery. Although no further changes in FMD were observed in the control group at 2 weeks after surgery, FMD was significantly increased in the raloxifene group. No remarkable changes in nitroglycerin or brachial-ankle pulse wave velocity were observed after surgery in either group. Conclusions:Raloxifene rapidly restored FMD that was impaired after surgical menopause. Therefore, raloxifene may be effective for ameliorating and maintaining endothelial function in premenopausal women who undergo ovariectomy.

Effect of dienogest on estrogen-induced nitric oxide production in human umbilical vein endothelial cells and endothelium-dependent vasodilatation in postmenopausal women.

Objective: We investigated the effects of dienogest (DNG), which has a profile similar to that of natural progesterone (P4), on the favorable effects of estrogen in endothelial function. Methods: (1) Human umbilical vein endothelial cells were treated with medroxyprogesterone acetate (MPA), DNG, or P4 with or without estradiol (E2), and then we examined nitric oxide (NO) production, phosphorylation of Akt, ERK, and endothelial NO synthase. (2) Twenty women with surgical menopause were randomly allocated to four groups: control (no treatment), E2 alone, E2 + MPA, and E2 + DNG. The treatment groups were treated with transdermal E2 (0.72 mg) for 2 days or E2 + MPA (2.5 mg/d) or E2 + DNG (2 mg/d) for a week starting 1 week after the operation; the control group did not use hormone. We examined the changes in the flow-mediated dilatation (FMD) of the brachial artery using ultrasonography. Results: (1) Although MPA attenuated E2-induced NO production and phosphorylation of Akt, extracellular signal-regulated kinase, and endothelial NO synthase, neither DNG nor P4 inhibited E2 effects. (2) A significant decrease in FMD was observed 1 week after the operation in all groups. E2 significantly ameliorated endothelial impairment (FMD, 3.4% ± 0.9% to 7.6% ± 1.3%) in the E2-alone group (P < 0.05), but E2 + MPA could not ameliorate endothelial impairment (3.3% ± 1.1% to 3.5% ± 1.0%). However, FMD in the E2 + DNG group significantly increased (2.9% ± 0.5% to 8.7% ± 1.0%; P < 0.05). Conclusions: These results suggest that DNG did not inhibit the restoration of vasodilatation by E2. DNG may have an advantage compared with MPA on the endothelial function in postmenopausal women receiving hormone therapy.