Shigehiko Mizutani

Functional Expression of the Angiotensin II type 1 receptor in human ovarian carcinoma cells and its blockade therapy resulting in suppression of tumor invasion, angiogenesis, and peritoneal dissemination

Purpose: Angiotensin II is a bioactive peptide of the renin-angiotensin system, acting not only as a vasoconstrictor but also as a growth promoter via angiotensin II type 1 receptors (AT1R). The present study examined AT1R expression in human ovarian carcinoma and attempted to determine whether AT1R blocker could suppress the tumor progression. Experimental Design: Expression of AT1R, vascular endothelial growth factor (VEGF), and CD34 was immunohistochemically analyzed in ovarian tumor tissues (n = 99). Effects of AT1R blocker on invasive potential and VEGF secretion in ovarian cancer cells were examined in vitro. Effects of AT1R blocker in vivo were evaluated in a mouse model of peritoneal carcinomatosis. Results: AT1R was expressed in 57 of 67 (85%) invasive ovarian adenocarcinomas and 12 of 18 (66%) borderline malignant tumors but in only 2 of 14 (14%) benign cystadenomas. In invasive carcinomas, VEGF expression intensity and intratumor microvessel density were significantly higher in cases that were strongly positive for AT1R (n = 37) compared with those in cases weakly positive (n = 20) or negative (n = 10) for AT1R. Angiotensin II significantly enhanced the invasive potential and VEGF secretion in AT1R-positive SKOV-3 ovarian cancer cells, both of which were completely inhibited by the AT1R blocker candesartan. Administration of candesartan into SKOV-3-transplanted athymic mice resulted in the reduction of peritoneal dissemination, decreased ascitic VEGF concentration, and suppression of tumor angiogenesis. Conclusions: AT1R is functionally expressed in ovarian carcinoma and involved in tumor progression and angiogenesis. AT1R blockade therapy may become a novel and promising strategy for ovarian cancer treatment.

Identification of human placental leucine aminopeptidase as oxytocinase

Human placental leucine aminopeptidase (P-LAP) was purified from retroplacental serum for the first time by serial chromatography on columns of Matrex Blue A, DEAE-Sepharose CL-6B, phenyl-Sepharose 4B, chelating-Sepharose, and Sepharose CL-6B. The purified P-LAP was apparently homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the apparent molecular weight (Mr) was estimated to be 210,000. By comparing P-LAP activity with cystine aminopeptidase activity, we concluded that both activities were shared by the same molecule. We also examined the hydrolytic activity of P-LAP using naturally occurring peptide hormones and found that the enzyme hydrolyzed oxytocin, vasopressin, and angiotensin III. These results suggest that P-LAP shows oxytocinase activity and plays an important role in the regulation of the plasma level of these hormones during pregnancy.

Female sterility in mice lacking the basigin gene, which encodes a transmembrane glycoprotein belonging to the immunoglobulin superfamily

Basigin (Bsg) is a transmembrane glycoprotein belonging to the immunoglobulin superfamily. Bsg knock‐out mice exhibit infertility of both sexes. Based on limited results, defective implantation has been considered to be the cause of the female infertility. We demonstrate here that disruption of the Bsg gene produces the failure of female reproductive processes including not only implantation but also fertilization. Bsg mRNA expression in cumulus cells and basolateral localization of the Bsg protein in the endometrial epithelium further support the importance of Bsg in these processes.

Pathways involved in the transition from hypertension to hypertrophy to heart failure. Treatment strategies

The renin-angiotensin-aldosterone system (RAAS) is critical in regulating systemic blood pressure, water and electrolyte balance, and pituitary gland hormones. These physiologies appear to be primarily mediated by the angiotensin II/AT1 receptor subtype system. Overstimulation of this system can predispose cardiovascular disease (CVD) characterized by excessive vasoconstriction, fibrosis, and cardiac remodeling. If untreated, the patient typically displays a continuum of pathophysiologic conditions progressing from atherosclerosis to left ventricle hypertrophy (LVH), coronary thrombosis, myocardial infarcts, with heart failure as an endpoint. Intervention with antihypertensive therapy is necessary to inhibit this progression. RAAS blocking drugs appear to be the most effective approach. Diastolic heart failure patients benefit from treatment with angiotensin converting enzyme (ACE) inhibitors and angiotensin AT1 receptor blockers (ARBs). Elderly CVD patients evidence age-related changes in body composition that alter the distribution and half-life of medications, thus presenting special challenges to treatment. The presence of comorbidities such as diabetes, renal dysfunction, liver insufficiency further complicates any therapeutic strategy. In addition, noncompliance because of cognitive impairment, depression, confusion due to the complexity of dose regimens, and lack of an appropriate social support system can disrupt positive outcome. The present review discusses the roles of an overactive RAAS and sympathetic nervous system as primary contributors to CVD. In addition, treatment strategies are discussed, focusing on middle aged and elderly hypertensive and heart failure patients.

Differential processing of substance P and neurokinin A by plasma dipeptidyl(amino)peptidase IV, aminopeptidase M and angiotensin converting enzyme

In addition to plasma metabolism of substance P (SP) by angiotensin converting enzyme (ACE; EC 3.4.15.1) (less than 1.0 nmol/min/ml), the majority of SP hydrolysis by rat and human plasma was due to dipeptidyl(amino)peptidase IV (DAP IV; EC 3.4.14.5) (3.15-5.91 nmol/min/ml), which sequentially converted SP to SP(3-11) and SP(5-11). In turn, the SP(5-11) metabolite was rapidly hydrolyzed by rat and human plasma aminopeptidase M (AmM; EC 3.4.11.2) (24.2-25.5 nmol/min/ml). The Km values of SP for DAP IV and of SP(5-11) for AmM ranged from 32.7 to 123 microM. In contrast, neurokinin A (NKA) was resistant to both ACE and DAP IV but was subject to N-terminal hydrolysis by AmM (3.76-10.8 nmol/min/ml; Km = 90.7 microM). These data demonstrate differential processing of SP and NKA by specific peptidases in rat and human plasma.

Placental leucine aminopeptidase/oxytocinase in maternal serum and placenta during normal pregnancy

Placental leucine aminopeptidase (P-LAP), which is identical with cystine aminopeptidase as oxytocinase, was found to be homologous with rat insulin-regulated membrane aminopeptidase (IRAP) by sequence comparison. In the current study, we determined the P-LAP levels in maternal serum and placenta during healthy pregnancy. P-LAP activities in maternal serum increased with gestation and rose to the peak of 80 IU/ml at 38 weeks of gestation. Northern blot analysis revealed the increase of P-LAP mRNA levels in placenta in the third trimester compared to the first trimester. P-LAP protein and related activities could be detected in the conditioned medium of placental tissue, while they could not be detected in that of human umbilical vein endothelial cells. Immunohistochemically P-LAP was positively stained in the apical membrane of syncytiotrophoblast cells throughout the gestation. These results established the normal range of serum and tissue P-LAP levels during pregnancy and the possible source of serum P-LAP, which will be helpful to elucidate the physiological and clinical roles of P-LAP/oxytocinase/IRAP.

Effects of 17β-estradiol and progesterone on growth-factor-induced proliferation and migration in human female aortic smooth muscle cells in vitro

Objective: The significantly low prevalence of atherosclerotic cardiovascular disease in premenopausal women has been noted, and postmenopausal hormone replacement therapy (HRT) is associated with protection from this disease. The aim of this study was to investigate the effects of estrogen and progestin on growth-factor-induced vascular smooth muscle cell (VSMC) proliferation and migration in vitro. Methods: Two cell lines of human female aortic smooth muscle cells (AOSMCs) were used for the study. DNA synthesis was evaluated by [3H]thymidine incorporation into cells. Migration assay was performed using modified Boyden chambers. Results: The presence of estrogen receptors was determined by Western and Northern blot analyses. [3H]Thymidine incorporation into AOSMCs was induced by 10 ng/ml EGF alone, the combination of 10 ng/ml EGF with 2 ng/ml b-FGF-induced and 1 nM PDGF-BB alone. Migration of AOSMCs was induced by PDGF-BB. Since 17β-estradiol (E2, 10−9 M~ 10−6 M) inhibited the [3H]thymidine incorporation into AOSMCs stimulated by above mitogens and the 1 nM PDGF-BB-induced AOSMC migration in a dose-dependent manner, estrone (E1), estriol (E3) and progesterone (P) had no significant effects. The combination of P (10−9 M ~ 10−6 M) did not show any effect on these inhibitory effects of 10−7 M E2. Preincubation of AOSMCs with the anti-estrogenic agent, tamoxifen (10−6 M), significantly antagonized these inhibitory effects of 10−7 M E2. Conclusions: These findings suggest that the inhibitory effect of E2 on VSMC proliferation and migration might be one of the factors involved in the decreased incidence of atherosclerotic cardiovascular disease in premenopausal women and postmenopausal HRT, and P might not affect these estrogenic responses.

BACE1 interacts with lipid raft proteins

A neuropathological hallmark of Alzheimers disease is the presence of amyloid plaques in the brain. Amyloid‐β peptide (Aβ) is the major constituent of the plaques and is generated by proteolytic cleavages of amyloid precursor protein (APP) by β‐ and γ‐secretases. Growing evidence shows that lipid rafts are critically involved in regulating the Aβ generation. In support of this, APP, Aβ, and presenilins have been found in lipid rafts. Although cholesterol plays a crucial role in maintaining lipid rafts, functions of other components in the generation of Aβ are unknown. Caveolins (CAVs) and flotillins (FLOTs) are principal proteins related to lipid rafts and have been suggested to be involved in APP processing. Here, we report that FLOT‐1 binds to BACE1 (beta‐site APP cleaving enzyme 1) and that overexpression of CAV‐1 or FLOT‐1 results in recruiting BACE1 into lipid rafts and influence on β‐secretase activity in cultured cells. Our results show that both CAV‐1 and FLOT‐1 may modulate β‐secretase activity by interacting with BACE1.

Increased mitochondrial damage by lipid peroxidation in trophoblast cells of preeclamptic placentas

Lipid peroxides and their related free radicals have been implicated in the pathogenesis of placental dysfunction in preeclampsia. Recent studies suggest that the placenta is a source of the increased lipid peroxides in the maternal circulation of women with preeclampsia. We examined intracellular localization of 4‐hydroxy‐2‐nonenal (HNE: a major aldehydic product of lipid peroxidation)‐modified proteins in human placentas by immunohistochemistry, and immunoblotting. The trophoblast layer of the chorionic villi showed intense immunoreactivity for HNE‐modified proteins in 4 of 12 preeclamptic placentas, whereas no staining was observed in 12 normal placentas. Immunoblotting revealed that three immunoreactive proteins with apparent molecular mass of 110 kDa, 75 kDa, and 70 kDa were localized in the mitochondrial fraction. The present results indicate that the damage to mitochondrial proteins by lipid peroxidation byproducts and subsequent dysfunction of trophoblasts contribute to the pathophysiology of preeclampsia.

Effects of placental proteases on maternal and fetal blood pressure in normal pregnancy and preeclampsia

Although many protease exist in human placenta, their physiologic roles are still unknown. Our study showed that placenta proteases metabolize vasoactive peptides possibly derived from the fetus. Because vasopressin and angiotensin are known to play an important role in normal and aberrant (preeclampsia) fetal-placental circulation, the clearance of these peptides in the placenta is important in controlling fetal blood pressure. Vasopressin and angiotensin act as a fetal-placental vasoconstrictor; therefore, placental proteases in human placenta are likely to work as a clearance factor for these peptides. Although human and animal pregnancy is normally associated with a refractory response to the pressor effect of exogenously infused angiotensin II, patients with preeclampsia, as well as nonpregnant women, are sensitive to the pressor effect of angiotensin II. Our study suggested that the decreased pressor responsiveness to angiotensin II in pregnancy is caused by increased inactivation of angiotensin II by angiotensinase in pregnant serum and the placenta. Although vasopressinase and angiotensinase activities increase with advancing gestation in normal pregnant sera, the activities of both enzymes in severe preeclampsia sera were clearly lower than those in normal pregnancy. Therefore, it is reasonable to speculate that the increased sensitivity to angiotensin II of preeclampsia is attributable to the decreased degradation of angiotensin II by placental angiotensinase. The negative correlations between the systolic to diastolic ratio obtained from pulsed Doppler measurement techniques and the activities of both enzymes in preeclampsia sera suggested that the systolic to diastolic ration, which reflected constriction of placental vessels, is influenced by the concentration of vasoactive peptides in the fetal-placental circulation due to changes in the activities of placental proteases. Placental proteases play important roles in controlling fetal and maternal blood pressure through regulation of the concentration of vasoactive peptides in the interface (placenta) between fetus and mother.