Kenji Onda

Effects of Pravastatin on Human Placenta, Endothelium, and Women With Severe Preeclampsia

&NA;Preeclampsia is a major pregnancy complication where excess placental release of soluble fms-like tyrosine kinase-1 (sFlt-1) and soluble endoglin causes maternal endothelial and multisystem organ injury. Clinical trials have commenced examining whether pravastatin can be used to treat preeclampsia. However, the preclinical evidence supporting pravastatin as a treatment is limited to animal models, with almost no studies in human tissues. Therefore, we examined whether pravastatin reduced sFlt-1 and soluble endoglin secretion and decreased endothelial dysfunction in primary human tissues. Pravastatin reduced sFlt-1 secretion from primary endothelial cells, purified cytotrophoblast cells, and placental explants obtained from women with preterm preeclampsia. It increased soluble endoglin secretion from endothelial cells but did not change secretion from placental explants. The regulation of sFlt-1 by pravastatin seemed to be mediated via the 3-hydroxy-3-methylglutaryl-coenzyme A reductase cholesterol synthesis pathway. Pravastatin also reduced markers of endothelial dysfunction, including vascular cell adhesion molecule-1 expression and leukocyte adhesion on endothelial cells and increased endothelial cell migration and invasion. We also treated 4 patients with preterm preeclampsia presenting at <30 weeks of gestation with daily pravastatin. Pravastatin seemed to stabilize blood pressure, proteinuria, and serum uric acid levels. Furthermore, serum sFlt-1 levels decreased. We collected the placentas at delivery and found that pravastatin reduced sFlt-1 secretion. These results indicate that pravastatin reduced sFlt-1 and soluble endoglin production and decreased endothelial dysfunction in primary human tissues. We also present pilot data, suggesting that pravastatin can stabilize clinical and biochemical features of preterm preeclampsia. Our data obtained in human tissues support the concept that pravastatin is a candidate therapeutic for preeclampsia. Clinical Trial Registration—URL: http://www.anzctr.org.au. Unique identifier: ACTRN12613000268741.

MDR1 mRNA Expressions in Peripheral Blood Mononuclear Cells of Patients with Ulcerative Colitis in Relation to Glucocorticoid Administration

Overexpression of multidrug resistance (MDR) protein, P‐glycoprotein (P‐gp), on lymphocytes has been suggested to be implicated in the failure of glucocorticoid (GC) therapy in patients with ulcerative colitis (UC). However, whether the overexpression of P‐gp in a class of patients with inflammatory bowel disease (IBD) is intrinsic or related to the administration of GC is unknown. Relative amounts of MDR1 mRNA expressed in peripheral blood mononuclear cells (PBMCs) were measured using the reverse‐transcriptase polymerase chain reaction (RT‐PCR) technique in 25 UC patients having no history of GC administration, 25 UC patients having experienced GC therapy, 19 patients with Crohns disease (CD) with no history of GC therapy, and 27 healthy subjects. Relative amounts of MDR1 mRNA expressed in PBMCs were compared among the groups. The relationship between the amounts of MDR1 mRNA expressed, as well as the total dose of GC administered or the period of GC therapy in UC patients, was examined. The relative amounts of MDR1 mRNA expressed in PBMCs were not significantly different between the healthy subjects and CD patients or UC patients having no history of GC therapy. However, the mean MDR1 mRNA amount in PBMCs of UC patients having experienced GC therapy was significantly greater than that in PBMCs of UC patients with no history of GC administration (p = 0.0375). The amounts of MDR1 mRNA in PBMCs of UC patients having experienced GC therapy significantly correlated with the total dose of GCs administered (p = 0.0175). Overexpression of MDR1 mRNA in PBMCs of IBD patients is not intrinsic. However, high‐dose administration of GCs for the treatment of UC may result in an increased expression of MDR1 mRNA, which may impair successful GC therapy in these patients.

Persistent reversal of P-glycoprotein-mediated daunorubicin resistance by tetrandrine in multidrug-resistant human T lymphoblastoid leukemia MOLT-4 cells.

Multidrug resistance (MDR) represents a major problem in cancer chemotherapy. P‐glycoprotein (P‐gp), the drug efflux pump that mediates this resistance, can be inhibited by compounds with a variety of pharmacological functions, thus circumventing the MDR phenotype. The present study was performed to evaluate a unique MDR‐reversal feature of a bisbenzylisoquinoline alkaloid tetrandrine (TET) in a P‐gp expressing MOLT‐4 MDR line (MOLT‐4/DNR) established in our laboratory. Cell viability was determined by an MTT assay. P‐gp function was characterized by determining the Rh123 accumulation/efflux capacity. P‐gp overexpression in resistant MOLT‐4/DNR cells was confirmed by flow cytometry analysis after staining with phycoerythrin‐conjugated anti‐P‐gp monoclonal antibody 17F9. Compared to ciclosporin A (CsA), TET exhibited stronger activity to reverse drug resistance to daunorubicin (DNR), vinblastine (VLB) and doxorubicin (DOX) in MOLT‐4/DNR cells. TET showed no cytotoxic effects on parental MOLT‐4 cells lacking P‐gp expression or on the resistant MOLT‐4/DNR cells. TET modulated DNR cytotoxicity even after it was washed with the medium for 24 h, while CsA almost completely lost its reversal capability 24 h after washing. TET and CsA similarly increased the accumulation of Rh123 in resistant MOLT‐4/DNR cells. However, TET inhibited Rh123 efflux from resistant cells even after washing with the medium, while CsA rapidly lost its ability to inhibit Rh123 efflux after washing. The current study suggests that TET enhances the cytotoxicity of anticancer drugs in the P‐gp expressing MDR cell line by modulating P‐gp in a different manner to the well‐known P‐gp inhibitor CsA.

Characterization of protocols for primary trophoblast purification, optimized for functional investigation of sFlt-1 and soluble endoglin

OBJECTIVES Soluble fms-like tyrosine kinase-1 (sFlt-1) and soluble endoglin (sEng) are the most studied molecules in preeclampsia. However, most trophoblast cell lines do not secrete both these factors. Thus, we set out to characterize protocols to functionally investigate sFlt-1 and sEng from primary trophoblast. STUDY DESIGN Primary trophoblasts were isolated from term placenta by percoll gradient, then negative selection using a CD9 antibody. Purity was assessed by cytokeratin 7 immunostaining. We first examined the effects of CD9 negative selection on sFlt-1, sEng and hCG secretion and the ability of forskolin to enhance syncytialization. We then examined the effects of hypoxia on sFlt-1 production and assessed gene knockdown using siRNA. RESULTS CD9 negative selection produced a pure population of primary trophoblasts. Secretion of sEng was 5-fold lower when CD9-positive cells were removed, sFlt1 was unchanged, and hCG was significantly increased. hCG analysis of the purified population indicated spontaneous syncytialization, which was not enhanced by forskolin. Forskolin similarly did not alter sFlt-1 secretion. Hypoxia significantly increased sFlt-1 secretion as expected. Importantly, high gene silencing efficiencies were readily achieved. CONCLUSION In conclusion, we present a protocol that yields primary trophoblasts of high purity that produce abundant sFlt-1 and low but detectable levels of sEng. Furthermore, these cells are readily amenable to gene silencing by siRNAs and hence suitable for functional studies.

Proton Pump Inhibitors Decrease Soluble fms-Like Tyrosine Kinase-1 and Soluble Endoglin Secretion, Decrease Hypertension, and Rescue Endothelial Dysfunction.

Preeclampsia is a severe complication of pregnancy. Antiangiogenic factors soluble fms-like tyrosine kinase-1 (sFlt-1) and soluble endoglin are secreted in excess from the placenta, causing hypertension, endothelial dysfunction, and multiorgan injury. Oxidative stress and vascular inflammation exacerbate the endothelial injury. A drug that can block these pathophysiological steps would be an attractive treatment option. Proton pump inhibitors (PPIs) are safe in pregnancy where they are prescribed for gastric reflux. We performed functional studies on primary human tissues and animal models to examine the effects of PPIs on sFlt-1 and soluble endoglin secretion, vessel dilatation, blood pressure, and endothelial dysfunction. PPIs decreased sFlt-1 and soluble endoglin secretion from trophoblast, placental explants from preeclamptic pregnancies, and endothelial cells. They also mitigated tumor necrosis factor-&agr;–induced endothelial dysfunction: PPIs blocked endothelial vascular cell adhesion molecule-1 expression, leukocyte adhesion to endothelium, and disruption of endothelial tube formation. PPIs decreased endothelin-1 secretion and enhanced endothelial cell migration. Interestingly, the PPI esomeprazole vasodilated maternal blood vessels from normal pregnancies and cases of preterm preeclampsia, but its vasodilatory effects were lost when the vessels were denuded of their endothelium. Esomeprazole decreased blood pressure in a transgenic mouse model where human sFlt-1 was overexpressed in placenta. PPIs upregulated endogenous antioxidant defenses and decreased cytokine secretion from placental tissue and endothelial cells. We have found that PPIs decrease sFlt-1 and soluble endoglin secretion and endothelial dysfunction, dilate blood vessels, decrease blood pressure, and have antioxidant and anti-inflammatory properties. They have therapeutic potential for preeclampsia and other diseases where endothelial dysfunction is involved.

Sofalcone Upregulates the Nuclear Factor (Erythroid-Derived 2)-Like 2/Heme Oxygenase-1 Pathway, Reduces Soluble fms–Like Tyrosine Kinase-1, and Quenches Endothelial Dysfunction Potential Therapeutic for Preeclampsia

Preeclampsia is a severe complication of pregnancy, characterized by hypertension, oxidative stress, and severe endothelial dysfunction. Antiangiogenic factors, soluble fms–like tyrosine kinase-1 (sFlt-1) and soluble endoglin, play key pathophysiological roles in preeclampsia. Heme oxygenase-1 (HO-1) is a cytoprotective, antioxidant enzyme reported to be downregulated in preeclampsia. Studies propose that inducing HO-1 may also decrease sFlt-1 production. Sofalcone, a gastric antiulcer agent in clinical use, is known to induce HO-1 in gastric epithelium. We aimed to investigate whether sofalcone induces HO-1 and reduces sFlt-1 release from primary human placental and endothelial cells and blocks endothelial dysfunction in vitro. We isolated human trophoblasts and endothelial cells (human umbilical vein endothelial cells) and also used uterine microvascular cells. We investigated the effects of sofalcone on (1) HO-1 production, (2) activation of the nuclear factor (erythroid-derived 2)–like 2 pathway, (3) sFlt-1 and soluble endoglin release, (4) tumor necrosis factor &agr;–induced monocyte adhesion and vascular cell adhesion molecule upregulation, and (5) endothelial tubule formation. Sofalcone potently increased HO-1 mRNA and protein in both primary trophoblasts and human umbilical vein endothelial cells. Furthermore, sofalcone treatment caused nuclear translocation of nuclear factor (erythroid-derived 2)–like 2 and transactivation of other nuclear factor (erythroid-derived 2)–like 2 responsive genes (NQO1, TXN, and GCLC). Importantly, sofalcone significantly decreased the secretion of sFlt-1 from primary human trophoblasts. Sofalcone potently suppressed endothelial dysfunction in 2 in vitro models, blocking tumor necrosis factor &agr;–induced monocyte adhesion and vascular cell adhesion molecule 1 expression in human umbilical vein endothelial cells. These results indicate that in primary human tissues, sofalcone can potently activate antioxidant nuclear factor (erythroid-derived 2)–like 2/HO-1 pathway, decrease sFlt-1 production, and ameliorate endothelial dysfunction. We propose that sofalcone is a novel therapeutic candidate for preeclampsia.

Protective effect of caffeine on streptozotocin‐induced beta‐cell damage in rats

Many epidemiological studies have shown that coffee consumption reduces the risk of type 2 diabetes mellitus (T2D), although the reasons as to why remain unclear. In this study we investigated the effect of caffeine on pancreatic beta‐cell damage in rats using the diabetogenic agent, streptozotocin (STZ). Wistar rats were given intraperitoneal injections of saline or caffeine (10, 50 or 100 mgkg−1). After 15 min, the rats were injected with a citrate buffer or 65 mgkg−1 STZ. Three days after injection, an oral glucose tolerance test (OGTT) was performed on the rats. Furthermore, three days after the OGTT, the pancreas was isolated and homogenized, followed by determination of insulin content. STZ treatment significantly increased the plasma glucose level compared with the control at all times during the OGTT, which was significantly diminished by caffeine pretreatment at all doses. STZ treatment significantly decreased the plasma insulin level, however, which was not recovered by caffeine pretreatment. Pancreatic insulin content was significantly reduced by STZ treatment compared with the control, which was significantly recovered by caffeine pretreatment at a dose of 100 mgkg−1 (P < 0.01). We showed that caffeine protects pancreatic beta‐cells against STZ toxicity. Further investigation will be required to understand the protective effect of caffeine against beta‐cell destruction in T2D.

Expression of c-fos, rather than c-jun or glucocorticoid-receptor mRNA, correlates with decreased glucocorticoid response of peripheral blood mononuclear cells in asthma

Resolution of the molecular mechanism(s) underlying glucocorticoid (GC) resistance is an important clinical problem when performing individualized GC therapy according to the GC response of peripheral cells in asthma. In order to investigate the mechanism(s) underlying the individual differences of lymphocyte GC response, we examined the relationship between lymphocyte sensitivity to GC in vitro and the expression of mRNAs for GC receptor (GR) alpha, GRbeta, c-fos and c-jun, which are reported to be implicated in the regulation of the pharmacological effects of GCs in asthma patients. Twenty-seven patients with bronchial asthma and 14 healthy subjects were included in the study. IC50s of prednisolone and methylprednisolone on blastogenesis of peripheral blood mononuclear cells (PBMCs) stimulated with concanavalin A in vitro were estimated. Transcripts for GRalpha, c-fos, c-jun and beta-actin genes in PBMCs were quantitatively determined by reverse transcription-competitive polymerase chain reaction (RT-cPCR) procedures. GRbeta mRNA expression was examined with an RT-PCR technique. A statistically significant positive correlation was observed between the IC50s for prednisolone (p <0.002) or methylprednisolone (p <0.001) and expression of c-fos mRNA in PBMCs of asthma patients (n = 27). Thus, the increased expression of c-fos mRNA correlated with the decreased responses of PBMCs to prednisolone and methylprednisolone in vitro. In contrast, the expression of GRalpha and c-jun mRNAs did not correlate with the IC50 for prednisolone and methylprednisolone in asthma patients. In addition, no statistically significant difference in IC50s of GCs between asthma patients with PBMCs exhibiting GRbeta mRNA and those without GRbeta mRNA expression was observed. The increased expression of c-fos mRNA suggests to attenuate PBMC response to GCs, which may contribute to progression of GC resistance in asthma. On the other hand, c-jun and GC receptor mRNA expression appears to have less influence on poor GC-response establishment.

Soluble endoglin production is upregulated by oxysterols but not quenched by pravastatin in primary placental and endothelial cells

INTRODUCTION Preeclampsia is a serious pregnancy complication. Soluble endoglin (sEng) is released from the placenta and contributes to the maternal endothelial dysfunction seen in preeclampsia. Recently oxysterols, which activate the Liver X Receptor (LXR), have been implicated in producing sEng, by upregulating matrix metalloproteinase-14 (MMP14; cleaves endoglin to produce sEng) and down-regulating tissue inhibitor of metalloproteinase-3 (TIMP-3; inhibitor of MMP14). The functional experiments in that study were performed on JAR cells (human choriocarcinoma cell line) and placental explants. METHODS We characterized LXR in severe preeclamptic placentas, and assessed whether oxysterols increase release of sEng from primary human umbilical vein endothelial cells (HUVECs), primary trophoblasts and placental explants. Given pravastatin is thought to block oxysterol production and inhibit the LXR, we examined whether pravastatin reduces sEng release. RESULTS LXRα and β were localized to the syncytiotrophoblast and villous tips and were significantly up-regulated in preeclamptic placenta. Oxysterols upregulated sEng production in HUVECs and placental explants although the increases were far more modest than that recently reported. Oxysterols did not upregulate sEng in primary trophoblasts. Furthermore, mRNA expression of MMP14 and TIMP-3 were not altered by oxysterols in any tissue. Surprisingly, pravastatin did not decrease oxysterol-induced upregulation of sEng. DISCUSSION LXR is up-regulated in preeclamptic placenta. Oxysterols upregulate sEng production from human tissues, but the increase is modest, suggesting this may not be the main mechanism for the very significant elevations in sEng seen in preeclampsia. Pravastatin does not decrease sEng production. CONCLUSION Oxysterols modestly up-regulate sEng production which is not quenched by pravastatin.

Expression of mRNA for glucocorticoid receptors in peripheral blood mononuclear cells of patients with Crohn's disease

Background: The amount of glucocorticoid (GC) receptors (GR) in immune cells might be critical for successful GC treatment of patients with Crohns disease (CD). However, little is known about the expression of GR in CD; therefore, we carried out quantitative analyses for GR messenger (m)RNA expression in peripheral‐blood mononuclear cells (PBMC) of CD.