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Objective: Marinobufagenin (MBG), a cardiotonic steroid, is distinctly elevated in preeclampsia (preE) and may directly contribute to pathogenesis, possibly through deleterious signaling in cytotrophoblast (CTB) cells. In this study, we evaluate the effects of anti-MBG human monoclonal antibodies on cellular signaling in CTB cells and in a rat model of preE. Methods: CTB cell proliferation in response to MBG with and without anti-MBG was measured using a Cell Titer assay. Pro-angiogenic factors (VEGF and PlGF) and anti-angiogenic factors (sFlt-1 and sEng) in response to MBG with and without antibody were measured. Finally, we evaluated the lead anti-MBG antibody in comparison with the parent murine antibody in a previously described rat model of preE which recapitulates the hypertension, proteinuria, and fetal growth effects of human preE. Results: CTB cells exposed to ≥ 1 nM MBG showed decreased ( p < 0.05) proliferation, decreased secretion of VEGF and PIGF, and increased secretion of sFlt-1 and sEng. Pretreatment with anti-MBG significantly ( p < 0.05) attenuated the MBG-induced modulation of cell proliferation and expression of VEGF, PIGF, sFlt-1, and sEng. In the rat model, anti-MBG treatment normalized blood pressure, reduced proteinuria, and eliminated fetal effects. Conclusions: MBG has the potential to be a causative agent for preE as it causes dysfunction in CTB cells due to the anti-angiogenic milieu. Our study suggests that anti-MBG antibodies can bind to MBG, neutralizing it, and preventing downstream signaling in vitro. In a saline-induced rat model of preE, treatment with anti-MBG antibody was effective at normalizing blood pressure, kidney function, and fetal birth weights. These data suggest that a human monoclonal antibody with high specificity and affinity for MBG has potential as a therapeutic for preE.

Background Melanoma is a malignant skin tumor and quercetin has been reported to inhibit metastasis. A quercetin glycoside and 7 methylquercetins were synthesized from rutin to investigate structure-activity relationships. Materials and methods We evaluated the anti-proliferative and anti-migration activity of quercetin glycoside and 7 methylquercetins in murine B16 melanoma cells by commercially available kits. We also examined the effect of these compounds on growth of human fibroblast cells to evaluate cytotoxicity. Results 3-O-methylquercetin (2) exhibited 30, 38, 45% migration activity at 25, 12.5, 6.25 μM respectively. Furthermore, 3,4',7-O-trimethylquercetin (5) and 3,4'-O-dimethylquercetin (3) inhibited migration more potent than 2 with no cytotoxicity. 3-hydroxymethylquercetin and quercetin glycoside exhibited no anti-migration activity. Furthermore, the 3 and 4 inhibited melanoma proliferation with no cytotoxicity. Conclusion The methoxyl group at the C-3 position plays an important role in inhibiting the migration of B16 melanoma cells.

Background: Preeclampsia (PreE) is a pregnancy disorder characterized by new onset of hypertension and reduced fetal weight. We have previously shown that marinobufagenin (MBG) and hyperglycemia impairs cytotrophoblasts (CTBs) function. The potential therapeutic role of H2 relaxin in PreE was reported, a novel H2 relaxin B-chain-only peptide B7-33 and its lipidated derivative have recently been developed. This study evaluates whether B7-33 and its lipidated derivative improve CTBs function and preE phenotype. Methods: A palmitic acid was attached at the N-terminus of B7-33. Human CTBs were treated with DMSO (vehicle) or 0.1, 1, 10 or 100 nM of MBG or with 100, 150, 200, 300, or 400 mg/dL glucose for 48h and were co-treated either with B7-33 (25 nM) or its lipidated derivative (25 nM) in the presence and absence of either MBG or hyperglycemia exposure. Some cells were pretreated with relaxin antagonist (1.0 μM RXFP1) prior to MBG or hyperglycemia exposure. Levels of vascular endothelial growth factor (VEGF), placental growth factor (PlGF), and soluble fms-like tyrosine kinase-1 (sFlt-1) were measured in culture media using ELISA kits. Statistical comparisons were performed using analysis of variance with Duncan’s post hoc test. Results: Lipidation of B7-33 results in a significant increase of half-life of B7-33 without altering its activity. Secretion of sFlt-1 was increased while VEGF and PIGF were decreased in CTBs treated with ≥1.0 nM MBG and ≥150 mg/dl of glucose (*p < 0.05 for each). Co-treatment with B7-33 (25 nM) and its lapidated derivative (25 nM) rescued CTBs from either MBG-induced and hyperglycemia-induced anti-angiogenic profile (p < 0.05 for each). B7-33 and its lapidated derivative cause an increase in the expression of VEGF in CTBs, however, they have no effect on other factors. The B7-33-induced upregulation of VEGF expression is attenuated by 1.0 μM RXFP1 antagonist. Conclusion: Both B7-33 and its lipidated derivative mitigate the MBG- and hyperglycemia-induced dysfunction of CTBs by attenuating anti-angiogenic phenotype similar to that seen in preE. Moreover, the B7-33 and its lipidated derivative induced effect on CTBs are attenuated by a relaxin antagonist. The preclinical study with B7-33 and its lipidated derivative are now underway.

Objective: Preeclampsia (preE), a syndrome of hypertension, and end organ damage, is a leading cause of maternal and fetal morbidity and mortality. Recently, we and other researchers have shown that circulating (pro)renin, soluble (pro)renin receptor (s(P)RR) and placental (P)RR were significantly higher during preE compared to normal pregnancy (NP). In this study, placental expression of (P)RR and serum s(P)RR were evaluated in preE human patients and various rat models of preE. Study Design: (1) Placenta and blood samples were collected from 40 NP and 30 consented preE patients; (2) expression of (P)RR in placenta and soluble (P)RR were evaluated in two established preE rat models: reduced uterine perfusion pressure (RUPP) and deoxycorticosterone acetate (DOCA; PDS) models of preE and were compared to normal pregnant (NP) (SD) rats (n=8). We measured the expression of (P)RR by western blotting (WB) and immunohistochemistry. Plasma (pro)renin and s(P)RR were detected using a commercially available ELISA Kit. Results: The placental expression of (P)RR was higher (p* Conclusions: These data suggest that increased expression of (P)RR in the placenta and s(P)RR are associated with placental ischemia related to the occurrence of preE in both human patients and animal models of disease.

Objective: Cinobufotalin (CINO), a cardiotonic steroid (CTS) or bufadienolide, is extracted from the skin secretions of giant toads and is utilized in traditional Chinese medicine (Chan Su). CINO has been used as a cardiotonic, diuretic and a hemostatic agent. Recently, CINO has been shown to inhibit lung cancer cell function and has been implicated in several other disease processes. In this study, we pursued the potential anticancer application of CINO using the ovarian tumor cell line SK-OV-3. Study Design: We evaluated the effect of CINO on cultures of SK-OV-3. Cells were treated with 0.1, 0.5, 1, 5, and 10 μM CINO. Cell proliferation, migration, invasion, and viability were measured using commercially available kits. Cell cycle progression was evaluated by a Cell Cycle Phase Determination Kit. Apoptosis was evaluated by an Apoptotic Blebs Assay Kit; cell cycle arrest and apoptotic signaling were determined by fluorescence-activated cell sorting (FACS) analysis. Results: CINO at ≥ 0.5 μM inhibited SKOV-3 cell proliferation, migration, and invasion (p < 0.05). There was a higher (p < 0.05) percentage of S phase cells in groups treated with CINO at 0.5 μM. CINO at ≥ 0.5 μM down regulated expression of Proliferating Cell Nuclear Antigen (PCNA) and caused cell death. Conclusion: This data demonstrates that CINO impairs SK-OV-3 cell function via cell cycle arrest and apoptotic signaling, suggesting CINO be further investigated as a novel anti-ovarian cancer agent.

Cinobufotalin (CINO), a cardiotonic steroid, has been used as an anticancer agent. This study assessed the cell-specific effect of CINO on SK-OV-3, CRL-1978 and CRL-11731 ovarian cancer cells which differ in terms of their respective karyotypes.Cell cultures were treated with CINO (0.1, 1, 5 and 10 μM) for 24, 48, and 72 h. Cell proliferation, migration, and invasion were measured using CellTiter, Cytoselect, and FluoroBlock assays, respectively. Expression of proliferating cell nuclear antigen (PCNA) was evaluated by western blot analysis. Cell viability was determined by fluorescence-activated cell sorting. Immunofluorescence was performed using Annexin-V staining and fluorescein isothiocyanate (FITC). Mitochondrial membrane potential (MMP) was measured using MitoTracker™ Red.CINO at 0.5 μM inhibited SK-OV-3, CRL-1978, and CRL-11731 proliferation, migration, and invasion. Each cell type differed in response to CINO doses for PCNA, Annexin-V expression and MMP.The antineoplastic property of CINO is consistent, but its mode of action varies among cell lines.

Background/aim 3,4',7-O-trimethylquercetin (34'7TMQ), a derivative of quercetin, inhibited ovarian cancer cell migration and invasion without affecting proliferation. In this study, the apoptotic effect of 34'7TMQ on three cancer cell lines (CRL-1978, CRL-11731, SK-OV-3) was evaluated. Materials and methods Expression of pro-apoptotic proteins such as Bax/Bcl-2 ratio, p38 MAP kinase, and caspase-9 were measured by western blot analysis. Annexin-V staining was performed to visualize apoptotic signaling. Results Caspase-9 was up-regulated in all three cell lines. Bax/Bcl-2 ratio was up-regulated in CRL-1978 and SK-OV-3 but down-regulated in CRL-11731. The p38 MAPK was down-regulated in CRL-1978, up-regulated in SK-OV-3, and had differential expression in CRL-11731. Annexin V staining indicated that 34'7TMQ at 6.25 μM induced apoptotic signaling in the CRL-1978 ovarian cancer cell line. Conclusion 34'7TMQ induced apoptosis in three types of cancer cell lines but it appears to have a different mechanism of action in each cell line.

Cholangiocyte proliferation is regulated in a coordinated fashion by many neuroendocrine factors through autocrine and paracrine mechanisms. The renin-angiotensin system (RAS) is known to play a role in the activation of hepatic stellate cells and blocking the RAS attenuates hepatic fibrosis. We investigated the role of the RAS during extrahepatic cholestasis induced by bile duct ligation (BDL). In this study, we used normal and BDL rats that were treated with control, angiotensin II (ANG II), or losartan for 2 wk. In vitro studies were performed in a primary rat cholangiocyte cell line (NRIC). The expression of renin, angiotensin-converting enzyme, angiotensinogen, and angiotensin receptor type 1 was evaluated by immunohistochemistry (IHC), real-time PCR, and FACs and found to be increased in BDL compared with normal rat. The levels of ANG II were evaluated by ELISA and found to be increased in serum and conditioned media of cholangiocytes from BDL compared with normal rats. Treatment with ANG II increased biliary mass and proliferation in both normal and BDL rats. Losartan attenuated BDL-induced biliary proliferation. In vitro, ANG II stimulated NRIC proliferation via increased intracellular cAMP levels and activation of the PKA/ERK/CREB intracellular signaling pathway. ANG II stimulated a significant increase in Sirius red staining and IHC for fibronectin that was blocked by angiotensin receptor blockade. In vitro, ANG II stimulated the gene expression of collagen 1A1, fibronectin 1, and IL-6. These results indicate that cholangiocytes express a local RAS and that ANG II plays an important role in regulating biliary proliferation and fibrosis during extraheptic cholestasis.

Background: Previous work has shown an increase in urinary excretion of marinobufagenin (MBG) in patients with preeclampsia (preE) compared to normal pregnancy at delivery. MBG is a novel urinary marker for preE; however, it is unknown how MBG levels fluctuate during gestation. Hypothesis: Shifts in MBG and angiogenic factors during early pregnancy identify cytotrophoblast’s dysfunction, contributing to reduced vascular development with impaired perfusion and preE. Methods: In this study, we prospectively enrolled two groups of patients; 50 patients in each group total of 100 patients for serial collection of urine and serum during each trimester and at delivery. These groups are: (1) Pregnant women at risk for development of preE; and (2) Pregnant women at low risk for development of preE. Risks were calculated for each pregnancy using the sum of relative risk values. Urine samples from 100 subjects (100 in first trimester, 67 in second trimester, 67 third trimester, and 67 at delivery) were assayed using ELISA for MBG (Panorama Research, Inc.), sENG, VEGF, PIGF, sFlt-1 expressed as pg/mg creatinine and the calculated uFP (log of 10x sFLT-1/PIGF ratio). The angiogenic factors were analyzed by commercially available kits. Results: Of 100 subjects, 50 had no risk for preE and 50 had relative risks on the average of 6.6 with a range of 1.4 to 16.6. There were no associations between relative risk and any of the 6 angiogenic measures during the first trimester (p > 0.36) and second trimester (p > 0.22) using linear regression. However, during the second trimester, the high risk cohort had lower PIGF levels than the no risk cohort (p < 0.05 using Mann-Whitney U test). Comparing the serial values of 6 angiogenic measures in 67 subjects during the first, second, third trimesters and at deliveries, only MBG and PIGF varied significantly (p < 0.05) with MBG. Conclusions: MBG increases with progression of pregnancy and the levels of MBG are the highest during delivery. We conclude that MBG levels increase during normal pregnancy, however, the differential levels of MBG in normal and preE pregnancies are now underway. Relationships to development of preE symptoms and MBG levels are pending with the addition of patients and additional sample.

Background/Aim: Methylquercetin, 3,4’,7-O-trimethylquercetin (34’7TMQ), has been reported to inhibit metastasis. Recently, we demonstrated that 34’7TMQ inhibited the in vitro melanoma B16 cell metastatic activity. We evaluated the effect of 34’7TMQ on three ovarian cancer cells (SK-OV-3, CRL11731 and CRL1978). Materials and Methods: Proliferation, migration and invasion were measured in 34’7TMQ-treated ovarian cancer cells by commercially available kits. We also evaluated the expression of proliferating cell nuclear antigen (PCNA), urokinase plasminogen activator (uPA), plasminogen activator inhibitor 1 (PAI-1) and matrix metalloproteinase (MMP)-2 by western blot analysis. Results: 34’7TMQ inhibited ovarian cancer cell migration and invasion without effecting proliferation. Furthermore, 34’7TMQ inhibited the expression of uPA and MMP-2; however, it had no effect on PAI-1 and PCNA. Conclusion: 34’7TMQ significantly regulates the expressions of protein to inhibit metastasis in ovarian cancers, while the regulatory effects of 34’7TMQ vary between different ovarian cancer cell lines.

Substance P (SP) promotes cholangiocyte growth during cholestasis by activating its receptor, NK1R. SP is a proteolytic product of tachykinin (Tac1) and is deactivated by membrane metalloendopeptidase (MME). This study aimed to evaluate the functional role of SP in the regulation of cholangiocarcinoma (CCA) growth. NK1R, Tac1, and MME expression and SP secretion were assessed in human CCA cells and nonmalignant cholangiocytes. The proliferative effects of SP (in the absence/presence of the NK1R inhibitor, L-733,060) and of L-733,060 were evaluated. In vivo, the effect of L-733,060 treatment or MME overexpression on tumor growth was evaluated by using a xenograft model of CCA in nu/nu nude mice. The expression of Tac1, MME, NK1R, PCNA, CK-19, and VEGF-A was analyzed in the resulting tumors. Human CCA cell lines had increased expression of Tac1 and NK1R, along with reduced levels of MME compared with nonmalignant cholangiocytes, resulting in a subsequent increase in SP secretion. SP treatment increased CCA cell proliferation in vitro, which was blocked by L-733,060. Treatment with L-733,060 alone inhibited CCA proliferation in vitro and in vivo. Xenograft tumors derived from MME-overexpressed human Mz-ChA-1 CCA cells had a slower growth rate than those derived from control cells. Expression of PCNA, CK-19, and VEGF-A decreased, whereas MME expression increased in the xenograft tumors treated with L-733,060 or MME-overexpressed xenograft tumors compared with controls. The study suggests that SP secreted by CCA promotes CCA growth via autocrine pathway. Blockade of SP secretion and NK1R signaling may be important for the management of CCA.

Background: Relaxin is a peptide hormone that allows vasodilation and plays an important role in the process of parturition. The literature suggests potential therapeutic role of H2 relaxin in preeclampsia (PreE), however, there is a controversy on hypotensive action of the peptide. Due to the complex insulin-like structure of relaxin (A- and B- chains, 53 amino acids, 3 disulfide bonds), a novel H2 relaxin B-chain-only peptide variant B7-33 (27 amino acids without any disulfide bonds) has recently been developed. This single-chain peptide displayed equivalent efficacy to the natural H2 relaxin in several functional assays both in vitro and in vivo . Importantly, B7-33 was shown to have H2 relaxin-like RXFP1 specific effects, particularly in endogenously expressing RXFP1 cells, thus we hypothesized that B7-33 could be an alternative and cost-effective treatment option for PreE compared with H2 relaxin. Methods: Human CTBs were treated with 100, 150, 200, 300, or 400 mg/dL glucose for 48h and were co-treated with B7-33 (25 nM) with glucose exposure, while some cells were treated with 5, 10, 25 and 50 nM B7-33 alone. Levels of vascular endothelial growth factor (VEGF), placental growth factor (PlGF), soluble fms-like tyrosine kinase-1 (sFlt-1), and soluble endoglin (sEng) were measured in culture media using ELISA kits. Cell lysates were utilized to evaluate the mTOR, pAKT and total AKT expression by western blotting. Statistical comparisons were performed using analysis of variance with Duncan’s post hoc test. Results: Secretion of sFlt-1 and sEng were increased while VEGF and PIGF were decreased in CTBs treated with ≥150 mg/dl of glucose (*p < 0.05 for each). B7-33 co-treatment significantly rescued CTBs from hyperglycemia-induced anti-angiogenic profile (p < 0.05 for each). There is no effect of B7-33 on sFLT-1, sEng and PlGF; however, it increases expression of VEGF, while CTBs were treated only with B7-33. B7-33 also causes increased mTOR and pAKT expression in CTBs without any change in total AKT. Conclusions: B7-33 mitigates the hyperglycemia-induced dysfunction of CTBs by attenuating anti-angiogenic phenotype similar to that seen in PreE. This study supports the importance of continuing research of B7-33 in preE prevention.

Background: Preeclampsia (PreE) is de novo development of hypertension and proteinuria after 20 weeks of gestation with multiple pathophysiologic triggers affecting 3-8% of pregnancies. PreE has a significant link to alterations of feto-placental stress that pass to the offspring causing detrimental effects. We assessed and compared the pregnancy outcomes between patients with and without PreE. Methods: We recruited 35 normal pregnant (NP) and 25 PreE consenting patients in an IRB approved prospective study at Scott & White Memorial Hospital, Temple, Texas. We evaluated maternal age, body mass index (BMI), blood pressures, proteinuria, and gestational age at delivery. We divided the PreE subjects into early (before 34 weeks) and late PreE (after 34 weeks) groups and compared their outcomes. Placental thickness and volumes were measured. We also evaluated neonates for intrauterine growth restriction (IUGR), gestational age at birth, anthropometric measurements including Ponderal Index (PI), length of hospitalization and other neonatal complications. Comparisons were performed using Student’s t test. Results: Maternal: The systolic blood pressure (SBP) and diastolic blood pressure (DBP) were higher in PreE (SBP 166 ± 11; DBP 93 ± 10) compared to normal pregnancies (SBP 122 ± 10; DBP 74 ± 9; p Placenta: The placental thickness in early PreE subjects was 25mm compared to 32mm in late PreE (p 3 compared to 393cm 3 (p Neonatal: The average GA at delivery was lower in PreE (34.8 wks. ± 4) compared to NP (39.2 weeks ± 0.3; p

Preeclampsia (PreE) is a hypertensive pregnancy disorder, which occurs in approximately 10% of all gestations. Recently, a digitalis-like factor, marinobufagenin (MBG) has been implicated as a caus...

Cigarette smoking contributes to the development of cancer, and pathogenesis of other diseases. Many chemicals have been identified in cigarettes that have potent biological properties. Nicotine is especially known for its role in addiction and plays a role in other physiological effects of smoking and tobacco use. Recent studies have provided compelling evidence that, in addition to promoting cancer, nicotine also plays a pathogenic role in systems, such as the lung, kidney, heart, and liver. In many organ systems, nicotine modulates fibrosis by altering the functions of fibroblasts. Understanding the processes modulated by nicotine holds therapeutic potential and may guide future clinical and research decisions. This review discusses the role of nicotine in the general fibrogenic process that governs fibrosis and fibrosis-related diseases, focusing on the cellular mechanisms that have implications in multiple organ systems. Potential research directions for the management of nicotine-induced fibrosis, and potential clinical considerations with regard to nicotine-replacement therapy (NRT) are presented.—Jensen, K., Nizamutdinov, D., Guerrier, M., Afroze, S., Dostal, D., Glaser, S. General mechanisms of nicotine-induced fibrogenesis.

Objective: Preeclampsia (PreE) and diabetes mellitus (DM) in pregnancy share many risk factors and consequences. Thus, the interactions between these two disease-processes need to be further examined. We compared normal pregnancies to those complicated with preE, gestational diabetes (GDM), and/or pre-existing DM to assess the effect of DM on placental development and outcomes when this condition is complicated by preE. Methods: Chart reviews were performed in an IRB approved retrospective case-control design with pregnancies resulting in live born singletons. Total 178 subjects with preE with and without DM or GDM and live born singletons were selected from deliveries in 2008 through 2011 at Scott & White Memorial Hospital, Temple, Texas. These were compared to 443 without preE and with and without DM or GDM. Statistical analysis was performed using ANOVA and Duncan’s post-hoc test. Results: Patients who developed preE had higher systolic and diastolic pressures compared to groups without preE (p < 0.05). Patients with DM or GDM were older (p < 0.05). There was no difference among groups for gravidity (p = 0.21) with the average gravidity of 2.7 (1.8 SD) for 621 subjects having a range of 1 to 14 pregnancies. Patients with preE delivered earlier in pregnancy than those without preE regardless of diabetes status. However, those with preE and DM delivered earlier at 35.0 ± 0.4 weeks than the other two preE groups (p < 0.05), suggesting a more severe condition. Patients with DM who developed preE delivered smaller (p < 0.05) babies (correcting for gestational age at delivery) than those with DM without preE (1.00 ± 0.03 versus 1.16 ± 0.04, respectively). Development of GDM did not result in smaller babies for those pregnancies with preE (1.07 versus 1.09). Conclusions: The development of preE in those with pre-existing DM led to growth restriction and more severe disease as evidenced by lower birth weights corrected for gestational age and earlier gestational ages at delivery. These differences were not seen in GDM pregnancies. This observation supports the concept that elevated glucose levels during first trimester placental development may alter the placenta and lead to restriction later in pregnancy when a second stimulus triggers preE.

Objective: Preeclampsia (preE), a syndrome of hypertension, proteinuria and edema, has many elusive triggers. The renin-angiotensin system (RAS) has been implicated in preE pathogenesis. Recently, we have demonstrated that (pro)renin levels are increased in preE patients and that levels of (pro)renin and (pro)renin receptor ((P)RR) are elevated in a rat model of preE. It has also been demonstrated that high circulating levels of soluble (P)RR at delivery is associated with preE. We evaluated the placental expression of (P)RR in preE patients as well as in a nonhuman primate. We also evaluated the serum levels of soluble (P)RR in preE patients and in a rat model of preE. Study Design: (1) Blood and placental samples were collected from 15 normal pregnant (NP) and 15 preE consenting patients in an IRB approved prospective study. (2) We used NP rats ( n=10) and pregnant rats receiving weekly injections of desoxycorticosterone acetate and whose drinking water was replaced with 0.9% saline (preE, n=10). (3) The placental samples from owl monkey (both early and term, NP, n= 2) were collected. The (P)RR expression was measured both by western blotting (WB) and Immunohistochemistry (IHC) with anti-ATP6IP2 (detect (P)RR). The extracellular-signal-regulated kinase ½ (ERK ½) expression was evaluated by WB. The levels of serum soluble (P)RR were measured by a commercially available ELISA kit. Results: The placental expression of (P)RR was higher (pConclusions: These data suggest that increased expression of (P)RR and ERK1/2 in preE placenta is related to the occurrence of preE. These data also reconfirmed that high level of circulatory soluble (P)RR is associated with preE. The higher expression of (P)RR in early pregnancy compared to term placenta in owl monkey suggests that the (P)RR is important for normal placental development. The expression of (P)RR in nonhuman primates reveals the approach of future studies on owl monkey preE model.

Objective: An increasing level of evidence supports the utility of pravastatin in prevention against preeclampsia (preE). We previously demonstrated that hyperglycemia induces cytotrophoblasts (CTBs) dysfunction characteristic of a preE-like phenotype. We sought to demonstrate the utility of pravastatin in rescuing CTBs from hyperglycemia induced dysfunction. Methods: Human CTBs were treated with 100, 150, 200, 300, or 400 mg/dL glucose for 48h. Some cells were pretreated with pravastatin (1ug/mL) for 2h, while others were co-treated with pravastatin (1ug/mL) prior to glucose treatment. Some cells were treated with D-Mannitol. Cell migration was performed by Matrigel migration assay kit according to manufacturer protocol. Cell lysates were utilized to evaluate the expression of urokinase plasminogen activator (uPA), plasminogen activator inhibitor 1 (PAI-1), proliferating cell nuclear antigen (PCNA) and p38 MAPK phosphorylation by western blot. Levels of vascular endothelial growth factor (VEGF), placental growth factor (PlGF), soluble fms-like tyrosine kinase-1 (sFlt-1), soluble endoglin (sEng) and interleukin 6 (IL-6) were measured in culture media using ELISA kits. Statistical comparisons were performed using analysis of variance with Duncan’s post hoc test. Results: Hyperglycemia inhibited CTBs migration, down-regulated uPA, PAI-1, PCNA and up-regulated p38 phosphorylation in CTBs treated with ≥150 mg/dL glucose compared to basal (100 mg/dL) (*p < 0.05 for each). Secretion of sFlt-1, sEng and IL-6 were increased while VEGF and PIGF were decreased in CTBs treated ≥150 mg/dl of glucose (*p < 0.05 for each). Both pravastatin pretreatment and co-treatment significantly rescued CTBs migration, up-regulated uPA, PAI-1, PCNA, down-regulated p38 phosphorylation, and corrected the angiogenic profile of CTBs (p < 0.05 for each). D-Mannitol had no effect on CTBs. Conclusions: Pravastatin mitigates the hyperglycemia-induced dysfunction of CTBs by attenuating the glucose-induced anti-proliferative, anti-invasive and anti-angiogenic phenotype. These data should alleviate critical concerns regarding pravastatin use on CTBs development early in pregnancy and support the current research to use of pravastatin in preE prevention.

The role of mechanical force as an important regulator of structure and function of mammalian cells, tissues, and organs has recently been recognized. However, mechanical overload is a pathogenesis or comorbidity existing in a variety of heart diseases, such as hypertension, aortic regurgitation and myocardial infarction. Physical stimuli sensed by cells are transmitted through intracellular signal transduction pathways resulting in altered physiological responses or pathological conditions. Emerging evidence from experimental studies indicate that β1-integrin and the angiotensin II type I (AT1) receptor play critical roles as mechanosensors in the regulation of heart contraction, growth and leading to heart failure. Integrin link the extracellular matrix and the intracellular cytoskeleton to initiate the mechanical signalling, whereas, the AT1 receptor could be activated by mechanical stress through an angiotensin-II-independent mechanism. Recent studies show that both Integrin and AT1 receptor and their downstream signalling factors including MAPKs, AKT, FAK, ILK and GTPase regulate heart function in cardiac myocytes. In this review we describe the role of mechanical sensors residing within the plasma membrane, mechanical sensor induced downstream signalling factors and its potential roles in cardiac contraction and growth.

The cytotrophoblast (CTB) cells of the human placenta have membrane receptors that bind certain cardiotonic steroids (CTS) found in blood plasma. One of these, marinobufagenin, is a key factor in the etiology of preeclampsia. Herein, we used synthetic receptors (SR) to study their effectiveness on the angiogenic profile of human first trimester CTB cells. The humanextravillous CTB cells (Sw.71) used in this study were derived from first trimester chorionic villus tissue. Culture media of CTB cells treated with ≥1 nM SR level revealed sFlt-1 (Soluble fms-like tyrosine kinase-1) was significantly increased while VEGF (vascular endothelial growth factor) was significantly decreased in the culture media (* p < 0.05 for each) The AT2 receptor (Angiotensin II receptor type 2) expression was significantly upregulated in ≥1 nM SR-treated CTB cells as compared to basal; however, the AT1 (Angiotensin II receptor, type 1) and VEGFR-1 (vascular endothelial growth factor receptor 1) receptor expression was significantly downregulated (* p < 0.05 for each). Our results show that the anti-proliferative and anti-angiogenic effects of SR on CTB cells are similar to the effects of CTS. The observed anti angiogenic activity of SR on CTB cells demonstrates that the functionalized-urea/thiourea molecules may be useful as potent inhibitors to prevent CTS-induced impairment of CTB cells.

Cholangiocarcinoma is a type of liver cancer arising from the neoplastic transformation of the epithelial cells that line the intraand extrahepatic bile ducts. Symptoms are usually evident only after blockage of the bile duct by the tumor. This is an extremely aggressive tumor, which has very poor prognosis and limited treatment options. Cholangiocarcinoma is relatively resistant to chemotherapy and radiation therapy leaving conventional treatment like surgery as the only option. Therefore, further understanding into the factors that are involved in tumor initiation, promotion and progression is required for designing alternate therapies to combat this devastating disease.

The bile duct system of the liver is lined by epithelial cells (i.e., cholangiocytes) that respond to a large number of neuroendocrine factors through alterations in their proliferative activities and the subsequent modification of the microenvironment. As such, activation of biliary proliferation compensates for the loss of cholangiocytes due to apoptosis and slows the progression of toxic injury and cholestasis. Over the course of the last three decades, much progress has been made in identifying the factors that trigger the biliary epithelium to remodel and grow. Because a large number of autocrine factors have recently been identified as relevant clinical targets, a compiled review of their contributions and function in cholestatic liver diseases would be beneficial. In this context, it is important to define the specific processes triggered by autocrine factors that promote cholangiocytes to proliferate, activate neighboring cells, and ultimately lead to extracellular matrix deposition. In this review, we discuss the role of each of the known autocrine factors with particular emphasis on proliferation and fibrogenesis. Because many of these molecules interact with one another throughout the progression of liver fibrosis, a model speculating their involvement in the progression of cholestatic liver disease is also presented.

A S L D A b st ra ct s increase in circulating Galanin in the serum. Galanin immunoreactivity was observed in both cholangiocytes and hepatocytes, whereas GalR1 was found predominantly in cholangiocytes. Systemic treatment of rats with M617 increased both CK-19 expression and IBDM in both sham and BDL-treated animals. In vitro, treatment of the mouse cholangiocyte cell line with M617 increased ERK1/2 and RSK-1 activity. There was a concomitant increase in cholangiocyte proliferation after M617 treatment that could be blocked by pretreatment with inhibitors for ERK1/2 and RSK-1. Conclusions: Data presented here demonstrate a direct stimulatory role for Galanin on cholangiocyte proliferation under physiological (sham) and pathological (BDL) conditions via a mechanism involving the activation of ERK1/2 and subsequent phosphorylation of RSK-1. Targeting Galanin or GalR1 may prove a useful strategy to regulate biliary mass during cholestatic liver injury.

A S L D A b st ra ct s increase in circulating Galanin in the serum. Galanin immunoreactivity was observed in both cholangiocytes and hepatocytes, whereas GalR1 was found predominantly in cholangiocytes. Systemic treatment of rats with M617 increased both CK-19 expression and IBDM in both sham and BDL-treated animals. In vitro, treatment of the mouse cholangiocyte cell line with M617 increased ERK1/2 and RSK-1 activity. There was a concomitant increase in cholangiocyte proliferation after M617 treatment that could be blocked by pretreatment with inhibitors for ERK1/2 and RSK-1. Conclusions: Data presented here demonstrate a direct stimulatory role for Galanin on cholangiocyte proliferation under physiological (sham) and pathological (BDL) conditions via a mechanism involving the activation of ERK1/2 and subsequent phosphorylation of RSK-1. Targeting Galanin or GalR1 may prove a useful strategy to regulate biliary mass during cholestatic liver injury.

The two isoforms of the Small GTP binding proteins of the ADP riboyslation factor (Arf1 and Arf6), which are involved in membrane trafficking events are extensively studied. As with most members of the ras superfamily, Arf proteins cycle between a GTP-bound and a GDP-bound form. This cycle is regulated by two groups of proteins: GEFs, which facilitate the exchange of GDP for GTP, and GTPase-activating proteins (GAPs), which promote the hydrolysis of bound GTP. Arf1 and Arf6 have distinct cellular functions despite sharing approximately 70% sequence identity. Arf1 regulates the recruitment of vesicle coat polymers, including COPI, to the Golgi apparatus. In contrast, Arf6 is located at the plasma membrane and implicated in endocytotic pathways and vesicle trafficking [1–3]. Recent studies, using mice injected with invasive melanoma cell lines (LOX) that express a dominantly inhibitory Arf6, have shown that Arf6 is an important regulator of tumor growth and metastasis [4]. Our goal is to study is the internalization of exogenous Arf6 and its effect on MDA-MB-231 breast cancer cells in order to explore Arf6 as a therapeutic agent. Methods and Results: MDA-MB-231 breast cancer cells were incubated for 24 h with either purified Arf6-GTPγS (GTPγS is the nonhydorlysable form of GTP) or Arf6-GDP at various concentrations (60–500 nM) at 37°C and 4°C and the internalization of the proteins were evaluated using western blot analysis and fluorescence microscopy. Both forms of Arf6 proteins were significantly internalized in MDA-MB-231 breast cancer cells, and the internalized proteins inhibited cell invasion, migration and proliferation when compared to PBS (vehicle)-treated cells (p°C and 4°C, internalization mechanisms other than endocytosis must have been involved. We have also observed the internalization of Arf6 in colon and lung cancer cells at 37°C. Conclusions and Perspectives: Our findings show that when MDA-MB-231 breast cancer cells are incubated with either Arf6-GTPγS or Arf6-GDP in the cell culture media, the proteins are internalized at both 37°C and 4°C. Though further studies are required in order to understand the internalization mechanisms of Arf6, it is known that direct membrane translocation of some peptides including TAT protein is observed in several cell types treated at 4°C. Arf6 is more basic protein (pls in the range of 8.5–9.5) than Arf1 (pls in the range of 6.0–7.0), which might explain why Arf6 is internalized, but not Arf1. The inhibition of proliferation, migration and invasion of the cells might have been induced by defective Arf6 cycling due to the internalized Arf6, which indicates the potential to exploit Arf6 as therapeutic agent for cancer. Citation Information: Clin Cancer Res 2010;16(7 Suppl):B38

Objective Preeclampsia (preE) has a significant link to alterations of placental function leading to stress and apoptotic signaling, which pass the placental barrier and leave persistent defect in the circulation of the offspring. We assessed apoptotic signaling in placentas and umbilical cords from patients with and without preE. Methods We collected placental and cord tissues from 27 normal pregnant (NP) women and 20 preE consenting patients after delivery in an IRB approved prospective study. p38 mitogen-activated protein kinase (p38 MAPK) phosphorylation, pro-apoptotic Bcl-2-associated X (Bax), anti-apoptotic Bcl-2, caspase-9, and pro-inflammatory cyclooxygenase-2 (Cox-2) were evaluated by western blot and immunohistochemistry. Comparisons were performed using Student's t-test. Results p38 phosphorylation (Placenta: 1.5 fold, Cord: 1.7 fold), ratio of Bax/Bcl-2 (Placenta: 1.7 fold, Cord: 2.2 fold), caspase-9 (Placenta: 1.5 fold, Cord: 1.8 fold) and Cox-2 (Placenta: 2.5 fold, Cord: 2.3 fold) were up-regulated (p