Search

OBJECTIVE This study evaluated urinary angiotensinogen in preeclampsia. METHODS Normal pregnant (n = 57) and preeclamptic patients (n = 31); Normal pregnant (n = 10) and preeclamptic rats (n = 10) were studied. Urinary angiotensinogen and plasma angiotensin II were assayed by enzyme-linked immunosorbent assay (ELISA). RESULTS Urinary angiotensinogen in preeclampsia patients (2.0 ± 1.1 ng/mg creatinine) was suppressed (*p

Edema is common in preeclampsia (preE), a hypertensive disorder of pregnancy. Cardiotonic steroids (CTSs) such as marinobufagenin (MBG) are involved in the pathogenesis of preE. To assess whether CTSs are involved in the leakage of lymphatic endothelial cell (LEC), we evaluated their effect on monolayer permeability of LECs (MPLEC) in culture. A rat mesenteric LECs were treated with DMSO (vehicle), and CTSs (MBG, CINO, OUB) at concentrations of 1, 10, and 100 nM. Some LECs were pretreated with 1 μM L-NAME (N-Nitro-l-Arginine Methyl Ester) before adding 100 nM MBG or cinobufotalin (CINO). Expression of β-catenin and vascular endothelial (VE)-cadherin in CTS-treated LECs was measured by immunofluorescence and MPLEC was quantified using a fluorescence plate reader. Western blot was performed to measure β-catenin and VE-cadherin protein levels and myosin light chain 20 (MLC20) phosphorylation. MBG (≥ 1 nM) and CINO (≥ 10 nM) caused an increase (p

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.

Preeclampsia (preE) is a hypertensive disorder of pregnancy. Cardiotonic steroids (CTS) are endogenous inhibitors of Na+/K+ ATPase, and at least one CTS, marinobufagenin (MBG), is elevated in a rat model of preE prior to the development of the syndrome. MBG and ouabain impair cytotrophoblast (CTB) cell function, which is critical for placental development. We evaluated the effect of a CTS, cinobufotalin (CINO), on CTB cell function in vitro. CINO at ≥1 nM inhibited CTB cell proliferation, migration, and invasion (p

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.

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.

The interaction between pre-eclampsia and diabetes mellitus (DM) is far from being completely understood. In this study, we compared normal pregnancies with those complicated with pre-eclampsia, gestational DM, and/or pre-existing diabetes to assess the effects of hyperglycemia on placental development. AnInstitutional Review Board (IRB) approved retrospective cross-sectional study with 621 subjects was performed. Statistical analysis was performed using Duncan's post hoc test and analysis of variance. Regardless of diabetes status, patients with pre-eclampsia delivered prematurely. Patients in the group with pre-eclampsia and pregestational diabetes delivered much earlier, at 35.0±0.4 weeks, when compared with the patients that had pre-eclampsia with gestational diabetes and pre-eclampsia with no diabetes (*P

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/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...

Preeclampsia (preE) is a hypertensive disorder that occurs 20% in diabetic pregnancy. We have shown that hyperglycemia impairs cytotrophoblast cell (CTB) function. In this study, we assess apoptotic and anti-angiogenic signaling in excess glucose-induced CTBs.Human extravillous CTBs (Sw. 71) were treated with 100, 150, 200, 300, or 400 mg/dL glucose for 48 h. Some cells were pretreated with a p38 inhibitor (SB203580) or a peroxisome proliferator-activated receptor gamma (PPARγ) ligand (rosiglitazone) or with D-mannitol. Cell lysates were utilized to measure p38 MAPK phosphorylation, PPARγ, Bcl-2-associated-X protein (Bax), anti-apoptotic Bcl-2, caspase-9, and cyclooxygenase-2 (Cox-2) expression by western blot. Levels of the 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.p38 phosphorylation and PPARγ were upregulated (p0.05) in CTBs treated with ≥150 mg/dL glucose compared to basal (100 mg/dL). Expressions of Bax/Bcl-2, Cox-2, and caspase-9 were upregulated (p0.05) in CTBs treated with ≥150 mg/dL glucose. Secretion of sFlt-1, sEng, and IL-6 was increased while VEGF and PIGF were decreased in CTB-treated ≥150 mg/dl of glucose (*p0.01 for each). SB203580 or rosiglitazone pretreatment significantly attenuated hyperglycemia-induced apoptotic and anti-angiogenic signaling. D-Mannitol had no effect.Hyperglycemia induced apoptotic and anti-angiogenic signaling in CTBs. The observed diminution of hyperglycemia-induced signaling by SB203580 or rosiglitazone pretreatment suggests the involvement of apoptotic and anti-angiogenic signaling in CTB dysfunction.

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.

Preeclampsia (preE) is characterized by abnormal placentation. Marinobufagenin (MBG), a cardiotonic steroid (CTS), inhibits the function of cytotrophoblast cells (CTBs). We demonstrated that CTSs induce anti-angiogenic and anti-proliferative effects in Sw-71 CTBs. This study tests that CTSs induce apoptotic and stress signaling.Human extravillous Sw-71 CTBs were incubated with 0, 0.1, 1, 10, and 100 nM of each of three CTSs (MBG, cinobufatalin (CINO) and ouabain (OUB)) for 48 h. Some cells were pretreated with 10 μM p38 mitogen-activated protein kinase (p38 MAPK) inhibitor (SB203580) for 2 h prior to CTSs treatment. We analyzed p38 MAPK phosphorylation, expression of pro-inflammatory protein cyclooxygenase-2 (Cox-2) and ratio of pro-apoptotic Bcl-2-associated X protein (Bax) to anti-apoptotic Bcl-2 protein by western blot in CTSs-treated CTBs lysates. 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. Statistical comparisons were performed using analysis of variance with Duncan's post hoc test.p38 MAPK phosphorylation, expression of Cox-2 and Bax/Bcl-2 was upregulated (*p0.05) in CTBs exposed to ≥ 0.1 nM CTSs. Secretion of sFlt-1 and sEng were increased while VEGF and PIGF were decreased in Sw-71 CTBs treated ≥1 nM of each CTSs (*p0.01 for each). The SB203580 pretreatment of CTBs significantly attenuated CTS-induced effects.Exposure of Sw-71 CTBs to CTSs induced apoptotic and stress signaling and causing anti-angiongenic effect. The observed diminution of CTS-induced signaling by SB203580 pretreatment implicates p38 MAPK as a regulator of these pathways.

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.

Objective Preeclampsia (preE), a syndrome of hypertension and proteinuria. Most recently it was demonstrated that high circulating levels of soluble (pro) renin receptor s(P)RR at delivery were associated with preE. In this study the placental expression of (P)RR were evaluated in preE patients and in a rat model of preE as well as in nonhuman primates. We also evaluated the circulatory levels of s(P)RR. Study Design (1) Placental samples were collected from 20 NP and 20 preE consenting patients in an IRB approved prospective study. (2) An established rat model of preE and NP rats (n=10 each) were used. (3) The placental samples from squirrel monkey (NP; n=10) and owl monkey (both early and term, NP, n=1) were collected. The (P)RR expression were measured both by western blotting (WB) and Immunohistochemistry (IHC) using anti-ATP6IP2. The levels of serum s(P)RR were measured by ELISA. Results The placental expression of (P)RR were higher (p Conclusions These data suggest that increased expression of (P)RR in the placenta are related to the occurrence of preE in both patients and rat models. These data also reconfirmed that the high level of circulatory s(P)RR is associated with preE. The higher expression of (P)RR in early owl monkey in compare to term placenta 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 and squirrel monkey preE models.

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 causative factor in preE. We demonstrated that MBG inhibits the proliferation, migration, and invasion of cytotrophoblast (CTB) cells. We have identified a novel human monoclonal antibody with higher specificity than Digibind for MBG. We assessed the attenuation of MBG-induced CTBs dysfunction by three anti-MBG antibodies: 206–208, H1L2, and 3e9. Methods A panel of anti-MBG antibodies with potential as human therapeutic agents was developed by Panorama Research, Inc.; Sunnyvale, CA (206–208, H1L2). H1L2 was a humanized version of previously described anti-MBG murine antibody 3e9. 206–208 was identified in a human phage antibody library. Human CTBs were treated with DMSO (vehicle) or 0.1, 1, 10 or 100 nM of MBG for 48 h. Some cells were pretreated with 206–208, H1L2, or 3e9 for 2h, while others were co-treated with these antibodies prior to MBG treatment. Culture media were collected for analysis of pro-angiogenic and anti-angiogenic factors. Cell viability was measured using a CellTiter Assay kit. Cell lysates were utilized to evaluate the expression of 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), and soluble endoglin (sEng) were measured in culture media using ELISA kits. Statistical comparisons were performed using analysis of variance with Duncan9s post hoc test. Results MBG down-regulated PCNA and up-regulated p38 phosphorylation in CTBs treated with ≥1 nM MBG compared to basal (DMSO treatment) (*p Conclusions We found that all 3 anti-MBG antibodies attenuate MBG-induced anti-proliferative and anti-angiogenic milieu in cultured CTBs. Here we describe for the first time two fully human anti-MBG antibodies, which can be targeted as therapeutic agents for the development of innovative treatment strategies for preE and potentially other disorders involving aberrant MBG signaling.

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. Our lab is familiar with CINO and has shown it to inhibit cytotrophoblast cells function. Recently, it has been shown that CINO also inhibits the lung cancer cell function, and has been further implicated in other disease processes. In the present study, we propose to pursue this potential application of CINO using ovarian tumor cell line SK-OV-3. Study Design We evaluated the in-vitro effect of CINO on ovarian cancer cell line SK-OV-3. Cells were treated with 0.1, 1, 5, and 10 µM CINO. Cell proliferation was measured using a CellTiter Assay (Promega), which is a colorimetric method for determining the number of viable cells. Cell migration was measured using a CytoSelect Assay (Cell Biolabs). Cell invasion was measured using a FluoroBlok Assay (BD). Cell viability was measure using a CellTiter Assay (Promega). Cell cycle progression was evaluated by a Cell Cycle Phase Determination Kit (Cayman Chemical) and apoptosis was evaluated by an Apoptotic Blebs Assay Kit (Cayman Chemical). Cell cycle arrest and apoptotic signaling was determined by fluorescence-activated cell sorting (FACS) analysis. Results CINO at ≥0.5 µM inhibited SKOV-3 cell proliferation, migration, and invasion (p Conclusion This data demonstrates that CINO impairs SK-OV-3 cell function via cell cycle arrest and apoptotic signaling. These findings demonstrate the complex nature of this compound. Not only is CINO directly modulating the actions of the Na/K ATPase through classic mechanism of cardiotonic steroids, but is also directly influencing the nuclear expression of proteins involved in cell cycle progression and DNA repair. Additional investigational studies looking into the molecular pathways involved in altering cell cycle and entry into apoptosis are warranted. In conclusion, we have shown CINO to impair SK-OV3 cell function via cell cycle arrest and apoptotic signaling and suggest that CINO might be further investigated as a novel anti-ovarian cancer agent.

Objective An increasing level of evidence supports the utility of pravastatin as prevention against preeclampsia (preE). We previously demonstrated a hyperglycemia induced cytotrophoblast (CTBs) dysfunction characteristic of a preE-like phenotype and sought to demonstrate the utility of pravastatin in rescuing CTBs from this hyperglycemia induced dysfunction. Methods Human CTBs were treated with 100, 150, 200, 300, or 400 mg/dL glucose for 48 hrs. Cells were treated with pravastatin (1 ug/mL) either alongside or 2 hrs prior to glucose exposure. Some cells were treated with D-Mannitol as a negative control for glucose exposure. Cell migration was performed by Matrigel migration assay kit according to manufacturer protocol. Cell lysates were utilized to evaluate the mRNA expression of urokinase plasminogen activator (uPA) and plasminogen activator inhibitor 1 (PAI-1), while also assessing 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 Duncan9s post hoc test. Results Hyperglycemia inhibited CTBs migration by down-regulating uPA, PAI-1, PCNA and up-regulating p38 phosphorylation in cells treated with >150 mg/dL glucose compared to basal (100 mg/dL) (*p Conclusions Pravastatin mitigates the hyperglycemia-induced dysfunction of CTBs by attenuating the glucose-induced anti-proliferative, anti-migratory, anti-invasive and anti-angiogenic phenotype similar to that seen in Preeclampsia. This study supports the potential for pravastatin use on CTBs development early in pregnancy and the importance of continuing research of pravastatin in preE prevention.

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.

The liver is well-known for its ability to regenerate in response to damage. While little is known about the precise molecular mechanism governing liver regeneration, much research has explored the signals and stem cells that hold the potential for liver regeneration. Research has uncovered stem cells isolated in various organs capable of becoming hepatic or hepatic-like cells. Furthermore, in some systems, the signals involved in their transformation have been described. This chapter will review the current knowledge and involvement of stem cells, both hepatic and extrahepatic, in the process of liver regeneration. The therapeutic potential of modulating the natural liver to regenerate during disease, and transplantation of extrahepatic cells will be discussed.

Objective Preeclampsia (preE) is a multifaceted complication found uniquely in the pregnant patient and one that has puzzled scientist for years. It has been demonstrated that preE is not a single disorder, but a complex syndrome that is produced by various pathophysiologic triggers and mechanisms affecting 3–8% of obstetrical patients worldwide. PreE, is a major cause of premature delivery and maternal and fetal death. It is characterized by de novo development of hypertension and proteinuria after 20 weeks gestation. 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 the comparison of pregnancy outcome between patients with and without preE. Methods We recruited 20 normal pregnant (NP) and 20 preE consenting patients after deliveries in an IRB approved prospective study from Scott & White Healthcare. We evaluated the following parameters for mothers BP, Proteinuria, BMI, Gestational age, Age, Placental factors: circumference, placental signaling proteins. The placental stress signaling proteins (p38 MAPK, COX-2 and Bax/Bcl-2) were measured. We also evaluated babies for IUGR and anthropometric measurements. Comparisons were performed using Student9s t test. Results Mothers in the PE group had significantly higher blood pressures (SBP p=0.0000001 and DBP p=0.001) and also higher urinary protein excretion (p=0.002). Average hospital stay for PreE babies were longer than NP babies (p=0.001879). No complications were reported for NP babies; however, preE babies had multiple complications like hypoglycemia, RDS etc though they were born at preterm. Many of the PreE babies were born premature (p=0.017). The birth weights of the PE babies were much lower than the NP babies with a p value of 0.027 and also the PE babies were significantly SGA when compare to the NP babies with significant difference in their Ponderal Index (PI) (p=0.0004). The placental stress signaling proteins p38 MAPK, COX-2 and Bax/Bcl-2 were up-regulated in preE compared to normal pregnancy (p Conclusions PreE alters the intrauterine environment and activates the detrimental signaling that is transported to fetus resulting in premature deliveries, IUGR babies and their related complications like extended hospitalization.

Objective Preeclampsia (preE) is a hypertensive disorder of pregnancy. Cardiotonic steroids (CTS) are endogenous inhibitors of Na + /K + ATPase and at least one CTS, marinobufagenin (MBG), is elevated in preE prior to the development of the syndrome in rats with preE. MBG and ouabain impair cytotrophoblast (CTB) function, which is critical for placental development. Study Design We evaluated the effect of a CTS, cinobufotalin (CINO), on CTB cell function in vitro . Results CINO at ≥1 nM inhibited CTB cell proliferation, migration, and invasion (p Conclusion This data demonstrates that CINO impairs CTB cell function via cell cycle arrest and apoptotic signaling.

Objective Cinobufotalin (CINO), a cardiotonic steroid (CTS) or bufadienolide, is extracted from the skin secretions of the traditional Chinese medicine giant toads (Chan su). CINO has been used as a cardiotonic, diuretic and a hemostatic agent. Previously we have shown that CINO inhibits the cytotrophoblast cell function. Recently other study has shown that CINO inhibits A549, a lung cancer cell function. In this study, we assessed the effect of CINO on three different ovarian cancer cell lines; SK-OV-3, CRL-1978 and CRL-11731 to confirm whether the effect of CINO is cell specific. Study Design We evaluated the effect of CINO on three ovarian cancer cells SK-OV-3, CRL-1978, and CRL-11731 function in vitro. Each Cell lines were treated with different concentrations of CINO (0.1, 1, 5 and 10 µM). For each cell line cell proliferation, migration and invasion were measured by using a CellTiter Assay (Promega), Cytoselect Assay (Cell Biolabs) and by using a FluoroBlock Assay (BD) respectively. Proliferating Cell Nuclear Antigen (PCNA) was also evaluated in cell lysates of CINO treated these 3 ovarian cancer cells by western blot analysis. Cell Cycle arrest and Cell viability were determined by fluorescence-activated cell sorting (FACS) analysis. We also performed Annexin V staining on CINO treated these 3 ovarian cancer cell lines by immunofluorescence to evaluate the pro-apoptotic protein expression. In addition mitochondrial membrane potential has also been measured for all these 3 ovarian cell lines after CINO treatment using MMP kit, by FACS analysis. Results Concentration of CINO at 0.5 µM inhibit SK-OV-3, CRL-1978, and CRL-11731 ovarian cancer cells proliferation, migration and invasion without cell death and loss of cell viability but cell viability differs for each cell line. Each cell lines differ in response to CINO doses for PCNA expression as well as Annexin V pro-apoptotic protein expression. CINO decreases mitochondrial membrane potential for SK-OV-3 but for CRL-1978 and CRL-11731 increases in response to CINO treatment. Conclusion CINO is cell specific, as each cancer cell line responds differently. These data demonstrate that the mode of action of CINO is different on these 3 types of ovarian cancer cells.

Objective Despite growing knowledge of the pathophysiology leading to the development of preeclampsia (PreE) and diabetes mellitus (DM), the interaction between the two disease processes needs to be further examined. This study compared normal pregnancies to those complicated with preE, gestational diabetes mellitus (GDM), and/or pre-existing diabetes in order to assess the effects of elevated glucose on placental development and its potential role in the pathogenesis of preE. Methods The chart review was performed in an IRB approved retrospective cross-sectional study of live born singleton deliveries. A total 621 subjects were randomly selected from deliveries occurring between 2008 to 2011 at Baylor Scott & White Memorial hospital. Statistical analysis was performed using Duncan9s post-hoc test and ANOVA. Results Patients who developed preE had higher systolic and diastolic blood pressures than those who did not develop preE (p Conclusions The development of preE in those with pre-existing diabetes led to growth restriction and more severe disease as evidenced by lower birth weights and earlier gestational ages at delivery. These differences were not seen in GDM pregnancies. This supports the concept that elevated glucose levels during placental development in the first trimester may alter the placenta and lead to restriction later in pregnancy when a second stimulus triggers preE.

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