Co-exposure effects of butyl benzyl phthalate and TiO2 nanomaterials (anatase) on Metaphire guillelmi gut health.

Phthalic acid esters (PAEs) and TiO2 nanomaterials (nTiO2) are commonly used as plastic additives, nano-fertilizers or nano-pesticides. Their excessive co-applications led to the co-occurrence, which can induce damage to soil organisms such as Metaphire guillelmi (an earthworm widespread in farmland). However, the co-exposure effects of butyl benzyl phthalate (BBP, a typical PAEs) and nTiO2 on Metaphire guillelmi at environmental-relevant concentrations remain unclear. In this study, 1 mg kg-1 BBP and 1 mg kg-1 nTiO2 (anatase) were added into the soil to assess: (1) their effects on oxidative damage, digestive system, and neurotoxicity in Metaphire guillelmi gut on days 14 and 28; and (2) whether BBP and nTiO2 affected Metaphire guillelmi gut health by disrupting intestinal microorganisms. The results demonstrated that BBP and nTiO2 had the potential to inhibit the activity of superoxide dismutase, cellulase, protease, Na+K+-ATPase, and Ca2+-ATPase, as well as cause oxidative damage by altering intestinal bacteria such as Marmoricola and Microvirga at genus levels after 28 d-exposure. However, the exposure did not cause disorders of the intestinal bacteria. The present study provides more evidence for the sustainable application and scientific management of BBP and nTiO2, thus providing better guidance for PAEs and engineered nanomaterials regulations in agroecosystems.

Metallic oxide nanomaterials act as antioxidant nanozymes in higher plants: Trends, meta-analysis, and prospect.

Improving plant resistance against various environmental stresses is crucial to gain higher agricultural productivity for meeting future food demands of the fast-growing global population. Nanozymes, nanomaterials (NMs) with enzyme-like activity, have shown the potential to defend environmental stresses via scavenging reactive oxygen species (ROS) and augmenting the inherent antioxidant functions of plants. However, several studies confirmed that NMs could cause oxidative damage triggered by excessive ROS. In this study, the conversion mechanism between antioxidant and oxidant activities of metallic oxidative nanozymes was systematically reviewed and evaluated using meta-analysis approach. Moreover, our work attempts to seek the optimal dose and physicochemical property of antioxidant-functionalized NMs and put forward future research directions. The meta-analysis results indicated that NMs at a low dose (below 20 ppm) exhibited antioxidant activity which could scavenge ROS and alleviate their deleterious impacts. Conversely, their oxidant activity was activated at the exposure dose above 200 ppm which might induce ROS overproduction and lead to oxidative stress. Further, root exposure tends to stimulate the oxidant activity of NMs, and the NMs modification is highly promising for improving their bioavailability. A SWOT analysis was conducted to evaluate the strengths, weaknesses, opportunities, and threats of agro-applied nanozymes. Therefore, the rational design and development of nanozymes for better antioxidant potential will be beneficial to their applications in agriculture.

High payload and targeted release of anthracyclines by aptamer-tethered DNA nanotrains - Thermodynamic and release kinetic study.

As one of the most promising drug delivery carriers, self-assembled DNA nanostructures are characterized of well-defined sizes, excellent biocompatibility, high drug loading and ability to control drug release. Studying the interactions between anticancer drugs and DNA nanostructures can help to associate microstructure-drug loading-release rate-therapeutic effect. Herein AS1411 aptamer-tethered DNA nanotrains (AS1411NTrs) were constructed and used as anthracyclines carrier with high payload for targeted delivery. The bindings of doxorubicin (DOX), epirubicin (EPI), and daunorubicin (DAU) to AS1411NTrs were investigated by isothermal titration calorimetry and fluorescence spectroscopy, and thermodynamic parameters were obtained. The high drug payload capacity of AS1411NTrs was verified by the large number of binding sites (~20). The binding mode was determined by differential scanning calorimetry and potassium iodide (KI) quenching experiments. The release experiment data showed that DNase I facilitated drug release and the release followed the first-order kinetic model. MTT cell viability assay demonstrated that the drug-loaded AS1411NTrs had significantly higher cytotoxicity against target HeLa cells than normal human liver L02 cells. These findings revealed that AS1411NTrs had high payload and targeted release capacity for DOX, EPI, and DAU. This result can provide a theoretical basis for constructing reasonable DNA nanostructures based on drug carriers.

A Flexible Reduced Logarithmic-X Family of Distributions with Biomedical Analysis.

Statistical distributions play a prominent role in applied sciences, particularly in biomedical sciences. The medical data sets are generally skewed to the right, and skewed distributions can be used quite effectively to model such data sets. In the present study, therefore, we propose a new family of distributions to model right skewed medical data sets. The proposed family may be named as a flexible reduced logarithmic-X family. The proposed family can be obtained via reparameterizing the exponentiated Kumaraswamy G-logarithmic family and the alpha logarithmic family of distributions. A special submodel of the proposed family called, a flexible reduced logarithmic-Weibull distribution, is discussed in detail. Some mathematical properties of the proposed family and certain related characterization results are presented. The maximum likelihood estimators of the model parameters are obtained. A brief Monte Carlo simulation study is done to evaluate the performance of these estimators. Finally, for the illustrative purposes, three applications from biomedical sciences are analyzed and the goodness of fit of the proposed distribution is compared to some well-known competitors.

Novel carrier-free nanoparticles composed of 7-ethyl-10-hydroxycamptothecin and chlorin e6: Self-assembly mechanism investigation and in vitro/in vivo evaluation.

The combination therapy strategy based on both chemotherapy and photodynamic therapy (PDT) exhibits great potential for advanced cancer treatment. Multimodal nanodrug delivery systems based on both chemotherapeutic drug and photodynamic agent have been proven to possess excellent synergistic efficacy. In this study, 7-ethyl-10-hydroxycamptothecin (SN38) and chlorin e6 (Ce6) were co-assembled into novel carrier-free nanoparticles (SN38/Ce6 NPs) via simple antisolvent precipitation method. As expected, SN38/Ce6 NPs exhibited uniform morphology with a particle size of around 150 nm and a zeta potential of about -30 mV, good stability in aqueous solution/at lyophilized state and high cellular uptake efficiency against murine mammary carcinoma (4T1) cell lines. Besides, enhanced singlet oxygen generation capacity of the nanoparticles was both observed in test-tube and in 4T1 cell lines in contrast with Ce6 injection. Moreover, a ∼85 % inhibition rate of SN38/Ce6 NPs with laser was detected, which was significantly higher (P < 0.05) than those without laser (∼65 %) and injections (less than 20 %), verified the excellent synergistic antitumor efficacy of the nanoparticles due to combined chemo-photodynamic therapy, enhanced tumor accumulation and higher cellular internalization. Notably, chemical thermodynamic method and molecular dynamics (MD) simulations supplied solid data and visual images to estimate the driving forces for the self-assembly process of the carrier-free nanoparticles as primary hydrophobic interactions (π-π stacking) and subordinate hydrogen bonds. Conclusively, the above self-assembled carrier-free nanoparticles represented a promising synergistic anticancer strategy capable of maximal therapeutic efficacy and minimal systemic toxicity. Moreover, the application of thermodynamic method together with MD simulations in the investigation of NPs self-assembly process also provided new ideas for the assembly mechanism exploration of more complicated nanodrug delivery system.

Delivery of anticancer drug using pH-sensitive micelles from triblock copolymer MPEG-b-PBAE-b-PLA.

To improve the drug release rate in well-controlled manner, a new pH-sensitive triblock amphiphilic copolymer methyl poly(ethylene glycol) ether-b-poly(ß-amino esters)-b-poly lactic acid (MPEG-b-PBAE-b-PLA) and its self-assembled micelles were developed for anticancer drug delivery. The average molecular weight and molecular structure of MPEG-b-PBAE-b-PLA were confirmed by gel permeation chromatography (GPC) and 1H NMR. The formation of self-assembled micelles, the microstructures at different pH values, and the distribution of doxorubicin (DOX) were investigated by dissipative particle dynamics (DPD) simulation combined with experimental techniques. The copolymers formed stable core-shell-type micelles in water. The critical micelle concentration (CMC) values, particle sizes and zeta potentials of the blank micelles increased along with globule-extended conformational transitions when the pH values decreased from 7.4 to 5.0, due to the protonation of amine groups of PBAE. Obvious increases in the particle sizes and the drug loading content of micelles were observed with increasing DOX. The in vitro release behavior of DOX from the micelles was pH-dependent. The DOX release rate was improved obviously as pH decreased from pH7.4 to pH5.0, with over 96% of DOX was released within 48h. The drug release mechanism under different conditions was also analyzed using theoretical formulas. All the results suggest that the pH-sensitive MPEG-b-PBAE-b-PLA micelles might be a prospective candidate as anticancer drug delivery carrier with well-controlled release behavior.

Early- or mid-trimester amniocentesis biomarkers for predicting preterm delivery: a meta-analysis.

OBJECTIVE: To determine the value of early- or mid-trimester amniotic fluid levels of interleukin-6 (IL-6), matrix metalloproteinase-8 (MMP-8), and glucose for predicting preterm delivery. METHODS: Randomized controlled trials and two-arm prospective, retrospective, cohorts, and case-controlled studies in which patients received early- or mid-trimester amniocentesis for karyotyping, and biomarker testing of the amniotic fluid was performed and delivery data were available were included in the analysis. RESULTS: Outcome measures were the associations of amniotic fluid IL-6, MMP-8, and glucose levels with preterm delivery. Differences in means with 95% confidence intervals (CIs) were calculated. Of 288 articles identified, 14 were included in the meta-analysis with a total of 675 patients who had preterm birth and 2518 patients who had term births. The preterm-delivery group had significantly higher amniotic fluid IL-6 and MMP-8 levels, and a significantly lower glucose level than the term delivery group (IL-6: difference in means = 0.32, 95% CI: 0.22-0.43, p < 0.001; MMP-8: difference in means = 4.47, 95% CI: 0.83-8.11), p = 0.016; glucose: difference in means = -5.22, 95% CI: -8.19 to -2.26, p = 0.001) Conclusion: Early- or mid-trimester amniotic fluid IL-6, MMP-8, and glucose levels are useful for predicting the risk of preterm delivery. KEY MESSAGES Median amniotic fluid ferritin and IL-6 levels, and mean amniotic fluid ALP levels were higher in the preterm group. The preterm-delivery group had significantly higher amniotic fluid IL-6 and MMP-8 levels, and a significantly lower glucose level than the term-delivery group.

Predictive value of serum ß-hCG for early pregnancy outcomes among women with recurrent spontaneous abortion.

OBJECTIVE: To examine peak serum levels of the ß-subunit of human chorionic gonadotropin (ß-hCG) for prediction of early pregnancy outcomes among women with recurrent spontaneous abortion (RSA). METHODS: In a retrospective study, the medical records of pregnant women with a history of RSA treated at Sun Yat-sen Memorial Hospital, China, between January 2011 and July 2013 were reviewed. Serum ß-hCG had been measured twice weekly from 5 to 13weeks of pregnancy, and pregnancy was monitored by transvaginal ultrasonography to 13(+6)weeks. Optimal cutoff for peak ß-hCG level was determined by receiver operator characteristic curve analysis and Youden index. Women were divided into four groups on the basis of optimal peak ß-hCG cutoff and pregnancy outcome (pregnancy at 13weeks or spontaneous abortion). Peak ß-hCG levels and length of pregnancy at this peak were examined. RESULTS: Overall, 1240 patients were included. The optimal cutoff value of peak ß-hCG was 88 468IU/L, with a sensitivity, specificity, positive predictive value, and negative predictive value for successful pregnancy of 95.6%, 88.0%, 95.6%, and 89.0%, respectively. A faster rise in ß-hCG, higher peak ß-hCG, and longer pregnancy length at peak ß-hCG were associated with successful early pregnancy. CONCLUSION: A cutoff value of serum ß-hCG of 88 000IU/L could be used to predict early pregnancy outcomes for women with a history of RSA.

Diagnostic value of amniotic fluid inflammatory biomarkers for subclinical chorioamnionitis.

OBJECTIVE: To determine the value of measuring amniotic fluid inflammatory biomarkers for diagnosis of subclinical chorioamnionitis. METHODS: A prospective study was conducted among pregnant women with cervical dilation, preterm premature rupture of membranes, threatened late abortion, or threatened premature labor who attended a tertiary care hospital in Guangzhou, China, between June 1, 2012, and January 31, 2014. Participants were divided into two groups according to the presence or absence of subclinical chorioamnionitis. Surface-enhanced laser desorption/ionization time-of-flight mass spectroscopy (SELDI-TOF-MS) was used to detect human neutrophil defensins (HNP-1 and HNP-2), calgranulins A (S100A8), and calgranulins C (S100A12) in amniocentesis samples. RESULTS: Overall, 22 patients had subclinical chorioamnionitis and 17 patients did not. Positive test results for HNP-2 were noted for more patients with subclinical chorioamnionitis than for those without for HNP-2 (19 [86%] vs 2 [12%]; P<0.001), HNP-1 (19 [86%] vs 5 [29%]; P=0.001), S100A12 (20 [91%] vs 9 [53%]; P=0.011), and S100A8 (12 [55%] vs 0; P<0.001). When three or four of these biomarkers were present, the accuracy for a diagnosis of subclinical chorioamnionitis was 89.7%. The sensitivity, specificity, positive predictive value, and negative predictive value were 81.8%, 100.0%, 100.0%, and 81.0%, respectively. CONCLUSION: Detection of inflammatory biomarkers in the amniotic fluid by SELDI-TOF-MS exhibited high diagnostic accuracy for subclinical chorioamnionitis.

MicroRNA-29b suppresses tumor angiogenesis, invasion, and metastasis by regulating matrix metalloproteinase 2 expression.

UNLABELLED: Hepatocellular carcinoma (HCC) is a highly vascularized tumor with frequent intrahepatic metastasis. Active angiogenesis and metastasis are responsible for rapid recurrence and poor survival of HCC. We previously found that microRNA-29b (miR-29b) down-regulation was significantly associated with poor recurrence-free survival of HCC patients. Therefore, the role of miR-29b in tumor angiogenesis, invasion, and metastasis was further investigated in this study using in vitro capillary tube formation and transwell assays, in vivo subcutaneous and orthotopic xenograft mouse models, and Matrigel plug assay, and human HCC samples. Both gain- and loss-of-function studies showed that miR-29b dramatically suppressed the ability of HCC cells to promote capillary tube formation of endothelial cells and to invade extracellular matrix gel in vitro. Using mouse models, we revealed that tumors derived from miR-29b-expressed HCC cells displayed significant reduction in microvessel density and in intrahepatic metastatic capacity compared with those from the control group. Subsequent investigations revealed that matrix metalloproteinase-2 (MMP-2) was a direct target of miR-29b. The blocking of MMP-2 by neutralizing antibody or RNA interference phenocopied the antiangiogenesis and antiinvasion effects of miR-29b, whereas introduction of MMP-2 antagonized the function of miR-29b. We further disclosed that miR-29b exerted its antiangiogenesis function, at least partly, by suppressing MMP-2 expression in tumor cells and, in turn, impairing vascular endothelial growth factor receptor 2-signaling in endothelial cells. Consistently, in human HCC tissues and mouse xenograft tumors miR-29b level was inversely correlated with MMP-2 expression, as well as tumor angiogenesis, venous invasion, and metastasis. CONCLUSION: miR-29b deregulation contributes to angiogenesis, invasion, and metastasis of HCC. Restoration of miR-29b represents a promising new strategy in anti-HCC therapy.

Licofelone-nitric oxide donors as anticancer agents.

Five licofelone ([2,2-dimethyl-6-(4-chlorophenyl)-7-phenyl-2,3-dihydro-1H-pyrrolizin-5-yl]acetic acid) nitric oxide donor conjugates were developed by a parallel synthesis approach. The biological screening revealed that compounds with a propyl (6b), butyl (6c), or octyl (6d) chain between licofelone and the nitric oxide donor exhibited high antiproliferative potency at MCF-7 and MDA-MB-231 breast cancer as well as at HT-29 colon cancer cells. Moreover, 6b-d possessed at least 2-fold higher cytotoxicity at MDA-MB-231 cells than the parent compound licofelone although they showed less inhibitory activity at COX-1 and COX-2. A correlation between COX inhibition and growth inhibitory properties is not visible. However, the high levels of nitric oxide production of the compounds may result in their high cytotoxic activity.

The Lbc Rho guanine nucleotide exchange factor α-catulin axis functions in serotonin-induced vascular smooth muscle cell mitogenesis and RhoA/ROCK activation.

Serotonin (5-hydroxytryptamine, 5-HT) is mitogenic for several cell types including pulmonary arterial smooth muscle cells (PASMC), and is associated with the abnormal vascular smooth muscle remodeling that occurs in pulmonary arterial hypertension. RhoA/Rho kinase (ROCK) function is required for 5-HT-induced PASMC mitogenesis, and 5-HT activates RhoA; however, the signaling steps are poorly defined. Rho guanine nucleotide exchange factors (Rho GEFs) transduce extracellular signals to Rho, and we found that 5-HT treatment of PASMC led to increased membrane-associated Lbc Rho GEF, suggesting modulation by 5-HT. Lbc knockdown by siRNA attenuated 5-HT-induced thymidine uptake in PASMC, indicating a role in PASMC mitogenesis. 5-HT triggered Rho-dependent serum response factor-mediated reporter activation in PASMC, and this was reduced by Lbc depletion. Lbc knockdown reduced 5-HT-induced RhoA/ROCK activation, but not p42/44 ERK MAP kinase activation, suggesting that Lbc is an intermediary between 5-HT and RhoA/ROCK, but not ERK. 5-HT stimulation of PASMC led to increased association between Lbc, RhoA, and the α-catulin scaffold. Furthermore, α-catulin knockdown attenuated 5-HT-induced PASMC thymidine uptake. 5-HT-induced PASMC mitogenesis was reduced by dominant-negative G(q) protein, suggesting cooperation with Lbc/α-catulin. These results for the first time define a Rho GEF involved in vascular smooth muscle cell growth and serotonin signaling, and suggest that Lbc Rho GEF family members play distinct roles. Thus, the Lbc/α-catulin axis participates in 5-HT-induced PASMC mitogenesis and RhoA/ROCK signaling, and may be an interventional target in diseases involving vascular smooth muscle remodeling.

JNK regulates serotonin-mediated proliferation and migration of pulmonary artery smooth muscle cells.

JNK is a member of the MAPK family and has essential roles in inflammation and cell differentiation and apoptosis. In recent years, there have been accumulating data indicating a novel role for JNK in cell growth and migration. In this report, we demonstrate that JNK activity is necessary for serotonin (5-HT)-induced proliferation and migration of bovine pulmonary artery smooth muscle cells (PASMCs). Stimulation with 5-HT was found to lead to activation of JNK with a maximal activation at 10 min. Inhibition of JNK with its specific inhibitor, SP-600125, or its dominant-negative form, DN-JNK, significantly reduced 5-HT-stimulated [(3)H]thymidine incorporation and cyclin D1 expression. A similar inhibitory effect on SMC migration produced by 5-HT, as detected by a wound healing assay, was observed with inhibition of JNK. Furthermore, inhibition of 5-HT receptors (1B) and (2A), but not inhibition of the 5-HT transporter, blocked 5-HT-induced JNK activation. Inhibition of phosphatidylinositol 3-kinase (PI3K) with LY-294002 and wortmannin had little or no effect on 5-HT-induced JNK phosphorylation, but JNK inhibitor SP-600125 and DN-JNK blocked 5-HT-stimulated phosphorylation of Akt and its downstream effectors, p70S6K1 and S6, indicating that Akt is a downstream effector of JNK. Activation of Akt by 5-HT was blocked only minimally, if at all, by inhibitors of ERK and p38 MAPK, indicating a uniqueness of JNK MAPK in this activation of Akt. Coimmunoprecipitation showed binding of Akt to JNK, further supporting the interaction of JNK and Akt. Thus JNK is a critical molecule in 5-HT-induced PASMC proliferation and migration and may act at an important point for cross talk of the MAPK and PI3K pathways. Its activation by 5-HT is initiated through 5-HT (1B) and (2A) receptors, and its stimulation of SMC proliferation and migration occurs through the Akt pathway.

Antiproliferative activity of the total saponin of Solanum lyratum Thunb in Hela cells by inducing apoptosis.

Total saponin of Solanum lyratum Thunb (TSSLT), a species of natural biologically active substances isolated from Solanum lyratum Thunb, possesses various bioactivities. It has been proposed that the induction of apoptosis may be the basis of its antitumor activity. However, the molecular mechanism underlying the total saponin-induced apoptotic process remains unknown. In the present study, we describe the anti-proliferative effect of TSSLT on human cervical cancer cells (Hela). The TSSLT induced apoptosis of Hela in a time-dependent manner with an IC50 for cell viability of 6 microg/ml. The TSSLT-induced cell death was characterized by changes in cell morphology, DNA fragmentation, activation of caspase-like activities, poly (ADP-ribose) polymerase (PARP) cleavage and release of cytochrome c (cyt c) into cytosol. TSSLT activated various caspases such as caspase-3, -8, and -9 (like) activities but not caspase-1 like activity. The cell death was completely prevented by the pan caspase inhibitor benzyloxy carbonyl-Val-Ala-Asp- fluoromethyl-ketone (Z-VAD-FMK). More than 80% cell survival was observed in the presence of a caspase-3 inhibitor. In addition, treatment with TSSLT induced the increase of Bax:Bcl-2 ratio in Hela cells. These results suggest that the induction of apoptosis by TSSLT involves multiple pathways antigen including death receptor and mitochondrial pathway and strongly suggest that the mitochondrial pathway was mediated by low expression of Bcl-2 and upregulation of Bax, release of cyt c and subsequent activation of caspase-3 followed by down stream events leading to apoptotic cell death.

Inhibition of serotonin-induced mitogenesis, migration, and ERK MAPK nuclear translocation in vascular smooth muscle cells by atorvastatin.

The HMG-CoA reductase inhibitors, statins, have pleiotropic effects which may include interference with the isoprenylation of Ras and Rho small GTPases. Statins have beneficial effects in animal models of pulmonary hypertension, although their mechanisms of action remain to be determined. Serotonin [5-hydroxytryptamine (5-HT)] is implicated in the process of pulmonary artery smooth muscle (PASM) remodeling as part of the pathophysiology of pulmonary hypertension. We examined the effect of atorvastatin on 5-HT-induced PASM cell responses. Atorvastatin dose dependently inhibits 5-HT-induced mitogenesis and migration of cultured bovine PASM cells. Inhibition by atorvastatin was reversed by mevalonate and geranylgeranylpyrophosphate (GGPP) supplement, suggesting that the statin targets a geranylgeranylated protein such as Rho. Concordantly, atorvastatin inhibits 5-HT-induced cellular RhoA activation, membrane localization, and Rho kinase-mediated phosphorylation of myosin phosphatase-1 subunit. Atorvastatin reduced activated RhoA-induced serum response factor-mediated reporter activity in HEK293 cells, indicating that atorvastatin inhibits Rho signaling, and this was reversed by GGPP. While 5-HT-induced ERK MAP and Akt kinase activation were unaffected by atorvastatin, 5-HT-induced ERK nuclear translocation was attenuated in a GGPP-dependent fashion. These studies suggest that atorvastatin inhibits 5-HT-induced PASM cell mitogenesis and migration through targeting isoprenylation which may, in part, attenuate the Rho pathway, a mechanism that may apply to statin effects on in vivo models of pulmonary hypertension.

The 5-HT transporter transactivates the PDGFbeta receptor in pulmonary artery smooth muscle cells.

Serotonin (5-HT) stimulates smooth muscle cell growth through 5-HT receptors and the 5-HT transporter (5-HTT), and has been associated with pulmonary hypertension (PH). Platelet-derived growth factor receptors (PDGFR) have also been associated with PH. We present evidence for the first time that 5-HT transactivates PDGFRbeta through the 5-HTT in pulmonary artery (PA) SMCs. Inhibition of PDGFR kinase with imatinib or AG1296 blocks 5-HT-stimulated PDGFRbeta phosphorylation. 5-HTT inhibitors and the Na+/K+-ATPase inhibitor ouabain, but not 5-HT2 and 5-HT1B/1D receptor inhibitors, block PDGFRbeta activation by 5-HT. Notably, 5-HTT binds the PDGFRbeta upon 5-HT stimulation and the 5-HTT inhibitor fluoxetine blocks both the binding and PDGDRbeta activation. Activation of PDGFRbeta may occur through oxidation of a catalytic cysteine of tyrosine phosphatase. 5-HT-activated PDGFRbeta phosphorylation is blocked by the antioxidant N-acetyl-L-cysteine and the NADPH oxidase inhibitor, DPI. Inhibition of PDGFR kinase with imatinib or AG1296 significantly inhibits SMC proliferation and migration induced by 5-HT in vitro. Infusion of 5-HT by miniosmotic pumps enhances PDGFRbeta activation in mouse lung in vivo. In summary, these results demonstrate that 5-HT transactivates PDGFRbeta in PASMCs leading to SMC proliferation and migration, and may be an important signaling pathway in the production of PH in vivo.

Serotonin-induced growth of pulmonary artery smooth muscle requires activation of phosphatidylinositol 3-kinase/serine-threonine protein kinase B/mammalian target of rapamycin/p70 ribosomal S6 kinase 1.

We have previously found that both mitogen-activated protein kinase (MAPK)- and Rho kinase (ROCK)-related signaling pathways are necessary for the induction of pulmonary artery smooth muscle cell (SMC) proliferation by serotonin (5-hydroxytryptamine [5-HT]). In the present study, we investigated the possible additional participation of a phosphatidylinositol 3-kinase (PI3K)/serine-threonine protein kinase B (Akt)/mammalian target of rapamycin (mTOR)/p70 ribosomal S6 kinase (S6K1) pathway in this growth response. We found transient activation of Akt (Ser473) and more prolonged activation of S6K1 by 5-HT. Inhibition of PI3K with Wortmannin and LY294002 completely blocked these activations, but not that of MAPK or the ROCK substrate myosin phosphatase targeting subunit. Similarly, inhibition of MAPK and ROCK failed to block the Akt activation. Inhibition of Akt with NL-71-101 and downregulation of Akt expression with Akt small interfering RNA blocked 5-HT-induced S6K1 phosphorylation. Wortmannin, LY294002, and NL-71-101 dose-dependently inhibited 5-HT-induced SMC proliferation. 5-HT stimulated mTOR phosphorylation and the mTOR inhibitor, rapamycin, blocked activations of S6K1 and S6 ribosomal protein, and inhibited 5-HT-induced SMC proliferation. Akt phosphorylation and cell proliferation were also blocked by the antioxidants, N-acetyl-l-cysteine, Ginko biloba 501, and tiron, the reduced nicotinamide adenine dinucleotide phosphate oxidase inhibitor, diphenyleneiodonium, and the 5-HT2 receptor antagonists ketanserin and mianserin, but not by the 5-HT serotonin transporter or 5-HT 1B/1D receptor antagonists. We conclude from these studies that a parallel PI3K- and reactive oxygen species-dependent Akt/mTOR/S6K1 pathway participates independently from MAPK and Rho/ROCK in the mitogenic effect of 5-HT on pulmonary artery SMCs. From these and other studies, we postulate that independent signaling pathways leading to 5-HT-induced SMC proliferation are initiated through multiple 5-HT receptors and serotonin transporter at the cell surface.

Isorhapontigenin and resveratrol suppress oxLDL-induced proliferation and activation of ERK1/2 mitogen-activated protein kinases of bovine aortic smooth muscle cells.

The objective of our study was to compare the inhibitory effect of isorhapontigenin (ISO) and resveratrol, two natural antioxidants, on oxidized low-density lipoprotein (oxLDL)-induced proliferation of bovine aortic smooth muscle cells (BASMCs) and its relation to reactive oxygen species (ROS) generation and extracellular signal-regulated kinase 1/2 activation. The results showed that stimulation of oxLDL (50-150 microg/mL) for 48 hr induced a dose-dependent increase in cell number and incorporation of [3H]thymidine into DNA of BASMCs. Western blot analysis demonstrated that oxLDL (150 microg/mL) stimulated an evident phosphorylation of p42/44 MAP kinases in BASMCs. Incubation of BASMCs with oxLDL induced significant increase in ROS detected by using an oxidant-sensitive fluorescent probe of 2',7'-dichlorofluorescin diacetate. The level of H2O2 in the medium of cultured BASMCs also increased markedly. Preincubation of BASMCs with ISO and resveratrol significantly inhibited oxLDL-induced cell proliferation and incorporation of [3H]thymidine, and the phosphorylation of p42/44 MAP kinases in BASMCs as well. Furthermore, preincubation of BASMCs with ISO and resveratrol attenuated oxLDL-induced increases in ROS and H2O2 levels. The results suggested that oxLDL-induced acute formation of ROS and subsequent activation of redox-sensitive extracellular signal-regulated kinase 1/2 MAPK pathways, which might be important for mitogenic signaling of oxLDL in vascular smooth muscle cells. The inhibitory effect of ISO and resveratrol on oxLDL-induced mitogenesis of BASMCs might be taken through blocking the generation of ROS and activation of the ERKs pathway.