Angiogenic, hyperpermeability and vasodilator network in utero-placental units along pregnancy in the guinea-pig (Cavia porcellus)

<p>Abstract</p> <p>Background</p> <p>The angiogenic and invasive properties of the cytotrophoblast are crucial to provide an adequate area for feto-maternal exchange. The present study aimed at identifying the localization of interrelated angiogenic, hyperpermeability and vasodilator factors in the feto-maternal interface in pregnant guinea-pigs.</p> <p>Methods</p> <p>Utero-placental units were collected from early to term pregnancy. VEGF, Flt-1, KDR, B2R and eNOS were analyzed by immunohistochemistry, and the intensity of the signals in placenta and syncytial streamers was digitally analysed. Flt1 and eNOS content of placental homogenates was determined by western blotting. Statistical analysis used one-way analysis of variance and Tukey's Multiple Comparison post-hoc test.</p> <p>Results</p> <p>In the subplacenta, placental interlobium and labyrinth VEGF, Flt-1, KDR, B2R and eNOS were expressed in all stages of pregnancy. Syncytial streamers in all stages of gestation, and cytotrophoblasts surrounding myometrial arteries in early and mid pregnancy – and replacing the smooth muscle at term – displayed immunoreactivity for VEGF, Flt-1, KDR, eNOS and B2R. In partly disrupted mesometrial arteries in late pregnancy cytotrophoblasts and endothelial cells expressed VEGF, Flt-1, KDR, B2R and eNOS. Sections incubated in absence of the first antibody, or in presence of rabbit IgG fraction and mouse IgG serum, yielded no staining. According to the digital analysis, Flt-1 increased in the placental interlobium in days 40 and 60 as compared to day 20 (P = 0.016), and in the labyrinth in day 60 as compared to days 20 and 40 (P = 0.026), while the signals for VEGF, KDR, B2R, and eNOS showed no variations along pregnancy. In syncytial streamers the intensity of VEGF immunoreactivity was increased in day 40 in comparison to day 20 (P = 0.027), while that of B2R decreased in days 40 and 60 as compared to day 20 (P = 0.011); VEGF, Flt-1, KDR, B2R and eNOS expression showed no variations. Western blots for eNOS and Flt-1 in placental homogenates showed no significant temporal differences along pregnancy.</p> <p>Conclusion</p> <p>The demonstration of different angiogenic, hyperpermeability and vasodilator factors in the same cellular protagonists of angiogenesis and invasion in the pregnant guinea-pig, supports the presence of a functional network, and strengthens the argument that this species provides an adequate model to understand human pregnancy.</p

Bradykinin promotes migration and invasion of human immortalized trophoblasts

Having demonstrated that the bradykinin B2 receptor (B2R) is expressed in cells that participate in trophoblast invasion in humans and guinea-pigs, we investigated the role of bradykinin (BK) on cell migration and invasion in the HTR-8/SVneo trophoblast cell line using wound healing and invasion assays. First, we documented that HTR-8/SVneo cells expressed kallikrein, B2R, B1R, MMP-2 and MMP-9 using immunocytochemistry. Incubation with BK (10.0 microMol/L) for 18 hours increased the migration index 3-fold in comparison to controls or to cells preincubated with the B2R antagonist HOE-140. BK (10.0 microMol/L) incubation yielded a similar number of proliferating and viable cells as controls, therefore the enhanced closure of the wound cannot be attributed to proliferating cells. Incubation with BK (10.0 microMol/L) for 18 hours increased the invasion index 2-fold in comparison to controls or to cells preincubated with the antagonist of the B2R. Neither the B1R ligand Lys-des-Arg9 BK, nor its antagonist Lys-(des-Arg9-Leu8), modified migration and invasion. Further support for the stimulatory effect of B2R activation on migration and invasion is provided by the 3-fold increase in the number of filopodia per cell versus controls or cells preincubated with the B2R antagonist. Bradykinin had no effect on the cellular protein content of the B2R, nor the MMP-9 and MMP-2 gelatinase activity in the culture media varied after incubation with BK. This study adds bradykinin-acting on the B2R-to the stimuli of trophoblast migration and invasion, an effect that should be integrated to other modifications of the kallikrein-kinin system in normal and pathological pregnancies

Breaking through an epigenetic wall: Re-activation ofOct4by KRAB-containing designer zinc finger transcription factors

The gene Oct4 encodes a transcription factor critical for the maintenance of pluripotency and self-renewal in embryonic stem cells. In addition, improper re-activation of Oct4 contributes to oncogenic processes. Herein, we describe a novel designer zinc finger protein (ZFP) capable of upregulating the endogenous Oct4 promoter in a panel of breast and ovarian cell lines carrying a silenced gene. In some ovarian tumor lines, the ZFP triggered a strong reactivation of Oct4, with levels of expression comparable with exogenous Oct4 cDNA delivery. Surprisingly, the reactivation of Oct4 required a KRAB domain for effective upregulation of the endogenous gene. While KRAB-containing ZFPs are traditionally described as transcriptional repressors, our results suggest that these proteins could, in certain genomic contexts, function as potent activators and, thus, outline an emerging novel function of KRAB-ZFPs. In addition, we document a novel ZFP that could be used for the epigenetic reprograming of cancer cells

Vasodilator factors in the systemic and local adaptations to pregnancy

We postulate that an orchestrated network composed of various vasodilatory systems participates in the systemic and local hemodynamic adaptations in pregnancy. The temporal patterns of increase in the circulating and urinary levels of five vasodilator factors/systems, prostacyclin, nitric oxide, kallikrein, angiotensin-(1–7) and VEGF, in normal pregnant women and animals, as well as the changes observed in preeclamptic pregnancies support their functional role in maintaining normotension by opposing the vasoconstrictor systems. In addition, the expression of these vasodilators in the different trophoblastic subtypes in various species supports their role in the transformation of the uterine arteries. Moreover, their expression in the fetal endothelium and in the syncytiotrophoblast in humans, rats and guinea-pigs, favour their participation in maintaining the uteroplacental circulation. The findings that sustain the functional associations of the various vasodilators, and their participation by endocrine, paracrine and autocrine regulation of the systemic and local vasoactive changes of pregnancy are abundant and compelling. However, further elucidation of the role of the various players is hampered by methodological problems. Among these difficulties is the complexity of the interactions between the different factors, the likelihood that experimental alterations induced in one system may be compensated by the other players of the network, and the possibility that data obtained by manipulating single factors in vitro or in animal studies may be difficult to translate to the human. In addition, the impossibility of sampling the uteroplacental interface along normal pregnancy precludes obtaining longitudinal profiles of the various players. Nevertheless, the possibility of improving maternal blood pressure regulation, trophoblast invasion and uteroplacental flow by enhancing vasodilation (e.g. L-arginine, NO donors, VEGF transfection) deserves unravelling the intricate association of vasoactive factors and the systemic and local adaptations to pregnancy

Autofagia en el diagnóstico y tratamiento de cáncer de ovario

Tesis (Doctor en Biotecnología)Beca de doctorado nacional CONICYT No. 21100327. Beca de apoyo de tesis No 24121406.Proyecto Consorcio de Biomedicina (Biomedical Research Consortiu) N°CTU06 Biomedicina área 1.Proyecto Fondecyt No.1100870El cáncer de ovario mata alrededor de 140.000 mujeres al año en el mundo, diagnosticándose más de 12 mil nuevos casos anuales en Sudamérica , con una mortalidad de aproximadamente 7000 casos . Este panorama desfavorable se debe principalmente a su detección tard ía y a la falta de tratamiento . Recientemente, el uso de metformina , medicamento utilizado para la diabetes mellitus tipo 2, ha mostrado ser beneficioso para pacientes con cáncer de ovario , aumentando su sobrevida libre de progresión . Estas observaciones sorprendentes dieron paso a estudios in vitro e in vivo para estudiar sus mecanismos de acción los cuales han mostrado su efecto potenciador de quimioterapia. Sin embargo, estos resultados se han originado del uso de concentraciones varias veces superiores a la dosis terapéutica de metformina. Este trabajo de tesis buscó explicar los beneficios mostrados por el uso de metformina, utilizando una concentración dentro de su rango terapéutico , estudiando además, la autofagia como un posible mecanismo de acción, mediante el cual aumentaría la respuesta a la quimioterapia. También se investigaron proteínas que regulan la autofagia para evaluar su potencial uso como biomarcadores de cáncer de ovario . Esta investigación se realizó utilizando líneas celulares de cáncer de ovario y cultivos primarios de muestras (ascitis) de pacientes con cáncer de ovario. Para evaluar las proteínas que regulan la autofagia , se utilizaron líneas celulares y muestras de cáncer con diferentes malignidades. Esto fue posible gracias a la colaboración de una red de hospitales en Chile y en Perú , la cual nos facilita las muestras. Este estudio mostró por primera vez que el uso de una concentración terapéutica de metformina entregado previo a carboplatino y en combinación con carboplatino (quimioterapia), actúan sinérgicamente disminuyendo la viabilidad celular. lnteresantemente, el 36% (9/25) de las ascitis de pacientes analizadas respondieron a esta combinación . Adicionalmente, se mostró que la autofagia forma parte del mecanismo de acción de metformina en células de cáncer de ovario, donde aparentemente una regulación positiva y sostenida de este proceso sería un factor importante para inducir muerte celular. Además se encontró que existe una expresión diferencial a nivel de ARNm de prote ínas que regulan la autofagia que podrían servir para el diagnóstico del cáncer de ovario avanzado. Más aún, se encontró que bajos niveles de ARNm de PEA-15 y Beclin1 se asociarían con una peor sobrevida libre de progresión de la enfermedad. Este trabajo deja de manifiesto nuevamente que comprender las implicancias clínicas de los factores moleculares involucrados en la desregulación de procesos, como en este caso la autofagia en el cáncer de ovario , podría entregar nuevas estrategias para el diagnóstico y terapia de esta malignidad que permitirían en un futuro entregar una medicina personalizada.Ovarían cancer kills approximately 140,000 women per year worldwide. In South America more than 12,000 new cases are diagnosed each year with a mortality of approximately 7,000. This unfavorable scenario is principally due to detection at advanced stage. Recently, metformin, a drug used to treat type 2 diabetes mellitus, has been shown to be beneficial in patients with ovarían cancer by increasing chemotherapy mediated progression-free survival. This surprising observation led to studies in vitro and in vivo to determine the mechanisms of action. However, these published studies used elevated and non-FDA approved concentrations of metformin. In this thesis , we sought to deliver a mechanism of action for metformin-enhanced response to chemotherapy , using a metformin concentration within the approved and clinically used therapeutic range . Utilizing cell lines of ovarían cancer and primary cultures of samples from ovarían cancer patients (ascites), this thesis centered on the study of autophagy in metformin action and the role of autophagy associated genes as potential biomarkers in ovarían cancer. For biomarker discovery we obtained ovarían cancer samples of varying type , grade and response to chemotherapy , to elucidate a RNA expression pattern that could be used for diagnosis, prediction of drug-response and progression. This was possible through collaborations with a network of hospitals in both Chile and Peru. Herein, we report for the first time that the use of a therapeutic concentration of metformin delivered continuously (prior to chemotherapy and in combination with chemotherapy) acts synergistically to increase chemotherapyinduced cytotoxicity (principally carboplatin). lnterestingly, 36% (9/25) of cultured ascites extracted from ovarían cancer patients responded to the combination of metformin and carboplatin. We demonstrate that the mechanism of action of metformin in ovarían cancer cells requires the process of autophagy. Our results suggest that the sustained up-regulation of this process may be a manner to induce cancer cell death. We further report a differential expression of autophagy-related genes that could serve as prognosis indicators (biomarkers) for ovarían cancer. Preliminary data raises the possibility that reduced mRNA levels of PEA-15 and Beclin1 associate with poor progression-free survival. This thesis demonstrates once again that each cancer is unique, irrespective of similar tumor type , stage and grade. We speculate that a better understanding of autophagy in ovarían cancer could provide new strategies for diagnosis and therapy of this malignancy, that in the future will permit the practice of personalized medicine

The immunoreactivity showed a granular pattern for interlobar Flt-1, and for interlobar and labyrinthine VEGF; labyrinthine Flt-1 displayed a diffuse cytoplasmic staining

Arrowheads highlight linear signal in endothelial cells. Bar = 100 μm.<p><b>Copyright information:</b></p><p>Taken from "Angiogenic, hyperpermeability and vasodilator network in utero-placental units along pregnancy in the guinea-pig (Cavia porcellus)"</p><p>http://www.rbej.com/content/6/1/13</p><p>Reproductive biology and endocrinology : RB&E 2008;6():13-13.</p><p>Published online 27 Mar 2008</p><p>PMCID:PMC2291058.</p><p></p

Purified human Flt-1 and eNOS yielded bands with approximate molecular weights of 180 and 140 kDa respectively

No significant differences were observed between the means of the 3 homogenates included in each study period, using one-way analysis of variance and Tukey's Multiple Comparison post-hoc test.<p><b>Copyright information:</b></p><p>Taken from "Angiogenic, hyperpermeability and vasodilator network in utero-placental units along pregnancy in the guinea-pig (Cavia porcellus)"</p><p>http://www.rbej.com/content/6/1/13</p><p>Reproductive biology and endocrinology : RB&E 2008;6():13-13.</p><p>Published online 27 Mar 2008</p><p>PMCID:PMC2291058.</p><p></p

Intramural cytotrophoblasts (arrowheads) near the vessel lumen also expressed a granular eNOS reactivity, and intense intracytoplasmatic cytokeratin-7 staining

Bar = 100 μm.<p><b>Copyright information:</b></p><p>Taken from "Angiogenic, hyperpermeability and vasodilator network in utero-placental units along pregnancy in the guinea-pig (Cavia porcellus)"</p><p>http://www.rbej.com/content/6/1/13</p><p>Reproductive biology and endocrinology : RB&E 2008;6():13-13.</p><p>Published online 27 Mar 2008</p><p>PMCID:PMC2291058.</p><p></p