Sorina Grisaru-Granovsky

Differential expression of Protease activated receptor 1 (Par1) and pY397FAK in benign and malignant human ovarian tissue samples

Protease activated receptors (PAR) form a family of G‐protein coupled receptors (GPCR) encoding their own ligands and uniquely activated via proteolytic cleavage. Although proteases in general have been implicated in the remodeling of the extracellular tumor microenvironment, the role of cell surface receptors activated by proteolysis is now emerging. In our present study we investigated the expression pattern of protease activated receptor 1 hPar1 in ovarian carcinoma tissue samples. Abundant hPar1 mRNA and protein were detected in “low malignant potential” and in invasive carcinomas, regardless of the histological subtype. In contrast, no hPar1 expression was detected on the cell surface of normal ovarian epithelium. The differential expression pattern of hPar1 was shown by in situ hybridization, immunohistochemistry and semi‐quantitative RT‐PCR analyses. In early stages of ovarian carcinoma (Ia), the contra lateral normal ovary showed strong PAR1 expression as opposed to the lack of expression in the ovarian epithelium obtained from normal individuals. In parallel, we analyzed the expression pattern of αvβ5 integrin and of activated focal adhesion kinase (FAK), a major focal contact protein, in these tissues. Although abundant expression of αvβ5 integrin was observed in all tissues specimens examined, regardless of either normal or malignant, the level of activated FAK was differentially expressed. Phosphorylated FAK was seen in invasive ovarian carcinoma, but not in the normal ovarian epithelium. The abundant hPar1 levels in pathological malignant ovarian carcinoma is likely to transmit signals leading to the phosphorylation of FAK and thereby alterations in the integrin functional state. Altogether our data suggest that hPar1 and FAK cooperate to promote ovarian cancer malignancy.

Effect of interpregnancy interval on adverse perinatal outcomes — a national study

BACKGROUND The interpregnancy interval (IPI) has been reported to influence the outcome of pregnancy and birth. We performed a national study in Israel to determine the impact of IPI on multiple adverse perinatal outcomes. STUDY DESIGN This longitudinal cohort study used birth certificates of siblings born to the same biological mother, with at least one previous birth and a subsequent singleton pregnancy. Adverse pregnancy outcomes included preterm delivery, very preterm birth, small for gestational age (SGA), very SGA (VSGA), early neonatal death and major congenital malformations. Multivariate logistic regression was performed for each outcome. RESULTS The study included 440,838 of a total of 846,845 reported live births in Israel over 5 years; excluded were primiparas (32%), multifetal births (4.9%) and those with incomplete data (10.9%). For IPIs shorter than 6 months, there were significantly increased risks for preterm birth (OR=1.23), SGA (OR=1.14), VSGA (OR=1.15), early neonatal death (OR=1.62) and congenital malformations (OR=1.14). Intervals of 60 months or longer had higher risks for preterm birth (OR=1.39) and VSGA (OR=1.16). CONCLUSION Optimal IPI recommendation of >11 months is an accessible and low-cost means to improve multiple adverse perinatal outcomes.

Human Protease-Activated Receptor 1 Expression in Malignant Epithelia: A Role in Invasiveness

While protease-activated receptors (PARs) play a traditional role in vascular biology, they emerge with surprisingly new assignments in tumor biology. PAR1 expression correlates with the invasion properties of breast carcinoma, whereas human PAR1 antisense reduces their ability to migrate through Matrigel. Part of the molecular mechanism of PAR1 invasion involves the formation of focal contact complexes on PAR1 activation. PAR1 induces angiogenesis in animal models in vivo and exhibits an oncogenic phenotype of enhanced ductal complexity when overexpressed in mouse mammary glands.

Expectant Management of Midtrimester Premature Rupture of Membranes: A Plea for Limits

OBJECTIVE: Our aim was to assess neonatal and maternal complications of the expectant management of pregnancies with preterm premature rupture of membranes (P-PROM) prior to 24 weeks of gestation and to delineate a patient consult strategy.STUDY DESIGN: We included all consecutive cases of early midtrimester P-PROM (16–24 weeks gestation). Information coded in our perinatal database was analyzed. Descriptive statistics, Students t-test and Mann–Whitney test, and a logistic regression model were built accordingly.RESULTS: A total of 28 women presented with P-PROM at 16–24 weeks (mean 22.7±1.0 weeks). Two patients declined conservative management and one was lost to follow-up (10.7%). In all, 25 (89.2%) were followed until the onset of labor or development of chorioamnionitis. Overall, 8/25 (32%) Of the neonates survived. Pulmonary hypoplasia accounted for three deaths (3/25, 12%). Of 10 pregnancies with P-PROM before 22 weeks gestation, two (20%) neonates survived. The amount of amniotic fluid and gestational age at the time of diagnosis were crucial independent factors determining overall survival. Pulmonary hypoplasia (12%) and skeletal deformities (0%) were infrequent. The 21-day mean maternal antenatal hospital stay was further complicated by a high cesarean rate delivery (33.7%) and by postpartum infectious morbidity (32%).CONCLUSION: In cases of early midtrimester P-PROM (<24 weeks) expectantly managed, neonatal survival is positively associated with the amount of amniotic fluid present and with the gestational age at the time of diagnosis. The mothers are at increased risk of prolonged antenatal hospitalization, cesarean delivery, preterm birth, and postpartum infection. In very early midtrimester P-PROM (<22 weeks), the maternal complication rate outweighs the poor neonatal outcome and expectant management should be reconsidered.

Protease activated receptor-1, PAR1, promotes placenta trophoblast invasion and β-catenin stabilization

Despite extensive efforts toward elucidation of the molecular pathway controlling cytotrophoblast (CTB) invasion to the uterine decidua, it remains poorly defined. There are striking similarities between tumor cell invasion and cytotrophoblast implantation to the deciduas whereby the role of Protease Activated Receptors (PARs) and wnt signaling is well recognized. We examine here consequences of modulation of PAR1 and PAR2 expression and function on CTB invasion and β‐catenin stabilization. Toward this end, we utilized a model system of extravillous trophoblast (EVT) organ culture and various placenta cell lines (e.g., JAR and HTR‐8/Svneo). Activation of PAR1 induces EVT invasion while hPar1‐SiRNA and PAR1 antagonist SCH79797—effectively inhibited it. In parallel, the Wnt inhibitor Dickkopf‐1 (Dkk1) similarly inhibited it. Nuclear localization of β‐catenin is seen only after PAR1 activation, and is markedly reduced following the application of hPar1‐SiRNA construct and PAR1 antagonist in CTBs. In contrast, PAR2 elicited a low cytoplasmic β‐catenin level as also proliferation and invasion. In the non‐activated CTBs in‐comparison, β‐catenin appeared limited to the membrane pools. Concomitantly, a temporal regulated pattern of Wnt‐4, 5a, 7b, 10a, 10b expression is seen along PAR1 appearance. Enforced expression of Wnt antagonists, Secreted Frizzled Related Proteins; SFRP2 & 5; into HTR‐8/Svneo, resulted with a markedly reduced nuclear β‐catenin levels, similar to the effect obtained by hPar1‐SiRNA treatment. Identification of PAR1 downstream target/s may nonetheless contribute to the formation of a future platform system for eliciting a firm placenta‐uterus interactions and to the definition of late pregnancy outcomes. J. Cell. Physiol. 218: 512–521, 2009.

The pattern of expression of protease-activated receptors (PARs) during early trophoblast development

Human fetal development depends on the ability of the embryo to gain access to the maternal circulation. Thus, specialized stem cells of the newly formed placenta, trophoblast, invade the uterus and its arterial network to establish an efficient feto‐maternal molecular exchange. To accomplish this task, trophoblast differentiation during the first trimester of pregnancy involves cell proliferation, invasion, and extracellular matrix (ECM) remodelling. Trophoblast invasion shares many features with tumour cell invasion, with the distinction that it is strictly spatially and temporally controlled. We have previously demonstrated that PAR1, the first member of the protease‐activated receptor (PAR) family, plays a central role in tumour cell invasion. In the present study we have examined the pattern of expression of PAR1 and other PAR family candidates during early human placental development. We show that PAR1 and PAR3 are highly and spatially expressed between the 7th and 10th weeks of gestation but not at the 12th week and thereafter. Likewise, high expression levels of PAR1 and PAR3 were observed in the cytotrophoblast cells of complete hydatidiform mole as compared to minimal levels in normal age‐matched placenta. Together, our data suggest the involvement of PAR1 and PAR3 in restricted and unrestricted pathological trophoblast invasion. Copyright

G Protein-Coupled Receptors in Cancer

Despite the fact that G protein-coupled receptors (GPCRs) are the largest signal-conveying receptor family and mediate many physiological processes, their role in tumor biology is underappreciated. Numerous lines of evidence now associate GPCRs and their downstream signaling targets in cancer growth and development. Indeed, GPCRs control many features of tumorigenesis, including immune cell-mediated functions, proliferation, invasion and survival at the secondary site. Technological advances have further substantiated GPCR modifications in human tumors. Among these are point mutations, gene overexpression, GPCR silencing by promoter methylation and the number of gene copies. At this point, it is imperative to elucidate specific signaling pathways of “cancer driver” GPCRs. Emerging data on GPCR biology point to functional selectivity and “biased agonism”; hence, there is a diminishing enthusiasm for the concept of “one drug per GPCR target” and increasing interest in the identification of several drug options. Therefore, determining the appropriate context-dependent conformation of a functional GPCR as well as the contribution of GPCR alterations to cancer development remain significant challenges for the discovery of dominant cancer genes and the development of targeted therapeutics.

PAR1 plays a role in epithelial malignancies: transcriptional regulation and novel signaling pathway.

Protease‐activated receptor1 (PAR1) is the first and prototype member of an established PAR family comprising four members. The role of PAR1 in tumor biology has been established, and is characterized by a consistent direct correlation between overexpression of its levels and epithelial tumor aggressiveness. We have found that high expression of the human Par1 (hPar1) gene in epithelial tumors is controlled largely at the transcriptional level. This led us to assign Egr‐1, a transcription activator, as an inducer of hPar1, and p53, a tumor suppressor gene, as an inhibitor, both acting to achieve fine tuning of hPar1 in prostate carcinoma. High PAR1 levels maintain prosurvival signals in tumor cells while silencing or ablation of the gene induce apoptosis. Studies of our hPar1 transgenic mice, which overexpress hPar1 in the mammary glands, revealed a novel PAR1‐induced β‐catenin stabilization function. The components connecting PAR1 to β‐catenin stabilization have been determined, assigning at first Gα13 as a selective immediate component. The PAR1‐Gα13 axis recruits disheveled (DVL), an upstream signaling partner of the canonical Wnt signaling pathway. Silencing of DVL by siRNA‐DVL potently abrogates PAR1‐induced β‐catenin stabilization, demonstrating its critical role in the process. We, thus, propose that transcriptional regulation of hPar1 gene over expression in epithelia malignancies initiates a novel signaling pathway, directly connecting to β‐catenin stabilization, a core event in both tumorigenesis and developmental processes.

Etk/Bmx Regulates Proteinase-Activated-Receptor1 (PAR1) in Breast Cancer Invasion: Signaling Partners, Hierarchy and Physiological Significance

Background While protease-activated-receptor 1 (PAR1) plays a central role in tumor progression, little is known about the cell signaling involved. Methodology/Principal Findings We show here the impact of PAR1 cellular activities using both an orthotopic mouse mammary xenograft and a colorectal-liver metastasis model in vivo, with biochemical analyses in vitro. Large and highly vascularized tumors were generated by cells over-expressing wt hPar1, Y397Z hPar1, with persistent signaling, or Y381A hPar1 mutant constructs. In contrast, cells over-expressing the truncated form of hPar1, which lacks the cytoplasmic tail, developed small or no tumors, similar to cells expressing empty vector or control untreated cells. Antibody array membranes revealed essential hPar1 partners including Etk/Bmx and Shc. PAR1 activation induces Etk/Bmx and Shc binding to the receptor C-tail to form a complex. Y/A mutations in the PAR1 C-tail did not prevent Shc-PAR1 association, but enhanced the number of liver metastases compared with the already increased metastases obtained with wt hPar1. We found that Etk/Bmx first binds via the PH domain to a region of seven residues, located between C378-S384 in PAR1 C-tail, enabling subsequent Shc association. Importantly, expression of the hPar1-7A mutant form (substituted A, residues 378-384), which is incapable of binding Etk/Bmx, resulted in inhibition of invasion through Matrigel-coated membranes. Similarly, knocking down Etk/Bmx inhibited PAR1-induced MDA-MB-435 cell migration. In addition, intact spheroid morphogenesis of MCF10A cells is markedly disrupted by the ectopic expression of wt hPar1. In contrast, the forced expression of the hPar1-7A mutant results in normal ball-shaped spheroids. Thus, by preventing binding of Etk/Bmx to PAR1 -C-tail, hPar1 oncogenic properties are abrogated. Conclusions/Significance This is the first demonstration that a cytoplasmic portion of the PAR1 C-tail functions as a scaffold site. We identify here essential signaling partners, determine the hierarchy of binding and provide a platform for therapeutic vehicles via definition of the critical PAR1 -associating region in the breast cancer signaling niche.

Peripartum anesthetic management of patients with Takayasu’s arteritis: case series and review

Takayasu or pulseless disease is a rare, chronic progressive inflammatory disease that causes thrombosis and occlusion of systemic and pulmonary arteries. Almost 80% of patients are women in their childbearing years. We present three patients with Takayasus disease who between them had six pregnancies and discuss them in the context of a review of previously published cases. Assessment of the parturient with Takayasus disease should include an evaluation of the extent of disease, including organ ischemia such as cardiac, renal, cerebral and limb. A multidisciplinary approach should be taken to optimize the parturients status and formulate a plan for delivery. Peripartum anesthetic management should include optimization of intravascular volume and appropriate monitoring, which may be difficult in the pulseless patient. A regional anesthetic technique may be preferred over general anesthesia to allow monitoring of the cerebral circulation. A slowly titrated regional technique may prevent hemodynamic instability. Patients should be closely monitored postpartum with attention to hypertensive or end organ complications.