Dual effect of nifedipine on pregnant human myometrium contractility: Implication of TRPC1.

Nifedipine, an L-type voltage-gated Ca2+ channel (L-VGCC) blocker, is one of the most used tocolytics to treat preterm labor. In clinical practice, nifedipine efficiently decreases uterine contractions, but its efficacy is limited over time, and repeated or maintained nifedipine-based tocolysis appears to be ineffective in preventing preterm birth. We aimed to understand why nifedipine has short-lasting efficiency for the inhibition of uterine contractions. We used ex vivo term pregnant human myometrial strips treated with cumulative doses of nifedipine. We observed that nifedipine inhibited spontaneous myometrial contractions in tissues with high and regular spontaneous contractions. By contrast, nifedipine appeared to increase contractions in tissues with low and/or irregular spontaneous contractions. To investigate the molecular mechanisms activated by nifedipine in myometrial cells, we used the pregnant human myometrial cell line PHM1-41 that does not express L-VGCC. The in vitro measurement of intracellular Ca2+ showed that high doses of nifedipine induced an important intracellular Ca2+ entry in myometrial cells. The inhibition or downregulation of the genes encoding for store-operated Ca2+ entry channels from the Orai and transient receptor potential-canonical (TRPC) families in PHM1-41 cells highlighted the implication of TRPC1 in nifedipine-induced Ca2+ entry. In addition, the use of 2-APB in combination with nifedipine on human myometrial strips tends to confirm that the pro-contractile effect induced by nifedipine on myometrial tissues may involve the activation of TRPC channels.

Linking unfolded protein response to ovarian cancer cell fusion.

BACKGROUND: Polyploid giant cancer cells (PGCCs) have been observed in epithelial ovarian tumors. They can resist antimitotic drugs, thus participating in tumor maintenance and recurrence. Although their origin remains unclear, PGCC formation seems to be enhanced by conditions that trigger the unfolded protein response (UPR) such as hypoxia or chemotherapeutic drugs like paclitaxel. Hypoxia has been shown to promote the formation of ovarian PGCCs by cell fusion. We thus hypothesized that the UPR could be involved in EOC cell fusion, possibly explaining the occurrence of PGCCs and the aggressiveness of EOC. METHODS: The UPR was induced in two ovarian cancer cell lines (SKOV3 and COV318). The UPR activation was assessed by Western blot and polyploidy indexes were calculated. Then, to confirm the implication of cell fusion in PGCC formation, two populations of SKOV3 cells were transfected with plasmids encoding for two distinct nuclear fluorescent proteins (GFP and mCherry) associated with different antibiotic resistance genes, and the two cell populations were mixed in co-culture. The co-culture was submitted to a double-antibiotic selection. The resulting cell population was characterized for its morphology, cyclicity, and proliferative and tumorigenic capacities, in addition to transcriptomic characterization. RESULTS: We demonstrated that cell fusion could be involved in the generation of ovarian PGCCs and this process was promoted by paclitaxel and the UPR activation. Double-antibiotic treatment of PGCCs led to the selection of a pure population of cells containing both GFP- and mCherry-positive nuclei. Interestingly, after 3 weeks of selection, we observed that these cells were no longer polynucleated but displayed a single nucleus positive for both fluorescent proteins, suggesting that genetic material mixing had occurred. These cells had reinitiated their normal cell cycles, acquired an increased invasive capacity, and could form ovarian tumors in ovo. CONCLUSIONS: The UPR activation increased the in vitro formation of PGCCs by cell fusion, with the newly generated cells further acquiring new properties. The UPR modulation in ovarian cancer patients could represent an interesting therapeutic strategy to avoid the formation of PGCCs and therefore limit cancer relapse and drug resistance.

Effect of local aromatase inhibition in endometriosis using a new chick embryo chorioallantoic membrane model.

Endometriosis is an oestrogen-dependent, inflammation-driven gynaecologic disorder causing severe disability. Endometriosis implants are characterized by unbalanced local oestrogen metabolism leading to hyperoestrogenism and aromatase up-regulation is one of main mechanism involved. Aromatase inhibitors such as letrozole or anastrozole use in young women are associated with severely side effects limiting their long-term clinical use. An endometriosis-targeted inhibition of local aromatase could be a viable alternative, although the role of the local inhibition of this enzyme is still unclear. Using a new chick embryo allantoic membrane (CAM) model incorporating xenografted human endometriosis cyst, we showed that topical treatment with anastrozole reduced lesion size, although oestrogens produced by CAM female embryo blunted this effect. Xenografted human endometriosis CAM is a new efficient model for the screening of new drugs targeting endometriosis tissue.

Oestradiol enhances apoptosis in bovine mammary epithelial cells in vitro.

Ovarian steroids, oestradiol and progesterone, are required for normal mammary growth at puberty and during pregnancy. They contribute to mammary parenchyma development by stimulating mammary epithelial cell (MEC) proliferation. However several studies demonstrate that oestradiol negatively affects milk production during the declining phase of lactation, but the oestradiol effect on MEC in lactating mammary gland remains unclear. The objective of this study was to investigate the differential effect of oestradiol on bovine MECs mimicking two physiological statuses: active and early apoptotic MECs. We demonstrated that oestradiol has a major effect on early apoptotic MECs and might accelerate MEC apoptosis by activation of caspases rather than by inducing apoptosis in active MECs. Early apoptotic MECs could be compared with senescent cells in the late-lactation mammary gland. These results suggest that the negative effect of oestradiol on milk production during the declining phase of lactation would be due to an enhancement of apoptotic processes in MECs.

Suppression of ovarian secretions before puberty strongly affects mammogenesis in the goat.

The objective of this study was to provide insight into the biological mechanisms underlying mammary development and the role of the ovaries in prepubertal caprine mammogenesis using a serial ovariectomy approach. Young Alpine goats were ovariectomized (Ovx) or sham-operated (Int) at three periods before puberty (G1=1 month, G2=2 month and G3=3 months of age) and one after puberty (G7=7 months of age). The goats were slaughtered at 9 months of age and mammary glands were removed. Ovariectomy performed at 1, 2 and 3 months of age caused a 50% reduction in DNA concentration, in mammary tissue taken from the parenchyma-stroma border region. Morphological analysis of mammary tissue sections indicated that the parenchymal structures of Ovx goats were negatively affected by ovariectomy. Goats ovariectomized before 2 months of age (Ovx-1 and Ovx-2) showed a significant decrease in the percent of cells proliferating in mammary glands of 9-month old goats (proliferating cell nuclear antigen expression and antigen Ki67-positive cell number). Also, goats ovariectomized at 1 and 2 months of age had reduced matrix metalloprotease 2 activity at 9 months of age. E-cadherin was strongly decreased in goats ovariectomized before 2 months of age (80 and 85% in Ovx-1 and Ovx-2 goats, respectively). Quantitative PCR analysis of transcripts encoding for oestrogen (ERα) and progesterone receptors (PR) and immunodetection of ERα showed that ovariectomy at 1 and 2 months of age strongly inhibited the transcription of ERα and PR in the mammary gland. We conclude that ovariectomy before 3 months of age markedly impaired parenchymal development. These findings suggest that prepubertal mammogenesis in goats depends on the ovaries to initiate mammary epithelial cell proliferation and mammary gland remodelling.

Telomerase is required for glomerular renewal in kidneys of adult mice.

Homeostatic renal filtration relies on the integrity of podocytes, which function in glomerular filtration. These highly specialized cells are damaged in 90% of chronic kidney disease, representing the leading cause of end-stage renal failure. Although modest podocyte renewal has been documented in adult mice, the mechanisms regulating this process remain largely unknown and controversial. Using a mouse model of Adriamycin-induced nephropathy, we find that the recovery of filtration function requires up-regulation of the endogenous telomerase component TERT. Previous work has shown that transient overexpression of catalytically inactive TERT (i-TERTci mouse model) has an unexpected role in triggering dramatic podocyte proliferation and renewal. We therefore used this model to conduct specific and stochastic lineage-tracing strategies in combination with high throughput sequencing methods. These experiments provide evidence that TERT drives the activation and clonal expansion of podocyte progenitor cells. Our findings demonstrate that the adult kidney bears intrinsic regenerative capabilities involving the protein component of telomerase, paving the way for innovative research toward the development of chronic kidney disease therapeutics.

Role of the Uteroplacental Renin-Angiotensin System in Placental Development and Function, and Its Implication in the Preeclampsia Pathogenesis.

Placental development and function implicate important morphological and physiological adaptations to thereby ensure efficient maternal-fetal exchanges, as well as pregnancy-specific hormone secretion and immune modulation. Incorrect placental development can lead to severe pregnancy disorders, such as preeclampsia (PE), which endangers both the mother and the infant. The implication of the systemic renin-angiotensin system (RAS) in the pregnancy-related physiological changes is now well established. However, despite the fact that the local uteroplacental RAS has been described for several decades, its role in placental development and function seems to have been underestimated. In this review, we provide an overview of the multiple roles of the uteroplacental RAS in several cellular processes of placental development, its implication in the regulation of placental function during pregnancy, and the consequences of its dysregulation in PE pathogenesis.

PLGA Nanoparticles for the Intraperitoneal Administration of CBD in the Treatment of Ovarian Cancer: In Vitro and In Ovo Assessment.

The intraperitoneal administration of chemotherapeutics has emerged as a potential route in ovarian cancer treatment. Nanoparticles as carriers for these agents could be interesting by increasing the retention of chemotherapeutics within the peritoneal cavity. Moreover, nanoparticles could be internalised by cancer cells and let the drug release near the biological target, which could increase the anticancer efficacy. Cannabidiol (CBD), the main nonpsychotropic cannabinoid, appears as a potential anticancer drug. The aim of this work was to develop polymer nanoparticles as CBD carriers capable of being internalised by ovarian cancer cells. The drug-loaded nanoparticles (CBD-NPs) exhibited a spherical shape, a particle size around 240 nm and a negative zeta potential (-16.6 ± 1.2 mV). The encapsulation efficiency was high, with values above 95%. A controlled CBD release for 96 h was achieved. Nanoparticle internalisation in SKOV-3 epithelial ovarian cancer cells mainly occurred between 2 and 4 h of incubation. CBD antiproliferative activity in ovarian cancer cells was preserved after encapsulation. In fact, CBD-NPs showed a lower IC50 values than CBD in solution. Both CBD in solution and CBD-NPs induced the expression of PARP, indicating the onset of apoptosis. In SKOV-3-derived tumours formed in the chick embryo model, a slightly higher-although not statistically significant-tumour growth inhibition was observed with CBD-NPs compared to CBD in solution. To sum up, poly-lactic-co-glycolic acid (PLGA) nanoparticles could be a good strategy to deliver CBD intraperitoneally for ovarian cancer treatment.

The fine-tuning of endoplasmic reticulum stress response and autophagy activation during trophoblast syncytialization.

The syncytiotrophoblast (STB) is a multinuclear layer forming the outer surface of the fetal part of the placenta deriving from villous cytotrophoblastic cell (vCTB) fusion and differentiation. This syncytialization process is characterized by morphological and biochemical alterations of the trophoblast, which probably require removal of pre-existing structures and proteins to maintain cell homeostasis and survival. Interestingly, autophagy, which allows degradation and recycling of cellular components, was shown to be activated in syncytiotrophoblast. Here we examined the involvement of endoplasmic reticulum stress (ERS) response in autophagy activation during vCTB syncytialization. We first demonstrated the activation of ERS response and autophagy during the time course of trophoblastic cell fusion and differentiation. Alteration of autophagy activation in vCTB by chemical treatments or Beclin-1 expression modulation leads to a decrease in trophoblastic syncytialization. Furthermore, ERS response inhibition by chemical treatment or siRNA strategy leads to a default in syncytialization, associated with alteration of autophagy markers and cell survival. From these data, we suggest that ERS response, by fine regulation of autophagy activation, may serve as an adaptive mechanism to promote cell survival during trophoblastic syncytialization.

Endoplasmic reticulum stress responses in placentation - A true balancing act.

The unfolded protein response (UPR) is recognized as a key mechanism to promote protein folding and processing in eukaryotes when endoplasmic reticulum stress (ERS) occurs. Some conditions such as hypoxia or glucose deprivation are factors that may elicit ERS response. Recent literature collectively proposes that ERS response is crucial for mammalian reproduction by allowing decidualization and placentation to occur. However, prolonged ERS and activation of UPR pathways can lead to apoptosis and autophagy, which in turn could pose adverse effects on pregnancy outcomes and placentation. ERS associated pregnancy pathologies include intrauterine growth restriction and early-onset preeclampsia. Given these findings, evidence suggests that overactivation of UPR may lead to harmful reproductive circumstances, whereas physiological regulation of ERS response is essential for mammalian reproduction and placental function. In this review, we discuss the dual role of UPR activation with respect to its contribution to placental development as well as pathologies caused by pathway overactivation. In addition, we suggest potential protein markers associated with the UPR, as circulating C-terminal GRP78 or anti-GRP78 autoantibodies which may prove to be of clinical interest.