synthesis-characterization-placental-chondroitin-sulfate-plcsa

Synthesis and Characterization of Placental Chondroitin Sulfate A (plCSA)-Targeting Lipid-Polymer Nanoparticles

An effective cancer therapeutic method reduces and eliminates tumors with minimal systemic toxicity. Actively targeting nanoparticles offer a promising ...

comprehensive-evaluation-effectiveness-safety-placenta-targeted-drug

Comprehensive Evaluation of the Effectiveness and Safety of Placenta-Targeted Drug Delivery Using Three Complementary Methods

No effective treatments currently exist for placenta-associated pregnancy complications, and developing strategies for the targeted delivery of drugs to ...

Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences (CAS)

Chinese Academy of Sciences

Placental choriocarcinoma, a reproductive system carcinoma in women, has about 0.81% occurrence frequency in China, which leads to over 90% lethality due to indistinct pathogenesis and the absence of efficient therapeutic treatment. In the present study, using immunostaining and reverse transcription PCR, we reported that Dickkopf-1 (Dkk-1) is prominently expressed in human cytotrophoblast (CTB) cell, but absent in the human placental choriocarcinoma cell line JAR and JEG3, implicating an unknown correlation between Dkk-1 and carcinogenesis of placental choriocarcinoma. Further, through exogenous introduction of Dkk-1, we found repressed proliferation in JAR and JEG3, induced apoptosis in JAR, and discovered significant tumor suppression effects of Dkk-1 in placental choriocarcinoma. Moreover we found that this function of Dkk-1 is achieved through c-Jun N-terminal kinase (JNK), whereas the canonical Wnt pathway may not have a great role. This discovery is not symphonic to previous functional understanding of Dkk-1, a canonical Wnt signaling antagonist. Together, our data indicate the possible correlation between Dkk-1 and human placental choriocarcinoma and suggest potential applications of Dkk-1 in treatment of human placental choriocarcinomas.

Dickkopf-1 induced apoptosis in human placental choriocarcinoma is independent of canonical Wnt signaling.

CXCL14, a member of chemokine family, was previously known to participate in many pathophysiological events, such as leukocytes recruitment and tumor suppression. However, it remained largely unknown whether CXCL14 is a physiological player during early pregnancy. In this regard, our recent global gene microarray analysis has observed an implantation-specific expression profile of CXCL14 mRNA during early pregnancy in mice, showing its higher levels at implantation sites compared to inter-implantation sites, implicating a potential role of CXCL14 in the periimplantation events. In the present investigation, using Northern blot, in situ hybridization and immunostaining, we further demonstrated that uterine CXCL14 expression was specifically induced at embryo implantation site and expanded with subsequent decidualization process in a spatiotemporal manner. The implanting embryo also showed a highlighted expression of CXCL14 in the blastocyst trophectoderm and its derived ectoplacental cones (EPCs) during postimplantation development. In vitro functional study revealed that CXCL14 could significantly inhibit both primary and secondary trophoblast attachment and outgrowth, correlated with a stage-dependant downregulation of MMP-2 and/or MMP-9 activity. Moreover, it was found that biotinylated CXCL14 could specifically bind to trophoblast cells in vitro and in vivo, suggesting trophoblast cell, perhaps expressing the unidentified CXCL14 receptor, is a bioactive target of CXCL14. Collectively, our findings provide evidences supporting the contention that CXCL14 is an important paracrine/autocrine modulator regulating trophoblast outgrowth at the maternal-fetal interface during the process of pregnancy establishment. This study is clinically related since CXCL14 is also highly expressed in human receptive endometrium and trophoblasts.

CXCL14 inhibits trophoblast outgrowth via a paracrine/autocrine manner during early pregnancy in mice.

In mouse, decidualization is characterized by the proliferation of stromal cells and their differentiation into specialized type of cells (decidual cells) with polyploidy, surrounding the implanting blastocyst. However, the mechanisms involved in these processes remain poorly understood. Using multiple approaches, we have examined the role of Adam12 in decidualization during early pregnancy in mice. Adam12 is spatiotemporally expressed in decidualizing stromal cells in intact pregnant females and in pseudopregnant mice undergoing artificially induced decidualization. In the ovariectomized mouse uterus, the expression of Adam12 is upregulated after progesterone treatment, which is primarily mediated by nuclear progesterone receptor. In a stromal cell culture model, the expression of Adam12 gradually rises with the progression of stromal decidualization, whereas the attenuated expression of Adam12 after siRNA knockdown significantly blocks the progression of decidualization. Our study suggests that Adam12 is involved in promoting uterine decidualization during pregnancy.

Adam12 plays a role during uterine decidualization in mice.

Airway smooth muscle cells (ASMCs) phenotype modulation, characterized by reversible switching between contractile and proliferative phenotypes, is considered to contribute to airway proliferative diseases such as allergic asthma. Nuclear Factor-κB (NF-κB) has been reported as a key regulator for the occurrence and development of asthma. However, little is known regarding its role in ASM cell phenotypic modulation. To elucidate the role of NF-κB in regulating ASM cells phenotypic modulation, we investigated the effects of NF-κB on ASM cells contractile marker protein expression, and its impact on proliferation and apoptosis. We found that chronic asthma increased the activation of NF-κB in the primary murine ASM cells with a concomitant marked decrease in the expression of contractile phenotypic marker protein including smooth muscle alpha-actin (α-SMA). Additionally, we used the normal ASM cells under different processing to build the phenotype switching when we found the activation of NF-κB. Meanwhile, the expression of α-SMA in asthma was significantly increased by the NF-κB blocker. NF-κB blocker also suppressed asthma mouse ASM cell proliferation and promoted apoptosis. These findings highlight a novel role for the NF-κB in murine ASM cell phenotypic modulation and provide a potential target for therapeutic intervention for asthma. 

Nuclear factor-κB mediates the phenotype switching of airway smooth muscle cells in a murine asthma model.

Oxygen nanocarrier (A/D-ONC) with a polymeric core entrapping hemoglobin and a cationic lipid shell absorbing a DOX-intercalating DNA duplex is developed. After endocytosis oxygenated A/D-ONC donates O2 to cancer cells that acts therapeutically by: (1) increasing intracellular ATP content that promotes DOX release, thereby converting ATP to the trigger of detrimental chemotherapy; (2) by synchronously increasing the ROS amount to amplify the lethality to cancer cells.

Oxygen Nanocarrier for Combined Cancer Therapy: Oxygen-Boosted ATP-Responsive Chemotherapy with Amplified ROS Lethality.

Placenta specific protein 1 (PLAC1) is thought to be important for murine and human placentation because of its abundant expression in placenta; however, the trophoblast subtypes that express PLAC1 at the fetomaternal interface and the major role of PLAC1 in placentation are still unclear. This study investigated the expression pattern of PLAC1 at the human fetomaternal interface and its involvement in trophoblast syncytialization. Localization of PLAC1 at the fetomaternal interface was studied using in situ hybridization (ISH) and immunohistochemistry (IHC) assays. Real time RT-PCR and Western Blot were employed to exhibit the expression pattern of PLAC1 during human spontaneous syncytialization of term primary cytotrophoblast cells (CTBs). Spontaneous syncytialization of a primary term CTBs model transfected with siRNA specific to PLAC1 was used to investigate the role of PLAC1 during human trophoblast syncytialization. The results showed that PLAC1 was mainly expressed in the human villous syncytiotrophoblast (STB) layer throughout gestation, and the expression level of PLAC1 was significantly elevated during human trophoblast syncytialization. Down-regulation of PLAC1 via specific PLAC1 siRNA transfection attenuated spontaneous syncytialization of primary term CTBs (p<0.05) as indicated by cell fusion index and the expression patterns of the corresponding markers. These data demonstrate the facilitative role of PLAC1 in normal human trophoblast syncytialization.

PLAC1 is involved in human trophoblast syncytialization.

Two-thirds of early pregnancy failures present with reduced trophoblast invasion, and SLIT2/ROBO1 signalling is considered to play an important role in trophoblast function during pregnancy. We investigated SLIT2/ROBO1 signalling associated with missed and threatened miscarriage during early gestation.

Alterted SLIT2/ROBO1 signalling is linked to impaired placentation of missed and threatened miscarriage in early pregnancy.

The aim of the present study was to investigate the effects of Icaritin on the proliferation and decidualization of endometrial stromal cells (ESCs). A total of 20 specimens of endometrium were collected during hysterectomy at the Gynecology Department of Shenzhen Nanshan People's Hospital (Shenzhen, China) between August 2014 and December 2015. The endometrium was digested with high concentrations of collagenase and DNase and filtered with meshes, and then the glandular epithelial and stromal cells were separated by the adhesion purification method. The purity of stromal cells was identified by vimetin and cytokeratin 7 immunostaining. The estradiol + progesterone (E2+P4) and/or cyclic adenosine monophosphate (cAMP) were added to induce an  decidualization model, which was used to analyze the effect of Icaritin on the decidualization ability of the human ESCs. The decidualization markers of human ESCs, prolactin (PRL) and insulin-like growth factor-binding protein 1 (IGFBP-1), was analyzed by reverse-transcription quantitative polymerase chain reaction measurements of the mRNA levels, PRL immunostaining and ELISA analysis of the IGFBP-1 protein levels in the cells or cell culture supernatant separately. The results demonstrated that treatment with E2+P4 and/or cAMP for 96 h was able to induce decidualization in ESCs, and that the cells demonstrated polygon-shaped epithelioid changes. The cell nuclei revealed multinuclear changes, and the cells were also observed to be large and round in shape. The PRL expression and upregulated IGFBP-1 mRNA and protein levels in the E2+P4+cAMP treatment group indicated successful decidualization of the  model. However, the addition of Icaritin inhibited the expression of PRL and IGFBP-1 mRNA, as well as IGFBP-1 protein in the induced ESCs compared with groups without Icaritin. These results suggest that Icaritin was able to inhibit the expression of decidualization-related genes in ESCs . However, the exact mechanisms require further investigation.

Icaritin inhibits decidualization of endometrial stromal cells.

Gestational trophoblastic neoplasia (GTN) can result from the over-proliferation of trophoblasts. Treatment of choriocarcinoma, the most aggressive GTN, currently requires high doses of systemic chemotherapeutic agents, which result in indiscriminate drug distribution and severe toxicity. To overcome these disadvantages and enhance the chemotherapeutic efficacy, chondroitin sulfate A (CSA)-binding nanoparticles were developed for the targeted delivery of doxorubicin (DOX) to choriocarcinoma cells using a synthetic CSA-binding peptide (CSA-BP), derived from malarial protein, which specifically binds to the CSA exclusively expressed in the placental trophoblast. CSA-BP-conjugated nanoparticles rapidly bonded to choriocarcinoma (JEG3) cells and were efficiently internalized into the lysosomes. Moreover, CSA-BP modification significantly increased the anti-cancer activity of the DOX-loaded nanoparticles in vitro. Intravenous injections of CSA-BP-conjugated nanoparticles loaded with indocyanine green (CSA-INPs) were rapidly localized to the tumor. The CSA-targeted nanoparticles loaded with DOX (CSA-DNPs) strongly inhibited primary tumor growth and, more importantly, significantly suppressed metastasis in vivo. Collectively, our results highlight the potential of the CSA-BP-decorated nanoparticles as an alternative targeted delivery system of chemotherapeutic agents for treating choriocarcinoma and for developing new GTN therapies based on drug targeting.

Targeted delivery of doxorubicin by CSA-binding nanoparticles for choriocarcinoma treatment.

Studies have shown that sFlt-1 overproduction stimulated by excess VEGF of deciduous origin in trophoblasts can cause preeclampsia. However, the mechanism underlying how VEGF regulates sFtl-1 expression in trophoblasts remains unknown. To address this issue, JEG3 and HTR-8/SV neo (HTR8) trophoblast cell lines were used to investigate the signaling pathways involved in the regulation of sFlt-1 production via VEGF overexpression in vitro. JEG3 (VEGF-GFP-JEG3, V-J) and HTR8 (VEGF-GFP-HTR8, V-H) cells overexpressing VEGF165 were established by infecting the JEG3 and HTR8 cell lines with lentivirus expressing VEGF165. Both the mRNA and protein levels of VEGF and sFlt-1 were dramatically up-regulated in the V-J and V-H cells compared to the JEG3 and HTR8 cells, and they were significantly decreased after treatment with an Flt-1 receptor inhibitor (MK-2461), a KDR receptor inhibitor (XL-184), or an Flt-1 and KDR receptor inhibitor (ABT-869). The mRNA levels of sFlt-1, Flt-1, and KDR were increased in V-H cells after treatment, and the VEGF-A mRNA levels were also elevated. The migration and invasion abilities of JEG3 and HTR8 cells were decreased after VEGF overexpression, and this reduction could be reversed with VEGF receptor inhibitor treatment. In addition, after the different treatments, the cell migration rates of V-J cells were significantly increased compared with the control treatment. Taken together, these results indicate that sFlt-1 up-regulation by VEGF may be mediated by the VEGF/Flt-1 and/or VEGF/KDR signaling pathways. However, elucidating which pathway plays this key role requires further investigation.

VEGF-A regulates sFlt-1 production in trophoblasts through both Flt-1 and KDR receptors.

 The availability of therapeutics to treat pregnancy complications is severely lacking, mainly due to the risk of harm to the fetus. In placental malaria, infected erythrocytes (IEs) accumulate in the placenta by adhering to chondroitin sulfate A (CSA) on the surfaces of trophoblasts. Based on this principle, we have developed a method for targeted delivery of payloads to the placenta using a synthetic placental CSA-binding peptide (plCSA-BP) derived from VAR2CSA, a CSA-binding protein expressed on IEs.  A biotinylated plCSA-BP was used to examine the specificity of plCSA-BP binding to mouse and human placental tissue in tissue sections . Different nanoparticles, including plCSA-BP-conjugated nanoparticles loaded with indocyanine green (plCSA-INPs) or methotrexate (plCSA-MNPs), were administered intravenously to pregnant mice to test their efficiency at drug delivery to the placenta . The tissue distribution and localization of the plCSA-INPs were monitored in live animals using an IVIS imaging system. The effect of plCSA-MNPs on fetal and placental development and pregnancy outcome were examined using a small-animal high-frequency ultrasound (HFUS) imaging system, and the concentrations of methotrexate in fetal and placental tissues were measured using high-performance liquid chromatography (HPLC).  plCSA-BP binds specifically to trophoblasts and not to other cell types in the placenta or to CSA-expressing cells in other tissues. Moreover, we found that intravenously administered plCSA-INPs accumulate in the mouse placenta, and  analysis of the fetuses and placentas confirmed placenta-specific delivery of these nanoparticles. We also demonstrate successful delivery of methotrexate specifically to placental cells by plCSA-BP-conjugated nanoparticles, resulting in dramatic impairment of placental and fetal development. Importantly, plCSA-MNPs treatment had no apparent adverse effects on maternal tissues.  These results demonstrate that plCSA-BP-guided nanoparticles could be used for the targeted delivery of payloads to the placenta and serve as a novel placenta-specific drug delivery option.

ArticleTotal : 2	Year
Publication title Cited by 12	2018
Publication title Cited by 10	2018

Article	Year
Dickkopf-1 induced apoptosis in human placental choriocarcinoma is independent of canonical Wnt signaling. Biochemical and biophysical research communications\| PubMed ID: 17026960	2006
CXCL14 inhibits trophoblast outgrowth via a paracrine/autocrine manner during early pregnancy in mice. Journal of cellular physiology\| PubMed ID: 19626669	2009
Adam12 plays a role during uterine decidualization in mice. Cell and tissue research\| PubMed ID: 19841944	2009
Nuclear factor-κB mediates the phenotype switching of airway smooth muscle cells in a murine asthma model. International journal of clinical and experimental pathology\| PubMed ID: 26722396	2015
Oxygen Nanocarrier for Combined Cancer Therapy: Oxygen-Boosted ATP-Responsive Chemotherapy with Amplified ROS Lethality. Advanced healthcare materials\| PubMed ID: 27253453	2016
PLAC1 is involved in human trophoblast syncytialization. Reproductive biology\| PubMed ID: 27692364	2016
Alterted SLIT2/ROBO1 signalling is linked to impaired placentation of missed and threatened miscarriage in early pregnancy. Histopathology\| PubMed ID: 28485101	2017
Icaritin inhibits decidualization of endometrial stromal cells. Experimental and therapeutic medicine\| PubMed ID: 29285144	2017
Targeted delivery of doxorubicin by CSA-binding nanoparticles for choriocarcinoma treatment. Drug delivery\| PubMed ID: 29426237	2018
VEGF-A regulates sFlt-1 production in trophoblasts through both Flt-1 and KDR receptors. Molecular and cellular biochemistry\| PubMed ID: 29497919	2018
Placenta-specific drug delivery by trophoblast-targeted nanoparticles in mice. Theranostics\| PubMed ID: 29774074	2018

Xiujun Fan

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