Detrimental actions of obesity-associated advanced glycation end-products on endometrial epithelial cell proliferation are alleviated by antioxidants.

RESEARCH QUESTION: Advanced glycation end-products (AGE) are elevated in the uterine environment of obese infertile women. Can the detrimental effects of AGE on endometrial epithelial cells be mitigated with therapeutics, and recapitulated in a more physiologically relevant primary model (organoids)? DESIGN: Human endometrial epithelial cells (ECC-1) were exposed to AGE at concentrations physiologically representative of uterine fluid in lean or obese individuals, and three potential therapeutics: 25 nmol/l receptor for AGE (RAGE) antagonist FPS-ZM1, 100 µmol/l metformin, or a combination of antioxidants (10 µmol/l N-acetyl-l-cysteine, 10 µmol/l N-acetyl-l-carnitine and 5 µmol/l α-lipoic acid). Real-time cell analysis (xCELLigence, ACEA Biosciences) determined the rate of adhesion and proliferation. The proliferation of organoid-derived cells and secretion of cytokines from organoids was characterized in the presence of AGE (n = 5). The uterine fluid of women undergoing assisted reproduction was profiled for AGE-associated inflammatory markers (n = 77). RESULTS: ECC-1 proliferation was reduced by AGE from obese versus lean conditions and vehicle control (P = 0.04 and P < 0.001, respectively), and restored to a proliferation corresponding to lean conditions by antioxidants. AGE influenced organoid derived primary endometrial epithelial cell proliferation in a donor-dependent manner. AGE increased the organoid secretion of the proinflammatory cytokine CXCL16 (P = 0.006). Clinically, CXCL16 correlated positively to maternal body mass index (R = 0.264, P = 0.021) and intrauterine glucose concentration (R = 0.736, P < 0.0001). CONCLUSIONS: Physiologically relevant concentrations of AGE alter endometrial epithelial cell function. Antioxidants restore the rate of proliferation of AGE-treated endometrial epithelial (ECC-1) cells. Primary endometrial epithelial cells, cultured as organoids, demonstrate altered proliferation and CXCL16 secretion in the presence of AGE equimolar with the uterine fluid from obese individuals.

Podocalyxin molecular characteristics and endometrial expression: high conservation between humans and macaques but divergence in mice†.

Podocalyxin (PODXL) is a newly identified key negative regulator of human endometrial receptivity, specifically down-regulated in the luminal epithelium at receptivity to permit embryo implantation. Here, we bioinformatically compared the molecular characteristics of PODXL among the human, rhesus macaque, and mouse, determined by immunohistochemistry and in situ hybridization (mouse tissues) whether endometrial PODXL expression is conserved across the three species and examined if PODXL inhibits mouse embryo attachment in vitro. The PODXL gene, mRNA, and protein sequences showed greater similarities between humans and macaques than with mice. In all species, PODXL was expressed in endometrial luminal/glandular epithelia and endothelia. In macaques (n = 9), luminal PODXL was significantly down-regulated when receptivity is developed, consistent with the pattern found in women. At receptivity, PODXL was also reduced in shallow glands, whereas endothelial expression was unchanged across the menstrual cycle. In mice, endometrial PODXL did not vary considerably across the estrous cycle (n = 16); however, around embryo attachment on d4.5 of pregnancy (n = 4), luminal PODXL was greatly reduced especially near the site of embryo attachment. Mouse embryos failed to attach or thrive when co-cultured on a monolayer of Ishikawa cells overexpressing PODXL. Thus, endometrial luminal PODXL expression is down-regulated for embryo implantation in all species examined, and PODXL inhibits mouse embryo implantation. Rhesus macaques share greater conservations with humans than mice in PODXL molecular characteristics and regulation, thus represent a better animal model for functional studies of endometrial PODXL for treatment of human fertility.

Progesterone suppresses podocalyxin partly by up-regulating miR-145 and miR-199 in human endometrial epithelial cells to enhance receptivity in in vitro models.

Establishment of endometrial surface receptivity is crucial for the initiation of embryo implantation yet the molecular mechanisms are not well understood, especially in humans. We have recently discovered that podocalyxin (PODXL) is a critical negative regulator of human endometrial surface receptivity. PODXL is highly expressed in all epithelial and endothelial cells in the non-receptive endometrium, but down-regulated specifically in the luminal epithelium at receptivity. We have further shown that PODXL inhibits embryo implantation, and that PODXL down-regulation is essential for endometrial surface receptivity. Our previous study also indicated that progesterone down-regulates PODXL; however, the exact molecular regulations are unknown. Here, we investigated whether progesterone suppresses PODXL via microRNAs (miRNAs). We first bioinformatically predicted 13 miRNAs that may potentially target human PODXL, then experimentally determined whether any of these 13 miRNAs are altered in primary human endometrial epithelial cells (HEECs) by progesterone, and whether the identified miRNAs can affect PODXL expression in Ishikawa cells without progesterone and alter receptivity to embryo implantation. Progesterone significantly up-regulated miR-145 and miR-199 while suppressing PODXL in HEECs. When these two miRNAs were transfected into Ishikawa cells, both significantly down-regulated PODXL mRNA and protein in the absence of progesterone. Moreover, both miR-145 and miR-199 significantly enhanced receptivity of the Ishikawa monolayer to embryo implantation in in vitro models. This study thus provides in vitro evidence that PODXL is down-regulated by progesterone partly via miR-145 and miR-199 during the development of human endometrial epithelial receptivity. These results also reveal the likely importance of hormonal regulation of miRNAs for embryo implantation.

MicroRNAs in the Regulation of Endometrial Receptivity for Embryo Implantation.

Development of endometrial receptivity is crucial for successful embryo implantation and pregnancy initiation. Understanding the molecular regulation underpinning endometrial transformation to a receptive state is key to improving implantation rates in fertility treatments such as IVF. With microRNAs (miRNAs) increasingly recognized as important gene regulators, recent studies have investigated the role of miRNAs in the endometrium. Studies on miRNAs in endometrial disorders such as endometriosis and endometrial cancer have been reviewed previously. In this minireview, we aim to provide an up-to-date knowledge of miRNAs in the regulation of endometrial receptivity. Since endometrial remodelling differs considerably between species, we firstly summarised the key events of the endometrial cycle in humans and mice and then reviewed the miRNAs identified so far in these two species with likely functional significance in receptivity establishment. To date, 29 miRNAs have been reported in humans and 15 miRNAs in mice within various compartments of the endometrium that may potentially modulate receptivity; miRNAs regulating the Wnt signalling and those from the let-7, miR-23, miR-30, miR-200 and miR-183 families are found in both species. Future studies are warranted to investigate miRNAs as biomarkers and/or therapeutic targets to detect/improve endometrial receptivity in human fertility treatment.

Podocalyxin is a key negative regulator of human endometrial epithelial receptivity for embryo implantation.

STUDY QUESTION: How is endometrial epithelial receptivity, particularly adhesiveness, regulated at the luminal epithelial surface for embryo implantation in the human? SUMMARY ANSWER: Podocalyxin (PCX), a transmembrane protein, was identified as a key negative regulator of endometrial epithelial receptivity; specific downregulation of PCX in the luminal epithelium in the mid-secretory phase, likely mediated by progesterone, may act as a critical step in converting endometrial surface from a non-receptive to an implantation-permitting state. WHAT IS KNOWN ALREADY: The human endometrium must undergo major molecular and cellular changes to transform from a non-receptive to a receptive state to accommodate embryo implantation. However, the fundamental mechanisms governing receptivity, particularly at the luminal surface where the embryo first interacts with, are not well understood. A widely held view is that upregulation of adhesion-promoting molecules is important, but the details are not well characterized. STUDY DESIGN, SIZE, DURATION: This study first aimed to identify novel adhesion-related membrane proteins with potential roles in receptivity in primary human endometrial epithelial cells (HEECs). Further experiments were then conducted to determine candidates' in vivo expression pattern in the human endometrium across the menstrual cycle, regulation by progesterone using cell culture, and functional importance in receptivity using in vitro human embryo attachment and invasion models. PARTICIPANTS/MATERIALS, SETTING, METHODS: Primary HEECs (n = 9) were isolated from the proliferative phase endometrial tissue, combined into three pools, subjected to plasma membrane protein enrichment by ultracentrifugation followed by proteomics analysis, which led to the discovery of PCX as a novel candidate of interest. Immunohistochemical analysis determined the in vivo expression pattern and cellular localization of PCX in the human endometrium across the menstrual cycle (n = 23). To investigate whether PCX is regulated by progesterone, the master driver of endometrial differentiation, primary HEECs were treated in culture with estradiol and progesterone and analyzed by RT-PCR (n = 5) and western blot (n = 4). To demonstrate that PCX acts as a negative regulator of receptivity, PCX was overexpressed in Ishikawa cells (a receptive line) and the impact on receptivity was determined using in vitro attachment (n = 3-5) and invasion models (n = 4-6), in which an Ishikawa monolayer mimicked the endometrial surface and primary human trophoblast spheroids mimicked embryos. Mann-Whitney U-test and ANOVA analyses established statistical significance at *P ≤ 0.05 and **P ≤ 0.01. MAIN RESULTS AND THE ROLE OF CHANCE: PCX was expressed on the apical surface of all epithelial and endothelial cells in the non-receptive endometrium, but selectively downregulated in the luminal epithelium from the mid-secretory phase coinciding with the establishment of receptivity. Progesterone was confirmed to be able to suppress PCX in primary HEECs, suggesting this hormone likely mediates the downregulation of luminal PCX in vivo for receptivity. Overexpression of PCX in Ishikawa monolayer inhibited not only the attachment but also the penetration of human embryo surrogates, demonstrating that PCX acts as an important negative regulator of epithelial receptivity for implantation. LIMITATIONS, REASONS FOR CAUTION: Primary HEECs isolated from the human endometrial tissue contained a mixture of luminal and glandular epithelial cells, as further purification into subtypes was not possible due to the lack of specific markers. Future study would need to investigate how progesterone differentially regulates PCX in endometrial epithelial subtypes. In addition, this study used primary human trophoblast spheroids as human embryo mimics and Ishikawa as endometrial epithelial cells in functional models, future studies with human blastocysts and primary epithelial cells would further validate the findings. WIDER IMPLICATIONS OF THE FINDINGS: The findings of this study add important new knowledge to the understanding of human endometrial remodeling for receptivity. The identification of PCX as a negative regulator of epithelial receptivity and the knowledge that its specific downregulation in the luminal epithelium coincides with receptivity development may provide new avenues to assess endometrial receptivity and individualize endometrial preparation protocols in assisted reproductive technology (ART). The study also discovered PCX as progesterone target in HEECs, identifying a potentially useful functional biomarker to monitor progesterone action, such as in the optimization of progesterone type/dose/route of administration for luteal support. STUDY FUNDING/COMPETING INTEREST(S): Study funding was obtained from ESHRE, Monash IVF and NHMRC. LR reports potential conflict of interests (received grants from Ferring Australia; personal fees from Monash IVF Group and Ferring Australia; and non-financial support from Merck Serono, MSD, and Guerbet outside the submitted work. LR is also a minority shareholder and the Group Medical Director for Monash IVF Group, a provider of fertility preservation services). The remaining authors have no potential conflict of interest to declare. TRIAL REGISTRATION NUMBER: NA.

Overview of Human HtrA Family Proteases and Their Distinctive Physiological Roles and Unique Involvement in Diseases, Especially Cancer and Pregnancy Complications.

The mammalian high temperature requirement A (HtrA) proteins are a family of evolutionarily conserved serine proteases, consisting of four homologs (HtrA1-4) that are involved in many cellular processes such as growth, unfolded protein stress response and programmed cell death. In humans, while HtrA1, 2 and 3 are widely expressed in multiple tissues with variable levels, HtrA4 expression is largely restricted to the placenta with the protein released into maternal circulation during pregnancy. This limited expression sets HtrA4 apart from the rest of the family. All four HtrAs are active proteases, and their specific cellular and physiological roles depend on tissue type. The dysregulation of HtrAs has been implicated in many human diseases such as cancer, arthritis, neurogenerative ailments and reproductive disorders. This review first discusses HtrAs broadly and then focuses on the current knowledge of key molecular characteristics of individual human HtrAs, their similarities and differences and their reported physiological functions. HtrAs in other species are also briefly mentioned in the context of understanding the human HtrAs. It then reviews the distinctive involvement of each HtrA in various human diseases, especially cancer and pregnancy complications. It is noteworthy that HtrA4 expression has not yet been reported in any primary tumour samples, suggesting an unlikely involvement of this HtrA in cancer. Collectively, we accentuate that a better understanding of tissue-specific regulation and distinctive physiological and pathological roles of each HtrA will improve our knowledge of many processes that are critical for human health.

High levels of HtrA4 detected in preeclamptic circulation may disrupt endothelial cell function by cleaving the main VEGFA receptor KDR.

Systemic endothelial dysfunction is a key characteristic of preeclampsia (PE), which is a serious disorder of human pregnancy. We have previously reported that high-temperature requirement factor (Htr)A4 is a placenta-specific protease that is secreted into the maternal circulation and significantly up-regulated in PE, especially early-onset PE. We have also demonstrated that high levels of HtrA4 detected in the early onset PE circulation induce endothelial dysfunction in HUVECs. In the current study, we investigated whether HtrA4 could cleave the main receptor of VEGFA, the kinase domain receptor (KDR), thereby inhibiting VEGFA signaling. We first demonstrated that HtrA4 cleaved recombinant KDR in vitro. We then confirmed that HtrA4 reduced the level of KDR in HUVECs and inhibited the VEGFA-induced phosphorylation of Akt kinase, which is essential for downstream signaling. Further functional studies demonstrated that HtrA4 prevented the VEGFA-induced tube formation in HUVECs and dose-dependently inhibited the VEGFA-induced angiogenesis in explants of mouse aortic rings. These data strongly suggest that high levels of HtrA4 in the maternal circulation could cleave the main receptor of VEGFA in endothelial cells to induce a wide-spread impairment of angiogenesis. Our studies therefore suggest that HtrA4 is a potential causal factor of early onset PE.-Wang, Y., La, M., Pham, T., Lovrecz, G. O., Nie, G. High levels of HtrA4 detected in preeclamptic circulation may disrupt endothelial cell function by cleaving the main VEGFA receptor KDR.

Low Serum Levels of HtrA3 at 15 Weeks of Gestation Are Associated with Late-Onset Preeclampsia Development and Small for Gestational Age Birth.

OBJECTIVE: The aim of this study was to investigate the potential utility of serum HtrA1 and HtrA3, serine proteases that are highly expressed in the developing placenta, at 15 and 20 weeks of gestation for predicting later development of adverse pregnancy outcomes of preeclampsia (PE), gestational hypertension (GHT), preterm birth (PTB), and small for gestational age (SGA) birth. METHODS: This is a nested case control study of 665 samples (330 controls, 335 cases) from the Adelaide SCOPE cohort. The cases included were 92 PE, 71 GHT, 56 PTB, and 116 SGA. Samples were assessed by ELISA and data adjusted for maternal age, BMI, socioeconomic index, hCG, and smoking status. Multivariate logistic regression was performed with other biochemical and biophysical parameters available for these samples. RESULTS: HtrA1 did not differ between the controls and cases. In contrast, HtrA3 was significantly lower at 15 weeks in pregnancies that later developed late-onset PE (LPE) or resulted in SGA birth, with an area under the ROC curve (AUC) of 0.716 and 0.790, respectively. The combination of HtrA3 with PAPP-A, uterine, and umbilical Doppler improved the AUC to 0.755 for LPE and 0.844 for SGA. CONCLUSION: HtrA3 at 15 weeks is associated with, and may be useful for, the early detection of LPE development and SGA birth.

Hypoxia is involved in the reduction of HtrA3 in patients with endometrial hyperplasia and cancer.

Endometrial cancer (EC) has recently become a major gynecological cancer and endometrial hyperplasia increases the risk for developing EC. Previous studies have reported that human high temperature requirement factor A3 (HtrA3), a member of ATP independent serine proteases family, is involved in endometrial carcinogenesis. However, the underlying mechanism of HtrA3 function is unclear in endometrial hyperplasia and cancer. In this study, we investigated that HtrA3 expression was reduced in endometrial hyperplasia as well as EC. The circulating levels of HtrA3 were also significantly reduced in both atypical hyperplasia and EC. Whether hypoxia is involved in the reduction of HtrA3 in EC was further investigated. Immunohistochemistry (IHC) scores of Glut1 and HtrA3 in type 1 and type 2 EC tissues showed the inverse correlation. And hypoxic condition reduced the expression of HtrA3. Furthermore, silencing HtrA3 promoted EC cell migration. Our study demonstrated the reduced levels of HtrA3 in endometrial hyperplasia including atypical hyperplasia which is a premalignant condition; and as the degree of hypoxia increases in EC, HtrA3 eventually loses its expression. Hypoxia is responsible for the reduction of HtrA3 which in turn promotes EC progression. These findings suggested that HtrA3 is an important adaptor in hypoxic regions that drives endometrial cancer development.

Serum levels of GDF15 are reduced in preeclampsia and the reduction is more profound in late-onset than early-onset cases.

BACKGROUND: Preeclampsia is a pregnancy specific disorder affecting 3-5% of pregnancies worldwide. It is clinically divided into early-onset and late-onset subtypes. Placental factors are involved in the pathogenesis of preeclampsia. Growth differentiation factor 15 (GDF15), a protein of the transforming growth factor beta superfamily, is highly expressed in the placenta. However, it is unclear whether the circulating levels of GDF15 are altered in preeclampsia at the time of or prior to disease presentation. METHODS: Serum samples across three trimesters from 29 healthy pregnancies, third trimester sera from 34 women presenting with preeclampsia (early-onset n=16, late-onset n=18) and 66 gestation-age-matched controls, and sera at 11-13weeks of pregnancy from women who later did (n=36) or did not (n=33) develop late-onset preeclampsia, were examined for GDF15 by ELISA. RESULTS: Serum GDF15 levels increased significantly with gestation in normal pregnancy. Serum GDF15 was significantly reduced in the third trimester in women presenting with preeclampsia compared to their gestation-age-matched controls. This reduction was apparent in both early-onset and late-onset subtypes, but it was more profound in late-onset cases. At 11-13weeks of gestation, however, serum levels of GDF15 were similar between women who subsequently did and did not develop late-onset preeclampsia. CONCLUSION: Serum GDF15 increased with gestation age, reaching the highest level in the third trimester. Serum GDF15 was significantly reduced in the third trimester in women presenting with preeclampsia, especially in late-onset cases. However, serum GDF15 was not altered in the first trimester in women destined to develop late-onset preeclampsia.

Posttranslational removal of α-dystroglycan N terminus by PC5/6 cleavage is important for uterine preparation for embryo implantation in women.

Embryo implantation requires a healthy embryo and a receptive endometrium (inner lining of the uterus); endometrial receptivity acquisition involves considerable epithelial surface remodeling. Dystroglycan (DG), a large cell surface glycoprotein, consists of α- and ß-subunits; ß-DG anchors within the plasma membrane whereas α-DG attaches extracellularly to ß-DG. The glycosylated central α-DG mediates adhesion, but it is obstructed by its large N terminus (α-DG-N); α-DG-N removal enables DG's adhesive function. We demonstrate here that full-length α-DG in the human endometrial epithelium is a barrier for embryo attachment and that removal of α-DG-N by proprotein convertase 5/6 (PC6; a protease critical for implantation) regulates receptivity. This was evidenced by: 1) α-DG contains a PC6-cleavage site near α-DG-N, and PC6 cleaves a peptide harboring such a site; 2) PC6 knockdown reduces α-DG-N removal from endometrial epithelial cell surface and blastocyst adhesion; 3) mutating the PC6-cleavage site prevents α-DG-N removal, causing cell surface retention of full-length α-DG and loss of adhesiveness; 4) α-DG-N is removed from endometrial tissue in vivo for receptivity and uterine fluid α-DG-N reflects tissue removal and receptivity. We thus identified α-DG-N removal as an important posttranslational control of endometrial receptivity and uterine fluid α-DG-N as a potential biomarker for receptivity in women.

Endometrial CRISP3 is regulated throughout the mouse estrous and human menstrual cycle and facilitates adhesion and proliferation of endometrial epithelial cells.

The endometrium (the mucosal lining of the uterus) is a dynamic tissue that undergoes extensive remodeling, secretory transformation in preparation for implantation of an embryo, inflammatory and proteolytic activity during menstruation, and rapid postmenstrual repair. A plethora of local factors influence these processes. Recently, a cysteine-rich protein, CRISP3, a clade of the CRISP, antigen 5, pathogenesis-related (CAP) protein superfamily, has been implicated in uterine function. The localization, regulation, and potential function of CRISP3 in both the human and mouse endometrium is described. CRISP3 localizes to the luminal and glandular epithelium of the endometrium within both species, with increased immunoreactivity during the proliferative phase of the human cycle. CRISP3 also localizes to neutrophils, particularly within the premenstrual human endometrium and during the postbreakdown repair phase of a mouse model of endometrial breakdown and repair. Endometrial CRISP3 is produced by primary human endometrial epithelial cells and secreted in vivo to accumulate in the uterine cavity. Secreted CRISP3 is more abundant in uterine lavage fluid during the proliferative phase of the menstrual cycle. Human endometrial epithelial CRISP3 is present in both a glycosylated and a nonglycosylated form in vitro and in vivo. Treatment of endometrial epithelial cells in vitro with recombinant CRISP3 enhances both adhesion and proliferation. These data suggest roles for epithelial and neutrophil-derived CRISP3 in postmenstrual endometrial repair and regeneration.

Proprotein convertase 5/6 cleaves platelet-derived growth factor A in the human endometrium in preparation for embryo implantation.

Establishment of endometrial receptivity is vital for successful embryo implantation. Proprotein convertase 5/6 (referred to as PC6) is up-regulated in the human endometrium specifically at the time of epithelial receptivity. PC6, a serine protease of the proprotein convertase family, plays an important role in converting precursor proteins into their active forms through specific proteolysis. The proform of platelet-derived growth factor A (pro-PDGFA) requires PC cleavage to convert to the active-PDGFA. We investigated the PC6-mediated activation of PDGFA in the human endometrium during the establishment of receptivity. Proteomic analysis identified that the pro-PDGFA was increased in the conditioned medium of HEC1A cells in which PC6 was stably knocked down by small interfering RNA (PC6-siRNA). Western blot analysis demonstrated an accumulation of the pro-PDGFA but a reduction in the active-PDGFA in PC6-siRNA cell lysates and medium compared with control. PC6 cleavage of pro-PDGFA was further confirmed in vitro by incubation of recombinant pro-PDGFA with PC6. Immunohistochemistry revealed cycle-stage-specific localization of the active-PDGFA in the human endometrium. During the non-receptive phase, the active-PDGFA was barely detectable. In contrast, it was localized specifically to the apical surface of the luminal and glandular epithelium in the receptive phase. Furthermore, the active-PDGFA was detected in uterine lavage with levels being significantly higher in the receptive than the non-receptive phase. We thus identified that the secreted PDGFA may serve as a biomarker for endometrial receptivity. This is also the first study demonstrating that the active-PDGFA localizes to the apical surface of the endometrium during receptivity.

Development of a high-throughput assay for human proprotein convertase 5/6 for detecting uterine receptivity.

Embryo implantation requires a healthy embryo and a receptive uterus. In women, the inner lining of the uterus, the endometrium, remains in a hostile state and becomes receptive for embryo implantation for only a short period during each menstrual cycle. Determining endometrial receptivity is vital in in vitro fertilization (IVF) treatment because the timing of embryo transfer needs to be synchronized with endometrial receptivity. We have previously demonstrated that proprotein convertase 5/6A (PC6) is highly expressed in the receptive endometrium and that PC6 is critical for receptivity establishment in women. Furthermore, endometrial PC6 is secreted into the uterine fluid, and levels correlate with receptivity status. Detection of PC6 in uterine fluids, therefore, would provide a nonsurgical assessment of endometrial receptivity. However, to date no assays are available for human PC6. In this study, we produced three PC6 monoclonal antibodies (mAbs) and developed a sandwich enzyme-linked immunosorbent assay (ELISA) for PC6 detection in human uterine fluids. The PC6 mAbs were confirmed to be highly specific to PC6, and the ELISA detected PC6 in human uterine fluids with a significantly higher level during the receptive phase. This newly established PC6 ELISA provides an important tool in the development of noninvasive strategies to detect endometrial receptivity in women.

HtrA4 is well conserved only in higher primates and functionally important for EVT differentiation.

INTRODUCTION: The placenta differs greatly among species, and deep extra-villous trophoblast (EVT) invasion is a unique feature of placentation of higher primates including humans. We reported serine protease HtrA4 being found predominantly in human placentas with aberrant expression linked to preeclampsia. However, it remains unclear where HtrA4 is produced in the placenta, how it is expressed in other species, and whether it is essential for human placentation. METHODS: We first compared HtrA4 protein sequences of over 100 species, then scrutinized the key characteristics of HtrA4 in the human, rhesus macaque and mouse, and determined cellular localization in the placenta. We next investigated functional significance of HtrA4 in EVT differentiation using human trophoblast stem cells (TSCs). RESULTS: Across broader species HtrA4 is well conserved only in higher primates. In humans, only the placenta expressed HtrA4, localising to trophoblasts of villous as well as extra-villous lineages. Rhesus macaques produced HtrA4 but again only in placentas, whereas mice showed no abundant HtrA4 expression anywhere including the placenta, yet it was an active protease if produced. The functional importance of HtrA4 in human EVT was demonstrated using TSCs, which expressed low levels of HtrA4 but significantly up-regulated it during EVT differentiation, and knockdown of HtrA4 severely inhibited the differentiation process. DISCUSSION: HtrA4 is expressed in placentas of humans and macaques but not mice; it is critical for human EVT differentiation. Together with previous reports showing HtrA4 is also indispensable for syncytialization, this study further revealed HtrA4 as a functionally important protease for human placentation.

HtrA4 is required for human trophoblast stem cell differentiation into syncytiotrophoblast.

INTRODUCTION: The syncytiotrophoblast (STB) of the human placenta facilitates vital maternal-fetal communication and is maintained by fusion (syncytialization) of cytotrophoblasts. Serine protease HtrA4 (high temperature requirement factor A4) is highly expressed only in the human placenta and was previously reported to be important for BeWo fusion. This study investigated whether HtrA4 is critical for differentiation of human trophoblast stem cells (TSCs) into STB. METHODS: Primary TSCs were isolated from first trimester placentas (n = 5) and validated by immunofluorescence (IF) for CD49f, CK7 and vimentin. TSCs were then differentiated into STB and the success of syncytialization was confirmed by RT-PCR, IF and ELISA of known markers. TSCs were next stably transfected with a HtrA4-targetting CRISPR/Cas9 plasmid, and cells with severe HtrA4 knockdown (HtrA4-KD) were analyzed to investigate the impact on STB differentiation. RESULTS: Primary TSCs were confirmed to be of high purity by staining positively for CD49f and CK7 but negatively for vimentin. These TSCs readily syncytialized when stimulated for STB differentiation, significantly increasing ß-hCG and syncytin-1, substantially decreasing E-cadherin, and markedly losing cell borders. While TSCs produced very low levels of HtrA4, upon stimulation for STB differentiation the cells drastically upregulated HtrA4 expression; secretion of HtrA4 protein also increased sharply, correlating positively and significantly with that of ß-hCG. The HtrA4-KD TSCs, however, failed to show this surge of HtrA4 production upon stimulation, and ultimately remained primarily mononucleated with no significant STB differentiation. DISCUSSION: This study demonstrates that HtrA4 plays a critical role in TSC differentiation into syncytiotrophoblast.

Podocalyxin promotes the formation of compact and chemoresistant cancer spheroids in high grade serous carcinoma.

High grade serous carcinoma (HGSC) metastasises primarily intraperitoneally via cancer spheroids. Podocalyxin (PODXL), an anti-adhesive transmembrane protein, has been reported to promote cancer survival against chemotherapy, however its role in HGSC chemoresistance is unclear. This study investigated whether PODXL plays a role in promoting chemoresistance of HGSC spheroids. We first showed that PODXL was expressed variably in HGSC patient tissues (n = 17) as well as in ovarian cancer cell lines (n = 28) that are more likely categorised as HGSC. We next demonstrated that PODXL-knockout (KO) cells proliferated more slowly, formed less compact spheroids and were more fragile than control cells. Furthermore, when treated with carboplatin and examined for post-treatment recovery, PODXL-KO spheroids showed significantly poorer cell viability, lower number of live cells, and less Ki-67 staining than controls. A similar trend was also observed in ascites-derived primary HGSC cells (n = 6)-spheroids expressing lower PODXL formed looser spheroids, were more vulnerable to fragmentation and more sensitive to carboplatin than spheroids with higher PODXL. Our studies thus suggests that PODXL plays an important role in promoting the formation of compact/hardy HGSC spheroids which are more resilient to chemotherapy drugs; these characteristics may contribute to the chemoresistant nature of HGSC.

Apelin-13: A Protective Role in Vascular Diseases.

Vascular disease is a common problem with high mortality all over the world. Apelin-13, a key subtype of apelin, takes part in many physiological and pathological responses via regulating many target genes and target molecules or participating in many signaling pathways. More and more studies have demonstrated that apelin-13 is implicated in the onset and progression of vascular disease in recent years. It has been shown that apelin-13 could ameliorate vascular disease by inhibiting inflammation, restraining apoptosis, suppressing oxidative stress, and facilitating autophagy. In this article, we sum up the progress of apelin-13 in the occurrence and development of vascular disease and offer some insightful views about the treatment and prevention strategies of vascular disease.

A Potential Role of NFIL3 in Atherosclerosis.

Nuclear factor interleukin-3 (NFIL3), a proline- and acidic-residue-rich (PAR) bZIP transcription factor, is called the E4 binding protein 4 (E4BP4) as well, which is relevant to regulate the circadian rhythms and the viability of cells. More and more evidence has shown that NFIL3 is associated with different cardiovascular diseases. In recent years, it has been found that NFIL3 has significant functions in the progression of atherosclerosis (AS) via the regulation of inflammatory response, macrophage polarization, some immune cells and lipid metabolism. In this overview, we sum up the function of NFIL3 during the development of AS and offer meaningful views how to treat cardiovascular disease related to AS.

The Potential Relationship Between Cardiovascular Diseases and Monkeypox.

Mpox, a novel epidemic disease, has broken out the period of coronavirus disease 2019 since May 2022, which was caused by the mpox virus. Up to 12 September 2023, there are more than 90,439 confirmed mpox cases in over 115 countries all over the world. Moreover, the outbreak of mpox in 2022 was verified to be Clade II rather than Clade I. Highlighting the significance of this finding, a growing body of literature suggests that mpox may lead to a series of cardiovascular complications, including myocarditis and pericarditis. It is indeed crucial to acquire more knowledge about mpox from a perspective from the clinical cardiologist. In this review, we would discuss the epidemiological characteristics and primary treatments of mpox to attempt to provide a framework for cardiovascular physicians.