Human umbilical cord mesenchymal stem cell derived exosomes (HUCMSC-exos) recovery soluble fms-like tyrosine kinase-1 (sFlt-1)-induced endothelial dysfunction in preeclampsia.

BACKGROUND: Preeclampsia is a unique multisystem disorder that affects 5-8% of pregnancies. A high level of soluble fms-like tyrosine kinase-1 (sFlt-1) is a hallmark of preeclampsia that causes endothelial dysfunction. Exosomes derived from mesenchymal stem cells (MSCs) have been indicated to improve endothelial performances by transporting signals to target cells. We hypothesized that exosomes derived from MSCs have potential effects against preeclampsia. METHODS: We collected human umbilical cord MSC-derived exosomes (HUCMSC-exos) by ultracentrifugation. The size and morphology of the exosomes were examined using a transmission electron microscope and nanoparticle tracking analysis. Pregnant mice were injected with murine sFlt-1 adenovirus to build the preeclampsia-like mouse model and then treated with HUCMSC-exos. Human umbilical vein endothelial cells (HUVECs) were infected with lentiviruses expressing tet-on-sFlt-1 to obtain cells overexpressing sFlt-1. Cell proliferation and migration assays were used to measure the endothelial functions. The exosomes enriched proteins underlying mechanisms were explored by proteomic analysis. RESULTS: In the current study, we successfully collected the cup-shaped HUCMSC-exos with diameters of 30-150 nm. In the sFlt-1-induced preeclampsia mouse model, HUCMSC-exos exhibited beneficial effects on adverse birth events by decreasing blood pressure and improving fetal birth weight. In addition, preeclamptic dams that were injected with HUCMSC-exos had rebuilt dense placental vascular networks. Furthermore, we observed that HUCMSC-exos partially rescued sFlt-1-induced HUVECs dysfunction in vitro. Proteomics analysis of HUCMSC-exos displayed functional enrichment in biological processes related to vesicle-mediated transport, cell communication, cell migration, and angiogenesis. CONCLUSION: We propose that exosomes derived from HUCMSCs contain abundant Versican and play beneficial roles in the birth outcomes of sFlt-1-induced preeclamptic mice by promoting angiogenesis.

Pregnancy-specific beta-1-glycoprotein 1-enriched exosomes are involved in the regulation of vascular endothelial cell function during pregnancy.

INTRODUCTION: Pregnancy is a dynamic time period associated with significant physiological changes in the cardiovascular system. It is well known that during pregnancy, the placenta secretes a variety of molecular signals, including exosomes, into the maternal circulation to adapt to increased blood volume and maintain blood pressure at normotensive levels. METHODS: In the present study, we compared the effects of exosomes derived from the peripheral blood serum of nonpregnant women (NP-Exo) and pregnant women with uncomplicated pregnancy (P-Exo) on endothelial cell function. We also analyzed the proteomic profiles of these two groups of exosomes and the molecular mechanisms underlying the effect of exosome cargoes on vascular endothelial cell function. RESULTS: We found that P-Exo were positively involved in regulating the function of human umbilical vein endothelial cell (HUVEC) and promoting the release of nitric oxide (NO). Furthermore, we revealed that trophoblast-derived pregnancy-specific beta-1-glycoprotein 1 (PSG1)-enriched exosomes treatment induced the promotion of HUVEC proliferation and migration as well as the release of NO. In addition, we found that P-Exo maintained blood pressure at normal levels in mice. DISCUSSION: These results suggested that PSG1-enriched exosomes derived from maternal peripheral blood regulate the function of vascular endothelial cells and play an important role in maintaining maternal blood pressure during pregnancy.

sFlt-1-enriched exosomes induced endothelial cell dysfunction and a preeclampsia-like phenotype in mice.

Preeclampsia (PE) is a hypertensive disorder of pregnancy characterized by maternal endothelial dysfunction and end-organ damage. Our previous work demonstrated that PE patient-derived exosomes contained higher levels of soluble FMS-like tyrosine kinase-1 (sFlt-1) and significantly induced endothelial dysfunction and PE development. However, the mechanisms underlying the effect of sFlt-1-enriched exosomes (sFlt-1-Exo) on PE development are poorly characterized. Here, we revealed that trophoblast-derived sFlt-1-Exo treatment induced significant inhibition of human umbilical vein endothelial cell (HUVEC) migration and tube formation, as well as an increase in sFlt-1 secretion. Mechanistically, we found that the increased sFlt-1 secretion in the cell culture medium was attributed to enhanced transcription of sFlt-1 in HUVECs. Importantly, we observed that treating pregnant mice with sFlt-1-Exo or recombinant mouse sFlt-1 triggered a preeclampsia-like phenotype, characterized by elevated blood pressure, proteinuria, increased plasma sFlt-1 and adverse pregnancy outcomes. These results strongly suggested that sFlt-1-Exo-induced endothelial dysfunction could be partially attributed to the upregulation of sFlt-1 in endothelial cells, potentially leading to the development of a preeclampsia-like phenotype in mice.

Valorization of Fish Processing By-Products: Microstructural, Rheological, Functional, and Properties of Silver Carp Skin Type I Collagen.

The objective of this study was to develop aquatic collagen production from fish processing by-product skin as a possible alternative to terrestrial sources. Silver carp skin collagen (SCSC) was isolated and identified as type I collagen, and LC-MS/MS analysis confirmed the SCSC as Hypophthalmichthys molitrix type I collagen, where the yield of SCSC was 40.35 ± 0.63% (dry basis weight). The thermal denaturation temperature (Td) value of SCSC was 30.37 °C, which was superior to the collagen of deep-sea fish and freshwater fish. Notably, SCSC had higher thermal stability than human placental collagen, and the rheological experiments showed that the SCSC was a shear-thinning pseudoplastic fluid. Moreover, SCSC was functionally superior to some other collagens from terrestrial sources, such as sheep, chicken cartilage, and pig skin collagen. Additionally, SCSC could provide a suitable environment for MC3T3-E1 cell growth and maintain normal cellular morphology. These results indicated that SCSC could be used for further applications in food, cosmetics, and biomedical fields.

PD-L1 enhances migration and invasion of trophoblasts by upregulating ARHGDIB via transcription factor PU.1.

As the main constituent cells of the human placenta, trophoblasts proliferate, differentiate, and invade the uterine endometrium via a series of processes, which are regulated exquisitely through intercellular signaling mediated by hormones, cytokines, and growth factors. Programmed cell death ligand 1 (PD-L1) is a biomarker of the response to immune checkpoint inhibitors and can regulate maternal-fetal immune tolerance during pregnancy progression. Recently, it was found that PD-L1 may regulate obstetric complications by affecting the function of trophoblasts. Therefore, we examined the expression and localization of PD-L1 in the human placenta and observed the effects of PD-L1 on trophoblasts migration and invasion in both the trophoblasts line HTR-8/SVneo and an extravillous explant culture model. Finally, we explored the molecular mechanisms underlying PD-L1-regulated trophoblasts migration and invasion through RNA sequencing and bioinformatics analysis. Our data showed that PD-L1 was mainly expressed in syncytiotrophoblasts and that its protein levels increased with gestational age. Interestingly, the protein expression of PD-L1 was significantly decreased in placentas from pregnancies with preeclampsia compared with normal placentas. Importantly, the migration and invasion abilities of trophoblasts were significantly changed after knockdown or overexpression of PD-L1 in HTR-8/SVneo cells and an extravillous explant culture model, which was partially mediated through the transcription factor PU.1 (encoded by Spi1)-regulated Rho GDP-dissociation inhibitor beta (ARHGDIB) expression. These results suggested that PD-L1 was highly involved in the regulation of trophoblasts migration and invasion, providing a potential target for the diagnosis and treatment of placenta-derived pregnancy disorders.

Human Placental Endothelial Cell and Trophoblast Heterogeneity and Differentiation Revealed by Single-Cell RNA Sequencing.

BACKGROUND: The placenta is an important organ for fetal and maternal health during pregnancy and impacts offspring health late in life. Defects in placental vasculature and trophoblast have been identified in several pregnancy complications. Thus, the detailed molecular profile and heterogeneity of endothelial cells and trophoblasts in placentas will aid us in better understanding placental behaviors and improving pregnancy outcomes. METHODS: Single-cell RNA sequencing (scRNA-seq) was performed to profile the transcriptomics of human placental villous tissues from eleven patients with normal pregnancies in the first and second trimesters (6-16 weeks of gestation). RESULTS: The transcriptomic landscape of 52,179 single cells was obtained, and the cells were classified as trophoblasts, fibroblasts, endothelial cells, erythroid cells, Hofbauer cells, and macrophages. Our analysis further revealed the three subtypes of placental endothelial cells, with distinct metabolic signatures and transcription factor regulatory networks. We also determined the transcriptomic features of the trophoblast subpopulations and characterized two distinct populations of progenitor cells in cytotrophoblasts, which were capable of differentiating to extravillous trophoblasts and syncytiotrophoblasts, respectively. CONCLUSIONS: Our study provided a high-resolution molecular profile of the human placenta between 6 and 16 weeks of gestation. Our data revealed the placental cell complexity and demonstrated the transcriptional networks and signaling involved in placental endothelial and trophoblast differentiation during early pregnancy, which will be a resource for future studies of the human placental development.

Risk factors influencing the prognosis of elderly patients infected with COVID-19: a clinical retrospective study in Wuhan, China.

The mortality rate of elderly patients with Coronavirus Disease 2019 (COVID-19) was significantly higher than the overall mortality rate. However, besides age, leading death risk factors for the high mortality in elderly patients remain unidentified. This retrospective study included 210 elderly COVID-19 patients (aged ≥ 65 years), of whom 175 patients were discharged and 35 died. All deceased patients had at least one comorbidity. A significantly higher proportion of patients in the deceased group had cardiovascular diseases (49% vs. 20%), respiratory diseases (51% vs. 11%), chronic kidney disease (29% vs. 5%) and cerebrovascular disease (20% vs. 3%) than that in the discharged group. The median levels of C-reactive protein (125.8mg/L vs. 9.3mg/L) and blood urea nitrogen (7.2mmol/L vs. 4.4mmol/L) were significantly higher and median lymphocyte counts (0.7×109/L vs. 1.1×109/L) significantly lower in the deceased group than those in the discharged group. The survival curve analysis showed that higher C-reactive protein (≥5mg/L) plus any other abnormalities of lymphocyte, blood urea nitrogen or lactate dehydrogenase significantly predicted poor prognosis of COVID-19 infected elderly patients. This study revealed that the risk factors for the death in these elderly patients included comorbidities, increased levels of C-reactive protein and blood urea nitrogen, and lymphopenia during hospitalization.

Downregulation of lysyl oxidase and lysyl oxidase-like protein 2 suppressed the migration and invasion of trophoblasts by activating the TGF-ß/collagen pathway in preeclampsia.

Preeclampsia is a pregnancy-specific disorder that is a major cause of maternal and fetal morbidity and mortality with a prevalence of 6-8% of pregnancies. Although impaired trophoblast invasion in early pregnancy is known to be closely associated with preeclampsia, the underlying mechanisms remain elusive. Here we revealed that lysyl oxidase (LOX) and LOX-like protein 2 (LOXL2) play a critical role in preeclampsia. Our results demonstrated that LOX and LOXL2 expression decreased in preeclamptic placentas. Moreover, knockdown of LOX or LOXL2 suppressed trophoblast cell migration and invasion. Mechanistically, collagen production was induced in LOX- or LOXL2-downregulated trophoblast cells through activation of the TGF-ß1/Smad3 pathway. Notably, inhibition of the TGF-ß1/Smad3 pathway could rescue the defects caused by LOX or LOXL2 knockdown, thereby underlining the significance of the TGF-ß1/Smad3 pathway downstream of LOX and LOXL2 in trophoblast cells. Additionally, induced collagen production and activated TGF-ß1/Smad3 were observed in clinical samples from preeclamptic placentas. Collectively, our study suggests that the downregulation of LOX and LOXL2 leading to reduced trophoblast cell migration and invasion through activation of the TGF-ß1/Smad3/collagen pathway is relevant to preeclampsia. Thus, we proposed that LOX, LOXL2, and the TGF-ß1/Smad3/collagen pathway can serve as potential markers and targets for clinical diagnosis and therapy for preeclampsia.

Lactic acid induced microRNA-744 enhances motility of SiHa cervical cancer cells through targeting ARHGAP5.

High-risk (hr) human papillomaviruses (HPV) infection and integration has caused the majority of cervical cancer, of which E6 and E7 oncogenes are invariably retained and expressed to immortalize cells probably via affecting cell migration and invasion, and tumor metastasis. However, the underlying mechanism that mediates the procedure such as motility of cervical cancer cells within the tumor microenvironment is not well understood. Herein, we examined one possible factor-extracellular lactic acid, an end up chemical in glycolytic tumor cells, on the motility in HPV16 positive SiHa cells. The results showed that lactic acid enhanced cell migration and invasion behavior via stimulating the expression of miR-744. ARHGAP5 was confirmed to be a target of miR-744, and silencing ARHGAP5 exhibited an inhibiting effect on cell migration and invasion as that observed by suppressing miR-744. In addition, lactic acid down-regulated E6 and E7 protein levels, and overexpression of either miR-744 or ARHGAP5 could also reduce E6 and E7 levels. Overall, our findings suggest that the miR-774/ARHGAP5 axis may provide a vital role in triggering lactic acid-induced migration and invasion in SiHa cells, regardless of the diminished effect due to the partial inhibition of E6 and E7 expression.

Diversity in human placental microvascular endothelial cells and macrovascular endothelial cells.

Angiogenesis is fundamental to normal placental development, and aberrant angiogenesis contributes substantially to placental pathologies. Placental angiogenesis is a pivotal process that plays a key mechanistic role in the elaboration of the placental villous tree, which is mainly taken by human placental microvascular endothelial cells (HPMECs), present in the fetal capillaries of chorionic villi, and macrovascular human umbilical vein endothelial cells (HUVECs) also play a role in this process. These are the two types of endothelial cells that form the placenta and differ in morphology and function. The placental vasculature represents a distinct territory that is highly specialized in structure and function. To distinguish the differences between HPMECs and HUVECs, we isolated HPMECs by paramagnetic particle separation and HUVECs through trypsinization and validated their characteristics. Then, we examined their response to fibroblast growth factor 2 (FGF2), vascular endothelial growth factor (VEGF) and endocrine-gland-derived vascular endothelial growth factor (EG-VEGF), as well as the underlying signaling mechanisms and their transcriptomes. We found that cultured HPMECs and HUVECs took up DiI-Ac-LDL and formed capillary-like tube structures on Matrigel. HPMECs and HUVECs had different expressions of eNOS, PROKR1 and PROKR2, and these characteristics substantiate the endothelial nature of cultured cells. FGF2 and VEGF stimulated the proliferation and migration of HPMECs and HUVECs via activation of PI3K/AKT1 and MEK1/MEK2/ERK1/ERK2. Interestingly, EG-VEGF increased the proliferation and migration of HPMECs via only MEK1/MEK2/ERK1/ERK2 and not PI3K/AKT1. Microarray analysis showed that there were some differentially expressed genes between HPMECs and HUVECs. Gene ontology analysis indicated that the differentially expressed genes were highly related to G-protein coupled receptor signaling pathway, angiogenesis, L-lysine transmembrane transport and blood vessel remodeling. These data provided evidence of heterogeneity between microvascular HPMECs and macrovascular HUVECs that most likely reflected significant differences in endothelial cell function in the two different cellular environments.

Syntenin-targeted peptide blocker inhibits progression of cancer cells.

The multidomain adaptor protein syntenin is known to mediate cancer cell metastasis and invasion through its tandem PDZ1 and PDZ2 domains, leading to the postulation that the PDZ tandem may serve as a potential drug target for cancer treatment. Here we report the development of high-affinity peptide blockers to target the syntenin tandem PDZ domain, and elucidate that blocking syntenin correlates with the inhibition of cell migration and spreading. Two strategies are employed to derive high-affinity blockers from the low-affinity natural binding peptides: first, dimerization of the C termini of natural syntenin-binding peptides confers dimer peptides with much higher affinity than the monomers; second, unnatural amino acid substitution at P-1 and P-2 positions of the PDZ-binding sequence increases the binding affinity. Through several rounds of optimization, we discovered a dimeric peptide that binds tightly to syntenin tandem PDZ domain, with a dissociation constant of 0.21 µM based on fluorescence polarization measurement. The peptide dimer inhibits the migration and invasion of syntenin high-expression human cancer cells through attenuating the ERK phosphorylation of the MAPK kinase pathway. This work showcases an effective strategy to derive high-affinity blocker of multidomain adaptor proteins, which resulted in a syntenin-targeted antagonist with potential pharmaceutical values for the treatment of syntenin over-expressing cancers.

Maternal and umbilical cord serum-derived exosomes enhance endothelial cell proliferation and migration.

We investigated the role of exosomes derived from maternal and umbilical cord blood in the regulation of angiogenesis. We report here that both maternal exosomes (MEs) and umbilical exosomes (UEs) significantly enhance HUVEC proliferation, migration, and tube formation. Importantly, ME-treated HUVECs (MEXs) displayed significantly increased migration, but not proliferation or tube formation, compared with UE-treated HUVECs (UEXs). We found that the expression of a subset of migration-related microRNAs (miRNAs), including miR-210-3p, miR-376c-3p, miR-151a-5p, miR-296-5p, miR-122-5p, and miR-550a-5p, among others, were significantly increased or decreased in UEs, and this altered expression was likely correlated with the differential regulation of HUVEC migration. We also found that the mRNA expression of hepatocyte growth factor (HGF) was up-regulated in MEXs and UEXs and, moreover, that inhibiting HGF partially abolished the enhanced cell migration induced by UEs. Our results suggest that both MEs and UEs greatly enhanced endothelial cell (EC) functions and differentially regulated EC migration, which was mostly attributed to the different expression profiles of exosomal miRNA. These findings highlight the importance of exosomes in the regulation of angiogenesis during pregnancy. Exosomal miRNAs, in particular, may be of great significance for the regulation of angiogenesis in maintaining normal pregnancy.-Jia, L., Zhou, X., Huang, X., Xu, X., Jia, Y., Wu, Y., Yao, J., Wu, Y., Wang, K. Maternal and umbilical cord serum-derived exosomes enhance endothelial cell proliferation and migration.

Dysregulated DNA Methyltransferase 3A Upregulates IGFBP5 to Suppress Trophoblast Cell Migration and Invasion in Preeclampsia.

Preeclampsia is a unique multiple system disorder during human pregnancy, which affects ≈5% to 8% of pregnancies. Its risks and complications have become the major causes of maternal and fetal morbidity and mortality. Although abnormal placentation to which DNA methylation dysregulation is always linked is speculated to be one of the reasons causing preeclampsia, the underlying mechanisms still remain elusive to date. Here we revealed that aberrant DNA methyltransferase 3A (DNMT3A) plays a critical role in preeclampsia. Our results show that the expression and localization of DNMT3A are dysregulated in preeclamptic placenta. Moreover, knockdown of DNMT3A obviously inhibits trophoblast cell migration and invasion. Mechanistically, IGFBP5 (insulin-like growth factor-binding protein 5), known as a suppressor, is upregulated by decreased DNMT3A because of promoter hypomethylation. Importantly, IGFBP5 downregulation can rescue the defects caused by DNMT3A knockdown, thereby, consolidating the significance of IGFBP5 in the downstream of DNMT3A in trophoblast. Furthermore, we detected low promoter methylation and high protein expression of IGFBP5 in the clinical samples of preeclamptic placenta. Collectively, our study suggests that dysregulation of DNMT3A and IGFBP5 is relevant to preeclampsia. Thus, we propose that DNMT3A and IGFBP5 can serve as potential markers and targets for the clinical diagnosis and therapy of preeclampsia.

FT-like NFT1 gene may play a role in flower transition induced by heat accumulation in Narcissus tazetta var. chinensis.

The low-temperature flowering-response pathway, used as an inductive stimulus to induce flowering in plant species from temperate regions in response to cold temperature, has been extensively studied. However, limited information is available on the flower transition of several bulbous species, which require high temperature for flower differentiation. Narcissus tazetta var. chinensis (Chinese narcissus) exhibits a 2 year juvenile phase, and flower initiation within its bulbs occurs during summer dormancy. The genetic factors that control flower initiation are mostly unknown in Chinese narcissus. In the present study, we found that a high storage temperature is necessary for flower initiation. Flower initiation was advanced in bulbs previously exposed to extended high temperature. The heat accumulation required for flower transition was also determined. High temperature treatment rescued the low flower percentage resulting from short storage duration under natural conditions. In addition, extended high storage temperature was found to increase the flowering percentage of 2-year-old plants, which can be applied in breeding. Narcissus FLOWERING LOCUS T1 (NFT1), a homolog of the Arabidopsis thaliana gene FLOWERING LOCUS T, was isolated in this study. NFT1 transcripts were abundant during flower initiation in mature bulbs and were up-regulated by high temperature. The genetic experiments, coupled with an expression profiling assay, suggest that NFT1 possibly takes part in flower transition control in response to high temperature.

Necessity of high temperature for the dormancy release of Narcissus tazetta var. chinensis.

Winter dormancy has been extensively studied in many plants, while much less information is available for summer dormancy. Narcissus tazetta var. chinensis is characterized by a prolonged period of summer dormancy during the annual cycle. In the present study, we characterized the nature of dormancy in the controlled growth conditions and investigated the effects of temperature and ethylene on dormancy release. Cessation of growth and senescence of aerial tissues occurred even under conditions favorable for growth, suggesting an endo-dormancy process. Bulbs failed to sprout when they were exposed to low storage temperatures, while high temperature treatment preceding low storage temperatures, or heating interruption during low storage temperatures, could make bulbs sprouting. These results suggest that high temperatures are necessary for endo-dormancy release. Ethylene application during a later storage stage showed an obvious accelerative effect on bulb sprouting, whereas ethylene application during the middle stage had no effect on sprouting. The biological progression of dormancy was further studied through cytological and physiological analyses. Under natural conditions, the ethylene level was reduced to trace amounts with the transition and progression of dormancy. A transient peak in ethylene release was found when the plugged plasmodesmata (PD) began to re-open and flower initiation began. The most common PD possessed electron-dense deposits in endo-dormancy. These data indicate that endo-dormancy ends when flower initiation begins and ethylene peak occurs. Ethylene application had no effect on dormancy release, while it hastened sprouting only after the release from endo-dormancy by high temperature.