Owing to the intrinsic polypharmacological nature of most small-molecule kinase inhibitors, there is a need for computational models that enable systematic exploration of the chemogenomic landscape underlying druggable kinome toward more efficient kinome-profiling strategies. We implemented VirtualKinomeProfiler, an efficient computational platform that captures distinct representations of chemical similarity space of the druggable kinome for various drug discovery endeavors. By using the computational platform, we profiled approximately 37 million compound-kinase pairs and made predictions for 151,708 compounds in terms of their repositioning and lead molecule potential, against 248 kinases simultaneously. Experimental testing with biochemical assays validated 51 of the predicted interactions, identifying 19 small-molecule inhibitors of EGFR, HCK, FLT1, and MSK1 protein kinases. The prediction model led to a 1.5-fold increase in precision and 2.8-fold decrease in false-discovery rate, when compared with traditional single-dose biochemical screening, which demonstrates its potential to drastically expedite the kinome-specific drug discovery process.

A murine embryonic mesenchymal cell line C3H/10T1/2 possesses the potential to differentiate into multiple cell phenotypes and has been recognized as multipotent mesenchymal stem cells, but no in vitro model of its endothelial differentiation has been established and the effect of angiogenic factors on the differentiation is unknown. The aim of the present study was to evaluate the role of angiogenic factors in inducing endothelial differentiation of C3H/10T1/2 cells in vitro. C3H/10T1/2 cells were treated with angiogenic factors, VEGF (10 ng/mL) and bFGF (5 ng/mL). At specified time points, cells were subjected to morphological study, immunofluorescence staining, RT-PCR, LDL-uptake tests and 3-D culture for the examination of the structural and functional characteristics of endothelial cells. Classic cobblestone-like growth pattern appeared at 6 day of the induced differentiation. Immunofluorescence staining and RT-PCR analyses revealed that the induced cells exhibited endothelial cell-specific markers such as CD31, von Willebrand factor, Flk1, Flt1, VE-cadherin, Tie2, EphrinB2 and Vezf1 at 9 day. The induced C3H/10T1/2 cells exhibited functional characteristics of the mature endothelial phenotype, such as uptake of acetylated low-density lipoproteins (Ac-LDL) and formation of capillary-like structures in three-dimensional culture. At 9 day, Weibel-Palade bodies were observed under a transmission electron microscope. This study demonstrates, for the first time, endothelial differentiation of C3H/10T1/2 cells induced by angiogenic factors, VEGF and bFGF, and confirms the multipotential differentiation ability. This in vitro model is useful for investigating the molecular events in endothelial differentiation of mesenchymal stem cells.

Preeclampsia is a multifactorial hypertensive disorder of pregnancy, with variable presentation in both maternal and fetal factors, such that no treatment or marker is currently universal to all cases. Here, we demonstrate that the prothrombinase and immunomodulatory secreted factor FGL-2 (fibrinogen-like protein 2) is differentially expressed across previously characterized gene expression clusters containing clinically relevant disease subtypes. FGL2 is low in a cluster consistent with the traditional paradigm of the pathology of preeclampsia (canonical preeclampsia) and high in a cluster exhibiting evidence of immune activation (immunological preeclampsia). We show that it is part of an immunoregulatory gene module integral to the transcriptional profile and placental pathology specific to immunological preeclampsia. We determine that FGL2 associates positively with chronic inflammation lesions of the placenta while associating negatively with maternal vascular malperfusion lesions. The transcriptional profiles of maternal vascular malperfusion lesions show downregulation of FGL2 and upregulation of previously investigated preeclampsia biomarkers, such as FLT1 (Fms Related Receptor Tyrosine Kinase 1) and ENG (endoglin). Conversely, the profiles of chronic inflammation lesions show an interesting downregulation of these genes, but an upregulation of FGL2 and of FGL2-correlated immunoregulatory genes, suggesting it is upregulated downstream of major inflammatory mediators such as TNF (tumor necrosis factor)-α and IFN (interferon)-γ, hallmarks of the immunological preeclampsia subtype. This work, overall, demonstrates that FGL-2 expression levels in the term placenta reflect the unique pathophysiology that leads to immunological preeclampsia, leading to its potential as a subtype-specific biomarker.

Keywords: chorionic villi; fibrin; histology; inflammation; placenta; preeclampsia.

Placental vascularity may be important in the development of fetal growth restriction (FGR). The overnourished adolescent ewe is a robust model of the condition, with ∼50% of offspring demonstrating FGR (birthweight >2 standard deviations below optimally-fed control mean). We studied whether placental vascularity, angiogenesis and glucose transport reflect FGR severity.

Singleton pregnancies were established in adolescent ewes either overnourished to putatively restrict fetoplacental growth (n = 27) or control-fed (n = 12). At 131d (term = 145d) pregnancies were interrupted and fetuses classified as FGR (n = 17, <4222 g, -2SD below control-fed mean) or non-FGR (n = 10). Placentome capillary area density (CAD), number density (CND), surface density (CSD), and area per capillary (APC) in the fetal cotyledon (COT) and maternal caruncle (CAR) were analysed using immunostaining. COT/CAR mRNA expression of angiogenic ligands/receptors and glucose transporters were measured by qRT-PCR.

Fetal weight was reduced in FGR vs. Non-FGR/Control groups. Total placentome weight was Control>> Non-FGR>> FGR and fetal:placental weight ratios were higher in overnourished versus Control groups. COT vascular indices were Non-FGR>> FGR>> Control. COT-CAD, CSD and APC were significantly greater in Non-FGR overnourished versus Control and intermediate in FGR groups. CAR vascularity did not differ. CAR-VEGFA/FLT1/KDR/ANGPT1/ANGPT2/SLC2A1/SLC2A3 mRNA was lower and COT-ANGPT2 higher in overnourished versus Control groups.

Relative to control-intake pregnancy, overnourished pregnancies are characterised by higher COT vascularity, potentially a compensatory response to reduced nutrient supply, reflected by higher fetal:placental weight ratios. Compared with overnourished pregnancies where fetal growth is relatively preserved, overnourished pregnancies culminating in marked FGR have less placental vascularity, suggesting incomplete adaptation to the prenatal insult.

OBJECTIVE

To study first and second/third trimester levels of soluble fms-like tyrosine kinase 1 (sFlt1), placental growth factor (PlGF) and soluble endoglin (sEng) in FINNPEC case-control cohort. The participants were further divided into subgroups based on parity and onset of the disease. Recommended cut-off values in aid of pre-eclampsia (PE) prediction and diagnosis were also tested.

METHODS

First trimester serum samples were available from 221 women who later developed PE and 239 women who did not develop PE. Second/third trimester serum samples were available from 175 PE and 55 non-PE women. sFlt-1 and PlGF were measured electro-chemiluminescence immunoassays and sEng by ELISA.

RESULTS

In all timepoints PlGF, endoglin and the sFlt-1/PlGF ratio were increased in the PE group compared to the non-PE group. The serum concentrations of sFlt-1 were increased only at second/third trimester in PE women. Higher concentrations of s-Flt1, endoglin and higher sFlt/PlGF ratio were found at the third trimester in primiparous women compared to multiparous women. Primiparous PE women also had lower concentrations of PlGF at the third trimester. The proportion of women exceeding all cut-offs of the sFlt-1/PlGF ratio (≥33, ≥38, ≥85 and ≥110) was greater in the PE group, but there were also pre-eclamptic women who met rule-out cut-off or did not meet rule-in cut-off.

CONCLUSIONS

Primiparous pregnancies have more anti-angiogenic profile during second/third trimester compared with multiparous pregnancies. Our findings also suggest that certain maternal characteristics, e.g. BMI, smoking and pre-existing diseases, should be taken into account when different sFlt-1/PlGF ratio cut-offs are utilized.

The objective was to examine uterine artery blood flow (UBF) as well as macroscopic and microscopic placentome vascular density in nutrient-restricted Angus and Brahman heifers. Angus (n = 6) and Brahman (n = 6) heifers were bred to a single sire and pregnancy confirmed at 30-d postbreeding. Heifers were randomly assigned to 1 of 2 dietary treatments consisting of 100% (control-fed; CON; n = 6) or 60% (total nutrient-restricted; RES; n = 6) based from net energy requirements for gestating heifers. Nutritional treatments were imposed from days 50 to 180 of gestation. On day 175 of gestation, UBF was collected ipsilateral and contralateral to the conceptus via Doppler ultrasonography. Heifers underwent Cesarean sections for collection of 2 adjacent placentomes on day 180 of gestation. The primary cotyledonary artery of 1 placentome was perfused with Alexa Fluor 647 Con A conjugate to examine macroscopic cotyledonary vascular density via an in vivo imaging system. The second placentome was fixed for microscopic immunofluorescence labeling of capillaries and separated into maternal (caruncle) and fetal (cotyledon) components for determination of angiogenic factor mRNA expression. Main effects of nutritional treatment and breed are reported in the absence of a significant nutritional treatment by breed interaction. Ipsilateral UBF was decreased (P < 0.05) by 48% in RES vs. CON, whereas breed did not influence ipsilateral UBF. Contralateral UBF was not different between nutritional treatments; however, contralateral UBF was decreased (P < 0.05) by 63% in Brahman vs. Angus cattle. Macroscopic cotyledonary vascular density was increased (P < 0.05) by 36% in RES vs. CON and 82% in Brahman vs. Angus heifers. Percent capillary area and capillary perimeter were increased (P < 0.05) in RES vs. CON and increased (P < 0.05) in Brahman vs. Angus heifers. Dietary treatments did not alter angiogenic factor expression; however, transcript abundance of caruncle and cotyledon ANGP1, FLT1, and KDR was increased (P < 0.05) in Brahman vs. Angus heifers. In summary, these data indicate compensatory responses in macroscopic and microscopic placentome blood vessel density during maternal nutrient restriction-induced reductions in UBF. Moreover, a greater macroscopic density of cotyledonary blood vessels was observed in Brahman vs. Angus heifers.

Aims were to (i) compare specific transcript abundance between endometrial samples collected by transcervical biopsy and cytobrush and (ii) measure the abundance of endometrial transcripts involved in PGF2α synthesis in samples collected by cytobrush. In Experiment 1, endometrial samples were taken transcervically by cytobrush and biopsy 10 days after ovulation. Compared to biopsy samples, abundance of transcripts for MSTN, AKR1C4 and PGR was similar, VIM, FLT1 and PTGES was lower (p < .05) and KRT18 and CD3D was greater in cytobrush samples (p < .05). Thus, there was an enrichment of epithelial and immune cells in the cytobrush samples. In Experiment 2, endometrial samples were collected by cytobrush on days 10, 13, 16 and 19 after ovulation. Abundance of PGR2 mRNA was maximum on day 10 then decreased (p < .05). Abundance of ESR1 decreased gradually from day 10 to day 16 then increased again on day 19. The greatest abundance of OXTR was noted on day 19. The sequential alterations in abundance of these transcripts are consistent with the release of PGF2α associated with luteolysis. In summary, cytobrush sampling provides representative, physiologically relevant samples of the luminal epithelium in cattle.

Pre-eclampsia is a serious heritable disorder that affects 5-8% of pregnancies worldwide. While classical genetic studies have identified several susceptibility genes they do not fully explain the heritability of pre-eclampsia. An additional contribution to risk can be quantified by examining the epigenome, in particular the methylome, which is a representation of interactions between environmental and genetic influences on the phenotype. Current array-based epigenetic studies only examine 2-5% of the methylome. Here, we used whole-genome bisulfite sequencing (WGBS) to determine the entire methylome of 13 individuals from two multiplex pre-eclampsia families, comprising one woman with eclampsia, six women with pre-eclampsia, four women with uncomplicated normotensive pregnancies and two male relatives. The analysis of WGBS profiles using two bioinformatics platforms, BSmooth and Bismark, revealed 18,909 differentially methylated CpGs and 4157 differentially methylated regions (DMRs) concordant in females. The methylation patterns support the involvement of previously reported candidate genes, including COL4A1, SLC2A4, PER3, FLT1, GPI, LCT, DDAH1, TGFB3, DLX5, and LRP1B. Statistical analysis of DMRs revealed three novel genes significantly correlated with pre-eclampsia: sorbitol dehydrogenase (SORD, p = 9.98 × 10^-6), diacylglycerol kinase iota (DGKI, p = 2.52 × 10^-5), and islet cell autoantigen 1 (ICA1, 7.54 × 10^-3), demonstrating the potential of WGBS in families for elucidating the role of epigenome in pre-eclampsia and other complex diseases.

OBJECTIVE

The role of F-18-fluorothymidine (FLT) PET-CT imaging in the evaluation of gynecologic cancers has not been established. We sought to evaluate (FLT) PET-CT imaging in gynecologic cancers by comparing standard uptake values (SUVs) of FLT with F-18-fluorodeoxyglucose (FDG) PET in the primary tumor at diagnosis, and assess FLT uptake immediately following concurrent chemoradiotherapy (chemoRT).

METHODS

In this pilot study, patients treated for cervical (5) or vaginal (1) cancer underwent FLT-PET and FDG-PET scanning at diagnosis (FLT1 and FDG1). Five patients (4 cervical and 1 vaginal) also underwent FLT-PET within 1-3 weeks after chemoRT before brachytherapy (FLT2). Wilcoxon rank-sum test was used to compare the FLT1 and FDG1 parameters.

RESULTS

Median age at diagnosis was 61-years (range, 33-72). Cervical cancers were staged as IB2 (n = 1, 20%), IIB (n = 1, 20%), IIIB (n = 1, 20%) and IVA (n = 2, 40%) and the single vaginal cancer was staged IIIB. The most common histology was squamous cell carcinoma (n = 3, 50%) followed by adenocarcinoma (n = 2, 33%) and clear-cell adenosquamous carcinoma (n = 1, 17%). Median tumor SUVmax at diagnosis was 7.8 on FLT1-PET (3.9-14.2) versus 11.6 (5.9-23.2) on FDG1-PET (p = 0.15). Tumor SUVmax of FLT declined 54%-100% after chemoRT.

CONCLUSIONS

The tumor SUV of FLT at diagnosis was lower than that of FDG-PET. FLT uptake was markedly decreased after chemoRT. Results indicate that there may not be a significant effect of inflammation on FLT uptake in gynecologic cancers. FLT may be a useful tool when assessing the effects of chemoRT on gynecologic malignancies and planning for postchemoRT brachytherapy treatments.

Background: The survival status of patients with breast cancer and brain metastasis (BCBM) receiving current treatments is poor.

Method: We designed a real-world study to investigate using patients' clinical and genetic aberrations to forecast the prognoses of BCBM patients. We recruited 146 BCBM patients and analyzed their clinical features to evaluate the overall survival (OS). For genetic testing, 30 BCBM and 165 non-brain-metastatic (BM) metastatic breast cancer (MBC) patients from Hunan Cancer Hospital, and 86 BCBM and 1416 non-BM MBC patients from the Geneplus database who received circulating tumor DNA testing, were compared and analyzed.

Results: Ki67 >14% and >3 metastatic brain tumors were significant risk factors associated with poor OS, while chemotherapy and brain radiotherapy were beneficial factors for better OS. Compared with non-BM MBC patients, BCBM patients had more fibroblast growth factor receptor (FGFR) aberrations. The combination of FGFR, TP53 and FLT1 aberrations plus immunohistochemistry HER2-positive were associated with an increased risk of brain metastasis (AUC = 77.13%). FGFR aberration alone was not only a predictive factor (AUC = 67.90%), but also a significant risk factor for poor progression-free survival (Logrank p = 0.029). FGFR1 aberration was more frequent than other FGFR family genes in BCBM patients, and FGFR1 aberration was significantly higher in BCBM patients than non-BM MBC patients. Most FGFR1-amplified MBC patients progressed within 3 months of the late-line (>2 lines) treatment.

Conclusion: A group of genetic events, including FGFR, TP53 and FLT1 genetic aberrations, and HER2-positivity, forecasted the occurrence of BM in breast cancers. FGFR genetic aberration alone predicted poor prognosis.

Keywords: FGFR aberrations; HER2-positive; PFS and OS; breast cancer with brain metastasis; circulating tumor DNA.

The aim of this work was to evaluate factors affecting ovum capture in superovulated buffaloes, by comparing the morphological features of pre-ovulatory follicles and oocytes, the intrafollicular and plasmatic steroid profile, as well as the expression of genes involved in cumulus expansion and steroid cascade in granulosa cells (GCs) and that of genes involved in contraction-relaxation of the oviduct between superovulated and synchronized buffaloes. Italian Mediterranean Buffalo cows were either synchronized by Ovsynch (n = 25) and superovulated (n = 10) with conventional FSH protocol and sacrificed 18 h after last GnRH. Antral follicular count, recovery rate and oocyte quality were recorded, and plasma and follicular fluid were collected for steroid profile determination. In addition, in 10 animals (5/group), GCs were collected to analyse the mRNA expression of gonadotropin receptors (LHR and FSHR) and genes involved in steroid synthesis, as the cytochrome P450 family 19 (CYP19A1) and the steroidogenic acute regulatory protein (STAR). Moreover, oviducts were collected to evaluate the mRNA expression of estrogen receptor 1 (ER1) and the progesterone receptor (PGR), the vascular endothelial growth factor (VEGF) and the VEGF receptors, i.e. the kinase insert domain receptor (FLK1) and the fms related tyrosine kinase 1 (FLT1). No differences were recorded in steroids plasma concentration between synchronized and superovulated animals whereas intrafollicular E2 and P4 concentrations decreased in superovulated group (63.2 ± 10.6 vs 30.3 ± 5.9 ng/mL of E2 and 130.1 ± 19.8 vs 71.6 ± 8.5 ng/mL of P4, respectively in synchronized and superovulated animals; P < 0.05). Interestingly, both the recovery rate (85.7% vs 56.6%, respectively in synchronized and in superovulated animals; P < 0.05) and the percentage of oocytes exhibiting proper cumulus expansion (75% vs 28.1%, respectively in synchronized and in superovulated animals; P < 0.01) decreased in superovulated animals. In addition, the expression of FSHR and CYP19A1 increased while the expression of STAR in GCs decreased (P < 0.05). Finally, in superovulated buffaloes a decreased expression of PGR, ER1, VEGF and its receptor FLK1 in the oviduct was observed. The results suggest that the exogenous FSH treatment impairs steroidogenesis, affecting both the oviduct and the ovarian function, accounting for the failure of ovum capture in superovulated buffaloes.

Intracellular Ca²⁺ signals are essential for stem cell function and play a significant role in the differentiation process. Dental pulp stem cells (DPSCs) are a potential source of stem cells; however, the mechanisms controlling cell differentiation remain largely unknown. Utilizing rat DPSCs, we examined the effect of adenosine triphosphate (ATP) on osteoblast differentiation and characterized its mechanism of action using real-time Ca ²⁺ imaging analysis. Our results revealed that ATP enhanced osteogenesis as indicated by Ca ²⁺ deposition in the extracellular matrix via Alizarin Red S staining. This was consistent with upregulation of osteoblast genes BMP2, Mmp13, Col3a1, Ctsk, Flt1, and Bgn. Stimulation of DPSCs with ATP (1-300 µM) increased intracellular Ca ²⁺ signals in a concentration-dependent manner, whereas histamine, acetylcholine, arginine vasopressin, carbachol, and stromal-cell-derived factor-1α failed to do so. Depletion of intracellular Ca ²⁺ stores in the endoplasmic reticulum by thapsigargin abolished the ATP responses which, nevertheless, remained detectable under extracellular Ca ²⁺ free condition. Furthermore, the phospholipase C (PLC) inhibitor U73122 and the inositol triphosphate (IP ₃ ) receptor inhibitor 2-aminoethoxydiphenyl borate inhibited the Ca ²⁺ signals. Our findings provide a better understanding of how ATP controls osteogenesis in DPSCs, which involves a Ca ²⁺ -dependent mechanism via the PLC-IP ₃ pathway. This knowledge could help improve osteogenic differentiation protocols for tissue regeneration of bone structures.

Preeclampsia is a hypertensive disorder of pregnancy that is a major cause of maternal-fetal morbidity and mortality worldwide. Severe preeclampsia (sPE) is mediated by pathology of the placental villi resulting in repressed PIGF (placental growth factor) production and hyper-secretion of sFLT1 (soluble fms-like tyrosine kinase 1), the net effect being wide-spread maternal endothelial dysfunction. Villous trophoblast differentiation is under control of the PPARγ (peroxisome proliferator-activated receptor γ) and GCM1 (glial cell missing 1) axis which is dysregulated in sPE. We hypothesized that disruption of trophoblast differentiation via the PPARγ-GCM1 axis is a major contribution to excess production of sFLT1 and pharmacological activation of PPARγ in the sPE placenta could reduce sFLT1 to normal levels. sPE, age-matched control placentas and first-trimester villous explants, were used to investigate the molecular relationships between PPARγ-GCM1 and sFLT1. We modulated this pathway by pharmacological activation/inhibition of PPARγ using Rosiglitazone and T0070907, respectively, and through siRNA repression of GCM1. PPARγ and GCM1 protein expressions are reduced in the sPE placenta while FLT1 protein and sFLT1 secretion are increased. GCM1 reduction in the first trimester explants significantly increased sFLT1 secretion, suggesting GCM1 as a key player in this pathway. Activation of PPARγ restored GCM1 and significantly reduced sFLT1 expression and release in first trimester and sPE placental villi. Functional integrity of the PPARγ-GCM1 axis in the villous trophoblast is critical for normal pregnancy development and is disrupted in the sPE placenta to favor excessive production of sFLT1. Pharmacological manipulation of PPARγ activity has the potential to rescue the antiangiogenic state of sPE and thereby prolong pregnancy and deliver improved clinical outcomes.

Keywords: arteries; placenta; preeclampsia; pregnancy; stillbirth; trophoblast.

A dense local vascular network is crucial for pancreatic endocrine cells to sense metabolites and secrete hormones, and understanding the interactions between the vasculature and the islets may allow for therapeutic modulation in disease conditions. Using live imaging in two models of vascular disruption in zebrafish, we identified two distinct roles for the pancreatic vasculature. At larval stages, expression of a dominant negative version of Vegfaa (dnVegfaa) in β-cells led to vascular and endocrine cell disruption with a minor impairment in β-cell function. In contrast, expression of a soluble isoform of Vegf receptor 1 (sFlt1) in β-cells blocked the formation of the pancreatic vasculature and drastically stunted glucose response while islet architecture was not affected. Notably, these effects of dnVegfaa or sFlt1 were not observed in animals lacking vegfaa, vegfab, kdrl, kdr, or flt1 function, indicating that they interfere with multiple ligands and/or receptors. In adults, disrupted islet architecture persisted in dnVegfaa expressing animals, while sFlt1 expressing animals displayed large sheets of β-cells along their pancreatic ducts, accompanied by impaired glucose tolerance in both models. Thus, our study reveals novel roles for the vasculature in patterning and function of the islet.

Blood vessels and nerve tissues are critical to the development and functionality of many vital organs. However, little is currently known about their interdependency during development and after injury. In this study, dual fluorescence transgenic reporter mice were utilized to observe blood vessels and nervous tissues in organs postnatally. Thy1-YFP and Flt1-DsRed (TYFD) mice were interbred to achieve dual fluorescence in the offspring, with Thy1-YFP yellow fluorescence expressed primarily in nerves, and Flt1-DsRed fluorescence expressed selectively in blood vessels. Using this dual fluorescent mouse strain, we were able to visualize the networks of nervous and vascular tissue simultaneously in various organ systems both in the physiological state and after injury. Using ex vivo high-resolution imaging in this dual fluorescent strain, we characterized the organizational patterns of both nervous and vascular systems in a diverse set of organs and tissues. In the cornea, we also observed the dynamic patterns of nerve and blood vessel networks following epithelial debridement injury. These findings highlight the versatility of this dual fluorescent strain for characterizing the relationship between nerve and blood vessel growth and organization.

Despite the indispensable role of the placenta in the successful course of pregnancy, regulation of its dynamic transcriptome is still underexplored. The purpose of this literature review was to give an overview and draw attention to the contribution of genetic variation in shaping the human placental gene expression. Studies of placental transcriptome shaped by chromosomal variants are limited and may be confounded by cellular mosaicism and somatic genomic rearrangements. Even in relatively simple cases, such as aneuploidies, the placental transcriptome appears to differ from the assumed systematically increased transcript levels of the involved chromosomes. Single nucleotide variants modulating placental gene expression referred to as expression quantitative trait loci (eQTLs) have been analyzed only in ten candidate gene and three genome-wide association studies (GWAS). The latter identified 417 confident placental eGenes, supported by at least two independent studies. Functional profiling of eGenes highlighted biological pathways important in pregnancy, such as immune response or transmembrane transport activity. A fraction of placental eQTLs (1-3%) co-localize with GWAS loci for adult disorders (metabolic, immunological, neurological), suggesting a co-contributory role of the placenta in the developmental programming of health. Some placental eQTLs have been identified as risk factors for adverse pregnancy outcomes, such as rs4769613 (C > T), located near the FLT1 gene and confidently associated with preeclampsia. More studies are needed to map genetic variants shaping gene expression in different placental cell types across three trimesters in normal and complicated gestations and to clarify to what extent these heritable factors contribute to maternal and offspring disease risks.

Keywords: Developmental programming; Gene expression; Genetic variation; Placenta; Pregnancy; eQTL.

Composite hemangioendothelioma (CHE) is considered a borderline malignant vascular tumor defined by an admixture of distinct vascular neoplastic components. A 21-year-old female is presented herein with a 1 cm painless mandibular vestibular mass of less than a year duration. The infiltrating tumor was characterized by dilated vascular channels lined by endothelial cells with bland ovoid or round nuclei exhibiting, occasionally, hobnail/matchstick-like arrangement. Intravascular cell proliferations with hyaline globular deposits were also present. Additionally, lobular spindle and epithelioid cell aggregates, as well as slit-like spaces exhibiting a retiform or angiosarcomatous morphology were observed. Intracytoplasmic signet-ring or lipoblast-like vacuolization was also noted. Mitotic activity was exceptionally rare. Vascular spaces and the stroma featured lymphocytes and plasma cells. Neoplastic cells were positive for CD31, CD34, D2-40 and ERG, negative for CAMTA1 and synaptophysin, while type IV collagen highlighted the plasmalemma of most vessels and hyaline globules. Fluorescence in situ hybridization revealed gene rearrangements in both YAP1 and MAML2 genes, in keeping with a YAP1-MAML2 fusion. Whole exome sequencing (WES) identified three missense mutations FLT1 [p.R1016G], PIK3CA [p.H1047L], and C11orf42 [p.A304P] and a mitochondrial frameshift insertion MT-ND4 [c.1107_1108insC; p.P370fs]. These WES results suggest that FLT1 and/or PIK3CA variants may contribute to tumor growth/transformation while the MT-ND4 variant may relate to proliferation, angiogenesis and/or inhibition of apoptosis.

Keywords: C11orf42; FLT1; MT-ND4; Oral composite hemangioendothelioma; PIK3CA; YAP1-MAML2 fusion.

OBJECTIVE

Soluble Flt1 (sFlt1) is an anti-angiogenic protein linked to the pathology of preeclampsia (PE). While the placenta serves as the major organ producing sFlt1 during normal pregnancy, peripheral blood mononuclear cells (PBMCs), endothelial cells, and stromal cells also produce sFlt1. The key question is 'what drives the overexpression of sFlt1 observed during PE?' In the present work we show evidence for sFlt1 over-expression in PBMCs due to interaction with placental villi from PE patients.

METHODS

sFlt1 production by PBMCs is estimated by using two blood collection methods with different coagulation chemistry. PBMCs were then cultured with homologous villous explants and heterologous villous explants to determine the effects of the interaction between the two tissues.

METHODS

sFlt1 levels were estimated using real time PCR, ELISA, and gel electrophoresis.

RESULTS

Plasma samples obtained using CTAD as anti-coagulant showed 16-23% less sFlt1 compared to plasma collected in EDTA. Preeclamptic PBMCs showed higher basal level of sFlt1 mRNA. In addition, we show evidence of placental interaction as a cause of sFlt1 overexpression in PBMCs using homologous and heterologous co-culture system. However, during co-culture, we observed that while the sFlt1 expression in PE PBMCs is increased, PE villous explants show reduced sFlt1 RNA expression.

CONCLUSIONS

sFlt1 was produced in significant amounts by preeclamptic PBMCs, and ex vivo studies show that the placenta induces this over-expression. In contrast, exposure to PBMCs appears to decrease sFlt1 production by preeclamptic placenta.

Intrauterine growth restriction (IUGR) and low birth weigth (LBW) are risk factors for neonatal chronic lung disease. However, maternal and fetal genetic factors and the molecular mechanisms remain unclear. We investigated the relationship between LBW and lung function with Mendelian randomisation analyses and studied angiogenesis in a low protein diet rat model of IUGR. Our data indicate a possible association between LBW and reduced FEV1 (p = 5.69E-18, MR-PRESSO) and FVC (6.02E-22, MR-PRESSO). Complimentary, we demonstrated two-phased perinatal programming after IUGR. The intrauterine phase (embryonic day 21) is earmarked by a reduction of endothelial cell markers (e.g. CD31) as well as mRNA expression of angiogenic factors (e.g., Vegfa, Flt1, Klf4). Protein analysis identified an activation of anti-angiogenic mTOR effectors. In the postnatal phase, lung capillaries (< 20 µm) were significantly reduced, expression of CD31 and VE-Cadherin were unaffected, whereas SMAD1/5/8 signaling and Klf4 protein were increased (p < 0.01). Moreover, elevated proteolytic activity of MMP2 and MMP9 was linked to a 50% reduction of lung elastic fibres. In conclusion, we show a possible link of LBW in humans and reduced lung function in adulthood. Experimental IUGR identifies an intrauterine phase with inhibition of angiogenic signaling, and a postnatal phase with proteolytic activity and reduced elastic fibres.

Chemokine (C-X-C motif) ligand 12 (CXCL12) and its receptor, chemokine (C-X-C motif) receptor 4 (CXCR4), are involved in significant biological processes associated with early pregnancy including increasing trophoblast invasion and stimulating placental vascularization. To further elucidate functions of CXCL12-CXCR4 signaling during early gestation, our objective was to inhibit CXCR4 in vivo using a CXCR4 antagonist, AMD3100. We hypothesized that inhibition of CXCR4 would negatively affect chemokine and angiogenic factor regulation imperative for placental development in sheep. Osmotic pumps containing PBS (control) or AMD3100 (CXCR4 antagonist) were surgically installed ipsilateral to the corpus luteum on d 12 of gestation and administered treatments directly into the uterine lumen. Maternal (caruncle and intercaruncle) and fetal membrane tissues were collected on d 23 of gestation and mRNA and protein expression were analyzed for vascular endothelial growth factor (VEGF), kinase insert domain receptor (KDR), fms related tyrosine kinase 1 (FLT1), fibroblast growth factor 2 (FGF2), angiopoietin 1 (ANGPT1), hypoxia inducible factor 1 ɑ subunit (HIF1A), CXCL12, and its corresponding receptors (CXCR4 and CXCR7). Immunohistochemical procedures were performed for analysis of CXCL12 and cell proliferation. In caruncle tissue ipsilateral to the pump, mRNA for KDR, ANGPT1, HIF1A, and CXCL12 increased (P < 0.05) in treated ewes compared to control, whereas caruncle tissue contralateral to the pump had increased expression (P < 0.05) of KDR, and CXCL12 in treated ewes. In fetal membrane, CXCR4 mRNA and protein decreased (P < 0.05), while VEGF protein decreased (P < 0.05) in caruncle and fetal membrane tissue from treated ewes. Results from this study highlight the importance of CXCL12-CXCR4 signaling at the fetal-maternal interface. Inhibiting this axis may disrupt typical regulation of angiogenic factors needed for placental development and embryo growth.

Conventional antiangiogenetic inhibitors suffered from poor delivery problems that result in unsatisfactory antitumor treatment efficacy. Although the liposomes or nanomaterial-based delivery systems can improve the therapeutic efficacy of antiangiogenic molecules, the assembly process is far too complex. Herein, a nanomaterial or a new nanodrug that could work without the help of a carrier and could be easily synthesized is needed. Au nanoclusters (AuNCs) are a kind of ideal nanostructures that could spontaneously enter into the cell and could be synthesized by a relatively easy one-pot method. Here, changing the traditional ligand glutathione (GSH) into an anti-Flt1 peptide (AF) has enriched the newly synthesized AF@AuNCs with targeted antiangiogenic properties. Based on the specific binding between AF and vascular endothelial growth factor receptor 1 (VEGFR1), the interaction between VEGFR1 and its ligands could be blocked. Furthermore, the expression of VEGFR2 could be downregulated. Compared with pure AF peptide- and GSH-participated AuNCs (GSH@AuNCs), AF@AuNCs were more effective in inhibiting both tube formation and migration of the endothelial cells in vitro. Furthermore, the in vivo chick embryo chorioallantoic membrane (CAM) experiment and antitumor experiment were conducted to further verify the enhanced antiangiogenesis and tumor inhibition effect of AF@AuNCs. Our findings provide promising evidence of a carrier-free nanodrug for tumors and other vascular hyperproliferative diseases.

Keywords: Au nanoclusters; anti-Flt1 peptide; antiangiogenesis; antitumor; nanomedicine.

Subcortical white matter infarction causes ischemic demyelination and loss of brain functions, as the result of disturbances of the blood flow. Although angiogenesis is one of the recovery processes after cerebral infarction, the dynamics of revascularization after white matter infarction still remains unclear. We induced white matter infarction in the internal capsule of Flk1-GFP::Flt1-tdsRed double transgenic mice by injection of endothelin-1 (ET-1), a vasoconstrictor peptide, together with N(G)-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor, and followed the changes in Flk1 and Flt1 expression in the vascular system in the infarct area. Reduction of Flt1-tdsRed-positive blood vessels 1 day after the injection and increase of Flk1-GFP-strongly-positive blood vessels 3 days after the injection were apparent. PDGFRβ-strongly-positive (PDGFRβ+) cells appeared in the infarct area 3 days after the injection and increased their number thereafter. Three days after the injection, most of these cells were in close contact with Flk1-GFP-positive endothelial cells, indicating these cells are bona fide pericytes. Seven days after the injection, the number of PDGFRβ+ cells increased dramatically, and the vast majority of these cells were not in close contact with Flk1-GFP-positive endothelial cells. Taken together, our results suggest revascularization begins early after the ischemic insult, and the emerging pericytes first ensheath blood vessels and then produce fibroblast-like cells not directly associated with blood vessels.

Objective: Autophagy influences a wide range of physiological and pathological processes in the human body. In this study, we aimed to investigate the role of autophagy in early-onset preeclampsia (EOPE); autophagy activation by hypoxia could rescue impaired angiogenesis and apoptosis in preeclampsia, leading by ox-LDL. Methods: Transmission electron microscopy was applied to identify autolysosomes in trophoblast cells of the placenta apical region. Quantitative real-time polymerase chain reaction, Western blot, flow cytometry, and wound-healing assays were adopted to determine autophagy activity, angiogenesis, and apoptosis in placenta tissues or HTR8/SVneo cells. Results: Autophagy activity was inhibited in the placenta of women who experienced EOPE; autophagy activation by hypoxia enhanced the migration ability, rescued ox-LDL-mediated impaired angiogenesis in HTR8/SVneo cells [vascular endothelial growth factor A (VEGFA) downregulation and FMS-like tyrosine kinase-1 (FLT1) upregulation], and protected against cell apoptosis (BAX downregulation). Conclusion: Autophagy could maintain the function of trophoblast cells by differentially regulating the expression of VEGFA and FLT1 and protecting against cell apoptosis at the maternal-fetal interface, potentially related to prevention of preeclampsia.

Keywords: apoptosis; autophagy activation; early-onset preeclampsia; hypoxia; oxidized low-density lipoprotein.

Fms-related tyrosine kinase 1 (Flt1), the receptor of VEGF/PIGF, is associated with cancer angiogenesis and tumorigenesis. Although the high expression of Flt1 in glioma is identified, its regulatory mechanism remains unclear. In the present study, we demonstrate that miR‑139‑5p inhibits Flt1 expression mediated by binding its 3' untranslated region (3'UTR) to regulate the progression of human glioma. We found miR‑139‑5p was downregulated in glioma tissues compared with normal brain tissues whereas a converse expression level of Flt1 was observed. Additionally we proved that miR‑139‑5p directly integrated with the 3'UTR of Flt1 via luciferase activity assay and cells transfected with miR‑139‑5p characterized with a low expression of Flt1 in mRNA and protein levels. Furthermore, we validated that miR‑139‑5p enforced its biological modulation via targeting Flt1 through rescue experiments. miR‑139‑5p suppressed and Flt1 stimulated the malignant activities of glioma cells. We demonstrated that miR‑139‑5p inhibited the Flt1-mediated Wnt/β-catenin signaling pathway in glioma cells. Conclusively, our study indicated that miR‑139‑5p can counteract the malignant phenotypes of glioma cells by the inhibitory effect of the Flt1-mediated Wnt/β-catenin signaling pathway.