Preeclamptic serum and soluble fms-like tyrosine kinase-1 suppress endothelial inward rectifier potassium currents.

INTRODUCTION: Inward rectifier K+ (Kir) channel, a major factor determining endothelial membrane potential, regulates Ca2+ influx and vasodilator release, which is impaired in preeclamptic blood vessels. Previously, human umbilical vein endothelial cell (HUVEC) Kir currents were shown to decrease after incubating in preeclamptic plasma. We aimed to demonstrate whether sFlt-1, which is high in preeclamptic blood, could inhibit Kir channel function and expression. METHODS: HUVECs were cultured in regular medium, regular medium with added sFlt-1, or serum from preeclampsia patients or normal pregnant women (Control, sFlt-1, PE, or NP, respectively). Using whole-cell patch clamp technique, we identified Kir currents with the Kir blocker 2 mM BaCl2 and compared the currents among groups. The expression of Kir 2.1 and 2.2 channels were determined using immunofluorescent staining. RESULTS: sFlt-1 and PE groups exhibited similar Kir currents, while NP group possessed significantly larger currents, similar to Control group currents. Moreover, sFlt-1 and sFlt-1/PlGF ratio showed strong negative correlation with Kir currents (r = -0.71 and -0.70, respectively; P < 0.05). There were no significant differences in mean fluorescence intensity representing Kir 2.1 and 2.2 channels expression in all four groups. DISCUSSION: This is the first report to demonstrate sFlt-1 inhibition against Kir currents, which could lead to maternal endothelial dysfunction and hypertension seen in preeclampsia. However, channel expression was unaffected by sFlt-1 incubation, suggesting dysfunctions of channel or other processes (e.g., membrane translocation). The present data could pave the way for novel therapies targeting sFlt-1 or Kir to alleviate hypertension in preeclampsia.

Procalcitonin (PCT)-guided antibiotic stewardship in Asia-Pacific countries: adaptation based on an expert consensus meeting.

Introduction Recently, an expert consensus on optimal use of procalcitonin (PCT)-guided antibiotic stewardship was published focusing mainly on Europe and the United States. However, for Asia-Pacific countries, recommendations may need adaptation due to differences in types of infections, available resources and standard of clinical care. Methods Practical experience with PCT-guided antibiotic stewardship was discussed among experts from different countries, reflecting on the applicability of the proposed Berlin consensus algorithms for Asia-Pacific. Using a Delphi process, the group reached consensus on two PCT algorithms for the critically ill and the non-critically ill patient populations. Results The group agreed that the existing evidence for PCT-guided antibiotic stewardship in patients with acute respiratory infections and sepsis is generally valid also for Asia-Pacific countries, in regard to proposed PCT cut-offs, emphasis on diagnosis, prognosis and antibiotic stewardship, overruling criteria and inevitable adaptations to clinical settings. However, the group noted an insufficient database on patients with tropical diseases currently limiting the clinical utility in these patients. Also, due to lower resource availabilities, biomarker levels may be measured less frequently and only when changes in treatment are highly likely. Conclusions Use of PCT to guide antibiotic stewardship in conjunction with continuous education and regular feedback to all stakeholders has high potential to improve the utilization of antibiotic treatment also in Asia-Pacific countries. However, there is need for adaptations of existing algorithms due to differences in types of infections and routine clinical care. Further research is needed to understand the optimal use of PCT in patients with tropical diseases.

Accuracy of anti-Müllerian hormone and total follicles count to diagnose polycystic ovary syndrome in reproductive women.

OBJECTIVE: Recently, there was a new recommendation of ultrasonographic criteria to diagnosis polycystic ovary syndrome (PCOS). In addition, serum anti-Müllerian hormone (AMH) was proposed as a surrogate marker for diagnosis of PCOS, but AMH cut-off level for diagnosis of PCOS is unclear. This study aimed to investigate the accuracy of serum AMH and evaluate new ultrasonographic criteria, follicle number per ovary (FNPO) threshold ≥ 25 follicles and ovarian volume (OV) > 10 mL, for diagnosis of PCOS. MATERIALS AND METHODS: A cross-sectional study was conducted. Fifty-five PCOS women and sixty-three normal ovulatory, non-hyperandrogenic women were recruited. Transvaginal or transrectal ultrasonography was performed in all participants to evaluate follicle number and OV. Serum AMH was evaluated in both study groups. RESULTS: The mean age of the participants was 25.1 ± 5.3 years old in PCOS group and 29.7 ± 7.2 years old in control group. Mean AMH, FNPO and OV in PCOS women were significantly higher than those in non-PCOS women. The area under the receiver-operating characteristic (ROC) curve of AMH was 0.903. The threshold of AMH at 4.7 ng/mL offered the best compromise between 80% sensitivity and 77.8% specificity. The appropriated threshold values for FNPO, follicle number per cross-section (FNPS) and OV were 15 follicles, 7 follicles and 6.5 mL, respectively. Serum AMH level was significantly positively correlated with FNPO, FNPS and OV in both PCOS and control groups. In PCOS women, serum AMH showed strongly correlation with FNPO (r = 0.53, p < 0.001) and weakly correlation with total testosterone (r = 0.283, p = 0.036). CONCLUSION: Serum AMH had a good diagnostic performance for diagnosis of PCOS presenting with oligo/anovulation and hyperandrogenism. AMH threshold at 4.7 ng/mL was the best compromise level for diagnosis of PCOS. FNPO ≥15, FNPS ≥7 and OV ≥ 6.5 mL were reliable threshold for detecting polycystic ovaries in women with frank manifestation of PCOS.

Serum-free placental growth factor isoform 1 at 11-13-week gestation: effects of maternal factors, mean arterial pressure, placental volume, and uterine artery pulsatility index.

INTRODUCTION: To define the effects of maternal factors, mean arterial pressure (MAP), placental volume (PV), and uterine artery Doppler pulsatility index (UtAPI) to serum level of free form of placental growth factor isoform 1 (free PlGF-1) measured with a novel automated assay. METHODS: We enrolled 200 Thai women singleton pregnancy from 11+0 to 13+6 weeks gestation with low prior risk maternal factors (age, parity, tobacco use, assisted reproductive technology, and body mass index). MAP was measured. Serum-free PlGF-1, PV, and UtAPI were measured with a new assay, transabdominal three-dimensional, and color Doppler ultrasounds, respectively. Effects of these variables to serum-free PlGF-1 level were assessed. RESULTS: Data from 195 eligible subjects showed an elevation of serum-free PlGF-1 from 11, 12, and 13 weeks (mean ± SD; 36.89 ± 24.92, 38.71 ± 17.44, and 49.68 ± 22.30 pg/mL, respectively (p < .05)). Serum-free PlGF-1 level showed positive correlation with PV (r = 0.290, p < .01), and negative correlation with right and left UtAPI (r = -0.717, p = .05 and r = -0.221, p < .05, respectively). PV showed negative correlation with right and left UtAPI (r = -0.243, p < .05 and r = -0.372, p < .05, respectively). Serum-free PlGF-1 level had no significant correlation with maternal factors or MAP (p > .05). There was no preeclampsia at <34 weeks in 161 subjects (82.6%) with known pregnancy outcomes. CONCLUSIONS: There was modest correlation of serum-free PlGF-1, PV, and UtAPI, but not with maternal factors or MAP. Adjustment of serum-free PlGF-1 in early preeclampsia screening algorithm should be considered.

Circulating soluble fms-like tyrosine kinase-1 and placental growth factor from 10 to 40 weeks' pregnancy in normotensive women.

INTRODUCTION: Circulating soluble fms-like tyrosine kinase-1 (sFlt-1) and placental growth factor (PlGF) are potential markers for preeclampsia. The objective was to construct and analyse the reference ranges of serum levels of sFlt-1 and PlGF throughout the course of pregnancy in low-risk Thai pregnant women. METHODS: We enrolled 110 low-risk, Thai women singleton pregnancy from 10 to 40 gestational weeks. Serum concentrations of sFlt-1 and PlGF were measured with an automated assay. The reference ranges of serum levels of sFlt-1, PlGF and sFlt-1/PlGF ratio were constructed and assessed for possible correlations with gestational age, maternal factors [age, parity, tobacco use, artificial reproductive technologies (ARTS) and body mass index (BMI)], and pregnancy outcomes (gestational age at delivery, development of preeclampsia, neonatal birth weight and placental weight). RESULTS: None of the subjects developed preeclampsia. Serum sFlt-1 concentrations significantly elevated from 20 to 40 gestational weeks (P=0.003). Significant elevation and dropping of serum PlGF levels and sFlt-1/PlGF ratios were observed at 10 to 29 and 30 to 40 weeks of gestation, respectively (P<0.001). There was an inversed correlation between serum PlGF levels at 20 to 29 gestational weeks and neonatal birth weights (r=-0.48, P<0.05). There were no associations between serum levels of sFlt-1, PlGF, or sFlt-1/PlGF ratios and maternal BMI, gestational age at delivery, or placental weight (P>0.05). Effects from parity, smoking and ARTS were inconclusive. CONCLUSION: Robust change of serum PlGF levels suggests for its broader clinical application compared to sFlt-1. Prediction of preeclampsia using serum analytes may be gestational period specific.

Circulating free soluble fms-like tyrosine kinase-1 during late first trimester in relation with placental volume as a surrogate for trophoblastic production: a physiology study in low-risk cohort.

BACKGROUND: Data on first-trimester circulating soluble fms-like tyrosine kinase-1 (sFlt-1) and ischemic placental disease is limited and conflicting. This study aimed to study its physiology in relation to trophoblastic mass as the source of production. METHODS: Low-risk (representing normal placentation) women from 11 0/7 to 13 6/7 weeks' gestation were prospectively enrolled. Selective measurement of serum free sFlt-1 using a new automated assay from 100 eligible subjects was analyzed with gestational age, maternal weight, fetal crown-rump length (CRL), and mean uterine artery Doppler pulsatility index (PI). Placental volume (surrogate for trophoblastic mass) was estimated using 3-dimensional ultrasound and was assessed for its association with serum free sFlt-1. RESULTS: There was no significant association between serum free sFlt-1 and placental volume in either arithmetic (r = 0.053, p = 0.600), logarithmic (r = 0.005, p = 0.963), or quartile (p = 0.703) scale. There was a significant negative correlation between free sFlt-1 level and maternal weight (r=-0.213, p = 0.033). No significant correlation was found between free sFlt-1 level and gestational age (r = 0.007, p = 0.947), CRL (r = 0.027, p = 0.788), and uterine artery Doppler mean PI (r = 0.020, p = 0.828). CONCLUSIONS: Lack of correlation between circulating free sFlt-1 level and placental volume suggests that trophoblasts are not its major source during first trimester with presumably physiologic placentation.

Isolation, characterization, and transplantation of cardiac endothelial cells.

Isolation and ex vivo expansion of cardiac endothelial cells have been a recurrent challenge due to difficulties in isolation, cell heterogeneity, lack of specific markers to identify myocardial endothelial cells, and inadequate conditions to maintain long-term cultures. Herein, we developed a method for isolation, characterization, and expansion of cardiac endothelial cells applicable to study endothelial cell biology and clinical applications such as neoangiogenesis. First, we dissociated the cells from murine heart by mechanical disaggregation and enzymatic digestion. Then, we used flow cytometry coupled with specific markers to isolate endothelial cells from murine hearts. CD45+ cells were gated out to eliminate the hematopoietic cells. CD31+/Sca-1+ cells were isolated as endothelial cells. Cells isolated from atrium grew faster than those from ventricle. Cardiac endothelial cells maintain endothelial cell function such as vascular tube formation and acetylated-LDL uptake in vitro. Finally, cardiac endothelial cells formed microvessels in dorsal matrigel plug and engrafted in cardiac microvessels following intravenous and intra-arterial injections. In conclusion, our multicolor flow cytometry method is an effective method to analyze and purify endothelial cells from murine heart, which in turn can be ex vivo expanded to study the biology of endothelial cells or for clinical applications such as therapeutic angiogenesis.

Isolation and characterization of neural crest-derived stem cells from dental pulp of neonatal mice.

Dental pulp stem cells (DPSCs) are shown to reside within the tooth and play an important role in dentin regeneration. DPSCs were first isolated and characterized from human teeth and most studies have focused on using this adult stem cell for clinical applications. However, mouse DPSCs have not been well characterized and their origin(s) have not yet been elucidated. Herein we examined if murine DPSCs are neural crest derived and determined their in vitro and in vivo capacity. DPSCs from neonatal murine tooth pulp expressed embryonic stem cell and neural crest related genes, but lacked expression of mesodermal genes. Cells isolated from the Wnt1-Cre/R26R-LacZ model, a reporter of neural crest-derived tissues, indicated that DPSCs were Wnt1-marked and therefore of neural crest origin. Clonal DPSCs showed multi-differentiation in neural crest lineage for odontoblasts, chondrocytes, adipocytes, neurons, and smooth muscles. Following in vivo subcutaneous transplantation with hydroxyapatite/tricalcium phosphate, based on tissue/cell morphology and specific antibody staining, the clones differentiated into odontoblast-like cells and produced dentin-like structure. Conversely, bone marrow stromal cells (BMSCs) gave rise to osteoblast-like cells and generated bone-like structure. Interestingly, the capillary distribution in the DPSC transplants showed close proximity to odontoblasts whereas in the BMSC transplants bone condensations were distant to capillaries resembling dentinogenesis in the former vs. osteogenesis in the latter. Thus we demonstrate the existence of neural crest-derived DPSCs with differentiation capacity into cranial mesenchymal tissues and other neural crest-derived tissues. In turn, DPSCs hold promise as a source for regenerating cranial mesenchyme and other neural crest derived tissues.