BMP inhibition stimulates WNT-dependent generation of chondrogenic mesoderm from embryonic stem cells

WNT and bone morphogenetic protein (BMP) signaling are known to stimulate hemogenesis from pluripotent embryonic stem (ES) cells. However, osteochondrogenic mesoderm was generated effectively when BMP signaling is kept to a low level, while WNT signaling was strongly activated. When mesoderm specification from ES cells was exogenous factor dependent, WNT3a addition supported the generation of cardiomyogenic cells expressing lateral plate/extraembryonic mesoderm genes, and this process involved endogenous BMP activities. Exogenous BMP4 showed a similar effect that depended on endogenous WNT activities. However, neither factor induced robust chondrogenic activity. In support, ES cell differentiation in the presence of either WNT3a or BMP4 was associated with elevated levels of both Bmp and Wnt mRNAs, which appeared to provide sufficient levels of active BMPs and WNTs to promote the nonchondrogenic mesoderm specification. The osteochondrogenic mesoderm expressed PDGFRα, which also expressed genes that mark somite and rostral presomitic mesoderm. A strong WNT signaling was required for generating the mesodermal progeny, while approximately 50- to 100-fold lower concentration of WNT3a was sufficient for specifying axial mes(end)oderm. Thus, depending on the dose and cofactor (BMP), WNT signaling stimulates the generation of different biological activities and specification of different types of mesodermal progeny from ES cells.     

Ongoing Pregnancy Rates in Women with Low and Extremely Low AMH Levels. A Multivariate Analysis of 769 Cycles

Background

The ideal test for ovarian reserve should permit the identification of women who have no real chance of pregnancy with IVF treatments consequent upon an extremely reduced ovarian reserve. The aim of the current study was to evaluate pregnancy rates in patients with low AMH levels (0.2–1 ng/ml) and extremely low AMH levels (<0.2 ng/ml) and to determine the cumulative pregnancy rates following consecutive IVF treatments.

Methods

We conducted an historical cohort analysis at a tertiary medical center. Serum AMH levels were measured at initial clinic visit and prior to all following treatment cycles in 181 women (769 cycles) with an initial AMH level ≤1 ng/ml, undergoing IVF-ICSI. Main outcome measures were laboratory outcomes and pregnancy rates.

Results

Seventy patients undergoing 249 cycles had extremely low AMH levels (≤0.2 ng/ml), whereas 111 patients undergoing 520 cycles had low AMH levels (0.21–1.0 ng/ml). Number of oocytes retrieved per cycle, fertilized oocytes and number of transferred embryos were significantly lower in the extremely low AMH levels group compared to the low AMH levels (P<0.003). Crude ongoing pregnancy rates were 4.4% for both groups of patients. Among 48 cycles of women aged ≥42 with AMH levels of ≤0.2 ng/ml no pregnancies were observed. But, in patients with AMH levels of 0.2–1.0 ng/ml, 3 ongoing pregnancies out of 192 cycles (1.6%) were observed. However, in a multivariate regression analysis adjusted for age and cycle characteristics, no significant differences in ongoing pregnancy rates per cycle between the two groups were evident. Cumulative pregnancy rates of 20% were observed following five cycles, for both groups of patients.

Conclusions

Patients with extremely low AMH measurements have reasonable and similar pregnancy rates as patients with low AMH. Therefore, AMH should not be used as the criterion to exclude couples from performing additional IVF treatments.     

Bmp signaling promotes intermediate mesoderm gene expression in a dose-dependent, cell-autonomous and translation-dependent manner

The intermediate mesoderm lies between the somites and the lateral plate and is the source of all kidney tissue in the developing vertebrate embryo. While bone morphogenetic protein (Bmp) signaling is known to regulate mesodermal cell type determination along the medio-lateral axis, its role in intermediate mesoderm formation has not been well characterized. The current study finds that low and high levels of Bmp ligand are both necessary and sufficient to activate intermediate and lateral mesodermal gene expression, respectively, both in vivo and in vitro. Dose-dependent activation of intermediate and lateral mesodermal genes by Bmp signaling is cell-autonomous, as demonstrated by electroporation of the avian embryo with constitutively active Bmp receptors driven by promoters of varying strengths. In explant cultures, Bmp activation of Odd-skipped related 1 (Odd-1), the earliest known gene expressed in the intermediate mesoderm, is blocked by cyclohexamide, indicating that the activation of Odd-1 by Bmp signaling is translation-dependent. The data from this study are integrated with that of other studies to generate a model for the role of Bmp signaling in trunk mesodermal patterning in which low levels of Bmp activate intermediate mesoderm gene expression by inhibition of repressors present in medial mesoderm, whereas high levels of Bmp repress both medial and intermediate mesoderm gene expression and activate lateral plate genes.     

The nodal precursor acting via activin receptors induces mesoderm by maintaining a source of its convertases and BMP4

During early mouse development, the subtilisin-like proprotein convertases (SPC) Furin and PACE4 pattern the primitive ectoderm and visceral endoderm, presumably by activating the TGFss-related Nodal precursor. Here, mutation of the SPC motif provides direct evidence that Nodal processing is essential to specify anterior visceral endoderm and mesendoderm. Surprisingly, however, the Nodal precursor binds and activates activin receptors to maintain expression of Furin, PACE4, and Bmp4 in extraembryonic ectoderm at a distance from the Nodal source. In return, Bmp4 induces Wnt3, which amplifies Nodal expression in the epiblast and mediates induction of mesoderm. We conclude that uncleaved Nodal sustains the extraembryonic source of proprotein convertases and Bmp4 to amplify Nodal signaling in two nonredundant feedback loops with dual timescales and to localize primitive streak formation at the posterior pole. Based on mathematical modeling, we discuss how these sequential loops control cell fate.     

High mobility group box 1 levels in large vessel vasculitis are not associated with disease activity but are influenced by age and statins

IntroductionTakayasu arteritis (TA) and giant cell arteritis (GCA) are large vessel vasculitides (LVV) that usually present as granulomatous inflammation in arterial walls. High mobility group box 1 (HMGB1) is a nuclear protein that acts as an alarmin when released by dying or activated cells. This study aims to evaluate whether serum HMGB1 can be used as a biomarker in LVV.MethodsTwenty-nine consecutive TA patients with 29 healthy controls (HC) were evaluated in a cross-sectional study. Eighteen consecutive GCA patients with 16 HC were evaluated at the onset of disease and some of them during follow-up. Serum HMGB1 levels were measured by enzyme-linked immunosorbent assay.ResultsIn GCA patients at disease onset mean serum HMGB1 levels did not differ from HC (5.74 ± 4.19 ng/ml vs. 4.17 ± 3.14 ng/ml; p = 0.230). No differences in HMGB1 levels were found between GCA patients with and without polymyalgia rheumatica (p = 0.167), ischemic manifestations (p = 0.873), systemic manifestations (p = 0.474) or relapsing disease (p = 0.608). During follow-up, no significant fluctuations on serum HMGB1 levels were observed from baseline to 3 months (n = 13) (p = 0.075), 12 months (n = 6) (p = 0.093) and at the first relapse (n = 4) (p = 0.202). Serum HMGB1 levels did not differ between TA patients and HC [1.19 (0.45–2.10) ng/ml vs. 1.46 (0.89–3.34) ng/ml; p = 0.181] and no difference was found between TA patients with active disease and in remission [1.31 (0.63–2.16) ng/ml vs. 0.75 (0.39–2.05) ng/ml; p = 0.281]. HMGB1 levels were significantly lower in 16 TA patients on statins compared with 13 patients without statins [0.59 (0.29–1.46) ng/ml vs. 1.93 (0.88–3.34) ng/ml; p = 0.019]. Age was independently associated with higher HMGB1 levels regardless of LVV or control status.ConclusionsPatients with TA and GCA present similar serum HMGB1 levels compared with HC. Serum HMGB1 is not useful to discriminate between active disease and remission. In TA, use of statins was associated with lower HMGB1 levels. HMGB1 is not a biomarker for LVV.     

Bmp and nodal independently regulate lefty1 expression to maintain unilateral nodal activity during left-right axis specification in zebrafish

In vertebrates, left-right (LR) axis specification is determined by a ciliated structure in the posterior region of the embryo. Fluid flow in this ciliated structure is responsible for the induction of unilateral left-sided Nodal activity in the lateral plate mesoderm, which in turn regulates organ laterality. Bmp signalling activity has been implied in repressing Nodal expression on the right side, however its mechanism of action has been controversial. In a forward genetic screen for mutations that affect LR patterning, we identified the zebrafish linkspoot (lin) mutant, characterized by cardiac laterality and mild dorsoventral patterning defects. Mapping of the lin mutation revealed an inactivating missense mutation in the Bmp receptor 1aa (bmpr1aa) gene. Embryos with a mutation in lin/bmpr1aa and a novel mutation in its paralogue, bmpr1ab, displayed a variety of dorsoventral and LR patterning defects with increasing severity corresponding with a decrease in bmpr1a dosage. In Bmpr1a-deficient embryos we observed bilateral expression of the Nodal-related gene, spaw, coupled with reduced expression of the Nodal-antagonist lefty1 in the midline. Using genetic models to induce or repress Bmp activity in combination with Nodal inhibition or activation, we found that Bmp and Nodal regulate lefty1 expression in the midline independently of each other. Furthermore, we observed that the regulation of lefty1 by Bmp signalling is required for its observed downregulation of Nodal activity in the LPM providing a novel explanation for this phenomenon. From these results we propose a two-step model in which Bmp regulates LR patterning. Prior to the onset of nodal flow and Nodal activation, Bmp is required to induce lefty1 expression in the midline. When nodal flow has been established and Nodal activity is apparent, both Nodal and Bmp independently are required for lefty1 expression to assure unilateral Nodal activation and correct LR patterning.     

Thalidomide induces apoptosis during early mesodermal differentiation of human induced pluripotent stem cells

Thalidomide was once administered to pregnant women as a mild sedative; however, it was subsequently shown to be strongly teratogenic. Recently, there has been renewed interest in thalidomide because of its curative effects against intractable diseases. However, the teratogenicity of thalidomide is manifested in various ways and is still not fully understood. In the present study, we evaluated the effects of thalidomide on early mesodermal differentiation by examining the differentiation of human induced pluripotent stem cells (hiPSCs). The most common symptom of thalidomide teratogenicity is limb abnormality, which led us to hypothesize that thalidomide prevents early mesodermal differentiation. Therefore, mesodermal differentiation of hiPSCs was induced over a 6-d period. To induce early mesoderm differentiation, 1 d after seeding, the cells were incubated with the small molecule compound CHIR99021 for 3 d. Thalidomide exposure was initiated at the same time as CHIR99021 treatment. After 5 d of thalidomide exposure, the hiPSCs began expressing a mesodermal marker; however, the number of viable cells decreased significantly as compared to that of control cells. We observed that the proportion of apoptotic and dead cells increased on day 2; however, the proportion of dead cells on day 5 had decreased, suggesting that the cells were damaged by thalidomide during early mesodermal differentiation (days 0–2). Our findings may help elucidate the mechanism underlying thalidomide teratogenicity and bring us closer to the safe use of this drug.     

Efficacy and Day 7 Plasma Piperaquine Concentrations in African Children Treated for Uncomplicated Malaria with Dihydroartemisinin-Piperaquine

Background

One promising new Artemisinin-based combination therapies (ACTs) is dihydroartemisinin-piperaquine (DHA-PQ). However, the pharmacokinetics of piperaquine and the relationship between drug levels and clinical efficacy are incompletely characterized, particularly in children.

Methods

We performed a single-arm open-label trial in Bobo-Dioulasso, Burkina Faso. A total of 379 participants aged 6 months or more with uncomplicated falciparum malaria were enrolled. Each participant received daily dose of DHA-PQ for three days and followed for 42 days. Parasitological efficacy was analyzed, considering rates of recrudescence and overall recurrence. PK was an exploratory endpoint and a priori, no sample size had been determined. Day 7 capillary and venous plasma concentrations of piperaquine were measured in children aged 2–10 years.

Results

Of the 379 participants, 365 (96.3%) completed 42 days of follow-up. The median daily dose of PQ was 18.5 mg/kg [6.5–24]. Treatment with DHA-PQ was well tolerated with fever and parasitemia resolution within 48 hours in nearly all children. Recurrent malaria within 42 days of follow-up occurred in 31.3% (10/34) of children less than 2 years old, 16.0% (16/106) of those aged 2–5 years, 9.4% (15/160) of those aged 5–10 years, and none (0/68) of those over 10 years old. After genotyping, 3 of 41 recurrent episodes were recrudescence. An exploratory analysis shows that children with successful treatment outcomes had significantly higher median plasma concentrations of PQ compared to those with recurrent malaria within 42 days after therapy, considering either capillary samples (68 ng/ml [50–85] compared to 48 ng/ml [36–55], p<0.001) or venous samples (42 ng/ml [29–59] compared to 25 ng/ml [19–44], p<0.001).

Conclusion

DHA-PQ was effective for uncomplicated P. falciparum malaria treatment and offers an alternative to other ACTs. Recurrent malaria was mainly due to new infections after treatment and was correlated with low day 7 PQ concentration in the youngest patients.

Trial Registration

Controlled-Trials.com ISRCTN59761234     

Impact of D-Dimer for Prediction of Incident Occult Cancer in Patients with Unprovoked Venous Thromboembolism

BackgroundUnprovoked venous thromboembolism (VTE) is related to a higher incidence of occult cancer. D-dimer is clinically used for screening VTE, and has often been shown to be present in patients with malignancy. We explored the predictive value of D-dimer for detecting occult cancer in patients with unprovoked VTE.MethodsWe retrospectively examined data from 824 patients diagnosed with deep vein thrombosis or pulmonary thromboembolism. Of these, 169 (20.5%) patients diagnosed with unprovoked VTE were selected to participate in this study. D-dimer was categorized into three groups as: <2,000, 2,000–4,000, and >4,000 ng/ml. Cox regression analysis was employed to estimate the odds of occult cancer and metastatic state of cancer according to D-dimer categories.ResultsDuring a median 5.3 (interquartile range: 3.4–6.7) years of follow-up, 24 (14%) patients with unprovoked VTE were diagnosed with cancer. Of these patients, 16 (67%) were identified as having been diagnosed with metastatic cancer. Log transformed D-dimer levels were significantly higher in those with occult cancer as compared with patients without diagnosis of occult cancer (3.5±0.5 vs. 3.2±0.5, P-value = 0.009, respectively). D-dimer levels >4,000 ng/ml was independently associated with occult cancer (HR: 4.12, 95% CI: 1.54–11.04, P-value = 0.005) when compared with D-dimer levels <2,000 ng/ml, even after adjusting for age, gender, and type of VTE (e.g., deep vein thrombosis or pulmonary thromboembolism). D-dimer levels >4000 ng/ml were also associated with a higher likelihood of metastatic cancer (HR: 9.55, 95% CI: 2.46–37.17, P-value <0.001).ConclusionElevated D-dimer concentrations >4000 ng/ml are independently associated with the likelihood of occult cancer among patients with unprovoked VTE.     

Extraembryonic proteases regulate Nodal signalling during gastrulation

During gastrulation, a cascade of inductive tissue interactions converts pre-existing polarity in the mammalian embryo into antero-posterior pattern. This process is triggered by Nodal, a protein related to transforming growth factor-beta (TFG-beta) that is expressed in the epiblast and visceral endoderm, and its co-receptor Cripto, which is induced downstream of Nodal. Here we show that the proprotein convertases Spc1 and Spc4 (also known as Furin and Pace4, respectively) are expressed in adjacent extraembryonic ectoderm. They stimulate Nodal maturation after its secretion and are required in vivo for Nodal signalling. Embryo explants deprived of extraembryonic ectoderm phenocopy Spc1(-/-); Spc4(-/-) double mutants in that endogenous Nodal fails to induce Cripto. But recombinant mature Nodal, unlike uncleaved precursor, can efficiently rescue Cripto expression. Cripto is also expressed in explants treated with bone morphogenetic protein 4 (BMP4). This indicates that Nodal may induce Cripto through both a signalling pathway in the embryo and induction of Bmp4 in the extraembryonic ectoderm. A lack of Spc1 and Spc4 affects both pathways because these proteases also stimulate induction of Bmp4.     

Distinct requirements for extra-embryonic and embryonic bone morphogenetic protein 4 in the formation of the node and primitive streak and coordination of left-right asymmetry in the mouse

In the mouse and chick embryo, the node plays a central role in generating left-right (LR) positional information. Using several different strategies, we provide evidence in the mouse that bone morphogenetic protein 4 (Bmp4) is required independently in two different sites for node morphogenesis and for LR patterning. Bmp4 expression in the trophoblast-derived extra-embryonic ectoderm is essential for the normal formation of the node and primitive streak. However, tetraploid chimera analysis demonstrates that Bmp4 made in epiblast-derived tissues is required for robust LR patterning, even when normal node morphology is restored. In the absence of embryonic Bmp4, the expression of left-side determinants such as Nodal and Lefty2 is absent in the left lateral plate mesoderm (LPM). Noggin-mediated inhibition of Bmp activity in cultured wild-type embryos results in suppression of Nodal expression in the LPM. Thus, unlike previous models proposed in the chick embryo in which Bmp4 suppresses left-sided gene expression, our results suggest that Bmp acts as a positive facilitator of the left-sided molecular cascade and is required for Nodal induction and maintenance in the left LPM.     

Temporal and contextual orchestration of cardiac fate by WNT‐BMP synergy and threshold

Cardiomyogenic development proceeds with a cascade of intricate signalling events that operate in a temporo‐spatial fashion to specify cardiac cell fate during early embryogenesis. In fact, conflicting reports exist regarding the role of Wnt/β‐catenin signalling during cardiomyogenesis. Here, we describe a dose‐dependent and temporal effect of Wnt/β‐catenin signalling on in vitro cardiomyogenesis using embryonic stem cells (ESCs) as a model system. We could demonstrate that canonical Wnt activation during early stage of differentiation either through ligand or by GSK3β inhibition helped in maintaining Oct4 and Nanog expressions, and in parallel, it promoted mesoderm and endoderm inductions. In contrast, it led to attenuation in cardiomyogenesis that was reversed by moderate concentration of DKK1, but not soluble Fz8. However, higher DKK1 could also block cardiomyogenesis, suggesting thereby governance of a particular signalling threshold underlying this developmental event. Interestingly, Wnt signalling activation at early stage modulated BMP4 expression in a stage‐specific manner. Wnt activation, synchronized with BMP4 and brachyury up‐regulation at early stage, correlated well with mesoderm induction. Conversely, Wnt activation led to BMP4 and Wnt5a down‐regulation at late stage culminating in cardiomyogenic attenuation. Our findings suggested the existence of precise regulatory machinery with context‐dependent role of Wnt for fine tuning mesoderm induction and its derivatives, through establishment of Wnt gradient during ESCs’ differentiation. Moreover, contrary to mere activation/inhibition, a specific threshold of Wnt and BMP and their synergy seemed necessary for providing the guiding cues in orchestrating mesoderm induction and subsequent cardiomyogenesis.     

Age-Specific Cutoff Value for the Application of Percent Free Prostate-Specific Antigen (PSA) in Chinese Men with Serum PSA Levels of 4.0–10.0 ng/ml

Objective

The influence of age on the performance of percent free prostate-specific antigen (%fPSA) in diagnosing prostate cancer (PCa) in East Asians is controversial. We tested the diagnostic performance of %fPSA in a multi-center biopsy cohort in China and identified the proper age-specific cutoff values to avoid unnecessary biopsies.

Methods

Consecutive patients with a prostate-specific antigen (PSA) level of 4.0–10.0 ng/ml or 10.1–20.0 ng/ml who underwent transrectal ultrasound-guided or transperineal prostate biopsy were enrolled from 22 Chinese medical centers from Jan 1, 2010 to Dec 31, 2013. The diagnostic accuracy of PSA and %fPSA was determined using the area under the receiver operating characteristic (ROC) curve (AUC). Age-specific cutoff values were calculated using ROC curve analysis.

Results

The median %fPSA was much lower in younger patients compared with older patients with a PSA level of 4.0–10.0 ng/ml or 10.1–20.0 ng/ml. The AUC of %fPSA was higher than PSA only in older patients. In patients aged 50 to 59 years, %fPSA failed to improve the diagnosis compared with PSA in these two PSA ranges. Age-specific cutoff values were 24%, 27% and 32% for patients aged 60–69, 70–79 and ≥80 years, respectively, to reduce unnecessary biopsies in men with PSA levels of 4.0–10.0 ng/ml to detect 90% of all PCa.

Conclusions

The effectiveness of %fPSA is correlated with age in the Chinese population. Age-specific cutoff values would help avoid unnecessary biopsies in the Chinese population.     

Myostatin and Insulin-Like Growth Factor I: Potential Therapeutic Biomarkers for Pompe Disease

Objective

Myostatin and insulin-like growth factor 1 (IGF-1) are serum markers for muscle growth and regeneration. However, their value in the clinical monitoring of Pompe disease – a muscle glycogen storage disease – is not known. In order to evaluate their possible utility for disease monitoring, we assessed the levels of these serum markers in Pompe disease patients receiving enzyme replacement therapy (ERT).

Design

A case-control study that included 10 patients with Pompe disease and 10 gender- and age-matched non-Pompe disease control subjects was performed in a referral medical center. Average follow-up duration after ERT for Pompe disease patients was 11.7 months (range: 6–23 months). Measurements of serum myostatin, IGF-1, and creatine kinase levels were obtained, and examinations of muscle pathology were undertaken before and after ERT in the patient group.

Results

Compared with control subjects, Pompe disease patients prior to undergoing ERT had significantly lower serum IGF-1 levels (98.6 ng/ml vs. 307.9 ng/ml, p = 0.010) and lower myostatin levels that bordered on significance (1.38 ng/ml vs. 3.32 ng/ml, p = 0.075). After ERT, respective myostatin and IGF-1 levels in Pompe disease patients increased significantly by 129% (from 1.38 ng/ml to 3.16 ng/ml, p = 0.047) and 74% (from 98.6 ng/ml to 171.1 ng/ml, p = 0.013); these values fall within age-matched normal ranges. In contrast, myostatin and IGF-1 serum markers did not increase in age-matched controls. Follistatin, a control marker unrelated to muscle, increased in both Pompe disease patients and control subjects. At the same time, the percentage of muscle fibers containing intracytoplasmic vacuoles decreased from 80.0±26.4% to 31.6±45.3%.

Conclusion

The increase in myostatin and IGF-1 levels in Pompe disease patients may reflect muscle regeneration after ERT. The role of these molecules as potential therapeutic biomarkers in Pompe disease and other neuromuscular diseases warrants further study.