Flow-induced protein kinase A–CREB pathway acts via BMP signaling to promote HSC emergence

Fluid shear stress promotes the emergence of hematopoietic stem cells (HSCs) in the aorta–gonad–mesonephros (AGM) of the developing mouse embryo. We determined that the AGM is enriched for expression of targets of protein kinase A (PKA)–cAMP response element-binding protein (CREB), a pathway activated by fluid shear stress. By analyzing CREB genomic occupancy from chromatin-immunoprecipitation sequencing (ChIP-seq) data, we identified the bone morphogenetic protein (BMP) pathway as a potential regulator of CREB. By chemical modulation of the PKA–CREB and BMP pathways in isolated AGM VE-cadherin⁺ cells from mid-gestation embryos, we demonstrate that PKA–CREB regulates hematopoietic engraftment and clonogenicity of hematopoietic progenitors, and is dependent on secreted BMP ligands through the type I BMP receptor. Finally, we observed blunting of this signaling axis using Ncx1-null embryos, which lack a heartbeat and intravascular flow. Collectively, we have identified a novel PKA–CREB–BMP signaling pathway downstream of shear stress that regulates HSC emergence in the AGM via the endothelial-to-hematopoietic transition.Embryonic hematopoietic development in the mouse proceeds through defined stages. The first hematopoietic cells of the erythroid lineage develop in the extraembryonic yolk sac at embryonic day 7.5 (E7.5; Moore and Metcalf, 1970). At E9, hematopoietic stem cell (HSC) activity from the yolk sac and paraaortic splanchnopleura (PSp) can be detected when transplanted into neonatal mice (Yoder et al., 1997; Arora et al., 2014). HSCs that engraft lethally irradiated adult recipients emerge in the aorta–gonad–mesonephros (AGM) region at E10-11 (Medvinsky and Dzierzak, 1996; North et al., 2002). These HSCs later colonize additional organs required for adult hematopoiesis and possess the ability to reconstitute multiple hematopoietic lineages.The development of the murine circulatory system at E8.5 coincides with the development of more definitive hematopoietic compartments, including HSCs and the lymphoid lineage. Accordingly, recent studies have linked biomechanical forces, such as blood flow–induced shear stress, to hematopoietic development (Adamo et al., 2009; North et al., 2009). In these studies, genetic mutants lacking intravascular circulation were used to demonstrate the reduction in hematopoietic emergence in the AGM and nitric oxide signaling was implicated in blood flow–dependent AGM hematopoiesis (Adamo et al., 2009; North et al., 2009; Wang et al., 2011). Although chimeric analysis demonstrated a cell autonomous requirement for nitric oxide signaling in zebrafish (North et al., 2009), whether this pathway directly promotes hematopoiesis remains an open question due to the vasodilatory effect of nitric oxide donors and their effects on smooth muscle. Apart from these observations, signaling pathways responsible for flow-dependent hematopoietic induction have not been characterized.Activation of protein kinase A (PKA) and its downstream target cAMP response element-binding protein (CREB) by exogenous shear stress has been observed in diverse cell types, including chondrocytes and osteocytes (Cherian et al., 2003; Ogawa et al., 2014). The classic mechanism of PKA activation involves the binding of a ligand to a G protein–coupled receptor and activation of adenylyl cyclase, which converts ATP into the second messenger cyclic AMP (cAMP). The binding of cAMP to PKA releases catalytic subunits that phosphorylate CREB in the nucleus. In differentiating mouse embryonic stem cells (mESCs), PKA–CREB has been linked to endothelial and hematopoietic differentiation via binding of CREB to the Etv2 promoter, which up-regulates pro-hematopoietic factors such as Gata2 and Scl/Tal1 (Yamamizu et al., 2012). Moreover, the PKA–CREB signaling pathway has been explored in the context of the prostaglandin E₂ signaling pathway in zebrafish, where it promotes AGM hematopoiesis via activation of the Wnt pathway (Goessling et al., 2009). However, whether this pathway is conserved in the mouse is unclear, especially given conflicting reports on Wnt signaling in AGM hematopoiesis (Ruiz-Herguido et al., 2012; Chanda et al., 2013). Prostaglandin E₂ also directly activates several pathways including PI3K–AKT and ERK–MAPK, which makes it difficult to conclude that PKA–CREB is the sole mediator of the pro-hematopoietic effects of this molecule (Alfranca et al., 2006). Given the shear-responsiveness of the PKA–CREB pathway and its implication in early embryonic hematopoiesis in other species, we investigated the possible role of shear stress–activated PKA–CREB signaling during AGM hematopoiesis in the mouse.We first verified that this pathway is activated by shear stress in VE-cadherin⁺ endothelial cells and present in the murine AGM, specifically in the cells lining the dorsal aorta. We then conducted a bioinformatics-based screen using microarray data on CREB overexpression and CREB chromatin immunoprecipitation-sequencing (ChIP-Seq) data using data available at Encyclopedia of DNA Elements (ENCODE) and elsewhere to identify regulators of CREB function in hematopoietic cells (Esparza et al., 2008; Jolma et al., 2010; Pencovich et al., 2011; Raney et al., 2011; Trompouki et al., 2011; Martens et al., 2012). Using insight gained from bioinformatics, we discover that the bone morphogenetic protein (BMP) signaling pathway acts downstream of PKA–CREB signaling in regulating AGM hematopoiesis. Finally, we show that this is a blood flow–dependent pathway by demonstrating the abrogation of PKA–CREB–BMP signaling axis in Ncx1-null embryos, which lack a heartbeat and intravascular flow. Our data thus document a blood-flow dependent pathway regulating hematopoietic development.     

Early Childhood Gut Microbiomes Show Strong Geographic Differences Among Subjects at High Risk for Type 1 Diabetes

OBJECTIVE

Gut microbiome dysbiosis is associated with numerous diseases, including type 1 diabetes. This pilot study determines how geographical location affects the microbiome of infants at high risk for type 1 diabetes in a population of homogenous HLA class II genotypes.

RESEARCH DESIGN AND METHODS

High-throughput 16S rRNA sequencing was performed on stool samples collected from 90 high-risk, nonautoimmune infants participating in The Environmental Determinants of Diabetes in the Young (TEDDY) study in the U.S., Germany, Sweden, and Finland.

RESULTS

Study site–specific patterns of gut colonization share characteristics across continents. Finland and Colorado have a significantly lower bacterial diversity, while Sweden and Washington state are dominated by Bifidobacterium in early life. Bacterial community diversity over time is significantly different by geographical location.

CONCLUSIONS

The microbiome of high-risk infants is associated with geographical location. Future studies aiming to identify the microbiome disease phenotype need to carefully consider the geographical origin of subjects.     

Mild Gestational Diabetes Mellitus and Long-Term Child Health

OBJECTIVETo evaluate whether treatment of mild gestational diabetes mellitus (GDM) confers sustained offspring health benefits, including a lower frequency of obesity.RESULTSFive hundred of 905 eligible offspring (55%) were enrolled. Maternal baseline characteristics were similar between the follow-up treated and untreated groups. The frequencies of BMI ≥95th (20.8% and 22.9%) and 85th (32.6% and 38.6%) percentiles were not significantly different in treated versus untreated offspring (P = 0.69 and P = 0.26). No associations were observed for BMI z score, log waist circumference, log triglycerides, HDL cholesterol, blood pressure, or log HOMA-estimated insulin resistance (HOMA-IR). The effect of treatment was different by sex for fasting glucose and log HOMA-IR (P for interaction = 0.002 and 0.02, respectively) but not by age-group (5–6 and 7–10 years) for any outcomes. Female offspring of treated women had significantly lower fasting glucose levels.CONCLUSIONSAlthough treatment for mild GDM has been associated with neonatal benefits, no reduction in childhood obesity or metabolic dysfunction in the offspring of treated women was found. However, only female offspring of women treated for mild GDM had lower fasting glucose.     

PCSK7 Genotype Modifies Effect of a Weight-Loss Diet on 2-Year Changes of Insulin Resistance: The POUNDS LOST Trial

OBJECTIVEA common variant rs236918 in the PCSK7 gene has the strongest association with iron homeostasis and is related to insulin resistance. Dietary carbohydrate (CHO) modulates the genetic effect on insulin resistance. We examined whether 2-year weight-loss diets modify the effect of PCSK7 genetic variants on changes in fasting insulin levels and insulin resistance in a randomized, controlled trial.RESULTSDuring the 6-month weight-loss phase, the PCSK7 rs236918 G allele was significantly associated with greater decreases in fasting insulin levels in the high–dietary CHO group (P for interaction = 0.04), while the interaction for changes in HOMA-insulin resistance (HOMA-IR) (P for interaction = 0.06) did not reach significant levels in white subjects. The G allele was significantly associated with a greater decrease in fasting insulin levels and HOMA-IR in response to high dietary CHO levels (P = 0.02 and P = 0.03, respectively). From 6 months to 2 years (weight-regain phase), the interactions became attenuated due to the regaining of weight (P for interactions = 0.08 and 0.06, respectively). In addition, we observed similar and even stronger results in the whole-study samples from the trial.CONCLUSIONSOur data suggest that PCSK7 genotypes may interact with dietary CHO intake on changes in insulin sensitivity in the white Americans.     

Biomaterials for promoting periodontal regeneration in human intrabony defects: a systematic review

Intrabony periodontal defects are a frequent complication of periodontitis and, if left untreated, may negatively affect long‐term tooth prognosis. The optimal outcome of treatment in intrabony defects is considered to be the absence of bleeding on probing, the presence of shallow pockets associated with periodontal regeneration (i.e. formation of new root cementum with functionally orientated inserting periodontal ligament fibers connected to new alveolar bone) and no soft‐tissue recession. A plethora of different surgical techniques, often including implantation of various types of bone graft and/or bone substitutes, root surface demineralization, guided tissue regeneration, growth and differentiation factors, enamel matrix proteins or various combinations thereof, have been employed to achieve periodontal regeneration. Despite positive observations in animal models and successful outcomes reported for many of the available regenerative techniques and materials in patients, including histologic reports, robust information on the degree to which reported clinical improvements reflect true periodontal regeneration does not exist. Thus, the aim of this review was to summarize, in a systematic manner, the available histologic evidence on the effect of reconstructive periodontal surgery using various types of biomaterials to enhance periodontal wound healing/regeneration in human intrabony defects. In addition, the inherent problems associated with performing human histologic studies and in interpreting the results, as well as certain ethical considerations, are discussed. The results of the present systematic review indicate that periodontal regeneration in human intrabony defects can be achieved to a variable extent using a range of methods and materials. Periodontal regeneration has been observed following the use of a variety of bone grafts and substitutes, guided tissue regeneration, biological factors and combinations thereof. Combination approaches appear to provide the best outcomes, whilst implantation of alloplastic material alone demonstrated limited, to no, periodontal regeneration.     

Nontraumatic rhabdomyolysis with short‐term alcohol intoxication – a case report

Alcohol‐induced rhabdomyolysis is a potentially life‐threatening condition due to the probability of progression to acute renal injury. Patients admitted to emergency department with acute alcohol intoxication should always undergo blood and urine tests for early recognition and treatment of rhabdomyolysis.