Embryonic Blood Research Articles

P-Selectin Expression and Platelet Function Are Developmentally Regulated

Platelets have numerous well-defined roles during adult hemostasis, however little is known about whether these functions are developmentally regulated. In the adult, P-selectin is constitutively expressed in megakaryocytes and localized to the membrane of alpha granules. Activation of platelets results in granule secretion and P-selectin surface exposure allowing for recruitment of leukocytes to sites of vascular injury and the formation of leukocyte-platelet aggregates. Here, we examined the expression and function of P-selectin in platelets during murine embryogenesis. Platelets were isolated from timed pregnant outbred-ICR mice at embryonic day 12.5 (E12.5), E15.5, postnatal day 1 (PN1), PN7, PN21, and were assayed for thrombin induced integrin activation and P-selectin translocation. While no significant differences were seen in the ability of embryonic platelets to be activated by thrombin, P-selectin surface expression after thrombin stimulation was significantly reduced in embryonic compared to adult platelets (E15.5 p=.0006, E12.5 p=.0004, PN1 p=.01, n=3, unpaired t-test). Surprisingly, P-selectin expression began to appear on the surface of activated platelets only after birth, approaching 50% and 75% of maternal expression at PN7 and PN21, respectively. No significant difference was seen in VEGF secretion after thrombin activation of E12.5, E15.5 and adult platelets (n=5), suggesting that the lack of P-selectin surface expression was not due to a loss of alpha granule secretion. To further characterize expression of P-selectin, real-time qPCR was performed on mRNA harvested from platelets at E12.5, E15.5, PN1 and adult stages. P-selectin transcript levels were significantly lower in embryonic and PN1 platelets, suggesting that P-selectin expression is transcriptionally regulated. Consistent with this concept, Imagestream analysis indicates that P-selectin protein is absent in megakaryocytes derived from the fetal liver. P-selectin expression on platelets is an important mediator of platelet-leukocyte interactions; therefore, we assayed the ability of platelets to bind to leukocytes in embryonic whole blood. Preliminary data suggest that embryonic platelets do not form leukocyte-platelet aggregates upon ex vivo stimulation with AYPGKF, a Par4 agonist. Taken together, our data indicate that P-selectin expression is developmentally regulated in primary mouse platelets. The absence of P-selectin expression in embryonic platelets correlates with an impaired ability to interact with leukocytes. DisclosuresNo relevant conflicts of interest to declare.

EMP Emergence from Hemogenic Endothelium in the Mammalian Yolk Sac Is Independent of Flow and Arterial Identity, but Is Regulated By Canonical Wnt Signaling

Hematopoietic stem cells (HSCs) emerge from arterial vessels of the mouse embryo through a Runx1-dependent process of endothelial-to-hematopoietic transition beginning at embryonic day 10.5 (E10.5). This arterial endothelial-to-hematopoietic transition is known to require embryonic circulation as well as beta-catenin signaling within the endothelial precursor, known as hemogenic endothelium. However, embryonic survival is dependent on the earlier emergence of a robust wave of yolk sac-derived definitive erythro-myeloid progenitors (EMPs), which have unilineage as well as multilineage potential, including high-proliferative potential colony forming cell (HPP-CFC) potential (Palis et al., PNAS, 2001). Like HSCs, EMP specification is dependent on Runx1, suggesting that they also emerge from a hemogenic endothelial precursor. However, the spatial localization of EMPs in the yolk sac and the mechanisms governing their emergence are not well understood. To visualize emerging EMPs in the yolk sac, we performed whole-mount immunohistochemistry for Kit, which we have demonstrated to contain nearly all EMP potential at E9.5. Kit+ cells coexpress Runx1 and CD31, and a subset have a polygonal/endothelial morphology, appear integrated into the vascular network, and are associated with rounded Kit+ cells in clusters, features consistent with an endothelial-to-hematopoietic transition. However, unlike HSCs, which emerge from major embryonic arteries, clusters of EMPs are located in larger and smaller caliber vessels in branches of both the arterial and venous vasculature, which is spatially organized within the yolk sac. To determine if EMP emergence from the vasculature is dependent on embryonic blood flow, which is required for HSC emergence, we analyzed the yolk sacs of Ncx1-null embryos, which fail to initiate heart contractions and subsequently lack embryonic circulation. Despite the lack of vascular remodeling in these circulation-deficient yolk sacs, Ncx1-null EMPs displayed normal cluster morphology, including both polygonal and rounded kit+ cells, indicating the endothelial-to-hematopoietic transition can occur without the mechanical influence of blood flow.To address whether EMP formation is responsive to other developmental signals, we utilized a yolk sac explant culture to evaluate the propensity of hemogenic endothelial cells to commit to hematopoiesis ex vivo. Culture of intact E8.5 yolk sacs for 48 hours with the canonical Wnt ligand Wnt3a resulted in an increase in both day 6-7 colony forming cells and day 13-14 HPP-CFC when compared with control yolk sacs. Preliminary treatment with Dkk1 alone did not adversely affect colony-forming activity when compared with untreated yolk sacs, and potentiation of endogenous canonical Wnt signaling with HLY78 did not augment colony production, suggesting that low levels of endogenous Wnt ligands are produced ex vivo. Despite the positive effect of Wnt3a on whole yolk sacs, treatment of isolated E9.5 Kit+CD41+CD16/32+ EMPs with Wnt3a did not increase colony formation, suggesting that Wnt signaling augments progenitor production at, or prior to, the hemogenic endothelial stage. Preliminary results utilizing imaging flow cytometry demonstrated increased beta-catenin intensity within the nuclear region in E9.5 Kit+VE-Cadherin/AA4.1+ endothelium following Wnt3a treatment, suggesting that hemogenic endothelial cells in the yolk sac are Wnt responsive. Consistent with this finding, in vitro Wnt3a treatment on primary E8.5-9.5 VE-Cadherin/AA4.1+CD16/32- endothelial cells resulted in upregulation of the beta-catenin target gene Axin2. To address whether Wnt signaling is endogenously active in vivo, we analyzed E8.5-E9 yolk sacs of BAT-gal reporter mice (Maretto et al., PNAS, 2003), and visualized a subset of cells with endothelial morphology expressing LacZ. Taken together, these data support the concept that EMPs, like HSCs, emerge from hemogenic endothelium. Surprisingly, this earlier endothelial-to-hematopoietic transition in the yolk sac is not dependent on blood flow or an arterial identity. However, similar to HSC emergence, EMP emergence from hemogenic endothelium is positively regulated by canonical Wnt signaling. These data highlight the presence of spatially, temporally, and functionally heterogeneous populations of hemogenic endothelium in the mammalian conceptus. DisclosuresNo relevant conflicts of interest to declare.

Embryonic blood-cerebrospinal fluid barrier formation and function.

During embryonic development and adult life, brain cavities and ventricles are filled with cerebrospinal fluid (CSF). CSF has attracted interest as an active signaling medium that regulates brain development, homeostasis and disease. CSF is a complex protein-rich fluid containing growth factors and signaling molecules that regulate multiple cell functions in the central nervous system (CNS). The composition and substance concentrations of CSF are tightly controlled. In recent years, it has been demonstrated that embryonic CSF (eCSF) has a key function as a fluid pathway for delivering diffusible signals to the developing brain, thus contributing to the proliferation, differentiation and survival of neural progenitor cells, and to the expansion and patterning of the brain. From fetal stages through to adult life, CSF is primarily produced by the choroid plexus. The development and functional activities of the choroid plexus and other blood–brain barrier (BBB) systems in adults and fetuses have been extensively analyzed. However, eCSF production and control of its homeostasis in embryos, from the closure of the anterior neuropore when the brain cavities become physiologically sealed, to the formation of the functional fetal choroid plexus, has not been studied in as much depth and remains open to debate. This review brings together the existing literature, some of which is based on experiments conducted by our research group, concerning the formation and function of a temporary embryonic blood–CSF barrier in the context of the crucial roles played by the molecules in eCSF.

Imaging of cardiovascular development in mammalian embryos using optical coherence tomography.

The cardiovascular system is the first functional organ system to develop within the mammalian embryo. During the early stages of cardiovascular development, the heart and blood vessels undergo rapid growth and remodeling required for embryo viability, proper morphogenesis, and the function of all organ systems. Live imaging of these dynamic events in early mouse embryos is critical to understanding when and how these morphological changes occur during normal development and how mutations and pharmacological agents affect cardiovascular structure and function in vivo. The use of optical coherence tomography (OCT) allows for rapid, three-dimensional structural and functional imaging of mouse embryos at cellular resolution without the aid of contrast agents. In this chapter, we will describe how OCT can be used to assess the morphology of vessels and the heart, dynamic analysis of cardiac function, and hemodynamics within extraembryonic and embryonic blood vessels.

PS216. Zizimin1 Overexpression Impairs Vascular Morphogenesis

Objectives: The Rho subfamily of small GTPases, including RhoA, Rac1, and Cdc42, regulates diverse cellular functions, including polarity, migration, and actin-based cytoskeleton dynamics. Our prior studies established an essential role for Cdc42 in vascular network assembly, demonstrating that the genetic inactivation of Cdc42 yields defective vascular morphogenesis due to impaired migration of endothelial precursor cells. We have further shown that protein kinase Ciota and glycogen synthase kinase-3b are downstream effectors of Cdc42 and are involved in mediating vascular network assembly. However, the guanine nucleotide exchange factors (GEFs) that activate Cdc42, remain unknown. Methods: We performed affinity pulldown assays using a nucleotide-free Cdc42 G15A mutant that specifically binds to Cdc42 GEFs. Mass spectrometric analysis identified Zizimin1, an upstream regulatory protein, as a candidate Cdc42 GEF. Results: During vasculogenesis in embryoid bodies (EBs) differentiated from embryonic stem cells, Zizimin1 is highly expressed in aggregated endothelial cell precursors before vascular network formation. Surprisingly, stable overexpression of Zizimin1 in EBs resulted in the inhibition of blood vessel formation compared with control, evidenced by immunohistochemistry demonstrating loss of vascular network development. Affinity pulldown assay helped to elucidate that overexpression of Zizimin1 increases Cdc42 activity; however, the activation of Rac1 and RhoA is significantly inhibited. Conclusions: Because Rac1 and RhoA signaling has been reported to play an essential role in embryonic blood vessel formation, our results suggest that the interplay betweenRhoGTPases guides vascular network assembly during development. Furthermore, these findings provide novel insights into the mechanisms of embryonic vasculogenesis and also important new information for the design of potential proangiogenic and/or antiangiogenic therapies.

Cell specificity and molecular mechanism of antibacterial and antitumor activities of carboxyl-terminal RWL-tagged antimicrobial peptides.

Antimicrobial peptides (AMPs) constitute a diverse class of naturally occurring or synthetic antimicrobial molecules that have potential for use in the treatment of drug-resistant infections. Several undesirable properties of AMPs, however, may ultimately hinder their development as antimicrobial agents. Thus, new synthetic strategies, including primarily the de novo design of AMPs, urgently need to be developed. In this study, a series of peptides, H-(RWL) n (n=1, 2, 3, 4, or 5), were designed. H represents GLRPKYS from the C-terminal sequence of AvBD-4. Our results showed that these RWL-tagged peptides can kill not only bacteria but also human hepatocellular carcinoma HepG2 cells. However, the peptide tagged with two repeats of RWL (GW13) showed less affinity to human embryonic lung fibroblast MRC-5 cells or human red blood cells (hRBCs) than HepG2 cells. These results demonstrated that GW13, with high amphiphilicity, exerted great selectivity toward bacteria and cancer cells, sparing host mammalian cells. The mechanism of action against bacteria was elucidated through combined studies of scanning electron microscopy (SEM) and fluorescence assays, showing that the peptide possessed membrane-lytic activities against microbial cells. The fluorescence assays illustrated that GW13 induced apoptosis in HepG2 cells. The cell morphology of HepG2 cells, observed by SEM, further illustrated that GW13 causes cell death by damaging the cell membrane. Our results indicate that GW13 has considerable potential for future development as an antimicrobial and antitumor agent.

Abstract 513: Endothelial Barrier Function is Maintained by the RNA-Binding Protein Quaking

Endothelial barrier function plays a major role in the onset of atherosclerosis. This barrier is maintained largely by adherens junctions. Remarkably, little is known about their regulation at the post–transcriptional level. We found that the RNA-binding protein Quaking (QKI), known for its function in embryonic blood vessel formation, is highly expressed in quiescent endothelial cells (EC) in vivo. In vitro, EC displayed increased levels of QKI when cultured under laminar, atheroprotective flow. Using KLF2 overexpression and a human QKI promoter reporter gene, we found that KLF2 mediates this increase in QKI expression. Subsequently, we aimed to investigate the role of QKI in EC vascular integrity. Interestingly, the mRNA of VE-cadherin, the prime adhesion protein in EC adherens junctions, contains a conserved QKI-binding site. We identified that the targeted reduction of QKI results in a reduction of VE-cadherin expression and organization at the cell periphery. These studies revealed a direct role for QKI in regulating VE-cadherin mRNA biology, as RNA-immunoprecipitation and luciferase-reporter assays revealed that QKI can directly bind to the VE-cadherin mRNA and induce transcript translation (4 fold ± 0.4; p<0.01), respectively. This effect was perturbed when the QKI-binding site was mutated. These results suggest that QKI acts to enhance barrier function. Overexpression of QKI markedly increased the capacity to form a high resistance endothelial monolayer (1.3 fold ± 0.96), while silencing of QKI markedly impaired EC barrier function (0.65 fold ± 0.13; p<0.05). To validate in vivo, we measured Bradykinin-induced vascular leakage in QKI viable mice (QKIv), which express decreased levels of the QKI protein. Indeed, QKIv mice displayed a 20% (p<0.05) increase in extravascular accumulation of Evans blue-labeled albumin compared to WT littermates. In conclusion, we show that QKI functions as a critical regulator of VE-cadherin, and the modulation of QKI expression affects endothelial monolayer integrity. These studies provide novel insight into the importance of post-transcriptional regulation on endothelial barrier function, and may have wide ranging implications for the preservation of vascular integrity in disease.

Probabilistic PCA of censored data: accounting for uncertainties in the visualization of high-throughput single-cell qPCR data

Motivation: High-throughput single-cell quantitative real-time polymerase chain reaction (qPCR) is a promising technique allowing for new insights in complex cellular processes. However, the PCR reaction can be detected only up to a certain detection limit, whereas failed reactions could be due to low or absent expression, and the true expression level is unknown. Because this censoring can occur for high proportions of the data, it is one of the main challenges when dealing with single-cell qPCR data. Principal component analysis (PCA) is an important tool for visualizing the structure of high-dimensional data as well as for identifying subpopulations of cells. However, to date it is not clear how to perform a PCA of censored data. We present a probabilistic approach that accounts for the censoring and evaluate it for two typical datasets containing single-cell qPCR data.Results: We use the Gaussian process latent variable model framework to account for censoring by introducing an appropriate noise model and allowing a different kernel for each dimension. We evaluate this new approach for two typical qPCR datasets (of mouse embryonic stem cells and blood stem/progenitor cells, respectively) by performing linear and non-linear probabilistic PCA. Taking the censoring into account results in a 2D representation of the data, which better reflects its known structure: in both datasets, our new approach results in a better separation of known cell types and is able to reveal subpopulations in one dataset that could not be resolved using standard PCA.Availability and implementation: The implementation was based on the existing Gaussian process latent variable model toolbox (https://github.com/SheffieldML/GPmat); extensions for noise models and kernels accounting for censoring are available at http://icb.helmholtz-muenchen.de/censgplvm.Contact: fbuettner.phys@gmail.comSupplementary information: Supplementary data are available at Bioinformatics online.

Comparative Anatomy of the Carotid-Basilar Arterial Trunk and Hindbrain Penetrating Arteries in Vertebrates

Despite a long history of anatomical studies, the vascular organization of the brainstem is poorly known in most vertebrates. This review examines results from the past 150 years and compares them with a series of new vascular microdissections to provide an overall assessment of vertebrate hindbrain vasculature. The gross anatomy and embryology of cyclostomes, elasmobranchs, actinopterygians and tetrapods reveal a conserved suite of hindbrain vascular features including the: 1) origin and branching of cerebral carotids; 2) ontogenetic origins of the basilar artery from paired precursors, often followed by midline fusion to form an unpaired basilar; and 3) presence of a quasi-segmental series of transverse basilar artery branches that extend circumferentially around the brainstem giving off penetrating vessels. Phylogenetic analysis suggests that paired basilars are primitive for jawed vertebrates, with unpaired basilars arising independently in chondrichthyes, teleosts and tetrapods. Teleosts present a major exception to the general pattern, as penetrating arteries are restricted to a series of midline segmental basilar arterial stems that branch only within the neuroepithelium. This is clearly derived relative to all other vertebrates. Given the dissimilar body plans/lifestyles of groups served by the general brainstem vascular design, the conservation of adult hindbrain macrovasculature likely results from the similarity of embryonic cerebral blood supply across groups, rather than from convergence towards similar neurovascular functional designs. With the high degree of conservation in vertebrate brainstem neuronal anatomy, vascular patterning cues provided by the neuroepithelium practically assure that homologous brain regions are supplied by roughly similar arteries across taxa.

A Review of Strategies for Producing Chimeric Birds

Avian chimeras have been shown to be very useful tools for studying embryonic development. Blastodermal cells form stage X embryos and primordial germ cells (PGCs) are extensively used in chimera production. Intraspecies and interspecies germline chimeras have been successfully produced by transferring blastodermal cells or PGCs into recipient blastoderm at stage X or into the early embryonic blood. The long-term culture system established using pluripotent cells from birds provides an alternative method for preserving the endangered species source and facilitates the manipulation of avian transgenesis. A high rate of donor cell-derived progeny production has always been pursued by germline chimera studies. However, the rate of progeny production was unstable. Different methods have been used in an attempt to improve the efficiency of germline chimera production. Several methods have been shown to greatly enhance the efficiency of donor cell-derived progeny production. In this review, we focus on the strategies used for germline chimera production in birds and discuss the factors and measures affecting the efficiency of obtaining donor cell-derived progeny.

Platelet-derived Growth Factor-C (PDGF-C) Induces Anti-apoptotic Effects on Macrophages through Akt and Bad Phosphorylation

PDGF-C, which is abundant in the malignant breast tumor microenvironment, plays an important role in cell growth and survival. Because tumor-associated macrophages (TAMs) contribute to cancer malignancy, macrophage survival mechanisms are an attractive area of research into controlling tumor progression. In this study, we investigated PDGF-C-mediated signaling pathways involved in anti-apoptotic effects in macrophages. We found that the human malignant breast cancer cell line MDA-MB-231 produced high quantities of PDGF-C, whereas benign MCF-7 cells did not. Recombinant PDGF-C induced PDGF receptor α chain phosphorylation, followed by Akt and Bad phosphorylation in THP-1-derived macrophages. MDA-MB-231 culture supernatants also activated macrophage PDGF-Rα. PDGF-C prevented staurosporine-induced macrophage apoptosis by inhibiting the activation of caspase-3, -7, -8, and -9 and cleavage of poly(ADP-ribose) polymerase. Finally, TAMs isolated from the PDGF-C knockdown murine breast cancer cell line 4T1 and PDGF-C knockdown MDA-MB-231-derived tumor mass showed higher rates of apoptosis than the respective WT controls. Collectively, our results suggest that tumor cell-derived PDGF-C enhances TAM survival, promoting tumor malignancy.

Heart field origin of great vessel precursors relies on nkx2.5-mediated vasculogenesis

The pharyngeal arch arteries (PAAs) are transient embryonic blood vessels that make indispensable contributions to the carotid arteries and great vessels of the heart, including the aorta and pulmonary artery1, 2. During embryogenesis, the PAAs appear in a craniocaudal sequence to connect pre-existing segments of the primitive circulation after de novo vasculogenic assembly from angioblast precursors3, 4. Despite the unique spatiotemporal characteristics of PAA development, the embryonic origins of PAA angioblasts and the genetic factors regulating their emergence remain unknown. Here, we identify the embryonic source of PAA endothelium as nkx2.5+ progenitors in lateral plate mesoderm long considered to adopt cell fates within the heart exclusively5, 6. Further, we report that PAA endothelial differentiation relies on Nkx2.5, a canonical cardiac transcription factor not previously implicated in blood vessel formation. Together, these studies reveal the heart field origin of PAA endothelium and attribute a novel vasculogenic function to the cardiac transcription factor nkx2.5 during great vessel precursor development.

Pulse propagation by a capacitive mechanism drives embryonic blood flow

Pulsatile flow is a universal feature of the blood circulatory system in vertebrates and can lead to diseases when abnormal. In the embryo, blood flow forces stimulate vessel remodeling and stem cell proliferation. At these early stages, when vessels lack muscle cells, the heart is valveless and the Reynolds number (Re) is low, few details are available regarding the mechanisms controlling pulses propagation in the developing vascular network. Making use of the recent advances in optical-tweezing flow probing approaches, fast imaging and elastic-network viscous flow modeling, we investigated the blood-flow mechanics in the zebrafish main artery and show how it modifies the heart pumping input to the network. The movement of blood cells in the embryonic artery suggests that elasticity of the network is an essential factor mediating the flow. Based on these observations, we propose a model for embryonic blood flow where arteries act like a capacitor in a way that reduces heart effort. These results demonstrate that biomechanics is key in controlling early flow propagation and argue that intravascular elasticity has a role in determining embryonic vascular function.

The Declaration of Independence of the Neurovascular Intimacy

In this issue of Neuron, Oh and Gu (2013) present a model in which intimately related embryonic nerves and blood vessels are patterned independently in response to different guidance cues from a central organizer: the whisker.

MicroRNA-200C and -150 play an important role in endothelial cell differentiation and vasculogenesis by targeting transcription repressor ZEB1

To investigate the role of miRNA in controlling human embryonic stem (hES) cell differentiation toward the endothelial lineage and chick embryonic blood vessel formation, undifferentiated hES cells were first cultured on Matrigel-coated flasks and in endothelial cell growth medium-2 (EGM-2) to initiate endothelial cell (EC) differentiation. CD146(+) cells were isolated from differentiating hES cells and expanded in vitro. The in vitro expanded CD146(+) cells were positive for EC markers, capable of Ac-LDL uptake, lectin binding, and the formation of vascular structures in vitro and in vivo. miRNA gain/loss-of-function analyses revealed that miR-150 and miR-200c were crucial in EC differentiation. Transcriptional repressor zinc finger E-box-binding homeobox 1 (ZEB1) was identified as the communal target gene of miRNA-200C and -150, and inhibition of ZEB1 was required for miRNA-200C or -150 mediated EC gene expressions. Moreover, we demonstrated that ZEB1 could transcriptionally repress EC gene expression through direct binding to promoters of EC genes. Finally, we also demonstrated that miRNA-200c and -150 played an important role in chick embryonic blood vessel formation by in vivo inhibition of miRNA-200C or -150 in developing chick embryos, and blocking ZEB1 signaling in CD146-positive cells could rescue the inhibitory effects of miR-200c inhibiton in in vivo vasculogenesis. Our findings revealed that miR-150 and miR-200c play an important role in human endothelial lineage specification and chick embryonic vasculogenesis by targeting ZEB1.

Uteroplacental Circulation and Fetal Vascular Function and Development

Although blood flow in the placental vasculature is governed by the same physiological forces of shear, pressure and resistance as in other organs, it is also uniquely specialized on the maternal and fetal sides. At the materno-fetal interface, the independent uteroplacental and umbilicoplacental circulations must coordinate sufficiently to supply the fetus with the nutrients and substrates it needs to grow and develop. Uterine arterial flow must increase dramatically to accommodate the growing fetus. Recent evidence delineates the hormonal and endothelial mechanisms by which maternal vessels dilate and remodel during pregnancy. The umbilical circulation is established de novo during embryonic development but blood does not flow through the placenta until late in the first trimester. The umbilical circulation operates in the interest of maintaining fetal oxygenation over the course of pregnancy, and is affected differently by mechanical and chemical regulators of vascular tone compared to other organs. The processes that match placental vascular growth and fetal tissue growth are not understood, but studies of compromised pregnancies provide clues. The subtle changes that cause the failure of the normally regulated vascular processes during pregnancy have not been thoroughly identified. Likewise, practical and effective therapeutic strategies to reverse detrimental placental perfusion patterns have yet to be investigated.

Quaking, an RNA-Binding Protein, Is a Critical Regulator of Vascular Smooth Muscle Cell Phenotype

RNA-binding proteins are critical post-transcriptional regulators of RNA and can influence pre-mRNA splicing, RNA localization, and stability. The RNA-binding protein Quaking (QKI) is essential for embryonic blood vessel development. However, the role of QKI in the adult vasculature, and in particular in vascular smooth muscle cells (VSMCs), is currently unknown. We sought to determine the role of QKI in regulating adult VSMC function and plasticity. We identified that QKI is highly expressed by neointimal VSMCs of human coronary restenotic lesions, but not in healthy vessels. In a mouse model of vascular injury, we observed reduced neointima hyperplasia in Quaking viable mice, which have decreased QKI expression. Concordantly, abrogation of QKI attenuated fibroproliferative properties of VSMCs, while potently inducing contractile apparatus protein expression, rendering noncontractile VSMCs with the capacity to contract. We identified that QKI localizes to the spliceosome, where it interacts with the myocardin pre-mRNA and regulates the splicing of alternative exon 2a. This post-transcriptional event impacts the Myocd_v3/Myocd_v1 mRNA balance and can be modulated by mutating the quaking response element in exon 2a of myocardin. Furthermore, we identified that arterial damage triggers myocardin alternative splicing and is tightly coupled with changes in the expression levels of distinct QKI isoforms. We propose that QKI is a central regulator of VSMC phenotypic plasticity and that intervention in QKI activity can ameliorate pathogenic, fibroproliferative responses to vascular injury.

Chorioallantoic Placentation in Viperid Snakes

Chorioallantoic Placentation in Viperid Snakes

De novo DNA demethylation and noncoding transcription define active intergenic regulatory elements

Deep sequencing of mammalian DNA methylomes has uncovered a previously unpredicted number of discrete hypomethylated regions in intergenic space (iHMRs). Here, we combined whole-genome bisulfite sequencing data with extensive gene expression and chromatin-state data to define functional classes of iHMRs, and to reconstruct the dynamics of their establishment in a developmental setting. Comparing HMR profiles in embryonic stem and primary blood cells, we show that iHMRs mark an exclusive subset of active DNase hypersensitive sites (DHS), and that both developmentally constitutive and cell-type-specific iHMRs display chromatin states typical of distinct regulatory elements. We also observe that iHMR changes are more predictive of nearby gene activity than the promoter HMR itself, and that expression of noncoding RNAs within the iHMR accompanies full activation and complete demethylation of mature B cell enhancers. Conserved sequence features corresponding to iHMR transcript start sites, including a discernible TATA motif, suggest a conserved, functional role for transcription in these regions. Similarly, we explored both primate-specific and human population variation at iHMRs, finding that while enhancer iHMRs are more variable in sequence and methylation status than any other functional class, conservation of the TATA box is highly predictive of iHMR maintenance, reflecting the impact of sequence plasticity and transcriptional signals on iHMR establishment. Overall, our analysis allowed us to construct a three-step timeline in which (1) intergenic DHS are pre-established in the stem cell, (2) partial demethylation of blood-specific intergenic DHSs occurs in blood progenitors, and (3) complete iHMR formation and transcription coincide with enhancer activation in lymphoid-specified cells.

Professor ya-ling han.

Professor Ya-Ling Han (Figure 1), a famous cardiologist in China, was born in Wuhan, Hubei province, in 1953. She graduated from Harbin Medical University in 1978. After getting her doctorate in 1994 from Shanghai Second Military Medical University, professor Han worked as the director and professor of the Department of Cardiology. She then worked as the vice president at Shenyang Northern Hospital since 1997. For the past two decades, she has been persistently leading the development of percutaneous coronary intervention (PCI) technique for complex coronary lesions and high risk patients. From 1998 to 1999 she received PCI Fellow training in Singapore National Heart Center for professional enhancement. Figure 1. Professor Ya-Ling Han Professor Han is a leading expert in the field of interventional therapy of coronary heart disease in China, especially in critical acute myocardial infarction, elderly coronary heart disease patients concomitant with multiple organ dysfunction, left main, chronic total occlusive and multi-vessel coronary diseases. In the past 20 years, professor Han and her colleagues have performed more than 30,000 cardiovascular interventional procedures with a successful rate over 99%. During the same period, many new techniques in coronary intervention have been established, including ‘improved tiny crush stent technology’ and ‘multi-wire plaque crushing technology’, etc. In addition, Professor Han has made contributions in improving PCI adjunctive pharmacology. She is the first to propose high dose clopidogrel, triple antiplatelet therapy and individualized antiplatelet strategy, which had been referenced and recommended by foreign expert and international conferences. Professor Han was in charge of many clinical studies such as the CREATE study, a 5-year large scale multicenter “real world” clinical study on efficacy and safety of domestic biodegradable DES in China, through which she found that 12-month dual antiplatelet was not inferior to 6-month treatment. The findings of the many clinical studies led by Professor Han have been published in leading international cardiology journals and contribute to the current management of coronary heart disease. Although an accomplished cardiovascular clinician who dedicated most of her energy and time on the diagnosis and treatment of her patients, Professor Han also committed to basic research of cardiology. She and her colleagues have investigated the biological role of CREG against vascular restenosis and atherosclerotic inflammation after PCI, which provide innovative insights into the early prevention of these diseases, the role and molecular mechanisms of CREG during embryonic blood vessel formation, and the regulation of CREG gene on iPS cell-based cardiogenesis. Professor Han, together with her colleagues, has obtained funding from Natural Science Foundation of China for 8 times, and one of which was the key project of national natural science funding. Professor Han have published over 400 research papers both in national and international journals. Professor Han now is the adjunct professors and doctoral supervisor for the Third Military Medical University, the Fourth Military Medical University and the Medical University of Dalian. She has been supervised research work for more than 120 post graduates. Professor Han has been awarded with numerous awards for her outstanding clinical, academic and social service work, such as National ‘Young Star in Medical Sciences’ award in 1996, ‘National Distinguished Woman’ and ‘Outstanding Award of Chinese Interventional Cardiology’ in 2005, ‘Second Prize of National Scientific and Technological Progress’ in 2008, ‘The 11th Five-Year Military Medical Major Science and Technology Achievement Award’ in 2010. Professor Han's achievements are recognized by medical community. She was elected to be the Vice President of Chinese Cardiovascular Society of Chinese Medical Association, the Vice President of Cardiovascular Branch in Chinese Medical Doctor Association. She also served in many editorial boards of medical journals. She is an Associate Editor of Chinese Journal of Cardiology and a vice Chief Editor of Cardiovascular Therapeutics. Professor Han is a member of FACC and FESC.