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Background: Familial hypertrophic cardiomyopathy (HCM) is the most common inherited cardiac disease and is typically caused by mutations in genes encoding sarcomeric proteins that regulate cardiac contractility. HCM manifestations include left ventricular hypertrophy and heart failure, arrythmias, and sudden cardiac death. How dysregulated sarcomeric force production is sensed and leads to pathological remodeling remains poorly understood in HCM, thereby inhibiting the efficient development of new therapeutics. Methods: Our discovery was based on insights from a severe phenotype of an individual with HCM and a second genetic alteration in a sarcomeric mechanosensing protein. We derived cardiomyocytes from patient-specific induced pluripotent stem cells and developed robust engineered heart tissues by seeding induced pluripotent stem cell–derived cardiomyocytes into a laser-cut scaffold possessing native cardiac fiber alignment to study human cardiac mechanobiology at both the cellular and tissue levels. Coupled with computational modeling for muscle contraction and rescue of disease phenotype by gene editing and pharmacological interventions, we have identified a new mechanotransduction pathway in HCM, shown to be essential in modulating the phenotypic expression of HCM in 5 families bearing distinct sarcomeric mutations. Results: Enhanced actomyosin crossbridge formation caused by sarcomeric mutations in cardiac myosin heavy chain ( MYH7 ) led to increased force generation, which, when coupled with slower twitch relaxation, destabilized the MLP (muscle LIM protein) stretch-sensing complex at the Z-disc. Subsequent reduction in the sarcomeric muscle LIM protein level caused disinhibition of calcineurin–nuclear factor of activated T-cells signaling, which promoted cardiac hypertrophy. We demonstrate that the common muscle LIM protein–W4R variant is an important modifier, exacerbating the phenotypic expression of HCM, but alone may not be a disease-causing mutation. By mitigating enhanced actomyosin crossbridge formation through either genetic or pharmacological means, we alleviated stress at the Z-disc, preventing the development of hypertrophy associated with sarcomeric mutations. Conclusions: Our studies have uncovered a novel biomechanical mechanism through which dysregulated sarcomeric force production is sensed and leads to pathological signaling, remodeling, and hypertrophic responses. Together, these establish the foundation for developing innovative mechanism-based treatments for HCM that stabilize the Z-disc MLP-mechanosensory complex.

Laser additive manufacturing (LAM) technique has unique advantages in producing geometrically complex metallic components. However, the poor low-cycle fatigue property (LCF) of LAM parts restricts its widely used. Here, the microstructural features of a Ti-6Al-4 V alloy manufactured via high power laser directed energy deposition subjected to low-cycle fatigue loading were studied. Before fatigue loading, the microstructure of the as-deposited parts was found to exhibit a non-homogeneous distribution of columnar prior-β grains (200–4000 μm) at various scanning velocities (300–1500 mm/min) and relatively coarse α-laths (1.0–4.5 μm). Under cyclic loading, fatigue microcracks typically initiated within the aligned α phases in the preferred orientation (∼45° to the loading direction) at the surface of the fatigue specimens. Fatigued Ti-6Al-4 V exhibited a single straight dislocation character at low strain amplitudes ( 1.1 %). In addition, dislocation substructure features, such as dislocation walls, stacking faults, and dislocation networks, were also observed. These findings may provide opportunities to understand the fatigue failure mechanism of additive manufactured titanium parts.

The failure of transpassive films on transition metal alloys was investigated. The results show that the failure of the transpassive film on 3D printing nickel-based superalloy in flowing NaNO3 solution is dominated by self-induced cracking of the film on niobium-segregated phases and the erosion-induced degradation on the film assisted by cation vacancy condensation at the metal/barrier layer interface. Secondary passivation induces the formation of a transpassive film, the nature of which has been debated for decades. The findings are consistent with a bi-layer structure comprising a point-defective barrier layer that grows into the substrate alloy and a precipitated outer layer.

The low cycle fatigue (LCF) properties of Ti-6Al-4V parts fabricated by high-power laser directed energy deposition were investigated. The results show that the parts after solution treatment and aging exhibit superior LCF lives to those of other reported AM Ti-6Al-4V parts, as well as are comparable to those of the wrought counterparts at intermediate strain amplitudes (from 0.8% to 1.1%). Cyclic softening behaviors were found at various strain amplitudes (from 0.65% to 1.7%). In addition, a microstructure-based multistage fatigue model was used to predict their LCF lives and shows good agreement with the experimental data.

Stress-relief annealing is one of the most frequently used heat treatments for additive manufactured components. The effects of the stress-relief annealing on anodic dissolution behaviour of laser solid formed Ti-6Al-4V in a 15wt.% NaCl electrolyte was investigated. The annealed Ti-6Al-4V exhibited comparatively improved resistance to dissolution compared with as-deposited one. The reduced micro-segregation resulted in the superior corrosion resistance for the annealed Ti-6Al-4V, owing to the formation of a more stable passive film and the weakened galvanic effect. The selective dissolution of α phase was lightened with the decreasing concentration difference of solutes between the refined α and β phases.

Coronary microvascular disease (MVD) remains a major clinical problem due to limited mechanistic understanding and a challenging diagnosis. In the present study we evaluated the utility of targeted imaging of active factor XIII (FXIII) for detection of coronary MVD associated with thrombus. We hypothesized that a high specificity and sensitivity FXIII targeted radiolabeled probe can serve as a biomarker for cross-linked thrombi in the microvasculature, and thus an indicator for underlying coronary MVD. To evaluate this approach, a coronary MVD model was established for local induction of singlet oxygen and reactive oxygen species (ROS) via a photochemical reaction (PCR). Methods: PCR was used to induce endothelial injury and microthrombi via focal over-production of ROS only in the coronary microvasculature. Oxidative stress was initially evaluated in primary coronary endothelial cells to optimize parameters of PCR, which were then translated to in vivo experiments. To develop the coronary MVD model, 64 mice were assigned to one of four groups after thoracotomy: 1) sham control; 2) rose bengal; 3) green light; or 4) their combination. Following interventions, the mice underwent transmission electron microscopy, fluorescent myocardial perfusion, coronary angiography, and immunohistochemical staining. Echocardiography (n = 12) and gene expression (n = 10) studies were also performed after MVD induction to monitor serial changes in cardiac function and explore possible mechanisms. To diagnose early onset MVD, FXIII radioactivity was assessed in 104 mice using ex vivo gamma well counting (GWC) and in 14 mice using in vivo serial single photon emission computed tomography / computed tomography (SPECT/CT) imaging of a FXIII targeted technetium-labeled probe (99mTc-NC100668). Results: In vitro experiments demonstrated that photosensitizer concentration and light illumination time were critical parameters for PCR. In vivo experiments demonstrated manifestations of clinical MVD, including endothelial damage, a “no flow zone,” arteriole rarefaction with patent epicardial coronary arteries, infiltration of inflammatory cells in the PCR-treated region, and preserved cardiac function. Gene expression also demonstrated a pro-thrombotic and impaired fibrinolytic status. In the early stages of MVD, enhanced FXIII activity was confirmed within the MVD region using GWC and in vivo SPECT/CT imaging. Conclusion: Our results demonstrate that molecular imaging of FXIII activity may allow for early detection of coronary MVD associated with thrombus, in a novel pre-clinical model.

Improvements to the surface of high-power laser solid formed Inconel 718 by use of electrochemical methods were systematically investigated. Formation features (surface microstructure characteristics and surface morphology) and the corresponding electrochemical anodic behavior (electrochemical dissolution behavior and surface levelling mechanism) were analysed. The electrochemical results show that the surface energy difference between the horizontal section and vertical section causes no difference to their transpassive current densities in 10 wt.% NaNO3 solution up to 2.5 V, owing to the existence of a passive film. Analysis of micro-morphologies indicates that the high current density leads to a smooth micro-morphology due to the higher detachment rates of the interdendritic phases and surface products. Numerical simulations suggest that surface peaks dissolve faster than depressions during the electrochemical levelling process owing to the higher local current density on the surface peaks. Importantly, a detailed microstructure selection map for as-deposited Inconel 718 and an anodic current-density-potential map were developed, and the electrochemical levelling mechanism of its wavy surfaces was proposed. This work has the potential to integrate understanding of laser solid forming-process conditions, formation features, and electrochemical anodic behavior.

An ionic liquid functionalised silane agent, 1-(3-triethoxysilylpropyl)-3-methylimidazolium chloride (TESPM-IL), was prepared and used to fabricate ionic liquid (IL)-grafted titanium dioxide (IL-TiO2) spheres via a sol-gel process. Thermogravimetric analysis and Fourier transform infrared spectroscopy confirmed that TESPM-IL was attached to TiO2 spheres. Morphologies of the IL-TiO2 spheres were observed by scanning electron microscopy and transmission electron microscopy, indicating the rough surface and smaller size of the IL-TiO2 particles caused by the modification of TESPM-IL. The IL-TiO2 particles were applied as a new dispersed-phase material of electrorheological (ER) fluids. The ER effect of IL-TiO2 particles was studied and compared with that of pure TiO2 particles. It was found that, upon the introduction of TESPM-IL, both yield stress and storage modulus of the IL-TiO2 ER fluid were enhanced significantly, thus improving the ER effect of TiO2- or inorganic-based ER materials effectively. Dielectric analysis was also used to observe the interfacial polarization process of the particles, which confirmed the effect of the IL layer on the surface of the TiO2 particles.

The high-cycle fatigue (HCF) properties of Inconel 718 (IN718) superalloy prepared using laser directed energy deposition (LDED) were investigated at room temperature (RT) and 650 °C. A specified heat treatment scheme, namely homogenization + solution + aging (HSA), was employed for the as-deposited LDED IN718 alloy. The results indicate that the grains of the HSA LDED IN718 alloy have a typical bimodal grain (BG) distribution consisting of coarse grains (CGs) and fine grains (FGs). The fatigue limit ( σ w ) of the HSA IN718 alloy at RT was higher than that at 650 °C (~385 and ~280 MPa, respectively), and their σ w values were only ~77% and ~63% those of wrought IN718, respectively. Fractographic analysis revealed that the fatigue failure model changed from surface slip failure to large crystallographic facet failure with increasing testing temperature. The HCF properties of the HSA IN718 alloy with BG microstructure are strongly affected by CGs in the material, which eclipses the deleterious effects of the initial pore defects in the deposit. Finally, the Basquin model was fitted to extract the stress-life curves of the HSA IN718 alloy. A certain degree of deviation was observed when predicting the σ w value of the HSA IN718 alloy at 650 °C using the Murakami model.

Development of autologous tissue-engineered vascular constructs using vascular smooth muscle cells (VSMCs) derived from human induced pluripotent stem cells (iPSCs) holds great potential in treating patients with vascular disease. However, preclinical, large animal iPSC-based cellular and tissue models are required to evaluate safety and efficacy prior to clinical application. Herein, swine iPSC (siPSC) lines were established by introducing doxycycline-inducible reprogramming factors into fetal fibroblasts from a line of inbred Massachusetts General Hospital miniature swine that accept tissue and organ transplants without immunosuppression within the line. Highly enriched, functional VSMCs were derived from siPSCs based on addition of ascorbic acid and inactivation of reprogramming factor via doxycycline withdrawal. Moreover, siPSC-VSMCs seeded onto biodegradable polyglycolic acid (PGA) scaffolds readily formed vascular tissues, which were implanted subcutaneously into immunodeficient mice and showed further maturation revealed by expression of the mature VSMC marker, smooth muscle myosin heavy chain. Finally, using a robust cellular self-assembly approach, we developed 3D scaffold-free tissue rings from siPSC-VSMCs that showed comparable mechanical properties and contractile function to those developed from swine primary VSMCs. These engineered vascular constructs, prepared from doxycycline-inducible inbred siPSCs, offer new opportunities for preclinical investigation of autologous human iPSC-based vascular tissues for patient treatment.

This work investigated the microstructure and tensile deformation behavior of Ti-6Al-4V alloy fabricated using a high-power laser solid forming (LSF) additive manufacturing. The results show that the post-fabricated heat-treated microstructure consists of coarse columnar prior-β grains (630–1000 μm wide) and α-laths (5–9 μm) under different scanning velocities (900 and 1500 mm/min), which caused large elongation (∼18%) superior to the conventional laser additive manufacturing Ti-6Al-4V alloy. The deformation behavior of the LSF Ti-6Al-4V alloy was investigated using in situ tensile test scanning electron microscopy. The results show that shear-bands appeared along the α/β interface and slip-bands occurred within the α-laths, which lead to cracks decaying in a zigzag-pattern in the LSF Ti-6Al-4V alloy with basket-weave microstructure. These results demonstrate that the small columnar prior-β grains and fine basket-weave microstructure exhibiting more α/β interfaces and α-laths can disperse the load and resist the deformation in the LSF Ti-6Al-4V components. In addition, a modified microstructure selection map of the LSF Ti-6Al-4V alloy was established, which can reasonably predict the microstructure evolution and relative grain size in the LSF process.

Arrhythmogenic cardiomyopathy (ACM) is an inherited disorder with variable genetic etiologies. Here, we focused on understanding the precise molecular pathology of a single clinical variant in DSP, the gene encoding desmoplakin. We initially identified a potentially novel missense desmoplakin variant (p.R451G) in a patient diagnosed with biventricular ACM. An extensive single-family ACM cohort was assembled, revealing a pattern of coinheritance for R451G desmoplakin and the ACM phenotype. An in vitro model system using patient-derived induced pluripotent stem cell lines showed depressed levels of desmoplakin in the absence of abnormal electrical propagation. Molecular dynamics simulations of desmoplakin R451G revealed no overt structural changes, but a significant loss of intramolecular interactions surrounding a putative calpain target site was observed. Protein degradation assays of recombinant desmoplakin R451G confirmed increased calpain vulnerability. In silico screening identified a subset of 3 additional ACM-linked desmoplakin missense mutations with apparent enhanced calpain susceptibility, predictions that were confirmed experimentally. Similar to R451G, these mutations are found in families with biventricular ACM. We conclude that augmented calpain-mediated degradation of desmoplakin represents a shared pathological mechanism for select ACM-linked missense variants. This approach for identifying variants with shared molecular pathologies may represent a powerful new strategy for understanding and treating inherited cardiomyopathies.

The fatigue crack growth (FCG) behavior of long cracks was studied in Ti–6Al–4V alloy fabricated by high-power laser directed energy deposition. The results show the deposited parts under heat treated conditions have superior FCG resistance than that of the other reported additive manufactured Ti–6Al–4V, owing to the specimens with coarse prior-β grains and interior thick α-laths. Using linear elastic fracture mechanics and two (von Mises and Tresca) yielding criterions, we calculated the monotonic and cyclic plastic zone sizes, and found the FCG rates as a function of cyclic plastic zone sizes ahead of the crack tip during FCG region. A method was proposed to determine the typical microstructural characteristic unit of the additive manufactured Ti–6Al–4V parts with basket-weave structure, which shows the same length-scale as plastic zone sizes at the transition point in FCG curves.

Summary In contrast to conventional human pluripotent stem cells (hPSCs) that are related to post-implantation embryo stages, naive hPSCs exhibit features of pre-implantation epiblast. Naive hPSCs are established by resetting conventional hPSCs, or are derived from dissociated embryo inner cell masses. Here we investigate conditions for transgene-free reprogramming of human somatic cells to naive pluripotency. We find that Wnt inhibition promotes RNA-mediated induction of naive pluripotency. We demonstrate application to independent human fibroblast cultures and endothelial progenitor cells. We show that induced naive hPSCs can be clonally expanded with a diploid karyotype and undergo somatic lineage differentiation following formative transition. Induced naive hPSC lines exhibit distinctive surface marker, transcriptome, and methylome properties of naive epiblast identity. This system for efficient, facile, and reliable induction of transgene-free naive hPSCs offers a robust platform, both for delineation of human reprogramming trajectories and for evaluating the attributes of isogenic naive versus conventional hPSCs., Highlights • Generation of transgene-free human naive iPSCs by RNA-mediated reprogramming • Wnt inhibition facilitates naive iPSC production and expansion • Naive iPSCs retain a diploid karyotype • Naive iPSCs are transcriptomically related to pre-implantation human epiblast, Molecular reprogramming can induce different states of pluripotency. In this paper, Ge Guo and colleagues report that naive stem cells related to the blastocyst stage human embryo can be generated reliably from human somatic cells using RNA delivery of reprogramming factors. Reprogramming is enhanced by inhibition of the Wnt pathway, which also stabilizes the human naive state.

Li, Hongge, Songchang Guo, Yongming Ren, Depeng Wang, Honghao Yu, Wenjing Li, Xinquan Zhao, and Zhijie Chang. VEGF(189) expression is highly related to adaptation of the plateau pika (Ochotona curzoniae) inhabiting high altitudes. High Alt Med Biol 14:395-404, 2013. The plateau pika (Ochotona curzonia) has adapted to high-altitude hypoxia during evolution. Higher microvessel density in specific tissues and a blunted hypoxic pulmonary vasoconstriction response are the critical components of this adaptation. VEGF, vascular endothelial growth factor, has proved to be a key regulator of angiogenesis in response to tissue hypoxia and to play an important role in vascular vasodilation. However, the role of VEGF in adaptation to high-altitude hypoxia in the plateau pika remains unknown. In this study, we cloned cDNAs for VEGF(165) and VEGF(189) and examined their expression in pikas inhabiting altitudes of 3200 and 4750m. Phylogenetic analysis reveals that pika VEGF(165) and VEGF(189) are evolutionarily conserved. Real-time PCR analysis demonstrates that VEGF(165) and VEGF(189) display tissue and altitude-specific expression patterns. Interestingly, we found that the levels of VEGF(189) mRNA are significantly higher than those of VEGF(165) in the brain and muscle tissues of the pika, which is different from what was previously observed in sea-level mammals. VEGF(189) mRNA levels in brain, muscle, and lung of the pika increased with increased habitat altitude, whereas VEGF(165) shows less change. Our study suggests an important role for VEGF(189) in adaptation to hypoxia by the plateau pika in the high-altitude environment.

We have developed an engineered heart tissue (EHT) system that uses laser-cut sheets of decellularized myocardium as scaffolds. This material enables formation of thin muscle strips whose biomechanical characteristics are easily measured and manipulated. To create EHTs, sections of porcine myocardium were laser-cut into ribbon-like shapes, decellularized and mounted in specialized clips for seeding and culture. Scaffolds were first tested by seeding with neonatal rat ventricular myocytes. EHTs beat synchronously by day five and exhibited robust length-dependent activation by day 21. Fiber orientation within the scaffold affected peak twitch stress, demonstrating its ability to guide cells toward physiologic contractile anisotropy. Scaffold anisotropy also made it possible to probe cellular responses to stretch as a function of fiber angle. Stretch that was aligned with the fiber direction increased expression of brain natriuretic peptide, but off-axis stretches (causing fiber shear) did not. The method also produced robust EHTs from cardiomyocytes derived from human embryonic stem cells and induced pluripotent stem cells (hiPSC). hiPSC-EHTs achieved maximum peak stress of 6.5 mN/mm2 and twitch kinetics approaching reported values from adult human trabeculae. We conclude that laser-cut EHTs are a viable platform for novel mechanotransduction experiments and characterizing the biomechanical function of patient-derived cardiomyoctyes.

Murine embryonic stem cell (mESC)-derived cardiomyocytes represent a promising source of cells for use in the development of models for studying early cardiac development as well as cell-based therapies in postnatal pathologies. Here, we report a highly efficient cardiac differentiation system in which high density embryoid body (EB) cultures leads to a marked increase of cardiomyocytes production from multiple mESC lines without the addition of any cardiogenic growth factors. Our results show that high density EB cultures significantly increase the yield of functional cardiomyocytes, which express typical cardiac markers, exhibit normal rhythmic Ca2+ transients, and respond to both β-adrenergic and electric stimulations. During the differentiation period, the inhibition of bone morphogenetic protein (BMP) signaling significantly attenuates the increase of cardiac differentiation as well as the increased expression of cardiac-specific genes, NK2 transcription factor related 5 (Nkx2.5) and myosin light chain 2v (Mlc2v) by high density EB cultures. Therefore, we believe that we offer a novel and efficient means of cardiomyocyte production for practical use of mESCs in cardiac regenerative medicine.

SNXs (sorting nexin), a family of proteins playing roles in cargo sorting and signaling from compartments within the endocytic network, regulate traffic of membrane proteins including TGF-β receptors. Here we report that the full length human and mouse SNX25, a SNX member with PX, PXA and RGS domains, co-localizes with TGF-β receptors, and forms internalized cytosolic punctae upon treatment with TGF-β. While overexpression of SNX25 inhibits TGF-β induced luciferase reporter activity, knocking down endogenous SNX25 by siRNA in NIH3T3 cells elevates the TGF-β receptor levels and facilitates TGF-β signaling. Immunoprecipitation experiments demonstrate that SNX25 interacts with TβRI. Western blot analyses indicate that SNX25 enhances the degradation of TGF-β receptors. SNX25 induced TGF-β receptor degradation is shown via the clathrin dependent endocytosis pathway into lysosome. We have characterized that PXA domain of SNX25 is required for the degradation of TβRI. Our findings demonstrate that SNX25 negatively regulates TGF-β signaling by enhancing the receptor degradation through lysosome pathway.

Sef (similar expression to fgf genes) was identified as a feedback antagonist of FGF signaling in zerbrafish, mouse and human. To construct recombinant adenoviral vectors expressing hSef-L and hSef-S, the coding sequences of the two isoforms were amplified and ligated into pAdTrack-CMV, forming shuttle vectors pAdTrack-CMV/hSef-L-Myc and pAdTrack-CMV/hSef-S-Myc. After sequence confirmation, these two shuttle vector plasmids were linearized by Pme I and then co-transformed respectively with the adenoviral genome vector pAdEasy-1 into E. coli BJ5183. The successful recombinants were selected by Kanamycin and confirmed by Pac I digestion. The recombinant vectors Ad-hSef-L-Myc and Ad-hSef-S-Myc were finally digested with Pac I and transfected into HEK293 cells to pack into viral particles. The virus were amplified in 293 cells and used to infect MEF cells. Western blotting analysis was used to demonstrate the expression of hSef-L-Myc and hSef-S-Myc proteins. The inhibitory effects of the adenovirus mediated Sef expression on FGF signaling was further evaluated by Elk luciferase reporter assay. Our results indicated the constructed virus could produce effectively the proteins and then inhibit FGF signaling in MEF cells.

Interleukin-17F (IL-17F), together with interleukin-17A (IL-17 or IL-17A), is a marker of T(H)17 cells, a new lineage of effector CD4(+) T cells to contribute to pathogenesis of a growing list of autoimmune and inflammatory diseases, such as experimental autoimmune encephalitis (EAE) and collagen-induced arthritis (CIA). IL-17F, similar to IL-17A, was reported to employ interleukin-17 receptor (IL-17R or IL-17RA) for signaling but the downstream cascades remain largely elusive. Here we report that TRAF6 interacts with IL-17R and mediates ubiquitination of the receptor. We observed that IL-17F and IL-17A could induce IL-17R ubiquitination and DN-TRAF6, a dominant-negative mutant, could block IL-17F- but not IL-17A-triggered ubiquitination of IL-17R. Moreover, we showed that the ubiquitination of IL-17R was positively correlated with the downstream signaling, as evaluated by a luciferase reporter driven by a putative native promoter of 24p3, an IL-17 targeted gene. Our results indicate that ubiquitination of IL-17R mediated by TRAF6 plays a critical role in IL-17F signaling. This study, for the first time, reveals a possible molecular mechanism that the initiation of the IL-17F/IL-17R signaling pathway requires the receptor ubiquitination by TRAF6.

Sef (similar expression to fgf genes) was identified as an inhibitor of FGF signaling. The regulation of this inhibitory effect was largely unknown. In this report we demonstrated that tyrosine 330 in hSef protein plays a critical role in the control of the protein localization and thereby in the regulation of Ras/MAPK signaling pathway. We found that the tyrosine 330 is in the form of the YXXcapital EF, Cyrillic signal context and mutation of this residue resulted in preferred plasma membrane localization of hSef. We also observed that both Sef and SefY330F (where tyrosine is substituted by phenylalanine) interacted and co-localized with FGFR in the co-immunoprecipitation assay, and immunostaining assay respectively. We further revealed that the increased amount of Sef localization in the plasma membrane was coupled with the enhanced inhibitory effect on the FGF signaling pathway, indicating that Sef might exert its inhibitory function on the plasma membrane. This paper revealed that tyrosine 330 is critical for the inhibitory function of Sef on FGF signaling.

Background: Coronary microvascular disease (MVD) remains a major clinical challenge, involving endothelial cell (ECs) dysfunction and microthrombosis. We hypothesize that active FXIII can be a specific biomarker for MVD and its activity can be evaluated by a FXIII targeted radiolabeled probe ( 99m Tc-NC100668). We also hypothesize that a murine MVD model can be established through photodynamic therapy (PDT). Methods: To optimize the PDT parameters, primary ECs from the heart were isolated and treated with various concentrations of rose bengal, different illumination times, multiple combinations of rose bengal and illumination times (PDT), or nothing to evaluate cell viability and production of reactive oxygen species (ROS). The optimal parameters were translated for in vivo MVD model establishment. 52 female mice were assigned to one of four groups: 1) rose bengal, 2) green light, 3) combination, or 4) nothing. Microvascular injury was evaluated by fluorescent myocardial perfusion, coronary microCT angiography, and electron microscopy. To validate the efficacy of this tracer for the diagnosis of coronary MVD, an additional 124 female mice with MVD were injected either 0.1mCi 99m Tc-NC100668 or 99m Tc-AH110563 (an inactive analog), at 3 hrs, 24 hrs, 3, 7, or 14 days, for ex vivo gamma well counting (GWC, n=100) to assess regional myocardial activity as %injection dose/mg (id/mg), or in vivo microSPECT/CT (n=24) to visualize active FXIII. Results: PDT were capable of injuring primary ECs by over-producting ROS in vitro . Coronary MVD developed only in PDT mice with focal “no flow phenomena”. microCT demonstrated arteriole rarefaction and Electron Microscopy showed luminal microthrombi and loss of endothelial integrity in PDT-treated myocardium. GWC demonstrated 99m Tc-NC100668 activity within endothelial-damaged myocardium, compared to an inactive tracer at early time points (3 hours: 9.33±1.98 vs. 2.84±1.97% id/mg, pIn vivo microSPECT/CT images confirmed focal myocardial uptake up to day 3. Conclusions: We successfully established a murine model of coronary MVD. Molecular imaging of the activated FXIII allowed visualization of acute and sub-acute coronary micro-thrombi, a hallmark of MVD.

Background— Supravalvular aortic stenosis (SVAS) is caused by mutations in the elastin ( ELN ) gene and is characterized by abnormal proliferation of vascular smooth muscle cells (SMCs) that can lead to narrowing or blockage of the ascending aorta and other arterial vessels. Having patient-specific SMCs available may facilitate the study of disease mechanisms and development of novel therapeutic interventions. Methods and Results— Here, we report the development of a human induced pluripotent stem cell (iPSC) line from a patient with SVAS caused by the premature termination in exon 10 of the ELN gene resulting from an exon 9 four-nucleotide insertion. We showed that SVAS iPSC-derived SMCs (iPSC-SMCs) had significantly fewer organized networks of smooth muscle α-actin filament bundles, a hallmark of mature contractile SMCs, compared with control iPSC-SMCs. The addition of elastin recombinant protein or enhancement of small GTPase RhoA signaling was able to rescue the formation of smooth muscle α-actin filament bundles in SVAS iPSC-SMCs. Cell counts and BrdU analysis revealed a significantly higher proliferation rate in SVAS iPSC-SMCs than control iPSC-SMCs. Furthermore, SVAS iPSC-SMCs migrated at a markedly higher rate to the chemotactic agent platelet-derived growth factor compared with the control iPSC-SMCs. We also provided evidence that elevated activity of extracellular signal-regulated kinase 1/2 is required for hyperproliferation of SVAS iPSC-SMCs. The phenotype was confirmed in iPSC-SMCs generated from a patient with deletion of elastin owing to Williams-Beuren syndrome. Conclusions— SVAS iPSC-SMCs recapitulate key pathological features of patients with SVAS and may provide a promising strategy to study disease mechanisms and to develop novel therapies.

The objective of this paper is to compare the dynamic response of bridge-vehicle coupled vibration between scale-model test and numerical analysis. In order to study the vibration response of a simple supported bridge subjected to a moving vehicle, three methods are introduced. Firstly, the equations of motion for bridge and vehicle are established respectively, an iteration solution method is presented to analyse the coupled vibration response of vehicle-bridge system based on the same displacement and coupling force at the interface. Nextly, the visualization approach to simulate the dynamic response of vehicle-bridge coupled vibration is put forward, through building 3-D FE models of vehicle and bridge using LS-DYNA. Lastly, based on the similitude law of bridge-vehicle interaction system, the simple girder bridge model test that the scale factor of geometry is 10.0, the static and dynamic response of model bridge are studied subjected to a moving vehicle. Comparative results between the testing of model bridge and numerical analysis of practical bridge conforms the feasibility of model text equipment and the validity of the text results. At the same time, the results also show efficiency and stabilization of the numerical simulation.

In order to study effectively vehicle-bridge interaction, one experimental method is presented in detail based on the scale-model system established through similarity theory in this paper. Firstly, the model materials are selected appropriately according to the characteristics of bridge and vehicle. Besides, the model parameters such as size, modulus and so on are determined according to the similarity theory. Finally, a new traction system was designed for dynamic model tests. Through model tests, the experimental results show that the proposed method is feasible and available to research the vehicle-bridge interaction‥

The similitude law of bridge-vehicle interaction system are presented by combining π theorem with dimension matrix analysis based on similarity theories of structure experimentation model. The ratios of similitude and similarity criteria of vibration test about the scale-model system for highway bridge-vehicle interaction are confirmed. Based on the simple girder bridge model test of perspex model that the scale factor of geometry is 10.0, the static and dynamic response of model bridge are studied subjected to a moving vehicle running on the smooth deck. The comparison of the testing results of model bridge and numerical calculation results of practical bridge conformed the similitude law of bridge-vehicle interaction model text. The results show the feasibility of model text equipment and the validity of the text results.

Human induced pluripotent stem (iPS) cells potentially provide a unique resource for generating patient-specific cardiomyocytes to study cardiac disease mechanisms and treatments. However, existing approaches to cardiomyocyte production from human iPS cells are inefficient, limiting the application of iPS cells in basic and translational cardiac research. Furthermore, strategies to accurately record changes in iPS cell-derived cardiomyocyte action potential duration (APD) are needed to monitor APD-related cardiac disease and for rapid drug screening. We examined whether modulation of the bone morphogenetic protein 4 (BMP-4) and Wnt/β-catenin signaling pathways could induce efficient cardiac differentiation of human iPS cells. We found that early treatment of human iPS cells with BMP-4 followed by late treatment with small molecule Wnt inhibitors led to a marked increase in production of cardiomyocytes compared to existing differentiation strategies. Using immunocytochemical staining and real-time intracellular calcium imaging, we showed that these induced cardiomyocytes expressed typical sarcomeric markers, exhibited normal rhythmic Ca(2+) transients, and responded to both β-adrenergic and electric stimulation. Furthermore, human iPS cell-derived cardiomyocytes demonstrated characteristic changes in action potential duration in response to cardioactive drugs procainamide and verapamil using voltage-sensitive dye-based optical recording. Thus, modulation of the BMP-4 and Wnt signaling pathways in human iPS cells leads to highly efficient production of cardiomyocytes with typical electrophysiological function and pharmacologic responsiveness. The use of human iPS cell-derived cardiomyocytes and the application of calcium- and voltage-sensitive dyes for the direct, rapid measurement of iPS cell-derived cardiomyocyte activity promise to offer attractive platforms for studying cardiac disease mechanisms and therapeutics.

Neuronal differentiation is tightly regulated by a variety of factors. In a search for neuron-specific genes, we identified a highly conserved novel zinc finger protein, ZNF536. We observed that ZNF536 is most abundant in the brain and, in particular, is expressed in the developing central nervous system and dorsal root ganglia and localized in the cerebral cortex, hippocampus, and hypothalamic area. During neuronal differentiation of P19 cells induced by retinoic acid (RA), ZNF536 expression is increased at an early stage, and it is maintained at a constant level in later stages. Overexpression of ZNF536 results in an inhibition of RA-induced neuronal differentiation, while depletion or mutation of the ZNF536 gene results in an enhancement of differentiation. We further demonstrated that ZNF536 inhibits expression of neuron-specific marker genes, possibly through the inhibition of RA response element-mediated transcriptional activity, as overexpression of RA receptor alpha can rescue the inhibitory role of ZNF536 in neuronal differentiation and neuron-specific gene expression. Our studies have identified a novel zinc finger protein that negatively regulates neuron differentiation.

The plateau pika (Ochotona curzoniae) is a high hypoxia-tolerant species living only at 3,000-5,000m above sea-level on the Qinghai-Tibetan plateau. Hypoxia-inducible factor-1 (HIF-1) is a key transcription factor that regulates a variety of cellular and systemic adaptations to hypoxia. To investigate how the plateau pika adapts to a high-altitude hypoxic environment at the molecular level, we examined the expression pattern of the HIF-1 alpha protein in the pika by Western blot and immunohistochemical analysis. We found that HIF-1 alpha protein is expressed at a significantly high level in the pika, which is higher in most tissues (particularly in the lung, liver, spleen and kidney) of the plateau pika than that of mice living at sea-level. Importantly, we found that the protein levels of HIF-1 alpha in the lung, liver, spleen and kidney of the pika were increased with increased habitat altitudes. We observed that the plateau pika HIF-1 alpha localized to the nucleus of cells by an immunostaining analysis, and enhanced HRE-driven gene expression by luciferase reporter assays. Our study suggests that the HIF-1 alpha protein levels are related to the adaptation of the plateau pika to the high-altitude hypoxic environment. J. Exp. Zool. 311A:134-141, 2009. (c) 2008 Wiley-Liss, Inc.

Ligand-dependent or independent oligomerization of receptor protein tyrosine kinase (RPTK) is often an essential step for receptor activation and intracellular signaling. The novel oncogene with kinase-domain (NOK) is a unique RPTK that almost completely lacks an ectodomain, expresses intracellularly and activates constitutively. However, it is unknown whether NOK can form oligomer or what function oligomerization would have. In this study, two NOK deletion mutants were generated by either removing the ectodomain (NOKDeltaECD) or including the endodomain (NOK-ICD). Co-immunoprecipitation demonstrated that the transmembrane (TM) domain of NOK was essential for its intermolecular interaction. The results further showed that NOK aggregated more closely as lower order oligomers (the dimer- and trimer-sized) than either deletion mutant did since NOK could be cross-linked by both Sulfo-EGS and formaldehyde, whereas either deletion mutant was only sensitive to Sulfo-EGS. Removing the NOK TM domain (NOK-ICD) not only markedly promoted higher order oligomerization, but also altered the subcellular localization of NOK and dramatically elevated the NOK-mediated constitutive activation of extracellular signal-regulated kinase (ERK). Moreover, NOK-ICD but not NOK or NOKDeltaECD was co-localized with the upstream signaling molecule RAS on cell membrane. Thus, TM-mediated intermolecular contacting may be mainly responsible for the constitutive activation of NOK and contribute to the autoinhibitory effect on RAS/MAPK signaling.

Sef (similar expression to fgf genes) was identified as an effective antagonist of fibroblast growth factor (FGF) in vertebrates. Previous reports have demonstrated that Sef interacts with FGF receptors (FGFRs) and inhibits FGF signaling, however, its role in regulating epidermal growth factor receptor (EGFR) signaling remains unclear. In this report, we found that hSef localizes to the plasma membrane (PM) and is subjected to rapid internalization and well localizes in early/recycling endosomes while poorly in late endosomes/lysosomes. We observed that hSef interacts and functionally colocalizes with EGFR in early endosomes in response to EGF stimulation. Importantly, we demonstrated that overexpression of hSef attenuates EGFR degradation and potentiates EGF-mediated mitogen-activated protein kinase (MAPK) signaling by interfering EGFR trafficking. Finally, our data showed that, with overexpression of hSef, elevated levels of Erk phosphorylation and differentiation of rat pheochromocytoma (PC12) cells occur in response to EGF stimulation. Taken together, these data suggest that hSef plays a positive role in the EGFR-mediated MAPK signaling pathway. This report, for the first time, reveals opposite roles for Sef in EGF and FGF signalings.

Receptor protein tyrosine kinases (RPTKs) are essential mediators of cell growth, differentiation, migration, and metabolism. Recently, a novel RPTK named NOK has been cloned and characterized. In current study, we investigated the role of the carboxyl terminal tyrosine 417 residue of NOK in the activations of different signaling pathways. A single tyrosine to phenylalanine point mutation at Y417 site (Y417 F) not only dramatically enhanced the NOK-induced activation of extracellular signal-regulated kinase (ERK), but also markedly promoted the NOK-mediated activation of both signal transducer and activator of transcription 1 and 3 (STAT1 and 3). Moreover, the proliferation potential of NIH3T3-NOK (Y417F) stable cells were significantly elevated as compared with that of NIH3T3-NOK. Overall, our results demonstrate that the tyrosine Y417 residue at the carboxyl tail of NOK exhibits an autoinhibitory role in NOK-mediated signaling transductions.