Substrate stiffness promotes vascular smooth muscle cell calcification by reducing the levels of nuclear actin monomers.

BACKGROUND: Vascular calcification (VC) is a prevalent independent risk factor for adverse cardiovascular events and is associated with diabetes, hypertension, chronic kidney disease, and atherosclerosis. However, the mechanisms regulating the osteogenic differentiation of vascular smooth muscle cells (VSMC) are not fully understood. METHODS: Using hydrogels of tuneable stiffness and lysyl oxidase-mediated stiffening of human saphenous vein ex vivo, we investigated the role of substrate stiffness in the regulation of VSMC calcification. RESULTS: We demonstrate that increased substrate stiffness enhances VSMC osteogenic differentiation and VSMC calcification. We show that the effects of substrate stiffness are mediated via a reduction in the level of actin monomer within the nucleus. We show that in cells interacting with soft substrate, elevated levels of nuclear actin monomer repress osteogenic differentiation and calcification by repressing YAP-mediated activation of both TEA Domain transcription factor (TEAD) and RUNX Family Transcription factor 2 (RUNX2). CONCLUSION: This work highlights for the first time the role of nuclear actin in mediating substrate stiffness-dependent VSMC calcification and the dual role of YAP-TEAD and YAP-RUNX2 transcriptional complexes.

Visualizing the coordination-spheres of photoexcited transition metal complexes with ultrafast hard X-rays.

The concept of coordination sphere (CS) is central to the rational development of hierarchical molecular assemblies in modern chemistry. Manipulating the organization around transition metal ions with covalent and supramolecular interactions is a general strategy that underlies most synthetic protocols. Achieving similar control for photoexcited molecular complexes is necessary to advance the design of light-driven functionalities. This objective calls for monitoring the ultrafast dynamics of the primary (1-CS) and the secondary (2-CS) coordination spheres on the atomic scale, which remains to date an important experimental challenge for short-lived species. In this work, transient wide-angle scattering of hard X-rays (25 keV) is employed with state-of-the-art AIMD simulations in order to visualize the 1-CS (solute-only) and the 2-CS (solvation cage) of the photoinduced high-spin (HS) state for [Fe(bpy)3]2+ (bpy = 2,2'-bipyridine) in aqueous solution. Correlating this structural information in real-space reveals the interlacing of the two CS, which in turn explains why solvation affects the photoinduced electronic and structural dynamics in this class of complexes. More generally, these results obtained for a prominent prototypical system in ultrafast X-ray sciences demonstrate the unique perspectives offered by this technique to gain the crucial knowledge about the multiscale solvation dynamics that is currently missing for controlling the solute-solvent interactions in advanced functional nano and biomaterials employed for photoconversion.

Protein kinase CK2 inhibition suppresses neointima formation via a proline-rich homeodomain-dependent mechanism.

Neointimal hyperplasia is a product of VSMC replication and consequent accumulation within the blood vessel wall. In this study, we determined whether inhibition of protein kinase CK2 and the resultant stabilisation of proline-rich homeodomain (PRH) could suppress VSMC proliferation. Both silencing and pharmacological inhibition of CK2 with K66 antagonised replication of isolated VSMCs. SiRNA-induced knockdown as well as ectopic overexpression of proline-rich homeodomain indicated that PRH disrupts cell cycle progression. Mutation of CK2 phosphorylation sites Ser163 and Ser177 within the PRH homeodomain enabled prolonged cell cycle arrest by PRH. Concomitant knockdown of PRH and inhibition of CK2 with K66 indicated that the anti-proliferative action of K66 required the presence of PRH. Both K66 and adenovirus-mediated gene transfer of S163C:S177C PRH impaired neointima formation in human saphenous vein organ cultures. Importantly, neither intervention had notable effects on cell cycle progression, cell survival or migration in cultured endothelial cells.

Spatiotemporal Coherent Control of Thermal Excitations in Solids.

X-ray reflectivity measurements of femtosecond laser-induced transient gratings (TG) are applied to demonstrate the spatiotemporal coherent control of thermally induced surface deformations on ultrafast time scales. Using grazing incidence x-ray diffraction we unambiguously measure the amplitude of transient surface deformations with sub-Å resolution. Understanding the dynamics of femtosecond TG excitations in terms of superposition of acoustic and thermal gratings makes it possible to develop new ways of coherent control in x-ray diffraction experiments. Being the dominant source of TG signal, the long-living thermal grating with spatial period Λ can be canceled by a second, time-delayed TG excitation shifted by Λ/2. The ultimate speed limits of such an ultrafast x-ray shutter are inferred from the detailed analysis of thermal and acoustic dynamics in TG experiments.

Glycerol prevents dehydration in lipid cubic phases.

Lipid cubic phase samples dry out and undergo phase transitions when exposed to air. We demonstrate experimentally and theoretically that adding glycerol controllably lowers the humidity at which cubic phases form. These results broaden the potential applications of cubic phases and open up the potential of a new humidity-responsive nanomaterial.

Self-assembly of PEGylated peptide conjugates containing a modified amyloid beta-peptide fragment.

The self-assembly of PEGylated peptides containing a modified sequence from the amyloid beta peptide, FFKLVFF, has been studied in aqueous solution. PEG molar masses PEG1k, PEG2k, and PEG10k were used in the conjugates. It is shown that the three FFKLVFF-PEG hybrids form fibrils comprising a FFKLVFF core and a PEG corona. The beta-sheet secondary structure of the peptide is retained in the FFKLVFF fibril core. At sufficiently high concentrations, FFKLVFF-PEG1k and FFKLVFF-PEG2k form a nematic phase, while PEG10k-FFKLVFF exhibits a hexagonal columnar phase. Simultaneous small angle neutron scattering/shear flow experiments were performed to study the shear flow alignment of the nematic and hexagonal liquid crystal phases. On drying, PEG crystallization occurs without disruption of the FFKLVFF beta-sheet structure leading to characteristic peaks in the X-ray diffraction pattern and FTIR spectra. The stability of beta-sheet structures was also studied in blends of FFKLVFF-PEG conjugates with poly(acrylic acid) (PAA). While PEG crystallization is only observed up to 25% PAA content in the blends, the FFKLVFF beta-sheet structure is retained up to 75% PAA.

Hydrogelation and self-assembly of Fmoc-tripeptides: unexpected influence of sequence on self-assembled fibril structure, and hydrogel modulus and anisotropy.

The self-assembly and hydrogelation properties of two Fmoc-tripeptides [Fmoc = N-(fluorenyl-9-methoxycarbonyl)] are investigated, in borate buffer and other basic solutions. A remarkable difference in self-assembly properties is observed comparing Fmoc-VLK(Boc) with Fmoc-K(Boc)LV, both containing K protected by N(epsilon)-tert-butyloxycarbonate (Boc). In borate buffer, the former peptide forms highly anisotropic fibrils which show local alignment, and the hydrogels show flow-aligning properties. In contrast, Fmoc-K(Boc)LV forms highly branched fibrils that produce isotropic hydrogels with a much higher modulus (G' > 10(4) Pa), and lower concentration for hydrogel formation. The distinct self-assembled structures are ascribed to conformational differences, as revealed by secondary structure probes (CD, FTIR, Raman spectroscopy) and X-ray diffraction. Fmoc-VLK(Boc) forms well-defined beta-sheets with a cross-beta X-ray diffraction pattern, whereas Fmoc-KLV(Boc) forms unoriented assemblies with multiple stacked sheets. Interchange of the K and V residues when inverting the tripeptide sequence thus leads to substantial differences in self-assembled structures, suggesting a promising approach to control hydrogel properties.

Superoxide from NADPH oxidase upregulates type 5 phosphodiesterase in human vascular smooth muscle cells: inhibition with iloprost and NONOate.

BACKGROUND AND PURPOSE: To determine whether there is an association between vascular NADPH oxidase (NOX), superoxide, the small GTPase Rac(1) and PDE type 5 (PDE5) in human vascular smooth muscle cell (hVSMCs). EXPERIMENTAL APPROACH: hVSMCs were incubated with xanthine-xanthine oxidase (X-XO; a superoxide generating system) or the thromboxane A(2) analogue, U46619 (+/-superoxide dismutase (SOD) or apocynin) for 16 h. The expression of PDE5 and NOX-1 was assessed using Western blotting and superoxide measured. The role of Rac(1) in superoxide generation was assessed by overexpressing either the dominant-negative or constitutively active Rac isoforms. The effects of iloprost, DETA-NONOate and the Rho-kinase inhibitor, Y27632, on PDE5 and NOX-1 expression were also studied. KEY RESULTS: Following 16 h incubation, U46619 and X-XO promoted the expression of PDE5 and NOX-1, an effect blocked by SOD or apocynin when co-incubated over the same time course. X-XO and U46619 both promoted the formation of superoxide. Overexpression of dominant-negative Rac(1) or addition of iloprost, DETA-NONOate or Y27632 completely blocked both superoxide release and PDE5 protein expression and activity. CONCLUSIONS AND IMPLICATIONS: These data demonstrate that superoxide derived from NOX upregulates the expression of PDE5 in human VSMCs. As PDE5 hydrolyses cyclic GMP, this effect may blunt the vasculoprotective actions of NO.

Pressure effects revealed by small angle neutron scattering on block copolymer gels.

We study the effects of hydrostatic pressure (P) on aqueous solutions and gels of the block copolymer B(20)E(610) (E, oxyethylene; B, oxybutylene; subscripts, number of repeats), by performing simultaneous small angle neutron scattering/pressure experiments. Micellar cubic gels were studied for 9.5 and 4.5 wt % B(20)E(610) at T = 20-80 and 35-55 degrees C, respectively, while micellar isotropic solutions where studied for 4.5 wt % B(20)E(610) at T > 55 degrees C. We observed that the interplanar distance d 110 (cubic unit cell parameter a = [see text for formula]) decreases while the correlation length of the cubic order (delta) increases, upon increasing P at a fixed T for 9.5 wt % B(20)E(610). The construction of master curves for d(110) and delta corresponding to 9.5 wt % B(20)E(610) proved the correlation between changes in T and P. Neither d(110) and delta nor the cubic-isotropic phase transition temperature was affected by the applied pressure for 4.5 wt % B(20)E(610). The dramatic contrast between the pressure-induced behavior observed for 9.5 and 4.5 wt % B(20)E(610) suggests that pressure induced effects might be more effectively transmitted through samples that present wider domains of cubic structure order (9.5 wt % compared to 4.5 wt % B(20)E(610)).

Acute inhibition of superoxide formation and Rac1 activation by nitric oxide and iloprost in human vascular smooth muscle cells in response to the thromboxane A2 analogue, U46619.

BACKGROUND: The over-production of superoxide (O(2)(-)) derived from NADPH oxidase (NOX) plays a central role in cardiovascular diseases. By contrast, nitric oxide (NO) and prostacyclin (PGI(2)) are vasculoprotective. The effect of the NO donor, NONOate and iloprost on O(2)(-) formation, p47(phox) and Rac(1) activation in human vascular smooth muscle cells (hVSMCs) was investigated. METHODS: hVSMCs were incubated with 10nM thromboxane A(2) analogue, U46619 for 16h, and then with apocynin (a NOX inhibitor), NONOate or iloprost for 1h and O(2)(-) measured spectrophometrically. The role of cyclic AMP and cyclic GMP was examined by co-incubation of drugs with protein kinase (PK) A and G inhibitors listed above. Rac(1) was studied using pull-down assays. RESULTS: NONOate and iloprost inhibited O(2)(-) formation, acutely, effects blocked by inhibition of PKG and PKA, respectively. Rac(1) and p47(phox) activation and translocation to the plasma membrane was completely inhibited by NONOate and iloprost, effects again reversed by co-incubation with PKG or PKA inhibitors. CONCLUSIONS: NO and PGI(2) block the acute activity of NOX in hVSMCs via the cGMP-PKG axis (for NO) and by the cAMP-PKA axis (for iloprost) through inhibition of Rac(1) and p47(phox) translocation. These findings have implications in the pathophysiology and treatment of CVD.

Identification of the prosurvival activity of nerve growth factor on cardiac myocytes.

Neurotrophins (NTs) control neuron survival and regeneration. Recent research showed that NTs possess cardiovascular actions. In this study, we investigated the hypothesis that the NT nerve growth factor (NGF) prevents cardiomyocyte apoptosis. We demonstrated that cultured rat neonatal cardiomyocytes (RNCMs) produce NGF and express its trkA (tropomyosin-related receptor A (NGF high-affinity receptor)) receptor. RNCMs given a neutralizing antibody for NGF or the trkA inhibitor K252a underwent apoptosis, thus suggesting that NGF is an endogenous prosurvival factor for cardiomyocytes. Adenovirus (Ad)-mediated NGF overexpression protected RNCMs from apoptosis induced by either hypoxia/reoxygenation or angiotensin II (AngII). Similarly, recombinant NGF inhibited AngII-induced apoptosis in isolated rat adult cardiomyocytes. Finally, in a rat model of myocardial infarction, NGF gene transfer promoted cardiomyocyte survival. In RNCMs, recombinant NGF induced trkA phosphorylation, followed by Ser473 phosphorylation and nuclear translocation of phospho-protein kinase B (Akt). In response to Akt activation, Forkhead transcription factors Foxo-3a and Foxo-1 were phosphorylated and excluded from the nucleus. The prosurvival effect of adenoviral vector carrying the human NGF gene was inhibited in vitro by K252a, LY294002 (a pan-phosphatidyl inositol 3-kinase - PI3K - inhibitor), an Akt small interfering RNA, and adenoviruses carrying a dominant negative mutant form of Akt (Ad.DN.Akt) or an Akt-resistant Foxo-3a (Ad.AAA-Foxo-3a). These results newly demonstrate the cardiac prosurvival action of NGF and provide mechanistic information on the signaling pathway, which encompasses trkA, PI3K-Akt, and Foxo.

Cloning of a mouse cytosolic 5'-nucleotidase-I identifies a new gene related to human autoimmune infertility-related protein.

Adenosine production catalysed by cytosolic 5'-nucleotidase (cN-I) regulates diverse physiological processes. We report here a mouse cN-I (mcN-I) cloned from heart and testis. The open reading frame contains several potential translation initiation sites, which yield similarly active 5'-nucleotidases. Using overexpression in COS-7 cells we showed that mcN-I, like the previously cloned pigeon cN-I, is activated by ADP and catalyses adenosine formation during ATP breakdown. The N- and C-termini of mcN-I and pcN-I are divergent. Deletion of the 12 C-terminal amino acids or the first 19 N-terminal amino acids of pcN-I does not diminish activity, although deletion of the first 31 N-terminal amino acids reduces activity by 70%. Overall mcN-I is only 66% identical to pcN-I or the recently cloned human cN-I (hcN-I), while hcN-I and pcN-I are 85% identical. We report here a partial hcN-I sequence that is only 70% identical with the published hcN-I amino acid sequence but is 87% identical with mcN-I. Both hcN-I sequences have perfect matches to distinct human genome sequences. Our data imply the existence of at least two genes for cN-I, cN-I(A), previously cloned from pigeon and human, and cN-I(B) that we report here from mouse and partially from human.

Distinct roles for recombinant cytosolic 5'-nucleotidase-I and -II in AMP and IMP catabolism in COS-7 and H9c2 rat myoblast cell lines.

Catabolism of AMP during ATP breakdown produces adenosine, which restores energy balance. Catabolism of IMP may be a key step regulating purine nucleotide pools. Two, cloned cytosolic 5'-nucleotidases (cN-I and cN-II) have been implicated in AMP and IMP breakdown. To evaluate their roles directly, we expressed recombinant pigeon cN-I or human cN-II at similar activities in COS-7 or H9c2 cells. During rapid (more than 90% in 10 min) or slower (30-40% in 10 min) ATP catabolism, cN-I-transfected COS-7 and H9c2 cells produced significantly more adenosine than cN-II-transfected cells, which were similar to control-transfected cells. Inosine and hypoxanthine concentrations increased only during slower ATP catabolism. In COS-7 cells, 5'-nucleotidase activity was not rate-limiting for inosine and hypoxanthine production, which was therefore unaffected by cN-II- and actually reduced by cN-I- overexpression. In H9c2 cells, in which 5'-nucleotidase activity was rate-limiting, only cN-II overexpression accelerated inosine and hypoxanthine formation. Guanosine formation from GMP was also increased by cN-II. Our results imply distinct roles for cN-I and cN-II. Under the conditions tested in these cells, only cN-I plays a significant role in AMP breakdown to adenosine, whereas only cN-II breaks down IMP to inosine and GMP to guanosine.

Cloning and expression of the bioluminescent photoprotein pholasin from the bivalve mollusc Pholas dactylus.

Pholasin is the photoprotein responsible for luminescence in the bivalve Pholas dactylus and consists of a luciferin tightly bound to a glycosylated protein. It is a sensitive indicator of reactive oxygen species. A full-length clone encoding apopholasin was isolated from a P. dactylus light organ cDNA library. The unprocessed apoprotein contained 225 amino acids, starting with a signal peptide of 20 amino acids, 3 predicted N-linked glycosylation sites, 1 O-linked site, no histidines, and 7 cysteines. The recombinant apoprotein was expressed in cell extracts and insect cells. The size of the apoprotein expressed in cell extracts and the cytosol of insect cells was 26 kDa but that of the fully processed protein was 34 kDa, as was native pholasin. Both the processed and unprocessed recombinant apoproteins were recognized by a polyclonal antibody raised against native pholasin. Acid methanol extracts from Pholas added to recombinant apoprotein resulted in chemiluminescence triggered by sodium hypochlorite but not photoprotein formation. These results have important implications in understanding the molecular evolution of bioluminescence and will allow the development of recombinant pholasin as an intracellular indicator of reactive oxygen species.

The mechanism of adenosine formation in cells. Cloning of cytosolic 5'-nucleotidase-I.

Adenosine increases blood flow and decreases excitatory nerve firing. In the heart, it reduces rate and force of contraction and preconditions the heart against injury by prolonged ischemia. Based on indirect kinetic arguments, an AMP-selective cytosolic 5'-nucleotidase designated cN-I has been implicated in adenosine formation during ATP breakdown. The molecular identity of cN-I is unknown, although an IMP/GMP-selective cytosolic 5'-nucleotidase (cN-II) and an ecto-5'-nucleotidase (e-N) have been cloned. We utilized the high abundance of cN-I in pigeon heart to purify a 40-kDa subunit for partial protein sequencing and subsequent cDNA cloning. We obtained a full-length clone encoding a novel 40-kDa peptide, unrelated to cN-II or e-N, that was most abundant in heart, brain, and breast muscle. Immunolocalization in heart showed a striated cytoplasmic location, suggesting association with contractile elements. Transient expression in COS-7 cells, generated a 5'-nucleotidase that catalyzed adenosine formation from AMP, which was increased during ATP catabolism. In conclusion, the cloning and expression of cN-I provides definitive evidence of its ability to produce adenosine during ATP breakdown.

Driving after severe head injury: the need for assessment.

This paper reports on a survey of return to driving after severe head injury. It highlights the lack of information provision, low rates of Driver and Vehicle Licensing Agency (DVLA) notification, and poor uptake of driving assessments. The findings highlight the need for liaison between head injury services and general practitioners (GPs) when assessing driving fitness.

Imaging bioluminescent indicators shows Ca2+ and ATP permeability thresholds in live cells attacked by complement.

A series of permeability thresholds to Ca2+ metabolites and macromolecules, occurring at different times when cells are attacked by complement, has been established by imaging HeLa cells transiently expressing a recombinant cytosolic fusion protein of firefly luciferase and aequorin (luciferase-aequorin) to measure changes in ATP and cytosolic free Ca2+. Nuclear fluorescence of propidium was used as a measure of permeability to small molecules, and luciferase activity imaged to assess lysis. The rise in cytosolic free Ca2+ observed after C9 attack preceded by at least 60 s both the increase in propidium fluorescence, measured in single cells, and the decrease in ATP monitored by luciferase light emission. These effects were dependent on the concentration of C9. At concentrations of C9 up to 4 micrograms/ml no loss of luciferase-aequorin protein was detected at the end of the experiment. Thus the membrane integrity of the cells remained intact, even though the cells were permeable to propidium. These results confirmed our earlier observations that propidium permeability in cells attacked by complement was not a reliable measure of cell death. They also show that it is vital to take account of cellular heterogeneity if the mechanisms by which cells respond to membrane pore former attack are to be correctly interpreted.

Recombinant apoaequorin acting as a pseudo-luciferase reports micromolar changes in the endoplasmic reticulum free Ca2+ of intact cells.

We describe a novel method to monitor the endoplasmic reticulum (ER) free Ca2+ in intact cells. Continuous perfusion of HeLa cells, expressing ER-targeted apoaequorin, with coelenterazine allowed the apoprotein to act as a pseudo-luciferase capable of reporting free Ca2+ from 0.1-100 microM. In intact HeLa cells, addition of ionomycin increased apoaequorin-generated light by 91%, indicating that resting ER free Ca2+ was approx. 2 microM. Agonist stimulation decreased the ER apoaequorin signal and proportionally increased cytosolic free Ca2+ consistent with agonist-induced release of Ca2+ from the ER.

Sequence and biochemical similarities between the luciferases of the glow-worm Lampyris noctiluca and the firefly Photinus pyralis.

A full-length clone encoding Lampyris noctiluca (British glow-worm) luciferase was isolated from a complementary DNA (cDNA) expression library constructed with MRNA extracted from light organs. The luciferase was a 547-residue protein, as deduced from the nucleotide sequence. The protein was closely related to those of other lampyrid beetles, the similarity to Photinus pyralis luciferase being 84% and to Luciola 67%. In contrast, Lampyris luciferase had less sequence similarity to the luciferases of the click beetle Pyrophorus, at 48%. Engineering Lampyris luciferase in vitro showed that the C-terminal peptide containing 12 amino acids in Photinus and 9 amino acids in Lampyris was essential for bioluminescence. The pH optimum and the Km values for ATP and luciferin were similar for both Photinus and Lampyris luciferases, although the light emitted by the latter shifted towards the blue and was less stable at 37 degrees C. It was concluded that the molecular and biochemical properties were not sufficient to explain the glowing or flashing of the two beetles Lampyris and Photinus.