Submicron focusing of high-energy X-rays with silicon saw-tooth refractive lenses - fabrication and aberrations: errata.

Three (3) items of errata are submitted for our recently published paper [Opt. Express28, 36505 (2020)10.1364/OE.405566]. One is a clarifying extension to the Fig. 3 caption. Two are typographical corrections. The scientific results and conclusions are unaffected.

Submicron focusing of high-energy X-rays with silicon saw-tooth refractive lenses: fabrication and aberrations.

Saw-tooth refractive lenses are extremely well-suited to focus high energy X-rays (>50 keV). These lenses have properties of being continuously tunable (in energy or focal length), effectively parabolic, in-line, and attenuation-free on-axis. Vertical focusing of 60 keV synchrotron X-rays to 690 nm at a focal length f = 1.3 m with silicon saw-tooth lenses at a high-energy undulator radiation beamine is demonstrated, with discussion of relevant fabrication and mounting considerations and of geometrical aberrations unique to these devices. Aberration corrections towards further progress into the diffraction-limited nanofocusing regime are suggested. The versatility of such optics, combined with the attainability of smaller spot sizes at these penetrating photon energies, should continue to enhance material microstructure investigations at increasingly higher spatial resolutions.

A heuristic computational model of basic cellular processes and oxygenation during spheroid-dependent biofabrication.

An emerging approach in biofabrication is the creation of 3D tissue constructs through scaffold-free, cell spheroid-only methods. The basic mechanism in this technology is spheroid fusion, which is driven by the minimization of energy, the same biophysical mechanism that governs spheroid formation. However, other factors such as oxygen and metabolite accessibility within spheroids impact on spheroid properties and their ability to form larger-scale structures. The goal of our work is to develop a simulation platform eventually capable of predicting the conditions that minimize metabolism-related cell loss within spheroids. To describe the behavior and dynamic properties of the cells in response to their neighbors and to transient nutrient concentration fields, we developed a hybrid discrete-continuous heuristic model, combining a cellular Potts-type approach with field equations applied to a randomly populated spheroid cross-section of prescribed cell-type constituency. This model allows for the description of: (i) cellular adhesiveness and motility; (ii) interactions with concentration fields, including diffusivity and oxygen consumption; and (iii) concentration-dependent, stochastic cell dynamics, driven by metabolite-dependent cell death. Our model readily captured the basic steps of spheroid-based biofabrication (as specifically dedicated to scaffold-free bioprinting), including intra-spheroid cell sorting (both in 2D and 3D implementations), spheroid defect closure, and inter-spheroid fusion. Moreover, we found that when hypoxia occurring at the core of the spheroid was set to trigger cell death, this was amplified upon spheroid fusion, but could be mitigated by external oxygen supplementation. In conclusion, optimization and further development of scaffold-free bioprinting techniques could benefit from our computational model which is able to simultaneously account for both cellular dynamics and metabolism in constructs obtained by scaffold-free biofabrication.

Development of optics for x-ray phase-contrast imaging of high energy density plasmas.

Phase-contrast or refraction-enhanced x-ray radiography can be useful for the diagnostic of low-Z high energy density plasmas, such as imploding inertial confinement fusion (ICF) pellets, due to its sensitivity to density gradients. To separate and quantify the absorption and refraction contributions to x-ray images, methods based on microperiodic optics, such as shearing interferometry, can be used. To enable applying such methods with the energetic x rays needed for ICF radiography, we investigate a new type of optics consisting of grazing incidence microperiodic mirrors. Using such mirrors, efficient phase-contrast imaging systems could be built for energies up to ∼100 keV. In addition, a simple lithographic method is proposed for the production of the microperiodic x-ray mirrors based on the difference in the total reflection between a low-Z substrate and a high-Z film. Prototype mirrors fabricated with this method show promising characteristics in laboratory tests.

Preventing nanoscale wear of atomic force microscopy tips through the use of monolithic ultrananocrystalline diamond probes.

Nanoscale wear is a key limitation of conventional atomic force microscopy (AFM) probes that results in decreased resolution, accuracy, and reproducibility in probe-based imaging, writing, measurement, and nanomanufacturing applications. Diamond is potentially an ideal probe material due to its unrivaled hardness and stiffness, its low friction and wear, and its chemical inertness. However, the manufacture of monolithic diamond probes with consistently shaped small-radius tips has not been previously achieved. The first wafer-level fabrication of monolithic ultrananocrystalline diamond (UNCD) probes with <5-nm grain sizes and smooth tips with radii of 30-40 nm is reported, which are obtained through a combination of microfabrication and hot-filament chemical vapor deposition. Their nanoscale wear resistance under contact-mode scanning conditions is compared with that of conventional silicon nitride (SiN(x)) probes of similar geometry at two different relative humidity levels (approximately 15 and approximately 70%). While SiN(x) probes exhibit significant wear that further increases with humidity, UNCD probes show little measurable wear. The only significant degradation of the UNCD probes observed in one case is associated with removal of the initial seed layer of the UNCD film. The results show the potential of a new material for AFM probes and demonstrate a systematic approach to studying wear at the nanoscale.

A subpopulation of peritoneal macrophages form capillarylike lumens and branching patterns in vitro.

OBJECTIVE: We have previously shown that monocytes/macrophages (MC/Mph) influence neovascularization by extracellular matrix degradation, and by direct incorporation into growing microvessels. To date, neither the phenotype of these cells, nor the stages of their capillary-like conversion were sufficiently characterized. METHODS: We isolated mouse peritoneal Mph from transgenic mice expressing fluorescent proteins either ubiquitously, or specifically in the myelocytic lineage. These Mph were embedded in Matrigel which contained fluorescent protease substrates, exposed to an MCP-1 chemotactic gradient, and then examined by confocal microscopy after various intervals. RESULTS: Within 3 hrs after gel embedding, we detected TIMP-1 and MMP-12 dependent proteolysis of the matrix surrounding Mph, mostly in the direction of high concentrations of MCP-1. After 2 days, Mph developed intracellular vacuoles containing degradation product. At 5 days these vacuoles were enlarged and/or fused to generate trans-cellular lumens in approximately 10% of cells or more (depending on animal's genetic background). At this stage, Mph became tubular, and occasionally organized in three-dimensional structures resembling branched microvessels. CONCLUSION: Isolated mouse peritoneal Mph penetrate Matrigel and form tunnels via a metalloprotease-driven proteolysis and phagocytosis. Following a morphological adjustment driven by occurrence, enlargement and/or fusion process of intracellular vacuoles, similar to that described in bona fide endothelium, a subpopulation of these cells end up by lining a capillary-like lumen in vitro. Thus we show that adult Mph, not only the more primitive 'endothelial progenitors', have functional properties until now considered defining of the endothelial phenotype.

Measurement of the spatial coherence function of undulator radiation using a phase mask.

A measurement of the horizontal coherence function of 7.9 keV radiation from an undulator beam line at the Advanced Photon Source is reported. X-ray diffraction from a phase-shifting mask was used, and the coherence function was measured as a function of the width of beam-conditioning slits in the beam line. The coherence distribution is found to be best described by a Lorentzian function.

Contribution of monocytes/macrophages to compensatory neovascularization: the drilling of metalloelastase-positive tunnels in ischemic myocardium.

In a transgenic model of ischemic cardiomyopathy in which monocytes are attracted to the myocardium by the targeted overexpression of monocyte chemoattractant protein-1 (MCP-1), we have observed the presence of endothelial NO synthase and platelet endothelial cell adhesion molecule-1-negative tunnels, occasionally containing blood-derived cells, that probe the cardiac tissue. Immunohistochemical data show that monocytes/macrophages (MCs/Mphs) drill tunnels using the broad-spectrum mouse macrophage metalloelastase. 5-Bromo-2'-deoxyuridine incorporation and neo-endothelial markers present in the microvasculature of MCP-1 mouse hearts suggest an active angiogenic process. Further studies will be required to establish that the MC-/Mph-drilled tunnels evolve to become capillaries, connected to the existing vessels and colonized by circulating endothelial cell progenitors. This possibility is supported by the availability of these cells, which is demonstrated by cell tagging with beta-galactosidase placed under an active endothelial Tie-2 promoter. This phenomenon might represent another mechanism, in addition to the secretion of the angiogenic factors, by which MCs/MPhs may participate in the elaboration of new blood vessels in adult tissues.

Endothelial dysfunction in a murine model of mild hyperhomocyst(e)inemia.

Homocysteine is a risk factor for the development of atherosclerosis and its thrombotic complications. We have employed an animal model to explore the hypothesis that an increase in reactive oxygen species and a subsequent loss of nitric oxide bioactivity contribute to endothelial dysfunction in mild hyperhomocysteinemia. We examined endothelial function and in vivo oxidant burden in mice heterozygous for a deletion in the cystathionine beta-synthase (CBS) gene, by studying isolated, precontracted aortic rings and mesenteric arterioles in situ. CBS(-/+) mice demonstrated impaired acetylcholine-induced aortic relaxation and a paradoxical vasoconstriction of mesenteric microvessels in response to superfusion of methacholine and bradykinin. Cyclic GMP accumulation following acetylcholine treatment was also impaired in isolated aortic segments from CBS(-/+) mice, but aortic relaxation and mesenteric arteriolar dilation in response to sodium nitroprusside were similar to wild-type. Plasma levels of 8-epi-PGF(2alpha) (8-IP) were somewhat increased in CBS(-/+) mice, but liver levels of 8-IP and phospholipid hydroperoxides, another marker of oxidative stress, were normal. Aortic tissue from CBS(-/+) mice also demonstrated greater superoxide production and greater immunostaining for 3-nitrotyrosine, particularly on the endothelial surface. Importantly, endothelial dysfunction appears early in CBS(-/+) mice in the absence of structural arterial abnormalities. Hence, mild hyperhomocysteinemia due to reduced CBS expression impairs endothelium-dependent vasodilation, likely due to impaired nitric oxide bioactivity, and increased oxidative stress apparently contributes to inactivating nitric oxide in chronic, mild hyperhomocysteinemia.

Leukotrienes and tyrosine phosphorylation mediate stretching-induced actin cytoskeletal remodeling in endothelial cells.

We studied actin cytoskeletal remodeling and the role of leukotrienes and tyrosine phosphorylation in the response of endothelial cells to different types of cyclic mechanical stretching. Human aortic endothelial cells were grown on deformable silicone membranes subjected to either cyclic one-directional (strip) stretching (10%, 0.5 Hz), or biaxial stretching. After 1 min of either type of stretching, actin cytoskeletons of the stretched cells were already disrupted. After stretching for 10 and 30 min, the percentage of the stretched cells that had disrupted actin cytoskeletons were significantly increased, compared with control cells without stretching. Also, at these two time points, biaxial stretching consistently produced higher frequencies of actin cytoskeleton disruption. At 3 h, strip stretching caused the formation of stress fiber bundles, which were oriented nearly perpendicular to the stretching direction. With biaxial stretching, however, actin cytoskeletons in many stretched cells were remodeled into three-dimensional actin structures protruding outside the substrate plane, within which cyclic stretching was applied. In both stretching conditions, actin filaments were formed in the direction without substrate deformation. Moreover, substantially inhibiting either leukotriene production with nordihydroguaiaretic acid or tyrosine phosphorylation with tyrphostin A25 did not block the actin cytoskeletal remodeling. However, inhibiting both leukotriene production and tyrosine phosphorylation completely blocked the actin cytoskeletal remodeling. Thus, the study showed that the remodeling of actin cytoskeletons of the stretched endothelial cells include rapid disruption first and then re-formation. The resulting pattern of the actin cytoskeleton after remodeling depends on the type of cyclic stretching applied, but under either type of cyclic stretching, the actin filaments are formed in the direction without substrate deformation. Finally, leukotrienes and tyrosine phosphorylation are necessary for actin cytoskeletal remodeling of the endothelial cells in response to mechanical stretching.

Redox changes of cultured endothelial cells and actin dynamics.

We studied the association between the production of reactive oxygen species, actin organization, and cellular motility. We have used an endothelial cell monolayer-wounding assay to demonstrate that the cells at the margin of the wound thus created produced significantly more free radicals than did cells in distant rows. The rate of incorporation of actin monomers into filaments was fastest at the wound margin, where heightened production of free radicals was detected. We have tested the effect of decreasing reactive oxygen species production on the migration of endothelial cells and on actin polymerization. The NADPH inhibitor diphenylene iodonium and the superoxide dismutase mimetic manganese (III) tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP) virtually abolished cytochalasin D-inhibitable actin monomer incorporation at the fast-growing barbed ends of filaments. Moreover, endothelial cell migration within the wound was significantly retarded in the presence of both diphenylene iodonium and MnTMPyP. We conclude that migration of endothelial cells in response to loss of confluence includes the intracellular production of reactive oxygen species, which contribute to the actin cytoskeleton reorganization required for the migratory behavior of endothelial cells.

Regulation of monocyte survival in vitro by deposited IgG: role of macrophage colony-stimulating factor.

IgG deposition at tissue sites characteristically leads to macrophage accumulation and organ injury. Although the mechanism by which deposited IgG induces tissue injury is not known, we have recently demonstrated that deposited IgG stimulates the release of IL-8 and monocyte chemoattractant protein-1 from normal human monocytes, which may drive inflammation. Since IgG also induces macrophage accumulation in these diseases, we hypothesized that deposited IgG protects monocytes from apoptosis. As an in vitro model of the effect of deposited IgG on monocyte survival, monocyte apoptosis was studied after FcgammaR cross-linking. Monocytes cultured on immobilized IgG, which induces FcgammaR cross-linking, were protected from apoptosis, whereas monocytes cultured with equivalent concentrations of F(ab')2 IgG or 50 times higher concentrations of soluble IgG, neither of which induces FcgammaR cross-linking, were not protected. Moreover, this protection was transferable, as supernatants from immobilized IgG-stimulated monocytes protected freshly isolated monocytes from apoptosis and contained functional M-CSF, a known monocyte survival factor. M-CSF mediated the monocyte survival induced by FcgammaR cross-linking, as neutralizing anti-human M-CSF Abs blocked the monocyte protection provided by either immobilized IgG or IgG-stimulated monocyte supernatants. These findings demonstrate a novel mechanism by which deposited IgG targets tissue macrophage accumulation through FcgammaR-mediated M-CSF release. This pathway may play an important role in promoting and potentiating IgG-mediated tissue injury.

The actin cytoskeleton reorganization induced by Rac1 requires the production of superoxide.

The small GTPase rac1 controls actin redistribution to membrane ruffles in fibroblasts and other cell types, as well as the activation of the NADPH oxidase in phagocytes. We explored the possibility that these two processes could be related. We used a replication-deficient adenoviral vector to overexpress the constitutively active form of rac1, racV12, in human and mouse aortic endothelial cells. We show here that, in addition to membrane ruffle formation, racV12 induced an increase in the total amount of F-actin within endothelial cells. Concurrently, racV12-overexpressing cells produced significantly higher amounts of free radicals, as detected by the fluorescent probe 5-(and-6)-chloromethyl-2',7'-dichloro-dihydrofluorescein diacetate, than cells infected with a control virus encoding the bacterial beta-galactosidase (Ad-betaGal). To assess the specific role of superoxide in racV12-induced actin reorganization, we co-expressed the human enzyme Cu,Zn-superoxide dismutase (SOD), by means of another adenoviral vector construct. Overexpressed SOD reduced the concentration of superoxide detected in Ad-racV12-transfected cells and reversed the effects of Ad-racV12 on the content of filamentous actin. MnTMPyP, an SOD mimetic, as well as the antioxidant N-acetyl cysteine, had similar effects, in that they reduced not only the free radicals production, but also ruffle formation and the concentration of F-actin within racV12-overexpressing endothelial cells. Our data support the hypothesis that superoxide is one of the important mediators acting downstream of rac1 on the pathway of actin cytoskeleton remodeling in endothelial cells.

Fas-mediated apoptosis in accelerated graft arteriosclerosis.

Pathological conditions have been recognized where vessel destruction is a prominent feature of the pathogenic process. One such condition consists of the chronic rejection of blood vessels in transplanted solid organs. Accelerated graft arteriosclerosis (AGA) is a multifactorial process characterized by the concentric proliferation of smooth muscle cells (SMCs) within the intima of the vessel wall of transplanted organs. Proliferation of SMCs within the intima corresponds to a response of these cells to injury. In situations like restenosis post-angioplasty, the mechanism of injury: the mechanical disruption of the tunica media, is evident. However, in the case of AGA, the mechanism of injury has remained elusive. In this report, we provide evidence that injury to SMCs in AGA vessels requires an intact Fas pathway. The resulting damage to the tunica media and internal elastic lamina, in turn, might trigger the proliferation of intimal smooth muscle cells that appear to be less sensitive to Fas mediated killing, particularly when supported by a favorable context of inflammatory cytokines and growth factors, as it is the case in AGA. This pathogenic process results in a absolute loss of functional blood vessels that is not being compensated by an efficient angiogenic response.

17beta-estradiol inhibits apoptosis of endothelial cells.

Endothelial cells provide an antithrombotic and anti-inflammatory barrier for the normal vessel wall. Dysfunction of endothelial cells has been shown to promote atherosclerosis, and normalization of previously dysfunctional endothelial cells can inhibit the genesis of atheroma. In normal arteries, endothelial cells are remarkably quiescent. Acceleration of the turnover rate of endothelial cells can lead to their dysfunction. Apoptosis is a physiological process that contributes to vessel homeostasis, by eliminating damaged cells from the vessel wall. However, increased endothelial cell turnover mediated through accelerated apoptosis may alter the function of the endothelium and therefore, promote atherosclerosis. Apoptotic endothelial cells can be detected on the luminal surface of atherosclerotic coronary vessels, but not in normal vessels. This finding links endothelial cell apoptosis and the process of atherosclerosis, although a causative role for apoptosis in this process remains hypothetical. Estrogen metabolites have been shown to be among the most potent anti-atherogenic agents available to date for post-menopausal women. The mechanism of estrogen's protective effect is currently incompletely characterized. Here we show that 17beta-estradiol, a key estrogen metabolite, inhibits apoptosis in cultured endothelial cells. Our data support the hypothesis that 17beta-estradiol's anti-apoptotic effect may be mediated via improved endothelial cell interaction with the substratum, increased tyrosine phosphorylation of pp125 focal adhesion kinase, and a subsequent reduction in programmed cell death of endothelial cells. Inhibition of apoptosis by estrogens may account for some of the anti-atherogenic properties of these compounds.

Regulation of endothelial cell adhesion by profilin.

BACKGROUND: Although profilin is believed to be an essential regulator of the actin cytoskeleton in most cells, its precise role in mammalian cells remains unknown. We have used replication-incompetent adenovirus carrying the human profilin I cDNA as a means rapidly to increase the concentration of profilin in human aortic endothelial cells 12-31-fold above baseline--levels never before achieved in mammalian cells. RESULTS: The concentration of filamentous actin was not detectably affected by profilin overexpression. Actin stress fibers were, however, absent from areas of high profilin content in overexpressing cells, and the bulk of filaments was located at the periphery of the cells. We observed a gradient in the distribution of overexpressed profilin in migrating endothelial cells, with most profilin molecules concentrated near the advancing edge where focal contacts are being formed and focal adhesion proteins are located. Profilin overexpression resulted in increased recruitment of fibronectin receptors to the plasma membrane. Adhesion of endothelial cells to fibronectin was markedly and selectively increased by profilin overexpression. CONCLUSIONS: We conclude that an important role for profilin in mammalian cells may be its contribution to the formation of focal contacts, particularly those involving the fibronectin receptor.

Ultrastructural evidence of differential solubility in Triton X-100 of endothelial vesicles and plasma membrane.

Endothelial plasmalemmal vesicles (EV) are distinct membrane-bound structures characteristic for all vascular endothelia, being involved in transcytosis of plasma macromolecules. EV are considered to be similar to the caveolae (characterized by a specific peptide called caveolin) found in other cell types. Caveolin-rich membrane domains were recently isolated from whole lung and chicken gizzard as a Triton X-100 (TX)-insoluble membrane fraction. However, ultrastructural data on the localization of these domains within cells have not yet been reported. We have examined whether EV are TX-insoluble structures. Cultured bovine aortic endothelial cells (BAEC) briefly fixed in paraformaldehyde (10 min, 37 degrees C) were exposed to 0.1% TX for 5 min at 22 degrees C and further subjected to standard electron microscopy procedure. The results showed an extensive solubilization of endothelial plasmalemma as well as other intracellular membranes. Individual or clusters of EV were not affected by TX extraction, retaining their trilaminar unit membrane appearance and dimensions. Moreover, a crude membrane fraction prepared from unfixed BAEC was also extracted with 1% TX for 20 min at 4 degrees C and the insoluble material was examined by electron microscopy. In this fraction clusters of about 10 membranous vesicles were found. These data suggest that EV and plasma membrane have a different lipid composition; the low TX solubility is a characteristic common to caveolin-rich domains (caveolae) of other cells types and EV, whereas the ultrastructural complexity and intracellular localization of the latter are specific for endothelia.

Binding of vascular anticoagulant alpha (annexin V) to the aortic intima of the hypercholesterolemic rabbit. An autoradiographic study.

Qualitative ultrastructural autoradiography was used to study the binding of the vascular anticoagulant alpha (annexin V) to normal and atherosclerotic (AS) rabbit aortic intima. Recombinant annexin V was labelled with 125I by the Iodogen method. Rabbits were fed a hypercholesterolemic (HC) diet up to 10 months. After laparotomy and exsanguination, the aortae were perfused with dilutions of 125I-annexin V (I-AV) for 10-15 min, either on ice or at 22 degrees C and then perfusion-fixed with aldehydes. Fragments of the labelled aortae were used for en face contact autoradiography, followed by Sudan Black staining of intimal lipid. Specimens were also included in Epon and sectioned for light- and electron-microscopic autoradiography. The binding of I-AV was increased on the AS aortae as compared with the normal ones, with an apparent preference for the lesioned areas. Microscopically, I-AV was found at the luminal front of aortic intima, on endothelial cells (EC), on macrophage foam cells, and on their disrupted remnants. The presence of the AV binding sites (reportedly known to interact with high affinity with phosphatidylserine) in the rabbit AS aortic intima, together with other known procoagulant conditions, may contribute to the initiation of coagulation events into the lesioned vascular wall, and may offer a rationale for the use of annexin V as an anticoagulant drug.