3D Imaging of a Dislocation Loop at the Onset of Plasticity in an Indented Nanocrystal.

Structural quality and stability of nanocrystals are fundamental problems that bear important consequences for the performances of small-scale devices. Indeed, at the nanoscale, their functional properties are largely influenced by elastic strain and depend critically on the presence of crystal defects. It is thus of prime importance to be able to monitor, by noninvasive means, the stability of the microstructure of nano-objects against external stimuli such as mechanical load. Here we demonstrate the potential of Bragg coherent diffraction imaging for such measurements, by imaging in 3D the evolution of the microstructure of a nanocrystal exposed to in situ mechanical loading. Not only could we observe the evolution of the internal strain field after successive loadings, but we also evidenced a transient microstructure hosting a stable dislocation loop. The latter is fully characterized from its characteristic displacement field. The mechanical behavior of this small crystal is clearly at odds with what happens in bulk materials where many dislocations interact. Moreover, this original in situ experiment opens interesting possibilities for the investigation of plastic deformation at the nanoscale.

Host bone-marrow cells are a source of donor intimal smooth- muscle-like cells in murine aortic transplant arteriopathy.

Long-term solid-organ allografts typically develop diffuse arterial intimal lesions (graft arterial disease; GAD), consisting of smooth-muscle cells (SMC), extracellular matrix and admixed mononuclear leukocytes. GAD eventually culminates in vascular stenosis and ischemic graft failure. Although the exact mechanisms are unknown, chronic low-level alloresponses likely induce inflammatory cells and/or dysfunctional vascular wall cells to secrete growth factors that promote SMC intimal recruitment, proliferation and matrix synthesis. Although prior work demonstrated that the endothelium and medial SMCs lining GAD lesions in cardiac allografts are donor-derived, the intimal SMC origin could not be determined. They are generally presumed to originate from the donor media, leading to interventions that target donor medial SMC proliferation, with limited efficacy. However, other reports indicate that allograft vessels may contain host-derived endothelium and SMCs (refs. 8,9). Moreover, subpopulations of bone-marrow and circulating cells can differentiate into endothelium, and implanted synthetic vascular grafts are seeded by host SMCs and endothelium. Here we used murine aortic transplants to formally identify the source of SMCs in GAD lesions. Allografts in beta-galactosidase transgenic recipients showed that intimal SMCs derived almost exclusively from host cells. Bone-marrow transplantation of beta-galactosidase--expressing cells into aortic allograft recipients demonstrated that intimal cells included those of marrow origin. Thus, smooth-muscle--like cells in GAD lesions can originate from circulating bone--marrow-derived precursors.

Functional small-diameter neovessels created using endothelial progenitor cells expanded ex vivo.

Arterial conduits are increasingly preferred for surgical bypass because of inherent functional properties conferred by arterial endothelial cells, especially nitric oxide production in response to physiologic stimuli. Here we tested whether endothelial progenitor cells (EPCs) can replace arterial endothelial cells and promote patency in tissue-engineered small-diameter blood vessels (4 mm). We isolated EPCs from peripheral blood of sheep, expanded them ex vivo and then seeded them on decellularized porcine iliac vessels. EPC-seeded grafts remained patent for 130 days as a carotid interposition graft in sheep, whereas non-seeded grafts occluded within 15 days. The EPC-explanted grafts exhibited contractile activity and nitric-oxide-mediated vascular relaxation that were similar to native carotid arteries. These results indicate that EPCs can function similarly to arterial endothelial cells and thereby confer longer vascular-graft survival. Due to their unique properties, EPCs might have other general applications for tissue-engineered structures and in treating vascular diseases.

Nickel nanoparticles set a new record of strength.

Material objects with micrometer or nanometer dimensions can exhibit much higher strength than macroscopic objects, but this strength rarely approaches the maximum theoretical strength of the material. Here, we demonstrate that faceted single-crystalline nickel (Ni) nanoparticles exhibit an ultrahigh compressive strength (up to 34 GPa) unprecedented for metallic materials. This strength matches the available estimates of Ni theoretical strength. Three factors are responsible for this record-high strength: the large Ni shear modulus, the smooth edges and corners of the nanoparticles, and the thin oxide layer on the particle surface. This finding is supported by molecular dynamics simulations that closely mimic the experimental conditions, which show that the mechanical failure of the strongest particles is triggered by homogeneous nucleation of dislocation loops inside the particle. The nucleation of a stable loop is preceded by multiple nucleation attempts accompanied by unusually large local atomic displacements caused by thermal fluctuations.

Targeted deletion of matrix metalloproteinase-9 attenuates left ventricular enlargement and collagen accumulation after experimental myocardial infarction.

Matrix metalloproteinase-9 (MMP-9) is prominently overexpressed after myocardial infarction (MI). We tested the hypothesis that mice with targeted deletion of MMP9 have less left ventricular (LV) dilation after experimental MI than do sibling wild-type (WT) mice. Animals that survived ligation of the left coronary artery underwent echocardiographic studies after MI; all analyses were performed without knowledge of mouse genotype. By day 8, MMP9 knockout (KO) mice had significantly smaller increases in end-diastolic and end-systolic ventricular dimensions at both midpapillary and apical levels, compared with infarcted WT mice; these differences persisted at 15 days after MI. MMP-9 KO mice had less collagen accumulation in the infarcted area than did WT mice, and they showed enhanced expression of MMP-2, MMP-13, and TIMP-1 and a reduced number of macrophages. We conclude that targeted deletion of the MMP9 gene attenuates LV dilation after experimental MI in mice. The decrease in collagen accumulation and the enhanced expression of other MMPs suggest that MMP-9 plays a prominent role in extracellular matrix remodeling after MI.

Neuroprotection mediated by changes in the endothelial actin cytoskeleton.

Cerebral blood flow is regulated by endothelium-derived nitric oxide (NO), and endothelial NO synthase-deficient (eNOS-deficient; eNOS(-/-)) mice develop larger cerebral infarctions following middle cerebral artery (MCA) occlusion. We report that disruption of Rho-mediated endothelial actin cytoskeleton leads to the upregulation of eNOS expression and reduces the severity of cerebral ischemia following MCA occlusion. Mice treated with the Rho inhibitor Clostridium botulinum C3 transferase (10 microgram/d) or the actin cytoskeleton disrupter cytochalasin D (1 mg/kg) showed a two- to fourfold increase in vascular eNOS expression and activity. This increase in eNOS expression was not due to increases in eNOS gene transcription, but to prolongation of eNOS mRNA half-life from 10 +/- 3 hours to 24 +/- 4 hours. Indeed, endothelial cells overexpressing a dominant-negative Rho mutant (N19RhoA) exhibited decreased actin stress fiber formation and increased eNOS expression. Inhibition of vascular Rho guanosine-5'-triphosphate binding activity by the 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor simvastatin increased cerebral blood flow to ischemic regions of the brain, and mice treated with simvastatin, C3 transferase, or cytochalasin D showed smaller cerebral infarctions following MCA occlusion. No neuroprotection was observed with these agents in eNOS(-/-) mice. These findings suggest that therapies which target the endothelial actin cytoskeleton may have beneficial effects in ischemic stroke.

Dietary lipid lowering reduces tissue factor expression in rabbit atheroma.

BACKGROUND: The mechanisms by which lipid lowering reduces the incidence of acute thrombotic complications of coronary atheroma in clinical trials remains unknown. Tissue factor (TF) overexpressed in atheroma may accelerate thrombus formation at the sites of plaque disruption. A cell surface cytokine CD40 ligand (CD40L) enhances TF expression in vitro. METHODS AND RESULTS: To test the hypothesis that lipid lowering reduces TF expression and activity, we produced atheroma in rabbit aortas by balloon injury and cholesterol feeding for 4 months (Baseline group, n=15), followed by either a chow diet (Low group, n=10) or a continued high-cholesterol diet for 16 months (High group, n=5). Immunolocalization of TF, CD40L, and its receptor CD40 was quantified by computer-assisted color image analysis. Macrophages in atheroma of the Baseline and High groups strongly expressed TF. Intimal smooth muscle cells and endothelial cells also contained immunoreactive TF. Regions of expression of CD40L and CD40 colocalized with TF. Protein expression of TF diminished substantially in the Low group in association with reduced expression of CD40L and CD40. In situ binding of TF to factors VIIa and X, detected by digoxigenin-labeled factors VIIa and X, colocalized with TF protein in atheroma and decreased after lipid lowering. We also determined reduced TF biological activity in the Low group by use of a chromogenic assay. The level of TF mRNA detected by reverse transcription-polymerase chain reaction also decreased after lipid lowering. CONCLUSIONS: These results suggest decreased expression and activity of TF as a novel mechanism of reduced incidence of thrombotic complications of atherosclerosis by lipid lowering.

Statins alter smooth muscle cell accumulation and collagen content in established atheroma of watanabe heritable hyperlipidemic rabbits.

BACKGROUND: Acute coronary syndromes often result from rupture of vulnerable plaques. The collagen content of plaques probably regulates their stability. This study tested whether HMG-CoA reductase inhibitors (statins) alter interstitial collagen gene expression or matrix metalloproteinase (MMP) levels in rabbit atheroma. METHODS AND RESULTS: We administered equihypocholesterolemic doses of pravastatin (a hydrophilic statin, 50 mg. kg(-1). d(-1), n=9), fluvastatin (a cell-permeant lipophilic statin, 20 mg. kg(-1). d(-1), n=10), or placebo (n=10) to mature Watanabe heritable hyperlipidemic rabbits for 52 weeks. The fluvastatin group achieved a much higher peak plasma concentration (23.7 micromol/L) than did the pravastatin group (1.3 micromol/L) under these conditions. Immunohistochemistry revealed that MMP-1, MMP-3, and MMP-9 expression by macrophages in the intima was lower in both the pravastatin and fluvastatin groups than in the placebo group, whereas there was no difference in macrophage numbers. Numbers of intimal smooth muscle cells (SMCs) (identified by immunohistochemistry) and expression of type I procollagen mRNA (detected by in situ hybridization), however, were significantly higher in the pravastatin group than in the fluvastatin group. Treatment with pravastatin, but not fluvastatin, preserved interstitial collagen content in vivo (detected by picrosirius red polarization). In vitro, fluvastatin, but not pravastatin, decreased numbers of rabbit and human aortic SMCs without altering procollagen I mRNA expression. CONCLUSIONS: This study showed that statins can reduce MMP expression in atheroma and that cell-permeant statins can decrease SMC number and collagen gene expression in vivo.

Activated interstitial myofibroblasts express catabolic enzymes and mediate matrix remodeling in myxomatous heart valves.

BACKGROUND: The mechanisms of extracellular matrix changes accompanying myxomatous valvular degeneration are uncertain. METHODS AND RESULTS: To test the hypothesis that valvular interstitial cells mediate extracellular matrix degradation in myxomatous degeneration by excessive secretion of catabolic enzymes, we examined the functional characteristics of valvular interstitial cells in 14 mitral valves removed for myxomatous degeneration from patients with mitral regurgitation and in 11 normal mitral valves obtained at autopsy. Immunohistochemical staining assessed (1) cell phenotype using antibodies to alpha-actin (microfilaments), vimentin and desmin (intermediate filaments), smooth muscle myosin (SM1), and SMemb (a nonmuscle myosin produced by activated mesenchymal cells) and (2) the expression of proteolytic activity using antibodies to collagenases (matrix metalloproteinase [MMP]-1, MMP-13), gelatinases (MMP-2, MMP-9), cysteine endoproteases (cathepsin S and K), and interleukin-1beta, a cytokine that can induce secretion of proteolytic enzymes. Although interstitial cells in normal valves stained positively for vimentin, but not alpha-actin or desmin, cells in myxomatous valves contained both vimentin and alpha-actin or desmin (characteristics of myofibroblasts). Moreover, cells in myxomatous valves strongly expressed SMemb, MMPs, cathepsins, and interleukin-1beta, which were weakly stained in controls. Nevertheless, interstitial cells in both groups strongly expressed procollagen-I mRNA (in situ hybridization), suggesting preserved ability to synthesize collagen in myxomatous valves. CONCLUSIONS: Interstitial cells in myxomatous valves have features of activated myofibroblasts and express excessive levels of catabolic enzymes, without altered levels of interstitial collagen mRNA. We conclude that valvular interstitial cells regulate matrix degradation and remodeling in myxomatous mitral valve degeneration.

Vegetable, animal, human : The perils and powers of transgressing (sociobiological) boundaries in narrative.

Bible myths, fairy tales, and science fictions all offer narratives that imply and sometimes question boundaries for human behavior. By subscription to certain narratives, individuals can enter and leave social groups; by evolving narratives, groups can adjust the realm of the allowable and the realm of the forbidden; and by selective transgression, individuals can gain power beyond that initially granted by the group. All these functions of narrative contribute to the sociobiological vigor of the individuals and groups that subscribe to them, suggesting that the creation and use of narratives has proadaptive functions.

[Phasic character of the process of color sensitivity in the visual analyzer]. / O faznosti protsessa tsvetovoi chuvstvitel'nosti zritel'nogo analizatora

In subjects with normal three-chromatic vision, during long-term uninterrupted adaptation (6--8 min) to spectral stimuli of different spectral areas (lambda=650+585 nm; lambda=535+585 nm), it was found that the state of color sensitivity undergoes changes in the form of alternating active (correct) and passive (wrong) phases of color discrimination. First, the phase of active color discrimination prevails, then both phases become sort of balanced, and in the end of the adaptation the phase of passive color discrimination increases considerably. The time of duration of these phases depends on the wave--length of the adapting stimulus.

[Quantitative assessment of cutaneous erythema]. / O kolichestvennoi otsenke éritemy kozhi

On the basis of general concepts on the objective nature of colour perception and colourimetric tables of the colour atlas special colourimetric rulers were elaborated for the assessment of skin erythema. These rulers can be used for the assessment of experimentally-reproduced common and allergic inflammations of the skin in acute and chronic diseases. The system of assesment take into consideration the experience of the grade characteristics of inflammation of the skin, with the description of erythema with the aid of clear physical colour parameters: brightness (p, %), the wave length (lambda, nm) and the saturation (P,%).

Acute rejection accelerates graft coronary disease in transplanted rabbit hearts.

BACKGROUND: The relation between episodes of acute rejection and the development of graft coronary arteriosclerosis remains controversial. We examined the hypothesis that acute rejection episodes accelerate graft coronary arteriosclerosis lesion formation in rabbit allografts. METHODS AND RESULTS: A control group (n = 5) received cyclosporine 5 mg.kg-1.d-1 for 6 weeks after heterotopic heart transplantation. In a rejection group (n = 5), cyclosporine was omitted for 4 days at 1 and 4 weeks after transplantation. We studied cross sections of grafted hearts at 6 weeks and evaluated myocardial rejection grade, incidence, and severity and cell composition of intimal lesions in multiple coronary artery profiles. Episodic withdrawal of cyclosporine augmented myocardial rejection (International Society for Heart and Lung Transplantation grades 0, 0, 0, 0, and 1A in the control group to grades 1A, 1B, 2, 3A, and 3B in the rejection group). Episodes of acute rejection significantly increased the incidence (7.8 +/- 2.7% to 49.7 +/- 1.9%) and severity (from grade 0.10 +/- 0.04 to 0.79 +/- 0.24) of intimal thickening in graft coronary arteries. Most intimal lesions consisted of smooth muscle cells and contained various degrees of T-lymphocyte infiltration but sparse macrophages. CONCLUSIONS: In this experimental model, episodes of acute rejection precipitated by cyclosporine withdrawal accelerated the development of graft vascular lesion formation. Activation of vascular cells and leukocyte recruitment during acute rejection may thus contribute to the pathogenesis of graft arteriosclerosis.

Lipid lowering promotes accumulation of mature smooth muscle cells expressing smooth muscle myosin heavy chain isoforms in rabbit atheroma.

Smooth muscle cells (SMCs) in the atherosclerotic intima characteristically differ from those in the arterial media, for example, by reduced expression of SMC differentiation/maturation markers such as smooth muscle myosin heavy chain isoforms (SM1 and SM2). This study tested the hypothesis that lipid lowering promotes maturation of intimal SMCs in 33 rabbits subjected to balloon injury and cholesterol feeding (0.3%) for 4 months (Baseline group, n=15); some of which then were switched to a low-cholesterol diet for 8 months (Low group at 8 months, n=3) or 16 months (Low group at 16 months, n=10). The remaining rabbits continued to consume a high-cholesterol diet for 16 months (High group, n=5). We monitored SMC phenotype by expression of immunoreactive alpha-smooth muscle actin, SM1, and SM2. alpha-Actin is an early marker, and SM1 and SM2 are late markers for SMC differentiation/maturation. Only fully differentiated or mature SMCs express SM2. Data are reported as the percentage of the alpha-actin-positive intimal area occupied by smooth muscle myosin-positive SMCs determined by color image analysis of immunostained sections. Levels of SM1 and SM2, highly expressed by SMCs in the normal aortic media (n=5) decreased in the aortic intima of the Baseline and High groups, indicating a less mature phenotype. In contrast, SM1 and SM2 increased in the Low (16 months) group, indicating that intimal SMCs exhibit a more mature phenotype after lipid lowering. Electron microscopy also showed the presence of mature intimal SMCs with abundant myofilaments. Furthermore, lipid lowering reduced levels of platelet-derived growth factor-B in the arterial intima, a factor known to suppress smooth muscle myosin expression. These data demonstrate that lipid lowering favors accumulation of mature SMCs in the atherosclerotic intima in association with reduced levels of platelet-derived growth factor-B expression. Intimal SMCs in the Low group also displayed reduced expression of matrix metalloproteinases-3 and -9 compared with the Baseline and High groups. These findings shed new light on the effects of lipid lowering at the level of the vascular wall, which may influence the biology of the atheroma.

Evidence for increased collagenolysis by interstitial collagenases-1 and -3 in vulnerable human atheromatous plaques.

BACKGROUND: Several recent studies attempted to classify plaques as those prone to cause clinical manifestations (vulnerable, atheromatous plaques) or those less frequently associated with acute thrombotic complication (stable, fibrous plaques). Defining the cellular and molecular mechanisms that underlie these morphological features remains a challenge. Because interstitial forms of collagen determine the biomechanical strength of the atherosclerotic lesion, this study investigated expression of the collagen-degrading matrix metalloproteinase (MMP) interstitial collagenase-3 (MMP-13) and the previously studied MMP-1 in human atheroma and used a novel technique to test the hypothesis that collagenolysis in atheromatous lesions exceeds that in fibrous human atherosclerotic lesions. METHODS AND RESULTS: Human carotid atherosclerotic plaques, similar in size, were separated by conventional morphological characteristics into fibrous (n=10) and atheromatous (n=10) lesions. Immunohistochemical and Western blot analysis demonstrated increased levels of MMP-1 and MMP-13 in atheromatous versus fibrous plaques. In addition, collagenase-cleaved type I collagen, demonstrated by a novel cleavage-specific antibody, colocalized with MMP-1- and MMP-13-positive macrophages. Macrophages, rather than endothelial or smooth muscle cells, expressed MMP-13 and MMP-1 on stimulation in vitro. Furthermore, Western blot analysis demonstrated loss of interstitial collagen type I and increased collagenolysis in atheromatous versus fibrous lesions. Finally, atheromatous plaques contained higher levels of proinflammatory cytokines, activators of MMPs. CONCLUSIONS: This report demonstrates that atheromatous rather than fibrous plaques might be prone to rupture due to increased collagenolysis associated with macrophages, probably mediated by the interstitial collagenases MMP-1 and MMP-13.

Aortic valve endothelial cells undergo transforming growth factor-beta-mediated and non-transforming growth factor-beta-mediated transdifferentiation in vitro.

Cardiac valves arise from endocardial cushions, specialized regions of the developing heart that are formed by an endothelial-to-mesenchymal cell transdifferentiation. Whether and to what extent this transdifferentiation is retained in mature heart valves is unknown. Herein we show that endothelial cells from mature valves can transdifferentiate to a mesenchymal phenotype. Using induction of alpha-smooth muscle actin (alpha-SMA), an established marker for this process, two distinct pathways of transdifferentiation were identified in clonally derived endothelial cell populations isolated from ovine aortic valve leaflets. alpha-SMA expression was induced by culturing clonal endothelial cells in medium containing either transforming growth factor-beta or low levels of serum and no basic fibroblast growth factor. Cells induced to express alpha-SMA exhibited markedly increased migration in response to platelet-derived growth factor-BB, consistent with a mesenchymal phenotype. A population of the differentiated cells co-expressed CD31, an endothelial marker, along with alpha-SMA, as seen by double-label immunofluorescence. Similarly, this co-expression of endothelial markers and alpha-SMA was detected in a subpopulation of cells in frozen sections of aortic valves, suggesting the transdifferentiation may occur in vivo. Hence, the clonal populations of valvular endothelial cells described here provide a powerful in vitro model for dissecting molecular events that regulate valvular endothelium.