Photoluminescence of Si nanocrystallites and amorphous oxygen-containing si nanoparticles: the reversible effect of ambient atmosphere on luminescence.

Si nanocrystallites of various sizes and oxygen-containing Si nanoparticles with different oxygen contents were prepared by vapor condensation. The Si nanocrystallites showed a visible light emission from 500 nm to 900 nm with the peak at 800 nm, and the intensity of photoluminescence increased with decreasing the particle size. This photoluminescence observed in vacuum could be quenched by air and hydrogen, and reappeared after the sample chamber was evacuated. The oxygen-containing Si nanoparticles consisting mainly of Si oxide were amorphous and had an average particle size of approximately 20 nm. Increasing the oxygen content of nanoparticles caused a blueshift of the absorption edge in the transmission spectra. A blue-green photoluminescence with two peaks at 500 nm and 800 nm was observed from these oxygen-containing Si nanoparticles. The luminescence intensity increased with the oxygen content of nanoparticles, and was very sensitive to the ambient atmosphere. Much lower intensity was observed in air, but higher intensity could be recovered in vacuum. Surface states and oxygen-induced luminescent centers were proposed to be responsible for the photoluminescence from the Si nanocrystallites and oxygen-containing Si nanoparticles, respectively. The reversible ambient effect in both cases could be explained by surface charge redistribution during the gas adsorption and desorption processes.

Interleukin 6 acts as a paracrine growth factor in human mammary carcinoma cell lines.

The effect of interleukin 6 (IL-6) on normal and human mammary carcinoma epithelial cells was studied. IL-6 inhibited the growth of estrogen receptor-positive [ER(+)] breast cancer cell lines, which underwent apoptosis with prolonged treatment. In contrast, ER(-) breast cancer cell lines were resistant to IL-6-mediated growth inhibition. By examining the components of the IL-6 receptor (IL-6R) system, we found that ER(+) breast cancer cells expressed predominantly soluble IL-6Ralpha, whereas the ER(-) breast cancer cells expressed primarily the transmembrane form of the IL-6R, gp130. In addition, detectable levels of IL-6 were secreted into the medium by ER(-) but not ER(+) breast cancer cells. Furthermore, the supernatant obtained from IL-6-secreting, ER(-) cells suppressed the growth of IL-6-sensitive, ER(+) breast cancer cells in a paracrine fashion. Although IL-6 is secreted by ER(-) breast cancer cells, this cytokine does not seem to stimulate the proliferation of these cells in an autocrine fashion. These studies indicate that IL-6 can regulate the growth of normal and transformed human mammary epithelial cells differentially, and that IL-6 secretion by some ER(-) breast cancer cells can function as a paracrine growth factor, suppressing the growth of ER(+) breast cancer cells in vitro.

Shear stress regulates endothelial cell autophagy via redox regulation and Sirt1 expression.

Disturbed cell autophagy is found in various cardiovascular disease conditions. Biomechanical stimuli induced by laminar blood flow have important protective actions against the development of various vascular diseases. However, the impacts and underlying mechanisms of shear stress on the autophagic process in vascular endothelial cells (ECs) are not entirely understood. Here we investigated the impacts of shear stress on autophagy in human vascular ECs. We found that shear stress induced by laminar flow, but not that by oscillatory or low-magnitude flow, promoted autophagy. Time-course analysis and flow cessation experiments confirmed that this effect was not a transient adaptive stress response but appeared to be a sustained physiological action. Flow had no effect on the mammalian target of rapamycin-ULK pathway, whereas it significantly upregulated Sirt1 expression. Inhibition of Sirt1 blunted shear stress-induced autophagy. Overexpression of wild-type Sirt1, but not the deacetylase-dead mutant, was sufficient to induce autophagy in ECs. Using both of gain- and loss-of-function experiments, we showed that Sirt1-dependent activation of FoxO1 was critical in mediating shear stress-induced autophagy. Shear stress also induced deacetylation of Atg5 and Atg7. Moreover, shear stress-induced Sirt1 expression and autophagy were redox dependent, whereas Sirt1 might act as a redox-sensitive transducer mediating reactive oxygen species-elicited autophagy. Functionally, we demonstrated that flow-conditioned cells are more resistant to oxidant-induced cell injury, and this cytoprotective effect was abolished after inhibition of autophagy. In summary, these results suggest that Sirt1-mediated autophagy in ECs may be a novel mechanism by which laminar flow produces its vascular-protective actions.

Intimal thickening under shear in a carotid bifurcation--a numerical study.

In the present study, a model for intimal thickening proposed by Friedman and his co-workers is employed to simulate the growth of intima in a carotid bifurcation with a steady flow. The computer code developed is a pressure-based, finite volume method in a boundary-fitted coordinate system. A model bifurcation is used to demonstrate the usefulness of the numerical tool. The predicted results are consistent with the available experimental observations. The localization of plaques is shown. It is found that thicker intima is formed in preferential regions near the junction and the carotid bulb due to relatively low wall shear stresses. The intimal thickening tends to reduce the size of low shear regions. It is also found that the new geometry resulting from the thickening yields a more even stress distribution over the bifurcation. The effect of the branching ratio on the distribution of intimal thickening is also studied. The computer code provides a potential tool for visualization of intimal thickening.

A numerical simulation of intimal thickening under shear in arteries.

A model of intima thickening proposed by Friedman and his coworkers (1,2) is incorporated in our computer code to simulate the growth of intima under shear. The computer code is based on a finite volume method in a boundary-fitted coordinate system. It is found that the model yields an evenly-distributed thickening over a straight, smooth vessel wall. However, in a stenosed or a curved artery, thicker intima can be formed in preferential regions due to unevenly-distributed wall shear stresses. The results clearly demonstrate the correlations among the geometry, wall shear rate and the plaque localization in arteries. The model is applied to a straight artery with a stenosis or sinus, a smooth curved artery and a stenosed curved artery. The effects of stenosis/sinus and lumen curvature on the flows and the intimal thickening are studied. The simulation provides a numerical visualization of the intimal thickening in a dynamic way.

BMP receptor-integrin interaction mediates responses of vascular endothelial Smad1/5 and proliferation to disturbed flow.

BACKGROUND: Vascular endothelial cells (ECs) are constantly exposed to blood flow-induced shear stress. Our previous study demonstrated that disturbed flow with low and oscillatory shear stress (OSS) induces bone morphogenetic protein receptor (BMPR)-specific Smad1/5 activation in ECs, but the underlying mechanisms and the in vivo functional role of Smad1/5 remain unclear. OBJECTIVES: Here we elucidated the molecular mechanisms by which OSS activates EC Smad1/5 and its in vivo functional role. METHODS: Lentiviral Smad5-specific short hairpin RNA (Lenti-shSmad5) was constructed and intra-arterially injected into the lumen of stenosed abdominal aorta in bromodeoxyuridine-infused rats. Co-immunoprecipitation and in situ proximity ligation assays were performed on ECs exposed to OSS (0.5 ± 4 dynes/cm(2) ) in a parallel-plate flow chamber to investigate BMPR-integrin interactions and their regulatory role in OSS-activation of EC Smad1/5. RESULTS: Intra-arterial administration of Lenti-shSmad5 inhibited bromodeoxyuridine uptake of ECs at post-stenotic sites, where disturbed flow with OSS occurs. OSS induced sustained BMPRIB-αv ß3 integrin association in ECs, which was mediated by the intracytoplasmic kinase domain of BMPRII and subsequently activated the Shc/focal adhesion kinase (FAK)/extracellular signal-regulated kinase (ERK) cascade, leading to Smad1/5 activation. This OSS-activation of Smad1/5 induced its association with and activation of runt-related transcription factor-2 (Runx2), leading to activations of mammalian target of rapamycin (mTOR) and p70S6 kinase (p70S6K), a pathway critical for EC proliferation in response to OSS. CONCLUSIONS: Oscillatory shear stress induces synergistic interactions between specific BMPRs and integrin to activate Smad1/5 through the Shc/FAK/ERK pathway, which leads to the activation of the Runx2/mTOR/p70S6K pathway to promote EC proliferation.

Computation of flow fields and shear rates in an aortic bifurcation.

A finite volume method in a boundary-fitted coordinate system together with a zonal grid method is employed to compute the flow fields and shear stresses in a two-dimensional aortic bifurcation. Eddy is found distal to plaques during pulsatile flow, whereas permanent eddies are observed only during steady flow. The computed flow fields are consistent with those visualized experimentally by other authors. It is also found that although the time averaged shear rates in a pulsatile flow are similar to those of a steady flow with mean Reynolds number in most regions, they are different in recirculation zones. This result implies that care should be taken if a steady flow shear rate were to be used in modeling shear-dependent physiological processes. The non-Newtonian viscosity has only a minor effect on the flows.

Effects of disturbed flow on endothelial cells.

Atherosclerotic lesions tend to localize at curvatures and branches of the arterial system, where the local flow is often disturbed and irregular (e.g., flow separation, recirculation, complex flow patterns, and nonuniform shear stress distributions). The effects of such flow conditions on cultured human umbilical vein endothelial cells (HUVECs) were studied in vitro by using a vertical-step flow channel (VSF). Detailed shear stress distributions and flow structures have been computed by using the finite volume method in a general curvilinear coordinate system. HUVECs in the reattachment areas with low shear stresses were generally rounded in shape. In contrast, the cells under higher shear stresses were significantly elongated and aligned with the flow direction, even for those in the area with reversed flow. When HUVECs were subjected to shearing in VSF, their actin stress fibers reorganized in association with the morphological changes. The rate of DNA synthesis in the vicinity of the flow reattachment area was higher than that in the laminar flow area. These in vitro experiments have provided data for the understanding of the in vivo responses of endothelial cells under complex flow environments found in regions of prevalence of atherosclerotic lesions.

Nitric oxide regulates shear stress-induced early growth response-1. Expression via the extracellular signal-regulated kinase pathway in endothelial cells.

Endothelial cells (ECs) subjected to shear stress constantly release nitric oxide (NO). The effect of NO on shear stress-induced endothelial responses was examined. ECs subjected to shear stress induced a transient and shear force-dependent increase in early growth response-1 (Egr-1) mRNA levels. Treatment of ECs with an NO donor, S-nitroso-N-acetylpenicillamine (SNAP) or 3-morpholinosydnonimine (SIN-1), inhibited this shear stress-induced Egr-1 expression. Conversely, an NO synthase inhibitor to ECs, N(G)-monomethyl-L-arginine, augmented this Egr-1 expression. NO modulation of Egr-1 expression was demonstrated by functional analysis of Egr-1 promoter activity using a chimera containing the Egr-1 promoter region (-698 bp) and reporter gene luciferase. In contrast to the enhanced promoter activity after N(G)-monomethyl-L-arginine treatment, shear stress-induced Egr-1 promoter activity was attenuated after ECs were treated with an NO donor. ECs cotransfected with a dominant negative mutant of Ras (RasN17), Raf-1 (Raf301), or a catalytically inactive mutant of extracellular signal-regulated kinase (ERK)-2 (mERK) inhibited shear stress-induced Egr-1 promoter activity. NO modulation of the signaling pathway was shown by its inhibitory effect on shear stress-induced ERK1/ERK2 phosphorylation and activity. This inhibitory effect was further substantiated by the inhibition of NO on both the shear stress-induced transcriptional activity of Elk-1 (an ERK substrate) and the promoter activity of a reporter construct containing serum response element. NO-treated ECs resulted in a reduction of binding of nuclear proteins to the Egr-1 binding sequences in the platelet-derived growth factor-A promoter region. These results indicate that shear stress-induced Egr-1 expression is modulated by NO via the ERK signaling pathway in ECs. Our findings support the importance of NO as a negative regulator in endothelial responses to hemodynamic forces.

Increase of reactive oxygen species (ROS) in endothelial cells by shear flow and involvement of ROS in shear-induced c-fos expression.

Intracellular reactive oxygen species (ROS) may participate in cellular responses to various stimuli including hemodynamic forces and act as signal transduction messengers. Human umbilical vein endothelial cells (ECs) were subjected to laminar shear flow with shear stress of 15, 25, or 40 dynes/cm2 in a parallel plate flow chamber to demonstrate the potential role of ROS in shear-induced cellular response. The use of 2',7'-dichlorofluorescin diacetate (DCFH-DA) to measure ROS levels in ECs indicated that shear flow for 15 minutes resulted in a 0.5- to 1.5-fold increase in intracellular ROS. The levels remained elevated under shear flow conditions for 2 hours when compared to unsheared controls. The shear-induced elevation of ROS was blocked by either antioxidant N-acetyl-cysteine (NAC) or catalase. An iron chelator, deferoxamine mesylate, also significantly reduced the ROS elevation. A similar inhibitory effect was seen with a hydroxyl radical (.OH) scavenger, 1,3-dimethyl-2-thiourea (DMTU), suggesting that hydrogen peroxide (H202), .OH, and possibly other ROS molecules in ECs were modulated by shear flow. Concomitantly, a 1.3-fold increase of decomposition of exogenously added H2O2 was observed in extracts from ECs sheared for 60 minutes. This antioxidant activity, abolished by a catalase inhibitor (3-amino-1,2,4-triazole), was primarily due to the catalase. The effect of ROS on intracellular events was examined in c-fos gene expression which was previously shown to be shear inducible. Decreasing ROS levels by antioxidant (NAC or catalase) significantly reduced the induction of c-fos expression in sheared ECs. We demonstrate for the first time that shear force can modulate intracellular ROS levels and antioxidant activity in ECs. Furthermore, the ROS generation is involved in mediating shear-induced c-fos expression. Our study illustrates the importance of ROS in the response and adaptation of ECs to shear flow.

Endothelial exposure to hypoxia induces Egr-1 expression involving PKCalpha-mediated Ras/Raf-1/ERK1/2 pathway.

Hypoxia induces endothelial dysfunction that results in a series of cardiovascular injuries. Early growth response-1 (Egr-1) has been indicated as a common theme in vascular injury. Here we demonstrates that in bovine aortic endothelial cells (ECs) subjected to hypoxia (PO(2) approximately 23 mmHg), rapidly increased Egr-1 mRNA expression which peaked within 30 min and decreased afterwards. Treatment of ECs with PD98059, a specific inhibitor to mitogen-activated protein kinase (MAPK/ERK), inhibited this hypoxia-induced Egr-1 expression. The involvement of ERK pathway was further substantiated by the inhibition of Egr-1 promoter activities when ECs were co-transfected with a dominant negative mutant of Ras (RasN17), Raf-1 (Raf 301), or a catalytically inactive mutant of ERK2 (mERK). In addition, the hypoxia-induced transcriptional activity of Elk-1, an ERK substrate, was abolished by administration of PD98059. Addition of calphostin C, a protein kinase C (PKC) inhibitor, completely blocked the hypoxia-augmented Egr-1 expression. The likewise occurred while exposing ECs to D609 to inhibit phospholipase C and BAPTA/AM to chelate intracellular calcium. Hypoxia to ECs increased ERK phosphorylation within 10 min and which was abolished by administration of PD98095, calphostin C, and BAPTA/AM. Hypoxia triggered a transient translocation of PKCalpha from cytosol to membrane fraction concurrent with the association of PKCalpha to Raf-1. Involvement of PKCalpha in mediating ERK activation was further confirmed by the inhibition of ERK and the subsequent Egr-1 gene induction with antisense oligonucleotides to PKCalpha. These results indicate that ECs under hypoxia induce Egr-1 expression and this induction requires calcium, phospholipase C activation, and PKCalpha-mediated Ras/Raf-1/ERK1/2 signaling pathway. Our finding support the importance of specific PKC isozyme linked to MAPK pathway in the regulation of endothelial responses to hypoxia.

Reactive oxygen species are involved in shear stress-induced intercellular adhesion molecule-1 expression in endothelial cells.

Vascular endothelial cells (ECs) are constantly subjected to flow-induced shear stress. Although the effects of shear stress on ECs are well known, the intracellular signal mechanisms remain largely unclear. Reactive oxygen species (ROS) have recently been suggested to act as intracellular second messengers. The potential role of ROS in shear-induced gene expression was examined in the present study by subjecting ECs to a shear force using a parallel-plate flow chamber system. ECs under shear flow increased their intracellular ROS as indicated by superoxide production. This superoxide production was maintained at an elevated level as shear flow remained. Sheared ECs, similar to TNF(alpha)-, PMA-, or H2O2-treated cells, increased their intercellular adhesion molecule-1 (ICAM-1) mRNA levels in a time-dependent manner. Pretreatment of ECs with an antioxidant, N-acetyl-cysteine (NAC) or catalase, inhibited this shear-induced or oxidant-induced ICAM-1 expression. ROS that were involved in the shear-induced ICAM-1 gene expression were further substantiated by functional analysis using a chimera containing the ICAM-1 promoter region (-850 bp) and the reporter gene luciferase. Shear-induced promoter activities were attenuated by pretreating sheared ECs with NAC and catalase. Flow cytometric analysis and monocytic adhesion assay confirmed the inhibitory effect of NAC and catalase on the shear-induced ICAM-1 expression on ECs. These results clearly demonstrate that shear flow to ECs can induce intracellular ROS generation that may result in an increase of ICAM-1 mRNA levels via transcriptional events. Our findings thus support the importance of intracellular ROS in modulating hemodynamically induced endothelial responses.