Abdul I. Barakat

Induction of Inflammation in Vascular Endothelial Cells by Metal Oxide Nanoparticles: Effect of Particle Composition

Background The mechanisms governing the correlation between exposure to ultrafine particles and the increased incidence of cardiovascular disease remain unknown. Ultrafine particles appear to cross the pulmonary epithelial barrier into the bloodstream, raising the possibility of direct contact with the vascular endothelium. Objectives Because endothelial inflammation is critical for the development of cardiovascular pathology, we hypothesized that direct exposure of human aortic endothelial cells (HAECs) to ultrafine particles induces an inflammatory response and that this response depends on particle composition. Methods To test the hypothesis, we incubated HAECs for 1–8 hr with different concentrations (0.001–50 μg/mL) of iron oxide (Fe2O3), yttrium oxide (Y2O3), and zinc oxide (ZnO) nanoparticles and subsequently measured mRNA and protein levels of the three inflammatory markers intra-cellular cell adhesion molecule-1, interleukin-8, and monocyte chemotactic protein-1. We also determined nanoparticle interactions with HAECs using inductively coupled plasma mass spectrometry and transmission electron microscopy. Results Our data indicate that nanoparticle delivery to the HAEC surface and uptake within the cells correlate directly with particle concentration in the cell culture medium. All three types of nanoparticles are internalized into HAECs and are often found within intracellular vesicles. Fe2O3 nanoparticles fail to provoke an inflammatory response in HAECs at any of the concentrations tested; however, Y2O3 and ZnO nanoparticles elicit a pronounced inflammatory response above a threshold concentration of 10 μg/mL. At the highest concentration, ZnO nanoparticles are cytotoxic and lead to considerable cell death. Conclusions These results demonstrate that inflammation in HAECs following acute exposure to metal oxide nanoparticles depends on particle composition.

Computational study of fluid mechanical disturbance induced by endovascular stents.

Arterial restenosis following stent deployment may be influenced by the local flow environment within and around the stent. We have used computational fluid dynamics to investigate the flow field in the vicinity of model stents positioned within straight and curved vessels. Our simulations have revealed the presence of flow separation and recirculation immediately downstream of stents. In steady flow within straight vessels, the extent of flow disturbance downstream of the stent increases with both Reynolds number and stent wire thickness but is relatively insensitive to stent interwire spacing. In curved vessels, flow disturbance downstream of the stent occurs along both the inner and outer vessel walls with the extent of disturbance dependent on the angle of vessel curvature. In pulsatile flow, the regions of flow disturbance periodically increase and decrease in size. Non-Newtonian fluid properties lead to a modest reduction in flow disturbance downstream of the stent. In more realistic stent geometries such as stents modeled as spirals or as intertwined rings, the nature of stent-induced flow disturbance is exquisitely sensitive to stent design. These results provide an understanding of the flow physics in the vicinity of stents and suggest strategies for stent design optimization to minimize flow disturbance.

Differential responsiveness of vascular endothelial cells to different types of fluid mechanical shear stress

Early atherosclerotic lesions localize preferentially, in arterial regions exposed to low flow, oscillatory flow, or both; however, the cellular basis of this observation remains to be determined. Atherogenesis involves dysfunction of the vascular endothelium, the cellular monolayer lining the inner surfaces of blood vessels. How low flow, oscillatory flow, or both may lead to endothelial dysfunction remains unknown. Over the past two decades, fluid mechanical shear (or frictional) stress has been shown to intricately regulate the structure and function of vascular endothelial cells (ECs). Furthermore, recent data indicate that beyond being merely responsive to shear stress, ECs are able to distinguish among and respond differently to different types of shear stress. This review focuses on EC differential responses to different types of steady and unsteady shear stress and discusses the implications of these responses for the localization of early atherosclerotic lesions. The mechanisms by which endothelial differential responsiveness to different types of flow may occur are also discussed.

Microchannel Platform for the Study of Endothelial Cell Shape and Function

Microfabrication technology is implemented to realize a versatile platform for the study of endothelial cell (EC) shape and function. The platform contains arrays of microchannels, 25–225 μm wide, that are fabricated by deep reactive ion etching (DRIE) of silicon and anodic bonding to glass and within which ECs are cultured. Silicon fluidic port modules, fabricated using a combination of silicon fusion bonding and anisotropic etching in KOH, provide a simple and reversible means of coupling, via standard tubing, between an individual microchannel and off-platform devices for flow monitoring and control. For flow experiments where a well-defined flow field is required, the channels are capped with either a glass lid or a thin, self-sealing elastomer membrane that can be punctured to provide direct access to cells within the microchannels. Under static culture conditions, bovine aortic ECs (BAECs) become progressively more elongated as the channel width decreases. The shape index, a dimensionless measure of cell roundness, decreases from 0.75±0.01 (mean±SEM) for BAECs cultured in 225 μm-wide microchannels to 0.31±0.02 in 25 μm-wide channels. When cuboidal BAECs are grown in 200 μm-wide microchannels and then subjected to a fluid shear stress of approximately 20 dyne/cm2 (2 Pa), they progressively elongate and align in the direction of flow in a similar manner to cells cultured on plain surfaces. To demonstrate the utility of the microfabricated platform for studying aspects of EC function, whole-cell patch-clamp recordings were performed under static conditions in open microchannels. The platform is demonstrated to be a versatile tool for studying relationships between EC shape and function and for probing the effect of flow on ECs of different shapes. Specific future applications and extensions of platform function are discussed.

Nesprin-3 regulates endothelial cell morphology, perinuclear cytoskeletal architecture, and flow-induced polarization

Nesprin-3, a protein that links intermediate filaments to the nucleus, plays a role in vascular endothelial cell (EC) function. Nesprin-3 regulates EC morphology, perinuclear cytoskeletal organization, centrosome–nuclear connectivity, and flow-induced cell polarization and migration.

Effect of cerium oxide nanoparticles on inflammation in vascular endothelial cells

Because vascular endothelial cell inflammation is critical in the development of cardiovascular pathology, we hypothesized that direct exposure of human aortic endothelial cells (HAECs) to ultrafine particles induces an inflammatory response. To test the hypothesis, we incubated HAECs for 4 h with different concentrations (0.001–50 μg/ml) of CeO2 nanoparticles and subsequently measured mRNA levels of the three inflammatory markers intercellular adhesion molecule 1 (ICAM-1), interleukin (IL)-8, and monocyte chemotactic protein (MCP-1) using real-time polymerase chain reaction (PCR). Ceria nanoparticles caused very little inflammatory response in HAECs, even at the highest dose. This material is apparently rather benign in comparison with Y2O3 and ZnO nanoparticles that we have studied previously. These results suggest that inflammation in HAECs following acute exposure to metal oxide nanoparticles depends strongly on particle composition.

Modulation of ATP/ADP concentration at the endothelial surface by shear stress: effect of flow-induced ATP release.

AbstractThe adenine nucleotides ATP and ADP induce the production of vasoactive compounds in vascular endothelial cells (ECs). Therefore, knowledge of how flow affects the concentration of ATP and ADP at the EC surface may be important for understanding shear stress-mediated vasoregulation. The concentration of ATP and ADP is determined by convective and diffusive transport as well as by hydrolysis of these nucleotides by ectonucleotidases at the EC surface. Previous mathematical modeling has demonstrated that for steady flow in a parallel plate flow chamber, the combined ATP+ADP concentration does not change considerably over a wide range of shear stress. This finding has been used to argue that the effect of flow on adenine nucleotide transport could not account for the dependence of endothelial responses to ATP on the magnitude of applied shear stress. The present study extends the previous modeling to include pulsatile flow as well as flow-induced endothelial ATP release. Our results demonstrate that flow-induced ATP release has a pronounced effect on nucleotide concentration under both steady and pulsatile flow conditions. While the combined ATP+ADP concentration at the EC surface in the absence of flow-induced ATP release changes by only ∼ 10% over the wall shear stress range 0.1-10 dyne/cm -2, inclusion of this release leads to a concentration change of ∼ 34% –106% over the same shear stress range, depending on how ATP release is modeled. These results suggest that the dependence of various endothelial responses to shear stress on the magnitude of the applied shear stress may be partially attributable to flow-induced changes in cell-surface adenine nucleotide concentration.

Modeling the transport of drugs eluted from stents: physical phenomena driving drug distribution in the arterial wall

Despite recent data that suggest that the overall performance of drug-eluting stents (DES) is superior to that of bare-metal stents, the long-term safety and efficacy of DES remain controversial. The risk of late stent thrombosis associated with the use of DES has also motivated the development of a new and promising treatment option in recent years, namely drug-coated balloons (DCB). Contrary to DES where the drug of choice is typically sirolimus and its derivatives, DCB use paclitaxel since the use of sirolimus does not appear to lead to satisfactory results. Since both sirolimus and paclitaxel are highly lipophilic drugs with similar transport properties, the reason for the success of paclitaxel but not sirolimus in DCB remains unclear. Computational models of the transport of drugs eluted from DES or DCB within the arterial wall promise to enhance our understanding of the performance of these devices. The present study develops a computational model of the transport of the two drugs paclitaxel and sirolimus eluted from DES in the arterial wall. The model takes into account the multilayered structure of the arterial wall and incorporates a reversible binding model to describe drug interactions with the constituents of the arterial wall. The present results demonstrate that the transport of paclitaxel in the arterial wall is dominated by convection while the transport of sirolimus is dominated by the binding process. These marked differences suggest that drug release kinetics of DES should be tailored to the type of drug used.

Serum proteins prevent aggregation of Fe2O3 and ZnO nanoparticles

Abstract Aggregation of metal oxide nanoparticles in aqueous media complicates interpretation of in vitro studies of nanoparticle–cell interactions. We used dynamic light scattering to investigate the aggregation dynamics of iron oxide and zinc oxide nanoparticles. Our results show that iron oxide particles aggregate more readily than zinc oxide particles. Pretreatment with serum stabilises iron oxide and zinc oxide nanoparticles against aggregation. Serum-treated iron oxide is stable only in pure water, while zinc oxide is stable in water or cell culture media. These findings, combined with zeta potential measurements and quantification of proteins adsorbed on particle surface, suggest that serum stabilisation of iron oxide particles occurs primarily through protein adsorption and resulting net surface charge. Zinc oxide stabilisation, however, also involves steric hindrance of particle aggregation. Fluid shear at levels used in flow experiments breaks up iron oxide particle aggregates. These results enhance our understanding of nanoparticle aggregation and its consequences for research on the biological effects of nanomaterials.

Flow-activated Chloride Channels in Vascular Endothelium SHEAR STRESS SENSITIVITY, DESENSITIZATION DYNAMICS, AND PHYSIOLOGICAL IMPLICATIONS

Although activation of outward rectifying Cl– channels is one of the fastest responses of endothelial cells (ECs) to shear stress, little is known about these channels. In this study, we used whole-cell patch clamp recordings to characterize the flow-activated Cl– current in bovine aortic ECs (BAECs). Application of shear stress induced rapid development of a Cl– current that was effectively blocked by the Cl– channel antagonist 5-nitro-2-(3-phenopropylamino)benzoic acid (100 μm). The current initiated at a shear stress as low as 0.3 dyne/cm2, attained its peak within minutes of flow onset, and saturated above 3.5 dynes/cm2 (∼2.5–3.5-fold increase over pre-flow levels). The Cl– current desensitized slowly in response to sustained flow, and step increases in shear stress elicited increased current only if the shear stress levels were below the 3.5 dynes/cm2 saturation level. Oscillatory flow with a physiological oscillation frequency of 1 Hz, as occurs in disturbed flow zones prone to atherosclerosis, failed to elicit the Cl– current, whereas lower oscillation frequencies led to partial recovery of the current. Nonreversing pulsatile flow, generally considered protective of atherosclerosis, was as effective in eliciting the current as steady flow. Measurements using fluids of different viscosities indicated that the Cl– current is responsive to shear stress rather than shear rate. Blocking the flow-activated Cl– current abolished flow-induced Akt phosphorylation in BAECs, whereas blocking flow-sensitive K+ currents had no effect, suggesting that flow-activated Cl– channels play an important role in regulating EC flow signaling.