Application of Shear Stress to Endothelial Cells Using a Parallel Plate Flow Chamber.

Endothelial cell response to shear stress from the flowing blood plays an important role in diseases such as atherosclerosis. It is therefore essential to study how different flow regimes, including both atheroprotective laminar flow and atheropromoting disturbed flow, impact endothelial cell function. Shear stress effects can be studied in vitro using a variety of techniques, each with their own advantages and disadvantages. In this chapter, we describe how to use a parallel plate flow chamber to study the impact of both laminar and disturbed flow on endothelial cell monolayers.

A Modified Parallel Plate Flow Chamber to Study Local Endothelial Response to Recirculating Disturbed Flow.

Atherosclerosis develops at arterial sites where endothelial cells (ECs) are exposed to low time-averaged shear stress, in particular in regions of recirculating disturbed flow. To understand how hemodynamics contributes to EC dysfunction in atheroma development, an in vitro parallel plate flow chamber gasket was modified with protruding baffles to produce large recirculating flow regions. Computational fluid dynamics (CFD) predicted that more than 60% of the flow surface area was below the 12 dynes/cm2 atheroprotective threshold. Bovine aortic endothelial cells (BAECs) were then seeded in the parallel plate flow chamber with either the standard laminar or the new disturbed flow gasket (DFG) and exposed to flow for 36 h. Cell morphology, nitric oxide (NO), proliferation, permeability, and monocyte adhesion were assessed by phase contrast and confocal microscopy. BAEC exposed to 20 dynes/cm2 shear stress in the laminar flow device aligned and elongated in the flow direction while increasing nitric oxide, decreasing permeability, and maintaining low proliferation and monocyte adhesion. BAEC in the recirculating flow and low shear stress disturbed flow device regions did not elongate or align, produced less nitric oxide, and showed higher proliferation, permeability, and monocyte adhesion than cells in the laminar flow device. However, cells in disturbed flow device regions exposed to atheroprotective shear stress did not consistently align or decrease permeability, and these cells demonstrated low nitric oxide levels. The new parallel plate DFG provides a means to study recirculating flow, highlighting the complex relationship between hemodynamics and endothelial function.

Antimicrobial efficacy and wound-healing property of a topical ointment containing nitric-oxide-loaded zeolites.

Topical delivery of nitric oxide (NO) through a wound dressing has the potential to reduce wound infections and improve healing of acute and chronic wounds. This study characterized the antibacterial efficacy of an ointment containing NO-loaded, zinc-exchanged zeolite A that releases NO upon contact with water. The release rate of NO from the ointment was measured using a chemiluminescence detection system. Minimum bactericidal concentration assays were performed using five common wound pathogens, including Gram-negative bacteria (Escherichia coli and Acinetobacter baumannii), Gram-positive bacteria (Staphylococcus epidermidis and meticillin-resistant Staphylococcus aureus) and a fungus (Candida albicans). The time dependence of antimicrobial activity was characterized by performing log-reduction assays at four time points after 1-8 h ointment exposure. The cytotoxicity of the ointment after 24 h was assessed using cultured 3T3 fibroblast cells. Minimum microbicidal concentrations (MMCs) for bacterial organisms (5×10(7) c.f.u.) ranged from 50 to 100 mg ointment (ml media)(-1); the MMC for C. albicans (5×10(4) c.f.u.) was 50 mg ointment (ml media)(-1). Five to eight log reductions in bacterial viability and three log reductions in fungal viability were observed after 8 h exposure to NO-zeolite ointment compared with untreated organisms. Fibroblasts remained viable after 24 h exposure to the same concentration of NO-zeolite ointment as was used in antimicrobial tests. In parallel studies, full-thickness cutaneous wounds on Zucker obese rats healed faster than wounds treated with a control ointment. These data indicate that ointment containing NO-loaded zeolites could potentially be used as a broad-spectrum antimicrobial wound-healing dressing.

Enhancement of tight junctional barrier function by micronutrients: compound-specific effects on permeability and claudin composition.

Amid an increasing number of reports in the literature concerning epithelial barrier enhancement by various nutrient compounds, there has never been a study performing side-by-side comparisons of these agents in a single epithelial model. We compare five nutrient compounds (previously reported in various epithelial models to enhance barrier function) regarding their ability to increase transepithelial electrical resistance (R(t)) and decrease transepithelial mannitol permeability (J(m)) across LLC-PK1 renal epithelial cell layers. The effects of these nutrients on the abundance of various tight junctional proteins are also compared. In the overall group of nutrients tested--zinc, indole, quercetin, butyrate and nicotine--only nicotine failed to improve barrier function by either parameter. Nicotine also was without effect on tight junctional proteins. Quercetin simultaneously increased R(t) and decreased J(m). Zinc, butyrate and indole only exhibited statistically significant enhancement of R(t). Each of these four effective nutrient compounds had unique patterns of effects on the panel of tight junctional proteins studied. No two compounds produced the same pattern of effects. This unique pattern of effects on tight junctional complex composition by each compound establishes the chance for additive or even synergistic improvement of barrier function by combinations of compounds. A synergistic effect of the combination of quercetin and zinc on R(t) is shown.