Mechanisms Of Shear-induced Stress And Enhancement On Epithelial-barrier

Mechanisms of Shear-Induced Stress and Enhancement on Epithelial-Barrier Function

Mackenzie (Max) Martin Eric Chau Ramana Sidhaye MD

General Background Respiratory Epithelium Ciliated

pseudostratified columnar cells ◦ Ciliated Cells ◦ Goblet Cells ◦ Basal Cells

Apical and

basolateral sides Moistens and protect airways as a barrier ◦ Permeability

General Background Nitric Oxide (NO)  Regulates the lungs,

mediates vasodilation, neurotransmisssion, inflammatory responses, and respiratory epithelium  Produced mainly by Nitric Oxide synthase (NOS) Inducible NOS (iNOS) Endothelial NOS (eNOS) Neuronal NOS (nNOS)

General Background Nitric Oxide  eNOS and nNOS

contribute mainly to smooth muscle relaxation in the airways and pulmonary circulation  iNOS is induced by bacterial endotoxin, inflammatory cytokines and pollutants

Objectives  Effects of shear in normal human bronchial

epithelial (NHBE) and airway epithelial cells (AW) on NO production and paracellular permeability  Determine which isoform of NO is most relevant/abundant in causing NO change in shear-induced NHBE and AW cells  Determine by what mechanisms do eNOS increase NO production and change in permeability in response to increased shear stress  Determine what are the effects of eNOS on permeability  Understand how asthmatic and smoker cells

Methods Cell Culture, In Vitro Shear Stress Immunoblotting Colormetric Nitrate/Nitrite assay kits

Results

Effects of shear in normal human

bronchial epithelial (NHBE) and airway epithelial cells (AW) on NO production and paracellular permeability

Sample NO Concentration  AW Static 5.791  AW Shear 11.648  Standard 1 0.0435 0.5  Standard 2 0.1065 1  Standard 3 0.0945 5  Standard 4 0.1435 10  Standard 5 0.223 25  Standard 6 0.399 50  Standard 7 0.701 100.000

Results

 Determine which isoform of NO is most

relevant/abundant in causing NO change in shear-induced NHBE and AW cells  Investigate its active form

Results Determine by what mechanisms do eNOS

and phospho eNOS (peNOS) increase NO production and change in permeability in response to increased shear stress Using different inhibitors  Inhibit other compounds

Results Determine what are the effects of eNOS

and peNOS on permeability Inhibit eNOS  Different permeability protein expression  FITC-Dextran experiment

Hypothesis Increasing peNOS decreases permeability Blocking peNOS increases permeability