Invitro Cigarette Smoke Exposure Studies Utilize the Vitrocell VC 1

Through grants, we were able to purchase a Vitrocell VC 1 manual smoking machine. The VC 1 is a very powerful tool that enables us to expose cells at an air-liquid interface to aersolized cigarette smoke. This is novel and important for cigarette smoke studies as previous studies utilizing cigarette smoke extract are limited by the  components of the cigarette smoke that can be solubilized in media. Moreover, it allows us to study what occurs when lung epithelial cells are exposed to cigarette smoke at an air-liquid interface rather than a liquid-liquid interface simulating more closely what occurs in vivo

Changes in Cortical Tension and Permeability

We have found that after long-term cigarette smoke exposure, the lung epithelium is unable to form strong cell-cell contacts. Moreover, we have observed that cells derived from patients with COPD have increased permeability, even when cultured several weeks in vitro. We have measured cortical tension in response to cigarette smoke and have found that after chronic cigarette smoke exposure, cortical tension increases. We hypothesize that this increase in cortical tension due to chronic cigarette smoke exposure causes a decrease in monolayer barrier function. As such, we are investigating  alterations in the actin-myosin cytoskeleton during cigarette smoke exposure in hopes of rescuing barrier function following cigarette smoke exposure.   Therefore, it is evident that the cells undergo a critical phenotypic changes, and we are interested identifying key regulatory proteins that could serve as novel targets to modify airway epithelial disease.  Most relevant for this proposal,  we are interested in identifying mechanisms that are altered in the lung epithelium of patients that promote the development of airway disease.

Changes in Cell Polarity Resulting in Altered Protein Localization and Activation

Loss of cell-cell adhesion proteins may also lead to loss of cellular polarity. Loss of cellular polarity may lead to altered apical-basal protein localization and even mislocalization of receptors and ligands. If ligands and receptors are not properly localized away from one another, aberrant activation may occur despite lack of signal activation. Patients with COPD have increased phosphorlyated EGFR compared to patients without COPD indicating that EGFR is may be aberrantly activated in the context of COPD. One possible explanation is the loss of cellular polarity and thus the loss of regulation. 

Epithelial to Mesenchymal Transition

With cigarette smoke exposure we also observe loss of E-cadherin, a common marker for epithelial cells. This effect alongside the loss of barrier function leads us to hypothesize that epithelial to mesenchymal transition (EMT) may be occurring. During EMT, epithelial cells that once were abundant in epithelial markers such as E-cadherin, ZO-1, and beta catenin, begin to lose expression of epithelial markers and begin to express more mesenchymal markers such as Vimentin, N-cadherin, Snail, and Fibronectin. Moreover, the epithelial cells begin to lose their epithelial cobblestone morphology and contact growth inhibition and begin to appear more spindle like and gain the ability to grow despite cell to cell contacts. This transition to a more mesenchymal state may manifest into the phenotypes that we observe-where increased EMT as a result of cigarette smoke may lead to increased cortical tension, decreased barrier function, and loss in cellular polarity.