The role of microvascular endothelial cells in the pathogenesis of emphysema

Abstract

COPD comprising small airways disease and emphysema is a chronic, debilitating often fatal lung condition that approximately 20% of smokers develop. Current therapies mostly target inflammation and airflow obstruction caused by small airways disease however there are no current therapies which treat emphysema, the pathogenesis of which remains poorly understood. The microvascular hypothesis of COPD is a credible alternative to the classical hypothesis of inflammation and protease driven lung destruction, whereby an initial insult to the microvasculature leads to loss of alveolar structure which typifies emphysema. I planned to investigate the role of the microvasculature in the pathogenesis of COPD by isolating susceptible lung microvascular endothelial cells (LMVECs) from individuals with emphysema in an attempt to mimic in vivo conditions more closely. LMVECs were isolated from explanted emphysematous lungs removed at transplantation. Following successful isolation (71%) and characterisation of emphysema LMVECs, I sought to study cellular responses to cigarette smoke injury, namely apoptosis and endothelial to mesenchymal transition. Apoptosis was investigated on tissue blocks via caspase 3 immunohistochemistry and by ex vivo methods including flow cytometry (annexin V), TUNEL and live cell imaging for activated caspase 3. Unfortunately cigarette smoke extract caused autofluorescence of cells and as all of these techniques employed the use of fluorescence for detection, any conclusions that can be made as to whether cells underwent apoptosis are limited. Endothelial to mesenchymal transition was investigated in response to TGFβ1 and cigarette smoke extract. While there was evidence of down regulation of endothelial markers in response to cigarette smoke on confocal imaging there was no convincing evidence of upregulation of mesenchymal markers with no corresponding change in protein expression via western blotting. One explanation may be that such changes in cell structure and endothelial cell expression may be more in keeping with endothelial activation rather than a true phenotypic switch. In summary, this study presents a new model of emphysema, with attempts to gain insight into endothelial injury in the pathogenesis of COPD, highlighting the challenges and limitations of working with primary diseased cells in response to cigarette smoke injury.