The response of human pulmonary microvascular endothelial cells to cigarette smoke injury and their role in emphysema.

Chronic Obstructive Pulmonary Disease (COPD) is the fifth leading cause of death worldwide and a major cause of chronic disability costing the NHS approximately £500 million per year. Current treatments improve symptoms but do not reverse or cure the disease. Cigarette smoking is the major cause of COPD. How smoking leads to this damage remains unclear. We urgently need to understand how smoking damages the lung in order to discover new treatments. In this study we plan to use tissue and cells obtained from the diseased lungs of patients with COPD undergoing lung transplantation at our centre. Patients will be asked for permission to use the tissue from their diseased lung, which would normally be discarded, for research. It is hoped that by studying in the laboratory how these cells behave when exposed to cigarette smoke compared to tissues and cells obtained from people with normal lungs, we may improve understanding of why they developed COPD. The study is being carried out at the lung transplant unit at Freeman hospital, Newcastle and Newcastle University with support from the pharmaceutical company AstraZeneca. We hope that by understanding how COPD occurs, new treatments may be developed to help patients with this condition.

Cigarette smoking is the major cause of Chronic Obstructive Pulmonary disease (COPD), which is characterised by inflammation of the airways (bronchitis) and lung parenchyma, with alveolar wall destruction and air space dilation (emphysema). The pathophysiological mechanisms by which smoking leads to these changes remains poorly understood. The pulmonary microvasculature was first implicated in the pathogenesis of emphysema from the histological observation of loss of alveolar septal capillaries and fibrosis of septal walls. Smoking may injure endothelial cells and alter levels of protective factors including Vascular Endothelial Growth Factor (VEGF). In support of this hypothesis, VEGF levels are reduced in emphysematous lung tissue specimens and rats treated with a VEGF receptor blocker develop emphysema that is prevented by addition of a caspase inhibitor, implicating apoptosis in the pathogenesis. Cells may undergo necrosis/ apoptosis or a phenotype change in response to chronic injury. Apoptosis may explain loss of endothelial cells while change in phenotype of endothelial cells into mesenchymal cells may account for both endothelial cell loss and appearance of alveolar septal fibrosis, with reduced elastic recoil, leading to dilation of air spaces. This study aims to investigate the fate of pulmonary microvascular endothelial cells in response to cigarette smoke injury and their role in the evolution of emphysema.

Objectives:
1. To isolate human pulmonary microvascular endothelial cells (HpMECs) from COPD lung tissue to investigate the response of these susceptible cells in contrast to normal cells. Isolation involves macerating fresh lung tissue obtained at transplantation, followed by a digest method with bead separation at the first passage.
2. To investigate apoptosis in response to cigarette smoke extract (CSE). Quantification of cell death will include the use of AnnexinV/ Propidium Iodide staining via FACS, caspase 3 measurements and LDH release. VEGF release in response to CSE shall also be investigated, in addition to the signalling pathways leading to cell death.
3. To investigate endothelial to mesenchymal transition (EnMT) in response to CSE and TGF-?1 via change in morphology, cellular expression and function, with deposition of collagen and production of tissue degrading matrix-metalloproteinases.
4. To perform immunohistochemical and morphological assessment of COPD lung tissue and correlate in vivo findings with in vitro work.

This unique research uses cells isolated from explanted COPD lungs and may afford considerable advances in understanding the basic pathophysiological mechanisms of this disease, as we witness how these susceptible cells behave in response to CSE.