Effects of hypoxia on activity of primary human lung fibroblast from COPD patients

Abstract

Chronic obstructive pulmonary disease (COPD) is a heterogeneous lung disease without curable treatment. COPD is divided into stages of increasing disease severity GOLD I – IV, with GOLD IV being most severe. Pathophysiological changes in COPD include chronic pulmonary inflammation, irreversible small airway remodelling and emphysema and found to be driven by hypoxia, inflammation and oxidative stress. Fibroblasts, the main producers of extracellular matrix proteins and angiogenic mediators, such as vascular endothelial growth factor (VEGF), have been regarded as key players in ongoing remodelling events in COPD. The aim of this study was to investigate the response of fibroblasts derived from distal lung to hypoxia and examine whether differences in COPD-fibroblasts could be observed. The activity of fibroblasts from patients with mild (GOLD II) and severe (GOLD IV) COPD (former smokers), healthy former smokers and non-smokers were investigated in normoxic and hypoxic (1% O2) conditions for 4 h and 24 h. Several genes and proteins associated with oxidative stress responses, remodelling and angiogenesis, inflammation and endoplasmic reticulum stress were analysed using RT-qPCR, immunostaining and ELISA. Our results indicated differences in the response to hypoxia on gene regulation involved in oxidative stress including mitochondria and ER stress in COPD-fibroblasts compared to healthy. The release of VEGF-A and VEGF-C were significantly altered (p<0.05) in COPD-fibroblasts compared to healthy. Increased release of prostaglandin F1α and E2 (p<0.05) was seen in mild COPD at normoxic conditions compared to severe COPD and healthy fibroblasts. In conclusion, our results suggest altered fibroblast activity of gene regulation involved in various stress responses and mediator release associated with remodelling events related to COPD and hypoxia, warranting further investigation. Future studies might contribute towards a better understanding of the pathology of COPD and could be helpful for treatment development.

Popular Abstract

Lack of oxygen in lung disease progression

The lung is one of the most important organs in our body. Its role is to provide organs and tissues with oxygen (O2) and remove carbon dioxide (CO2). The lung is a complex structure subdivided into several compartments to allow for efficient gas exchange. The gas exchange occurs in air sacs, called alveoli, that are located throughout the lung. The alveoli sit around bronchioles, which are small air tubes that spread out from the two major airway branches, the bronchi. The air travels through the bronchi and bronchioles to the alveoli, where O2 is taken up by small blood vessel with thin walls, the capillaries, at the same time as CO2 is released. Many lung diseases exist where the airflow and gas exchange are limited and cause difficulties breathing which can reduces the quality of life dramatically. Drugs to treat symptoms are available but no cure exist.

One common lung disease is chronic obstructive pulmonary disease (COPD). In COPD, the airflow limitation is often caused by exposure to cigarette smoke or other air pollutants. Particles in smoke can irritate and damage the cells in the lung, which activates the immune system and repair responses. Most of the time, these responses go as they should, and the damage is repaired. However, if the exposure to smoke is prolonged, and the irritation becomes chronic, these responses can cause irreversible changes in the lung structure. Chronic inflammation causes the airways to constrict and produce more mucus which blocks airflow and is known as chronic bronchitis. This obstruction reduces the gas exchange efficiency in the alveoli as less air arrives in the air sacs. Another problem in COPD is the permanent destruction of alveoli due to inflammation, which is called emphysema. In emphysema and chronic bronchitis, remodelling of the structural composition of the lung takes place. Fibroblasts are specialised cells that produce the elements (proteins) that make up the structural environment in which cells live, called the extracellular matrix, and are involved in repair and remodelling processes.

Another side effect of airway obstruction is the lack of O2. When less O2 than needed is available, an area is called hypoxic. Hypoxia is a strong inducer of remodelling and leads to the formation of new blood vessels to provide oxygen to this location.

We were interested in how lung fibroblasts from individuals with COPD would respond to hypoxia. We exposed COPD fibroblasts to hypoxia and analysed their response by looking at gene regulation (mRNA), as well as staining the cells with antibodies for microscopic imaging and measuring the release of mediators. Our focus was on proteins and genes involved in remodelling and stress responses. This was also done with fibroblasts from healthy individuals for comparison. Our data implicate differences in the response to hypoxia between fibroblasts from healthy subjects and COPD patients. Differences in gene expression were also seen in normal culture conditions. Our results could help to understand a little bit more about the various processes in COPD which ultimately could lead to better treatment or even a cure for this disease.

Master’s degree project in Molecular Biology, 30 credits, 2021
Department of Biology, Lund University
Advisor: Anna-Karin Larsson-Callerfelt
Lung Biology, Faculty of Medicine, Lund University

• author: Marek, Nora
• supervisor: Anna-Karin Larsson Callerfelt
• organization: Degree Projects in Molecular Biology
• course: MOBM02 20211
• year: 2021
• type: H2 - Master's Degree (Two Years)
• subject: Biology and Life Sciences
• language: English
• id: 9064737
• date added to LUP: 2021-09-03 13:49:40
• date last changed: 2021-09-03 13:49:40