Hypoxic conditions promote a proliferative, poorly differentiated, and pro-secretory phenotype in COPD lung tissue progenitor cells in vitro

Abstract

Chronic obstructive pulmonary disease patients experience variable symptoms dependent on the presence of an emphysematous versus a chronic bronchitis phenotype. Both presentations can be associated with lung tissue and systemic hypoxia, at its most severe leading to Cor pulmonale. Despite this, minimal attention has been given to the effects of hypoxia at the cellular disease level.We isolated and cultured progenitor cells from the distal lung tissue of a 64 year-old, male, emphysematous donor in ambient (21%) and hypoxic (2%) oxygen conditions. Proliferative capacity was determined on collagen coated culture plastic and growth-inactivated 3T3-J2 co-cultures. Epithelial (E-cadherin and pan-cytokeratin) and progenitor (TP63, cytokeratin 5) marker expression were examined. Expanded cells were differentiated at air-liquid interface and ciliated, mucous producing, and club cell populations identified.Isolated cells were positive for the epithelial, pan-cytokeratin and E-cadherin, and progenitor, TP63 and cytokeratin 5, cell markers at isolation and again at passage 5. Passage 5 expanded cells in hypoxia had increased the proportion of TP63 expressing cells by 10% from 51.6 ± 1.2% to 62.6 ± 2.3% (p ≤ 0.01). Proliferative capacity was greater in 3T3J2 co-cultured cells overall and in 2% oxygen this supported the emergence of a proliferation unrestricted population with a limited differentiation capacity. Cells expanded on collagen I in either oxygen underwent differentiation having been expanded with the production of ciliated cells positive for βIV tubulin, and mucin 5ac, mucin 5b and CC10 positive secretory cells. Epithelial barrier formation was reduced significantly (p ≤ 0.0001) in hypoxia-expanded cells compared to normoxia. qRT-PCR showed higher expression of mucins in 2% expanded cells, significantly so with MUC5B (P ≤ 0.05) although mucin protein secretion was greater in 21% expanded cells.Concomitantly these results demonstrate that hypoxia promotes a proliferative phenotype while reducing the overall differentiation capacity of the cells. Further, the retained differentiation potential becomes skewed to a more secretory phenotype demonstrating that hypoxia may be contributing to disease symptom and severity in COPD patients.

Molecular and Functional Characteristics of Airway Epithelium under Chronic Hypoxia

“…Goblet cell hyperplasia is a common feature of epithelium in individuals with other chronic respiratory conditions with reduced lung function, such as COPD and asthma, as well as in ALI cultures subjected to hypoxia using the submersion method [ 17 , 56 , 57 ]. The exact mechanism of this change in phenotype is not well understood but is reported to be attributed to the Notch, HIF-1α and HIF-2α signaling [ 17 , 58 , 59 ].…”

“…Media change and passaging of both normoxic and hypoxic cultures for each participant were performed simultaneously. Airway basal stem cell expansion was assessed over one passage only in this study since the characteristics of airway basal stem cells expanded over serial passages, e.g., expansion capacity and population doubling rate, have already been extensively studied [ 15 , 56 ].…”