BPD is a chronic lung disease commonly observed in preterm infants, characterised by vascularisation disorders and pulmonary vascular dysplasia. In this study, we successfully established a mouse BPD model through hyperoxia exposure. The observed changes in lung histomorphology, such as pulmonary septal thickening, alveolar simplification, and variations in size, were consistent with classical BPD pathology, confirming the success of our model.
PVECs play a decisive role in pulmonary angiogenesis and contribute to the endothelial barrier function. The pulmonary vasculature actively promotes alveolar growth during lung development, which helps to maintain alveolar structure and promote lung development (Surate et al. 2017). However, hyperoxia exposure can lead to oxidative stress in PVECs, resulting in cellular oedema, dysfunction, and reduced survival and growth (Zhang et al. 2018). PECAM-1, a cell adhesion molecule expressed in endothelial cells, promotes PVEC migration and pulmonary angiogenesis and serves as a proliferation marker for PVECs (Woodfin et al. 2007). In this study, we observed decreased PECAM-1 protein level and reduced pulmonary vascular density in the lung tissues of BPD model mice exposed to hyperoxia. This suggests that hyperoxia inhibits PVEC proliferation, impacting pulmonary vascular development and vascularisation. VEGFA, a potent PVEC-specific mitogen and survival factor, promotes PVEC proliferation and differentiation, thereby facilitating pulmonary vasculature growth and remodelling. It serves as a key regulator of early pulmonary vascular development, contributing to neonatal hyperoxic lung injury (Wiszniak et al. 2021). Additionally, Ang-1, a vascular growth factor acting specifically on PVECs, cooperates with VEGFA to induce neonatal pulmonary vascularisation in vivo and maintain vascular stability (Kamp et al. 2022). In the present study, we found significant reductions in VEGFA and Ang-1 protein levels in the lung tissues of mice exposed to hyperoxia compared to those in the control group. This reduction in VEGFA and Ang-1 protein levels could hinder PVEC migration and tube formation, thus impeding pulmonary vasculature development under hyperoxia exposure conditions. Sudhadevi et al found that a decrease in Ang-1 protein level was associated with the activity of PVECs and the impairment of alveolisation both in vitro and in mice (Sudhadevi et al. 2021). Therefore, together with our results, it is suggested that the decrease in VEGFA and Ang-1 protein levels, related to the proliferation of PVECs, represents impaired pulmonary vascular development under hyperoxia exposure.
Tregs are a group of T cells with immunosuppressive functions that play a crucial role in immune activation and developmental control of tissue damage in preterm infants (Pagel et al. 2020). Tregs have protective effects against PVECs in diseases such as pulmonary hypertension (Tamosiuniene et al. 2011). Our study revealed a decrease in Treg numbers in the lung tissues of mice exposed to hyperoxia, suggesting that hyperoxia inhibits Treg proliferation. Moreover, Treg count was positively correlated with PECAM-1 protein level, consistent with the trends observed in VEGFA and Ang-1 protein levels. This indicates that Tregs may protect the pulmonary vasculature by promoting PVEC proliferation. Thus, the impaired pulmonary vasculature development in BPD model mice may be linked to the attenuated protective effect of Tregs.
FOXP3, a nuclear transcription factor, is an important marker for Tregs (along with other classical markers such as CD4+, CD25+, and FOXP3+), which exert their immunosuppressive functions under its control (Wang et al. 2020). Kurebayashi et al. demonstrated that antagonism of FOXP3 expression in tumour vasculature decreases vascular density (Kurebayashi et al. 2021). Another study showed that in pulmonary hypertension, FOXP3 has also been associated with promoting pulmonary angiogenesis and development (Tian et al. 2021). Our study revealed consistent FOXP3 protein level and Treg proliferation in both control and hyperoxia-exposed C57 mice, suggesting that the protective effects of Tregs on the pulmonary vasculature are mediated through FOXP3.
IRF4, a member of the IRF family, primarily expressed in immune cells, serves as a co-transcription factor for Tregs. It plays roles in various immune functions, including cell development, differentiation, proliferation, and apoptosis, as well as the regulation of intrinsic and adaptive immune responses induced by pathogens (Nam et al. 2016). IRF4 is involved in the pathological process of a variety of inflammatory diseases, including the promotion of interleukin(IL)-6 production in inflammatory bowel disease, which induces STAT3 expression in T lymphocytes to promote apoptosis followed by intestinal inflammation (Zhu et al. 2016). Furthermore, IRF4 deficiency reduces inflammation and renal fibrosis following acute kidney injury induced by folic acid (Sasaki et al. 2021). Studies have also shown that IRF4 is involved in regulating immune responses such as oxidative stress in the lungs, and lung intrinsic lymphocytes depend on IRF4 to exert pro-inflammatory effects. For instance, IRF4 knockdown in mice markedly attenuates pro-inflammatory responses in response to external environmental stimuli, suggesting the involvement of IRF4 in inflammatory responses in the lungs (Liu et al. 2018). In addition, IRF4 can be expressed in vascular smooth muscle cells, regulating vascular wall integrity and functions. In our hyperoxia-induced BPD model, we observed significantly increased IRF4 protein levels in lung tissues, especially after 14 vs. 7 days of hyperoxia exposure. This suggests that upregulation of IRF4 may be involved in BPD development.
FOXP3 and IRF4, as two co-transcription factors of Tregs, are jointly involved in the signalling pathway to regulate their expression. It has been observed that FOXP3 is significantly upregulated in IRF4−/− Th cells cultured in the co-presence of IL-21 and transforming growth factor-β (Huber et al. 2008). Decreased FOXP3 expression and enhanced IRF4 expression were also detected in a mouse model of allergic asthma (Übel et al. 2014). On this basis, we also investigated the interaction between IRF4 and FOXP3 in the lung tissues of BPD model mice. We found a significant negative correlation between IRF4 and FOXP3 protein levels, suggesting that IRF4 exerts an inhibitory effect on FOXP3. This inhibition may influence the protective effect of Tregs on PVEC proliferation and pulmonary vascular development, contributing to BPD development(Fig. 5).
To explore this further, we used CRISPR/Cas9 technology to knock down the IRF4 gene in mice and conducted hyperoxia exposure experiments. Knocking down IRF4 resulted in increased FOXP3 protein level, promoting Treg proliferation. Additionally, IRF4−/− mice exposed to hyperoxia exhibited improved lung histopathology, enhanced lung structure organisation, increased PECAM-1 protein level, higher lung vascular density, and elevated levels of angiogenesis-related proteins VEGFA and Ang-1. These results indicate that IRF4 knockdown mitigates lung injury and impaired lung vascular development under hyperoxia conditions.