Hypoxia and FOXF1 Regulation in Lung Endothelial Cells

Hypoxia Represses FOXF1 in Lung Endothelial Cells Through HIF-1α

• Transcription factor Forkhead Box F1 (FOXF1) is important in lung angiogenesis during development and repair after injury
• FOXF1 expression decreases in endothelial cells after lung injury, but the mechanisms are not well understood
• Hypoxia is associated with decreased FOXF1 expression
• Inducing hypoxia in vitro helps identify the molecular mechanism

Methods

• Used immunostaining of injured mouse lung tissues
• Used FACS-sorted lung endothelial cells from hypoxia-treated mice
• Used data from patients diagnosed with hypoxemic respiratory failure
• Endothelial cell cultures were used to induce hypoxia in vitro
• Goal was to identify upstream molecular mechanism through which hypoxia inhibits FOXF1 gene expression

Results

• Bleomycin-induced lung injury induced hypoxia and decreased Foxf1 expression in mouse lung tissue
• Human FOXF1 mRNA was decreased in the lungs of patients diagnosed with hypoxemic respiratory failure
• Mice exposed to hypoxia exhibited reduced Foxf1 expression in lung tissue and FACS-sorted lung endothelial cells
• In vitro, hypoxia (1% of O2) or treatment with cobalt (II) chloride increased HIF-1a protein levels and inhibited FOXF1 expression in three endothelial cell lines
• Overexpression of HIF-1a in cultured endothelial cells was sufficient to inhibit Foxf1 expression
• siRNA-mediated depletion of HIF-1a prevented the downregulation of Foxf1 gene expression after hypoxia or cobalt (II) chloride treatment

Conclusion

• Hypoxia inhibits FOXF1 expression in endothelial cells in a HIF-1a dependent manner
• Endothelial cell-specific inhibition of HIF-1a via gene therapy might restore FOXF1 and improve lung repair in patients with severe lung injury

Introduction

• The lung is normally well-oxygenated but various conditions lead to hypoxia
• Hypoxic conditions include acute respiratory distress syndrome (ARDS), acute lung injury (ALI), COVID-19, influenza, bacterial pneumonia, and chronic obstructive pulmonary disease (COPD)
• Hypoxia is correlated with disease severity and poor outcomes in IPF and COPD patients
• FOXF1 is essential for angiogenesis during embryonic development
• Heterozygous deletions and loss-of-function mutations in FOXF1 are associated with alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV)

Materials and methods

Bleomycin-induced Lung Injury

• Mice administered bleomycin sulfate intratracheally to induce pulmonary fibrosis

Detection of Hypoxia in Bleomycin Model of Lung Injury

• Pimonidazole hydrochloride injected into mice with bleomycin-induced injury
• Lungs were analyzed to detect pimonidazole adducts, indicating hypoxia

In vivo Model of Hypoxia

• Mice were exposed to 10% oxygen in a hypoxia chamber
• Oxygen concentration was monitored
• Lungs were harvested and analyzed after 3, 7, and 10 days of hypoxia exposure

Cell lines and Reagents

• Human endothelial HUVEC, HPAEC, and mouse endothelial MFLM-91U cells cultured in specific growth mediums

In vitro Models of Hypoxia

• Cells exposed to 1% oxygen in a Whitley H35 Hypoxystation or treated with 150 µM of cobalt (II) chloride to mimic hypoxia

Flow Cytometry

• Enzyme-digested whole lungs were used for flow cytometry
• Cells were labeled with 7-AAD, CD45, and CD31 to identify and sort endothelial cells

qRT-PCR and Western Blot

• MFLM-91U cells transfected with siHifla or control siRNAs
• Cells lysed for RNA and protein for qRT-PCR and Western blot analyses
• TaqMan probes used for mouse and human genes

Human RNA seq

• Human samples from Pulmonary Hypertension Breakthrough Initiative (PHBI) Biobank were analyzed
• RNA sequencing performed using an Illumina sequencer

Results

• Bleomycin-injured mouse lungs showed increased hypoxia
• Human lung tissue from hypoxemic patients expressed decreased FOXF1
• Hypoxia alone, in vivo, decreased Foxf1 expression in lung endothelial cells

Hypoxia Inhibits Foxf1 in Lung Endothelial Cells in vitro

• 3 different endothelial cell lines (MFLM-91U, HUVECs, HPAECs) were used for time-course studies
• Cells placed under hypoxia (1% oxygen)
• Hypoxia inhibits Foxf1 expression in mouse lung endothelial cells in vivo

Hypoxia Represses Foxf1 Expression in Lung Endothelial Cells Through Hif-1α

• Relationship between hypoxia, increased HIF-1a protein levels, and reduced Foxf1 gene expression noted
• Overexpression of HIF-1a sufficient to inhibit Foxf1 even under normoxic conditions in endothelial cell cultures
• Inhibition of HIF-1a can restore the FOXF1 expression
• Restores FOXF1 mRNA and protein expression
• Results indicates hypoxia inhibits Foxf1 gene expression in endothelial cells

Discussion

• Maintenance of endothelial barrier function is crucial for maintaining normal lung homeostasis and repair
• Hypoxia plays a role in the downregulation of FOXF1 in endothelial cells