Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2024 Jan 11:14:1309155.
doi: 10.3389/fphys.2023.1309155. eCollection 2023.

Hypoxia represses FOXF1 in lung endothelial cells through HIF-1α

Anusha Acharya  1 , Fenghua Bian  1 , Jose Gomez-Arroyo  1   2 , Kimberly A Wagner  1 , Vladimir V Kalinichenko  3   4 , Tanya V Kalin  1   3
Affiliations

Hypoxia represses FOXF1 in lung endothelial cells through HIF-1α

Anusha Acharya et al. Front Physiol. .

Abstract

Introduction: Forkhead Box F1 (FOXF1) transcription factor plays a critical role in lung angiogenesis during embryonic development and lung repair after injury. FOXF1 expression is decreased in endothelial cells after lung injury; however, molecular mechanisms responsible for the FOXF1 transcript changes in injured lung endothelium remain unknown. Methods: We used immunostaining of injured mouse lung tissues, FACS-sorted lung endothelial cells from hypoxia-treated mice, and data from patients diagnosed with hypoxemic respiratory failure to demonstrate that hypoxia is associated with decreased FOXF1 expression. Endothelial cell cultures were used to induce hypoxia in vitro and identify the upstream molecular mechanism through which hypoxia inhibits FOXF1 gene expression. Results: Bleomycin-induced lung injury induced hypoxia in the mouse lung tissue which was associated with decreased Foxf1 expression. Human FOXF1 mRNA was decreased in the lungs of patients diagnosed with hypoxemic respiratory failure. Mice exposed to hypoxia exhibited reduced Foxf1 expression in the lung tissue and FACS-sorted lung endothelial cells. In vitro, hypoxia (1% of O2) or treatment with cobalt (II) chloride increased HIF-1α protein levels but inhibited FOXF1 expression in three endothelial cell lines. Overexpression of HIF-1α in cultured endothelial cells was sufficient to inhibit Foxf1 expression. siRNA-mediated depletion of HIF-1α 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-1α dependent manner. Our data suggest that endothelial cell-specific inhibition of HIF-1α via gene therapy can be considered to restore FOXF1 and improve lung repair in patients with severe lung injury.

Keywords: FOXF1; HIF-1α; endothelial cells; hypoxia; lung.

PubMed Disclaimer

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Hypoxia is associated with decreased Foxf1 expression in injured mouse and human lungs. (A,B) Immunohistochemical staining of mouse lung sections for hypoxyprobe-1-mAb1 shows bleomycin-injured lungs have increased hypoxia at day 3 post-injury induction. Hypoxyprobe area was calculated as % of hypoxyprobe-1-mAb1 positive area per field in 5 random fields using NIS elements software version 4.5. Each dot represents a single field. N = 3 mice per group. Both male and female mice were included. Scale bar = 50 µm. Values are shown as mean ± SD. *, p < 0.05; **, p < 0.01; ***, p < 0.001 from Student’s t test (two-tailed). (C) Hypoxemic patients had a P/F value of less than 400. The P/F ratio of patients was calculated as the partial pressure of oxygen in blood divided by the ratio of the inspired fraction of oxygen (P/F ratio) provided by the mechanical ventilator. N = total 46 patients. Values are shown as mean ± SD. *, p < 0.05; **, p < 0.01; ***, p < 0.001 from non-parametric Mann-Whitney U test. (D) Hypoxemic patients have decreased FOXF1 expression compared to non-hypoxemic patients. Normalized counts of FOXF1 expression were obtained from the RNA seq data set. N = total 46 patients. Each dot represents a patient. Values are shown as mean ± SD. *, p < 0.05; **, p < 0.01; ***, p < 0.001 from non-parametric Mann-Whitney U test.
FIGURE 2
FIGURE 2
Hypoxia inhibits Foxf1 in lung endothelial cells in vivo (A) Experimental schematic showing timeline of exposure of 8–12 weeks old Wild-type C57BL/6 mice to hypoxia or normoxia and lung harvest. (B) Foxf1 mRNA expression was decreased in mouse lungs at day 3, day 7 and day 10 after exposure to hypoxia. qRT-PCR was performed on total RNA isolated from harvested lung tissue. β-Actin (Actb) was used as the housekeeping gene. N = 4 mice per group. (C) The gating strategy utilized to FACS sort CD45-/CD31+ endothelial cells from mouse lungs exposed to hypoxia or normoxia. (D) Foxf1 mRNA expression was decreased in CD45−/CD31+ endothelial cells FACS sorted from mouse lungs at day 3, day 7 and day 10 post hypoxic exposure. qRT-PCR analysis was performed in triplicate on pooled RNA samples from each group. β-Actin (Actb) was used as the housekeeping gene. N = 4 mice per group. (E) FOXF1 protein expression was decreased in CD45-/CD31+ endothelial cells FACS sorted from mouse lungs at day 3, day 7 and day 10 post hypoxic exposure. Values on the western blot image represent FOXF1 protein expression relative to normoxic condition. Densitometric analysis was done using ImageJ software. Values were normalized to housekeeping protein β-Actin. Western blot was performed on pooled protein samples from each group. N = 4 mice per group. All values in this figure are shown as mean ± SD. *, p < 0.05; **, p < 0.01; ***, p < 0.001 from one-way ANOVA followed by Dunnett’s test.
FIGURE 3
FIGURE 3
Hypoxia inhibits Foxf1 in lung endothelial cells in vitro. (A) Foxf1 mRNA expression was decreased in mouse endothelial MFLM 91U cells under hypoxic exposure (1% O2) for 8 hours, 12 hours, and 24 hours (B) FOXF1 mRNA expression was decreased in human endothelial cells HUVEC (left) and HPAEC (right) under hypoxic exposure (1% O2) for 8 hours, 12 hours, and 24 hours (C) HIF-1α protein was induced in MFLM 91U cells under hypoxia (1% O2) from 8 hours to 24 hours (D) HIF-1α protein was induced in HUVEC (left) and HPAEC (right) under hypoxia (1% O2) from 8 hours to 24 hours (E) HIF-1α protein was induced in MFLM 91U cells upon treatment with 150 µM of cobalt (II) chloride for 8 hours, 12 hours, and 24 hours (F) HIF-1α was induced in HUVEC (left) and HPAEC (right) upon treatment with 150 µM of cobalt (II) chloride for 8 hours, 12 hours and 24 hours (G) Foxf1 mRNA expression was decreased in MFLM 91U upon treatment with 150 µM of cobalt (II) chloride for 8 hours, 12 hours and 24 hours (H) FOXF1 mRNA expression was decreased in HUVEC (left) and HPAEC (right) upon treatment with 150 µM of cobalt (II) chloride for 8 hours, 12 hours, and 24 hours. All qRT-PCR analyses employed β-Actin (Actb) as the housekeeping gene. Experiments were conducted three times. All values in this figure are shown as mean ± SD. *, p < 0.05; **, p < 0.01; ***, p < 0.001 from one-way ANOVA followed by Dunnett’s test.
FIGURE 4
FIGURE 4
Hypoxia represses Foxf1 expression in lung endothelial cells through HIF-1α. (A,B) HIF-1α protein was overexpressed in MFLM 91U cells transfected with CMV-HIF-1α under normoxic conditions. Protein expression is represented relative to control cells transfected with a CMV-empty vector. (C) Foxf1 mRNA expression was decreased in MFLM-91U cells with overexpression of HIF-1α under normoxic conditions. (D,E) FOXF1 protein expression was decreased in MFLM-91U cells with overexpression of HIF-1α under normoxic conditions. Protein expression is represented relative to control cells transfected with a CMV-empty vector. (F) Hif1α was knocked down at an efficiency of 75% under normoxic (left) and 67% under hypoxic (right) conditions. MFLM-91U cells were transfected with non-targeting siRNA (siNT) or siHif1α and subsequently exposed to normoxia (21% O2) or hypoxia (1% O2). (G,H) Knockdown of Hif1α prevents induction of HIF-1α protein under hypoxia (1% O2). MFLM-91U cells transfected with non-targeting siRNA (siNT) or siHif1α were exposed to normoxia (21% O2) or hypoxia (1% O2) for 12 hours. Protein expression is represented relative to control cells transfected with non-targeting siRNA (siNT) in normoxia. (I) Foxf1 mRNA expression is restored under hypoxic conditions upon knockdown of Hif1α. qRT-PCR was performed on MFLM-91U cells transfected with non-targeting siRNA (siNT) or siHif1α and exposed to normoxia (21% O2) or hypoxia (1% O2) for 12 h (J,K) FOXF1 protein expression is restored under hypoxic conditions upon knockdown of Hif1α. Protein expression is represented relative to control cells transfected with non-targeting siRNA (siNT) in normoxia. (L) Hif1α was knocked down at an efficiency of 73% under cobalt (II) chloride treatment. MFLM-91U cells were transfected with non-targeting siRNA (siNT) or siHif1α and subsequently treated with cobalt (II) chloride. (M,N) Knockdown of Hif1α prevents induction of HIF-1α protein under cobalt (II) chloride treatment. MFLM-91U cells were transfected with non-targeting siRNA (siNT) or siHif1α followed by treatment with cobalt (II) chloride for 12 h. Protein expression is represented relative to control cells transfected with non-targeting siRNA (siNT) and no cobalt (II) chloride treatment. (O) Foxf1 mRNA expression is restored under cobalt (II) chloride treatment upon knockdown of Hif1α. qRT-PCR was performed on MFLM-91U cells transfected with non-targeting siRNA (siNT) or siHif-1α and subsequently treated with cobalt (II) chloride for 12 h (P,Q) FOXF1 protein expression is restored under cobalt (II) chloride treatment upon knockdown of Hif1α. Protein expression is represented relative to control cells transfected with non-targeting siRNA (siNT) and no cobalt (II) chloride treatment. All qRT-PCR analyses employed β-Actin (Actb) as the housekeeping gene. Western blot quantifications were performed using densitometric analysis on ImageJ software. Experiments were conducted three times. All values in this figure are shown as mean ± SD. *, p < 0.05; **, p < 0.01; ***, p < 0.001 from Student’s t test (two-tailed).
See this image and copyright information in PMC

Similar articles

See all similar articles

References

    1. Akahori D., Inui N., Inoue Y., Yasui H., Hozumi H., Suzuki Y., et al. (2022). Effect of hypoxia on pulmonary endothelial cells from bleomycin-induced pulmonary fibrosis model mice. Int. J. Mol. Sci. 23, 8996. 10.3390/ijms23168996 - DOI - PMC - PubMed
    1. Aquino-Gálvez A., González-Ávila G., Jiménez-Sánchez L. L., Maldonado-Martínez H. A., Cisneros J., Toscano-Marquez F., et al. (2019). Dysregulated expression of hypoxia inducible factors augments myofibroblasts differentiation in idiopathic pulmonary fibrosis. Respir. Res. 20, 130. 10.1186/s12931-019-1100-4 - DOI - PMC - PubMed
    1. Ball M. K., Waypa G. B., Mungai P. T., Nielsen J. M., Czech L., Dudley V. J., et al. (2014). Regulation of hypoxia-induced pulmonary hypertension by vascular smooth muscle hypoxia-inducible factor-1a. Am. J. Respir. Crit. Care Med. 189, 314–324. 10.1164/rccm.201302-0302OC - DOI - PMC - PubMed
    1. Bian F., Lan Y.-W., Zhao S., Deng Z., Shukla S., Acharya A., et al. (2023). Lung endothelial cells regulate pulmonary fibrosis through FOXF1/R-Ras signaling. Nat. Commun. 14, 2560. 10.1038/s41467-023-38177-2 - DOI - PMC - PubMed
    1. Bishop N. B., Stankiewicz P., Steinhorn R. H. (2011). Alveolar capillary dysplasia. Am. J. Respir. Crit. Care Med. 184, 172–179. 10.1164/rccm.201010-1697CI - DOI - PMC - PubMed

LinkOut - more resources