FoxM1 transcription factor (previously called HFH-11B, Trident, FoxM1b, Win, and MPP2) is expressed in actively dividing cells and critical for cell cycle progression. FoxM1 expression is induced in a variety of tissues during embryogenesis, and Foxm1-/- mice exhibit embryonic lethal phenotype due to multiple abnormalities in the liver, heart, lung and blood vessels. FoxM1 levels are dramatically decreased in adult tissues, but FoxM1 expression is re-activated during organ injury and numerous cancers. In this review, we discussed the role of FoxM1 in different cell lineages using recent data from transgenic mouse models with conditional “gain-of-function” and “loss-of-function” of FoxM1, as well as tissue samples from human patients. In addition, we provided experimental data showing additional sites of FoxM1 expression in the mouse embryo. Novel cell-autonomous roles of FoxM1 in embryonic development, organ injury and cancer formation in vivo were analyzed. Potential application of these findings for the diagnosis and treatment of human diseases were discussed.

Rationale:: Inactivating mutations in the Forkhead Box transcription factor F1 ( FOXF1 ) gene locus are frequently found in patients with alveolar capillary dysplasia with misalignment of pulmonary veins, a lethal congenital disorder, which is characterized by severe abnormalities in the respiratory, cardiovascular, and gastrointestinal systems. In mice, haploinsufficiency of the Foxf1 gene causes alveolar capillary dysplasia and developmental defects in lung, intestinal, and gall bladder morphogenesis. Objective:: Although FOXF1 is expressed in multiple mesenchyme-derived cell types, cellular origins and molecular mechanisms of developmental abnormalities in FOXF1-deficient mice and patients with alveolar capillary dysplasia with misalignment of pulmonary veins remain uncharacterized because of lack of mouse models with cell-restricted inactivation of the Foxf1 gene. In the present study, the role of FOXF1 in endothelial cells was examined using a conditional knockout approach. Methods and Results:: A novel mouse line harboring Foxf1 -floxed alleles was generated by homologous recombination. Tie2-Cre and Pdgfb-CreER transgenes were used to delete Foxf1 from endothelial cells. FOXF1-deficient embryos exhibited embryonic lethality, growth retardation, polyhydramnios, cardiac ventricular hypoplasia, and vascular abnormalities in the lung, placenta, yolk sac, and retina. Deletion of FOXF1 from endothelial cells reduced endothelial proliferation, increased apoptosis, inhibited vascular endothelial growth factor signaling, and decreased expression of endothelial genes critical for vascular development, including vascular endothelial growth factor receptors Flt1 and Flk1, Pdgfb, Pecam1, CD34, integrin β3, ephrin B2, Tie2, and the noncoding RNA Fendrr . Chromatin immunoprecipitation assay demonstrated that Flt1, Flk1, Pdgfb, Pecam1, and Tie2 genes are direct transcriptional targets of FOXF1. Conclusions:: FOXF1 is required for the formation of embryonic vasculature by regulating endothelial genes critical for vascular development and vascular endothelial growth factor signaling.

Pulmonary fibrosis results from dysregulated lung repair and involves multiple cell types. The role of endothelial cells (EC) in lung fibrosis is poorly understood. Using single cell RNA-sequencing we identified endothelial transcription factors involved in lung fibrogenesis, including FOXF1, SMAD6, ETV6 and LEF1. Focusing on FOXF1, we found that FOXF1 is decreased in EC within human idiopathic pulmonary fibrosis (IPF) and mouse bleomycin-injured lungs. Endothelial-specific Foxf1 inhibition in mice increased collagen depositions, promoted lung inflammation, and impaired R-Ras signaling. In vitro, FOXF1-deficient EC increased proliferation, invasion and activation of human lung fibroblasts, and stimulated macrophage migration by secreting IL-6, TNFα, CCL2 and CXCL1. FOXF1 inhibited TNFα and CCL2 through direct transcriptional activation of Rras gene promoter. Transgenic overexpression or endothelial-specific nanoparticle delivery of Foxf1 cDNA decreased pulmonary fibrosis in bleomycin-injured mice. Nanoparticle delivery of FOXF1 cDNA can be considered for future therapies in IPF.

Respiratory disorders are among the most important medical problems threatening human life. The conventional therapeutics for respiratory disorders are hindered by insufficient drug concentrations at pathological lesions, lack of cell-specific targeting, and various biobarriers in the conducting airways and alveoli. To address these critical issues, various nanoparticle delivery systems have been developed to serve as carriers of specific drugs, DNA expression vectors, and RNAs. The unique properties of nanoparticles, including controlled size and distribution, surface functional groups, high payload capacity, and drug release triggering capabilities, are tailored to specific requirements in drug/gene delivery to overcome major delivery barriers in pulmonary diseases. To avoid off-target effects and improve therapeutic efficacy, nanoparticles with high cell-targeting specificity are essential for successful nanoparticle therapies. Furthermore, low toxicity and high degradability of the nanoparticles are among the most important requirements in the nanoparticle designs. In this review, we provide the most up-to-date research and clinical outcomes in nanoparticle therapies for pulmonary diseases. We also address the current critical issues in key areas of pulmonary cell targeting, biosafety and compatibility, and molecular mechanisms for selective cellular uptake.

Multiple signaling pathways, structural proteins, and transcription factors are involved in the regulation of endothelial barrier function. The forkhead protein FOXF1 is a key transcriptional regulator of embryonic lung development, and we used a conditional knockout approach to examine the role of FOXF1 in adult lung homeostasis, injury, and repair. Tamoxifen-regulated deletion of both Foxf1 alleles in endothelial cells of adult mice (Pdgfb-iCreER/Foxf1^−/−) caused lung inflammation and edema, leading to respiratory insufficiency and death. Deletion of a single Foxf1 allele made heterozygous Pdgfb-iCreER/Foxf1^+/− mice more susceptible to acute lung injury. FOXF1 abundance was decreased in pulmonary endothelial cells of human patients with acute lung injury. Gene expression analysis of pulmonary endothelial cells with homozygous FOXF1 deletion indicated reduced expression of genes critical for maintenance and regulation of adherens junctions. FOXF1 knockdown in vitro and in vivo disrupted adherens junctions, enhanced lung endothelial permeability, and increased the abundance of the mRNA and protein for sphingosine 1-phosphate receptor 1 (S1PR1), a key regulator of endothelial barrier function. Chromatin immunoprecipitation and luciferase reporter assays demonstrated that FOXF1 directly bound to and induced the transcriptional activity of the S1pr1 promoter. Pharmacological administration of S1P to injured Pdgfb-iCreER/Foxf1^+/− mice restored endothelial barrier function, decreased lung edema, and improved survival. Thus, FOXF1 promotes normal lung homeostasis and repair, in part, by enhancing endothelial barrier function through activation of the S1P/S1PR1 signaling pathway.

Forkhead Box M1 (Foxm1) is a transcription factor essential for organ morphogenesis and development of various cancers. Although complete deletion of Foxm1 in Foxm1^−/− mice caused embryonic lethality due to severe abnormalities in multiple organ systems, requirements for Foxm1 in cardiomyocytes remain to be determined. This study was designed to elucidate the cardiomyocyte-autonomous role of Foxm1 signaling in heart development. We generated a new mouse model in which Foxm1 was specifically deleted from cardiomyocytes (Nkx2.5-Cre/Foxm1^fl/f mice). Deletion of Foxm1 from cardiomyocytes was sufficient to disrupt heart morphogenesis and induce embryonic lethality in late gestation. Nkx2.5-Cre/Foxm1^fl/fl hearts were dilated with thinning of the ventricular walls and interventricular septum, as well as disorganization of the myocardium which culminated in cardiac fibrosis and decreased capillary density. Cardiomyocyte proliferation was diminished in Nkx2.5-Cre/Foxm1^fl/fl hearts owing to altered expression of multiple cell cycle regulatory genes, such as Cdc25B, Cyclin B₁, Plk-1, nMyc and p21^cip1. In addition, Foxm1 deficient hearts displayed reduced expression of CaMKIIδ, Hey2 and myocardin, which are critical mediators of cardiac function and myocardial growth. Our results indicate that Foxm1 expression in cardiomyocytes is critical for proper heart development and required for cardiomyocyte proliferation and myocardial growth.