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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. Pulmonary fibrosis results from dysregulated lung repair, but the role of endothelial cells (EC) in fibrosis is unclear. Here, the authors show that FOXF1/R-Ras signalling in EC inhibits profibrotic mediators and that ECspecific nanoparticle FOXF1 gene therapy decreases lung fibrosis in mice.

Background Alveolar capillary dysplasia with misalignment of pulmonary veins (ACD/MPV) is a fatal congenital lung disorder strongly associated with genomic alterations in the Forkhead box F1 (FOXF1) gene and its regulatory region. However, little is known about how FOXF1 genomic alterations cause ACD/MPV and what molecular mechanisms are affected by these mutations. Therefore, the effect of ACD/MPV patient-specific mutations in the FOXF1 gene on the molecular function of FOXF1 was studied. Methods Epitope-tagged FOXF1 constructs containing one of the ACD/MPV-associated mutations were expressed in mammalian cell lines to study the effect of FOXF1 mutations on protein function. EMSA binding assays and luciferase assays were performed to study the effect on target gene binding and activation. Immunoprecipitation followed by SDS‒PAGE and western blotting were used to study protein‒protein interactions. Protein phosphorylation was studied using phos-tag western blotting. Results An overview of the localization of ACD/MPV-associated FOXF1 mutations revealed that the G91-S101 region was frequently mutated. A three-dimensional model of the forkhead DNA-binding domain of FOXF1 showed that the G91-S101 region consists of an α-helix and is predicted to be important for DNA binding. We showed that FOXF1 missense mutations in this region differentially affect the DNA binding of the FOXF1 protein and influence the transcriptional regulation of target genes depending on the location of the mutation. Furthermore, we showed that some of these mutations can affect the FOXF1 protein at the posttranscriptional level, as shown by altered phosphorylation by MST1 and MST2 kinases. Conclusion Missense mutations in the coding region of the FOXF1 gene alter the molecular function of the FOXF1 protein at multiple levels, such as phosphorylation, DNA binding and target gene activation. These results indicate that FOXF1 molecular pathways may be differentially affected in ACD/MPV patients carrying missense mutations in the DNA-binding domain and may explain the phenotypic heterogeneity of ACD/MPV.

Cancer cells re-program normal lung endothelial cells (EC) into tumor-associated endothelial cells (TEC) that form leaky vessels supporting carcinogenesis. Transcriptional regulators that control the reprogramming of EC into TEC are poorly understood. We identified Forkhead box F1 (FOXF1) as a critical regulator of EC-to-TEC transition. FOXF1 was highly expressed in normal lung vasculature but was decreased in TEC within non-small cell lung cancers (NSCLC). Low FOXF1 correlated with poor overall survival of NSCLC patients. In mice, endothelial-specific deletion of FOXF1 decreased pericyte coverage, increased vessel permeability and hypoxia, and promoted lung tumor growth and metastasis. Endothelial-specific overexpression of FOXF1 normalized tumor vessels and inhibited the progression of lung cancer. FOXF1 deficiency decreased Wnt/β-catenin signaling in TECs through direct transcriptional activation of Fzd4 . Restoring FZD4 expression in FOXF1-deficient TECs through endothelial-specific nanoparticle delivery of Fzd4 cDNA rescued Wnt/β-catenin signaling in TECs, normalized tumor vessels and inhibited the progression of lung cancer. Altogether, FOXF1 increases tumor vessel stability, and inhibits lung cancer progression by stimulating FZD4/Wnt/β-catenin signaling in TECs. Nanoparticle delivery of FZD4 cDNA has promise for future therapies in NSCLC. Synopsis Angiogenesis supports tumor growth. Current antiangiogenic therapies have not significantly improved survival; there is a critical need for new approaches. Mechanisms that regulate transition from normal to tumor-associated endothelial cells and drive pathological angiogenesis are poorly understood. FOXF1 is decreased in tumor-associated endothelial cells of NSCLC patients and mouse model of lung cancer. Genetic deletion of FOXF1 gene from endothelial cells increases lung tumor growth and metastasis, whereas transgenic over-expression of FOXF1 decreases lung tumor growth and metastasis. FOXF1-deficient tumor-associated vasculature is structurally and functionally abnormal. Loss of FOXF1 decreases Wnt/β-catenin signaling in endothelial cells through direct transcriptional activation of Fzd4. Endothelial-specific nanoparticle delivery of FZD4 cDNA normalizes tumor vessels and inhibits lung cancer progression. Angiogenesis supports tumor growth. Current antiangiogenic therapies have not significantly improved survival; there is a critical need for new approaches. Mechanisms that regulate transition from normal to tumor-associated endothelial cells and drive pathological angiogenesis are poorly understood.

Abstract Introduction/Objective Alveolar Capillary Dysplasia with Misalignment of Pulmonary Veins (ACD/MPV) is a rare disorder characterized by abnormal development of the pulmonary vasculature resulting in intractable pulmonary hypertension in neonates. Affected newborns present with progressive hypoxemia that is nearly always fatal. ACD/MPV is typically seen in the setting of other congenital anomalies, including gastrointestinal, genitourinary, and cardiac systems. Although the pathogenesis of ACD/MPV is poorly understood, inactivating mutations in the FOXF1 gene have been implicated. We present a case of ACD/MPV with associated gastrointestinal/cardiac anomalies and a novel heterozygous mutation of FOXF1. Methods/Case Report A complete autopsy was perfomed. Rapid whole exome sequencing of whole blood was done via reference laboratory. Results (if a Case Study enter NA) A 36-week gestational age male was born via cesarean delivery following a pregnancy course complicated by polyhydramnios. The neonate developed respiratory failure requiring intubation and Extracorporeal Membranous Oxygenation (ECMO). Further clinical evaluation revealed a patent foremen ovale and ductus arteriosus with right to left shunting. After 18 days on ECMO, he was transferred to comfort care where he later died. The body was that of a small for gestational age, phenotypic male neonate without gross dysmorphia noted on external examination. Internal examination revealed duodenal atresia, a patent ductus arteriosus and foramen ovale, and heavy, edematous lungs. Histologic examination of the lungs demonstrated prominent congested veins abutting small arteries with medial hypertrophy within shared adventitial sheaths. The interlobar septa contained dilated lymphatic spaces. Airspaces showed a dense neutrophilic exudate. Rapid whole exome sequencing revealed heterozygous mutations in FOXF1 (c.182T>C, p.Ile61Thr), NOTCH1 (c.7495A>C, p.Ser2499Arg) and a hemizygous mutation in SSR4 (c.20G>T, p.Gly7Val). The FOXF1 mutation was predicted to be damaging and has not yet been reported in the literature; however, its clinical significance was interpreted as uncertain. The significance of the NOTCH1 and SSR4 mutations were considered unknown. Given these findings, a diagnosis of ACD/MPV was given as the cause of death. Conclusion This case illustrates the classic clinical, gross and histologic findings described in ACD/MPV. Additionally, a novel heterozygous mutation of FOXF1 was identified and predicted to be damaging, supporting the role of FOXF1 mutations in the pathogenesis of ACD/MPV.

p53 is an established tumor suppressor that can activate the transcription of multiple target genes. Recent evidence suggests that p53 may contribute to the regulation of cell invasion and migration. In this study, we show that the forkhead box transcription factor FOXF1 is a novel target of the p53 family because FOXF1 is upregulated by p53, TAp73 and TAp63. We show that FOXF1 is induced upon DNA damage in a p53-dependent manner. Furthermore, we identified a response element located within the FOXF1 gene that is responsive to wild-type p53, TAp73β and TAp63γ. The ectopic expression of FOXF1 inhibited cancer cell invasion and migration, whereas the inactivation of FOXF1 stimulated cell invasion and migration. We also show that FOXF1 regulates the transcriptional activity of E-cadherin ( CDH1 ) by acting on its FOXF1 consensus binding site located upstream of the E-cadherin gene. Collectively, our results show that FOXF1 is a p53 family target gene, and our data suggest that FOXF1 and p53 form a portion of a regulatory transcriptional network that appears to have an important role in cancer cell invasion and migration.

Alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV) is a rare, often fatal congenital disorder characterized by severe neonatal respiratory distress and associated with complex multisystem malformations. In approximately 90% of cases, the condition is linked to deletions or mutations affecting the FOXF1 gene or its upstream enhancer region on chromosome 16q24.1. This review analyzes reported prenatal cases with 16q24.1 deletion involving FOXF1, aiming to identify recurrent sonographic features and elucidate the underlying genomic and epigenetic mechanisms. We reviewed prenatal cases reported in the literature involving deletions of the 16q24.1 region, including the FOXF1 gene. Here, we expand the case series by reporting a fetus with increased nuchal translucency measuring 8 mm and a de novo 16q24.1 deletion. We identified nine prenatal cases with a 16q24.1 deletion, all involving the FOXF1 gene or its enhancer region. The main ultrasound findings included increased nuchal translucency and cystic hygroma during the first trimester, and cardiac, renal, and intestinal malformations from 20 weeks of gestation onward. Prenatal diagnosis of ACDMPV based solely on ultrasound findings is challenging. In most reported cases, the pregnancy was carried to term, with the diagnosis being confirmed by post-mortem histopathological examination. In the only case in which the pregnancy was terminated at 14 weeks’ gestation, histological examination of the fetal lungs, despite them being in the early stages of development, revealed misaligned pulmonary veins in close proximity to the pulmonary arteries and bronchioles. Evidence highlights the significance of non-coding regulatory regions in the regulation of FOXF1 expression. Differential methylation patterns, and possible contributions of parental imprinting, highlight the complexity of FOXF1 regulation. Early detection through array comparative genomic hybridization (array CGH) or next-generation sequencing to identify point mutations in the FOXF1 gene, combined with increased awareness of ultrasound markers suggestive of the condition, could improve the accuracy of prenatal diagnosis and genetic counseling. Further research into the epigenetic regulation of FOXF1 is crucial for refining recurrence risk estimates and improving genetic counseling practices.

Background Alveolar capillary dysplasia with or without misalignment of the pulmonary veins (ACD/MPV) is a lethal congenital lung disorder associated with a variety of heterozygous genomic alterations in the FOXF1 gene or its 60 kb enhancer. Cases without a genomic alteration in the FOXF1 locus have been described as well. The mechanisms responsible for FOXF1 haploinsufficiency and the cause of ACD/MPV in patients without a genomic FOXF1 variant are poorly understood, complicating the search for potential therapeutic targets for ACD/MPV. To investigate the contribution of aberrant DNA methylation, genome wide methylation patterns of ACD/MPV lung tissues were compared with methylation patterns of control lung tissues using the recently developed technique Methylated DNA sequencing (MeD-seq). Results Eight ACD/MPV lung tissue samples and three control samples were sequenced and their mutual comparison resulted in identification of 319 differentially methylated regions (DMRs) genome wide, involving 115 protein coding genes. The potentially upregulated genes were significantly enriched in developmental signalling pathways, whereas potentially downregulated genes were mainly enriched in O-linked glycosylation. In patients with a large maternal deletion encompassing the 60 kb FOXF1 enhancer, DNA methylation patterns in this FOXF1 enhancer were not significantly different compared to controls. However, two hypermethylated regions were detected in the 60 kb FOXF1 enhancer of patients harbouring a FOXF1 point mutation. Lastly, a large hypermethylated region overlapping the first FOXF1 exon was found in one of the ACD/MPV patients without a known pathogenic FOXF1 variation. Conclusion This is the first study providing genome wide methylation data on lung tissue of ACD/MPV patients. DNA methylation analyses in the FOXF1 locus excludes maternal imprinting of the 60 kb FOXF1 enhancer. Hypermethylation at the 60 kb FOXF1 enhancer might contribute to FOXF1 haploinsufficiency caused by heterozygous mutations in the FOXF1 coding region. Interestingly, DNA methylation analyses of patients without a genomic FOXF1 variant suggest that abnormal hypermethylation of exon 1 might play a role in some ACD/MPV in patients.

Mutations in the (forkhead box F1) gene, encoding the mesenchymal ( ) transcription factor, are linked to alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV), a severe congenital disorder associated with the loss of alveolar capillaries and lung hypoplasia. Although proangiogenic functions of FOXF1 have been extensively studied, the role of FOXF1 in mesenchymal-epithelial signaling during lung development remains uncharacterized. Herein, we used murine lung organoids to demonstrate that the mutation (found in patients with ACDMPV) stimulates canonical WNT/β-catenin signaling in type 2 alveolar epithelial cells (AEC2s), leading to increased proliferation of AEC2s and decreased differentiation of AEC2s into type 1 alveolar epithelial cells (AEC1s). Alveolar organoids containing lung fibroblasts and wild-type epithelial cells grew faster on Matrigel and exhibited AEC2 hyperplasia. AEC2 hyperplasia and loss of AEC1s were found in the lungs of embryos, a mouse model of ACDMPV. Activation of canonical WNT/β-catenin signaling in AEC2s of lung organoids and mice was associated with decreased expression of noncanonical WNT5A (Wnt family member 5A) ligand in lung fibroblasts. Mechanistically, FOXF1 directly activates the gene transcription through an evolutionarily conserved +6320/+6326 region located in the first intron of the gene. Site-directed mutagenesis of the +6320/+6326 region prevented the transcriptional activation of the enhancer by FOXF1. Treatment with exogenous WNT5A ligand inhibited the effects of the mutation on canonical WNT/β-catenin signaling in alveolar organoids, preventing aberrant AEC2 expansion and restoring differentiation of AEC1s. Activation of either FOXF1 or WNT5A may provide an attractive strategy to improve lung function in patients with ACDMPV.

Background Epithelial-mesenchymal transition (EMT) increases cell migration and is implicated in cancer cell invasion and metastasis. We have previously described the involvement of the transcription factors, nuclear factor I-C2 (NFI-C2) and Forkhead box F1 (FoxF1), in the regulation of EMT and invasion during breast tumor progression. NFI-C2 counteracts these processes and FoxF1 is a directly repressed target of NFI-C2. FoxF1 induces EMT and invasiveness and enhances xenograft tumorigenicity in nude mice. Here we identify oppositely regulated targets of NFI-C2 and FoxF1 involved in these processes and further study a possible role for FoxF1 in tumorigenesis. Methods We used Affymetrix microarray to detect changes in the transcriptome of a mouse mammary epithelial cell line upon overexpression of NFI-C2 or FoxF1. To elucidate the effects and signaling events following FoxF1 overexpression we investigated in vitro invasion capacity and changes in transcription and protein expression resulting from RNAi and inhibitor treatment. Results The extracellular matrix enzyme lysyl oxidase (LOX) was negatively regulated by NFI-C2 and positively regulated by FoxF1, and upregulation of LOX following FoxF1 overexpression in mouse mammary epithelial cells increased in vitro cell invasion. In the nuclei of FoxF1-overexpressing cells, the phosphorylation of Smad2 decreased, while that of p38 increased. Depletion of LOX by RNAi enhanced phosphorylation of Smad2 by a focal adhesion kinase (FAK)-dependent mechanism. In addition, induced expression of FoxF1 in a non-malignant human mammary epithelial cell line showed that the increase in LOX transcription and the suppression of Smad2 activity are early effects of FoxF1. Conclusion These data show that FoxF1 enhances invasion in a LOX-dependent manner, is involved in the regulation of Smad2 signaling, and that FoxF1 overexpression ultimately leads to activation of p38 MAPK signaling. These findings provide new insights into the regulation of signaling pathways known to be important during breast tumor progression.

Viral carrier transport efficiency of gene delivery is high, depending on the type of vector. However, viral delivery poses significant safety concerns such as inefficient/unpredictable reprogramming outcomes, genomic integration, as well as unwarranted immune responses and toxicity. Thus, non-viral gene delivery methods are more feasible for translation as these allow safer delivery of genes and can modulate gene expression transiently both in vivo , ex vivo , and in vitro . Based on current studies, the efficiency of these technologies appears to be more limited, but they are appealing for clinical translation. This review presents a summary of recent advancements in orthopedics, where primarily bone and joints from the musculoskeletal apparatus were targeted. In connective tissues, which are known to have a poor healing capacity, and have a relatively low cell-density, i.e., articular cartilage, bone, and the intervertebral disk (IVD) several approaches have recently been undertaken. We provide a brief overview of the existing technologies, using nano-spheres/engineered vesicles, lipofection, and in vivo electroporation. Here, delivery for microRNA (miRNA), and silencing RNA (siRNA) and DNA plasmids will be discussed. Recent studies will be summarized that aimed to improve regeneration of these tissues, involving the delivery of bone morphogenic proteins (BMPs), such as BMP2 for improvement of bone healing. For articular cartilage/osteochondral junction, non-viral methods concentrate on targeted delivery to chondrocytes or MSCs for tissue engineering-based approaches. For the IVD, growth factors such as GDF5 or GDF6 or developmental transcription factors such as Brachyury or FOXF1 seem to be of high clinical interest. However, the most efficient method of gene transfer is still elusive, as several preclinical studies have reported many different non-viral methods and clinical translation of these techniques still needs to be validated. Here we discuss the non-viral methods applied for bone and joint and propose methods that can be promising in clinical use.