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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.

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.

Mutations in the FOXF1 gene, a key transcriptional regulator of pulmonary vascular development, cause Alveolar Capillary Dysplasia with Misalignment of Pulmonary Veins, a lethal lung disease affecting newborns and infants. Identification of new FOXF1 upstream regulatory elements is critical to explain why frequent non-coding FOXF1 deletions are linked to the disease. Herein, we use multiome single-nuclei RNA and ATAC sequencing of mouse and human patient lungs to identify four conserved endothelial and mesenchymal FOXF1 enhancers. We demonstrate that endothelial FOXF1 enhancers are autoactivated, whereas mesenchymal FOXF1 enhancers are regulated by EBF1 and GLI1. The cell-specificity of FOXF1 enhancers is validated by disrupting these enhancers in mouse embryonic stem cells using CRISPR/Cpf1 genome editing followed by lineage-tracing of mutant embryonic stem cells in mouse embryos using blastocyst complementation. This study resolves an important clinical question why frequent non-coding FOXF1 deletions that interfere with endothelial and mesenchymal enhancers can lead to the disease. Mutations in FOXF1 , a key transcriptional regulator of pulmonary vascular development, cause Alveolar Capillary Dysplasia with Misalignment of Pulmonary Veins. Here, the authors discovered four genomic regions that control cell type-specific activity of Foxf1 during lung development and show that disrupting these regions via genetic deletions leads to alveolar capillary dysplasia.

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.

The PAX3-FOXO1 fusion protein is the key oncogenic driver in fusion positive rhabdomyosarcoma (FP-RMS), an aggressive soft tissue malignancy with a particularly poor prognosis. Identifying key downstream targets of PAX3-FOXO1 will provide new therapeutic opportunities for treatment of FP-RMS. Herein, we demonstrate that Forkhead Box F1 (FOXF1) transcription factor is uniquely expressed in FP-RMS and is required for FP-RMS tumorigenesis. The PAX3-FOXO1 directly binds to FOXF1 enhancers and induces FOXF1 gene expression. CRISPR/Cas9 mediated inactivation of either FOXF1 coding sequence or FOXF1 enhancers suppresses FP-RMS tumorigenesis even in the presence of PAX3-FOXO1 oncogene. Knockdown or genetic knockout of FOXF1 induces myogenic differentiation in PAX3-FOXO1-positive FP-RMS. Over-expression of FOXF1 decreases myogenic differentiation in primary human myoblasts. In FP-RMS tumor cells, FOXF1 protein binds chromatin near enhancers associated with FP-RMS gene signature. FOXF1 cooperates with PAX3-FOXO1 and E-box transcription factors MYOD1 and MYOG to regulate FP-RMS-specific gene expression. Altogether, FOXF1 functions downstream of PAX3-FOXO1 to promote FP-RMS tumorigenesis.

Background Pneumonia constitutes a major health challenge in sheep, severely compromising growth rates and overall productivity, and resulting in considerable economic losses to the sheep industry. To address this issue, the development of disease-resistant breeding programs based on the identification of genetic markers associated with pneumonia susceptibility is of critical importance. This study investigated a sheep population on a farm where pneumonia was endemic. The purpose was to use multi-omics methods to rapidly identify the principal pathogens responsible for pneumonia outbreaks, and to screen for genetic loci and key genes related to pneumonia resistance, thereby providing a scientific basis for the implementation of targeted breeding strategies for pneumonia resistance. Results Here, we assessed the impact of pneumonia on sheep growth by evaluating the pneumonia phenotypes of 912 sheep. High-throughput transcriptome sequencing of 40 lungs was conducted to obtain exogenous RNA fragments for microbial sequence alignment. Additionally, 16S rRNA sequencing was performed on lung tissues from 10 healthy and 10 diseased sheep to identify biomarkers associated with phenotypic differences. Mycoplasma ovipneumoniae was identified as the primary pneumonia pathogen, and its presence was further validated by load quantification and immunohistochemical analysis. Integration of genome-wide association study (GWAS) data from 266 lung pathological scores with transcriptome-based differentially expressed genes analysis enabled the identification of five single nucleotide polymorphisms (SNPs) and three potential candidate genes associated with Mycoplasma pneumonia. Subsequent genotyping and phenotype association analyses confirmed the significance of two SNPs and established a strong association between the FOXF1 gene and resistance to Mycoplasma pneumonia. Conclusions High-throughput sequencing technologies have enabled the rapid and accurate identification of the causative pathogen of sheep pneumonia. By integrating multi-omics data, two genomic loci significantly associated with Mycoplasma pneumonia were screened, as well as an anti- Mycoplasma pneumonia key gene, FOXF1 .

Background Epigenetic modifications such as DNA methylation in the CpG islands of genes occur at a high rate. In this study, we measured the methylation level of the promoter region of the FOXF1 gene as a new blood biomarker for the detection of colorectal cancer in the early stages. Methods The methylation level of the promoter region of the FOXF1 gene was measured in the plasma samples of 50 colorectal cancer patients and 50 normal individuals. DNA was extracted after exposure to sodium bisulfite by the MethyLight polymerase chain reaction (PCR) method. The percentage of promoter region was measured in all samples, and statistical analysis was done using SPSS v24 software. Results The average promoter region between the plasma samples of colorectal cancer patients and healthy individuals had a significant difference (P < 0.001). The average promoter region of the FOXF1 gene in tumor plasma samples was 7.1 and in the control samples was 0.48. The sensitivity and specificity of the sample plasma levels were 78% and 89.5%, respectively. Conclusion The promoter region value of the FOXF1 gene in plasma samples using the MethyLight PCR method had high sensitivity and specificity as a non-invasive method for colorectal cancer diagnosis. This research is the first report that has been presented regarding the investigation of FOXF1 gene methylation in plasma samples in colorectal cancer. Therefore, it is necessary to conduct more studies with larger size samples to evaluate the efficiency of the gene under investigation.

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.

BackgroundAlveolar capillary dysplasia with misalignment of the pulmonary veins (ACDMPV) is a lethal disorder of lung development. ACDMPV is associated with haploinsufficiency of the transcription factor FOXF1, which plays an important role in the development of the lung and intestine. CNVs upstream of the FOXF1 gene have also been associated with an ACDMPV phenotype, but mechanism(s) by which these deletions disrupt lung development are not well understood. The objective of our study is to gain insights into the mechanisms by which CNVs contribute to an ACDMPV phenotype.MethodsWe analysed primary lung tissue from an infant with classic clinical and histological findings of ACDMPV and harboured a 340 kb deletion on chromosome 16q24.1 located 250 kb upstream of FOXF1.ResultsIn RNA generated from paraffin-fixed lung sections, our patient had lower expression of FOXF1 than age-matched controls. He also had an abnormal pattern of FOXF1 protein expression, with a dramatic loss of FOXF1 expression in the lung. To gain insights into the mechanisms underlying these changes, we assessed the epigenetic landscape using chromatin immunoprecipitation, which demonstrated loss of histone H3 lysine 27 acetylation (H3K27Ac), an epigenetic mark of active enhancers, in the region of the deletion.ConclusionsTogether, these data suggest that the deletion disrupts an enhancer responsible for directing FOXF1 expression in the developing lung and provide novel insights into the mechanisms underlying a fatal developmental lung disorder.