Fibroblast Growth Factor Signaling Research Articles

Feasibility and antitumour activity of the FGFR inhibitor erdafitnib in three paediatric CNS tumour patients.

Alterations of the fibroblast growth factor (FGF) signalling pathway are increasingly recognized as frequent oncogenic drivers of paediatric brain tumours. We report on three patients treated with the selective FGFR1-4 inhibitor erdafitinib. Two patients were diagnosed with a posterior fossa ependymoma group A (PFA EPN) and one with a low-grade glioma (LGG), harbouring FGFR3/FGFR1 overexpression and an FGFR1 internal tandem duplication (ITD), respectively. While both EPN patients did not respond to erdafitinib treatment, the FGFR1-ITD-harbouring tumour showed a significant decrease in tumour volume and contrast enhancement throughout treatment. The tumour remained stable 6months after treatment discontinuation.

FGF2/HGF priming facilitates adipose-derived stem cell-mediated bone formation in osteoporotic defects

AimsThe activity of adipose-derived stem cells (ADSCs) is susceptible to the physiological conditions of the donor. Therefore, employing ADSCs from donors of advanced age or with diseases for cell therapy necessitates a strategy to enhance therapeutic efficacy before transplantation. This study aims to investigate the impact of supplementing Fibroblast Growth Factor 2 (FGF2) and Hepatocyte Growth Factor (HGF) on ADSC-mediated osteogenesis under osteoporotic conditions and to explore the underlying mechanisms of action. Main methodsAdipose-derived stem cells (ADSCs) obtained from ovariectomized (OVX) rats were cultured ex vivo. These cells were cultured in an osteogenic medium supplemented with FGF2 and HGF and subsequently autologously transplanted into osteoporotic femur defects using Hydroxyapatite-Tricalcium Phosphate. The assessment of bone formation was conducted four weeks post-transplantation. Key findingsOsteoporosis detrimentally affects the viability and osteogenic differentiation potential of ADSCs, often accompanied by a deficiency in FGF2 and HGF signaling. However, priming with FGF2 and HGF facilitated the formation of immature osteoblasts from OVX ADSCs in vitro, promoting the expression of osteoblastogenic proteins, including Runx-2, osterix, and ALP, during the early phase of osteogenesis. Furthermore, FGF2/HGF priming augmented the levels of VEGF and SDF-1α in the microenvironment of OVX ADSCs under osteogenic induction. Importantly, transplantation of OVX ADSCs primed with FGF2/HGF for 6 days significantly enhanced bone formation compared to non-primed cells. The success of bone regeneration was confirmed by the expression of type-1 collagen and osteocalcin in the bone tissue of the deficient area. SignificanceOur findings corroborate that priming with FGF2/HGF can improve the differentiation potential of ADSCs. This could be applied in autologous stem cell therapy for skeletal disease in the geriatric population.

Diverse Responses of Oligodendrocytes to Different FGF-Family Members: Uncoupling Structure-Function Relationship Within FGF Subfamilies.

The fifteen canonical paracrine fibroblast growth factors (FGFs) are organized in five subfamilies that interact with four FGF-receptors (FGFRs) and heparan sulfate proteoglycan (HSPG) co-receptors. Many of these FGFs are expressed in CNS regions where oligodendrocyte (OL) progenitors originate, migrate or differentiate. FGF2 (basic FGF) is considered a prototype FGF and the information about the effects of FGF signaling on OL-lineage cells has evolved largely from the study of FGF2. However, other FGFs from four subfamilies ((FGF1 (FGF1,-2), FGF4 (FGF4,-5,-6), FGF8 (FGF8,-17,-18) and FGF9 (FGF9,-16,-20)) that can interact with the isoforms of FGFRs expressed in OL-lineage cells may also play important roles. We previously reported OL-responses to FGF8 family members. Here, we investigate the effects of members of the FGF1,-4, and -9 subfamilies on proliferation and differentiation of OL progenitors (OPCs), and on cell cycle re-entry and down-regulation of myelin proteins by mature OLs. We found that while FGF2 induced all these responses strongly, FGF4,-6,-9 could do so only transiently and in the presence of exogenous HSPGs, and that FGF5,-16,-20 could not do so even in the presence of heparin or at higher concentrations. Furthermore, we noted that structurally similar FGFs within subfamilies did not always show similarities in their biological effects on OL-lineage cells. Taken together, these studies reveal that FGFs differ in the way they regulate the OL-lineage cells, emphasizes the selectivity and importance of HSPGs as FGF co-receptors in OL-lineage cells and suggests that structural similarity among FGF-subfamily members may not always predict their overlapping biological functions.

Efficient and reproducible generation of human induced pluripotent stem cell-derived expandable liver organoids for disease modeling

Genetic liver disease modeling is difficult because it is challenging to access patient tissue samples and to develop practical and relevant model systems. Previously, we developed novel proliferative and functional liver organoids from pluripotent stem cells; however, the protocol requires improvement for standardization and reproducible mass production. Here, we improved the method such that it is suitable for scalable expansion and relatively homogenous production, resulting in an efficient and reproducible process. Moreover, three medium components critical for long-term expansion were defined. Detailed transcriptome analysis revealed that fibroblast growth factor signaling, the essential pathway for hepatocyte proliferation during liver regeneration, was mainly enriched in proliferative liver organoids. Short hairpin RNA-mediated knockdown of FGFR4 impaired the generation and proliferation of organoids. Finally, glycogen storage disease type Ia (GSD1a) patient-specific liver organoids were efficiently and reproducibly generated using the new protocol. They well maintained disease-specific phenotypes such as higher lipid and glycogen accumulation in the liver organoids and lactate secretion into the medium consistent with the main pathologic characteristics of patients with GSD1a. Therefore, our newly established liver organoid platform can provide scalable and practical personalized disease models and help to find new therapies for incurable liver diseases including genetic liver diseases.

The expression level of fibroblast growth factor gene in serum samples of lung cancer patients.

Lung cancer, one of the most deadly and dangerous types of cancer in the world, kills many men and women every year. Activation of fibroblast growth factor signals plays a role in the pathogenesis of several cancers, including lung cancer. Also, this factor may indicate prognosis and is related to the survival rate in patients with NSCLC. Therefore, this research investigated the level of fibroblast growth factor gene expression in the serum of people with lung cancer. In this research, 60 serum samples of healthy people and 60 serum samples of people with NSCLC were prepared, and personal and clinicopathological information of the studied people were collected by questionnaires. Then, plasma isolation, RNA extraction, cDNA synthesis, primers design, implementation, and changes in fibroblast growth factor gene expression in the serum of healthy and lung cancer patients were evaluated by the Real-time PCR method. REST software was used to analyze the results. The findings showed no significant difference in the expression of the fibroblast growth factor gene in the serum of people with the first to third stages of metastasis. However, in the serum of patients with the fourth stage of metastasis, the expression level of this gene was significantly decreased by 3.92 times compared to normal samples (P<0.05). According to the results of this study, it is possible to use the expression level of the fibroblast growth factor gene in people's serum to predict the metastasis stage of lung cancer.

Suppression of BMP signaling restores mitral cell development impaired by FGF signaling deficits in mouse olfactory bulb

Fibroblast growth factors (FGFs) and bone morphogenic proteins (BMPs) play various important roles in the development of the central nervous system. However, the roles of FGF and BMP signaling in the development of the olfactory bulb (OB) are largely unknown. In this study, we first showed the expression of FGF receptors (FGFRs) and BMP receptors (BMPRs) in OB RGCs, radial glial cells (RGCs) in the developing OB, which generate the OB projection neurons, mitral and tufted cells. When the FGF signaling was inhibited by a dominant-negative form of FGFR1 (dnFGFR1), OB RGCs accelerated their state transition to mitral cell precursors without affecting their transcription cascade and fate. However, the mitral cell precursors could not radially migrate to form the mitral cell layer (MCL). In addition, FGF signaling inhibition reduced the expression of a BMP antagonist, Noggin, in the developing OB. When BMP signaling was suppressed by the ectopic expression of Noggin or a dominant-negative form of BMPR1a (dnBMPR1a) in the developing OB, the defect in MCL formation caused by the dnFGFR1 was rescued. However, the dnBMPR1a did not rescue the accelerated state transition of OB RGCs. These results demonstrate that FGF signaling is important for OB RGCs to maintain their self-renewal state and MCL formation. Moreover, the suppression of BMP signaling is required for mitral cells to form the MCL. This study sheds new light on the roles of FGFs and BMPs in OB development.

Development of Physiologically Relevant Skin Organoids from Human Induced Pluripotent Stem Cells.

The development of skin organs for studying developmental pathways, modeling diseases, or regenerative medicine purposes is a major endeavor in the field. Human induced pluripotent stem cells (hiPSCs) are successfully used to deriveskin cells, but the field is still far from meeting the goal of creating skin containing appendages, such as hair follicles and sweat glands. Here, the goal is to generate skin organoids (SKOs) from human skin fibroblast or placental CD34+ cell-derived hiPSCs. With all three hiPSC lines, complex SKOs with stratified skin layers and pigmented hair follicles are generated with different efficacies. In addition, the hiPSC-derived SKOs develop sebaceous glands, touch-receptive Merkel cells, and more importantly eccrine sweat glands. Together, physiologically relevant skin organoids are developed by direct induction of embryoid body formation, along with simultaneous inactivation of transforming growth factor beta signaling, activation of fibroblast growth factor signaling, and inhibition of bone morphogenetic protein signaling pathways. The skin organoids created in this study can be used as valuable platforms for further research into human skin development, disease modeling, or reconstructive surgeries.

Fibroblast growth factor 10

Fibroblast growth factor 10 (FGF10) is a major morphoregulatory factor that plays essential signaling roles during vertebrate multiorgan development and homeostasis. FGF10 is predominantly expressed in mesenchymal cells and signals though FGFR2b in adjacent epithelia to regulate branching morphogenesis, stem cell fate, tissue differentiation and proliferation, in addition to autocrine roles. Genetic loss of function analyses have revealed critical requirements for FGF10 signaling during limb, lung, digestive system, ectodermal, nervous system, craniofacial and cardiac development. Heterozygous FGF10 mutations have been identified in human genetic syndromes associated with craniofacial anomalies, including lacrimal and salivary gland aplasia. Elevated Fgf10 expression is associated with poor prognosis in a range of cancers. In addition to developmental and disease roles, FGF10 regulates homeostasis and repair of diverse adult tissues and has been identified as a target for regenerative medicine.

FGF23 Inhibition Improves Hematopoietic Stem Cell Transplantation in β-Thalassemia

Clinical evidence has established a link between anemias and skeletal abnormalities, although the underlying molecular mechanisms remain elusive. β-thalassemia (βThal) represents a valuable model for studying congenital anemia and associated bone and bone marrow (BM) microenvironment defects. Osteoporosis has a high morbidity in βThal patients, and we demonstrated that impaired hematopoietic stem cell (HSC) function is due to the prolonged persistence in an altered βThal BM niche ( Aprile et al, 2020). The correction of the genetic cause of βThal, mutations in the β-globin gene, is achieved by transplantation of HSCs from healthy donors or autologous HSCs from patients upon gene therapy. In both scenarios, a comprehensive understanding of HSCs and the BM niche is crucial to obtain successful outcomes. Our research focused on the role of fibroblast growth factor 23 (FGF23), a phosphaturic hormone at the crossroads of bone and erythropoiesis. We demonstrated that the elevated erythropoietin characteristic of the disease, leads to increased FGF23 production in both the βThal mouse model ( th3) and βThal patients, negatively affecting bone homeostasis and the interaction between HSCs and the stromal niche. Notably, in vivo FGF23 inhibition by cFGF23 peptide in th3 mice fully rescued bone defects, BM niche and HSC function ( Aprile, Raggi et al, 2023). To mimic the allogenic transplantation, we transplanted wt and th3 cells in a competitive setting into untreated th3 or th3+cFGF23 recipients. We observed partially enhanced engraftment in the cFGF23-treated mice indicating that FGF23 inhibition improves the supportive capacity of the damaged niche. We hypothesized that prolonged cFGF23 administration to recipient mice after transplantation could further restore the BM niche and boost the graft, thus achieving therapeutic outcomes. To establish the proper dose regimen with withdrawal periods for prolonged cFGF23 treatment, we analyzed bone mineral density (BMD) and HSC cell cycle at 1 and 2 weeks after discontinuing cFGF23 administration. Our results showed that rescued BMD was sustained after the discontinuation of treatment for 1 and 2 weeks ( th3 vs. th3+cFGF23 _1wk vs. th3+cFGF23_2wks: 125.9±3.8 vs. 181±19.9 vs. 142±1.6 mg/cm 3, p&amp;lt;0.01). Ongoing histomorphometric analysis will provide a better characterization of bone quality. Additionally, we observed the restoration of defective quiescence in th3 HSCs after 1- and 2 weeks ( th3 vs. th3+cFGF23 _1wk vs. th3+cFGF23_2wks: 77.8±1,4 vs. 92.1±1.8 vs. 87.8±0.9 on Lin neg BM cells, p&amp;lt;0.001). To test autologous transplantation upon gene therapy, we transduced th3 Lin neg BM cells with a lentiviral vector carrying the β-globin gene (GLOBE LV) at MOI 5 resulted in a mean vector copy number of 1 and a transduction efficiency of 50%. We performed competitive transplantation using 1x10 5 (CD45.1) transduced donor th3 cells and 1x10 5 (CD45.2) competitor mock-transduced th3 cells into busulfan-conditioned (CD45.2) untreated th3 or th3+cFGF23 recipient mice. The treated recipient mice received two doses of cFGF23 every 2 weeks as a maintenance regimen. We observed higher engraftment of transduced cells in the peripheral blood of cFGF23-treated mice at 8 weeks post-transplant ( th3 vs. th3+cFGF23: 4.9±1.1 vs. 10.7±1.9 % of CD45.1 engrafted cells, p&amp;lt;0.05). Comparative evaluation of hematological parameters and molecular analysis of transduced cells are ongoing. Targeting FGF23 signaling provides a promising strategy to improve bone defects and HSC-niche alterations in βThal. Here, we show that the use of FGF23 inhibition in the transplantation setting in β-thalassemia results in superior engraftment of donor cells in treated recipients, indicating a potential application to ameliorate the clinical outcome.

Nkx2.3 is responsible for posterior pharyngeal cartilage formation by inhibiting Fgf signaling

Nkx2.3, a transcription factor, plays important roles in various developmental processes. However, the mechanisms underlying nkx2.3's regulation of pouch and pharyngeal arch development in zebrafish remain unclear. In this study, we demonstrated that knockdown or knockout of nkx2.3 resulted in the absence of posterior ceratobranchial cartilages in zebrafish. The absence of posterior pharyngeal cartilages is a consequence of the compromised proliferation and differentiation and survival of cranial neural crest cells (CNCCs). Notably, we found that nkx2.3 was not involved in endoderm pouch formation. Additionally, our findings suggested that nkx2.3 negatively regulated Fibroblast growth factor (Fgf) signaling, as overexpression of fgf8 could mimic the phenotype observed in nkx2.3 morphants, suppressing CNCC differentiation. Moreover, inhibiting Fgf signaling restored the abnormalities in posterior cartilages induced by nkx2.3 knockdown. These findings establish the essential role of nkx2.3 in the development of posterior ceratobranchial cartilages through the inhibition of fgf8.

Fibroblast growth factor 9 (FGF9)-mediated neurodegeneration: Implications for progressive multiple sclerosis?

Fibroblast growth factor (FGF) signalling is dysregulated in multiple sclerosis (MS) and other neurological and psychiatric conditions, but there is little or no consensus as to how individual FGF family members contribute to disease pathogenesis. Lesion development in MS is associated with increased expression of FGF1, FGF2 and FGF9, all of which modulate remyelination in a variety of experimental settings. However, FGF9 is also selectively upregulated in major depressive disorder (MDD), prompting us to speculate it may also have a direct effect on neuronal function and survival. Transcriptional profiling of myelinating cultures treated with FGF1, FGF2 or FGF9 was performed, and the effects of FGF9 on cortical neurons investigated using a combination of transcriptional, electrophysiological and immunofluorescence microscopic techniques. The in vivo effects of FGF9 were explored by stereotactic injection of adeno-associated viral (AAV) vectors encoding either FGF9 or EGFP into the rat motor cortex. Transcriptional profiling of myelinating cultures after FGF9 treatment revealed a distinct neuronal response with a pronounced downregulation of gene networks associated with axonal transport and synaptic function. In cortical neuronal cultures, FGF9 also rapidly downregulated expression of genes associated with synaptic function. This was associated with a complete block in the development of photo-inducible spiking activity, as demonstrated using multi-electrode recordings of channel rhodopsin-transfected rat cortical neurons in vitro and, ultimately, neuronal cell death. Overexpression of FGF9 in vivo resulted in rapid loss of neurons and subsequent development of chronic grey matter lesions with neuroaxonal reduction and ensuing myelin loss. These observations identify overexpression of FGF9 as a mechanism by which neuroaxonal pathology could develop independently of immune-mediated demyelination in MS. We suggest targeting neuronal FGF9-dependent pathways may provide a novel strategy to slow if not halt neuroaxonal atrophy and loss in MS, MDD and potentially other neurodegenerative diseases.

Structural asymmetry in FGF23 signaling

Chen et al. have derived cryogenic electron microscopy (cryo-EM) structures of signaling complexes of the endocrine hormone fibroblast growth factor 23 (FGF23) with fibroblast growth factor receptor (FGFR), α-Klotho, and heparin sulfate. These structures are asymmetric, leading to questions concerning in vivo function, and will facilitate structure-based drug design to modulate FGF23 signaling.

Zbtb16 mediates a switch between Fgf signalling regimes in the developing hindbrain.

Developing tissues are sequentially patterned by extracellular signals that are turned on and off at specific times. In the zebrafish hindbrain, fibroblast growth factor (Fgf) signalling has different roles at different developmental stages: in the early hindbrain, transient Fgf3 and Fgf8 signalling from rhombomere 4 is required for correct segmentation, whereas later, neuronal Fgf20 expression confines neurogenesis to specific spatial domains within each rhombomere. How the switch between these two signalling regimes is coordinated is not known. We present evidence that the Zbtb16 transcription factor is required for this transition to happen in an orderly fashion. Zbtb16 expression is high in the early anterior hindbrain, then gradually upregulated posteriorly and confined to neural progenitors. In mutants lacking functional Zbtb16, fgf3 expression fails to be downregulated and persists until a late stage, resulting in excess and more widespread Fgf signalling during neurogenesis. Accordingly, the spatial pattern of neurogenesis is disrupted in Zbtb16 mutants. Our results reveal how the distinct stage-specific roles of Fgf signalling are coordinated in the zebrafish hindbrain.

An improved Erk biosensor detects oscillatory Erk dynamics driven by mitotic erasure during early development

Extracellular signal-regulated kinase (Erk) signaling dynamics elicit distinct cellular responses in a variety of contexts. The early zebrafish embryo is an ideal model to explore the role of Erk signaling dynamics invivo, as a gradient of activated diphosphorylated Erk (P-Erk) is induced by fibroblast growth factor (Fgf) signaling at the blastula margin. Here, we describe an improved Erk-specific biosensor, which we term modified Erk kinase translocation reporter (modErk-KTR). We demonstrate the utility of this biosensor invitro and in developing zebrafish and Drosophila embryos. Moreover, we show that Fgf/Erk signaling is dynamic and coupled to tissue growth during both early zebrafish and Drosophila development. Erk activity is rapidly extinguished just prior to mitosis, which we refer to as mitotic erasure, inducing periods of inactivity, thus providing a source of heterogeneity in an asynchronously dividing tissue. Our modified reporter and transgenic lines represent an important resource for interrogating the role of Erk signaling dynamics invivo.

Genome-wide open reading frame profiling identifies fibroblast growth factor signaling as a driver of PD-L1 expression in head and neck squamous cell carcinoma

Head and neck squamous cell carcinomas (HNSCC) are associated with significant treatment-related morbidity and poor disease-free and disease-specific survival, especially in the recurrent and metastatic (R/M HNSCC) setting. Inhibition of the programmed death-1/ligand-1 (PD-1/PD-L1) immune checkpoint is accepted as a first-line treatment strategy for R/M HNSCC and has expanded into the neoadjuvant, definitive, and adjuvant settings. To understand cellular signals modulating the PD-L1 in HNSCC, we profiled a HNSCC cell-line with a genome-wide open reading frame (ORF) library of 17,000 individual constructs (14,000 unique genes). We identified 335 ORFs enriched in PD-L1high cells and independently validated five of these ORFs (FGF6, IL17A, CD300C, KLR1C and NFKBIA) as drivers of PD-L1 upregulation. We showed that exogenous FGF ligand is sufficient to induce PD-L1 expression in multiple HNSCC cell lines and human immature dendritic cells. Accordingly, overexpression of FGFR1, FGFR3 or the FGFR3 S249C and D786N mutants common to HNSCC tumors also induced PD-L1 overexpression on tumor cells. Small molecule inhibition of FGF signaling abrogated PD-L1 upregulation in these models and also blocked “classical” IFNγ-regulated PD-L1 expression in a STAT1-independent manner. Finally, we found that FGF specifically upregulated a glycosylated form of PD-L1 in our study, and exogenous FGF led to concomitant upregulation of glycosyltransferases that may stabilize PD-L1 on the surface of HNSCC cells. Taken together, our study supports a potential role for FGF/FGFR pathway signaling as a mechanism driving immune escape and rationalizes further exploration of novel combination therapies to improve clinical responses to PD-1/PD-L1 axis inhibition in HNSCC.

Preferential FGF18/FGFR activity in pseudoglandular versus canalicular stage human lung fibroblasts.

Fibroblast growth factor (FGF) signaling is necessary for proper lung branching morphogenesis, alveolarization, and vascular development. Dysregulation of FGF activity has been implicated in various lung diseases. Recently, we showed that FGF18 promotes human lung branching morphogenesis by regulating mesenchymal progenitor cells. However, the underlying mechanisms remain unclear. Thus, we aimed to determine the role of FGF18 and its receptors (FGFR) in regulating mesenchymal cell proliferation, migration, and differentiation from pseudoglandular to canalicular stage. We performed siRNA assays to identify the specific FGFR(s) associated with FGF18-induced biological processes. We found that FGF18 increased proliferation and migration in human fetal lung fibroblasts (HFLF) from both stages. FGFR2/FGFR4 played a significant role in pseudoglandular stage. HFLF proliferation, while FGFR3/FGFR4 were involved in canalicular stage. FGF18 enhanced HFLF migration through FGFR2 and FGFR4 in pseudoglandular and canalicular stage, respectively. Finally, we provide evidence that FGF18 treatment leads to reduced expression of myofibroblast markers (ACTA2 and COL1A1) and increased expression of lipofibroblast markers (ADRP and PPARγ) in both stages HFLF. However, the specific FGF18/FGFR complex involved in this process varies depending on the stage. Our findings suggest that in context of human lung development, FGF18 tends to associate with distinct FGFRs to initiate specific biological processes on mesenchymal cells.

Fibroblast growth factor 2 acts as an upstream regulator of inhibition of pulmonary fibroblast activation.

Fibroblast growth factor (FGF) signaling plays a crucial role in lung development and repair. Fibroblast growth factor 2 (FGF2) can inhibit fibrotic gene expression and suppress the differentiation of pulmonary fibroblasts (PFs) into myofibroblasts in vitro, suggesting that FGF2 is a potential target for inhibiting pulmonary fibrosis. To gain deeper insights into the molecular mechanism underlying FGF2-mediated regulation of PFs, we performed mRNA sequencing analysis to systematically and globally uncover the regulated genes and biological functions of FGF2 in PFs. Gene Ontology analysis revealed that the differentially expressed genes regulated by FGF2 were enriched in multiple cellular functions including extracellular matrix (ECM) organization, cytoskeleton formation, β-catenin-independent Wnt signaling pathway, supramolecular fiber organization, epithelial cell proliferation, and cell adhesion. Gene Set Enrichment Analysis and cellular experiments confirmed that FGF2 can suppress ECM and actin filament organization and increase PFs proliferation. Taken together, these findings indicate that FGF2 acts as an upstream regulator of the inhibition of PFs activation and may play a regulatory role in pulmonary fibrosis.

Abstract P3002: Role Of Vascular Fibroblast Growth Factor Signaling In Group 3 Hypoxia-induced Pulmonary Hypertension

Pulmonary hypertension (PH) is a debilitating disease characterized by pathologic changes in the pulmonary vasculature that increases pulmonary vascular resistance and leads to right heart failure. Group 3 PH is caused by lung disease and/or hypoxia, including bronchopulmonary dysplasia (BPD) and chronic obstructive lung disease (COPD), and is the second largest cause of mortality in PH patients. PH further increases the morbidity and mortality in both COPD and BPD patients. Fibroblast Growth Factor 2 (FGF2) and FGF Receptor 1 (FGFR1) expression is elevated in lung tissue of PH patients. To study the role of FGF signaling in the pathogenesis of group 3 PH, we developed a physiologically relevant mouse model in which mice are challenged with 10% hypoxia for 2 weeks. Following hypoxia exposure, cardiac catheterization is used to measure right ventricular (RV) pressure that mimics Group 3 PH. Lung tissue is then analyzed for histological and molecular changes. Our initial work showed that inactivation of FGFRs in endothelial cells (EC) worsened hypoxia-induced PH while expression of a constitutively active FGFR1 (caFGFR1) in ECs protected against hypoxia-induced PH. Here we hypothesize that FGF signaling may also affect the pathogenic response to hypoxia of vascular smooth muscle (VSMC), potentially by limiting vascular muscularization in response to hypoxia. We show that in vitro inhibition of FGF2 signaling in human pulmonary artery VSMCs reduced hypoxia-induced proliferation while promoting a contractile phenotype, and in vivo inactivation of FGFRs 1 and 2 worsened hypoxia-induced vascular remodeling. These data suggest the potential for a dual beneficial effect of activation of FGF signaling, both in regulating EC-VSMC interactions and directly preventing VSMC remodeling.

Decreased left heart flow in fetal lambs causes left heart hypoplasia and pro-fibrotic tissue remodeling

Low blood flow through the fetal left heart is often conjectured as an etiology for hypoplastic left heart syndrome (HLHS). To investigate if a decrease in left heart flow results in growth failure, we generate left ventricular inflow obstruction (LVIO) in mid-gestation fetal lambs by implanting coils in their left atrium using an ultrasound-guided percutaneous technique. Significant LVIO recapitulates important clinical features of HLHS: decreased antegrade aortic valve flow, compensatory retrograde perfusion of the brain and ascending aorta (AAo) from the arterial duct, severe left heart hypoplasia, a non-apex forming LV, and a thickened endocardial layer. The hypoplastic AAo have miRNA-gene pairs annotating to cell proliferation that are inversely differentially expressed by bulk RNA-seq. Single-nucleus RNA-seq of the hypoplastic LV myocardium shows an increase in fibroblasts with a reciprocal decrease in cardiomyocyte nuclei proportions. Fibroblasts, cardiomyocytes and endothelial cells from hypoplastic myocardium have increased expression of extracellular matrix component or fibrosis genes with dysregulated fibroblast growth factor signaling. Hence, a severe sustained ( ~ 1/3 gestation) reduction in fetal left heart flow is sufficient to cause left heart hypoplasia. This is accompanied by changes in cellular composition and gene expression consistent with a pro-fibrotic environment and aberrant induction of mesenchymal programs.

Influence of FGF4 and BMP4 on FGFR2 dynamics during the segregation of epiblast and primitive endoderm cells in the pre-implantation mouse embryo.

Specification of the epiblast (EPI) and primitive endoderm (PE) in the mouse embryo involves fibroblast growth factor (FGF) signaling through the RAS/MAP kinase pathway. FGFR1 and FGFR2 are thought to mediate this signaling in the inner cell mass (ICM) of the mouse blastocyst and BMP signaling can also influence PE specification. In this study, we further explored the dynamics of FGFR2 expression through an enhanced green fluorescent protein (eGFP) reporter mouse line (FGFR2-eGFP). We observed that FGFR2-eGFP is present in the late 8-cell stage; however, it is absent or reduced in the ICM of early blastocysts. We then statistically correlated eGFP expression with PE and EPI markers GATA6 and NANOG, respectively. We detected that eGFP is weakly correlated with GATA6 in early blastocysts, but this correlation quickly increases as the blastocyst develops. The correlation between eGFP and NANOG decreases throughout blastocyst development. Treatment with FGF from the morula stage onwards did not affect FGFR2-eGFP presence in the ICM of early blastocysts; however, late blastocysts presented FGFR2-eGFP in all cells of the ICM. BMP treatment positively influenced FGFR2-eGFP expression and reduced the number of NANOG-positive cells in late blastocysts. In conclusion, FGFR2 is not strongly associated with PE precursors in the early blastocyst, but it is highly correlated with PE cells as blastocyst development progresses, consistent with the proposed role for FGFR2 in maintenance rather than initiating the PE lineage.