Control Fibroblasts Research Articles

B-170 Identifying Metabolic Phenotyping Variability in a Cellular Model of Barth Syndrome

Abstract Introduction Barth Syndrome is an X-linked multisystem disease caused by the mutation in the TAZ gene (TAZ, G 4.5, OMIM 300394) that encodes for acyltransferase taffazin. Taffazin is a mitochondrial protein highly expressed in heart and skeletal muscle and has a central role in cardiolipin remodeling process. TAZ is the only known single gene defect in cardiolipin remodeling, with more than a hundred TAZ mutations identified. However, no systematic correlation has been established between TAZ genotypes and clinical or biochemical phenotypes in Barth syndrome. There is a large phenotypic heterogeneity among affected individuals sometimes even within the same family. This raises a possibility of genetic and metabolic modifiers involved in TAZ clinical and metabolic phenotypes. The aim of this study is to apply LC-MS/MS untargeted and targeted metabolic analyses to explore possible correlations between two TAZ genotypes and metabolome in a cellular model of Barth Syndrome. Methods Mutant TAZ 3(c.647G>T), TAZ 4(c.778-24_778-7delinsA) fibroblast and matching by donors’ age and gender control fibroblast lines (healthy#1, healthy #2) were cultured under identical conditions. Cells (n = 6 for each genotype) were harvested and extracted followed Bligh and Dyer protocol. Extracted metabolites were analyzed by Agilent 6545 Q-TOF coupled with 1290 Infinity II HPLC. Chromatographic separation was achieved by using a Water Premier BEH Z-HILIC Column (2.5 µM, 2.1 × 150 mm) and Waters X-select HSS T3 (2.5 µM, 2.1 × 100 mm) columns for polar and non-polar metabolites respectively. Raw data was normalized with internal standard and total cellular protein amounts. Statistical and pathway enrichment analyses were performed with a chemometric platform Mass Profiler Professional (Agilent) and Metaboanalyst webtool. Results Data was treated first as for two separate genotypes (healthy vs TAZ) followed and compared to the analysis with four separate genotypes (healthy#1, healthy #2 TAZ c.647G>T, TAZ c.778-24_778-7delinsA). Metabolites contributing to discrimination of the genotypes based on metabolic profiles were identified by using a univariate analysis (P < 0.05) and a supervised PLSDA (VIP>1 for small polar metabolites and VIP > 3 for non-polar molecules). The differentially expressed metabolites included amino acids, acylcarnitine, Krebs cycle intermediates, ATP, 3-methylglutaconic acid, fatty acids and a variety of phospholipids. Pathway enrichment analysis revealed that several metabolic pathways were involved. Conclusions Our analyses reveal that genetic variations in TAZ gene cause the differences in the abundance of certain metabolites and affected pathways. This leads us to the conclusion that from the point of view of metabolic pathophysiology, TAZ should be considered beyond a single-gene defect and a more personalized approach is needed in search for potential therapies.

Enhanced Efficiency of the Basal and Induced Apoptosis Process in Mucopolysaccharidosis IVA and IVB Human Fibroblasts.

Morquio disease, also called mucopolysaccharidosis IV (MPS IV), belongs to the group of lysosomal storage diseases (LSD). Due to deficiencies in the activities of galactose-6-sulfate sulfatase (in type A) or β-galactosidase (in type B), arising from mutations in GALNS or GLB1, respectively, keratan sulfate (one of glycosaminoglycans, GAGs) cannot be degraded efficiently and accumulates in lysosomes. This primary defect leads to many cellular dysfunctions which then cause specific disease symptoms. Recent works have indicated that different secondary effects of GAG accumulation might significantly contribute to the pathomechanisms of MPS. Apoptosis is among the cellular processes that were discovered to be affected in MPS cells on the basis of transcriptomic studies and some cell biology experiments. However, Morquio disease is the MPS type which is the least studied in light of apoptosis dysregulation, while RNA-seq analyses suggested considerable changes in the expression of genes involved in apoptosis in MPS IVA and IVB fibroblasts. Here we demonstrate that cytochrome c release from mitochondria is more efficient in MPS IVA and IVB fibroblasts relative to control cells, both under the standard cultivation conditions and after treatment with staurosporine, an apoptosis inducer. This indication of apoptosis stimulation was corroborated by measurements of the levels of caspases 9, 3, 6, and 7, as well as PARP, cleaved at specific sites, in Morquio disease and control fibroblasts. The more detailed analyses of the transcriptomic data revealed which genes related to apoptosis are down- and up-regulated in MPS IVA and IVB fibroblasts. We conclude that apoptosis is stimulated in Morquio disease under both standard cell culture conditions and after induction with staurosporine which may contribute to the pathomechanism of this disorder. Dysregulation of apoptosis in other MPS types is discussed.

Proteomic and functional characterisation of extracellular vesicles from collagen VI deficient human fibroblasts reveals a role in cell motility

Extracellular vesicles (EVs) are key mediators of cell-to-cell communication. Their content reflects the state of diseased cells representing a window into disease progression. Collagen-VI Related Muscular Dystrophy (COL6-RD) is a multi-systemic disease involving different cell types. The role of EVs in this disease has not been explored. We compared by quantitative proteomics the protein cargo of EVs released from fibroblasts from patients with COL6-RD and controls. Isolated EVs contained a significant proportion of the most frequently reported proteins in EVs according to Exocarta and Vesiclepedia. We identified 67 differentially abundant proteins associated with vesicle transport and exocytosis, actin remodelling and the cytoskeleton, hemostasis and oxidative stress. Treatment of control fibroblasts with EVs from either patient or healthy fibroblasts altered significantly the motility of cells on a cell migration assay highlighting the functional relevance of EVs. In parallel, we analysed the secretome from the same cells and found a distinctly different set of 48 differentially abundant proteins related to extracellular matrix organisation and remodelling, growth factor response, RNA metabolism and the proteasome. The EVs and secretome sets of proteins only shared two identifiers indicating that the sorting of proteins towards EVs or the secretory pathway is tightly regulated for different functions.

Persistent hypoxia promotes myofibroblast differentiation via GPR-81 and differential regulation of LDH isoenzymes in normal and idiopathic pulmonary fibrosis fibroblasts.

Hypoxia, a state of insufficient oxygen availability, promotes cellular lactate production. Lactate levels are increased in lungs from patients with idiopathic pulmonary fibrosis (IPF), a disease characterized by excessive scar formation, and lactate is implicated in the pathobiology of lung fibrosis. However, the mechanisms underlying the effects of hypoxia and lactate on fibroblast phenotype are poorly understood. We exposed normal and IPF lung fibroblasts to persistent hypoxia and found that increased lactate generation by IPF fibroblasts was driven by the FoxM1-dependent increase of lactate dehydrogenase A (LDHA) coupled with decreased LDHB that was not observed in normal lung fibroblasts. Importantly, hypoxia reduced α-smooth muscle actin (α-SMA) expression in normal fibroblasts but had no significant impact on this marker of differentiation in IPF fibroblasts. Treatment of control and IPF fibroblasts with TGF-β under hypoxic conditions did not significantly change LDHA or LDHB expression. Surprisingly, lactate directly induced the differentiation of normal, but not IPF fibroblasts under hypoxic conditions. Moreover, while expression of GPR-81, a G-protein-coupled receptor that binds extracellular lactate, was increased by hypoxia in both normal and IPF fibroblasts, its inhibition or silencing only suppressed lactate-mediated differentiation in normal fibroblasts. These studies show that hypoxia differentially affects normal and fibrotic fibroblasts, promoting increased lactate generation by IPF fibroblasts through regulation of the LDHA/LDHB ratio and promoting normal lung fibroblast responsiveness to lactate through GPR-81. This supports a novel paradigm in which lactate may serve as a paracrine intercellular signal in oxygen-deficient microenvironments.

ABCD1 Transporter Deficiency Results in Altered Cholesterol Homeostasis.

X-linked adrenoleukodystrophy (X-ALD), the most common peroxisomal disorder, is caused by mutations in the peroxisomal transporter ABCD1, resulting in the accumulation of very long-chain fatty acids (VLCFA). Strongly affected cell types, such as oligodendrocytes, adrenocortical cells and macrophages, exhibit high cholesterol turnover. Here, we investigated how ABCD1 deficiency affects cholesterol metabolism in human X-ALD patient-derived fibroblasts and CNS tissues of Abcd1-deficient mice. Lipidome analyses revealed increased levels of cholesterol esters (CE), containing both saturated VLCFA and mono/polyunsaturated (V)LCFA. The elevated CE(26:0) and CE(26:1) levels remained unchanged in LXR agonist-treated Abcd1 KO mice despite reduced total C26:0. Under high-cholesterol loading, gene expression of SOAT1, converting cholesterol to CE and lipid droplet formation were increased in human X-ALD fibroblasts versus healthy control fibroblasts. However, the expression of NCEH1, catalysing CE hydrolysis and the cholesterol transporter ABCA1 and cholesterol efflux were also upregulated. Elevated Soat1 and Abca1 expression and lipid droplet content were confirmed in the spinal cord of X-ALD mice, where expression of the CNS cholesterol transporter Apoe was also elevated. The extent of peroxisome-lipid droplet co-localisation appeared low and was not impaired by ABCD1-deficiency in cholesterol-loaded primary fibroblasts. Finally, addressing steroidogenesis, progesterone-induced cortisol release was amplified in X-ALD fibroblasts. These results link VLCFA to cholesterol homeostasis and justify further consideration of therapeutic approaches towards reducing VLCFA and cholesterol levels in X-ALD.

Treprostinil Reconstitutes Mitochondrial Organisation and Structure in Idiopathic Pulmonary Fibrosis Cells.

Idiopathic pulmonary fibrosis (IPF) presents as an incurable change in the lung tissue and mitochondrial dysfunction of unknown origin. Treprostinil, a prostacyclin analogue, has been suggested for IPF therapy. This study assessed the effect of treprostinil on the cAMP signalling and mitochondrial activity in healthy lung fibroblasts and fibroblast-like cells from IPF patients. Six control fibroblast strains and six fibroblast-like IPF cell strains were isolated and expanded from freshly resected lung tissue. The cells were grown to confluence before being treated with either transforming growth factor (TGF)-β1, treprostinil, their combination, or a vehicle for up to 2 days. Mitochondria-regulating proteins were analysed using Western blotting and immunofluorescence, and the mitochondria were analysed using cytochrome C, mitochondrial cytochrome C oxidase II (MTCO2), and MTCO4. The IPF cells showed an increased rate of damaged mitochondria, which were significantly reduced when the cells were treated with treprostinil over 24 h. In the control cells, treprostinil prevented TGF-β-induced mitochondrial damage. Treatment with treprostinil modified the expression of several mitochondria-regulating proteins. In both cell types, treprostinil upregulated the expression of PTEN, p21(Waf1/Cip1), beclin1, LC3 II, parkin, PINK1, MTCO2, and MTCO4. In contrast, treprostinil downregulated the phosphorylation of mTOR and the expression of p62, mitofusin1, and mtiofusin2 in IPF cells. This might explain the reduced mitochondrial damage observed in treprostinil-treated IPF cells and suggest an improvement in the mitochondrial function in IPF. In this study, treprostinil improved mitochondrial impairment in vitro, which might, in part, explain the beneficial clinical effects documented in patients.

PPAR-gamma agonist pioglitazone recovers mitochondrial quality control in fibroblasts from PITRM1-deficient patients.

Introduction: Biallelic variants in PITRM1 are associated with a slowly progressive syndrome characterized by intellectual disability, spinocerebellar ataxia, cognitive decline and psychosis. The pitrilysin metallopeptidase 1 (PITRM1) is a mitochondrial matrix enzyme, which digests diverse oligopeptides, including the mitochondrial targeting sequences (MTS) that are cleaved from proteins imported across the inner mitochondrial membrane by the mitochondrial processing peptidase (MPP). Mitochondrial peptidases also play a role in the maturation of Frataxin, the protein affected in Friedreich's ataxia. Recent studies in yeast indicated that the mitochondrial matrix protease Ste23, which is a homologue of the human insulin-degrading enzyme (IDE), cooperates with Cym1 (homologue of PITRM1) to ensure the proper functioning of the preprotein processing machinery. In humans, IDE could be upregulated by Peroxisome Proliferator-Activated Receptor Gamma (PPARG) agonists. Methods: We investigated preprotein processing, mitochondrial membrane potential and MTS degradation in control and patients' fibroblasts, and we evaluated the pharmacological effect of the PPARG agonist Pioglitazone on mitochondrial proteostasis. Results: We discovered that PITRM1 dysfunction results in the accumulation of MTS, leading to the disruption and dissipation of the mitochondrial membrane potential. This triggers a feedback inhibition of MPP activity, consequently impairing the processing and maturation of Frataxin. Furthermore, we found that the pharmacological stimulation of PPARG by Pioglitazone upregulates IDE and also PITRM1 protein levels restoring the presequence processing machinery and improving Frataxin maturation and mitochondrial function. Discussion: Our findings provide mechanistic insights and suggest a potential pharmacological strategy for this rare neurodegenerative mitochondrial disease.

A Diagnostic Gene-Expression Signature in Fibroblasts of Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal, progressive neurodegenerative disease with limited treatment options. Diagnosis can be difficult due to the heterogeneity and non-specific nature of the initial symptoms, resulting in delays that compromise prompt access to effective therapeutic strategies. Transcriptome profiling of patient-derived peripheral cells represents a valuable benchmark in overcoming such challenges, providing the opportunity to identify molecular diagnostic signatures. In this study, we characterized transcriptome changes in skin fibroblasts of sporadic ALS patients (sALS) and controls and evaluated their utility as a molecular classifier for ALS diagnosis. Our analysis identified 277 differentially expressed transcripts predominantly involved in transcriptional regulation, synaptic transmission, and the inflammatory response. A support vector machine classifier based on this 277-gene signature was developed to discriminate patients with sALS from controls, showing significant predictive power in both the discovery dataset and in six independent publicly available gene expression datasets obtained from different sALS tissue/cell samples. Taken together, our findings support the utility of transcriptional signatures in peripheral cells as valuable biomarkers for the diagnosis of ALS.

De novo PHF5A variants are associated with craniofacial abnormalities, developmental delay, and hypospadias

PurposeThe SF3B splicing complex is composed of SF3B1-6 and PHF5A. We report a developmental disorder caused by de novo variants in PHF5A. MethodsClinical, genomic, and functional studies using subject-derived fibroblasts and a heterologous cellular system were performed. ResultsWe studied 9 subjects with congenital malformations, including preauricular tags and hypospadias, growth abnormalities, and developmental delay who had de novo heterozygous PHF5A variants, including 4 loss-of-function (LOF), 3 missense, 1 splice, and 1 start-loss variant. In subject-derived fibroblasts with PHF5A LOF variants, wild-type and variant PHF5A mRNAs had a 1:1 ratio, and PHF5A mRNA levels were normal. Transcriptome sequencing revealed alternative promoter use and downregulated genes involved in cell-cycle regulation. Subject and control fibroblasts had similar amounts of PHF5A with the predicted wild-type molecular weight and of SF3B1-3 and SF3B6. SF3B complex formation was unaffected in 2 subject cell lines. ConclusionOur data suggest the existence of feedback mechanisms in fibroblasts with PHF5A LOF variants to maintain normal levels of SF3B components. These compensatory mechanisms in subject fibroblasts with PHF5A or SF3B4 LOF variants suggest disturbed autoregulation of mutated splicing factor genes in specific cell types, that is, neural crest cells, during embryonic development rather than haploinsufficiency as pathomechanism.

Identification of Paired-related Homeobox Protein 1 as a key mesenchymal transcription factor in pulmonary fibrosis.

Matrix remodeling is a salient feature of idiopathic pulmonary fibrosis (IPF). Targeting cells driving matrix remodeling could be a promising avenue for IPF treatment. Analysis of transcriptomic database identified the mesenchymal transcription factor PRRX1 as upregulated in IPF. PRRX1, strongly expressed by lung fibroblasts, was regulated by a TGF-β/PGE2 balance in vitro in control and IPF human lung fibroblasts, while IPF fibroblast-derived matrix increased PRRX1 expression in a PDGFR-dependent manner in control ones. PRRX1 inhibition decreased human lung fibroblast proliferation by downregulating the expression of S phase cyclins. PRRX1 inhibition also impacted TGF-β driven myofibroblastic differentiation by inhibiting SMAD2/3 phosphorylation through phosphatase PPM1A upregulation and TGFBR2 downregulation, leading to TGF-β response global decrease. Finally, targeted inhibition of Prrx1 attenuated fibrotic remodeling in vivo with intra-tracheal antisense oligonucleotides in bleomycin mouse model of lung fibrosis and ex vivo using human and mouse precision-cut lung slices. Our results identified PRRX1 as a key mesenchymal transcription factor during lung fibrogenesis.

Metformin protects fibroblasts from patients with GNE myopathy by restoring autophagic flux via an AMPK/mTOR-independent pathway

UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) myopathy is an autosomal recessive disease characterized by rimmed vacuoles (RVs). Previous studies have shown that metformin protects against several neuromuscular disorders. In the present study, we summarize the clinical features of three GNE patients with the p.D207V mutation. The pathogenesis of GNE myopathy is described, and the significance of metformin in this disease is observed. Skin biopsy-derived fibroblasts from patients with GNE myopathy, carrying a D207V mutation in GNE, were cultured. GNE fibroblasts and control fibroblasts were treated under normal culture conditions, serum starvation conditions, or serum starvation + metformin conditions. Histopathological and immunohistochemical analyses of muscle samples showed that autophagy was involved in the formation of RVs in the muscle of patients. Starved GNE fibroblasts showed decreased autophagy-related proteins and impaired autophagic flow (p < 0.05). The mRFP-GFP-LC3 assay showed that the fusion of autophagosomes with lysosomes was partially blocked in GNE cells. Notably, metformin treatment upregulated the expression of autophagy proteins, increased the number of autolysosomes (p < 0.001), and influenced the viability of GNE cells (p < 0.001). Furthermore, adenosine 5′-monophosphate (AMP)-activated protein kinase (AMPK) and phosphorylated (p)-AMPK expression levels were upregulated in serum-starved GNE fibroblasts, while the mammalian target of rapamycin (mTOR) and p-mTOR expression levels were downregulated in both groups. Metformin treatment inhibited the AMPK–mTOR signaling pathway. Our results suggest that metformin plays a protective role in the GNE fibroblast by restoring autophagic flux and through the AMPK/mTOR-independent pathway.

Accelerated replicative senescence of ataxia-telangiectasia skin fibroblasts is retained at physiologic oxygen levels, with unique and common transcriptional patterns.

The genetic disorder, ataxia-telangiectasia (A-T), is caused by loss of the homeostatic protein kinase, ATM, and combines genome instability, tissue degeneration, cancer predisposition, and premature aging. Primary fibroblasts from A-T patients exhibit premature senescence when grown at ambient oxygen concentration (21%). Here, we show that reducing oxygen concentration to a physiological level range (3%) dramatically extends the proliferative lifespan of human A-T skin fibroblasts. However, they still undergo senescence earlier than control cells grown under the same conditions and exhibit high genome instability. Comparative RNA-seq analysis of A-T and control fibroblasts cultured at 3% oxygen followed by cluster analysis of differentially expressed genes and functional enrichment analysis, revealed distinct transcriptional dynamics in A-T fibroblasts senescing in physiological oxygen concentration. While some transcriptional patterns were similar to those observed during replicative senescence of control cells, others were unique to the senescing A-T cells. We observed in them a robust activation of interferon-stimulated genes, with undetected expression the interferon genes themselves. This finding suggests an activation of a non-canonical cGAS-STING-mediated pathway, which presumably responds to cytosolic DNA emanating from extranuclear micronuclei detected in these cells. Senescing A-T fibroblasts also exhibited a marked, intriguely complex alteration in the expression of genes associated with extracellular matrix (ECM) remodeling. Notably, many of the induced ECM genes encode senescence-associated secretory phenotype (SASP) factors known for their paracrine pro-fibrotic effects. Our data provide a molecular dimension to the segmental premature aging observed in A-T patients and its associated symptoms, which develop as the patients advance in age.

Understanding of arthrofibrosis: New explorative insights into extracellular matrix remodeling of synovial fibroblasts.

Arthrofibrosis following total knee arthroplasty is a fibroproliferative joint disorder marked by dysregulated biosynthesis of extracellular matrix proteins, such as collagens and proteoglycans. The underlying cellular events remain incompletely understood. Myofibroblasts are highly contractile matrix-producing cells characterized by increased alpha-smooth muscle actin expression and xylosyltransferase-I (XT-I) secretion. Human XT-I has been identified as a key mediator of arthrofibrotic remodeling. Primary fibroblasts from patients with arthrofibrosis provide a useful in vitro model to identify and characterize disease regulators and potential therapeutic targets. This study aims at characterizing primary synovial fibroblasts from arthrofibrotic tissues (AFib) regarding their molecular and cellular phenotype by utilizing myofibroblast cell culture models. Compared to synovial control fibroblasts (CF), AFib are marked by enhanced cell contractility and a higher XT secretion rate, demonstrating an increased fibroblast-to-myofibroblast transition rate during arthrofibrosis. Histochemical assays and quantitative gene expression analysis confirmed higher collagen and proteoglycan expression and accumulation in AFib compared to CF. Furthermore, fibrosis-based gene expression profiling identified novel modifier genes in the context of arthrofibrosis remodeling. In summary, this study revealed a unique profibrotic phenotype in AFib that resembles some traits of other fibroproliferative diseases and can be used for the future development of therapeutic interventions.

Perturbations in fatty acid metabolism and collagen production infer pathogenicity of a novel MBTPS2 variant in Osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is a heritable and chronically debilitating skeletal dysplasia. Patients with OI typically present with reduced bone mass, tendency for recurrent fractures, short stature and bowing deformities of the long bones. Mutations causative of OI have been identified in over 20 genes involved in collagen folding, posttranslational modification and processing, and in bone mineralization and osteoblast development. In 2016, we described the first X-linked recessive form of OI caused by MBTPS2 missense variants in patients with moderate to severe phenotypes. MBTPS2 encodes site-2 protease, a Golgi transmembrane protein that activates membrane-tethered transcription factors. These transcription factors regulate genes involved in lipid metabolism, bone and cartilage development, and ER stress response. The interpretation of genetic variants in MBTPS2 is complicated by the gene's pleiotropic properties; MBTPS2 variants can also cause the dermatological conditions Ichthyosis Follicularis, Atrichia and Photophobia (IFAP), Keratosis Follicularis Spinulosa Decalvans (KFSD) and Olmsted syndrome (OS) without skeletal abnormalities typical of OI. Using control and patient-derived fibroblasts, we previously identified gene expression signatures that distinguish MBTPS2-OI from MBTPS2-IFAP/KFSD and observed stronger suppression of genes involved in fatty acid metabolism in MBTPS2-OI than in MBTPS2-IFAP/KFSD; this was coupled with alterations in the relative abundance of fatty acids in MBTPS2-OI. Furthermore, we observed a reduction in collagen deposition in the extracellular matrix by MBTPS2-OI fibroblasts. Here, we extrapolate our observations in the molecular signature unique to MBTPS2-OI to infer the pathogenicity of a novel MBTPS2 c.516A>C (p.Glu172Asp) variant of unknown significance in a male proband. The pregnancy was terminated at gestational week 21 after ultrasound scans showed bowing of femurs and tibiae and shortening of long bones particularly of the lower extremity; these were further confirmed by autopsy. By performing transcriptional analyses, gas chromatography-tandem mass spectrometry-based quantification of fatty acids and immunocytochemistry on fibroblasts derived from the umbilical cord of the proband, we observed perturbations in fatty acid metabolism and collagen production similar to what we previously described in MBTPS2-OI. These findings support pathogenicity of the MBTPS2 variant p.Glu172Asp as OI-causative and highlights the value of extrapolating molecular signatures identified in multiomics studies to characterize novel genetic variants.

Maintenance of chronicity signatures in fibroblasts isolated from recessive dystrophic epidermolysis bullosa chronic wound dressings under culture conditions

BackgroundRecessive Dystrophic Epidermolysis Bullosa (RDEB) is a rare inherited skin disease caused by variants in the COL7A1 gene, coding for type VII collagen (C7), an important component of anchoring fibrils in the basement membrane of the epidermis. RDEB patients suffer from skin fragility starting with blister formation and evolving into chronic wounds, inflammation and skin fibrosis, with a high risk of developing aggressive skin carcinomas. Restricted therapeutic options are limited by the lack of in vitro models of defective wound healing in RDEB patients.ResultsIn order to explore a more efficient, non-invasive in vitro model for RDEB studies, we obtained patient fibroblasts derived from discarded dressings) and examined their phenotypic features compared with fibroblasts derived from non-injured skin of RDEB and healthy-donor skin biopsies. Our results demonstrate that fibroblasts derived from RDEB chronic wounds (RDEB-CW) displayed characteristics of senescent cells, increased myofibroblast differentiation, and augmented levels of TGF-β1 signaling components compared to fibroblasts derived from RDEB acute wounds and unaffected RDEB skin as well as skin from healthy-donors. Furthermore, RDEB-CW fibroblasts exhibited an increased pattern of inflammatory cytokine secretion (IL-1β and IL-6) when compared with RDEB and control fibroblasts. Interestingly, these aberrant patterns were found specifically in RDEB-CW fibroblasts independent of the culturing method, since fibroblasts obtained from dressing of acute wounds displayed a phenotype more similar to fibroblasts obtained from RDEB normal skin biopsies.ConclusionsOur results show that in vitro cultured RDEB-CW fibroblasts maintain distinctive cellular and molecular characteristics resembling the inflammatory and fibrotic microenvironment observed in RDEB patients’ chronic wounds. This work describes a novel, non-invasive and painless strategy to obtain human fibroblasts chronically subjected to an inflammatory and fibrotic environment, supporting their use as an accessible model for in vitro studies of RDEB wound healing pathogenesis. As such, this approach is well suited to testing new therapeutic strategies under controlled laboratory conditions.

The Effect of Glucocorticoids on TAF1 Gene Transcription in X-linked Dystonia Parkinsonism.

X-linked Dystonia Parkinsonism (XDP) is associated with a SINE-VNTR- Alu (SVA) retrotransposon insertion in an intron of the TAF1 gene that alters gene transcription and splicing. In this study, we determined if the SVA insertion introduces glucocorticoid (GC)-responsive cis-regulatory elements that may contribute to dysregulated TAF1 transcription and XDP disease progression. We performed in silico analysis to identify potential GC receptor (GR) binding sites within the XDP-SVA. We also conducted promoter-reporter assays on HeLa and HEK293T cells to assess the intrinsic promoter activity of three XDP-SVA variants representing different hexameric repeat lengths associated with differences in disease onset. We treated XDP fibroblast cell models with GR agonist (CORT) or antagonist (RU486), then subjected TAF1 and the XDP-associated aberrant transcript, TAF1-32i to gene expression analysis. A transcription factor binding site search revealed three binding sites for GR within the XDP-SVA-two within the SINE region and one in the Alu region. Promoter-reporter assays showed induction of XDP-SVA promoter activity upon CORT treatment that was dependent on the cell line and XDP-SVA hexamer repeat length. Gene expression analysis showed that baseline TAF1 levels differed between control and patient fibroblast cell lines, and treatment with CORT led to an increasing trend in the expression of the aberrant TAF1-32i transcript but did not reach statistical significance. Treatment with RU486 increased TAF1 mRNA expression only in the control cell lines. Using reporter assays, the XDP-SVA was shown to exhibit CORT-dependent transcriptional activation. Gene expression analysis also showed that GC signaling may influence TAF1 and TAF1-32i expression, possibly through interaction with the XDP-SVA. Our data provide a potential link between stress and XDP progression.

Evidence of inter- and intra-keloid heterogeneity through analysis of dermal fibroblasts: A new insight in deciphering keloid physiopathology.

Keloid scars are hypertrophic and proliferating pathological scars extending beyond the initial lesion and without tendency to regression. Usually, keloids are considered and treated as a single entity but clinical observations suggest heterogeneity in keloid morphologies with distinction of superficial/extensive and nodular entities. Within a keloid, heterogeneity could also be detected between superficial and deep dermis or centre and periphery. Focusing on fibroblasts as main actors of keloid formation, we aimed at evaluating intra- and inter-keloid fibroblast heterogeneity by analysing their gene expression and functional capacities (proliferation, migration, traction forces), in order to improve our understanding of keloid pathogenesis. Fibroblasts were obtained from centre, periphery, papillary and reticular dermis from extensive or nodular keloids and were compared to control fibroblasts from healthy skin. Transcriptional profiling of fibroblasts identified a total of 834 differentially expressed genes between nodular and extensive keloids. Quantification of ECM-associated gene expression by RT-qPCR brought evidence that central reticular fibroblasts of nodular keloids are the population which synthesize higher levels of mature collagens, TGFβ, HIF1α and αSMA as compared to control skin, suggesting that this central deep region is the nucleus of ECM production with a centrifuge extension in keloids. Although no significant variations were found for basal proliferation, migration of peripheral fibroblasts from extensive keloids was higher than that of central ones and from nodular cells. Moreover, these peripheral fibroblasts from extensive keloids exhibited higher traction forces than central cells, control fibroblasts and nodular ones. Altogether, studying fibroblast features demonstrate keloid heterogeneity, leading to a better understanding of keloid pathophysiology and treatment adaptation.

Single-nucleus chromatin accessibility identifies a critical role for TWIST1 in idiopathic pulmonary fibrosis myofibroblast activity.

In idiopathic pulmonary fibrosis (IPF), myofibroblasts are key effectors of fibrosis and architectural distortion by excessive deposition of extracellular matrix and their acquired contractile capacity. Single-cell RNA-sequencing (scRNA-seq) has precisely defined the IPF myofibroblast transcriptome, but identifying critical transcription factor activity by this approach is imprecise. We performed single-nucleus assay for transposase-accessible chromatin sequencing on explanted lungs from patients with IPF (n=3) and donor controls (n=2) and integrated this with a larger scRNA-seq dataset (10 IPF, eight controls) to identify differentially accessible chromatin regions and enriched transcription factor motifs within lung cell populations. We performed RNA-sequencing on pulmonary fibroblasts of bleomycin-injured Twist1-overexpressing COL1A2 Cre-ER mice to examine alterations in fibrosis-relevant pathways following Twist1 overexpression in collagen-producing cells. TWIST1, and other E-box transcription factor motifs, were significantly enriched in open chromatin of IPF myofibroblasts compared to both IPF nonmyogenic (log2 fold change (FC) 8.909, adjusted p-value 1.82×10-35) and control fibroblasts (log2FC 8.975, adjusted p-value 3.72×10-28). TWIST1 expression was selectively upregulated in IPF myofibroblasts (log2FC 3.136, adjusted p-value 1.41×10- 24), with two regions of TWIST1 having significantly increased accessibility in IPF myofibroblasts. Overexpression of Twist1 in COL1A2-expressing fibroblasts of bleomycin-injured mice resulted in increased collagen synthesis and upregulation of genes with enriched chromatin accessibility in IPF myofibroblasts. Our studies utilising human multiomic single-cell analyses combined with in vivo murine disease models confirm a critical regulatory function for TWIST1 in IPF myofibroblast activity in the fibrotic lung. Understanding the global process of opening TWIST1 and other E-box transcription factor motifs that govern myofibroblast differentiation may identify new therapeutic interventions for fibrotic pulmonary diseases.

Abstract 288: Development Of A Phenotypic Screen With 56 Marfan Syndrome Patient-derived Fibroblasts

Marfan syndrome (MFS) is a connective tissue disorder caused by mutations in the extracellular matrix gene coding for fibrillin-1 (FBN1). MFS leads to aortic aneurysms as key feature. Due to the high genetic diversity within the MFS patient population, genotype-phenotype predictions on disease progression and response to therapy remain challenging. Furthermore, current preclinical murine models represent only a subset of FBN1 mutations, which limits translational studies. In this study, we developed a preclinical model using primary cells of MFS patients that represent genetic and clinical variability in order to develop improved drug strategies. Human primary fibroblasts were isolated from skin biopsies derived from MFS patients (n=56) participating in the COMPARE trial. Human aortic smooth muscle cells (AoSMCs) were isolated from aortic tissue derived from MFS patients undergoing prophylactic aortic surgery (n=4). Long-term cultures in 384-well plates were assessed for the extracellular matrix and disease associated pathways. Analysis of all MFS fibroblasts showed either a reduced or abolished fibrillin-1 extracellular matrix network. Moreover, substantially reduced secretion of fibrillin-1 and asprosin in the culture medium was measured, as compared to healthy control fibroblasts. MFS fibroblasts with an abolished fibrillin-1 matrix were associated with patients with more rapid aortic root diameter growth. Exploring disease-relevant pathways revealed reduced nuclear localization of transcription factor KLF4 in MFS fibroblasts, which associated with the level of fibrillin-1 matrix. As a marker for myofibroblasts, α-smooth muscle actin (ASMA) staining was increased in these MFS fibroblasts. These findings were validated in AoSMC cultures, showing similar MFS fibrillin-1 matrix, nuclear KLF4, and ASMA expression profiles. In conclusion, phenotypic screening of patient-derived cells contributes to improved understanding of disease development and is valuable as preclinical tool for risk assessment and drug development for MFS and related conditions.

OA07 Clinical and pathogenic significance of S100A4 overexpression in systemic sclerosis

Abstract Background/Aims Preclinical studies suggest that the S100A4, a Damage Associated Molecular Pattern (DAMP) protein upregulated upon stress or injury, may be a target for antifibrotic therapy. We have studied expression and function of S100A4 in systemic sclerosis (SSc) to further explore its relevance as a driver of fibroblast activation. Methods S100A4 protein concentration was measured by ELISA (Cusabio, USA) in serum of SSc (n = 94) and healthy controls (n = 15). Association of serum level with major organ complications and skin score (mRSS) was determined. S100A4 protein expression in supernatant and cell layer of skin fibroblast cultures from diffuse cutaneous SSc (SScF, n = 6) and healthy controls (NF, n = 6) was assessed. Recombinant S100A4 and a high affinity anti-S100A4 neutralising monoclonal antibody (AX-202) were used to investigate activation of profibrotic molecular and functional phenotype in SSc and control fibroblasts. Results Median [range] S100A4 (ng/ml) was higher in serum of SSc (89.9 [15.0-240.0]) than healthy controls (71.4 [7.9-131.8]; p = 0.027). There was significant association with SSc-ILD (p = 0.025, n = 55), scleroderma renal crisis (p = 0.026, n = 4) and trend of increase in pulmonary hypertension (p = 0.075, n = 7). There was a trend for S100A4 to fall in longer disease duration (r=-0.18, p = 0.09) and a weak (r = 0.28, p = 0.07) correlation with current mRSS but not peak mRSS in dcSSc. Median [range] S100A4 (ng/ml) was higher in culture supernatants of SScF (4.19 [0.52-8.42]) than NF controls (0.28 [0.02-3.29]; p &amp;lt; 0.0001) and SSc cell layer had increased levels by Western blot analysis (mean [sd] RDU NF = 0.50 [0.91]; SScF 8.16 [2.49]; p &amp;lt; 0.0001). S100A4 induced a fibrotic phenotype in NF with concentration dependent increased in Col1, SMA and CTGF expression, and promotion of migration (scratch wound assay) and 3D type I collagen gel contraction that was attenuated by AX-202. AX-202 also reduced the constitutive profibrotic gene and protein expression phenotype of SScF. The effect of S100A4 was significantly attenuated by SB431524, a specific inhibitor of ALK5 (p &amp;lt; 0.0001 ANOVA) using qPCR assays across treatment groups for Col1, SMA and CTGF. Gene expression data (qPCR) confirmed protein expression. Genome-wide RNAseq analysis identified an S100A4 activated signature in NF overlapping the hallmark gene expression signature of SScF. Thus, 464 differentially expressed genes (FDR&amp;lt;0.001 and FC &amp;gt; 1.5) induced in NF by S100A4 were also constitutively overexpressed in SScF, and significantly downregulated by AX-202. Pathway mapping of these S100A4 dependent transcripts in SSc showed the most significant enriched Kegg® pathways (FDR&amp;lt;0.001) were regulation of stem cell pluripotency (4.6-fold) and metabolic pathways (1.9-fold). Conclusion Our findings provide compelling evidence supporting a profibrotic role for S100A4 in fibroblast activation in SSc and suggest that serum level may be a biomarker of major organ manifestations and disease severity. Studies to elucidate the therapeutic potential of targeting S100A4 in SSc are warranted. Disclosure C.P. Denton: None. S. Xu: None. R.H. Maclean: None. K.E. Clark: None. S. Borchert: Corporate appointments; Employee of ARXX Therapeutics. R. Hussain: Corporate appointments; Employee of ARXX Therapeutics. J. Klingelhöfer: Corporate appointments; Employee of ARXX Therapeutics. J. Hallén: Corporate appointments; Employee of ARXX Therapeutics. V.H. Ong: None.