Fibroblast Phenotype Research Articles

FGF21 Signaling Exerts Anti-Fibrotic Properties During Pulmonary Fibrosis.

Idiopathic Pulmonary Fibrosis (IPF) is a lethal disease with limited therapeutic options. FGF21, an endocrine fibroblast growth factor that acts through the FGFR1/KLB pathway, mitigates liver fibrosis. We hypothesized that FGF21 could exert anti-fibrotic properties in the lung. The concentrations of FGF21 and KLB in the plasma of IPF patients and control subjects were assessed. Pulmonary fibrosis development was assessed in Fgf21-deficient mice as compared to Wild Type littermates, at Day 14 after intra-tracheal injection of bleomycin. We determined the effect of repeated subcutaneous injections of a PEGylated FGF21 analog (PEG-FGF21) at D7, 10, 14 and 17 after bleomycin on the development of pulmonary fibrosis. Mice were sacrificed at D21. The effects of FGF21, alone or with KLB, on apoptosis in MLE15 cells and on the phenotype of human lung fibroblasts were assessed in vitro. In the plasma of IPF patients, FGF21 concentration was increased, while KLB levels were decreased. Fgf21 deficient mice presented an increased sensitivity to bleomycin, in comparison to their Wild Type littermate. Treatment with PEGylated FGF21 mitigated lung fibrogenesis, as evidenced by a lower injury score, decreased fibrosis markers and pro-fibrotic mediators expression as compared to the control group receiving the diluent. In MLE15 cells, stimulation with FGF21 and KLB inhibited apoptosis, through the decrease of BAX and BIM. Fibroblastic phenotype remained unaltered. Our data indicate a possible anti-fibrotic effect of FGF21 in the lung achieved through the inhibition of alveolar type 2 cells apoptosis.

The orphan nuclear receptor Nr4a1 contributes to interstitial cardiac fibrosis via modulation of cardiac fibroblast and macrophage phenotype

The orphan nuclear receptor Nr4a1 has complex biological functions and has been implicated in numerous diseases, including cardiovascular disease. While protective in atherosclerosis and myocardial ischemia, Nr4a1 has been shown to cause cardiac fibrosis in non-ischemic adverse remodeling of the heart. However, mechanisms underlying these actions are still poorly understood. Accordingly, we sought to: (1) understand the contribution of Nr4a1 to the inflammatory environment including macrophage phenotype; and (2) determine the contribution of Nr4a1 to cardiac fibroblast phenotype in the fibrotic heart. Wild type and Nr4a1−/− mice were infused with angiotensin II (1500 ng/kg/min) to induce cardiac fibrosis and diastolic dysfunction. Nr4a1 deletion prevented cardiac fibrosis and maintained normal diastolic function. We determined that macrophages lacking Nr4a1 had distinctly different phenotypes to wild type macrophages, with Nr4a1 deletion preventing the induction of a pro-inflammatory macrophage phenotype, instead promoting an anti-inflammatory phenotype. This had functional consequences in that macrophages lacking Nr4a1 showed a reduced ability to induce cardiac fibroblast migration. Interestingly, deletion of Nr4a1 in isolated cardiac fibroblasts also had profound effects on their phenotype and function, with these cells not able to produce excess extracellular matrix proteins, convert to a myofibroblast phenotype, or respond to macrophage stimuli. Nr4a1 causes cardiac fibrosis and subsequent diastolic dysfunction by inducing a pro-inflammatory phenotype in macrophages and by pushing cardiac fibroblasts towards a pro-fibrotic phenotype in response to pro-fibrotic stimuli. Nr4a1 is also critical for macrophage/fibroblast interactions.

Targeting Epigenetic Alterations Linked to Cancer-Associated Fibroblast Phenotypes in Lung Cancer

The focus in cancer research and treatment has recently shifted from being primarily tumor-centric to emphasizing the tumor microenvironment (TME) [...]

Targeting fibroblast phenotype switching in cardiac remodelling as a promising antifibrotic strategy.

Myocardial fibrosis, a common feature of heart disease, remains an unsolved clinical challenge. Fibrosis resolution requires activation of cardiac fibroblasts exhibiting context-dependent beneficial and detrimental dichotomy. Here, we explored the hypothesis of fibroblast reversible transition between quiescence and activated myofibroblastic states as a manifestation of cell phenotypic switching in myocardial remodelling. In support, gene regulatory networks executing conversion of cardiac fibroblasts to myofibroblasts and vice versa in fibrosis resolution are reconstructed using TRANSPATH database. In a scenario of fibroblast activation triggered by transforming growth factor β, a cardinal mediator of tissue fibrosis, signalling cascades governing entry into or exit from specific fibroblast statures in cardiac fibrotic remodelling were dissected. It is suggested that fibroblast phenotypic switching constitutes the central gait toward guiding cell state-gating strategies to counteract adverse cardiac fibrosis, a devastating disorder with no approved therapeutic option.

Inhaled exogenous thymosin beta 4 suppresses bleomycin-induced pulmonary fibrosis in mice via TGF-β1 signalling pathway.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and fibrotic interstitial lung disease. The two drugs indicated for IPF have limited efficacy and there is an urgent need to develop new drugs. Thymosin β4 (Tβ4) is a natural endogenous repair factor whose antifibrotic effects have been reported. This study aimed to evaluate the effect of exogenous recombinant human thymosin beta 4 (rhTβ4) on pulmonary fibrosis. Pulmonary fibrosis was induced in mice with bleomycin, and rhTβ4 was administrated by nebulization following three strategies: early dosing, mid-term dosing, and late dosing. The rhTβ4 efficacy was assessed by hydroxyproline, lung function, and lung histopathology. In vitro, the effects of rhTβ4 on fibroblast and lung epithelial cell phenotypes, as well as the TGF-β1 pathway, were evaluated. Aerosol administration of rhTβ4 could alleviate bleomycin-induced pulmonary fibrosis in mice at different stages of fibrosis. Studies conducted in vitro suggested that rhTβ4 could suppress lung fibroblasts from proliferating, migrating, and activation via regulating the TGF-β1 signalling pathway. In vitro, rhTβ4 also inhibited the epithelial-mesenchymal transition-like process of pulmonary epithelial cells. This study suggests that nebulized rhTβ4 is a potential treatment for IPF.

Adipocyte associated glucocorticoid signaling regulates normal fibroblast function which is lost in inflammatory arthritis

Fibroblasts play critical roles in tissue homeostasis, but in pathologic states they can drive fibrosis, inflammation, and tissue destruction. Little is known about what regulates the homeostatic functions of fibroblasts. Here, we perform RNA sequencing and identify a gene expression program in healthy synovial fibroblasts characterized by enhanced fatty acid metabolism and lipid transport. We identify cortisol as the key driver of the healthy fibroblast phenotype and that depletion of adipocytes, which express high levels of Hsd11b1, results in loss of the healthy fibroblast phenotype in mouse synovium. Additionally, fibroblast-specific glucocorticoid receptor Nr3c1 deletion in vivo leads to worsened arthritis. Cortisol signaling in fibroblasts mitigates matrix remodeling induced by TNF and TGF-β1 in vitro, while stimulation with these cytokines represses cortisol signaling and adipogenesis. Together, these findings demonstrate the importance of adipocytes and cortisol signaling in driving the healthy synovial fibroblast state that is lost in disease.

Restored Collagen VI Microfilaments Network in the Extracellular Matrix of CRISPR-Edited Ullrich Congenital Muscular Dystrophy Fibroblasts.

Collagen VI is an essential component of the extracellular matrix (ECM) composed by α1, α2 and α3 chains and encoded by COL6A1, COL6A2 and COL6A3 genes. Dominant negative pathogenic variants in COL6A genes result in defects in collagen VI protein and are implicated in the pathogenesis of muscular diseases, including Ullrich congenital muscular dystrophy (UCMD). Here, we designed a CRISPR genome editing strategy to tackle a dominant heterozygous deletion c.824_838del in exon 9 of the COL6A1 gene, causing a lack of secreted collagen VI in a patient's dermal fibroblasts. The evaluation of efficiency and specificity of gene editing in treating patient's fibroblasts revealed the 32% efficiency of editing the mutated allele but negligible editing of the wild-type allele. CRISPR-treated UCMD skin fibroblasts rescued the secretion of collagen VI in the ECM, which restored the ultrastructure of the collagen VI microfibril network. By using normal melanocytes as surrogates of muscle cells, we found that collagen VI secreted by the corrected patient's skin fibroblasts recovered the anchorage to the cell surface, pointing to a functional improvement of the protein properties. These results support the application of the CRISPR editing approach to knock out COL6A1 mutated alleles and rescue the UCMD phenotype in patient-derived fibroblasts.

Inhibition of Small Extracellular Vesicles by GW4869 Does not Disrupt the Paracrine Regulation of Adipose-Derived Mesenchymal Stem Cells Over Keloid Fibroblasts.

Keloid, scar caused by atypical wound repair, represents a significant difficulty for specialists in plastic surgery and dermatology. Adipose-derived mesenchymal stem cells (ADSCs) can regulate fibrotic phenotypes of keloid fibroblasts (KFs) in a paracrine fashion, but whether small extracellular vesicles (SEVs) are the key functional carrier in ADSC paracrine regulation of KFs remains unknown. This study aims to explore whether the regulatory effects of conditioned medium (CM) obtained from ADSCs on KFs can be impaired by decreased SEV content in the ADSC-CM. Clinical specimens were utilized to extract keloid fibroblasts (KFs), normal fibroblasts (NFs), and adipose-derived stem cells (ADSCs). Fibroblasts were cultured with CM obtained from ADSCs untreated or treated with the sphingomyelinase inhibitor GW4869. The features of SEVs derived from ADSC-CM were characterized, and fibroblast proliferation, migration, apoptosis, and expression of ECM proteins were analyzed. The sphingomyelinase inhibitor GW4869 successfully reduced the SEV content in ADSC-CM, and both control ADSC-CM and ADSC-CM with reduced SEV content significantly inhibited KF proliferation, migration, and α-SMA synthesis but not KF apoptosis, whereas only NF proliferation was inhibited by ADSC-CM. The reduced SEV content only affected the inhibition of KF proliferation induced by ADSC-CM. ADSC-CM inhibits various fibrotic phenotypes of KFs, but decreasing the SEV content in ADSC-CM did not significantly alter the antifibrotic effects of ADSC-CM. Thus, SEVs may not be the key mediator of ADSCs paracrine regulation of KFs. This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors . www.springer.com/00266 .

Inhibiting YAP1 reduced abdominal aortic aneurysm formation by suppressing adventitial fibroblast phenotype transformation and migration.

The adventitial fibroblast (AF) is the most abundant cell in the vascular adventitia, a few studies had confirmed that AF contributed to abdominal aortic aneurysm (AAA) development; YAP1 involved in several vascular diseases by promoting AF transformed to myofibroblast, the role of YAP1 in AAA is not clear yet. This study aims to determine whether YAP1 play a role in AAA process by regulating AF function. We found the expression of YAP1was significantly increased in aneurysm tissues of AAA patients compared to normal adjacent vascular tissues and mainly in adventitia. YAP1 also upregulated in elastase-induced and CaCl2-induced mice AAA model. Suppressed YAP1 function with YAP1 inhibitor-Verteporfin declined AAA incident rate remarkably in mice, and the collagen deposition in the adventitia was alleviated obviously. Afterwards, we studied the effect of YAP1 on the function of AF, Verteporfin was used to block YAP1 invitro, the process of AF transforming to myofibroblast and migration were almost completely eliminated after inhibiting YAP1 expression. This study demonstrated that YAP1 may play a key role in AAA development, inhibiting YAP1 significantly reduced AAA formation through suppressed the process of AF transformed to myofibroblast and migration.

Hyperuricemia suppresses lumican, exacerbating adverse remodeling after myocardial infarction by promoting fibroblast phenotype transition

BackgroundHyperuricemia is independently associated with a poor prognosis in patients with myocardial infarction (MI). Furthermore, MI induces activation of the repair response in local fibroblasts, resulting in extracellular matrix accumulation that generates a stable fibrotic scar in the infarcted area. However, researchers have not determined whether hyperuricemia affects fibroblast activation and its involvement in postinfarction cardiac remodeling.ObjectivesWe aimed to trigger hyperuricemia by administering potassium oxonate in a mouse model of MI to evaluate the role of hyperuricemia in MI pathogenesis.MethodsMicroarray datasets and single-cell sequencing data from gout patients, heart failure patients, and model mice were used to identify the underlying mechanisms responsible for the effect of hyperuricemia on MI progression. A hyperuricemia-related MI mouse model was established. Cardiac function was assessed, followed by sample collection and a uric acid assay. We conducted an enzyme-linked immunosorbent assay, histological detection, immunofluorescence, sequencing data processing, single-cell RNA-seq, and functional enrichment analysis. We then isolated and cultured cardiac fibroblasts and performed Western blotting, quantitative real-time polymerase chain reaction, and shRNA-mediated lumican knockdown assays.ResultsHyperuricemia decreased cardiac function, increased mortality, and aggravated adverse fibrosis remodeling in mice after MI. These outcomes were closely related to reduced levels of fibroblast-derived lumican. This reduction activated the TGF-β/SMAD signaling pathway to induce aberrant myofibroblast activation and extracellular matrix deposition in the infarcted area. Furthermore, lumican supplementation or uric acid-lowering therapy with allopurinol alleviated hyperuricemia-mediated abnormal cardiac remodeling.ConclusionHyperuricemia aggravates postinfarction cardiac remodeling by reducing lumican expression and promoting fibroblast phenotype transition. We highlight the clinical importance of lowering uric acid levels in hyperuricemia-related MI to prevent adverse ventricular remodeling.

Bone mineral density affects tumor growth by shaping microenvironmental heterogeneity

Breast cancer bone metastasis is a major cause of mortality in patients with advanced breast cancer. Although decreased mineral density is a known risk factor for bone metastasis, the underlying mechanisms remain poorly understood because studying the isolated effect of bone mineral density on tumor heterogeneity is challenging with conventional approaches. Moreover, mineralized biomaterials are commonly utilized for clinical bone defect repair, but how mineralized biomaterials affect the foreign body response and wound healing is unclear. Here, we investigate how bone mineral affects tumor growth and microenvironmental complexity in vivo by combining single-cell RNA-sequencing with mineral-containing or mineral-free decellularized bone matrices. We discover that the absence of bone mineral significantly influences fibroblast and immune cell heterogeneity, promoting phenotypes that increase tumor growth and alter the response to injury or disease. Importantly, we observe that the stromal response to bone mineral content depends on the murine tumor model used. While lack of bone mineral induces tumor-promoting microenvironments in both immunocompromised and immunocompetent animals, these changes are mediated by altered fibroblast phenotype in immunocompromised mice and macrophage polarization in immunocompetent mice. Collectively, our findings suggest that bone mineral density affects tumor growth by impacting microenvironmental complexity in an organism-dependent manner.

Integrin alpha1 beta1 promotes interstitial fibrosis in a mouse model of polycystic kidney disease.

Fibrosis is the cause of end-stage kidney failure in patients with Autosomal Dominant Polycystic Kidney Disease (ADPKD). The molecular and cellular mechanisms involved in fibrosis are complex and anti-fibrotic therapies have so far failed to make an impact on patient welfare. Using unbiased proteomics analysis on the Pkd1 nl/nl mouse, we found that expression of the integrin α1 subunit is increased in this model of ADPKD. In human ADPKD tissue and two single cell RNA kidney disease datasets, ITGA1 was also upregulated. To investigate the functional role of this integrin subunit in ADPKD, we generated a Pkd1 nl/nl Itga1 -/- mouse. We observed a significant reduction in kidney volume and kidney dysfunction in mice lacking the integrin α1 subunit. Kidneys from Pkd1 nl/nl Itga1 -/- mice had smaller cysts and reduced interstitial expansion and tubular atrophy. Picrosirius red staining identified a restriction in collagen staining in the interstitium and the myofibroblast marker α smooth muscle actin was also downregulated. Myofibroblast cell proliferation was reduced in Pkd1 nl/nl Itga1 -/- mice and primary fibroblast cultures demonstrated an abrogated fibrogenic phenotype in integrin α1-depleted fibroblasts. These results highlight a previously unrecognised role for the integrin α1 subunit in kidney fibrosis.

CALB2 drives pancreatic cancer metastasis through inflammatory reprogramming of the tumor microenvironment

BackgroundEarly dissemination to distant organs accounts for the dismal prognosis of patients with pancreatic ductal adenocarcinoma (PDAC). Chronic, dysregulated, persistent and unresolved inflammation provides a preferred tumor microenvironment (TME) for tumorigenesis, development, and metastasis. A better understanding of the key regulators that maintain inflammatory TME and the development of predictive biomarkers to identify patients who are most likely to benefit from specific inflammatory-targeted therapies is crucial for advancing personalized cancer treatment.MethodsThis study identified cell-specific expression of CALB2 in human PDAC through single-cell RNA sequencing analysis and assessed its clinicopathological correlations in tissue microarray using multi-color immunofluorescence. Co-culture systems containing cancer-associated fibroblasts (CAFs) and patient-derived organoids (PDOs) in vitro and in vivo were employed to elucidate the effects of CALB2-activated CAFs on PDAC malignancy. Furthermore, CUT&RUN assays, luciferase reporter assays, RNA sequencing, and gain- or loss-of-function assays were used to unravel the molecular mechanisms of CALB2-mediated inflammatory reprogramming and metastasis. Additionally, immunocompetent KPC organoid allograft models were constructed to evaluate CALB2-induced immunosuppression and PDAC metastasis, as well as the efficacy of inflammation-targeted therapy.ResultsCALB2 was highly expressed both in CAFs and cancer cells and correlated with an unfavorable prognosis and immunosuppressive TME in PDAC patients. CALB2 collaborated with hypoxia to activate an inflammatory fibroblast phenotype, which promoted PDAC cell migration and PDO growth in vitro and in vivo. In turn, CALB2-activated CAFs upregulated CALB2 expression in cancer cells through IL6-STAT3 signaling-mediated direct transcription. In cancer cells, CALB2 further activated Ca2+-CXCL14 inflammatory axis to facilitate PDAC metastatic outgrowth and immunosuppression. Genetic or pharmaceutical inhibition of CXCL14 significantly suppressed CALB2-mediated metastatic colonization of PDAC cells in vivo and extended mouse survival.ConclusionsThese findings identify CALB2 as a key regulator of inflammatory reprogramming to promote PDAC metastatic progression. Combination therapy with αCXCL14 monoclonal antibody and gemcitabine emerges as a promising strategy to suppress distant metastasis and improve survival outcomes in PDAC with CALB2 overexpression.

Cardiac and skeletal muscle manifestations in the G608G mouse model of Hutchinson-Gilford progeria syndrome.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare premature aging disorder resulting from de novo mutations in the lamin A gene. Children with HGPS typically pass away in their teenage years due to cardiovascular diseases such as atherosclerosis, myocardial infarction, heart failure, and stroke. In this study, we characterized the G608G HGPS mouse model and explored cardiac and skeletal muscle function, along with senescence-associated phenotypes in fibroblasts. Homozygous G608G HGPS mice exhibited cardiac dysfunction, including decreased cardiac output and stroke volume, and impaired left ventricle relaxation. Additionally, skeletal muscle exhibited decreased isometric tetanic torque, muscle atrophy, and increased fibrosis. HGPS fibroblasts showed nuclear abnormalities, decreased proliferation, and increased expression of senescence markers. These findings provide insights into the pathophysiology of the G608G HGPS mouse model and inform potential therapeutic strategies for HGPS.

Astragaloside IV alleviates skin fibrosis by modulating fibroblast phenotype in keloids.

Keloids are fibroproliferative diseases featuring abnormal fibroblast proliferation and extracellular matrix (ECM) deposition. Current therapeutic methods for keloids are unsatisfactory, and the recurrence rates of keloids are high. Astragaloside IV (AS-IV) is a key active component of Astragalus membranaceus Bunge, and has been reported to exert potent anti-fibrotic effects. Accordingly, our research aimed to explore whether AS-IV suppresses fibroblast dysfunction and skin fibrosis during the development of keloids. Human keloid-derived fibroblasts (KFs) were stimulated by TGF-β1 to evaluate the influence of AS-IV on abnormal proliferation, migration, and accumulation of ECM in vitro. A bleomycin (BLM)-induced skin fibrosis model was established to assess the influence of AS-IV on ECM deposition and skin fibrosis in vivo. TGF-β1 stimulation enhanced the proliferation, migration, and accumulation of ECM in KFs, which were abolished by AS-IV treatment. The in vivo assay revealed that AS-IV administration restrained ECM accumulation and skin fibrosis in mouse models. AS-IV plays an anti-fibrotic role in keloids by suppressing fibroblast dysfunction and reducing ECM deposition.

ETS1 Expression in Diabetic Foot Ulcers: Implications for Fibroblast Phenotype and Wound Healing Through the PP2A/YAP Pathway.

Diabetic foot ulcers (DFUs) are a serious complication of diabetes, characterized by impaired wound healing and high morbidity and mortality risks. While ETS1 is known to influence fibroblast pathological remodeling, its specific role in DFU and fibroblast wound healing remains unclear. Skin tissue samples from DFU patients were categorized by Wagner grades to analyze ETS1 expression. Primary fibroblasts derived from diabetes mellitus wound (DMFBs) were collected from wound margins to test migration ability and analyze cell phenotype by immunofluorescence; they were further treated with siETS1 and the ETS1 inhibitor YK-4-279. Techniques including Western blotting, quantitative Real-Time PCR (qRT-PCR), and immunofluorescence were used to assess the expressionof ETS1, Collagen I, and phenotype in DMFBs. Additionally, the binding sites between human ETS1 and the PP2A promoter were predicted by the UCSC and JASPAR databases. It intended to explore the negative transcriptional regulation of PP2A by ETS1 and its implications in fibroblast function and wound healing. Fibroblasts derived from Wagner Grades II-IV exhibit differences in cell morphology, migratory ability, and phenotype. Our findings indicate a significant upregulation of ETS1 in Wagner III and IV. The downregulation of ETS1 was observed to enhance DMFB migration and increase the expression of Collagen I and α-SMA. These changes suggest a potential mechanism by which PP2A regulates the YAP/Hippo pathway in diabetic wound healing. ETS1 appears to impede the repair processes in DFUs, likely through the negative regulation of PP2A, affecting fibroblast function and wound healing.

Growth defect of domain III glycoprotein B mutants of human cytomegalovirus reverted by compensatory mutations co-localizing in post-fusion conformation.

Cell entry is a crucial step for a virus to infect a host cell. Human cytomegalovirus utilizes glycoprotein B (gB) to fuse the viral and host cell membranes upon receptor binding of gH/gL-containing complexes. Fusion is mediated by major conformational changes of gB from a metastable pre-fusion to a stable post-fusion state whereby the central trimeric coiled-coils, formed by domain (Dom)III α helices, remain structurally nearly unchanged. To better understand the role of the stable core, we individually introduced three potentially helix-breaking or one disulfide bond-breaking mutation in the DIII α3 to study different aspects of the viral behavior upon long-term culturing. Two of the three helix-breaking mutations, gB_Y494P and gB_I495P, were lethal for the virus in either fibroblasts or epithelial cells. The third substitution, gB_G493P, on the other hand, displayed a delayed replication and spread, which was more pronounced in epithelial cells, hinting at an impaired fusion. Interestingly, the disulfide bond-breaker mutation, gB_C507S, performed strikingly differently in the two cell types - lethal in epithelial cells and an atypical phenotype in fibroblasts, respectively. Replication curve analyses paired with the infection efficiency, the spread morphology, and the cell-cell fusogenicity suggest a dysregulated fusion process, which could be reverted by second-site mutations mapping predominantly to gB DomV. Our findings underline the functional importance of a stable DomIII core for a well-regulated DomV rearrangement during fusion.IMPORTANCEHuman cytomegalovirus (HCMV) can establish a lifelong infection. In most people, the infection follows an asymptomatic course; however, it is a major cause of morbidity and mortality in immunocompromised patients or neonates. HCMV has a very broad cell tropism, ranging from fibroblasts to epi- and endothelial cells. The virus uses different entry pathways utilizing the core fusion machinery consisting of glycoprotein complexes gH/gL and glycoprotein B (gB). The fusion protein gB undergoes fundamental rearrangements from a metastable pre-fusion to a stable post-fusion conformation. Here, we characterized the viral behavior after the introduction of four single-point mutations in the gB central core. These led to various cell type-specific atypical phenotypes and the emergence of compensatory mutations, demonstrating an important interaction between domains III and V. We provide a new basis for the development of a structurally and functionally altered gB, which can further serve as a tool for drug and vaccine development.

Extracellular vesicles from cancer cell lines of different origins drive the phenotype of normal oral fibroblasts in a CAF-like direction.

Normal oral fibroblasts (NOFs) are located in the connective tissue of the oral mucosa. The NOFs play an important role in wound healing, tumor progression, and metastasis. They are subjected to influence by external and internal stimuli, among them extracellular vesicles (EVs), that are considered as important players in cell to cell communication, especially in carcinogenesis and metastatic processes. During tumorigenesis, stromal NOFs may undergo activation into cancer-associated fibroblasts (CAFs) that modify their phenotype to provide pro-oncogenic signals that in turn facilitate tumor initiation, progression, and metastasis. The aim of the study was to reveal the effect of EVs derived from local (oral squamous cell carcinoma - OSCC) and distant (pancreatic adenocarcinoma - PDAC; malignant melanoma brain metastasis - MBM) cancer origin on NOFs and their possible change into a CAF-like direction. The effect of each of the cancer EV types on NOFs proliferation, viability, and migration was tested. Also, changes in gene expression of the well-established CAF biomarkers ACTA2, FAP, PDGFR, and two putative CAF biomarkers, the Ca2+- activated ion channels ANO1 and KCNMA, were studied. Obtained results indicate that NOFs receive and process signals transmitted by EVs originating from both OSCC, PDAC, and MBM. The fibroblast response was dependent on EV origin and concentration, and duration of EV exposure. The present results indicate that the molecular cargo of the EVs direct NOFs towards a pro-tumorigenic phenotype.

Single-cell transcriptomics reveal distinctive patterns of fibroblast activation in heart failure with preserved ejection fraction

Inflammation, fibrosis and metabolic stress critically promote heart failure with preserved ejection fraction (HFpEF). Exposure to high-fat diet and nitric oxide synthase inhibitor N[w]-nitro-l-arginine methyl ester (L-NAME) recapitulate features of HFpEF in mice. To identify disease-specific traits during adverse remodeling, we profiled interstitial cells in early murine HFpEF using single-cell RNAseq (scRNAseq). Diastolic dysfunction and perivascular fibrosis were accompanied by an activation of cardiac fibroblast and macrophage subsets. Integration of fibroblasts from HFpEF with two murine models for heart failure with reduced ejection fraction (HFrEF) identified a catalog of conserved fibroblast phenotypes across mouse models. Moreover, HFpEF-specific characteristics included induced metabolic, hypoxic and inflammatory transcription factors and pathways, including enhanced expression of Angiopoietin-like 4 (Angptl4) next to basement membrane compounds, such as collagen IV (Col4a1). Fibroblast activation was further dissected into transcriptional and compositional shifts and thereby highly responsive cell states for each HF model were identified. In contrast to HFrEF, where myofibroblast and matrifibrocyte activation were crucial features, we found that these cell states played a subsidiary role in early HFpEF. These disease-specific fibroblast signatures were corroborated in human myocardial bulk transcriptomes. Furthermore, we identified a potential cross-talk between macrophages and fibroblasts via SPP1 and TNFɑ with estimated fibroblast target genes including Col4a1 and Angptl4. Treatment with recombinant ANGPTL4 ameliorated the murine HFpEF phenotype and diastolic dysfunction by reducing collagen IV deposition from fibroblasts in vivo and in vitro. In line, ANGPTL4, was elevated in plasma samples of HFpEF patients and particularly high levels associated with a preserved global-longitudinal strain. Taken together, our study provides a comprehensive characterization of molecular fibroblast activation patterns in murine HFpEF, as well as the identification of Angiopoietin-like 4 as central mechanistic regulator with protective effects.

TRIM35 triggers cardiac remodeling by regulating SLC7A5-mediated amino acid transport and mTORC1 activation in fibroblasts

BackgroundCardiac maladaptive remodeling is one of the leading causes of heart failure with highly complicated pathogeneses. The E3 ligase tripartite motif containing 35 (TRIM35) has been identified as a crucial regulator governing cellular growth, immune responses, and metabolism. Nonetheless, the role of TRIM35 in fibroblasts in cardiac remodeling remains elusive.MethodsHeart tissues from human donors were used to verify tissue-specific expression of TRIM35. Fibroblast-specific Trim35 gene knockout mice (Trim35cKO) were used to investigate the function of TRIM35 in fibroblasts. Cardiac function, morphology, and molecular changes in the heart tissues were analyzed after transverse aortic constriction (TAC) surgery. The mechanisms by which TRIM35 regulates fibroblast phenotypes were elucidated using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and RNA sequencing (RNA-Seq). These findings were further validated through the use of adenoviral and adeno-associated viral transfection systems, as well as the mTORC1 inhibitor Rapamycin.ResultsTRIM35 expression is primarily up-regulated in cardiac fibroblasts in both murine and human fibrotic hearts, and responds to TGF-β1 stimulation. Specific deletion of TRIM35 in cardiac fibroblasts significantly improves cardiac fibrosis and hypertrophy. Consistently, the overexpression of TRIM35 promotes fibroblast proliferation, migration, and differentiation. Through paracrine signaling, it induces hypertrophic growth of cardiomyocytes. Mechanistically, we found that TRIM35 interacts with, ubiquitinates, and up-regulates the amino acid transporter SLC7A5, which enhances amino acid transport and activates the mTORC1 signaling pathway. Furthermore, overexpression of SLC7A5 significantly reverses the reduced cardiac fibrosis and hypertrophy caused by conditional knockout of TRIM35.ConclusionOur findings demonstrate a novel role of fibroblast-TRIM35 in cardiac remodeling and uncover the mechanism underlying SLC7A5-mediated amino acid transport and mTORC1 activation. These results provide a potential novel therapeutic target for treating cardiac remodeling.