[Regulatory effects of bio-intensity electric field on transformation of human skin fibroblasts].

Objective: To investigate the regulatory effects of bio-intensity electric field on the transformation of human skin fibroblasts (HSFs). Methods: The experimental research methods were used. HSFs were collected and divided into 200 mV/mm electric field group treated with 200 mV/mm electric field for 6 h and simulated electric field group placed in the electric field device without electricity for 6 h. Changes in morphology and arrangement of cells were observed in the living cell workstation; the number of cells at 0 and 6 h of treatment was recorded, and the rate of change in cell number was calculated; the direction of cell movement, movement velocity, and trajectory velocity within 3 h were observed and calculated (the number of samples was 34 in the simulated electric field group and 30 in 200 mV/mm electric field group in the aforementioned experiments); the protein expression of α-smooth muscle actin (α-SMA) in cells after 3 h of treatment was detected by immunofluorescence method (the number of sample was 3). HSFs were collected and divided into simulated electric field group placed in the electric field device without electricity for 3 h, and 100 mV/mm electric field group, 200 mV/mm electric field group, and 400 mV/mm electric field group which were treated with electric fields of corresponding intensities for 3 h. Besides, HSFs were divided into simulated electric field group placed in the electric field device without electricity for 6 h, and electric field treatment 1 h group, electric field treatment 3 h group, and electric field treatment 6 h group treated with 200 mV/mm electric field for corresponding time. The protein expressions of α-SMA and proliferating cell nuclear antigen (PCNA) were detected by Western blotting (the number of sample was 3). Data were statistically analyzed with Mann-Whitney U test, one-way analysis of variance, independent sample t test, and least significant difference test. Results: After 6 h of treatment, compared with that in simulated electric field group, the cells in 200 mV/mm electric field group were elongated in shape and locally adhered; the cells in simulated electric field group were randomly arranged, while the cells in 200 mV/mm electric field group were arranged in a regular longitudinal direction; the change rates in the number of cells in the two groups were similar (P>0.05). Within 3 h of treatment, the cells in 200 mV/mm electric field group had an obvious tendency to move toward the positive electrode, and the cells in simulated electric field group moved around the origin; compared with those in simulated electric field group, the movement velocity and trajectory velocity of the cells in 200 mV/mm electric field group were increased significantly (with Z values of -5.33 and -5.41, respectively, P<0.01), and the directionality was significantly enhanced (Z=-4.39, P<0.01). After 3 h of treatment, the protein expression of α-SMA of cells in 200 mV/mm electric field group was significantly higher than that in simulated electric field group (t=-9.81, P<0.01). After 3 h of treatment, the protein expressions of α-SMA of cells in 100 mV/mm electric field group, 200 mV/mm electric field group, and 400 mV/mm electric field group were 1.195±0.057, 1.606±0.041, and 1.616±0.039, respectively, which were significantly more than 0.649±0.028 in simulated electric field group (P<0.01). Compared with that in 100 mV/mm electric field group, the protein expressions of α-SMA of cells in 200 mV/mm electric field group and 400 mV/mm electric field group were significantly increased (P<0.01). The protein expressions of α-SMA of cells in electric field treatment 1 h group, electric field treatment 3 h group, and electric field treatment 6 h group were 0.730±0.032, 1.561±0.031, and 1.553±0.045, respectively, significantly more than 0.464±0.020 in simulated electric field group (P<0.01). Compared with that in electric field treatment 1 h group, the protein expressions of α-SMA in electric field treatment 3 h group and electric field treatment 6 h group were significantly increased (P<0.01). After 3 h of treatment, compared with that in simulated electric field group, the protein expressions of PCNA of cells in 100 mV/mm electric field group, 200 mV/mm electric field group, and 400 mV/mm electric field group were significantly decreased (P<0.05 or P<0.01); compared with that in 100 mV/mm electric field group, the protein expressions of PCNA of cells in 200 mV/mm electric field group and 400 mV/mm electric field group were significantly decreased (P<0.05 or P<0.01); compared with that in 200 mV/mm electric field group, the protein expression of PCNA of cells in 400 mV/mm electric field group was significantly decreased (P<0.01). Compared with that in simulated electric field group, the protein expressions of PCNA of cells in electric field treatment 1 h group, electric field treatment 3 h group, and electric field treatment 6 h group were significantly decreased (P<0.01); compared with that in electric field treatment 1 h group, the protein expressions of PCNA of cells in electric field treatment 3 h group and electric field treatment 6 h group were significantly decreased (P<0.05 or P<0.01); compared with that in electric field treatment 3 h group, the protein expression of PCNA of cells in electric field treatment 6 h group was significantly decreased (P<0.01). Conclusions: The bio-intensity electric field can induce the migration of HSFs and promote the transformation of fibroblasts to myofibroblasts, and the transformation displays certain dependence on the time and intensity of electric field.

Acquisition of cellular properties during alveolar formation requires differential activity and distribution of mitochondria.

Alveolar formation requires coordinated movement and interaction between alveolar epithelial cells, mesenchymal myofibroblasts, and endothelial cells/pericytes to produce secondary septa. These processes rely on the acquisition of distinct cellular properties to enable ligand secretion for cell-cell signaling and initiate morphogenesis through cellular contraction, cell migration, and cell shape change. In this study, we showed that mitochondrial activity and distribution play a key role in bestowing cellular functions on both alveolar epithelial cells and mesenchymal myofibroblasts for generating secondary septa to form alveoli in mice. These results suggest that mitochondrial function is tightly regulated to empower cellular machineries in a spatially specific manner. Indeed, such regulation via mitochondria is required for secretion of ligands, such as platelet-derived growth factor, from alveolar epithelial cells to influence myofibroblast proliferation and contraction/migration. Moreover, mitochondrial function enables myofibroblast contraction/migration during alveolar formation. Together, these findings yield novel mechanistic insights into how mitochondria regulate pivotal steps of alveologenesis. They highlight selective utilization of energy in cells and diverse energy demands in different cellular processes during development. Our work serves as a paradigm for studying how mitochondria control tissue patterning.

The lungs display an intricate, tree-shaped structure which enables the complex gas exchanges required for life. The end of each tiny 'branch' hosts delicate air sacs, or alveoli, which are further divided by internal walls called septa. In mammals, this final structure is acquired during the last stage of lung development. Then, many different types of cells in the immature alveoli multiply and reach the right location to start constructing additional septa. While the structural changes underlining alveoli maturation are well-studied, the energy requirements for that process remain poorly understood. In particular, the exact role of the mitochondria, the cellular compartments that power most life processes, is still unclear. Zhang et al. therefore set out to map, in detail, the role of mitochondria in alveolar development. Microscope imaging revealed how mitochondria were unevenly distributed within the lung cells of newborn mice. Mitochondria accumulated around the machinery that controls protein secretion in the epithelial cells that line the air sacs, and around the contractile apparatus in the underlying cells (the 'myofibroblasts'). Genetically altering the mice to reduce mitochondrial activity or perturb mitochondrial location in these two cell types produced defective alveoli with fewer septa, but it had no effect on lung development before alveoli formation. This suggests that the formation of alveoli requires more energy than other steps of lung development. Disrupting mitochondrial activity or location also compromised how epithelial cells produced chemical signals necessary for the contraction or migration of the myofibroblasts. Together, these results highlight the importance of tightly regulating mitochondrial activity and location during lung patterning. In the future, this insight could lay the groundwork to determine how energy requirements in various tissues shape other biological processes in health and disease.

Network modeling predicts personalized gene expression and drug responses in valve myofibroblasts cultured with patient sera.

Aortic valve stenosis (AVS) patients experience pathogenic valve leaflet stiffening due to excessive extracellular matrix (ECM) remodeling. Numerous microenvironmental cues influence pathogenic expression of ECM remodeling genes in tissue-resident valvular myofibroblasts, and the regulation of complex myofibroblast signaling networks depends on patient-specific extracellular factors. Here, we combined a manually curated myofibroblast signaling network with a data-driven transcription factor network to predict patient-specific myofibroblast gene expression signatures and drug responses. Using transcriptomic data from myofibroblasts cultured with AVS patient sera, we produced a large-scale, logic-gated differential equation model in which 11 biochemical and biomechanical signals were transduced via a network of 334 signaling and transcription reactions to accurately predict the expression of 27 fibrosis-related genes. Correlations were found between personalized model-predicted gene expression and AVS patient echocardiography data, suggesting links between fibrosis-related signaling and patient-specific AVS severity. Further, global network perturbation analyses revealed signaling molecules with the most influence over network-wide activity, including endothelin 1 (ET1), interleukin 6 (IL6), and transforming growth factor ß (TGFß), along with downstream mediators c-Jun N-terminal kinase (JNK), signal transducer and activator of transcription (STAT), and reactive oxygen species (ROS). Lastly, we performed virtual drug screening to identify patient-specific drug responses, which were experimentally validated via fibrotic gene expression measurements in valvular interstitial cells cultured with AVS patient sera and treated with or without bosentan-a clinically approved ET1 receptor inhibitor. In sum, our work advances the ability of computational approaches to provide a mechanistic basis for clinical decisions including patient stratification and personalized drug screening.

Morin inhibits the transformation of fibroblasts towards myofibroblasts through regulating "PPAR-γ-glutaminolysis-DEPTOR" pathway in pulmonary fibrosis.

Morin, a natural flavonoid exists in many foods and dietary plants, owns good bioactivities. Herein, we investigated its effect on pulmonary fibrosis (PF), and further explored the mechanisms. Results showed that morin remarkably improved the pathologic alterations, and inhibited the transformation of fibroblasts towards myofibroblasts in lungs of mice with bleomycin-induced PF as well as TGF-ß1 or hypoxia-stimulated NIH-3T3 cells. Mechanistic studies revealed that morin activated peroxisome proliferator activated receptor-gamma (PPAR-γ), and GW9662 or siPPAR-γ significantly weakened the inhibition of morin on the transformation of NIH-3T3 cells. Furthermore, morin restricted glutaminolysis by down-regulating the level of glutaminase 1 (GLS1), which was confirmed by glutamine deprivation, and GLS1 overexpression. Replenishment of metabolite α-ketoglutarate (α-KG) and 2-hydroxyglutarate (2-HG) inhibited morin-prevented transformation of fibroblasts, but neither TGF-ß1 nor hypoxia could induce the transformation of IDH2-knockdown fibroblasts, suggesting 2-HG was directly involved in the action of morin. Then, ubiquitination of DEPTOR was demonstrated to be prevented by morin, which was attributed to KDM4A, an enzyme inactivated by 2-HG, and leucine as well as KDM4A inhibitor obstructed the effect of morin. Finally, the mechanisms of morin were further confirmed in vivo. Collectively, morin inhibited PF through intervening in "PPAR-γ-glutaminolysis-DEPTOR" signals, and subsequent restriction on the transformation of fibroblasts towards myofibroblasts.

Integrative analysis of lung molecular signatures reveals key drivers of systemic sclerosis-associated interstitial lung disease.

OBJECTIVES: Interstitial lung disease is a significant comorbidity and the leading cause of mortality in patients with systemic sclerosis. Transcriptomic data of systemic sclerosis-associated interstitial lung disease (SSc-ILD) were analysed to evaluate the salient molecular and cellular signatures in comparison with those in related pulmonary diseases and to identify the key driver genes and target molecules in the disease module. METHODS: A transcriptomic dataset of lung tissues from patients with SSc-ILD (n=52), idiopathic pulmonary fibrosis (IPF) (n=549), non-specific interstitial pneumonia (n=49) and pulmonary arterial hypertension (n=81) and from normal healthy controls (n=331) was subjected to filtration of differentially expressed genes, functional enrichment analysis, network-based key driver analysis and kernel-based diffusion scoring. The association of enriched pathways with clinical parameters was evaluated in patients with SSc-ILD. RESULTS: SSc-ILD shared key pathogenic pathways with other fibrosing pulmonary diseases but was distinguishable in some pathological processes. SSc-ILD showed general similarity with IPF in molecular and cellular signatures but stronger signals for myofibroblasts, which in SSc-ILD were in a senescent and apoptosis-resistant state. The p53 signalling pathway was the most enriched signature in lung tissues and lung fibroblasts of SSc-ILD, and was significantly correlated with carbon monoxide diffusing capacity of lung, cellular senescence and apoptosis. EEF2, EFF2K, PHKG2, VCAM1, PRKACB, ITGA4, CDK1, CDK2, FN1 and HDAC1 were key regulators with high diffusion scores in the disease module. CONCLUSIONS: Integrative transcriptomic analysis of lung tissues revealed key signatures of fibrosis in SSc-ILD. A network-based Bayesian approach provides deep insights into key regulatory genes and molecular targets applicable to treating SSc-ILD.

Regulatory effects of bio-intensity electric field on transformation of human skin fibroblasts / 中华烧伤杂志

Objective: To investigate the regulatory effects of bio-intensity electric field on the transformation of human skin fibroblasts (HSFs). Methods: The experimental research methods were used. HSFs were collected and divided into 200 mV/mm electric field group treated with 200 mV/mm electric field for 6 h and simulated electric field group placed in the electric field device without electricity for 6 h. Changes in morphology and arrangement of cells were observed in the living cell workstation; the number of cells at 0 and 6 h of treatment was recorded, and the rate of change in cell number was calculated; the direction of cell movement, movement velocity, and trajectory velocity within 3 h were observed and calculated (the number of samples was 34 in the simulated electric field group and 30 in 200 mV/mm electric field group in the aforementioned experiments); the protein expression of α-smooth muscle actin (α-SMA) in cells after 3 h of treatment was detected by immunofluorescence method (the number of sample was 3). HSFs were collected and divided into simulated electric field group placed in the electric field device without electricity for 3 h, and 100 mV/mm electric field group, 200 mV/mm electric field group, and 400 mV/mm electric field group which were treated with electric fields of corresponding intensities for 3 h. Besides, HSFs were divided into simulated electric field group placed in the electric field device without electricity for 6 h, and electric field treatment 1 h group, electric field treatment 3 h group, and electric field treatment 6 h group treated with 200 mV/mm electric field for corresponding time. The protein expressions of α-SMA and proliferating cell nuclear antigen (PCNA) were detected by Western blotting (the number of sample was 3). Data were statistically analyzed with Mann-Whitney U test, one-way analysis of variance, independent sample t test, and least significant difference test. Results: After 6 h of treatment, compared with that in simulated electric field group, the cells in 200 mV/mm electric field group were elongated in shape and locally adhered; the cells in simulated electric field group were randomly arranged, while the cells in 200 mV/mm electric field group were arranged in a regular longitudinal direction; the change rates in the number of cells in the two groups were similar (P>0.05). Within 3 h of treatment, the cells in 200 mV/mm electric field group had an obvious tendency to move toward the positive electrode, and the cells in simulated electric field group moved around the origin; compared with those in simulated electric field group, the movement velocity and trajectory velocity of the cells in 200 mV/mm electric field group were increased significantly (with Z values of -5.33 and -5.41, respectively, P<0.01), and the directionality was significantly enhanced (Z=-4.39, P<0.01). After 3 h of treatment, the protein expression of α-SMA of cells in 200 mV/mm electric field group was significantly higher than that in simulated electric field group (t=-9.81, P<0.01). After 3 h of treatment, the protein expressions of α-SMA of cells in 100 mV/mm electric field group, 200 mV/mm electric field group, and 400 mV/mm electric field group were 1.195±0.057, 1.606±0.041, and 1.616±0.039, respectively, which were significantly more than 0.649±0.028 in simulated electric field group (P<0.01). Compared with that in 100 mV/mm electric field group, the protein expressions of α-SMA of cells in 200 mV/mm electric field group and 400 mV/mm electric field group were significantly increased (P<0.01). The protein expressions of α-SMA of cells in electric field treatment 1 h group, electric field treatment 3 h group, and electric field treatment 6 h group were 0.730±0.032, 1.561±0.031, and 1.553±0.045, respectively, significantly more than 0.464±0.020 in simulated electric field group (P<0.01). Compared with that in electric field treatment 1 h group, the protein expressions of α-SMA in electric field treatment 3 h group and electric field treatment 6 h group were significantly increased (P<0.01). After 3 h of treatment, compared with that in simulated electric field group, the protein expressions of PCNA of cells in 100 mV/mm electric field group, 200 mV/mm electric field group, and 400 mV/mm electric field group were significantly decreased (P<0.05 or P<0.01); compared with that in 100 mV/mm electric field group, the protein expressions of PCNA of cells in 200 mV/mm electric field group and 400 mV/mm electric field group were significantly decreased (P<0.05 or P<0.01); compared with that in 200 mV/mm electric field group, the protein expression of PCNA of cells in 400 mV/mm electric field group was significantly decreased (P<0.01). Compared with that in simulated electric field group, the protein expressions of PCNA of cells in electric field treatment 1 h group, electric field treatment 3 h group, and electric field treatment 6 h group were significantly decreased (P<0.01); compared with that in electric field treatment 1 h group, the protein expressions of PCNA of cells in electric field treatment 3 h group and electric field treatment 6 h group were significantly decreased (P<0.05 or P<0.01); compared with that in electric field treatment 3 h group, the protein expression of PCNA of cells in electric field treatment 6 h group was significantly decreased (P<0.01). Conclusions: The bio-intensity electric field can induce the migration of HSFs and promote the transformation of fibroblasts to myofibroblasts, and the transformation displays certain dependence on the time and intensity of electric field.

Local Renin-Angiotensin System Signaling Mediates Cellular Function of Aortic Valves.

The renin-angiotensin system (RAS) is activated in aortic valve disease, yet little is understood about how it affects the acute functional response of valve interstitial cells (VICs). Herein, we developed a gelatin-based valve thin film (vTF) platform to investigate whether the contractile response of VICs can be regulated via RAS mediators and inhibitors. First, the impact of culture medium (quiescent, activated, and osteogenic medium) on VIC phenotype and function was assessed. Contractility of VICs was measured upon treatment with angiotensin I (Ang I), angiotensin II (Ang II), angiotensin-converting enzyme (ACE) inhibitor, and Angiotensin II type 1 receptor (AT1R) inhibitor. Anisotropic cell alignment on gelatin vTF was achieved independent of culture conditions. Cells cultured in activated and osteogenic conditions were found to be more elongated than in quiescent medium. Increased α-SMA expression was observed in activated medium and no RUNX2 expression were observed in cells. VIC contractile stress increased with increasing concentrations (from 10-10 to 10-6 M) of Ang I and Ang II. Moreover, cell contraction was significantly reduced in all ACE and AT1R inhibitor-treated groups. Together, these findings suggest that local RAS is active in VICs, and our vTF may provide a powerful platform for valve drug screening and development.

The serine proteases dipeptidyl-peptidase 4 and urokinase are key molecules in human and mouse scar formation.

Despite recent advances in understanding skin scarring, mechanisms triggering hypertrophic scar formation are still poorly understood. In the present study, we investigate mature human hypertrophic scars and developing scars in mice at single cell resolution. Compared to normal skin, we find significant differences in gene expression in most cell types present in scar tissue. Fibroblasts show the most prominent alterations in gene expression, displaying a distinct fibrotic signature. By comparing genes upregulated in murine fibroblasts during scar development with genes highly expressed in mature human hypertrophic scars, we identify a group of serine proteases, tentatively involved in scar formation. Two of them, dipeptidyl-peptidase 4 (DPP4) and urokinase (PLAU), are further analyzed in functional assays, revealing a role in TGFß1-mediated myofibroblast differentiation and over-production of components of the extracellular matrix in vitro. Topical treatment with inhibitors of DPP4 and PLAU during scar formation in vivo shows anti-fibrotic activity and improvement of scar quality, most prominently after application of the PLAU inhibitor BC-11. In this study, we delineate the genetic landscape of hypertrophic scars and present insights into mechanisms involved in hypertrophic scar formation. Our data suggest the use of serine protease inhibitors for the treatment of skin fibrosis.

PGE2 and Thrombin Induce Myofibroblast Transdifferentiation via Activin A and CTGF in Endometrial Stromal Cells.

Endometriosis is characterized by inflammation and fibrotic changes. Our previous study using a mouse model showed that proinflammatory factors present in peritoneal hemorrhage exacerbated inflammation in endometriosis-like grafts, at least in part through the activation of prostaglandin (PG) E2 receptor and protease-activated receptor (PAR). In addition, menstruation-related factors, PGE2 and thrombin (P/T), a PAR1 agonist induced epithelial-mesenchymal transition (EMT) of endometrial cells under hypoxia. However, the molecular mechanisms by which P/T induce development of endometriosis have not been fully characterized. To investigate the effects of P/T, RNA extracted from endometrial stromal cells (ESCs) treated with P/T were subjected to RNA sequence analysis, and identified activin A, FOS, and GATA2 as upregulated genes. Activin A increased the expression of connective tissue growth factor (CTGF) and mesenchymal marker genes in ESCs. CTGF induced the expression of fibrosis marker type I collagen, fibronectin, and α-smooth muscle actin (αSMA), indicating fibroblast to myofibroblast transdifferentiation (FMT) of ESCs. In addition, activin A, FOS, GATA2, CTGF, and αSMA were localized in endometriosis lesions. Taken together, our data show that P/T induces changes resembling EMT and FMT in ectopic ESCs derived from retrograde menstruation, and that these are associated with fibrotic changes in the lesions. Pharmacological means that block P/T-induced activin A and CTGF signaling may be strategies to inhibit fibrosis in endometriotic lesions.

Pyruvate kinase M2 regulates fibrosis development and progression by controlling glycine auxotrophy in myofibroblasts.

Rationale: Fibrosis is a pathologic condition of abnormal accumulation of collagen fibrils. Collagen is a major extracellular matrix (ECM) protein synthesized and secreted by myofibroblasts, composing mainly (Gly-X-Y)n triplet repeats with >30% Gly residue. During fibrosis progression, myofibroblasts must upregulate glycine metabolism to meet the high demands of amino acids for collagen synthesis. Method: Expression of PKM2 in myofibroblasts was analyzed in cultured fibroblasts and fibrosis disease tissues. Functional roles of PKM2 and PKM2 activator in biosynthesis of serine → glycine and production of collagen from glycolysis intermediates were assayed in cultured activated LX-2 and human primary lung fibroblast cells. Mouse models of Liver, lung, and pancreas fibrosis were employed to analyze treatment effects of PKM2 activator in organ tissue fibrosis. Results: We report here that myofibroblast differentiation upregulates pyruvate kinase M2 (PKM2) and promotes dimerization of PKM2. Dimer PKM2 slows the flow rate of glycolysis and channels glycolytic intermediates to de novo glycine synthesis, which facilitates collagen synthesis and secretion in myofibroblasts. Our results show that PKM2 activator that converts PKM2 dimer to tetramer, inhibits fibrosis progression in mouse models of liver, lung, and pancreatic fibrosis. Furthermore, metabolism alteration by dimer PKM2 increases NADPH production, which consequently protects myofibroblasts from apoptosis. Conclusion: Our study uncovers a novel role of PKM2 in tissue/organ fibrosis, suggesting a possible strategy for treatment of fibrotic diseases using PKM2 activator.

Mechanisms of Endothelial-to-Mesenchymal Transition Induction by Extracellular Matrix Components in Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis can be caused by different factors, including accumulation of pathological extracellular matrix (ECM) with abnormal composition, stiffness, and architecture in the lung tissue. We studied the effect of ECM produced by lung fibroblasts of healthy mice or mice with bleomycin-induced pulmonary fibrosis on the process of endothelialto- mesenchymal transition, one of the main sources of effector myofibroblasts in fibrosis progression. Despite stimulation of spontaneous and TGFß-1-induced differentiation of fibroblasts into myofibroblasts by fibrotic ECM, the appearance of α-SMA, the main marker of myofibroblasts, and its integration in stress fibrils in endotheliocytes were not observed under similar conditions. However, the expression of transcription factors SNAI1 and SNAI2/Slug and the production of components of fibrotic ECM (specific EDA-fibronectin splice form and collagen type I) were increased in endotheliocytes cultured on fibrotic ECM. Endothelium also demonstrated increased cell velocity in the models of directed cell migration. These data indicate activation of the intermediate state of the endothelial-to-mesenchymal transition in endotheliocytes upon contact with fibrotic, but not normal stromal matrix. In combination with the complex microenvironment that develops during fibrosis progression, it can lead to the replenishment of myofibroblasts pool from the resident endothelium.

Compromised Biomechanical Properties, Cell-Cell Adhesion and Nanotubes Communication in Cardiac Fibroblasts Carrying the Lamin A/C D192G Mutation.

Clinical effects induced by arrhythmogenic cardiomyopathy (ACM) originate from a large spectrum of genetic variations, including the missense mutation of the lamin A/C gene (LMNA), LMNA D192G. The aim of our study was to investigate the biophysical and biomechanical impact of the LMNA D192G mutation on neonatal rat ventricular fibroblasts (NRVF). The main findings in mutated NRVFs were: (i) cytoskeleton disorganization (actin and intermediate filaments); (ii) decreased elasticity of NRVFs; (iii) altered cell-cell adhesion properties, that highlighted a strong effect on cellular communication, in particular on tunneling nanotubes (TNTs). In mutant-expressing fibroblasts, these nanotubes were weakened with altered mechanical properties as shown by atomic force microscopy (AFM) and optical tweezers. These outcomes complement prior investigations on LMNA mutant cardiomyocytes and suggest that the LMNA D192G mutation impacts the biomechanical properties of both cardiomyocytes and cardiac fibroblasts. These observations could explain how this mutation influences cardiac biomechanical pathology and the severity of ACM in LMNA-cardiomyopathy.

Developmental lineage of human pluripotent stem cell-derived cardiac fibroblasts affects their functional phenotype.

Cardiac fibroblasts (CFBs) support heart function by secreting extracellular matrix (ECM) and paracrine factors, respond to stress associated with injury and disease, and therefore are an increasingly important therapeutic target. We describe how developmental lineage of human pluripotent stem cell-derived CFBs, epicardial (EpiC-FB), and second heart field (SHF-FB) impacts transcriptional and functional properties. Both EpiC-FBs and SHF-FBs exhibited CFB transcriptional programs and improved calcium handling in human pluripotent stem cell-derived cardiac tissues. We identified differences including in composition of ECM synthesized, secretion of growth and differentiation factors, and myofibroblast activation potential, with EpiC-FBs exhibiting higher stress-induced activation potential akin to myofibroblasts and SHF-FBs demonstrating higher calcification and mineralization potential. These phenotypic differences suggest that EpiC-FBs have utility in modeling fibrotic diseases while SHF-FBs are a promising source of cells for regenerative therapies. This work directly contrasts regional and developmental specificity of CFBs and informs CFB in vitro model selection.

Hepatic Perisinusoidal Myofibroblast Proliferation and Systemic Inflammatory Response Precedes Sep/Tox Hepatitis in Broilers.

Septicemia-toxemia (sep/tox) falls under U.S. Department of Agriculture (USDA) food safety Category 1 and is the most common and economically significant cause of broiler carcass condemnations. Hepatic lesions are considered a possible consequence of septicemia and associated bacterial contamination of the carcass. Thus, these lesions are considered an indicator of sep/tox (sep/tox hepatitis). This study was undertaken to analyze the histologic lesions preceding grossly visible liver lesions leading to condemnation because of sep/tox at the processing plant. Livers from carcasses of broilers condemned by USDA inspectors for sep/tox were used to establish microscopic and gross criteria of end-stage sep/tox hepatitis. Following the characterization of sep/tox hepatitis, broilers from a farm with a history of sep/tox condemnations were submitted for postmortem examination and bacteriologic investigation at four intervals during the final 20 days of production. Five healthy and five clinically ill chickens were submitted from four houses at 18, 25, 32, and 38 days of production (160 total). Microscopic lesions representing hepatic perisinusoidal myofibroblast proliferation (HPMP), periportal extramedullary granulopoiesis (PEMG), splenic follicular histiocytosis, and bone marrow cellularity (BMC) were graded subjectively for each bird, and subjective grading was evaluated with digital quantitative techniques. Perisinusoidal hepatic stellate cell morphology and progressive transformation of these cells into myofibroblasts was confirmed by immunohistochemistry for smooth muscle actin and desmin. Aerobic cultures of livers and gall bladders from sep/tox birds yielded no growth of bacteria associated with septicemia. Mild to severe HPMP was observed in all age groups, representing 28% of examined birds. Increases in inflammatory cells observed by PEMG and BMC were positively correlated with progressive HPMP and end-stage sep/tox hepatitis in broiler chickens.

Artículo regular­Proliferación de miofibroblastos perisinusoidales hepáticos y respuesta inflamatoria sistémica que precede a la hepatitis por septicemia y toxemia (sep/tox) en pollos de engorde. La septicemia-toxemia (sep/tox) se incluye en la Categoría 1 de seguridad alimentaria del Departamento de Agricultura de los Estados Unidos. (USDA) y es la causa más común y económicamente significativa de decomisos de canales de pollos de engorde. Las lesiones hepáticas se consideran una posible consecuencia de la septicemia y de la contaminación bacteriana asociada con la canal. Por lo tanto, estas lesiones se consideran un indicador de septicemia/toxemia (hepatitis sep/tox). Este estudio se llevó a cabo para analizar las lesiones histológicas que preceden a las lesiones hepáticas muy visibles que conducen a los decomisos debido a septicemia/toxemia en la planta de procesamiento. Se utilizaron hígados de canales de pollos de engorde decomisados por los inspectores del USDA por septicemia/toxemia para establecer criterios microscópicos y generales de hepatitis en etapa terminal de la septicemia/toxemia. Después de la caracterización de la hepatitis por septicemia/toxemia, los pollos de engorde de una granja con un historial de decomisos por septicemia/toxemia se sometieron a examen post mortem e investigación bacteriológica en cuatro intervalos durante los últimos 20 días de producción. Se enviaron cinco pollos sanos y cinco clínicamente enfermos de cuatro casetas a los 18, 25, 32 y 38 días de producción (160 en total). Las lesiones microscópicas que representan la proliferación de miofibroblastos perisinusoidales hepáticos (HPMP), la granulopoyesis extramedular periportal (PEMG), la histocitosis folicular esplénica y la celularidad de la médula ósea (BMC) se clasificaron subjetivamente para cada ave, y la clasificación subjetiva se evaluó con técnicas cuantitativas digitales. La morfología de las células estrelladas hepáticas perisinusoidales y la transformación progresiva de estas células en miofibroblastos se confirmó mediante inmunohistoquímica para actina y desmina del músculo liso. Los cultivos aeróbicos de hígados y vesícula biliar de aves con septicemia/toxemia no produjeron crecimiento de bacterias asociadas con la septicemia. Se observó proliferación de miofibroblastos perisinusoidales hepáticos de leve a severa en todos los grupos de edad, lo que representa el 28% de las aves examinadas. Los aumentos en las células inflamatorias observados por granulopoyesis extramedular periportal y celularidad de la médula ósea se correlacionaron positivamente con proliferación progresiva de miofibroblastos perisinusoidales hepáticos y con hepatitis por septicemia/toxemia en etapa terminal en pollos de engorde.

Mechanical and Thermodynamic Properties of Non-Muscle Contractile Tissues: The Myofibroblast and the Molecular Motor Non-Muscle Myosin Type IIA.

Myofibroblasts are contractile cells found in multiple tissues. They are physiological cells as in the human placenta and can be obtained from bone marrow mesenchymal stem cells after differentiation by transforming growth factor-ß (TGF-ß). They are also found in the stroma of cancerous tissues and can be located in non-muscle contractile tissues. When stimulated by an electric current or after exposure to KCl, these tissues contract. They relax either by lowering the intracellular Ca2+ concentration (by means of isosorbide dinitrate or sildenafil) or by inhibiting actin-myosin interactions (by means of 2,3-butanedione monoxime or blebbistatin). Their shortening velocity and their developed tension are dramatically low compared to those of muscles. Like sarcomeric and smooth muscles, they obey Frank-Starling's law and exhibit the Hill hyperbolic tension-velocity relationship. The molecular motor of the myofibroblast is the non-muscle myosin type IIA (NMIIA). Its essential characteristic is the extreme slowness of its molecular kinetics. In contrast, NMIIA develops a unitary force similar to that of muscle myosins. From a thermodynamic point of view, non-muscle contractile tissues containing NMIIA operate extremely close to equilibrium in a linear stationary mode.

The dual distinct role of telomerase in repression of senescence and myofibroblast differentiation.

Many aging related diseases such as cancer implicate the myofibroblast in disease progression. Furthermore genesis of the myofibroblast is associated with manifestation of cellular senescence of unclear significance. In this study we investigated the role of a common regulator, namely telomerase reverse transcriptase (TERT), in order to evaluate the potential significance of this association between both processes. We analyzed the effects of TERT overexpression or deficiency on expression of CDKN2A and ACTA2 as indicators of senescence and differentiation, respectively. We assess binding of TERT or YB-1, a repressor of both genes, to their promoters. TERT repressed both CDKN2A and ACTA2 expression, and abolished stress-induced expression of both genes. Conversely, TERT deficiency enhanced their expression. Altering CDKN2A expression had no effect on ACTA2 expression. Both TERT and YB-1 were shown to bind the CDKN2A promoter but only YB-1 was shown to bind the ACTA2 promoter. TERT overexpression inhibited CDKN2A promoter activity while stimulating YB-1 expression and activation to repress ACTA2 gene. TERT repressed myofibroblast differentiation and senescence via distinct mechanisms. The latter was associated with TERT binding to the CDKN2A promoter, but not to the ACTA2 promoter, which may require interaction with co-factors such as YB-1.

Engrailed 1 coordinates cytoskeletal reorganization to induce myofibroblast differentiation.

Transforming growth factor-ß (TGFß) is a key mediator of fibroblast activation in fibrotic diseases, including systemic sclerosis. Here we show that Engrailed 1 (EN1) is reexpressed in multiple fibroblast subpopulations in the skin of SSc patients. We characterize EN1 as a molecular amplifier of TGFß signaling in myofibroblast differentiation: TGFß induces EN1 expression in a SMAD3-dependent manner, and in turn, EN1 mediates the profibrotic effects of TGFß. RNA sequencing demonstrates that EN1 induces a profibrotic gene expression profile functionally related to cytoskeleton organization and ROCK activation. EN1 regulates gene expression by modulating the activity of SP1 and other SP transcription factors, as confirmed by ChIP-seq experiments for EN1 and SP1. Functional experiments confirm the coordinating role of EN1 on ROCK activity and the reorganization of cytoskeleton during myofibroblast differentiation, in both standard fibroblast culture systems and in vitro skin models. Consistently, mice with fibroblast-specific knockout of En1 demonstrate impaired fibroblast-to-myofibroblast transition and are partially protected from experimental skin fibrosis.

HE4 Predicts Progressive Fibrosis and Cardiovascular Events in Patients With Dilated Cardiomyopathy.

Background Cardiac fibrosis plays a crucial role in the pathogenesis of dilated cardiomyopathy (DCM). HE4 (human epididymis protein 4) is a secretory protein expressed in activated fibroblasts that exacerbates tissue fibrosis. In the present study, we investigated the clinical utility of HE4 measurement in patients with DCM and its pathophysiological role in preclinical experiments in vivo and in vitro. Methods and Results We measured serum HE4 levels of 87 patients with DCM. Endomyocardial biopsy expressed severe fibrosis only in the high HE4 group (P<0.0001). Echocardiography showed that left ventricular end-diastolic diameter tends to decrease over time (58±7.3 to 51±6.6 mm; P<0.0001) in the low HE4 group (<59.65 pmol/L [median value]). HE4 was significantly associated with risk reduction of mortality and cardiovascular hospitalization in multivariate Cox model. In vivo, HE4 was highly expressed in kidney and lung tissue of mouse, and scarcely expressed in heart. In genetically induced DCM mouse model, HE4 expression increased in kidney but not in heart and lung. In vitro, supernatant from HE4-transfected human embryonic kidney 293T cells enhanced transdifferentiation of rat neonatal fibroblasts and increased expression of fibrosis-related genes, and this was accompanied by the activation of extracellular signal-regulated kinase signaling in cardiac fibroblasts. Treatment with an inhibitor of upstream signal of extracellular signal-regulated kinase or a neutralizing HE4 antibody canceled the profibrotic properties of HE4. Conclusions HE4 functions as a secretory factor, activating cardiac fibroblasts, thereby inducing cardiac interstitial fibrosis. HE4 could be a promising biomarker for assessing ongoing fibrosis and a novel therapeutic target in DCM. Registration URL: https://upload.umin.ac.jp/cgi-open-bin/ctr; Unique identifier: UMIN000043062.

G-MDSCs promote aging-related cardiac fibrosis by activating myofibroblasts and preventing senescence.

Aging is one of the most prominent risk factors for heart failure. Myeloid-derived suppressor cells (MDSCs) accumulate in aged tissue and have been confirmed to be associated with various aging-related diseases. However, the role of MDSCs in the aging heart remains unknown. Through RNA-seq and biochemical approaches, we found that granulocytic MDSCs (G-MDSCs) accumulated significantly in the aging heart compared with monocytic MDSCs (M-MDSCs). Therefore, we explored the effects of G-MDSCs on the aging heart. We found that the adoptive transfer of G-MDSCs of aging mice to young hearts resulted in cardiac diastolic dysfunction by inducing cardiac fibrosis, similar to that in aging hearts. S100A8/A9 derived from G-MDSCs induced inflammatory phenotypes and increased the osteopontin (OPN) level in fibroblasts. The upregulation of fibroblast growth factor 2 (FGF2) expression in fibroblasts mediated by G-MDSCs promoted antisenescence and antiapoptotic phenotypes of fibroblasts. SOX9 is the downstream gene of FGF2 and is required for FGF2-mediated and G-MDSC-mediated profibrotic effects. Interestingly, both FGF2 levels and SOX9 levels were upregulated in fibroblasts but not in G-MDSCs and were independent of S100A8/9. Therefore, a novel FGF2-SOX9 signaling axis that regulates fibroblast self-renewal and antiapoptotic phenotypes was identified. Our study revealed the mechanism by which G-MDSCs promote cardiac fibrosis via the secretion of S100A8/A9 and the regulation of FGF2-SOX9 signaling in fibroblasts during aging.

Ca2+/calmodulin kinase II-dependent regulation of ßIV-spectrin modulates cardiac fibroblast gene expression, proliferation, and contractility.

Fibrosis is a pronounced feature of heart disease and the result of dysregulated activation of resident cardiac fibroblasts (CFs). Recent work identified stress-induced degradation of the cytoskeletal protein ßIV-spectrin as an important step in CF activation and cardiac fibrosis. Furthermore, loss of ßIV-spectrin was found to depend on Ca2+/calmodulin-dependent kinase II (CaMKII). Therefore, we sought to determine the mechanism for CaMKII-dependent regulation of ßIV-spectrin and CF activity. Computational screening and MS revealed a critical serine residue (S2250 in mouse and S2254 in human) in ßIV-spectrin phosphorylated by CaMKII. Disruption of ßIV-spectrin/CaMKII interaction or alanine substitution of ßIV-spectrin Ser2250 (ßIV-S2254A) prevented CaMKII-induced degradation, whereas a phosphomimetic construct (ßIV-spectrin with glutamic acid substitution at serine 2254 [ßIV-S2254E]) showed accelerated degradation in the absence of CaMKII. To assess the physiological significance of this phosphorylation event, we expressed exogenous ßIV-S2254A and ßIV-S2254E constructs in ßIV-spectrin-deficient CFs, which have increased proliferation and fibrotic gene expression compared with WT CFs. ßIV-S2254A but not ßIV-S2254E normalized CF proliferation, gene expression, and contractility. Pathophysiological targeting of ßIV-spectrin phosphorylation and subsequent degradation was identified in CFs activated with the profibrotic ligand angiotensin II, resulting in increased proliferation and signal transducer and activation of transcription 3 nuclear accumulation. While therapeutic delivery of exogenous WT ßIV-spectrin partially reversed these trends, ßIV-S2254A completely negated increased CF proliferation and signal transducer and activation of transcription 3 translocation. Moreover, we observed ßIV-spectrin phosphorylation and associated loss in total protein within human heart tissue following heart failure. Together, these data illustrate a considerable role for the ßIV-spectrin/CaMKII interaction in activating profibrotic signaling.