Myofibroblast Transdifferentiation Research Articles

Fibrosis signaling in endometrial cells and endometriosis development

In endometriosis, the tissues similar to the endometrial tissue attaches outside the uterine cavity, causing inflammation and fibrosis. The retrograde menstruation theory is the most plausible mechanism, though the detailed pathogenesis remains unclear. Our observations suggest that endometriosis-like lesions occur more often at sites of ovarian excision causing bleeding in mouse models. Additionally, prostaglandin E2 (PGE2) and thrombin, a protease-activated receptor (PAR) agonist in menstrual blood exacerbate inflammation in these lesions. Focusing on the hypoxic conditions of menstrual blood, we investigated the effects of PGE2/thrombin on inflammation and fibrosis using primary cultured endometrial stromal cells (ESCs) and glandular epithelial cells (EECs) under low oxygen conditions. Chemokine CXCL12 secreted by endometrial stromal cells under hypoxia acts on CXCR4 receptors on glandular epithelial cells, inducing epithelial-mesenchymal transition (EMT), suggesting a possible role in endometriosis progression. RNA-seq analysis of PGE2/thrombin effects on endometrial stromal cells revealed activation of the transforming growth factor (TGF)-β pathway, particularly increased production and secretion of activin A, a member of the TGFβ family. Activin A, via increased connective tissue growth factor (CTGF) expression, promotes differentiation of endometrial stromal cells from fibroblast-like to myofibroblast transdifferentiation (FMT) of ESCs. In conclusion, targeting the CXCL12/CXCR4 and activin A/CTGF signaling pathways holds promise for improving fibrosis in endometriosis lesions.

MicroRNA-145-5p restricts cardiac scar resolution during cardiac regeneration

Abstract Background Human hearts have a very limited capacity to renew cardiomyocytes following injury. The lost myocardium is permanently replaced with noncontractile fibrotic tissue, consequently leading to heart failure. In contrast, zebrafish retain cardiac regenerative capacity during their lifetime. After injury, zebrafish hearts undergo transient fibrotic scarring that subsequently resolves and is replaced with new cardiomyocytes over time. Thus, understanding the molecular mechanisms governing cardiac scar resolution in zebrafish could offer insights into enhancing the regenerative capacities of human hearts. Objective We aimed to decipher the molecular pathways associated with zebrafish cardiac scar resolution. Methods We investigated the mRNA and miRNA responses to injury at 14 days post-injury by high-throughput transcriptome profiling of RNAs isolated from healthy or cryoinjured hearts. The prediction of miRNA-mRNA interactions was performed to identify the most differentially expressed miRNAs and their target genes. These miRNAs and genes were validated in zebrafish regenerating hearts and in the left ventricles of ischemic cardiomyopathy or dilated cardiomyopathy patients. The potential role of identified miRNAs in cardiac scar resolution was investigated in human cardiac fibroblasts. Results Principal component analyses identify two distinct clusters in both the mRNAome and miRNAome corresponding to healthy and injured hearts, indicating significant changes in transcriptome profiles during zebrafish cardiac regeneration. Gene set enrichment analyses indicate that molecular pathways involved in extracellular matrix organization and inflammatory response are markedly activated whereas genes associated with mitochondrial organization and oxidative phosphorylation are downregulated in injured hearts. We observed the downregulation of miR-145-5p and the upregulation of procollagen V in the injured hearts. In contrast to zebrafish, in the left ventricles of ischemic cardiomyopathy patients, miR-145-5p is significantly upregulated and procollagen V α1 is downregulated. Theoretical prediction of miRNA-mRNA interactions indicates procollagen V α1 is negatively correlated with miR-145-5p. Overexpression of miR-145-5p in human cardiac fibroblasts downregulates collagen V α1 and alpha smooth muscle actin, upregulates procollagen I and matrix metalloproteinase 9, and inhibits cell proliferation and migration. In contrast, inhibition of miR-145-5p upregulates collagen V α1 and alpha smooth muscle actin, downregulates procollagen I and matrix metalloproteinase 9, and stimulates cell proliferation and migration. Conclusion Our study suggests that miR-145-5p may play a role in the suppression of myofibroblast transdifferentiation via collagen V α1, which restricts scar resolution and cardiac repair.

Key role for inflammation-related signaling in the pathogenesis of myopia based on evidence from proteomics analysis

The mechanisms underlying myopia pathogenesis are not well understood. Using publicly-available human and animal datasets, we expound on the roles of known, implicated proteins, and new myopia-related signaling pathways were hypothesized. Proteins identified from human serum or ocular fluids, and from ocular tissues in myopic animal models, were uploaded and analyzed with the QIAGEN Ingenuity Pathway Analysis (IPA) software (March 2023). With each IPA database update, more potentially-relevant proteins and signaling pathways previously unavailable during data acquisition are added, allowing extraction of novel conclusions from existing data. Canonical pathway analysis was used to analyze these data and calculate an IPA activation z-score—which indicates not only whether an association is significant, but also whether the pathway is likely activated or inhibited. Cellular immune response and cytokine signaling were frequently found to be affected in both human and animal myopia studies. Analysis of two publicly-available proteomic datasets highlighted a potential role of the innate immune system and inflammation in myopia development, detailing specific signaling pathways involved such as Granzyme A (GzmA) and S100 family signaling in the retina, and activation of myofibroblast trans-differentiation in the sclera. This perspective in myopia research may facilitate development of more effective and targeted therapeutic agents.

Dexamethasone induces trabecular meshwork cell myofibroblast transdifferentiation through ARHGEF26.

Glucocorticoid use may cause elevated intraocular pressure, leading to the development of glucocorticoid-induced glaucoma (GIG). However, the mechanism of GIG development remains incompletely understood. In this study, we subjected primary human trabecular meshwork cells (TMCs) and mice to dexamethasone treatment to mimic glucocorticoid exposure. The myofibroblast transdifferentiation of TMCs was observed in cellular and mouse models, as well as in human trabecular mesh specimens. This was demonstrated by the cytoskeletal reorganization, alterations in cell morphology, heightened transdifferentiation markers, increased extracellular matrix deposition, and cellular dysfunction. Knockdown of Rho guanine nucleotide exchange factor 26 (ARHGEF26) expression ameliorated dexamethasone-induced changes in cell morphology and upregulation of myofibroblast markers, reversed dysfunction and extracellular matrix deposition in TMCs, and prevented the development of dexamethasone-induced intraocular hypertension. And, this process may be related to the TGF-β pathway. In conclusion, glucocorticoids induced the myofibroblast transdifferentiation in TMCs, which played a crucial role in the pathogenesis of GIG. Inhibition of ARHGEF26 expression protected TMCs by reversing myofibroblast transdifferentiation. This study demonstrated the potential of reversing the myofibroblast transdifferentiation of TMCs as a new target for treating GIG.

Manipulating mitochondrial pyruvate carrier function causes metabolic remodeling in corneal myofibroblasts that ameliorates fibrosis

Myofibroblasts are key cellular effectors of corneal wound healing from trauma, surgery, or infection. However, their persistent deposition of disorganized extracellular matrix can also cause corneal fibrosis and visual impairment. Recent work showed that the PPARγ agonist Troglitazone can mitigate established corneal fibrosis, and parallel in vitro data suggested this occurred through inhibition of the mitochondrial pyruvate carrier (MPC) rather than PPARγ. In addition to oxidative phosphorylation (Ox-Phos), pyruvate and other mitochondrial metabolites provide carbon for the synthesis of biological macromolecules. However, it is currently unclear how these roles selectively impact fibrosis. Here, we performed bioenergetic, metabolomic, and epigenetic analyses of corneal fibroblasts treated with TGF-β1 to stimulate myofibroblast trans-differentiation, with further addition of Troglitazone or the MPC inhibitor UK5099, to identify MPC-dependencies that may facilitate remodeling and loss of the myofibroblast phenotype. Our results show that a shift in energy metabolism is associated with, but not sufficient to drive cellular remodeling. Metabolites whose abundances were sensitive to MPC inhibition suggest that sustained carbon influx into the Krebs’ cycle is prioritized over proline synthesis to fuel collagen deposition. Furthermore, increased abundance of acetyl-CoA and increased histone H3 acetylation suggest that epigenetic mechanisms downstream of metabolic remodeling may reinforce cellular phenotypes. Overall, our results highlight a novel molecular target and metabolic vulnerability that affects myofibroblast persistence in the context of corneal wounding.

Diverse roles of SARS-CoV-2 Spike and Nucleocapsid proteins in EndMT stimulation through the TGF-β-MRTF axis inhibited by aspirin

BackgroundThe SARS-CoV-2 virus causes severe COVID-19 in one-fifth of patients. In addition to high mortality, infection may induce respiratory failure and cardiovascular complications associated with inflammation. Acute or prolonged inflammation results in organ fibrosis, the cause of which might be endothelial disorders arising during the endothelial-mesenchymal transition (EndMT).MethodsHUVECs and HMEC-1 cells were stimulated with SARS-CoV-2 S (Spike) and N (Nucleocapsid) proteins, and EndMT induction was evaluated by studying specific protein markers via Western blotting. Wound healing and tube formation assays were employed to assess the potential of SARS-CoV-2 to stimulate changes in cell behaviour. MRTF nuclear translocation, ROS generation, TLR4 inhibitors, TGF-β-neutralizing antibodies, and inhibitors of the TGF-β-dependent pathway were used to investigate the role of the TGF-β-MRTF signalling axis in SARS-CoV-2-dependent EndMT stimulation.ResultsBoth viral proteins stimulate myofibroblast trans-differentiation. However, the N protein is more effective at EndMT induction. The TGF-β-MRTF pathway plays a critical role in this process. The N protein preferentially favours action through TGF-β2, whose secretion is induced through TLR4-ROS action. TGF-β2 stimulates MRTF-A and MRTF-B nuclear translocation and strongly regulates EndMT. In contrast, the Spike protein stimulates TGF-β1 secretion as a result of ACE2 downregulation. TGF-β1 induces only MRTF-B, which, in turn, weakly regulates EndMT. Furthermore, aspirin, a common nonsteroidal anti-inflammatory drug, might prevent and reverse SARS-CoV-2-dependent EndMT induction through TGF-β-MRTF pathway deregulation.ConclusionThe reported study revealed that SARS-CoV-2 infection induces EndMT. Moreover, it was demonstrated for the first time at the molecular level that the intensity of the EndMT triggered by SARS-CoV-2 infection may vary and depend on the viral protein involved. The N protein acts through TLR4-ROS-TGF-β2-MRTF-A/B, whereas the S protein acts through ACE2-TGF-β1-MRTF-B. Furthermore, we identified aspirin as a potential anti-fibrotic drug for treating patients with SARS-CoV-2 infection.

S100A4 makes two appearances in mechanisms leading to fibrosis

Non-muscle myosin 2 (NM2) is known to play an important role in myofibroblast transdifferentiation, a hallmark of fibrotic disorders. In a recent JBC article, Southern etal. demonstrate that endogenous S100A4, a calcium- and NM2-binding protein acts as a mechanoeffector in this process. Since extracellular S100A4 is also involved in fibrogenesis by triggering the inflammatory response, this small protein appears to contribute to fibrosis via at least two distinct mechanisms.

Exploring unconventional targets in myofibroblast transdifferentiation outside classical TGF- signaling in renal fibrosis.

We propose that the key initiators of renal fibrosis are myofibroblasts which originate from four predominant sources-fibroblasts, pericytes, endothelial cells and macrophages. Increased accumulation of renal interstitial myofibroblasts correlates with an increase in collagen, fibrillar proteins, and fibrosis severity. The canonical TGF- pathway, signaling via Smad proteins, is the central molecular hub that initiates these cellular transformations. However, directly targeting these classical pathway molecules has proven challenging due their integral roles in metabolic process, and/or non-sustainable effects involving compensatory cross-talk with TGF-β. This review explores recently discovered alternative molecular targets that drive transdifferentiation into myofibroblasts. Discovering targets outside of the classical TGF-β/Smad pathway is crucial for advancing antifibrotic therapies, and strategically targeting the development of myofibroblasts offers a promising approach to control excessive extracellular matrix deposition and impede fibrosis progression.

Group2 innate lymphoid cells ameliorate renal fibrosis and dysfunction associated with adenine-induced CKD

Renal fibrosis is a common pathway of chronic kidney disease (CKD) progression involving primary kidney injury and kidney diseases. Group 2 innate lymphoid cells (ILC2s) mediate type 2 immune responses irrespective of antigen presentation and play a reno-protective role in kidney injury and disease. In the present study, we observed a decrease in kidney-resident ILC2s in CKD and found that enrichment of ILC2s in the kidney ameliorates renal fibrosis. In CKD kidney, ILC2s preferentially produced IL-13 over IL-5 in response to IL-33 stimulation, regardless of ST2L expression. Moreover, GATA3 expression was decreased in ILC2s, and T-bet+ ILC1s and RORγt+ ILC3s were increased in CKD kidney. Adoptive transfer of kidney ILC2s into adenine-induced CKD model mouse improved renal function and fibrosis. Renal fibroblasts cultured with IL33-activated kidney ILC2s suppressed myofibroblast trans-differentiation through Acta2 and Fn-1 regulation. These results suggest that kidney ILC2s prevent CKD progression via improvement of renal fibrosis. Our findings also suggest that ILC2s may contribute to the development of new therapeutic agents and strategies for tissue fibroses.

Aberrantly downregulated FENDRR by arecoline elevates ROS and myofibroblast activation via mitigating the miR-214/MFN2 axis

Long non-coding RNA FENDRR possesses both anti-fibrotic and anti-cancer properties, but its significance in the development of premalignant oral submucous fibrosis (OSF) remains unclear. Here, we showed that FENDRR was downregulated in OSF specimens and fibrotic buccal mucosal fibroblasts (fBMFs), and overexpression of FENDRR mitigated various myofibroblasts hallmarks, and vice versa. In the course of investigating the mechanism underlying the implication of FENDRR in myofibroblast transdifferentiation, we found that FENDRR can directly bind to miR-214 and exhibit its suppressive effect on myofibroblast activation via titrating miR-214. Moreover, we showed that mitofusin 2 (MFN2), a protein that is crucial to the fusion of mitochondria, was a direct target of miR-214. Our data suggested that FENDRR was positively correlated with MFN2 and MFN2 was required for the inhibitory property of FENDRR pertaining to myofibroblast phenotypes. Additionally, our results showed that the FENDRR/miR-214 axis participated in the arecoline-induced reactive oxygen species (ROS) accumulation and myofibroblast transdifferentiation. Building on these results, we concluded that the aberrant downregulation of FENDRR in OSF may be associated with chronic exposure to arecoline, leading to upregulation of ROS and myofibroblast activation via the miR-214-mediated suppression of MFN2.

Defining Transcriptomic Heterogeneity between Left and Right Ventricle-Derived Cardiac Fibroblasts.

Cardiac fibrosis is a key aspect of heart failure, leading to reduced ventricular compliance and impaired electrical conduction in the myocardium. Various pathophysiologic conditions can lead to fibrosis in the left ventricle (LV) and/or right ventricle (RV). Despite growing evidence to support the transcriptomic heterogeneity of cardiac fibroblasts (CFs) in healthy and diseased states, there have been no direct comparisons of CFs in the LV and RV. Given the distinct natures of the ventricles, we hypothesized that LV- and RV-derived CFs would display baseline transcriptomic differences that influence their proliferation and differentiation following injury. Bulk RNA sequencing of CFs isolated from healthy murine left and right ventricles indicated that LV-derived CFs may be further along the myofibroblast transdifferentiation trajectory than cells isolated from the RV. Single-cell RNA-sequencing analysis of the two populations confirmed that Postn+ CFs were more enriched in the LV, whereas Igfbp3+ CFs were enriched in the RV at baseline. Notably, following pressure overload injury, the LV developed a larger subpopulation of pro-fibrotic Thbs4+/Cthrc1+ injury-induced CFs, while the RV showed a unique expansion of two less-well-characterized CF subpopulations (Igfbp3+ and Inmt+). These findings demonstrate that LV- and RV-derived CFs display baseline subpopulation differences that may dictate their diverging responses to pressure overload injury. Further study of these subpopulations will elucidate their role in the development of fibrosis and inform on whether LV and RV fibrosis require distinct treatments.

Modulation of canonical Wnt signaling regulates peribiliary mesenchymal identity during homeostasis and injury.

The matrix and associated mesenchyme of the extrahepatic bile ducts are distinct, which could drive diseases with a predilection for these ducts, such as primary sclerosing cholangitis. We aimed to understand the molecular drivers of peribiliary mesenchymal cell (PMC) identity in the extrahepatic bile ducts and dissect how this changed in the context of injury using an entirely in vivo approach with transcriptomic analysis. Single-cell sequencing with a receptor-ligand analysis showed that PMCs had the most interactions with surrounding cells. Wnt4, Wnt5a, and Wnt7b were identified as the major ligands secreted from PMCs and cholangiocytes that interacted in both paracrine and autocrine fashion. Bile duct ligation caused an increase in all 3 Wingless/Integrated ligands and Axin2 with an associated increase in the transcription factors T-box transcription factor (Tbx)2 and Tbx3. Conversely, Indian hedgehog secretion decreased without an associated decrease in hedgehog signaling effectors. Loss of smoothened within PMCs did not impact hedgehog signaling effectors or cellular identity, whereas smoothened gain of function led to myofibroblast transdifferentiation with upregulation of Tbx2 and Tbx3 without injury. Loss of β-catenin caused a decrease in expression of all 3 Gli transcription factors and associated mesenchymal gene expression, which was phenocopied with compound Gli2 and Gli3 loss in uninjured PMCs. With injury, loss of β-catenin resulted in decreased myofibroblast transdifferentiation with reduced Tbx2 and Tbx3 expression. Our results show how modulation of canonical Wingless/Integrated signaling in PMCs is important for regulating basal mesenchymal gene expression and initiating a myogenic gene transcriptional program during injury. They also highlight reciprocating interactions between the hedgehog and Wingless/Integrated signaling pathways within PMCs.

Ddx5 Targeted Epigenetic Modification of Pericytes in Pulmonary Hypertension After Intrauterine Growth Restriction.

Newborns with intrauterine growth restriction (IUGR) have a higher likelihood of developing pulmonary arterial hypertension (PAH) in adulthood. Although there is increasing evidence suggesting that pericytes play a role in regulating myofibroblast transdifferentiation and angiogenesis in malignant and cardiovascular diseases, their involvement in the pathogenesis of IUGR-related pulmonary hypertension and the underlying mechanisms remain incompletely understood. To address this issue, a study was conducted using a Sprague-Dawley rat model of IUGR-related pulmonary hypertension. Our investigation revealed increased proliferation and migration of pulmonary microvascular pericytes in IUGR-related pulmonary hypertension, accompanied by weakened endothelial-pericyte interactions. Through whole-transcriptome sequencing, Ddx5 (DEAD-box protein 5) was identified as one of the hub genes in pericytes. DDX5, a member of the RNA helicase family, plays a role in the regulation of ATP-dependent RNA helicase activities and cellular function. MicroRNAs have been implicated in the pathogenesis of PAH, and microRNA-205 (miR-205) regulates cell proliferation, migration, and angiogenesis. The results of dual-luciferase reporter assays confirmed the specific binding of miR-205 to Ddx5. Mechanistically, miR-205 negatively regulates Ddx5, leading to the degradation of β-catenin by inhibiting the phosphorylation of Gsk3β at serine 9. In vitro experiments showed the addition of miR-205 effectively ameliorated pericyte dysfunction. Furthermore, invivo experiments demonstrated that miR-205 agomir could ameliorate pulmonary hypertension. Our findings indicated that the downregulation of miR-205 expression mediates pericyte dysfunction through the activation of Ddx5. Therefore, targeting the miR-205/Ddx5/p-Gsk3β/β-catenin axis could be a promising therapeutic approach for IUGR-related pulmonary hypertension.

MiR-125b-5p delivered by adipose-derived stem cell exosomes alleviates hypertrophic scarring by suppressing Smad2.

Hypertrophic scarring is the most serious and unmet challenge following burn and trauma injury and often leads to pain, itching and even loss of function. However, the demand for ideal scar prevention and treatment is difficult to satisfy. We aimed to discover the effects and mechanisms of adipose-derived stem cell (ADSC) exosomes in hypertrophic scarring. ADSC exosomes were isolated from the culture supernatant of ADSCs and identified by nanoparticle tracking analysis, transmission electron microscopy and western blotting. The effect of ADSC exosomes on wound healing and scar formation was detected by the wound model of BALB/c mice. We isolated myofibroblasts from hypertrophic scar tissue and detected the cell viability, proliferation and migration of myofibroblasts. In addition, collagen formation and fibrosis-related molecules were also detected. To further disclose the mechanism of ADSC exosomes on fibrosis in myofibroblasts, we detected the expression of Smad2 in hypertrophic scar tissue and normal skin and the regulatory mechanism of ADSC exosomes on Smad2. Injection of bleomycin was performed in male BALB/c mice to establish an in vivo fibrosis model while ADSC exosomes were administered to observe their protective effect. The tissue injury of mice was observed via hematoxylin and eosin and Masson staining and related testing. In this study, we found that ADSC exosomes could not only speed up wound healing and improve healing quality but also prevent scar formation. ADSC exosomes inhibited expression of fibrosis-related molecules such as α-smooth muscle actin, collagen I (COL1) and COL3 and inhibited the transdifferentiation of myofibroblasts. In addition, we verified that Smad2 is highly expressed in both hypertrophic scar tissue and hypertrophic fibroblasts, while ADSC exosomes downregulated the expression of Smad2 in hypertrophic fibroblasts. Further regulatory mechanism analysis revealed that microRNA-125b-5p (miR-125b-5p) is highly expressed in ADSC exosomes and binds to the 3' untranslated region of Smad2, thus inhibiting its expression. In vivo experiments also revealed that ADSC exosomes could alleviate bleomycin-induced skin fibrosis and downregulate the expression of Smad2. We found that ADSC exosomes could alleviate hypertrophic scars via the suppression of Smad2 by the specific delivery of miR-125b-5p.

ECM-based bioadhesive hydrogel for sutureless repair of deep anterior corneal defects.

Corneal transplantation is the gold standard treatment for corneal-related blindness; however, this strategy faces challenges such as limited donor cornea, graft rejection, suture-related complications, and the need for specialized equipment and advanced surgical skills. Development of tissue adhesives for corneal regeneration is of great clinical value. However, currently available corneal tissue sealants pose challenges, such as lack of safety, biocompatibility, and desired mechanical properties. To meet these requirements simultaneously, a bovine stromal corneal extracellular matrix (dCor) was used to design a bioadhesive photocurable hydrogel based on gelatin methacrylate (GelMA) and polyethylene glycol diacrylate (PEGDA) hydrogels (dCor/Gel-PEG). Integration of dCor into the dual networks of GelMA and PEGDA (Gel-PEG) led to a bioadhesive hydrogel for curing corneal defects, which could be crosslinked by Irgacure 2959 within 5 min ultraviolet irradiation. The viability of corneal stromal stem cells (CSSCs) was improved on the dCor/Gel-PEG hydrogel in comparison to the Gel-PEG hydrogel. The gene expression profile supported the keratocyte differentiation of CSSCs seeded on dCor/Gel-PEG via increased KERA and ALDH, with inhibited myofibroblast transdifferentiation via decreased α-SMA due to the presence of dCor. Interestingly, the dCor/Gel-PEG hydrogel exhibited favorable mechanical performance in terms of elasticity and bioadherence to the host corneal stroma. Ex vivo and in vivo examinations proved the feasibility of this hydrogel for the sutureless reconstruction of deep anterior corneal defects with promising histopathological results.

Genome-wide RNA sequencing of ocular fibroblasts from glaucomatous and normal eyes: Implications for glaucoma management.

Primary open angle glaucoma is a leading cause of visual impairment and blindness which is commonly treated with drugs or laser but may require surgery. Tenon's ocular fibroblasts are involved in wound-healing after glaucoma filtration surgery and may compromise a favourable outcome of glaucoma surgery by contributing to fibrosis. To investigate changes in gene expression and key pathways contributing to the glaucomatous state we performed genome-wide RNA sequencing. Human Tenon's ocular fibroblasts were cultured from normal and glaucomatous human donors undergoing eye surgery (n = 12). mRNA was extracted and RNA-Seq performed on the Illumina platform. Differentially expressed genes were identified using a bioinformatics pipeline consisting of FastQC, STAR, FeatureCounts and edgeR. Changes in biological functions and pathways were determined using Enrichr and clustered using Cytoscape. A total of 5817 genes were differentially expressed between Tenon's ocular fibroblasts from normal versus glaucomatous eyes. Enrichment analysis showed 787 significantly different biological functions and pathways which were clustered into 176 clusters. Tenon's ocular fibroblasts from glaucomatous eyes showed signs of fibrosis with fibroblast to myofibroblast transdifferentiation and associated changes in mitochondrial fission, remodeling of the extracellular matrix, proliferation, unfolded protein response, inflammation and apoptosis which may relate to the pathogenesis of glaucoma or the detrimental effects of topical glaucoma therapies. Altered gene expression in glaucomatous Tenon's ocular fibroblasts may contribute to an unfavourable outcome of glaucoma filtration surgery. This work presents a genome-wide transcriptome of glaucomatous versus normal Tenon's ocular fibroblasts which may identify genes or pathways of therapeutic value to improve surgical outcomes.

A novel mechanoeffector role of fibroblast S100A4 in myofibroblast transdifferentiation and fibrosis

Fibroblast to myofibroblast transdifferentiation mediates numerous fibrotic disorders, such as idiopathic pulmonary fibrosis (IPF). We have previously demonstrated that non-muscle myosin II (NMII) is activated in response to fibrotic lung extracellular matrix, thereby mediating myofibroblast transdifferentiation. NMII-A is known to interact with the calcium-binding protein S100A4, but the mechanism by which S100A4 regulates fibrotic disorders is unclear. In this study, we show that fibroblast S100A4 is a calcium-dependent, mechanoeffector protein that is uniquely sensitive to pathophysiologic-range lung stiffness (8-25kPa) and thereby mediates myofibroblast transdifferentiation. Re-expression of endogenous fibroblast S100A4 rescues the myofibroblastic phenotype in S100A4 KO fibroblasts. Analysis of NMII-A/actin dynamics reveals that S100A4 mediates the unraveling and redistribution of peripheral actomyosin to a central location, resulting in a contractile myofibroblast. Furthermore, S100A4 loss protects against murine invivo pulmonary fibrosis, and S100A4 expression is dysregulated in IPF. Our data reveal a novel mechanosensor/effector role for endogenous fibroblast S100A4 in inducing cytoskeletal redistribution in fibrotic disorders such as IPF.

Hydrogen sulfide inhibits alveolar type II cell senescence and limits pulmonary fibrosis via promoting MDM2-mediated p53 degradation.

Senescence of alveolar type II (AT2) cells is an important driver of pulmonary fibrosis. This study aimed to investigate whether and how dysregulation of hydrogen sulfide (H2 S) production affected AT2 cell senescence, and then explored the effect of H2 S on the communication between AT2 and fibroblasts. ICR mice were intratracheally administered with bleomycin (3 mg/kg). Sodium hydrosulfide (NaHS, 28 μmol/kg/d) was intraperitoneally injected for 2 weeks. The H2 S-generating enzyme cystathionine-β-synthase (CBS) knockout heterozygous (CBS+/- ) mice were used as a low H2 S production model. Analysis of microarray datasets revealed downregulation of H2 S-generating enzymes in lung tissues of patients with pulmonary fibrosis. Decreased H2 S production was correlated with higher levels of cell senescence markers p53 and p21 in bleomycin-induced lung fibrosis. CBS+/- mice exhibited increased levels of p53 and p21. The numbers of AT2 cells positive for p53 and p21 were increased in CBS+/- mice as compared to control mice. H2 S donor NaHS attenuated bleomycin-induced AT2 cell senescence both invivo and invitro. H2 S donor suppressed bleomycin-induced senescence-associated secretory phenotype (SASP) of AT2 cells via inhibiting p53/p21 pathway, consequently suppressing proliferation and myofibroblast transdifferentiation of fibroblasts. Mechanically, H2 S suppressed p53 expression by enhancing the mouse double-minute 2 homologue (MDM2)-mediated ubiquitination and degradation of p53. H2 S inactivated p53-p21 pathway, consequently suppressing AT2 cell senescence as well as cell communication between senescent AT2 cells and fibroblasts. Aberrant H2 S synthesis may contribute to the development of pulmonary fibrosis through promoting the activation loop involving senescent AT2 cells and activated fibroblasts.

Foxo3a and cardiac myofibroblast transdifferentiation after myocardial infarction

Abstract Background Fibroblast/ myofibroblast differentiation following acute myocardial infarction (MI) is important for myocardial scar formation but also causes adverse remodeling and stiffening of the myocardium leading to cardiac dysfunction and heart failure. Transdifferentiation of myofibroblasts are characterized by periostin, and extracellular matrix protein expression. The transcription factor forkhead box O 3 (FOXO3a) has been recently shown to inhibit hypertrophic cardiac remodeling. Purpose We hypothesized that FOXO3a, a key regulator of cell size, immunity, and cell differentiation, might inhibit transdifferentiation of fibroblasts into myofibroblasts and therefore cardiac fibrosis after MI. Methods Acute MI was induced in Foxo3a-/- mice and wild-type littermates by permanent LAD ligation. Cardiac function was determined by transthoracic echocardiography (TTE) at baseline as well as on days 4 and 14 post-MI. Immune histochemistry was performed with collagen and periostin. Finally, fibroblast differentiation was investigated by single nucleus RNA-sequencing (snRNA-seq). Results Foxo3a-/- mice showed significantly improved survival despite similar infarct size post-MI (p<0.01). The survival advantage was in part due to reduced cardiac ruptures on autopsy. TTE showed reduced LV function, cardiac output, and global strain 4 days post-MI that did not differ between both groups of animals. However, systolic LV function was significantly attenuated in Foxo3a-/- mice 14 days post-MI indicating enhanced wall stiffness. In line with these findings, sn-RNAseq on day 4 post-MI revealed significantly increased numbers of periostin positive myofibroblasts (Myo) (p<0.05) important for stabilizing matrix interactions that transdifferentiated mainly from progenitor-like state fibroblasts (PLS) and homeostatic epicardial derived fibroblasts (HEpiD) in Foxo3a-/- mice. Myo differentially expressed, i.e. upregulated genes important for extracellular matrix structure and organization, cell migration and collagen organization. Moreover, an upregulation of pro-fibrotic genes was observed in other fibroblast clusters such as in HEpiD (i.e., Col1a1, Col1a2, Col3a1), PLS (i.e., fgf10, il1r1), late response fibroblasts (LR-F) (i.e., Col14a1, Rin2) and in activated fibroblasts (F-ACT) (i.e., Gpc6, Slt2) in Foxo3a-/- mice. These changes were corroborated by significantly elevated collagen type 1 and 3 as well as periostin protein expression in Foxo3a-/- mice. Cell chat analysis indicated enhanced number of interactions and interaction strength of myeloid cells with fibroblasts in Foxo3a-/- mice. Conclusion Our data identify Foxo3a as a master regulator of cardiac remodeling attenuating myofibroblast transdifferentiation after acute MI. These findings suggest that deficiency of Foxo3a promotes early scar formation preventing rupture but also contributes to adverse remodeling in later stages resulting in impaired cardiac function and enhanced fibrosis after MI.

Polyenylphosphatidylcholine as bioactive excipient in tablets for the treatment of liver fibrosis

Liver fibrosis is a condition characterized by the accumulation of extracellular matrix (ECM) arising from the myofibroblastic transdifferentiation of hepatic stellate cells (HSCs) occurring as the natural response to liver damage. To date, no pharmacological treatments have been specifically approved for liver fibrosis. We recently reported a beneficial effect of polyenylphosphatidylcholines (PPCs)-rich formulations in reverting fibrogenic features of HSCs. However, unsaturated phospholipids’ properties pose a constant challenge to the development of tablets as preferred patient-centric dosage form. Profiting from the advantageous physical properties of the PPCs-rich Soluthin® S 80 M, we developed a tablet formulation incorporating 70% w/w of this bioactive lipid. Tablets were characterized via X-ray powder diffraction, thermogravimetry, and Raman confocal imaging, and passed the major compendial requirements. To mimic physiological absorption after oral intake, phospholipids extracted from tablets were reconstituted as protein-free chylomicron (PFC)-like emulsions and tested on the fibrogenic human HSC line LX-2 and on primary cirrhotic rat hepatic stellate cells (PRHSC). Lipids extracted from tablets and reconstituted in buffer or as PFC-like emulsions exerted the same antifibrotic effect on both activated LX-2 and PRHSCs as observed with plain S 80 M liposomes, showing that the manufacturing process did not interfere with the bioactivity of PPCs.