Transformation Of Fibroblasts Research Articles

Arecoline induces activation of human oral fibroblasts by promoting macrophage secretion of exosomes containing miR-155-5p

To investigate the mechanism by which arecoline regulates the level of miR-155-5p in macrophage-secreted exosomes to induce the transformation of human oral mucosal fibroblasts (HOMFs) into fibroblast phenotype. Exosomes were harvested from human monocytic cell line THP-1 with or without arecoline treatment. The effects of arecoline-treated THP-1 cell culture supernatant (CS), THP-1-derived exosomes (EXO), exosome-depleted THP-1 cell supernatant (NES), miR-155-5p overexpression, and miR-155-5p inhibitor on migration ability of arecoline-treated HOMF cells were examined using Transwell migration assay. The polarization of THP-1 cells was detected using flow cytometry. DCFH-DA was used to detect the level of oxidative stress in the cells with different treatments. The mRNA and protein expressions of α- SMA, type I collagen and SOCS1 in the cells were detected with qRT-PCR and Western blotting. Flow cytometry showed that arecoline-treated THP-1 cells exhibited obvious polarization from M0 to M1. Both the supernatant and exosomes from arecoline-treated THP-1 cells significantly enhanced the migration ability of HOMF cells, increased intracellular oxidative stress, up-regulated the expressions of miR-155- 5p and the mRNA and protein levels of α-SMA and type I collagen, and lowered the mRNA and protein expressions of SOCS1. In HOMF cells treated with exosomes from arecoline- treated THP-1 cells, overexpression of miR-155-5p significantly enhanced cell migration ability and increased cellular expressions of α-SMA and type I collagen, and miR-155-5p inhibitor caused the opposite changes. Arecoline can up-regulate miR-155-5p expression in THP-1 cells and inhibit the expression of SOCS1 protein in HOMF cells via the exosome pathway, thus promoting the fibrotic phenotype transformation of HOMF cells.

The Antibiotic Kitasamycin-A Potential Agent for Specific Fibrosis Preventing Therapy after Fistulating Glaucoma Surgery?

One major complication after fistulating glaucoma surgeries are fibroblast-mediated scarring processes and their specific prevention is key in the development of novel pharmaceutical concepts. Within this study a possible antifibrotic potential of kitasamycin (KM) in a transforming growth factor (TGF)-β1-mediated fibroblast model was evaluated in vitro. Primary ocular fibroblasts were isolated, cultivated and a dose-response test including determination of the half maximal effective concentration (EC50) for KM was conducted. Transformation of fibroblasts into myofibroblasts was induced by TGF-β1and immunofluorescence (IF), and Western blot (WB) analyses were performed with fibroblasts and myofibroblasts. IF analyses were carried out using antibodies against α-smooth muscle actin (α-SMA) and fibronectin, and protein detection of intracellular and extracellular proteins was performed by WB. Using the dose-response test, the viability, cytotoxicity and EC50 of KM after 24 and 48 h were determined. Fibroblasts exposed to various KM concentrations showed no increase in α-SMA and extracellular matrix expression. In TGF-ß1-stimulated myofibroblasts, KM inhibited the expression of α-SMA and fibronectin in a concentration-dependent manner. These findings demonstrate that KM could impair the transformation of fibroblasts into myofibroblasts and the expression of proteins involved in fibrotic processes, representing a potential agent for specific fibrosis prevention in future therapeutic concepts.

IGFBP-6 Alters Mesenchymal Stromal Cell Phenotype Driving Dasatinib Resistance in Chronic Myeloid Leukemia.

Chronic myeloid leukemia (CML), BCR-ABL1-positive, is classified as a myeloproliferative characterized by Philadelphia chromosome/translocation t(9;22) and proliferating granulocytes. Despite the clinical success of tyrosine kinase inhibitors (TKi) agents in the treatment of CML, most patients have minimal residual disease contained in the bone marrow microenvironment, within which stromal cells assume a pro-inflammatory phenotype that determines their transformation in cancer-associated fibroblasts (CAF) which, in turn can play a fundamental role in resistance to therapy. Insulin-like Growth Factor Binding Protein-6 (IGFBP-6) is expressed during tumor development, and is involved in immune-escape and inflammation as well, providing a potential additional target for CML therapy. Here, we aimed at investigating the role of IGFBP-6/SHH/TLR4 axis in TKi response. We used a CML cell line, LAMA84-s, and healthy bone marrow stromal cells, HS-5, in mono- or co-culture. The two cell lines were treated with Dasatinib and/or IGFBP-6, and the expression of inflammatory markers was tested by qRT-PCR; furthermore, expression of IGFBP-6, TLR4 and Gli1 were evaluated by Western blot analysis and immumocytochemistry. The results showed that both co-culture and Dasatinib exposure induce inflammation in stromal and cancer cells so that they modulate the expression of TLR4, and these effects were more marked following IGFBP-6 pre-treatment suggesting that this molecule may confer resistance through the inflammatory processes. This phenomenon was coupled with sonic hedgehog (SHH) signaling. Indeed, our data also demonstrate that HS-5 treatment with PMO (an inducer of SHH) induces significant modulation of TLR4 and overexpression of IGFPB-6 suggesting that the two pathways are interconnected with each other and with the TLR-4 pathway. Finally, we demonstrated that pretreatment with IGFBP-6 and/or PMO restored LAMA-84 cell viability after treatment with Dasatinib, suggesting that both IGFBP-6 and SHH are involved in the resistance mechanisms induced by the modulation of TLR-4, thus indicating that the two pathways may be considered as potential therapeutic targets.

Lactoferrin attenuates cardiac fibrosis and cardiac remodeling after myocardial infarction via inhibiting mTORC1/S6K signaling pathway.

Rationale: Myocardial infarction (MI) causes a severe injury response that eventually leads to adverse cardiac remodeling and heart failure. Lactoferrin (Ltf), as a secreted protein, bears multi-pharmacological properties. Present study aims to establish the cardioprotective function and corresponding mechanism of Ltf in MI process. Methods and results: We performed proteomic analysis in Tregs derived from MI heart, and identified Ltf as a remarkably upregulated secreted protein. However, Ltf was decreased in circulation and positively correlated with cardiac function both in mice and patients after MI. Ltf administration remarkably alleviated cardiac fibrosis and remodeling, improved cardiac function, and reduced incidence of heart failure in mice post-MI. In vitro, Ltf suppressed fibroblast to myofibroblast conversion induced by transforming growth factor-β (TGF-β). Mechanistically, phosphoproteomic landscape analysis revealed that Ltf repressed the activation of mTORC1/S6K/eIF-4B signaling pathway via interaction with CD74 receptor. Administration of mTORC1/S6K/eIF-4B axis agonist MHY1485 abolished the cardioprotective effects of Ltf. Besides, MHY1485 also markedly reversed the effects of Ltf on suppressing the transformation of fibroblast to myofibroblast mediated by TGF-β. Conclusion: Our study established the cardiac protective role of Ltf in attenuating cardiac remodeling and improving cardiac function by inhibiting the activation of myofibroblasts through suppressing mTORC1/S6K/eIF-4B signaling pathway post-MI. Treatment with Ltf may serve as a potential novel therapeutic intervention in patients with MI.

Hybrid computational modeling highlights reverse warburg effect in breast cancer-associated fibroblasts

Cancer-associated fibroblasts (CAFs) are amongst the key players of the tumor microenvironment (TME) and are involved in cancer initiation, progression, and resistance to therapy. They exhibit aggressive phenotypes affecting extracellular matrix remodeling, angiogenesis, immune system modulation, tumor growth, and proliferation. CAFs phenotypic changes appear to be associated with metabolic alterations, notably a reverse Warburg effect that may drive fibroblasts transformation. However, its precise molecular mechanisms and regulatory drivers are still under investigation. Deciphering the reverse Warburg effect in breast CAFs may contribute to a better understanding of the interplay between TME and tumor cells, leading to new treatment strategies. In this regard, dynamic modeling approaches able to span multiple biological layers are essential to capture the emergent properties of various biological entities when complex and intertwined pathways are involved. This work presents the first hybrid large-scale computational model for breast CAFs covering major cellular signaling, gene regulation, and metabolic processes. It was generated by combining a cell- and disease-specific asynchronous Boolean model with a generic core metabolic network leveraging both data-driven and manual curation approaches. This model reproduces the experimentally observed reverse Warburg effect in breast CAFs and further identifies Hypoxia-Inducible Factor 1 (HIF-1) as its key molecular driver. Targeting HIF-1 as part of a TME-centered therapeutic strategy may prove beneficial in the treatment of breast cancer by addressing the reverse Warburg effect. Such findings in CAFs, in light of our previously published results in rheumatoid arthritis synovial fibroblasts, point to a common HIF-1-driven metabolic reprogramming of fibroblasts in breast cancer and rheumatoid arthritis.

Inhibition of MLL1-menin interaction attenuates renal fibrosis in obstructive nephropathy.

Mixed lineage leukemia 1 (MLL1), a histone H3 lysine 4 (H3K4) methyltransferase, exerts its enzymatic activity by interacting with menin and other proteins. It is unclear whether inhibition of the MLL1-menin interaction influences epithelial-mesenchymal transition (EMT), renal fibroblast activation, and renal fibrosis. In this study, we investigated the effect of disrupting MLL1-menin interaction on those events and mechanisms involved in a murine model of renal fibrosis induced by unilateral ureteral obstruction (UUO), in cultured mouse proximal tubular cells and renal interstitial fibroblasts. Injury to the kidney increased the expression of MLL1 and menin and H3K4 monomethylation (H3K4me1); MLL1 and menin were expressed in renal epithelial cells and renal interstitial fibroblasts. Inhibition of the MLL1-menin interaction by MI-503 administration or siRNA-mediated silencing of MLL1 attenuated UUO-induced renal fibrosis, and reduced expression of α-smooth muscle actin (α-SMA) and fibronectin. These treatments also inhibited UUO-induced expression of transcription factors Snail and Twist and transforming growth factor β1 (TGF-β1) while expression of E-cadherin was preserved. Moreover, treatment with MI-503 and transfection with either MLL siRNA or menin siRNA inhibited TGF-β1-induced upregulation of α-SMA, fibronectin and Snail, phosphorylation of Smad3 and AKT, and downregulation of E-cadherin in cultured renal epithelial cells. Finally, MI-503 was effective in abrogating serum or TGFβ1-induced transformation of renal interstitial fibroblasts to myofibroblasts in vitro. Taken together, these results suggest that targeting disruption of the MLL1-menin interaction attenuates renal fibrosis through inhibition of partial EMT and renal fibroblast activation.

Loss of KDM5B ameliorates pathological cardiac fibrosis and dysfunction by epigenetically enhancing ATF3 expression

Excessive cardiac fibrosis is central to adverse cardiac remodeling and dysfunction leading to heart failure in many cardiac diseases. Histone methylation plays a crucial role in various pathophysiological events. However, the role of histone methylation modification enzymes in pathological cardiac fibrosis needs to be fully elucidated. Here, we identified lysine demethylase 5B (KDM5B), a histone H3K4me2/me3 demethylase, as a key epigenetic mediator of pathological cardiac fibrosis. KDM5B expression was upregulated in cardiac fibroblasts and myocardial tissues in response to pathological stress. KDM5B deficiency markedly ameliorated cardiac fibrosis, improved cardiac function, and prevented adverse cardiac remodeling following myocardial infarction (MI) or pressure overload. KDM5B knockout or inhibitor treatment constrained the transition of cardiac fibroblasts to profibrogenic myofibroblasts and suppressed fibrotic responses. KDM5B deficiency also facilitated the transformation of cardiac fibroblasts to endothelial-like cells and promoted angiogenesis in response to myocardial injury. Mechanistically, KDM5B bound to the promoter of activating transcription factor 3 (Atf3), an antifibrotic regulator of cardiac fibrosis, and inhibited ATF3 expression by demethylating the activated H3K4me2/3 modification, leading to the enhanced activation of TGF-β signaling and excessive expression of profibrotic genes. Our study indicates that KDM5B drives pathological cardiac fibrosis and represents a candidate target for intervention in cardiac dysfunction and heart failure.

616 Investigating if hydroxypyridone anti-fungals can target already established myofibroblasts in an in vitro model of hypertrophic scarring

Hypertrophic scars are a common complication of burn injury, yet there are no medications to prevent their formation. Myofibroblasts, a key cell involved in scar formation, are resistant to apoptosis. We have previously shown that hydroxypyridone anti-fungals can inhibit myofibroblast transformation in burn scar-derived fibroblasts. This study aimed to investigate if these drugs can target established myofibroblasts. Primary human dermal fibroblasts, derived from burn patient scars, were exposed to transforming growth factor beta-1 (TGF-β1) for 72 h to induce myofibroblast transformation.

S-allylmercapto-N-acetylcysteine ameliorates pulmonary fibrosis in mice via Nrf2 pathway activation and NF-κB, TGF-β1/Smad2/3 pathway suppression

Pulmonary fibrosis (PF) is a chronic lung disease characterised by alveolar inflammatory injury, alveolar septal thickening, and eventually fibrosis. Patients with severe Coronavirus Disease 2019 (COVID-19) may have left a certain degree of pulmonary fibrosis. PF is commonly caused by oxidative imbalance and inflammatory damage. S-allylmercapto-N-acetylcysteine (ASSNAC) exhibits anti-oxidative and anti-inflammatory effects in other diseases. However, the pharmacodynamics of ASSNAC remain unclear for PF. This investigation aimed to evaluate the efficacy and mechanism of ASSNAC against PF. The PF model was established by TGF-β1 stimulating HFL-1 cells in vitro. ASSNAC exhibited the potential to inhibit fibroblast transformation into myofibroblasts. Also, in the PF mice model with bleomycin (BLM), the sodium salt of ASSNAC (ASSNAC-Na) inhalation was treated. ASSNAC remarkably improved mice's lung tissue structure and collagen deposition. The important indicator proteins of PF, collagen Ⅰ, collagen Ⅲ, and α-SMA significantly decreased in the ASSNAC treated groups. Besides, ASSNAC attenuated oxidative stress by reversing glutathione (GSH), superoxide dismutase (SOD) levels and interfering with Nrf2/NOX4 signaling pathways. ASSNAC showed an anti-inflammatory effect by reducing the number of inflammatory cells and inflammatory cytokines, such as TNF-α and IL-6, and blocking the NF-κB signaling pathway. ASSNAC inhibited fibroblast differentiation by blocking the TGF-β1/Smad2/3 signaling pathway. This study implicates that ASSNAC alleviates pulmonary fibrosis through fighting against oxidative stress, reducing inflammation and inhibiting fibroblast differentiation.

Cardiac Overexpression of Chil1 Improves Wound Healing to Prevent Cardiac Rupture After Myocardial Infarction.

Timely formation of collagen-rich-scar is of importance to prevent ventricular rupture after myocardial infarction (MI). Chil1 (Chitinase 3-like 1) is a secreted protein associated with tissue remodeling response. However, its function in MI progression remains elusive. Chil1 was downregulated in the injured area overall post-MI. Overexpression of Chil1 markedly reduced cardiac rupture, increased wall thickness, and improved cardiac function post-MI due to collagen-rich-scar formation and extracellular matrix remodeling. In vitro, Chil1 induced the transformation of fibroblasts to myofibroblasts. Mechanistically, a phosphoproteomics study revealed that Chil1 binded to the EGFR enhancing RAF/MEK1/ERK signaling pathway to exert cardiac protection function. The effects of Chil1 on fibroblasts transformation and cardiac protections after MI were partially abolished by co-treated with RAF inhibitor. Together, our findings identify Chil1 as a protection factor in MI progression through binding to EGFR which further activates RAF/MEK1/ERK signaling pathway.

Phenotypic screening identifies hydroxypyridone anti-fungals as novel medicines for the prevention of hypertrophic scars

Although hypertrophic scarring affects ∼91% of burn patients annually, there is no drug to prevent this common complication. Hypertrophic scars are a result of dysregulated wound healing, characterised by persistent myofibroblast transformation and the excessive accumulation of extracellular matrix (ECM). Due to the multi-mechanistic nature of the scarring process, target-based approaches for identifying novel drugs have failed.Primary human dermal fibroblasts, derived from burn scar tissue, were exposed to transforming growth factor-beta 1 (TGF-β1) to induce myofibroblast transformation. A phenotypic screening assay, measuring alpha-smooth muscle actin (α-SMA) expression, was developed to screen 1,954 approved drugs. Drugs that elicited >80% inhibition of α-SMA expression, and >80% cell viability were progressed as candidate drugs. Anti-myofibroblast activity of the candidates was confirmed before investigating their effects on extracellular matrix (ECM) production and keratinocyte epithelial-mesenchymal transition (EMT).TGF-β1 induced myofibroblast transformation in primary human dermal fibroblasts (Emax = >3 ng/mL). The assay was optimised and validated (Z’ = 0.59), before screening 1,954 approved drugs. 90 drugs were identified as hits and hydroxypyridone anti-fungals selected for further testing. Concentration-response curves for these drugs confirmed their concentration-dependent anti-myofibroblast activity (IC50 = 1.4 – 16.7 μM). Hydroxypyridone anti-fungals were also found to successfully reduce ECM production and keratinocyte EMT.This is the first study to screen approved drugs in primary human dermal fibroblasts. Hydroxypyridone anti-fungals were found to prevent myofibroblast transformation, ECM production and keratinocyte EMT suggesting they could be repurposed to prevent hypertrophic scarring.

Inhibition of Wdr5 Attenuates Ang-II-Induced Fibroblast-to-Myofibroblast Transition in Cardiac Fibrosis by Regulating Mdm2/P53/P21 Pathway.

Cardiac fibrosis is an important pathological process in many diseases. Wdr5 catalyzes the trimethylation of lysine K4 on histone H3. The effects of Wdr5 on the cardiac fibrosis phenotype and the activation or transformation of cardiac fibroblasts were investigated by Ang-II-infused mice by osmotic mini-pump and isolated primary neonatal rat cardiac fibroblasts. We found that the Wdr5 expression and histone H3K4me3 modification were significantly increased in Ang-II-infused mice. By stimulating primary neonatal rat cardiac fibroblasts with Ang II, we detected that the expression of Wdr5 and H3K4me3 modification were also significantly increased. Two Wdr5-specific inhibitors, and the lentivirus that transfected Sh-Wdr5, were used to treat primary mouse cardiac fibroblasts, which not only inhibited the histone methylation by Wdr5 but also significantly reduced the activation and migration ability of Ang-II-treated fibroblasts. To explore its mechanism, we found that the inhibition of Wdr5 increased the expression of P53, P21. Cut&Tag-qPCR showed that the inhibition of Wdr5 significantly reduced the enrichment of H3K4me3 in the Mdm2 promoter region. For in vivo experiments, we finally proved that the Wdr5 inhibitor OICR9429 significantly reduced Ang-II-induced cardiac fibrosis and increased the expression of P21 in cardiac fibroblasts. Inhibition of Wdr5 may mediate cardiac fibroblast cycle arrest through the Mdm2/P53/P21 pathway and alleviate cardiac fibrosis.

Assessment of Mitochondrial Health in Cancer-Associated Fibroblasts Isolated from 3D Multicellular Lung Tumor Spheroids.

Cancer-associated fibroblasts (CAFs) are among the most abundant stromal cells present in the tumor microenvironment, facilitating tumor growth and progression. Complexity within the tumor microenvironment, including tumor secretome, low-grade inflammation, hypoxia, and redox imbalance, fosters heterotypic interaction and allows the transformation of inactive resident fibroblasts to become active CAFs. CAFs are metabolically distinguished from normal fibroblasts (NFs) as they are more glycolytically active, produce higher levels of reactive oxygen species (ROS), and overexpress lactate exporter MCT-4, leading to the opening of the mitochondrial permeability transition pore (MPTP). Here a method has been described to analyze the mitochondrial health of activated CAFs isolated from the multicellular 3D tumor spheroids comprising of human lung adenocarcinoma cells (A549), human monocytes (THP-1), and human lung fibroblast cells (MRC5). Tumor spheroids were disintegrated at different time intervals and through magnetic-activated cell sorting, CAFs were isolated. The mitochondrial membrane potential of CAFs was assessed using JC-1 dye, ROS production by 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) staining, and enzyme activity in the isolated CAFs. Analyzing the mitochondrial health of isolated CAFs provides a better understanding of the reverse Warburg effect and can also be applied to study the consequences of CAF mitochondrial changes, such as metabolic fluxes and the corresponding regulatory mechanisms on lung cancer heterogeneity. Thus, the present study advocates an understanding of tumor-stroma interactions on mitochondrial health. It would provide a platform to check mitochondrial-specific drug candidates for their efficacies against CAFs as potential therapeutics in the tumor microenvironment, thereby preventing CAF involvement in lung cancer progression.

Morphological features of regenerative processes after dacryocystorinostomy with the use of mitomycin C

ABSTRACT Purpose To study the morphological features of regenerative processes in dynamics after dacryocystorinostomy (DCR) with the use of Mitomycin C (MMC). Methods The study includes 31 cases. All patients underwent endonasal endoscopic DCR. Group 1 included 16 cases who received MMC injections. Group 2 included 15 cases who were treated with MMC on collagen sponge into the area of formed anastomosis. Biopsies for histological examination were taken on the 2nd, 5th, 7th, 14th, 21st, 28th and 60th days after the surgery. The samples were subjected to histological examination. Results Activated fibroblasts were detected in patients of group 2 on the 5th day after the surgery which became the main cellular elements by the 14th day, and incomplete fibroblast mitoses were observed in group 1 by the 14th day. On the 28th day after the surgery, the transformation of the extracellular matrix into loose connective tissue was determined in patients of group 2, while single cellular elements represented by fibroblasts were preserved in biopsies obtained from patients of group 1. By the 60th day after the surgery, the restoration of a full-fledged epithelial lining was observed in all specimens. Conclusion With the injection of MMC, inhibition of collagenogenesis by depression of extracellular collagen matrix formation, production of abortive mitoses in cells and inhibition of maturation and transformation of fibroblasts occur. The regeneration process takes place after MMC injection under a relative decrease in fibroblast impact.

Inhibition of ferroptosis and iron accumulation alleviates pulmonary fibrosis in a bleomycin model

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive disease characterized by excessive proliferation of fibroblasts and excessive accumulation of extracellular matrix (ECM). Ferroptosis is a novel form of cell death characterized by the lethal accumulation of iron and lipid peroxidation, which is associated with many diseases. Our study addressed the potential role played by ferroptosis and iron accumulation in the progression of pulmonary fibrosis. We found that the inducers of pulmonary fibrosis and injury, namely, bleomycin (BLM) and lipopolysaccharide (LPS), induced ferroptosis of lung epithelial cells. Both the ferroptosis inhibitor liproxstatin-1 (Lip-1) and the iron chelator deferoxamine (DFO) alleviated the symptoms of pulmonary fibrosis induced by bleomycin or LPS. TGF-β stimulation upregulated the expression of transferrin receptor protein 1 (TFRC) in the human lung fibroblast cell line (MRC-5) and mouse primary lung fibroblasts, resulting in increased intracellular Fe2+, which promoted the transformation of fibroblasts into myofibroblasts. Mechanistically, TGF-β enhanced the expression and nuclear localization of the transcriptional coactivator tafazzin (TAZ), which combined with the transcription factor TEA domain protein (TEAD)-4 to promote the transcription of TFRC. In addition, elevated Fe2+ failed to induce the ferroptosis of fibroblasts, which might be related to the regulation of iron export and lipid metabolism. Finally, we specifically knocked out TFRC expression in fibroblasts in mice, and compared with those in the control mice, the symptoms of pulmonary fibrosis were reduced in the knockout mice after bleomycin induction. Collectively, these findings suggest the therapeutic potential of ferroptosis inhibitors and iron chelators in treating pulmonary fibrosis.

Parabolic relationship between SMAD3 expression level and the reprogramming efficiency of goat induced mammary epithelial cells.

Mammary epithelial cells are the only cells of mammary glands with lactation capacity. They are closely related to mammary development and milk yield. Our earlier studies showed that the transformation of goat fibroblasts into induced mammary epithelial cells (iMECs) was closely correlated with SMAD3 overexpression. Therefore, we further explored the role of SMAD3 on iMECs reprogramming in this study. The SMAD3 gene was overexpressed in goat ear fibroblasts using the tetracycline-induced expression method. The outcomes demonstrated that goat ear fibroblasts can be converted into iMECs by overexpressing the SMAD3 gene. In contrast, it was discovered that SMAD3 downregulation by RNA interference significantly decrease the reprogramming efficiency of iMECs. These results show that SMAD3 plays a key regulatory role in the reprogramming of iMECs. Surprisingly, we also found a parabolic relationship between SMAD3 expression level and iMECs reprogramming efficiency, and that the reprogramming efficiency was maximum when the addition of doxycycline concentration was 5 μg/ml. In light of this, our findings may offer new perspectives on the regulatory mechanism governing mammary epithelial cell fate in goats as well as a fresh approach to studying mammary development and differentiation in vitro.

Copper promotes cardiac functional recovery via suppressing the transformation of fibroblasts to myofibroblasts in ischemia-infarcted monkey hearts

Myocardial ischemia leads to cardiac fibrosis along with copper (Cu) loss. Cu repletion diminishes myocardial fibrosis and improves cardiac function. The transformation of fibroblasts to myofibroblasts is highly responsible for the pathogenesis of cardiac fibrosis. This study was undertaken to test the hypothesis that Cu inhibition of cardiac fibrosis results from suppression of myofibroblasts. Rhesus monkeys 4–5 years old were subjected to coronary artery ligation to induce myocardial infarction (MI). At the end of the fourth week after the surgery, an ultrasound-directed Cu-albumin microbubble organ-specific Cu delivery technique was used to treat the ischemia-infarcted monkey hearts twice a week for 4 weeks. This treatment increased Cu concentrations in the infarct area, loosened the collagen cross-linking network, restored blood vessel density, and improved cardiac contractility. Total fibroblasts labeled with vimentin were increased in the infarct area, and Cu repletion did not alter this increase. Myofibroblasts, dually labeled with vimentin and α-smooth muscle actin (α-SMA), were also significantly increased in the infarct area but were significantly reduced by Cu repletion. Correspondingly, the products of myofibroblasts, type I and III collagens and inhibitors of collagenases were significantly reduced. In contrast, metalloproteinase-1 (MMP-1) and MMP-1 producing fibroblasts (vimentin+ and MMP-1+ cells) were significantly increased. These results suggest that Cu inhibits the transformation of fibroblasts to myofibroblasts, leading to a pro-fibrinolytic switch and an improvement in cardiac function.

Extracellular Vesicles Derived from Lung Cancer Cells Induce Transformation of Normal Fibroblasts into Lung Cancer-Associated Fibroblasts and Promote Metastasis of Lung Cancer by Delivering lncRNA HOTAIR.

Human lung cancer (LC) cells A549/H358, normal lung epithelial cells BEAS-2B, and lung normal fibroblasts (NFs) were cultured, followed by transfection of H358 cells with HOTAIR shRNA. Extracellular vesicles (EVs) extracted from H358 cells were identified. The internalization of Dil-labeled-EVs by NFs was tested, and protein levels of cancer-associated fibroblast (CAF) surface markers, inflammatory cytokines, cell proliferation, invasion, and migration, and lncRNA HOTAIR levels were determined. A549 cells were cultured in an H358-EVs-treated conditioned medium of NFs (NFCM), followed by intravenous injection of A549 cells into nude mice. The lesions and Ki-67-positive cells in lung tissues were measured. The results showed that tumor cell-derived EVs (T-EVs) motivated the transformation of NFs into CAFs. Specifically, EVs can be internalized by NFs, and the protein levels of CAF surface markers and inflammation levels were elevated in H358-EVs-treated NFs. The proliferation, invasion, and migration of A549 cells cultured in T-EVs-treated NFCM were increased. H358-EVs carried HOTAIR into NFs and promoted the transformation of NFs into CAFs. Inhibition of HOTAIR partially reversed the promoting effect of H358-EVs on the transformation of NFs into CAFs and invasion and migration of LC cells. T-EVs promoted metastasis of LC in vivo by transforming NFs into CAFs.

Immuno-inflammatory response in patients after myocardial infarction and involvement in fibrosis in vitro

Abstract Background Pathophysiology of acute myocardial infarction (AMI) has been described in mice and is based on a three-stage model involving temporal modifications of immune cells and fibroblasts. The acute inflammatory phase is followed by a reparative phase and fibrous scar maturation phase. Purinergic signaling, particularly P2Y11 receptor, has been reported to be involved in the regulation of inflammation after ischemia and could be an actor of resolution of inflammation after AMI. Purpose The aims of our study were: (1) to characterize the immune and P2Y11R profiles of AMI patients and (2) to analyze interactions between patients' immune cells and cardiac fibroblasts in vitro. Methods We collected peripheral blood mononuclear cells (PBMC) of 182 patients at various times (H0, H4, H24, H48, D3, M1, M6, M12) after reperfused ST-segment elevation AMI, and of 30 healthy donors. Expression level of genes involved in tolerogenicity profile of dendritic cells (HMOX1, STAT3, IDO1), in T cell polarization (CD4, FOXP3, TBX21/GATA3) as well as P2RY11 were evaluated by RT-PCR. P2Y11R expression was analyzed using flow cytometry. PBMC and human cardiac fibroblasts (HCF) were co-cultured during one day (5PBMC/1 HCF) and gene level expression (ACTA2/VIM, COL1A1), phenotype (α-SMA/vimentin) and secretory (soluble collagen) profiles were analyzed by RT-PCR, flow cytometry and Sircol assay. Results In the first 48 hours after AMI, the expression level of HMOX1 (fold-change = 8.478 at H48, p<0.0001), STAT3 (2.856 at H0, p=0.0027) and CD4 increased (2.451 at H48, p=0.0052); IDO1 (0.2055 at H24, p<0.0001) and TBX21/GATA3 ratio decreased (0.4498 at H48, p=0.0026); FOXP3 did not vary significantly. In the same time, the expression level of P2RY11 increased (2.124 at H0, p=0.0015) as well as protein expression in T cells (median MFI = 845.5 at H0 vs 229.5 for healthy donors, p=0.0375). In vitro, we observed a non-significant increase of ACTA2/VIM ratio in HCF co-cultured with H24, M1, M6 PBMC; of α-SMA/vimentin with H48, M12 PBMC; of COL1A1 with H24, H48, M12 PBMC; and of soluble collagen in coculture supernatant with M1 PBMC. Conclusion Our results suggest that P2Y11R could be involved in evolution of immune response after AMI, and that, in the first 2 days, circulating immune cells have a reparative profile and could participate in the transformation of fibroblast into myofibroblasts. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): Fondation pour la recherche médicale

Role of Nintedanib in COVID-19-Related Lung Fibrosis

In December 2020, Indonesia was introduced to the long Coronavirus disease 2019 (COVID-19) phenomenon. The Centers for Disease Control and Prevention (CDC) introduced the term "post-COVID condition" as a health problem that persists after four weeks from the first exposure to COVID-19. The National Institute for Healthcare and Care Excellence (NICE) classifies COVID-19 infections into three categories based on disease duration: (1) acute infection for up to 4 weeks; (2) ongoing infection within 4-12 weeks; and (3) post-COVID-19 syndrome for more than 12 weeks and not associated with an alternative diagnosis. One of these phenomena is lung fibrosis. About 80% of COVID-19 survivors had mild to severe chest X-rays in 6 months of follow-up with decreasing lung function. COVID-19-related lung fibrosis is still not widely researched. COVID-19 survivors who develop lung fibrosis usually recover independently, but some develop persistent lung fibrosis. The use of antifibrotic agents, such as nintedanib, has long been approved for idiopathic pulmonary fibrosis (IPF). However, its use in the cases of lung fibrosis due to COVID-19 has not been widely studied. Nintedanib is a tyrosine kinase inhibitor. It inhibits receptor activity of vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and fibroblast growth factor (FGF). Those actions will eventually inhibit the proliferation, migration, and transformation of fibroblasts into myofibroblasts in lung fibrogenesis. Therefore, an antifibrotic agent is potentially needed to inhibit COVID-19-related lung fibrosis to improve quality of life and prevent further lung damage.