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Myofibroblast buildup and prostatic fibrosis play a crucial role in the development of benign prostatic hyperplasia (BPH). Treatments specifically targeting myofibroblasts could be a promising approach for treating BPH. Tadalafil, a phosphodiesterase type 5 (PDE5) inhibitor, holds the potential to intervene in this biological process. This study employs prostatic stromal fibroblasts to induce myofibroblast differentiation through TGFß1 stimulation. As a result, tadalafil significantly inhibited prostatic stromal fibroblast proliferation and fibrosis process, compared to the control group. Furthermore, our transcriptome sequencing results revealed that tadalafil inhibited FGF9 secretion and simultaneously improved miR-3126-3p expression via TGFß1 suppression. Overall, TGFß1 can trigger pro-fibrotic signaling through miR-3126-3p in the prostatic stroma, and the use of tadalafil can inhibit this process.

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Pulmonary fibrosis is a fatal and chronic lung disease that is characterized by accumulation of thickened scar in the lungs and impairment of gas exchange. The cases with unknown etiology are referred as idiopathic pulmonary fibrosis (IPF). There are currently no effective therapeutics to cure the disease; thus, the investigation of the pathogenesis of IPF is of great importance. Recent studies on bone morphogenic proteins (BMPs) and their receptors have indicated that reduction of BMP signaling in lungs may play a significant role in the development of lung fibrosis. BMPs are members of TGF-ß superfamily, and they have been shown to play an anti-fibrotic role in combating TGF-ß-mediated pathways. The impact of BMP receptors, in particular BMPR2, on pulmonary fibrosis is growing attraction to researchers. Previous studies on BMPR2 have often focused on pulmonary arterial hypertension (PAH). Given the strong clinical association between PAH and lung fibrosis, understanding BMPs/BMPR2-mediated signaling pathway is important for development of therapeutic strategies to treat IPF. In this review, we comprehensively review recent studies regarding the biological functions of BMPs and their receptors in lungs, especially focusing on their roles in the pathogenesis of pulmonary fibrosis and fibrosis resolution.

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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-ß 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.

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BACKGROUND: Extended contact with silica particles can lead to Silicosis, a chronic lung condition lacking established treatment protocols or clear mechanisms of development. The urgency for innovative treatments arises from the unavailability of effective treatment methodologies. The origin of silica-induced pulmonary fibrosis includes essential processes such as macrophage activation and the conversion of fibroblasts into myofibroblasts, with oxidative stress playing a pivotal role. Shionone (SHI), a triterpenoid extracted from the Aster tataricus plant, is recognized for its extensive health benefits. This study explores the capability of SHI to alleviate the effects of silica-induced lung fibrosis in mice. METHODS: This investigation explored the impact of SHI on lung inflammation and fibrosis at different stages (early and late) triggered by silica in mice, focusing specifically on the initial and more developed phases. It comprised an analysis of isolated peritoneal macrophages and fibroblasts extracted from mice to elucidate SHI's therapeutic potential and its underlying mechanism. The methodology employed encompassed quantitative PCR, immunofluorescence, flow cytometry, and western blotting to examine macrophage activity and their transition into myofibroblasts. The activation of the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway by SHI was confirmed via immunofluorescence and western blot studies. SHI's antioxidative properties were evidenced by the measurement of reactive oxygen species (ROS) and mitochondrial ROS within both macrophages and fibroblasts, using 2', 7'-dichlorodihydrofluorescein diacetate and MitoSOX, respectively. The relevance of SHI was further underscored by applying ML385 and Nrf2 siRNA to gauge its effectiveness. RESULTS: Starting SHI treatment early countered the harmful effects of lung inflammation and fibrosis caused by silica, while initiating SHI at a later phase decelerated the advancement of fibrosis. SHI's action was linked to the activation of the Nrf2 signaling pathway, a boost in antioxidant enzyme levels, and a decrease in oxidative stress and inflammation in macrophages affected by silica. Furthermore, SHI prevented the conversion of fibroblasts into myofibroblasts prompted by TGF-ß, along with the resultant oxidative stress. The beneficial outcomes of SHI were negated when ML385 and Nrf2 siRNA were applied, highlighting the pivotal role of the Nrf2 pathway in SHI's efficacy. CONCLUSION: SHI plays a significant role in stimulating the Nrf2 pathway, thereby defending against silica-induced oxidative stress and inflammatory reactions in macrophages, and inhibiting the conversion of fibroblasts to myofibroblasts due to TGF-ß. This suggests that SHI is a viable option for treating lung inflammation and fibrosis in mice suffering from silicosis.

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BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a debilitating interstitial lung disorder characterized by its limited therapeutic interventions. Macrophages, particularly the alternatively activated macrophages (M2 subtype), have been acknowledged for their substantial involvement in the development of pulmonary fibrosis. Hence, targeting macrophages emerges as a plausible therapeutic avenue for IPF. Icariside II (ISE II) is a natural flavonoid glycoside molecule known for its excellent anti-tumor and anti-fibrotic activities. Nevertheless, the impact of ISE II on pulmonary fibrosis and the intricate mechanisms through which it operates have yet to be elucidated. OBJECTIVE: To scrutinize the impact of ISE II on the regulation of M2 macrophage polarization and its inhibitory effect on pulmonary fibrosis, as well as to delve deeper into the underlying mechanisms of its actions. METHODS: The effect of ISE II on proliferation and apoptosis in RAW264.7 cells was assessed through the use of EdU-488 labeling and the Annexin V/PI assay. Flow cytometry, western blot, and qPCR were employed to detect markers associated with the M2 polarization phenotype. The anti-fibrotic effects of ISE II in NIH-3T3 cells were investigated in a co-culture with M2 macrophages. Si-Ctnnb1 and pcDNA3.1(+)-Ctnnb1 plasmid were used to investigate the mechanism of targeted intervention. The murine model of pulmonary fibrosis was induced by intratracheal administration of bleomycin (BLM). Pulmonary function, histopathological manifestations, lung M2 macrophage infiltration, and markers associated with pulmonary fibrosis were evaluated. Furthermore, in vivo transcriptomics analysis was employed to elucidate differentially regulated genes in lung tissues. Immunofluorescence, western blot, and immunohistochemistry were conducted for corresponding validation. RESULTS: Our investigation demonstrated that ISE II effectively inhibited the proliferation of RAW264.7 cells and mitigated the pro-fibrotic characteristics of M2 macrophages, exemplified by the downregulation of CD206, Arg-1, and YM-1, Fizz1, through the inhibition of the PI3K/Akt/ß-catenin signaling pathway. This impact led to the amelioration of myofibroblast activation and the suppression of nuclear translocation of ß-catenin of NIH-3T3 cells in a co-culture. Consequently, it resulted in decreased collagen deposition, reduced infiltration of profibrotic macrophages, and a concurrent restoration of pulmonary function in mice IPF models. Furthermore, our RNA sequencing results showed that ISE II could suppress the expression of genes related to M2 polarization, primarily by inhibiting the PI3K/Akt and ß-catenin signaling pathway. In essence, our findings suggest that ISE II holds potential as an anti-fibrotic agent by orchestrating macrophage polarization. This may have significant implications in clinical practice. CONCLUSION: This study has provided evidence that ISE II exerts a significant anti-fibrotic effect by inhibiting macrophage M2 polarization through the suppression of the PI3K/Akt/ß-catenin signaling pathway. These findings underscore the potential of ISE II as a promising candidate for the development of anti-fibrotic pharmaceuticals in the future.

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Idiopathic pulmonary fibrosis treatments are limited, often with severe side effects, highlighting the need for novel options. Taraxerone has diverse biomedical properties, but its mechanism remains unclear. This study investigates taraxerone's impact and the mechanisms involved in bleomycin-induced pulmonary fibrosis in mice. After establishing a pulmonary fibrosis mouse model, taraxerone was intraperitoneally injected continuously for 14-28 days. The in vivo antifibrotic and antioxidative stress effects of taraxerone were assessed. In vitro, the influence of taraxerone on transforming growth factor-ß1-induced myofibroblast transformation and oxidative stress was investigated. Subsequently, quantitative polymerase chain reaction screened the histone deacetylase and Sirtuin family, and taraxerone's effects on SIRT1 were assessed. After SIRT1 siRNA treatment, changes in myofibroblast transformation and antioxidant capacity in response to taraxerone were observed. Acetylation and phosphorylation levels of Smad3 were evaluated. We also examined the binding levels of SIRT1 with Pho-Smad3 and Smad3, as well as the nuclear localization of Smad2/3. EX527 confirmed SIRT1's in vivo action in response to taraxerone. In vitro experiments suggested that taraxerone inhibited myofibroblast differentiation by activating SIRT1 and reducing oxidative stress. We also observed a new interaction between SIRT1 and the Smad complex. Taraxerone activates SIRT1, enabling it to bind directly to Smad3. This leads to reduced Smad complex phosphorylation and limited nuclear translocation. As a result, the transcription of fibrotic factors is reduced. In vivo validation confirms taraxerone's SIRT1-mediated antifibrotic effectiveness. This suggests that targeting SIRT1-mediated inhibition of myofibroblast differentiation could be a key strategy in taraxerone-based therapy for pulmonary fibrosis.

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Desbuquois dysplasia type 2 (DBQD2) and spondylo-ocular syndrome (SOS) are autosomal recessive disorders affecting the extracellular matrix (ECM) and categorized as glycosaminoglycan (GAG) linkeropathies. Linkeropathies result from mutations within glycosyltransferases involved in the synthesis of the tetrasaccharide linker, a linker between the core protein of proteoglycan (PG) and GAG. DBQD2 and SOS are caused by the isolated mutations of the xylosyltransferase (XT) isoforms. In this work, we successfully generated XYLT1- as well as XYLT2-deficient GAG linkeropathy model systems in human dermal fibroblasts using a ribonucleoprotein-based CRISPR/Cas9-system. Furthermore, it was possible to generate a complete XYLT-knockdown. Short- and long-term XT activity deficiency led to the mutual reduction in all linker transferase-encoding genes, suggesting a potential multienzyme complex with mutual regulation. Fibroblasts compensated for ECM misregulation initially by overexpressing ECM through the TGFß1 signaling pathway, akin to myofibroblast differentiation patterns. The long-term reduction in one XT isoform induced a stress response, reducing ECM components. The isolated XYLT1-knockout exhibited α-smooth muscle actin overexpression, possibly partially compensated by unaltered XT-II activity. XYLT2-knockout leads to the reduction in both XT isoforms and a strong stress response with indications of oxidative stress, induced senescence and apoptotic cells. In conclusion, introducing XYLT-deficiency revealed temporal and isoform-specific regulatory differences.

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Corneal stromal injury is a common surgical disease. With the development of tissue engineering materials, many artificial corneal scaffolds have been developed to replace allograft corneal transplantation and solve the problem of corneal donor shortage. However, few researchers have paid attention to corneal stromal wound healing. Herein, a nanocomposite of amino modified mesoporous bioactive glass (MBG-NH2) and microRNA-133b (miR-133b) was introduced into the patterned collagen films to achieve corneal stromal injury repair. MBG-NH2nanoparticles as a nano delivery carrier could efficiently load miR-133b and achieve the slow release of miR-133b. The physicochemical properties of collagen films were characterized and found the microgrooved collagen films loaded with miR-133b@MBG-NH2nanoparticles possessed similar swelling properties, optical clarity, and biodegradability to the natural cornea.In vitrocell experiments were also conducted and proved that the patterned collagen films with miR-133b@MBG-NH2possessed good biocompatibility, and miR-133b@MBG-NH2nanoparticles could be significantly uptake by rabbit corneal stromal cells (RCSCs) and have a significant impact on the orientation, proliferation, migration, and gene expression of RCSCs. More importantly, the patterned collagen films with miR-133b@MBG-NH2could effectively promote the migration of RCSCs and accelerate wound healing process, and down-regulate the expression levels ofα-SMA, COL-I, and CTGF genes associated with myofibroblast differentiation of corneal stromal cells, which has a potential application prospect in the repair of corneal stromal injury.

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BACKGROUND: At present, the treatment options available for idiopathic pulmonary fibrosis are both limited and often come with severe side effects, emphasizing the pressing requirement for innovative therapeutic alternatives. Myofibroblasts, which hold a central role in pulmonary fibrosis, have a close association with the Smad signaling pathway induced by transforming growth factor-ß1 (TGF-ß1) and the transformation of myofibroblasts driven by oxidative stress. Liquiritigenin, an active compound extracted from the traditional Chinese herb licorice, boasts a wide array of biomedical properties, such as anti-fibrosis and anti-oxidation. The primary objective of this study was to examine the impact of liquiritigenin on bleomycin-induced pulmonary fibrosis in mice and the underlying mechanisms. METHODS: The anti-pulmonary fibrosis and anti-oxidant effects of liquiritigenin in vivo were tested by HE staining, Masson staining, DHE staining and bio-chemical methods. In vitro, primary mouse lung fibroblasts were treated with TGF-ß1 with or without liquiritigenin, the effects of liquiritigenin in inhibiting differentiation of myofibroblasts and facilitating the translocation of Nrf2 were valued using Quantitative real-time polymerase chain reaction (Q-PCR), western blotting and immunofluorescence. Nrf2 siRNA and SIRT1 siRNA were used to investigate the mechanism underlies liquiritigenin's effect in inhibiting myofibroblast differentiation. RESULTS: Liquiritigenin displayed a dose-dependent reduction effect in bleomycin-induced fibrosis. In laboratory experiments, it was evident that liquiritigenin possessed the ability to enhance and activate sirtuin1 (SIRT1), thereby facilitating the nuclear translocation of Nrf2 and mitigating the oxidative stress-induced differentiation of primary mouse myofibroblasts. Moreover, our investigation unveiled that SIRT1 not only regulated myofibroblast differentiation via Nrf2-mediated antioxidant responses against oxidative stress but also revealed liquiritigenin's activation of SIRT1, enabling direct binding to Smad. This led to decreased phosphorylation of the Smad complex, constrained nuclear translocation, and suppressed acetylation of the Smad complex, ultimately curtailing the transcription of fibrotic factors. Validation in live subjects provided substantial evidence for the anti-fibrotic efficacy of liquiritigenin through the SIRT1/Nrf2 signaling pathway. CONCLUSIONS: Our findings imply that targeting myofibroblast differentiation via the SIRT1/Nrf2 signaling pathway may constitute a pivotal strategy for liquiritigenin-based therapy against pulmonary fibrosis.

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This study aims to explore the potential inhibition effects of staurosporine isolated from a Streptomyces sp. SNC087 strain obtained from seawater on nasal polyps. Staurosporine possesses antimicrobial and antihypertensive activities. This research focuses on investigating the effects of staurosporine on suppressing the growth and development of nasal polyps and elucidating the underlying mechanisms involved. The experimental design includes in vitro and ex vivo evaluations to assess the inhibition activity and therapeutic potential of staurosporine against nasal polyps. Nasal polyp-derived fibroblasts (NPDFs) were stimulated with TGF-ß1 in the presence of staurosporine. The levels of α-smooth muscle actin (α-SMA), collagen type-I (Col-1), fibronectin, and phosphorylated (p)-Smad 2 were investigated using Western blotting. VEGF expression levels were analyzed in nasal polyp organ cultures treated with staurosporine. TGF-ß1 stimulated the production of Col-1, fibronectin, and α-SMA and was attenuated by staurosporine pretreatment. Furthermore, these inhibitory effects were mediated by modulation of the signaling pathway of Smad 2 in TGF-ß1-induced NPDFs. Staurosporine also inhibits the production of VEGF in ex vivo NP tissues. The findings from this study will contribute to a better understanding of staurosporine's role in nasal polyp management and provide insights into its mechanisms of action.

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PURPOSE: To investigate whether or not intermittent hypoxia (IH), the main characteristic of obstructive sleep apnea (OSA) may affect the myofibroblast differentiation and extracellular matrix (ECM) production of lung fibroblast through the HIF-1α-TGF-ß/Smad pathway and assess the interventional role of a HIF-1α inhibitor, 2-methoxyestradiol (2-ME2). METHOD: The human lung fibroblast MRC5 cells were exposed to normoxia or IH conditions, and the expression of myofibroblast differentiation marker α-smooth muscle actin (α-SMA) and ECM protein collagen I were evaluated. To clarify the underlying mechanism, the expression level of HIF-1α, TGF-ß, and p-Smads/Smads were measured and the effects of inhibiting HIF-1α with 2-ME2 on the α-SMA expression level and ECM production through the TGF-ß/Smad pathway were assessed. Si HIF-1α was applied to genetically inhibit HIF-1α in MRC5 cells, and the related proteins were assessed. RESULTS: IH increased the protein and mRNA expression of Collagen I and α-SMA of MRC5 cells in a time-dependent manner. IH activated the protein and mRNA level of HIF-1α and TGF-ß and increased the phosphorylation of Smad2/Smad3 of MRC5 cells in a time-dependent manner. 2-ME2 inhibited the activation of HIF-1α induced by IH and decreased overexpression of TGF-ß, p-Smad2/Smad2, and p-Smad3/Smad3, which in turn partially reversed the upregulation of α-SMA and Collagen I induced by IH in MRC5 cells. When HIF-1α was successfully silenced by si-HIF-1α, upregulation of TGF-ß induced by intermittent hypoxia was partially decreased. CONCLUSIONS: This study showed that IH contributes to myofibroblast differentiation and excessive ECM production of MRC5 cells through activation of the HIF-1α-TGF-ß/Smad pathway. 2-ME2 partially attenuated myofibroblast differentiation induced by IH by inhibiting the HIF-1α-TGF-ß/Smad pathway.

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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 (8-25 kPa) 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 in vivo 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.

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Fibrosis and the associated decline in organ functionality lead to an almost 50% mortality rate in developed countries. Multipotent mesenchymal stromal cells (MSC) were shown to suppress the development and progression of fibrosis through secreted factors including specific non-coding RNAs transferred within extracellular vesicles (EV). However, age-associated chronic inflammation can provoke MSC senescence and change secretome composition, thereby affecting their antifibrotic properties. Alternatively activated macrophages (M2-type) are key players in chronic inflammation that may interact with MSC through paracrine mechanisms and decrease their antifibrotic functions. To confirm this hypothesis, we evaluated the M2-macrophage conditioned medium (CM-M2) effect on human adipose-tissue-derived MSC senescence in vitro. We found that CM-M2, as well as a pro-senescence agent, hydrogen peroxide (H2O2), increased p21+-MSC number and secretion of IL-6 and MCP-1, which are considered main senescence-associated secretory phenotype (SASP) components. Thus, both exposures led to the senescent phenotype acquisition of MSC. EV from both CM-M2 and H2O2-exposed MSC, which showed a decreased effect on the suppression of TGFß-induced fibroblast-to-myofibroblast differentiation compared to EV from control MSC according to αSMA level and the αSMA+-stress fiber reduction. After two weeks of subsequent cultivation under standard conditions, MSC demonstrated a decrease in senescence hallmarks and fibroblast differentiation suppression via EV. These results suggest that M2-macrophage-induced chronic inflammation can reversibly induce MSC senescence, which reduces the MSC's ability to inhibit fibroblast-to-myofibroblast differentiation.

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Mechanomimetic materials are particularly attractive for modeling in vitro fibroblast to myofibroblast (Myof) transition, a key process in the physiological repair of damaged tissue, and recognized as the core cellular mechanism of pathological fibrosis in different organs. In vivo, mechanical stimuli from the extracellular matrix (ECM) are crucial, together with cell-cell contacts and the pro-fibrotic transforming growth factor (TGF)-ß1, in promoting fibroblast differentiation. Here, we explore the impact of hydrogels made by polyacrylamide with different composition on fibroblast behavior. By appropriate modulation of the hydrogel composition (e.g. adjusting the crosslinker content), we produce and fully characterize three kinds of scaffolds with different Young modulus (E). We observe that soft hydrogels (E < 1 kPa) induced fibroblast differentiation better than stiffer ones, also in the absence of TGF-ß1. This study provides a readily accessible biomaterial platform to promote Myof generation. The easy approach used and the commercial availability of the monomers make these hydrogels suitable to a wide range of biomedical applications combined with high reproducibility and simple preparation protocols.

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Background: Urethral stricture is a common disorder of the lower urinary tract in men. A complex network of pathways and interactions are involved in the pathogenesis of urethral fibrosis. However, the mechanisms underlying urethral fibrosis remain poorly understood. Objectives: To investigate the critical role of the canonical Wnt pathway in development of urethral fibrosis and explore DKK1, the endogenous inhibitor of Wnt pathway, as a potential target to prevent urethral fibrosis in vitro and in vivo. Methods: Urethral fibrosis tissue derived from patients and rat models were harvested to assess the activation of the canonical Wnt pathway by using western blot, qRT-PCR and immunohistochemistryWe performed histological staining, western blot, qRT-PCR and immunohistochemistry to examine the effects of DKK1 treatment on in vivo rat urethral fibrosis models. In vitro, human urethral fibroblasts (HUFs) were cultured to examine the effects of DKK1 in TGFß1-induced HUFs by CCK-8 assay, hydroxyproline assay, flow cytometry, cell migration assay, western blot, qRT-PCR and immunofluorescence. Results: The key components of Wnt signaling were upregulated in urethral fibrosis tissue derived from patients and rat models while DKK 1 was downregulated. DKK1 ameliorated TGFß1-induced urethral fibrosis in rats. TGFß1 induced myofibroblast differentiation by upregulating collagen I, collagen III, α-SMA, ß-catenin and p-GSK-3ß, while DKK1 was decreased. DKK1 significantly inhibited cell proliferation, collagen content, cell migration and promoted cell apoptosis in TGFß1-induced HUFs. DKK1 significantly suppressed myofibroblast differentiation of TGFß1-induced HUFs by downregulating collagen I, collagen III, α-SMA, ß-catenin and p-GSK-3ß with a mechanism independent of Smad2/3. Conclusions: Our study demonstrated that canonical Wnt pathway may be an essential mechanism underlying the pathogenesis of urethral fibrosis and explored the potential role of DKK1 participation in the development of urethral fibrosis.

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SMAD3 downregulation is documented in transforming growth factor ß1 (TGF-ß1)-induced corneal fibroblasts differentiation to myofibroblasts ("fibroTOmyoDiff") or corneal wound healing. However, the exact regulatory mechanism of TGF-ß1/SMAD3 pathway in this context remains unclear. Here, we investigated the role and related mechanism of SMAD3 down-regulation in TGF-ß1-induced human corneal fibroTOmyoDiff. By detecting expression changes of SMAD family during this process, we demonstrated that SMAD3 protein expression was dramatically decreased in the process and the decrease occurred mainly in SMAD3 gene transcription. Furthermore, SMAD3 overexpression using lentivirus infection and knockdown using sgRNA lentivirus infection or siRNAs revealed that SMAD3 overexpression enhanced TGF-ß1-induced corneal fibroTOmyoDiff and vice versa. In addition, specific siRNAs and inhibitors targeting particular signaling pathway were used to figure out the intracellular signaling pathway regulating SMAD3, and the result showed that the decease of SMAD3 induced by TGF-ß1 stimulation in human corneal fibroblasts (HCFs) was strikingly prevented by SMAD4 knockdown or p38 signaling inhibitor SB203580 treatment. Collectively, these results demonstrate that, in TGF-ß1 induced corneal fibroTOmyoDiff, down-regulation of SMAD3 expression regulated by SMAD4 and p38 signaling pathways forms a negative feedback loop of TGFß signaling to avoid excessive activation of the signaling, which suggest that SMAD3 may be a key target for corneal fibrosis treatment.

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Angiotensin II (Ang II) upregulates transforming growth factor-beta1 (TGF-ß1) and endothelin-1 (ET-1) in various types of cells, and all of them act as profibrotic mediators. However, the signal transduction of angiotensin II receptor (ATR) for upregulation of TGF-ß1 and ET-1, and their effectors that play an essential role in myofibroblast differentiation, are not fully understood. Therefore, we investigated the ATR networking with TGF-ß1 and ET-1 and identified the signal transduction of these mediators by measuring the mRNA expression of alpha-smooth muscle actin (α-SMA) and collagen I using qRT-PCR. Myofibroblast phenotypes were monitored by α-SMA and stress fiber formation with fluorescence microscopy. Our findings suggested that Ang II induced collagen I and α-SMA synthesis and stress fiber formation through the AT1R/Gαq axis in adult human cardiac fibroblasts (HCFs). Following AT1R stimulation, Gαq protein, not Gßγ subunit, was required for upregulation of TGF-ß1 and ET-1. Moreover, dual inhibition of TGF-ß and ET-1 signaling completely inhibited Ang II-induced myofibroblast differentiation. The AT1R/Gαq cascade transduced signals to TGF-ß1, which in turn upregulated ET-1 via the Smad- and ERK1/2-dependent pathways. ET-1 consecutively bound to and activated endothelin receptor type A (ETAR), leading to increases in collagen I and α-SMA synthesis and stress fiber formation. Remarkably, dual blockade of TGF-ß receptor and ETR exhibited the restorative effects to reverse the myofibroblast phenotype induced by Ang II. Collectively, TGF-ß1 and ET-1 are major effectors of AT1R/Gαq cascade, and therefore, negative regulation of TGF-ß and ET-1 signaling represents a targeted therapeutic strategy for the prevention and restoration of cardiac fibrosis.

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BACKGROUND: Capsular contracture is a critical complication of silicone implantation caused by fibrotic tissue formation from excessive foreign body responses. Various approaches have been applied, but targeting the mechanisms of capsule formation has not been completely solved. Myofibroblast differentiation through the transforming growth factor beta (TGF-ß)/p-SMADs signaling is one of the key factors for capsular contracture development. In addition, biofilm formation on implants may result chronic inflammation promoting capsular fibrosis formation with subsequent contraction. To date, there have been no approaches targeting multi-facted mechanisms of capsular contracture development. METHODS: In this study, we developed a multi-targeting nitric oxide (NO) releasing bionanomatrix coating to reduce capsular contracture formation by targeting myofibroblast differentiation, inflammatory responses, and infections. First, we characterized the bionanomatrix coating on silicon implants by conducting rheology test, scanning electron microcsopy analysis, nanoindentation analysis, and NO release kinetics evaluation. In addition, differentiated monocyte adhesion and S. epidermidis biofilm formation on bionanomatrix coated silicone implants were evaluated in vitro. Bionanomatrix coated silicone and uncoated silicone groups were subcutaneously implanted into a mouse model for evaluation of capsular contracture development for a month. Fibrosis formation, capsule thickness, TGF-ß/SMAD 2/3 signaling cascade, NO production, and inflammatory cytokine production were evaluated using histology, immunofluorescent imaging analysis, and gene and protein expression assays. RESULTS: The bionanomatrix coating maintained a uniform and smooth surface on the silicone even after mechanical stress conditions. In addition, the bionanomatrix coating showed sustained NO release for at least one month and reduction of differentiated monocyte adhesion and S. epidermidis biofilm formation on the silicone implants in vitro. In in vivo implantation studies, the bionanomatrix coated groups demonstrated significant reduction of capsule thickness surrounding the implants. This result was due to a decrease of myofibroblast differentiation and fibrous extracellular matrix production through inhibition of the TGF-ß/p-SMADs signaling. Also, the bionanomatrix coated groups reduced gene expression of M1 macrophage markers and promoted M2 macrophage markers which indicated the bionanomatrix could reduce inflammation but promote healing process. CONCLUSIONS: In conclusion, the bionanomatrix coating significantly reduced capsular contracture formation and promoted healing process on silicone implants by reducing myfibroblast differentiation, fibrotic tissue formation, and inflammation. A multi-targeting nitric oxide releasing bionanomatrix coating for silicone implant can reduce capsular contracture and improve healing process. The bionanomatrix coating reduces capsule thickness, α-smooth muscle actin and collagen synthesis, and myofibroblast differentiation through inhibition of TGF-ß/SMADs signaling cascades in the subcutaneous mouse models for a month.

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Endothelin-1 (ET-1) has been implicated in the pathogenesis of cardiac fibrosis. Stimulation of endothelin receptors (ETR) with ET-1 leads to fibroblast activation and myofibroblast differentiation, which is mainly characterized by an overexpression of α-smooth muscle actin (α-SMA) and collagens. Although ET-1 is a potent profibrotic mediator, the signal transductions and subtype specificity of ETR contributing to cell proliferation, as well as α-SMA and collagen I synthesis in human cardiac fibroblasts are not well clarified. This study aimed to evaluate the subtype specificity and signal transduction of ETR on fibroblast activation and myofibroblast differentiation. Treatment with ET-1 induced fibroblast proliferation, and synthesis of myofibroblast markers, α-SMA, and collagen I through the ETAR subtype. Inhibition of Gαq protein, not Gαi or Gßγ, inhibited these effects of ET-1, indicating the essential role of Gαq protein-mediated ETAR signaling. In addition, ERK1/2 was required for ETAR/Gαq axis-induced proliferative capacity and overexpression of these myofibroblast markers. Antagonism of ETR with ETR antagonists (ERAs), ambrisentan and bosentan, inhibited ET-1-induced cell proliferation and synthesis of α-SMA and collagen I. Furthermore, ambrisentan and bosentan promoted the reversal of myofibroblasts after day 3 of treatment, with loss of proliferative ability and a reduction in α-SMA synthesis, confirming the restorative effects of ERAs. This novel work reports on the ETAR/Gαq/ERK signaling pathway for ET-1 actions and blockade of ETR signaling with ERAs, representing a promising therapeutic strategy for prevention and restoration of ET-1-induced cardiac fibrosis.

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The transdifferentiation from cardiac fibroblasts to myofibroblasts is an important event in the initiation of cardiac fibrosis. However, the underlying mechanism is not fully understood. Circ-sh3rf3 (circular RNA SH3 domain containing Ring Finger 3) is a novel circular RNA which was induced in hypertrophied ventricles by isoproterenol hydrochloride, and our work has established that it is a potential regulator in cardiac hypertrophy, but whether circ-sh3rf3 plays a role in cardiac fibrosis remains unclear, especially in the conversion of cardiac fibroblasts into myofibroblasts. Here, we found that circ-sh3rf3 was down-regulated in isoproterenol-treated rat cardiac fibroblasts and cardiomyocytes as well as during fibroblast differentiation into myofibroblasts. We further confirmed that circ-sh3rf3 could interact with GATA-4 proteins and reduce the expression of GATA-4, which in turn abolishes GATA-4 repression of miR-29a expression and thus up-regulates miR-29a expression, thereby inhibiting fibroblast-myofibroblast differentiation and myocardial fibrosis. Our work has established a novel Circ-sh3rf3/GATA-4/miR-29a regulatory cascade in fibroblast-myofibroblast differentiation and myocardial fibrosis, which provides a new therapeutic target for myocardial fibrosis.

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