Midkine promotes renal fibrosis by stabilizing C/EBPß to facilitate endothelial-mesenchymal transition.

Numerous myofibroblasts are arisen from endothelial cells (ECs) through endothelial to mesenchymal transition (EndMT) triggered by TGF-ß. However, the mechanism of ECs transforms to a different subtype, or whether there exists an intermediate state of ECs remains unclear. In present study, we demonstrate Midkine (MDK) mainly expressed by CD31 + ACTA2+ECs going through partial EndMT contribute greatly to myofibroblasts by spatial and single-cell transcriptomics. MDK is induced in TGF-ß treated ECs, which upregulates C/EBPß and increases EndMT genes, and these effects could be reversed by siMDK. Mechanistically, MDK promotes the binding ability of C/EBPß with ACTA2 promoter by stabilizing the C/EBPß protein. In vivo, knockout of Mdk or conditional knockout of Mdk in ECs reduces EndMT markers and significantly reverses fibrogenesis. In conclusion, our study provides a mechanistic link between the induction of EndMT by TGF-ß and MDK, which suggests that blocking MDK provides potential therapeutic strategies for renal fibrosis.

Noncanonical WNT5A controls the activation of latent TGF-ß to drive fibroblast activation and tissue fibrosis.

Transforming growth factor ß (TGF-ß) signaling is a core pathway of fibrosis, but the molecular regulation of the activation of latent TGF-ß remains incompletely understood. Here, we demonstrate a crucial role of WNT5A/JNK/ROCK signaling that rapidly coordinates the activation of latent TGF-ß in fibrotic diseases. WNT5A was identified as a predominant noncanonical WNT ligand in fibrotic diseases such as systemic sclerosis, sclerodermatous chronic graft-versus-host disease, and idiopathic pulmonary fibrosis, stimulating fibroblast-to-myofibroblast transition and tissue fibrosis by activation of latent TGF-ß. The activation of latent TGF-ß requires rapid JNK- and ROCK-dependent cytoskeletal rearrangements and integrin αV (ITGAV). Conditional ablation of WNT5A or its downstream targets prevented activation of latent TGF-ß, rebalanced TGF-ß signaling, and ameliorated experimental fibrosis. We thus uncovered what we believe to be a novel mechanism for the aberrant activation of latent TGF-ß in fibrotic diseases and provided evidence for targeting WNT5A/JNK/ROCK signaling in fibrotic diseases as a new therapeutic approach.

Assessment of the association of myofibroblasts and structural components of the extracellular matrix with histopathological parameters of actinic cheilitis and lower lip squamous cell carcinoma.

BACKGROUND: To evaluate the presence of myofibroblasts (MFs) in the development of lip carcinogenesis, through the correlation of clinical, histomorphometric and immunohistochemical parameters, in actinic cheilitis (ACs) and lower lip squamous cell carcinomas (LLSCCs). METHODS: Samples of ACs, LLSCCs, and control group (CG) were prepared by tissue microarray (TMA) for immunohistochemical TGF-ß, α-SMA, and Ki-67 and histochemical hematoxylin and eosin, picrosirius red, and verhoeff van gieson reactions. Clinical and microscopic data were associated using the Mann-Whitney, Kruskal-Wallis/Dunn, and Spearman correlation tests (SPSS, p < 0.05). RESULTS: ACs showed higher number of α-SMA+ MFs when compared to CG (p = 0.034), and these cells were associated with the vertical expansion of solar elastosis (SE) itself (p = 0.027). Areas of SE had lower deposits of collagen (p < 0.001), immunostaining for TGF-ß (p < 0.001), and higher density of elastic fibers (p < 0.05) when compared to areas without SE. A positive correlation was observed between high-risk epithelial dysplasia (ED) and the proximity of SE to the dysplastic epithelium (p = 0.027). LLSCCs showed a higher number of α-SMA+ MFs about CG (p = 0.034), as well as a reduction in the deposition of total collagen (p = 0.009) in relation to ACs and CG. There was also a negative correlation between the amount of α-SMA+ cells and the accumulation of total collagen (p = 0.041). Collagen and elastic density loss was higher in larger tumors (p = 0.045) with nodal invasion (p = 0.047). CONCLUSIONS: Our findings show the possible role of MFs, collagen fibers, and elastosis areas in the lip carcinogenesis process.

Salzmann's Nodular Degeneration in Refractive Surgery: The Earlier Hit Hypothesis of EBM Injury-Related Fibrosis of the Subepithelial Space and Deeper Corneal Extension.

PURPOSE: To review the atypical development of Salzmann's nodular degeneration (SND) after two cases of laser in situ keratomileusis (LASIK) and one case of photorefractive keratomileusis (PRK), and to highlight the pathophysiology of SND and its treatment. METHODS: Three cases of SND (two following LASIK performed with microkeratomes and one following PRK) were reviewed and Pubmed.gov and internet searches were performed. RESULTS: SND is myofibroblast-generated fibrosis in the subepithelial space between the epithelium and Bowman's layer that develops years or decades after traumatic, surgical, infectious, or inflammatory injuries to the cornea in which the epithelial basement membrane is damaged in one or more locations and does not fully regenerate. It is hypothesized based on these cases, and the previous immunohistochemistry of other investigators, that myofibroblast precursors, such as fibrocytes or corneal fibroblasts, that enter the subepithelial space are driven to develop into myofibroblasts, which slowly proliferate and extend the fibrosis, by transforming growth factor-beta from epithelium and tears that passes through the defective epithelial basement membrane. These myofibroblasts and the disordered collagens, and other extracellular matrix components they produce, make up the subepithelial opacity characteristic of SND. Nodules are larger accumulations of myofibroblasts and disordered extracellular matrix. If the injury is associated with damage to the underlying Bowman's layer and stroma, as in LASIK flap generation, then the myofibroblasts and fibrosis can extend into Bowman's layer and the underlying anterior stroma. CONCLUSIONS: SND fibrosis often extends into Bowman's layer and the anterior stroma if there are associated Bowman's defects, such as incisions or lacerations. In the latter cases, SND frequently cannot be removed by simple scrape and peel, as typically performed for most common SND cases, but can be trimmed to remove the offending tissue. This condition is more accurately termed Salzmann's subepithelial fibrosis. [J Refract Surg. 2024;40(5):e279-e290.].

Eplerenone reduces lymphangiogenesis in the contralateral kidneys of UUO rats.

Inflammation and fibrosis often occur in the kidney after acute injury, resulting in chronic kidney disease and consequent renal failure. Recent studies have indicated that lymphangiogenesis can drive renal inflammation and fibrosis in injured kidneys. However, whether and how this pathogenesis affects the contralateral kidney remain largely unknown. In our study, we uncovered a mechanism by which the contralateral kidney responded to injury. We found that the activation of mineralocorticoid receptors and the increase in vascular endothelial growth factor C in the contralateral kidney after unilateral ureteral obstruction could promote lymphangiogenesis. Furthermore, mineralocorticoid receptor activation in lymphatic endothelial cells resulted in the secretion of myofibroblast markers, thereby contributing to renal fibrosis. We observed that this process could be attenuated by administering the mineralocorticoid receptor blocker eplerenone, which, prevented the development of fibrotic injury in the contralateral kidneys of rats with unilateral ureteral obstruction. These findings offer valuable insights into the intricate mechanisms underlying kidney injury and may have implications for the development of therapeutic strategies to mitigate renal fibrosis in the context of kidney disease.

PBX/Knotted 1 homeobox-2 (PKNOX2) is a novel regulator of myocardial fibrosis.

Much effort has been made to uncover the cellular heterogeneities of human hearts by single-nucleus RNA sequencing. However, the cardiac transcriptional regulation networks have not been systematically described because of the limitations in detecting transcription factors. In this study, we optimized a pipeline for isolating nuclei and conducting single-nucleus RNA sequencing targeted to detect a higher number of cell signal genes and an optimal number of transcription factors. With this unbiased protocol, we characterized the cellular composition of healthy human hearts and investigated the transcriptional regulation networks involved in determining the cellular identities and functions of the main cardiac cell subtypes. Particularly in fibroblasts, a novel regulator, PKNOX2, was identified as being associated with physiological fibroblast activation in healthy hearts. To validate the roles of these transcription factors in maintaining homeostasis, we used single-nucleus RNA-sequencing analysis of transplanted failing hearts focusing on fibroblast remodelling. The trajectory analysis suggested that PKNOX2 was abnormally decreased from fibroblast activation to pathological myofibroblast formation. Both gain- and loss-of-function in vitro experiments demonstrated the inhibitory role of PKNOX2 in pathological fibrosis remodelling. Moreover, fibroblast-specific overexpression and knockout of PKNOX2 in a heart failure mouse model induced by transverse aortic constriction surgery significantly improved and aggravated myocardial fibrosis, respectively. In summary, this study established a high-quality pipeline for single-nucleus RNA-sequencing analysis of heart muscle. With this optimized protocol, we described the transcriptional regulation networks of the main cardiac cell subtypes and identified PKNOX2 as a novel regulator in suppressing fibrosis and a potential therapeutic target for future translational studies.

Taurine attenuates activation of hepatic stellate cells by inhibiting autophagy and inducing ferroptosis.

BACKGROUND: Liver fibrosis is a compensatory response during the tissue repair process in chronic liver injury, and finally leads to liver cirrhosis or even hepatocellular carcinoma. The pathogenesis of hepatic fibrosis is associated with the progressive accumulation of activated hepatic stellate cells (HSCs), which can transdifferentiate into myofibroblasts to produce an excess of the extracellular matrix (ECM). Myofibroblasts are the main source of the excessive ECM responsible for hepatic fibrosis. Therefore, activated hepatic stellate cells (aHSCs), the principal ECM producing cells in the injured liver, are a promising therapeutic target for the treatment of hepatic fibrosis. AIM: To explore the effect of taurine on aHSC proliferation and the mechanisms involved. METHODS: Human HSCs (LX-2) were randomly divided into five groups: Normal control group, platelet-derived growth factor-BB (PDGF-BB) (20 ng/mL) treated group, and low, medium, and high dosage of taurine (10 mmol/L, 50 mmol/L, and 100 mmol/L, respectively) with PDGF-BB (20 ng/mL) treated group. Cell Counting Kit-8 method was performed to evaluate the effect of taurine on the viability of aHSCs. Enzyme-linked immunosorbent assay was used to estimate the effect of taurine on the levels of reactive oxygen species (ROS), malondialdehyde, glutathione, and iron concentration. Transmission electron microscopy was applied to observe the effect of taurine on the autophagosomes and ferroptosis features in aHSCs. Quantitative real-time polymerase chain reaction and Western blot analysis were performed to detect the effect of taurine on the expression of α-SMA, Collagen I, Fibronectin 1, LC3B, ATG5, Beclin 1, PTGS2, SLC7A11, and p62. RESULTS: Taurine promoted the death of aHSCs and reduced the deposition of the ECM. Treatment with taurine could alleviate autophagy in HSCs to inhibit their activation, by decreasing autophagosome formation, downregulating LC3B and Beclin 1 protein expression, and upregulating p62 protein expression. Meanwhile, treatment with taurine triggered ferroptosis and ferritinophagy to eliminate aHSCs characterized by iron overload, lipid ROS accumulation, glutathione depletion, and lipid peroxidation. Furthermore, bioinformatics analysis demonstrated that taurine had a direct targeting effect on nuclear receptor coactivator 4, exhibiting the best average binding affinity of -20.99 kcal/mol. CONCLUSION: Taurine exerts therapeutic effects on liver fibrosis via mechanisms that involve inhibition of autophagy and trigger of ferroptosis and ferritinophagy in HSCs to eliminate aHSCs.

Two-phase mechanism in the treatment of corneal stromal fibrosis with topical losartan.

Recent studies in rabbits and case reports in humans have demonstrated the efficacy of topical losartan in the treatment of corneal scarring fibrosis after a wide range of injuries, including chemical burns, infections, surgical complications, and some diseases. It is hypothesized that the effect of losartan on the fibrotic corneal stroma occurs through a two-phase process in which losartan first triggers the elimination of myofibroblasts by directing their apoptosis via inhibition of extracellular signal-regulated kinase (ERK)-mediated signal transduction, and possibly through signaling effects on the viability and development of corneal fibroblast and fibrocyte myofibroblast precursor cells. This first step likely occurs within a week or two in most corneas with fibrosis treated with topical losartan, but the medication must be continued for much longer until the epithelial basement membrane (EBM) is fully regenerated or new myofibroblasts will develop from precursor cells. Once the myofibroblasts are eliminated from the fibrotic stroma, corneal fibroblasts can migrate into the fibrotic tissue and reabsorb/reorganize the disordered extracellular matrix (ECM) previously produced by the myofibroblasts. This second stage is longer and more variable in different eyes of rabbits and humans, and accounts for most of the variability in the time it takes for the stromal opacity to be markedly reduced by topical losartan treatment. Eventually, keratocytes reemerge in the previously fibrotic stromal tissue to fine-tune the collagens and other ECM components and maintain the normal structure of the corneal stroma. The efficacy of losartan in the prevention and treatment of corneal fibrosis suggests that it acts as a surrogate for the EBM, by suppressing TGF beta-directed scarring of the wounded corneal stroma, until control over TGF beta action is re-established by a healed EBM, while also supporting regeneration of the EBM by allowing corneal fibroblasts to occupy the subepithelial stroma in the place of myofibroblasts.

Evaluation of stromal myofibroblasts in oral submucous fibrosis and its malignant transformation: An immunohistochemical study.

BACKGROUND: Oral submucous fibrosis (OSF) is a precancerous lesion, with oral squamous cell carcinoma (OSCC) being the most prevalent malignancy affecting the oral mucosa. The malignant transformation of OSF into OSCC is estimated to occur in 7-13% of cases. Myofibroblasts (MFs) play pivotal roles in both physiological and pathological processes, such as wound healing and tumorigenesis, respectively. This study aimed to explore the involvement of MFs in the progression of OSF and its malignant transformation. MATERIALS AND METHODS: In total, 94 formalin-fixed paraffin-embedded tissue blocks were collected, including normal oral mucosa (NOM; n = 10), early-moderate OSF (EMOSF; n = 29), advanced OSF (AOSF; n = 29), paracancerous OSF (POSF; n = 21), and OSCC (n = 5) samples. Alpha-smooth muscle actin was used for the immunohistochemical identification of MFs. RESULTS: NOM exhibited infrequent expression of MFs. A higher staining index of MFs was found in AOSF, followed by EMOSF and NOM. Additionally, a significant increase in the staining index of MFs was found from EMOSF to POSF and OSCC. The staining index of MFs in NOM, EMOSF, AOSF, POSF, and OSCC was 0.14 ± 0.2, 1.69 ± 1.4, 2.47 ± 1.2, 3.57 ± 2.6, and 8.86 ± 1.4, respectively. All results were statistically significant (P < 0.05). CONCLUSIONS: The expression of MFs exhibited a gradual increase as the disease progressed from mild to malignant transformation, indicating the contributory role of MFs in the fibrogenesis and potential tumorigenesis associated with OSF.

The c-Abl-RACK1-FAK signaling axis promotes renal fibrosis in mice through regulating fibroblast-myofibroblast transition.

BACKGROUND: Renal fibrosis is a prevalent manifestation of chronic kidney disease (CKD), and effective treatments for this disease are currently lacking. Myofibroblasts, which originate from interstitial fibroblasts, aggregate in the renal interstitium, leading to significant accumulation of extracellular matrix and impairment of renal function. The nonreceptor tyrosine kinase c-Abl (encoded by the Abl1 gene) has been implicated in the development of renal fibrosis. However, the precise role of c-Abl in this process and its involvement in fibroblast-myofibroblast transition (FMT) remain poorly understood. METHODS: To investigate the effect of c-Abl in FMT during renal fibrosis, we investigated the expression of c-Abl in fibrotic renal tissues of patients with CKD and mouse models. We studied the phenotypic changes in fibroblast or myofibroblast-specific c-Abl conditional knockout mice. We explored the potential targets of c-Abl in NRK-49F fibroblasts. RESULTS: In this study, fibrotic mouse and cell models demonstrated that c-Abl deficiency in fibroblasts mitigated fibrosis by suppressing fibroblast activation, fibroblast-myofibroblast transition, and extracellular matrix deposition. Mechanistically, c-Abl maintains the stability of the RACK1 protein, which serves as a scaffold for proteins such as c-Abl and focal adhesion kinase at focal adhesions, driving fibroblast activation and differentiation during renal fibrosis. Moreover, specifically targeting c-Abl deletion in renal myofibroblasts could prove beneficial in established kidney fibrosis by reducing RACK1 expression and diminishing the extent of fibrosis. CONCLUSIONS: Our findings suggest that c-Abl plays a pathogenic role in interstitial fibrosis through the regulation of RACK1 protein stabilization and myofibroblast differentiation, suggesting a promising strategy for the treatment of CKD.

MAPK phosphatase 1 inhibition of p38α within lung myofibroblasts is essential for spontaneous fibrosis resolution.

Fibrosis following tissue injury is distinguished from normal repair by the accumulation of pathogenic and apoptosis-resistant myofibroblasts (MFs), which arise primarily by differentiation from resident fibroblasts. Endogenous molecular brakes that promote MF dedifferentiation and clearance during spontaneous resolution of experimental lung fibrosis may provide insights that could inform and improve the treatment of progressive pulmonary fibrosis in patients. MAPK phosphatase 1 (MKP1) influences the cellular phenotype and fate through precise and timely regulation of MAPK activity within various cell types and tissues, yet its role in lung fibroblasts and pulmonary fibrosis has not been explored. Using gain- and loss-of-function studies, we found that MKP1 promoted lung MF dedifferentiation and restored the sensitivity of these cells to apoptosis - effects determined to be mainly dependent on MKP1's dephosphorylation of p38α MAPK (p38α). Fibroblast-specific deletion of MKP1 following peak bleomycin-induced lung fibrosis largely abrogated its subsequent spontaneous resolution. Such resolution was restored by treating these transgenic mice with the p38α inhibitor VX-702. We conclude that MKP1 is a critical antifibrotic brake whose inhibition of pathogenic p38α in lung fibroblasts is necessary for fibrosis resolution following lung injury.

Naringenin nanoparticles targeting cyclin B1 suppress the progression of rheumatoid arthritis-associated lung cancer by inhibiting fibroblast-to-myofibroblast transition.

There is growing evidence of an elevated risk of lung cancer in patients with rheumatoid arthritis. The poor prognosis of rheumatoid arthritis-associated lung cancer and the lack of therapeutic options pose an even greater challenge to the clinical management of patients. This study aimed to identify potential molecular targets associated with the progression of rheumatoid arthritis-associated lung cancer and examine the efficacy of naringenin nanoparticles targeting cyclin B1. Mendelian randomizatio analysis revealed that rheumatoid arthritis has a positive correlation with the risk of lung cancer. Cyclin B1 was significantly upregulated in patients with rheumatoid arthritis-associated lung cancer and was significantly overexpressed in synovial tissue fibroblasts. Furthermore, the overexpression of cyclin B1 in rheumatoid arthritis fibroblast-like synoviocytes, which promotes their proliferation and fibroblast-to-myofibroblast transition, can significantly contribute to the growth and infiltration of lung cancer cells. Importantly, our prepared naringenin nanoparticles targeting cyclin B1 effectively attenuated proliferation and fibroblast-to-myofibroblast transition by blocking cells at the G2/M phase. In vivo experiments, naringenin nanoparticles targeting cyclin B1 significantly alleviated the development of collagen-induced arthritis and lung orthotopic tumors. Collectively, our results reveal that naringenin nanoparticles targeting cyclin B1 can suppress the progression of rheumatoid arthritis-associated lung cancer by inhibiting fibroblast-to-myofibroblast transition. These findings provide new insights into the treatment of rheumatoid arthritis-associated lung cancer therapy.

Characterization of Vascular Niche in Systemic Sclerosis by Spatial Proteomics.

BACKGROUND: Systemic sclerosis (SSc) is a connective tissue disease that can serve as a model to study vascular changes in response to inflammation, autoimmunity, and fibrotic remodeling. Although microvascular changes are the earliest histopathologic manifestation of SSc, the vascular pathophysiology remains poorly understood. METHODS: We applied spatial proteomic approaches to deconvolute the heterogeneity of vascular cells at the single-cell level in situ and characterize cellular alterations of the vascular niches of patients with SSc. Skin biopsies of patients with SSc and control individuals were analyzed by imaging mass cytometry, yielding a total of 90 755 cells including 2987 endothelial cells and 4096 immune cells. RESULTS: We identified 7 different subpopulations of blood vascular endothelial cells (VECs), 2 subpopulations of lymphatic endothelial cells, and 3 subpopulations of pericytes. A novel population of CD34+;αSMA+ (α-smooth muscle actin);CD31+ VECs was more common in SSc, whereas endothelial precursor cells were decreased. Co-detection by indexing and tyramide signal amplification confirmed these findings. The microenvironment of CD34+;αSMA+;CD31+ VECs was enriched for immune cells and myofibroblasts, and CD34+;αSMA+;CD31+ VECs expressed markers of endothelial-to-mesenchymal transition. The density of CD34+;αSMA+;CD31+ VECs was associated with clinical progression of fibrosis in SSc. CONCLUSIONS: Using spatial proteomics, we unraveled the heterogeneity of vascular cells in control individuals and patients with SSc. We identified CD34+;αSMA+;CD31+ VECs as a novel endothelial cell population that is increased in patients with SSc, expresses markers for endothelial-to-mesenchymal transition, and is located in close proximity to immune cells and myofibroblasts. CD34+;αSMA+;CD31+ VEC counts were associated with clinical outcomes of progressive fibrotic remodeling, thus providing a novel cellular correlate for the crosstalk of vasculopathy and fibrosis.

Cardiac and perivascular myofibroblasts, matrifibrocytes, and immune fibrocytes in hypertension; commonalities and differences with other cardiovascular diseases.

Hypertension is a major cause of cardiovascular diseases such as myocardial infarction and stroke. Cardiovascular fibrosis occurs with hypertension and contributes to vascular resistance, aortic stiffness, and cardiac hypertrophy. However, the molecular mechanisms leading to fibroblast activation in hypertension remain largely unknown. There are two types of fibrosis: replacement fibrosis and reactive fibrosis. Replacement fibrosis occurs in response to the loss of viable tissue to form a scar. Reactive fibrosis occurs in response to an increase in mechanical and neurohormonal stress. Although both types of fibrosis are considered adaptive processes, they become maladaptive when the tissue loss is too large, or the stress persists. Myofibroblasts represent a subpopulation of activated fibroblasts that have gained contractile function to promote wound healing. Therefore, myofibroblasts are a critical cell type that promotes replacement fibrosis. Although myofibroblasts were recognized as the fibroblasts participating in reactive fibrosis, recent experimental evidence indicated there are distinct fibroblast populations in cardiovascular reactive fibrosis. Accordingly, we will discuss the updated definition of fibroblast subpopulations, the regulatory mechanisms, and their potential roles in cardiovascular pathophysiology utilizing new knowledge from various lineage tracing and single-cell RNA sequencing studies. Among the fibroblast subpopulations, we will highlight the novel roles of matrifibrocytes and immune fibrocytes in cardiovascular fibrosis including experimental models of hypertension, pressure overload, myocardial infarction, atherosclerosis, aortic aneurysm, and nephrosclerosis. Exploration into the molecular mechanisms involved in the differentiation and activation of those fibroblast subpopulations may lead to novel treatments for end-organ damage associated with hypertension and other cardiovascular diseases.

TEA domain transcription factor 1(TEAD1) induces cardiac fibroblasts cells remodeling through BRD4/Wnt4 pathway.

Cardiac fibroblasts (CFs) are the primary cells tasked with depositing and remodeling collagen and significantly associated with heart failure (HF). TEAD1 has been shown to be essential for heart development and homeostasis. However, fibroblast endogenous TEAD1 in cardiac remodeling remains incompletely understood. Transcriptomic analyses revealed consistently upregulated cardiac TEAD1 expression in mice 4 weeks after transverse aortic constriction (TAC) and Ang-II infusion. Further investigation revealed that CFs were the primary cell type expressing elevated TEAD1 levels in response to pressure overload. Conditional TEAD1 knockout was achieved by crossing TEAD1-floxed mice with CFs- and myofibroblasts-specific Cre mice. Echocardiographic and histological analyses demonstrated that CFs- and myofibroblasts-specific TEAD1 deficiency and treatment with TEAD1 inhibitor, VT103, ameliorated TAC-induced cardiac remodeling. Mechanistically, RNA-seq and ChIP-seq analysis identified Wnt4 as a novel TEAD1 target. TEAD1 has been shown to promote the fibroblast-to-myofibroblast transition through the Wnt signalling pathway, and genetic Wnt4 knockdown inhibited the pro-transformation phenotype in CFs with TEAD1 overexpression. Furthermore, co-immunoprecipitation combined with mass spectrometry, chromatin immunoprecipitation, and luciferase assays demonstrated interaction between TEAD1 and BET protein BRD4, leading to the binding and activation of the Wnt4 promoter. In conclusion, TEAD1 is an essential regulator of the pro-fibrotic CFs phenotype associated with pathological cardiac remodeling via the BRD4/Wnt4 signalling pathway.

Hydroxypyridone anti-fungals selectively induce myofibroblast apoptosis in an in vitro model of hypertrophic scars.

Hypertrophic scars are a common complication of burn injuries, yet there are no medications to prevent their formation. During scar formation, resident fibroblasts are transformed to myofibroblasts which become resistant to apoptosis. Previously, we have shown that hydroxypyridone anti-fungals can inhibit transformation of fibroblasts, isolated from hypertrophic scars, to myofibroblasts. This study aimed to investigate if these drugs can also target myofibroblast persistence. Primary human dermal fibroblasts, derived from burn scar tissue, were exposed to transforming growth factor beta-1 (TGF-ß1) for 72 h to induce myofibroblast transformation. The cells were then incubated with three hydroxypyridone anti-fungals (ciclopirox, ciclopirox ethanolamine and piroctone olamine; 0.03-300 µM) for a further 72 h. The In-Cell ELISA method was utilised to quantify myofibroblast transformation by measuring alpha-smooth muscle actin (α-SMA) expression and DRAQ5 staining, to measure cell viability. TUNEL staining was utilised to assess if the drugs could induce apoptosis. When given to established myofibroblasts, the three hydroxypyridones did not reverse myofibroblast transformation, but instead elicited a concentration-dependent decrease in cell viability. TUNEL staining confirmed that the hydroxypyridone anti-fungals induced apoptosis in established myofibroblasts. This is the first study to show that hydroxypyridone anti-fungals are capable of inducing apoptosis in established myofibroblasts. Together with our previous results, we suggest that hydroxypyridone anti-fungals can prevent scar formation by preventing the formation of new myofibroblasts and by reducing the number of existing myofibroblasts.

Myofibroblast and pro-fibrotic cytokines in fibrosis of IgG4-related disease (IgG4-RD) patients from South Asia: preliminary data.

INTRODUCTION: Fibrosis is a typical pathological characteristic in IgG4-RD patients and often irreversible. There exists a lack of suitable markers for detection of earlier onset of fibrosis in various organs in IgG4-RD patients. Hence, this study aims at analysing ambispectively the myofibroblasts and the pro-fibrotic cytokines, IFN gamma and IL-33 involved in IgG4-RD associated fibrosis in South Asian patients. METHOD: Archived biopsy samples of definite/probable/possible cases of IgG4-RD, classified according to diagnostic criteria, taken from patients who attended the OPD and IPD of our tertiary care centre during January 2015-January 2020 were chosen for this study. The paraffin sections were examined qualitatively for fibrosis and the excessive collagen deposition by Hematoxylin & Eosin and Masson's Trichrome staining. Also, the presence of alpha-Smooth muscle actin (α-SMA) expressing myofibroblasts and the involvement of pro-fibrotic cytokines (IFN-gamma, IL-33) were assessed by Immunohistochemistry and scored semi-quantitatively (+mild, ++moderate, +++ severe). Serum IL-33 levels were analysed by indirect Elisa (R & D Systems). RESULTS: Myofibroblasts were present in 10/12 biopsy samples, in moderate levels in 4 (33%) and very high levels (+++) in 3 (25%) of the patients. IFN-gamma was expressed at low levels in 6 (50%) and absent in 6 (50%). All patients showed IL-33 expression with very high levels in tissue (6, 50%), as well as in serum samples. CONCLUSION: The findings of this study reinforce the role of myofibroblasts and profibrotic cytokines like IL-33 in fibrosis of Ig4-RD patients, pointing to their potential as earlier predictive markers of onset and extent of fibrosis.

Ocular inflammatory myofibroblastic tumor in a pediatric patient.

Inflammatory myofibroblastic tumor (IMT) is a mesenchymal neoplasm that rarely metastasizes and is more commonly seen in children, adolescents, and young adults than older adults. These tumors, composed of myofibroblasts and inflammatory cells, are often confused for a local infection due to the inflammatory cell infiltration, and they form in mucosal surfaces but rarely arise in the orbit. We present the case of a 6-year-old girl with excisional biopsy-confirmed conjunctival stromal IMT. There was no evidence of recurrence 2 years following resection with no subsequent medical therapy.

Colonic mucosa barrier defects in collagenous and ischemic colitis.

AIMS: The subepithelial myofibroblasts (SEMFs) and the subepithelial band of macrophages (SEBM) are major components of the colonic mucosa barrier. Although their role in homeostasis is widely recognized, their contribution to disease states is largely unknown. Our aim was to explore histological characteristics of SEMFs and SEBM in collagenous and ischemic colitis in order to identify specific changes in distinct mucosa backgrounds lacking significant inflammation. METHODS: SEMFs, SEBM and lamina propria (LP) macrophages were identified immunohistochemically by alpha smooth muscle Actin and Cluster of Differentiation 68 respectively in 38 colonic biopsies [14 collagenous colitis (CC), 14 ischemic colitis (IC), 10 normal mucosa]. RESULTS: In CC, SEMFs were rarely detectable in the collagenous band while aSMA-negative pericryptal fibroblast-like cells appeared. In lower LP interconnecting SEMFs processes were formed. SEBM was preserved in areas with a collagenous layer up to 20 µm. In thicker layers, it was fragmented and gradually disappeared in parallel with engulfment of enlarged macrophages. LP macrophages were usually increased. In IC, slight SEMFs changes preceded discernible epithelial alterations. Rounding, disintegration and extinction of SEMFs constituted successive alterations coinciding with crypt shrinkage and denudation. SEBM displayed total or almost total abolishment in areas with crypt damage but also in sites with minimal changes and in adjacent normal mucosa. CONCLUSION: Our findings provide evidence of impairment of both mucosa barrier constituents in CC and IC. In CC, histological alterations are closely related to the collagenous layer which seems to affect SEMFs differentiation and migration as well as SEBM integrity. The early extinction of SEBM in IC is indicative of its high sensitivity to hypoxia and hypoperfusion.

Integrin ß8 prevents pericyte-myofibroblast transition and renal fibrosis through inhibiting the TGF-ß1/TGFBR1/Smad3 pathway in diabetic kidney disease.

Diabetic kidney disease (DKD) is one of the leading causes to develop end-stage kidney disease worldwide. Pericytes are implicated in the development of tissue fibrosis. However, the underlying mechanisms of pericytes in DKD remain largely unknown. We isolated and cultured primary pericytes and rat mesangial cells (HBZY-1). Western blot and qRT-PCR analysis were used to explore the role and regulatory mechanism of Integrin ß8/transforming growth factor beta 1 (TGF-ß1) pathway. We also constructed pericyte-specific Integrin ß8 knock-in mice as the research objects to determine the role of Integrin ß8 in vivo. We discovered that reduced Integrin ß8 expression was closely associated with pericyte transition in DKD. Overexpressed Integrin ß8 in pericytes dramatically suppressed TGF-ß1/TGF beta receptor 1 (TGFBR1)/Smad3 signaling pathway and protected glomerular endothelial cells (GECs) in vitro. In vivo, pericyte-specific Integrin ß8 knock-in ameliorated pericyte transition, endothelium injury and renal fibrosis in STZ-induced diabetic mice. Mechanistically, Murine double minute 2 (MDM2) was found to increase the degradation of Integrin ß8 and caused TGF-ß1 release and activation. Knockdown MDM2 could partly reverse the decline of Integrin ß8 and suppress pericytes transition. In conclusion, the present findings suggested that upregulated MDM2 expression contributes to the degradation of Integrin ß8 and activation of TGF-ß1/TGFBR1/Smad3 signaling pathway, which ultimately leads to pericyte transition during DKD progression. These results indicate MDM2/Integrin ß8 might be considered as therapeutic targets for DKD.