Evaluation of CNPase and TGFß1/Smad Signalling Pathway Molecule Expression in Sinus Epithelial Tissues of Patients with Chronic Rhinosinusitis with (CRSwNP) and without Nasal Polyps (CRSsNP).

Chronic rhinosinusitis with and without nasal polyps (CRSwNP and CRSsNP, respectively) is a chronic inflammatory disease affecting almost 5 to 12% of the population and exhibiting high recurrence rates after functional endoscopic sinus surgery (FESS). TGFß1-related pathways contribute to tissue remodelling, which is one of the key aspects of CRS pathogenesis. Additionally, adenosine signalling participates in inflammatory processes, and CNPase was shown to elevate adenosine levels by metabolizing cyclic monophosphates. Thus, the aim of this study was to assess the expression levels of Smad2, pSmad3, TGFß1, and CNPase protein via immunohistochemistry in sinus epithelial tissues from patients with CRSwNP (n = 20), CRSsNP (n = 23), and non-CRS patients (n = 8). The expression of Smad2, pSmad3, TGFß1, and CNPase was observed in the sinus epithelium and subepithelial area of all three groups of patients, and their expression correlated with several clinical symptoms of CRS. Smad2 expression was increased in CRSsNP patients compared to CRSwNP patients and controls (p = 0.001 and p < 0.001, respectively), pSmad3 expression was elevated in CRSwNP patients compared to controls (p = 0.007), TGFß1 expression was elevated in CRSwNP patients compared to controls (p = 0.009), and CNPase was decreased in CRSsNP patients compared to controls (p = 0.03). To the best of our knowledge, we are the first to demonstrate CNPase expression in the upper airway epithelium of CRSwNP, CRSsNP, and non-CRS patients and point out a putative synergy between CNPase and TGFß1/Smad signalling in CRS pathogenesis that emerges as a novel still undiscovered aspect of CRS pathogenesis; further studies are needed to explore its function in the course of the chronic inflammation of the upper airways.

Human Antigen R -mediated modulation of Transforming Growth Factor Beta 1 expression in retinal pathological milieu.

The fate and stability of messenger RNA (mRNA), from transcription to degradation is regulated by a dynamic shuttle of epigenetic modifications and RNA binding proteins in maintaining healthy cellular homeostasis and disease development. While Transforming Growth Factor Beta 1 (TGFß1) has been implicated as a key regulator for diabetic retinopathy, a microvascular complication of diabetes, the RNA binding proteins post-transcriptionally regulating its expression remain unreported in the ocular context. Further, dysfunction of TGFß1 signalling is also strongly associated with angiogenesis, inflammatory responses and tissue fibrosis in many eye conditions leading to vision loss. In this study, computational and molecular simulations were initially carried out to identify Human Antigen R (HuR) binding sites in TGFß1 mRNA and predict the structural stability of these RNA-protein interactions. These findings were further validated through in vitro experiments utilizing Cobalt Chloride (CoCl2) as a hypoxia mimetic agent in human retinal microvascular endothelial cells (HRMVEC). In silico analysis revealed that HuR preferentially binds to the 5'-UTR of TGFß1 and displayed more stable interaction than the 3'UTR. Consistent with in silico analysis, RNA immunoprecipitation demonstrated a robust association between HuR and TGFß1 mRNA specifically under hypoxic conditions. Further, silencing of HuR significantly reduced TGFß1 protein expression upon CoCl2 treatment. Thus, for the first time in ocular pathological milieu, direct evidence of HuR- TGFß1 mRNA interaction under conditions of hypoxia has been reported in this study providing valuable insights into RNA binding proteins as therapeutic targets for ocular diseases associated with TGFß1 dysregulation.

Evidence for platelet-derived transforming growth factor ß1 as an early inducer of liver regeneration after hepatectomy in mice.

Platelets play a crucial role in tissue regeneration, and their involvement in liver regeneration is well-established. However, the specific contribution of platelet-derived Transforming Growth Factor Beta 1 (TGFß1) to liver regeneration remains unexplored. This study investigated the role of platelet-derived TGFß1 in initiating liver regeneration following 2/3 liver resection. Using platelet-specific TGFß1 knockout (Plt.TGFß1 KO) mice and wild-type littermates (Plt.TGFß1 WT) as controls, the study assessed circulating levels and hepatic gene expression of TGFß1, Platelet Factor 4 (PF4), and Thrombopoietin (TPO) at early time points post-hepatectomy (post-PHx). Hepatocyte proliferation was quantified through Ki67 staining and PCNA expression in total liver lysates at various intervals, and phosphohistone-H3 (PHH3) staining was employed to mark mitotic cells. Circulating levels of hepatic mitogens, Hepatocyte Growth Factor (HGF), and Interleukin-6 (IL6) were also assessed. Results revealed that platelet-TGFß1 deficiency significantly reduced total plasma TGFß1 levels at 5 h post-PHx in Plt.TGFß1 KO mice compared to controls. While circulating PF4 levels, liver platelet recruitment and activation appeared normal at early time points, Plt.TGFß1 KO mice showed more stable circulating platelet numbers with higher numbers at 48 h post-PHx. Notably, hepatocyte proliferation was significantly reduced in Plt.TGFß1 KO mice. The results show that a lack of TGFß1 in platelets leads to an unbalanced expression of IL6 in the liver and to strongly increased HGF levels 48 h after liver resection, and yet liver regeneration remains reduced. The study identifies platelet-TGFß1 as a regulator of hepatocyte proliferation and platelet homeostasis in the early stages of liver regeneration.

Insights into epithelial-mesenchymal transition from cystic fibrosis rat models.

BACKGROUND: Molecular pathways contributing to Cystic Fibrosis pathogenesis remain poorly understood. Epithelial-mesenchymal transition (EMT) has been recently observed in CF lungs and certain CFTR mutation classes may be more susceptible than others. No investigations of EMT processes in CF animal models have been reported. AIM: The aim of this study was to assess the expression of EMT-related markers in Phe508del and knockout (CFTR-KO) rat lung tissue and tracheal-derived basal epithelial stem cells, to determine whether CFTR dysfunction can produce an EMT state. METHOD: The expression of EMT-related markers in lung tissue and cultured tracheal basal epithelial stem cells from wildtype (WT), Phe508del, and CFTR-KO rats were assessed using qPCR and Western blots. Cell responses were evaluated in the presence of Rho-associated protein kinase (ROCK) inhibitor Y27632, which blocks EMT-pathways, or after treatment with TGFß1 to stimulate EMT. RESULTS: Different gene expression profiles were observed between Phe508del and CFTR-KO rat models compared to wild type. There was lower expression of type 1 collagen in KO lungs and primary cell cultures, while Phe508del lungs and cells had higher expression, particularly when treated with TGFß1. The addition of Y27632 rescued changes in EMT related genes in Phe508del cells but not in KO cells. CONCLUSION: Our findings show the first evidence of upregulated EMT pathways in the lungs and airway cells of any CF animal model. Differences in the regulation of the EMT genes and proteins in the Phe508del and CFTR-KO cells suggest that the signalling pathways underlying EMT are CFTR mutation dependent.

TMEM176B Prevents and alleviates bleomycin-induced pulmonary fibrosis via inhibiting transforming growth factor ß-Smad signaling.

Pulmonary fibrosis is a severe and progressive lung disease characterized by the abnormal accumulation of extracellular matrix, leading to scarring and loss of normal lung function. Recent bioinformatics analysis through the Gene Expression Omnibus (GEO) database identified a significant downregulation of Transmembrane Protein 176B (TMEM176B), previously unexplored in the context of fibrotic lung tissues. To investigate the functional role of TMEM176B, we induced pulmonary fibrosis in mice using bleomycin, TGFß1, and silica, which consistently resulted in a marked decrease in TMEM176B expression. Intriguingly, overexpression of TMEM176B via adenoviral vectors prior to the induction of fibrosis led to significant improvements in fibrotic manifestations and lung function. Mechanistically, TMEM176B appears to mitigate pulmonary fibrosis by inhibiting the TGFß1-SMAD signaling pathway, which is a critical mediator of fibroblast proliferation and differentiation and promotes extracellular matrix production. These findings suggest that TMEM176B plays an inhibitory role in the pathophysiological processes of pulmonary fibrosis, highlighting its potential as a therapeutic target.

Targeting alternative splicing of fibronectin in human renal proximal tubule epithelial cells with antisense oligonucleotides to reduce EDA+ fibronectin production and block an autocrine loop that drives renal fibrosis.

TGFß1 is a powerful regulator of fibrosis; secreted in a latent form, it becomes active after release from the latent complex. During tissue fibrosis, the EDA + isoform of cellular fibronectin is overexpressed. In pulmonary fibrosis it has been proposed that the fibronectin splice variant including an EDA domain (FN EDA+) activates latent TGFß. Our work investigates the potential of blocking the 'splicing in' of EDA with antisense oligonucleotides to inhibit TGFß1-induced EDA + fibronectin and to prevent the cascade of events initiated by TGFß1 in human renal proximal tubule cells (PTEC). Human primary PTEC were treated with TGFß1 for 48 h, medium removed and the cells transfected with RNase H-independent antisense oligonucleotides (ASO) designed to block EDA exon inclusion (ASO5). The efficacy of ASO to block EDA exon inclusion was assessed by EDA + fibronectin RNA and protein expression; the expression of TGFß, αSMA (α smooth muscle actin), MMP2 (matrix metalloproteinse-2), MMP9 (matrix metalloproteinse-9), Collagen I, K Cadherin and connexin 43 was analysed. Targeting antisense oligonucleotides designed to block EDA exon inclusion in fibronectin pre mRNA were effective in reducing the amount of TGFß1 -induced cellular EDA + fibronectin RNA and secreted EDA + fibronectin protein (assessed by western immunoblotting and immunocytochemistry) in human proximal tubule cells in an in vitro cell culture model. The effect was selective for EDA + exon with no effect on EDB + fibronectin RNA and total fibronectin mRNA. Exogenous TGFß1 induced endogenous TGFß, αSMA, MMP2, MMP9 and Col I mRNA. TGFß1 treatment for 48h reduced the expression of K-Cadherin and increased the expression of connexin-43. These TGFß1-induced pro-fibrotic changes were attenuated by ASO5 treatment. 48 h after the removal of exogenous TGFß, further increases in αSMA, MMP2, MMP9 was observed; ASO5 significantly inhibited this subsequent increase. ASO5 treatment also significantly inhibited ability of the cell culture medium harvested at the end of the experiment (96h) to stimulate SMAD3 reporter cells. The role of endogenous TGFß1 was confirmed by the use of a TGFß receptor inhibitor. Our results demonstrate a critical role of FN EDA+ in a cycle of TGFß driven pro-fibrotic responses in human PTEC and blocking its production with ASO technology offers a potential therapy to interrupt this vicious circle and hence limit the progression of renal fibrosis.

Signalling of the neuropeptide calcitonin gene-related peptide (CGRP) through RAMP1 promotes liver fibrosis via TGFß1/Smad2 and YAP pathways.

The liver is innervated by primary sensory nerve fibres releasing the neuropeptide calcitonin gene-related peptide (CGRP). Elevated plasma levels of CGRP have been found in patients with liver fibrosis or cirrhosis. We hypothesised that signalling of CGRP and its receptors might regulate liver fibrosis and propose a novel potential target for the treatment. In this study, hepatic expression of CGRP and its receptor component, the receptor activity-modifying protein 1 (RAMP1), was dramatically increased in diseased livers of patients. In a murine liver fibrosis model, deficiency of RAMP1 resulted in attenuated fibrogenesis characterized by less collagen deposition and decreased activity of hepatic stellate cells (HSC). Mechanistically, activity of the TGFß1 signalling core component Smad2 was severely impaired in the absence of RAMP1, and Yes-associated protein (YAP) activity was found to be diminished in RAMP1-deficient liver parenchyma. In vitro, stimulation of the HSC line LX-2 cells with CGRP induces TGFß1 production and downstream signalling as well as HSC activation documented by increased α-SMA expression and collagen synthesis. We further demonstrate in LX-2 cells that CGRP promotes YAP activation and its nuclear translocation subsequent to TGFß1/Smad2 signals. These data support a promotive effect of CGRP signalling in liver fibrosis via stimulation of TGFß1/Smad2 and YAP activity.

TGFß1, SNAIL2, and PAPP-A Expression in Placenta of Gestational Diabetes Mellitus Patients.

Background: Gestational diabetes mellitus (GDM) is a pregnancy-related diabetic condition that may cause serious complications. However, its pathogenesis remains unclear. Placental damage due to GDM may lead to several health issues that cannot be ignored. Thus, we aimed to identify the mechanisms underlying GDM by screening differentially expressed genes (DEGs) related to vascular endothelial cells in the GDM databases and verify the expression of these DEGs in the placentas of women afflicted by GDM. Methods: We used GDM microarray datasets integrated from the Gene Expression Omnibus (GEO) database. Functional annotation and protein-protein interaction (PPI) analyses were used to screen DEGs. Placental tissues from 20 pregnant women with GDM and 20 healthy pregnant women were collected, and differential gene expression in the placental tissues was verified via qRT-PCR, western blotting, and immunofluorescence. Results: Bioinformatics analysis revealed three significant DEGs: SNAIL2, PAPP-A, and TGFß1. These genes were all predicted to be underexpressed in patients with GDM. The results of qRT-PCR, western blot, and immunofluorescence analyses indicated that SNAIL2 and PAPP-A in the placenta tissue of patients with GDM were significantly underexpressed. However, TGFß1 in the placenta tissues of GDM was significantly overexpressed. Conclusion: SNAIL2, TGFß1, and PAPP-A may affect the placentas of pregnant women with GDM, warranting further investigation.

Anaplastic thyroid cancer cell-secreted TGFß1 plays a key role in inducing macrophage polarization of human monocytes.

Anaplastic thyroid cancer (ATC) is a clinically aggressive form of undifferentiated thyroid cancer with limited treatment options. Tumor-associated macrophages (TAMs) constitute over 50% of ATC-infiltrating cells, and their presence is associated with a poor prognosis. We have previously shown that paracrine signals released by ATC cells induced pro-tumor M2-like polarization of human monocytes. However, which soluble factors derived from ATC cells drive monocyte activation, are largely unknown. In this study we investigated the participation of transforming growth factor ß1 (TGFß1) on the phenotype of macrophage activation induced by ATC cell-derived conditioned media (CM). THP-1 cells exposed to CM derived from ATC cells and recombinant human TGFß1 induced M2-like macrophage polarization, showing high CD163 and Dectin1 expression. Moreover, we showed that TGFß1 induced the messenger RNA (mRNA) and protein expression of the transcription factors SNAIL and SLUG. Accordingly, increased TGFß1 secretion from ATC cells was confirmed by enzyme-linked immunosorbent assay (ELISA). Addition of SB431542, a TGFß receptor inhibitor, significantly decreased the Dectin1, CD163, SNAIL and SLUG expression stimulated by ATC cell-derived CM. We validated the clinical significance of the expression of TGFß ligands, their receptors, as well as SNAIL and SLUG in human ATC by analyzing public microarray datasets. We found that the expression of the main TGFß ligands, TGFß1 and TGFß3, along with their receptors, TGFR1 and TGFR2, as well as SLUG, was significantly higher in human ATC tissue samples than in normal thyroid tissues. Our findings indicate that ATC cell-secreted TGFß1 may play a key role in M2-like macrophage polarization of human monocytes and in the up-regulation of SNAIL and SLUG transcription factors. Thus, ours results uncovered a novel mechanism involved in the activation of TAMs by soluble factors released by ATC cells, which suggest potential therapeutic targets for ATC.

Increased Prorenin Expression in the Kidneys May Be Involved in the Abnormal Renal Function Caused by Prolonged Environmental Exposure to Microcystin-LR.

Toxic algae in eutrophic lakes produce cyanotoxic microcystins. Prior research on the effect of microcystin-LR in the kidney utilized intraperitoneal injections, which did not reflect natural exposure. Oral microcystin-LR research has focused on renal function and histopathology without examining the molecular mechanisms. The present study aimed to evaluate the mechanism of microcystin-LR in the kidneys via oral administration in WKAH/HkmSlc rats over 7 weeks, alongside stimulation of the proximal tubular cells. Although there were no differences in the concentrations of plasma albumin, blood urea nitrogen, and creatinine, which are parameters of renal function, between the control and microcystin-LR-administrated rats, prorenin expression was significantly increased in the renal cortex of the rats administered microcystin-LR and the microcystin-LR-treated proximal tubular cells. The expression levels of (pro)renin receptor (PRR), transforming growth factor-ß1 (TGFß1), and α-smooth muscle actin (α-SMA) in the renal cortex did not differ significantly between the control and microcystin-LR-administered rats. However, the expression levels of prorenin were significantly positively correlated with those of PRR, TGFß1, and α-SMA in the renal cortex of rats administered microcystin-LR. Additionally, a significant positive correlation was observed between the expression levels of TGFß1 and α-SMA. Collectively, increased prorenin expression caused by the long-term consumption of microcystin-LR may initiate a process that influences renal fibrosis and abnormal renal function by regulating the expression levels of PRR, TGFß1, and α-SMA.

Metformin relieves bone cancer pain by reducing TGFßRI-TRPV1 signaling in rats.

Common cancer complications include bone cancer pain (BCP), which was not sufficiently alleviated by traditional analgesics. More safe and effective therapy was urgent needed. Metformin relieved osteoarthritis pain, but the analgesia of Metformin in BCP was not well studied. The study aimed to explore the Metformin-mediated analgesic effect and its molecular mechanisms in BCP rats. We demonstrated that Walker 256 cell transplantation into the medullary cavity of the tibia worsened mechanical allodynia in BCP rats, increased the expression of TGFß1 in the metastatic bone tissue, and raised the expression of TGFßRI and TRPV1 in the L4-6 dorsal root ganglion (DRG) of BCP rats. While, selectively blockade of TGFßRI by SD208 could obviously elevated the paw withdraw threshold (PWT) of BCP rats, together with decreased TRPV1 expression in L4-6 DRG. Notably, continuous Metformin treatment reduced TGFß1, TGFßRI and TRPV1 expression, and relieved mechanical allodynia of BCP rats in a long-term effect. In conclusion, these results illustrated that Metformin ameliorated bone cancer pain, and the downregulation of TGFß1-TGFßRI-TRPV1 might be a potential mechanism of Metformin-mediated analgesia in BCP.

Unveiling stem-like traits and chemoresistance mechanisms in ovarian cancer cells through the TGFß1-PITX2A/B signaling axis.

Ovarian cancer (OC) is the deadliest gynecological malignancy, having a high mortality rate due to its asymptomatic nature, chemoresistance, and recurrence. However, the proper mechanistic knowledge behind these phenomena is still inadequate. Cancer recurrence is commonly observed due to cancer stem cells which also show chemoresistance. We aimed to decipher the molecular mechanism behind chemoresistance and stemness in OC. Earlier studies suggested that PITX2, a homeobox transcription factor and, its different isoforms are associated with OC progression upon regulating different signaling pathways. Moreover, they regulate the expression of drug efflux transporters in kidney and colon cancer, rendering chemoresistance properties in the tumor cell. Considering these backgrounds, we decided to look for the role of PITX2 isoforms in promoting stemness and chemoresistance in OC cells. In this study, PITX2A/B has been shown to promote stemness and to enhance the transcription of ABCB1. PITX2 has been discovered to augment ABCB1 gene expression by directly binding to its promoter. To further investigate the regulatory mechanism of PITX2 gene expression, we found that TGFß signaling could augment the PITX2A/B expression through both SMAD and non-SMAD signaling pathways. Collectively, we conclude that TGFß1-activated PITX2A/B induces stem-like features and chemoresistance properties in the OC cells.

High Glucose Increases Lactate and Induces the Transforming Growth Factor Beta-Smad 1/5 Atherogenic Pathway in Primary Human Macrophages.

Hundreds of millions of people worldwide are expected to suffer from diabetes mellitus. Diabetes is characterized as a dynamic and heterogeneous disease that requires deeper understanding of the pathophysiology, genetics, and metabolic shaping of this disease and its macro/microvascular complications. Macrophages play an essential role in regulating local immune responses, tissue homeostasis, and disease pathogenesis. Here, we have analyzed transforming growth factor beta 1 (TGFß1)/Smad signaling in primary human macrophages grown in normal (NG) and high-glucose (HG; +25 mM glucose) conditions. Cell culture lactate concentration and cellular phosphofructokinase (PFK) activity were increased in HG concentrations. High glucose levels in the growth media led to increased macrophage mRNA expression of TGFß1, and TGFß-regulated HAMP and PLAUR mRNA levels, while the expression of TGFß receptor II remained unchanged. Stimulation of cells with TGFß1 protein lead to Smad2 phosphorylation in both NG and HG conditions, while the phosphorylation of Smad1/5 was detected only in response to TGFß1 stimulation in HG conditions. The use of the specific Alk1/2 inhibitor dorsomorphin and the Alk5 inhibitor SB431542, respectively, revealed that HG conditions led TGFß1 to activation of Smad1/5 signaling and its downstream target genes. Thus, high-glucose activates TGFß1 signaling to the Smad1/5 pathway in primary human macrophages, which may contribute to cellular homeostasis in a harmful manner, priming the tissues for diabetic complications.

Core needle biopsy changes the expression of TGFß1 and TGFßRII at protein level, and the distribution of CD4 and CD8 positive T cells in primary breast cancer.

Core needle biopsy (CNB) has become a paradigm in preoperative breast cancer (BC) diagnosis. Although considered safe, it is an invasive procedure, which changes the tumor microenvironment. It facilitates a tumor supportive immune response, induces epithelial-mesenchymal transition (EMT), and enables the release of circulating tumor cells. The cytokine Transforming Growth Factor ß (TGFß) with its pleiotropic immunologic functions has an important role in this process. The aim of this study was to clarify the specific impact of CNB on the activity of the TGFß pathway in early BC. We compared formalin fixed paraffin embedded samples from CNBs to the corresponding surgical resection specimens (SRSs) of 49 patients with BC. We found that the expression of TGFß1 at protein level was significantly higher in both tumor epithelial and benign stromal cells in the SRSs (p=0.001), whereas the expression of TGFßRII in tumor cells was lower (p=0.001). The frequency of intra tumoral CD8 and CD4 positive T lymphocytes was lower in SRSs (p=0081 and p=0001, respectively), while in the peripheral stroma their prevalence was increased (p=0001 and p=0012, respectively). Our results show that CNB changes the hallmarks of the TGFß path way in early BC. These CNB-induced changes in the tumor and in its microenvironment suggest that the procedure may change the immunological anti-tumor response of the host.

GPR176 promotes fibroblast-to-myofibroblast transition in organ fibrosis progression.

Fibrosis is characterized by excessive deposition of extracellular matrix proteins, particularly collagen, caused by myofibroblasts in response to chronic inflammation. Although G protein-coupled receptors (GPCRs) are among the targets of current antifibrotic drugs, no drug has yet been approved to stop fibrosis progression. Herein, we aimed to identify GPCRs with profibrotic effects. In gene expression analysis of mouse lungs with induced fibrosis, eight GPCRs were identified, showing a >2-fold increase in mRNA expression after fibrosis induction. Among them, we focused on Gpr176 owing to its significant correlation with a myofibroblast marker α-smooth muscle actin (αSMA), the profibrotic factor transforming growth factor ß1 (TGFß1), and collagen in a human lung gene expression database. Similar to the lung fibrosis model, increased Gpr176 expression was also observed in other organs affected by fibrosis, including the kidney, liver, and heart, suggesting its role in fibrosis across various organs. Furthermore, fibroblasts abundantly expressed Gpr176 compared to alveolar epithelial cells, endothelial cells, and macrophages in the fibrotic lung. GPR176 expression was unaffected by TGFß1 stimulation in rat renal fibroblast NRK-49 cells, whereas knockdown of Gpr176 by siRNA reduced TGFß1-induced expression of αSMA, fibronectin, and collagen as well as Smad2 phosphorylation. This suggested that Gpr176 regulates fibroblast activation. Consequently, Gpr176 acts in a profibrotic manner, and inhibiting its activity could potentially prevent myofibroblast differentiation and improve fibrosis. Developing a GPR176 inverse agonist or allosteric modulator is a promising therapeutic approach for fibrosis.

Beta cells deficient for Renalase counteract autoimmunity by shaping natural killer cell activity.

Type 1 diabetes (T1D) arises from autoimmune-mediated destruction of insulin-producing pancreatic beta cells. Recent advancements in the technology of generating pancreatic beta cells from human pluripotent stem cells (SC-beta cells) have facilitated the exploration of cell replacement therapies for treating T1D. However, the persistent threat of autoimmunity poses a significant challenge to the survival of transplanted SC-beta cells. Genetic engineering is a promising approach to enhance immune resistance of beta cells as we previously showed by inactivating the Renalase (Rnls) gene. Here, we demonstrate that Rnls loss of function in beta cells shapes autoimmunity by mediating a regulatory natural killer (NK) cell phenotype important for the induction of tolerogenic antigen-presenting cells. Rnls-deficient beta cells mediate cell-cell contact-independent induction of hallmark anti-inflammatory cytokine Tgfß1 in NK cells. In addition, surface expression of regulatory NK immune checkpoints CD47 and Ceacam1 is markedly elevated on beta cells deficient for Rnls. Altered glucose metabolism in Rnls mutant beta cells is involved in the upregulation of CD47 surface expression. These findings are crucial to better understand how genetically engineered beta cells shape autoimmunity, giving valuable insights for future therapeutic advancements to treat and cure T1D.

TGFß1 Regulates Cellular Composition of In Vitro Cardiac Perivascular Niche Based on Cardiospheres.

The cardiac perivascular niche is a cellular microenvironment of a blood vessel. The principles of niche regulation are still poorly understood. We studied the effect of TGFß1 on cells forming the cardiac perivascular niche using 3D cell culture (cardiospheres). Cardiospheres contained progenitor (c-Kit), endothelial (CD31), and mural (αSMA) cells, basement membrane proteins (laminin) and extracellular matrix proteins (collagen I, fibronectin). TGFß1 treatment decreased the length of CD31+ microvasculature, VE cadherin protein level, and proportion of NG2+ cells, and increased proportion of αSMA+ cells and transgelin/SM22α protein level. We supposed that this effect is related to the stabilizing function of TGFß1 on vascular cells: decreased endothelial cell proliferation, as shown for HUVEC, and activation of mural cell differentiation.

Blockade of Sialylation with Decrease in Polysialic Acid Levels Counteracts Transforming Growth Factor ß1-Induced Skin Fibroblast-to-Myofibroblast Transition.

Aberrant sialylation with overexpression of the homopolymeric glycan polysialic acid (polySia) was recently reported in fibroblasts from fibrotic skin lesions. Yet, whether such a rise in polySia levels or sialylation in general may be functionally implicated in profibrotic activation of fibroblasts and their transition to myofibroblasts remains unknown. Therefore, we herein explored whether inhibition of sialylation could interfere with the process of skin fibroblast-to-myofibroblast transition induced by the master profibrotic mediator transforming growth factor ß1 (TGFß1). Adult human skin fibroblasts were pretreated with the competitive pan-sialyltransferase inhibitor 3-Fax-peracetyl-Neu5Ac (3-Fax) before stimulation with recombinant human TGFß1, and then analyzed for polySia expression, cell viability, proliferation, migratory ability, and acquisition of myofibroblast-like morphofunctional features. Skin fibroblast stimulation with TGFß1 resulted in overexpression of polySia, which was effectively blunted by 3-Fax pre-administration. Pretreatment with 3-Fax efficiently lessened TGFß1-induced skin fibroblast proliferation, migration, changes in cell morphology, and phenotypic and functional differentiation into myofibroblasts, as testified by a significant reduction in FAP, ACTA2, COL1A1, COL1A2, and FN1 gene expression, and α-smooth muscle actin, N-cadherin, COL1A1, and FN-EDA protein levels, as well as a reduced contractile capability. Moreover, skin fibroblasts pre-administered with 3-Fax displayed a significant decrease in Smad3-dependent canonical TGFß1 signaling. Collectively, our in vitro findings demonstrate for the first time that aberrant sialylation with increased polySia levels has a functional role in skin fibroblast-to-myofibroblast transition and suggest that competitive sialyltransferase inhibition might offer new therapeutic opportunities against skin fibrosis.

Inhibition of endogenous hydrogen sulfide production reduces activation of hepatic stellate cells via the induction of cellular senescence.

In chronic liver injury, quiescent hepatic stellate cells (HSCs) transdifferentiate into activated myofibroblast-like cells and produce large amounts of extracellular matrix components, e.g. collagen type 1. Cellular senescence is characterized by irreversible cell-cycle arrest, arrested cell proliferation and the acquisition of the senescence-associated secretory phenotype (SASP) and reversal of HSCs activation. Previous studies reported that H2S prevents induction of senescence via its antioxidant activity. We hypothesized that inhibition of endogenous H2S production induces cellular senescence and reduces activation of HSCs. Rat HSCs were isolated and culture-activated for 7 days. After activation, HSCs treated with H2S slow-releasing donor GYY4137 and/or DL-propargylglycine (DL-PAG), an inhibitor of the H2S-producing enzyme cystathionine γ-lyase (CTH), as well as the PI3K inhibitor LY294002. In our result, CTH expression was significantly increased in fully activated HSCs compared to quiescent HSCs and was also observed in activated stellate cells in a in vivo model of cirrhosis. Inhibition of CTH reduced proliferation and expression of fibrotic markers Col1a1 and Acta2 in HSCs. Concomitantly, DL-PAG increased the cell-cycle arrest markers Cdkn1a (p21), p53 and the SASP marker Il6. Additionally, the number of ß-galactosidase positive senescent HSCs was increased. GYY4137 partially restored the proliferation of senescent HSCs and attenuated the DL-PAG-induced senescent phenotype. Inhibition of PI3K partially reversed the senescence phenotype of HSCs induced by DL-PAG. Inhibition of endogenous H2S production reduces HSCs activation via induction of cellular senescence in a PI3K-Akt dependent manner. Our results show that cell-specific inhibition of H2S could be a novel target for anti-fibrotic therapy via induced cell senescence.

Intra-articular sustained-release of pirfenidone as a disease-modifying treatment for early osteoarthritis.

Osteoarthritis (OA) is a major clinical challenge, and effective disease-modifying drugs for OA are still lacking due to the complicated pathology and scattered treatment targets. Effective early treatments are urgently needed to prevent OA progression. The excessive amount of transforming growth factor ß (TGFß) is one of the major causes of synovial fibrosis and subchondral bone sclerosis, and such pathogenic changes in early OA precede cartilage damage. Herein we report a novel strategy of intra-articular sustained-release of pirfenidone (PFD), a clinically-approved TGFß inhibitor, to achieve disease-modifying effects on early OA joints. We found that PFD effectively restored the mineralization in the presence of excessive amount of TGFß1 (as those levels found in patients' synovial fluid). A monthly injection strategy was then designed of using poly lactic-co-glycolic acid (PLGA) microparticles and hyaluronic acid (HA) solution to enable a sustained release of PFD (the "PLGA-PFD + HA" strategy). This strategy effectively regulated OA progression in destabilization of the medial meniscus (DMM)- induced OA mice model, including preventing subchondral bone loss in early OA and subchondral bone sclerosis in late OA, and reduced synovitis and pain with cartilage preservation effects. This finding suggests the promising clinical application of PFD as a novel disease-modifying OA drug.