Fibrotic Gene Expression Research Articles

RUNX2 promotes fibrosis via an alveolar-to-pathological fibroblast transition.

A hallmark of pulmonary fibrosis is the aberrant activation of lung fibroblasts into pathological fibroblasts that produce excessive extracellular matrix1-3. Thus, the identification of key regulators that promote the generation of pathological fibroblasts can inform the development of effective countermeasures against disease progression. Here we use two mouse models of pulmonary fibrosis to show that LEPR+ fibroblasts that arise during alveologenesis include SCUBE2+ alveolar fibroblasts as a major constituent. These alveolar fibroblasts in turn contribute substantially to CTHRC1+POSTN+ pathological fibroblasts. Genetic ablation of POSTN+ pathological fibroblasts attenuates fibrosis. Comprehensive analyses of scRNA-seq and scATAC-seq data reveal that RUNX2 is a key regulator of the expression of fibrotic genes. Consistently, conditional deletion of Runx2 with LeprcreERT2 or Scube2creERT2 reduces the generation of pathological fibroblasts, extracellular matrix deposition and pulmonary fibrosis. Therefore, LEPR+ cells that include SCUBE2+ alveolar fibroblasts are a key source of pathological fibroblasts, and targeting Runx2 provides a potential treatment option for pulmonary fibrosis.

Folate alleviated skin inflammation and fibrosis resulting from impaired homocysteine metabolism.

Skin fibrosis, characterized by uncontrolled secretion of extracellular matrix (ECM) proteins such as collagen, can lead to excessive scarring and compromised tissue function. Despite the widespread occurrence of fibrotic diseases, effective therapies are lacking. Recent clinical studies have demonstrated a positive correlation between serum homocysteine (Hcy) levels and the severity of systemic sclerosis. However, it remains unclear whether Hcy accumulation plays a pathogenic role in skin fibrosis. Here, we report that Hcy metabolism in fibroblasts plays a crucial role in regulating the pathogenesis of skin fibrosis. Fibrotic skin fibroblasts exhibited elevated levels of Hcy due to the downregulation of catabolism genes CBS and MTR. Experimental skin fibrosis was induced and exacerbated in mouse skin fibroblasts and tissues through adenoviral knockdown of Cbs or Mtr, whereas overexpression of these catabolic genes mitigated the pathogenesis. Furthermore, exogenous Hcy supplementation induced and aggravated the expression of inflammatory and fibrotic genes, promoting both spontaneous and BLM-induced skin fibrosis. Notably, folate administration enhanced Hcy catabolism and ameliorated skin inflammation and fibrosis by inhibiting JAK2/STAT3 signaling pathway. Collectively, these results indicate that skin fibrosis is associated with Hcy metabolic disorders and suggest that targeting Hcy metabolism or supplementing folate may provide a novel strategy for skin fibrosis.

Inhibition of PKM2 by shikonin impedes TGF-β1 expression by repressing histone lactylation to alleviate renal fibrosis.

Macrophage-myofibroblast transition (MMT) plays a significant role in the progression of renal fibrosis in chronic kidney disease (CKD), making inhibition of MMT a promising therapeutic strategy. Pyruvate kinase M2 (PKM2) and its metabolite lactate are implicated in the pathogenesis of renal fibrosis; however, the mechanisms through which they contribute to this process remain poorly understood. To investigate the effects of PKM2 inhibition by shikonin on renal fibrosis and the underly mechanisms. Mice were subjected to unilateral ureteral obstruction (UUO) to establish a CKD model. Renal fibrosis was assessed using histochemistry and western blotting. The MMT and histone lactylation levels were evaluated by immunofluorescence and western blotting. The interaction between the Tgfb1 promoter and lactylated histone H3 (K18) was examined using chromatin Immunoprecipitation (ChIP). PKM2 expression was significantly elevated in the renal tubular cells of UUO mouse kidneys, resulting in increased pyruvate and lactate production. Similarly, lactate levels were elevated in TGF-β1-treated TCMK-1 cells and in the serum of CKD patients. In UUO mice, treatment with shikonin, a potent PKM2 inhibitor, effectively reduced lactate production, alleviated renal fibrosis, decreased TGF-β1 expression, and suppressed the MMT process. Mechanistic studies revealed that lactate treatment stimulates Tgfb1 expression in TCMK-1 cells. Consequently, TGF-β1 in conditioned media from lactate-treated TCMK-1 cells promoted M2 macrophage polarization and upregulated fibrotic gene expression in RAW264.7 cells. Pharmacological intervention demonstrated that TGF-β1 activates the Smad3 pathway to drive the MMT process. In TCMK-1 cells, both lactate treatment and PKM2 overexpression induced Tgfb1 expression by promoting histone H3K18 lactylation. Our findings indicate that PKM2-induced excessive lactate production renal tubular cells contributes to renal fibrosis. Lactate promotes histone lactylation, leading to TGF-β1 expression in these cells, which subsequently activates the Smad3 pathway in macrophages, driving the MMT and fibrosis in the kidney. Therefore, targeting PKM2, as with shikonin treatment, may represent an effective therapeutic strategy for managing renal fibrosis in CKD.

Coleus vettiveroides Root Extract Protects Against Thioacetamide-Induced Chronic Liver Injury by Inhibiting NF-κB Signaling Pathway.

The roots of Coleus vettiveroides (CV) have been traditionally used in Indian medicinal systems such as Ayurveda and Siddha for its antioxidant, anti-inflammatory, and antidiabetic effects. This study examines the antifibrotic potential of CV ethanolic root extract (CVERE) against thioacetamide (TAA)-induced liver fibrosis in Wistar rats. TAA was administered via i.p., thrice weekly for 11 weeks to induce liver fibrosis in rats. In separate groups, rats were administered with TAA and were concurrently treated with CVERE 125 mg/kg, CVERE 250 mg/kg, and silymarin (SIL) 100 mg/kg. Liver marker enzymes of hepatotoxicity, oxidative stress markers, proinflammatory marker gene expression (TNF-α, NF-κB, COX, and ILs), fibrotic marker gene expression (collagen I and III), immune histochemical expression of fibrosis marker proteins, and histopathologic changes were analyzed. TAA administration led to a significant (p < 0.001) increase in the serum level of hepatotoxic marker enzymes. The TAA-treated group showed higher levels (p < 0.001) of MDA and reduced activities of SOD and CAT in the liver. TAA administration increased CYP2E1 expression, proinflammatory, and fibrotic marker gene expressions in rat liver. The histopathology of the liver confirms TAA-induced architectural distortion and fibrotic changes. CVERE and SIL simultaneous treatments significantly protected against TAA-induced oxidative stress, inflammation, and liver fibrosis. In conclusion, CVERE inhibited TAA-induced liver fibrosis through downregulation of TAA metabolic activation, redox imbalance, and inflammation through repression of the NF-κB pathway.

Infiltrative classical monocyte-derived and SPP1 lipid-associated macrophages mediate inflammation and fibrosis in ANCA-associated glomerulonephritis.

Kidney macrophage infiltration is a histological hallmark of vasculitic lesions and is strongly linked to disease activity in anti-neutrophil cytoplasmic antibodies (ANCA)-associated glomerulonephritis (AGN). The precise mechanisms by which kidney macrophages influence local inflammation and long-term damage remain largely unknown. Here, we investigate kidney macrophage diversity using single-cell transcriptome analysis of 25 485 freshly retrieved unfrozen, high-quality kidney CD45+ immune cells from five AGN patients during active disease, a lupus nephritis and nephrectomy control. Detailed subclustering of myeloid cells was performed to identify disease-specific macrophage subtypes. Next, transcriptome differences between macrophage subsets and disease serotypes were assessed. Findings were validated by immunostainings of an extended cohort of kidney biopsies and flow cytometric analysis of peripheral blood monocytes. Four main macrophage subsets were identified, including a classical monocyte-derived macrophage (MDM) subset expressing a chemotactic (CXCL2, CXCL3, CXCL8, CCL3) and pro-inflammatory (IL1β, TNF) set of markers and a osteopontin/SPP1+ lipid-associated macrophage (SPP1 LAMs) subtype exhibiting distinctive upregulation of fibrotic genesets. AGN samples revealed a markedly increased proportion of CD163+ macrophages, predominantly composed of classical MDMs, accompanied by resident-like C1Q macrophages, and SPP1 LAMs An analogous trend was observed in the expansion of peripheral blood classical monocytes during active disease. The proteinase 3 (PR3)-AGN subtype exhibited heightened classical MDM and SPP1 LAM infiltration and markers of acute inflammation, while interferon signaling and markers of chronicity were reduced compared to myeloperoxidase (MPO)-AGN. Our findings highlight the expression of inflammatory and fibrotic genes by kidney macrophage subsets in AGN. Classical monocyte dysregulation might contribute to inflammation in the pathogenesis of AGN. Targeting these specific monocyte/macrophage subsets may potentially control the inflammatory cascade and attenuate resulting fibrosis in AGN and kidney disease in general.

Sulfur Amino Acid Restriction Mitigates High-Fat Diet-Induced Molecular Alterations in Cardiac Remodeling Primarily via FGF21-Independent Mechanisms.

Background/Objectives: Dietary sulfur amino acid restriction (SAAR) elicits various health benefits, some mediated by fibroblast growth factor 21 (FGF21). However, research on SAAR's effects on the heart is limited and presents mixed findings. This study aimed to evaluate SAAR-induced molecular alterations associated with cardiac remodeling and their dependence on FGF21. Methods: Male C57BL/6J wild-type and FGF21 knockout mice were randomized into four dietary regimens, including normal fat and high-fat diets (HFDs) with and without SAAR, over five weeks. Results: SAAR significantly reduced body weight and visceral adiposity while increasing serum FGF21 levels. In the heart, SAAR-induced molecular metabolic alterations are indicative of enhanced lipid utilization, glucose uptake, and mitochondrial biogenesis. SAAR also elicited opposing effects on the cardiac gene expression of FGF21 and adiponectin. Regarding cellular stress responses, SAAR mitigated the HFD-induced increase in the cardiac expression of genes involved in oxidative stress, inflammation, and apoptosis, while upregulating antioxidative genes. Structurally, SAAR did not induce alterations indicative of cardiac hypertrophy and it counteracted HFD-induced fibrotic gene expression. Overall, most alterations induced by SAAR were FGF21-independent, except for those related to lipid utilization and glucose uptake. Conclusions: Altogether, SAAR promotes cardiac alterations indicative of physiological rather than pathological remodeling, primarily through FGF21-independent mechanisms.

Clinical implications of mineralocorticoid receptor overactivation.

The mineralocorticoid receptor (MR) is a nuclear transcription factor that plays a critical role in regulating fluid, electrolytes, blood pressure, and hemodynamic stability. In conditions such as chronic kidney disease (CKD) and heart failure (HF), MR overactivation leads to increased salt and water retention, inflammatory and fibrotic gene expression, and organ injury. The MR is essential for transcriptional regulation and is implicated in metabolic, proinflammatory, and pro-fibrotic pathways. It is widely expressed in various cell types throughout the body, including the gastrointestinal tract, heart, brain, kidneys, immune cells, and vasculature. Animal studies suggest that MR activation induces oxidative stress in the kidneys and mediates renal inflammation and fibrosis. Immune cell-specific deletion of MR has shown protection against cardiac fibrosis, indicating the MR's role in pathological remodeling. In vascular smooth muscle cells, the MR regulates vascular tone and vasoconstriction. Mineralocorticoid receptor antagonists (MRAs) can be categorized based on their chemical structure as either steroidal or nonsteroidal. Steroidal MRAs (sMRA), such as spironolactone and eplerenone, have demonstrated cardiovascular benefits but are limited by hyperkalemia, gynecomastia, and sexual dysfunction. Nonsteroidal MRAs (nsMRA) have shown promise in preclinical studies and clinical trials. They offer a promising alternative by effectively blocking MR without hormone-like effects, potentially improving cardiovascular and renal disease management. Further education is necessary regarding the significance of MRA utilization in CKD and HF, balancing benefits with the risk of hyperkalemia. This risk could be mitigated by combining MRAs with potassium-binding agents. Studies are underway to explore the synergistic effects between nsMRAs and other agents, such as SGLT-2i inhibitors and Glucagon-like peptide-1 agonists, to optimize cardiorenal outcomes. Overall, MR overactivation remains a significant therapeutic target, with nsMRAs showing promise as pivotal therapies in CKD and HF management. This review highlights the evolving landscape of MR-targeted therapies, their molecular mechanisms, and clinical implications in cardiorenal diseases.

MiR-192–5p targets cell cycle regulation in diabetic kidney disease via cyclin-dependent kinase inhibitor 3

Diabetic kidney disease (DKD), a.k.a diabetic nephropathy, is a leading cause of end-stage renal disease. However, in a fair percentage of patients with type-2 diabetes, renal involvement also occurs due to non-diabetic reasons (non-diabetic kidney disease, NDKD). In this study, we identified miRNA-mRNA regulatory networks specific to human DKD pathogenesis. miRNA profiling of the renal biopsy from cases (DKD, n = 5), disease controls (T2DM with NDKD, n = 6), and non-diabetic, non-CKD controls (patients undergoing nephrectomy for renal cancer, n = 3) revealed 68 DKD-specific miRNA regulation. Sixteen target mRNAs of these DKD-miRNAs were found to have a negative association with the estimated glomerular filtration rate (eGFR) in patients with DKD. The renal gene expression and eGFR data of DKD patients (n = 10–18) in the NephroSeq database were used. Based on these findings, 11 miRNA-mRNA regulatory networks were constructed for human DKD pathogenesis. Of these, in-vitro validation of miR-192-5p- CDKN3 (Cell cycle-dependent kinase inhibitor 3) network was done as miR-192–5p exhibited a maximum number of target genes in the identified DKD regulatory networks, and CDKN3 appeared as a novel target of miR-192–5p in our study. We demonstrated that miR-192–5p overexpression or knockdown of CDKN3 attenuated high glucose-induced apoptosis, fibrotic gene expression, cell hypertrophy, and cell cycle dysregulation and improved viability of proximal tubular cells. Moreover, miR-192–5p overexpression significantly inhibited CDKN3 mRNA and protein expression in proximal tubular cells. Overall, 11 miRNA-mRNA regulatory networks were predicted for human DKD pathogenesis; among these, the association of miR-192-5p- CDKN3 network DKD pathogenesis was confirmed in proximal tubular cell culture.

Chemokine receptor CXCR7 antagonism ameliorates cardiac and renal fibrosis induced by mineralocorticoid excess

Cardiorenal fibrosis is a common feature of chronic cardiovascular disease and recent data suggests that cytokines and chemokines may also drive fibrosis. Here we tested the hypothesis that CXCR7, a highly conserved chemokine receptor, contributes to cardiac and renal fibrosis. We generated an anti-mouse CXCR7-specific monoclonal antibody (CXCR7 mAb) and tested its anti-fibrotic actions in cardiorenal fibrosis induced using the deoxycorticosterone acetate/uni-nephrectomy (DOCA-UNX) model. CXCR7 mAb treatment (10 mg/kg, twice weekly for 6 weeks) significantly attenuated the development of cardiac and renal fibrosis, and reduced fibrotic and inflammatory gene expression levels, in the absence of an effect on blood pressure. Immunohistochemical analysis demonstrated an increase in the vascular expression of CXCR7 in DOCA-UNX-treated mice. This study demonstrated that a CXCR7 mediated pathway plays a significant role in cardiac and renal fibrosis induced by DOCA-UNX treatment. Accordingly, antagonism of CXCR7 may provide a therapeutic opportunity to mitigate against fibrosis in the setting of mineralocorticoid excess.

The critical role of endoplasmic reticulum stress and the stimulator of interferon genes (STING) pathway in kidney fibrosis

Endoplasmic reticulum (ER) stress is a condition in which the ER is overwhelmed and unable to manage its protein load properly. The precise activation mechanisms and role of ER stress in kidney disease remain unclear. To study this, we performed unbiased transcriptomics analysis to demonstrate ER stress in kidneys of patients with chronic kidney disease and in mouse models of acute and chronic kidney injury (cisplatin and unilateral ureteral obstruction and reanalyzed previously published data on folic acid and mitochondrial transcription factor A(TFAM) knockout mice). Inhibiting the protein kinase RNA-like ER kinase (PERK) arm of ER stress but not activating transcription factor 6 or inositol-requiring enzyme 1, protected mice from kidney fibrosis. The stimulator of interferon genes (STING) was identified as an important upstream activator of ER stress in kidney tubule cells. STING and PERK were found to physically interact, and STING agonists induced PERK activation in kidney tubule cells. Mice with a STING activating mutation presented with ER stress and kidney fibroinflammation. We also generated mice with a tubule specific STING deletion that were resistant to ER stress and kidney fibrosis. Human kidney spatial transcriptomics highlighted a spatial correlation between STING, ER stress and fibrotic gene expression. Thus, our results indicate that STING is an important upstream regulator of PERK and ER stress in tubule cells during kidney fibrosis development.

Time-Restricted Feeding Attenuates Adipose Tissue Inflammation and Fibrosis in Mice Under Chronic Light Exposure.

Time-restricted feeding (TRF) has emerged as a promising dietary approach for improving metabolic parameters associated with obesity. However, it remains largely unclear whether TRF offers benefits for obesity related to exposure to light at night. This study examined whether lean and obese mice under chronic light exposure could benefit from TRF intervention. Six-week-old C57BL/6 male mice were fed either a low-fat diet or a high-fat diet under a 12 h light/12 h dark cycle for 6 weeks. They were then divided into three subgroups: control light, chronic 24 h light, and chronic light with a daily 10 h TRF. Chronic light exposure led to increased weight gain and higher expression of inflammatory and fibrotic markers in the adipose tissue of both lean and obese mice. It also increased hepatic triglyceride content in mice, regardless of their weight status. TRF protected both lean and obese mice from weight gain, normalized inflammatory and fibrotic gene expression, and reduced adipose tissue collagen and liver triglyceride accumulation caused by light exposure alone or in combination with obesity. These results suggest that TRF could have clinical implications for preventing obesity associated with night shift work, regardless of current weight status.

Multiscale drug screening for cardiac fibrosis identifies MD2 as a therapeutic target

Cardiac fibrosis impairs cardiac function, but no effective clinical therapies exist. To address this unmet need, we employed a high-throughput screening for antifibrotic compounds using human induced pluripotent stem cell (iPSC)-derived cardiac fibroblasts (CFs). Counter-screening of the initial candidates using iPSC-derived cardiomyocytes and iPSC-derived endothelial cells excluded hits with cardiotoxicity. This screening process identified artesunate as the lead compound. Following profibrotic stimuli, artesunate inhibited proliferation, migration, and contraction in human primary CFs, reduced collagen deposition, and improved contractile function in 3D-engineered heart tissues. Artesunate also attenuated cardiac fibrosis and improved cardiac function in heart failure mouse models. Mechanistically, artesunate targeted myeloid differentiation factor 2 (MD2) and inhibited MD2/Toll-like receptor 4 (TLR4) signaling pathway, alleviating fibrotic gene expression in CFs. Our study leverages multiscale drug screening that integrates a human iPSC platform, tissue engineering, animal models, in silico simulations, and multiomics to identify MD2 as a therapeutic target for cardiac fibrosis.

Diminished nuclear-localized β-adrenoceptor signalling activates YAP to promote kidney fibrosis in diabetic nephropathy.

Diabetic nephropathy (DN) is a leading cause of chronic kidney disease (CKD), which is characterized by mesangial matrix expansion that involves dysfunctional mesangial cells (MCs). However, the underlying mechanisms remain unclear. This study aims to delineate the spatiotemporal contribution of adrenergic signalling in diabetic kidney fibrosis to reveal potential therapeutic targets. A model of diabetic nephropathy was induced by in db/db mice. Gene expression in kidneys was profiled by RNA-seq analyses, western blot and immunostaining. Subcellular-localized fluorescence resonance energy transfer (FRET) biosensors determined adrenergic signalling microdomains in MCs. Effects of oral rolipram, a phosphodiesterase 4 (PDE4) inhibitor, on the model were measured. Our model exhibited impaired kidney function with elevated expression of adrenergic and fibrotic genes, including Adrb1, PDEs, Acta2 and Tgfβ. RNA-seq analysis revealed that MCs with dysregulated YAP pathway were crucial to the extracellular matrix secretion in kidneys from diabetic nephropathy patients. In cultured MCs, TGF-β promoted profibrotic gene transcription, which was regulated by nuclear-localized β-adrenoceptor signalling. Mechanistically, TGF-β treatment diminished nuclear-specific cAMP signalling in MCs and reduced PKA-dependent phosphorylation of YAP, leading to its activation. In parallel, db/db mouse kidneys showed increased expressions of PDE4B and PDE4D. Treatment with oral rolipram alleviated kidney fibrosis in db/db mice. Diabetic nephropathy impaired nuclear-localized β1-adrenoceptor-cAMP signalling microdomain through upregulating PDE4 expression, promoting fibrosis in MCs via PKA dephosphorylation-dependent YAP activation. Our results suggest PDE4 inhibition as a promising strategy for alleviating kidney fibrosis in diabetic nephropathy.

Biochanin A suppresses Klf6-mediated Smad3 transcription to attenuate renal fibrosis in UUO mice

BackgroundRenal fibrosis is a hallmark of chronic kidney disease (CKD). Smad3 serves as the principal transcription factor mediating the pro-fibrosis effects of TGF-β signaling in renal fibrosis. Biochanin A (BCA), a natural isoflavone, has been shown to attenuate renal fibrosis by inhibiting TGF-β signaling but the detailed mechanisms remain unresolved. This study aimed to elucidate the specific mechanisms by which BCA modulates TGF-β signaling. MethodsRenal fibrosis models were established both in vitro, using TGF-β1-stimulated mouse renal tubular TCMK1 cells, and in vivo, employing mice with unilateral ureter obstruction (UUO). RNA-seq was conducted to identify BCA-regulated genes. The AnimalTFDB4.0 database was utilized to predict transcription factors with potential binding to Smad3 promoter. The activities of TGF-β signaling and the cloned mouse Smad3 promoter were assessed using luciferase reporter assays. Plasmid transfection was performed using polyethylenimine in TCMK1 cells or ultrasound microbubbles in UUO kidneys. Gene expression was analyzed by RT-PCR, western blot, and immunohistochemistry assays. ResultsBCA significantly inhibited TGF-β signaling activity and suppressed TGF-β1-induced fibrotic gene expression in TCMK1 cells. RNA-seq and in silico analyses identified Smad3 as the key gene downregulated by BCA, while leaving Smad2 unaffected. This selective transcriptional suppression of Smad3 by BCA was validated by luciferase reporter assays using the cloned Smad3 promoter. Furthermore, transcription factor binding prediction identified that Klf6, a transcription factor downregulated by BCA, has binding potential to the Smad3 promoter and promotes Smad3 transcription. Klf6 expression was induced in TGF-β1-stimulated TCMK1 cells and UUO kidneys, but this induction was abolished upon BCA treatment. Importantly, Klf6 overexpression restored Smad3 expression and counteracted the anti-fibrosis effects of BCA in both TGF-β1-stimulated TCMK1 cells and UUO kidneys. ConclusionTGF-β-responsive Klf6 transcriptionally transactivates Smad3 expression. BCA exerts anti-renal fibrosis effects by inhibiting the Klf6-Smad3 signaling axis, underscoring its therapeutic potential in the treatment of CKD.

SP6.7 - Exploring the Mechanistic Basis of the Adipose Derived Stem Cell Secretome on Extracellular Matrix Remodelling in Radiation Induced Fibrosis

Abstract Background Radiation-induced fibrosis (RIF) is a debilitating complication following radiotherapy, characterised by fibroblast proliferation and excessive extracellular matrix (ECM) deposition, leading to functional and aesthetic impairments. Although autologous fat grafting (AFG) has shown promise in reversing RIF, the mechanisms, largely attributed to adipose-derived stem cells (ADSCs), remain elusive. This study aimed to investigate ADSCs’ and their secretome on ECM remodelling in RIF management. Methods The anti-fibrotic potential of the ADSC secretome on HDFs subjected to ionising radiation was assessed via a series of in vitro experiments. Control HDFs were compared with irradiated HDFs to assess any phenotypical changes. Irradiated HDFs were treated with ADSC pre-conditioned media (CM) or ADSC co-culture (CC). Gene expression (COL1A1, TGF-β1, CCN-2, MMP-1, MMP-3, TIMP-1, ACTA2, TNC) was assessed via real-time quantitative PCR (RT-qPCR). ProCollagen1a1 and CCN2 protein concentrations were assessed by ELISA. Results Irradiated HDFs demonstrated changes in fibrotic gene expression. ADSC-CM treatment significantly reduced COL1A1 expression, although protein levels remained unchanged. TGF-β1, CCN2, MMP-3, TIMP-1 and ACTA-2 exhibited reduced expression post-ADSC-CM treatment whereas TNC expression was found to be significantly increased. Co-culture with ADSCs led to a general, but not statistically significant, decrease in pro-fibrotic gene expression. Notably, CCN-2 protein expression significantly increased. Conclusion This study demonstrates that ionising radiation results in a pathological pro-fibrotic phenotype in HDFs. The ADSC secretome potentially mitigates this, reducing pro-fibrotic gene expression. Unexpectedly, co-culture experiments highlighted a complex interaction with upregulation of fibrosis-associated genes. Further research is needed to clarify these interactions to improve therapeutic interventions for RIF.

Desmoplakin mutations in cardiac fibroblasts cause TGFβ1-mediated pathological fibrogenesis in desmoplakin cardiomyopathy via beclin-1 regulation.

Pathological fibrosis is a major finding in cardiovascular diseases and can result in arrhythmia and heart failure. Desmosome gene mutations can lead to arrhythmogenic cardiomyopathy (ACM). Among ACM, pathogenic desmoplakin ( DSP ) variants cause a distinctive cardiomyopathy with excessive cardiac fibrosis that could precede ventricular dysfunction. DSP variants are also linked to other fibrotic diseases. Whether DSP plays any role in pathological fibrosis remain unknown. Mesenchymal stromal cells (MSCs) are resident fibroblast-like cells that are responsible for fibrogenesis in most organs, including hearts. We first used unbiased genome-wide analyses to generate cardiac fibroblasts-like, induced pluripotent stem cell-derived MSCs from normal donors and ACM patients with DSP mutations. We then studied the fibrogenic responses of cardiac MSCs to transforming growth factor beta-1 (TGF-β1) using Western/Co-IP, autophagy assay, gene knockdowns/over-expressions, genomic analyses, mouse DSP knockdown models, immunostaining, and qPCR. TGFβ1 induced excessive accumulations of vimentin (VIM)/fibrillar collagens, and over-activated fibrotic genes in DSP- mutant MSCs when compared to normal MSCs. In normal MSCs, VIMs bind to wild-type DSP during normal fibrogenesis after TGFβ1. DSP- mutant MSCs exhibited a haplo-insufficient phenotype with increased DSP-unbound VIMs that sequestered beclin-1 (BECN1) from activating autophagy and caveolin-1 (CAV1)-mediated endocytosis. Decreased autophagy caused collagen accumulations and diminished CAV1 endocytosis resulted in abnormal CAV1 plaque formation that over-activated fibrotic genes [ COL1A1, COL3A1, and fibronectin ( FN )] via heightened p38 activities after TGFβ1. Genome-wide analysis and DSP knockdown in mouse fibroblasts confirmed this novel role of DSP mutations in pathological fibrosis. Overexpression of VIM-binding domains of DSP could suppress pathological fibrosis by increasing collagen autophagic degradation and decreasing fibrotic gene expressions. Our data reveal that DSP deficiency in MSCs/fibroblasts leads to exaggerated fibrogenesis in DSP-cardiomyopathy by decreasing BECN1 availability for autophagy and CAV1-endocytosis. Overexpression of VIM binding domains of DSP could be a new strategy to treat pathological fibrosis.

Hepatocyte MMP14 mediates liver and inter-organ inflammatory responses to diet-induced liver injury.

The matrix metalloproteinase MMP14 is a ubiquitously expressed, membrane-bound, secreted endopeptidase that proteolyzes substrates to regulate development, signaling, and metabolism. However, the spatial and contextual events inciting MMP14 activation and its metabolic sequelae are not fully understood. Here, we introduce an inducible, hepatocyte-specific MMP14-deficient model (MMP14LKO mice) to elucidate cell-intrinsic and systemic MMP14 function. We show that hepatocyte MMP14 mediates diet-induced body weight gain, peripheral adiposity, and impaired glucose homeostasis and drives diet-induced liver triglyceride accumulation and induction of hepatic inflammatory and fibrotic gene expression. Single-nucleus RNA sequencing revealed that hepatocyte MMP14 mediates Kupffer cell and T-cell accumulation and promotes diet-induced hepatocellular subpopulation shifts toward protection against lipid absorption. MMP14 co-immunoprecipitation and proteomic analyses revealed MMP14 substrate binding across both inflammatory and cytokine signaling, as well as metabolic pathways. Strikingly, hepatocyte MMP14 loss-of-function suppressed skeletal muscle and adipose inflammation in vivo, and in a reductionist adipose-hepatocyte co-culture model. Finally, we reveal that trehalose-type glucose transporter inhibitors decrease hepatocyte MMP14 gene expression and nominate these inhibitors as translatable therapeutic metabolic agents. We conclude that hepatocyte MMP14 drives liver and inter-organ inflammatory and metabolic sequelae of obesogenic dietary insult. Modulating MMP14 activation and blockade thus represents a targetable node in the pathogenesis of hepatic inflammation.

Titanium nanoparticles released from orthopedic implants induce muscle fibrosis via activation of SNAI2

Titanium alloys represent the prevailing material employed in orthopedic implants, which are present in millions of patients worldwide. The prolonged presence of these implants in the human body has raised concerns about possible health effects. This study presents a comprehensive analysis of titanium implants and surrounding tissue samples obtained from patients who underwent revision surgery for therapeutic reasons. The surface of the implants exhibited nano-scale corrosion defects, and nanoparticles were deposited in adjacent samples. In addition, muscle in close proximity to the implant showed clear evidence of fibrotic proliferation, with titanium content in the muscle tissue increasing the closer it was to the implant. Transcriptomics analysis revealed SNAI2 upregulation and activation of PI3K/AKT signaling. In vivo rodent and zebrafish models validated that titanium implant or nanoparticles exposure provoked collagen deposition and disorganized muscle structure. Snai2 knockdown significantly reduced implant-associated fibrosis in both rodent and zebrafish models. Cellular experiments demonstrated that titanium dioxide nanoparticles (TiO2 NPs) induced fibrotic gene expression at sub-cytotoxic doses, whereas Snai2 knockdown significantly reduced TiO2 NPs-induced fibrotic gene expression. The in vivo and in vitro experiments collectively demonstrated that Snai2 plays a pivotal role in mediating titanium-induced fibrosis. Overall, these findings indicate a significant release of titanium nanoparticles from the implants into the surrounding tissues, resulting in muscular fibrosis, partially through Snai2-dependent signaling.Graphical

The Kv4 potassium channel modulator NS5806 attenuates cardiac hypertrophy in vivo and in vitro

The compound NS5806 is a Kv4 channel modulator. This study investigated the chronic effects of NS5806 on cardiac hypertrophy induced by transverse aortic constriction (TAC) in mice in vivo and on neonatal rat ventricular cardiomyocyte hypertrophy induced by endothelin-1 (ET-1) in vitro. Four weeks after TAC, NS5806 was administered by gavage for 4 weeks. Echocardiograms revealed pronounced left ventricular (LV) hypertrophy in TAC-treated mice compared with sham mice. NS5806 attenuated LV hypertrophy, as manifested by the restoration of LV wall thickness and weight and the reversal of contractile dysfunction in TAC-treated mice. NS5806 also blunted the TAC-induced increases in the expression of cardiac hypertrophic and fibrotic genes, including ANP, BNP and TGF-β. Electrophysiological recordings revealed a significant prolongation of action potential duration and QT intervals, accompanied by an increase in susceptibility to ventricular arrhythmias in mice with cardiac hypertrophy. However, NS5806 restored these alterations in electrical parameters and thus reduced the incidence of mouse sudden death. Furthermore, NS5806 abrogated the downregulation of the Kv4 protein in the hypertrophic myocardium but did not influence the reduction in Kv4 mRNA expression. In addition, NS5806 suppressed in vitro cardiomyocyte hypertrophy. The results provide novel insight for further ion channel modulator development as a potential treatment option for cardiac hypertrophy.

Hic-5 antisense oligonucleotide inhibits advanced hepatic fibrosis and steatosis in vivo

Background & AimsChronic liver diseases, including metabolic dysfunction-associated steatohepatitis (MASH), pose a significant global health burden. Progressive liver fibrosis can lead to severe outcomes; however, there is a lack of effective therapies targeting advanced fibrosis. Hydrogen peroxide-inducible clone-5 (Hic-5), an adaptor protein in focal adhesion, is critical for promoting liver fibrosis in hepatic stellate cells. This study investigated its clinical applicability by examining hepatic Hic-5 expression in human fibrotic tissues, exploring its association with MASH, and assessing the therapeutic potential of antisense oligonucleotides (ASOs) targeting Hic-5 in a MASH mouse model. MethodsHepatic Hic-5 expression in human fibrotic tissues underwent pathological image analysis and single-cell RNA sequencing. ASOs targeting Hic-5 were developed and tested using in vitro cell models. An in vivo MASH mouse model was used to evaluate the effects of anti-Hic-5 ASOs on advanced fibrosis and steatosis. ResultsHepatic Hic-5 expression increased with the progression of fibrosis, particularly in advanced stages. Single-cell RNA sequencing revealed Hic-5 expression primarily in hepatic stellate cells. In MASH-associated fibrosis, Hic-5 expression correlated with the expression of fibrotic genes. In the MASH mouse model, hepatic Hic-5 expression increased with disease progression. Anti-Hic-5 ASOs effectively suppressed Hic-5 expression in vitro and attenuated advanced fibrosis and steatosis in vivo, indicating their therapeutic potential. ConclusionsHepatic Hic-5 expression is associated with advanced liver fibrosis and MASH. Anti-Hic-5 ASOs are promising therapeutic interventions for MASH accompanied by advanced fibrosis. These findings provide valuable insights into potential clinical treatments for advanced liver fibrosis. Impact and implicationsThis study investigated the role of Hic-5 in liver fibrosis and steatohepatitis, highlighting its potential as a therapeutic target. We developed an antisense oligonucleotide (ASO) that was particularly transportable to the liver, and targeted Hic-5. Anti-Hic-5 ASO exhibited therapeutic efficacy for liver fibrosis and steatosis in vivo, indicating its therapeutic potential for liver fibrosis and steatosis. ASOs have already achieved dramatic therapeutic effects as approved nucleic acid drugs. Thus, anti-Hic-5 ASO is expected to lead the direct generation of seed compounds for the clinical development of drugs for liver fibrosis and steatosis.