Profibrotic Responses Research Articles

Takotsubo syndrome induced oxidative stress and endothelial dysfunction in left ventricle of rat model: association with NADPH oxidases/SGLT2 pro- oxidant pathway

Abstract Background Takotsubo syndrome (TTS) is characterized by apical ballooning predominantly affecting post-menopausal women following extreme physical and/or emotional stress. The phenotype of TTS is associated with akinetic apex and hyperkinetic base of the left ventricle (LV). Presently, there are no targeted therapies for TTS that can effectively reduce LV impairment. Empagliflozin (EMPA), an SGLT2 inhibitor, has shown considerable clinical benefits in managing heart failure. However, its specific effects and underlying mechanisms in treating TTS remain unclear. Purpose To explore the pathophysiology of TTS in the LV of rats, with a particular focus on the potential role of SGLT2, and to elucidate the underlying mechanisms involved. Methods Female Sprague Dawley rats (250-300 g) were allocated into four groups: a control group (n=8), a group treated with EMPA (30 mg/kg/day) (n=8), a group receiving ISO (Isoproterenol) at 100 mg/kg/day (n=18), and a group receiving both ISO and EMPA (IE) at 30 mg/kg/day (n=18). EMPA was administered orally through food two weeks prior to ISO injection. Echocardiography was conducted at 6 and 24 h, and on day 3. On day 3, rats were euthanized, and their organs were collected. Gene expression of markers was assessed using RT-qPCR, protein expression via immunofluorescence (IF) staining, and oxidative stress using dihydroethidium. Results In the ISO group, the success rate of induced Takotsubo syndrome (TTS) was 53% (10 out of 18 rats), with a mortality rate of 5%. Conversely, in the EMPA-treated group, TTS occurred in 22% of rats (4 out of 18) with no mortality observed. Significant reductions in the area of left ventricular (LV) ballooning and ejection fraction were noted in the IE group compared to the ISO group. Additionally, increased levels of reactive oxygen species (ROS) were observed in the LV of ISO rats, which were normalized by EMPA treatment in the IE rats. Furthermore, the ISO group exhibited higher protein expressions of CD68, SGLT2, and VCAM-1, which were attenuated by EMPA treatment. The mRNA expression of CYBA (p22phox) and NOX4, both representing subunits of NADPH oxidases, along with VCAM1, ICAM1, NOS2 (iNOS) and SELE (e-selectin) gene, endothelial activation marker, and TGF-β1, a fibrotic marker, showed elevated levels in the apex part of the left ventricle (LV) in the ISO group; these levels were significantly lower in the IE group. Conclusions These findings suggest that TTS is associated with oxidative stress-mediated endothelial dysfunction, inflammation, and pro-fibrotic responses. Moreover, SGLT2 inhibition appears to mitigate the pathology and progression of the syndrome. Therefore, SGLT2 inhibition emerges as an appealing and novel therapeutic approach for the treatment of TTS.

Chromatin remodelling drives immune cell-fibroblast communication in heart failure.

Chronic inflammation and tissue fibrosis are common responses that worsen organ function, yet the molecular mechanisms governing their cross-talk are poorly understood. In diseased organs, stress-induced gene expression changes fuel maladaptive cell state transitions1 and pathological interaction between cellular compartments. Although chronic fibroblast activation worsens dysfunction in the lungs, liver, kidneys and heart, and exacerbates many cancers2, the stress-sensing mechanisms initiating transcriptional activation of fibroblasts are poorly understood. Here we show that conditional deletion of the transcriptional co-activator Brd4 in infiltrating Cx3cr1+ macrophages ameliorates heart failure in mice and significantly reduces fibroblast activation. Analysis of single-cell chromatin accessibility and BRD4 occupancy in vivo in Cx3cr1+ cells identified a large enhancer proximal to interleukin-1β (IL-1β, encoded by Il1b), and a series of CRISPR-based deletions revealed the precise stress-dependent regulatory element that controls Il1b expression. Secreted IL-1β activated a fibroblast RELA-dependent (also known as p65) enhancer near the transcription factor MEOX1, resulting in a profibrotic response in human cardiac fibroblasts. In vivo, antibody-mediated IL-1β neutralization improved cardiac function and tissue fibrosis in heart failure. Systemic IL-1β inhibition or targeted Il1b deletion in Cx3cr1+ cells prevented stress-induced Meox1 expression and fibroblast activation. The elucidation of BRD4-dependent cross-talk between a specific immune cell subset and fibroblasts through IL-1β reveals how inflammation drives profibrotic cell states and supports strategies that modulate this process in heart disease and other chronic inflammatory disorders featuring tissue remodelling.

Structure-function analysis of time-resolved immunological phases in metabolic dysfunction-associated fatty liver disease (MASH) comparing the NIF mouse model to human MASH

Metabolic dysfunction-associated steatohepatitis (MASH) is a common but frequently unrecognized complication of obesity and type 2 diabetes. The association between these conditions is multifaceted and involves complex interactions between metabolic, inflammatory, and genetic factors. Here we assess the underlying structural and molecular processes focusing on the immunological phase of MASH in the nonobese inflammation and fibrosis (NIF) mouse model and compare it to the human disease as well as other murine models. Histopathology together with synchrotron-radiation-based x-ray micro-computed tomography (SRµCT) was used to investigate structural changes within the hepatic sinusoids network in the NIF mouse in comparison to patients with different severities of MASH. A time-course, bulk RNA-sequencing analysis of liver tissue from NIF mice was performed to identify the dynamics of key processes associated with the pathogenesis. Transcriptomics profiling of the NIF mouse revealed a gradual transition from an initially reactive inflammatory response to a regenerative, pro-fibrotic inflammatory response suggesting new avenues for treatment strategies that focus on immunological targets. Despite the lack of metabolic stress induced liver phenotype, a large similarity between the NIF mouse and the immunological phase of human MASH was detected. The translational value was further supported by the comparative analyses with MASH patients and additional animal models. Finally, the impact of diets known to induce metabolic stress, was explored in the NIF mouse. An obesogenic diet was found to induce key physiological, metabolic, and histologic changes akin to those observed in human MASH.

Targeting CD13/aminopeptidase N as a novel therapeutic approach for scleroderma fibrosis.

Systemic sclerosis (SSc) is an autoimmune multisystem disease with poorly understood pathogenesis and ineffective treatment options. Soluble CD13 (sCD13), generated by cleavage of cell surface CD13 via matrix metalloproteinase 14 (MMP14), signals through the bradykinin receptor B1 (B1R) to elicit pro-inflammatory, pro-arthritic, and pro-angiogenic responses. In this study we explored the anti-fibrotic potential of targeting the sCD13-B1R axis in SSc. The expression of CD13, B1R and MMP14 was examined in SSc skin and explanted dermal fibroblasts. The efficacy of B1R antagonists in the inhibition on fibrosis was determined in vitro and in vivo. Expression of the genes for CD13, B1R and MMP14 was elevated in skin biopsies from patients with diffuse cutaneous (dc)SSc. Notably, single cell analysis of SSc skin biopsies revealed the highest BDKRB1 expression in COL8A1-positive myofibroblasts, a population exclusively seen in SSc. TGF-β induced the expression of BDKRB1 and production of sCD13 by dcSSc skin fibroblasts. Treatment of dcSSc fibroblasts with sCD13 promoted fibrotic gene expression, signaling, cell proliferation, migration, and gel contraction. The profibrotic sCD13 or TGFβ responses were prevented by a B1R antagonist. Mice lacking Cd13 or Bdkrb1 were resistant to bleomycin-induced skin fibrosis and inflammation. Pharmacological B1R inhibition had a comparable antifibrotic effect. These results are the first to demonstrate a key role for sCD13 in SSc skin fibrosis, and suggest that targeting the sCD13-B1R signaling axis is a promising novel therapeutic approach for SSc.

Abstract P100: TRPM7 deficiency amplifies aldosterone signalling cardiac fibroblasts

Introduction: Transient Receptor Potential Melastatin 7 (TRPM7) channel is a channel bound to a kinase domain that influences cellular Mg 2+ with an important role in inflammatory response and cell metabolism. TRPM7 is regulated by aldosterone. Here, we investigated the importance of TRPM7-Mg2+ in pro-fibrotic responses and oxidative stress in cardiac fibroblasts (CF) in the context of hypertension and organ damage induced by aldosterone. Methods: Wild-type (WT) and TRPM7-deficient (M7+/Δ) mice were treated with aldosterone (600µg/Kg/day) plus NaCl (1% drinking water), 4 weeks. Mechanisms were investigated in primary culture of CF. Ca 2+ and Mg 2+ influx was assessed by fluorescence microscopy and flow cytometry. Gene and protein expression was assessed by real-time PCR and immunoblotting. Proteomic analysis was performed in two-dimensional liquid chromatography and mass spectrometry (LC/LC-MS/MS). Results: M7+/Δ mice exhibited reduced tissue [Mg 2+ ] (28%) and increased cardiac collagen deposition (2-fold). Aldosterone-induced fibrosis and hypertension were enhanced in M7+/Δ. CF M7+/Δ had reduced aldosterone-induced calcium influx (136±6 vs WT:166±7) and Mg 2+ influx (1.05±0.05 vs WT 1.14±0.02). CF M7+/Δ exhibit increased expression of calpain-1(300%), α-SMA(80%), collagen-1 (50%), mineralocorticoid receptor target proteins ENaC(70%) and SGK1(30%) and reduced proliferation(33%) vs WT(p<0.05). Aldosterone reduced TRPM7 (44%) and increased expression of ENaC (150%) and collagen-1 (93%) in CF WT to similar levels observed in CF M7+/Δ. CF M7+/Δ exhibit increased Nox2(70%), Nox4(120%) and ROS production (1,362 vs WT: 899 RLU/mg protein). Increased mRNA for the antioxidant catalase, NQO1 and Prdx1 (1.7-2.6-fold) was found in M7+/Δ CF. Mg 2+ supplementation normalised Ca 2+ and Mg 2+ influx, proliferation and expression of SGK1, Nox4, catalase and NQO1, whereas other effects might be mediated by the kinase domain. Proteomic analysis showed that CF M7+/Δ exhibit reduced expression of proteins related to cell migration and proliferation (Tnc, Cdcc80, Crip1, Ggt5) cell cycle and differentiation (AnxA8, Rpl22, Prickle2) and cell metabolism (Marcks, Mtab, Ces1d, Serpina3n, Afap1l1). Conclusion: Our findings demonstrate that in aldosterone-treated mice TRPM7 expression and tissue Mg 2+ levels are decreased. These processes are associated with cardiac fibrosis and oxidative stress. Our findings suggest a cardio-protective role for TRPM7/Mg2+ in aldosterone-induced hypertension.

Knockdown of nicotinamide N-methyltransferase ameliorates renal fibrosis caused by ischemia-reperfusion injury and remodels sphingosine metabolism.

CKD currently affects 8.2% to 9.1% of the global population and the CKD mortality rate has increased during recent decades, making it necessary to identify new therapeutic targets. This study investigated the role of nicotinamide N-methyltransferase (NNMT) in renal fibrosis following ischemia-reperfusion injury (IRI), a key factor in chronic kidney disease (CKD) progression. We established a mouse model with a knockdown of NNMT to investigate the impact of this enzyme on renal fibrosis after unilateral IRI. We then utilized histology, immunohistochemistry, and metabolomic analyses to investigate fibrosis markers and sphingolipid metabolism in NNMT-deficient mice. We also utilized an Nnmt lentivirus interference vector or an Nnmt overexpression plasmid to transfect mouse kidney proximal tubule cells, stimulated these cells with TGF-β1, and then measured the pro-fibrotic response and the expression of the methylated and unmethylated forms of Sphk1. The results demonstrated that reducing NNMT expression mitigated fibrosis, inflammation, and lipid deposition, potentially through the modulation of sphingolipid metabolism. Histology, immunohistochemistry, and metabolomic analyses provided evidence of decreased fibrosis and enhanced sphingolipid metabolism in NNMT-deficient mice. NNMT mediated the TGF-β1-induced pro-fibrotic response, knockdown of Nnmt decreased the level of unmethylated Sphk1 and increased the level of methylated Sphk1 in renal tubular epithelial cells. Our findings suggest that NNMT functions in sphingolipid metabolism and has potential as a therapeutic target for CKD. Further research is needed to elucidate the mechanisms linking NNMT to sphingolipid metabolism and renal fibrosis.

Meis1 Targets Protein Tyrosine Phosphatase Receptor J in Fibroblast to Retard Chronic Kidney Disease Progression.

Renal fibrosis is a common pathological feature of chronic kidney disease (CKD) with the proliferation and activation of myofibroblasts being definite effectors and drivers. Here, increased expression of Meis1 (myeloid ecotropic viral integration site 1) is observed, predominantly in the nucleus of the kidney of CKD patients and mice, and negatively correlates with serum creatinine. Fibroblast-specific knock-in of Meis1 inhibits myofibroblast activation and attenuates renal fibrosis and kidney dysfunction in CKD models. Overexpression of Meis1 in NRK-49F cells suppresses the pro-fibrotic response induced by transforming growth factor-β1 but accelerates by its knockdown. Mechanistically, Meis1 targets protein tyrosine phosphatase receptor J (Ptprj) to block renal fibrosis by inhibiting the proliferation and activation of fibroblasts. Finally, a new activator of Ptprj is identified through computer-aided virtual screening, which has the effect of alleviating renal fibrosis. Collectively, these results illustrate that the Meis1/Ptprj axis has therapeutic potential for clinically treating CKD.

Generation of a conditional cellular senescence model using proximal tubule cells and fibroblasts from human kidneys

Emerging evidence highlights cellular senescence’s pivotal role in chronic kidney disease (CKD). Proximal tubule epithelial cells (PTECs) and fibroblasts are major players in CKD and serve as cellular sources of senescence. The generation of a conditionally immortalized human kidney cell model would allow to better understand the specific mechanisms and factors associated with cellular senescence in a controlled setting, devoid of potential confounding factors such as age and comorbidities. In addition, the availability of human kidney cell lines for preclinical research is sparse and most cell lines do not reflect their in vivo counterparts due to their altered behavior as immortalized cancer-like cells. In this study, PTECs and fibroblasts from human kidneys were isolated and transduced with doxycycline-inducible simian virus 40 large T antigen (SV40LT) vector. By comparing their gene expression with single-cell RNA sequencing data from human kidneys, the newly produced human kidney cell lines demonstrated significant resemblances to their in vivo counterparts. As predicted, PTECs showed functional activity and fibroblasts responded to injury with fibrosis. Withdrawal of the immortalizing factor doxycycline led to p21+ cell-cycle arrest and the key hallmarks of senescence. The obtained senescence gene set largely overlapped between both cell lines and with the previously published SenMayo set of senescence-associated genes. Furthermore, crosstalk experiments showed that senescent PTECs can cause a profibrotic response in fibroblasts by paracrine actions. In 76 human kidney sections, the number of p21+ cells correlated with the degree of fibrosis, age and reduced glomerular filtration, validating the role of senescence in CKD. In conclusion, we provide a novel cellular ex vivo model to study kidney senescence which can serve as a platform for large scale compounds testing.

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

TGFβ1 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 TGFβ. Our work investigates the potential of blocking the ‘splicing in’ of EDA with antisense oligonucleotides to inhibit TGFβ1-induced EDA + fibronectin and to prevent the cascade of events initiated by TGFβ1 in human renal proximal tubule cells (PTEC).Human primary PTEC were treated with TGFβ1 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 TGFβ, 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 TGFβ1 -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 TGFβ1 induced endogenous TGFβ, αSMA, MMP2, MMP9 and Col I mRNA. TGFβ1 treatment for 48h reduced the expression of K-Cadherin and increased the expression of connexin-43. These TGFβ1-induced pro-fibrotic changes were attenuated by ASO5 treatment. 48 h after the removal of exogenous TGFβ, 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 TGFβ1 was confirmed by the use of a TGFβ receptor inhibitor.Our results demonstrate a critical role of FN EDA+ in a cycle of TGFβ 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.

Mechanosensitive Piezo1 channels promote neurogenic bladder fibrosis via regulating TGF-β1/smad and Hippo/YAP1 pathways

Bladder fibrosis is the final common pathway of neurogenic bladder (NB), and its underlying mechanisms are not fully understood. The current study aims to evaluate the involvement of Piezo1, a mechanosensitive channel, in bladder fibrosis. A full-thickness bladder specimen was taken during ileocystoplasty or ureteral reimplantation from the surgical cut's edge. By chopping off the bilateral lumbar 6 (L6) and sacral 1 (S1) spinal nerves, NB rat models were produced. Utilizing both pharmacological inhibition and Piezo1 deletion, the function of Piezo1 in the TGF-β1-induced fibrosis model of SV-HUC-1 cells was delineated. RNA-seq, immunofluorescence, immunohistochemistry (IHC), and Western blotting were used to evaluate the degrees of fibrosis and biochemical signaling pathways. Piezo1 protein expression was noticeably elevated in the human NB bladder. The abundance of Piezo1 protein in bladder of NB rats was significantly increased. RNA-seq analysis revealed that the ECM-receptor interaction signaling pathway and collagen-containing ECM were increased in spinal cord injury (SCI)-induced bladder fibrosis. Moreover, the bladder of the NB rat model showed activation of YAP1 and TGF-β1/Smad. In SV-HUC-1 cells, siRNA suppression of Piezo1 led to profibrotic responses and activation of the TGF-β1/Smad pathway. However, Yoda1, a Piezo1-specific agonist, significantly reduced these effects. TGF-β1 increased Piezo1 activation and profibrotic responses in SV-HUC-1 cells. In the TGF-β1-induced fibrosis model of SV-HUC-1 cells, the TGF-β1/Smad pathway was activated, whereas the Hippo/YAP1 signal pathway was blocked. Inhibition of Piezo1 further prevented this process. Piezo1 is involved in the progression of NB bladder fibrosis and profibrotic alterations in SV-HUC-1 cells, likely through regulating the TGF-β1/Smad and Hippo/YAP1 pathways.

Perinatal asphyxia leads to acute kidney damage and increased renal susceptibility in adulthood.

Perinatal asphyxia (PA) poses a significant threat to multiple organs, particularly the kidneys. Diagnosing PA-associated kidney injury remains challenging, and treatment options are inadequate. Furthermore, there is a lack of long-term follow-up data regarding the renal implications of PA. In this study, 7-day-old male Wistar rats were exposed to PA using a gas mixture (4% O2; 20% CO2 in N2 for 15 min) to investigate molecular pathways linked to renal tubular damage, hypoxia, angiogenesis, heat shock response, inflammation, and fibrosis in the kidney. In a second experiment, adult rats with a history of PA were subjected to moderate renal ischemia-reperfusion (IR) injury to test the hypothesis that PA exacerbates renal susceptibility. Our results revealed an increased gene expression of renal injury markers (kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin), hypoxic and heat shock factors (hypoxia-inducible factor-1α, heat shock factor-1, and heat shock protein-27), proinflammatory cytokines (interleukin-1β, interleukin-6, tumor necrosis factor-α, and monocyte chemoattractant protein-1), and fibrotic markers (transforming growth factor-β, connective tissue growth factor, and fibronectin) promptly after PA. Moreover, a machine learning model was identified through random forest analysis, demonstrating an impressive classification accuracy (95.5%) for PA. Post-PA rats showed exacerbated functional decline and tubular injury and more intense hypoxic, heat shock, proinflammatory, and profibrotic response after renal IR injury compared with controls. In conclusion, PA leads to subclinical kidney injury, which may increase the susceptibility to subsequent renal damage later in life. In addition, the parameters identified through random forest analysis provide a robust foundation for future biomarker research in the context of PA.NEW & NOTEWORTHY This article demonstrates that perinatal asphyxia leads to subclinical kidney injury that permanently increases renal susceptibility to subsequent ischemic injury. We identified major molecular pathways involved in perinatal asphyxia-induced renal complications, highlighting potential targets of therapeutic approaches. In addition, random forest analysis revealed a model that classifies perinatal asphyxia with 95.5% accuracy that may provide a strong foundation for further biomarker research. These findings underscore the importance of multiorgan follow-up for perinatal asphyxia-affected patients.

Adverse cardiac events of hypercholesterolemia are enhanced by sitagliptin in sprague dawley rats

BackgroundCardiovascular disease (CVD) affects millions worldwide and is the leading cause of death among non-communicable diseases. Western diets typically comprise of meat and dairy products, both of which are rich in cholesterol (Cho) and methionine (Met), two well-known compounds with atherogenic capabilities. Despite their individual effects, literature on a dietary combination of the two in the context of CVD are limited. Therefore, studies on the combined effects of Cho and Met were carried out using male Sprague Dawley rats. An additional interest was to investigate the cardioprotective potential of sitagliptin, an anti-type 2 diabetic drug. We hypothesized that feeding a dietary combination of Cho and Met would result in adverse cardiac effects and would be attenuated upon administration of sitagliptin.MethodsAdult male Sprague-Dawley rats were fed either a control (Con), high Met (1.5%), high Cho (2.0%), or high Met (1.5%) + high Cho (2.0%) diet for 35 days. They were orally gavaged with an aqueous preparation of sitagliptin (100 mg/kg/d) or vehicle (water) from day 10 through 35. On day 36, rats were euthanized, and tissues were collected for analysis.ResultsHistopathological evaluation revealed a reduction in myocardial striations and increased collagen deposition in hypercholesterolemia (HChol), responses that became exacerbated upon sitagliptin administration. Cardiac pro-inflammatory and pro-fibrotic responses were adversely impacted in similar fashion. The addition of Met to Cho (MC) attenuated all adverse structural and biochemical responses, with or without sitagliptin.ConclusionsAdverse cardiac outcomes in HChol were enhanced by the administration of sitagliptin, and such effects were alleviated by Met. Our findings could be significant for understanding or revisiting the risk-benefit evaluation of sitagliptin in type 2 diabetics, and especially those who are known to consume atherogenic diets.

Chemically Defined Organoid Culture System for Cholangiocyte Differentiation.

Cholangiocyte organoids provide a powerful platform for applications ranging from in vitro modeling to tissue engineering for regenerative medicine. However, their expansion and differentiation are typically conducted in animal-derived hydrogels, which impede the full maturation of organoids into functional cholangiocytes. In addition, these hydrogels are poorly defined and complex, limiting the clinical applicability of organoids. In this study, a novel medium composition combined with synthetic polyisocyanopeptide (PIC) hydrogels to enhance the maturation of intrahepatic cholangiocyte organoids (ICOs) into functional cholangiocytes is utilized. ICOs cultured in the presence of sodium butyrate and valproic acid, a histone deacetylase inhibitor, and a Notch signaling activator, respectively, in PIC hydrogel exhibit a more mature phenotype, as evidenced by increased expression of key cholangiocyte markers, crucial for biliary function. Notably, mature cholangiocyte organoids in PIC hydrogel display apical-out polarity, in contrast to the traditional basal-out polarization of ICOs cultured in Matrigel. Moreover, these mature cholangiocyte organoids effectively model the biliary pro-fibrotic response induced by transforming growth factor beta. Taken together, an animal-free, chemically defined culture system that promotes the ICOs into mature cholangiocytes with apical-out polarity, facilitating regenerative medicine applications and in vitro studies that require access to the apical membrane, is developed.

Comparative single-cell analysis reveals IFN-γ as a driver of respiratory sequelae after acute COVID-19.

Postacute sequelae of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection (PASC) represent an urgent public health challenge and are estimated to affect more than 60 million individuals globally. Although a growing body of evidence suggests that dysregulated immune reactions may be linked with PASC symptoms, most investigations have primarily centered around blood-based studies, with few focusing on samples derived from affected tissues. Furthermore, clinical studies alone often provide correlative insights rather than causal mechanisms. Thus, it is essential to compare clinical samples with relevant animal models and conduct functional experiments to understand the etiology of PASC. In this study, we comprehensively compared bronchoalveolar lavage fluid single-cell RNA sequencing data derived from clinical PASC samples and a mouse model of PASC. This revealed a pro-fibrotic monocyte-derived macrophage response in respiratory PASC, as well as abnormal interactions between pulmonary macrophages and respiratory resident T cells, in both humans and mice. Interferon-γ (IFN-γ) emerged as a key node mediating the immune anomalies in respiratory PASC. Neutralizing IFN-γ after the resolution of acute SARS-CoV-2 infection reduced lung inflammation and tissue fibrosis in mice. Together, our study underscores the importance of performing comparative analysis to understand the cause of PASC and suggests that the IFN-γ signaling axis might represent a therapeutic target.

Depleting profibrotic macrophages using bioactivated in vivo assembly peptides ameliorates kidney fibrosis.

Managing renal fibrosis is challenging owing to the complex cell signaling redundancy in diseased kidneys. Renal fibrosis involves an immune response dominated by macrophages, which activates myofibroblasts in fibrotic niches. However, macrophages exhibit high heterogeneity, hindering their potential as therapeutic cell targets. Herein, we aimed to eliminate specific macrophage subsets that drive the profibrotic immune response in the kidney both temporally and spatially. We identified the major profibrotic macrophage subset (Fn1+Spp1+Arg1+) in the kidney and then constructed a 12-mer glycopeptide that was designated as bioactivated in vivo assembly PK (BIVA-PK) to deplete these cells. BIVA-PK specifically binds to and is internalized by profibrotic macrophages. By inducing macrophage celldeath, BIVA-PK reshaped the renal microenvironment and suppressed profibrotic immune responses. The robust efficacy of BIVA-PK in ameliorating renal fibrosis and preserving kidney function highlights the value of targeting macrophage subsets as a potential therapy for patients with CKD.

KLF4 in smooth muscle cell-derived progenitor cells is essential for angiotensin II-induced cardiac inflammation and fibrosis.

Cardiac fibrosis is defined by the excessive accumulation of extracellular matrix (ECM) material resulting in cardiac tissue scarring and dysfunction. While it is commonly accepted that myofibroblasts are the major contributors to ECM deposition in cardiac fibrosis, their origin remains debated. By combining lineage tracing and RNA sequencing, our group made the paradigm-shifting discovery that a subpopulation of resident vascular stem cells residing within the aortic, carotid artery, and femoral aartery adventitia (termed AdvSca1-SM cells) originate from mature vascular smooth muscle cells (SMCs) through an in situ reprogramming process. SMC-to-AdvSca1-SM reprogramming and AdvSca1-SM cell maintenance is dependent on induction and activity of the transcription factor, KLF4. However, the molecular mechanism whereby KLF4 regulates AdvSca1-SM phenotype remains unclear. In the current study, leveraging a highly specific AdvSca1-SM cell reporter system, single-cell RNA-sequencing (scRNA-seq), and spatial transcriptomic approaches, we demonstrate the profibrotic differentiation trajectory of coronary artery-associated AdvSca1-SM cells in the setting of Angiotensin II (AngII)-induced cardiac fibrosis. Differentiation was characterized by loss of stemness-related genes, including Klf4 , but gain of expression of a profibrotic phenotype. Importantly, these changes were recapitulated in human cardiac hypertrophic tissue, supporting the translational significance of profibrotic transition of AdvSca1-SM-like cells in human cardiomyopathy. Surprisingly and paradoxically, AdvSca1-SM-specific genetic knockout of Klf4 prior to AngII treatment protected against cardiac inflammation and fibrosis, indicating that Klf4 is essential for the profibrotic response of AdvSca1-SM cells. Overall, our data reveal the contribution of AdvSca1-SM cells to myofibroblasts in the setting of AngII-induced cardiac fibrosis. KLF4 not only maintains the stemness of AdvSca1-SM cells, but also orchestrates their response to profibrotic stimuli, and may serve as a therapeutic target in cardiac fibrosis.

Primary cilia suppress the fibrotic activity of atrial fibroblasts from patients with atrial fibrillation in vitro

Atrial fibrosis serves as an arrhythmogenic substrate in atrial fibrillation (AF) and contributes to AF persistence. Treating atrial fibrosis is challenging because atrial fibroblast activity is multifactorial. We hypothesized that the primary cilium regulates the profibrotic response of AF atrial fibroblasts, and explored therapeutic potentials of targeting primary cilia to treat fibrosis in AF. We included 25 patients without AF (non-AF) and 26 persistent AF patients (AF). Immunohistochemistry using a subset of the patients (non-AF: n = 10, AF: n = 10) showed less ciliated fibroblasts in AF versus non-AF. Acetylated α-tubulin protein levels were decreased in AF, while the gene expressions of AURKA and NEDD9 were highly increased in AF patients’ left atrium. Loss of primary cilia in human atrial fibroblasts through IFT88 knockdown enhanced expression of ECM genes, including FN1 and COL1A1. Remarkably, restoration or elongation of primary cilia by an AURKA selective inhibitor or lithium chloride, respectively, prevented the increased expression of ECM genes induced by different profibrotic cytokines in atrial fibroblasts of AF patients. Our data reveal a novel mechanism underlying fibrotic substrate formation via primary cilia loss in AF atrial fibroblasts and suggest a therapeutic potential for abrogating atrial fibrosis by restoring primary cilia.

#894 Dynamic multi-omics and mechanistic modelling approach provides deeper insight into kidney fibrosis progression

Abstract Background and Aims Chronic kidney disease (CKD) affects more than 10% of the world's population and causes millions of deaths annually. Organ fibrosis is the main driver of CKD, leading to excessive accumulation of extracellular matrix (ECM). The presence of myofibroblasts correlates with disease progression. The pro-fibrotic responses of these cells, such as ECM production, are driven by the master regulator TGF-β. Despite progress in understanding the disease, there are no treatments available, and current diagnostic indicators neither facilitate early detection of CKD nor correlate with actual renal damage [1–4] Using human kidney-derived PDGFRβ+ mesenchymal cells we aim to provide time-resolved mechanistic insight into CKD progression. Method Our goal is to understand and model how these cells contribute to fibrosis by studying their kinetic response to TGF-β. Therefore, we use multiple omics measurements: transcriptomics, secretomics and phospho-/proteomics coupled with quantitative imaging of the ECM. For each time point, we quantified differential changes in omics and estimated the activities of transcription factors, kinases and phosphatases [5]. Furthermore, we used causal reasoning to draw mechanistic relationships between omics layers within and across time points. Results For validation, we focused on a set of transcription factors that also showed importance in the network context that considers several data modalities. Using knock-down experiments, we show the relevance of these transcription factors for the ECM deposition as well as the fibrotic response to TGF-β. Conclusion Taken together, by applying mechanistic modeling and the integration of multiple datasets, we are contributing to a better understanding of the factors and mechanisms driving kidney fibrosis. Furthermore, we will publish this dataset and thus provide a valuable resource for researchers in the field of CKD.

The increase of long noncoding RNA Fendrr in hepatocytes contributes to liver fibrosis by promoting IL-6 production

Liver fibrosis/cirrhosis is a pathological state caused by excessive extracellular matrix deposition. Sustained activation of hepatic stellate cells (HSC) is the predominant cause of liver fibrosis, but the detailed mechanism is far from clear. In this study, we found that long noncoding RNA Fendrr is exclusively increased in hepatocytes in the murine model of CCl4- and bile duct ligation-induced liver fibrosis, as well as in the biopsies of liver cirrhosis patients. In vivo, ectopic expression of Fendrr aggravated the severity of CCl4-induced liver fibrosis in mice. In contrast, inhibiting Fendrr blockaded the activation of HSC and ameliorated CCl4-induced liver fibrosis. Our mechanistic study showed that Fendrr binds to STAT2 and enhances its enrichment in the nucleus, which then promote the expression of IL-6, and, ultimately, activates HSC in a paracrine manner. Accordingly, disrupting the interaction between Fendrr and STAT2 by ectopic expression of a STAT2 mutant attenuated the pro-fibrotic response inspired by Fendrr in the CCl4-induced liver fibrosis. Notably, the increase of Fendrr in patient fibrotic liver is positively correlated with the severity of fibrosis and the expression of IL-6. Meanwhile, hepatic IL-6 positively correlates with the extent of liver fibrosis and HSC activation as well, thus suggesting a causative role of Fendrr in HSC activation and liver fibrosis. In conclusion, these observations identify an important regulatory crosstalk between hepatocyte Fendrr and HSC activation in the progression of liver fibrosis, which might represent a potential strategy for therapeutic intervention.

Activated factor X stimulates atrial endothelial cells and tissues to promote remodelling responses through AT1R/NADPH oxidases/SGLT1/2.

Atrial fibrillation (AF), the most common cardiac arrhythmia favouring ischemic stroke and heart failure involves left atrial remodelling, fibrosis and a complex interplay between cardiovascular risk factors. This study examined whether activated factor X (FXa) induces pro-remodelling and pro-fibrotic responses in atrial endothelial cells (AECs) and human atrial tissues and determined the underlying mechanisms. AECs collected from porcine hearts and human right atrial appendages (RAA) from patients undergoing heart surgery. Protein expression levels were assessed by Western blot and immunofluorescence staining, mRNA levels by RT-qPCR, formation of reactive oxygen species (ROS) and NO using fluorescent probes, thrombin and angiotensin II generation by specific assays, fibrosis by Sirius red staining and senescence by senescence-associated beta-galactosidase (SA-β-gal) activity. In AECs, FXa increased ROS formation, senescence (SA-β-gal activity, p53, p21), angiotensin II generation and the expression of pro-inflammatory (VCAM-1, MCP-1), pro-thrombotic (tissue factor), pro-fibrotic (TGF-β and collagen-1/3a) and pro-remodelling (MMP-2/9) markers whereas eNOS levels and NO formation were reduced. These effects were prevented by inhibitors of FXa but not thrombin, protease-activated receptors antagonists (PAR-1/2) and inhibitors of NADPH oxidases, ACE, AT1R, SGLT1/SGLT2. FXa also increased expression levels of ACE1, AT1R, SGLT1/2 proteins which were prevented by SGLT1/2 inhibitors. Human RAA showed tissue factor mRNA levels that correlated with markers of endothelial activation, pro-remodelling and pro-fibrotic responses and SGLT1/2 mRNA levels. They also showed protein expression levels of ACE1, AT1R, p22phox, SGLT1/2, and immunofluorescence signals of nitrotyrosine and SGLT1/2 colocalized with those of CD31. FXa increased oxidative stress levels which were prevented by inhibitors of the AT1R/NADPH oxidases/SGLT1/2 pathway. FXa promotes oxidative stress triggering premature endothelial senescence and dysfunction associated with pro-thrombotic, pro-remodelling and pro-fibrotic responses in AECs and human RAA involving the AT1R/NADPH oxidases/SGLT1/2 pro-oxidant pathway. Targeting this pathway may be of interest to prevent atrial remodelling and the progression of atrial fibrillation substrate.