Fibrosis-related Pathways Research Articles

SUMO: A new perspective to decipher fibrosis.

Fibrosis is characterized by excessive extracellular matrix (ECM) deposition resulting from dysregulated wound healing and connective tissue repair mechanisms. Excessive accumulation of ECM leads to fibrous tissue formation, impairing organ function and driving the progression of various fibrotic diseases. Recently, the role of small ubiquitin-like modifiers (SUMO) in fibrotic diseases has attracted significant attention. SUMO-mediated SUMOylation, a highly conserved posttranslational modification, participates in a variety of biological processes, including nuclear-cytosolic transport, cell cycle progression, DNA damage repair, and cellular metabolism. Conversely, SUMO-specific proteases cleave the isopeptide bond of SUMO conjugates, thereby regulating the deSUMOylation process. Mounting evidence indicates that SUMOylation and deSUMOylation regulate the functions of several proteins, such as Smad3, NF-κB, and promyelocytic leukemia protein, which are implicated in fibrotic diseases like liver fibrosis, myocardial fibrosis, and pulmonary fibrosis. This review summarizes the role of SUMO in fibrosis-related pathways and explores its pathological relevance in various fibrotic diseases. All evidence suggest that the SUMO pathway is important targets for the development of treatments for fibrotic diseases.

Alternative splicing and intron retention: Their profiles and roles in cutaneous fibrosis of systemic sclerosis

BackgroundAlternative splicing (AS) and intron retention (IR) implicated in multiple pathophysiological processes, have rarely been reported in systemic sclerosis (SSc). MethodsWe integrated bulk RNA-seq and 4D label-free mass spectrometry to perform a multi-omics analysis of AS and IR in SSc skin tissue and fibroblasts. RMATS and iREAD were used to identify AS and IR, which were validated by real-time PCR. Spearman correlation and the LASSO method were employed to assess correlations among clinical features, introns, splicing factors (regulators of AS) and proteins. FindingsAS profiles showed distinct alterations in SSc skin tissue, with the most pronounced changes occurring in IR. AS and IR were associated with total modified Rodnan skin score (mRSS) and local skin score. Upon TGF-β stimulation, fibroblasts exhibited significant alterations in IR profiles, affecting genes related to fibroblast proliferation and collagen fibril organization. A comprehensive integrated analysis of introns, exons, and proteome profiles revealed that IR exerted a negative impact on protein expression, with certain changes being under intronic control. RT-PCR confirmed the presence of intron and exon-derived sequences of CTTN, OGA, MED16 and PHYKPL. Additionally, notable changes were observed in the regulatory network of splicing factors in SSc skin tissues. These factors are also involved in fibrosis pathways and correlated with clinical features. ConclusionTotally, abnormal AS, IR profiles and splicing factors were identified in SSc, altered IRs and splicing factors participated in fibrosis-related pathways. IR exerted a negative impact on protein expression in TGF-β-stimulated fibroblasts. Clarification of the IR mechanisms will provide new insights into the pathophysiology of SSc.

Low-Dose Metformin and Profibrotic Signature in Central Centrifugal Cicatricial Alopecia

Central centrifugal cicatricial alopecia (CCCA) is a scarring alopecia predominantly affecting Black female individuals. Current conventional treatments target inflammation but not the underlying fibrotic processes, often leading to permanent hair loss. To investigate the associations of low-dose oral metformin, an antidiabetic medication with antifibrotic properties, with clinical symptoms and scalp gene expression patterns in patients with CCCA. This retrospective clinical case series and transcriptomic analysis included patients treated at a single tertiary academic medical center between January 2023 and March 2024. All patients had biopsy-confirmed CCCA refractory to standard treatments. Transcriptomic analysis was performed on patients with previously banked, paired scalp biopsies before and after treatment with adjuvant metformin for at least 6 weeks. Extended-release metformin, 500 mg, once daily was added to participants' baseline CCCA treatment regimens. Clinical assessments included pruritus, inflammation, scalp resistance, and hair regrowth. Gene expression profiling via bulk RNA sequencing analysis evaluated differential gene expression and pathway enrichment. A total of 12 Black female participants were included in the study, and transcriptomic analysis was performed in 4 participants. After at least 6 months of metformin treatment, 9 participants experienced improvement in disease, including scalp pain, inflammation, and/or pruritus, and 6 demonstrated clinical evidence of hair regrowth. The addition of metformin led to reversal of many prominent gene pathways previously identified in CCCA. Transcriptomic analysis revealed upregulation of pathways and genes (keratin-associated proteins [KRTAPs]) involved in keratinization, epidermis development, and the hair cycle (absolute log2-fold change > 4), with concomitant downregulation of fibrosis-related pathways and genes (eg, MMP7, COL6A1) (fold change >1.5; all false discovery rate <.05). Gene set analysis showed reduced expression of helper T cell 17 and epithelial-mesenchymal transition pathways and elevated adenosine monophosphate kinase signaling and KRTAPs after metformin treatment. In this case series of patients with treatment-refractory CCCA, low-dose oral metformin was associated with symptomatic improvement and dual modulation of gene expression, stimulating hair growth pathways while suppressing fibrosis and inflammation markers. These findings provide a rationale for future clinical trials studying metformin as a targeted therapy for CCCA and other cicatricial alopecias.

Ferroptosis-Associated Extracellular Matrix Remodeling in Radiation-Induced Lung Fibrosis Progression.

Background: Radiation-induced lung fibrosis (RILF) is a life-threatening complication of thoracic radiotherapy. Ferroptosis, a recently discovered type of cell death, is believed to contribute to RILF, though the associated mechanisms are unknown. This study aimed to investigate the potential mechanism of ferroptosis in RILF and examine the contribution of different cell types to ferroptosis during RILF progression. Methods: Histopathological changes in RILF lung tissue were assessed through H&E and Masson staining. IHC staining investigated ferroptosis markers (GPX4, ACSL4, NCOA4). Ferroptosis-related genes (FRG) and pathway scores were derived from RILF transcriptome microarray data. The sc-RNAseq analysis detected FRG score dynamics across cell types, validated by IF staining for PDGFR-α and ACSL4. Results: ACSL4 and NCOA4 protein levels were significantly higher and GPX4 lower in IR than control. FRG scores were positively correlated with fibrosis-related pathway scores in the RILF transcriptome data. FRG and ECM scores were concurrently upregulated in myofibroblasts. Enhanced co-staining of PDGFR-α and ACSL4 were observed in the fibrotic areas of RILF lungs. Conclusions: Our research indicated that in RILF, fibroblasts undergoing ferroptosis may release increased levels of ECM, potentially accelerating the progression of lung fibrosis. This finding presents ferroptosis as a potential therapeutic target in RILF.

SFTPB in serum extracellular vesicles as a biomarker of progressive pulmonary fibrosis.

Progressive pulmonary fibrosis (PPF), defined as the worsening of various interstitial lung diseases (ILDs), currently lacks useful biomarkers. To identify novel biomarkers for early detection of patients at risk of PPF, we performed a proteomic analysis of serum extracellular vesicles (EVs). Notably, the identified candidate biomarkers were enriched for lung-derived proteins participating in fibrosis-related pathways. Among them, pulmonary surfactant-associated protein B (SFTPB) in serum EVs could predict ILD progression better than the known biomarkers, serum KL-6 and SP-D, and it was identified as an independent prognostic factor from ILD-gender-age-physiology index. Subsequently, the utility of SFTPB for predicting ILD progression was evaluated further in 2 cohorts using serum EVs and serum, respectively, suggesting that SFTPB in serum EVs but not in serum was helpful. Among SFTPB forms, pro-SFTPB levels were increased in both serum EVs and lungs of patients with PPF compared with those of the control. Consistently, in a mouse model, the levels of pro-SFTPB, primarily originating from alveolar epithelial type 2 cells, were increased similarly in serum EVs and lungs, reflecting pro-fibrotic changes in the lungs, as supported by single-cell RNA sequencing. SFTPB, especially its pro-form, in serum EVs could serve as a biomarker for predicting ILD progression.

Identifying hub genes in response to ustekinumab and the impact of ustekinumab treatment on fibrosis in Crohn's disease.

Crohn's disease (CD) is a chronic inflammatory disease. Approximately 50% of patients with CD progressed from inflammation to fibrosis. Currently, there are no effective drugs for treating intestinal fibrosis. Biologic therapies for CD such as ustekinumab have benefited patients; however, up to 30% of patients with CD have no response to initial treatment, and the effect of ustekinumab on intestinal fibrosis is still uncertain. Therefore, it is of great significance to explore the predictive factors of ustekinumab treatment response and the effect of ustekinumab on intestinal fibrosis. Public datasets-GSE207465 (blood samples) and GSE112366 and GSE207022 (intestinal samples)-were downloaded and analyzed individually (unmerged) based on the treatment response. Differentially expressed genes (DEGs) were identified by the "limma" R package and changes in immune cell infiltration were determined by the "CIBERSORT" R package in both blood and intestinal samples at week 0 (before treatment). To find predictive factors of ustekinumab treatment response, the weighted gene co-expression network analysis (WGCNA) R package was used to identify hub genes in GSE112366. Hub genes were then verified in GSE207022, and a prediction model was built by random forest algorithm. Furthermore, fibrosis-related gene changes were analyzed in ileal samples before and after treatment with ustekinumab. (1) Our analysis found that MUC1, DUOX2, LCN2, and PDZK1IP1 were hub genes in GSE112366. GSE207022 revealed that MUC1 (AUC:0.761), LCN2 (AUC:0.79), and PDZK1IP1 (AUC:0.731) were also lower in the response group. Moreover, the random forest model was shown to have strong predictive capabilities in identifying responders (AUC = 0.875). To explore the relationship between intestinal tissue and blood, we found that ITGA4 had lower expression in the intestinal and blood samples of responders. The expression of IL18R1 is also lower in responders' intestines. IL18, the ligand of IL18R1, was also found to have lower expression in the blood samples from responders vs. non-responders. (2) GSE112366 revealed a significant decrease in fibrosis-related module genes (COL4A1, TUBB6, IFITM2, SERPING1, DRAM1, NAMPT, MMP1, ZEB2, ICAM1, PFKFB3, and ACTA2) and fibrosis-related pathways (ECM-receptor interaction and PI3K-AKT pathways) after ustekinumab treatment. MUC1, LCN2, and PDZK1IP1 were identified as hub genes in intestinal samples, with lower expression indicating a positive prediction of ustekinumab treatment response. Moreover, ITGA4 and IL18/IL18R1 may be involved in the treatment response in blood and intestinal samples. Finally, ustekinumab treatment was shown to significantly alter fibrotic genes and pathways.

Multi-omics integration highlights the role of ubiquitination in endometriosis fibrosis

BackgroundEndometriosis, characterized by the presence of active endometrial-like tissues outside the uterus, causes symptoms like dysmenorrhea and infertility due to the fibrosis of endometrial cells, which involves excessive deposition of extracellular matrix (ECM) proteins. Ubiquitination, an important post-transcriptional modification, regulates various biological processes in human diseases. However, its role in the fibrosis process in endometriosis remains unclear.MethodsWe employed multi-omics approaches on two cohorts of endometriosis patients with 39 samples. GO terms and KEGG pathways enrichment analyses were used to investigate the functional changes involved in endometriosis. Pearson’s correlation coefficient analysis was conducted to explore the relationship between global proteome and ubiquitylome in endometriosis. The protein expression levels of ubiquitin-, fibrosis-related proteins, and E3 ubiquitin-protein ligase TRIM33 were validated via Western blot. Transfecting human endometrial stroma cells (hESCs) with TRIM33 small interfering RNA (siRNA) in vitro to explore how TRIM33 affects fibrosis-related proteins.ResultsIntegration of proteomics and transcriptomics showed genes with concurrent change of both mRNA and protein level which involved in ECM production in ectopic endometria. Ubiquitylomics distinguished 1647 and 1698 ubiquitinated lysine sites in the ectopic (EC) group compared to the normal (NC) and eutopic (EU) groups, respectively. Further multi-omics integration highlighted the essential role of ubiquitination in key fibrosis regulators in endometriosis. Correlation analysis between proteome and ubiquitylome showed correlation coefficients of 0.32 and 0.36 for ubiquitinated fibrosis proteins in EC/NC and EC/EU groups, respectively, indicating positive regulation of fibrosis-related protein expression by ubiquitination in ectopic lesions. We identified ubiquitination in 41 pivotal proteins within the fibrosis-related pathway of endometriosis. Finally, the elevated expression of TGFBR1/α-SMA/FAP/FN1/Collagen1 proteins in EC tissues were validated across independent samples. More importantly, we demonstrated that both the mRNA and protein levels of TRIM33 were reduced in endometriotic tissues. Knockdown of TRIM33 promoted TGFBR1/p-SMAD2/α-SMA/FN1 protein expressions in hESCs but did not significantly affect Collagen1/FAP levels, suggesting its inhibitory effect on fibrosis in vitro.ConclusionsThis study, employing multi-omics approaches, provides novel insights into endometriosis ubiquitination profiles and reveals aberrant expression of the E3 ubiquitin ligase TRIM33 in endometriotic tissues, emphasizing their critical involvement in fibrosis pathogenesis and potential therapeutic targets.

Regulation of cardiac fibrosis in mice with TAC/DOCA-induced HFpEF by resistin-like molecule gamma and adenylate cyclase 1.

Heart failure with preserved ejection fraction (HFpEF) is one of the major subtypes of heart failure (HF) and no effective treatments for this common disease exist to date. Cardiac fibrosis is central to the pathology of HF and a potential avenue for the treatment of HFpEF. To explore key fibrosis-related genes and pathways in the pathophysiological process of HFpEF, a mouse model of HFpEF was constructed. The relevant gene expression profiles were downloaded from the Gene Expression Omnibus database, and single-sample Gene Set Enrichment Analysis (ssGSEA) was performed targeting fibrosis-related pathways to explore differentially expressed genes (DEGs) in healthy control and HFpEF heart tissues with cross-tabulation analysis of fibrosis-related genes. Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed on the identified fibrosis-related genes. The two most significant DEGs were selected, and further validation was conducted in HFpEF mice. The results indicated that myocardial fibrosis was significantly upregulated in HFpEF mice compared to healthy controls, while the ssGSEA results revealed significant differences in the enrichment of nine fibrosis-related pathways in HFpEF myocardial tissue, with 112 out of 798 DEGs being related to fibrosis. The in vivo results demonstrated that expression levels of resistin-like molecule gamma (Relmg) and adenylate cyclase 1 (Adcy1) in the heart tissues of HFpEF mice were significantly higher and lower, respectively, compared to healthy controls. Taken together, these results suggest that Relmg and Acdy1 as well as the fibrosis process may be potential targets for HFpEF treatment.

TFCP2L1 as a potential diagnostic gene biomarker of Keloid given its association with immune cells-a study based on machine learning and RNA sequence

Keloids represent a specific type of dermal tumor, where delayed treatment greatly heighten risks of recurrence. This study thus seeks to identify key genes for early keloid diagnosis and compare how immune cell infiltration between keloid lesion skin (KLS) and keloid adjacent non-lesional skin (KNL) differ. Utilizing two GEO datasets, which encompassed KLS (n=8) and KNL (n=6) samples, 564 differentially expressed genes (DEGs) were identified, with a primary emphasis on inflammation- and fibrosis-related pathways. The gene TFCP2L1 was pinpointed from the intersection of LASSO and SVM-RFE algorithms, exhibiting downregulation in KLS when juxtaposed with KNL. To aid prognosis and identification of potential immunotherapy targets, the CIBERSORT algorithm was employed to assess prelative proportions of immune cells. In the KNL samples, there was an observed increase in follicular helper T cells, activated NK cells, and activated dendritic cells. Conversely, M1 macrophages were elevated in KLS samples. TFCP2L1 exhibited a positive correlation with activated NK cells and activated dendritic cells, while it showed a negative correlation with M1macrophages. These findings highlight the intertwined roles of TFCP2L1 and immune cell infiltration, lending to TFCP2L1’s potential as a diagnostic biomarker for keloids and highlights how TFCP2L1 might influence keloid development through immune cell populations.

Elevated ITGA1 levels in type 2 diabetes: implications for cardiac function impairment

Aims/hypothesisType 2 diabetes mellitus is known to contribute to the development of heart failure with preserved ejection fraction (HFpEF). However, identifying HFpEF in individuals with type 2 diabetes early on is often challenging due to a limited array of biomarkers. This study aims to investigate specific biomarkers associated with the progression of HFpEF in individuals with type 2 diabetes, for the purpose of enabling early detection and more effective management strategies.MethodsBlood samples were collected from individuals with type 2 diabetes, both with and without HFpEF, for proteomic analysis. Plasma integrin α1 (ITGA1) levels were measured and compared between the two groups. Participants were further categorised based on ITGA1 levels and underwent detailed transthoracic echocardiography at baseline and during a median follow-up period of 30 months. Multivariable linear and Cox regression analyses were conducted separately to assess the associations between plasma ITGA1 levels and changes in echocardiography indicators and re-hospitalisation risk. Additionally, proteomic data for the individuals’ left ventricles, from ProteomeXchange database, were analysed to uncover mechanisms underlying the change in ITGA1 levels in HFpEF.ResultsIndividuals with type 2 diabetes and HFpEF showed significantly higher plasma ITGA1 levels than the individuals with type 2 diabetes without HFpEF. These elevated ITGA1 levels were associated with left ventricular remodelling and impaired diastolic function. Furthermore, during a median follow-up of 30 months, multivariable analysis revealed that elevated ITGA1 levels independently correlated with deterioration of both diastolic and systolic cardiac functions. Additionally, higher baseline plasma ITGA1 levels independently predicted re-hospitalisation risk (HR 2.331 [95% CI 1.387, 3.917], p=0.001). Proteomic analysis of left ventricular myocardial tissue provided insights into the impact of increased ITGA1 levels on cardiac fibrosis-related pathways and the contribution made by these changes to the development and progression of HFpEF.Conclusions/interpretationITGA1 serves as a biomarker for monitoring cardiac structural and functional damage, can be used to accurately diagnose the presence of HFpEF, and can be used to predict potential deterioration in cardiac structure and function as well as re-hospitalisation for individuals with type 2 diabetes. Its measurement holds promise for facilitating risk stratification and early intervention to mitigate the adverse cardiovascular effects associated with diabetes.Data availabilityThe proteomic data of left ventricular myocardial tissue from individuals with type 2 diabetes, encompassing both those with and without HFpEF, is available from the ProteomeXchange database at http://proteomecentral.proteomexchange.org.Graphical

Proteomic analysis reveals activation of platelet- and fibrosis-related pathways in hearts of ApoE−/− mice exposed to diesel exhaust particles

Air pollution is an environmental risk factor linked to multiple human diseases including cardiovascular diseases (CVDs). While particulate matter (PM) emitted by diesel exhaust damages multiple organ systems, heart disease is one of the most severe pathologies affected by PM. However, the in vivo effects of diesel exhaust particles (DEP) on the heart and the molecular mechanisms of DEP-induced heart dysfunction have not been investigated. In the current study, we attempted to identify the proteomic signatures of heart fibrosis caused by diesel exhaust particles (DEP) in CVDs-prone apolipoprotein E knockout (ApoE−/−) mice model using tandem mass tag (TMT)-based quantitative proteomic analysis. DEP exposure induced mild heart fibrosis in ApoE−/− mice compared with severe heart fibrosis in ApoE−/− mice that were treated with CVDs-inducing peptide, angiotensin II. TMT-based quantitative proteomic analysis of heart tissues between PBS- and DEP-treated ApoE−/− mice revealed significant upregulation of proteins associated with platelet activation and TGFβ-dependent pathways. Our data suggest that DEP exposure could induce heart fibrosis, potentially via platelet-related pathways and TGFβ induction, causing cardiac fibrosis and dysfunction.

Genetic deletion of MMP12 ameliorates cardiometabolic disease by improving insulin sensitivity, systemic inflammation, and atherosclerotic features in mice

BackgroundMatrix metalloproteinase 12 (MMP12) is a macrophage-secreted protein that is massively upregulated as a pro-inflammatory factor in metabolic and vascular tissues of mice and humans suffering from cardiometabolic diseases (CMDs). However, the molecular mechanisms explaining the contributions of MMP12 to CMDs are still unclear.MethodsWe investigated the impact of MMP12 deficiency on CMDs in a mouse model that mimics human disease by simultaneously developing adipose tissue inflammation, insulin resistance, and atherosclerosis. To this end, we generated and characterized low-density lipoprotein receptor (Ldlr)/Mmp12-double knockout (DKO) mice fed a high-fat sucrose- and cholesterol-enriched diet for 16–20 weeks.ResultsDKO mice showed lower cholesterol and plasma glucose concentrations and improved insulin sensitivity compared with LdlrKO mice. Untargeted proteomic analyses of epididymal white adipose tissue revealed that inflammation- and fibrosis-related pathways were downregulated in DKO mice. In addition, genetic deletion of MMP12 led to alterations in immune cell composition and a reduction in plasma monocyte chemoattractant protein-1 in peripheral blood which indicated decreased low-grade systemic inflammation. Aortic en face analyses and staining of aortic valve sections demonstrated reduced atherosclerotic plaque size and collagen content, which was paralleled by an improved relaxation pattern and endothelial function of the aortic rings and more elastic aortic sections in DKO compared to LdlrKO mice. Shotgun proteomics revealed upregulation of anti-inflammatory and atheroprotective markers in the aortas of DKO mice, further supporting our data. In humans, MMP12 serum concentrations were only weakly associated with clinical and laboratory indicators of CMDs.ConclusionWe conclude that the genetic deletion of MMP12 ameliorates obesity-induced low-grade inflammation, white adipose tissue dysfunction, biomechanical properties of the aorta, and the development of atherosclerosis. Therefore, therapeutic strategies targeting MMP12 may represent a promising approach to combat CMDs.

Construction of predictive model of interstitial fibrosis and tubular atrophy after kidney transplantation with machine learning algorithms.

Background: Interstitial fibrosis and tubular atrophy (IFTA) are the histopathological manifestations of chronic kidney disease (CKD) and one of the causes of long-term renal loss in transplanted kidneys. Necroptosis as a type of programmed death plays an important role in the development of IFTA, and in the late functional decline and even loss of grafts. In this study, 13 machine learning algorithms were used to construct IFTA diagnostic models based on necroptosis-related genes. Methods: We screened all 162 "kidney transplant"-related cohorts in the GEO database and obtained five data sets (training sets: GSE98320 and GSE76882, validation sets: GSE22459 and GSE53605, and survival set: GSE21374). The training set was constructed after removing batch effects of GSE98320 and GSE76882 by using the SVA package. The differentially expressed gene (DEG) analysis was used to identify necroptosis-related DEGs. A total of 13 machine learning algorithms-LASSO, Ridge, Enet, Stepglm, SVM, glmboost, LDA, plsRglm, random forest, GBM, XGBoost, Naive Bayes, and ANNs-were used to construct 114 IFTA diagnostic models, and the optimal models were screened by the AUC values. Post-transplantation patients were then grouped using consensus clustering, and the different subgroups were further explored using PCA, Kaplan-Meier (KM) survival analysis, functional enrichment analysis, CIBERSOFT, and single-sample Gene Set Enrichment Analysis. Results: A total of 55 necroptosis-related DEGs were identified by taking the intersection of the DEGs and necroptosis-related gene sets. Stepglm[both]+RF is the optimal model with an average AUC of 0.822. A total of four molecular subgroups of renal transplantation patients were obtained by clustering, and significant upregulation of fibrosis-related pathways and upregulation of immune response-related pathways were found in the C4 group, which had poor prognosis. Conclusion: Based on the combination of the 13 machine learning algorithms, we developed 114 IFTA classification models. Furthermore, we tested the top model using two independent data sets from GEO.

Targeted delivery of galunisertib using machine perfusion reduces fibrogenesis in an integrated ex vivo renal transplant and fibrogenesis model.

Fibrosis in kidney allografts is a major post-transplant complication that contributes to graft failure. Lately, multiple potent inhibitors of fibrosis-related pathways have been developed such as galunisertib, an inhibitor of the transforming growth factor-beta (TGF-β/TGFβ1) signalling pathway. This drug, however, poses risks for adverse effects when administered systemically. Therefore, we devised a new repurposing strategy in which galunisertib is administered ex vivo. We combined machine perfusion and tissue slices to explore the antifibrotic effects of galunisertib in renal grafts. Porcine kidneys were subjected to 30 min of warm ischaemia, 24 h of oxygenated hypothermic machine perfusion and 6 h of normothermic machine perfusion with various treatments (i.e. untreated control, TGFβ1, galunisertib or TGFβ1 + galunisertib; n = 8 kidneys per group). To determine whether effects persisted upon ceasing treatment, kidney slices were prepared from respective kidneys and incubated for 48 h. Galunisertib treatment improved general viability without negatively affecting renal function or elevating levels of injury markers or by-products of oxidative stress during perfusion. Galunisertib also reduced inflammation and, more importantly, reduced the onset of fibrosis after 48 h of incubation. Our findings demonstrate the value of using machine perfusion for administering antifibrotic drugs such as galunisertib, proving it to be an effective example of repurposing.

Contribution of common and rare variants to Asian neovascular age-related macular degeneration subtypes

Neovascular age-related macular degeneration (nAMD), along with its clinical subtype known as polypoidal choroidal vasculopathy (PCV), are among the leading causes of vision loss in elderly Asians. In a genome-wide association study (GWAS) comprising 3,128 nAMD (1,555 PCV and 1,573 typical nAMD), and 5,493 controls of East Asian ancestry, we identify twelve loci, of which four are novel (P , < , 1.19times {10}^{-8}). Substantial genetic sharing between PCV and typical nAMD is noted (rg = 0.666), whereas collagen extracellular matrix and fibrosis-related pathways are more pronounced for PCV. Whole-exome sequencing in 259 PCV patients revealed functional rare variants burden in collagen type I alpha 1 chain gene (COL1A1; P=1.05times {10}^{-6}) and potential enrichment of functional rare mutations at AMD-associated loci. At the GATA binding protein 5 (GATA5) locus, the most significant GWAS novel loci, the expressions of genes including laminin subunit alpha 5 (Lama5), mitochondrial ribosome associated GTPase 2 (Mtg2), and collagen type IX alpha 3 chain (Col9A3), are significantly induced during retinal angiogenesis and subretinal fibrosis in murine models. Furthermore, retinoic acid increased the expression of LAMA5 and MTG2 in vitro. Taken together, our data provide insights into the genetic basis of AMD pathogenesis in the Asian population.

Immune landscape in rejection of renal transplantation revealed by high-throughput single-cell RNA sequencing.

Background: The role of the cellular level in kidney transplant rejection is unclear, and single-cell RNA sequencing (scRNA-seq) can reveal the single-cell landscape behind rejection of human kidney allografts at the single-cell level. Methods: High-quality transcriptomes were generated from scRNA-seq data from five human kidney transplantation biopsy cores. Cluster analysis was performed on the scRNA-seq data by known cell marker genes in order to identify different cell types. In addition, pathways, pseudotime developmental trajectories and transcriptional regulatory networks involved in different cell subpopulations were explored. Next, we systematically analyzed the scoring of gene sets regarding single-cell expression profiles based on biological processes associated with oxidative stress. Results: We obtained 81,139 single cells by scRNA-seq from kidney transplant tissue biopsies of three antibody-mediated rejection (ABMR) patients and two acute kidney injury (AKI) patients with non-rejection causes and identified 11 cell types, including immune cells, renal cells and several stromal cells. Immune cells such as macrophages showed inflammatory activation and antigen presentation and complement signaling, especially in rejection where some subpopulations of cells specifically expressed in rejection showed specific pro-inflammatory responses. In addition, patients with rejection are characterized by an increased number of fibroblasts, and further analysis of subpopulations of fibroblasts revealed their involvement in inflammatory and fibrosis-related pathways leading to increased renal rejection and fibrosis. Notably, the gene set score for response to oxidative stress was higher in patients with rejection. Conclusion: Insight into histological differences in kidney transplant patients with or without rejection was gained by assessing differences in cellular levels at single-cell resolution. In conclusion, we applied scRNA-seq to rejection after renal transplantation to deconstruct its heterogeneity and identify new targets for personalized therapeutic approaches.

Cellular Distribution of Secreted Phospholipase A2 in Lungs of IPF Patients and Its Inhibition in Bleomycin-Induced Pulmonary Fibrosis in Mice.

Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease with a very poor prognosis as it has a 2.5 to 5 years mean survival after proper diagnosis. Even nintedanib and pirfenidone cannot halt the progression, though they slow the progression of IPF. Hence, there is a need to understand the novel pathophysiology. Phospholipase A2 (PLA2) could be the ideal candidate to study in IPF, as they have a role in both inflammation and fibrosis. In the present study, we have shown the expression profile of various secretory Phospholipase A2 (PLA2) isoforms by analyzing publicly available transcriptome data of single cells from the lungs of healthy individuals and IPF patients. Among 11 members of sPLA2, PLA2G2A is found to be increased in the fibroblasts and mesothelial cells while PLA2G5 is found to be increased in the fibroblasts of IPF patients. We identified a subset of fibroblasts expressing high PLA2G2A with moderate expression of PLA2G5 and which are specific to IPF only; we named it as PLA2G2A+ IPF fibroblast. Pathway analysis revealed that these PLA2G2A+ IPF fibroblast have upregulation of both inflammatory and fibrosis-related pathways like the TGF-β signaling pathway, IL-17 signaling, the arachidonic acid metabolism pathway and ECM-receptor interaction. In addition to this, we found elevated levels of sPLA2-IIA in plasma samples of IPF patients in our cohort. PLA2G3, PLA2G10 and PLA2G12B are found in to be increased in certain epithelial cells of IPF patients. Thus, these findings indicate that these five isoforms have a disease-dominant role along with innate immune roles as these isoforms are found predominantly in structural cells of IPF patients. Further, we have targeted sPLA2 in mice model of bleomycin-induced lung fibrosis by pBPB, a known sPLA2 inhibitor. pBPB treatment attenuated lung fibrosis induced by bleomycin along with a reduction in TGF-β and deposition of extracellular matrix in lung. Thus, these findings indicate that these sPLA2 isoforms especially PLA2G2A may serve as a therapeutic target in lung fibrosis.

Gene expression patterns and related pathways in the hearts of rhesus monkeys subjected to prolonged myocardial ischemia.

After myocardial infarction (MI) occurs, progressive pathological cardiac remodeling results in heart dysfunction and even heart failure during the following months or years. The present study explored the molecular mechanisms underlying the late phase of MI at the global transcript level. A rhesus monkey model of myocardial ischemia induced by left anterior descending (LAD) artery ligation was established, and the heart tissue was collected eight weeks after ligation for transcriptome analysis by DNA microarray technology. Differentially expressed genes in the core infarcted area and remote infarcted area of the ischemic heart were detected with significance analysis of microarray (SAM), and related pathways were detected by Gene Ontology (GO)/pathway analysis. We found that compared to the sham condition, prolonged ischemia increased the levels of 941 transcripts, decreased the levels of 380 transcripts in the core infarcted area, and decreased the levels of 8 transcripts in the remote area in monkey heart tissue. Loss of coordination between the expression of genes, including natriuretic peptide A (NPPA), NPPB, and corin (Corin, serine peptidase), may aggravate cardiac remodeling. Furthermore, imbalance in the enriched significantly changed pathways, including fibrosis-related pathways, cardioprotective pathways, and the cardiac systolic pathway, likely also plays a key role in regulating the development of heart remodeling.

Identifying novel mechanisms of abdominal aortic aneurysm via unbiased proteomics and systems biology.

BackgroundAbdominal aortic aneurysm (AAA), characterized by a continued expansion of the aorta, leads to rupture if not surgically repaired. Mice aid the study of disease progression and its underlying mechanisms since sequential studies of aneurysm development are not feasible in humans. The present study used unbiased proteomics and systems biology to understand the molecular relationship between the mouse models of AAA and the human disease.Methods and resultsAortic tissues of developing and established aneurysms produced by either angiotensin II (AngII) infusion in Apoe−/− and Ldlr−/− mice or intraluminal elastase incubation in wildtype C57BL/6J mice were examined. Aortas were dissected free and separated into eight anatomical segments for proteomics in comparison to their appropriate controls. High-dimensional proteome cluster analyses identified site-specific protein signatures in the suprarenal segment for AngII-infused mice (159 for Apoe−/− and 158 for Ldlr−/−) and the infrarenal segment for elastase-incubated mice (173). Network analysis revealed a predominance of inflammatory and coagulation factors in developing aneurysms, and a predominance of fibrosis-related pathways in established aneurysms for both models. To further substantiate our discovery platform, proteomics was performed on human infrarenal aortic aneurysm tissues as well as aortic tissue collected from age-matched controls. Protein processing and inflammatory pathways, particularly neutrophil-associated inflammation, dominated the proteome of the human aneurysm abdominal tissue. Aneurysmal tissue from both mouse and human had inflammation, coagulation, and protein processing signatures, but differed in the prevalence of neutrophil-associated pathways, and erythrocyte and oxidative stress-dominated networks in the human aneurysms.ConclusionsIdentifying changes unique to each mouse model will help to contextualize model-specific findings. Focusing on shared proteins between mouse experimental models or between mouse and human tissues may help to better understand the mechanisms for AAA and establish molecular bases for novel therapies.

Comparative Analysis of Gene Expression in Fibroblastic Foci in Patients with Idiopathic Pulmonary Fibrosis and Pulmonary Sarcoidosis.

Background: Fibroblastic foci (FF) are characteristic features of usual interstitial pneumonia (UIP)/idiopathic pulmonary fibrosis (IPF) and one cardinal feature thought to represent a key mechanism of pathogenesis. Hence, FF have a high impact on UIP/IPF diagnosis in current guidelines. However, although less frequent, these histomorphological hallmarks also occur in other fibrotic pulmonary diseases. Currently, there is therefore a gap in knowledge regarding the underlying molecular similarities and differences of FF in different disease entities. Methods: In this work, we analyzed the compartment-specific gene expression profiles of FF in IPF and sarcoidosis in order to elucidate similarities and differences as well as shared pathomechanisms. For this purpose, we used laser capture microdissection, mRNA and protein expression analysis. Biological pathway analysis was performed using two different gene expression databases. As control samples, we used healthy lung tissue that was donated but not used for lung transplantation. Results: Based on Holm Bonferroni corrected expression data, mRNA expression analysis revealed a significantly altered expression signature for 136 out of 760 genes compared to healthy controls while half of these showed a similar regulation in both groups. Immunostaining of selected markers from each group corroborated these results. However, when comparing all differentially expressed genes with the fdr-based expression data, only 2 of these genes were differentially expressed between sarcoidosis and IPF compared to controls, i.e., calcium transport protein 1 (CAT1) and SMAD specific E3 ubiquitin protein ligase 1 (SMURF1), both in the sarcoidosis group. Direct comparison of sarcoidosis and IPF did not show any differentially regulated genes independent from the statistical methodology. Biological pathway analysis revealed a number of fibrosis-related pathways pronounced in IPF without differences in the regulatory direction. Conclusions: These results demonstrate that FF of end-stage IPF and sarcoidosis lungs, although different in initiation, are similar in gene and protein expression, encouraging further studies on the use of antifibrotic agents in sarcoidosis.