Fibrotic Progression Research Articles

Prevalence of Second Cancers in Patients with Polycythemia Vera (PV): A Retrospective Analysis of US Real-World Claims Data

Background:Polycythemia vera (PV) is a chronic myeloproliferative neoplasm (MPN) associated with risk of leukemic or fibrotic progression (Tefferi and Barbui. Am J Hematol. 2017;92;94-108). In addition to the known risk of disease transformation, patients with MPNs have a 60% higher risk of developing second non-hematologic cancers compared to matched controls. Skin cancers are among the most prevalent second cancers, with a reported 2.8-fold increase in risk of non-melanoma skin cancer (NMSC) (Landtblom et al., Leukemia. 2018;32:2203-10). The inherent risk associated with MPNs is exacerbated by cytoreductive treatments, as patients treated with hydroxyurea (HU) were found to have a two-fold higher risk of NMSC compared to patients who were not treated with HU (Barbui et al., Leukemia. 2019;33:1996-2005). Previous studies examined fewer than 2000 patients; the study populations may have included patients with other MPNs and PV. Our objective was to evaluate the frequency of second cancers in patients with a primary PV diagnosis who were treated with HU compared to phlebotomy only using a large US real-world claims database. Methods:This real-world, retrospective cohort study used Optum's deidentified Market Clarity Data consisting of electronic health records (EHR) on 105 million US patients and linked medical and prescription claims between 1/1/2007 to 12/31/2019. More than 82,000 patients with ≥1 PV diagnosis at any time were identified within the extracted dataset. For this analysis, eligible patients were ≥18 years old and required ≥2 PV diagnosis codes with ≥60 days between the first and last diagnosis code in the data period. Patients were indexed at the first diagnosis code of PV preceded by ≥1 year of EHR data (“index”) and required ≥1 year of post-index data or death prior to data period end. Patients with a pre-index diagnosis of secondary polycythemia, leukemia, myelodysplastic syndrome, myelofibrosis, or stem cell transplant were excluded. Second cancers were identified at first occurrence of a diagnosis code for a defined cancer in EHR during specified time periods. Analysis of post-treatment cancer rates used a subset of patients with pre-index medication data and excluded cancer types identified prior to treatment. Results:The total study cohort included 20,089 patients with PV (mean age, 63.0 years; 57.8% male; 88.6% Caucasian). Some patients (15.5%; n=3112) had evidence of a pre-index cancer. Median duration of follow-up was 4.3 years (IQR, 2.4-5.9). Among the total cohort, 35.7% (7181 patients) reported at least one second cancer in the post-index period (“post-index period prevalence”). The rate of second cancers in the post-index period was 109.7 events per 1000 patient years (PY). The most common non-hematologic malignancies were skin cancers (22.1/1000 PY) followed by prostate cancer (8.1/1000 PY) and breast cancer (6.7/1000 PY) (Figure 1). Within the total cohort, 9.1% (1830 patients) reported at least one skin cancer in the post-index period, with 8.3% (1659 patients) experiencing NMSC and 1.4% (286 patients) experiencing melanoma (Figure 1). The post-index period prevalence rate of NMSC was 6 times higher vs. the rate of melanoma (19.8 vs. 3.3/1000 PY). Among NMSCs, the rate of basal cell carcinoma was higher than the rate of squamous cell carcinoma (8.7 vs. 6.9/1000 PY). However, the highest rate was observed for non-specific or unspecified malignant neoplasms (8.9/1000 PY). Within a subset of 17,402 patients with pre-index medication data, 20.2% (n=3512) were exposed to at least 4 weeks of HU (without ruxolitinib) at any time; 19.0% (n=3314) were treated with phlebotomy (PHL) only. The rate of any second malignancy subsequent to treatment was >2.5-times higher in the HU vs. PHL group (140.4 vs. 55.3/1000 PY) (Table 1). Rates of skin cancers were nearly two times higher in the HU vs. PHL group for any skin cancer (31.6 vs. 17.7/1000 PY), NMSC (28.9 vs. 15.7/1000 PY), and melanoma (4.5 vs. 2.9/1000 PY). Conclusion:To our knowledge, this is the largest retrospective study evaluating the frequency of second cancers in a cohort of US patients with PV to-date. We found that patients with PV had high rates of malignancies associated with their primary PV diagnosis. The highest rates were observed for skin cancers, driven by the frequency of NMSC. Patients treated with HU had nearly twice the rate of skin cancers (including NMSC and melanoma) compared to PHL-only treated patients.

Upregulation of Platelet Transcripts As Biomarkers of Fibrotic Progression in Myeloproliferative Neoplasms

Myeloproliferative neoplasms (MPN) are a group of clonal hemopoietic disorders that have a proliferative phase (including essential thrombocythemia (ET) and polycythemia vera (PV)), and which can progress to advanced disease such as myelofibrosis (MF) or acute leukemia. This is thought to be due in part to progressively dysfunctional megakaryocytes driving reticulin deposition in the bone marrow. In previous work, we demonstrated that the platelet transcriptome in MPN differs from normal, and the difference is most marked with increased reticulin (Guo et al., 2020). A putative 3-gene signature ( CCND1, H2AFX and CEP55) could discriminate between patients with and without increased reticulin fibrosis. To progress this, we have profiled the transcriptome of platelets from a further 173 MPN patients were analyzed using next generation sequencing. CCND1, H2AFX and CEP55 were upregulated in platelets from patients with MF compared to ET (4.6-fold, padj<0.001, 9.0-fold, padj<0.001 and 10.5-fold, padj<0.001, respectively) and to PV (3.9-fold, padj<0.001, 9.2-fold, padj<0.001, 12.0-fold, padj<0.001). Application of the established 3-gene signature had a positive predictive value of 78%, a negative prediction value of 90% and accuracy of 88% for MF. To assess the temporal changes in these gene transcripts in platelets and the potential utility to predict fibrotic progression, we then performed longitudinal analyses in two MPN mouse models. Jak2V617F knock-in mice ( n=25) with stable serially transplantable disease, which resembles PV (Mullally et al., 2010), were sacrificed at 4, 8, 12, 16 and 20 weeks post-transplant, and controls ( n=5) at 20 weeks. Blood counts were performed, and spleen and sternum harvested and stained with hematoxylin and eosin, and for reticulin. By 20-weeks, the platelet and leucocyte counts and hemoglobin had increased as expected. There was splenomegaly with extramedullary hematopoiesis and a mild increase in marrow reticulin. There was no significant up-regulation of Ccnd1, H2afx or Cep55 in platelet transcripts at any timepoint compared to the control mice. This data showed stability of the mouse PV phenotype and platelet transcripts, in keeping with the chronic phase of MPN. Mice with MPLW515L ( n=8) (Pikman et al., 2006) were sacrificed at 3 and 6 weeks post-transplant, and control mice ( n=4) at 6 weeks. At 3-weeks post-transplant Cep55 (131.6-fold, padj<0.001) and H2afx (1.7-fold, padj<0.01) had increased in the MPLW515L mice and there was no increase in reticulin in the bone marrow. At 6-weeks platelet and leucocyte counts had increased, there was splenomegaly with extramedullary hematopoiesis including marked megakaryocytosis and marrow reticulin. On platelet transcript analysis Cep55 (154.6-fold, padj<0.001) and H2afx (2.4-fold, padj<0.001) were significantly increased; there was no significant change for Ccnd1. Upregulation of Cep55 and H2afx in the MPLW515L mouse occurred prior to the accumulation of bone marrow reticulin and development of MF phenotype. This suggests that up-regulation of Cep55 and H2afx in platelets may be a predictor of disease progression from ET and PV to MF. For MPN patients, a deviation from steady-state platelet transcript levels, specifically for H2AFX and CEP55, may infer a biological transition towards the fibrotic phenotype. This novel approach utilizing platelet transcript analysis may therefore have potential for serially monitoring MPN patients and the early identification of genomic changes that may herald disease transformation to MF.

Proteomic Screening Identifies Megakaryocyte Derived PF4/Cxcl4 As a Critical Driver of Myelofibrosis

Background: Despite advances in understanding the genomic landscape of Myeloproliferative Neoplasms (MPNs), the precise pathological mechanisms driving fibrotic progression in MPNs remain incompletely understood. Previous research has shown that Megakaryocytes (Mks) play a role in MPN pathology by promoting inflammation and extracellular matrix (ECM) deposition by activated stromal cells. However, the specific factors derived from Mks that contribute to the inflammatory milieu and myelofibrosis progression are not well defined. Aims: To investigate the events that drive bone marrow (BM) fibrosis due to abnormal megakaryocyte-stromal crosstalk, we employed mass spectrometry-based proteomics in a myelofibrosis-like mouse model. Our goal was to identify differentially expressed proteins involving Mks, platelets and BM cells in fibrotic versus non-fibrotic conditions. Methods: We induced myelofibrosis in mice through repeated injections of supra-pharmacological doses of the thrombopoietin-mimetic Romiplostim (TPO high). Subsequently, we isolated Mks, BM cells and platelets from TPO high and control mice (injected with saline) and analyzed protein extracts using LC-MS/MS assay. Cytokine levels in plasma and BM cell-free fluids of treated mice were assessed using cytokine arrays. To evaluate fibrosis-related markers, we analyzed the expression of myofibroblasts markers (a-SMA, Vimentin) and ECM synthesis (Fibronectin, Type III Collagen) in mouse embryonic fibroblasts (mEFs) and human BM mesenchymal stromal cells after stimulation with recombinant PF4 using western blot. mEFs were co-cultured with BM-derived Mks in direct and indirect co-culture systems in the presence of increasing TPO concentrations. In some experiments, Mks were transfected with siRNA specifically targeting CXCL4 gene or control siRNA before co-culture with mEFs. Additionally, we quantified plasma PF4/Cxcl4 levels using ELISA assay in a large cohort of MPN patients and normal controls. We also sorted CD41/CD61 Mks from BM aspirates of MPN patients and evaluated CXCL4 mRNA levels through qPCR. Results: We observed that signaling pathways related to cytoskeletal reorganization, cell adhesion and inflammation, were commonly activated in Mks, platelets and BM cells of TPO high mice compared to control mice. The MSP-RON signaling pathway and the Unfolded Protein Response were specifically activated in Mks and platelets, respectively. Among the differentially expressed proteins,the chemokine PF4/Cxcl4 was up regulated exclusively in the proteasomes of the TPO high mice. We confirmed Increased levels of PF4/Cxcl4 in plasma and BM cell-free fluids of fibrotic mice using cytokine arrays. In vitro stimulation of BM-derived Mks with high TPO concentrations resulted in increased synthesis and secretion of PF4/Cxcl4. Recombinant PF4/Cxcl4 was rapidly internalized through surface glycosaminoglycans by mEFs and human BM mesenchymal stromal cells, leading to myofibroblast differentiation. Importantly, we demonstrated that these mechanisms were interconnected during Mk-mEF cross-talk in co-culture systems with high TPO concentrations. Genetic down-regulation of CXCL4 in Mks, prior to co-culture in TPO-saturated conditions, mitigated the profibrotic phenotype of mEFs in terms of ECM synthesis and myofibroblast markers expression. Finally, we found higher plasma levels of circulating PF4/Cxcl4 and increased expression in BM-sorted Mks in patients with MPNs. Conclusions: Our findings identify a crucial role of Mk derived PF4 in the fibrosis progression of MPNs, providing further support for the potential therapeutic strategy of neutralizing PF4.

Cooperation between SF3B1 and JAK2V617F Mutations Accelerates Fibrotic Progression in Myeloproliferative Neoplasms By Enhancing STAT1 Signaling

Myeloproliferative neoplasms (MPN) are heterogenous clonal hematologic neoplasms where current therapies show limited disease-modification. We previously reported that the splicing factor, SF3B1, is mutated in 5-10% of MPN correlating with myelofibrotic progression in essential thrombocythemia. This adverse phenotype contrasts with SF3B1 mutationsin myelodysplasia where it is associated with milder disease. Moreover, SF3B1 K666 is the dominant hotspot in MPN in contrast to K700E in MDS (abstract #185043). The mechanism by which SF3B1 mutations accelerate myelofibrotic progression in JAK2V617F-mutated MPN is not understood and will be a key step towards the development of disease-modifying therapies. To address this, we applied two complimentary single-cell multiomic methods to study 15 individuals (healthy donor [HD, n=5], JAK2V617F-single mutated [J+, n=5] and JAK2V617F- SF3B1K666double mutated MPN cases [JS+, n=5]). First, we characterised the cellular landscape of JS+ MPN using CITE-seq/10X genomics platform. Then, we analyzed SF3B1-mutant ( SF3B1mut) disease-driving hematopoietic stem and progenitor cells (HSPCs) and their associated aberrant splicing signatures using TARGET-seq, which permitted single-cell genotyping with allelic resolution and intra-patient comparison of mutant versus wild-type (wt) cells. In an analysis of 109,498 cells, we observed abnormally expanded erythroid (ERP), megakaryocyte (MK) and unexpectedly, eosinophil-basophil-mast cell (EBM) progenitors in JS+ as compared with J+ or HD HSPCs (Fig 1A). Two HSC clusters were present, one of which (HSC_mpp2) principally comprised of JS+ cells and had a distinct molecular program (Fig 1A); upregulation in MK (eg. PF4, VWF) and EBM lineage signature genes (eg. TPSB2), interferon signaling genes but downregulation in apoptosis genes and canonical stemness markers (eg. CD133). These signatures suggest the JS+ driven HSC cluster may be MK/EBM-transcriptionally primed and may therefore, promote expansion of these abnormal progenitors which are implicated in driving fibrosis development. Through genotyping of >5000 HSPCs, we resolved the clonal architecture of JS+ MPN, observing that in most cases double mutant cells were present and clonally dominant, with mutant SF3B1 the initiating event. Mutations arose from the HSC level and genotypes were equally distributed in the HSPC compartment. Highlighting the importance of single-cell analysis, and not apparent at the bulk level, JAK2V617F and SF3B1K666mutationsresided in separate clones for one case with a WHO fibrosis score 0 in contrast to the other 4 cases with double mutant cells and fibrosis scores 1-3. This reinforces the cooperative effect of thesemutationsto drive fibrosis. We performed full-length transcriptomics of select HSPCs to study SF3B1mut-specific aberrant splicing events (ASE). In a global splicing analysis, more events were detected in JAK2V617F- SF3B1K666 double mutant than single mutant HSPCs. Retained intron (RI) events were the most prevalent ASE, followed by skipped exon and alternative 3' splice site events. We identified previously described SF3B1mut-specific ASE ( MAP3K7, ERGIC3 and SEPTIN6) confirming the validity of our dataset to study new ASE. Inter- and intra-patient comparison of SF3B1mut versus SF3B1wt cells uncovered a number of novel biologically relevant ASE, including two RI events in STAT1. These RI events were reduced in SF3B1mut HSPCs with the predicted consequence of increased STAT1 expression and activation, of direct relevance to MPN pathobiology since STAT1 is known to play a key role in enhancing megakaryopoiesis in MPN (Fig 1B). We validated these events using long-read Nanopore sequencing of single-cell cDNA libraries and have developed a cell model system with transient expression of SF3B1K666 mutation versus SF3B1wt in HEK293T to corroborate STAT1 ASE occurring as a direct consequence of mutant SF3B1. In further support of upregulated STAT1 activation, we observed upregulation of STAT1 downstream targets in abnormally expanded JS+ HSPCs. In conclusion, this analysis resolved the clonal architecture and hematopoietic cellular composition of JS+ MPN, identifying abnormally expanded and transcriptionally primed HSPC populations with abnormal splicing of STAT1 enhancing JAK-STAT signaling, uncovering the mechanism by which these mutations promote an accelerated fibrotic phenotype.

BMAL1 inhibits renal fibrosis and renal interstitial inflammation by targeting the ERK1/2/ELK-1/Egr-1 axis

RationaleRenal fibrosis and renal interstitial inflammation due to hydronephrosis are associated with progressive chronic kidney disease (CKD). The clock gene BMAL1 is thought to be involved in various diseases, including hypertension, diabetes, etc. However, little is known about how BMAL1 regulates renal fibrosis and renal interstitial inflammation in obstructed kidneys. MethodsThe expression level of BMAL1 in UUO was examined using the GEO database. Lentivirus, siRNA and adeno-associated virus were used to modulate BMAL1 levels in HK-2 cells and mouse kidney. qRT-PCR, immunofluorescence staining, histological analysis, ELISA and Western blot were used to determine the level of fibrin deposition and the release of inflammatory factors. Immunofluorescence staining and western blotting were used to examine the interaction between BMAL1 and the ERK1/2/ELK-1/Egr-1 axis. ResultsBioinformatics analysis and in vivo experiments in this study showed that the expression level of BMAL1 in UUO model kidneys was higher than that in normal kidneys. We then found that downregulation of BMAL1 promoted the production of extracellular matrix (ECM) proteins and proinflammatory factors in vivo and in vitro, whereas upregulation inhibited this process. In addition, we demonstrated that the ERK1/2/ELK-1/Egr-1 axis is an important pathway for BMAL1 to play a regulatory role, and the use of PD98059 abolished the promoting effect of down-regulation of BMAL1 on fibrosis and inflammation. ConclusionsOur findings suggest that BAML1 can target the ERK1/2/ELK-1/Egr-1 axis to suppress fibrotic progression and inflammatory events in obstructed kidneys, thereby inhibiting the development of CKD.

The Upregulation of the FBLN1 and HS3ST4 Genes May Represent a Potential Mechanism Driving the Fibrotic Progression in Primary Myelofibrosis and Leading to Adverse Prognosis

Myelofibrosis is a group of diseases characterized by increased proliferation of bone marrow fibrous connective tissue and a decrease in hematopoietic cells, leading to impaired bone marrow hematopoietic function. Myelofibrosis includes primary myelofibrosis（PMF）, myelofibrosis secondary to polycythemia vera（post-PV MF）, and myelofibrosis secondary to essential thrombocythemia（post-ET MF）. Studies suggest that certain genes may be responsible for driving the development of myelofibrosis and causing adverse prognosis. By conducting RNA-Seq analysis, we investigated the differentially expressed genes and their impact on prognosis in primary myelofibrosis and secondary myelofibrosis. Furthermore, we analyzed the potential mechanisms driving the progression of fibrosis. Additionally, we performed a comparative analysis of the clinical features among various types of myelofibrosis. In this study, we performed transcriptome sequencing to analyze gene expression differences in 14 overt-PMF samples, 5 post-ET MF samples, and 3 post-PV MF samples.Functional and pathway analyses were conducted on these differentially expressed genes, and cluster heatmaps were used to visualize their expression patterns.Simultaneously, we performed a comparative analysis of clinical characteristics among 370 overt-PMF patients, 96 post-ET MF patients, and 52 post-PV MF patients.The study revealed that compared to post-PV MF, overt-PMF showed 81 upregulated and 2 downregulated differentially expressed genes, while compared to post-ET MF, overt-PMF displayed 19 upregulated and 3 downregulated differentially expressed genes (Figure a) (Figure b). Based on the similarity of gene expression profiles among the samples, a clustering analysis was performed (Figure c).The results demonstrated that in overt-PMF, the FBLN1 gene was significantly upregulated compared to post-PV MF and was enriched in pathways related to extracellular matrix formation and cell adhesion among the differentially expressed genes (Figure d). Additionally, the HS3ST4 gene was significantly upregulated in overt-PMF compared to post-ET MF and was enriched in pathways related to interleukin-8 generation and regulation of cytokine production involved in inflammatory responses.In the analysis of clinical characteristics among different types of myelofibrosis patients, it was observed that overt-PMF was more prone to transform into acute leukemia compared to post-PV MF, had a higher tendency to develop transfusion dependency (Figure e), and exhibited lower hemoglobin levels and hematocrit at the time of diagnosis (Figure f). Moreover, compared to post-ET MF, overt-PMF had a higher likelihood of transforming into acute leukemia and lower platelet counts at the time of diagnosis. The research findings indicate that the expressions of FBLN1 and HS3ST4 genes are significantly upregulated in overt-PMF, and patients with overt-PMF have a worse prognosis compared to those with post-PV MF and post-ET MF. Previous studies have shown that FBLN1 is involved in extracellular matrix assembly, promoting the formation and stabilization of collagen fibers. It can also interact with integrin receptors and other cell adhesion molecules, participating in cell-extracellular matrix signaling processes. On the other hand, HS3ST4 is involved in processes such as cell adhesion, cell migration, and cell signaling. Based on these findings, it is speculated that the upregulation of FBLN1 and HS3ST4 gene expressions may potentially drive the progression of fibrosis and lead to an adverse prognosis in patients with myelofibrosis. Acknowledgement: This research was funded by the Key R&D Program of Zhejiang, No. 2022C03137; Public Technology Application Research Program of Zhejiang, China, No. LGF21H080003; Zhejiang Medical Association Clinical Medical Research special fund project, No. 2022ZYC-D09. Correspondence to: Dr Jian Huang, Department of Hematology, The First Affiliated Hospital of Zhejiang University School of Medicine. No. 79 Qingchun Road. Hangzhou, Zhejiang, PR China. househuang@zju.edu.cn

Myopathologic trajectory in Duchenne muscular dystrophy (DMD) reveals lack of regeneration due to senescence in satellite cells

Duchenne muscular dystrophy (DMD) is a devastating X-linked muscular disease, caused by mutations in the DMD gene encoding Dystrophin and affecting 1:5000 boys worldwide. Lack of Dystrophin leads to progressive muscle wasting and degeneration resulting in cardiorespiratory failure. Despite the absence of a definitive cure, innovative therapeutic avenues are emerging. Myopathologic studies are important to further understand the biological mechanisms of the disease and to identify histopathologic benchmarks for clinical evaluations. We conducted a myopathologic analysis on twenty-four muscle biopsies from DMD patients, with particular emphasis on regeneration, fibro-adipogenic progenitors and muscle stem cells behavior. We describe an increase in content of fibro-adipogenic progenitors, central orchestrators of fibrotic progression and lipid deposition, concurrently with a decline in muscle regenerative capacity. This regenerative impairment strongly correlates with compromised activation and expansion of muscle stem cells. Furthermore, our study uncovers an early acquisition of a senescence phenotype by DMD-afflicted muscle stem cells. Here we describe the myopathologic trajectory intrinsic to DMD and establish muscle stem cell senescence as a pivotal readout for future therapeutic interventions.

TMT-based proteomics analysis identifies the interventional mechanisms of Qijia Rougan decoction in improving hepatic fibrosis

Ethnopharmacological relevanceQijia Rougan decoction (QJ), consisting of eight herbs and two animal drugs, is an effective traditional Chinese medicine with hepatoprotective and antifibrotic effects. However, its underlying action mechanism remains unclear. Aim of the studyTo explore the mechanism underlying the treatment of liver fibrosis in rats by QJ. Materials and methodsRats with fibrosis were constructed using carbon tetrachloride (CCl4). The QJ was orally administered to fibrotic rats. Hepatic pathological changes were evaluated using hematoxylin and eosin and Masson's trichrome staining. The differentially expressed proteins (DEPs) in QJ were analyzed using quantitative proteomics. Subsequently, the underlying mechanisms in liver fibrosis after QJ treatment were validated using Western blotting. ResultsThe QJ markedly improved liver function and attenuated fibrotic progression. Based on the tandem mass-tag based (TMT) proteomics, we identified 818 common DEPs between QJ vs Model and Model vs Control, including 296 upregulated and 522 downregulated DEPs, which mostly participate in metabolic pathways, oxidation-reduction reactions, and collagen biosynthetic processes. In addition, we found that QJ reduced hepatocellular death by inhibiting the expression of caspase proteins, repressing pro-apoptotic proteins, and promoting anti-apoptotic proteins. We further demonstrated that QJ suppressed the Akt/mTOR pathway. ConclusionQJ exerted hepatoprotective effects in CCl4-induced rats through multi-pathway regulation. This study provides protein information on liver fibrosis treated with QJ.

Macrophage-derived MMP12 promotes fibrosis through sustained damage to endothelial cells

Macrophages are essential for the maintenance of endothelial cell function. However, the potential impact and mechanisms of crosstalk between macrophages and endothelial cells during silicosis progression remain unexplored. To fill this knowledge gap, a mouse model of silicosis was established. Single cell sequencing, spatial transcriptome sequencing, western blotting, immunofluorescence staining, tube-forming and wound healing assays were used to explore the effects of silicon dioxide on macrophage-endothelial interactions. To investigate the mechanism of macrophage-mediated fibrosis, MMP12 was specifically inactivated using siRNA and pharmacological approaches, and macrophages were depleted using disodium chlorophosphite liposomes. Compared to the normal saline group, the silica dust group showed altered macrophage-endothelial interactions. Matrix metalloproteinase family member MMP12 was identified as a key mediator of the altered function of macrophage-endothelial interactions after silica exposure, which was accompanied by pro-inflammatory macrophage activation and fibrotic progression. By using ablation strategies, macrophage-derived MMP12 was shown to mediate endothelial cell dysfunction by accumulating on the extracellular matrix. During the inflammatory phase of silicosis, MMP12 secreted by pro-inflammatory macrophages caused decreased endothelial cell viability, reduced migration, decreased trans-endothelial resistance and increased permeability; while during the fibrotic phase, macrophage-derived MMP12 sustained endothelial cell injury through accumulation on the extracellular matrix.

Myeloid Heterogeneity Mediates Acute Exacerbations of Pulmonary Fibrosis.

Epidemiological evidence indicates that exposure to particulate matter is linked to the development of idiopathic pulmonary fibrosis (IPF) and increases the incidence of acute exacerbations of IPF. In addition to accelerating the rate of lung function decline, exposure to fine particulate matter (particulate matter smaller than 2.5 μm [PM2.5]) is a risk factor for increased mortality in subjects with IPF. In this article, we show that exposure to PM2.5 mediates monocyte recruitment and fibrotic progression in mice with established fibrosis. In mice with established fibrosis, bronchoalveolar lavage cells showed monocyte/macrophage heterogeneity after exposure to PM2.5. These cells had a significant inflammatory and anti-inflammatory signature. The mixed heterogeneity of cells contributed to the proinflammatory and anti-inflammatory response. Although monocyte-derived macrophages were recruited to the lung in bleomycin-injured mice treated with PM2.5, recruitment of monocytes expressing Ly6Chi to the lung promoted progression of fibrosis, reduced lung aeration on computed tomography, and impacted lung compliance. Ly6Chi monocytes isolated from PM2.5-exposed fibrotic mice showed enhanced expression of proinflammatory markers compared with fibrotic mice exposed to vehicle. Moreover, IPF bronchoalveolar lavage cells treated ex vivo with PM2.5 showed an exaggerated inflammatory response. Targeting Ly6Chi monocyte recruitment inhibited fibrotic progression in mice. Moreover, the adoptive transfer of Ly6Chi monocytes exacerbated established fibrosis. These observations suggest that enhanced recruitment of Ly6Chi monocytes with a proinflammatory phenotype mediates acute exacerbations of pulmonary fibrosis, and targeting these cells may provide a potential novel therapeutic target to protect against acute exacerbations of IPF.

Antler stem cell exosomes alleviate pulmonary fibrosis via inhibiting recruitment of monocyte macrophage, rather than polarization of M2 macrophages in mice

Pulmonary fibrosis (PF), a chronic interstitial lung disease, is characterized by over-abundant deposition of extracellular matrix consisting mainly of collagen I. In previous studies, we demonstrated that deer antler stem cells (AnSCs), a novel type of adult stem cell, are capable of significantly down-regulating collagen formation in different organs and tissues and speculated that they could effectively treat PF via reducing collagen deposition in the lung tissue. In the present study, we found that administration of AnSCs improved the survival rate of PF mice and reduced lung fibrosis, collagen deposition and myofibroblast differentiation. The effects of AnSC treatment were significantly better than the positive control (adipose-derived stem cells). Interestingly, AnSC-Exos were almost equally effective as AnSCs in treating PF, suggesting that the effects of AnSCs on reduction of PF may be mainly through a paracrine mechanism. Further, AnSC-Exos reduced the number of M2 macrophages, a type of macrophage that secrets pro-fibrotic factors to accelerate fibrotic progression, in the lung tissues. In vitro experiments showed that the effects of AnSC-Exos on macrophage modulation were likely achieved via inhibition of the recruitment of circulating monocyte-derived macrophages (reducing the number of macrophages), rather than via inhibition of M2 polarization of macrophages. Inhibition of macrophage recruitment by AnSCs may be achieved indirectly via inhibiting CCL7 expression in fibroblasts; both let-7b and let-7a were highly enriched in AnSC-Exos and may play a critical role in the inhibition of CCL7 expression of fibroblasts. Collectively, the use of antler stem cells or their exosomes opens up a novel strategy for PF treatment in the clinical setting.

Identification of Fibrotic Biomarkers Associated with Macrophages in Diabetic Nephropathy.

BACKGROUND Diabetic nephropathy (DN) is the main cause of end-stage renal disease. Renal fibrosis is an important pathological feature of kidney injury, and the therapeutic means are very limited. The functions of macrophages play important roles in renal fibrosis. There is a complicated link between altered immune metabolism and oxidative stress. Hence, we designed this study to identify the oxidative stress- and macrophage-relevant biomarkers reflecting fibrosis in DN. MATERIAL AND METHODS Differential expression analysis was performed based on the GSE96804 dataset. xCell and weighted gene co-expression network analysis were used to determine the distinctions in infiltrating immune cells between DN and control specimens. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses were conducted. A protein-protein interaction network was constructed to identify the hub genes. Hub genes were validated in an external dataset, GSE30528, and cell models. RESULTS MMP2, CASP3, and HIF-1alpha were identified as biomarkers, which were upregulated in the DN group and positively correlated with the infiltration of macrophages and M1 macrophages. In vitro, the 3 genes were highly expressed in murine MPC5 cells treated with high glucose and human THP-1 macrophages treated with advanced glycation end products. CONCLUSIONS Our results provided biomarkers for predicting the fibrotic progression of DN and confirmed that MMP2, CAPS3, and HIF-1alpha have good diagnostic value. They might be involved in the progression of DN fibrosis by regulating oxidative stress and macrophage recruitment or polarization.

Progression of radiographic fibrosis in rheumatoid arthritis-associated interstitial lung disease.

Preclinical interstitial lung disease (pILD) may represent the early stages of rheumatoid arthritis-associated interstitial lung disease (RA-ILD). However, the characteristics, clinical outcomes, and risk factors associated with fibrosis progression in RA-ILD, including pILD and ILD, remain poorly understood. Baseline data were compared between patients with RA-ILD and those with RA alone. Multivariate logistic regression and Cox regression analyses were performed to identify risk factors associated with the prevalence and imaging progression of RA-ILD, respectively. Among the 371 enrolled RA patients, 32.3% had RA-ILD. Multiple logistic regression analyses identified age over 60.0 years (OR 2.22), smoking (OR 2.09), diabetes mellitus (DM) (OR 3.09), mixed connective tissue disease (MCTD) (OR 2.98), serum lactate dehydrogenase (LDH) levels exceeding 250.0 U/L (OR 6.73), and positive anti-cyclic citrullinated peptide (anti-CCP) antibody (OR 2.06) as independent risk factors for RA-ILD (p< 0.05 or 0.01). Among the 98 RA-ILD patients who underwent follow-up for a median duration of 19.1 months, 51.0% demonstrated fibrotic progression on high-resolution computed tomography (HRCT). Multiple Cox regression analysis identified DM (HR 2.03), Disease Activity Score in 28 joints-Erythrocyte Sedimentation Rate (DAS28-ESR) greater than 5.1 (HR 2.21), and baseline HRCT scores exceeding 5.0 (HR 2.30) as independent risk factors for fibrosis progression in RA-ILD (p< 0.05 or 0.01). Nearly one-third of RA patients in this cohort had prevalent pILD or ILD, and half of them demonstrated imaging progression during follow-up. DM, higher DAS28-ESR, and advanced HRCT scores were identified as independent risk factors for progressive fibrosis in RA-ILD.

Concise review: Breastfeeding, lactation, and NAFLD. An updated view of cross-generational disease transmission and prevention

Evidence suggests that breastfeeding protects the mother-infant dyad against the development and progression of nonalcoholic fatty liver disease (NAFLD). In this context, we aim to provide insight into the most notable and representative epidemiological studies published in the literature. Furthermore, we will delve into the potential underlying pathomechanisms that might be involved in this relationship. The current definitions of breastfeeding, lactation, mother-infant dyad, and nonalcoholic fatty liver disease (NAFLD) are provided. Next, the epidemiological evidence supporting potential benefits for the (long-term) lactating mother in terms of protection from the development and progression of NAFLD is reviewed. The putative mechanisms underlying this protection are also analyzed. Similarly, clinical and epidemiological studies evaluating the benefits of breastfeeding for the offspring are examined, together with a discussion of the putative underlying mechanisms. In conclusion, our understanding of breastfeeding (for the offspring) and lactation (for the mother) as protective factors from NAFLD development and fibrotic progression will provide further insight into unprecedented disease mechanisms shared by the mother-infant dyad promising to interrupt the vicious cycle of NAFLD transmission across generations.

Efficient intervention for pulmonary fibrosis via mitochondrial transfer promoted by mitochondrial biogenesis

The use of exogenous mitochondria to replenish damaged mitochondria has been proposed as a strategy for the treatment of pulmonary fibrosis. However, the success of this strategy is partially restricted by the difficulty of supplying sufficient mitochondria to diseased cells. Herein, we report the generation of high-powered mesenchymal stem cells with promoted mitochondrial biogenesis and facilitated mitochondrial transfer to injured lung cells by the sequential treatment of pioglitazone and iron oxide nanoparticles. This highly efficient mitochondrial transfer is shown to not only restore mitochondrial homeostasis but also reactivate inhibited mitophagy, consequently recovering impaired cellular functions. We perform studies in mouse to show that these high-powered mesenchymal stem cells successfully mitigate fibrotic progression in a progressive fibrosis model, which was further verified in a humanized multicellular lung spheroid model. The present findings provide a potential strategy to overcome the current limitations in mitochondrial replenishment therapy, thereby promoting therapeutic applications for fibrotic intervention.

Increased expression of TNFRSF14 and LIGHT in biliary epithelial cells of patients with primary sclerosing cholangitis

Background and aimsThere is a lack of biliary epithelial molecular markers for primary sclerosing cholangitis (PSC). We analyzed candidates from disease susceptibility genes identified in recent genome-wide association studies (GWAS). MethodsExpression levels of GWAS genes were analyzed in archival liver tissues of patients with PSC and controls. Immunohistochemical analysis was performed to evaluate expression levels in the biliary epithelia of PSC (N = 45) and controls (N = 12). Samples from patients with primary biliary cholangitis (PBC) were used as disease controls (N = 20). ResultsHepatic expression levels of ATXN2, HHEX, PRDX5, MST1, and TNFRSF14 were significantly altered in the PSC group. We focused on the immune-related receptor, TNFRSF14. Immunohistochemistry revealed that high expression of TNFRSF14 in biliary epithelial cells was observed only in the PSC group. In addition, the expression of LIGHT, which encodes a TNFRSF14-activating ligand, was increased in PSC liver. Immunohistochemistry showed that high expression of LIGHT was more common in PSC biliary epithelia (53%) than in the PBC (15%) or control (0%) groups; moreover, it was positively associated with fibrotic progression, although it was not an independent prognostic factor. ConclusionsTNFRSF14 and LIGHT are promising candidate markers for PSC.

Inhibition of galectin-3 post-infarction impedes progressive fibrosis by regulating inflammatory profibrotic cascades.

Acute myocardial infarction (MI) causes inflammation, collagen deposition, and reparative fibrosis in response to myocyte death and, subsequently, a pathological myocardial remodelling process characterized by excessive interstitial fibrosis, driving heart failure (HF). Nonetheless, how or when to limit excessive fibrosis for therapeutic purposes remains uncertain. Galectin-3, a major mediator of organ fibrosis, promotes cardiac fibrosis and remodelling. We performed a preclinical assessment of a protein inhibitor of galectin-3 (its C-terminal domain, Gal-3C) to limit excessive fibrosis resulting from MI and prevent ventricular enlargement and HF. Gal-3C was produced by enzymatic cleavage of full-length galectin-3 or by direct expression of the truncated form in Escherichia coli. Gal-3C was intravenously administered for 7 days in acute MI models of young and aged rats, starting either pre-MI or 4 days post-MI. Echocardiography, haemodynamics, histology, and molecular and cellular analyses were performed to assess post-MI cardiac functionality and pathological fibrotic progression. Gal-3C profoundly benefitted left ventricular ejection fraction, end-systolic and end-diastolic volumes, haemodynamic parameters, infarct scar size, and interstitial fibrosis, with better therapeutic efficacy than losartan and spironolactone monotherapies over the 56-day study. Gal-3C therapy in post-MI aged rats substantially improved pump function and attenuated ventricular dilation, preventing progressive HF. Gal-3C in vitro treatment of M2-polarized macrophage-like cells reduced their M2-phenotypic expression of arginase-1 and interleukin-10. Gal-3C inhibited M2 polarization of cardiac macrophages during reparative response post-MI. Gal-3C impeded progressive fibrosis post-MI by down-regulating galectin-3-mediated profibrotic signalling cascades including a reduction in endogenous arginase-1 and inducible nitric oxide synthase (iNOS). Gal-3C treatment improved long-term cardiac function post-MI by reduction in the wound-healing response, and inhibition of inflammatory fibrogenic signalling to avert an augmentation of fibrosis in the periinfarct region. Thus, Gal-3C treatment prevented the infarcted heart from extensive fibrosis that accelerates the development of HF, providing a potential targeted therapy.

Combining the age-male-albumin-bilirubin-platelets score and shear wave elastography stratifies carcinogenic risk in hepatitis C patients after viral clearance.

The treatment of hepatitis C with direct-acting antiviral agents (DAAs) produces a high rate of sustained virological response (SVR) with fewer adverse events than interferon (IFN) therapy with a similar effect in inhibiting carcinogenesis as IFN therapy. The age-male-albumin-bilirubin-platelets (aMAP) score is useful for stratifying the risk of hepatocellular carcinoma in chronic hepatitis patients, and the velocity of shear waves (Vs) measured by shear wave elastography has also been shown to be useful for diagnosing the level of fibrotic progression in hepatitis C and predicting carcinogenic risk. Combining these two may improve the prediction of carcinogenic risk. To determine whether combining the aMAP score with Vs improves carcinogenic risk stratification in medium-to-high-risk hepatitis C patients. This retrospective, observational study involved hepatitis C patients treated with DAAs who achieved SVR. Vs was measured before treatment (baseline), at the end of treatment (EOT), and 12 wk (follow-up 12) and 24 wk (follow-up 24) after treatment. The patients were followed for at least six months after EOT to determine whether cancer developed. Multiple regression analysis was used to identify factors contributing to hepatic carcinogenesis. The diagnostic performances of clinical parameters for predicting the presence of hepatocellular carcinoma were evaluated using receiver-operating characteristic (ROC) curve analyses. A total of 279 patients (mean age 65.9 years, 118 males, 161 females) were included in the analysis. Multiple regression analysis was performed with carcinogenesis as the target variable and alanine aminotransferase, platelets, α-fetoprotein, Vs, and the Fib-4 index as explanatory variables; only Vs was found to be significant (P = 0.0296). The cut-off value for Vs for liver carcinogenesis calculated using the ROC curve was 1.53 m/s. Carcinoma developed in 2.0% (3/151) of those with Vs < 1.53 m/s and in 10.5% (9/86) of those with Vs ≥ 1.53 m/s. In hepatitis C patients after SVR, combining the aMAP score and Vs to stratify the risk of carcinogenesis is more efficient than uniform surveillance of all patients.

Cell Adhesion Molecules in Fibrotic Diseases.

Mechanisms underlying the pathogenesis of tissue fibrosis remain incompletely understood. Emerging evidence suggests that cell adhesion molecules (CAMs) are critical in fibrotic progression in many organs, including lung, kidney, skin, and liver. CAMs promote cell-cell and cell-extracellular matrix (ECM) interactions to maintain tissue architecture and normal function in homeostasis. However, dysregulated expression and function of CAMs can lead to chronic inflammation and tissue fibrosis. The major families of CAMs include integrins, cadherins, selectins, and immunoglobulins. Here, we review the role of the CAMs in fibrosis development across various organs with a focus on integrins and cadherins, and discuss their respective roles in the development of pulmonary fibrosis.

Spermidine from arginine metabolism activates Nrf2 and inhibits kidney fibrosis

Kidney metabolism may be greatly altered in chronic kidney disease. Here we report that arginine metabolism is the most altered in unilateral ureteral obstruction (UUO)-induced fibrosis of the kidneys in metabolomic analysis. Spermidine is the most increased metabolite of arginine. In human glomerulonephritis, the amount of spermidine shown by immunostaining is associated with the amount of fibrosis. In human proximal tubule cells, spermidine induces nuclear factor erythroid 2-related factor 2 (Nrf2). Subsequently, fibrotic signals, such as transforming growth factor β1 secretion, collagen 1 mRNA, and oxidative stress, represented by a decrease in the mitochondrial membrane potential is suppressed by spermidine. UUO kidneys of Arg2 knockout mice show less spermidine and significantly exacerbated fibrosis compared with wild-type mice. Nrf2 activation is reduced in Arg2 knockout UUO kidneys. Spermidine treatment prevents significant fibrotic progression in Arg2 knockout mice. Spermidine is increased in kidney fibrosis, but further increases in spermidine may reduce fibrosis.