Microvesicles from Mesenchymal Stem Cells Overexpressing MiR-34a Ameliorate Renal Fibrosis In Vivo.

INTRODUCTION: We recently discovered that microvesicles (MVs)  derived from mesenchymal stem cells (MSCs) overexpressing  miRNA-34a can alleviate experimental kidney injury in mice. In  this study, we further explored the effects of miR34a-MV on renal  fibrosis in the unilateral ureteral obstruction (UUO) models.  Methods. Bone marrow MSCs were modified by lentiviruses  overexpressing miR-34a, and MVs were collected from the  supernatants of MSCs. C57BL6/J mice were divided into control,  unilateral ureteral obstruction (UUO), UUO + MV, UUO + miR-34aMV and UUO + miR-34a-inhibitor-MV groups. MVs were injected  to mice after surgery. The mice were then euthanized on day 7  and 14 of modeling, and renal tissues were collected for further  analyses by Hematoxylin and eosin, Masson's trichrome,  and Immunohistochemical (IHC) staining.  Results. The UUO + MV group exhibited a significantly reduced  degree of renal interstitial fibrosis with inflammatory cell infiltration,  tubular epithelial cell atrophy, and vacuole degeneration compared  with the UUO group. Surprisingly, overexpressing miR-34a enhanced  these effects of MSC-MV on the UUO mice.  Conclusion. Our study demonstrates that miR34a further enhances  the effects of MSC-MV on renal fibrosis in mice through the  regulation of epithelial-to-mesenchymal transition (EMT) and  Notch pathway. miR-34a may be a candidate molecular therapeutic  target for the treatment of renal fibrosis. DOI: 10.52547/ijkd.7673.

PBX/Knotted 1 homeobox-2 (PKNOX2) is a novel regulator of myocardial fibrosis.

Much effort has been made to uncover the cellular heterogeneities of human hearts by single-nucleus RNA sequencing. However, the cardiac transcriptional regulation networks have not been systematically described because of the limitations in detecting transcription factors. In this study, we optimized a pipeline for isolating nuclei and conducting single-nucleus RNA sequencing targeted to detect a higher number of cell signal genes and an optimal number of transcription factors. With this unbiased protocol, we characterized the cellular composition of healthy human hearts and investigated the transcriptional regulation networks involved in determining the cellular identities and functions of the main cardiac cell subtypes. Particularly in fibroblasts, a novel regulator, PKNOX2, was identified as being associated with physiological fibroblast activation in healthy hearts. To validate the roles of these transcription factors in maintaining homeostasis, we used single-nucleus RNA-sequencing analysis of transplanted failing hearts focusing on fibroblast remodelling. The trajectory analysis suggested that PKNOX2 was abnormally decreased from fibroblast activation to pathological myofibroblast formation. Both gain- and loss-of-function in vitro experiments demonstrated the inhibitory role of PKNOX2 in pathological fibrosis remodelling. Moreover, fibroblast-specific overexpression and knockout of PKNOX2 in a heart failure mouse model induced by transverse aortic constriction surgery significantly improved and aggravated myocardial fibrosis, respectively. In summary, this study established a high-quality pipeline for single-nucleus RNA-sequencing analysis of heart muscle. With this optimized protocol, we described the transcriptional regulation networks of the main cardiac cell subtypes and identified PKNOX2 as a novel regulator in suppressing fibrosis and a potential therapeutic target for future translational studies.

DNMT3A clonal hematopoiesis-driver mutations induce cardiac fibrosis by paracrine activation of fibroblasts.

Hematopoietic mutations in epigenetic regulators like DNA methyltransferase 3 alpha (DNMT3A), play a pivotal role in driving clonal hematopoiesis of indeterminate potential (CHIP), and are associated with unfavorable outcomes in patients suffering from heart failure (HF). However, the precise interactions between CHIP-mutated cells and other cardiac cell types remain unknown. Here, we identify fibroblasts as potential partners in interactions with CHIP-mutated monocytes. We used combined transcriptomic data derived from peripheral blood mononuclear cells of HF patients, both with and without CHIP, and cardiac tissue. We demonstrate that inactivation of DNMT3A in macrophages intensifies interactions with cardiac fibroblasts and increases cardiac fibrosis. DNMT3A inactivation amplifies the release of heparin-binding epidermal growth factor-like growth factor, thereby facilitating activation of cardiac fibroblasts. These findings identify a potential pathway of DNMT3A CHIP-driver mutations to the initiation and progression of HF and may also provide a compelling basis for the development of innovative anti-fibrotic strategies.

Exploring the role of ITGB6: fibrosis, cancer, and other diseases.

Integrin ß6 (ITGB6), a member of the integrin family of proteins, is only present in epithelial tissues and frequently associates with integrin subunit αv to form transmembrane heterodimers named integrin αvß6. Importantly, ITGB6 determines αvß6 expression and availability. In addition to being engaged in organ fibrosis, ITGB6 is also directly linked to the emergence of cancer, periodontitis, and several potential genetic diseases. Therefore, it is of great significance to study the molecular-biological mechanism of ITGB6, which could provide novel insights for future clinical diagnosis and therapy. This review introduces the structure, distribution, and biological function of ITGB6. This review also expounds on ITGB6-related diseases, detailing the known biological effects of ITGB6.

The Long Noncoding RNA H19 Promotes Fibrotic Processes in Lens Epithelial Cells.

Purpose: The purpose of this study was to investigate the role of lncRNA H19 in epithelial-mesenchymal transition (EMT) and its molecular mechanism in fibrotic cataracts. Methods: TGF-ß2-induced EMT was induced in human lens epithelial cell line (HLECs) and rat lens explants to mimic posterior capsular opacification (PCO) in vitro and in vivo. Anterior subcapsular cataract (ASC) was induced in C57BL/6J mice. The long noncoding RNA (lncRNA) H19 (H19) expression was detected by RT-qPCR. Whole-mount staining of lens anterior capsule was used to detect α-SMA and vimentin. Lentiviruses carrying shRNA or H19 vector were transfected in HLECs to knockdown or overexpress H19. Cell migration and proliferation were characterized by EdU, Transwell, and scratch assay. EMT level was detected by Western blotting and immunofluorescence. The rAAV2 carrying mouse H19 shRNA was injected into ASC model mouse anterior chambers as a gene therapy to determine its therapeutic potential. Results: PCO and ASC models were built successfully. We found H19 upregulation in PCO and ASC models in vivo and in vitro. Overexpression of H19 by lentivirus transfection increased cell migration, proliferation, and EMT. In addition, H19 knockdown by lentivirus suppressed cell migration, proliferation, and EMT levels in HLECs. Moreover, transfection of rAAV2 H19 shRNA alleviated fibrotic area in ASC mouse lens anterior capsules. Conclusions: Excessive H19 participates in lens fibrosis. Overexpression of H19 increases, whereas knockdown of H19 ameliorates HLECs migration, proliferation, and EMT. These results demonstrate H19 might be a potential target for fibrotic cataracts.

COL28 promotes proliferation, migration, and EMT of renal tubular epithelial cells.

Type XXVIII collagen (COL28) is involved in cancer and lung fibrosis. COL28 polymorphisms and mutations might be involved in kidney fibrosis, but the exact role of COL28 in renal fibrosis is unknown. This study explored the function of COL28 in renal tubular cells by examining the expression of COL28 mRNA and the effects of COL28 overexpression in human tubular cells. COL28 mRNA expression and localization were observed in normal and fibrotic kidney tissues from humans and mice using real-time PCR, western blot, immunofluorescence, and immunohistochemistry. The consequences of COL28 overexpression on cell proliferation, migration, cell polarity, and epithelial-to-mesenchymal transition (EMT) induced by TGF-ß1 were examined in human tubular HK-2 cells. COL28 expression was low in human normal renal tissues, mainly observed in the renal tubular epithelial cells and especially in proximal renal tubules. COL28 protein expression in human and mouse obstructive kidney disease was higher than in normal tissues (p < 0.05) and more significant in the UUO2-Week than the UUO1-Week group. The overexpression of COL28 promoted HK-2 cell proliferation and enhanced their migration ability (all p < 0.05). TGF-ß1 (10 ng/ml) induced COL28 mRNA expression in HK-2 cells, decreased E-cadherin and increased α-SMA in the COL28-overexpression group compared with controls (p < 0.05). ZO-1 expression decreased while COL6 increased in the COL28-overexpression group compared with controls (p < 0.05). In conclusion, COL28 overexpression promotes the migration and proliferation of renal tubular epithelial cells. The EMT could also be involved. COL28 could be a therapeutic target against renal- fibrotic diseases.

Hepatic fibrosis: Targeting peroxisome proliferator-activated receptor alpha from mechanism to medicines.

Liver fibrosis is the result of sustained chronic liver injury and inflammation leading to hepatocyte cell death followed by the formation of fibrous scars, which is the hallmark of NASH and alcoholic steatohepatitis and can lead to cirrhosis, HCC, and liver failure. Although progress has been made in understanding the pathogenesis and clinical consequences of hepatic fibrosis, therapeutic strategies for this disease are limited. Preclinical studies suggest that peroxisome proliferator-activated receptor alpha plays an important role in preventing the development of liver fibrosis by activating genes involved in detoxifying lipotoxicity and toxins, transrepressing genes involved in inflammation, and inhibiting activation of hepatic stellate cells. Given the robust preclinical data, several peroxisome proliferator-activated receptor alpha agonists have been tested in clinical trials for liver fibrosis. Here, we provide an update on recent progress in understanding the mechanisms by which peroxisome proliferator-activated receptor alpha prevents fibrosis and discuss the potential of targeting PPARα for the development of antifibrotic treatments.

miR-31-5p-Modified RAW 264.7 Macrophages Affect Profibrotic Phenotype of Lymphatic Endothelial Cells In Vitro.

Cardiac lymphatic vessel (LyV) remodeling as a contributor to heart failure has not been extensively evaluated in metabolic syndrome (MetS). Our studies have shown structural changes in cardiac LyV in MetS that contribute to the development of edema and lead to myocardial fibrosis. Tissue macrophages may affect LyV via secretion of various substances, including noncoding RNAs. The aim of the study was to evaluate the influence of macrophages modified by miR-31-5p, a molecule that regulates fibrosis and lymphangiogenesis, on lymphatic endothelial cells (LECs) in vitro. The experiments were carried out on the RAW 264.7 macrophage cell line and primary dermal lymphatic endothelial cells. RAW 264.7 macrophages were transfected with miR-31-5p and supernatant from this culture was used for LEC stimulation. mRNA expression levels for genes associated with lymphangiogenesis and fibrosis were measured with qRT-PCR. Selected results were confirmed with ELISA or Western blotting. miR-31-5p-modified RAW 264.7 macrophages secreted increased amounts of VEGF-C and TGF-ß and a decreased amount of IGF-1. The supernatant from miR-31-5p-modified RAW 264.7 downregulated the mRNA expression for genes regulating endothelial-to-mesenchymal transition (EndoMT) and fibrosis in LECs. Our results suggest that macrophages under the influence of miR-31-5p show the potential to inhibit LEC-dependent fibrosis. However, more studies are needed to confirm this effect in vivo.

Salmonella pathogenicity island 1 knockdown confers protection against myocardial fibrosis and inflammation in uremic cardiomyopathy via down-regulation of S100 Calcium Binding Protein A8/A9 transcription.

BACKGROUND/AIM: Uremic cardiomyopathy (UCM) is a characteristic cardiac pathology that is commonly found in patients with chronic kidney disease. This study dissected the mechanism of SPI1 in myocardial fibrosis and inflammation induced by UCM through S100A8/A9. METHODS: An UCM rat model was established, followed by qRT-PCR and western blot analyses of SPI1 and S100A8/A9 expression in myocardial tissues. After alterations of SPI1 and S100A8/A9 expression in UCM rats, the blood specimens were harvested from the cardiac apex of rats. The levels of creatine phosphokinase-MB (CK-MB), blood creatinine, blood urea nitrogen (BUN), and inflammatory cytokines (interleukin [IL]-6, IL-1ß, and tumor necrosis factor-α [TNF-α]) were examined in the collected blood. Collagen fibrosis was assessed by Masson staining. The expression of fibrosis markers [transforming growth factor (TGF)-ß1, α-smooth muscle actin (SMA), Collagen 4a1, and Fibronectin], IL-6, IL-1ß, and TNF-α was measured in myocardial tissues. Chromatin immunoprecipitation and dual-luciferase reporter gene assays were conducted to test the binding relationship between SPI1 and S100A8/A9. RESULTS: S100A8/A9 and SPI1 were highly expressed in the myocardial tissues of UCM rats. Mechanistically, SPI1 bound to the promoter of S100A8/A9 to facilitate S100A8/A9 transcription. S100A8/A9 or SPI1 knockdown reduced myocardial fibrosis and inflammation and the levels of CK-MB, blood creatinine, and BUN, as well as the expression of TGF-ß1, α-SMA, Collagen 4a1, Fibronectin, IL-6, TNF-α, and IL-1ß in UCM rats. CONCLUSION: SPI1 knockdown diminished S100A8/A9 transcription, thus suppressing myocardial fibrosis and inflammation caused by UCM.

Consecutive dosing of UVB irradiation induces loss of ABCB5 expression and activation of EMT and fibrosis proteins in limbal epithelial cells similar to pterygium epithelium.

Pterygium pathogenesis is often attributed to a population of altered limbal stem cells, which initiate corneal invasion and drive the hyperproliferation and fibrosis associated with the disease. These cells are thought to undergo epithelial to mesenchymal transition (EMT) and to contribute to subepithelial stromal fibrosis. In this study, the presence of the novel limbal stem cell marker ABCB5 in clusters of basal epithelial pterygium cells co-expressing with P63α and P40 is reported. ABCB5-positive pterygium cells also express EMT-associated fibrosis markers including vimentin and α-SMA while their ß-catenin expression is reduced. By using a novel in vitro model of two-dose UV-induced EMT activation on limbal epithelial cells, we could observe the dysregulation of EMT-related proteins including an increase of vimentin and α-SMA as well as downregulation of ß-catenin in epithelial cells correlating to downregulation of ABCB5. The sequential irradiation of limbal fibroblasts also induced an increase in vimentin and α-SMA. Taken together, these data demonstrate for the first time the expression of ABCB5 in pterygium stem cell activity and EMT-related events while the involvement of limbal stem cells in pterygium pathogenesis is exhibited via sequential irradiation of limbal epithelial cells. The later in vitro approach can be used to further study the involvement of limbal epithelium UV-induced EMT in pterygium pathogenesis and help identify novel treatments against pterygium growth and recurrence.

Expression of cartilage oligomeric matrix protein in periampullary adenocarcinoma is associated with pancreatobiliary-type morphology, higher levels of fibrosis and immune cell exclusion.

Cartilage oligomeric matrix protein (COMP) is an emerging regulator of tumor progression. The aim of this study was to evaluate the expression of COMP in periampullary adenocarcinoma with respect to prognostic value for survival and relapse, levels of fibrosis and infiltrating immune cells. COMP expression was evaluated using immunohistochemistry in primary tumors and subsets of paired lymph node metastases in tissue microarrays including 175 patients with periampullary adenocarcinoma. Collagen content was assessed with Sirius Red-Fast Green staining. High COMP levels were detected in cancer cells and in stroma, in 46% and 57% of the patients, respectively. High COMP expression was strongly associated with more aggressive pancreatobiliary-type (PB-type) compared to intestinal-type tumors (p < .0001). Importantly, high expression of COMP correlated with the exclusion of cytotoxic T-cells from the cancer cell compartment of the tumors, particularly in PB-type tumors. Higher levels of fibrosis measured by the density of collagen fibers correlated with high COMP levels in both cancer cells and stroma. This in turn could lead to exclusion of cytotoxic T-cells from accessing the cancer cells, a recognized immunotherapy resistance mechanism. Targeting COMP could therefore be considered as a novel therapeutic strategy in PB-type periampullary adenocarcinoma.

Increased expression and accumulation of GDF15 in IPF extracellular matrix contribute to fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic disease of unmet medical need. It is characterized by formation of scar tissue leading to a progressive and irreversible decline in lung function. IPF is associated with repeated injury, which may alter the composition of the extracellular matrix (ECM). Here, we demonstrate that IPF patient-derived pulmonary ECM drives profibrotic response in normal human lung fibroblasts (NHLF) in a 3D spheroid assay. Next, we reveal distinct alterations in composition of the diseased ECM, identifying potentially novel associations with IPF. Growth differentiation factor 15 (GDF15) was identified among the most significantly upregulated proteins in the IPF lung-derived ECM. In vivo, GDF15 neutralization in a bleomycin-induced lung fibrosis model led to significantly less fibrosis. In vitro, recombinant GDF15 (rGDF15) stimulated α smooth muscle actin (αSMA) expression in NHLF, and this was mediated by the activin receptor-like kinase 5 (ALK5) receptor. Furthermore, in the presence of rGDF15, the migration of NHLF in collagen gel was reduced. In addition, we observed a cell type-dependent effect of GDF15 on the expression of cell senescence markers. Our data suggest that GDF15 mediates lung fibrosis through fibroblast activation and differentiation, implicating a potential direct role of this matrix-associated cytokine in promoting aberrant cell responses in disease.

A LIGHT-HVEM/LTßR axis contributes to the fibrosis of intrauterine adhesion.

Intrauterine adhesion (IUA) is a fibrotic disease, with complex and multifactorial process, causing menstrual disorders, pregnancy loss or infertility. LIGHT (also named TNFSF14), mainly expressed by immune cells, has been reported to be associated with tissue fibrosis. However, the features of immunocyte subsets, the expression and roles of LIGHT and its receptor HVEM (herpes virus entry mediator) and LTßR (lymphotoxin beta receptor) in IUA remain largely unknown. Compared with the control group, we observed increased ratios of CD45+ cells, neutrophils, T cells, macrophages and decreased natural killer cells proportion, and high LIGHT expression on CD4+ T cells and macrophages in IUA endometrium. Further analysis showed there was a positive correlation between upregulated profibrotic factors (e.g., ɑ-smooth muscle actin, transforming growth factor ß1) and HVEM in IUA endometrial tissue. More importantly, recombinant human LIGHT protein directly up-regulated the expression of HVEM, LTßR, profibrotic and proinflammatory factors expression in human endometrial stromal cells. These findings reveal abnormal changes of immune cell subsets proportion and the overexpression of LIGHT-HVEM/LTßR axis in IUA endometrium, should contribute to inflammation and fibrosis formation of IUA.

Targeting TGF-ß signal transduction for fibrosis and cancer therapy.

Transforming growth factor ß (TGF-ß) has long been identified with its intensive involvement in early embryonic development and organogenesis, immune supervision, tissue repair, and adult homeostasis. The role of TGF-ß in fibrosis and cancer is complex and sometimes even contradictory, exhibiting either inhibitory or promoting effects depending on the stage of the disease. Under pathological conditions, overexpressed TGF-ß causes epithelial-mesenchymal transition (EMT), extracellular matrix (ECM) deposition, cancer-associated fibroblast (CAF) formation, which leads to fibrotic disease, and cancer. Given the critical role of TGF-ß and its downstream molecules in the progression of fibrosis and cancers, therapeutics targeting TGF-ß signaling appears to be a promising strategy. However, due to potential systemic cytotoxicity, the development of TGF-ß therapeutics has lagged. In this review, we summarized the biological process of TGF-ß, with its dual role in fibrosis and tumorigenesis, and the clinical application of TGF-ß-targeting therapies.

Macrophage interferon regulatory factor 4 deletion ameliorates aristolochic acid nephropathy via reduced migration and increased apoptosis.

Aristolochic acid (AA) is the causative nephrotoxic alkaloid in AA nephropathy, which results in a tubulointerstitial fibrosis. AA causes direct proximal tubule damage as well as an influx of macrophages, although the role of macrophages in pathogenesis is poorly understood. Here, we demonstrate that AA directly stimulates migration, inflammation, and ROS production in macrophages ex vivo. Cells lacking interferon regulatory factor 4 (IRF4), a known regulator of macrophage migration and phenotype, had a reduced migratory response, though effects on ROS production and inflammation were preserved or increased relative to WT cells. Macrophage-specific IRF4-knockout mice were protected from both acute and chronic kidney effects of AA administration based on functional and histological analysis. Renal macrophages from kidneys of AA-treated macrophage-specific IRF4-knockout mice demonstrated increased apoptosis and ROS production compared with WT controls, indicating that AA directly polarizes macrophages to a promigratory and proinflammatory phenotype. However, knockout mice had reduced renal macrophage abundance following AA administration. While macrophages lacking IRF4 can adopt a proinflammatory phenotype upon AA exposure, their inability to migrate to the kidney and increased rates of apoptosis upon infiltration provide protection from AA in vivo. These results provide evidence of direct AA effects on macrophages in AA nephropathy and add to the growing body of evidence that supports a key role of IRF4 in modulating macrophage function in kidney injury.

Association between the Genetic Polymorphisms of CCL2, CCL5, CCL8, CCR2, and CCR5 with Chronic Hepatitis C Virus Infection in the Chinese Han Population.

Hepatitis C virus (HCV) infection is a global public health burden. Chronic HCV infection leads to the development of fibrosis, cirrhosis, liver cancer, and liver failure over time. A total of 250 patients with chronic HCV infection and 299 healthy blood donors were recruited. Sixteen candidate single nucleotide polymorphisms (SNPs) in chemokine (C-C motif) ligand 2 (CCL2), CCL5, CCL8, C-C chemokine receptor 2 (CCR2), and CCR5 were genotyped in all participants. The rs1024610 AA genotype was significantly associated with decreased susceptibility to chronic HCV infection. Aspartate aminotransferase (AST) levels, AST/platelet ratio index, and the fibrosis 4 score were significantly lower in the CCL2 rs1024610 T allele and haplotype ATGC carriers. Moreover, expression levels of collagen IV (C-IV) and laminin (LN) were significantly higher in patients with the CCL5 rs2280788 C allele compared to the non-carriers. Similarly, the expression levels of C-IV, LN, and hyaluronic acid were significantly higher in patients with the CCL5 haplotype, TGCT. No significant differences were identified between the SNPs/haplotypes and plasma levels of CCL2, CCL5, CCL8, CCR2, and CCR5 in the healthy controls, and the rs1024610 allele alteration had no effect on CCL2 promoter activity. This is the first study to report an association between CCL2 rs1024610 and the risk of chronic HCV infection in the Chinese Han population. rs1024610 and ATGC haplotype in CCL2 were reasonable candidate markers of liver abnormalities. rs2280788 and TGCT haplotype in CCL5 are likely to play a significant role in liver fibrosis during chronic HCV infection.

GPR65 (TDAG8) inhibits intestinal inflammation and colitis-associated colorectal cancer development in experimental mouse models.

GPR65 (TDAG8) is a proton-sensing G protein-coupled receptor predominantly expressed in immune cells. Genome-wide association studies (GWAS) have identified GPR65 gene polymorphisms as an emerging risk factor for the development of inflammatory bowel disease (IBD). Patients with IBD have an elevated risk of developing colorectal cancer when compared to the general population. To study the role of GPR65 in intestinal inflammation and colitis-associated colorectal cancer (CAC), colitis and CAC were induced in GPR65 knockout (KO) and wild-type (WT) mice using dextran sulfate sodium (DSS) and azoxymethane (AOM)/DSS, respectively. Disease severity parameters such as fecal score, colon shortening, histopathology, and mesenteric lymph node enlargement were aggravated in GPR65 KO mice compared to WT mice treated with DSS. Elevated leukocyte infiltration and fibrosis were observed in the inflamed colon of GPR65 KO when compared to WT mice which may represent a cellular mechanism for the observed exacerbation of intestinal inflammation. In line with high expression of GPR65 in infiltrated leukocytes, GPR65 gene expression was increased in inflamed intestinal tissue samples of IBD patients compared to normal intestinal tissues. Moreover, colitis-associated colorectal cancer development was higher in GPR65 KO mice than WT mice when treated with AOM/DSS. Altogether, our data demonstrate that GPR65 suppresses intestinal inflammation and colitis-associated tumor development in murine colitis and CAC models, suggesting potentiation of GPR65 with agonists may have an anti-inflammatory therapeutic effect in IBD and reduce the risk of developing colitis-associated colorectal cancer.

Deciphering Mesenchymal Drivers of Human Dupuytren's Disease at Single-Cell Level.

Dupuytren's disease (DD) is a common, progressive fibroproliferative disease affecting the palmar fascia of the hands, causing fingers to irreversibly flex toward the palm with significant loss of function. Surgical treatments are limited; therefore, effective new therapies for DD are urgently required. To identify the key cellular and molecular pathways driving DD, we employed single-cell RNA sequencing, profiling the transcriptomes of 35,250 human single cells from DD, nonpathogenic fascia, and healthy dermis. We identify a DD-specific population of pathogenic PDPN+/FAP+ mesenchymal cells displaying an elevated expression of fibrillar collagens and profibrogenic genes. In silico trajectory analysis reveals resident fibroblasts to be the source of this pathogenic population. To resolve the processes governing DD progression, genes differentially expressed during fibroblast differentiation were identified, including upregulated TNFRSF12A and transcription factor SCX. Knockdown of SCX and blockade of TNFRSF12A inhibited the proliferation and altered the profibrotic gene expression of cultured human FAP+ mesenchymal cells, demonstrating a functional role for these genes in DD. The power of single-cell RNA sequencing is utilized to identify the major pathogenic mesenchymal subpopulations driving DD and the key molecular pathways regulating the DD-specific myofibroblast phenotype. Using this precision medicine approach, inhibition of TNFRSF12A has shown potential clinical utility in the treatment of DD.

Circulating inflammatory cytokines alter transcriptional activity within fibrotic tissue of Dupuytren's disease patients.

Dupuytren's disease is a benign fibroproliferative disorder of the hand that results in disabling digital contractures that impair function and diminish the quality of life. The incidence of this disease has been correlated with chronic inflammatory states, but any direct association between inflammatory cytokines and Dupuytren's disease is not known. We hypothesized that advanced fibroproliferation is associated with increased levels of circulating inflammatory cytokines. Blood and fibrotic cord tissue were collected preoperatively from patients with severe contracture and control patients. Blood plasma concentrations of known inflammatory cytokines were evaluated using a multiplex immunoassay. Proteins from the cord tissue were analyzed by RNA sequencing and immunohistochemistry. Moreover, collagen-rich cords were analyzed using Fourier-transform infrared spectroscopy. The results indicate that patients exhibited significantly elevated circulating inflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin (IL)-2, and IL-12p70, as compared with controls. Similarly, IL-4 and IL-13 were detected significantly more frequently in Dupuytren's disease as compared with control. RNA sequencing revealed 5311 differentially expressed genes and distinct clustering between diseased and control samples. In addition to increased expression of genes associated with fibroproliferation, we also observed upregulation of transcripts activated by inflammatory cytokines, including prolactin inducible protein and keratin intermediate filaments. IL-2, but not TNF-α, was detected in fibrotic cord tissue by immunohistochemistry. Finally, spectroscopic assays revealed a significant reduction of the collagen content and alterations of collagen cross-linking within the Dupuytren's disease tissues. In total, our results illustrate that patients with severe Dupuytren's disease exhibit substantially elevated circulating inflammatory cytokines that may drive fibroproliferation. Clinical Significance: The results from this study establish the basis for a specific cytokine profile that may be useful for diagnostic testing and therapeutic intervention in Dupuytren's disease.