Profibrotic Cells Research Articles

Single-cell Analysis of Subcutaneous Fat Reveals Pro-fibrotic Cells that Correlate with Visceral Adiposity in HIV.

Cardiometabolic diseases are common in persons with HIV (PWH) on antiretroviral therapy (ART), which has been attributed to preferential lipid storage in visceral adipose tissue (VAT) compared with subcutaneous adipose tissue (SAT). However, the relationship of SAT-specific cellular and molecular programs with VAT volume is poorly understood in PWH. We characterized SAT cell-type specific composition and transcriptional programs that are associated with greater VAT volume in PWH on contemporary ART. We enrolled PWH on long-term ART with a spectrum of metabolic health. Ninety-two participants underwent SAT biopsy for bulk RNA sequencing and 43 had single-cell RNA sequencing. Computed tomography quantified VAT volume and insulin resistance was calculated using HOMA2-IR. VAT volume was associated with HOMA2-IR (p < 0.001). Higher proportions of SAT intermediate macrophages (IMs), myofibroblasts, and MYOC + fibroblasts were associated with greater VAT volume using partial Spearman's correlation adjusting for age, sex, and body mass index (ρ=0.34-0.49, p < 0.05 for all). Whole SAT transcriptomics showed PWH with greater VAT volume have increased expression of extracellular matrix (ECM)- and inflammation-associated genes, and reduced expression of lipolysis- and fatty acid metabolism-associated genes. In PWH, greater VAT volume is associated with higher proportion of SAT IMs and fibroblasts, and a SAT ECM and inflammatory transcriptome, which is similar to findings in HIV-negative persons with obesity. These data identify SAT cell-type specific changes associated with VAT volume in PWH that could underlie the high rates of cardiometabolic diseases in PWH, though additional longitudinal studies are needed to define directionality and mechanisms.

CREEPING FAT-DERIVED FIBROBLASTS PARTICIPATE IN INTESTINAL FIBROSIS IN A NOVEL MOUSE MODEL OF INTESTINAL STRICTURES

Abstract INTRODUCTION An intestinal stricture is a common morbidity in Crohn's disease, but its pathogenesis is poorly understood. CF forms adjacent to strictures and is associated with stricture recurrence, but whether it promotes stricture formation is unclear. Adipocytes display remarkable plasticity, and adipocyte-derived fibroblasts (ADFs) play important roles in tissue wound healing and fibrosis. Here, we show that CF ADFs may promote intestinal stricture formation. METHODS We developed a novel mouse model of CF and intestinal fibrosis by creating anti-mesenteric colotomies. We then created colotomies in Adipoq-Cre; mTmG mice and harvested the mice at post-operative day (POD) 14. This system exclusively labels adipocytes and their derivatives GFP+, whereas all other cells are TdTomato+ (Figure 1A-B). We immunostained sham and colotomy bowel for mature adipocyte marker PLIN1. We quantified GFP+ lineage-negative ADFs (CD45-CD31-Terr119-EPCAM-ECadherin-) isolated from either sham or colotomy bowel at POD 14. We then isolated ADFs (GFP+) and non-ADFs (nADFs) (TdTomato+) by flow cytometry and performed single-cell RNA-seq (scRNA-seq) (Figure 1F). RESULTS The creation of a colotomy is sufficient to induce localized mesenteric adipose tissue (MAT) expansion around the colotomy site, consistent with CF formation (Figure 1C). We observed the presence of GFP+ cells with a fibroblast morphology that lost the expression of adipocyte markers and acquired the expression of fibroblast markers. (Figure 1D). Furthermore, we observed that GFP+ ADFs expanded in colotomy bowel compared to sham (Figure 1E). scRNA-seq of ADFs and nADFs revealed 5 major fibroblast clusters, including a Clu+ pro-fibrotic population and a Pi16+ progenitor population, which were enriched in bowel GFP+ ADFs (Figure 1G-H). Finally, quantification of gene expression revealed that GFP+ ADFs lacked mature adipocyte markers, expressed fibroblast markers, and were enriched for various ECM proteins. CONCLUSIONS An anti-mesenteric model of intestinal strictures and CF in combination with immunostaining and scRNA-seq revealed that ADFs from CF infiltrate the bowel wall and expand at the site of fibrosis. ADFs exhibit fibroblast heterogeneity and are enriched for pro-fibrotic and stromal progenitor populations. Finally, ADFs produce collagen and fibronectin, suggesting that they may be pro-fibrotic. Altogether, these data highly suggest that CF contains pro-fibrotic stromal cells that infiltrate the stricture site to promote fibrosis.

The complex heterogeneity of immune cell signatures across wasting tissues with C26 and 5-fluorouracil-induced cachexia.

Immune cell-driven pathways are linked to cancer cachexia. Tumor presence is associated with immune cell infiltration whereas cytotoxic chemotherapies reduce immune cell counts. Despite these paradoxical effects, both cancer and chemotherapy can cause cachexia; however, our understanding of immune responses in the cachexia condition with cancer and chemotherapy is largely unknown. We sought to advance our understanding of the immunology underlying cancer and cancer with chemotherapy-induced cachexia. CD2F1 mice were given 106 C26 cells, followed by five doses of 5-fluorouracil (5FU; 30 mg/kg LM, ip) or PBS. Indices of cachexia and tumor (TUM), skeletal muscle (SKM), and adipose tissue (AT) immune cell populations were examined using high-parameter flow cytometry. Although 5FU was able to stunt tumor growth, % body weight loss and muscle mass were not different between C26 and C26 + 5FU. C26 increased CD11b+Ly6g+ and CD11b+Ly6cInt inflammatory myeloid cells in SKM and AT; however, both populations were reduced with C26 + 5FU. tSNE analysis revealed 24 SKM macrophage subsets wherein 8 were changed with C26 or C26 + 5FU. C26 + 5FU increased SKM CD11b-CD11c+ dendritic cells, CD11b-NK1.1+ NK-cells, and CD11b-B220+ B-cells, and reduced Ly6cHiCX3CR1+CD206+CD163IntCD11c-MHCII- infiltrated macrophages and other CD11b+Ly6cHi myeloid cells compared with C26. Both C26 and C26 + 5FU had elevated CD11b+F480+CD206+MHCII- or more specifically Ly6cLoCX3CR1+CD206+CD163IntCD11c-MHCII- profibrotic macrophages. 5FU suppressed tumor growth and decreased SKM and AT inflammatory immune cells without protecting against cachexia suggesting that these cells are not required for wasting. However, profibrotic cells and muscle inflammatory/atrophic signaling appear consistent with cancer- and cancer with chemotherapy-induced wasting and remain potential therapeutic targets.NEW & NOTEWORTHY Despite being an immune-driven condition, our understanding of skeletal muscle and adipose tissue immune cells with cachexia is limited. Here, we identified immune cell populations in tumors, skeletal muscle, and adipose tissue in C26 tumor-bearing mice with/without 5-fluorouracil (5FU). C26 and C26 + 5FU had increased skeletal muscle profibrotic macrophages, but 5FU reduced inflammatory myeloid cells without sparing mass. Tumor presence and chemotherapy have contrasting effects on certain immune cells, which appeared not necessary for wasting.

Single-Cell RNA Sequencing of Coronary Perivascular Adipose Tissue From End-Stage Heart Failure Patients Identifies SPP1+ Macrophage Subpopulation as a Target for Alleviating Fibrosis.

Perivascular adipose tissue (PVAT) is vital for vascular homeostasis, and PVAT dysfunction is associated with increased atherosclerotic plaque burden. But the mechanisms underlining coronary PVAT dysfunction in coronary atherosclerosis remain elusive. We performed single-cell RNA sequencing of the stromal vascular fraction of coronary PVAT from 3 groups of heart transplant recipients with end-stage heart failure, including 3 patients with nonobstructive coronary atherosclerosis, 3 patients with obstructive coronary artery atherosclerosis, and 4 nonatherosclerosis control subjects. Bioinformatics was used to annotate the cellular populations, depict the cellular developmental trajectories and interactions, and explore the differences among 3 groups of coronary PVAT at the cellular and molecular levels. Pathological staining, quantitative real-time polymerase chain reaction, and in vitro studies were performed to validate the key findings. Ten cell types were identified among 67 936 cells from human coronary PVAT. Several cellular subpopulations, including SPP1+ (secreted phosphoprotein 1) macrophages and profibrotic fibroadipogenic progenitor cells, were accumulated in PVAT surrounding atherosclerotic coronary arteries compared with nonatherosclerosis coronary arteries. The fibrosis percentage was increased in PVAT surrounding atherosclerotic coronary arteries, and it was positively associated with the grade of coronary artery stenosis. Cellular interaction analysis suggested OPN (osteopontin) secreted by SPP1+ macrophages interacted with CD44 (cluster of differentiation 44)/integrin on fibroadipogenic progenitor cells. Strikingly, correlation analyses uncovered that higher level of SPP1 in PVAT correlates with a more severe fibrosis degree and a higher coronary stenosis grade. In vitro studies showed that conditioned medium from atherosclerotic coronary PVAT promoted the migration and proliferation of fibroadipogenic progenitor cells, while such effect was prevented by blocking CD44 or integrin. SPP1+ macrophages accumulated in the PVAT surrounding atherosclerotic coronary arteries, and they promoted the migration and proliferation of fibroadipogenic progenitor cells via OPN-CD44/integrin interaction and thus aggravated the fibrosis of coronary PVAT, which was positively correlated to the coronary stenosis burden. Therefore, SPP1+ macrophages in coronary PVAT may participate in the progression of coronary atherosclerosis.

PTX3 shapes profibrotic immune cells and epithelial/fibroblast repair and regeneration in a murine model of pulmonary fibrosis

The long pentraxin 3 (PTX3) is protective in different pathologies but was not analyzed in-depth in Idiopathic Pulmonary Fibrosis (IPF). Here, we have explored the influence of PTX3 in the bleomycin (BLM)-induced murine model of IPF by looking at immune cells (macrophages, mast cells, T cells) and stemness/regenerative markers of lung epithelium (SOX2) and fibro-blasts/myofibroblasts (CD44) at different time points that retrace the progression of the disease from onset at day 14, to full-blown disease at day 21, to incomplete regression at day 28. We took advantage of transgenic PTX3 overexpressing mice (Tie2-PTX3) and Ptx3 null ones (PTX3-KO) in which pulmonary fibrosis was induced. Our data have shown that PTX3 overexpression in Tie2-PTX3 compared to WT or PTX3-KO: reduced CD68+ and CD163+ macrophages and the Tryptase+ mast cells during the whole experimental time; on the contrary, CD4+ T cells are consistently present on day 14 and dramatically decreased on day 21; CD8+ T cells do not show significant differences on day 14, but are significantly reduced on day 21; SOX2 is reduced on days 14 and 21; CD44 is reduced on day 21. Therefore, PTX3 could act on the proimmune and fibrogenic microenvironment to prevent fibrosis in BLM-treated mice.

Role of the epithelial barrier in intestinal fibrosis associated with inflammatory bowel disease: relevance of the epithelial-to mesenchymal transition.

In inflammatory bowel disease (IBD), chronic inflammation in the gastrointestinal tract can lead to tissue damage and remodelling, which can ultimately result in fibrosis. Prolonged injury and inflammation can trigger the activation of fibroblasts and extracellular matrix (ECM) components. As fibrosis progresses, the tissue becomes increasingly stiff and less functional, which can lead to complications such as intestinal strictures, obstructive symptoms, and eventually, organ dysfunction. Epithelial cells play a key role in fibrosis, as they secrete cytokines and growth factors that promote fibroblast activation and ECM deposition. Additionally, epithelial cells can undergo a process called epithelial-mesenchymal transition, in which they acquire a more mesenchymal-like phenotype and contribute directly to fibroblast activation and ECM deposition. Overall, the interactions between epithelial cells, immune cells, and fibroblasts play a critical role in the development and progression of fibrosis in IBD. Understanding these complex interactions may provide new targets for therapeutic interventions to prevent or treat fibrosis in IBD. In this review, we have collected and discussed the recent literature highlighting the contribution of epithelial cells to the pathogenesis of the fibrotic complications of IBD, including evidence of EMT, the epigenetic control of the EMT, the potential influence of the intestinal microbiome in EMT, and the possible therapeutic strategies to target EMT. Finally we discuss the pro-fibrotic interactions epithelial-immune cells and epithelial-fibroblasts cells.

Autocrine TGF-β-positive feedback in profibrotic AT2-lineage cells plays a crucial role in non-inflammatory lung fibrogenesis

The molecular etiology of idiopathic pulmonary fibrosis (IPF) has been extensively investigated to identify new therapeutic targets. Although anti-inflammatory treatments are not effective for patients with IPF, damaged alveolar epithelial cells play a critical role in lung fibrogenesis. Here, we establish an organoid-based lung fibrosis model using mouse and human lung tissues to assess the direct communication between damaged alveolar type II (AT2)-lineage cells and lung fibroblasts by excluding immune cells. Using this in vitro model and mouse genetics, we demonstrate that bleomycin causes DNA damage and activates p53 signaling in AT2-lineage cells, leading to AT2-to-AT1 transition-like state with a senescence-associated secretory phenotype (SASP). Among SASP-related factors, TGF-β plays an exclusive role in promoting lung fibroblast-to-myofibroblast differentiation. Moreover, the autocrine TGF-β-positive feedback loop in AT2-lineage cells is a critical cellular system in non-inflammatory lung fibrogenesis. These findings provide insights into the mechanism of IPF and potential therapeutic targets.

Targeting CD301+ macrophages inhibits endometrial fibrosis and improves pregnancy outcome

Macrophages are a key and heterogeneous cell population involved in endometrial repair and regeneration during the menstrual cycle, but their role in the development of intrauterine adhesion (IUA) and sequential endometrial fibrosis remains unclear. Here, we reported that CD301+ macrophages were significantly increased and showed their most active interaction with profibrotic cells in the endometria of IUA patients compared with the normal endometria by single‐cell RNA sequencing, bulk RNA sequencing, and experimental verification. Increasing CD301+ macrophages promoted the differentiation of endometrial stromal cells into myofibroblasts and resulted in extracellular matrix accumulation, which destroyed the physiological architecture of endometrial tissue, drove endometrial fibrosis, and ultimately led to female infertility or adverse pregnancy outcomes. Mechanistically, CD301+ macrophages secreted GAS6 to activate the AXL/NF‐κB pathway, upregulating the profibrotic protein synthesis. Targeted deletion of CD301+ macrophages or inhibition of AXL by Bemcentinib blunted the pathology and improved the outcomes of pregnancy in mice, supporting the therapeutic potential of targeting CD301+ macrophages for treating endometrial fibrosis.

The Role of CCL24 in Systemic Sclerosis.

Systemic sclerosis (SSc) is a chronic immune-mediated disease characterized by microangiopathy, immune dysregulation, and progressive fibrosis of the skin and internal organs. Though not fully understood, the pathogenesis of SSc is dominated by microvascular injury, endothelial dysregulation, and immune response that are thought to be associated with fibroblast activation and related fibrogenesis. Among the main clinical subsets, diffuse SSc (dSSc) is a progressive form with rapid and disseminated skin thickening accompanied by internal organ fibrosis and dysfunction. Despite recent advances and multiple randomized clinical trials in early dSSc patients, an effective disease-modifying treatment for progressive skin fibrosis is still missing, and there is a crucial need to identify new targets for therapeutic intervention. Eotaxin-2 (CCL24) is a chemokine secreted by immune cells and epithelial cells, which promotes trafficking of immune cells and activation of pro-fibrotic cells through CCR3 receptor binding. Higher levels of CCL24 and CCR3 were found in the skin and sera of patients with SSc compared with healthy controls; elevated levels of CCL24 and CCR3 were associated with fibrosis and predictive of greater lung function deterioration. Growing evidence supports the potency of a CCL24-blocking antibody as an anti-inflammatory and anti-fibrotic modulating agent in multiple preclinical models that involve liver, skin, and lung inflammation and fibrosis. This review highlights the role of CCL24 in orchestrating immune, vascular, and fibrotic pathways, and the potential of CCL24 inhibition as a novel treatment for SSc.

Sirolimus-eluting airway stent reduces profibrotic Th17 cells and inhibits laryngotracheal stenosis.

Laryngotracheal stenosis (LTS) is pathologic fibrotic narrowing of the larynx and trachea characterized by hypermetabolic fibroblasts and CD4+ T cell-mediated inflammation. However, the role of CD4+ T cells in promoting LTS fibrosis is unknown. The mTOR signaling pathways have been shown to regulate the T cell phenotype. Here we investigated the influence of mTOR signaling in CD4+ T cells on LTS pathogenesis. In this study, human LTS specimens revealed a higher population of CD4+ T cells expressing the activated isoform of mTOR. In a murine LTS model, targeting mTOR with systemic sirolimus and a sirolimus-eluting airway stent reduced fibrosis and Th17 cells. Selective deletion of mTOR in CD4+ cells reduced Th17 cells and attenuated fibrosis, demonstrating CD4+ T cells' pathologic role in LTS. Multispectral immunofluorescence of human LTS revealed increased Th17 cells. In vitro, Th17 cells increased collagen-1 production by LTS fibroblasts, which was prevented with sirolimus pretreatment of Th17 cells. Collectively, mTOR signaling drove pathologic CD4+ T cell phenotypes in LTS, and targeting mTOR with sirolimus was effective at treating LTS through inhibition of profibrotic Th17 cells. Finally, sirolimus may be delivered locally with a drug-eluting stent, transforming clinical therapy for LTS.

Cyp4a12-mediated retinol metabolism in stellate cells is the antihepatic fibrosis mechanism of the Chinese medicine Fuzheng Huayu recipe

BackgroundHepatic stellate cells (HSCs), which contain multiple retinol-containing lipid droplets, are important profibrotic cells in liver fibrosis. Under Cyp4a12a/b oxidation, HSC activation was accompanied by the downregulation of genes involved in retinol metabolism, inducing RAE-1 production. By eliminating activated HSCs, NK cells expressing the activating receptor NKG2D are recruited to alleviate fibrosis. FZHY was found to significantly reduce the severity of liver fibrosis by inhibiting the activation and proliferation of HSCs. The molecular processes that govern retinol metabolism, on the other hand, are largely unexplored. This study focused on the regulation of Cyp4a12a/b by FZHY to elucidate the antifibrotic molecular mechanisms underlying the effect of FZHY on retinol metabolism.MethodsTo investigate mechanisms and altered pathways of FZHY against carbon tetrachloride (CCl4)-induced liver fibrosis based on transcriptomics data. Bioinformatics analysis was used to screen its pharmacological targets. The predicted targets were confirmed by a series of in vitro and in vivo experiments, including mass spectrometry, in situ hybridization, immunofluorescence assays and real-time PCR. Then, the results were further characterized by recombinant adenovirus vectors that were constructed and transfected into the cultured primary HSCs.ResultsTranscriptomics revealed that Cyp4a12a/b is nearly completely lost in liver fibrosis, and these effects might be partially reversed by FZHY therapy recovery. In vitro and in vivo studies indicated that Cyp4a12a/b deletion disrupted retinol metabolism and lowered Rae-1 expression. Activated HSCs successfully escape recognition and elimination by natural killer (NK) cells as a result of reduced Rae-1. Notablely, we discovered that FZHY may restore the Cyp4a12a/b capability, allowing the recovery of the cytotoxic function of NK cells against HSCs, and thereby reducing hepatic fibrosis by suppressing HSC activation.ConclusionOur findings revealed a new role for Cyp4a in retinol metabolism in the development of hepatic fibrosis, and they highlight Cyp4a12/Rae-1 signals as possible therapeutic targets for antifibrotic medicines.

The Influence of Design Modifications and Microstructured Surface Topographies on Bleb Survival after Glaucoma Tube Shunt Implantation.

Lowering intraocular pressure (IOP) by placement of a glaucoma shunt is an effective treatment for glaucoma. However, fibrosis of the outflow site can hamper surgical outcome. In this study, the antifibrotic effect of adding an endplate (with or without microstructured surface topographies) to a microshunt made of poly(styrene-block-isobutylene-block-styrene) is investigated. New Zealand white rabbits are implanted with a control implant (without endplate) and modified implants. Afterward, bleb morphology and IOP is recorded for 30 days. After killing of the animals, eyes are collected for histology, Addition of an endplate extended bleb survival, Topography-990 has the longest recorded bleb-survival time. Histology reveals that the addition of an endplate increases the presence of myofibroblasts, macrophages, polymorphonuclear cells, and foreign body giant cells compared to the control. However, an increased capsule thickness and inflammatory response are observed in the groups with surface topographies, The addition of an endplate results in prolonged bleb survival, demonstrating that engineering of the shape of glaucoma implants could prolong bleb functionality. Future research should further elaborate the effect of surface topographies on long-term bleb survival, since an increased presence of pro-fibrotic cells and increased capsule thickness are observed compared to the control.

A POLY(SBMA) ZWITTERIONIC HYDROGEL COATING OF POLYIMIDE SURFACES TO REDUCE THE FOREIGN BODY REACTION TO INVASIVE NEURAL INTERFACES

Intraneural electrodes can be harnessed to control neural prosthetic devices in human amputees. However, in chronic implants we witness a gradual loss of device functionality and electrode isolation due to a nonspecific inflammatory response to the implanted material, called foreign body reaction (FBR). FBR may eventually lead to a fibrous encapsulation of the electrode surface. Poly(ethylene glycol) (PEG) is one of the most common low-fouling materials used to coat and protect electrode surfaces. Yet, PEG can easily undergo encapsulation and oxidative damage in long-term in vivo applications. Poly(sulfobetaine methacrylate) - poly(SBMA) - zwitterionic hydrogels may represent more promising alternatives to minimize the FBR due to their ultra-low fouling features. Here, we tested and compared the poly(SBMA) zwitterionic hydrogel coating with the PEG coating in reducing adhesion and activation of pro-inflammatory and pro-fibrotic cells to polyimide surfaces, which are early hallmarks of FBR. We aimed to coat polyimide surfaces with a hydrogel thin film and analysed the release of a model drug from the hydrogel.We performed hydrogel synthesis, mechanical characterization and biocompatibility analysis. Cell adhesion, viability and morphology of human myofibroblasts cultured on PEG- and hydrogel-coated surfaces were evaluated through confocal microscopy-based high-content analysis (HCA). Reduced activation of pro-inflammatory human macrophages cultured on hydrogels was assessed as well as the hydrogel drug release profile.Because of its high hydration, biocompatibility, low stiffness and ultra-low fouling characteristics the hydrogel enabled lower adhesion and activation of pro-inflammatory and pro-fibrotic cells vs. polystyrene controls, and showed a long-term release of the anti-fibrotic drug Everolimus. Furthermore, a polyimide surface was successfully coated with a hydrogel thin film.Our soft zwitterionic hydrogel could outperform PEG as more suitable coating material of neural electrodes for mitigating the FBR. Such poly(SBMA)-based biomaterial could also be envisioned as long-term delivery system for a sustained release of anti-inflammatory and anti-fibrotic drugs in vivo.

Neprilysin-dependent neuropeptide Y cleavage in the liver promotes fibrosis by blocking NPY-receptor 1.

Development of liver fibrosis is paralleled by contraction of hepatic stellate cells (HSCs), the main profibrotic hepatic cells. Yet, little is known about the interplay of neprilysin (NEP) and its substrate neuropeptide Y (NPY), a potent enhancer of contraction, in liver fibrosis. We demonstrate that HSCs are the source of NEP. Importantly, NPY originates majorly from the splanchnic region and is cleaved by NEP in order to terminate contraction. Interestingly, NEP deficiency (Nep-/-) showed less fibrosis but portal hypertension upon liver injury in two different fibrosis models in mice. We demonstrate the incremental benefit of Nep-/- in addition to AT1R blocker (ARB) or ACE inhibitors for fibrosis and portal hypertension. Finally, oral administration of Entresto, a combination of ARB and NEP inhibitor, decreased hepatic fibrosis and portal pressure in mice. These results provide a mechanistic rationale for translation of NEP-AT1R-blockade in human liver fibrosis and portal hypertension.

The non-selective Rho-kinase inhibitors Y-27632 and Y-33075 decrease contraction but increase migration in murine and human hepatic stellate cells.

The Rho-kinase ROCK II plays a major role in the activation of hepatic stellate cells (HSC), which are the key profibrotic and contractile cells contributing to the development of chronic liver disease. Inhibition of ROCK II ultimately blocks the phosphorylation of the myosin light chain (MLC) and thus inhibits stress fibre assembly and cell contraction. We investigated the effects of the ROCK inhibitors Y-33075 as well as Y-27632 in murine and human hepatic stellate cells. Primary isolated HSC from FVB/NJ mice and the immortalized human HSC line TWNT-4 were culture-activated and incubated with Y-27632 and Y-33075 (10nM to 10μM) for 24h. Protein expression levels were analyzed by Western Blots and transcriptional levels of pro-fibrotic markers and proliferative markers were evaluated using real-time qPCR. Migration was investigated by wound-healing assay. Proliferation was assessed by BrdU assay. Contraction of HSC was measured using 3D collagen matrices after incubation with Y-27632 or Y-33075 in different doses. Both Rho-kinase inhibitors, Y-27632 and Y-33075, reduced contraction, fibrogenesis and proliferation in activated primary mouse HSC (FVB/NJ) and human HSC line (TWNT-4) significantly. Y-33075 demonstrated a 10-times increased potency compared to Y-27632. Surprisingly, both inhibitors mediated a substantial and unexpected increase in migration of HSC in FVB/NJ. ROCK inhibition by the tested compounds decreased contraction but increased migration. Y-33075 proved more potent than Y27632 in the inhibition of contraction of HSCs and should be further evaluated in chronic liver disease.

Anti-inflammatory and antifibrotic effects of CBP/β-catenin inhibitor for hepatocytes: small molecular inhibitor, OP-724 possibly improves liver function.

Wnt/β-catenin signals are associated with several functions, including organ fibrosis. A synthetic small molecule, OP-724 (prodrug of C-82), an inhibitor of cyclic AMP response element-binding protein (CREB)-binding protein (CBP)/β-catenin, has demonstrated antifibrotic activity in mouse models of hepatic fibrosis. OP-724 is mediated by profibrotic and antifibrotic cells, such as hepatic stellate cells, macrophages, and neutrophils. In this study, the direct effects of C-82 on hepatocytes in hepatic inflammation were investigated. Immortalized human hepatocytes were pretreated with inflammatory cytokines. Moreover, the alteration of mRNA and protein expressions of cytokines and chemokines associated with hepatic inflammation and fibrosis, and of mitochondria-related molecules after C-82 treatment were analyzed in this study. The mRNA expression of several proinflammatory and profibrotic chemokines was upregulated by the stimulation of these inflammatory cytokines. In addition, this increase was prevented by C-82. In particular, the protein secretion of CCL2, CCL5, CXCL1, CXCL9, and CXCL10 was noticeably upregulated by TNFα and prevented by additional C-82. Moreover, C-82 increased the VEGF-A and FGF-2 proteins, categorized as anti-inflammatory and antifibrotic molecules, respectively. It also increased the expression of mitochondrial components and mitochondrial membrane potential. In conclusion, C-82 inhibits hepatocyte-mediated proinflammation and fibrogenesis. It also directly activates the mitochondrial function, thus improving liver dysfunction.

Vaccination-based immunotherapy to target profibrotic cells in liver and lung.

Fibrosis is the final path of nearly every form of chronic disease, regardless of the pathogenesis. Upon chronic injury, activated, fibrogenic fibroblasts deposit excess extracellular matrix, and severe tissue fibrosis can occur in virtually any organ. However, antifibrotic therapies that target fibrogenic cells, while sparing homeostatic fibroblasts in healthy tissues, are limited. We tested whether specific immunization against endogenous proteins, strongly expressed in fibrogenic cells but highly restricted in quiescent fibroblasts, can elicit an antigen-specific cytotoxic Tcell response to ameliorate organ fibrosis. In silico epitope prediction revealed that activation of the genes Adam12 and Gli1 in profibrotic cells and the resulting "self-peptides" can be exploited for Tcell vaccines to ablate fibrogenic cells. We demonstrate the efficacy of a vaccination approach to mount CD8+ Tcell responses that reduce fibroblasts and fibrosis in the liver and lungs in mice. These results provide proof of principle for vaccination-based immunotherapies to treat fibrosis.

Modulation of inflammatory M1-macrophages phenotype by valvular interstitial cells

BackgroundAortic valve stenosis involves inflammation, excess deposition of a collagen-rich extracellular matrix, and calcification. Recent studies have shown that M1 or inflammatory macrophages derived from infiltrating monocytes promote calcification of valvular interstitial cells, the most prevalent cell type of the aortic valve. We hypothesized that valvular interstitial cells could modulate inflammatory macrophages phenotype. MethodsWe first assessed macrophage phenotype in human aortic valve stenosis and control aortic valves from donors. Then, we examined profibrotic and inflammatory-related gene expression in valves and valvular interstitial cells. Finally, we investigated whether valvular interstitial cells can modify the phenotype of inflammatory macrophages. ResultsCirculating monocytes and plasma transforming growth factor beta-1 levels of patients with aortic valve stenosis were significantly higher compared with patients without aortic valve stenosis. Histologic analysis of thickened spongiosa of the aortic valve from patients with aortic valve stenosis showed a high macrophage infiltration but a low matrix metalloproteinase-9 expression compared with control aortic valves. On the other hand, valvular interstitial cell culture of aortic valve stenosis exhibited a profibrotic phenotype with a high expression of transforming growth factor beta-1 and transforming growth factor beta-1/transforming growth factor beta-3 ratio but a decreased expression of the peroxisome proliferator-activated receptor gamma nuclear receptor. Valvular interstitial cell–conditioned media of aortic valve stenosis led to a decrease in enzymatic activity of matrix metalloproteinase-9 and an increase in production of collagen in inflammatory macrophages compared with valvular interstitial cell–conditioned media from control aortic valve donors. ConclusionsThese findings indicate that profibrotic valvular interstitial cells promote the imbalance of extracellular matrix remodeling by reducing matrix metalloproteinase-9 production on inflammatory macrophages that lead to excessive collagen deposition observed in aortic valve stenosis. Further investigation is needed to clarify the role of transforming growth factor beta-1/proliferator-activated receptor gamma nuclear receptor/matrix metalloproteinase-9 in aortic valve stenosis.

The fate of erythropoietin-producing cells: another piece of the puzzle

In this issue, Kaneko etal. reported the generation of a mouse line that allows for the labeling of cells under control of the erythropoietin (Epo) gene promotor. The authors show that Epo-producing cells become proliferating, profibrotic cells after kidney injury and lose their ability to produce Epo. Furthermore, they show that the fluorescent-labeled cells can recover their Epo synthesis capability subsequently to a recovery period.

Single-cell analysis highlights differences in druggable pathways underlying adaptive or fibrotic kidney regeneration

The kidney has tremendous capacity to repair after acute injury, however, pathways guiding adaptive and fibrotic repair are poorly understood. We developed a model of adaptive and fibrotic kidney regeneration by titrating ischemic injury dose. We performed detailed biochemical and histological analysis and profiled transcriptomic changes at bulk and single-cell level (> 110,000 cells) over time. Our analysis highlights kidney proximal tubule cells as key susceptible cells to injury. Adaptive proximal tubule repair correlated with fatty acid oxidation and oxidative phosphorylation. We identify a specific maladaptive/profibrotic proximal tubule cluster after long ischemia, which expresses proinflammatory and profibrotic cytokines and myeloid cell chemotactic factors. Druggability analysis highlights pyroptosis/ferroptosis as vulnerable pathways in these profibrotic cells. Pharmacological targeting of pyroptosis/ferroptosis in vivo pushed cells towards adaptive repair and ameliorates fibrosis. In summary, our single-cell analysis defines key differences in adaptive and fibrotic repair and identifies druggable pathways for pharmacological intervention to prevent kidney fibrosis.