Major Source Of Myofibroblasts Research Articles

Serum response factor activates peroxidasin transcription to block senescence of hepatic stellate cells

AimsAberrant liver fibrosis is a hallmark event in end-stage liver diseases. Hepatic stellate cells (HSCs) are considered the major source of myofibroblasts in the liver that produce extracellular matrix proteins to promote liver fibrosis. HSCs undergo senescence in response to various stimuli, a process that can be exploited to dampen liver fibrosis. We investigated the role of serum response factor (SRF) in this process. Methods and materialsSenescence was induced HSCs by serum withdrawal or progressive passage. DNA-protein interaction was evaluated by chromatin immunoprecipitation (ChIP). ResultsSRF expression was down-regulated in HSCs entering into senescence. Coincidently, SRF depletion by RNAi accelerated HSC senescence. Of note, treatment of an anti-oxidant (N-acetylcysteine or NAC) blocked HSC senescence by SRF deficiency suggesting that SRF may antagonize HSC senescence by eliminating excessive reactive oxygen species (ROS). PCR-array based screening identified peroxidasin (PXDN) as a potential target for SRF in HSCs. PXDN expression was inversely correlated with HSC senescence whereas PXDN knockdown accelerated HSC senescence. Further analysis reveals that SRF directly bound to the PXDN promoter and activated PXDN transcription. Consistently, PXDN over-expression protected whereas PXDN depletion amplified HSC senescence. Finally, PXDN knockout mice displayed diminished liver fibrosis compared to wild type mice when subjected to bile duct ligation (BDL). SignificanceOur data suggest that SRF, via its downstream target PXDN, plays a key role in regulating HSC senescence.

Regulation of pericyte metabolic reprogramming restricts the AKI to CKD transition

Background and aimsAcute kidney injury (AKI) is associated with high morbidity and mortality and is recognized as a long-term risk factor for progression to chronic kidney disease (CKD). The AKI to CKD transition is characterized by interstitial fibrosis and the proliferation of collagen-secreting myofibroblasts. Pericytes are the major source of myofibroblasts in kidney fibrosis. However, the underlying mechanism of pericyte-myofibroblast transition (PMT) is still unclear. Here we investigated the role of metabolic reprogramming in PMT. MethodsUnilateral ischemia/reperfusion-induced AKI to CKD mouse model and TGF-β-treated pericyte-like cells were used to detect the levels of fatty acid oxidation (FAO) and glycolysis, and the critical signaling pathways during PMT under the treatment of drugs regulating metabolic reprogramming. ResultsPMT is characterized by a decrease in FAO and an increase in glycolysis. Enhancement of FAO by the peroxisome proliferator-activated receptor gamma coactivator-1α (PGC1α) activator ZLN-005 or suppression of glycolysis by the hexokinase 2 (HK2) inhibitor 2-DG can inhibit PMT, preventing the transition of AKI to CKD. Mechanistically, AMPK modulates various pathways involved in the metabolic switch from glycolysis to FAO. Specifically, the PGC1α-CPT1A pathway activates FAO, while inhibition of the HIF1α-HK2 pathway drives glycolysis inhibition. The modulations of these pathways by AMPK contribute to inhibiting PMT. ConclusionsMetabolic reprogramming controls the fate of pericyte transdifferentiation and targets the abnormal metabolism of pericytes can effectively prevent AKI to CKD transition.

Inhibition of HIF-1α Attenuates Silica-Induced Pulmonary Fibrosis.

Background: Excessive accumulation of extracellular matrix is a key feature of pulmonary fibrosis (PF), and myofibroblasts are the main producers of extracellular matrix. Fibroblasts are the major source of myofibroblasts, but the mechanisms of transdifferentiation are unclear. Methods: In vitro, transforming growth factor-β1 was used to induce NIH-3T3 cell transdifferentiation. DMOG was used to increase hypoxia-inducible factor-1α subunit (HIF-1α) expression. KC7F2 and siRNA decreased HIF-1α expression. In vivo, silica particles were used to induce PF in C57BL/6N mice, and KC7F2 was used to reduce HIF-1α expression in C57BL/6N mice. Western blot was used to detect the expression of collagen type 1 alpha 1(COL1A1), α-smooth muscle actin (α-SMA), SMAD family member (SAMD) 3, Phospho-SMAD3 (PSMAD3), and HIF-1α. PCR was used to detect the expression of COL1A1, α-SMA, and HIF-1α. Immunohistochemistry was used to detect the expression of COL1A1 and HIF-1α. Results: In vitro, compared to the control group, COL1A1, α-SMA, PSMAD3, and HIF-1α expression were elevated in the DMOG group, and COL1A1, α-SMA, PSMAD3, and HIF-1α expression were decreased in the KC7F2 group and siRNA group. Compared to the DMOG group, COL1A1, α-SMA, and PSMAD3 expression were decreased in the DMOG + SIS3 group. In vivo, compared to the saline group, COL1A1, α-SMA, PSMAD3, and HIF-1α expression were increased in the pulmonary tissue of C57BL/6N mice in the silica group. Compared to the silica group, COL1A1, α-SMA, PSMAD3, and HIF-1α expression and the degree of PF were decreased in the silica + KC7F2 group. Conclusion: Inhibition of HIF-1α reduced α-SMA, decreased COL1A1 expression, and attenuated the degree of PF in C57BL/6N mice. Therefore, HIF-1α may be a new target for the treatment of silica-induced PF.

The Role of Mesenchymal Stromal Cells in Tissue Repair and Fibrosis.

Significance: The present review covers an overview of the current understanding of biology of mesenchymal stromal cells (MSCs) and suggests an important role of their differential potential for clinical approaches associated with tissue repair and fibrosis. Recent Advances: Genetic lineage tracing technology has enabled the delineation of cellular hierarchies and examination of MSC cellular origins and myofibroblast sources. This technique has led to the characterization of perivascular MSC populations and suggests that pericytes might provide a local source of tissue-specific MSCs, which can differentiate into tissue-specific cells for tissue repair and fibrosis. Autologous adipose tissue MSCs led to the advance in tissue engineering for regeneration of damaged tissues. Critical Issues: Recent investigation has revealed that perivascular MSCs might be the origin of myofibroblasts during fibrosis development, and perivascular MSCs might be the major source of myofibroblasts in fibrogenic disease. Adipose tissue MSCs combined with cytokines and biomaterials are available in the treatment of soft tissue defect and skin wound healing. Future Directions: Further investigation of the roles of perivascular MSCs may enable new approaches in the treatment of fibrogenic disease; moreover, perivascular MSCs might have potential as an antifibrotic target for fibrogenic disease. Autologous adipose tissue MSCs combined with cytokines and biomaterials will be an alternative method for the treatment of soft tissue defect and skin wound healing.

Down-Regulation of CXXC5 De-Represses MYCL1 to Promote Hepatic Stellate Cell Activation.

Liver fibrosis is mediated by myofibroblasts, a specialized cell type involved in wound healing and extracellular matrix production. Hepatic stellate cells (HSC) are the major source of myofibroblasts in the fibrotic livers. In the present study we investigated the involvement of CXXC-type zinc-finger protein 5 (CXXC5) in HSC activation and the underlying mechanism. Down-regulation of CXXC5 was observed in activated HSCs compared to quiescent HSCs both in vivo and in vitro. In accordance, over-expression of CXXC5 suppressed HSC activation. RNA-seq analysis revealed that CXXC5 influenced multiple signaling pathways to regulate HSC activation. The proto-oncogene MYCL1 was identified as a novel target for CXXC5. CXXC5 bound to the proximal MYCL1 promoter to repress MYCL1 transcription in quiescent HSCs. Loss of CXXC5 expression during HSC activation led to the removal of CpG methylation and acquisition of acetylated histone H3K9/H3K27 on the MYCL1 promoter resulting in MYCL1 trans-activation. Finally, MYCL1 knockdown attenuated HSC activation whereas MYCL1 over-expression partially relieved the blockade of HSC activation by CXXC5. In conclusion, our data unveil a novel transcriptional mechanism contributing to HSC activation and liver fibrosis.

Single-Cell Transcriptomic Analysis Reveals a Hepatic Stellate Cell-Activation Roadmap and Myofibroblast Origin During Liver Fibrosis in Mice.

Background and AimsHSCs and portal fibroblasts (PFs) are the major sources of collagen‐producing myofibroblasts during liver fibrosis, depending on different etiologies. However, the mechanisms by which their dynamic gene expression directs the transition from the quiescent to the activated state—as well as their contributions to fibrotic myofibroblasts—remain unclear. Here, we analyze the activation of HSCs and PFs in CCL4‐induced and bile duct ligation–induced fibrosis mouse models, using single‐cell RNA sequencing and lineage tracing.Approach and ResultsWe demonstrate that HSCs, rather than PFs, undergo dramatic transcriptomic changes, with the sequential activation of inflammatory, migrative, and extracellular matrix–producing programs. The data also reveal that HSCs are the exclusive source of myofibroblasts in CCL4‐treated liver, while PFs are the major source of myofibroblasts in early cholestatic liver fibrosis. Single‐cell and lineage‐tracing analysis also uncovers differential gene‐expression features between HSCs and PFs; for example, nitric oxide receptor soluble guanylate cyclase is exclusively expressed in HSCs, but not in PFs. The soluble guanylate cyclase stimulator Riociguat potently reduced liver fibrosis in CCL4‐treated livers but showed no therapeutic efficacy in bile duct ligation livers.ConclusionsThis study provides a transcriptional roadmap for the activation of HSCs during liver fibrosis and yields comprehensive evidence that the differential transcriptomic features of HSCs and PFs, along with their relative contributions to liver fibrosis of different etiologies, should be considered in developing effective antifibrotic therapeutic strategies.

Targeting Follistatin like 1 ameliorates liver fibrosis induced by carbon tetrachloride through TGF-\u03b21-miR29a in mice

BackgroundHepatic fibrosis is a pathological response of the liver to a variety of chronic stimuli. Hepatic stellate cells (HSCs) are the major source of myofibroblasts in the liver. Follistatin like 1 (Fstl1) is a secreted glycoprotein induced by transforming growth factor-β1 (TGF-β1). However, the precise functions and regulation mechanisms of Fstl1 in liver fibrogenesis remains unclear.MethodsHepatic stellate cell (HSC) line LX-2 stimulated by TGF-β1, primary culture of mouse HSCs and a model of liver fibrosis induced by CCl4 in mice was used to assess the effect of Fstl1 in vitro and in vivo.ResultsHere, we found that Fstl1 was significantly up regulated in human and mouse fibrotic livers, as well as activated HSCs. Haplodeficiency of Fstl1 or blockage of Fstl1 with a neutralizing antibody 22B6 attenuated CCl4-induced liver fibrosis in vivo. Fstl1 modulates TGF-β1 classic Samd2 and non-classic JNK signaling pathways. Knockdown of Fstl1 in HSCs significantly ameliorated cell activation, cell migration, chemokines C-C Motif Chemokine Ligand 2 (CCL2) and C-X-C Motif Chemokine Ligand 8 (CXCL8) secretion and extracellular matrix (ECM) production, and also modulated microRNA-29a (miR29a) expression. Furthermore, we identified that Fstl1 was a target gene of miR29a. And TGF-β1 induction of Fstl1 expression was partially through down regulation of miR29a in HSCs.ConclusionsOur data suggests TGF-β1-miR29a-Fstl1 regulatory circuit plays a key role in regulation the HSC activation and ECM production, and targeting Fstl1 may be a strategy for the treatment of liver fibrosis.3WAK3Ucq24Ug3VSsxSRHPQVideo Graphical abstract

Intraglomerular Monocyte/Macrophage Infiltration and Macrophage-Myofibroblast Transition during Diabetic Nephropathy Is Regulated by the A2B Adenosine Receptor.

Diabetic nephropathy (DN) is considered the main cause of kidney disease in which myofibroblasts lead to renal fibrosis. Macrophages were recently identified as the major source of myofibroblasts in a process known as macrophage–myofibroblast transition (MMT). Adenosine levels increase during DN and in vivo administration of MRS1754, an antagonist of the A2B adenosine receptor (A2BAR), attenuated glomerular fibrosis (glomerulosclerosis). We aimed to investigate the association between A2BAR and MMT in glomerulosclerosis during DN. Kidneys/glomeruli of non-diabetic, diabetic, and MRS1754-treated diabetic (DM+MRS1754) rats were processed for histopathologic, transcriptomic, flow cytometry, and cellular in vitro analyses. Macrophages were used for in vitro cell migration/transmigration assays and MMT studies. In vivo MRS1754 treatment attenuated the clinical and histopathological signs of glomerulosclerosis in DN rats. Transcriptomic analysis demonstrated a decrease in chemokine-chemoattractants/cell-adhesion genes of monocytes/macrophages in DM+MRS1754 glomeruli. The number of intraglomerular infiltrated macrophages and MMT cells increased in diabetic rats. This was reverted by MRS1754 treatment. In vitro cell migration/transmigration decreased in macrophages treated with MRS1754. Human macrophages cultured with adenosine and/or TGF-β induced MMT, a process which was reduced by MRS1754. We concluded that pharmacologic blockade of A2BAR attenuated some clinical signs of renal dysfunction and glomerulosclerosis, and decreased intraglomerular macrophage infiltration and MMT in DN rats.

The effect of core fucosylation-mediated regulation of multiple signaling pathways on lung pericyte activation and fibrosis.

The main event in the progression of pulmonary fibrosis is the appearance of myofibroblasts. Recent evidence supports pericytes as a major source of myofibroblasts. TGFβ/Smad2/3 and PDGF/Erk signaling pathways are important for regulating pericyte activation. Previous studies have demonstrated that PDGFβR and TGFβR are modified by core fucosylation (CF) catalyzed by α-1,6-fucosyltransferase (FUT8). The aim of this study was to compare the effect of inhibiting CF versus the PDGFβR and TGFβR signaling pathways on pericyte activation and lung fibrosis. FUT8shRNA was used to knock down FUT8-mediated CF both in vivo and in isolated lung pericytes. The small molecule receptor antagonists, ST1571 (imatinib) and LY2109761, were used to block the PDGFβ/pErk and TGFβ/pSmad2/3 signaling pathways, respectively. Pericyte detachment and myofibroblastic transformation were assessed by immunofluorescence and Western blot. Histochemical and immunohistochemical staining were used to evaluate the effect of the intervention on pulmonary fibrosis. Our findings demonstrate that FUT8shRNA significantly blocked pericyte activation and the progression of pulmonary fibrosis, achieving intervention effects superior to the small molecule inhibitors. The PDGFβ and TGFβ pathways were simultaneously affected by the CF blockade. FUT8 expression was upregulated with the transformation of pericytes into myofibroblasts, and silencing FUT8 expression inhibited this transformation. In addition, there is a causal relationship between CF modification catalyzed by FUT8 and pulmonary fibrosis. Our findings suggest that FUT8 may be a novel therapeutic target for pulmonary fibrosis.

LPS-Binding Protein Modulates Acute Renal Fibrosis by Inducing Pericyte-to-Myofibroblast Trans-Differentiation through TLR-4 Signaling.

During sepsis, the increased synthesis of circulating lipopolysaccharide (LPS)-binding protein (LBP) activates LPS/TLR4 signaling in renal resident cells, leading to acute kidney injury (AKI). Pericytes are the major source of myofibroblasts during chronic kidney disease (CKD), but their involvement in AKI is poorly understood. Here, we investigate the occurrence of pericyte-to-myofibroblast trans-differentiation (PMT) in sepsis-induced AKI. In a swine model of sepsis-induced AKI, PMT was detected within 9 h from LPS injection, as evaluated by the reduction of physiologic PDGFRβ expression and the dysfunctional α-SMA increase in peritubular pericytes. The therapeutic intervention by citrate-based coupled plasma filtration adsorption (CPFA) significantly reduced LBP, TGF-β, and endothelin-1 (ET-1) serum levels, and furthermore preserved PDGFRβ and decreased α-SMA expression in renal biopsies. In vitro, both LPS and septic sera led to PMT with a significant increase in Collagen I synthesis and α-SMA reorganization in contractile fibers by both SMAD2/3-dependent and -independent TGF-β signaling. Interestingly, the removal of LBP from septic plasma inhibited PMT. Finally, LPS-stimulated pericytes secreted LBP and TGF-β and underwent PMT also upon TGF-β receptor-blocking, indicating the crucial pro-fibrotic role of TLR4 signaling. Our data demonstrate that the selective removal of LBP may represent a therapeutic option to prevent PMT and the development of acute renal fibrosis in sepsis-induced AKI.

Liver repair and regeneration after ischemia-reperfusion injury is associated with prolonged fibrosis.

Liver recovery after hepatic ischemia-reperfusion (I/R) injury is characterized by clearance of dead tissue and its replacement with functional liver parenchyma. Previous reports have observed fibrosis after liver I/R. To determine whether liver fibrosis after I/R was a pathologic consequence of the injury response, we assessed the development of liver fibrosis after I/R and its impact on subsequent insult. A murine model of partial I/R was used to induce liver injury and study the reparative response. During liver remodeling after I/R, expression of the profibrotic genes increased in the ischemic liver. Histologically, α-smooth muscle actin (α-SMA)-positive hepatic stellate cells (HSCs)/myofibroblasts increased, and collagen deposition was enhanced along the injured site. Selective staining experiments showed that HSCs, not portal fibroblasts, were the major source of myofibroblasts. During liver repair after I/R, liver fibrosis was readily observed at the interface between necrotic tissue and regenerating liver in association with HSCs/myofibroblasts. The number of HSCs/myofibroblasts decreasing shortly after the full resolution of necrotic injury and restoration are normal liver architecture. However, liver fibrosis persisted for several more weeks before gradually resolving. Resolution of liver fibrosis was accompanied by upregulated expression of matrix metalloproteinase-13. After resolution of fibrosis, the administration of CCl4 did not result in exacerbated liver injury, suggesting that I/R injury does not predispose the liver to future fibrotic insults. The data suggest that liver fibrosis is a component of tissue repair after I/R, is caused by myofibroblasts derived from HSC, and does not increase susceptibility of the liver to subsequent hepatic injury. NEW & NOTEWORTHY This study is the first to assess pathology of liver fibrosis during the reparative process after ischemia-reperfusion (I/R) injury. Here we show that profibrotic gene expression increased in the liver after I/R, and collagen accumulation produced by hepatic stellate cells (HSCs)/myofibroblasts enhanced at the interface between necrotic tissue and regenerating liver. Liver fibrosis gradually resolved concomitant with decreasing activation of HSC and upregulating matrix metalloproteinase-13. After resolution of fibrosis, the liver was not more susceptible to subsequent hepatic injury.

Novel Mechanism of the Pericyte-Myofibroblast Transition in Renal Interstitial Fibrosis: Core Fucosylation Regulation

Pericytes have been identified as a major source of myofibroblasts in renal interstitial fibrosis (RIF). The overactivation of several signaling pathways, mainly the TGF-β and PDGF pathways, initiates the pericyte-myofibroblast transition during RIF. Key receptors in these two pathways have been shown to be modified by fucosyltransferase 8 (FUT8), the enzyme that catalyzes core fucosylation. This study postulated that core fucosylation might play an important role in regulating the pericyte transition in RIF. The data showed that core fucosylation increased with the extent of RIF in patients with IgA nephropathy (IgAN). Similarly, core fucosylation of pericytes increased in both a unilateral ureteral occlusion (UUO) mouse model and an in vitro model of pericyte transition. Inhibition of core fucosylation by adenoviral-mediated FUT8 shRNA in vivo and FUT8 siRNA in vitro significantly reduced pericyte transition and RIF. In addition, the activation of both the TGF-β/Smad and PDGF/ERK pathways was blocked by core fucosylation inhibition. In conclusion, core fucosylation may regulate the pericyte transition in RIF by modifying both the TGF-β/Smad and PDGF/ERK pathways. Glycosylation might be a novel “hub” target to prevent RIF.

Liver inflammation and fibrosis.

Chronic liver inflammation leads to fibrosis and cirrhosis, which is the 12th leading cause of death in the United States. Hepatocyte steatosis is a component of metabolic syndrome and insulin resistance. Hepatic steatosis may be benign or progress to hepatocyte injury and the initiation of inflammation, which activates immune cells. While Kupffer cells are the resident macrophage in the liver, inflammatory cells such as infiltrating macrophages, T lymphocytes, neutrophils, and DCs all contribute to liver inflammation. The inflammatory cells activate hepatic stellate cells, which are the major source of myofibroblasts in the liver. Here we review the initiation of inflammation in the liver, the liver inflammatory cells, and their crosstalk with myofibroblasts.

Role of mesenchymal stem cells in kidney injury and fibrosis.

Multiple studies have established the beneficial role of mesenchymal stem cell (MSC) therapy for kidney injury. In this review we will discuss the recent identification of Gli1 as a marker for perivascular MSC and the role of this cell population in kidney fibrosis. Recent studies demonstrate that expression of the hedgehog transcriptional activator Gli1 specifically marks perivascular MSC. Genetic fate tracing of MSC in kidney injury revealed their contribution to the myofibroblast pool whereas ablation of MSC reduced kidney fibrosis. Furthermore, strong evidence suggests that pharmacologically targeting Gli proteins inhibits cell-cycle progression of myofibroblasts in kidney fibrosis and is a promising therapeutic strategy in chronic kidney disease. Although there is tremendous excitement about MSC as cellular therapy in kidney injury it has been shown that resident perivascular MSC are a major source of myofibroblasts and a novel therapeutic target in kidney fibrosis. While resident kidney MSC might also be involved in capillary rarefaction after injury and during fibrosis progression their potential role in kidney repair, angiogenesis, and regeneration remains unclear. Further studies are needed to identify the molecular pathways that control the profibrotic versus proregenerative role of resident MSC in kidney injury and repair.

Epithelial-to-Mesenchymal Transition in Diabetic Nephropathy: Fact or Fiction?

The pathophysiology of diabetic nephropathy (DN), one of the most serious complications in diabetic patients and the leading cause of end-stage renal disease worldwide, is complex and not fully elucidated. A typical hallmark of DN is the excessive deposition of extracellular matrix (ECM) proteins in the glomerulus and in the renal tubulointerstitium, eventually leading to glomerulosclerosis and interstitial fibrosis. Although it is obvious that myofibroblasts play a major role in the synthesis and secretion of ECM, the origin of myofibroblasts in DN remains the subject of controversial debates. A number of studies have focused on epithelial-to-mesenchymal transition (EMT) as one source of matrix-generating fibroblasts in the diseased kidney. EMT is characterized by the acquisition of mesenchymal properties by epithelial cells, preferentially proximal tubular cells and podocytes. In this review we comprehensively review the literature and discuss arguments both for and against a function of EMT in renal fibrosis in DN. While the precise extent of the contribution to nephrotic fibrosis is certainly arduous to quantify, the picture that emerges from this extensive body of literature suggests EMT as a major source of myofibroblasts in DN.

Isolation and epithelial co-culture of mouse renal peritubular endothelial cells.

BackgroundEndothelial-mesenchymal transition (EndoMT) has been shown to be a major source of myofibroblasts, contributing to kidney fibrosis. However, in vitro study of endothelial cells often relies on culture of isolated primary endothelial cells due to the unavailability of endothelial cell lines. Our recent study suggested that peritubular endothelial cells could contribute to kidney fibrosis through EndoMT. Therefore, successful isolation and culture of mouse peritubular endothelial cells could provide a new platform for studying kidney fibrosis. This study describes an immunomagnetic separation method for the isolation of mouse renal peritubular endothelial cells using anti-CD146 MicroBeads, followed by co-culture with mouse renal proximal tubular epithelial cells to maintain endothelial phenotype.ResultsFlow cytometry showed that after isolation and two days of culture, about 95% of cells were positive for endothelial-specific marker CD146. The percentage of other cells, including dendritic cells (CD11c) and macrophages (F4/80), was less than 1%. Maintenance of endothelial cell phenotype required vascular endothelial growth factor (VEGF) and co-culture with mouse proximal tubular epithelial cells.ConclusionIn this study, we established a method for the isolation of mouse renal peritubular endothelial cells by using immunomagnetic separation with anti-CD146 MicroBeads, followed by co-culture with mouse renal proximal tubular epithelial cells to maintain phenotype.Electronic supplementary materialThe online version of this article (doi:10.1186/s12860-014-0040-6) contains supplementary material, which is available to authorized users.

The types of hepatic myofibroblasts contributing to liver fibrosis of different etiologies.

Liver fibrosis results from dysregulation of normal wound healing, inflammation, activation of myofibroblasts, and deposition of extracellular matrix (ECM). Chronic liver injury causes death of hepatocytes and formation of apoptotic bodies, which in turn, release factors that recruit inflammatory cells (neutrophils, monocytes, macrophages, and lymphocytes) to the injured liver. Hepatic macrophages (Kupffer cells) produce TGFβ1 and other inflammatory cytokines that activate Collagen Type I producing myofibroblasts, which are not present in the normal liver. Secretion of TGFβ1 and activation of myofibroblasts play a critical role in the pathogenesis of liver fibrosis of different etiologies. Although the composition of fibrogenic myofibroblasts varies dependent on etiology of liver injury, liver resident hepatic stellate cells and portal fibroblasts are the major source of myofibroblasts in fibrotic liver in both experimental models of liver fibrosis and in patients with liver disease. Several studies have demonstrated that hepatic fibrosis can reverse upon cessation of liver injury. Regression of liver fibrosis is accompanied by the disappearance of fibrogenic myofibroblasts followed by resorption of the fibrous scar. Myofibroblasts either apoptose or inactivate into a quiescent-like state (e.g., stop collagen production and partially restore expression of lipogenic genes). Resolution of liver fibrosis is associated with recruitment of macrophages that secrete matrix-degrading enzymes (matrix metalloproteinase, collagenases) and are responsible for fibrosis resolution. However, prolonged/repeated liver injury may cause irreversible crosslinking of ECM and formation of uncleavable collagen fibers. Advanced fibrosis progresses to cirrhosis and hepatocellular carcinoma. The current review will summarize the role and contribution of different cell types to populations of fibrogenic myofibroblasts in fibrotic liver.

Integrins, myofibroblasts, and organ fibrosis

Myofibroblasts are the major source of extracellular matrix components that accumulate during tissue fibrosis, and hepatic stellate cells (HSCs) are believed to be the major source of myofibroblasts in the liver. To date, robust systems to genetically manipulate these cells have not been developed. We report that Cre under control of the promoter of Pdgfrb (Pdgfrb-Cre) inactivates loxP-flanked genes in mouse HSCs with high efficiency. We used this system to delete the gene encoding αV integrin subunit because various αV-containing integrins have been suggested as central mediators of fibrosis in multiple organs. Such depletion protected mice from carbon tetrachloride–induced hepatic fibrosis, whereas global loss of β3, β5 or β6 integrin or conditional loss of β8 integrins in HSCs did not. We also found that Pdgfrb-Cre effectively targeted myofibroblasts in multiple organs, and depletion of the αV integrin subunit using this system was protective in other models of organ fibrosis, including pulmonary and renal fibrosis. Pharmacological blockade of αV-containing integrins by a small molecule (CWHM 12) attenuated both liver and lung fibrosis, including in a therapeutic manner. These data identify a core pathway that regulates fibrosis and suggest that pharmacological targeting of all αV integrins may have clinical utility in the treatment of patients with a broad range of fibrotic diseases.

Mesodermal mesenchymal cells give rise to myofibroblasts, but not epithelial cells, in mouse liver injury

Hepatic stellate cells (HSCs) and portal fibroblasts (PFs) are believed to be the major source of myofibroblasts that participate in fibrogenesis by way of synthesis of proinflammatory cytokines and extracellular matrices. Previous lineage tracing studies using MesP1(Cre) and Rosa26lacZ(flox) mice demonstrated that MesP1+ mesoderm gives rise to mesothelial cells (MCs), which differentiate into HSCs and PFs during liver development. In contrast, several in vivo and in vitro studies reported that HSCs can differentiate into other cell types, including hepatocytes, cholangiocytes, and progenitor cell types known as oval cells, thereby acting as stem cells in the liver. To test whether HSCs give rise to epithelial cells in adult liver, we determined the hepatic lineages of HSCs and PFs using MesP1(Cre) and Rosa26mTmG(flox) mice. Genetic cell lineage tracing revealed that the MesP1+ mesoderm gives rise to MCs, HSCs, and PFs, but not to hepatocytes or cholangiocytes, in the adult liver. Upon carbon tetrachloride injection or bile duct ligation surgery-mediated liver injury, mesodermal mesenchymal cells, including HSCs and PFs, differentiate into myofibroblasts but not into hepatocytes or cholangiocytes. Furthermore, differentiation of the mesodermal mesenchymal cells into oval cells was not observed. These results indicate that HSCs are not sufficiently multipotent to produce hepatocytes, cholangiocytes, or oval cells by way of mesenchymal-epithelial transition in vivo. Cell lineage tracing demonstrated that mesodermal mesenchymal cells including HSCs are the major source of myofibroblasts but do not differentiate into epithelial cell types such as hepatocytes, cholangiocytes, and oval cells.

Targeting of αv integrin identifies a core molecular pathway that regulates fibrosis in several organs

Myofibroblasts are the major source of extracellular matrix components that accumulate during tissue fibrosis, and hepatic stellate cells (HSCs) are the major source of myofibroblasts in the liver. To date, robust systems to genetically manipulate these cells have not existed. We report that Pdgfrb-Cre inactivates genes in murine HSCs with high efficiency. We used this system to delete the αv integrin subunit because of the suggested role of multiple αv integrins as central mediators of fibrosis in multiple organs. Depletion of the αv integrin subunit in HSCs protected mice from CCl4-induced hepatic fibrosis, whereas global loss of αvβ3, αvβ5 or αvβ6 or conditional loss of αvβ8 on HSCs did not. Pdgfrb-Cre effectively targeted myofibroblasts in multiple organs, and depletion of αv integrins using this system was also protective in models of pulmonary and renal fibrosis. Critically, pharmacological blockade of αv integrins by a novel small molecule (CWHM 12) attenuated both liver and lung fibrosis, even when administered after fibrosis was established. These data identify a core pathway that regulates fibrosis, and suggest that pharmacological targeting of all αv integrins may have clinical utility in the treatment of patients with a broad range of fibrotic diseases.