Fibrotic Activation Research Articles

Interfering with local fibrotic platelet activation significantly inhibits fibrosis in multiple animal models: suggestions of the importance of the platelet-wound healing axis for fibrosis

Interfering with local fibrotic platelet activation significantly inhibits fibrosis in multiple animal models: suggestions of the importance of the platelet-wound healing axis for fibrosis

Crocin mediated amelioration of oxidative burden and inflammatory cascade suppresses diabetic nephropathy progression in diabetic rats

Diabetic Nephropathy (DN) is one of the main complications associated with diabetes mellitus. Persistently elevated blood glucose level drives histopathological changes in renal tissues that hinder normal kidney functions. In the current study, crocin; the main bioactive constituent of Crocus sativus was investigated as a reno-protective agent against DN by virtue of its numerous pharmacological activities. Diabetes was induced in male Sprague-Dawely rats through intravenous injection of streptozocin (STZ) (50 mg/kg), DN was confirmed eight weeks post diabetes induction. Daily oral crocin for eight weeks (20 mg/kg) significantly reduced blood glucose level with a significant increase in insulin level. Moreover, crocin improved impaired kidney functions as manifested in reduction of serum creatinine levels, blood urea nitrogen and proteinuria with concomitant increase in urinary creatinine clearance. Furthermore, biomarkers of cell injury and tissue necrosis like LDH activity was significantly reduced, kidney content of NOS significantly declined likewise. In addition, renal antioxidants such as SOD, GSH and serum catalase activity significantly increased with concomitant reduction of kidney MDA; biomarker of oxidative load. Kidney content of toll-like receptors 4 and IL-6 significantly declined with simultaneous suppression of nuclear factor kappa-B (NF-κB/p65) protein expression and immuno-staining in rat renal cortex. Furthermore, crocin inhibited progression of renal fibrosis as seen with reduction of renal hydroxyproline and collagen content, TGF-β immuno-staining and Masson's Trichrome positive tissue. Histopathologically, crocin pretreatment was associated with minimal renal damage with fewer fibrotic lesions. There was a concomitant restoration of renal tubules integrity with preservation of glomerular space area. In conclusion, crocin's ameliorative impact on DN may be attributed to its free radicals scavenging properties, its ability to enhance host antioxidant defense system and its ability to inhibit inflammatory and fibrotic cascades activation.

Densification of Type I Collagen Matrices as a Model for Cardiac Fibrosis.

Cardiac fibrosis is a disease state characterized by excessive collagenous matrix accumulation within the myocardium that can lead to ventricular dilation and systolic failure. Current treatment options are severely lacking due in part to the poor understanding of the complexity of molecular pathways involved in cardiac fibrosis. To close this gap, in vitro model systems that recapitulate the defining features of the fibrotic cellular environment are in need. Type I collagen, a major cardiac extracellular matrix protein and the defining component of fibrotic depositions, is an attractive choice for a fibrosis model, but demonstrates poor mechanical strength due to solubility limits. However, plastic compression of collagen matrices is shown to significantly increase its mechanical properties. Here, confined compression of oligomeric, type I collagen matrices is utilized to resemble defining hallmarks seen in fibrotic tissue such as increased collagen content, fibril thickness, and bulk compressive modulus. Cardiomyocytes seeded on compressed matrices show a strong beating abrogation as observed in cardiac fibrosis. Gene expression analysis of selected fibrosis markers indicates fibrotic activation and cardiomyocyte maturation with regard to the existing literature. With these results, a promising first step toward a facile heart-on-chip model is presented to study cardiac fibrosis.

Exosomes derived from palmitic acid-treated hepatocytes induce fibrotic activation of hepatic stellate cells

Non-alcoholic fatty liver disease (NAFLD) is a dominant cause of chronic liver disease, but the exact mechanism of progression from simple steatosis to nonalcoholic steatohepatitis (NASH) remains unknown. Here, we investigated the role of exosomes in NAFLD progression. Exosomes were isolated from a human hepatoma cell line treated with palmitic acid (PA) and their miRNA profiles examined by microarray. The human hepatic stellate cell (HSC) line (LX-2) was then treated with exosome isolated from hepatocytes. Compared with controls, PA-treated hepatocytes displayed significantly increased CD36 and exosome production. The microarray analysis showed there to be distinctive miRNA expression patterns between exosomes from vehicle- and PA-treated hepatocytes. When LX-2 cells were cultured with exosomes from PA-treated hepatocytes, the expression of genes related to the development of fibrosis were significantly amplified compared to those treated with exosomes from vehicle-treated hepatocytes. In conclusion, PA treatment enhanced the production of exosomes in these hepatocytes and changed their exosomal miRNA profile. Moreover, exosomes derived from PA-treated hepatocytes caused an increase in the expression levels of fibrotic genes in HSCs. Therefore, exosomes may have important roles in the crosstalk between hepatocytes and HSCs in the progression from simple steatosis to NASH.

Mechanosensitive transient receptor potential vanilloid 4 regulates Dermatophagoides farinae-induced airway remodeling via 2 distinct pathways modulating matrix synthesis and degradation.

Contributions of mechanical signals to airway remodeling during asthma are poorly understood. Transient receptor potential vanilloid 4 (TRPV4), a mechanosensitive ion channel, has been implicated in cardiac and pulmonary fibrosis; however, its role in asthma remains elusive. Employing a Dermatophagoides farinae-induced asthma model, we report here that TRPV4-knockout mice were protected from D. farinae-induced airway remodeling. Furthermore, lung fibroblasts that were isolated from TRPV4-knockout mice showed diminished differentiation potential compared with wild-type mice. Fibroblasts from asthmatic lung exhibited increased TRPV4 activity and enhanced differentiation potential compared with normal human lung fibroblasts. Of interest, TGF-β1 treatment enhanced TRPV4 activation in a PI3K-dependent manner in normal human lung fibroblasts in vitro Mechanistically, TRPV4 modulated matrix remodeling in the lung via 2 distinct but dependent pathways: one enhances matrix deposition by fibrotic gene activation, whereas the other slows down matrix degradation by increased plasminogen activator inhibitor 1. Of importance, both pathways are regulated by Rho/myocardin-related transcription factor-A and contribute to fibroblast differentiation and matrix remodeling in the lung. Thus, our results support a unique role for TRPV4 in D. farinae-induced airway remodeling and warrant further studies in humans for it to be used as a novel therapeutic target in the treatment of asthma.-Gombedza, F., Kondeti, V., Al-Azzam, N., Koppes, S., Duah, E., Patil, P., Hexter, M., Phillips, D., Thodeti, C. K., Paruchuri, S. Mechanosensitive transient receptor potential vanilloid 4 regulates Dermatophagoides farinae-induced airway remodeling via 2 distinct pathways modulating matrix synthesis and degradation.

Mechanistic Target of Rapamycin Complex 1 (mTORC1) and mTORC2 as Key Signaling Intermediates in Mesenchymal Cell Activation

Fibrotic diseases display mesenchymal cell (MC) activation with pathologic deposition of matrix proteins such as collagen. Here we investigate the role of mTOR complex 1 (mTORC1) and mTORC2 in regulating MC collagen expression, a hallmark of fibrotic disease. Relative to normal MCs (non-Fib MCs), MCs derived from fibrotic human lung allografts (Fib-MCs) demonstrated increased phosphoinositide-3kinase (PI3K) dependent activation of both mTORC1 and mTORC2, as measured by increased phosphorylation of S6K1 and 4E-BP1 (mTORC1 substrates) and AKT (an mTORC2 substrate). Dual ATP-competitive TORC1/2 inhibitor AZD8055, in contrast to allosteric mTORC1-specific inhibitor rapamycin, strongly inhibited 4E-BP1 phosphorylation and collagen I expression in Fib-MCs. In non-Fib MCs, increased mTORC1 signaling was shown to augment collagen I expression. mTORC1/4E-BP1 pathway was identified as an important driver of collagen I expression in Fib-MCs in experiments utilizing raptor gene silencing and overexpression of dominant-inhibitory 4E-BP1. Furthermore, siRNA-mediated knockdown of rictor, an mTORC2 partner protein, reduced mTORC1 substrate phosphorylation and collagen expression in Fib-, but not non-Fib MCs, revealing a dependence of mTORC1 signaling on mTORC2 function in activated MCs. Together these studies suggest a novel paradigm where fibrotic activation in MCs increases PI3K dependent mTORC1 and mTORC2 signaling and leads to increased collagen I expression via the mTORC1-dependent 4E-BP1/eIF4E pathway. These data provide rationale for targeting specific components of mTORC pathways in fibrotic states and underscore the need to further delineate mTORC2 signaling in activated cell states.

High inorganic phosphate causes DNMT1 phosphorylation and subsequent fibrotic fibroblast activation

Phosphate is an essential constituent of critical cellular functions including energy metabolism, nucleic acid synthesis and phosphorylation-dependent cell signaling. Increased plasma phosphate levels are an independent risk factor for lowered life-expectancy as well as for heart and kidney failure. Nevertheless, direct cellular effects of elevated phosphate concentrations within the microenvironment are poorly understood and have been largely neglected in favor of phosphor-regulatory hormones. Because interstitial fibrosis is the common determinant of chronic progressive kidney disease, and because fibroblasts are major mediators of fibrogenesis, we here explored the effect of high extracellular phosphate levels on renal fibroblasts. We demonstrate that high inorganic phosphate directly induces fibrotic fibroblast activation associated with increased proliferative activity, increased expression of α-smooth muscle actin and increased synthesis of type I collagen. We further demonstrate that such fibroblast activation is dependent on phosphate influx, aberrant phosphorylation of DNA methyltransferase DNMT1 and aberrant CpG island promoter methylation. In summary, our studies demonstrate that elevated phosphate concentrations induce pro-fibrotic fibroblast activation independent of phospho-regulatory hormones.

A human liver microphysiology platform for investigating physiology, drug safety, and disease models.

This paper describes the development and characterization of a microphysiology platform for drug safety and efficacy in liver models of disease that includes a human, 3D, microfluidic, four-cell, sequentially layered, self-assembly liver model (SQL-SAL); fluorescent protein biosensors for mechanistic readouts; as well as a microphysiology system database (MPS-Db) to manage, analyze, and model data. The goal of our approach is to create the simplest design in terms of cells, matrix materials, and microfluidic device parameters that will support a physiologically relevant liver model that is robust and reproducible for at least 28 days for stand-alone liver studies and microfluidic integration with other organs-on-chips. The current SQL-SAL uses primary human hepatocytes along with human endothelial (EA.hy926), immune (U937) and stellate (LX-2) cells in physiological ratios and is viable for at least 28 days under continuous flow. Approximately, 20% of primary hepatocytes and/or stellate cells contain fluorescent protein biosensors (called sentinel cells) to measure apoptosis, reactive oxygen species (ROS) and/or cell location by high content analysis (HCA). In addition, drugs, drug metabolites, albumin, urea and lactate dehydrogenase (LDH) are monitored in the efflux media. Exposure to 180 μM troglitazone or 210 μM nimesulide produced acute toxicity within 2-4 days, whereas 28 μM troglitazone produced a gradual and much delayed toxic response over 21 days, concordant with known mechanisms of toxicity, while 600 µM caffeine had no effect. Immune-mediated toxicity was demonstrated with trovafloxacin with lipopolysaccharide (LPS), but not levofloxacin with LPS. The SQL-SAL exhibited early fibrotic activation in response to 30 nM methotrexate, indicated by increased stellate cell migration, expression of alpha-smooth muscle actin and collagen, type 1, alpha 2. Data collected from the invitro model can be integrated into a database with access to related chemical, bioactivity, preclinical and clinical information uploaded from external databases for constructing predictive models.

Serotonin drives the activation of pulmonary artery adventitial fibroblasts and TGF-β1/Smad3-mediated fibrotic responses through 5-HT(2A) receptors.

Pulmonary arterial remodeling is characterized by excessive proliferation, migration, and pro-differentiation and fibrotic activation of adventitial fibroblasts in pulmonary arterial hypertension (PAH) process. Several lines of evidence indicate that serotonin (5-HT) plays a central role in the pathogenesis of pulmonary arterial remodeling. In the present study, we investigated whether 5-HT is directly involved in the functional regulation of pulmonary artery adventitial fibroblasts (PAFs). Incubation of cultured rat PAFs with 5-HT caused a dose-dependent stimulation of cell proliferation, migration activity, and a time-dependent increase of α-SMA expression, a marker of fibroblast differentiation into myofibroblasts, and adventitia fibrosis, evaluating connective tissue growth factor (CTGF) and extracellular matrix (ECM) mRNAs and proteins. These effects were attenuated by the 5-HT2A receptor antagonist, ketanserin and mimicked by the 5-HT2A receptor agonist DOI. 5-HT-induced fibroblasts phenotypic alterations and ECM accumulation were dependent on stimulation of transforming growth factor (TGF)-β1 as demonstrated using a neutralizing antibody. 5-HT also caused Smad3 phosphorylation and ketanserin diminished 5-HT-induced Smad3 activation. These results demonstrated that 5-HT can directly activate PAFs through 5-HT2A receptor and promote fibroblasts phenotypic alterations and adventitia fibrosis depending on the signaling of the TGF-β1/Smad3 pathway.

Biomechanics of TGFβ-induced epithelial-mesenchymal transition: implications for fibrosis and cancer.

Fibrosis, a disease that results in loss of organ function, contributes to a significant number of deaths worldwide and sustained fibrotic activation has been suggested to increase the risk of developing cancer in a variety of tissues. Fibrogenesis and tumor progression are regulated in part through the activation and activity of myofibroblasts. Increasing evidence links myofibroblasts found within fibrotic lesions and the tumor microenvironment to a process termed epithelial-mesenchymal transition (EMT), a phenotypic change in which epithelial cells acquire mesenchymal characteristics. EMT can be stimulated by soluble signals, including transforming growth factor (TGF)-β, and recent studies have identified a role for mechanical cues in directing EMT. In this review, we describe the role that EMT plays in fibrogenesis and in the progression of cancer, with particular emphasis placed on biophysical signaling mechanisms that control the EMT program. We further describe specific TGFβ-induced intracellular signaling cascades that are affected by cell- and tissue-level mechanics. Finally, we highlight the implications of mechanical induction of EMT on the development of treatments and targeted intervention strategies for fibrosis and cancer.

DNA methylation in cardiac fibrosis: new advances and perspectives.

Cardiac fibrosis is characterized by net accumulation of extracellular matrix (ECM) proteins in the cardiac interstitium, and contributes to both systolic and diastolic dysfunction in many cardiac pathophysiologic conditions. More specifically, cardiac fibroblasts are activated by a variety of pathological stimuli, thereby undergoing proliferation, differentiation to myofibroblasts, and production of various cytokines and ECM proteins. Thus, understanding the biological processes of cardiac fibroblasts will provide novel insights into the underlying mechanisms of cardiac fibrosis. DNA methylation is an important epigenetic mechanism, which often occurs in response to environmental stimuli and is crucial in regulating gene expression. The aberrant methylation of CpG island promoters of selected genes is the prominent epigenetic mechanism by which gene transcription can be effectively silenced. Aberrant hypermethylation of a few selected genes such as RASSF1A plays an important role in facilitating fibrotic fibroblast activation and in driving fibrosis. In this review we will discuss the mechanisms of DNA methylation and their implications for cardiac fibroblasts activation and fibrosis. Control of DNA methylation may serve as a new strategy for anti-fibrotic therapy.

Upregulation of cannabinoid receptor‐1 and fibrotic activation of mouse hepatic stellate cells during Schistosoma J. infection: role of NADPH oxidase (693.13)

The endocannabinoid system (CS) has been implicated in schistosomiasis‐associated liver fibrosis (SSLF). However, the molecular mechanisms mediating the action of the CS in SSLF are largely unknown. The present study hypothesized that Schistosoma J. infection upregulates cannabinoid receptor (CB) signaling via activation of NADPH oxidase leading to a fibrotic phenotype in hepatic stellate cells (HSCs). A SSLF model was developed by infecting mice with Schistosoma J. cercariae in the skin. HSCs from uninfected (control) or Schistosoma J.‐infected (SSLF) mice were isolated, cultured and identified by analyzing HSC markers α‐SMA and desmin. In HSCs isolated from control or SSLF mice, both CB1 and CB2 receptors were detected, however, only the CB1 significantly increased in HSCs from SSLF mice compared to that control mice. Such increase of CB1 in HSCs was accompanied by enhanced expression of fibrotic markers α‐SMA, collagen I, and TIMP‐1. Similarly, CB1 upregulation and enhanced fibrotic changes were observed in HSCs isolated from control mice when these cells were treated with soluble egg antigen (SEA) from Schistosoma J. Interestingly, HSCs from SSLF mice or SEA‐treated HSCs from control mice exhibited enhanced superoxide (O2.‐) production as analyzed by electron spin resonance. Importantly, inhibition of O2.‐ production by knocking down NADPH oxidase genes (Nox1 or Nox4) prevented SEA‐induced upregulation of CB1 and fibrotic markers in HSCs. Further, CB1 gene silencing blocked SEA‐induced fibrotic changes in HSCs but had no effect on SEA‐induced O2.‐ production in these cells indicating a triggering role of O2.‐ production in CB1‐mediated fibrotic signaling. Taken together, these data suggest that the fibrotic activation of HSCs upon Schistosoma J. infection or SEA stimulation is associated with NADPH oxidase‐mediated redox regulation of CB1 expression, which may be a triggering mechanism for SSLF.Grant Funding Source: Supported by NIH grants HL057244, HL091464 and HL075316

Upregulation of cannabinoid receptor-1 and fibrotic activation of mouse hepatic stellate cells during Schistosoma J. infection: Role of NADPH oxidase

The endocannabinoid system (CS) has been implicated in the development of hepatic fibrosis such as schistosomiasis-associated liver fibrosis (SSLF). However, the mechanisms mediating the action of the CS in hepatic fibrosis are unclear. The present study hypothesized that Schistosoma J. infection upregulates cannabinoid receptor 1 (CB1) due to activation of NADPH oxidase leading to a fibrotic phenotype in hepatic stellate cells (HSCs). The SSLF model was developed by infecting mice with Schistosoma J. cercariae in the skin, and HSCs from control and infected mice were then isolated, cultured, and confirmed by analysis of HSC markers α-SMA and desmin. CB1 significantly increased in HSCs isolated from mice with SSLF, which was accompanied by a greater expression of fibrotic markers α-SMA, collagen I, and TIMP-1. CB1 upregulation and enhanced fibrotic changes were also observed in normal HSCs treated with soluble egg antigen (SEA) from Schistosoma J. Electron spin resonance (ESR) analysis further demonstrated that superoxide (O2−) production was increased in infected HSCs or normal HSCs stimulated with SEA. Both Nox4 and Nox1 siRNA prevented SEA-induced upregulation of CB1, α-SMA, collagen I, and TIMP-1 by inhibition of O2− production, while CB1 siRNA blocked SEA-induced fibrotic changes without effect on O2− production in these HSCs. Taken together, these data suggest that the fibrotic activation of HSCs on Schistosoma J. infection or SEA stimulation is associated with NADPH oxidase-mediated redox regulation of CB1 expression, which may be a triggering mechanism for SSLF.

Regulatory RNAs and paracrine networks in the heart

Cardiac hypertrophy and fibrosis are two closely related adaptive response mechanisms of the myocardium to mechanical, metabolic, and genetic stress that finally contribute to the development of heart failure (HF). This relation is based on a dynamic interplay between many cell types including cardiomyocytes and fibroblasts during disease progression. Both cell types secrete a variety of growth factors, cytokines, and hormones that influence hypertrophic cardiomyocyte growth and fibrotic fibroblast activation in a paracrine and autocrine manner. It has become evident that, aside proteinous signals, microRNAs (miRNAs) and possible other RNA species such as long non-coding RNAs are potential players in such a cell-to-cell communication. By directly acting as paracrine signals or by modulating downstream intercellular signalling mediators, miRNAs can act as moderators of the intercellular crosstalk. These small regulators can potentially be secreted in a 'mircrine' fashion, so that miRNAs can be assumed as the message itself. This review will summarize the recent findings about the paracrine crosstalk between cardiac fibroblasts and cardiomyocytes and addresses how miRNAs may be involved in this interplay. It also highlights therapeutic strategies targeting factors of pathological communication for the treatment of HF.

The role of promoter hypermethylation in fibroblast activation and fibrogenesis

The aberrant methylation of CpG island promoters of selected genes is the prominent epigenetic mechanism by which gene transcription can be effectively silenced. Aberrant hypermethylation of a few selected genes plays an important role in facilitating fibrotic fibroblast activation and in driving fibrogenesis. Here we review mechanisms of DNA methylation and demethylation and their implications for fibroblast activation and tissue fibrosis.

TGF-beta 1 induced fibroblast proliferation is mediated by the FGF-2/ERK pathway

Pulmonary fibrosis, defined as the accumulation of connective tissue in the lungs, is a severe and often fatal form of interstitial lung disease. Transforming growth factor-beta (TGF-beta) is a powerful activator of connective tissue synthesis and fibroblast proliferation in the lung, and a critical paracrine signal for the development of pulmonary fibrosis. To investigate signaling pathways downstream of TGF- beta that contribute to lung fibrosis, TGF- beta stimulation of fibroblasts was replicated by treating NIH3T3 fibroblasts with conditioned medium (CM) from TGF- beta -treated type II alveolar epithelial cells (ATII cells). The data showed that fibroblast growth factor 2 (FGF-2) signaling is responsible for TGF-beta 1 CM-induced fibroblast proliferation, while it does not affect TGF-beta 1 CM-induced fibrotic differentiation. Moreover, fibroblast proliferation and differentiation induced by TGF- beta CM was totally abrogated by pretreatment of NIH3T3 cells with the specific ERK1/2 inhibitor, PD98059. These findings indicate that FGF-2 secreted by alveolar epithelial cells in response to TGF- beta 1 induces fibroblast proliferation and fibrotic activation through the ERK kinase pathway.

Transcriptome Changes of Chronic Tubulointerstitial Damage in Early Kidney Transplantation

Tubulointerstitial damage (TID) is a key feature of chronic kidney transplant failure; however, the associated gene expression changes are poorly defined. This pilot study used RNA from 59 protocol kidney transplant biopsies at implantation, 1, 3, and 12 months (n=18 patients), processed into cDNA and hybridized to 8K human cDNA microarrays. Gene expression was correlated with graft histology categorized by the Banff schema. Gene and pathway expression were differentially activated according to the time after transplantation. Immune pathway activity peaked at 1 month, fibrotic expression at 3 months, wound healing-remodelling and cell proliferation-repair processes were activated between 3 and 12 months, whereas macrophage-related gene expression occurred late by 12 months. Forty percent of genes and 50% pathways initially activated persisted to 3 months. Biopsies with TID displayed 262 differentially expressed genes (P<0.001, B>2 compared with implantation), dominated by upregulated fibrogenic and immune-related genes reflecting unique immune (10% to 15% of genes) and fibrotic (15% vs. 4% in normal) pathway activation. Profibrotic genes were expressed before interstitial fibrosis was observed by sequential microscopic analysis. Kidneys progressing to TID by 3 months demonstrated 30 unique genes (B>1, P<0.05) versus nonprogressors with 95 genes (B>1, P<0.009). Fourteen of these progressor genes also occurred in the top decile from an independent validation set. Allografts display predictable immune and fibrotic gene expression profiles, with patterns of expression gradually varying by time after transplantation. The pathology reflects differential activation of intrinsic pathways. Gene expression predated histologic damage, suggesting its possible use in early diagnostic testing.

Single Administration of Thrombopoietin Prevents Progression of Liver Fibrosis and Promotes Liver Regeneration After Partial Hepatectomy in Cirrhotic Rats

To evaluate the effect of thrombopoietin on liver regeneration after hepatectomy and antifibrosis under conditions of liver cirrhosis in rats. We revealed that platelets induced by thrombopoietin administration promote liver regeneration after hepatectomy in the normal liver. Seventy percent hepatectomy was carried out in rats, which were subsequently divided into 4 groups: (1) normal group without any treatment, (2) liver cirrhosis (LC) group, (3) combined thrombopoietin and liver cirrhosis (LC+TPO) group, and (4) combined thrombopoietin, antiplatelet serum and liver cirrhosis (LC+TPO+APS) group. Growth kinetics in the liver regeneration and growth factors were analyzed. Liver fibrotic area and activation of hepatic stellate cells were also investigated. In LC group, liver regeneration was significantly delayed compared with normal group 24 hours after hepatectomy. On the other hand, liver regeneration of LC+TPO group increased significantly compared with LC group, to a level that was the same as that recorded in normal group. In LC group, liver fibrotic area before hepatectomy was significantly higher compared with the normal group. Liver fibrosis of LC+TPO group was significantly reduced compared with LC group. The antifibrotic and liver regeneration promoting effects of LC+TPO group were inhibited by antiplatelet serum in LC+TPO+APS group. The administration of thrombopoietin reduces liver fibrosis and stimulates regeneration after hepatectomy through increment and accumulation of platelets in the cirrhotic liver. This could be a potentially useful treatment for liver cirrhosis.

Cu/Zn superoxide dismutase modulates phenotypic changes in cultured fibroblasts from human skin with chronic radiotherapy damage

Purpose: As we previously observed that bovine liposomal Cu/Zn SOD (LipSOD) reduces cutaneous radiation-induced fibrosis (RIF) in human therapeutic assays the mechanisms involved were investigated here by an in vitro study of the LipSOD effects on cellular antioxidant metabolism and regulation of matrix degradation. Methods: Primary cultures of human fibroblasts harvested from normal or RIF skin were treated with various doses of LipSOD. Catalase, Cu/Zn and Mn SOD endogenous cell enzyme activities and protein amounts were assayed by polyacrylamide gel electrophoresis and western blotting. Gene expressions of tissue inhibitor of metalloproteinases (TIMP) and TGF-β1 was investigated by northern blot analysis. Results: A deficiency of endogenous Mn SOD, considered to favour cell proliferation, was observed in cultured RIF cell. The present study showed that bovine Cu/Zn SOD entered the cells. Exposure to LipSOD (a) enhanced endogenous Mn SOD activity and protein level, without changes of endogenous Cu/Zn SOD and catalase, and (b) significantly reduced TIMP and TGF-β1 gene expression, in RIF cells. No changes in these parameters were noted in treated control skin fibroblasts. Conclusion: Modulation of RIF skin fibroblasts by LipSOD seems effective via indirect endogenous Mn SOD activation, which might explain the cell phenotype reversion observed. TIMP reduction accounts for the elimination of collagenase activity inhibition and the subsequent digestion of excess extracellular matrix deposition, as well as RIF reversibility in vivo. The reduction of TGF-β1 expression might explain the breaking of maintaining fibrotic cell activation connected with this growth factor.