Fibrogenesis & Tissue Repair最新文献

Fibrosis is a progressive and potentially fatal process that can occur in numerous organ systems. Characterised by the excessive deposition of extracellular matrix proteins such as collagens and fibronectin, fibrosis affects normal tissue architecture and impedes organ function. Although a considerable amount of research has focused on the mechanisms underlying disease pathogenesis, current therapeutic options do not directly target the pro-fibrotic process. As a result, there is a clear unmet clinical need to develop new agents. Novel findings implicate a role for epigenetic modifications contributing to the progression of fibrosis by alteration of gene expression profiles. This review will focus on DNA methylation; its association with fibroblast differentiation and activation and the consequent buildup of fibrotic scar tissue. The potential use of therapies that modulate this epigenetic pathway for the treatment of fibrosis in several organ systems is also discussed.

Background: Hepatic fibrosis, which is the excessive accumulation of extracellular matrices (ECMs) produced mainly from activated hepatic stellate cells (HSCs), develops to cirrhosis over several decades. There are no validated biomarkers that can non-invasively monitor excessive production of ECM (i.e., fibrogenesis). Transforming growth factor (TGF)-β, a key driver of fibrogenesis, is produced as an inactive latent complex, in which active TGF-β is enveloped by its pro-peptide, the latency-associated protein (LAP). Thus, active TGF-β must be released from the complex for binding to its receptor and inducing ECM synthesis. We recently reported that during the pathogenesis of liver fibrosis, plasma kallikrein (PLK) activates TGF-β by cleavage between R(58) and L(59) residues within LAP and that one of its by-products, the N-terminal side LAP degradation products ending at residue R(58) (R(58) LAP-DPs), can be detected mainly around activated HSCs by specific antibodies against R(58) cleavage edges and functions as a footprint of PLK-dependent TGF-β activation. Here, we describe a sandwich enzyme-linked immunosorbent assay (ELISA) that detects the other by-products, the C-terminal side LAP-DPs starting from residue L(59) (L(59) LAP-DPs). We demonstrated that the L(59) LAP-DPs are a potentially novel blood biomarker reflecting hepatic fibrogenesis.

Results: We established a specific sandwich ELISA to quantify L(59) LAP-DPs as low as 2 pM and measured L(59) LAP-DP levels in the culture media of a human activated HSC line, TWNT-4 cells. L(59) LAP-DPs could be detected in their media, and after treatment of TWNT-4 cells with a TGF-β receptor kinase inhibitor, SB431542, a simultaneous reduction was observed in both L(59) LAP-DP levels in the culture media and the mRNA expression levels of collagen type (I) α1. In carbon tetrachloride- and bile duct ligation-induced liver fibrosis models in mice, plasma L(59) LAP-DP levels increased prior to increase of hepatic hydroxyproline (HDP) contents and well correlated with α-smooth muscle actin (αSMA) expression in liver tissues. At this time, αSMA-positive cells as well as R(58) LAP-DPs were seen in their liver tissues.

Conclusions: L(59) LAP-DPs reflect PLK-dependent TGF-β activation and the increase in αSMA-positive activated HSCs in liver injury, thereby serving as a novel blood biomarker for liver fibrogenesis.

Based on extensive pre-clinical achievements over the past decades, it appears to be due time for a successful clinical translation in the renal fibrosis field-but what is the quickest road to get there? In light of the recent launch of the Precision Medicine Initiative and success of molecularly informed drugs in oncology, we here discuss what it may take to bring molecularly targeted anti-fibrotic to clinical use in chronic progressive kidney disease.

Background: Fibrosis is a physiological response to cellular injury in the liver and is mediated by the activation of hepatic stellate cells resulting in the replacement of hepatocytes with extracellular matrix comprised principally of collagen 1 to form a hepatic scar. Although the novel hexaco-ordinated globin cytoglobin was identified in activated hepatic stellate cells more than 10 years ago, its role in stellate cell biology and liver fibrosis remains enigmatic.

Results: In the current study, we investigated the role of different extracellular matrix proteins in stellate cell proliferation, activation (alpha smooth muscle actin expression and retinoic acid uptake) and cytoglobin expression. Our results demonstrate that cytoglobin expression is correlated with a more quiescent phenotype of stellate cells in culture and that cytoglobin is regulated by the extracellular matrix through integrin signalling dependent on activation of focal adhesion kinase.

Conclusions: Although further studies are required, we provide evidence that cytoglobin is a negative regulator of stellate cell activation and therefore may represent a novel target for anti-fibrotic treatments in the future.

Background: Liver fibrosis is characterized by the excessive formation and accumulation of matrix proteins as a result of wound healing in the liver. A main event during fibrogenesis is the activation of the liver resident quiescent hepatic stellate cell (qHSC). Recent studies suggest that reversion of the activated HSC (aHSC) phenotype into a quiescent-like phenotype could be a major cellular mechanism underlying fibrosis regression in the liver, thereby offering new therapeutic perspectives for the treatment of liver fibrosis. Whether human HSCs have the ability to undergo a similar reversion in phenotype is currently unknown. The aim of the present study is to identify experimental conditions that can revert the in vitro activated phenotype of primary human HSCs and consequently to map the molecular events associated with this reversion process by gene expression profiling.

Results: We find that epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF2) synergistically downregulate the expression of ACTA2 and LOX in primary human aHSCs. Their combination with oleic acid, palmitic acid, and retinol further potentiates a more quiescent-like phenotype as demonstrated by the abundant presence of retinyl ester-positive intra-cytoplasmic lipid droplets, low expression levels of activation markers, and a reduced basal as well as cytokine-stimulated proliferation and matrix metalloproteinase activity. Gene expression profiling experiments reveal that these in vitro reverted primary human HSCs (rHSCs) display an intermediary phenotype that is distinct from qHSCs and aHSCs. Interestingly, this intermediary phenotype is characterized by the increased expression of several previously identified signature genes of in vivo inactivated mouse HSCs such as CXCL1, CXCL2, and CTSS, suggesting also a potential role for these genes in promoting a quiescent-like phenotype in human HSCs.

Conclusions: We provide evidence for the ability of human primary aHSCs to revert in vitro to a transitional state through synergistic action of EGF, FGF2, dietary fatty acids and retinol, and provide a first phenotypic and genomic characterization of human in vitro rHSCs.

Background: Chronic kidney disease (CKD) is an independent risk factor for the development and severity of coronary artery disease (CHD) and endothelial dysfunction. There is an increase in the circulating angiogenesis inhibitors endostatin (END), thrombospondin-2 (TSP), angiopoietin-2 (ANG) and the nitric oxide (NO) inhibitor asymmetric dimethyl arginine (ADMA) in CKD patients. The aim of this study was to evaluate associations of the serum level of these factors and of the related angiogenesis inhibitor, endoglin (ENG), with burden of coronary atherosclerosis.

Methods: One hundred twenty-two patients undergoing coronary angiography were recruited from the cardiac catheterization lab at a single center. The total burden of coronary plaque (mm(2)) and the presence of coronary collaterals were quantified using quantitative coronary angiography (QCA). Serum levels of angiogenesis inhibitors were measured by ELISA (ENG, END, and ANG), Luminex assay (TSP), or HLPC (ADMA), respectively. Associations with plaque burden and coronary collateral supply were analyzed in multi-variable linear and logistic regression models.

Results: There was no significant association found between levels of circulating ADMA, ENG, END, ANG, or TSP and coronary plaque burden or collateral formation.

Conclusions: Our findings suggest that associations of circulating END, ENG, TSP, and ANG with cardiovascular mortality are unlikely to be mediated via direct effects on coronary plaque formation or by inhibition of collateral formation. Whether associations of these factors with mortality are mediated via local concentrations, myocardial tissue, or intra-plaque expression of these factors or by an effect on plaque vulnerability merits additional investigation.

Background: The process of epithelial-mesenchymal transition (EMT), which is generally defined by phenotypic changes of injured tubules such as loss of epithelial markers or acquisition of mesenchymal markers, implies various activating steps, including proliferation, migration, and ability to produce extracellular matrix proteins. We established here a novel approach for the detection of tubular cell migration into the interstitium during renal fibrosis in vivo.

Results: Using an osmotic pump, bromodeoxyuridine (BrdU) was continuously given to 7-week-old Wistar rats for 4 weeks, and BrdU-positive cells were detected by immunostaining. BrdU-positive cells were present in aquaporin-1-positive proximal tubules, but not in the interstitium of BrdU-treated rat kidneys. After unilateral ureteral obstruction (UUO), some BrdU-positive tubular cells protruded from the basement membrane and migrated into the interstitium. Interstitial BrdU-positive cells were co-localized with alpha-smooth muscle actin, fibroblast specific protein-1, vimentin, and type I collagen, but not with CD68 or CD3. No BrdU-positive cells were observed in the interstitium of sham-operated kidneys. The number of BrdU-positive cells migrating into the interstitium significantly increased and peaked at 8 days after UUO.

Conclusions: Long-term BrdU labeling marked some of the proximal tubular cells and enabled us to detect tubular cell migration into the interstitium after UUO. This simple method might be useful to detect EMT in vivo.

Background: A major health disparity suffered by African Americans (AA) is a predisposition toward fibrotic diseases of the skin, lung, and other organs. We previously showed that healthy AA and scleroderma (systemic sclerosis (SSc)) patient monocytes share biochemical and functional differences from control Caucasian (C) monocytes that may predispose AA to SSc. The central difference is a decrease in caveolin-1. Low caveolin-1 levels promote monocyte migration, their differentiation into fibrocytes, and fibrocyte recruitment into fibrotic tissues. Here we have greatly expanded our studies on the mechanism of action in fibrosis of caveolin-1 in AA and SSc monocytes.

Results: Expression of chemokine receptors (CCR1, CCR2, CCR3) is enhanced in healthy AA monocytes compared to healthy C monocytes and further increased in SSc monocytes. A parallel increase in function occurs assessed by migration toward chemokines MCP-1 and MCP-3. Chemokine-receptor expression and function are inhibited by the caveolin-1 scaffolding domain peptide (CSD) via its action as a surrogate for caveolin-1. Cells bearing chemokine receptors accumulate to high levels in fibrotic lung and skin tissue from SSc patients and from mice treated with bleomycin. This accumulation is almost completely blocked in mice treated with CSD. In signaling studies, Src activation is enhanced in AA monocytes compared to C monocytes and further increased in SSc monocytes. Lyn is also highly activated in SSc monocytes. Src and Lyn activation are inhibited by CSD. Src and Lyn's roles in monocyte migration were demonstrated using specific inhibitors.

Conclusions: To the best of our knowledge, this is the first report that the expression and function of CCR1, CCR2, and CCR3 are upregulated in monocytes from healthy AA and from SSc patients via molecular mechanisms involving caveolin-1, Src/Lyn, and MEK/ERK. The results suggest that the migration/recruitment of monocytes and fibrocytes into fibrotic tissues, mediated at least in part by CCR1, CCR2, and CCR3, plays a major role in the progression of lung and skin fibrosis and in the predisposition of AA to fibrotic diseases. Our findings further suggest that chemokine receptors and signaling molecules, particularly caveolin-1, that control their expression/function are promising targets for treating fibrotic diseases.

Background: Pentoxifylline (PTX) is a methylxanthine compound with immunomodulatory and antifibrotic properties. The simultaneous use of PTX and antifungal therapy (itraconazole) has previously been evaluated in an experimental model of pulmonary paracoccidioidomycosis (PCM), a systemic fungal disease caused by the fungus Paracoccidioides brasiliensis (Pb) and characterized by chronic inflammation and lung fibrosis that appears even after a successful course of antifungal therapy. The results revealed prompt and statistically significant reductions in inflammation and fibrosis when compared to itraconazole alone. However, the effect of monotherapy with PTX on the host response to PCM has not been well-documented. Our aim was to determine the effect of PTX on the course of pulmonary lesions and on the local immune response.

Results: At the middle and end of treatment, the Pb-infected-PTX-treated mice exhibited significant reductions in lung density compared to the Pb-infected-non-treated mice as assessed by the quantification of Hounsfield units on high-resolution computed tomography (HRCT) (p <0.05 by Kruskal-Wallis test); additionally, at the end of therapy, the lung areas involved in the inflammatory reactions were only 3 vs. 22 %, respectively, by histomorphometry (p <0.05 by Mann-Whitney test), and this reduction was associated with a lower fungal burden and limited collagen increment in the pulmonary lesions. PTX treatment restored the levels of IFN-γ, MIP-1β, and IL-3 that had been down-regulated by Pb infection. Additionally, IL-12p70, IL-10, IL-13, and eotaxin were significantly increased, whereas Regulated upon Activation, Normal T cell Expressed and Secreted (RANTES) levels were decreased in the lungs of the Pb-infected-PTX-treated mice compared to the non-treated group.

Conclusions/significance: This study showed that PTX therapy administered at an "early" stage of granulomatous inflammation controlled the progress of the PCM by diminishing the pulmonary inflammation and the fungal burden and avoiding the appearance of collagen deposits in the pulmonary lesions.

Since the discovery of induced pluripotent stem cells (iPSC) in 2006, the symptoms of many human diseases have been reversed in animal models with iPSC therapy, setting the stage for future clinical development. From the animal data it is clear that iPSC are rapidly becoming the lead cell type for cell replacement therapy and for the newly developing field of iPSC-derived body organ transplantation. The first human pathology that might be treated in the near future with iPSC is age-related macular degeneration (AMD), which has recently passed the criteria set down by regulators for phase I clinical trials with allogeneic human embryonic stem cell-derived cell transplantation in humans. Given that iPSC are currently in clinical trial in Japan (RIKEN) to treat AMD, the establishment of a set of international criteria to make clinical-grade iPSC and their differentiated progeny is the next step in order to prepare for future autologous cell therapy clinical trials. Armed with clinical-grade iPSC, we can then specifically test for their threat of cancer, for proper and efficient differentiation to the correct cell type to treat human disease and then to determine their immunogenicity. Such a rigorous approach sets a far more relevant paradigm for their intended future use than non-clinical-grade iPSC. This review focuses on the latest developments regarding the first possible use of iPSC-derived retinal pigment epithelial cells in treating human disease, covers data gathered on animal models to date and methods to make clinical-grade iPSC, suggests techniques to ensure quality control and discusses possible clinical immune responses.