Thursday, December 9, 2010

Hinz and Gabbiani: Fibrosis: recent advances in myofibroblast

From F1000 Biology Reports

Fibrosis: recent advances in myofibroblast biology and new therapeutic perspectives

Boris Hinz1 and Giulio Gabbiani2
Laboratory of Tissue Repair and Regeneration, Matrix Dynamics Group, Faculty of Dentistry, University of Toronto, 150 College Street, Toronto, ON M5S 3E2, Canada
Department of Pathology and Immunology, CMU, University of Geneva, Rue Michel-Servet 1, 1211, Switzerland
Corresponding author
F1000 Biol Reports2010, 2:78 (doi: 10.3410/B2-78)
Published: 11 Nov 2010
The electronic version of this article is the complete one and can be found at: http://f1000.com/reports/b/2/78

Abstract

The crucial role of the myofibroblast in wound healing and fibrosis development is well established. This review discusses the mechanisms of myofibroblast action and the new findings that may develop into therapeutic strategies during the next few years.

Introduction and context

Tissue destruction by organ fibrosis contributes to the lethal outcomes associated with heart, lung, liver, kidney, and skin diseases. The cell responsible for the detrimental fibrotic tissue contractures is the myofibroblast, which has a phenotype characterized by excessive production of collagenous extracellular matrix (ECM) and tensile force [1]. The concept that the myofibroblast plays a pivotal role in the establishment of fibrotic conditions has paved the way for a new approach in the understanding of the mechanisms of these pathologic situations [2]. In particular, it has become accepted that mechanical force generation by myofibroblasts, which in turn depends on the neo-expression of α-smooth muscle actin (α-SMA) in stress fibers of these cells, regulates essential phenomena for tissue remodeling, such as cytokine synthesis and ECM component production [2]. The myofibroblast participates in a variety of phenomena, including embryologic development, organ fibrosis, and the stroma reaction to epithelial tumors [1]. This widespread occurrence suggests that the term myofibroblast describes a functional status rather than a fixed cell type. This assumption has been supported by recent findings indicating that myofibroblasts originate from a spectrum of cellular sources depending on the physiological or pathological situation [1]. Myofibroblast origin and its tissue environment should be considered when planning new therapeutic strategies that aim at decreasing myofibroblast number or activity.

Major recent advances

The list of cells from which myofibroblasts can derive has grown impressively during the last years. It includes local fibroblasts, epithelial cells, endothelial cells, smooth muscle cells, pericytes, hepatic perisinusoidal cells, mesenchymal stem cells, and bone marrow-derived cells known as fibrocytes [1,3]. Most attention has been given to the fibrocyte as a possible myofibroblast precursor [4] and the phenomena of epithelial- and endothelial-mesenchymal transition as myofibroblast sources, particularly during lung and kidney fibrosis [5,6]. Transition of epithelial cells all the way to the myofibroblast phenotype is inducible in culture and regulated by different signaling pathways [7,8]. However, the relative contribution of myofibroblast precursors remains to be determined. As one would intuitively expect, it appears likely that in most situations local fibroblasts represent the major source of myofibroblasts [1]. The local derivation of myofibroblasts from mesenchymal rather than epithelial or endothelial cells has recently been documented in a model of renal interstitial fibrosis by means of genetic lineage tracing [9].
At present there is no accepted therapy for fibrotic diseases [10]. A number of previous and recent antifibrotic strategies attempt to interfere with myofibroblast formation by targeting key factors in the differentiation process (Figure 1). It is well established that myofibroblast differentiation and organ fibrosis are predominantly controlled by transforming growth factor β1 (TGF-β1) [11] and the ED-A (extra domain A) found in cellular fibronectin [12]. Moreover, several cytokines and chemokines (and their receptors), as well as coagulation factors and ECM components, have been implicated in this process [13-18]. It is likely that the heterogeneity of the myofibroblast origin requires specific factors and specific mechanical conditions in each situation.


Figure 1. Targeting the myofibroblast Potential antifibrotic therapies can interfere with the chemical and mechanical factors that lead to myofibroblast formation from different precursor cells (shown here for fibroblasts). Alternatively, or additionally, specific features of the differentiated myofibroblast can be targeted to induce myofibroblast regression and/or apoptosis. α-SMA, alpha-smooth muscle actin; ECM, extracellular matrix; ED-A, extra domain A; IST-9; fibronectin antibody; TGFβ1, transforming growth factor β1. 
Fibrosis is usually diagnosed when tissue destruction is already progressing, and it is possible that therapies will have to target the resident myofibroblast population. For this, aiming at the contractile apparatus is allegedly the most straight forward and promising strategy to inhibit myofibroblast function (Figure 1). A new direction, which has proven experimentally successful, is based on the observation that intracellular delivery of the α-SMA amino-terminal sequence Ac-EEED inhibits the incorporation of this protein in myofibroblast stress fibers, thus reducing force production as well as collagen type I synthesis by myofibroblasts in vitro; moreover, it significantly inhibits experimental wound contraction in vivo [19]. Due to its relative specificity for α-SMA-expressing myofibroblast stress fibers, this peptide appears as a good candidate for topical (e.g., burn scars) and systemic (e.g., organ fibrosis) administration.
Another promising strategy to induce myofibroblast disappearance is to stimulate them to go into apoptosis. Two major intracellular pathways have been identified that act pro-survival (or anti-apoptotic) for the myofibroblast: focal adhesion kinase signaling in cell ECM adhesions and phosphatidylinositol 3-kinase (PI3K)-AKT signaling. Focal adhesion kinase activation protects myofibroblasts from going into apoptosis in response to the loss of cell adhesion, a phenomenon called anoikis [20]. TGF-β1 and endothelin-1 have been shown to independently activate the PI3K-AKT pathway and thereby render myofibroblasts apoptosis-resistant [21]. Development of protein kinase inhibitors as specific inducers of myofibroblast apoptosis is an exciting new avenue in fighting fibrosis [22]. Other strategies could include myofibroblast-specific delivery of apoptosis-inducing drugs as applied in a mouse model of liver fibrosis [23].
Inducing myofibroblast disappearance does not necessarily include their killing; interrupting the auto/paracrine production of active TGF-β1 leads to myofibroblast de-differentiation, at least in vitro [24]. However, attempts to use general inhibitors of TGF-β1 have been relatively unsuccessful, showing that fibrosis development is a more complex phenomenon than expected [25-28]. The limitation of such global strategies is the interference of the beneficial effects of the pleiotrophic TGF-β1, such as controlling homeostasis of epithelial, vascular, endothelial, and immune cells. Therefore, more promising strategies may be to prevent latent TGF-β1 activation in a cell-type-specific manner rather than blocking already active TGF-β1. TGF-β1 is secreted together with LAP (latency-associated peptide), forming a large complex with latent TGF-β1 binding protein 1 (LTBP-1) in the ECM [29]. Epithelial cells activate latent TGF-β1 via integrin αvβ6 [30,31], which requires ECM binding mediated by LTBP-1 [29]. Inhibition of the epithelium-specific αvβ6 integrin protects from lung, kidney, and bile duct fibrosis [32-35]. In addition, integrins αvβ3, αvβ8, and αvβ5 play a role in latent TGF-β1 activation by fibroblastic cells, either directly or in a process involving proteases [11,36-40]. We have described a novel mechanical mechanism of latent TGF-β1 activation for myofibroblasts that, literally, pulls on the large latent complex using the integrin αvβ5 [41].

Future directions

Blocking specific integrins is a promising future strategy to control the development of myofibroblasts in fibrotic disorders (Figure 1). In addition to blocking the latent TGF-β1-activating integrin αvβ5 [41-44], inhibition of integrin α3 [8,45], α11, [46] αvβ3 [44,47], and β1 [48] were shown to block myofibroblast development and may be developed into future therapies. The latter integrins are all implicated in myofibroblast mechanoperception and transduction. It becomes increasingly clear that myofibroblast differentiation crucially depends on mechanical factors such as ECM stiffness and intracellular tension. Mechanical stress determines the stress fiber localization of α-SMA [49], modulates α-SMA promoter activity and protein expression in a process that involves the myocardin-related transcription factor [7,48,50], and modulates the bioactivity of TGF-β1 [41]. Hence, releasing myofibroblasts from stress – for example, using the Ac-EEED peptide [51] – will have a profound and long-term effect on myofibroblast persistence and may even induce myofibroblast apoptosis [52].

Abbreviations

α-SMA, alpha-smooth muscle actin; ECM, extracellular matrix; PI3K, phosphatidylinositol 3-kinase; LTBP-1, latent transforming growth factor β1 binding protein 1; TGF-β1, transforming growth factor β1.

Competing Interests

The authors declare that they have no competing interests.

Acknowledgements

The work of BH is supported by grants from the Swiss National Science Foundation (grant# 3100A0-113733/1), the Heart and Stroke Foundation Ontario (grant# NA7086), and the Canadian Institutes of Health Research (grant# 210820).

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Scar-reducing peptide

A recent study by Dr. Erkki Ruoslahti published in PNAS,  using a wound-homing peptide, CARSKNKDC, to tag decorin, in vivo, showed that the peptide-tagged decorin significantly reduced scar formation in mice.


Proc Natl Acad Sci U S A. 2010 Nov 24. [Epub ahead of print]
Target-seeking antifibrotic compound enhances wound healing and suppresses scar formation in mice.
Järvinen TA, Ruoslahti E.

Vascular Mapping Laboratory, Center for Nanomedicine, Sanford-Burnham Medical Research Institute, University of California, Santa Barbara, CA 93106 and Cancer Research Center, Sanford-Burnham Medical Research Institute, La Jolla, CA 92037.
Abstract
Permanent scars form upon healing of tissue injuries such as those caused by ischemia (myocardial infarction, stroke), trauma, surgery, and inflammation. Current options in reducing scar formation are limited to local intervention. We have designed a systemically administered, target-seeking biotherapeutic for scar prevention. It consists of a vascular targeting peptide that specifically recognizes angiogenic blood vessels and extravasates into sites of injury, fused with a therapeutic molecule, decorin. Decorin prevents tissue fibrosis and promotes tissue regeneration by inhibiting TGF-β activity and by other regulatory activities. The decorin-targeting peptide fusion protein had substantially increased neutralizing activity against TGF-β1 in vitro compared with untargeted decorin. In vivo, the fusion protein selectively accumulated in wounds, and promoted wound healing and suppressed scar formation at doses where nontargeted decorin was inactive. These results show that selective targeting yields a tissue-healing and scar-reducing compound with enhanced specificity and potency. This approach may help make reducing scar formation by systemic drug delivery a feasible option for surgery and for the treatment of pathological processes in which scar formation is a problem.

PMID: 21106754 [PubMed - as supplied by publisher]

Monday, December 6, 2010

Treg Depletion Attenuated Silica-Induced Lung Fibrosis

A recent study published in PlosOne by Dr. Chen and associates suggested that depletion of Tregs with anti-CD25 mAb attenuated silica-induced lung fibrosis. Depletion of CD4+CD25+Foxp3+ Treg cells enhanced Th1 response and decelerated Th1/Th2 balance toward a Th2 phenotype in silica-induced lung fibrosis. These suggested that Treg cells regulated the inflammation against silica via suppression of inflammatory cells in the early stage and Treg cells may modulate Th1/Th2 polarization toward a Th2 dominant response by suppressing Th1 response in experimental model of silica-induced lung fibrosis.

Unfortunately, the study did not show collagen changes.

http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015404

CD4+CD25+Foxp3+ Regulatory T Cells Depletion May Attenuate the Development of Silica-Induced Lung Fibrosis in Mice

Fangwei Liu, Jie Liu, Dong Weng, Ying Chen, Laiyu Song, Qincheng He, Jie Chen*

Division of Pneumoconiosis, School of Public Health, China Medical University, Shenyang, People's Republic of China

Abstract
Background

Silicosis is an occupational lung disease caused by inhalation of silica dust characterized by lung inflammation and fibrosis. Previous study showed that Th1 and Th2 cytokines are involved in silicosis, but Th1/Th2 polarization during the development of silicosis is still a matter of debate. Regulatory T cells (Treg cells) represent a crucial role in modulation of immune homeostasis by regulating Th1/Th2 polarization, but their possible implication in silicosis remains to be explored.
Methodology/Principal Findings

To evaluate the implication of Treg cells in the development of silicosis, we generated the Treg-depleted mice model by administration of anti-CD25 mAbs and mice were exposed to silica by intratracheal instillation to establish experimental model of silica-induced lung fibrosis. The pathologic examinations show that the Treg-depleted mice are susceptive to severer inflammation in the early stage, with enhanced infiltration of inflammatory cells. Also, depletion of Treg cells causes a delay of the progress of silica-induced lung fibrosis in mice model. Further study of mRNA expression of cytokines reveals that depletion of Tregs leads to the increased production of Th1-cytokines and decreased production of Th2-cytokine. The Flow Cytometry and realtime PCR study show that Treg cells exert the modulation function both directly by expressing CTLA-4 at the inflammatory stage, and indirectly by secreting increasing amount of IL-10 and TGF-β during the fibrotic stage in silica-induced lung fibrosis.
Conclusion/Significance

Our study suggests that depletion of Tregs may attenuate the progress of silica-induced lung fibrosis and enhance Th1 response and decelerate Th1/Th2 balance toward a Th2 phenotype in silica-induced lung fibrosis. The regulatory function of Treg cells may depend on direct mechanism and indirect mechanism during the inflammatory stage of silicosis.

Sildenafil Has No Benefit for Advanced Idiopathic Pulmonary Fibrosis

 A new controlled clinic trial showed that Sildenafil, a phosphodiesterase-5 inhibitor, commonly known as Viagra, had no benefit for the advanced IPF patients.  The trial enrolled 180 patients. The study was published in N Engl J Med. 2010 Aug 12;363(7):620-8.


Recently, Dr. Rossi and Dr. Seccia of University of Padua, Padua, Italy, suggested that the conclusion of the study represents an example of a type II statistical error.

"The primary outcome was the proportion of patients who improved at least 20% on the 6-minute walk distance after 12 weeks of receiving sildenafil, as compared with placebo. The investigators based their power calculation on a study involving 14 patients in which there was no increase among those who had a baseline 6-minute walk distance that was similar to the walk distance for patients in STEP-IPF. Thus, we think that the investigators' power calculation was undersized for the primary outcome. A potential benefit for sildenafil is suggested by patients' improvement in several less variable secondary outcomes."
Dr. Flaherty replied that "we agree with Rossi and Seccia that our study was underpowered to detect response rates of 20% versus 10% for patients in the sildenafil group, as compared with those in the placebo group". A sample size of approximately 580 patients would have been required for the study to be adequately powered to detect response rates of 20% versus 10%. On the basis of our preliminary data and discussions about clinically meaningful effects, we powered the study for response rates of 30% versus 10%. As noted by Rossi and Seccia, the positive response in several secondary end points provides some evidence for a benefit of sildenafil for the treatment of patients with advanced idiopathic pulmonary fibrosis, even though there was no significant between-group difference in the primary end point.

Arosh: PGE2 and invasion

Mol Cell Endocrinol. 2010 Nov 24. [Epub ahead of print]

Selective inhibition of prostaglandin E2 receptors EP2 and EP4 inhibits invasion of human immortalized endometriotic epithelial and stromal cells through suppression of metalloproteinases.

Reproductive Endocrinology and Cell Signaling Laboratory.

Abstract

Prostaglandin E2 (PGE2) plays an important role in the pathogenesis of endometriosis. We recently reported that inhibition COX-2 decreased migration as well as invasion of human endometriotic epithelial and stromal cells. Results of the present study indicates that selective inhibition of PGE2 receptors EP2 and EP4 suppresses expression and/or activity of MMP1, MPP2, MMP3, MMP7 and MMP9 proteins and increases expression of TIMP1, TIMP2, TIMP3, and TIMP4 proteins and thereby
decreases migration and invasion of human immortalized endometriotic epithelial and stromal cells into matrigel. The interactions between EP2/EP4 and MMPs are mediated through Src and β-arrestin 1 protein complex involving MT1-MMP and EMMPRIN in human endometriotic cells. These novel findings provide an important molecular and cellular framework for further evaluation of selective inhibition of EP2 and EP4 as potential nonsteroidal therapy for endometriosis in child-bearing age women.
Copyright © 2010. Published by Elsevier Ireland Ltd.
 
PMID: 21111772 [PubMed - as supplied by publisher]