Regression of Allograft Airway Fibrosis: The Role of MMP-Dependent Tissue Remodeling in Obliterative Bronchiolitis after Lung Transplantation

Obliterative bronchiolitis after lung transplantation is a chronic inflammatory and fibrotic condition of small airways. The fibrosis associated with obliterative bronchiolitis might be reversible. Matrix metalloproteinases (MMPs) participate in inflammation and tissue remodeling. MMP-2 localized to myofibroblasts in post-transplant human obliterative bronchiolitis lesions and to allograft fibrosis in a rat intrapulmonary tracheal transplant model. Small numbers of infiltrating T cells were also observed within the fibrosis. To modulate inflammation and tissue remodeling, the broad-spectrum MMP inhibitor SC080 was administered after the allograft was obliterated, starting at post-transplant day 21. The allograft lumen remained obliterated after treatment. Only low-dose (2.5 mg/kg per day) SC080 significantly reduced collagen deposition, reduced the number of myofibroblasts and the infiltration of T cells in association with increased collagenolytic activity, increased MMP-2 gene expression, and decreased MMP-8, MMP-9, and MMP-13 gene expression. In in vitro experiments using cultured myofibroblasts, a relatively low concentration of SC080 increased MMP-2 activity and degradation of type I collagen. Moreover, coculture with T cells facilitated persistence of myofibroblasts, suggesting a role for T-cell infiltration in myofibroblast persistence in fibrosis. By combining low-dose SC080 with cyclosporine in vivo at post-transplant day 28, partial reversal of obliterative fibrosis was observed at day 42. Thus, modulating MMP activity might reverse established allograft airway fibrosis by regulating inflammation and tissue remodeling.Chronic allograft dysfunction after lung transplantation is manifested by obliterative bronchiolitis (OB), a fibroproliferative obstructive lesion in small airways, and its clinical correlate, bronchiolitis obliterans syndrome (BOS).^1,2 Once the fibrotic process of OB is initiated, conventional immunosuppression is usually ineffective.³ The traditional pathological perspective is that fibrosis is the end result of damage: scar tissue, with no possibility of return to the pre-existing structure.⁴ However, increasing evidence suggests that fibrosis still undergoes dynamic remodeling and is potentially a reversible process. For example, the resolution of liver fibrosis is well documented both clinically and experimentally. In animal experiments, up-regulation or overexpression of matrix metalloproteinases (MMPs) capable of degrading interstitial type I and type III collagen (including MMP-1,⁵ MMP-8,⁶ MMP-13,⁷and MMP-2 and MMP-14^8,9) is associated with the regression of liver fibrosis. Pulmonary fibrosis has also been shown to be conditionally reversible.¹⁰One possible mechanism rendering fibrosis unlikely to resolve is the aberrant persistence of myofibroblasts, an active form of fibroblasts positive for α-smooth muscle actin (α-SMA), which leads to production of extracellular matrix (ECM) in excess of MMP-dependent ECM degradation.¹¹ Unresolved inflammation can be an important contributor to this mechanism.¹⁰ Accumulating evidence suggests that chronic fibrotic conditions are mediated by complex interactions between immune and nonimmune cells, in which the persistence of a relatively low grade of inflammation continuously stimulates resident stromal cells^12,13 and provides survival signals to myofibroblasts.¹⁴ For instance, the resolution of liver fibrosis encountered in alcohol-induced and virus-related fibrosis occurs only after remedy of the underlying cause.^15,16 Moreover, in experimental models of fibrosis, reversal of fibrosis has occurred in one-hit injury models such as bleomycin-induced pulmonary fibrosis,¹⁷ in which the initial tissue injury leads to fibrosis but the tissue injury or inflammation is not continuous.^8,9Along those lines, OB after lung transplantation is a fibrotic and chronic inflammatory condition¹⁸ in which myofibroblasts persist.¹⁹ The intrapulmonary tracheal transplant model of OB is a unique animal model in which persistent alloantigen from the donor trachea within the pulmonary milieu causes continuous alloantigen-induced inflammation and results in robust fibrosis in the allograft lumen.²⁰ We have previously demonstrated that myofibroblasts expressing high levels of collagen and MMP-2 and MMP-14 play a central role in the remodeling of established allograft airway fibrosis.²⁰ Given that MMPs also play important but complex roles in the trafficking of immune responsive cells,²⁰ MMPs involved in both tissue remodeling and inflammation may play key roles in the reversal of fibrosis.We therefore hypothesized that allograft airway fibrosis is a potentially reversible process involving MMPs. Here, we demonstrate expression patterns of MMPs in established human OB lesions and describe the roles of MMPs in the remodeling of collagen matrix, myofibroblasts, and immune responsive cells using in vivo and in vitro models with SC080, a general MMP inhibitor. Finally, we demonstrate for the first time reversibility of allograft airway fibrosis by combining immunosuppression with a low dose of SC080.     

Fate Tracing Reveals the Pericyte and Not Epithelial Origin of Myofibroblasts in Kidney Fibrosis

Understanding the origin of myofibroblasts in kidney is of great interest because these cells are responsible for scar formation in fibrotic kidney disease. Recent studies suggest epithelial cells are an important source of myofibroblasts through a process described as the epithelial-to-mesenchymal transition; however, confirmatory studies in vivo are lacking. To quantitatively assess the contribution of renal epithelial cells to myofibroblasts, we used Cre/Lox techniques to genetically label and fate map renal epithelia in models of kidney fibrosis. Genetically labeled primary proximal epithelial cells cultured in vitro from these mice readily induce markers of myofibroblasts after transforming growth factor β₁ treatment. However, using either red fluorescent protein or β-galactosidase as fate markers, we found no evidence that epithelial cells migrate outside of the tubular basement membrane and differentiate into interstitial myofibroblasts in vivo. Thus, although renal epithelial cells can acquire mesenchymal markers in vitro, they do not directly contribute to interstitial myofibroblast cells in vivo. Lineage analysis shows that during nephrogenesis, FoxD1-positive(⁺) mesenchymal cells give rise to adult CD73⁺, platelet derived growth factor receptor β⁺, smooth muscle actin-negative interstitial pericytes, and these FoxD1-derivative interstitial cells expand and differentiate into smooth muscle actin⁺ myofibroblasts during fibrosis, accounting for a large majority of myofibroblasts. These data indicate that therapeutic strategies directly targeting pericyte differentiation in vivo may productively impact fibrotic kidney disease.Understanding the origin and differentiation pathways of myofibroblasts in vivo is critical for identifying new therapeutic strategies for fibrosing disease. Myofibroblasts, contractile cells that deposit pathological extracellular matrix, were first believed to derive from a specialized perivascular cell known as the hepatic stellate cell when studied in the liver. In health these cells store retinoic acid in intracellular vesicles and cultured stellate cells possess all of the hallmarks of myofibroblasts in vitro.¹ In other organ systems, similar perivascular cells have been postulated to be the source of myofibroblasts, but have been hard to define.^2,3 Mesoderm-derived cells, when cultured in vitro, differentiate into cells with hallmarks of myofibroblasts, including most notably mesenchymal stem cells from bone marrow, as well as mesangial cells of the kidney, and cultured monocyte-derived macrophages.^4–6 Whether mesoderm-derived pericytes (also called perivascular fibroblasts) give rise to kidney myofibroblasts remains controversial, partly because primary epithelial cells when cultured in vitro can be induced to express some genes that are also expressed in myofibroblasts.^7–9 During carcinogenesis phenotypic alterations termed epithelial-to-mesenchymal transition (EMT) have been well characterized and promote cell migration, invasion, and metastasis.¹⁰ Further, a recent report suggests that other terminally differentiated cells such as endothelial cells can develop a myofibroblast phenotype in vitro and in vivo.^11,12It has been postulated that during kidney injury in vivo, epithelial cells undergo a phenotypic transition or can transdifferentiate into interstitial myofibroblasts by this same process of EMT.^13,14 Subsequent studies both in vivo and in vitro support this hypothesis.^15,16 The implication from these observations is that if the molecular mechanisms by which epithelial cells traverse the basement membrane and differentiate into myofibroblasts can be understood, novel antifibrotic strategies will be identified.Epithelial cells are known to respond to injury in several ways. They undergo morphological changes, lose polarity, acquire stress fibers, and migrate along the basement membrane.¹⁷ They up-regulate inflammatory genes and genes that enhance their ability to survive in a hostile environment.^18,19 In addition, they express some genes shared by embryonic mesenchymal cells transitioning to epithelium during nephrogenesis.^20–22 Thus it has been suggested that in response to injury epithelial cells undergo EMT, recapitulating primitive mesenchymal cells of the intermediate mesoderm.⁹ This, however, is misleading since intermediate mesoderm cells do not express inflammatory and cell-survival genes that injured adult epithelial cells up-regulate, and expression of a limited number of genes shared by embryonic mesenchyme such as α smooth muscle actin (SMA), by itself, does not define injured epithelial cells as mesenchymal.^23–25 Neoplastic epithelial cells have the capacity to metastasize, share some characteristics with myofibroblasts, and express or down-regulate key regulators of metastasis such as mts1 (S100A4 or FSP-1), Twist, Snail, and β-catenin, genes whose expression can also be activated in cultured epithelial cells.^26–28 Proponents of the hypothesis that myofibroblasts in inflammation and scarring derive from epithelial cells have drawn on these observations to extend the term EMT to mean epithelial-to-myofibroblast transition.Interstitial myofibroblasts are the principle source of interstitial collagens, including fibrillar collagens I and III. They are widely held to be the primary cell in the injured kidney that lays down the interstitial matrix that becomes fibrotic (For review see²⁹). Many myofibroblasts express the actin fiber, αSMA that correlates with contractile and activated morphology, and recent studies confirmed that in the fibrotic kidney more than 80% of these produce fibrillary collagen.³⁰ Although this is not specific to interstitial myofibroblasts (αSMA is also expressed by vascular smooth muscle cells), αSMA has long been used as a marker of myofibroblasts.Although it is widely accepted that primary epithelial cells cultured in vitro up-regulate genes that result in a myofibroblast phenotype,^9,25 and generate fibrillar collagens, the evidence that this occurs in vivo is less well-established. There are some published examples of epithelial cells transgressing intact or disrupted basement membrane or cells co-expressing established epithelial and fibroblast markers in vivo,^31–33 but histological snapshots do not prove a lineage relationship, and cells may express a variety of antigens during injury. In our own extensive studies of injured epithelial cells in kidney repair, we concluded that non-epithelial cells do not migrate from interstitium into the tubule.³⁴ Similarly, we have never observed a cell outside of the confines of the epithelial basement membrane that was positive for markers of epithelial injury. Explanations for a failure to make these observations in fixed tissues include the hypothesis that a cell exiting the confines of the basement membrane rapidly loses epithelial markers and only subsequently gains myofibroblast markers.³⁵ However, in vitro, epithelial cells can express both fibroblast markers and epithelial markers simultaneously.³⁶Because efforts to design new antifibrotic therapies require a rigorous understanding of the cellular origin of myofibroblasts in vivo, we have performed lineage analysis of both renal epithelial cells and interstitial stromal cells during fibrosis in vivo. Transgenic or knock-in mice with lineage-restricted expression of bacterial Cre recombinase were used for genetic tracking of three cell populations. The HoxB7-Cre driver is expressed exclusively in the mesonephric duct and its derivatives, resulting in labeling of collecting duct epithelium and ureteral epithelium of adult kidney.³⁷ In the Six2-Cre transgenic mouse, expression of Cre occurs in cap-mesenchyme and labels all non-ureteric, bud-derived, nephron epithelia, including podocytes, proximal tubule, loop of Henle, and connecting segment, but it does not label any interstitial cell population.^34,38 FoxD1 is a well characterized marker of renal stromal cells, but not epithelia, during development, and we used FoxD1-Cre knock-in mice to genetically label renal stroma.³⁹ We crossed these three Cre drivers against two different reporter lines to permanently and heritably label all epithelial cells of the entire nephron in adult mouse kidney or all stromal cells.^34,38 We demonstrate that, contrary to the prevailing model, kidney epithelial cells do not become myofibroblasts in vivo during fibrotic disease. Rather, we show by genetic tracing that myofibroblasts derive from interstitial pericytes/perivascular fibroblasts.     

Pleural Mesothelial Cell Differentiation and Invasion in Fibrogenic Lung Injury

The origin of the myofibroblast in fibrotic lung disease is uncertain, and no effective medical therapy for fibrosis exists. We have previously demonstrated that transforming growth factor-β1 (TGF-β1) induces pleural mesothelial cell (PMC) transformation into myofibroblasts and haptotactic migration in vitro. Whether PMC differentiation and migration occurs in vivo, and whether this response can be modulated for therapeutic benefit, is unknown. Here, using mice recombinant for green fluorescent protein (GFP) driven by the Wilms tumor-1 (WT-1) promoter, we demonstrate PMC trafficking into the lung and differentiation into myofibroblasts. Carbon monoxide or the induction of heme oxygenase-1 (HO-1) inhibited the expression of myofibroblast markers, contractility, and haptotaxis in PMCs treated with TGF-β1. Intrapleural HO-1 induction inhibited PMC migration after intratracheal fibrogenic injury. PMCs from patients with idiopathic pulmonary fibrosis (IPF) exhibited increased expression of myofibroblast markers and enhanced contractility and haptotaxis, compared with normal PMCs. Carbon monoxide reversed this IPF PMC profibrotic phenotype. WT-1–expressing cells were present within fibrotic regions of the lungs in IPF subjects, supporting a role for PMC differentiation and trafficking as contributors to the myofibroblast population in lung fibrosis. Our findings also support a potential role for pleural-based therapies to modulate pleural mesothelial activation and parenchymal fibrosis progression.Idiopathic pulmonary fibrosis (IPF), the most common idiopathic interstitial pneumonia, is characterized by cellular and structural changes in the parenchyma associated with the proliferation of myofibroblasts and deposition of extracellular matrix components.¹ IPF begins in the subpleural region and extends centrally, resulting in a progressive decline in lung function. The origin of the pathogenic myofibroblast is uncertain. The hallmark lesions of IPF, the fibroblastic foci seen on two-dimensional histopathological slides, were thought of as discrete sites of epithelial injury and repair.² Cool et al³ used three-dimensional reconstruction to demonstrate that these foci are part of a complex, highly interconnected reticulum and suggested the leading edge of the fibroblastic invasion extends from the pleura to the underlying parenchyma like a wave of fibrosis.The pleura is a metabolically active monolayer of mesothelial cells that intimately approximates the lung parenchyma. The close proximity of PMCs to the underlying lung ideally positions them to respond to cytokines, chemokines, and growth factors released during parenchymal stress, injury, infection, or inflammation. The cytokine transforming growth factor-β1 (TGF-β1) is a crucial mediator of epithelial–mesenchymal transition (EMT) and acts as a master switch for induction of fibrosis in many organs, including the lung.^4–6 PMC transformation into myofibroblasts and haptotactic migration occur in vitro in response to TGF-β1.⁷ The concept of pleural mesothelial–mesenchymal transition (MMT) was investigated in a recent study demonstrating PMC migration into the lung and expression of myofibroblast phenotypic markers. This study demonstrated the presence of PMCs in the lung parenchyma of patients with IPF and a correlation with disease severity and the degree of fibrosis.⁸ PMC differentiation into myofibroblasts and subsequent migration may play a crucial role in the development of fibrotic lung disease.Heme oxygenase-1 (HO-1) is the inducible form of the rate-limiting enzyme involved in the degradation of heme with the generation of equimolar quantities of carbon monoxide (CO), iron, and biliverdin.⁹ HO-1 induction with subsequent CO production is highly sensitive to numerous stimuli and cellular insults that cause oxidative stress.¹⁰ Such induction represents a beneficial response to injurious stimuli in diverse diseases, including atherosclerosis, sepsis, and fibrosis. The protective effects of HO-1 and CO are due to anti-inflammatory, antiapoptotic, antioxidant, and antiproliferative properties.¹¹ Indeed, HO-1 deficiency has been associated with increased fibrosis, tubular TGF-β1 expression, inflammation, and enhanced EMT in a model of obstructive kidney disease.¹² Up-regulation of HO-1 provides protection against renal injury after unilateral ureteral obstruction and suppression of tubulointerstitial fibrosis via anti-apoptotic pathway modulation.¹³ Additionally, adenoviral transfer of the HO-1 gene, as well as administration of CO and bilirubin, has been associated with suppression of fibrosis in animal models of fibrotic lung disease.^14,15 Here, we present definitive evidence for the presence of PMCs in the lung parenchyma of patients with IPF and show that the fibrotic disposition of these cells can be reversed by CO and HO-1 pathway modulation in vitro. Furthermore, using animal models of lung injury, we demonstrate PMC differentiation into myofibroblasts and parenchymal invasion that is inhibited by intrapleural HO-1 induction.     

Mesenchymal Deficiency of Notch1 Attenuates Bleomycin-Induced Pulmonary Fibrosis

Notch signaling pathway is involved in the regulation of cell fate, differentiation, proliferation, and apoptosis in development and disease. Previous studies suggest the importance of Notch1 in myofibroblast differentiation in lung alveogenesis and fibrosis. However, direct in vivo evidence of Notch1-mediated myofibroblast differentiation is lacking. In this study, we examined the effects of conditional mesenchymal-specific deletion of Notch1 on pulmonary fibrosis. Crossing of mice bearing the floxed Notch1 gene with α2(I) collagen enhancer-Cre-ER(T)–bearing mice successfully generated progeny with a conditional knockout (CKO) of Notch1 in collagen I–expressing (mesenchymal) cells on treatment with tamoxifen (Notch1 CKO). Because Notch signaling is known to be activated in the bleomycin model of pulmonary fibrosis, control and Notch1 CKO mice were analyzed for their responses to bleomycin treatment. The results showed significant attenuation of pulmonary fibrosis in CKO relative to control mice, as examined by collagen deposition, myofibroblast differentiation, and histopathology. However, there were no significant differences in inflammatory or immune cell influx between bleomycin-treated CKO and control mouse lungs. Analysis of isolated lung fibroblasts confirmed absence of Notch1 expression in cells from CKO mice, which contained fewer myofibroblasts and significantly diminished collagen I expression relative to those from control mice. These findings revealed an essential role for Notch1-mediated myofibroblast differentiation in the pathogenesis of pulmonary fibrosis.Notch signaling is known to play critical roles in development, tissue homeostasis, and disease.^{1, 2, 3, 4, 5, 6, 7, 8, 9, 10} Notch signaling is mediated via four known receptors, Notch 1, 2, 3, and 4, which serve as receptors for five membrane-bound ligands, Jagged 1 and 2 and Delta 1, 3, and 4.^{1, 11, 12, 13} The Notch receptors differ primarily in the number of epidermal growth factor-like repeats and C-terminal sequences.¹³ For instance, Notch 1 contains 36 of epidermal growth factor-like repeats, is composed of approximately 40 amino acids, and is defined largely by six conserved cysteine residues that form three conserved disulfide bonds.^{1, 13, 14, 15} These epidermal growth factor-like repeats can be modified by O-linked glycans at specific sites, which is important for their function.^{1, 14, 15} Modulation of Notch signaling by Fringe proteins,^{16, 17, 18} which are N-acetylglucosamine transferases, illustrates the importance of these carbohydrate residues.^{16, 18} Moreover, mutation of the GDP-4-keto-6-deoxymannose-3,5-epimerase-4-reductase causes defective fucosylation of Notch1, resulting in impairment of the Notch1 signaling pathway and myofibroblast differentiation.^{19, 20, 21} Because myofibroblasts are important in both lung development and fibrosis, elucidation of the role of Notch signaling in their genesis in vivo will provide insight into the significance of this signaling pathway in either context.The importance of Notch signaling in tissue fibrosis is suggested in multiple studies.^{10, 21, 22, 23, 24} As in other organs or tissues, pulmonary fibrosis is characterized by fibroblast proliferation and de novo emergence of myofibroblasts, which is predominantly responsible for the increased extracellular matrix production and deposition.^{25, 26, 27, 28, 29, 30, 31} Animal models, such as bleomycin-induced pulmonary fibrosis, are characterized by both acute and chronic inflammation with subsequent myofibroblast differentiation that mainly originated from the mesenchymal compartment.^{21, 25, 26, 27, 28} In vitro studies of cultured cells implicate Notch signaling in myofibroblast differentiation,²¹ which is mediated by induction of the Notch1 ligand Jagged1 when lung fibroblasts are treated with found in inflammatory zone 1.²¹ Moreover, GDP-4-keto-6-deoxymannose-3,5-epimerase-4-reductase knockout mice with defective fucosylation of Notch1 exhibit consequent impairment of Notch signaling and attenuated pulmonary fibrosis in studies using the bleomycin model.²¹ The in vivo importance of Notch signaling in myofibroblast differentiation during lung development has also been suggested by demonstration of impaired alveogenesis in mice deficient in lunatic fringe³² or Notch receptors.^{10, 33, 34, 35} These in vivo studies, however, do not pinpoint the cell type in which deficient Notch signaling is causing the observed impairment of myofibroblast differentiation. This is further complicated by the extensive evidence showing that, in addition to myofibroblast differentiation, Notch1 mediates multiple functional responses in diverse cell types, including inflammation and the immune system.^{21, 36, 37, 38} In the case of tissue injury and fibrosis, including the bleomycin model, the associated inflammation and immune response as well as parenchymal injury can affect myofibroblast differentiation via paracrine mechanisms.^{39, 40} Thus, although global impairment of Notch signaling can impair myofibroblast differentiation in vivo, it does not necessarily indicate a specific direct effect on the mesenchymal precursor cell. Furthermore, understanding the importance of Notch signaling in these different cell compartments is critical for future translational studies to develop effective drugs targeting this signaling pathway with minimal off-target or negative adverse effects.In this study, the effects of conditional selective Notch1 deficiency in the mesenchymal compartment on myofibroblast differentiation and bleomycin-induced pulmonary fibrosis were examined using a Cre-Lox strategy. The transgenic Cre mice bore the Cre-ER(T) gene composed of Cre recombinase and a ligand-binding domain of the estrogen receptor⁴¹ driven by a minimal promoter containing a far-upstream enhancer from the α2(I) collagen gene. When activated by tamoxifen, this enhancer enabled selective Cre expression only in type I collagen-expressing (mesenchymal) cells, such as fibroblasts and other mesenchymal cells,⁴² leading to excision of LoxP consensus sequence flanked target gene DNA fragment (floxed gene) of interest.^{41, 43, 44, 45, 46} To evaluate the importance of Notch1 in the mesenchymal compartment and discriminate its effects from those in the inflammatory and immune system and other compartments, the transgenic Cre-ER(T) mice [Col1α2-Cre-ER(T)^+/0] were crossed with mice harboring the floxed (containing loxP sites) Notch1 gene (Notch1^fl/fl). The resulting progeny mice [Notch1 conditional knockout (CKO)] that were homozygous for the floxed Notch1 allele and hemizygous for the Col1α2-Cre-ER(T) allele with genotype [Notch1^fl/fl,Col1α2-Cre-ER(T)^+/0] were Notch1 deficient in the mesenchymal compartment when injected with tamoxifen. Control Notch1 wild-type (WT) mice exhibited the expected pulmonary fibrosis along with induction of Jagged1 and Notch1 on treatment with bleomycin, consistent with previous observation of Notch signaling activation in this model.²¹ Isolated and cultured Notch1 CKO mouse lung fibroblasts were deficient in Notch1 and exhibited diminished myofibroblast differentiation compared with cells from the corresponding WT control mice. Most important, compared with WT control mice, the CKO mice exhibited diminished bleomycin-induced pulmonary fibrosis that was accompanied by significant reduction in α-smooth muscle actin (α-SMA) and type I collagen gene expression, consistent with defective myofibroblast differentiation. In contrast, enumeration of lung inflammatory and immune cells failed to show a significant difference in bleomycin-induced recruitment of these cells between control and CKO mice. Thus, selective Notch1 deficiency in mesenchymal cells caused impairment of fibrosis that is at least, in part, because of deficient myofibroblast differentiation, and without affecting the inflammatory and immune response in this animal model.     

Local Origin of Mesenchymal Cells in a Murine Orthotopic Lung Transplantation Model of Bronchiolitis Obliterans

Bronchiolitis obliterans is the leading cause of chronic graft failure and long-term mortality in lung transplant recipients. Here, we used a novel murine model to characterize allograft fibrogenesis within a whole-lung microenvironment. Unilateral left lung transplantation was performed in mice across varying degrees of major histocompatibility complex mismatch combinations. B6D2F1/J (a cross between C57BL/6J and DBA/2J) (Haplotype H2b/d) lungs transplanted into DBA/2J (H2d) recipients were identified to show histopathology for bronchiolitis obliterans in all allogeneic grafts. Time course analysis showed an evolution from immune cell infiltration of the bronchioles and vessels at day 14, consistent with acute rejection and lymphocytic bronchitis, to subepithelial and intraluminal fibrotic lesions of bronchiolitis obliterans by day 28. Allografts at day 28 showed a significantly higher hydroxyproline content than the isografts (33.21 ± 1.89 versus 22.36 ± 2.33 μg/mL). At day 40 the hydroxyproline content had increased further (48.91 ± 7.09 μg/mL). Flow cytometric analysis was used to investigate the origin of mesenchymal cells in fibrotic allografts. Collagen I–positive cells (89.43% ± 6.53%) in day 28 allografts were H2Db positive, showing their donor origin. This novel murine model shows consistent and reproducible allograft fibrogenesis in the context of single-lung transplantation and represents a major step forward in investigating mechanisms of chronic graft failure.Bronchiolitis obliterans (BO), a fibroproliferative process targeting the small airways of the lung, is the predominant cause of chronic graft failure and poor long-term outcomes after lung transplantation.^1–3 BO is also a common complication after allogeneic hematopoietic stem-cell transplantation. At present, no therapeutic options are available to prevent the development of or slow the progression of BO.^1–3Airway remodeling of BO, marked by mesenchymal cell infiltration and collagen deposition, evolves in a complex milieu marked by interactions of infiltrating recipient-derived cells and graft-resident somatic cells. Peribronchiolar mononuclear inflammation (also known as lymphocytic bronchiolitis)^4–6 and episodes of acute rejection (AR) marked by perivascular inflammation^7–11 precede the development of BO. Both T and B lymphocytes are important, suggesting a role for cell-mediated and humoral immunity.^12–15 Allo-immune injury also is implicated, with evidence of collagen V–specific cellular immunity noted before BO development.¹⁶ The epithelium is an important target of these immune responses^17–20 and epithelial cell injury precedes the ensuing mesenchymal cell recruitment and activation.²¹ However, in vivo investigations into the mechanisms of allograft fibrogenesis in a whole-lung milieu are hampered by the lack of a robust and reproducible murine model of BO and allograft fibrosis.^22–24 The commonly used heterotopic tracheal transplantation model relies on the investigation of fibrosis in an isolated trachea placed in an extrapulmonary environment.²³ A significant concern here is the applicability of findings from this tracheal transplant model to a whole-lung microenvironment and the need to target the mesenchymal cell population specifically responsible for matrix deposition and fibrotic remodeling in the transplanted lung. Human investigations, although limited by technical aspects, suggest that locally resident cells are the primary mesenchymal cell populations in a transplanted lung and contribute to fibrogenesis,^25,26 mesenchymal cells in the tracheal transplant model show a recipient origin and focus attention on cells such as fibrocytes.^27–29 Thus, a whole-lung transplant model, which allows investigation into the origin of mesenchymal cells at the single-cell level in a fibrotic lung allograft and mimics human disease, is needed.In this study we investigated fibrogenesis in whole-lung allografts transplanted across varying degrees of major histocompatibility complex (MHC) mismatch. We show a model using a transplant from F1 hybrid into a parent mouse that reproducibly shows BO with, evolution from moderate AR and lymphocytic bronchitis to airway and vascular fibrosis. Furthermore, we investigated the origin of the mesenchymal cell population in whole-lung allografts at a single-cell level and show that the collagen I–positive population in a fibrotic lung allograft is predominantly of donor origin.     

Macrophage Matrix Metalloproteinase-9 Mediates Epithelial-Mesenchymal Transition in Vitro in Murine Renal Tubular Cells

As a rich source of pro-fibrogenic growth factors and matrix metalloproteinases (MMPs), macrophages are well-placed to play an important role in renal fibrosis. However, the exact underlying mechanisms and the extent of macrophage involvement are unclear. Tubular cell epithelial−mesenchymal transition (EMT) is an important contributor to renal fibrosis and MMPs to induction of tubular cell EMT. The aim of this study was to investigate the contribution of macrophages and MMPs to induction of tubular cell EMT. The murine C1.1 tubular epithelial cell line and primary tubular epithelial cells were cultured in activated macrophage-conditioned medium (AMCM) derived from lipopolysaccharide-activated J774 macrophages. MMP-9, but not MMP-2 activity was detected in AMCM. AMCM-induced tubular cell EMT in C1.1 cells was inhibited by broad-spectrum MMP inhibitor (GM6001), MMP-2/9 inhibitor, and in AMCM after MMP-9 removal by monoclonal Ab against MMP-9. AMCM-induced EMT in primary tubular epithelial cells was inhibited by MMP-2/9 inhibitor. MMP-9 induced tubular cell EMT in both C1.1 cells and primary tubular epithelial cells. Furthermore, MMP-9 induced tubular cell EMT in C1.1 cells to an extent similar to transforming growth factor-β. Transforming growth factor-β-induced tubular cell EMT in C1.1 cells was inhibited by MMP-2/9 inhibitor. Our in vitro study provides evidence that MMPs, specifically MMP-9, secreted by effector macrophages can induce tubular cell EMT and thereby contribute to renal fibrosis.Interstitial macrophage infiltration is a hallmark of all progressive renal diseases regardless of the initial cause of the injury.^1,2 Macrophages have long been known to play an important role in renal fibrosis,³ which is a central component of the final common pathway leading to renal failure. Previous studies have demonstrated a close association between macrophage infiltrate and excessive extracellular matrix protein accumulation in diseased human kidney as well as in experimental models.^4–6 In addition, the number of infiltrating macrophages has been shown to correlate well with the number of myofibroblasts,^7,8 the effector cells responsible for secretion of extracellular matrix proteins. A recent study revealed that blockade of macrophage recruitment in obstructive renal injury resulted in a reduction in renal fibrosis via tubular cell epithelial−mesenchymal transition (EMT),⁹ which has been recognized as an important source of myofibroblasts in renal fibrosis. However, the exact mechanism underlying the contribution of macrophages to renal fibrosis via tubular cell EMT remains undefined. As a major source of pro-fibrogenic growth factors and matrix metalloproteinases (MMPs), macrophages may be major determinants of the outcome of renal fibrosis.Tubular cell EMT is a process by which tubular epithelial cells lose their epithelial characteristics and acquire a mesenchymal phenotype. This process has been recognized as one of several pathways contributing to the myofibroblast population in renal fibrosis.¹⁰ Despite emerging and conflicting evidence about the relative importance of various sources of myofibroblasts,^11,12 it is generally accepted that tubular cell EMT plays an important role in renal fibrosis. Since the concept of tubular cell EMT was first proposed, numerous studies have provided evidence for tubular cell EMT in various experimental models as well as in human biopsies.¹⁰ Furthermore, the importance of tubular cell EMT has been demonstrated by Iwano et al¹³ using transgenic mice and direct genetic tagging of tubular epithelial cells to show that more than a third of myofibroblasts in kidneys with unilateral ureteral obstruction are derived from tubular epithelial cells via tubular cell EMT. Moreover, blockade of tubular EMT has been shown to attenuate renal fibrosis in obstructive nephropathy.¹⁴ However, some controversy remains as to whether tubular cell EMT plays a consistent role in other experimental models, and its exact contribution in renal fibrosis is yet to be established.Although pro-fibrogenic growth factors are well known as inducers of tubular cell EMT, cumulative evidence suggests an important role for MMPs. Traditionally, MMPs were thought to be antifibrogenic due to their ability to degrade extracellular matrix proteins, yet MMPs—in particular MMP-2 and MMP-9—have been recognized as promoters of tubular cell EMT via basement membrane disruption. In fact, induction of tubular cell EMT in vitro¹⁵ and in vivo¹⁴ has been shown to be associated with increased expression of MMP-2 and MMP-9. Earlier studies have demonstrated that tubular epithelial cells undergoing mesenchymal transition are closely associated with damaged tubular basement membrane and that complete transition requires tubular basement membrane damage.¹⁶ Later studies have shown directly that MMPs can disrupt basement membrane integrity; loss of MMP-9 expression lead to preservation of basement membrane integrity and inhibition of tubular cell EMT in obstructed kidney of tissue type plasminogen activator knockout mice.¹⁴ Despite this evidence supporting induction of tubular cell EMT by MMPs, the precise contribution of MMPs may have been underestimated. In cancer research, MMPs are well known to directly induce EMT in tumor cells of epithelial origin and to promote tumor progression via basement membrane disruption.¹⁷ MMP-2 has been shown consistently to be necessary and sufficient to induce tubular cell EMT in a rat tubular epithelial cell line (NRK52e).¹⁸ In addition, recent studies from our laboratory have demonstrated that MMP-3 and MMP-9 are also capable of inducing tubular cell EMT in NRK52e cells via the disruption of the cell adhesion molecule E-cadherin. Finally, the fact that transforming growth factor (TGF)-β-induced tubular cell EMT in NRK52e was inhibited by a broad spectrum MMP inhibitor suggests a primary role of MMP in TGF-β-induced tubular cell EMT.¹⁹ Together, these data suggest that MMPs from macrophages may play a major role in induction of tubular cell EMT. Therefore the aim of this study was to investigate the contribution of macrophages and their secreted MMPs to the induction of tubular cell EMT.     

αB-Crystallin Regulates Subretinal Fibrosis by Modulation of Epithelial-Mesenchymal Transition

Subretinal fibrosis is an end stage of neovascular age-related macular degeneration, characterized by fibrous membrane formation after choroidal neovascularization. An initial step of the pathogenesis is an epithelial-mesenchymal transition (EMT) of retinal pigment epithelium cells. αB-crystallin plays multiple roles in age-related macular degeneration, including cytoprotection and angiogenesis. However, the role of αB-crystallin in subretinal EMT and fibrosis is unknown. Herein, we showed attenuation of subretinal fibrosis after regression of laser-induced choroidal neovascularization and a decrease in mesenchymal retinal pigment epithelium cells in αB-crystallin knockout mice compared with wild-type mice. αB-crystallin was prominently expressed in subretinal fibrotic lesions in mice. In vitro, overexpression of αB-crystallin induced EMT, whereas suppression of αB-crystallin induced a mesenchymal-epithelial transition. Transforming growth factor-β2–induced EMT was further enhanced by overexpression of αB-crystallin but was inhibited by suppression of αB-crystallin. Silencing of αB-crystallin inhibited multiple fibrotic processes, including cell proliferation, migration, and fibronectin production. Bone morphogenetic protein 4 up-regulated αB-crystallin, and its EMT induction was inhibited by knockdown of αB-crystallin. Furthermore, inhibition of αB-crystallin enhanced monotetraubiquitination of SMAD4, which can impair its nuclear localization. Overexpression of αB-crystallin enhanced nuclear translocation and accumulation of SMAD4 and SMAD5. Thus, αB-crystallin is an important regulator of EMT, acting as a molecular chaperone for SMAD4 and as its potential therapeutic target for preventing subretinal fibrosis development in neovascular age-related macular degeneration.Age-related macular degeneration (AMD) is a leading cause of blindness because of progressive degeneration of the macular region of the retina that is responsible for visual acuity and color vision. The natural history of AMD is a progression from its early stage to the two forms of late stage of AMD: geographic atrophy and neovascular AMD (nAMD).¹The visual prognosis for nAMD is poor; the condition progresses rapidly with the development of choroidal neovascularization (CNV) and subsequent subretinal fibrosis. Although the commonly used treatment with anti-vascular endothelial growth factor (VEGF) drugs improves visual acuity in nAMD patients, the subretinal scarring (fibrosis) that may develop in approximately half of all anti–VEGF-treated eyes within 2 years has been identified as a cause of unsuccessful outcomes.² Thus, therapeutic strategies for the inhibition of subretinal fibrosis are currently an active area of investigation. Among the critical growth factors involved in subretinal fibrosis, platelet-derived growth factor (PDGF) is a potential therapeutic target. Currently, several clinical trials for the treatment for nAMD have been evaluating the efficacy of dual VEGF/PDGF inhibitors, such as the following: {"type":"entrez-nucleotide","attrs":{"text":"E10030","term_id":"22026652","term_text":"E10030"}}E10030 (Ophthotech, New York, NY), an anti-PDGF pegylated aptamer as an adjunct to anti-VEGF therapy; sorafenib, an inhibitor of VEGF receptor, PDGF receptor, and Raf kinases; and pazopanib, an inhibitor of VEGF receptor, PDGF receptor, and c-kit.³ In addition, it has been shown that nucleotide-binding oligomerization domain-, leucine-rich repeat domain-, and pyrin domain–containing 3 inflammasome activation is implicated in the pathogenesis of nAMD.⁴ The recent implication of inflammasome activation in the pathogenesis of hepatic fibrosis⁵ suggests that the inflammasome should be further evaluated for its potential role in subretinal fibrosis. Important inflammasome effector cytokines, IL-1β and IL-18, can be potential therapeutic targets for the treatment of subretinal fibrosis in nAMD. In support of this contention, inhibition of IL-1β has been shown to inhibit the development of experimental CNV in mice.⁶Subretinal fibrosis results from an excessive wound healing response, characterized by fibrous membrane formation after CNV. In fibrous membranes, the main cellular components are myofibroblasts, the cells immunoreactive for α-smooth muscle actin (α-SMA). Previous histological studies imply that the source of myofibroblasts can be both bone marrow–derived cells and retinal pigment epithelium (RPE) cells.^{7, 8} After injury to RPE, the cells undergo epithelial-mesenchymal transition (EMT), which enables transdifferentiation, resulting in the conversion of epithelial cells to myofibroblasts. CNV induction can result in the recruitment of more inflammatory cells and fibroblasts, which can be a direct or indirect source of additional myofibroblasts. Myofibroblasts play important roles in the development of subretinal fibrosis, such as proliferation, migration, and extracellular matrix remodeling.⁸ Although previous studies have indicated the involvement of several growth factors and cytokines in EMT,⁸ the precise molecular mechanism and the critical regulators of this process remain to be determined.The soluble cytoplasmic protein αB-crystallin is a prominent member of the small heat shock protein family. The small heat shock proteins exert diverse biological activities in both normal and stressed cells. They can act as molecular chaperones by binding misfolded proteins to prevent their denaturation and aggregation.⁹ αB-crystallin can bind to and stabilize cytoskeleton proteins, such as desmin and actin, and help to maintain cytoskeletal integrity.¹⁰ The role of αB-crystallin in EMT in liver and lung fibrosis has been recently reported.^{11, 12}Our previous work has suggested an important role for αB-crystallin in both the early and late stages of AMD. The early stage of AMD is characterized by the accumulation of drusen between the RPE and Bruch''s membrane, accompanied by RPE cell death and synaptic dysfunction.¹³ Geographic atrophy is caused by extensive atrophy and loss of the RPE and the overlying photoreceptors that rely on the RPE for trophic support.¹ αB-crystallin can be seen in RPE, associated with drusen and identified as one of the components of drusen.^{14, 15, 16} Our laboratory has shown that RPE cells lacking αB-crystallin are more susceptible to oxidative and endoplasmic reticulum stress compared with normal RPE.^{17, 18, 19, 20} Furthermore, RPE-overexpressing αB-crystallin shows resistance to apoptosis.²¹ These findings suggest that αB-crystallin plays a cytoprotective role against multiple stress stimuli that can cause RPE cell death, resulting in drusen formation and geographic atrophy. In addition, we previously demonstrated that αB-crystallin plays a regulatory role by functioning as a chaperone for VEGF in ocular angiogenesis and may play a part in CNV formation in nAMD.²² However, the involvement of αB-crystallin in subretinal fibrosis in nAMD has not been studied.Herein, we examined the pathogenesis of subretinal fibrosis in αB-crystallin^−/− and wild-type (WT) mice; we further investigated the role of αB-crystallin in EMT and fibrotic process in cultured RPE cells.     

Role of the Urokinase-Fibrinolytic System in Epithelial–Mesenchymal Transition during Lung Injury

Alveolar type II epithelial (ATII) cell injury precedes development of pulmonary fibrosis. Mice lacking urokinase-type plasminogen activator (uPA) are highly susceptible, whereas those deficient in plasminogen activator inhibitor (PAI-1) are resistant to lung injury and pulmonary fibrosis. Epithelial–mesenchymal transition (EMT) has been considered, at least in part, as a source of myofibroblast formation during fibrogenesis. However, the contribution of altered expression of major components of the uPA system on ATII cell EMT during lung injury is not well understood. To investigate whether changes in uPA and PAI-1 by ATII cells contribute to EMT, ATII cells from patients with idiopathic pulmonary fibrosis and chronic obstructive pulmonary disease, and mice with bleomycin-, transforming growth factor β–, or passive cigarette smoke–induced lung injury were analyzed for uPA, PAI-1, and EMT markers. We found reduced expression of E-cadherin and zona occludens-1, whereas collagen-I and α-smooth muscle actin were increased in ATII cells isolated from injured lungs. These changes were associated with a parallel increase in PAI-1 and reduced uPA expression. Further, inhibition of Src kinase activity using caveolin-1 scaffolding domain peptide suppressed bleomycin-, transforming growth factor β–, or passive cigarette smoke–induced EMT and restored uPA expression while suppressing PAI-1. These studies show that induction of PAI-1 and inhibition of uPA during fibrosing lung injury lead to EMT in ATII cells.Idiopathic pulmonary fibrosis (IPF) and other interstitial lung diseases are characterized by destruction of lung architecture due to excessive deposition of extracellular matrix proteins by activated fibroblasts or myofibroblasts, leading to progressive dyspnea and loss of lung function.^1–3 The origins of myofibroblasts participating in the pathological remodeling of IPF lungs are not clear. Histopathological evaluation demonstrates that myofibroblasts accumulate in fibroblastic foci. Emerging evidence suggests that polarized type II alveolar epithelial (ATII) cells undergo epithelial–mesenchymal transitions (EMT) after lung injury. The ATII cells assume phenotypic changes such as increased migration, invasion, resistance to apoptosis, and production of elevated levels of extracellular matrix proteins^4,5 and therefore serve as a source of myofibroblasts. Understanding the possible mechanisms contributing to EMT in ATII cells may help identify new targets to treat or at least limit fibrogenesis after lung injury.A number of molecular processes are involved in the initiation of EMT in ATII cells.⁵ Components of the fibrinolytic system such as urokinase-type plasminogen activator (uPA), uPA plasma membrane receptor (uPAR), and its major inhibitor, plasminogen activator inhibitor (PAI-1) are all elaborated by ATII cells. These proteins independently influence a broad range of biological processes germane to lung injury and its repair.⁶ However, their role in fibrogenesis via EMT is unclear. Recent publications using bleomycin (BLM)⁷ and a passive cigarette smoke (PCS)⁸ or adenovirus expressing constitutively active transforming growth factor β (Ad-TGF-β)^1,9 exposure model of lung injury indicate that a coordinate increase in PAI-1 and a decrement in uPA by ATII cells promote lung injury and subsequent pulmonary fibrosis (PF). We also found that caveolin-1 scaffolding domain peptide (CSP) acts as a competitor to caveolin-1, restores expression of uPA and uPAR, and inhibits PAI-1 in ATII cells after lung injury. These changes prevent development of PF after lung injury.⁷ Recent literature suggests that up to 30% to 50% of myofibroblasts may be derived via EMT during fibrogenesis.^10–12 However, an in vivo genetic lineage tracing study reported by Rock et al¹³ contradicts these findings. Our objective in the current study is to elucidate the role of altered expression of uPA, uPAR, and PAI-1 after lung injury in EMT, and further evaluate whether reinstatement of baseline expression of uPA, uPAR, and PAI-1 by CSP intervention after lung injury reduces EMT in ATII cells.     

Transforming Growth Factor β-1 Stimulates Profibrotic Epithelial Signaling to Activate Pericyte-Myofibroblast Transition in Obstructive Kidney Fibrosis

Pericytes have been identified as the major source of precursors of scar-producing myofibroblasts during kidney fibrosis. The underlying mechanisms triggering pericyte-myofibroblast transition are poorly understood. Transforming growth factor β-1 (TGF-β1) is well recognized as a pluripotent cytokine that drives organ fibrosis. We investigated the role of TGF-β1 in inducing profibrotic signaling from epithelial cells to activate pericyte-myofibroblast transition. Increased expression of TGF-β1 was detected predominantly in injured epithelium after unilateral ureteral obstruction, whereas downstream signaling from the TGF-β1 receptor increased in both injured epithelium and pericytes. In mice with ureteral obstruction that were treated with the pan anti–TGF-β antibody (1D11) or TGF-β receptor type I inhibitor (SB431542), kidney pericyte-myofibroblast transition was blunted. The consequence was marked attenuation of fibrosis. In addition, epithelial cell cycle G2/M arrest and production of profibrotic cytokines were both attenuated. Although TGF-β1 alone did not trigger pericyte proliferation in vitro, it robustly induced α smooth muscle actin (α-SMA). In cultured kidney epithelial cells, TGF-β1 stimulated G2/M arrest and production of profibrotic cytokines that had the capacity to stimulate proliferation and transition of pericytes to myofibroblasts. In conclusion, this study identified a novel link between injured epithelium and pericyte-myofibroblast transition through TGF-β1 during kidney fibrosis.Pericytes are mesenchyme-derived perivascular cells attached to the abluminal surface of capillaries.¹ They share developmental origins with fibroblasts, and there may be plasticity between pericytes attached to capillaries and fibroblasts embedded in adjacent collagenous matrix; however, unlike fibroblasts, pericytes have vital functions in regulating microvascular stability, angiogenesis, capillary permeability, capillary flow, and capillary basement membrane synthesis.¹ We have previously shown that pericytes are the major sources of scar-producing myofibroblasts during kidney injury, and we have identified adult kidney pericytes and perivascular fibroblasts are derived from Foxd1-expressing progenitors, positive for collagen I(α1)-GFP (Coll-GFP⁺), platelet-derived growth factor receptor β (PDGFR-β⁺), and CD73 (CD73⁺) and negative for α smooth muscle actin (α-SMA⁻) and CD45 (CD45⁻).^2–4 Recently, spinal cord pericytes were identified as major progenitors of scar tissue in the central nervous system, intestinal pericytes as a source of myofibroblasts in models of colitis, and hepatic stellate cells, the major precursor of myofibroblasts in liver disease, have been determined to be specialized pericytes of the hepatic sinusoid,^5–8 indicating that pericytes may represent myofibroblast precursors in many organs. Many independent studies support the notion of perivascular resident mesenchymal cells, not injured tubular epithelial cells, as the major source of myofibroblasts in kidneys.^9–12Prompted by the newly identified role for these perivascular cells in the pathogenesis of kidney fibrosis, we earlier investigated the cellular crosstalk that regulates pericyte detachment from capillaries and regulates the transition of pericytes to myofibroblasts.^13–15 Our investigations so far have focused on pericyte-endothelial crosstalk, because pericytes form direct communications with endothelial cells of peritubular capillaries at peg and socket junctions, where direct cell-cell signaling has been thought to occur.^13–20 We have recently shown that Coll-GFP⁺ kidney pericytes function identically to brain pericytes in migrating to and stabilizing capillary networks, functions that require expression of tissue inhibitor of metalloproteinase 3 (TIMP-3).¹⁵ These pericyte functions are lost when Coll-GFP⁺ pericytes transition to myofibroblasts.¹⁵ Furthermore, we reported that endothelial activation at vascular endothelial cell growth factor (VEGF) receptor 2 and PDGFR-β signaling by pericytes are two critical signaling pathways that link endothelial activation with pericyte transition to myofibroblasts.¹⁴ Our studies showed that these signaling events alone are sufficient to drive microvascular rarefaction, inflammation, and fibrosis in models of kidney disease.¹⁴ These findings are striking, because during embryonic and fetal microvascular development these same signaling pathways are critical in normal formation of the vasculature, indicating that dysregulation of signaling pathways between endothelium and pericytes is central to kidney pathogenesis.Nonetheless, studies unequivocally show that the injured tubular epithelium can directly trigger interstitial fibrosis. For example, overexpression of VEGF-A in adult kidney epithelium is sufficient to drive fibrosis, and cell cycle arrest of the kidney proximal epithelium at the G2/M checkpoint is also sufficient to drive fibrosis.^21,22 Therefore, epithelial signaling events must somehow be transmitted across the tubular basement membrane to pericytes to drive interstitial fibrosis. These obscure molecular signaling events are the focus of the studies we report here.In previous investigations of embryonic microvascular development, endothelial cells have been shown to be a source of both PDGF and transforming growth factor β-1 (TGF-β1), cytokines that regulate pericyte attachment, differentiation, and angiogenesis.^17,23,24 Moreover, genetic inactivation of either TGFB1 or of genes encoding its receptors in mice leads to vascular defects and embryonic lethality.^17–19 TGF-β1 is thus a cytokine with a profound effect on microvascular development and angiogenesis.In adult kidney injury, although endothelial cells produce PDGF and TGF-β1 in fibrosing kidneys, injured epithelial cells are a major source of these cytokines, and the TGF-β1 activator integrin αvβ6 is restricted to kidney epithelium.^13,25–29 Increased TGF-β1 expression by epithelium is accompanied by activation of intracellular signaling pathways and downstream effectors in the epithelium itself.^30,31 Blocking TGF-β1 and its downstream effectors can attenuate kidney injury and fibrosis,^30–33 whereas transgenic overexpression of TGF-β1 in kidney epithelial cells is sufficient to trigger interstitial kidney fibrosis in the absence of migration of epithelial-derived cells into the interstitium.^34,35 Therefore, epithelial transgenic overexpression of TGF-β1, which stimulates epithelial cell dedifferentiation and autophagy, must stimulate pericyte to myofibroblast transition by epithelial cell to pericyte crosstalk.³⁴ Our aim in the present study was to identify the mechanism by which TGF-β1 signaling from injured tubular epithelial cells can activate pericytes to drive progressive kidney fibrosis.     

Radiation-Induced Lung Injury Is Mitigated by Blockade of Gastrin-Releasing Peptide

Gastrin-releasing peptide (GRP), secreted by pulmonary neuroendocrine cells, mediates oxidant-induced lung injury in animal models. Considering that GRP blockade abrogates pulmonary inflammation and fibrosis in hyperoxic baboons, we hypothesized that ionizing radiation triggers GRP secretion, contributing to inflammatory and fibrotic phases of radiation-induced lung injury (RiLI). Using C57BL/6 mouse model of pulmonary fibrosis developing ≥20 weeks after high-dose thoracic radiation (15 Gy), we injected small molecule 77427 i.p. approximately 1 hour after radiation then twice weekly for up to 20 weeks. Sham controls were anesthetized and placed in the irradiator without radiation. Lung paraffin sections were immunostained and quantitative image analyses performed. Mice exposed to radiation plus PBS had increased interstitial CD68⁺ macrophages 4 weeks after radiation and pulmonary neuroendocrine cells hyperplasia 6 weeks after radiation. Ten weeks later radiation plus PBS controls had significantly increased pSmad2/3⁺ nuclei/cm². GRP blockade with 77427 treatment diminished CD68⁺, GRP⁺, and pSmad2/3⁺ cells. Finally, interstitial fibrosis was evident 20 weeks after radiation by immunostaining for α-smooth muscle actin and collagen deposition. Treatment with 77427 abrogated interstitial α-smooth muscle actin and collagen. Sham mice given 77427 did not differ significantly from PBS controls. Our data are the first to show that GRP blockade decreases inflammatory and fibrotic responses to radiation in mice. GRP blockade is a novel radiation fibrosis mitigating agent that could be clinically useful in humans exposed to radiation therapeutically or unintentionally.Radiation fibrosis is a serious complication that affects normal lung following unintentional exposure or due to therapeutic ionizing radiation of thoracic tumors. Despite advances in radiobiology, precise mechanisms by which radiation induces lung injury remain controversial.¹ Classically, radiation-induced lung injury (RiLI) is characterized by a latent period that can last for weeks to months after radiation exposure, followed by 2 stages of overt lung injury that can lead to life-threatening and debilitating pulmonary toxic effects.^2,3 Acute inflammatory lung injury arises 1 to 6 months after radiation exposure, with diffuse alveolar damage, similar to acute respiratory distress syndrome. Later, chronic interstitial and intra-alveolar fibrosis develops, predominantly in irradiated segments, with myofibroblast proliferation and collagen deposition. It is unclear why only approximately 15% of radiation-exposed patients develop RiLI.^1,4 General cytoprotective agents, such as a catalytic antioxidant metalloporphyrin (AEOL10113), can reduce the severity of RiLI by decreasing free radical injury after radiation.⁵Our novel paradigm links gastrin-releasing peptide (GRP) to radiation lung injury. We hypothesized that GRP is a mediator of RiLI: promoting both macrophage accumulation and fibrosis. We propose that ionizing radiation triggers pulmonary neuroendocrine cell (PNEC) hyperplasia, leading to GRP secretion, which then mediates chronic lung injury. GRP receptor (GRPR) gene expression is detected and functional in pulmonary epithelial cells, fibroblasts, endothelial cells, and macrophages.^6–10 GRP is a proinflammatory neuropeptide that functions as an inflammatory cell activator, mitogen, and cell differentiation factor.^8,10,11 GRP is expressed at the highest levels in PNEC in fetal lung,¹² where it can promote lung development.¹³ After birth, GRP production normally decreases, but elevated levels are associated with many inflammatory lung conditions, including chronic lung disease of newborns (bronchopulmonary dysplasia).^14–17 PNEC hyperplasia can be triggered by inflammation or exposure to oxygen or ozone^10,16,18 and can take weeks to reach peak levels.¹⁹The present investigation tests the hypothesis that GRP contributes to radiation-induced pulmonary fibrosis in C57BL/6 mice. One hour post exposure to thoracic radiation (15 Gy), we treated mice i.p. with either PBS or GRP blockade by using small molecule 77427. We have quantified results of immunohistochemistry (IHC) by using image analysis with ImageJ version 1.46e (NIH, Bethesda, MD) to determine whether GRP contributes to radiation-induced inflammatory responses and/or fibrosis, specifically including assessment of active transforming growth factor (TGF)-β signaling.     

α2-Antiplasmin Is Associated with the Progression of Fibrosis

Systemic sclerosis results in tissue fibrosis due to the activation of fibroblasts and the ensuing overproduction of the extracellular matrix. We previously reported that the absence of α2-antiplasmin (α2AP) attenuated the process of dermal fibrosis; however, the detailed mechanism of how α2AP affects the progression of fibrosis remained unclear. The goal of the present study was to examine the role of α2AP in fibrotic change. We observed significantly higher levels of α2AP expression in the skin of bleomycin-injected systemic sclerosis model mice in comparison with the levels seen in control mice. We also demonstrated that α2AP induced myofibroblast differentiation, and the absence of α2AP attenuated the induction of myofibroblast differentiation. Moreover, we found that connective tissue growth factor induced the expression of α2AP through both the extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK) pathways in fibroblasts. Interestingly, α2AP also induced transforming growth factor-β expression through the same pathways, and the inhibition of ERK1/2 and JNK slowed the progression of bleomycin-induced fibrosis. Our findings suggest that α2AP is associated with the progression of fibrosis, and regulation of α2AP expression by the ERK1/2 and JNK pathways may be an effective antifibrotic therapy for the treatment of systemic sclerosis.Systemic sclerosis (SSc) affects the skin and the internal organs, resulting in tissue fibrosis. Although the disease process involves immunological mechanisms, vascular damage, and activation of fibroblasts, the pathogenesis of SSc remains to be further elucidated. Fibrotic diseases are characterized by excessive scarring due to excessive production, deposition, and contraction of the extracellular matrix (ECM). This process usually occurs over many months and years, and can lead to organ dysfunction or death. Connective tissue growth factor (CTGF) is constitutively overexpressed in fibrotic lesions such as in scleroderma,¹ liver,² renal,^3,4 lung,⁵ and pancreatic fibrosis.⁵ CTGF acts as a downstream effecter of at least some of the profibrotic effects of transforming growth factor-β (TGF-ß),⁶ and promotes fibroblast proliferation, myofibroblasts differentiation, matrix production, and granulation tissue formation.^7,8Human and murine α2-antiplasmin (α2AP) are serpins (serine protease inhibitors) with a molecular weight of 65 to 70 kd,⁹ which rapidly inactivate plasmin, resulting in the formation of a stable inactive complex, plasmin-α2AP.¹⁰ Tissue fibrosis is generally considered to arise due to a failure of the normal wound healing response to terminate.¹¹ Previous our studies show that α2AP is associated with the wound healing and the fibrosis.^12,13 In addition, it has been reported that the level of plasmin-α2AP complex in plasma is elevated in SSc patients.¹⁴ These findings suggest that α2AP may be associated with the progression of fibrotic disease, but the physiological roles of α2AP are not precisely understood. We herein report that α2AP plays an important role in the progression of fibrosis.     

Absence of Nogo-B (Reticulon 4B) Facilitates Hepatic Stellate Cell Apoptosis and Diminishes Hepatic Fibrosis in Mice

Nogo-B (reticulon 4B) accentuates hepatic fibrosis and cirrhosis, but the mechanism remains unclear. The aim of this study was to identify the role of Nogo-B in hepatic stellate cell (HSC) apoptosis in cirrhotic livers. Cirrhosis was generated by carbon tetrachloride inhalation in wild-type (WT) and Nogo-A/B knockout (Nogo-B KO) mice. HSCs were isolated from WT and Nogo-B KO mice and cultured for activation and transformation to myofibroblasts (MF-HSCs). Human hepatic stellate cells (LX2 cells) were used to assess apoptotic responses of activated HSCs after silencing or overexpressing Nogo-B. Livers from cirrhotic Nogo-B KO mice showed significantly reduced fibrosis (P < 0.05) compared with WT mice. Apoptotic cells were more prominent in fibrotic areas of cirrhotic Nogo-B KO livers. Nogo-B KO MF-HSCs showed significantly increased levels of apoptotic markers, cleaved poly (ADP-ribose) polymerase, and caspase-3 and -8 (P < 0.05) compared with WT MF-HSCs in response to staurosporine. Treatment with tunicamycin, an endoplasmic reticulum stress inducer, increased cleaved caspase-3 and -8 levels in Nogo-B KO MF-HSCs compared with WT MF-HSCs (P < 0.01). In LX2 cells, Nogo-B knockdown enhanced apoptosis in response to staurosporine, whereas Nogo-B overexpression inhibited apoptosis. The absence of Nogo-B enhances apoptosis of HSCs in experimental cirrhosis. Selective blockade of Nogo-B in HSCs may represent a potential therapeutic strategy to mitigate liver fibrosis.Liver fibrosis and its end-stage manifestation of cirrhosis represent clinical challenges worldwide. Hepatic stellate cell (HSC) activation is the cardinal feature that results in hepatic fibrosis. When stimulated by reactive oxygen species or cytokines in response to various hepatic insults, quiescent HSCs are transformed to myofibroblasts (MF-HSCs) that proliferate and secrete collagen.^1–4 Studies have shown that apoptosis of activated HSCs can reverse fibrosis.^5–13 Thus, the mechanisms that control MF-HSC apoptosis may represent potential therapeutic targets that result in reduced fibrosis.^14–16Nogo-B, also known as reticulon 4B, is a member of the reticulon protein family that is localized primarily to the endoplasmic reticulum (ER).^17,18 Four groups of reticulons (1, 2, 3, and 4) exist, and each has multiple isoforms. Reticulon 4 has three isoforms, Nogo-A, B, and C. The most recognized isoform, Nogo-A (200 kDa), a potent neural outgrowth inhibitor,^19–21 is expressed mainly in the nervous system.^22–24 Nogo-C (25 kDa) is highly expressed in the differentiated muscle fibers and somewhat in the brain,^17,18,22 however, its function remains unclear.Nogo-B (55 kDa), a splice variant of Nogo-A, is expressed in most tissues and has been reported for its role in modulating endothelial and smooth muscle cellular responses after injury in a variety of organs/tissues, including blood vessels,^25,26 lung,^27,28 kidney,²⁹ and liver.³⁰ We previously showed that the absence of Nogo-B in a murine model blocks the progression of fibrosis/cirrhosis and the development of portal hypertension.³⁰ Further, we showed that lack of Nogo-B decreases the levels of α-smooth muscle actin (α-SMA), a marker of MF-HSCs, in murine cholestatic livers. These findings led us to hypothesize that absence of Nogo-B may increase the susceptibility of MF-HSCs to apoptosis, thereby reducing fibrosis/cirrhosis in mice. In this study, we investigated the role of Nogo-B in MF-HSC apoptosis in vivo and in vitro.