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The caveolin-1 scaffolding domain (CSD, amino acids 82-101 of caveolin-1) has been shown to suppress bleomycin-induced lung and skin fibrosis and angiotensin II (AngII)-induced myocardial fibrosis. To identify active subregions within CSD, we split its sequence into three slightly overlapping 8-amino acid subregions (82-89, 88-95, and 94-101). Interestingly, all three peptides showed activity. In bleomycin-treated mice, all three subregions suppressed the pathological effects on lung and skin tissue morphology. In addition, while bone marrow monocytes isolated from bleomycin-treated mice showed greatly enhanced migration in vitro toward CXCL12, treatment in vivo with CSD and its subregions almost completely suppressed this enhanced migration. In AngII-induced heart failure, both 82-89 and 88-95 significantly suppressed fibrosis (both Col I and HSP47 levels), microvascular leakage, and heart weight/ body weight ratio (HW/BW) while improving ventricular function. In contrast, while 94-101 suppressed the increase in Col I, it did not improve the other parameters. The idea that all three subregions can be active depending on the assay was further supported by experiments studying the in vitro migration of human monocytes in which all three subregions were extremely active. These studies are very novel in that it has been suggested that there is only one active region within CSD that is centered on amino acids 90-92. In contrast, we demonstrate here the presence of other active regions within CSD., Competing Interests: This study was funded in part by a Sponsored Research Agreement from Lung Therapeutics, Inc., to Dr. Stanley Hoffman. Lung Therapeutics has also licensed a use patent (no. 8,058,227) issued to the Medical University of South Carolina for the caveolin-1 scaffolding domain peptide as a treatment for fibrotic diseases on which Drs. Hoffman and Tourkina are named coinventors. Lung Therapeutics played no role in the study design; collection, analysis, and interpretation of data; writing of the paper; and/or decision to submit for publication and only provided financial support in the form of authors’ salaries [SH, ET, DK, PC] and research materials. Our partial funding by Lung Therapeutics does not affect our adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.

The potential value of mesenchymal stromal/stem cell therapy in treating skin fibrosis in scleroderma (systemic sclerosis) and of the caveolin-1 scaffolding domain peptide in treating lung, skin, and heart fibrosis is known. To understand how these observations may relate to differences between mesenchymal stromal/stem cells from healthy subjects and subjects with fibrosis, we have characterized the fibrogenic and adipogenic potential of adipose-derived mesenchymal stromal/stem cells from systemic sclerosis patients, from mice with fibrotic lung and skin disease induced by systemic bleomycin treatment, and from healthy controls. Early passage systemic sclerosis adipose-derived mesenchymal stromal/stem cells have a profibrotic/anti-adipogenic phenotype compared to healthy adipose-derived mesenchymal stromal/stem cells (low caveolin-1, high α-smooth muscle actin, high HSP47, low pAKT, low capacity for adipogenic differentiation). This phenotype is mimicked by treating healthy adipose-derived mesenchymal stromal/stem cells with transforming growth factor beta or caveolin-1 small interfering RNA and is reversed in systemic sclerosis adipose-derived mesenchymal stromal/stem cells by treatment with caveolin-1 scaffolding domain peptide, but not scrambled caveolin-1 scaffolding domain peptide. Similar results were obtained with adipose-derived mesenchymal stromal/stem cells from systemic sclerosis patients and from bleomycin-treated mice, indicating the central role of caveolin-1 in mesenchymal stromal/stem cell differentiation in fibrotic disease., Competing Interests: Declaration of conflicting interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s) 2019.)

Dysregulation of the renin-angiotensin system leads to systemic hypertension and maladaptive fibrosis in various organs. We showed recently that myocardial fibrosis and the loss of cardiac function in mice with transverse aortic constriction (TAC) could be averted by treatment with the caveolin-1 scaffolding domain (CSD) peptide. Here, we used angiotensin II (AngII) infusion (2.1 mg/kg/day for 2 wk) in mice as a second model to confirm and extend our observations on the beneficial effects of CSD on heart and kidney disease. AngII caused cardiac hypertrophy (increased heart weight to body weight ratio (HW/BW) and cardiomyocyte cross-sectional area); fibrosis in heart and kidney (increased levels of collagen I and heat shock protein-47 (HSP47)); and vascular leakage (increased levels of IgG in heart and kidney). Echocardiograms of AngII-infused mice showed increased left ventricular posterior wall thickness (pWTh) and isovolumic relaxation time (IVRT), and decreased ejection fraction (EF), stroke volume (SV), and cardiac output (CO). CSD treatment (i.p. injections, 50 μg/mouse/day) of AngII-infused mice significantly suppressed all of these pathological changes in fibrosis, hypertrophy, vascular leakage, and ventricular function. AngII infusion increased β1 and β3 integrin levels and activated Pyk2 in both heart and kidney. These changes were also suppressed by CSD. Finally, bone marrow cell (BMC) isolated from AngII-infused mice showed hyper-migration toward SDF1. When AngII-infused mice were treated with CSD, BMC migration was reduced to the basal level observed in cells from control mice. Importantly, CSD did not affect the AngII-induced increase in blood pressure (BP), indicating that the beneficial effects of CSD were not mediated via normalization of BP. These results strongly indicate that CSD suppresses AngII-induced pathological changes in mice, suggesting that CSD can be developed as a treatment for patients with hypertension and pressure overload-induced heart failure., Competing Interests: This study was funded in part by a Sponsored Research Agreement from Lung Therapeutics, Inc., to Dr. Stanley Hoffman. Lung Therapeutics has also licensed a use patent (no. 8,058,227) issued to the Medical University of South Carolina for the caveolin-1 scaffolding domain peptide as a treatment for fibrotic diseases on which Drs. Hoffman and Tourkina are named coinventors. Lung Therapeutics played no role in the study design; collection, analysis, and interpretation of data; writing of the paper; and/or decision to submit for publication. Our partial funding by Lung Therapeutics does not affect our adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.

Monocytes from systemic sclerosis (SSc, scleroderma) patients and healthy African Americans (AA) are deficient in the regulatory protein caveolin-1 leading to enhanced migration toward chemokines and fibrogenic differentiation. While dermal fibrosis is the hallmark of SSc, loss of subcutaneous adipose tissue is a lesser-known feature. To better understand the etiology of SSc and the predisposition of AA to SSc, we studied the adipogenic potential of SSc and healthy AA monocytes. The ability of SSc and healthy AA monocytes to differentiate into adipocyte-like cells (ALC) is inhibited compared to healthy Caucasian (C) monocytes. We validated that monocyte-derived ALCs are distinct from macrophages by flow cytometry and immunocytochemistry. Like their enhanced fibrogenic differentiation, their inhibited adipogenic differentiation is reversed by the caveolin-1 scaffolding domain peptide (CSD, a surrogate for caveolin-1). The altered differentiation of SSc and healthy AA monocytes is additionally regulated by peroxisome proliferator-activated receptor γ (PPARγ) which is also present at reduced levels in these cells. In vivo studies further support the importance of caveolin-1 and PPARγ in fibrogenesis and adipogenesis. In SSc patients, healthy AA, and mice treated systemically with bleomycin, adipocytes lose caveolin-1 and PPARγ and the subcutaneous adipose layer is diminished. CSD treatment of these mice leads to a reappearance of the caveolin-1+/PPARγ+/FABP4+ subcutaneous adipose layer. Moreover, many of these adipocytes are CD45+, suggesting they are monocyte derived. Tracing experiments with injected EGFP+ monocytes confirm that monocytes contribute to the repair of the adipose layer when it is damaged by bleomycin treatment. Our observations strongly suggest that caveolin-1 and PPARγ work together to maintain a balance between the fibrogenic and adipogenic differentiation of monocytes, that this balance is altered in SSc and in healthy AA, and that monocytes make a major contribution to the repair of the adipose layer.

Chronic ventricular pressure overload (PO) results in congestive heart failure (CHF) in which myocardial fibrosis develops in concert with ventricular dysfunction. Caveolin-1 is important in fibrosis in various tissues due to its decreased expression in fibroblasts and monocytes. The profibrotic effects of low caveolin-1 can be blocked with the caveolin-1 scaffolding domain peptide (CSD, a caveolin-1 surrogate) using both mouse models and human cells. We have studied the beneficial effects of CSD on mice in which PO was induced by trans-aortic constriction (TAC). Beneficial effects observed in TAC mice receiving CSD injections daily included: improved ventricular function (increased ejection fraction, stroke volume, and cardiac output; reduced wall thickness); decreased collagen I, collagen chaperone HSP47, fibronectin, and CTGF levels; decreased activation of non-receptor tyrosine kinases Pyk2 and Src; and decreased activation of eNOS. To determine the source of cells that contribute to fibrosis in CHF, flow cytometric studies were performed that suggested that myofibroblasts in the heart are in large part bone marrow-derived. Two CD45+ cell populations were observed. One (Zone 1) contained CD45+/HSP47-/macrophage marker+ cells (macrophages). The second (Zone 2) contained CD45 moderate /HSP47+/macrophage marker- cells often defined as fibrocytes. TAC increased the number of cells in Zones 1 and 2 and the level of HSP47 in Zone 2. These studies are a first step in elucidating the mechanism of action of CSD in heart fibrosis and promoting the development of CSD as a novel treatment to reduce fibrosis and improve ventricular function in CHF patients.

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.

Objective: Interstitial lung disease (ILD) is the leading cause of death in patients with systemic sclerosis (SSc; scleroderma). Although SSc-related ILD is more common and severe in African Americans than in Caucasians, little is known about factors underlying this significant health disparity. The aim of this study was to examine the role that low expression of caveolin-1 might play in susceptibility to ILD among African Americans., Methods: Assays of monocyte migration toward stromal cell-derived factor 1 (SDF-1) were performed using monocytes from Caucasian and African American healthy donors and patients with SSc. For fibrocyte differentiation studies, total peripheral blood mononuclear cells were incubated on fibronectin-coated plates. Protein expression was evaluated by immunohistochemistry and Western blotting., Results: Monocytes from healthy African American donors and those from patients with SSc had low caveolin-1 levels, enhanced migration toward the CXCR4 ligand SDF-1, and enhanced differentiation to fibrocytes. Enhanced migration and differentiation of monocytes from African Americans and patients with SSc appeared to be attributable to the lack of caveolin-1, because restoring caveolin-1 function using a caveolin-1 scaffolding domain peptide inhibited these processes. Although they differed from monocytes from Caucasians, monocytes from both African Americans and patients with SSc were not identical, because SSc monocytes showed major increases from baseline in ERK, JNK, p38, and Smad2/3 activation, while monocytes from African Americans showed only limited ERK activation and no activation of JNK, p38, or Smad2/3. In contrast, SDF-1 exposure caused no additional ERK activation in SSc monocytes but did cause significant additional activation in monocytes from African Americans., Conclusion: African Americans may be predisposed to SSc-related ILD due to low baseline caveolin-1 levels in their monocytes, potentially affecting signaling, migration, and fibrocyte differentiation. The monocytes of African Americans may lack caveolin-1 due to high levels of transforming growth factor β in their blood., (Copyright © 2014 by the American College of Rheumatology.)

Fibrocytes are bone marrow hematopoietic-derived cells that also express a mesenchymal cell marker (commonly collagen I) and participate in fibrotic diseases of multiple organs. Given their origin, they or their precursors must be circulating cells before recruitment into target tissues. While most previous studies focused on circulating fibrocytes, here we focus on the fibrocyte phenotype in fibrotic tissue. The study's relevance to human disease is heightened by use of a model in which bleomycin is delivered systemically, recapitulating several features of human scleroderma including multi-organ fibrosis not observed when bleomycin is delivered directly into the lungs. Using flow cytometry, we find in the fibrotic lung a large population of CD45(high) fibrocytes (called Region I) rarely found in vehicle-treated control mice. A second population of CD45+ fibrocytes (called Region II) is observed in both control and fibrotic lung. The level of CD45 in circulating fibrocytes is far lower than in either Region I or II lung fibrocytes. The chemokine receptors CXCR4 and CCR5 are expressed at higher levels in Region I than in Region II and are present at very low levels in all other lung cells including CD45+/collagen I- leucocytes. The collagen chaperone HSP47 is present at similar high levels in both Regions I and II, but at a higher level in fibrotic lung than in control lung. There is also a major population of HSP47(high)/CD45- cells in fibrotic lung not present in control lung. CD44 is present at higher levels in Region I than in Region II and at much lower levels in all other cells including CD45+/collagen I- leucocytes. When lung fibrosis is inhibited by restoring caveolin-1 activity using a caveolin-1 scaffolding domain peptide (CSD), a strong correlation is observed between fibrocyte number and fibrosis score. In summary, the distinctive phenotype of fibrotic lung fibrocytes suggests that fibrocyte differentiation occurs primarily within the target organ.

In fibrotic diseases caveolin-1 underexpression in fibroblasts results in collagen overexpression and in monocytes leads to hypermigration. These profibrotic behaviors are blocked by the caveolin-1 scaffolding domain peptide (CSD) which compensates for caveolin-1 deficiency. Monocytes and fibroblasts are related in that monocytes are the progenitors of fibrocytes (CD45+/Collagen I+ cells) that, in turn, are the progenitors of many fibroblasts in fibrotic tissues. In an additional anti-fibrotic activity, CSD blocks monocyte differentiation into fibrocytes. We studied a mouse fibrosis model (Pump Model) involving systemic bleomycin delivery that closely models scleroderma (SSc) in several ways, the most important of which for this study is that fibrosis is observed in the lungs, skin, and internal organs. We show here that dermal thickness is increased 2-fold in the Pump Model and that this effect is almost completely blocked by CSD (p < 0.001). Concomitantly, the subcutaneous fat layer becomes >80% thinner. This effect is also blocked by CSD (p < 0.001). Even in mice receiving vehicle instead of bleomycin, CSD increases the thickness of the fat layer. To study the mechanisms of action of bleomycin and CSD, we examined the accumulation of the chemokine receptor CCR5 and its ligands MIP1α and MIP1β in fibrotic tissue and their roles in monocyte migration. Fibrocytes and other leukocytes expressing CCR5 and its ligands were present at high levels in the fibrotic dermis of SSc patients and Pump Model mice while CSD blocked their accumulation in mouse dermis. Migration toward CCR5 ligands of SSc monocytes and Pump Model bone marrow cells was 3-fold greater than cells from control subjects. This enhanced migration was almost completely blocked by CSD. These results suggest that low monocyte caveolin-1 promotes fibrosis by enhancing the recruitment of fibrocytes and their progenitors into affected tissue.

The interstitial lung diseases (ILD) include a large number of chronic, progressive, irreversible respiratory disorders involving pulmonary fibrosis, the most common of which are idiopathic pulmonary fibrosis and scleroderma lung disease (SSc ILD). Because bleomycin causes lung fibrosis when used in cancer chemotherapy, it is used to model human ILD in rodents. In most studies, bleomycin has been delivered directly into the lung by intratracheal or intraoral administration. Here we have compared the effects in mice of bleomycin delivered directly into the lungs (direct model) or systemically using osmotic minipumps (pump model) to determine which more closely resembles human ILD. The pump model is more similar to human SSc ILD in that: 1) lung injury/fibrosis is limited to the subpleural portion of the lung in the pump model and in SSc ILD, whereas the entire lung is affected in the direct model; 2) conversely, there is massive inflammation throughout the lung in the direct model, whereas inflammation is limited in the pump model and in SSc ILD; 3) hypertrophic type II alveolar epithelial cells are present at high levels in SSc ILD and in the pump model but not in the direct model; and 4) lung fibrosis is accompanied by dermal fibrosis. The pump model is also move convenient and humane than the direct model because there is less weight loss and mortality.

Background: In fibrotic lung diseases, expression of caveolin-1 is decreased in fibroblasts and monocytes. The effects of this deficiency are reversed by treating cells or animals with the caveolin-1 scaffolding domain peptide (CSD, amino acids 82-101 of caveolin-1) which compensates for the lack of caveolin-1. Here we compare the function of CSD subdomains (Cav-A, Cav-B, Cav-C, Cav-AB, and Cav-BC) and mutated versions of CSD (F92A and T90A/T91A/F92A)., Methods: Migration toward the chemokine CXCL12 and the associated expression of F-actin, CXCR4, and pSmad 2/3 were studied in monocytes from healthy donors and SSc patients. Fibrocyte differentiation was studied using PBMC from healthy donors and SSc patients. Collagen I secretion and signaling were studied in fibroblasts derived from the lung tissue of healthy subjects and SSc patients., Results: Cav-BC and CSD at concentrations as low as 0.01 μM inhibited the hypermigration of SSc monocytes and TGFβ-activated Normal monocytes and the differentiation into fibrocytes of SSc and Normal monocytes. While CSD also inhibited the migration of poorly migrating Normal monocytes, Cav-A (and other subdomains to a lesser extent) promoted the migration of Normal monocytes while inhibiting the hypermigration of TGFβ-activated Normal monocytes. The effects of versions of CSD on migration may be mediated in part via their effects on CXCR4, F-actin, and pSmad 2/3 expression. Cav-BC was as effective as CSD in inhibiting fibroblast collagen I and ASMA expression and MEK/ERK signaling. Cav-C and Cav-AB also inhibited collagen I expression, but in many cases did not affect ASMA or MEK/ERK. Cav-A increased collagen I expression in scleroderma lung fibroblasts. Full effects on fibroblasts of versions of CSD required 5 μM peptide., Conclusions: Cav-BC retains most of the anti-fibrotic functions of CSD; Cav-A exhibits certain pro-fibrotic functions. Results obtained with subdomains and mutated versions of CSD further suggest that the critical functional residues in CSD depend on the cell type and readout being studied. Monocytes may be more sensitive to versions of CSD than fibroblasts and endothelial cells because the baseline level of caveolin-1 in monocytes is much lower than in these other cell types.

Background: Caveolin-1 has emerged as a critical regulator of signaling pathways involved in lung fibrosis and inflammation., Methods: Therefore, we investigated whether caveolin-1 is deficient in asthmatic patients and in a murine model of asthma., Results: Immunohistochemical analyses of endobronchial biopsies showed a remarkable loss of caveolin-1 in the lungs of asthmatic patients compared with controls. This loss was most evident in bronchial epithelial cells and associated with an increase in the expression of extracellular matrix proteins: collagen I, tenascin, and periostin. Cultured primary bronchial epithelial cells of asthmatics had lower caveolin-1 expression compared with control cells. In addition, caveolin-1 expression was significantly decreased in peripheral blood monocytes from asthma patients. The loss of caveolin-1 was also observed in a mouse model for asthma (mice sensitized and challenged with aspergillus fumigatus)., Conclusions: To our knowledge, this is the first demonstration that the regulatory protein caveolin-1 is reduced in patients with asthma., (© 2012 John Wiley & Sons A/S.)

Purpose of Review: Racial disparities appear to exist in the susceptibility and severity of systemic sclerosis (SSc, scleroderma) and are responsible for a greater health burden in blacks as compared with whites. Disparities in socioeconomic status and access to healthcare do not sufficiently explain the observed differences in prevalence and mortality. It is important to determine whether there might be a biologic basis for the racial disparities observed in SSc., Recent Findings: We present data to suggest that the increased susceptibility and severity of SSc in blacks may result in part from an imbalance of profibrotic and antifibrotic factors. Racial differences in the expression of transforming growth factor-β1 (TGF-β1) and caveolin-1, as well as differences in the expression of hepatocyte growth factor and PPAR-γ, have been demonstrated in blacks with SSc, as well as in normal black individuals. A genetic predisposition to fibrosis may account for much of the racial disparities between black and white patients with SSc., Summary: A better understanding of the biologic basis for the racial disparities observed in SSc may lead to improved therapies, along with the recognition that different therapies may need to be adapted for different groups of patients.

Caveolin-1 is a master regulator of several signaling cascades because it is able to bind to and thereby inhibit members of a variety of kinase families. While associated with caveolae and involved in their generation, caveolin-1 is also present at other sites. A variety of studies have suggested that caveolin-1 may be a useful therapeutic target in fibrotic diseases of the lung and other tissues because in these diseases a low level of caveolin-1 expression is associated with a high level of collagen expression and fibrosis. Reduced caveolin-1 expression is observed not only in the fibroblasts that secrete collagen, but also in epithelial cells and monocytes. This is intriguing because both epithelial cells and monocytes have been suggested to be precursors of fibroblasts. Likely downstream effects of loss of caveolin-1 in fibrosis include activation of TGF-β signaling and upregulation of CXCR4 in monocytes resulting in their enhanced migration into damaged tissue where its ligand CXCL12 is produced. Finally, it may be possible to target caveolin-1 in fibrotic diseases without the use of gene therapy. A caveolin-1 peptide (caveolin-1 scaffolding domain) has been identified that retains the function of the full-length molecule to inhibit kinases and that can be modified by addition of the Antennapedia internalization sequence to allow it to enter cells both in vitro and in vivo.

Interstitial lung disease (ILD) is a major cause of morbidity and mortality in scleroderma (systemic sclerosis, or SSc). Fibrocytes are a monocyte-derived cell population implicated in the pathogenesis of fibrosing disorders. Given the recently recognized importance of caveolin-1 in regulating function and signaling in SSc monocytes, in the present study we examined the role of caveolin-1 in the migration and/or trafficking and phenotype of monocytes and fibrocytes in fibrotic lung disease in human patients and an animal model. These studies fill a gap in our understanding of how monocytes and fibrocytes contribute to SSc-ILD pathology. We found that C-X-C chemokine receptor type 4-positive (CXCR4+)/collagen I-positive (ColI+), CD34+/ColI+ and CD45+/ColI+ cells are present in SSc-ILD lungs, but not in control lungs, with CXCR4+ cells being most prevalent. Expression of CXCR4 and its ligand, stromal cell-derived factor 1 (CXCL12), are also highly upregulated in SSc-ILD lung tissue. SSc monocytes, which lack caveolin-1 and therefore overexpress CXCR4, exhibit almost sevenfold increased migration toward CXCL12 compared to control monocytes. Restoration of caveolin-1 function by administering the caveolin scaffolding domain (CSD) peptide reverses this hypermigration. Similarly, transforming growth factor β-treated normal monocytes lose caveolin-1, overexpress CXCR4 and exhibit 15-fold increased monocyte migration that is CSD peptide-sensitive. SSc monocytes exhibit a different phenotype than normal monocytes, expressing high levels of ColI, CD14 and CD34. Because ColI+/CD14+ cells are prevalent in SSc blood, we looked for such cells in lung tissue and confirmed their presence in SSc-ILD lungs but not in normal lungs. Finally, in the bleomycin model of lung fibrosis, we show that CSD peptide diminishes fibrocyte accumulation in the lungs. Our results suggest that low caveolin-1 in SSc monocytes contributes to ILD via effects on cell migration and phenotype and that the hyperaccumulation of fibrocytes in SSc-ILD may result from the altered phenotype and migratory activity of their monocyte precursors.

Objectives: Reduced caveolin-1 levels in lung fibroblasts from patients with scleroderma and the lungs of bleomycin-treated mice promote collagen overexpression and lung fibrosis. This study was undertaken to determine whether caveolin-1 is deficient in leucocytes from bleomycin-treated mice and patients with scleroderma and to examine the consequences of this deficiency and its reversal., Methods: Mice or cells received the caveolin-1 scaffolding domain (CSD) peptide to reverse the pathological effects of reduced caveolin-1 expression. In bleomycin-treated mice, the levels of caveolin-1 in leucocytes and the effect of CSD peptide on leucocyte accumulation in lung tissue were examined. To validate the results in human disease and to identify caveolin-1-regulated molecular mechanisms, monocytes and neutrophils were isolated from patients with scleroderma and control subjects and caveolin-1, extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK), p38, CXC chemokine receptor 4 (CXCR4) and matrix metalloproteinase 9 (MMP-9) expression/activation were evaluated. These parameters were also studied in monocytes treated with cytokines or CSD peptide., Results: Leucocyte caveolin-1 is important in lung fibrosis. In bleomycin-treated mice, caveolin-1 expression was diminished in monocytes and CSD peptide inhibited leucocyte recruitment into the lungs. These observations are relevant to human disease. Monocytes and neutrophils from patients with scleroderma contained less caveolin-1 and more activated ERK, JNK and p38 than those from control subjects. Treatment with CSD peptide reversed ERK, JNK and p38 hyperactivation. Scleroderma monocytes also overexpressed CXCR4 and MMP-9, which was inhibited by the CSD peptide. Cytokine treatment of normal monocytes caused adoption of the scleroderma phenotype (low caveolin-1, high CXCR4 and MMP-9 and signalling molecule hyperactivation)., Conclusions: Caveolin-1 downregulation in leucocytes contributes to fibrotic lung disease, highlighting caveolin-1 as a promising therapeutic target in scleroderma.

The roles of MEK, ERK, the epsilon and alpha isoforms of protein kinase C (PKC), and caveolin-1 in regulating collagen expression were studied in normal lung fibroblasts. Knocking down caveolin-1 gave particularly striking results. A 70% decrease caused a 5-fold increase in MEK/ERK activation and collagen expression. The combined data reveal a branched signaling pathway. In its central portion MEK activates ERK, leading to increased collagen expression. Two branches converge on MEK/ERK. In one, increased PKCepsilon leads to MEK/ERK activation. In another, increased PKCalpha induces caveolin-1 expression, which in turn inhibits MEK/ERK activation and collagen expression. Lung fibroblasts from scleroderma patients with pulmonary fibrosis showed altered signaling. Consistent with their overexpression of collagen, scleroderma lung fibroblasts contain more activated MEK/ERK and less caveolin-1 than normal lung fibroblasts. Because cutaneous fibrosis is the hallmark of scleroderma, we also studied dermal fibroblasts. As in lung, there was more activated MEK/ERK in cells from scleroderma patients than in control cells, and MEK inhibition decreased collagen expression. However, the distinctive levels of PKCepsilon, PKCalpha, and caveolin-1 in lung and dermal fibroblasts from scleroderma patients and control subjects indicate that the links between these signaling proteins and MEK/ERK must function differently in the four cell types. Finally, we confirmed the relevance of these signaling cascades in vivo. The combined results demonstrate that a branched signaling pathway involving MEK, ERK, PKCepsilon, PKCalpha, and caveolin-1 regulates collagen expression in normal lung tissue and is perturbed during fibrosis.

A method to detect and quantify curcumin and two curcuminoid metabolites in biological matrices, including mouse serum and mouse lung cell cultures, was developed. Standard curves between 0.04 and 10.00 nmol curcumin were prepared in serum, giving correlation coefficients of 0.94-0.99. Alcoholic extraction, concentration, and addition of dilute hydrochloric acid to stabilize the curcumin were essential to the reproducibility of the protocol. Untreated and curcumin-treated mouse lung fibrotic and nonfibrotic cell cultures were analyzed by matrix-assisted laser desorption ionization time of flight mass spectrometry utilizing this method. Curcumin uptake was calculated to be 7.0-11.6% for the saline-treated cells and 7.4-11.9% for the bleomycin-treated cultures. Curcumin was not detected in untreated cells. Two additional peaks (m/z=399 and 429) were observed in the curcumin-treated cells. These may be curcumin-derived products resulting from HCl treatment of the tissue samples.

Scleroderma, a disease involving excessive collagen deposition, can be studied using fibroblasts cultured from affected tissues. We find that curcumin, the active component of the spice turmeric, causes apoptosis in scleroderma lung fibroblasts (SLF), but not in normal lung fibroblasts (NLF). This effect is likely to be linked to the fact that although curcumin induces the expression of the phase 2 detoxification enzymes heme oxygenase 1 and glutathione S-transferase P1 (GST P1) in NLF, SLF are deficient in these enzymes, particularly after curcumin treatment. The sensitivity of cells to curcumin-induced apoptosis and the expression of GST P1 (but not heme oxygenase 1) are regulated by the epsilon isoform of protein kinase C (PKCepsilon). SLF, which contain less PKCepsilon and less GST P1 than NLF, become less sensitive to curcumin-induced apoptosis and express higher levels of GST P1 when transfected with wild-type PKCepsilon, but not with dominant-negative PKCepsilon. Conversely, NLF become sensitive to curcumin-induced apoptosis and express lower levels of GST P1 when PKCepsilon expression or function is inhibited. The subcellular distribution of PKCepsilon also differs in NLF and SLF. PKCepsilon is predominantly nuclear or perinuclear in NLF but is associated with stress fibers in SLF. Just as PKCepsilon levels are lower in SLF than in NLF in vitro, PKCepsilon expression is decreased in fibrotic lung tissue in vivo. In summary, our results suggest that a signaling pathway involving PKCepsilon and phase 2 detoxification enzymes provides protection against curcumin-induced apoptosis in NLF and is defective in SLF. These observations suggest that curcumin may have therapeutic value in treating scleroderma, just as it has already been shown to protect rats from lung fibrosis induced by a variety of agents.

Activated fibroblasts, or myofibroblasts, are crucial players in tissue remodeling, wound healing, and various fibrotic disorders, including interstitial lung fibrosis associated with scleroderma. Here we characterize the signaling pathways in normal lung fibroblasts exposed to thrombin as they acquire two of the main features of myofibroblasts: smooth muscle (SM) alpha-actin organization and collagen gel contraction. Our results show that the small G protein Rho is involved in lung myofibroblast differentiation. Thrombin induces Rho-35S-labeled guanosine 5'-O-(3-thiotriphosphate) binding in a dose-dependent manner. It potently stimulates Rho activity in vivo and initiates protein kinase C (PKC)-epsilon-Rho complex formation. Toxin B, which inactivates Rho by ADP ribosylation, inhibits thrombin-induced SM alpha-actin organization, collagen gel contraction, and PKC-epsilon-SM alpha-actin and PKC-epsilon-RhoA coimmunoprecipitation. However, it has no effect on PKC-epsilon activation or translocation of PKC-epsilon to the membrane. Overexpression of constitutively active PKC-epsilon and constitutively active RhoA induces collagen gel contraction or SM alpha-actin organization, whereas, individually, they do not perform these functions. We therefore conclude that the contractile activity of myofibroblasts induced by thrombin is mediated via PKC-epsilon- and RhoA-dependent pathways and that activation of both of these molecules is required. We postulate that PKC-epsilon-RhoA complex formation is an early event in thrombin activation of lung fibroblasts, followed by PKC-epsilon-SM alpha-actin coimmunoprecipitation, which leads to the PKC-epsilon-RhoA-SM alpha-actin ternary complex formation.

Myofibroblasts are ultrastructurally and metabolically distinctive fibroblasts that express smooth muscle (SM)-alpha actin and are associated with various fibrotic lesions. The present study was undertaken to investigate the myofibroblast phenotype that appears after activation of normal lung fibroblasts by thrombin. We demonstrate that thrombin induces smooth muscle-alpha actin expression and rapid collagen gel contraction by normal lung fibroblasts via the proteolytically activated receptor-1 and independent of transforming growth factor-beta pathway. Using antisense oligonucleotides we demonstrate that a decreased level of PKCepsilon abolishes SM-alpha actin expression and collagen gel contraction induced by thrombin in normal lung fibroblasts. Inhibition of PKCepsilon translocation also abolishes thrombin-induced collagen gel contraction, SM-alpha actin increase, and its organization by normal lung fibroblasts, suggesting that activation of PKCepsilon is required for these effects. In normal lung fibroblasts PKCepsilon binds to SM-alpha actin after thrombin treatment, but in activated fibroblasts derived from scleroderma lung they associate even in untreated cells. This suggests that SM-alpha actin may serve as a substrate for PKCepsilon in lung fibroblasts when activated by thrombin. We propose that thrombin differentiates normal lung fibroblasts to a myofibroblast phenotype via a PKC-dependent pathway. Thrombin-induced differentiation of normal lung fibroblasts to a myofibroblast phenotype resembles the phenotype observed in scleroderma lung fibroblasts. Therefore, we conclude that chronic exposure to thrombin after microvascular injury leads to activation of normal lung fibroblasts and to the appearance of a myofibroblast phenotype in vivo. Our study provides novel, compelling evidence that thrombin is an important mediator of the interstitial lung fibrosis associated with scleroderma.

Objective: To determine whether the extracellular matrix protein tenascin-C (TN-C) is overexpressed in lung fibroblasts from systemic sclerosis (SSc) patients, the molecular mechanisms regulating TN-C secretion in SSc and normal lung fibroblasts, and how these processes might contribute to lung fibrosis in SSc patients., Methods: TN-C secretion by SSc and normal fibroblasts was compared in vivo (in bronchoalveolar lavage [BAL] fluid) and in vitro (in culture medium). The ability of thrombin to induce TN-C was confirmed at both the protein and the messenger RNA (mRNA) level. The role of protein kinase Cepsilon (PKCepsilon) in the expression of TN-C was evaluated by determining the effects of thrombin on PKCepsilon levels and by directly manipulating PKCepsilon levels via the use of antisense oligonucleotides., Results: BAL fluid from SSc patients contained high levels of TN-C, whereas that from normal subjects contained little or no TN-C. In vitro, SSc lung fibroblasts expressed much higher amounts of TN-C than did normal lung fibroblasts. Consistent with the idea that thrombin is a physiologic inducer of TN-C, thrombin stimulated TN-C mRNA and protein expression in both SSc and normal lung fibroblasts by a mechanism that required proteolytic cleavage of the thrombin receptor. Surprisingly, thrombin treatment and antisense oligonucleotide-mediated depletion of PKCepsilon indicated that TN-C expression is regulated via opposite signaling mechanisms in SSc and normal cells. In SSc lung fibroblasts, thrombin decreased PKCepsilon levels, and the decreased PKCepsilon induced TN-C secretion; in normal fibroblasts, thrombin increased PKCepsilon levels, and the increased PKCepsilon induced TN-C secretion. Normal and SSc lung fibroblasts also differed in the subcellular localization of PKCepsilon, both before and after thrombin treatment., Conclusion: These studies are the first to demonstrate that thrombin is a potent simulator of TN-C in lung fibroblasts and that PKCepsilon is a critical regulator of TN-C protein levels in these cells. Furthermore, our results indicate that both the regulation of PKCepsilon levels by thrombin and the regulation of TN-C levels by PKCepsilon are defective in SSc lung fibroblasts.

Acute and chronic interstitial lung diseases are accompanied by evidence of inflammation and vascular injury. Thrombin activity in bronchoalveolar lavage fluid from such conditions is often increased, as well as interleukin (IL)-8. We observed that conditioned medium from lung fibroblasts exposed to thrombin has chemotactic activity for polymorphonuclear cells, and that this activity can be abolished by antibody to IL-8. We report that thrombin stimulates expression of IL-8 in human lung fibroblasts on both the messenger RNA and protein levels in a time- and dose-dependent manner. Stimulation of IL-8 expression by thrombin is inhibited by specific thrombin inhibitors. Synthetic thrombin receptor agonist peptide-14 mimics thrombin's stimulation of IL-8 expression in a dose-dependent manner consistent with the idea that upregulation of IL-8 by thrombin in human lung fibroblasts requires cleavage of proteolytically activated receptor-I. We demonstrate further that thrombin-induced IL-8 synthesis is regulated by protein kinase (PK) C. PKC-gamma may be involved in the upregulation of lung fibroblast IL-8 by thrombin because stimulation of lung fibroblasts with thrombin caused significant upregulation of PKC-gamma and because PKC-gamma antisense oligonucleotides inhibited the accumulation of PKC-gamma protein and IL-8 protein. Our data suggest that the PKC-gamma isoform increase observed after thrombin stimulation is required for thrombin-induced IL-8 formation by human lung fibroblasts.

The eosinophilia-myalgia syndrome was associated with the ingestion of L-tryptophan products containing a number of contaminants, one of which has been identified as 1,1'-ethylidene-bis-(L-tryptophan) (EBT), also known as peak E or peak 97. In earlier studies, we demonstrated that EBT induces inflammation and fibrosis in dermal and subcutaneous tissue of C57BL/6 mice. Others have shown EBT to be a potent stimulus for fibroblast activation and collagen synthesis in vitro, and dermal tissue from EMS patients reveals evidence of enhanced collagen gene expression. In the present study using Northern blot analysis and in situ hybridization, we demonstrate enhanced expression of genes for types I, III, and VI collagen in the dermis and subcutis of C57BL/6 mice treated with EBT for 3-21 days. Increased type I procollagen mRNA was noted on day 6 of EBT treatment and was followed by enhanced expression of type III and VI procollagen mRNA at day 21. L-Tryptophan, free of contaminants associated with the eosinophilia-myalgia syndrome epidemic, increased dermal collagen mRNA to a lesser extent than did EBT. Increased procollagen gene expression was accompanied by evidence of enhanced TGF-beta 1 expression in the dermis and subcutis. This animal model provides additional evidence for EBT as a causal agent of the eosinophilia-myalgia syndrome and should prove useful in the study of the pathogenesis of that syndrome.

Taxol-resistant sublines of HL-60 myeloid leukemia cells (HL-60/TAX100 and HL-60/TAX1000) have been isolated in vitro by subculturing in progressively higher concentrations of taxol. HL-60/TAX100 and HL-60/TAX1000 cells are capable of continuous growth in the presence of 0.1 microM and 1.0 microM taxol, respectively, and the IC50 (50% growth inhibitory dose) values for taxol for the two sublines are 0.34 and 2.44 microM as compared to 3.1 nM for the parent HL-60 cells. HL-60/TAX100 and HL-60/TAX1000 cells display a variable degree of cross-resistance to taxotere, vincristine and doxorubicin, but are sensitive to the antimetabolite Ara-C. Both HL-60/TAX100 and HL-60/TAX1000 cells over-express MDR-1 m-RNA and the membrane efflux multidrug transporter P-glycoprotein (PGP), as determined by Western blot and immunofluorescence labeling with anti-PGP antibodies. Consequently, exposure of the taxol-resistant cells to [3H]taxol or daunomycin results in the accumulation of significantly lower levels of the two drugs. Co-treatment with cyclosporine (0.5 microgram/ml) or verapamil (10 microM) partially overcomes taxol resistance in HL-60/TAX1000 cells. Following treatment with clinically relevant concentration of taxol (1.0 microM for 24 h), HL-60 but not HL-60/TAX1000 cells display intracellular microtubular bundling, markedly enhanced accumulation of the cells in G2/M phase of cell-cycle and internucleosomal DNA fragmentation associated with apoptosis which is independent of bcl-2 gene expression. These taxol-resistant myeloid leukemia cells may serve as in vitro experimental models for examinating strategies which may have potential applicability for overcoming taxol resistance.

In autologous bone marrow transplantation in patients with acute myeloid leukemia (AML), 4-hydroperoxycyclophosphamide (4-HC) is a commonly used ex vivo purging agent for leukemic blasts. In the present report, we demonstrate that exposure to high concentrations of 4-HC for 1 hour, as used in ex vivo bone marrow purging, produces internucleosomal DNA fragmentation characteristic of apoptosis, or programmed cell death (PCD), in human myeloid leukemia HL60 cells. Lower concentrations of 4-HC (10, 20, or 50 microM/L) failed to cause this effect, while higher concentrations (> or = 200 microM/L) produced random DNA fragmentation. 4-HC-mediated internucleosomal DNA fragmentation was associated with a marked induction in c-jun and significant reductions in bcl-2 and c-myc oncogene expressions. A combined treatment with interleukin-3 (IL-3) plus IL-6 for 18 hours before an additional, 1-hour concurrent treatment with 4-HC (100 microM/L) significantly increased 4-HC-induced DNA fragmentation as well as colony growth inhibition of HL60 cells. The effects of cotreatment with IL-3 plus IL-6 were also associated with a further, modest decrease in bcl-2 and c-myc and augmentation of c-jun expression. These findings highlight an alternative mechanism of 4-HC-induced leukemic cell death that can be potentially enhanced by cotreatment with IL-3 plus IL-6.

Mitoxantrone has been shown in vitro to exhibit a steep dose-response relationship with respect to the clonogenic survival of acute myeloid leukemia cells. In this report, we show that 1-hour exposure of human myeloid leukemia HL-60 and KG-1 cells to mitoxantrone concentrations ranging between 0.1 and 10.0 mumol/L induced internucleosomal DNA fragmentation of approximately 200-bp integer multiples, characteristic of cells undergoing programmed cell death (PCD) or apoptosis. Mitoxantrone-mediated PCD was associated with a steep inhibition of the clonogenic survival of the leukemic cells. In addition, intracellularly, mitoxantrone-induced PCD was associated with a marked induction of c-jun and significant repression of c-myc and BCL-2 oncogenes. Pretreatment with the protein kinase C stimulator phorbol myristate acetate enhanced mitoxantrone-induced internucleosomal DNA fragmentation, whereas protein kinase C inhibitors staurosporine and H7 had no effect. These findings suggest that PCD is a potential mechanism underlying the steep dose-response relationship of mitoxantrone to the inhibition of clonogenic survival of acute myeloid leukemia cells.

The present results demonstrate that the exposure of human myeloid leukemia HL-60 and KG-1 cells to clinically achievable concentrations of taxol produced internucleosomal DNA fragmentation of approximately 200 base-pair multiples, and the morphologic changes characteristic of cells undergoing programmed cell death (PCD) or apoptosis. Taxol-induced PCD was associated with a marked inhibition of suspension culture growth and clonogenic survival of HL-60 cells. In addition, taxol treatment decreased BCL-2 oncogene expression, which is known to block PCD. The exposure to taxol moderately decreased c-myc expression, but did not induce c-jun expression--which has been previously noted for a variety of DNA interactive, antileukemic drugs. These findings indicate that taxol may induce leukemic cell death partly by the alternative but gene-directed and active mechanism of PCD.

Recently, high dose Ara-C (HIDAC) has been shown to induce leukemic cell death in vitro by the alternative process of programmed cell death (PCD) or apoptosis which correlates with the inhibition of their clonogenic survival. Since co-treatment with hemopoietic growth facts (HGFs) GM-CSF and IL-3 have been demonstrated to enhance the metabolism and cytotoxic effects of HIDAC against leukemic progenitor cells, we examined the effect of HGFs pIXY 321 (a GM-CSF/IL3 fusion protein) and G-CSF on HIDAC induced PCD and related gene expressions as well as HIDAC mediated colony growth inhibition of human myeloid leukemia cells. Treatment with G-CSF or pIXY 321 alone for up to 24 hours neither suppressed nor induced PCD in HL-60 or KG-1 cells. However, exposure to either of the HGFs for 20 hours followed by a combined treatment for 4 hours with HIDAC plus either of the HGFs versus HIDAC alone significantly enhanced the intracellular Ara-CTP accumulation and the oligonucleosomal DNA fragmentation characteristic of PCD. This was temporally associated with a marked induction of C-jun expression but a significant repression in BCL-2 and c-myc expressions. In addition, the treatment with either of the HGFs plus HIDAC versus HIDAC alone produced a significantly greater inhibition of the clonogenic survival of the myeloid leukemia cells. These findings underscore an additional mechanism of leukemic cell death induced by HIDAC which can be modulated by the HGFs to improve the antileukemic activity of HIDAC.

High dose Ara-C (HIDAC) induces programmed cell death (PCD) or apoptosis in vitro in human myeloid leukemia cells, which correlates with the inhibition of their clonogenic survival. Hematopoietic growth factors (HGFs) granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3) have been demonstrated to enhance the metabolism and cytotoxic effects of HIDAC against leukemic progenitor cells. We examined the effect of pIXY 321 (a GM-CSF/IL-3 fusion protein) on HIDAC-induced PCD and related gene expressions as well as HIDAC-mediated colony growth inhibition of human myeloid leukemia cells. Unlike the previously described effects of HGFs on normal bone marrow progenitor cells, exposure to pIXY 321 alone for up to 24 hours did not suppress PCD in HL-60 or KG-1 cells. However, exposure to pIXY 321 for 20 hours followed by a combined treatment with Ara-C plus pIXY 321 for 4 or 24 hours versus treatment with Ara-C alone significantly enhanced the oligonucleosomal DNA fragmentation characteristic of PCD. This was temporally associated with a marked induction of c-jun expression and a significant decrease in BCL-2. In addition, the treatment with pIXY 321 plus HIDAC versus HIDAC alone produced a significantly greater inhibition of HL-60 colony growth. These findings highlight an additional mechanism of HIDAC-induced leukemic cell death that is augmented by cotreatment with pIXY 321 and may contribute toward an improved antileukemic activity of HIDAC.