Lee R, Reese C, Bonner M, Tourkina E, Hajdu Z, Riemer EC, Silver RM, Visconti RP, Hoffman S. Bleomycin delivery by osmotic minipump: similarity to human scleroderma interstitial lung disease.

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.

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 mono-nuclear cells were incubated on fibronectin-coated plates. Protein expression was evaluated by immuno-histochemistry 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.

BackgroundA 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.ResultsExpression 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.ConclusionsTo 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13069-015-0028-7) contains supplementary material, which is available to authorized users.

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 CD45moderate/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.