Sem H. Phan

Bone marrow–derived progenitor cells in pulmonary fibrosis

The origin of fibroblasts in pulmonary fibrosis is assumed to be intrapulmonary, but their extrapulmonary origin and especially derivation from bone marrow (BM) progenitor cells has not been ruled out. To examine this possibility directly, adult mice were durably engrafted with BM isolated from transgenic mice expressing enhanced GFP. Induction of pulmonary fibrosis in such chimera mice by endotracheal bleomycin (BLM) injection caused large numbers of GFP(+) cells to appear in active fibrotic lesions, while only a few GFP(+) cells could be identified in control lungs. Flow-cytometric analysis of lung cells confirmed the BLM-induced increase in GFP(+) cells in chimera mice and revealed a significant increase in GFP(+) cells that also express type I collagen. GFP(+) lung fibroblasts isolated from chimera mice expressed collagen and telomerase reverse transcriptase but not alpha-smooth muscle actin. Treatment of isolated GFP(+) fibroblasts with TGF-beta failed to induce myofibroblast differentiation. Cultured lung fibroblasts expressed the chemokine receptors CXCR4 and CCR7 and responded chemotactically to their cognate ligands, stromal cell-derived factor-1 alpha and secondary lymphoid chemokine, respectively. Thus the collagen-producing lung fibroblasts in pulmonary fibrosis can also be derived from BM progenitor cells.

Recent developments in myofibroblast biology: paradigms for connective tissue remodeling.

The discovery of the myofibroblast has opened new perspectives for the comprehension of the biological mechanisms involved in wound healing and fibrotic diseases. In recent years, many advances have been made in understanding important aspects of myofibroblast basic biological characteristics. This review summarizes such advances in several fields, such as the following: i) force production by the myofibroblast and mechanisms of connective tissue remodeling; ii) factors controlling the expression of α-smooth muscle actin, the most used marker of myofibroblastic phenotype and, more important, involved in force generation by the myofibroblast; and iii) factors affecting genesis of the myofibroblast and its differentiation from precursor cells, in particular epigenetic factors, such as DNA methylation, microRNAs, and histone modification. We also review the origin and the specific features of the myofibroblast in diverse fibrotic lesions, such as systemic sclerosis; kidney, liver, and lung fibrosis; and the stromal reaction to certain epithelial tumors. Finally, we summarize the emerging strategies for influencing myofibroblast behavior in vitro and in vivo, with the ultimate goal of an effective therapeutic approach for myofibroblast-dependent diseases.

Costimulation of Fibroblast Collagen and Transforming Growth Factor β1 Gene Expression by Monocyte Chemoattractant Protein-1 via Specific Receptors

Recent studies indicate potential roles of monocyte chemotactic protein-1 (MCP-1) in recruitment of monocytes to sites of inflammation. However, their increased expression does not always correlate with monocyte influx, suggesting other possible biological activities for this member of the C-C chemokine family. In view of its potential role in regulating extracellular matrix expression in fibrotic disorders, the effects of MCP-1 on lung fibroblast collagen expression were evaluated. Isolated rat lung fibroblasts were treated with increasing doses of MCP-1 for variable periods of time and examined for effects on collagen synthesis and expression of procollagen α1(I) mRNA expression. The results show that MCP-1 was able to stimulate collagen expression in these cells in a dose-dependent manner but required over 24 h for significant elevation to occur. In view of this delayed time course, the possibility of mediation via endogenous transforming growth factor β (TGFβ) was tested by the ability of anti-TGFβ antibody to inhibit this MCP-1 stimulation of collagen expression. Significant but incomplete inhibition by this antibody was observed. Pretreatment of the cells with antisense but not by sense or missense TGFβ1 oligodeoxyribonucleotides caused essentially complete inhibition of this MCP-1 stimulatory effect. Furthermore, MCP-1 treatment was found to also stimulate TGFβ secretion and mRNA expression, which was also abolished by pretreatment with antisense TGFβ1 oligodeoxyribonucleotides. The kinetics of TGFβ expression indicates that significant increase preceded that for collagen expression. Binding studies using 125I-labeled MCP-1 indicated the presence of specific and saturable binding sites with a dissociation constant consistent with the dose response curves for stimulation of fibroblast collagen synthesis and TGFβ activity by MCP-1. These results taken together suggest that MCP-1 stimulates fibroblast collagen expression via specific receptors and endogenous up-regulation of TGFβ expression. The latter then results in autocrine and/or juxtacrine stimulation of collagen gene expression.

Lung cytokine production in bleomycin-induced pulmonary fibrosis.

In bleomycin-induced pulmonary fibrosis, lung injury is accompanied with inflammation and subsequent fibrosis. In this study, lung mRNA for several cytokines was measured in bleomycin-treated mice to evaluate their roles in lung fibrosis. Significant increases in tumor necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta (TGF-beta) mRNA were found in lungs of bleomycin-treated responder CBA mice but not in nonresponder BALB/c mice. Increases in responder animals peaked on day 7 after bleomycin administration, and subsequently returned toward control levels. This time course paralleled that for the increase in beta-actin mRNA, but preceded the peak increase in mRNA for collagens I and III. When lung macrophages were analyzed for cytokine secretion, differences were observed between alveolar macrophages and interstitial cells, and between cells from bleomycin-responsive CBA and nonresponsive BALB/c mice. Only alveolar macrophages from CBA mice secreted increased amounts of IL-1. TNF-alpha activity was increased in conditioned media of alveolar and interstitial cells of CBA mice, while only alveolar macrophages of nonresponder BALB/c mice secreted any activity. The kinetics of the increased secretion of TNF-alpha was dissimilar for these different cells. These results are consistent with the conclusion that increased production of TNF-alpha and TGF-beta is an important component of the fibrotic process.

Nephrogenic fibrosing dermopathy: a novel cutaneous fibrosing disorder in patients with renal failure

BACKGROUND Nephrogenic fibrosing dermopathy is a newly recognized cutaneous fibrosing disorder marked by the acute onset of induration involving the upper and lower limbs in patients with acute or chronic renal failure. The etiology, pathogenesis, associated clinical conditions (other than renal failure), and ultimate course have not been defined in the few cases studied. Presently, there is no effective treatment, and the condition persists in most patients. METHODS Clinical and histopathologic data on 13 patients from our institution with the diagnosis of nephrogenic fibrosing dermopathy were reviewed. Several clinical and laboratory parameters were examined to see if any were consistently associated with the disease. Biopsy specimens were analyzed to determine if there was a pattern to the evolution of fibrosis in these patients. RESULTS All 13 patients had renal failure before disease onset: 8 were undergoing chronic hemodialysis, 2 were undergoing chronic peritoneal dialysis, and 3 with acute renal failure had never undergone dialysis before the development of dermopathy. Most patients had other serious underlying medical conditions. Many patients were taking erythropoietin, cyclosporine, or both before the onset of disease. In transplant patients, no histocompatibility antigens were found to be associated with the disease. There were various laboratory abnormalities, but none were consistently associated with the condition. In skin biopsy specimens taken 7 to 180 days after disease onset, there were histopathologic changes suggestive of a tissue reaction to injury, as well as the development of smooth muscle actin-positive myofibroblasts. CONCLUSION Nephrogenic fibrosing dermopathy is a novel cutaneous fibrosing disorder that is distinguished from other sclerosing or fibrosing skin disorders by distinctive clinical and histopathologic findings occurring in the setting of renal failure. There were no additional clinical risk factors or laboratory findings common to the 13 patients studied, other than renal failure. The resemblance to a tissue injury reaction and the presence of myofibroblasts in the tissue specimens suggest that fibrogenic cytokines may be involved in the evolution of the disease.

Endothelial–Mesenchymal Transition in Bleomycin-Induced Pulmonary Fibrosis

The pathological hallmark lesions in idiopathic pulmonary fibrosis are the fibroblastic foci, in which fibroblasts are thought to be involved in the tissue remodeling, matrix deposition, and cross-talk with alveolar epithelium. Recent evidence indicates that some fibroblasts in fibrosis may be derived from bone marrow progenitors as well as from epithelial cells through epithelial-mesenchymal transition. To evaluate whether endothelial cells could represent an additional source for fibroblasts, bleomycin-induced lung fibrosis was established in Tie2-Cre/CAG-CAT-LacZ double-transgenic mice, in which LacZ was stably expressed in pan-endothelial cells. Combined X-gal staining and immunocytochemical staining for type I collagen and alpha-smooth muscle actin revealed the presence of X-gal-positive cells in lung fibroblast cultures from bleomycin-treated mice. To explore the underlying mechanisms, by which loss of endothelial-specific markers and gain of mesenchymal phenotypes could be involved in microvascular endothelial cells, the effects of activated Ras and TGF-beta on the microvascular endothelial cell line MS1 were analyzed. Combined treatment with activated Ras and TGF-beta caused a significant loss of endothelial-specific markers, while inducing de novo mesenchymal phenotypes. The altered expression of these markers in MS1 cells with activated Ras persisted after withdrawal of TGF-beta in vitro and in vivo. These findings are the first to show that lung capillary endothelial cells could give rise to significant numbers of fibroblasts through an endothelial-mesenchymal transition in bleomycin-induced lung fibrosis model.

Dual Roles of IL-4 in Lung Injury and Fibrosis

Increased lung IL-4 expression in pulmonary fibrosis suggests a potential pathogenetic role for this cytokine. To dissect this role, bleomycin-induced pulmonary inflammation and fibrosis were analyzed and compared in wild type (IL-4+/+) vs IL-4-deficient (IL-4−/−) mice. Lethal pulmonary injury after bleomycin treatment was higher in IL-4−/− vs IL-4+/+ mice. By administration of anti-CD3 Abs, we demonstrated that this early response was linked to the marked T lymphocyte lung infiltration and to the overproduction of the proinflammatory mediators such as TNF-α, IFN-γ, and NO in IL-4−/− mice. In contrast to this early anti-inflammatory/immunosuppressive role, during later stages of fibrosis, IL-4 played a profibrotic role since IL-4−/− mice developed significantly less pulmonary fibrosis relative to IL-4+/+ mice. However, IL-4 failed to directly stimulate proliferation, α-smooth muscle actin, and type I collagen expression in lung fibroblasts isolated from the wild-type mice. Upon appropriate stimulation with other known fibrogenic cytokines, fibroblasts from IL-4−/− mice were relatively deficient in the studied parameters in comparison to fibroblasts isolated from IL-4+/+ mice. Taken together, these data suggest dual effects of IL-4 in this model of lung fibrosis: 1) limiting early recruitment of T lymphocytes, and 2) stimulation of fibrosis chronically.

FIZZ1 Stimulation of Myofibroblast Differentiation

Bleomycin-induced pulmonary fibrosis is characterized by inflammation, emergence of myofibroblasts, and deposition of extracellular matrix. In an attempt to identify genes that may be involved in fibrosis, we used a 10,000 element (10 K) rat cDNA microarray to analyze the lung gene expression profiles in this model in the rat. Cluster analysis showed 628 genes were more than or equal to twofold up- or down-regulated, many of which were known to be involved in fibrosis. However, the most dramatic increase was observed with FIZZ1 (found in inflammatory zone; also known as RELM-alpha or resistin-like molecule-alpha), which was induced 17-fold to approximately 25-fold at the peak of expression. In situ hybridization analysis revealed FIZZ1 expression to localize primarily to alveolar and airway epithelium, which was confirmed in vitro by analysis of isolated type II alveolar epithelial cells. However FIZZ1 expression was not detected in isolated lung fibroblasts. Co-culture of FIZZ1-expressing type II cells with fibroblasts stimulated alpha-smooth muscle actin and type I collagen expression independent of transforming growth factor-beta. Transfection of a FIZZ1-expressing plasmid into fibroblasts or treatment with glutathione S-transferase-FIZZ1 fusion protein stimulated alpha-smooth muscle actin and collagen I production. These results suggest a novel role for FIZZ1 in myofibroblast differentiation in pulmonary fibrosis.

Kielin/chordin-like protein, a novel enhancer of BMP signaling, attenuates renal fibrotic disease

The bone morphogenetic proteins (BMPs) profoundly affect embryonic development, differentiation and disease. BMP signaling is suppressed by cysteine-rich domain proteins, such as chordin, that sequester ligands from the BMP receptor. We describe a novel protein, KCP, with 18 cysteine-rich domains. Unlike chordin, KCP enhances BMP signaling in a paracrine manner. Smad1-dependent transcription and phosphorylated Smad1 (P-Smad1) levels are increased, as KCP binds to BMP7 and enhances binding to the type I receptor. In vivo, Kcp−/− mice are viable and fertile. Because BMPs have a pivotal role in renal disease, we examined the phenotype of Kcp−/− mice in two different models of renal injury. Kcp−/− animals show reduced levels of P-Smad1, are more susceptible to developing renal interstitial fibrosis, are more sensitive to tubular injury and show substantial pathology after recovery. The data indicate an important role for KCP in attenuating the pathology of renal fibrotic disease.

Regulation of Found in Inflammatory Zone 1 Expression in Bleomycin-Induced Lung Fibrosis: Role of IL-4/IL-13 and Mediation via STAT-6

Found in inflammatory zone (FIZZ)1, also known as resistin-like molecule α, belongs to a novel class of cysteine-rich secreted protein family, named FIZZ/resistin-like molecule, with unique tissue expression patterns. FIZZ1 is induced in alveolar type II epithelial cells (AECs) in bleomycin (BLM)-induced lung fibrosis, and found to induce myofibroblast differentiation in vitro. The objective of this study was to elucidate the regulation of AEC FIZZ1 expression in pulmonary fibrosis. AECs were isolated from rat lungs and the effects of a number of cytokines on FIZZ1 expression were evaluated by RT-PCR. Of all cytokines examined, only IL-4 and IL-13 were effective in stimulating FIZZ1 expression in AECs. Stimulation by IL-4/IL-13 was accompanied by increases in phosphorylated STAT6 and JAK1. FIZZ1 expression was also stimulated by transfection with a STAT6 expression plasmid, but was inhibited by antisense oligonucleotides directed against STAT6. In vivo studies showed that compared with wild-type controls, both IL-4- and IL-13-deficient mice showed reduced BLM-induced lung FIZZ1 expression and fibrosis, which were essentially abolished in IL-4 and IL-13 doubly deficient mice. Furthermore, STAT6-deficient mice showed marked reduction in BLM-induced lung FIZZ1 expression. Thus, IL-4 and IL-13 are potent inducers of AEC FIZZ1 expression via STAT6 and play key roles in BLM-induced lung FIZZ1 expression and fibrosis. This represents a potential mechanism by which IL-4/IL-13 could play a role in the pathogenesis of lung fibrosis.