A genome-wide analysis of the response to inhaled ß2-agonists in chronic obstructive pulmonary disease.

Short-acting ß2-agonist bronchodilators are the most common medications used in treating chronic obstructive pulmonary disease (COPD). Genetic variants determining bronchodilator responsiveness (BDR) in COPD have not been identified. We performed a genome-wide association study (GWAS) of BDR in 5789 current or former smokers with COPD in one African-American and four white populations. BDR was defined as the quantitative spirometric response to inhaled ß2-agonists. We combined results in a meta-analysis. In the meta-analysis, single-nucleotide polymorphisms (SNPs) in the genes KCNK1 (P=2.02 × 10(-7)) and KCNJ2 (P=1.79 × 10(-7)) were the top associations with BDR. Among African Americans, SNPs in CDH13 were significantly associated with BDR (P=5.1 × 10(-9)). A nominal association with CDH13 was identified in a gene-based analysis in all subjects. We identified suggestive association with BDR among COPD subjects for variants near two potassium channel genes (KCNK1 and KCNJ2). SNPs in CDH13 were significantly associated with BDR in African Americans.The Pharmacogenomics Journal advance online publication, 27 October 2015; doi:10.1038/tpj.2015.65.

Sphingosylphosphorylcholine induces α-smooth muscle actin expression in human lung fibroblasts and fibroblast-mediated gel contraction via S1P2 receptor and Rho/Rho-kinase pathway.

Chronic airway diseases like COPD and asthma are usually accompanied with airway fibrosis. Myofibroblasts, which are characterized by expression of smooth muscle actin (α-SMA), play an important role in a variety of developmental and pathological processes, including fibrosis and wound healing. Sphingosylphosphorylcholine (SPC), a sphingolipid metabolite, has been implicated in many physiological and pathological conditions. The current study tested the hypothesis that SPC may modulate tissue remodeling by affecting the expression of α-SMA in human fetal lung fibroblast (HFL-1) and fibroblast mediated gel contraction. The results show that SPC stimulates α-SMA expression in HFL-1 and augments HFL-1 mediated collagen gel contraction in a time- and concentration-dependent manner. The α-SMA protein expression and fibroblast gel contraction induced by SPC was not blocked by TGF-ß1 neutralizing antibody. However, it was significantly blocked by S1P2 receptor antagonist JTE-013, the Rho-specific inhibitor C3 exoenzyme, and a Rho-kinase inhibitor Y-27632. These findings suggest that SPC stimulates α-SMA protein expression and HFL-1 mediated collagen gel contraction via S1P2 receptor and Rho/Rho kinase pathway, and by which mechanism, SPC may be involved in lung tissue remodeling.

Impact of non-linear smoking effects on the identification of gene-by-smoking interactions in COPD genetics studies.

BACKGROUND: The identification of gene-by-environment interactions is important for understanding the genetic basis of chronic obstructive pulmonary disease (COPD). Many COPD genetic association analyses assume a linear relationship between pack-years of smoking exposure and forced expiratory volume in 1 s (FEV(1)); however, this assumption has not been evaluated empirically in cohorts with a wide spectrum of COPD severity. METHODS: The relationship between FEV(1) and pack-years of smoking exposure was examined in four large cohorts assembled for the purpose of identifying genetic associations with COPD. Using data from the Alpha-1 Antitrypsin Genetic Modifiers Study, the accuracy and power of two different approaches to model smoking were compared by performing a simulation study of a genetic variant with a range of gene-by-smoking interaction effects. RESULTS: Non-linear relationships between smoking and FEV(1) were identified in the four cohorts. It was found that, in most situations where the relationship between pack-years and FEV(1) is non-linear, a piecewise linear approach to model smoking and gene-by-smoking interactions is preferable to the commonly used total pack-years approach. The piecewise linear approach was applied to a genetic association analysis of the PI*Z allele in the Norway Case-Control cohort and a potential PI*Z-by-smoking interaction was identified (p=0.03 for FEV(1) analysis, p=0.01 for COPD susceptibility analysis). CONCLUSION: In study samples of subjects with a wide range of COPD severity, a non-linear relationship between pack-years of smoking and FEV(1) is likely. In this setting, approaches that account for this non-linearity can be more powerful and less biased than the more common approach of using total pack-years to model the smoking effect.

Candidate genes for COPD in two large data sets.

Lack of reproducibility of findings has been a criticism of genetic association studies on complex diseases, such as chronic obstructive pulmonary disease (COPD). We selected 257 polymorphisms of 16 genes with reported or potential relationships to COPD and genotyped these variants in a case-control study that included 953 COPD cases and 956 control subjects. We explored the association of these polymorphisms to three COPD phenotypes: a COPD binary phenotype and two quantitative traits (post-bronchodilator forced expiratory volume in 1 s (FEV1) % predicted and FEV1/forced vital capacity (FVC)). The polymorphisms significantly associated to these phenotypes in this first study were tested in a second, family-based study that included 635 pedigrees with 1,910 individuals. Significant associations to the binary COPD phenotype in both populations were seen for STAT1 (rs13010343) and NFKBIB/SIRT2 (rs2241704) (p<0.05). Single-nucleotide polymorphisms rs17467825 and rs1155563 of the GC gene were significantly associated with FEV1 % predicted and FEV1/FVC, respectively, in both populations (p<0.05). This study has replicated associations to COPD phenotypes in the STAT1, NFKBIB/SIRT2 and GC genes in two independent populations, the associations of the former two genes representing novel findings.

Antibodies to laminin in Chagas' disease.

We have found that sera from humans with Chagas' disease and Rhesus monkeys infected with Trypanosoma cruzi contain IgM and IgG antibodies, which react with structures in a variety of connective tissues. These antibodies react with laminin but not with various other purified connective tissue components like collagen types I, III, IV, and V, fibronectin, heparan sulfate (BM-1) proteoglycan, or chondronectin. The tissue-reacting antibodies were isolated by absorption to a laminin-Sepharose column. The bound fraction contained all the tissue-reacting antibodies. These antibodies strongly stained trypomastigotes and amastigotes, but weakly stained epimastigotes. These studies show that sera from T. cruzi-infected primates contain antilaminin antibodies, which may be produced by those host in response to a laminin-like molecule present in the parasite.

Statins inhibit matrix metalloproteinase release from human lung fibroblasts.

Pleiotropic effects of statins have been reported to include inhibition of matrix metalloproteinase (MMP) release from macrophages and endothelial cells. We evaluated whether statins would inhibit MMP release from human lung fibroblasts, which play a major role in remodelling processes. Monolayer and three-dimensional (3D) collagen gel cultures of fibroblasts were used. Cytokines (tumour necrosis factor-alpha and interleukin-1alpha) were used to induce MMP release and mRNA expression. Collagen degradation induced by cytokines and neutrophil elastase (NE) was evaluated by quantifying hydroxyproline. Atorvastatin inhibited MMP-1 and -3 release and mRNA expression in both culture systems. Similar results were obtained with simvastatin and fluvastatin. In 3D cultures where cytokines also stimulated MMP-9 release, atorvastatin also inhibited MMP-9 release. In 3D cultures, cytokines together with NE induced collagen degradation, which was also inhibited by atorvastatin. The effect of atorvastatin was reversed by mevalonate and geranylgeranyl-pyrophosphate but not by farnesyl-pyrophosphate. The current data suggest that statins may modulate remodelling processes mediated by fibroblasts by inhibiting MMP release.

Heritability of lung function in severe alpha-1 antitrypsin deficiency.

Severe alpha-1 antitrypsin (AAT) deficiency is a proven genetic risk factor for COPD, but there is marked variation in the development of COPD among AAT deficient subjects. To investigate familial aggregation of lung function in subjects with AAT deficiency, we estimated heritability for forced expiratory volume in 1 s (FEV1) and FEV1/forced vital capacity (FVC) in 378 AAT deficient subjects from 167 families in the AAT Genetic Modifiers Study; all subjects were verified homozygous for the Z AAT deficiency allele. Heritability was evaluated for models that included and excluded an ascertainment correction, as well as for models that excluded, included and were stratified by a cigarette smoking covariate. In models without an ascertainment correction, and in all models without a covariate for smoking, no evidence for familial aggregation of lung function was observed. In models conditioned on the index proband with covariates for smoking, post-bronchodilator FEV1/FVC demonstrated significant heritability (0.26 +/- 0.14, p = 0.03). When we limited the analysis to subjects with a smoking history, post-bronchodilator FEV1 demonstrated significant heritability (0.47 +/- 0.21, p = 0.02). Severity rate phenotypes were also assessed as potential phenotypes for genetic modifier studies. Significant heritability was found with all age-of-onset threshold models that included smoking and ascertainment adjustments. Using the t-distribution, the heritability estimates ranged from 0.43 to 0.64, depending on the age-of-onset of FEV1 decline used for the severity rate calculation. Correction for ascertainment and consideration of gene-by-smoking interactions will be crucial for the identification of genes that may modify susceptibility for COPD in families with AAT deficiency.

Development of predictive models for airflow obstruction in alpha-1-antitrypsin deficiency.

Alpha-1-antitrypsin deficiency is a genetic condition associated with severe, early-onset chronic obstructive pulmonary disease (COPD). However, there is significant variability in lung function impairment among persons with the protease inhibitor ZZ genotype. Early identification of persons at highest risk of developing lung disease could be beneficial in guiding monitoring and treatment decisions. Using a multicenter, family-based study sample (2002-2005) of 372 persons with the protease inhibitor ZZ genotype, the authors developed prediction models for forced expiratory volume in 1 second (FEV(1)) and the presence of severe COPD using demographic, clinical, and genetic variables. Half of the data sample was used for model development, and the other half was used for model validation. In the training sample, variables found to be predictive of both FEV(1) and severe COPD were age, sex, pack-years of smoking, bronchodilator responsiveness, chronic bronchitis symptoms, and index case status. In the validation sample, the predictive model for FEV(1) explained 50% of the variance in FEV(1), and the model for severe COPD exhibited excellent discrimination (c statistic = 0.88).

3-Nitrotyrosine inhibits fibroblast-mediated collagen gel contraction and chemotaxis.

Reactive nitrogen species induce tissue inflammation and nitrate tyrosine residues of various kinds of proteins. Recent studies have established that the free amino acid form of 3-nitrotyrosine induces cytotoxity and growth inhibition and alters the cellular function in cultured cells. The aim of this study was to evaluate whether 3-nitrotyrosine could affect tissue remodelling in fibroblasts. To accomplish this, human fetal lung fibroblasts (HFL-1) were used to assess the fibroblast-mediated contraction of floating gels and chemotaxis towards fibronectin. In addition, the ability of fibroblasts to release fibronectin, transforming growth factor (TGF)-beta1, fibronectin and vascular endothelial growth factor (VEGF) was assessed. 3-Nitrotyrosine significantly inhibited gel contraction (p<0.01) compared with control and this inhibition was abolished by nitric oxide synthase (NOS) inhibitor. 3-Nitrotyrosine did not affect TGF-beta1 and VEGF but significantly decreased fibronectin release (p<0.01) into the media. 3-Nitrotyrosine significantly inhibited chemotaxis towards fibronectin through suppression of alpha(5)beta(1) integrin expression (p<0.01). NOS inhibitor also reversed 3-nitrotyrosine-inhibited chemotaxis (p<0.01). Finally, 3-nitrotyrosine enhanced the expression of the inducible type of NOS (p<0.01) and nitric oxide release (p<0.01) through nuclear factor-kappaB activation. These results suggest that the free amino acid form of 3-nitrotyrosine can affect the tissue repair process by modulating nitric oxide production.

Role of fibronectin as a growth factor for fibroblasts.

Fibroblast replication is regulated by exogenous signals provided by growth factors, mediators that interact with the target cell surface and signal the cell to proliferate. A useful model of growth regulation, the "dual control model," suggests that growth factors can be grouped either as competence factors or as progression factors, and that optimal replication of fibroblasts requires the presence of both types of growth factors. Although most growth factors are soluble mediators, recent studies have demonstrated that, for some cell types, the extracellular matrix can replace the requirement for a competence factor. Since fibronectin is an important constituent of the extracellular matrix that interacts with specific domains on the fibroblast surface, we examined the ability of fibronectin to act as a competence factor to promote the growth of human diploid fibroblasts. To accomplish this, fibronectins purified from two sources, human plasma and human alveolar macrophages, were tested for their ability to (a) stimulate fibroblast replication in serum-free medium containing characterized progression factors (insulin or alveolar macrophage-derived growth factor); (b) provide a growth-promoting signal early in G1. Fibronectin stimulated fibroblast replication in a dose-dependent manner in the presence of a fixed dose of a progression factor. Conversely, fibronectin conferred on previously unresponsive fibroblasts the ability to replicate in a dose-dependent manner when cultured with increasing amounts of a progression factor. Moreover, fibronectin signaled growth-arrested fibroblasts to traverse G1 approximately 4 h closer to S phase. No differences were observed in the ability of plasma or macrophage fibronectins to provide a competence signal for fibroblast replication. Since fibronectin is a major component of the extracellular matrix, these observations suggest that it may provide at least one of the signals by which the matrix conveys the "competence" that permits fibroblasts to replicate in the presence of an appropriate progression signal.

Elastin fragments attract macrophage precursors to diseased sites in pulmonary emphysema.

This study suggests one mechanism by which alveolar macrophages accumulate in the lung in pulmonary emphysema: elastin fragments generated at the diseased sites are potent chemoattractants for monocytes, the precursors of the macrophages. The most chemotactic elastin fragments have a molecular weight between 10,000 and 50,000 and are active at concentrations as low as 3 nanograms per milliliter. By comparison, elastin fragments with higher molecular weights and desmosines are active at concentrations greater than 0.3 microgram per milliliter. In addition, preincubation of monocytes with the 10,000- to 50,000-dalton elastin impairs the ability of the cells to migrate toward elastin fragments but not toward activated serum. Fragments of tropoelastin are not chemotactic for monocytes. Because elastin, but not tropoelastin, contains lysyl-derived cross-links, these structures may be the active chemotactic site on the elastin fragments.

Alternative mechanisms for tiotropium.

Tiotropium is commonly used in the treatment of chronic obstructive pulmonary disease. Although largely considered to be a long-acting bronchodilator, its demonstrated efficacy in reducing the frequency of exacerbations and preliminary evidence from early studies indicating that it might slow the rate of decline in lung function suggested mechanisms of action in addition to simple bronchodilation. This hypothesis was examined in the recently published UPLIFT study and, although spirometric and other clinical benefits of tiotropium treatment extended to four years, the rate of decline in lung function did not appear to be reduced by the addition of tiotropium in this study. This article summarizes data from a variety of investigations that provide insights into possible mechanisms to account for the effects of tiotropium. The report summarizes the discussion on basic and clinical research in this field.

Outcomes for COPD pharmacological trials: from lung function to biomarkers.

The American Thoracic Society/European Respiratory Society jointly created a Task Force on "Outcomes for COPD pharmacological trials: from lung function to biomarkers" to inform the chronic obstructive pulmonary disease research community about the possible use and limitations of current outcomes and markers when evaluating the impact of a pharmacological therapy. Based on their review of the published literature, the following document has been prepared with individual sections that address specific outcomes and markers, and a final section that summarises their recommendations.

Fibronectin in human bronchopulmonary lavage fluid. Elevation in patients with interstitial lung disease.

Fibronectin is a major adhesive and opsonic glycoprotein found in plasma and tissues. Because this molecule appears to mediate a number of interactions between cells and extracellular matrix, and because the interstitial lung disease are characterized by marked derangements of the pulmonary extracellular matrix, we evaluated fibronectin in the lower respiratory tract in patients with these disorders. Fibronectin could be detected in the bronchoalveolar lavage fluid of normals (11/11), as well as those with noninterstitial lung diseases (18/18), idiopathic pulmonary fibrosis (21/21), sarcoidosis (20/20), and other interstitial lung diseases (22/22). Compared with normal and those with noninterstitial lung disease, the levels in bronchoalveolar lavage of patients with interstitial disease were significantly higher (P less than 0.01), all comparisons). This was true only for bronchoalveolar lavage fibronectin; plasma levels were similar in all study groups (P greater than 0.2, all comparisons). The lavage fluid fibronectin was intact antigenically and retained collagen binding capability, although in some cases of interstitial disease, the presence of lower molecular weight fragments suggested some degradation. Thus, fibronectin is a normal constituent of the epithelial fluid of the lower respiratory tract and is present in increased amounts in a significant number of individuals with interstitial lung disease.

Human alveolar macrophage growth factor for fibroblasts. Regulation and partial characterization.

The number of fibroblasts composing the alveolar structures in controlled within narrow limits by a strictly modulated rate of fibroblast replication. One possible source of growth-modulating signals for alveolar fibroblasts is the alveolar macrophage, a member of the mononuclear phagocyte family of cells, which collectively are known to be important sources of growth factors for a variety of target cells. To evaluate the role of alveolar macrophages in the control of alveolar fibroblast replication, macrophages from normal individuals obtained by bronchoalveolar lavage were maintained in suspension culture with and without added stimuli, and supernates were evaluated for fibroblast growth-promoting effect. Supernates from unstimulated macrophages contained no growth factor activity. In marked contrast, supernates from macrophages stimulated with particulates and immune complexes contained a growth factor that caused a significant increase in fibroblast replication rate. Maximum growth factor activity was observed 3-4 h after macrophage stimulation, at a concentration of 1-2 x 10(6) macrophages/ml. The alveolar macrophagederived growth factor eluted from DEAE-cellulose at 0.27 M NaCl at neutral pH had an apparent molecular weight of 18,000, and appeared to be distinct from other characterized growth factors. The alveolar macrophage-derived growth factor stimulated lung fibroblast DNA synthesis within 12 h, with cell division apparent within 48 h. In serum-free culture, the alveolar macrophage-derived growth factor by itself did not promote fibroblast replication, but rather acted as a progression factor causing a synergistic increase in fibroblast replication rate in the presence of competence factors such as fibroblast growth factor or platelet-derived growth factor. These studies suggest that when stimulated, human alveolar macrophages may modulate, in part, the replication rate of alveolar fibroblasts by releasing a growth factor within the alveolar microenvironment.

Antielastases of the human alveolar structures. Implications for the protease-antiprotease theory of emphysema.

The current concepts of the pathogenesis of emphysema hold that progressive, chronic destruction of the alveolar structures occurs because there was in imbalance between the proteases and antiproteases in the lower respiratory tract. In this context, proteases, particularly neutrophil elastase, work unimpeded to destroy the alveolar structures. This concept has evolved from consideration of patients with alpha 1-antitrypsin deficiency, who have decreased levels of serum alpha 1-antitrypsin and who have progressive panacinar emphysema. To directly assess the antiprotease side of this equation, the lower respiratory tract of non-smoking individuals with normal serum antiproteases and individuals with PiZ homozygous alpha 1-antitrypsin deficiency underwent bronchoalveolar lavage to evaluate the antiprotease screen of their lower respiratory tract. These studies demonstrated that: (a) alpha 1-antitrypsin is the major antielastase of the normal human lower respiratory tract; (b) alpha 2-macroglobulin, a large serum antielastase, and the bronchial mucous inhibitor, an antielastase of the central airways, do not contribute to the antielastase protection of the human alveolar structures; (c) individuals with PiZ alpha 1-antitrypsin deficiency have little or no alpha 1-antitrypsin in their lower respiratory tract and have no alternative antiprotease protection against neutrophil elastase; and (d) the lack of antiprotease protection of the lower respiratory tract of PiZ individuals is a chronic process, suggesting their vulnerability to neutrophil elastase is always present.

Modulation of alveolar macrophage-driven fibroblast proliferation by alternative macrophage mediators.

Tissue fibrosis results, in part, from an interaction between growth regulatory molecules released by mononuclear phagocytes and fibroblasts. In the chronic interstitial lung disorders, alveolar macrophages, the mononuclear phagocytes of the lung, are known to spontaneously release two growth factors for fibroblasts, fibronectin and alveolar macrophage-derived growth factor (AMDGF) that together stimulate nonreplicating lung fibroblasts to divide. In addition to these two primary growth promoting signals, alveolar macrophages are able to release other mediators that may have a potential role in modulating lung fibroblast replication in response to these primary signals, including interferon gamma (IFN gamma), prostaglandin E2 (PGE2), and interleukin 1 (IL-1). To evaluate this possibility, we examined the effect of each of these other mediators on lung fibroblast replication in response to fibronectin and AMDGF in serum-free, defined medium. IFN gamma had no effect on fibroblast replication. In contrast, PGE2 resulted in a dose-dependent inhibition of fibroblast replication in response to fibronectin and AMDGF with 50% of the maximum inhibition observed at a PGE2 concentration of less than 10 ng/ml. IL-1, while not active as a primary growth promoting signal, at concentrations of 4-10 U/ml, augmented fibroblast replication in response to fibronectin and AMDGF by 10 to 15%. Temporally, the growth augmenting effect of IL-1 occurred early in the G1 phase of the cell cycle. These data indicate that lung fibroblast replication in response to two of the primary growth promoting signals spontaneously released by alveolar macrophages in the interstitial lung disorders, while uninfluenced by IFN gamma, can be inhibited by PGE2 and modestly augmented by IL-1. Understanding the relevant fibroblast growth modulatory signals within the alveolar microenvironment in the chronic interstitial disorders may lead to rational therapeutic strategies designed to interrupt the fibrotic process.

Spontaneous production of transforming growth factor-beta 2 by primary cultures of bronchial epithelial cells. Effects on cell behavior in vitro.

The ability of airway epithelial cells to produce transforming growth factor-beta (TGF-beta) may be an important mechanism for the control of growth, differentiation, and repair of the airway epithelium. To determine whether airway epithelial cells are capable of producing TGF-beta, we examined primary cultures of bovine bronchial epithelial cells. Using a bioassay, TGF-beta activity was detected readily in media conditioned by bovine bronchial epithelial cells. Neutralizing antisera to TGF-beta 1 and TGF-beta 2 were used to demonstrate that the majority of the activity was of the TGF-beta 2 isoform. Interestingly, some of the TGF-beta activity was present in the conditioned media as "active" TGF-beta, not requiring acid activation. The production of TGF-beta was variable, depending on cell density and the presence of retinoic acid. The presence of endogenously produced active TGF-beta in the culture media was shown to modulate the behavior of the cell cultures as evidenced by the effects of TGF-beta-neutralizing antisera on cell size and fibronectin production. Our results suggest that active TGF-beta produced by airway epithelial cells may function in an autocrine or paracrine manner to modulate epithelial cell behavior.

Normal human alveolar macrophages obtained by bronchoalveolar lavage have a limited capacity to release interleukin-1.

Interleukin-1 (IL-1) is a mediator released by stimulated mononuclear phagocytes that is thought to play an important role in modulating T and B lymphocyte activation as well as in contributing to the febrile response and other inflammatory processes. Circulating mononuclear phagocytes, blood monocytes, readily release IL-1 when stimulated. However, the ability of lung mononuclear phagocytes, alveolar macrophages, to dispose of the large daily burden of inhaled antigens without stimulating an inflammatory response suggests that the release of IL-1 by alveolar macrophages may differ significantly from that of blood monocytes. To evaluate this hypothesis, normal autologous alveolar macrophages, obtained by bronchoalveolar lavage, were compared with blood monocytes for their ability to release IL-1 in response to a standard stimulus, lipopolysaccharide (LPS). Alveolar macrophages were found to be at least 1,000 times less sensitive to LPS than blood monocytes. Furthermore, alveolar macrophages released significantly less IL-1 than blood monocytes (26 +/- 11 vs. 128 +/- 21 U/10(6) cells X 24 h, respectively, after stimulation with 10 micrograms/ml of LPS, P less than 0.001). This difference was not due to the release of substances by macrophages, which inhibited lymphocyte proliferation in response to IL-1, or to degradation of IL-1 by macrophages. Culturing macrophages in the presence of indomethacin and dialysis of macrophage supernatants did not affect the difference, and culturing macrophages with monocytes did not decrease detectable IL-1 activity from the monocytes. The IL-1 produced by the two cell types was indistinguishable by anion-exchange chromatography, gel filtration, and isoelectric focusing. In addition, consistent with the findings for alveolar macrophages, macrophages generated by the in vitro maturation of blood monocytes were also deficient in their ability to release IL-1. These findings suggest that if the population of alveolar macrophages obtained by bronchoalveolar lavage represents the total in vivo population of alveolar macrophages, although normal human macrophages are capable of IL-1 release, they are relatively limited in this ability, and this limitation seems to be linked to the maturational state of the mononuclear phagocyte. These observations may explain, in part, the ability of alveolar macrophages to clear the airspaces of foreign antigens without extensive activation of other pulmonary inflammatory and immune effector cells.

Adhesion characteristics of murine metastatic and nonmetastatic tumor cells in vitro.

We have studied the attachment of mouse fibroblasts, transformed nonmetastatic fibroblasts, and metastatic fibrosarcoma cells to various substrates. The metastatic cells attach preferentially to type IV (basement membrane) collagen in the absence of serum, compared to type I collagen and plastic. In the presence of fibronectin, these cells attach well to both type I and type IV collagens. The normal and transformed fibroblasts attach to all these substrates, although the transformed fibroblasts attach more slowly. The ability to attach to type I collagen and plastic is correlated with the levels of fibronectin and collagen produced by these cells. The data indicate that the transformed and metastatic cells differ from normal cells in their attachment properties and ability to synthesize matrix proteins. The metastatic cells possess a unique system for binding to type IV collagen that could be important in enabling these cells to penetrate tissues bounded by basement membranes.