Dynamic change in respiratory resistance during inspiratory and expiratory phases of tidal breathing in patients with chronic obstructive pulmonary disease

Yasuhiro Yamauchi1,2, Tadashi Kohyama2, Taisuke Jo2, Takahide Nagase21Division of Health Promotion Center, 2Department of Respiratory Medicine, University of Tokyo, Tokyo, JapanBackground and objective: Chronic obstructive pulmonary disease (COPD) is characterized by persistent airflow limitation consisting of airway obstruction and parenchymal emphysema, with loss of elastic recoil. The forced oscillation technique can detect impairment of lung function by measuring lung impedance during normal tidal breathing. Respiratory resistance (Rrs) in COPD has been well-studied, but the differences in Rrs in the inspiratory and expiratory phases between mild and moderate COPD remain poorly understood. Since airway obstruction in COPD is known to change dynamically during tidal breathing and might affect Rrs, the differences in Rrs during tidal breathing between mild and moderate COPD were evaluated.Methods: Mild (n = 13) and moderate (n = 13) COPD patients were recruited at Tokyo University Hospital (Tokyo, Japan). Rrs was measured using MostGraph-01 (Chest MI, Inc, Tokyo, Japan), which depicted Rrs in a frequency- and respiratory cycle-dependent manner in three-dimensional graphics. Rrs was evaluated at 4–35 Hz during tidal breathing.Results: Rrs changed dynamically during tidal breathing in COPD. The mean Rrs values were significantly greater in the moderate COPD group than in the mild group. The maximal and minimal Rrs values at higher frequencies in the respiratory cycle were significantly greater in moderate COPD. In inspiratory–expiratory breath analysis, the maximal and minimal Rrs values at 20 Hz and 35 Hz were significantly greater in the moderate group, whereas at 4 Hz they did not differ significantly between the groups.Conclusion: Rrs changed dynamically during tidal breathing in patients with COPD. The Rrs values at higher frequencies were greater in moderate COPD than in mild COPD. Rrs at higher frequencies might reflect the degree of airway obstruction in tidal breathing in patients with COPD and might be a useful marker for evaluation of airway obstruction at an early stage of COPD.Keywords: COPD, airflow limitation, respiratory resistance, forced oscillation techniqu

Fibroblasts and monocyte macrophages contract and degrade three-dimensional collagen gels in extended co-culture

BACKGROUND: Inflammatory cells are believed to play a prominent role during tissue repair and remodeling. Since repair processes develop and mature over extended time frames, the present study was designed to evaluate the effect of monocytes and fibroblasts in prolonged culture in three-dimensional collagen gels. METHODS: Blood monocytes from healthy donors and human fetal lung fibroblasts were cast into type I collagen gels and maintained in floating cultures for three weeks. RESULTS: Fibroblast-mediated gel contraction was initially inhibited by the presence of monocytes (P < 0.01). However, with extended co-culture, contraction of the collagen gels was greatly augmented (P < 0.01). In addition, with extended co-culture, degradation of collagen in the gels occurred. The addition of neutrophil elastase to the medium augmented both contraction and degradation (P < 0.01). Prostaglandin E(2) production was significantly increased by co-culture and its presence attenuated collagen degradation. CONCLUSION: The current study, therefore, demonstrates that interaction between monocytes and fibroblasts can contract and degrade extracellular matrix in extended culture

Collaborative interactions between neutrophil elastase and metalloproteinases in extracellular matrix degradation in three-dimensional collagen gels

BACKGROUND: Extended culture of monocytes and fibroblasts in three-dimensional collagen gels leads to degradation of the gels (see linked study in this issue, "Fibroblasts and monocytes contract and degrade three-dimensional collagen gels in extended co-culture"). The current study, therefore, was designed to evaluate production of matrix-degrading metalloproteinases by these cells in co-culture and to determine if neutrophil elastase could collaborate in the activation of these enzymes. Since co-cultures produce prostaglandin E(2) (PGE(2)), the role of PGE(2) in this process was also evaluated. METHODS: Blood monocytes from healthy donors and human fetal lung fibroblasts were cast into type I collagen gels and maintained in floating cultures for three weeks. Matrix metalloproteinases (MMPs) were assessed by gelatin zymography (MMPs 2 and 9) and immunoblotting (MMPs 1 and 3). The role of PGE(2) was explored by direct quantification, and by the addition of exogenous indomethacin and/or PGE(2). RESULTS: Gelatin zymography and immunoblots revealed that MMPs 1, 2, 3 and 9 were induced by co-cultures of fibroblasts and monocytes. Neutrophil elastase added to the medium resulted in marked conversion of latent MMPs to lower molecular weight forms consistent with active MMPs, and was associated with augmentation of both contraction and degradation (P < 0.01). PGE(2) appeared to decrease both MMP production and activation. CONCLUSION: The current study demonstrates that interactions between monocytes and fibroblasts can mediate tissue remodeling

TGF-β1 and serum both stimulate contraction but differentially affect apoptosis in 3D collagen gels

Apoptosis of fibroblasts may be key for the removal of cells following repair processes. Contraction of three-dimensional collagen gels is a model of wound healing and remodeling. Here two potent inducers of contraction, TGF-β1 and fetal calf serum (FCS) were evaluated for their effect on fibroblast apoptosis in contracting collagen gels. Human fetal lung fibroblasts were cultured in floating type I collagen gels, exposed to TGF-β1 or FCS, and allowed to contract for 5 days. Apoptosis was evaluated using TUNEL and confirmed by DNA content profiling. Both TGF-β1 and serum significantly augmented collagen gel contraction. TGF-β1 also increased apoptosis assessed by TUNEL positivity and DNA content analysis. In contrast, serum did not affect apoptosis. TGF-β1 induction of apoptosis was associated with augmented expression of Bax, a pro-apoptotic member of the Bax/Bcl-2 family, inhibition of Bcl-2, an anti-apoptotic member of the same family, and inhibition of both cIAP-1 and XIAP, two inhibitors of the caspase cascade. Serum was associated with an increase in cIAP-1 and Bcl-2, anti-apoptotic proteins. Interestingly, serum was also associated with an apparent increase in Bax, a pro-apoptotic protein. Blockade of Smad3 with either siRNA or by using murine fibroblasts deficient in Smad3 resulted in a lack of TGF-β induction of augmented contraction and apoptosis. Contraction induced by different factors, therefore, may be differentially associated with apoptosis, which may be related to the persistence or resolution of the fibroblasts that accumulate following injury

Differentiation of embryonic stem cells into fibroblast-like cells in three-dimensional type I collagen gel cultures

Fibroblasts are heterogeneous mesenchymal cells that play important roles in the production and maintenance of extracellular matrix. Although their heterogeneity is recognized, progenitor progeny relationships among fibroblasts and the factors that control fibroblast differentiation are poorly defined. The current study was designed to develop a reliable method that would permit in vitro differentiation of fibroblast-like cells from human and murine embryonic stem cells (ESCs). Undifferentiated ESCs were differentiated into embryoid bodies (EBs) with differentiation media. EBs were then cast into type I collagen gels and cultured for 21 d with basal media. The spindle-shaped cells that subsequently grew from the EBs were released from the gels and subsequently cultured as monolayers in basal media supplemented with serum. Differentiated cells showed a characteristic spindle-shaped morphology and had ultrastructural features consistent with fibroblasts. Immunocytochemistry showed positive staining for vimentin and alpha-smooth muscle actin but was negative for stage-specific embryonic antigens and cytokeratins. Assays of fibroblast function, including proliferation, chemotaxis, and contraction of collagen gels demonstrated that the differentiated cells, derived from both human and murine ESCs, responded to transforming growth factor-β1 and prostaglandin E2 as would be expected of fibroblasts, functions not expected of endothelial or epithelial cells. The current study demonstrates that cells with the morphologic and functional features of fibroblasts can be reliably derived from human and murine ESCs. This methodology provides a means to investigate and define the mechanisms that regulate fibroblast differentiation