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Cell Density Determines Extent Of Damage Caused By Cigarette Smoke Exposure


New findings may offer roadmap to predicting how the body will respond to a deadly habit

First- or second-hand exposure to cigarettes can lead to a variety of diseases, including tissue destruction found in pulmonary emphysema and osteoporosis. Also included among cigarette smoking-induced diseases are disorders in which an excessive deposition of fibrotic scar occurs, such as with atherosclerosis and idiopathic pulmonary fibrosis.

Collagen is the major protein of the white fibers found in connective tissue, cartilage, and bone. It comprises a family of genetically distinct molecules, all of which have a unique triple helix configuration of three polypeptide subunits known as “chains.” At least 13 types of collagen have been identified, each with a different polypeptide chain. Fibroblasts, spindle-shaped cells with cytoplasmic processes present in connective tissue, are capable of forming collagen fibers. The effect of smoking on this physiological process is undetermined.

The Study

A new study has sought to determine whether the effects of cigarette smoke on the contraction of fibroblast-populated collagen gels is dependent on cell density. This research attempted to demonstrate density-dependent effects, and explore the mechanisms by which smoke exerts differential effects by determining the effect of cigarette smoke exposure (CSE) on the release of transforming growth factor and PGE2, mediators that possibly function as local regulators of collagen gel contraction.

The authors of the study, “Effect of Cigarette Smoke on Fibroblast-mediated Gel Contraction is Dependent on Cell Density,” are Hangjun Wang, from Mount Sinai Hospital, Toronto, Canada; Xiangde Liu, Fu-Qiang Wen, Debra J. Romberger, John R. Spurzem, and Stephen I. Rennard, from the University of Nebraska Medical Center, Omaha, NE; Takeshi Umino from Tokyo Medical and Dental University, Japan; Tadashi Kohyama, Department of Respiratory Medicine, University of Tokyo, Japan; Yun Kui Zhu, Department of Respiratory Diseases, Jincheng Hospital, Lanzhou, China; and Hui Jung Kim, Department of Internal Medicine, Seoul Adventist Hospital, Seoul, Korea. Their findings appear in the January 2003 edition of the American Journal of Physiology—Lung Cellular and Molecular Physiology.


The experiment consisted of the following elements:

The researchers selected type I collagen gels made from collagen extracted from rat tail tendons. Tendons were excised from rat tails, and the tendon sheath and other connective tissues were carefully removed. Type I collagen was extracted; protein concentration was determined by weighing a lyophilized aliquot from each lot of collagen solution. Anti-TGF-â neutralizing antibody, which showed greater than two percent cross-reactivity with human TGF-â2 and TGF-â3 and did not cross-react with other growth factors, and anti-immunoglobulin were used.

CSE was obtained by combusting one cigarette without filter with a modified syringe-driven device. The smoke was bubbled through 25 ml of serum-free DMEM glucose. Human fibroblasts were obtained, and cells were cultured on tissue culture dishes with DMEM supplemented. Cells were cultured at 37°C in a humidified atmosphere of 5 percent CO2 and passaged once a week at a 1:3 ratio. Fibroblasts were used between the 14th and 20th passages. Cell suspensions, routinely added last, were added to achieve several fibroblast cell densities.

To investigate the effect of anti-TGF antibody on fibroblast-mediated gel contraction, the researchers added TGF antibody (10 g/ml) to the culture media after gels were released. Antihuman IgG antibody was used as control. To measure TGF-â1, samples were assayed both with and without acidification and neutralization to convert the latent form of TGF-â1 to active forms. TGF-â1 was quantified by an ELISA test.


Five percent CSE inhibited the contraction of collagen gels populated by fibroblasts at low density but augmented contraction of those populated by fibroblasts at high density. The inhibitory effects of 10 percent CSE were greater than that of five percent, but much more notably so in the low-density cells than in the high-density cells. CSE inhibited production of fibronectin in low-density cultures but stimulated fibronectin production in high-density cultures. Similarly, TGF-â1 release was inhibited in low-density cultures but trended toward stimulation in high-density cultures. Perhaps more importantly, five percent CSE appeared to augment the release of active TGF-â in high-density cultures, while having no detectable effect on active TGF-â in low-density cultures. The effects on TGF-â production were paralleled by effects on TGF-â mRNA.

The augmented contraction observed in high density cultures is likely due to activity of TGF as antibodies to TGF blocked this response. Contraction of gels composed of native collagen fibers in which fibroblasts are cultured has been used as a model of wound repair and tissue fibrosis. Like both scars and fibrotic tissues, fibroblast-populated collagen gels contract. The degree of contraction depends on a number of factors, including the concentration of collagen in the gel, the presence of serum or exogenous growth factors, and, importantly, the density of fibroblasts within the gels. Gels cultured with a higher density of fibroblasts contract to a greater degree.


The findings suggest that multiple components of cigarette smoke may have interacting toxic effects. The extent to which these components reach fibroblasts depends on their interaction with a variety of components present between the inhaled air stream and the tissue cells. This includes the surface layer, the epithelial cells, and components in the interstitial matrix, including factors derived from the circulation system. These lung structures have considerable capacity to detoxify cigarette smoke. Airway epithelial cells, for example, are capable of metabolizing xenobiotics. The toxicity of smoke on fibroblasts in vivo, therefore, depends not only on the ability of smoke-derived toxins to injure fibroblasts, but also on the defense mechanisms present in the lung that can serve to mitigate the effects of smoke.

Remodeling of tissues is a process that likely requires collaborative interaction among cells distributed within a tissue. Such processes are ideally suited for regulation and coordination through a variety of cell-cell communication mechanisms. As the reduction of these mediators will vary with cell density, it seems likely that paracrine modulation of tissue repair will be highly dependent on cell density. Finally, the fibroblasts are not the only source of TGF within the lung, and fibroblasts can be modulated by many factors in addition to TGF. The effect of cigarette smoke within the tissue, therefore, will depend not only on the effect of cigarette smoke on fibroblasts but also on the actions of other cells within the lung.

This study demonstrates that CSE modulation of contraction of three-dimensional collagen gels populated by fibroblasts depends on cell density. Inhibition of contraction occurs at low cell density due to inhibition of fibronectin production. In contrast, in high-density cultures, CSE augments contraction likely through increased release of active TGF. These density-dependent effects may account for the varied types of pathology that can result from cigarette smoke exposure.

Source: January 2003 edition of the American Journal of Physiology—Lung Cellular and Molecular Physiology.

The American Physiological Society (APS) was founded in 1887 to foster basic and applied science, much of it relating to human health. The Bethesda, MD-based Society has more than 10,000 members and publishes 3,800 articles in its 14 peer-reviewed journals every year.

Contact: Donna Krupa: 703.527.7357
Cell: 703.967.2751 or

Donna Krupa | The American Physiological Socie
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