Search

Transforming growth factor-beta (TGF-beta) is believed to play a central role in fibroblastic wound repair and subsequent scar formation. Fibroblasts undergo apoptosis as granulation tissue evolves into a scar. To determine whether TGF-beta influences fibroblast apoptosis, human lung fibroblasts (IMR-90) were treated with TGF-beta (2 to 10 ng/mL) and then exposed to H(2)O(2) (50 to 150 microM), an inducer of fibroblast apoptosis. Apoptosis was evaluated by nuclear staining with Hoechst33342 and terminal deoxynucleotidyl transferase (TdT)-mediated nucleotide nick-end labeling. TGF-beta alone did not induce fibroblast apoptosis, but it dose-dependently augmented the apoptosis induced by exposure to H(2)O(2). TGF-beta also increased the intracellular level of peroxides measured with carboxydichlorodihydrofluorescein, but it did not affect the protein levels of Bcl-2, Bax, Bad, or p53 proteins. These results suggest that TGF-beta promotes lung fibroblast apoptosis induced by H(2)O(2), probably by increasing intracellular peroxides. Thus, TGF-beta may promote the elimination of fibroblasts from wounds, particularly under conditions of exposure to enhanced oxidative stress in the lung.

The purpose of this study was to examine how lungs in early postnatal animals that are administered bromodeoxyuridine (BrdU), a thymidine analog known to be a cytogenetically toxic agent, change in adult life. BrdU was injected (1 mg/g body weight) intraperitoneally in suckling rats on day 6 after birth. Immunohistochemical studies revealed that BrdU was incorporated in alveolar cells, and infrequently in the mesothelium, endothelium, and bronchiolar epithelium. The number (per high-power field) of alveolar cells incorporating BrdU was 8.76 +/- 0.50 in the interstitium and 1.66 +/- 0.25 in the epithelium. The animals were sacrificed at 2 and 8 weeks and 12 months of age. Control animals were treated the same as the BrdU-treated animals except for the injection of BrdU. Body weight, body size (nose-tail length), and lung weight were identical between BrdU-treated animals and control animals at all experimental periods. The histopathology and physiology of these lungs were studied. At 2 weeks of age, the BrdU-treated animals appeared morphologically and functionally similar to control animals. At 8 weeks and 12 months of age, the lungs in the BrdU-treated animals showed abnormal enlargement of the airspace, few alveoli, and less complexity in pulmonary architecture with or without increased lung compliance. These findings suggest that lung injured cytogenetically in early postnatal life may develop functional and structural abnormalities in later life.

Irreversible lung parenchymal injury is usually healed by fibrosis, which depends on the abilities of fibroblasts to proliferate, migrate into the wound, and survive. Because the lung is frequently exposed to increased oxidative stress, which is thought to mediate apoptosis, we examined whether oxidative stress induces apoptosis in fibroblasts during wound healing. We performed an in vitro scratch wound assay where cultured fibroblast monolayers were exposed to H2O2 (10-500 microM) after artificial wounding. Apoptosis was evaluated by nuclear staining with Hoechst33342 or terminal deoxynucleotidyl transferase (TdT)-mediated nucleotide nick end-labeling (TUNEL). Intracellular oxidants were assessed with the peroxide-sensitive fluorochrome carboxydichlorodihydrofluorescein (CDCF). We found that repopulating fibroblasts at the wound margin, but not quiescent fibroblasts at the intact site, selectively underwent oxidant accumulation and apoptosis in response to H2O2 exposure. Some of the apoptotic cells had incorporated bromodeoxyuridine (BrdU), an indicator of proliferating cells. These results suggest that oxidative stress selectively induces apoptosis in fibroblasts that are stimulated to proliferate and/or migrate into the wound. Fibroblast apoptosis induced by oxidative stress during wound repopulation may be relevant to intractable wound healing.

Circulating neutrophils are exposed to widely varying levels of hemodynamic stress induced by blood flow conditions. This study examined the effect of hemodynamic stress on the functional responsiveness of neutrophils obtained from healthy humans to chemotactic stimuli. To expose neutrophils to hemodynamic stress in vitro, isolated neutrophils were agitated under artificial flow conditions induced by a rotary tube apparatus. Although such hemodynamic stress produced no spontaneous or random migration of neutrophils, it enhanced neutrophil migration in response to the chemotactic peptide f-methionyl-leucyl-phenylalanine (FMLP) by as much as 200%. Hemodynamic stress also enhanced polarization in response to FMLP, producing a change in shape characteristic of migration. Polarization was reversible when neutrophils were transferred to quiescent conditions after being exposed to hemodynamic stress. Hemodynamic stress also enhanced O2.- production and granular beta-glucuronidase release in response to FMLP and enhanced polarization and O2.- production in response to phorbol myristate acetate (PMA), a direct activator of protein kinase C. Extracellular Ca2+ was not required for the enhancement of chemotactic responsiveness by hemodynamic stress, and the stress produced no detectable change in intracellular Ca2+, intracellular cyclic AMP, or activated protein kinase C levels in neutrophils. The results show that hemodynamic stress enhances the functional responsiveness of neutrophils to chemotactic stimuli and provide insights into interpretation of in vitro data usually obtained from quiescent conditions.