Hepoxilin Analogs Inhibit Bleomycin-Induced Pulmonary Fibrosis in the Mouse

Abstract

Bleomycin has been suggested to incite plasma extravasation and influx of inflammatory cells leading to pulmonary fibrosis. We hypothesized that stable analogs of the 12-lipoxygenase product, hepoxilin, may attenuate these effects. We initially investigated the effects of the four hepoxilin analogs (PBT-1 to -4) coadministered intradermally with bleomycin and found that PBT-1 and -2 significantly opposed the vascular permeability effects of bleomycin in rat skin. We subsequently tested the hepoxilin analogs for their actions in opposing the intratracheal bleomycin-evoked acute inflammatory phase of lung fibrosis in the mouse, characterized by a marked accumulation of macrophages and an increase in the rate of collagen synthesis and deposition. We found that the bleomycin-evoked effects on macrophage influx were inhibited by all the hepoxilin analogs (PBT-1, -3, and -4 Ͼ PBT-2) administered i.p. for 8 days. Increased total lung collagen was completely abrogated by PBT-1 and -2, whereas PBT-3 and -4 had little effect. A doseresponse study with PBT-1 indicated that the effective dose for inhibition of bleomycin-induced inflammatory and histological changes was below 10 g/day. These studies demonstrate an in vivo action of stable analogs of hepoxilin and support an effect on inflammation and vascular permeability from these novel compounds, especially for PBT-1.Idiopathic pulmonary fibrosis is a devastating disorder that is poorly understood and resistant to treatment (Cooper, 2000). The observation that the antibiotic bleomycin sulfate (BL), a potent cancer chemotherapeutic agent (Adamson, 1984;Nici et al., 1998), may cause interstitial lung fibrosis in humans (Yagoda et al., 1972) led to the development of animal models in which a single dose of BL administered intratracheally induced changes resembling human idiopathic pulmonary fibrosis histopathologically (Kelley et al., 1980). The acute phase of this response is characterized by a marked accumulation of inflammatory cells and an increase in the rate of collagen synthesis and deposition (Cooper et al., 1988;Gurujeyalakshmi and Giri, 1995;Giri and Hollinger, 1996).Previous animal studies have demonstrated that BLevoked lung fibrosis is exacerbated with nordihydroguaiaretic acid, a lipoxygenase inhibitor, suggesting that a lipoxygenase product may be involved in endogenous mechanisms controlling lung fibrosis (Giri and Hollinger, 1996). The hepoxilins (HXs) may be candidates for the control of lung fibrosis as they are formed through the 12-lipoxygenase pathway of metabolism of arachidonic acid (Pace-Asciak et al., 1983;Pace-Asciak, 1984;Pace-Asciak and Martin, 1984). These compounds have previously been shown to have significant biological actions (Pace-Asciak, 1984;Dho et al., 1990;Laneuville et al., 1992;Pace-Asciak et al., 1995;Reynaud et al., 1996Reynaud et al., , 1999Sutherland et al., 2000). HXs raise free intracellular calcium in human neutrophils ex vivo through the release from stores and vascular tissue in vitro and block the intracellular calc...

Arginase inhibition prevents bleomycin-induced pulmonary hypertension, vascular remodeling, and collagen deposition in neonatal rat lungs

“…Bleomycin-induced lung injury is characterized by induction of proinflammatory cytokines (18), influx of macrophages and other inflammatory cells (23), "emphysematous" lung morphology, and severe PHT (49,60). Herein, we report that arginase expression is greatly increased in the lungs of bleomycin-exposed neonatal rats and that concomitant treatment with an arginase inhibitor, amino-2-borono-6-hexanoic acid (ABH), prevented bleomycin-induced development of PHT and deposition of collagen.…”

“…Paraffin-embedded inflated lung tissue sections were stained with hematoxylin and eosin (for lung morphometry), Hart's elastin (for the arterial medial wall area), or Picrosirius Red (to identify collagen), as previously described (23,26,33,42,43). To localize NOS2 or to assess an indicator of oxidative/ nitrative stress, sections were immunostained for NOS2 or 3-nitrotyrosine, respectively (22,23). For 3-nitrotyrosine, positive control slides were generated by preincubation of sections with 4 mM peroxynitrite for 5 min before application of primary antibody.…”

Therapeutic hypercapnia prevents bleomycin-induced pulmonary hypertension in neonatal rats by limiting macrophage-derived tumor necrosis factor-α

“…The early phases of bleomycin-induced lung injury are characterized by increased expression of proinflammatory cytokines (19), a marked inflammatory cell influx that is dominated by macrophages (27), increased apoptosis (20), changes in growth factors, such as transforming growth factor-␤ (3, 12), "emphysematous" lung morphology, and severe PHT (63,77). In neonatal rats, selective decrease in lung growth, arrested alveolarization and vascular hypoplasia are prominent features (47,74).…”

Hepoxilins in cancer and inflammation—use of hepoxilin antagonists

“…2 HxA3 is an early signal to produce the inflammatory response through the recruitment of neutrophils to the site of infection/injury. Blockade of HxA3 action by the selective antagonists, PBTs, may lead to early resolution of inflammation by blocking the neutrophil recruitment process [2,5,97]. The PGs and LTs are formed AFTER the HxA3 effect and serve to support the inflammatory response initiated by HxA3 [2] 6.1.4 Inflammatory hyperalgesia Hyperalgesia is an increased sensitivity to pain brought about by nerve damage.…”

“…A rodent model resembling the pathology of idiopathic pulmonary fibrosis has been described with a single intratracheal administration of bleomycin [96]. The potential involvement of the hepoxilin analogs (PBTs) in antagonizing the bleomycin-evoked causation of lung fibrosis was investigated [97], as the native hepoxilin (HxA3) is known to cause migration of inflammatory cells (neutrophils) to sites of infection leading to an inflammatory response (see above). The PBTs (500-5,000 μg/kg i.p.)…”