Total Lung Hydroxyproline Research Articles

Total sesquiterpenoids from Eupatorium lindleyanum DC. attenuate bleomycin-induced lung fibrosis by suppressing myofibroblast transition

Eupatorium lindleyanum DC. has been used as a functional food in China for a long time. However, the antifibrotic activity of total sesquiterpenoids from Eupatorium lindleyanum DC. (TS-EL) is still unknown. In this study, we discovered that TS-EL reduced the increase in α-smooth muscle actin (α-SMA), type I collagen and fibronectin content, the formation of cell filaments and collagen gel contraction in transforming growth factor-β1-stimulated human lung fibroblasts. Intriguingly, TS-EL did not change the phosphorylation of Smad2/3 and Erk1/2. TS-EL decreased the levels of serum response factor (SRF), a critical transcription factor of α-SMA, and SRF knockdown alleviated the transition of lung myofibroblasts. Furthermore, TS-EL significantly attenuated bleomycin (BLM)-induced lung pathology and collagen deposition and reduced the levels of two profibrotic markers, total lung hydroxyproline and α-SMA. TS-EL also decreased the levels of SRF protein expression in BLM-induced mice. These results suggested that TS-EL attenuates pulmonary fibrosis by inhibiting myofibroblast transition via the downregulation of SRF.

Intracellular hydroxyproline imprinting following resolution of bleomycin-induced pulmonary fibrosis.

BackgroundIdiopathic pulmonary fibrosis (IPF) is a fatal lung disease with few treatment options. The poor success in developing anti-IPF strategies has impelled researchers to reconsider the importance of the choice of animal model and assessment methodologies. Currently, it is still not settled whether the bleomycin-induced lung fibrosis mouse model finally returns to resolution.MethodsThis study aimed to follow the dynamic fibrotic features of bleomycin-treated mouse lungs over extended durations through a combination of the latest technologies (micro-computed tomography imaging and histological detection of degraded collagens) and traditional methods. In addition, we also applied immunohistochemistry to explore the distribution of all hydroxyproline-containing molecules.ResultsAs determined by classical biochemical methods, total lung hydroxyproline contents reached a peak at 4 weeks after bleomycin injury and maintained a steady high level thereafter until the end of the experiments (16 weeks). This result seemed to partially contradict with the changes of other fibrosis evaluation parameters, which indicated a gradual degradation of collagens and a recovery of lung aeration after the fibrosis peak. This inconsistency was well reconciled by our data from immunostaining against hydroxyproline and fluorescent peptide staining against degraded collagen, together showing large amounts of hydroxyproline-rich degraded collagen fragments detained and enriched within the intracellular regions at 10 or 16 weeks rather than at 4 weeks after bleomycin treatment.ConclusionsOur present data not only offer respiratory researchers a new perspective towards the resolution nature of mouse lung fibrosis, but also remind them to be cautious when using the hydroxyproline content assay to evaluate the severity of fibrosis.

Pan-PPAR agonist IVA337 is effective in experimental lung fibrosis and pulmonary hypertension

ObjectiveTo evaluate the antifibrotic effects of the pan-peroxisome proliferator-activated receptor (PPAR) agonist IVA337 in preclinical mouse models of pulmonary fibrosis and related pulmonary hypertension (PH).MethodsIVA337 has been evaluated in the...

P70 ribosomal S6 kinase regulates subpleural fibrosis following transforming growth factor-α expression in the lung.

The p70 ribosomal S6 kinase (S6K) is a downstream substrate that is phosphorylated and activated by the mammalian target of rapamycin complex and regulates multiple cellular processes associated with fibrogenesis. Recent studies demonstrate that aberrant mTORC1-S6K signaling contributes to various pathological conditions, but a direct role in pulmonary fibroproliferation has not been established. Increased phosphorylation of the S6K pathway is detected immediately following transforming growth factor-α (TGF-α) expression in a transgenic model of progressive lung fibrosis. To test the hypothesis that the S6K directly regulates pulmonary fibroproliferative disease we determined the cellular sites of S6K phosphorylation during the induction of fibrosis in the TGF-α model and tested the efficacy of specific pharmacological inhibition of the S6K pathway to prevent and reverse fibrotic disease. Following TGF-α expression increased phosphorylation of the S6K was detected in the airway and alveolar epithelium and the mesenchyme of advanced subpleural fibrotic regions. Specific inhibition of the S6K with the small molecule inhibitor LY-2584702 decreased TGF-α and platelet-derived growth factor-β-induced proliferation of lung fibroblasts in vitro. Administration of S6K inhibitors to TGF-α mice prevented the development of extensive subpleural fibrosis and alterations in lung mechanics, and attenuated the increase in total lung hydroxyproline. S6K inhibition after fibrosis was established attenuated the progression of subpleural fibrosis. Together these studies demonstrate targeting the S6K pathway selectively modifies the progression of pulmonary fibrosis in the subpleural compartment of the lung.

Innate Immune Activation by Inhaled Lipopolysaccharide, Independent of Oxidative Stress, Exacerbates Silica-Induced Pulmonary Fibrosis in Mice

Acute exacerbations of pulmonary fibrosis are characterized by rapid decrements in lung function. Environmental factors that may contribute to acute exacerbations remain poorly understood. We have previously demonstrated that exposure to inhaled lipopolysaccharide (LPS) induces expression of genes associated with fibrosis. To address whether exposure to LPS could exacerbate fibrosis, we exposed male C57BL/6 mice to crystalline silica, or vehicle, followed 28 days later by LPS or saline inhalation. We observed that mice receiving both silica and LPS had significantly more total inflammatory cells, more whole lung lavage MCP-1, MIP-2, KC and IL-1β, more evidence of oxidative stress and more total lung hydroxyproline than mice receiving either LPS alone, or silica alone. Blocking oxidative stress with N-acetylcysteine attenuated whole lung inflammation but had no effect on total lung hydroxyproline. These observations suggest that exposure to innate immune stimuli, such as LPS in the environment, may exacerbate stable pulmonary fibrosis via mechanisms that are independent of inflammation and oxidative stress.

Gender influences the response to experimental silica-induced lung fibrosis in mice

Accumulating evidence suggests that gender can have a profound effect on incidence and severity of a variety of pulmonary diseases. To address the influence of gender on the development of silica-induced pulmonary fibrosis, we instilled 0.2 g/kg silica into male and female C57BL/6 mice and examined the fibrotic and inflammatory response at 14 days postexposure. Both silica-exposed male and female mice had significant increases in total lung hydroxyproline compared with saline controls. However, silica-exposed female mice had significantly less total lung hydroxyproline than silica-exposed male mice. This observation was confirmed by color thresholding image analysis. Interestingly, silica-exposed female mice had significantly more inflammatory cells, the majority of which were macrophages, as well as higher levels of the macrophage-specific chemokines MCP-1 and CCL9 in whole lung lavage compared with silica-exposed male mice. We also show that at baseline, estrogen receptor α (ERα) mRNA expression is lower in female mice than in males and that ERα mRNA expression is decreased by silica exposure. Finally, we show that the response of ovariectomized female mice to silica instillation is similar to that of male mice. These observations together show that gender influences the lung response to silica.

Hydrogen sulfide attenuates the pathogenesis of pulmonary fibrosis induced by bleomycin in rats

The present study investigated the role of the endogenous cystathionine gamma-lyase (CSE) / hydrogen sulfide pathway in the pathogenesis of pulmonary fibrosis. Rats treated with intratracheal bleomycin were exposed either to the H2S donor NaHS or to saline. The results on day 7 showed that plasma H2S concentration and pulmonary CSE activity (H2S production rate) were significantly lower in rats treated with bleomycin and saline (fibrosis-alone) than in controls, whereas on day 28 plasma H2S concentration was higher and pulmonary CSE activity was the same as that of controls. The relative CSE mRNA level in the lungs of rats treated with bleomycin was significantly higher than control values on days 7 and 28. After exposure to NaHS, the total lung hydroxyproline content and the malondialdehyde (MDA) content were both significantly lower, with no difference observed between NaHS high-dose and low-dose treatments. Further, MDA formation stimulated by the free radical-generating system (FRGS) in vitro was lower in lung tissue incubated with NaHS than it was in tissue incubated with FRGS alone. These results suggest that NaHS administration ameliorated the pulmonary fibrosis induced by bleomycin in rats and that this protective effect of H2S may be mediated by its antioxidative action.

Effects of Pirfenidone on Prevention of Radiation-induced Lung Toxicity - Results of an Animal Experiment

Effects of Pirfenidone on Prevention of Radiation-induced Lung Toxicity - Results of an Animal Experiment

Role of interferon-gamma in the evolution of murine bleomycin lung fibrosis.

IFN-gamma production is upregulated in lung cells (LC) of bleomycin-treated C57BL/6 mice. The present study characterizes the time course, cellular source, and regulation of IFN-gamma expression in bleomycin-induced lung injury. IFN-gamma mRNA in LC from bleomycin-treated mice peaked 3 days after intratracheal instillation. IFN-gamma protein levels were increased at 6 days, as was the percentage of LC expressing IFN-gamma. CD4+, CD8+, and natural killer cells each contributed significantly to IFN-gamma production. IL-12 mRNA levels were increased at 1 day in LC of bleomycin-treated mice. Anti-IL-12 and anti-IL-18 antibodies decreased IFN-gamma production by these cells. To define the role of endogenous IFN-gamma in the evolution of bleomycin lung injury, we compared the effect of bleomycin in mice with a targeted knockout mutation of the IFN-gamma gene (IFN-gamma knockout) and wild-type mice. At 14 days after intratracheal bleomycin, total bronchoalveolar lavage cell counts and lung hydroxyproline were decreased in IFN-gamma knockouts compared with wild-type animals. There was no difference in morphometric parameters of fibrosis. Our data show that enhanced IFN-gamma production in the lungs of bleomycin-treated mice is at least partly IL-12 and IL-18 dependent. Absence of IFN-gamma in IFN-gamma knockout mice does not increase pulmonary fibrosis. Endogenous IFN-gamma may play a proinflammatory or profibrotic role in bleomycin-induced lung fibrosis.

Quantitative image analysis of drug-induced lung fibrosis using laser scanning confocal microscopy.

Pulmonary fibrosis is a serious lung disorder that in certain cases may be difficult to quantify. It was our objective to evaluate the use of laser scanning confocal microscopy (LSCM) in quantifying fibrosis after exposure to amiodarone (AD) and bleomycin (BLM), two commonly used therapeutic drugs known to cause debilitating lung fibrosis in humans. Male F344 rats were intratracheally dosed with AD (6.25 mg/kg on days 0 and 2), BLM (0.25 and 1.0 mg/kg on day 0), or their respective vehicle controls. The right lung was assayed for hydroxyproline, a biochemical measure of collagen, at day 21 for the BLM groups and day 28 for the AD groups. The left lung was fixed, sectioned into blocks, dehydrated, stained with Lucifer yellow (LY, 0.1 mg/ml), and embedded in Spurr resin. The area of lung tissue stained by LY was quantified by LSCM. A fibrotic response in the AD and BLM groups was confirmed by histopathological assessment and a significant increase (p < 0.05) in total right lung hydroxyproline above control values. The area of connective tissue stained by LY of the two drug-treated groups appeared as bright linear bands in the alveolar septae and was significantly increased (p < 0.05) as measured by image analysis when compared with their respective controls. LSCM, with its advanced image analysis system, is an alternate method to quantify fibrotic lung disease. LSCM could be particularly useful when tissue quantity is limited, such as when tissue has been archived from previous studies, or when analyzing human lung biopsy samples for disease diagnosis, where biochemical analysis is difficult.

PG490-88, a Derivative of Triptolide, Blocks Bleomycin-Induced Lung Fibrosis

In this study we evaluate the antifibrotic properties of PG-490-88, a water-soluble derivative of triptolide. Triptolide is an oxygenated diterpene that is derived from a traditional Chinese herb that has potent immunosuppressive and antitumor activity. We used the intratracheal bleomycin mouse model and found that PG490-88 inhibits fibrosis in the bleomycin group when given the same day or 5 days after bleomycin. PG490-88 also markedly reduced the number of myofibroblasts in the bleomycin treatment group. An enzyme-linked immunosorbent assay of transforming growth factor (TGF)-beta in the bronchoalveolar lavage fluid showed a significant decrease in TGF-beta in the PG490-88-treated groups compared to the bleomycin-treated group. Additionally, triptolide blocked bleomycin-induced increase in TGF-beta mRNA in cultured normal human lung fibroblasts. The efficacy of PG490-88 when administered late after bleomycin installation suggests a potential role in the treatment of idiopathic pulmonary fibrosis.

Curcumin inhibition of bleomycin-induced pulmonary fibrosis in rats.

Curcumin, an anti-inflammatory, antioxidant, was evaluated for its ability to suppress bleomycin (BLM)-induced pulmonary fibrosis in rats. A single intratracheal instillation of BLM (0.75 U 100(-1) g, sacrificed 3, 5, 7, 14 and 28 days post-BLM) resulted in significant increases in total cell numbers, total protein, and angiotensin-converting enzyme (ACE), and alkaline phosphatase (AKP) activities in bronchoalveolar lavage fluid. Animals with fibrosis had a significant increase in lung hydroxyproline content. Alveolar macrophages from BLM-administered rats elaborated significant increases in tumour necrosis factor (TNF)-alpha release, and superoxide and nitric oxide production in culture medium. Interestingly, oral administration of curcumin (300 mg kg(-1) 10 days before and daily thereafter throughout the experimental time period) inhibited BLM-induced increases in total cell counts and biomarkers of inflammatory responses in BALF. In addition, curcumin significantly reduced the total lung hydroxyproline in BLM rats. Furthermore, curcumin remarkably suppressed the BLM-induced alveolar macrophage production of TNF-alpha, superoxide and nitric oxide. These findings suggest curcumin as a potent anti-inflammatory and anti-fibrotic agent against BLM-induced pulmonary fibrosis in rats.

IFN-γ-Inducible Protein-10 Attenuates Bleomycin-Induced Pulmonary Fibrosis Via Inhibition of Angiogenesis

AbstractFew studies have addressed the importance of vascular remodeling in the lung during the development of bleomycin-induced pulmonary fibrosis (BPF). For fibroplasia and deposition of extracellular matrix to occur, there must be a geometric increase in neovascularization. We hypothesized that net angiogenesis during the pathogenesis of fibroplasia and deposition of extracellular matrix during BPF are dependent in part on a relative deficiency of the angiostatic CXC chemokine, IFN-γ-inducible protein-10 (IP-10). To test this hypothesis, we measured IP-10 by specific ELISA in whole lung homogenates in either bleomycin-treated or control mice and correlated these levels with lung hydroxyproline. We found that lung tissue from mice treated with bleomycin, compared with that from saline-treated controls, demonstrated a decrease in the presence of IP-10 that was correlated to a greater angiogenic response and total lung hydroxyproline content. Systemic administration of IP-10 significantly reduced BPF without any alteration in lung lymphocyte or NK cell populations. This was also paralleled by a reduction in angiogenesis. Furthermore, IP-10 had no direct effect on isolated pulmonary fibroblasts. These results demonstrate that the angiostatic CXC chemokine, IP-10, inhibits fibroplasia and deposition of extracellular matrix by regulating angiogenesis.

Neutralization of the CXC Chemokine, Macrophage Inflammatory Protein-2, Attenuates Bleomycin-Induced Pulmonary Fibrosis

Few studies have addressed the importance of vascular remodeling in the lung during the development of bleomycin-induced pulmonary fibrosis. For fibroplasia and deposition of extracellular matrix to occur, there must be a geometric increase in neovascularization. We hypothesized that net angiogenesis during the pathogenesis of fibroplasia and deposition of extracellular matrix during bleomycin-induced pulmonary fibrosis are dependent in part upon an overexpression of the angiogenic CXC chemokine, macrophage inflammatory protein-2 (MIP-2). To test this hypothesis, we measured MIP-2 by specific ELISA in whole lung homogenates in either bleomycin-treated or control CBA/J mice and correlated these levels with lung hydroxyproline. We found that lung tissue from mice treated with bleomycin, compared with that from saline-treated controls, demonstrated a significant increase in the presence of MIP-2 that was correlated to a greater angiogenic response and total lung hydroxyproline content. Neutralizing anti-MIP-2 Abs inhibited the angiogenic activity of day 16 bleomycin-treated lung specimens using an in vivo angiogenesis bioassay. Furthermore, when MIP-2 was depleted in vivo by passive immunization, bleomycin-induced pulmonary fibrosis was significantly reduced without a change in the presence of pulmonary neutrophils, fibroblast proliferation, or collagen gene expression. This was also paralleled by a reduction in angiogenesis. These results demonstrate that the angiogenic CXC chemokine, MIP-2, is an important factor that regulates angiogenesis/fibrosis in pulmonary fibrosis.

Pirfenidone attenuates bleomycin-induced changes in pulmonary functions in hamsters.

The antifibrotic potential of pirfenidone (5-methyl-1-phenyl-2-[1H]-pyridone) was examined in a single intratracheal bleomycin dose hamster model of pulmonary fibrosis. Bleomycin-induced fibrosis and the effectiveness of pirfenidone treatment were assessed by measuring pulmonary functions (Cqst, TLC, VC, IC, FRC, RV) and the level of hydroxyproline in whole lung homogenates. Thirty-five male golden Syrian hamsters were randomized into four experimental groups: saline instilled and fed a control diet of rat chow (SCD, n = 8); saline instilled and fed the control diet containing 0.5% (w/w) pirfenidone (SPD, n = 8); bleomycin instilled and fed the control diet (BCD, n = 7); and bleomycin instilled and fed the control diet containing 0.5% pirfenidone (BPD, n = 10). Twenty-one days following bleomycin instillation Cqst/TLC, TLC, VC, and IC were significantly reduced and total lung hydroxyproline levels were significantly increased in the BCD and BPD groups as compared with the SCD and SPD groups, respectively. Pirfenidone ingestion significantly attenuated these bleomycin-induced pertubations in pulmonary functions and lung hydroxyproline levels (BCD versus BPD). The data obtained in this study provide evidence of the benefical effects of pirfenidone in the hamster model of bleomycin-induced pulmonary fibrosis both at the functional and biochemical level.

Pirfenidone diminishes cyclophosphamide-induced lung fibrosis in mice

The deposition of excess or abnormal collagen characteristic of pulmonary fibrosis can disrupt gas exchange resulting in severe respiratory impairment. There currently are no effective pharmacologic agents available that inhibit the fibrotic process. Pirfenidone (5-methyl-1-phenyl-2-(1H)-pyridone) is an investigational drug that, when administered at 0.5% (w/w) of the diet, decreases both histologic and biochemical evidence of lung fibrosis in hamsters treated intratracheally with bleomycin. The effectiveness of pirfenidone against lung fibrosis initiated by a systemically administered agent was investigated in mice treated intraperitoneally with 200 mg/kg cyclophosphamide (CP). Control and treated animals were fed a diet containing 0.277% (w/w) pirfenidone beginning 1 day after CP. Despite anorexia in the CP-treated mice the first day after treatment, they ingested a greater average pirfenidone dose over 20 days than saline-treated control mice (717±44 versus 564±30 mg/kg per day, respectively). Total lung hydroxyproline content, an index of fibrosis, was significantly lower 21 days after treatment with CP plus pirfenidone as compared to mice treated with CP alone. Although microscopic lung fibrosis scores were not significantly decreased by pirfenidone in CP-treated mice, the overall incidence of fibrosis was significantly decreased. Histologically, mice treated with CP showed fibrosis while mice treated with CP plus pirfenidone exhibited fewer abnormalities. The rate of hydroxyproline synthesis by lung tissue 9 days after treatment with CP was significantly elevated. This rate was not affected by pirfenidone treatment. Overall, these data support an antifibrotic effect of pirfenidone against CP-induced lung fibrosis in mice. The mechanism of its effect is not known, but appears to be unrelated to an inhibition of collagen synthesis.

Effect of gestational age on the increase in fetal lung growth following tracheal obstruction.

The growth response of the fetal lung to increased expansion was compared at two gestational ages. In fetal sheep, lung expansion was increased by occluding the trachea for 48 h at either 112-114 days (younger fetuses) or 125-127 days (older fetuses) of gestation (term is approximately 145 days). After 24 h of tracheal occlusion, the volumes of liquid that could be drained from the lungs were increased by 64.7 and 158% above control in younger and older fetuses respectively; the volumes were not increased further after 48 h. In younger fetuses, 48 h of tracheal occlusion increased (p < .05) fetal lung wet weights (21% above control) and protein contents (43% above control) but not DNA contents. In older fetuses, 48 h of tracheal occlusion increased (p < .05) fetal lung wet weights (61% above control), protein contents (41% above control), and DNA contents (22% above control). However, 48 h of tracheal occlusion did not alter total lung hydroxyproline content at either age, resulting in a reduction in the hydroxyproline/protein ratio of the fetal lungs. The results suggest that the lung growth response to tracheal occlusion is greater at 125-127 days of gestation than at 112-114 days of gestation, probably due to a greater accumulation of lung liquid and hence a greater increase in lung expansion, in older fetuses.

Trichloroethylene induces pulmonary fibrosis in mice

Trichloroethylene elicits acute pulmonary cytotoxicity in mice, which involves Clara cells of bronchioles. In this study, we have examined the effects of an acute dose of trichloroethylene in lungs of mice over 3 months. Pulmonary fibrosis was first detected at 15 days and was progressive with time elapsed after trichloroethylene exposure. Diffuse interstitial fibrosis was observed in the alveolar zone, resulting in thickening of alveolar septa and distortion of lung structure. The fibrosis was most pronounced at 90 days after treatment, resulting in deposition of connective tissue in the alveolar septa. Levels of total lung hydroxyproline were not significantly different in control and treated mice at 30 and 60 days after trichloroethylene treatment, but were significantly increased at 90 days. Proline content remained unchanged during the course of this study. The increase in collagen deposition at 90 days coincided with a significant increase in lung elastic recoil. Our results show that a single acute dose of trichloroethylene causes structural and functional abnormalities that are progressive for at least 3 months.

Effect of cytotoxic monoclonal antibody depletion of T-lymphocyte subpopulations on bleomycin-induced lung damage in C57BL/6J mice

Treatment of cancer patients with the antitumor antibiotic bleomycin (BLM) is associated with lung damage which can progress to pulmonary fibrosis. Shortly after intratracheal (it) administration of BLM to experimental animals there is an influx of inflammatory cells into the lung. These inflammatory cells, consisting primarily of polymorphonuclear cells, monocytes and lymphocytes, are believed to modulate the pathogenesis of pulmonary fibrosis. The objective of the present study was to determine the role of specific T-lymphocyte subpopulations in this disease process following a single it administration of BLM to C57BL/6J mice. Specific in vivo T-lymphocyte subpopulation depletion was accomplished by multiple intraperitoneal administrations of cytotoxic monoclonal antibodies to mice prior to and following BLM administration. Acute lung damage was assessed by measuring levels of angiotensin-converting enzyme and total protein in the bronchoalveolar lavage fluid 7 days after BLM treatment while chronic fibrosis was assessed by total lung hydroxyproline 28 days after BLM. Although we were able to deplete lymph nodes and BAL of specific T-lymphocyte subpopulations we were unable to detect a difference in the extent or severity of either the acute or chronic stage of BLM-induced lung damage. These results suggest that BLM lung disease progresses unabated in C57BL/6J mice despite virtually complete depletion of either L3T4 + or Lyt-2 + T-lymphocytes. Although a > 80% decrease in Thy-1.2 + T-lymphocytes was accomplished, there was a residual population of Thy-1.2 + lymphocytes resistant to the cytotoxic antibody. It is possible, therefore, that this population of cells does play a role in the development of BLM lung disease.

Tumor necrosis factor/cachectin plays a key role in bleomycin-induced pneumopathy and fibrosis.

The role of TNF-alpha/cachectin in the pneumopathy elicited by bleomycin has been investigated. After a single intratracheal bleomycin instillation, an increase of the lung TNF-alpha mRNA level was evident, from days 5 to 15, as shown by Northern gel analysis of whole lung RNA. In contrast, lung IL-1-alpha and GM-CSF mRNA were not detectable. In mice passively immunized with rabbit anti-mouse TNF-alpha IgG, the bleomycin-induced collagen deposition, evaluated by the total lung hydroxyproline assay on day 15, was prevented. Depletion of the CD4 and CD8 T lymphocytes by an in vivo treatment with mAb prevented the bleomycin-induced increase of TNF mRNA level and fibrosis. After an administration of bleomycin in continuous intraperitoneal perfusion, the diffuse alveolar damage observed by light and electron microscopy was almost completely prevented by anti-TNF antibody. These results indicate that in response to bleomycin, the T lymphocytes induce, by an undefined mechanism, an increase of the pulmonary TNF production, which leads to alveolar damage, growth of fibroblast, and collagen deposition.