Lung Fibrosis In Rats Research Articles

Animal models of pulmonary fibrosis: how far from effective reality?

idiopathic pulmonary fibrosis (IPF) is a chronic and progressive lung disease, associated with high mortality rates and unresponsive to currently available treatments. Animal models are critical to identify and validate new therapeutic drug targets, a concept that is nicely highlighted by the review

Comparative anti-fibrotic effect between ACE inhibitors on paraquat-induced lung fibrosis in rats

Comparative anti-fibrotic effect between ACE inhibitors on paraquat-induced lung fibrosis in rats

Effect of intramuscular injection of hepatocyte growth factor plasmid DNA with electroporation on bleomycin-induced lung fibrosis in rats

So far, there is no efficient treatment for pulmonary fibrosis. The objective of this study was to determine whether intramuscular injection of the hepatocyte growth factor (HGF) plasmid DNA by in vivo electroporation could prevent bleomycin-induced pulmonary fibrosis in rats, and to investigate the possible mechanisms. Twenty male Wistar rats were randomly divided into four groups: control group (group C), model group (group M), early intervention group (group I) and late intervention group (group II). Groups M, I and II were intratracheally infused with bleomycin, then injected the plasmid pcDNA3.1-hHGF to group I on day 7, 14 and 21. Group II received the same treatment like Group I on day 14 and 21. All the rats were killed on day 28 after bleomycin injection. We detected Homo HGF expression in the rats with ELISA method and estimated the pathological fibrosis score of lung tissue using hematoxylin eosin (HE) and Massion staining. The mRNA expression of transforming growth factor-beta1 (TGF-beta1), cycloxygenase-2 (COX-2), and rat HGF in rat pulmonary parenchyma were evaluated by RT-PCR. Immunohistochemistry and Western blotting were performed to determine the protein expression of transforming TGF-beta1 and COX-2 in lung parenchyma. The plasmid pcDNA3.1-hHGF could express hHGF in NIH3T3 cells and the hHGF protein is secreted into the culture medium. The expression of hHGF protein could be monitored in quadriceps muscle, plasma and lung in Groups I and II. Pulmonary fibrosis levels of Groups I and II were obviously lower than that of group M (P < 0.05). Expression of TGF-beta1 protein and mRNA in lung tissue was markedly decreased in Groups I and II compared with Group M (P < 0.05). The level of expression of HGF and COX-2 mRNA was higher in Groups I and II than in Group M (P < 0.05). Injection of the plasmid pcDNA3.1-hHGF into skeletal muscle with electroporation has a potential role in the treatment of bleomycin-induced lung fibrosis. Exogenous HGF may inhibit the expression of TGF-beta1 and regulate the crosstalk between AECs and mesenchymal fibroblasts.

Antifibrotic effect of captopril and enalapril on paraquat‐induced lung fibrosis in rats

Although different treatment modalities have been implemented for pulmonary fibrosis, the results have not been promising and these conditions have been considered untreatable and irreversible. Thus, a plethora of new drugs has been tried for the control of this condition in recent years. This study examined the effects of two angiotensin-converting enzyme inhibitors, captopril and enalapril, on paraquat-induced pulmonary fibrosis in rats, through biochemical and histopathological parameters. Male albino Wistar rats were divided into eight groups (n = 4-5 each), including control, paraquat, captopril alone, captopril treatment and pre-treatment, enalapril alone, enalapril treatment and pre-treatment. After 21 days of treatment, the lungs were removed and the levels of hydroxyproline, glutathione and lipid peroxidation were determined. Angiotensin-converting enzyme inhibitors showed no effect on glutathione and lipid peroxidation. The results also demonstrated that captopril and enalapril improved pulmonary fibrosis as shown by histopathology, as well as a decreased content of hydroxyproline (P < 0.001) in the lung tissue. In conclusion, the present findings suggest that the antifibrotic effect of these drugs may be related to the inhibition of angiotensin-converting enzyme.

Extravascular sources of lung angiotensin peptide synthesis in idiopathic pulmonary fibrosis

Previous work from this laboratory demonstrated de novo synthesis of angiotensin (ANG) peptides by apoptotic pulmonary alveolar epithelial cells (AEC) and by lung myofibroblasts in vitro and in bleomycin-treated rats. To determine whether these same cell types also synthesize ANG peptides de novo within the fibrotic human lung in situ, we subjected paraffin sections of normal and fibrotic (idiopathic pulmonary fibrosis, IPF) human lung to immunohistochemistry (IHC) and in situ hybridization to detect ANG peptides and angiotensinogen (AGT) mRNA. These were analyzed both alone and in combination with cell-specific markers of AEC [monoclonal antibody (MAb) MNF-116] and myofibroblasts [alpha-smooth muscle actin (alpha-SMA) MAb] and an in situ DNA end labeling (ISEL) method to detect apoptosis. In normal human lung, IHC detected AGT protein in smooth muscle underlying normal bronchi and vessels, but not elsewhere. Real-time RT-PCR and Western blotting revealed that AGT mRNA and protein were 21-fold and 3.6-fold more abundant, respectively, in IPF lung biopsies relative to biopsies of normal human lung (both P < 0.05). In IPF lung, both AGT protein and mRNA were detected in AEC that double-labeled with MAb MNF-116 and with ISEL, suggesting AGT expression by apoptotic epithelia in situ. AGT protein and mRNA also colocalized to myofibroblast foci detected by alpha-SMA MAb, but AGT mRNA was not detected in smooth muscle. These data are consistent with earlier data from isolated human lung cells in vitro and bleomycin-induced rat lung fibrosis models, and they suggest that apoptotic AEC and myofibroblasts constitute key sources of locally derived ANG peptides in the IPF lung.

Short courses of low dose dexamethasone delay bleomycin-induced lung fibrosis in rats

After comparing mortality and clinical signs in rats receiving different dexamethasone treatments, we investigated whether 0.5 mg/kg/d dexamethasone could delay pulmonary fibrosis induced by bleomycin and its time course (1, 3, 7, 14, 21 and 28 days). Tissue injury was assessed by apoptosis, lactate dehydrogenase (LDH) release, malondialdehyde content, and protein content; and inflammation was measured in terms of myeloperoxidase (MPO) activity, inflammatory cell count, and the mRNA expression of pro/inflammatory cytokines. Fibrogenic activity was analyzed by measuring the mRNA expression of fibrotic cytokines in tissue, and the promotion of fibroproliferation and synthesis of collagen type I by bronchoalveolar lavage fluids in vitro; and fibrosis was assessed by measuring the hydroxyproline content and collagen-I mRNA expression, and by histology. Bleomycin treatment induced tissue injury, inflammation and fibrogenic activity in lung, and led to fibrosis. Treatment with dexamethasone diminished the extent of fibrosis by strongly reducing inflammation, lung damage, and fibrogenic activity. These results demonstrate that the progression of bleomycin-induced pulmonary fibrosis in rats can be delayed by dexamethasone treatment, which appeared to alleviate not only inflammation but also lung damage and fibrogenic activity, indicating a possible new role for dexamethasone in the treatment of fibrosis.

Ginkgo biloba inhibits bleomycin-induced lung fibrosis in rats

Oxidative stress has been implicated in the pathogenesis of bleomycin-induced lung fibrosis and many antioxidant agents have been studied for prevention and treatment of the disease in animals and humans. We therefore examined whether Ginkgo biloba (Gb), a flavonoid-rich antioxidant, inhibits bleomycin-induced lung fibrosis in rats. Male Spraque–Dawley rats were given a single dose of bleomycin (2.5 mg/kg, intratracheally) in pulmonary fibrosis groups and saline in controls. First dose of Gb was given a day before the bleomycin injection and continued until sacrifice. At day 14, fibrotic changes in lung were estimated to occur by Aschoft's criteria and lung hydroxyproline content. Bleomycin challenge provoked severe pulmonary fibrosis with marked increase in hydroxyproline content of lung tissue and typical histological findings, which is prevented by Gb. Hydroxyproline level was significantly higher (13.51 ± 0.87 mg/g dried tissue) in bleomycin treated rats than controls (9.2 ± 1.33), and its level was remained to the control levels (7.38 ± 0.76) in rats treated with prophylactic Gb. On the other hand, bleomycin injection significantly reduced activities of glutathione peroxidase, superoxide dismutase and catalase in lung tissue which is prevented by Gb. Also, bleomycin injection resulted in a marked increase of malondialdehyde and nirite level which is attenuated by Gb. The data suggest that Gb has a potent antioxidant activity in the model of bleomycin-induced lung fibrosis in rats, and therefore has a potent antifibrotic activity against bleomycin-induced lung fibrosis model in rats.

Effects of erdosteine on bleomycin-induced lung fibrosis in rats

This study was designed to examine the effects of erdosteine on bleomycin (BLM)-induced lung fibrosis in rats. Thirty-three Sprague-Dawley rats were divided randomly into three groups, bleomycin alone (BLM), bleomycin + erdosteine (BLM + ERD), and saline alone (control). The BLM and BLM + ERD groups, were given 2.5 mg/kg BLM intratracheally. The first dose of oral erdosteine (10 mg/kg/day) in the BLM + ERD group was started 2 days before BLM administration and continued until animals were sacrificed. Animals were sacrificed 14 days after intratracheal instillation of BLM. The effect of erdosteine on pulmonary fibrosis was studied by analysis of bronchoalveolar lavage (BAL) fluid, histopathology, and biochemical measurements of lung tissue superoxide dismutase (SOD) and glutathione (GSH) as antioxidants, malondialdehyde (MDA) as an index for lipid peroxidation, and nitrite/nitrate levels. Bleomycin-induced lung fibrosis as determined by lung histology was prevented with erdosteine (grades of fibrosis were 4.9, 2.3, and 0.2 in BLM, BLM + ERD, and control groups, respectively). Erdosteine also prevented bleomycin-induced increase in MDA (MDA levels were 0.50 +/- 0.15, 0.11 +/- 0.02, and 0.087+/- 0.03 nmol/mg protein in BLM, BLM + ERD, and control groups, respectively) and nitrite/nitrate (nitrite/nitrate levels were 0.92 +/- 0.06, 0.60 +/- 0.09, and 0.56+/- 0.1 micromol/mg protein in BLM, BLM + ERD, and control groups respectively) levels. Bleomycin-induced decrease in GSH and SOD levels in the lung tissue also prevented by erdosteine [(GSH levels were 213.5 +/- 12.4, 253.2+/- 25.2, and 287.9+/- 34.4 nmol/mg protein) (SOD levels were 1.42+/- 0.12, 1.75+/- 0.17, and 1.89+/- 0.09 U/mg protein) in BLM, BLM + ERD, and control groups respectively]. Erdosteine prevented bleomycin-induced increases in total cell number and neutrophil content of the BAL fluid. In conclusion, oral erdosteine is effective in prevention of BLM-induced lung fibrosis in rats possibly via the repression of neutrophil accumulation, inhibition of lipid peroxidation, and maintenance of antioxidant and free radical scavenger properties.

TGF-β and Smad3 Signaling Link Inflammation to Chronic Fibrogenesis

Transient adenovirus-mediated gene transfer of IL-1beta (AdIL-1beta), a proinflammatory cytokine, induces marked inflammation and severe and progressive fibrosis in rat lungs. This is associated with an increase in TGF-beta1 concentration in bronchoalveolar lavage (BAL) fluid. TGF-beta1 is a key cytokine in the process of fibrogenesis, using intracellular signaling pathways involving Smad2 and Smad3. In this study we investigate whether inflammation induced by IL-1beta is able to independently induce lung fibrosis in mice deficient in the Smad3 gene. Seven days after AdIL-1beta administration, similar levels of IL-1beta transgene are seen in BAL in both wild-type (WT) and knockout (KO) mice, and BAL cell profiles demonstrated a similar marked neutrophilic inflammation. Phospho-Smad2 staining was positive in areas of inflammation in both WT and KO mice at day 7. By day 35 after transient IL-1beta expression, WT mice showed marked fibrosis in peribronchial areas, quantified by picrosirius red staining and morphometry. However, there was no evidence of fibrosis or collagen accumulation in IL-1beta-treated KO mice, and peribronchial areas were not different from KO mice treated with the control adenovector. TGF-beta1 and phospho-Smad2 were strongly positive at day 35 in fibrotic areas observed in WT mice, but no such staining was detectable in KO mice. The IL-1beta-induced chronic fibrotic response in mouse lungs is dependent on Smad3. KO and WT animals demonstrated a similar inflammatory response to overexpression of IL-1beta indicating that inflammation must link to the Smad3 pathway, likely through TGF-beta, to induce progressive fibrosis.

Preventive effect of melatonin on bleomycin‐induced lung fibrosis in rats

Oxidative stress has an important role in the pathogenesis of idiopathic pulmonary fibrosis. Melatonin has direct and indirect free radical-detoxifying activity. The present study investigated whether melatonin treatment attenuates bleomycin-induced lung fibrosis in rats. A group of rats was given one dose of bleomycin while the control animals were given saline. The first dose of melatonin (4 mg/kg/day) was given 2 days before the bleomycin injection. At day 14, fibrotic changes were evaluated using Aschoft's criteria and lung hydroxyproline content. Bleomycin produced a 2.7-fold rise in the fibrosis score that was decreased 65% by melatonin (P < 0.05) and a 1.4-fold increase in hydroxyproline content which was completely prevented by melatonin. Protein carbonyl and thiobarbituric acid reactive substances levels, which were significantly elevated in the bleomycin treated rats, were significantly attenuated by melatonin. Bleomycin administration significantly reduced the activities of catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in lung tissue. The reduction in CAT activity was prevented by melatonin but SOD and GSH-Px were not influenced. These results revealed that melatonin may prevent the development of bleomycin-induced lung fibrosis via the repression of protein and lipid peroxidation.

Attenuation of bleomycin-induced lung fibrosis in rats by mesna

Lung fibrosis is a common side effect of the chemotherapeutic agent, bleomycin. Current evidence suggests that reactive oxygen species may play a key role in the development of lung fibrosis. The present study examined the effect of mesna on bleomycin-induced lung fibrosis in rats. Animals were divided into three groups: (1) saline control group; (2) Bleomycin group in which rats were injected with bleomycin (15 mg/kg, i.p.) three times a week for four weeks; (3) Bleomycin and mesna group, in which mesna was given to rats (180 mg/kg/day, i.p.) a week prior to bleomycin and daily during bleomycin injections for 4 weeks until the end of the treatment. Bleomycin treatment resulted in a pronounced fall in the average body weight of animals. Bleomycin-induced pulmonary injury and lung fibrosis was indicated by increased lung hydroxyproline content, and elevated nitric oxide synthase, myeoloperoxidase, platelet activating factor, and tumor necrosis factor-α in lung tissues. On the other hand, bleomycin induced a reduction in reduced glutathione concentration and angiotensin converting enzyme activity in lung tissues. Moreover, bleomycin-induced severe histological changes in lung tissues revealed as lymphocytes and neutrophils infiltration, increased collagen deposition and fibrosis. Co-administration of bleomycin and mesna reduced bleomycin-induced weight loss and attenuated lung injury as evaluated by the significant reduction in hydroxyproline content, nitric oxide synthase activity, and concentrations of myeoloperoxidase, platelet activating factor, and tumor necrosis factor-α in lung tissues. Furthermore, mesna ameliorated bleomycin-induced reduction in reduced glutathione concentration and angiotensin activity in lung tissues. Finally, histological evidence supported the ability of mesna to attenuate bleomycin-induced lung fibrosis and consolidation. Thus, the findings of the present study provide evidence that mesna may serve as a novel target for potential therapeutic treatment of lung fibrosis.

Effects of aminoguanidine and antioxidant erdosteine on bleomycin-induced lung fibrosis in rats

Reactive oxygen and nitrogen species have been implicated in the pathogenesis of bleomycin-induced lung fibrosis. The effects of aminoguanidine and erdosteine on the bleomycin-induced lung fibrosis were evaluated in rats. The animals were placed into five groups: Vehicle + vehicle, vehicle + bleomycin (2.5 U/kg), bleomycin + aminoguanidine (200 mg/kg), bleomycin + erdosteine (10 mg/kg), and bleomycin + erdosteine + aminoguanidine. Bleomycin administration resulted in prominent lung fibrosis as measured by lung hydroxyproline content and lung histology, which is completely prevented by erdosteine and aminoguanidine. A strong staining for nitro tyrosine antibody in lung tissue and increased levels of lung NO were found in bleomycin group, that were significantly reduced by aminoguanidine and erdosteine. Aminoguanidine and erdosteine significantly prevented depletion of superoxide dismutase and glutathione peroxidase and elevated myeloperoxidase activities, malondialdehyde level in lung tissue produced by bleomycin. Data presented here indicate that aminoguanidine and erdosteine prevented bleomycin-induced lung fibrosis and that nitric oxide mediated tyrosine nitration of proteins plays a significant role in the pathogenesis of bleomycin-induced lung fibrosis. Also our data suggest that antifibrotic affect of antioxidants may be due to their inhibitory effect on nitric oxide generation in this model.

Inhibitory effect of caffeic acid phenethyl ester on bleomycine-induced lung fibrosis in rats

Background: Pulmonary fibrosis (PF) induced by anticancerogenic bleomycin (BLM) is one of the more common side effects encountered during cancer treatment. It has been suggested in the last decades that the main responsible agent in PF is reactive oxygen species which were generated also in normal physiological conditions in the human body. In this experimental study, we investigated the preventive or attenuating effects of caffeic acid phenethyl ester (CAPE) that has been demonstrated to have anti-inflammatory, cytocytatic, anticancerogenic, antiprolipherative and antioxidant effects on BLM-induced PF. Methods: Thirty-six Sprague–Dawley rats were divided randomly into four groups as sham operation, BLM, BLM+vitamin E (vit E), and BLM+CAPE groups. BLM (7.5 mg/kg, single dose) was applied intratracheally, and CAPE and vit E intraperitoneally in the appropriate groups. At the end of the fibrosis processes, lung tissues were removed and the levels of tissues hydroxyproline (OH-proline), malondialdehyde (MDA) and NO as well as the activities of superoxide dismutase (SOD), catalase (CAT) and myeloperoxidase (MPO) were determined. Also, the weights of the rats were recorded at 7th and 14th days of the experiments. Results: BLM application to the rats resulted in a significant increase in the OH-proline level as compared to the controls. Administration of CAPE and vit E led to the remarkable reduction of total lung OH-proline levels compared to the rats treated with BLM alone ( p<0.0001). There were a decreases in antioxidant enzyme (SOD and CAT) activities while an increase in MPO activity in BLM group was found vs. the control group ( p<0.0001). CAPE had a regulator effect on these parameters: the increase in CAT and SOD activities and the decrease in MPO activity were seen after CAPE application. NO, MDA and OH-proline levels were increased in BLM group vs. the control group. CAPE was more effective in decreasing the tissue levels of NO, MDA and OH-proline than vit E. MPO activity, as a good marker of neutrophil sequestration to the tissues, in the BLM group was decreased by CAPE approximately to the control group. Conclusion: We suggest that CAPE is more effective on the prevention of BLM-induced fibrosis via antioxidant and free radical scavenger properties than vit E at the doses used in the present study. CAPE has some attenuating effects on BLM-induced PF affecting both oxidant and antioxidant systems as well as neutrophils sequestration.

Protective effects of curcumin against amiodarone-induced pulmonary fibrosis in rats.

(1) We have studied whether curcumin prevents amiodarone-induced lung fibrosis in rats. Intratracheal instillation of amiodarone (6.25 mg kg(-1) on days 0 and 2, and then killed on day 3, day 5, week 1, week 3 and week 5 after amiodarone administration) induced increases in total protein and lactate dehydrogenase (LDH) activity on days 3 and 5 in bronchoalveolar lavage fluid (BALF). Total cell counts, alveolar macrophages, neutrophils and eosinophils recovered by BAL, and lung myeloperoxidase (MPO) activity were significantly higher in amiodarone rats. (2) Tumor necrosis factor-alpha (TNF-alpha) release after lipopolysaccharide (LPS) stimulation and superoxide anion generation after phorbol myristate acetate (PMA) stimulation were higher in the alveolar macrophages of amiodarone rats at 3 and 5 weeks postamiodarone instillation than in controls. Amiodarone also induced increases in transforming growth factor-beta1 (TGF-beta1) expression, collagen deposition, type I collagen expression and c-Jun protein in lungs. (3) Curcumin (200 mg kg(-1) body weight after first amiodarone instillation and daily thereafter for 5 weeks)-treated amiodarone rats had reduced levels of protein, LDH activity, total cell numbers and differential cell counts in BALF. LPS-stimulated TNF-alpha release and PMA-stimulated superoxide generation were significantly suppressed by curcumin. Furthermore, curcumin inhibited the increases in lung MPO activity, TGF-beta1 expression, lung hydroxyproline content, expression of type I collagen and c-Jun protein in amiodarone rats. Our results have important implications for the treatment of amiodarone-induced lung fibrosis.

Relationship of jute dust to interstitial fibrosis in rat lung.

The relationship between jute dust and lung interstitial fibrosis was studied by instilling groups of rats, via trachea, with jute dust and comparing the results with those for positive (quartz) and negative (saline) controls. The rats were sacrificed at regular intervals and their lungs and hilar lymph nodes were analyzed for collagen content and morphologic changes. The earliest changes consisted of alveolar edema, increased numbers of intraalveolar macrophages, and marked thickening of the interalveolar septa, with mixed cellular infiltrates. Moderate thickening of the alveolar walls and the zones around the peribronchioles was seen in the test groups at 6 mo. After 12 mo, some fibrosis of the alveoli walls and peribronchiole zones occurred. Interstitial cellular nodules were observed occasionally, composed mainly of dust particles, fibroblasts, reticular fibers, and collagen fibers. The collagen content in the lungs of the jute dust groups was significantly higher than for the saline control group for all test periods. The authors conclude that jute dust may induce lung interstitial fibrosis.

Attenuation by oral N-acetylcysteine of bleomycin-induced lung injury in rats.

Antioxidant therapy may be useful in diseases with impaired oxidant-antioxidant balance such as pulmonary fibrosis. This study examines the effect of N-acetylcysteine (NAC) on bleomycin-induced lung fibrosis in rats. NAC (3 mmol x kg(-1); oral) was given daily from 1 week prior to a single intratracheal instillation of bleomycin (2.5 U x kg(-1)) or saline, until 14 days postinstillation. NAC partially decreased the augmented collagen deposition in bleomycin-exposed rats (hydroxyproline content was 4,354+/-386 and 3,416+/-326 microg x lung(-1) in vehicle-treated and NAC-treated rats, respectively; p < 0.05). The histological assessment using a semiquantitative score showed less collagen deposition and inflammatory cells in NAC-treated rats compared to those receiving bleomycin alone. NAC failed to inhibit the bleomycin-induced increases in lung wet weight and in cell counts and protein levels of bronchoalveolar lavage fluid, but significantly increased total glutathione and taurine levels in bronchoalveolar lavage fluid. These results indicate that oral N-acetylcysteine improves the pulmonary antioxidant protection and may be useful in reducing lung damage produced by bleomycin.

Irradiation-induced expression of hyaluronan (HA) synthase 2 and hyaluronidase 2 genes in rat lung tissue accompanies active turnover of HA and induction of types I and III collagen gene expression.

Hyaluronan (HA) is a linear glycosaminoglycan that accumulates in the interstitium of injured lung and inhibits gas exchange between air and blood. In the present study we investigated the molecular mechanisms behind the local turnover of HA during the early phase of irradiation-evoked lung fibrosis in rats. Irradiation with a single dose of 30 Gy to the lower part of the right lung of rats induced an accumulation of HA in bronchoalveolar lavage fluid 6 wk after irradiation, followed by return to almost normal levels at 10 wk after irradiation. This was parallelled with a transient downregulation of HA receptors on alveolar macrophages (AMs); 4 and 6 wk after irradiation the binding of [(3)H]HA to AMs was decreased to about 50% of that of AMs from nonirradiated control rats, returning to almost normal level at 10 wk after irradiation. Analysis of the expression of rat HA synthase (HAS) isoforms (rHAS1, rHAS2, and rHAS3) and rat hyaluronidases (rHYAL1 and rHYAL2) by Northern blotting revealed an upregulation of rHAS2 messenger RNA at 4, 6, and 10 wk after irradiation, but a progressive decrease in the constitutive expression of rHYAL2 at 6 and 10 wk after irradiation; rHAS1 was undetectable, whereas rHAS3 and rHYAL1 were faintly detectable. Although transforming growth factor-beta1 stimulated HA production by normal lung fibroblasts, it inhibited HYAL activity in lysosomes and HYAL activity released into the culture media. Another interesting observation was that HA fragments, which likely result from the action of HYAL, induced expression of types I and III collagen genes. Our results indicate that rHAS2 and rHYAL2 are involved in the turnover of HA during the early phase of lung injury and that rHAS2 and rHYAL2 as well as HA fragments may play important roles in the pathogenesis of lung fibrosis.

LU135252, an endothelin(A) receptor antagonist did not prevent pulmonary vascular remodelling or lung fibrosis in a rat model of myocardial infarction.

The early intervention with endothelin(A) (ET(A)) receptor antagonists following coronary artery ligation has been shown to reduce the development of pulmonary hypertension, despite a lack of improvement in left ventricular function. The present study examined the contribution of pulmonary vascular remodelling and the progression of lung fibrosis in the development of pulmonary hypertension and the subsequent role of endothelin-1 in these processes in a rat model of myocardial infarction (MI). The administration of 60 mg kg(-1) per day of the specific ET(A) receptor antagonist LU135253 ((+)-(S)-2-(4, 6-dimethoxy-pyrimidin-2-yloxy)-3-methoxy-3,3-diphenyl-propionic acid) 24 h following coronary artery ligation, failed to improve left ventricular contractile indices, but reduced the extent of pulmonary hypertension, as reflected by the significant decrease in right ventricular systolic pressure. The medial wall thickness of small pulmonary arteries (50 - 200 microm) was significantly increased 4 weeks following MI, albeit LU135253 treatment did not ameliorate this pattern of vascular remodelling. The steady-state mRNA levels of collagen, fibronectin, transforming growth factor-beta(1), and -beta(3) were significantly increased in the lungs of MI rats. The treatment with LU135252 did not alter this pattern of gene expression. Thus, these data demonstrate pulmonary vascular remodelling and the increased expression of extracellular matrix proteins represent underlying mechanisms implicated in the development of pulmonary hypertension in the MI rat. Despite the amelioration of the pulmonary hypertensive state, ET(A) receptor blockade was insufficient to reverse pulmonary vascular remodelling, or the development of lung fibrosis in the MI rat.

Alterations in the immunohistochemical distribution patterns of vascular endothelial growth factor receptors Flk1 and Flt1 in bleomycin-induced rat lung fibrosis.

To investigate the role of vascular endothelial growth factor (VEGF) in fibrogenesis, the distribution patterns of the VEGF receptors Flt1 and Flk1 were studied by immunohistochemistry, double immunofluorescence, and immunoelectron microscopy in normal (n=2) and bleomycin-treated (n=21) adult rats. Lungs were studied at 5, 24, 28, 35, and 42 days after treatment (p.t.). Flt1, Flk1, and VEGF immunoreactivity localised predominantly to the pulmonary epithelium. In control lungs, Flt1 immunoreactivity was present in ciliated bronchial epithelium and type 2 pneumocytes, Flk1 in Clara cells, and VEGF in Clara cells and type 2 pneumocytes. Flk1 localised to mast cells, present in the peribronchovascular and pleural interstitium only. Flt1- and Flk1-mRNAs were observed in Clara cells and type 2 pneumocytes. Bleomycin-induced fibrogenesis was characterised by a decrease in Flk1 immunoreactivity of Clara cells, and an increase in VEGF-immunoreactive myofibroblasts and type 2 pneumocytes by day 5 p.t., followed by a progressive accumulation of Flk1-immunoreactive mast cells by day 24 p.t. in fibrotic lesions containing VEGF-immunoreactive myofibroblasts. After 42 days, fibrotic regions were densely populated by mast cells. Since mast cells are known to be chemotactically attracted by VEGF, we suggest that VEGF/Flk1 represents the molecular link between proliferation of myofibroblasts, accumulation of mast cells, and the burst of fibrosis at sites of initial lesions in bleomycin-induced fibrosis.

Differential immunolocalization of VEGF in rat and human adult lung, and in experimental rat lung fibrosis: Light, fluorescence, and electron microscopy

Vascular endothelial growth factor (VEGF) is a cytokine with main angiogenetic functions in embryonic development and tumor-formation. In the adult lung, reports of the localization of VEGF were controversial. A precise cell typing of VEGF-positive pulmonary cells is still lacking. Nothing is known about a potential role in pulmonary fibrosis. Immunohistochemistry (IH), double immunofluorescence microscopy (DIF), and immunoelectron microscopy (IEM) were used to study the differential distribution of VEGF in paraffin-embedded (IH, DIF) and in cryo-substituted, Lowicryl-embedded (IEM) specimens of normal rat and human lungs and fibrotic rat lungs. Fibrosis was induced by intratracheal bleomycin treatment. IH and DIF showed that VEGF was present in surfactant protein (SP) D-positive alveolar type II pneumocytes, bronchiolar Clara cells, smooth muscle (SM) cells, and α-SM actin-positive myofibroblasts of normal rat and human lungs. Fibrotic lesions in bleomycin-treated rat lungs were rich in VEGF-positive cells presenting with a heterogeneous phenotype (mainly SP-D-positive type II pneumocytes, α-SM actin-positive myofibroblasts). There were no signs of angiogenesis. Post-embedding immunogold labeling using protein A-gold and IgG-gold technique revealed a specific localization of VEGF to mitochondria, Clara cell secretory granules, and capillary interendothelial cell junctions. The predominant localization of VEGF to bronchiolar and alveolar epithelial and α-SM actin-positive cells, and the marked increase of VEGF-positive type II pneumocytes and myofibroblasts in fibrotic lung lesions, indicate that in adult lungs VEGF is involved in processes other than angiogenesis. Anat Rec 254:61–73, 1999. © 1999 Wiley-Liss, Inc.