Local activation of the pulmonary extravascular angiotensin system induces epithelial apoptosis and lung fibrosis.

Previous work suggests that a local extravascular angiotensin system plays an important role in the development of pulmonary fibrosis through stimulation of alveolar epithelial cell (AEC) apoptosis and collagen deposition. To demonstrate a causative role for the local tissue angiotensin (ANG) system in lung fibrosis, we hypothesize that overexpression of the angiotensinogen (AGT) gene or pharmacologic elevation of lung tissue ANG II levels might cause apoptosis of AECs and lung fibrosis. ANGII levels were elevated in rat or mouse lung tissue by intratracheal instillation of either purified ANGII or an adenovirus expressing AGT, or by ubiquitous overexpression of AGT in transgenic mice. Intratracheal instillation of purified ANGII caused significant collagen accumulation in lung tissue, both ex vivo and in vivo. Ubiquitous overexpression of AGT enhanced the profibrotic effect of bleomycin given at suboptimal doses. Intratracheal delivery of an adenoviral vector expressing mouse AGT (Ad-AGT) overexpressed AGT primarily in AECs and caused both apoptosis of AECs and pulmonary fibrosis. The lung collagen accumulation and AEC apoptosis caused by Ad-AGT was blocked by the caspase inhibitor ZVAD-fmk, by the ANG receptor AT1 antagonist Losartan or by the non-selective ANGII receptor antagonist Saralasin. Together, these data support the hypothesis that elevated pulmonary expression of AGT and its conversion to angiotensin II plays a causative role in the development of lung fibrosis through its induction of AEC apoptosis.

[Effect of iron overload on experimental immunological liver injury in rats and the role of angiotensin].

OBJECTIVE: To investigate the effect of iron overload on experimental immunological liver injury in rats and the roles of losartan (LOS), which is the selective antagonist of angiotensin II receptor subtype AT1. METHODS: Fifty male Wistar rats were divided by random into five groups (control, liver injury, liver injury + LOS, liver injury + ID and liver injury + ID + LOS). Immunological liver injury model was reproduceed by intravenous injection of BCG (Bacilli Calmette Guein) and then lipopolysaccharide (LPS). Iron overload model was created by intraperitoneal injection of iron dextran (ID). Serum iron (SI), transferrin (TRF), total protein (TP), the activity of asparatate aminotransferase (AST) and malondialdehyde (MDA) and liver iron (HIC) were tested. The expression of bcl-2 and Bax and the bax/bcl-2 ratio in hepatocyte were tested by flow cytometric analysis. Apoptotic index (AI) and proliferative index were also calculated. RESULTS: (1) In comparison with blank control group, the activity of serum AST was higher. Serum TP and TRF were lower in liver injury animals. Liver MDA increased significantly and along with a lower SOD activity. The expression of Bax in liver injury group was significantly higher than that in control group. The bax/ bcl-2 ratio and AI increased significantly in liver injury group. (2) Compared with liver injury group, the animals treated with ID showed an increase of serum AST activity, increased MDA and the expression of bax, the bax/bcl-2 ratio and AI. HIC was higher than the control group. (3) Compared with liver injury group, the activity of serum AST was lower and TRF was higher, MDA was reduced and SOD activity increased in animals treated with LOS. The expression of bcl-2 was increased, bax/bcl-2 ratio and AI decreased in this group. (4) In comparison with ID treated liver injury animals, the activity of AST and the content of MDA, and TRF increased in the animals treated with ID plus LOS. CONCLUSION: The immunological liver injury could be aggravated by iron overload through catalyzing lipid peroxidation and facilitating the apoptotic process of hepatocyte. Angiotensin faciliates in this kind of liver damage.

Apoptosis-dependent acute pulmonary injury after intratracheal instillation of angiotensin II.

To test the hypothesis that exogenous purified angiotensin II (ANG) might cause apoptosis of alveolar epithelial cells (AECs) and acute lung injury, male Wistar rats were intratracheally instilled with purified ANG (10 mumol/L), ANG plus the caspase inhibitor ZVAD-fmk (60 mumol/L), ANG plus the ANG receptor AT1 antagonist losartan (LOS, 100 mumol/L) or sterile phosphate-buffered saline (PBS) vehicle alone. Six or 20 h later, the lungs were lavaged in situ for determination of bronchoalveolar lavage (BAL) fluid content of hemoglobin (Hb) and fluorescent (BODIPY)-albumin, a bolus of which was injected intravenously 15 min prior to BAL. Terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) revealed that instillation of ANG, but not PBS alone, increased labeling of fragmented DNA in bronchiolar epithelial cells and in AECs (P<0.05) at 6 h post-ANG. Increased TUNEL was abrogated by concurrent instillation of ZVAD-fmk or LOS. Significant increased numbers of caspase-positive cells were observed by anti-caspase 3 immunolabeling after instillation of ANG (P<0.01); the same doses of LOS or ZVAD-fmk that blocked TUNEL also blocked the activation of caspase 3 (P<0.01). Intratracheal instillation of ANG also remarkably increased BAL BODIPY-albumin (P< 0.01) and Hb (P<0.05), both of which were eliminated by ZVAD-fmk or LOS. These data indicate that exposure of AECs to ANG in vivo is sufficient to induce apoptosis and alveolar epithelial barrier injury mediated by ANG receptor AT1.

Attenuation of bleomycin-induced pulmonary fibrosis by intratracheal administration of antisense oligonucleotides against angiotensinogen mRNA.

Apoptosis of alveolar epithelial cells (AECs) is believed to be critical for the development of bleomycin (BLEO)-induced pulmonary fibrosis. Previous studies showed that apoptosis of alveolar epithelial cells in response to BLEO could be abrogated by antisense oligonucleotides against angiotensinogen (AGT) mRNA and requires angiotensin II (ANG II) synthesis de novo [17]. In this study we hypothesized that blockade of local pulmonary ANG II synthesis by intratracheal (I.T.) administration of antisense oligonucleotides against AGT mRNA might attenuate BLEO-induced apoptosis of AECs and prevent pulmonary fibrosis. In a BLEO-induced rat model of lung fibrosis, endogenous lung AGT was upregulated in vivo as early as 3 hours after BLEO instillation, as detected by RT-PCR, in situ hybridization and immunohistochemistry. AGT mRNA and angiotensin peptides were localized in type II alveolar epithelial cells and also colocalized with alpha-smooth muscle actin (alpha-SMA), a marker of myofibroblasts. Tagged antisense administered I.T. was specifically accumulated by the lung relative to liver and kidney, and localized primarily in the epithelium of airways and cells within alveolar walls. The intratracheal AGT antisense reduced BLEO-induced pulmonary fibrosis measured by lung hydroxyproline assay, decreased lung AGT and active caspase-3 proteins, and reduced the number of apoptotic epithelial cells but had no effect on the serum ANG II concentration. These data are consistent with the hypothesis that lung-derived AGT and local pulmonary ANG II are required for BLEO-induced pulmonary fibrosis, and suggest the possibility of antisense-based manipulation of the local angiotensin system as a potential treatment of fibrotic lung diseases.

[Study on the roles of the expression of bcl-2 and bax in experimental immunological liver injury in rats].

OBJECTIVE: To elucidate the roles of Bcl-2 and Bax in experimental immunological liver injury in rat and the effect of lowering serum levels on the expression and the injury. METHODS: 48 male Wistar rats were divided into six groups randomly. The animal model of iron low-load was created by intravenation of deferoxamine (DFO) or phlebotomy respectively, and immunological liver damage model was reproduced by injection of BCG (Bacilli Calmette Guein) and lipopolysaccharide (LPS). Then the following parameters were determined such as serum iron (SI) concentration, transferrin (TRF) concentration, total proteins (TP) volume, the serum activity of aspartate aminotransferase (AST), malondialdehyde (MDA) content, iron content (HIC), the expression of Bcl-2 and Bax proteins in liver tissue were assayed; the ratio of Bax to Bcl-2, apoptotic index (AI), and proliferative index (PI) were also calculated. RESULTS: (1) The amount of Bax expression in liver injury group was significantly higher than that of control one, but no change in Bcl-2 expression. The ratio of Bax to Bcl-2 and AI augmented significantly, along with increased serum activities of AST and level of MDA, reduced volume of TP in liver injury animals. (2) The serum activity of AST and TP volume in both of the control groups with DFO and phlebotomy pretreatment remained at control level. Although the expression of Bax and Bcl-2, Bax/Bcl-2 ratio and AI were all higher than those of blank controls, the increased magnitudes of Bax/Bcl-2 ratio and Al were significantly lower than those of the liver injury animals. (3) The expression amounts of Bcl-2 and Bax increased in the injury animals induced after injecting DFO or phlebotomy, thus Bax/Bcl-2 ratio and Al increased. However, Bax/Bcl-2 ratio and AI of them were less than those of the injuries without lowered SI, and the magnitude of increased serum activity of AST was lower than that of the injuries, but no change in TP volume in the rats with lowered SI. The MDA levels in the injuries with lower value of serum iron were lower than those of the animals without lower SI. CONCLUSION: The results show that the apoptotic process of hepatocyte accelerates in immunological liver injury, apoptosis may facilitate hepatocyte damage. Effect of iron on the expression of apoptosis regulating proteins have played an important role in apoptosis of immunological hepatic injury.

Essential role for cathepsin D in bleomycin-induced apoptosis of alveolar epithelial cells.

Our earlier studies showed that bleomycin-induced apoptosis of type II alveolar epithelial cells (AECs) requires the autocrine synthesis and proteolytic processing of angiotensinogen into ANG II and that inhibitors of ANG-converting enzyme (ACEis) block bleomycin-induced apoptosis (Li X, Zhang H, Soledad-Conrad V, Zhuang J, and Uhal BD. Am J Physiol Lung Cell Mol Physiol 284: L501-L507, 2003). Given the documented role of cathepsin D (CatD) in apoptosis of other cell types, we hypothesized that CatD might be the AEC enzyme responsible for the conversion of angiotensinogen into ANG I, the substrate for ACE. Primary cultures of rat type II AECs challenged with bleomycin in vitro showed upregulation and secretion of CatD enzymatic activity and immunoreactive protein but no increases in CatD mRNA. The aspartyl protease inhibitor pepstatin A, which completely blocked CatD enzymatic activity, inhibited bleomycin-induced nuclear fragmentation by 76% and reduced bleomycin-induced caspase-3 activation by 47%. Antisense oligonucleotides against CatD mRNA reduced CatD-immunoreactive protein and inhibited bleomycin-induced nuclear fragmentation by 48%. A purified fragment of angiotensinogen (F1-14) containing the CatD and ACE cleavage sites, when applied to unchallenged AEC in vitro, yielded mature ANG II peptide and induced apoptosis. The apoptosis induced by F1-14 was inhibited 96% by pepstatin A and 77% by neutralizing antibodies specific for CatD (both P < 0.001). These data indicate a critical role for CatD in bleomycin-induced apoptosis of cultured AEC and suggest that the role(s) of CatD in AEC apoptosis include the conversion of newly synthesized angiotensinogen to ANG II.

[Putative mechanism of experimental immunological liver injury and influence of iron low-load on the injury in rat].

OBJECTIVE: To investigate the effects of superoxide dismutase (SOD) and reducing iron load on experimental immunological liver injury in rat. METHODS: 48 male Wistar rats were divided into six groups randomly. The animal model of iron low-load was created by intravasation of deferoxamine (DFO) or phlebotomy respectively. Immunological liver damage model was reproduced by injection of lipopolysaccharide (LPS) and BCG (Bacilli Calmette Guerin). The following parameters were determined such as serum iron (SI), transferrin (TRF) concentration, total proteins (TP) volume, the serum activities of aspartate aminotransferase (AST), and malondialdehyde (MDA) content, the activities of superoxide dismutase and iron deposition (HIC) in liver tissue extracts. RESULTS: In comparison with blank control group, the SI levels lowered (P < 0.05) in pure DFO group, pure phlebotomy group and liver injury group. Compared with each own control groups, the serum activities of AST in all injury groups augmented and the levels of TP in them reduced significantly. The increased magnitude of the activities of AST and the decreased magnitude of TP levels in DFO injury group and phlebotomy injury group were less than those of pure injury group (P < 0.05). In comparison with the control group, the MDA levels in pure DFO and phlebotomy groups declined significantly and the activities of SOD in them had no significant difference. Compared with each own control groups, the MDA levels augmented and the activities of SOD reduced significantly in all injury groups. CONCLUSION: SOD activity reduces in immunological liver injury in rat. Iron low-load can mitigate the severity of liver injury.

Inhibition of amiodarone-induced lung fibrosis but not alveolitis by angiotensin system antagonists.

Earlier work in this laboratory showed that amiodarone induces apoptosis in alveolar epithelial cells by a mechanism inhibitable by angiotensin system antagonists. A variety of recent studies suggests a critical role for alveolar epithelial cell apoptosis in the pathogenesis of lung fibrosis. On this basis we hypothesized that amiodarone-induced alveolar epithelial cell apoptosis and lung fibrosis in vivo might be inhibitable by the angiotensin converting enzyme inhibitor captopril or the angiotensin receptor antagonist losartan. Amiodarone-induced lung fibrosis was induced in male Wistar rats by oral administration over six months. Replicate groups of rats received captopril or losartan in addition to amiodarone. Apoptosis was detected by increased total lung activity of caspase 3 and in situ end labeling (ISEL) of fragmented DNA. Collagen was localized and quantitated by the picrosirius red technique. Alveolar epithelial cell apoptosis was detected in amiodarone-treated animals as early as three weeks after the start of amiodarone administration; by six months exposure, the incidence of alveolar epithelial cell apoptosis was significantly reduced by coadministration of captopril or losartan. Alveolar wall collagen accumulation also was significantly attenuated by captopril (100%) or losartan (74%), but neither agent blunted the accumulation of alveolar macrophages evoked by amiodarone (5.3-fold at 6 months). Lung neutrophil content was unchanged by amiodarone treatment for three weeks or six months. These results indicate that amiodarone induces alveolar epithelial cell apoptosis in vivo that is inhibitable by angiotensin antagonists. They also support the hypothesis that blockade of angiotensin formation or function attenuates amiodarone-induced lung fibrosis irrespective of the severity of alveolitis.

Apoptosis-dependent acute lung injury and repair after intratracheal instillation of noradrenaline in rats.

Earlier work in this laboratory showed that noradrenaline (NA) induces apoptosis in primary cultures of alveolar epithelial cells (AECs). Apoptosis of alveolar epithelial cells may promote the collapse of lung barrier function. On this basis we hypothesized that exogenous NA, administered by intratracheal (I.T.) instillation, might induce AEC apoptosis in vivo followed by acute lung injury. Delivery of NA (10 microM) I.T. into male Wistar rats increased labelling of both fragmented DNA, measured by in situ end labelling (ISEL), and the active form of caspase 3 (anti-Casp3) 6 and 20 h after administration (P < 0.05), but instillation of the vehicle alone (PBS) had no effect. Both ISEL and anti-Casp3 labelling were attenuated by concurrent I.T. delivery of the broad-spectrum caspase inhibitor ZVADfmk. After 6 h, most ISEL- and Casp3-positive cells were located in the surfaces of alveolar walls, but after 20 h more were found in alveolar spaces (P < 0.05). Instillation of NA also increased the bronchoalveolar lavage (BAL) content of fluorescent albumin (BODIPY-alb), which had previously been injected intravenously; the increase was reversed by concurrent ZVADfmk administration. These data suggest that NA-induced apoptosis of AECs in vivo is sufficient to invoke transient collapse of AEC barrier function that is rapidly repaired.

Bleomycin-induced apoptosis of alveolar epithelial cells requires angiotensin synthesis de novo.

Primary cultures of rat type II alveolar epithelial cells (AECs) or human AEC-derived A549 cells, when exposed to bleomycin (Bleo), exhibited concentration-dependent apoptosis detected by altered nuclear morphology, fragmentation of DNA, activation of caspase-3, and net cell loss over time. In both cell culture models, exposure to Bleo caused time-dependent increases in angiotensinogen (ANGEN) mRNA. Antisense oligonucleotides against ANGEN mRNA inhibited Bleo-induced apoptosis of rat AEC or A549 cells by 83 and 84%, respectively (P < 0.01 and P < 0.05), and prevented Bleo-induced net cell loss. Apoptosis of rat AECs or A549 cells in response to Bleo was inhibited 91% by the ANG-converting enzyme inhibitor captopril or 82%, respectively, by neutralizing antibodies specific for ANG II (both P < 0.01). Antagonists of ANG receptor AT(1) (losartan, L-158809, or saralasin), but not an AT(2)-selective blocker (PD-123319), inhibited Bleo-induced apoptosis of either rat AECs (79%, P < 0.01) or A549 cells (83%, P < 0.01) and also reduced the activity of caspase-3 by 52% (P < 0.05). These data indicate that Bleo, like Fas(L) or TNF-alpha, induces transactivation of ANG synthesis de novo that is required for AEC apoptosis. They also support the theory that ANG system antagonists have potential for the blockade of AEC apoptosis in situ.

Angiotensin receptor subtype AT(1) mediates alveolar epithelial cell apoptosis in response to ANG II.

Previous work from this laboratory demonstrated induction of apoptosis in lung alveolar epithelial cells (AEC) by purified angiotensin II (ANG II) and expression of mRNAs for both ANG II receptor subtypes AT(1) and AT(2) (Wang R, Zagariya A, Ibarra-Sunga O, Gidea C, Ang E, Deshmukh S, Chaudhary G, Baraboutis J, Filippatos G, and Uhal BD. Am J Physiol Lung Cell Mol Physiol 276: L885-L889, 1999.). The present study was designed to determine the ANG II receptor subtype mediating AEC apoptosis in response to ANG II. Apoptosis was induced with purified ANG II applied to the human lung AEC-derived carcinoma cell line A549 or to primary AEC isolated from Wistar rats. In both cell types, the AT(1)-selective receptor antagonists L-158809 or losartan inhibited ANG II-induced apoptosis by 90% at concentrations of 10(-8) M and 10(-7) M, respectively. The inhibition was concentration dependent with IC(50) of 10(-12) M and 10(-11) M on the primary rat AEC. In contrast, the AT(2)-selective antagonists PD-123319 or PD-126055 could not block ANG II-induced apoptosis in either cell type. In primary rat AEC, apoptosis in response to ANG II was blunted in a dose-dependent manner by the protein kinase C inhibitor chelerythrine but not by the tyrosine phosphatase inhibitor sodium orthovanadate. Together, these data indicate that AEC apoptosis in response to ANG II is mediated by receptor subtype AT(1), despite the expression of mRNAs for both AT(1) and AT(2).