Amal Abdul-Hafez

Angiotensin converting enzyme-2 is protective but downregulated in human and experimental lung fibrosis.

Earlier work from this laboratory showed that local generation of angiotensin (ANG) II is required for the pathogenesis of experimental pulmonary fibrosis and that ANG peptides are expressed robustly in the lungs of patients with idiopathic pulmonary fibrosis (IPF). Angiotensin converting enzyme-2 (ACE-2) degrades the octapeptide ANG II to form the heptapeptide ANG1-7 and thereby limits ANG II accumulation. On this basis, we hypothesized that ACE-2 would be protective against experimental lung fibrogenesis and might be downregulated in human and experimental lung fibrosis. In lung biopsy specimens from patients with IPF, ACE-2 mRNA and enzyme activity were decreased by 92% (P<0.01) and 74% (P<0.05), respectively. ACE-2 mRNA and activity were also decreased similarly in the lungs of bleomycin-treated rats and C57-BL6 mice. In mice exposed to low doses of bleomycin, lung collagen accumulation was enhanced by intratracheal administration of either ACE-2-specific small interfering RNAs (siRNAs) or the peptide DX(600), a competitive inhibitor of ACE-2 (P<0.05). Administration of either ACE-2 siRNA or DX(600) significantly increased the ANG II content of mouse lung tissue above the level induced by bleomycin alone. Coadministration of the ANG II receptor antagonist saralasin blocked the DX(600)-induced increase in lung collagen. Moreover, purified recombinant human ACE-2, delivered to mice systemically by osmotic minipump, attenuated bleomycin-induced lung collagen accumulation. Together, these data show that ACE-2 mRNA and activity are severely downregulated in both human and experimental lung fibrosis and suggest that ACE-2 protects against lung fibrogenesis by limiting the local accumulation of the profibrotic peptide ANG II.

Regulation of alveolar epithelial cell survival by the ACE-2/angiotensin 1–7/Mas axis

Earlier work from this laboratory demonstrated that apoptosis of alveolar epithelial cells (AECs) requires autocrine generation of angiotensin (ANG) II. More recent studies showed that angiotensin converting enzyme-2 (ACE-2), which degrades ANGII to form ANG1-7, is protective but severely downregulated in human and experimental lung fibrosis. Here it was theorized that ACE-2 and its product ANG1-7 might therefore regulate AEC apoptosis. To evaluate this hypothesis, the AEC cell line MLE-12 and primary cultures of rat AECs were exposed to the profibrotic apoptosis inducers ANGII or bleomycin (Bleo). Markers of apoptosis (caspase-9 or -3 activation and nuclear fragmentation), steady-state ANGII and ANG1-7, and JNK phosphorylation were measured thereafter. In the absence of Bleo, inhibition of ACE-2 by small interfering RNA or by a competitive inhibitor (DX600 peptide) caused a reciprocal increase in autocrine ANGII and corresponding decrease in ANG1-7 in cell culture media (both P < 0.05) and, moreover, induced AEC apoptosis. At baseline (without inhibitor), ANG1-7 in culture media was 10-fold higher than ANGII (P < 0.01). Addition of purified ANGII or bleomycin-induced caspase activation, nuclear fragmentation, and JNK phosphorylation in cultured AECs. However, preincubation with ANG1-7 (0.1 μM) prevented JNK phosphorylation and apoptosis. Moreover, pretreatment with A779, a specific blocker of the ANG1-7 receptor mas, prevented ANG1-7 blockade of JNK phosphorylation, caspase activation, and nuclear fragmentation. These data demonstrate that ACE-2 regulates AEC survival by balancing the proapoptotic ANGII and its antiapoptotic degradation product ANG1-7. They also suggest that ANG1-7 inhibits AEC apoptosis through the ANG1-7 receptor mas.

JunD and HIF-1α mediate transcriptional activation of angiotensinogen by TGF-β1 in human lung fibroblasts

Earlier work showed that TGF‐β 1 potently increases angiotensinogen (AGT) gene mRNA in primary human lung fibroblasts. Here the mechanism of TGF‐β 1‐induced AGT expression was studied in the IMR90 human lung fibroblast cell line. The increase in AGT mRNA induced by TGF‐β 1 was completely blocked by actinomycin‐D. TGF‐β 1 increased the activity of a full‐length human AGT promoter‐luciferase reporter (AGT‐LUC) but did not alter AGT mRNA half‐life. Serial deletion analyses revealed that 67% of TGF‐β‐inducible AGT‐LUC activity resides in a small domain of the AGT core promoter;this domain contains binding sites for hypoxia‐inducible factor (HIF)‐1 and activationprotein‐1 (AP‐1) transcription factors. TGF‐β1 increasedHIF‐1α protein abundance and the activity of a hypoxia‐responsive element reporter;overexpression ofHIF‐1 increased basal AGT‐LUC activity. Both oligonucleotide pulldown and chromatin immunoprecipita‐tion assays revealed increased binding of JunD andHIF‐1 α to the AGT core promoter in response to TGF‐β1. TGF‐β1‐inducible AGT‐LUC was reduced by anAP‐1 dominant negative or by mutation of theAP‐1 site. Knockdown of either JunD orHIF‐1α individually by siRNA partially reduced AGT‐LUC. In contrast, simultaneous knockdown of both JunD andHIF‐1α completely eliminated TGF‐β 1‐inducible AGT‐LUC activity. These data suggest that TGF‐β 1 up‐regulates AGT transcription in human lung fibroblasts through a mechanism that requires both JunD andHIF‐1α binding to the AGT core promoter. They also suggest a molecular mechanism linking hypoxia signaling and fibrogenic stimuli in the lungs.—Abdul‐Hafez, A.,Shu, R., Uhal, B.D. JunD andHIF‐1a mediate transcriptional activation of angiotensinogen by TGF‐P 1 in human lung fibroblasts. FASEB J. 23, 1655–1662 (2009)

Angiotensinogen gene G-6A polymorphism influences idiopathic pulmonary fibrosis disease progression

Angiotensin II is a growth factor that plays a key role in the physiopathology of idiopathic pulmonary fibrosis (IPF). A nucleotide substitution of an adenine instead of a guanine (G-6A) in the proximal promoter region of angiotensinogen (AGT), the precursor of angiotensin II, has been associated with an increased gene transcription rate. In order to investigate whether the G-6A polymorphism of the AGT gene is associated with IPF development, severity and progression, the present study utilised a case–control study design and genotyped G-6A in 219 patients with IPF and 224 control subjects. The distribution of G-6A genotypes and alleles did not significantly differ between cases and controls. The G-6A polymorphism of the AGT gene was not associated with disease severity at diagnosis. The presence of the A allele was strongly associated with increased alveolar arterial oxygen tension difference during follow-up, after controlling for the confounding factors. Higher alveolar arterial oxygen tension changes over time were observed in patients with the AA genotype (0.37±0.7 mmHg (0.049±0.093 kPa) per month) compared to GA genotype (0.12±1 mmHg (0.016±0.133 kPa) per month) and GG genotype (0.2±0.6 mmHg (0.027±0.080 kPa) per month). G-6A polymorphism of the angiotensinogen gene is associated with idiopathic pulmonary fibrosis progression but not with disease predisposition. This polymorphism could have a predictive significance in idiopathic pulmonary fibrosis patients.

Cell cycle dependence of ACE-2 explains downregulation in idiopathic pulmonary fibrosis.

Alveolar epithelial type II cells, a major source of angiotensin-converting enzyme (ACE)-2 in the adult lung, are normally quiescent but actively proliferate in lung fibrosis and downregulate this protective enzyme. It was, therefore, hypothesised that ACE-2 expression might be related to cell cycle progression. To test this hypothesis, ACE-2 mRNA levels, protein levels and enzymatic activity were examined in fibrotic human lungs and in the alveolar epithelial cell lines A549 and MLE-12 studied at postconfluent (quiescent) versus subconfluent (proliferating) densities. ACE-2 mRNA, immunoreactive protein and enzymatic activity were all high in quiescent cells, but were severely downregulated or absent in actively proliferating cells. Upregulation of the enzyme in cells that were progressing to quiescence was completely inhibited by the transcription blocker actinomycin D or by SP600125, an inhibitor of c-Jun N-terminal kinase (JNK). In lung biopsy specimens obtained from patients with idiopathic pulmonary fibrosis, immunoreactive enzyme was absent in alveolar epithelia that were positive for proliferation markers, but was robustly expressed in alveolar epithelia devoid of proliferation markers. These data explain the loss of ACE-2 in lung fibrosis and demonstrate cell cycle-dependent regulation of this protective enzyme by a JNK-mediated transcriptional mechanism.

Angiotensinogen Gene Transcription in Pulmonary Fibrosis

An established body of literature supports the hypothesis that activation of a local tissue angiotensin (ANG) system in the extravascular tissue compartment of the lungs is required for lung fibrogenesis. Transcriptional activation of the angiotensinogen (AGT) gene is believed to be a critical and necessary step in this activation. This paper summarizes the data in support of this theory and discusses transcriptional regulation of AGT, with an emphasis on lung AGT synthesis as a determinant of fibrosis severity. Genetic data linking AGT polymorphisms to the severity of disease in Idiopathic Pulmonary Fibrosis are also discussed.

Angiotensin II in apoptotic lung injury: potential role in meconium aspiration syndrome.

Meconium aspiration injures a number of cell types in the lung, most notably airway and alveolar epithelial lining cells. Recent data show that at least some of the cell death induced by meconium occurs by apoptosis, and therefore has the potential for pharmacologic inhibition through the use of apoptosis blockers or other strategies. Related work in adult animal models of lung injury has shown that apoptosis of lung epithelial cells induces a local (that is, entirely lung tissue specific) renin-angiotensin system (RASL). Furthermore, this inducible RASL is required for the apoptotic response and affects other adjacent cell types through the release of angiotensin II and related peptides. This manuscript reviews the published data supporting this viewpoint as well as more recent works that suggest the involvement of a RASL in the perinatal lung damage associated with meconium aspiration syndrome (MAS). The implications of these findings regarding their potential for the clinical management of MAS are also discussed.

Prior hypoxia prevents downregulation of ACE-2 by hyperoxia in fetal human lung fibroblasts

ABSTRACT Purpose/Aim of Study: The renin angiotensin system is involved in experimentally induced lung fibrosis. Angiotensin (ANG)-II is profibrotic. Angiotensin converting enzyme-2 (ACE-2) cleaves ANG-II and is thus protective. ACE-2 has recently been reported to be significantly decreased under hyperoxic conditions. Hyperoxia is linked to Bronchopulmonary Dysplasia and lung fibrosis. Fetal lung cells normally do not undergo fibrotic changes with physiologic hypoxemia. We hypothesized that hypoxia prior to hyperoxic exposure in fetal lung fibroblasts (IMR-90 cell line) might be protective by preventing ACE-2 downregulation. Materials and Methods: IMR-90 cells were exposed to hypoxia (1%O2/99%N2) followed by hyperoxia (95%O2/5%CO2) or normoxia (21%O2) in vitro. Cells and culture media were recovered separately for assays of ACE-2, TNF-α-converting enzyme (TACE), αSmooth muscle actin (αSMA)—myofibroblast marker-, N-cadherin, and β-catenin immunoreactive protein. Results: ACE-2 significantly increased when IMR-90 were hypoxic prior to hyperoxic exposure with no recovery. In contrast to hyperoxia alone, ACE-2 did not decrease when IMR-90 were hypoxic prior to hyperoxic exposure with recovery. TACE/ADAM17 protein and mRNA were significantly decreased under these conditions. αSMA N-cadherin, and β-catenin proteins were significantly decreased with or without normoxic recovery. Conclusions: Hypoxia prior to hyperoxic exposure of fetal lung fibroblasts prevented ACE-2 downregulation and decreased ADAM17/TACE protein and mRNA. αSMA, N-cadherin, and β-catenin were also significantly decreased under these conditions.