Human lung myofibroblast-derived inducers of alveolar epithelial apoptosis identified as angiotensin peptides

Abstract

Earlier work from this laboratory found that fibroblasts isolated from fibrotic human lung [human interstitial pulmonary fibrosis (HIPF)] secrete a soluble inducer(s) of apoptosis in alveolar epithelial cells (AECs) in vitro [B. D. Uhal, I. Joshi, A. True, S. Mundle, A. Raza, A. Pardo, and M. Selman. Am. J. Physiol. 269 (Lung Cell. Mol. Physiol. 13): L819-L828, 1995]. The cultured human fibroblast strains most active in producing the apoptotic activity contained high numbers of stellate cells expressing alpha-smooth muscle actin, a myofibroblast marker. The apoptotic activity eluted from gel-filtration columns only in fractions corresponding to proteins. Western blotting of the protein fraction identified immunoreactive angiotensinogen (ANGEN), and two-step RT-PCR revealed expression of ANGEN by HIPF fibroblasts but not by normal human lung fibroblasts. Specific ELISA detected angiotensin II (ANG II) at concentrations sixfold higher in HIPF-conditioned medium than in normal fibroblast-conditioned medium. Pretreatment of the concentrated medium with purified renin plus purified angiotensin-converting enzyme (ACE) further increased the ELISA-detectable ANG II eightfold. Apoptosis of AECs in response to HIPF-conditioned medium was completely abrogated by the ANG II receptor antagonist saralasin (50 microg/ml) or anti-ANG II antibodies. These results identify the protein inducers of AEC apoptosis produced by HIPF fibroblasts as ANGEN and its derivative ANG II. They also suggest a mechanism for AEC death adjacent to HIPF myofibroblasts [B. D. Uhal, I. Joshi, C. Ramos, A. Pardo, and M. Selman. Am. J. Physiol. 275 (Lung Cell. Mol. Physiol. 19): L1192-L1199, 1998].

Evolving Concepts of Apoptosis in Idiopathic Pulmonary Fibrosis

“…Several pericellular factors may contribute to this dysregulated communication. AEC apoptosis/necrosis adjacent to underlying ␣-smooth muscle actin-expressing myofibroblasts has been demonstrated (20); this effect may involve the secretion of angiotensin-II by myofibroblasts (36). In addition, myofibroblastdifferentiated cells are potent generators of extracellular ROS, specifically hydrogen peroxide (37).…”

Angiotensin-II-induced apoptosis requires regulation of nucleolin and Bcl-xL by SHP-2 in primary lung endothelial cells

“…The local synthesis of Ang II has been demonstrated in lung fibrotic plaques, where it is produced by activated myofibroblasts and probably impacts the survival of other neighboring cells (Uhal, 2002;Wang et al, 1999). We investigated the effect of Ang II on bovine PAECs using the neutral comet assay, which detects chromosomal breakdown as a function of apoptosis.…”

“…Blockade of Ang-II signaling, either by using inhibitors of angiotensinconverting enzyme to prevent Ang-II maturation or using antagonists of Ang-II cellular receptors, can abrogate the development of fibrosis in animal models in the lung, liver, kidney and heart (Bataller et al, 2005;Dendorfer et al, 2005;Konigshoff et al, 2007;Sun, 2009;Wolf, 2008). In a number of fibrotic diseases, local synthesis of Ang II has been observed, and fibroblasts from the diseased tissues of human patients were found to generate Ang II (Bader, 2002;Sun et al, 2000;Wang et al, 1999). Two primary events are associated with the development and progression of fibrosis: (1) the transdifferentiation of fibroblasts into the activated fibroblast (myofibroblast) phenotype that secretes abnormal extracellular-matrix proteins such as collagens I and III (Wynn, 2008); and (2) apoptosis of the normal epithelial and endothelial cells of the tissues (Marshall, 2003;Wang et al, 2000).…”

Epithelial apoptosis in the initiation of lung fibrosis

“…1), noradrenaline (NA) [18] and angiotensin (ANG)II [19,20]. The latter is particularly important because in vitro studies have demonstrated that ANGII, synthesised de novo by the AEC, is required as a mediator of the apoptotic response to Fas activation, to TNF-a, amiodarone, NA [15][16][17][18], and possibly other inducers [9].…”

“…With regard to human lung, the author has also shown that myofibroblasts derived from lung biopsies obtained from patients with IPF produce and secrete ANG peptides, and on this basis it was hypothesised that local ANG production is key in both initiating and "propogating" the fibrotic focus [20].…”