The present study demonstrates that adventitial fibroblasts of hypoxia-stimulated remodelled PA acquire the following three characteristics not observed in control fibroblasts: (i) elevated expression and activation of PDGFβ-R, (ii) marked increase in PDGF-BB-induced ROS generation, and (iii) greatly increased proliferation with PDGF-BB-stimulation. We are also reporting for the first time that fibroblasts from the remodelled PA obtain the unique capacity for selective activation of JNK1 in a response to PDGFβ-R activation with PDGF-BB. Furthermore, we show that interference with the signalling via this PDGFβ-R-JNK1 axis with either SP600125 or siRNA-targeting JNK1 selectively blocks replication of remodelled PA adventitial cells. Therefore, our data strongly suggest that PDGFβ-R-stimulated JNK1 might be the key intracellular mediator of fibroproliferative responses in chronic PH. Therapeutic blockade of JNK1 selectively in fibroblasts might consequently reduce or even prevent marked adventitial remodelling characteristic of hypoxia-induced PH and serve as a stepping stone toward restoration of vascular homeostasis in the PA.
Although increased levels of PDGFβ-R have been reported in PA adventitial cells of PH patients and experimental models of PH,12,13
the molecular mechanisms underlying adventitial fibroblast activation by PDGFβ-R remain unexplored. However, the knowledge regarding the intracellular signalling pathways engaged in PDGFβ-R-mediated adventitial cell responses is absolutely critical for the development of new ‘reverse remodelling’ strategies for PH as adventitial fibroblasts have been shown to be the cells in the PA wall which are first to proliferate in the earliest stages of hypoxia-induced PH. Findings of the current study are further supported by our earlier reports,7,27
demonstrating that chronic hypoxia exposure induces phenotypic alteration in PA adventitial fibroblasts. It is conceivable that up-regulation in PDGFβ-R phosphorylation in PA adventitial cells might be in the future used as a marker of hypoxia-stimulated modification of adventitial fibroblast phenotype. This in turn encourages questioning whether chronic hypoxia exposure stimulates PDGFβ-R activation in adventitial cells by either up-regulating tyrosine kinase activity or down-regulating protein tyrosine phosphatase activity, or affecting both. How actions of such receptor-regulatory moieties might influence PH pathophysiology remains to be explored. Thus, heightened PDGFβ-R phosphorylation in adventitial cells might underscore the presence of highly-activated specialized cell populations in hypoxia-induced remodelled PA adventitia.
Our findings that the PDGFβ-R-ROS signalling is important for excessive cell proliferation in the PA of calves exposed to chronic hypoxia are supported by a study in a murine model of PH, in which chronic intermittent hypoxia stimulates generation of NADPH oxidase-derived ROS, increases the activity of PDGFβ-R, and induces proliferation of smooth muscle cells.15
Our work, however, provides novel progress toward understanding of the mechanisms governing these responses with a focus on an adventitial fibroblast. Furthermore, we present here a novel downstream molecular target of the PDGFβ-R-ROS signalling axis, JNK1, a kinase that is selectively responsible for major increases in fibroblast proliferation observed in this model of severe PH.
In the present studies, we have also found that ROS production upon PDGF-BB stimulation is significantly greater in Neo-PH cells compared with that in the control fibroblasts. However, the magnitude of differences in ROS levels between Neo-PH and Neo-C fibroblasts is rather small. In contrast, the degree of differences in the proliferative responses between these two fibroblast populations is fairly large (Figure
). In this case, the concentrations of ROS achieved might dictate the type of corresponding cellular response. In support of this notion is the fact that excessive production of ROS has been shown to induce oxidative stress, a detrimental process that can lead to cellular damage, whereas beneficial effects of ROS occur at low/moderate concentrations and control healthy physiological responses in cells.28
This report strongly supports our findings showing that a modest increase in ROS generation might elicit a sufficient signal, which in turn mediates replicative responses in Neo-PH cells with a greater magnitude than the elevation in ROS itself. Therefore, the small elevation in ROS production in Neo-PH cells might be the key driver of heightened proliferative phenotype of adventitial cells during the remodelling process in PH.
ERK1/2 phosphorylation is a major downstream signal activated by stimulating PDGFβ-R.29
In our study focusing on fibroblasts, we have found that PDGFβ-R activation induces conventional ERK1/2 phosphorylation via ROS generation in both Neo-C and Neo-PH cells, a process that leads to replication of these cells. However, the most striking finding of the current report points to JNK1 as the pro-proliferative kinase in Neo-PH cells. JNK pathways have established roles in apoptotic signalling, but might under certain circumstances, including responses to hypoxic stimulation described here, also contribute to cell proliferation and migration.25,30
In the present study, we have found that inhibition of JNK1 with JNK1-targeting siRNA blocks selectively PDGF-BB-stimulated increase in cell numbers in Neo-PH cells, suggesting that JNK1 functions as a replication regulator for these cells. Transient JNK1 phosphorylation induced by activating PDGFβ-R in Neo-PH cells might be due to the phenotypic modification caused by hypoxia, which in this model functions as a stimulator of proliferation rather than an inducer of apoptosis as seen in other cell types. In contrast, in Neo-C cells, JNK1 is not phosphorylated by PDGFβ-R. In the future studies, it will be important to investigate the pathways involved in differential PDGFβ-R-JNK1 activation patterns in adventitial cells to attempt to find a more selective target to be pursued for therapeutic development of novel PH treatments. Recent studies in JNK-deficient mice demonstrating that JNK1 acts as a profibrogenic kinase during hepatic fibrosis31
also support our data that identify JNK1 as a fibroblast activator. Since we have reported earlier that JNK regulates hypoxia-induced differentiation of fibroblasts to myofibroblasts in PA adventitia,26
we will next explore whether mice deficient in JNK1 will develop blunted hypoxia-induced PH due to the interference with the adventitial remodelling process.
Within the complex pattern of vessel remodelling in PH, adventitial fibroblasts expressing increased levels of activated PDGFβ-R contribute significantly to the observed pathology. Signalling events centred on selective activation of JNK1 make this kinase an important molecular target for interventional strategies in PH. Both experimental and clinical evidence of therapeutic efficacy of imatinib, a PDGFβ-R inhibitor, represent one novel and promising approach for the treatment of PH.10,32
However, imatinib treatment may be complicated by significant cardiotoxicity, which may result in overt heart failure.33
Therefore, it is crucial to thoroughly characterize PDGF signalling pathways specifically in adventitial cells of the vascular wall in order to identify more specific targets for pharmacological intervention in PH. Our present data strongly suggest that modulating the activity of JNK1 selectively in fibroblasts is a case in point.