Impact of HIF‐1α and HIF‐2α on proliferation and migration of human pulmonary artery fibroblasts in hypoxia

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Abstract
Prolonged alveolar hypoxia, as occurs at high altitude, in chronic obstructive pulmonary disease and in various restrictive lung diseases, results in a remodeling of the pulmonary vasculature, which is characterized by proliferative changes in the intima, media, and adventitia of the pulmonary artery. These changes may cause chronic pulmonary arterial hypertension and subsequent cor pulmonale. Several findings indicate that the adventitial layer of the pulmonary arteries is centrally involved in the hypoxia-driven remodeling process. In this study, we investigated the hypothesis that hypoxic adaptive processes are mainly related to the induction of hypoxia-inducible factors (HIF). We followed the aim to analyze relevant cellular adaptive responses in human adventitial fibroblasts in relation to hypoxia and HIF induction. Using RNA interference for specific inhibition of HIF-1α and HIF-2α, we analyzed the dependency of cellular adaptive processes such as cell proliferation, apoptosis and cell migration, and the expression of smooth muscle α-actin (α-SMA) in adventitial fibroblasts (FBPA). On the other hand, addition of transforming growth factor β (TGF-β), a known stimulus of α-SMA expression, provoked the typical increase of α-SMA in FBPA. Thus, we conclude that α-SMA is apparently not directly controlled by hypoxia. The cellular localization of the HIF isoforms was monitored by immunocytochemistry. HIF-1α, almost not detectable in normoxia, appeared under hypoxic conditions in the nucleus. HIF-2α illustrated a cytosolic presence with weak staining around the nucleus under normoxic conditions, with strong translocation response into the nucleus occurring under hypoxia. HIF-1α and HIF-2α could be specifically inhibited by using RNA interference for either HIF-1α or HIF-2α as proven by HIF-1α- and HIF-2α mRNA and protein analyses and by expression analysis of the common target genes VEGF, PGK, as well as the specific HIF-2 target gene KDR. This study investigated hypoxia-induced responses of human pulmonary artery adventitial fibroblasts, which are assumed to play a major role in the remodeling process of the lung vasculature under conditions of oxygen deprivation. Hypoxia induced a strong up-regulation of proliferation and migration of the FBPA whereas apoptosis was down-regulated, which is well in line with the phenotypic changes observed in hypoxia induced pulmonary hypertension in vivo. In contrast, the expression of α-SMA was apparently not substantially affected by the hypoxic conditions. Focusing on the underlying molecular mechanisms, expression analysis of the hypoxia-inducible transcription factor subtypes was performed. Hypoxia induced a marked up-regulation (protein level) of both HIF-1α and HIF-2α, alongside with nuclear translocation of these transcription factors. Specific inhibition of either HIF-1α or HIF-2α was achieved by RNA interference technology, as proven by HIF-1α- and HIF-2α mRNA and protein analyses and by expression analysis of the common target genes VEGF, PGK, as well as the specific HIF-2 target gene KDR. The hypoxia induced proliferative response of the FBPA was found to be solely HIF-2α dependent, whereas the migratory response was significantly reduced by both HIF-1α and HIF-2α interference. We propose the hypothesis that HIF-1α and HIF-2α act in a cell specific manner on both common and specific targets (Fig. 3 ⤻ ). We suggest that the differences in HIF-1α and HIF-2α targets may contribute to the differential effects of these factors on cell proliferation and migration. HIF-1α and HIF-2α targets may comprise the target gene level, dependent on the respective DNA binding elements or directly interacting proteins that still have to be identified. We have shown that HIF-1 and HIF-2 are essential for hypoxic cellular responses such as proliferation and migration in human pulmonary artery adventitial fibroblasts. Differential HIF subtype dependency was noted, with HIF-2α playing a predominant role, which may offer for future intervention strategies. 1These authors contribute equally to this work. To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4104fje;
Funding Information
  • Deutsche Forschungsgemeinschaft (SFB 547, B9)