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Hypoxic pulmonary artery fibroblasts trigger proliferation of vascular smooth muscle cells: role of hypoxia-inducible transcription factors¹

Department of Internal Medicine/Pulmonary and Critical Care Medicine, Justus-Liebig-University, D-35385 Giessen, Germany; and
^* Institute of Veterinary Physiology, University of Zürich, CH-8057 Zürich, Switzerland

²Correspondence: Department of Internal Medicine 2, Klinikstr. 36, D-35392 Giessen, Germany. E-mail: Joerg.Haenze{at}innere.med.uni-giessen.de

SPECIFIC AIMS

Sustained generalized alveolar hypoxia as it occurs in chronic obstructive and restrictive lung disease, as well as in association with alveolar hypoventilation due to neuromuscular disorders, leads to pulmonary hypertension and cor pulmonale. This phenomenon is the predominant disease of the lung vasculature and the leading cause of early invalidity and death due to right heart failure. It is characterized by vascular smooth muscle cell (SMC) hyperplasia; media thickening and de novo muscularization of small precapillary vessels are the key features of a vascular remodeling process with progressive narrowing of the vascular lumen.

The intra- and intercellular molecular mechanisms of hypoxia sensing and signaling that underlie this remodeling response in hypoxic pulmonary vessels, are still under investigation. We tested the hypothesis that besides vascular SMC, adventitial fibroblasts (FB) may substantially contribute to the hypoxia-driven proliferative vascular response and analyzed the putative role of hypoxia-inducible factors (HIF). Three subtypes of HIF are currently known (HIF-1, HIF-2, HIF-3); they all affect gene regulation via a conserved hypoxia-responsive element (HRE).

PRINCIPAL FINDINGS

In a hypoxic atmosphere, strong proliferation of pulmonary artery fibroblasts (FB_PA) was noted whereas isolated SMC_PA did not display a proliferative response to hypoxia. However, the coculture of SMC_PA with FB_PA under conditions of hypoxia markedly amplified SMC_PA growth (Fig. 1 ). This effect was fully reproduced by the use of conditioned serum-free cell supernatant from FB_PA incubated under conditions of hypoxia, which was then transferred to normoxic SMC_PA.

View larger version (24K): [in this window] [in a new window]   Figure 1. Proliferation of human pulmonary artery fibroblasts (FBPA) and human smooth muscle cells (SMCPA) measured by BrdU incorporation (the photometric absorption at 450 nm as a value for BrdU incorporation is shown). A) Left: proliferation of FBPA and SMCPA in normoxia and hypoxia in serum-free medium (1% O2, 24 h). Right: proliferation of SMCPA cocultured with FBPA in normoxia and hypoxia in serum-free medium (1% O2, 24 h). The proliferation of SMCPA cultured in 0% and 10% fetal-calf-serum (FCS) containing medium during normoxia is shown for comparison (mean±SD, n=4 independent cell preparations, *P<0.05, paired t test). B) Hypoxia/normoxia BrdU ratio of FBPA and SMCPA cultured under conditions of normoxia and hypoxia (1% O2, 24 h) in various cell densities from 40% to 100% confluence (mean±SD, n=4 independent cell preparations).

Complete reproduction of the proliferative SMC_PA response by serum-free supernatant originating from hypoxic fibroblasts and transferred to normoxic SMC strongly suggests hypoxia sensing by the fibroblasts and intercellular signaling to the adjacent SMC as an underlying mechanism. On the molecular level, the currently known hypoxia-inducible transcription factors HIF-1, HIF-2, and HIF-3 subtypes were all found to be up-regulated in the hypoxic fibroblasts, HIF-1 being documented at the protein level and HIF-2 and HIF-3 being analyzed at the mRNA level (Fig. 2 a, b). Moreover, an HRE-dependent reporter gene assay showed strong activation in pulmonary artery fibroblasts in response to hypoxia (Fig. 2c ). Use of the transcription decoy technique, currently applied to HIF as a novel approach, was found to fully block the capacity of hypoxic fibroblasts to elicit SMC_PA growth, thus providing further strong evidence that the hypoxia-induced gene expression in the fibroblasts and the proliferative SMC response were causally linked. Since the HIF decoy fragment carries the same HRE sequence as in the HRE-TK and HRE-SV40 reporter vector experiments, we suggest a specific HIF decoy effect on the suppression of hypoxia-dependent target genes controlled by consensus HRE sequences.

View larger version (55K): [in this window] [in a new window]   Figure 2. a) Expression of the hypoxia-inducible transcription factor subtypes HIF-1, HIF-2, and HIF-3 mRNA in human lung adventitial fibroblasts (FB) and smooth muscle cells (SMC), each being cultured under normoxic (-) or hypoxic conditions (+) (1% O2, 24 h) vs. hypoxanthine-guanine phosphoribosyltransferase (HPRT) mRNA. Autoradiography of the ethidium bromide-stained gel electrophoresis after performance of RT-PCR is shown (M, size marker; kb, kilobases). b) Analysis of HIF-1 protein by Western blot using a polyclonal HIF-1 antibody. Nuclear extracts originated from rabbit pulmonary artery SMC and FB incubated under normoxia (-) or hypoxia (+) (1% O2) for 12 h (kd, kilodalton). c) Raw data (RLU relative light units) of the hypoxia-responsive element (HRE) reporter gene assays performed on rabbit pulmonary artery SMC and FB using different HRE promoter reporter vector constructs. Cells were incubated under normoxia or hypoxia (1% O2, 24 h) (HRE-SV40, HRE coupled to simian virus 40 promoter; HRE-TK, HRE coupled to herpes simplex thymidine-kinase promoter; VEGF, vascular endothelial growth factor promoter; mean±SD, n=4 independent cell preparations, *P<0.05, paired t test).

CONCLUSIONS

The experimental data suggest that 1) pulmonary vascular adventitial fibroblasts respond to hypoxia by secreting soluble factors that induce proliferation of adjacent smooth muscle cells; 2) these soluble factors are shown to be controlled or encoded by the expression of genes carrying a hypoxia-responsive element (DNA binding domain for HIF transcription factors) in their regulatory regions; 3) HIF transcription factor decoy, a strategy for inhibition of HIF-dependent gene regulation used for the first time in this study, suppresses fibroblast-dependent, hypoxia-induced SMC_PA proliferation and thus may be considered as a new therapeutic tool for treatment of pulmonary hypertension (Fig. 3 ).

View larger version (35K): [in this window] [in a new window]   Figure 3. Schematic diagram. Hypoxic pulmonary artery fibroblasts (FBPA) secrete soluble factors in an HIF-dependent manner. These factors trigger the growth of adjacent SMCPA cells. Use of HIF transcription decoy by transfection of FBPA with synthetic DNA fragments carrying the hypoxia-responsive element blocks the release of these factors and prevents SMCPA from undergoing hypoxic-induced proliferation.

The findings are related to the state of the field with respect to the recent exploration of hypoxic-dependent gene regulation by three hypoxia-inducible transcription factors (HIF-1, HIF-2, HIF3). These factors have been identified on the molecular level and shown to be functionally involved in the induction of angiogenesis, anaerobic glycolysis, and polycythemia. Here we describe a new organ specific role of these factors in the lung vasculature and offer a new molecular to interfere with HIF-dependent gene regulation. We also show a specific role of adventitial fibroblasts in pulmonary vascular remodeling that confirms suggestions from previous studies that these cells are involved in the pathogenetic sequelae of pulmonary hypertension.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0420fje; to cite this article, use FASEB J. 10.1096/fj.02-0420fje.

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