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Hypoxia-driven proliferation of human pulmonary artery fibroblasts: cross-talk between HIF-1 and an autocrine angiotensin system

Stefanie Krick^*,1, Jörg Hänze^*, Bastian Eul^*, Rajkumar Savai^*, Ulrike Seay^*, Friedrich Grimminger^*, Jürgen Lohmeyer^*, Walter Klepetko, Werner Seeger^* and Frank Rose^*

^* Department of Internal Medicine, Pulmonary and Critical Care Medicine, Justus-Liebig-University Giessen, Giessen, Germany; and
Department of Cardio-Thoracic Surgery, Vienna University Hospital, Vienna, Austria

¹ Correspondence: Department of Internal Medicine II, Klinikstr. 36, Giessen D-35392, Germany. E-mail: stefanie krick{at}innere.med.uni-giessen.de

SPECIFIC AIMS

Pulmonary artery adventitial fibroblasts (FB_PA) may play a central role in lung vascular remodeling under conditions of hypoxia and imbalance of mitogenic and antimitogenic mediators. This is characterized by structural and functional changes in the adventitia of the vascular walls finally resulting in chronic pulmonary arterial hypertension and subsequent cor pulmonale. We investigated the interaction between the hypoxia-induced signaling pathway and its impact on a local angiotensin system in promoting pulmonary artery fibroblast proliferation.

PRINCIPAL FINDINGS

1. Strong pro-proliferative and antiapoptotic effects of hypoxia and/or angiotensin (Ang II) on human FB_PA
Ang II (100 nM) and hypoxia (1% O₂) significantly enhanced the proliferation of FB_PA compared with control cells (Fig. 1 A). Incubation with Ang II under hypoxic conditions further increased this mitogenic response. Flow cytometric analysis showed that hypoxia and Ang II induced cell cycle progression by an increase in the number of cells in the S/M-phase (Fig. 1B ). Application of both stimuli further elevated the percentage of cells in S/M-phase (P<0.001). Exposure to Ang II or hypoxia reduced the percentage of annexin-V-positive cells in a significant manner which was synergistic in combination.

View larger version (29K): [in this window] [in a new window]   Figure 1. Effect of Ang II and hypoxia on HIF-1 expression in pulmonary adventitial fibroblasts. A) Immunocytochemistry of HIF-1 expression in FBPA after application of hypoxia, Ang II, or both. B) Western blot of hypoxia- and Ang II-induced HIF-1 expression (nuclear and cytosolic fractions) after 24 h. The histogram represents relative intensity of the bands in the Western blot. Data indicate mean values ± SE (n=5); *P < 0.05 for direct comparison between groups. ß-Actin levels were used as control for equal loading. Right histogram: hypoxia-responsive element (HRE) reporter gene assay performed on adventitial fibroblasts, without any treatment, or treated with hypoxia and/or Ang II for 24 h. Shown is the ratio of hypoxia-dependent Firefly luciferase and hypoxia-independent Renilla luciferase values. Data indicate mean values ± SE (n=5); *P < 0.05, **P < 0.01 for direct comparison between groups. C) Western blot of HIF-1 showing the time course of hypoxia- and Ang II- induced increase of protein expression with ß-actin as loading control.

2. Activation of PI-3Kinase mediates hypoxia and Ang II-induced proliferation of FB_PA
Western blot analysis of cell extracts indicated that hypoxia and Ang II induced a biphasic phosphorylation of Akt, a downstream effector molecule of PI-3 kinase. This was inhibited by preincubation of the cells with LY 294002, a specific inhibitor of PI-3 kinase. Assessment of proliferation and apoptosis clearly demonstrated that LY 294002 significantly attenuated Ang II and hypoxia mediated effects (P<0.01), indicating a signaling pathway common to both stimuli.

3. Activation of the HIF-1/HRE-axis in hypoxic and Ang II treated FB_PA
Immunocytochemistry and Western blot revealed a marked up-regulation of HIF-1 under conditions of hypoxia. This was observed after treatment with Ang II, but did not last as long as the hypoxic up-regulation (Fig. 2 A, C). In the presence of Ang II, the hypoxia-induced expression of HIF-1 protein was significantly increased (P<0.05, Fig. 2B ). Using a dual reporter gene assay, hypoxia-treated FB_PA (24 h) showed a marked increase of HRE activity (467±16%). Incubation of FB_PA with Ang II under hypoxic conditions further enhanced HRE activation up to 785 ± 83% (Fig. 2B , right).

View larger version (18K): [in this window] [in a new window]   Figure 2. Suppression of the hypoxic response by siRNA targeting HIF-1. Cells were transiently transfected with siRNA targeting HIF-1 by liposomal transfection. After 24 h hypoxia, immunocytochemistry, and apoptosis experiments were performed. As control, random small interfering RNA (siRAN) was used. A) As transfection control, siRNA conjugated to FITC was used. LM = light microscopic picture of FBPA. To assess transfection efficiency, immunocytochemistry for HIF-1 was used indicating that siHIF-1 transfected cells showed no HIF-1 signal compared with control cells transfected with siRAN. B) Assessment of AT-1 and ACE levels by immunostaining, compared with control siRNA-transfected cells. C) FACS scan analysis of hypoxia-induced up-regulation of AT-1 and ACE, impact of siHIF-1 compared with siRAN. (5 independent experiments; each mean ± SE).

4. Blockade of angiotensin II receptor type 1 (AT-1) and angiotensin-converting enzyme (ACE) attenuated the hypoxia-induced antiapoptosis and HIF-1 expression
To analyze the involvement of angiotensin-converting enzyme and distinct Ang II receptor subtypes, we preincubated adventitial fibroblasts with Enalapril (10^–8 g/mL), an inhibitor of ACE, the specific AT-1inhibitor Losartan (1 µM) and the AT-2 antagonist PD-123,319 (1 µM) for 25 min before exposure to hypoxia for 24 h. Enalapril and Losartan significantly suppressed hypoxia-induced HIF-1 expression, cell proliferation, and the antiapoptotic effects. In contrast, the AT-2 antagonist PD-123,319 did not abrogate these effects.

5. Hypoxia enhanced the expression of AT-1 surface density on FB_PA and activity and expression of ACE
Exposure of adventitial fibroblasts to hypoxia led to an increased AT-1 receptor density on the surface of FB_PA. We found an increase in ACE expression on protein and mRNA levels. FB_PA maintained under hypoxic conditions exhibited significant up-regulation of ACE activity compared with control cells.

6. Use of RNA interference technique targeting HIF-1 suppressed the hypoxic response, while transfection with HIF-1 cDNA reproduced the hypoxia-driven effect on FB_PA proliferation
We transfected primary FB_PA with siRNA targeting HIF-1 and random small interfering RNA as control. Immunocytochemistry and flow cytometry experiments demonstrated that hypoxia-induced up-regulation of ACE and AT-1 expression were blocked by siHIF-1, while overexpression by transient transfection with HIF-1 cDNA significantly enhanced the hypoxia-induced antiapoptosis. Transfection of HIF-1 cDNA induced an increase in AT-1 receptor surface density of FB_PA compared with control cells.

CONCLUSIONS AND SIGNIFICANCE

The potency of Ang II to foster cell cycle entry and to suppress the rate of apoptosis has previously been demonstrated for smooth muscle cells and for fibroblasts of different origins. Our study is the first to document this profile for human pulmonary artery fibroblasts. This profile is associated with a strong up-regulation of HIF-1 protein in accordance with the notion that this hypoxia-driven transcription factor is predominantly regulated on the protein level. Further extending this concept, exogenous stimulation of the Ang II system augmented the activation of HIF-1 and the hypoxia-induced proliferation. This was evident from the enhancement of hypoxia-induced up-regulation of HIF-1 protein in the presence of Ang II.

Possible explanations for this are: 1) that inhibition of ACE or AT-1 favors the bradykinin-NO pathway, thereby attenuating the hypoxic response, and 2) that hypoxia induces the production of Ang precursor peptides. These peptides then act via ACE and AT-1 promoting proliferation of FB_PA. This mechanism has been observed in lung fibrosis where interstitial fibroblasts can produce Ang II precursors for paracrine signaling, and is well characterized in the pancreatic system after hypoxia. Notwithstanding these putative aspects of hypoxia-angiotensin crosstalk, the present studies discovered an important role of oxygen-dependent regulation of AT-1 and ACE in adventitial fibroblasts.

The present study demonstrates that adventitial fibroblasts are strongly responsive to hypoxia and Ang II, resulting in cell cycle entry, suppression of apoptosis, and thereby marked increase in cell number. Crosstalk between HIF-1 and the ACE/Ang II/AT-1 axis was observed in these cells: exogenous Ang II enhances the stimulation of the HIF/HRE axis in hypoxia, and hypoxia results in a HIF-mediated activation of the autocrine ACE/Ang II/AT-1 loop, further promoting the proliferative response.

These findings lend further support to the concept that pulmonary adventitial fibroblasts are important contributors to the vascular remodeling processes and the development of pulmonary hypertension under conditions of hypoxia. Elucidation of the molecular mechanisms mediating this phenomenon may open up new strategies for therapeutic intervention.

View larger version (21K): [in this window] [in a new window]   Figure 3. Scheme of hypoxia-driven growth of pulmonary artery fibroblasts. Hypoxia sensing via HIF/HRE activates an autocrine loop of ACE up-regulation, Ang II formation, and signaling via AT-1, exerting positive feedback of the HIF/HRE axis and further fostering cell growth and antiapoptotic effects; HIF, hypoxia-inducible factor, ACE, angiotensin-converting enzyme; Ang II, angiotensin II; AT-1, angiotensin II receptor type 1; PI-3K, phosphoinositol-3-kinase.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2890fje;

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