Cyclic stretch stimulates mitochondrial reactive oxygen species and Nox4 signaling in pulmonary artery smooth muscle cells

Abstract

This study was designed to determine whether cyclic stretch induces a persistent pulmonary hypertension of the newborn (PPHN) phenotype of increased NADPH oxidase (Nox) 4 signaling in control pulmonary artery smooth muscle cells (PASMC), and to identify the signal transduction molecules involved. To achieve this, PPHN was induced in lambs by antenatal ligation of the ductus arteriosus at 128 days gestation. After 9 days, lungs and PASMC were isolated from control (twin) and PPHN lambs. Control PASMC were exposed to cyclic stretch at 1 Hz and 15% elongation for 24 h. Stretch-induced Nox4 expression was attenuated by inhibition of mitochondrial complex III and NF-κB, and stretch-induced protein thiol oxidation was attenuated by Nox4 small interfering RNA and complex III inhibition. NF-κB activity was increased by stretch in a complex III-dependent fashion, and stretch-induced cyclin D1 expression was attenuated by complex III inhibition and Nox4 small interfering RNA. This is the first study to show that cyclic stretch increases Nox4 expression via mitochondrial complex III-induced activation of NF-κB in fetal PASMC, resulting in ROS signaling and increased cyclin D1 expression. Targeting these signaling molecules may attenuate pulmonary vascular remodeling associated with PPHN.

Keywords: NADPH oxidase; pulmonary hypertension; reactive oxygen species.

Fig. 1.

Cyclic stretch increases cytosolic protein thiol oxidation via mitochondrial complex III to induce a persistent pulmonary hypertension of the newborn (PPHN) pulmonary artery smooth muscle cell (PASMC) phenotype. Control and PPHN PASMC were infected with an adenovirus expressing reduction-oxidation-sensitive green fluorescent protein (roGFP) in the cytosol (A) or mitochondrial matrix (B). After 48 h, control cells were subjected to 24-h cyclic stretch at 1 Hz and 15% elongation. The extent of roGFP oxidation in lysates was determined by flow cytometry, and the percent oxidation determined by fully oxidizing and fully reducing the probe. Between 5,000 and 20,000 roGFP-positive cells were quantified for each sample. A and B: RoGFP oxidation in static and stretch PPHN PASMC is included for comparison. C: RoGFP-infected control PASMC were treated with vehicle (Veh) or 1 μM myxothiazol (Myx) to inhibit complex III and then exposed to 24-h stretch (Str), as above. Stat, static. D: PASMC were treated with Veh or 1 μM Myx for 24 h and then fixed and nuclei stained with DAPI. Values are means ± SE; n ≥ 3. *P < 0.05 vs. control static (A and B) or control Stat Veh (C). †P < 0.05 vs. Str Veh. UTD, ???.

Fig. 2.

Stretch induces NADPH oxidase (Nox) 4 expression and increases cytosolic reactive oxygen species (ROS) in PASMC. A: control cells were subjected to 24-h cyclic stretch at 1 Hz and 15% elongation, and protein levels were analyzed by Western blotting. B: Nox isoform band intensities were normalized to β-actin and expressed relative to static controls. C: control PASMC were transfected with a scrambled (Sc) small interfering RNA (siRNA) or a siRNA specific to ovine Nox4 (N4). After 48 h, cells were subjected to 24-h cyclic stretch (Str) at 1 Hz and 15% elongation, and Nox4 protein levels were quantified by Western blotting as in A and B to determine the level of knockdown. D: control PASMC were transfected with a Sc siRNA (Sc) or a siRNA specific to ovine Nox4 (N4) and infected with an adenovirus expressing roGFP in the cytosol. After 48 h, cells were subjected to 24-h cyclic Str at 1 Hz and 15% elongation, and roGFP oxidation was determined by flow cytometry. Values are means ± SE; n ≥ 3. *P < 0.05 vs. static (B) or Stat Sc siRNA (C and D). †P < 0.05 vs. Str Sc siRNA (C and D).

Fig. 3.

Inhibition of mitochondrial complex III attenuates stretch-induced Nox4 expression. Control PASMC were treated with Veh, 1 μM Myx, or 1 μM antimycin A (AntA) to inhibit complex III and subjected to 24-h cyclic stretch at 1 Hz and 15% elongation. Protein and mRNA were analyzed for Nox4 expression. A: real-time PCR using primers to ovine Nox4. Expression levels were normalized to β-actin mRNA levels and expressed relative to controls. B: representative Western blots for Nox4 from PASMC treated with Myx and stretched for 24 h. C: Nox4 band intensities in B were normalized to β-actin and expressed relative to static controls. D: representative Western blots for Nox4 from PASMC treated with AntA and stretched for 24 h. E: Nox4 band intensities in D were normalized to β-actin and expressed relative to static controls. Values are means ± SE; n ≥ 3. *P < 0.05 vs. static Veh. †P < 0.05 vs. stretch Veh.

Fig. 4.

Stretch activates NF-κB via mitochondrial complex III, resulting in increased expression of Nox4. Control PASMC were transfected with a plasmid containing 5 consensus κB sites in tandem upstream of a luciferase reporter, treated with Veh or 1 μM Myx and subjected to 24-h cyclic stretch at 1 Hz and 15% elongation. A: relative light units were determined in a luminometer and expressed relative to static Veh cells. Real-time PCR was performed on mRNA from PASMC treated with helenalin (hel) and stretched for 24 h using primers to ovine Nox4. B: expression levels were normalized to β-actin mRNA levels and expressed relative to static Veh cells. C: representative Western blots for Nox4 from PASMC treated with hel and stretched for 24 h. D: Nox4 band intensities in C were normalized to β-actin and expressed relative to static controls. Values are means ± SE; n ≥ 3. *P < 0.05 vs. static Veh. †P < 0.05 vs. stretch Veh.

Fig. 5.

Inhibition of Nox4 and mitochondrial complex III attenuates stretch-induced cyclin D1 expression in PASMC. A and B: PASMC were treated with Veh or with 1 μM Myx, and subjected to 24-h cyclic stretch at 1 Hz and 15% elongation. Protein was analyzed for Nox4 expression. A: representative Western blot for Nox4 from PASMC. B: Nox4 band intensities were normalized to β-actin and expressed relative to static controls. PASMC were transfected with a plasmid containing the human cyclin D1 promoter region, treated with Veh or 1 μM Myx (C), or with Sc (scram) or Nox4 siRNA (Nox4) and subjected to 24-h cyclic stretch at 1 Hz and 15% elongation (C and D). Relative light units were determined in a luminometer and expressed relative to static controls. Values are means ± SE; n ≥ 3. *P < 0.05 vs. static Veh or static Sc siRNA. †P < 0.05 vs. stretch Veh or stretch Sc siRNA.

Fig. 6.

Proposed pathway based on data from the present study showing increased Nox4 expression in response to stretch. Stretch increases cytosolic ROS via mitochondrial complex III, triggering a feed-forward mechanism via NF-κB, resulting in increased Nox4 expression and a sustained increase in cytosolic ROS. Nox4 increases cyclin D1 expression, leading to PASMC proliferation and vascular remodeling. Dashed lines highlight other potential pathways, as evidenced by the literature, but not investigated in the present study. Increased cyclin D1 expression may contribute to the positive feedback mechanism via the release of E2F, which have been shown to upregulate Nox4 expression. There is increasing evidence of cross talk between Nox isoforms and mitochondrial ROS, and increased Nox4 expression may augment ROS generation at complex III.