rBMSC/Cav-1F92A Mediates Oxidative Stress in PAH Rat by Regulating SelW/14-3-3 η and CA1/Kininogen Signal Transduction

Abstract

Background/objectives: Carbonic anhydrase 1 (CA1)/kininogen and selenoprotein W (SelW)/14-3-3η signal transduction orchestrate oxidative stress, which can also be regulated by nitric oxide (NO). The mutated caveolin-1 (Cav-1^F92A) gene may enhance NO production. This study explored the effect of Cav-1^F92A-modified rat bone marrow mesenchymal stem cells (rBMSC/Cav-1^F92A) on oxidative stress regulation through CA1/kininogen and SelW/14-3-3η signal transduction in a rat model of monocrotaline- (MCT-) induced pulmonary arterial hypertension (PAH).

Method: PAH was induced in rats through the subcutaneous injection of MCT. Next, rBMSC/Vector (negative control), rBMSC/Cav-1, rBMSC/Cav-1^F92A, or rBMSC/Cav-1^F92A+L-NAME were administered to the rats. Changes in pulmonary hemodynamic and vascular morphometry and oxidative stress levels were evaluated. CA1/kininogen and SelW/14-3-3η signal transduction, endothelial nitric oxide synthase (eNOS) dimerization, and eNOS/NO/sGC/cGMP pathway changes were determined through real-time polymerase chain reaction, Western blot, or immunohistochemical analyses.

Results: In MCT-induced PAH rats, rBMSC/Cav-1^F92A treatment reduced right ventricular systolic pressure, vascular stenosis, and oxidative stress; downregulated CA1/kininogen signal transduction; upregulated SelW/14-3-3η signal transduction; and reactivated the NO pathway.

Conclusions: In a rat model of MCT-induced PAH, rBMSC/Cav-1^F92A reduced oxidative stress by regulating CA1/kininogen and SelW/14-3-3η signal transduction.

Figure 1

Transduction efficiency and the change of RVSP. (a) Lentiviral vector transduction efficiency at 5 days postinfection (bars = 100 μm). (b, c) The effect of rBMSC/Cav-1^F92A on RVSP in MCT-induced PAH rats. RVSP: right ventricular systolic pressure. ^∗∗p < 0.01.

Figure 2

The effects of rBMSC/Cav1^F92A on pulmonary artery stenosis. Photomicrographs of serial sections of the peripheral rat lung containing small arteries from control animals or rats exposed to MCT. (a) H&E staining (bar = 50 μm). (b) Masson staining. The staining showed the difference between different groups in the morphology of the small pulmonary arteries (bar = 50 μm). (c) The percentage of the wall area of the total area (%). (d) The positive rate of Masson staining. The arrow indicates the pulmonary artery. ^∗∗p < 0.01.

Figure 3

The effects of rBMSC/Cav1^F92A on inflammation and oxidative stress in PAH rats. (a) The level of proinflammatory cytokines INF-γ, IL-1α, and TNF-α. (b, c) The production of ROS and GSH. ^∗∗p < 0.01.

Figure 4

Immunohistology detection of CA1 and SelW expression in lung tissue. Photomicrographs of serial sections of the rat lung containing small arteries from control animals or rats exposed to MCT with saline vehicle or modulated rBMSCs. (a) Sections were immunostained for CA1 (bar = 50 μm). (b) Sections were immunostained for SelW (bar = 50 μm). (c) The protein of CA1 and SelW expression. The arrow indicates the pulmonary vascular. Positive expression is dark brown. ^∗∗p < 0.01.

Figure 5

rBMSC/Cav1^F92A downregulated CA1/kininogen signal transduction and upregulated SelW/14-3-3η signal transduction. (a) Immunoblots of lung CA1, kininogen, SelW, and 14-3-3η expression in control animals or monocrotaline- (MCT-) treated rats with saline vehicle or modulated rBMSCs. (b–e) Densitometry of immunoblots showing quantification of changes in CA1, kininogen, SelW, and 14-3-3η expression. ^∗p < 0.05; ^∗∗p < 0.01.

Figure 6

rBMSC/Cav1^F92A reactivated NO signal in MCT-induced PAH rats. (a) The changes of NO and BH₄ production in serum. (c, d) Densitometry of immunoblots showing quantification of changes in eNOS, ratio of eNOS dimerization, and sGC in the lung of control animals or MCT-treated rats with saline vehicle or modulated rBMSCs. (e) Lung cGMP mRNA expression determined by real-time qPCR in control animals or MCT-exposed rats with saline vehicle or modulated rBMSCs. ^∗p < 0.05; ^∗∗p < 0.01.