Chloroquine Restores eNOS Signaling in Shunt Endothelial Cells via Inhibiting eNOS Uncoupling

Area of Science:

• Cardiovascular Research
• Cell Biology
• Pharmacology

Background:

• Pulmonary arterial hypertension (PAH) involves vascular stiffness and impaired relaxation due to reduced nitric oxide (NO) production.
• Endothelial dysfunction in PAH is linked to decreased tetrahydrobiopterin (BH4) and GTP cyclohydrolase 1 (GCH1) expression.
• Congenital heart defect (CHD)-associated PAH models exhibit reduced NO bioavailability.

Purpose of the Study:

• To investigate the mechanisms by which chloroquine restores NO bioavailability in endothelial cells from a CHD-associated PAH model.
• To elucidate the role of autophagy and specific protein interactions in regulating NO synthesis in PAH.
• To evaluate autophagy inhibition as a potential therapeutic strategy for PAH.

Main Methods:

• Utilized pulmonary artery endothelial cells (PAECs) from aortopulmonary shunt lambs (a PAH model).
• Assessed nitric oxide (NO) production, BH4 levels, GCH1 expression, and endothelial nitric oxide synthase (eNOS) levels.
• Investigated the interaction between GCH1, Hsp70, and CHIP (carboxyl terminus of Hsp70-interacting protein) using chloroquine and bafilomycin A1 (autophagy inhibitors).

Main Results:

• Shunt PAECs showed significantly reduced NO production, BH4, and GCH1 expression, despite increased eNOS.
• Chloroquine treatment restored NO production, increased BH4 levels, and upregulated GCH1 expression.
• Chloroquine disrupted the GCH1-HSP70-CHIP complex, preventing GCH1 degradation and enhancing NO synthesis. Bafilomycin A1 showed similar effects.

Conclusions:

• Autophagy inhibition effectively enhances endothelial NO synthesis in conditions of depleted NO bioavailability, such as PAH.
• Targeting the GCH1-HSP70-CHIP complex via autophagy inhibition presents a promising therapeutic avenue for PAH.
• Chloroquine's ability to restore NO bioavailability offers potential for managing PAH, particularly CHD-associated forms.