Nitric oxide and superoxide generation from endothelial NOS: modulation by HSP90

Area of Science:

• Cardiovascular Biology
• Endothelial Cell Function
• Molecular Medicine

Background:

• Pulmonary arterial endothelial cells (PAECs) show altered nitric oxide (NO) and superoxide production during postnatal development, suggesting endothelial NO synthase (eNOS) uncoupling.
• Heat shock protein 90 (HSP90) is a known chaperone protein with potential roles in protein complex formation and function.

Purpose of the Study:

• To investigate the role of HSP90 in regulating eNOS coupling and activity in PAECs.
• To determine if HSP90 influences NO and superoxide production by eNOS.
• To examine the association between HSP90-eNOS interactions and endothelial dysfunction in a lamb model of pulmonary hypertension.

Main Methods:

• Western blot analysis to assess HSP90 expression and HSP90-eNOS interaction.
• In vitro studies using recombinant human eNOS and HSP90.
• Adenoviral overexpression of HSP90 in PAECs.
• Treatment with HSP90 inhibitor radicicol.
• Analysis of PAECs from fetal and 4-week-old lambs, and lambs with pulmonary hypertension (shunt model).

Main Results:

• HSP90 expression was higher in fetal PAECs compared to 4-week-old PAECs.
• In vitro, HSP90 augmented NO production and inhibited superoxide generation by eNOS.
• HSP90 overexpression in PAECs increased NO production and reduced superoxide generation.
• Radicicol treatment decreased HSP90-eNOS interaction, reduced NO, and increased superoxide production, indicating eNOS uncoupling.
• In shunt lambs, decreased HSP90-eNOS interaction correlated with reduced NO and increased superoxide generation.

Conclusions:

• HSP90 plays a critical role in maintaining eNOS coupling, promoting NO generation, and inhibiting superoxide production.
• Disruption of HSP90-eNOS interactions contributes to endothelial dysfunction observed in pulmonary hypertension.
• Targeting HSP90-eNOS interactions may offer a therapeutic strategy for pulmonary hypertension.