Taking nitroglycerin for a long time can make your blood vessels produce too many harmful molecules, which stops the drug from working properly and makes your blood vessels less healthy—this is why the medicine stops helping over time.

In patients with coronary artery disease or heart failure, chronic GTN therapy (vs. placebo or non-nitrate control) over 4–8 weeks would be tested for changes in vascular oxidative stress markers (e.g., superoxide, H2O2), ALDH-2 activity, soluble guanylyl cyclase sensitivity, and endothelial function (e.g., flow-mediated dilation), with direct measurement of nitrate tolerance via hemodynamic response to GTN challenge.

A prospective cohort of patients with heart failure or CAD initiating chronic GTN therapy would be followed for 6–12 months to observe temporal associations between rising oxidative stress markers, declining ALDH-2 activity, reduced sGC responsiveness, and the onset of clinical nitrate tolerance (e.g., reduced angina relief).

Patients with documented nitrate tolerance (cases) would be compared to GTN-treated patients without tolerance (controls) to assess differences in baseline and on-treatment levels of mitochondrial/NADPH oxidase ROS, ALDH-2 expression/activity, and sGC sensitivity.

Exposure of human vascular tissue from GTN-treated patients to GTN ex vivo would test whether prior chronic GTN exposure reduces ALDH-2-mediated bioactivation and increases ROS production, leading to diminished cGMP response.

Chronic GTN administration in a rodent model of heart failure would test whether it reproduces increased vascular ROS, ALDH-2 inhibition, sGC desensitization, and loss of vasodilatory response.