Enhancing Parasympathetic Activity to Improve Endothelial Dysfunction, Vascular Oxidative Stress in African Americans

Overview

Specific Aim 1: To test the hypothesis that prolonged (3-month) treatment with galantamine inhibits NADPH IsoLG-protein adducts formation and improves markers of endothelial cell (EC) dysfunction in AAs. Aim 1a: The investigators will determine if galantamine inhibits NADPH IsoLG-protein adducts formation, superoxide production, and immune cell activation compared to placebo. For this purpose, the investigators will study peripheral blood mononuclear cell (PBMC), a critical source of systemic oxidative stress, collected from study participants. Aim 1b: The investigators will determine if galantamine reduces intracellular Iso-LGs, ICAM-1, and 3-nitrotyrosine, a marker of vascular oxidative stress, in ECs harvested from study participants. Specific Aim 2: To determine if prolonged (3-month) treatment with galantamine improves endothelial dysfunction as measured by vascular reactivity in AAs. The investigators will measure vascular reactivity in response to ischemia in two vascular beds: (a) in conduit arteries (brachial artery) using brachial artery diameter flow-mediated dilation (FMD), and (b) in the microvasculature (MBV) using contrast-enhanced ultrasonography in skeletal muscle. Sub-study (optional) Will study the effect of trans-auricular vagus nerve stimulation (TaVNS) during a period of enhanced vascular oxidative stress This proposal will study a novel mechanism that could alter the oxidative and immunogenic responses that contributes to endothelial dysfunction in AAs and will offer a potential pathway for the development of more effective therapies aimed at decreasing the progression of endothelial dysfunction to cardiovascular disease in this population.

Endothelial dysfunction, a pro-thrombotic, inflammatory condition that causes impaired vascular reactivity is an early reversible step in the development of atherosclerosis and cardiovascular disease (CVD). Multiple studies consistently shown that African Americans (AAs) have impaired endothelial function compared to whites. African Americans also experience disproportionately higher CV morbidity and 20% higher mortality than whites or Hispanics. Endothelial dysfunction is caused by the overproduction of reactive oxygen species (ROS), particularly superoxide which interferes with endothelial-derived nitric oxide signaling pathways. One of the major sources of superoxide is NADPH oxidase; our previous work found that activation of NADPH oxidase contributes to vascular oxidation through the formation of highly immunogenic isolevuglandins (IsoLG-protein adducts) in peripheral mononuclear cells (PBMCs), which stimulates antigen presenting cells (APC) and inflammatory mediators. Inflammation and oxidative stress are modulated by the parasympathetic nervous system (PNS). The investigators and others found that AAs have reduced PNS activity compared with whites. Studies in animal models have shown that parasympathetic nervous stimulation with either acetylcholinesterase inhibitors or direct vagus nerve stimulation protect against oxidative stress and inflammation There is Sub-study (optional) attached to the above main study. It is to evaluate the effect of trans-auricular vagus nerve stimulation (TaVNS) during a period of enhanced vascular oxidative stress. It is an ancillary, mechanistic, proof-of-concept study to determine if acute vagal stimulation with a Transcutaneous Electrical Nerve Stimulation device (TENS) inhibits immune cell activation and improves markers of endothelial cell dysfunction. The investigators preliminary data in obese AA women found that stimulation of the PNS cholinergic transmission with the acetylcholinesterase inhibitor, galantamine, blocked the production of oxidative stress and inflammatory cytokines induced by lipids. The overall goal of the current proposal is to determine if prolonged treatment with galantamine improves endothelial dysfunction and vascular oxidative stress in AAs. For this purpose, the investigators will conduct a proof-of-concept, blinded, randomized, placebo-controlled study to test the effect of 3-month treatment with galantamine (16 mg/day) on vascular oxidative stress and impaired vascular reactivity in AAs. A total of 160 participants enrolled and will be randomized, so that we can complete 88 AAs (44 per treatment arm) in a 1:1 ratio to 8 mg p.o. twice a day (16 mg/day) of galantamine Screening visit: After the informed consent process, subjects will undergo a history and physical examination. Other tests include EKG, collection of fasting blood samples: cell blood count, lipid profile, comprehensive metabolic panel and HbA1c. Randomization (V1): Two antecubital IVs will be placed for EC harvesting, blood collection, and drug infusion; we will monitor continuous blood pressure and ECG throughout the study to evaluate for cardiovascular autonomic parameters. Subjects will then be challenged with an intralipid infusion to stimulate oxidative stress, and the assessment of endpoints will be performed. After completing visit 1 procedures, and prior to being discharged, the subjects will be randomized to 8 mg p.o. twice a day (bid) (16 mg/day) galantamine or matching placebo in a 1:1 ratio using a block randomization algorithm with random block size and the assignments will be blinded to the investigator and the study subject. Telemedicine visits: The study nurse will conduct telemedicine visits with the study subject at 1, 3 and 5 days after the initiation of the study drug to evaluate for drug's side effects. Safety follow-up visits (V2, V3, V4): Subjects will be asked to attend three outpatient clinic visits at 2, 4 and 8 weeks that coincide with the dose-titration schedule. The first dose will be 4 mg a day for 2 weeks (visit 1, V1), then they will increase it to 8 mg a day for 2 weeks (visit 2, V2) and then 8 mg twice a day (visit 3, V3) for the remainder of the study. Final assessment of endpoints Visit 5 (V5): The blinded medication will be administered at 7:00 AM before any procedure is performed. The subject will repeat the procedures outlined in V1. Sub study (Optional): Mentoring in cholinergic regulation of vascular oxidation This pilot study is an open-label, before-after design. The total sample size for Aims 1 and 2 is 12 subjects. Subjects of those completing the parent trial will meet the inclusion criteria, will return for an additional visit (V6) Aim 1: To test the hypothesis that acute vagal stimulation with TaVNS inhibits immune cell activation. For this purpose, we will determine NADPH IsoLG-protein adducts formation, superoxide production, and immune cell activation in peripheral blood mononuclear cells (PBMC), a critical source of systemic oxidative stress, before and after lipid-induced ROS production, during continuous TaVNS. Aim 2: We will determine if acute vagus nerve stimulation with TaVNS inhibits endothelial cell dysfunction. For this purpose, we will assess intracellular Iso-LGs, ICAM- 1, and 3-nitrotyrosine, a marker of vascular oxidative stress, in harvested ECs before and after lipid-induced ROS production during continuous TaVNS. Subjects of those completing the parent trial, will return for an additional visit (V6). This visit will be scheduled at least 4 weeks after V5 (end of parent trial) to allow wash-out of the blinded study medication and maintain the blind of the parent study. Subjects will be instrumented for TaVNS, before been challenged with an intralipid infusion to stimulate oxidative stress, and the assessment of endpoints will be performed. PBMCs will be collected before, at 2 and 4 hours after lipid infusion to assess for NADPH activation (aim 1) and will also harvest ECs before and after lipid infusion to assess for intracellular IsoLG-protein adducts, ICAM-1, and 3-nitrotyrosine in harvested ECs. ECs will be harvested using four sterile J-wires. Primary Intervention (TaVNS) The FDA-approved TENS 7000 device for the treatment of pain will be used for TaVNS. This device will be supplemented with ear clip electrodes. The site of the stimulation for such electrodes are the tragus or concha Specifically, the investigators will evaluate whether 3 month of galantamine treatment inhibits the activation of NADPH-IsoLG formation and the subsequent immunogenic responses in PBMCs. Furthermore, the investigators will determine if galantamine decreases markers of oxidative stress and inflammation in harvested endothelial cells (ECs) and improves vascular reactivity in the same study subjects. The planned studies will provide a comprehensive assessment of the mechanism underlying the effect of increased PNS cholinergic transmission on endothelial dysfunction. If the investigators' hypothesis is correct, and galantamine improves endothelial dysfunction in AAs, a population with a high risk for CVD, they will discover a novel mechanism that could alter the oxidative and immunogenic responses in this population and will offer a potential pathway for the development of more effective therapies aimed at decreasing CVD. The parent study along with the Sub study offers a novel research approach with advanced skills in using both a pharmacological intervention that enhances cholinergic activity and direct vagus nerve stimulation. These approaches could generate more effective therapies aimed at decreasing the progression of endothelial dysfunction to cardiovascular disease African Americans.