Effect of an Isolevuglandin Scavenger on Salt Sensitivity of Blood Pressure and Immune Cell Activation in Humans

Overview

Hypertension is the leading cause of preventable deaths globally, driven by complications such as myocardial infarction, stroke, heart failure, and kidney disease. Recent updates in hypertension classification by the American Heart Association (AHA) place nearly half of the U.S. population in the hypertensive category. Excess dietary salt is a major risk factor for hypertension, with 50% of hypertensive individuals exhibiting salt-sensitivity of blood pressure (SSBP). SSBP is an independent predictor of cardiovascular events and death. While kidney mechanisms in salt-sensing have been extensively studied, emerging evidence suggests that immune cells can also sense sodium (Na+). This trial hypothesizes that myeloid cell-derived isolevuglandins (IsoLGs) drive endothelial dysfunction, perpetuating the salt-sensitive phenotype. Preliminary data indicate that targeting IsoLGs with the IsoLG scavenger 2-hydroxybenzylamine (2-HOBA) may interrupt this immune-vascular axis, reducing salt sensitivity and associated cardiovascular risks. This phase 2 clinical trial aims to investigate the role of 2-HOBA in modulating immune cell function within blood vessels in hypertensive patients. The study will explore the impact of immunity on salt sensitivity and assess 2-HOBA's potential to reduce endothelial dysfunction, improve immune cell activation, and alleviate SSBP.

Investigators will perform a 12-week randomized, double-blind, placebo-controlled, 2x2 crossover proof-of-concept phase II study with treatment of 2-HOBA in participants previously phenotyped for SSBP. Investigators will randomize people with known SSBP (i.e., 10 mmHg drop in systolic blood pressure from salt-loading to salt-depletion) to treatment with 2-HOBA (500mg) three times a day for 4 weeks, followed by a washout period of 4 weeks, and then placebo for 4 weeks versus placebo then washout then 2-HOBA. Assessments will be obtained at week 0, 4, 8 and 12 per cross-over design above. The investigators will employ the Weinberger protocol in 20 patients as previously reported. Investigators will advise participants to maintain their usual diet and salt consumption until the onset of the protocol and provide the participant with a container and appropriate instructions to collect a 24-hour urine specimen the day before admission to the hospital. Admission will take place two weeks later, for washout of the effect of the medications, and on an evening, for the patient to rest overnight in the hospital before onset on the protocol. During these two weeks, untreated blood pressure will be monitored either by the patients at home or in interim scheduled visits, to assure that it does not exceed 180/110 mmHg, in which case the protocol will be discontinued for safety. On admission, volume of the baseline urine will be measured, and aliquots sent to the lab or frozen at -80ºC for research analytes. On the morning after admission (6 am) an ambulatory blood pressure monitor (SpaceLabs 90207 or 90210) will be set up for continuous blood pressure recording throughout the study (every 15 minutes from 6 am until 10 pm and every 30 minutes from 10 pm to 6 am), body weight will be recorded with the patient wearing a gown (same scale as future days), blood samples will be obtained for the analytes of the study described below (baseline tests), and a new 24-hour collection will be started for the period of salt loading at 8 am. Salt loading will be achieved by the combination of a high-salt diet (isocaloric, with 160 mEq Na and 70 mEq K), which the patient will consume in its entirety, and an infusion of 2L of saline (300 mEq Na+). Patients will have free access to water but their food will be limited to that provided by the protocol. Participants will retire to bed at 10 pm. On the following morning (6 am) body weight and blood samples will again be obtained, and the 24-hr collection will be completed with an intentional voiding at 8 am. These laboratory data will reflect the effect of salt loading. At 8 am, subjects will be subjected to salt depletion. This is accomplished by administering an isocaloric diet containing 10 mEq Na and 70 mEq K and continued unlimited water intake. At 8 am, 12 noon and 4 pm, subjects will be given 40 mg of furosemide orally. An additional 12-hour urine collection will be started at 8 am and ended with an intentional void at 8 pm (reflecting the period of furosemide natriuresis). A new 12-hour collection will start at 8 pm, to be closed with intentional voiding the next morning at 8 am (reflecting the period of salt depletion). The next morning (6 am) the last recording of body weight and drawing of blood samples will take place, and by 8 am the 12-hour urine collection for the period of salt depletion will be completed. The patients will be given breakfast from a regular hospital diet, which may be supplemented with salty food or fluids if the salt depletion intervention produced dizziness or documented orthostasis. Once the patient is stable and exhibits tolerance to the upright posture, the participant will be discharged home with instructions to resume antihypertensive medications, unless the presence of orthostasis requires a modification in the regimen, which will be advised by a physician member of the research team. Laboratory blood measurements: Tests to be conducted in the 3 daily sets of blood samples will include CBCs and CMP at the central laboratory of VUMC. Mass spectrometry will be performed at baseline, after salt-loading and depletion to confirm if SSBP is associated with IsoLG-adducts in isolated human monocytes and plasma by Core B. Isolated monocytes and plasma fractions will be immediately stored at -80ºC upon collection until analyzed by mass spectrometry as previously described.25 Serum and urine creatinine, Na+ and K+ will be analyzed at the central laboratory of Vanderbilt. Plasma renin activity and aldosterone will be measured by radioimmunoassay. Plasma and urine eicosatrienoic acids (EETs) and 20-hydroxyeicosatetraenoic acid (20-HETE) will be measured at the Vanderbilt eicosanoid core by liquid chromatography/tandem mass spectrometry. Assessing activity of the renin-angiotensin-aldosterone system and eicosanoids is part of the usual phenotyping of SS and SR subjects. Tests to be conducted in the 4 urine specimens (24-hr baseline, 24-hr salt loading, 12-hr furosemide natriuresis, and 12-hr salt depletion) will include: urinalysis, Na+, K+, and creatinine concentration at the central laboratory of VUMC; urine eicosatrienoic acids (EETs) and 20-hydroxyeicosatetraenoic acid (20-HETE) at the eicosanoid core of VUMC by liquid chromatography/tandem mass spectrometry. Urine pregnancy tests will be performed in women of childbearing potential. Definition of salt sensitivity of blood pressure: Blood pressure data from the monitors will be downloaded and the average of the systolic blood pressure of the day of salt loading, from 12 noon (end of the saline infusion) until 10 pm will be used as the BP for the salt-loading period of study. The average of the systolic blood pressure of the day of salt depletion, from 12 noon (second dose of furosemide) until 10 pm will be used as the BP for the salt-depleted period of study. A fall in systolic BP ≥10 mm Hg from the salt-loading to the salt-depletion periods will be used to classify a subject as salt sensitive. Immune Cell Activation Studies: The investigators will determine if salt sensitivity is associated with the activation state of human monocytes. Heparinized blood samples (40 ml) are obtained and PBMCs are isolated by Ficoll-gradient. Monocytes are isolated from the PBMC by magnetic labeling and negative selection using the Miltenyi monocyte isolation kit and analyzed using flow cytometry. Investigators will identify monocytes as CD45+/CD14+ cells and can further examine intermediate (CD14+/CD16+ and non-classical monocytes (CD14-/CD16+). The CD14+/CD16+ cells which comprise about 10% of the circulation, are of particular interest they produce increased levels of TNFalpha and promote T cell activation. These are increased in humans with hypertension and in response to in vitro elevated Na+ exposure. Although subtypes of DCs are relatively rare in number, the investigators will quantify them by measuring CD45+/CD1c+, CD45+/CD141+, CD45+/CD209+, CD45+/CD83+ and the recently identified Axl/SICLEC6 cells. 7-AAD is used to exclude dead cells. Intracellular staining with the single chain antibody D-11 to detect IsoLG-protein adducts will be used. Surface expression of CD80 and CD86, which are expressed on mature antigen-presenting cells allowing them to participate in T cell co-stimulation will be measured. General characterization of peripheral blood mononuclear cells by flow cytometry for other inflammatory cells including total leukocytes (CD45+ cells), B cells (CD45+/CD19+), total T cells (CD45+/CD3+ cells), and the T cell subtype (CD3+/CD8+ and CD4+) cells will also be performed. These experiments will be performed in freshly-isolated cells and also in monocytes exposed to normal salt or high salt for 48 hours to determine if salt-sensitivity is reflected in the monocytes. Medium from monocytes cultured for 48 hours is analyzed for cytokine release including IL-1 beta, TNF-alpha, IL-6, and IL-23 using luminex. The investigators will also obtain plasma to assess for cytokines including GM-CSF, IL-4, and Flt3 lig and that are responsible for conversion of monocytes to DCs. To ensure reproducibility, the samples will be submitted to the Vanderbilt Endocrinology and Metabolism Core Facility to perform a blinded Luminex analysis. Measurements of O2·- production and activation of NADPH oxidase including phosphorylation of p47phox and association of p47phox with gp91phox by immunoprecipitation and western blot both at baseline and in response elevated Na+ will be done previously described.Flow mediated vasodilatation (FMD) procedure: For Aim 2, Investigators will perform FMD studies with co-investigator Dr. Shibao, following published guidelines.30,31 Briefly, the brachial artery diameter will be measured using B-mode ultrasonography equipped with a high-resolution linear array transducer (10-14 mHz). A longitudinal image (parallel to the artery), proximal to the antecubital fossa will be obtained with the transducer positioned to optimize images of the near and far wall interfaces. A simultaneous ECG signal is recorded, and images are digitally acquired at end-diastole, synchronized to the R wave on the ECG. To assess endothelium-dependent vasodilation, brachial artery diameter will be measured under basal conditions and during reactive hyperemia. Reactive hyperemia results from a five-minute vascular occlusion produced by rapidly (0.3 seconds) inflating a blood pressure cuff placed distal to the imaging site up to supra-systolic pressures (200-250 mm Hg). Following cuff deflation, the maximal increase in brachial artery diameter occurs at approximately 1 minute of reactive hyperemia, a response mediated by endothelium-derived nitric oxide. Additionally, Investigators will determine the true peak diameter on an individual basis and will report the peak vasodilation. After a rest period to reestablish basal conditions, endothelium-independent vasodilation will be assessed by imaging the brachial artery under basal conditions and following the administration of sublingual nitroglycerin (0.4 mg). The half-life of nitroglycerin is three minutes; however, its effects on the vasculature could last for up to 30 minutes; therefore, Investigators will delay performing any measurements or procedure during this period. The video output and electrocardiographic signal of the ultrasound machine is connected to a computer equipped with a Data Translation frame grabber video card. The R wave on the electrocardiogram is used as a trigger to acquire (digitize) frames. Digitized images at baseline and after intervention (e.g., reactive hyperemia, nitroglycerin) will be stored on the hard drive and backed up on removable media. Acquisition and analysis of the stored images is performed using software designed for this purpose by Medical Imaging Applications as previously published by our group. 32 The vessel wall lumen interface is determined by derivative based edge detection following the identification of the region of the anterior and posterior walls by the investigator. The maximum diameter of the vessel is determined and the percent change in diameter (% vasodilation) can be calculated. Endothelial Cell Harvesting and Analysis: This procedure will be performed in the contralateral arm of the FMD, and before FMD. Investigators will harvest endothelial cells using 4 sterile J-wires. The J-wires will be briefly advanced (\~5 cm beyond the tip of the catheter) and retracted through an 18-gauge catheter. The wires will be transferred to a dissociation buffer solution and washed 10 times to detach the endothelial cells. Harvested endothelial cells will undergo surface staining using markers: Live/Dead, CD45+, CD31+, CD54+, and CD144+. The cells will then be fixed, permeabilized and intracellular staining will be performed using D-11 for IsoLG formation and with anti-3-nitrotyrosine. Endothelial cells will be characterized by CD31+ CD45- expression using flow cytometry. Investigators will use fluorescence minus one (FMOs) and isotype controls to accurately adjust gates for all fluorophores.