Date on Master's Thesis/Doctoral Dissertation

5-2020

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Pharmacology and Toxicology

Degree Program

Pharmacology and Toxicology, PhD

Committee Chair

Conklin, Daniel

Committee Co-Chair (if applicable)

Bhatnagar, Aruni

Committee Member

Bhatnagar, Aruni

Committee Member

Srivastava, Sanjay

Committee Member

Wise, John

Committee Member

Cai, Lu

Committee Member

Hoyle, Gary

Author's Keywords

transient receptor potential ankyrin-1; aldehydes; EDRF; nitric oxide; mesenteric artery; aorta

Abstract

Cardiovascular diseases (CVD) are the leading cause of death in most countries and are significant health and financial burden worldwide. Smoking contributes to more than 480,000 premature deaths annually -- accounting for one of every five deaths in the United States each year. Numerous studies have shown that smoking increases the risk of factors including endothelial dysfunction, thrombosis, and atherosclerosis, which may lead to an incidence of myocardial infarction, coronary artery disease, and stroke. Electronic cigarettes (e-cigarettes) are a new kind of tobacco product, and some studies show that e-cigarettes also could induce CVD since both e-cigarettes and conventional tobacco products release many aldehydes. Extensive epidemiological and experimental data show that aldehydes contribute to cardiovascular morbidity and mortality. However, whether these aldehydes contribute to the adverse effects of emerging tobacco-products such as e-cigarettes are unknown. The transient receptor potential ankyrin1 (TRPA1) channel is a receptor of unsaturated aldehydes that mediates tobacco smoke-induced lung injury in some animal models. Thus, our central hypothesis is that aldehydes derived from e-cigarettes or conventional tobacco contribute to vascular toxicity through the activation of the TRPA1 pathway. Both in vivo and in vitro studies were performed using male and female C57BL/6J mice. For in vivo studies, mice were exposed to aldehyde gas (1-5 ppm, 6 h/day) for 4 days or 12 weeks (6 h/day, 5 days/week). Non-invasive blood pressure and heart rate were measured weekly for 12 weeks by the tail-cuff system. Measure real-time blood pressure and heart rate by telemetry system. After exposures, we collected aortas and examined vascular function ex vivo, specifically, testing for any changes in sensitivity and efficacy of the aorta in response to pharmacological agents: phenylephrine (PE; contractions), acetylcholine (ACh; relaxation) and sodium nitroprusside (SNP; relaxation). PE-induced isometric contractions (mN) were normalized by segment length and/or volume to compare aortic contractility. Relaxations were calculated as a percentage reduction in PE-induced tension. The sensitivity of each agent was calculated as the effective concentration producing a 50% response (EC50). In our in vitro studies, we examined the effects and underlying mechanisms of action (relaxation) of aldehydes in both isolated mouse superior mesenteric artery (SMA) and aorta. To test specific pathways, we used pharmacological inhibitors of nitric oxide synthase (NOS), guanylyl cyclase, TRPA1, and K+ channels as well as the mechanical injury of the endothelium. In our in vivo studies, C57BL/6J (wild type, WT) mice were exposed to HEPA-filtered room air (control), formaldehyde (FA; 5 ppm) or acetaldehyde (AA; 5ppm) for 4 days. There were no differences in the relaxations of ACh and SNP or the PE-induced contractility of between control and aldehyde-exposed groups. So, short-term exposure to FA or AA at levels present in tobacco product aerosols did not alter aortic function. Mice were exposed to crotonaldehyde (CR) or filtered room air for 12 weeks (1 ppm, 6h/day, 5 d/week). Non-invasive blood pressure was measured by tail-cuff (once a week) for 12 weeks. Blood pressures (systolic, mean, diastolic) were significantly lower in the CR group than in the air group over the last several weeks, yet there was no difference in heart rate between these two groups. Blood pressure changes were accompanied by changes in aortic vasoreactivity ex vivo. Following 12-wk CR chronic exposure, the sensitivity of ACh-induced relaxation was significantly increased in the CR group, however, there was no change in aortic response to PE. Similarly, aortic sensitivity to SNP relaxation was enhanced by CR compared with the air control group. To dissect the mechanism of CR-induced vascular changes, we performed acute 4-day exposures (1 and 3 ppm). Similarly, CR exposure also altered aortic responses to ACh and slightly to SNP. Because a 4-day CR exposure was sufficient to alter aortic reactivity, we tested whether the transient receptor potential ankyrin 1 (TRPA1) channel was involved. In TRPA1-null mice exposed to CR (4-days, 1ppm), there were no changes in aortic reactivity to any agonist (PE, ACh, and SNP) compared with the air group, indicating a distinct role of TRPA1 in CR-induced changes. Interestingly, there also were no differences in any aortic responses in CR- (1 ppm 4 days) versus air-exposed female WT mice. For the in vitro studies, we explored the direct effects of FA, AA, and CR in the isolated murine superior mesenteric artery (SMA) and aorta. FA (30-1,200 mM) and AA (1-100 mM) strongly and reversibly relaxed agonist-induced contractions of SMA including phenylephrine (PE), thromboxane A2 analog (U46,619) and high potassium (high K+, 60 mM) without toxicity. The sensitivity but not the efficacy of AA-, FA- and CR-induced relaxations was dependent on blood vessels (wherein SMA was more sensitive than aorta) and contractile agonist (PE; U46,619; high K+) yet independent of the circadian cycle and sex. All three aldehyde-induced relaxations were significantly inhibited (at low concentrations) in the presence of 1) mechanically-impaired endothelium; 2) Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME), and 3) guanylyl cyclase (GC) inhibitor (ODQ). Positive immunofluorescent staining for TRPA1 was co-localized in the endothelium of isolated SMA and the TRPA1-specific gene sequence was detected in SMA. The relaxations of FA and CR but not AA at low concentrations were all inhibited in the presence of TRPA1 antagonist (A967079). These studies indicate that short-term exposure of mice to FA or AA has limited direct toxicity in the aorta, however, CR exposure (acute and chronic) leads to altered vascular function that may lead to vascular disease. Moreover, we describe the direct effects of FA, AA, and CR that are dependent on an EDRF/NO and VSMC GC/PKG pathway in SMA, with FA- and CR-induced relaxations also dependent on an endothelium-localized TRPA1 receptor. From our vitro study, we conclude that aldehydes play a key role in the regulation of blood flow in the gastrointestinal (GI) tract to augment digestion and nutrient absorption and induce postprandial hyperemia, and TRPA1 receptor plays an important role in the aldehyde-induced vasodilation in the vasculature. Future studies are required to better understand how CR exposure modifies cardiovascular pathophysiology and the specific roles of TRPA1 and sex play in contributing to the burden of tobacco-related CVD including pathophysiological processes of atherosclerosis.

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