Novel therapies for hypertension and associated cardiovascular risk

University of Minnesota Ph.D. dissertation. August 2018. Major: Biomedical Engineering. Advisor: Alena Talkachova. 1 computer file (PDF); xvii, 134 pages.This thesis is comprised of two parts. The first part investigates a novel therapy, vagus nerve stimulation, for hypertension and hypertension-induced heart disease. Hypertension impacts over 1 billion people worldwide, and clinical management is challenging. Left uncontrolled, high blood pressure can significantly increase the risk of cardiovascular events. The majority of hypertensive patients are treated with anti-hypertensive drugs to control blood pressure, but many limitations exist including resistant hypertension, inability to tolerate therapy, and non-compliance with the medication regime. For these patients, an alternative approach is needed to control blood pressure. Recently, the imbalance in the autonomic nervous system, evident in hypertension, has been the target of novel device-based therapies such as vagus nerve stimulation. The main goal of this research is to evaluate the efficacy of vagus nerve stimulation to treat hypertension and hypertension-induced heart disease. This thesis investigates the impact of vagus nerve stimulation on disease progression, survival, and cardiovascular remodeling in Dahl salt-sensitive hypertensive rats. Overall, the results of this work provide evidence for the beneficial therapeutic effects of vagus nerve stimulation in hypertension and motivate future studies to optimize therapy parameters and further understand therapeutic mechanisms. The second part of this thesis focuses on atrial fibrillation and the evaluation of new mapping techniques for improving rotor localization for ablation procedures. Currently, success rates for ablation procedures for non-paroxysmal atrial fibrillation are low and require repeat procedures or a lifetime of pharmacological agents to reduce the risk of stroke. Improved signal processing techniques for mapping electrical activity in the atrium can help further our understanding of the generation and maintenance of atrial fibrillation and ultimately improve ablation procedure success rates and terminate the arrhythmia. The main goal of this work was to validate new signal processing techniques – multiscale frequency, kurtosis, Shannon entropy, and multiscale entropy – to identify regions of abnormal electrical activity. The results of this work demonstrate improved accuracy of these novel techniques in mapping rotors in cardiac arrhythmias and motivates further studies evaluating more complex arrhythmias and human intracardiac electrograms

Chronic Low-Level Vagus Nerve Stimulation Improves Long-Term Survival in Salt-Sensitive Hypertensive Rats

Chronic hypertension (HTN) affects more than 1 billion people worldwide, and is associated with an increased risk of cardiovascular disease. Despite decades of promising research, effective treatment of HTN remains challenging. This work investigates vagus nerve stimulation (VNS) as a novel, device-based therapy for HTN treatment, and specifically evaluates its effects on long-term survival and HTN-associated adverse effects. HTN was induced in Dahl salt-sensitive rats using a high-salt diet, and the rats were randomly divided into two groups: VNS (n = 9) and Sham (n = 8), which were implanted with functional or non-functional VNS stimulators, respectively. Acute and chronic effects of VNS therapy were evaluated through continuous monitoring of blood pressure (BP) and ECG via telemetry devices. Autonomic tone was quantified using heart rate (HR), HR variability (HRV) and baroreflex sensitivity (BRS) analysis. Structural cardiac changes were quantified through gross morphology and histology studies. VNS significantly improved the long-term survival of hypertensive rats, increasing median event-free survival by 78% in comparison to Sham rats. Acutely, VNS improved autonomic balance by significantly increasing HRV during stimulation, which may lead to beneficial chronic effects of VNS therapy. Chronic VNS therapy slowed the progression of HTN through an attenuation of SBP and by preserving HRV. Finally, VNS significantly altered cardiac structure, increasing heart weight, but did not alter the amount of fibrosis in the hypertensive hearts. These results suggest that VNS has the potential to improve outcomes in subjects with severe HTN

A cholinergic-sympathetic pathway primes immunity in hypertension and mediates brain-to-spleen communication

The crucial role of the immune system in hypertension is now widely recognized. We previously reported that hypertensive challenges couple the nervous drive with immune system activation, but the physiological and molecular mechanisms of this connection are unknown. Here, we show that hypertensive challenges activate splenic sympathetic nerve discharge to prime immune response. More specifically, a vagus-splenic nerve drive, mediated by nicotinic cholinergic receptors, links the brain and spleen. The sympathetic discharge induced by hypertensive stimuli was absent in both coeliac vagotomized mice and in mice lacking α7nAChR, a receptor typically expressed by peripheral ganglionic neurons. This cholinergic-sympathetic pathway is necessary for T cell activation and egression on hypertensive challenges. In addition, we show that selectively thermoablating the splenic nerve prevents T cell egression and protects against hypertension. This novel experimental procedure for selective splenic denervation suggests new clinical strategies for resistant hypertension

New perspectives on the melanocortins and their cardiovascular effects: Potential implications for the treatment of cardiovascular diseases with melanocortin analogues

The melanocortin peptides, including melanocyte-stimulating hormones, α-, β- and γ-MSH, are derived from the precursor peptide proopiomelanocortin and mediate their biological actions via five different melanocortin receptors, named from MC1 to MC5. Melanocortins have been implicated in the central regulation of energy balance and cardiovascular functions, but their local effects, via yet unidentified sites of action, in the vasculature, and their therapeutic potential in major vascular pathologies remain unclear. Therefore, the main aim of this thesis was to characterise the role of melanocortins in circulatory regulation, and to investigate whether targeting of the melanocortin system by pharmacological means could translate into therapeutic benefits in the treatment of cardiovascular diseases such as hypertension. In experiments designed to elucidate the local effects of α-MSH on vascular tone, it was found that α-MSH improved blood vessel relaxation via a nitric oxide (NO)-dependent mechanism without directly contracting or relaxing blood vessels. Furthermore, α-MSH was shown to regulate the expression and function of endothelial NO synthase in cultured human endothelial cells via melanocortin 1 receptors. In keeping with the vascular protective role, pharmacological treatment of mice with α-MSH analogues displayed therapeutic efficacy in conditions associated with vascular dysfunction such as obesity. Furthermore, α-MSH analogues elicited marked diuretic and natriuretic responses, which together with their vascular effects, seemed to provide protection against sodium retention and blood pressure elevation in experimental models of hypertension. In conclusion, the present results identify novel effects for melanocortins in the local control of vascular function, pointing to the potential future use of melanocortin analogues in the treatment of cardiovascular pathologies.Melanokortiinien sydän- ja verisuonivaikutukset: Mahdollisuudet melanokortiinianalogien käytölle sydän- ja verisuonisairauksien hoidossa. Melanokortiinit, joihin lukeutuvat α-, β- ja γ-melanosyyttejä stimuloiva hormoni (MSH), ovat pääasiassa aivoissa ilmenevän esiasteen pro-opiomelanokortiinin pilkkoutumisiessa syntyviä, pienempiä peptidejä, jotka vaikuttavat melanokortiinireseptoreiden välityksellä elimistön fysiologisiin toimintoihin. Melanokortiinit osallistuvat merkittävälla tavalla elimistön energiatasapainon sekä sydämen ja verenkiertoelimistön keskushermostoperäiseen säätelyyn, mutta niiden paikalliset vaikutukset ja vaikutusmekanismit verenkierron säätelyssä ovat pitkälti tuntemattomia. Lisäksi, melanokortiinien mahdollisista, terapeuttisista vaikutuksista sydän- ja verisuonisiarauksien hoidossa tiedetään hyvin vähän. Tämän väitöskirjatutkimuksen keskeisimpänä tavoitteena oli tutkia melanokortiinien vaikutuksia verisuonten toiminnan säätelyssä sekä arvioida löydösten merkitystä uusien hoitomahdollisuuksien kannalta. Suonten toimintakykyä mittaavissa kokeissa havaitsimme, että α-MSH tehosti verisuonten sisäpintaa verhoavan endoteelin kykyä laajentaa verisuonia ilman, että se itse suoraan vaikutti suonten supistumistilaan. Tämä vaikutus oli yhteydessä verisuonten lisääntyneeseen typpioksidin (NO) tuottoon ja herkkyyteen NO:n verisuonia laajentavalle vaikutukselle. Viljelemällä ihmisperäisiä endoteelisoluja osoitimme melanokortiini 1 reseptoreiden välittävän α-MSH vaikutuksia NO:n tuotantoa sääteleviin tekijöihin kuten endoteliaalisen NO syntaasin määrään ja aktiivisuuteen. Näiden verisuonten toimintaa parantavien vaikutusten ansiosta, α-MSH-analogit paransivat verisuonten toimintaa kokeellisissa tautimalleissa, joihin liittyy erityisesti endoteelin toiminnan häiriö kuten lihavuuden yhteydessä. Lisäksi havaitsimme, että α-MSH:n synteettinen analogi lisää veden ja natriumin erittymistä elimistöstä sekä vaikuttaa terapeuttisesti kohonneen verenpaineen hoidossa hiirillä. Nämä löydökset laajentavat ymmärrystämme melanokortiinien vaikutuksista verenkierron säätelyssä tuoden samalla näyttöä uusista hoitomahdollisuuksista sydän- ja verisuonisairauksissa.Siirretty Doriast

Mechanisms Contributing to the Generation of Mayer Waves

Mayer waves may synchronize overlapping propriobulbar interneuronal microcircuits constituting the respiratory rhythm and pattern generator, sympathetic oscillators, and cardiac vagal preganglionic neurons. Initially described by Sir Sigmund Mayer in the year 1876 in the arterial pressure waveform of anesthetized rabbits, authors have since extensively observed these oscillations in recordings of hemodynamic variables, including arterial pressure waveform, peripheral resistance, and blood flow. Authors would later reveal the presence of these oscillations in sympathetic neural efferent discharge and brainstem and spinal zones corresponding with sympathetic oscillators. Mayer wave central tendency proves highly consistent within, though the specific frequency band varies extensively across, species. Striking resemblance of the Mayer wave central tendency to the species-specific baroreflex resonant frequency has led the majority of investigators to comfortably presume, and generate computational models premised upon, a baroreflex origin of these oscillations. Empirical interrogation of this conjecture has generated variable results and derivative interpretations. Sinoaortic denervation and effector sympathectomy variably reduces or abolishes spectral power contained within the Mayer wave frequency band. Refractorines of Mayer wave generation to barodeafferentation lends credence to the hypothesis these waves are chiefly generated by brainstem propriobulbar and spinal cord propriospinal interneuronal microcircuit oscillators and likely modulated by the baroreflex. The presence of these waves in unitary discharge of medullary lateral tegmental field and rostral ventrolateral medullary neurons (contemporaneously exhibiting fast sympathetic rhythms [2-6 and 10 Hz bands]) in spectral variability in vagotomized pentobarbital-anesthetized and unanesthetized midcollicular (i.e., intercollicular) decerebrate cats supports genesis of Mayer waves by supraspinal sympathetic microcircuit oscillators. Persistence of these waves following high cervical transection in vagotomized unanesthetized midcollicular decerebrate cats would seem to suggestspinalsympathetic microcircuit oscillators generate these waves. The widespread presence of Mayer waves in brainstem sympathetic-related and non-sympathetic-related cells would seem to betray a general tendency of neurons to oscillate at this frequency. We have thus presented an extensive and, hopefully cohesive, discourse evaluating, and evolving the interpretive consideration of, evidence seeking to illumine our understanding of origins of, and insight into mechanisms contributing to, the genesis of Mayer waves. We have predicated our arguments and conjectures in the substance and matter of empirical data, though we have occasionally waxed philosophical beyond these traditional confines in suggesting interpretations exceeding these limits. We believe our synthesis and interpretation of the relevant literature will fruitfully inspire future studies from the perspective of a more intimate appreciation and conceptualization of network mechanisms generating oscillatory variability in neuronal and neural outputs. Our evaluation of Mayer waves informs a novel set of disciplines we term quantum neurophysics extendable to describing subatomic reality. Beyond informing our appreciation of mechanisms generating sympathetic oscillations, Mayer waves may constitute an intrinsic property of neurons extant throughout the cerebrum, brainstem, and spinal cord or reflect an emergent property of interactions between arteriogenic and neuronal oscillations.Peer reviewe

The Role of Central ACE2 and Nrf2 in Sympatho-Excitation: Responses to Central Angiotensin II

Sympatho-excitation is a key characteristic in cardiovascular diseases such as chronic heart failure (CHF) and primary Hypertension (HTN). Evidence suggests that increased sympathetic tone is closely related to activation of the Renin-Angiotensin-Aldosterone system (RAAS) in the central nervous system. An underlying mechanism for sympatho-excitation is thought to be oxidative stress resulting from Angiotensin II (AngII) type 1 receptor (AT1R) activation. Over the past several decades, pharmacological targeting of components of the RAAS have been used as standard therapy in CHF and HTN. However, additional therapeutic strategies are necessary to control these diseases. Oxidative stress is regulated, in part, by the balance between components of the RAAS and the ability of the system to scavenge oxygen radicals. Over the past decade, Nuclear factor E2-related factor 2 (Nrf2) has emerged as an important transcriptional regulator that maintains redox homeostasis by governing a broad array of antioxidant genes in response to oxidant stress. Central Nrf2 dysregulation has been found in animals with CHF and HTN. To determine if Nrf2 contributes to decreased antioxidant defense and increased sympathetic nerve activity (SNA) in CHF, we upregulated Nrf2 in the rostral ventrolateral medulla (RVLM) in C57BL/6 mice and evaluated their hemodynamic and sympathetic function in the CHF state. We found that (1) Nrf2 and two target proteins, NAD(P)H dehydrogenase [quinone] 1 (NQO1) and Heme oxygenase (HO-1) in the RVLM were significantly lower in CHF compared to Sham mice; (2) Urinary norepinephrine (NE) excretion in CHF mice was markedly reduced following Nrf2 upregulation; (3) CHF mice overexpressing Nrf2 exhibited an enhancement in spontaneous baroreflex gain and a decrease in basal renal SNA. In an attempt to understand the antioxidant function of the RAAS we examined the role of Angiotensin converting enzyme 2 (ACE2) in a model of central AngII-induced HTN. Despite its direct enzymatic effect on AngII, ACE2 has been shown to reduce oxidative stress and to be sympatho-inhibitory. It has been demonstrated that animals with CHF exhibit increased Angiotensin converting enzyme (ACE) and decreased ACE2 in the RVLM. We hypothesized that overexpression of ACE2 in the brain reduces the sympathetic and blood pressure (BP) responses to central AngII by activation of Nrf2 and enhancing antioxidant enzyme expression. To illuminate the role of Nrf2 in the central regulation of SNA in response to central AngII, we assessed Nrf2 changes in the RVLM in SynhACE2 mice treated with ICV AngII infusion. Mice with central overexpression of ACE2 inhibited the pressor and sympathetic responses to central AngII. We found that Nrf2 was upregulated in the RVLM in SynhACE2 mice, and that pharmacological upregulation of central Nrf2 had a significant impact on BP in response to central AngII. Overall, the experiments described in this dissertation showed that selectively upregulating Nrf2 in the RVLM attenuates sympatho-excitation in CHF mice. We also describe a novel role of interplay between central AngII, ACE2 and Nrf2 in the regulation of sympatho-excitation in central HTN. While not definitive, these studies suggest a role for ACE2 and Nrf2 as targets for therapy in CHF and HTN

Time course of the hemodynamic responses to aortic depressor nerve stimulation in conscious spontaneously hypertensive rats

The time to reach the maximum response of arterial pressure, heart rate and vascular resistance (hindquarter and mesenteric) was measured in conscious male spontaneously hypertensive (SHR) and normotensive control rats (NCR; Wistar; 18-22 weeks) subjected to electrical stimulation of the aortic depressor nerve (ADN) under thiopental anesthesia. The parameters of stimulation were 1 mA intensity and 2 ms pulse length applied for 5 s, using frequencies of 10, 30, and 90 Hz. The time to reach the hemodynamic responses at different frequencies of ADN stimulation was similar for SHR (N = 15) and NCR (N = 14); hypotension = NCR (4194 +/- 336 to 3695 +/- 463 ms) vs SHR ( 3475 +/- 354 to 4494 +/- 300 ms); bradycardia = NCR (1618 +/- 152 to 1358 +/- 185 ms) vs SHR (1911 +/- 323 to 1852 +/- 431 ms), and the fall in hindquarter vascular resistance = NCR (6054 +/- 486 to 6550 +/- 847 ms) vs SHR (4849 +/- 918 to 4926 +/- 646 ms); mesenteric = NCR (5574 +/- 790 to 5752 +/- 539 ms) vs SHR (5638 +/- 648 to 6777 +/- 624 ms). In addition, ADN stimulation produced baroreflex responses characterized by a faster cardiac effect followed by a vascular effect, which together contributed to the decrease in arterial pressure. Therefore, the results indicate that there is no alteration in the conduction of the electrical impulse after the site of baroreceptor mechanical transduction in the baroreflex pathway (central and/or efferent) in conscious SHR compared to NCR.FAPESPFAPESP [02/09406-5]CNPqCNPq [134480/2006-6]CAPESCAPES [1681/07

The neonatal sepsis is diminished by cervical vagus nerve stimulation and tracked non-invasively by ECG: a preliminary report in the piglet model

In adults, vagus nerve stimulation (VNS) reduces inflammation. In neonates, the effects of VNS are not known. An electrocardiogram (ECG)-derived heart rate variability (HRV) index reliably tracks the inflammatory response induced by low-dose lipopolysaccharide (LPS) in near-term sheep fetuses. We evaluated the VNS effect on the systemic inflammatory response induced by a high dose of LPS in neonatal piglets to mimic late-onset neonatal sepsis. Next, we tested if our HRV inflammatory index tracks inflammation in piglets. Following anesthesia, electrodes were attached to the left vagal nerve; ECG and blood pressure (BP) were recorded throughout the experiment. Following baseline, the piglets were administered LPS as 2mg/kg IV bolus. In the VNS treated piglet, the vagus nerve was stimulated for 10 minutes prior to and 10 min after the injection of LPS. In both groups, every 15 min post LPS, the arterial blood sample was drawn for blood gas, metabolites, and inflammatory cytokines. At the end of the experiment, the piglets were euthanized. BP and HRV measures were calculated. The piglets developed a potent inflammatory response to the LPS injection with TNF-alpha, IL-1beta, IL-6 and IL-8 peaking between 45 and 90 min post-injection. VNS diminished the LPS-induced systemic inflammatory response varying across the measured cytokines from two to ten-fold. The HRV index tracked accurately the cytokines' temporal profile. This novel model allows manipulating and tracking neonatal sepsis: The HRV inflammatory index 1) applies across species pre- and postnatally and 2) performs well at different degrees of sepsis (i.e., nanogram and milligram doses of LPS); 3) the present VNS paradigm effectively suppresses LPS-induced inflammation, even at high doses of LPS. The potential of early postnatal VNS to counteract sepsis and of HRV monitoring to early detect and track it deserve further study