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    Multiparity has beneficial effects on vascular function in an experimental model of type 2 diabetes
    (2016-03-23) Hannan, Johanna; Webb, R.; Goulopoulou, Styliani PhD; Posey, Rachel
    Background: Correlational studies have showed that parity is associated with risk for cardiovascular disease (CVD). According to these correlations, multiparous women have reduced risk of developing CVD compared to nulliparous women. Experimental studies examining the effects of parity on vascular function are lacking and most importantly, how parity affects women with established cardiovascular risk factors is unknown. Women with type 2 diabetes have a greater risk of developing vascular complications compared to their non-diabetic counterparts. This study focuses on the influence of parity on contractile arterial responses of multiparous and nulliparous rats with type 2 diabetes. Hypothesis: Old multiparous rats with type 2 diabetes (Goto-Kakizaki rats, GK) were hypothesized to have a decreased contractile response to phenylephrine (PE) and endothelin-1 (ET-1) compared to nulliparous GK young and age-matched rats. Materials and Methods: Three experimental groups were used: GK young nulliparous rats, 20 weeks old (GK Young NP); GK old nulliparous rats, 60 weeks old (GK Old NP); and GK old multiparous rats, 60 weeks old (GK Old MP). Mesenteric resistance arteries (MA) were mounted in a wire myograph to measure the contractile responses to PE (10-9 – 3x10-5 M) and ET-1 (10-11 – 10-7 M). Results: GK Old MP rats were heavier compared to GK Old and GK Young NP rats (297 ± 7 g vs. 255 ± 11 g vs. 232 ± 4 g, p0.05). Mesenteric arteries from GK Old MP rats had reduced sensitivity (-logEC50) to ET-1 compared to arteries from GK Old NP and GK Young NP groups (7.82 ± 0.11 vs. 8.45 ± 0.08 vs. 8.24 ± 0.07, p Conclusions: Nulliparity may have adverse effects on vascular function in women with type 2 diabetes. Taking a parity history may be beneficial when evaluating women with type 2 diabetes for risk of developing CVD.
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    Role of Median Preoptic AT1a Receptors in Control of Thirst Regulation
    (2016-03-23) Cunningham, J. Thomas; Farmer, Gef; Shell, Brent
    Purpose Thirst is an essential mechanism for organisms to maintain osmotic and blood volume homeostasis and is therefore tightly regulated. Angiotensin II (Ang II) has long been known to stimulate thirst by both its action peripherally as a circulating hormone, and centrally acting as a peptide neurotransmitter. Peripherally circulating Ang II can elicit thirst by stimulating the Subfornical Organ (SFO), which in turn stimulates other hypothalamic nuclei regulating blood volume homeostasis such as the Median Preoptic Nucleus (MnPO) and Paraventricular Nucleus presumably through Ang II signaling. These hypothalamic nuclei then activate higher cognitive centers resulting in thirst. Central Ang II administered via intracerbroventricular (ICV) infusion also induces thirst by directly stimulating regions such as the MnPO through the Angiotensin Type 1 receptor and following a similar signaling pathway as peripheral Ang II only ignoring the circumventricular organs. Methods Male Sprague-Dawley rats are microinjected in the MnPO with either a virus to knockdown AT1aR expression (shAT1a) or a scramble virus (SCR) and instrumented with radio telemetry a week later. Our lab microinjected an adeno-associated virus with short hairpin RNA matched to the Angiotensin Type 1a receptor (At1aR) into the MnPO to test whether Ang II was necessary for thirst signaling in the MnPO. We hypothesized that Ang II was necessary for the thirst response to peripherally circulating Ang II as well as ICV injected central Ang II. Sprague Dawley rats were separated into subcutaneous (SC) and ICV Ang II administration groups. The SC group was pretested for their drinking response to 2mg/kg Ang II, and those animals that drank in response to the SC Ang II administration were utilized in the study. All animals were injected with the shAt1aR virus on Day 0, and were allowed to recover before drinking tests on day 14 and day 18. ICV animals were microinjected with the virus on Day 0 as well and instrumented with a chronic lateral ventricle cannula. These animals were administered 2ng Ang II in 1ul aCSF. Results Unexpectedly, we found that knockdown of AT1aR in the MnPO did not reduce drinking in the subcutaneous animals (p [greater than] .05), but did significantly reduce drinking in the ICV animals (P Conclusions Peripheral generation of thirst through Ang II may be predominantly mediated through glutamatergic neurotransmission from the SFO which only uses Ang II as a co-transmitter. Mechanistic understanding of thirst generation in the hypothalamus is critical for pharmacological manipulation with currently available pharmaceuticals as well as for development of future compounds.
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    Energy Expenditure and Substrate Utilization with Intermittent Blood Flow Restriction Aerobic Exercise
    (2016-03-23) Sprick, Justin; Colby, Hannah; Rickards, Caroline; Schaefer, Travis
    Blood flow restriction (BFR) training is characterized by the use of compressive occlusive devices proximal to the active muscle during exercise. We hypothesized that an acute bout of aerobic BFR exercise would result in equivalent caloric expenditure and substrate utilization when compared with conventional aerobic exercise (CE). Six human volunteers (3M/3F; age, 30.2±2.6 years; BMI, 23.9± 1.0 kg/m2) performed 40-min of treadmill exercise at 65-70% of maximal heart rate (HR) with and without intermittent BFR (220 mmHg thigh cuff pressure applied over 4x5-min intervals followed by 5-min reperfusion periods). HR and metabolic parameters were measured and analyzed in 5-min time blocks. Oxygen consumption (VO2) and CO2 production (VCO2) were used to calculate caloric expenditure between conditions, and the respiratory exchange ratio (RER) was used as an index of substrate utilization. Treadmill speed remained constant at 2.5 mph, while treadmill incline (%) was modified to elicit the target HR response. Treadmill incline was subsequently assessed to determine the absolute intensity of the exercise bouts. VO2 and VCO2 increased at the onset of exercise in both conditions (p2 and VCO2 versus the BFR condition for minutes 10-40 of the exercise bout (p≤0.05 for VO2, p≤0.09 for VCO2). Additionally, a lower treadmill incline (%) was required to elicit the target HR response for BFR exercise compared to CE from minutes 10-40 (Range: 4.0-5.0 % for BFR vs. 5.5-6.8% for CE, p≤0.025). There was no difference in overall caloric expenditure (230.3±22.8 kcal for CE vs. 204.3±22.5 kcal for BFR, P=0.44) or RER (0.87±0.01 for CE vs. 0.88±0.01 for BFR, P=0.55) between conditions. These findings suggest that aerobic exercise with intermittent BFR results in similar caloric expenditure and fuel utilization as CE. Aerobic exercise with BFR could be used as an alternative exercise modality for individuals who cannot exercise at the same absolute workloads as healthy individuals, such as those with musculoskeletal limitations.
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    Cardioprotective Intermittent Hypoxia Conditioning Induces Glyoxalase-1 in Rat Left Ventricle
    (2016-03-23) Scott, Gary; Williams, Arthur Jr.; Ryou, Myoung-Gwi; Mallet, Robert T.; Ramani, Azaan
    Intermittent, normobaric hypoxia (IH) conditioning provides significant cardioprotection, including dramatically increasing myocardial resistance to ischemia-reperfusion. The cardioprotective mechanisms of IH are unknown. Myocardial ischemia-reperfusion generates methylglyoxal, a potent and toxic glycating agent. Our previous studies1 demonstrated that reactive oxygen species (ROS) generated during IH cycles contribute to cardioprotection. We hypothesize that the generation of ROS during IH cycles induce the transcription factor Nrf2 to activate expression of genes encoding cytoprotective proteins. Here we evaluated IH induction of glyoxylase 1 (GLO-1), a major enzyme responsible for methylglyoxal detoxification. Sprague-Dawley rats were conditioned by a 20 day IH program consisting of 5-8 daily, 5-10 min cycles of hypoxia (9.5-10% inspired O2) with intervening 4 min room air exposures, previously shown to produce robust cardioprotection.1 Control rats were sham-conditioned using 21% O2. After completion of the conditioning protocol, the left ventricle was isolated and extracted for enzyme analysis. The activities of cytoprotective enzymes were analyzed by spectrophotometric assays. GLO-1 activity (U/mg protein) increased threefold in IH conditioned (1.05±0.16) vs. sham (0.35±0.11) rats. Glutathione reductase activity (U/mg protein) was unchanged between IH conditioned (0.015±0.0047) vs. sham rats (0.023±0.016). IH augmentation of the anti-glycation enzyme GLO-1 may contribute to the heart’s increased resistance to ischemic injury.
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    Is Testosterone a Risk Factor for Ischemic Stroke and Neurodegeneration in Men?
    (2016-03-23) Cunningham, Rebecca; Holmes, Shaletha S.
    In the aging population, cardiovascular disease (i.e. stroke) is a common cause of mortality that affects 1 in 3 men. An ischemic event is characterized by oxidative stress (OS) and neuroinflammation. Testosterone is an oxidative stressor, which can be protective or detrimental depending on the environment. In aging males that have high levels of OS, testosterone can increase the risk for ischemic stroke. However, it is unknown if testosterone-induced OS can increase inflammation, such as COX2, a prominent mediator of neuroinflammation and oxidative stress, leading to neuronal death. Therefore, we hypothesize that testosterone, under OS conditions, will further increase OS, induce COX2 inflammation, and increase apoptosis. To test this hypothesis, we exposed a neuronal cell line (N27 cells) to a sublethal concentration of the pro-oxidant, tert-butyl hydrogen peroxide (H2O2) for 24 hours followed by the exposure to physiological levels of testosterone to assess oxidative stress and inflammatory signaling. In addition, N27 cells were exposed to ibuprofen prior to OS (H2O2) and hormone (testosterone) treatment. Under OS conditions, testosterone increased COX2 signaling and apoptosis in neurons. Further, ibuprofen attenuated the effects of testosterone in an OS environment. Our data shows testosterone promotes COX2 inflammation, which contributes to neurodegeneration in an OS environment. Notably, ibuprofen is a common and inexpensive over the counter anti-inflammatory therapeutic. Thus, ibuprofen may be a preventative intervention against ischemic events and neurodegeneration.
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    Chronic Vagus Nerve Stimulation Attenuates Renal Inflammation in Autoimmune-Induced Hypertension
    (2016-03-23) Fairley, Amber; Maloy, Charles; Mathis, Keisa W.; Pham, Grace
    Chronic renal inflammation has been implicated in the pathogenesis of hypertension, which identifies the autoimmune disease systemic lupus erythematosus (SLE) as a compelling disease model. The cholinergic anti-inflammatory pathway (CAP) is a vagally mediated neuroimmune pathway that, when impaired, may facilitate the development of chronic inflammation. While stimulation of the CAP attenuates inflammation in various models of inflammatory disease, it is unclear whether the pathway is impaired in SLE, or whether its stimulation is protective. We hypothesized that pharmacological activation of the CAP at the level of the vagus nerve would decrease renal inflammation and therefore blood pressure in a hypertensive SLE mouse model. Initially, female SLE (NZWF1) and control (NZW) mice were administered the efferent vagal stimulant, CNI-1493 (Semapimod hydrochloride; 8 mg/kg IP) or saline twice per week for 4 weeks starting at 30 weeks of age. SLE mice had 1143±439% (p
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    Low-dose Aspirin During Gestation Promotes Vascular Dysfunction and does not Ameliorate Maternal Hypertension in Rats Exposed to Innate Immune System Activation
    (2016-03-23) Nguyen, An; Valdes, Melissa; Osikoya, Oluwatobiloba; Goulopoulou, Styliani PhD; Jaini, Paresh
    Background: Daily low-dose aspirin after 12 weeks of gestation is recommended as a preventive intervention for women at high risk for preeclampsia, a hypertensive disorder of pregnancy with high rates of maternal and fetal mortality and morbidity. Activation of the innate immune system during pregnancy is implicated in the development of preeclampsia. Maternal exposure to synthetic CpG oligonucleotides (CpG ODN, specific ligand of the innate immune receptor Toll-like receptor 9) induces maternal hypertension, vascular dysfunction, and upregulation of cyclooxygenase enzymes in pregnant rats. Hypothesis: We hypothesized that maternal treatment with low-dose aspirin during gestation would ameliorate TLR9-induced hypertension and vascular dysfunction in pregnant rats. Methods: Pregnant Sprague-Dawley rats were treated with a synthetic CpG ODN (ODN2395) or vehicle on gestational day (GD) 14, 16, and 18. Aspirin treatment (or control) started on GD10 and continued throughout gestation for all groups [control (no treatment), ODN2395 (300 μg), aspirin (1.5 mg/kgBW), aspirin+ODN2395]. Blood pressure was measured on GD19 using the tail cuff method and mesenteric resistance artery (MES) function was assessed on GD21 using wire myography. Results: ODN2395-treated rats had higher blood pressure on GD19 compared to vehicle-treated dams and aspirin did not ameliorate ODN2395-induced hypertension (control: 97 ± 0.4 mmHg, ODN2395: 121 ± 7 mmHg, aspirin: 101 ± 5 mmHg, aspirin+ODN2395: 121 ± 7 mmHg, p Aspirin treatment increased MES sensitivity to PE (pEC50, ODN2395: 5.6 ± 0.1 vs. aspirin+ODN2395: 5.9 ± 0.1, ppEC50, ODN2395: 7.6 ± 0.1 vs. aspirin+ODN2395: 7.0 ± 0.1, pConclusion: Treatment with low-dose aspirin throughout gestation did not prevent the development of TLR9-induced maternal hypertension, augmented vascular sensitivity to α1-adrenergic receptor activation and attenuated endothelium-dependent dilation in rats exposed to innate immune system activation. The use of aspirin during gestation should be considered with caution in clinical cases associated with innate immune system-induced pregnancy complications.
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    Effect of Pain on Cardiac Twist Mechanics: A Preliminary Study
    (2016-03-23) Lawrence, Julie; Starwalt, John; Jouett, Noah; Blesson, John; Smith, Michael PhD; Miller, Richard
    Introduction: The sympathetic nervous system (SNS) is known to respond to various stresses that the body encounters. Stimulation of the SNS is associated with increases in heart rate, blood pressure, myocardial contractility and work of the heart. While it is universally accepted that pain causes excitation of the SNS, the relationship between the two is not completely understood due to its complicated nature. Moreover, in patients with impaired pump function, pain may further compromise pump function of the heart, yet this has not been previously investigated. As a preliminary study, we hypothesized that acute pain will increase the demand of the pump, and hence decrease myocardial strain indices, with a resultant increase in myocardial stiffness in healthy subjects. Methods: IRB approval was obtained and each subject gave informed consent according to the Declaration of Helsinki. Each subject (N=3) underwent a standard medical history and physical prior to participation. Subjects each completed two cold pressor stimuli (CPS) by submerging a hand in two different water temperatures (4° C, and 16°) in duplicate for a total of 4 CPS exposures. After the subject’s hand had been submerged for one minute, echocardiographic measurements were recorded. Images taken during this time included a 4-chamber, 2-chamber, and AP long axis view. A period of 20 minutes between each test was allotted to allow the subject’s physiologic variables to return to baseline values. After completion of each study, echocardiographic data, including strain and strain rate was analyzed to calculate cardiac strain indices. Results: It was found that the 4°C CPS was associated with a decreased in cardiac strain (5% change in strain from baseline), correlating with increased chamber stiffness and increased sympathetic stimulation. There was little change in cardiac strain indices during the 16°C CPT trials (2% change in strain from baseline). Conclusions: These preliminary results suggest that perceived acute pain is at least partially responsible for increasing myocardial wall stiffness, as evidenced by cardiac strain indices. This study will be continued to fully address the hypotheses and eventually be implemented in a patient population to determine whether the effects are more significant in a diseased heart.
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    Contractile Differences In Left And Right Ventricles Of Healthy Human Hearts
    (2016-03-23) Nagwekar, Janhavi; Raut, Sangram; Rich, Ryan; Das, Hriday; Gryczysnki, Ignacy; Fudala, Rafal; Gryczysnki, Zygmunt; Blair, Cheavar; Campbell, Kenneth; Borejdo, Julian; Guglin, Maya; Duggal, Divya
    Left ventricle (LV) and right ventricle (RV) differ in embryology, structure and function. Left ventricle originates from the primary heart field while right ventricle originates from the secondary heart field. The two heart fields express different sets of transcription factors and signaling molecules. At the molecular level, the gene expression in response to the pressure loading and failure is different in both ventricles. They also exhibit differences in response to adrenergic stimulation. Adrenergic agonist increase LV contractility but RV contractility may be reduced. Although numerous studies have been performed at macroscopic (whole organ) level previously, none (to my knowledge) address the differences at a mesoscopic level, where only a few molecules are investigated. This becomes important, as in macroscopic studies there may be differences due to basic fiber structures, differences in orientation of fibers as well as molecular crowding. These differences were investigated at the level of few molecules by sparsely labeling the myosin lever arm with SeTau 647 Maleimide. The kinetics and steady-state distribution of cross-bridges were examined in ex-vivo myofibrils isolated from the ventricles of human non-failing and failing hearts and differences were compared in LV and RV. We show that the kinetics and the steady-state distribution of orientations of myosin were different in contracting LV and RV of the non-failing human heart. In contrast, kinetics and the steady-state distribution of myosin in the failing hearts were the same. These results suggest that there is a difference in the way actin interacts with myosin cross-bridges in ventricles of non-failing hearts. We compare the clinical parameters of the failing heart with the kinetics and the distribution of the non-failing heart, and suggest molecular effectors of the function of myosin.
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    Accentuated Antagonism in Cold Induced Sympathetic Activation
    (2016-03-23) Jouett, Noah; Sanchez, Amani; Miller, Richard; Lawrence, Julie; Smith, Michael PhD; Thomas, Sibi; McDaniel, Charles
    Title: Accentuated Antagonism in Cold Induced Sympathetic Activation Introduction: Accentuated antagonism (AA) is a physiological phenomenon where sympathetic nerve activity (SNA) potentiates the action of the vagus nerve on heart rate slowing. This concept has been thoroughly investigated in animals but has not sufficiently been studied in humans. Exploring AA has significant public health relevance because in states when SNA is high, any given activation of the vagus may slow heart rate to a dangerous degree. Therefore, giving cardio-selective sympathetic blocking agents may have significant clinical utility in these settings. Hence, we hypothesize that R-R interval (RRI) will be higher when background SNA is high during 4 degree Celsius cold pressor test (CPT) compared to a room temperature control, and that this difference will be mitigated by giving intravenous metoprolol. Methods 4 healthy human subjects were recruited and informed consent was obtained according to the Declaration of Helsinki. Subjects first underwent baseline -60 mm Hg neck suctions to stimulate vagal nerve-mediated heart rate slowing through the baroreflex. These suctions were repeated after submersion of the hand at wrist level in 4°C water and then 23°C water. These conditions were repeated with infusion of 10 mL saline placebo and up to 10 mg of intravenous metoprolol. We were able to differentiate between the vagal and sympathetic effects on the heart by either infusing saline or blocking the SNA with metoprolol at both temperatures. Results: In comparison of the placebo group with the Metoprolol group in 25°C water, we did not see a significant different in the RR interval (Difference of Means=58.007 milliseconds; P=0.344). In addition we did not see a statistical difference when comparing the RR interval of the placebo group with the Metoprolol group in 4°C water (Difference of Means=72.073 milliseconds; P=0.247). However, there are significant trends within the data that deserve further study. Conclusions: We believe the lack in statistical significance of the data presented is due to the small number of participants in the study. This study warrants further elucidation of the concept of accentuated antagonism in human subjects.
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    Improvement in Blood Pressure and Renal Injury Following Vagal Nerve Stimulation
    (2016-03-23) Fairley, Amber; Mathis, Keisa W.; Pham, Grace; Maloy, Charles
    Purpose: The concept that autoimmunity and its subsequent inflammation could play a role in the pathogenesis of hypertension and its end-organ damage has recently been supported by many studies. Recent studies have proposed a novel pathway, the cholinergic anti-inflammatory pathway, which may combat this inflammation and prevent the subsequent damage. Stimulation of the vagus nerve has been shown to reduce inflammation via this pathway. Systemic Lupus Erythematosus (SLE), an autoimmune disease with a high prevalence of hypertension, provides an appropriate model to test whether stimulation of this pathway can combat chronic inflammation and hypertension. We tested the hypothesis that pharmacological stimulation of the vagus nerve reduces blood pressure and improves renal injury and function in SLE mice (NZBWF1). Methods: Starting at 30 weeks of age, female SLE and control (NZW) mice were treated twice per week over four weeks with CNI­1493 (Semapimod hydrochloride; 8 mg/kg, IP), which has been shown to stimulate the vagus nerve. Saline was used as the vehicle treatment. After treatment, a catheter was placed in the carotid artery to measure mean arterial pressure. Renal blood flow (mL/min*g kidney weight) was measured using a flow probe and renal vascular resistance was calculated (mmHg*min*g kidney weight/mL) as indices of renal function. Results: Mean arterial pressure (mmHg) was higher in SLE mice compared to controls (143±11 vs. 116±4; p=0.003; n=3­ 5/group). Treatment with CNI­1493 attenuated the development of hypertension in the SLE mice (130 ± 4), but did not alter mean arterial pressure in controls (119 ± 5). Renal injury in the form of albuminuria (albumin excretion rate; μg/day) was increased in SLE mice compared to controls (4409±2519 vs. 4 ± 1; p=0.002; n =3­6/group). Albumin excretion rate was diminished in CNI­ 1493­treated SLE mice (420±410; p = 0.008). CNI­1493 ­treated SLE mice had higher renal blood flow (3.0 vs. 2.5±0.1mL/min*g; n=1­ 2/group) and lower renal vascular resistance (21.9 vs. 23.9±1mmHg*min*g/mL; n=1­2/group) compared to SLE mice treated with saline. Conclusion: These data suggest that stimulation of the vagus nerve may protect the kidney and prevent SLE hypertension; however, future studies are warranted to determine how this occurs. Taken together, our findings suggest that the vagally­mediated cholinergic anti­inflammatory pathway plays a mechanistic role in the development hypertension in the setting of chronic inflammation.
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    Can we use Decision Tools to predict Emergent Cardiac Testing and Outcomes among Chest Pain Patients placed in Emergency Department Observation Unit?
    (2016-03-23) Watson, Katherine; Umeijiego, Johnbosco; Hamblin, Layton; Hoang, Steven; Domanski, Kristina; Overstreet, Sterling; Akin, Amanda; Robinson, Richard; Krech, Ryan; Schrader, Chet; Stroder, Ryan; Leuck, JoAnna; Zenarosa, Nestor R.; Wang, Hao
    Chest pain decision tools (HEART, GRACE, and TIMI) have been used to predict the risk of acute coronary syndrome and their major cardiac event outcomes (MACE) in many studies. However, their use to predict emergent cardiac testing is limited in Emergency Medicine Observation Unit (EDOU) patients. This study aims to 1) identify the role of chest pain decision tools in determining emergent cardiac testing; and 2) further validate their use in the prediction of MACE among EDOU chest pain patients. This is a prospective observational study and included patients placed to EDOU due to chest pain. Cardiac testing included any exercise/dobutamine stress echo, exercise/regadenoson nuclear stress test, treadmill, or invasive coronary angiography. Objective stress testing were ordered by EDOU physician and invasive coronary angiography by cardiologists. All providers were blinded and ordered any cardiac testing at their own discretions. HEART, GRACE, and TIMI scores were calculated and categorized as low or above-low risks. Patients followed up at 6 months upon the index discharge. Results of cardiac testing, EDOU length of stay (LOS), and MACE at 6 months were analyzed and compared with different decision tools. ANOVA was used to compare groups with continuous data and Chi square test was used for categorical data. From 01-2014 until 06-2015, 986 total patients were enrolled. Emergent cardiac testing was performed on 62% of patients. Majority of patients placed to EDOU were deemed low risk by any of the decision tools (85% by HEART, 81% by GRACE, and 80% by TIMI) which also had low abnormal cardiac testing results than patients with above-low risks, especially when HEART tool was used. Average LOS was 33h in patients with emergent cardiac testing versus 26h without. In all low risk patients, the occurrence of MACE in 6 month showed no significant difference regardless of whether cardiac testing was done emergently or not (MACE of 0.31% in low risk HEART patients without versus 0.97% with emergent cardiac testing during index admission, 0.27% versus 0.95% in TIMI, and 0% versus 0.81% in GRACE, p [greater than] 0.05). Using chest pain decision tools may minimize the emergent cardiac testing need among EDOU patients with
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    Alteration of Inflammatory Profile following Activation of a Neuroimmune Pathway
    (2016-03-23) Mathis, Keisa W.; Fairley, Amber; Patel, Maitryben
    Abstract Hypertension is estimated to cause 7.5 million deaths worldwide and is a major risk factor for stroke, renal failure, and heart failure, all of which may be potentially fatal. Systemic lupus erythematosus (SLE) is a chronic autoimmune inflammatory disorder with a high prevalence of hypertension. Inflammatory cytokines have been shown to promote the development of hypertension in recent studies. Other studies suggest that acute stimulation of an innate, vagally-mediated, neuro-immune pathway (the cholinergic anti-inflammatory pathway) suppresses the production of inflammatory mediators in the spleen. It is unknown whether chronic stimulation of this pathway is capable of reducing inflammation, and ultimately blood pressure during SLE. Purpose (a): The purpose of this study was to evaluate the effectiveness of stimulation of the cholinergic anti-inflammatory pathway on reducing inflammation in SLE. We hypothesized that pharmacological stimulation of the vagus nerve with CNI-1493 would suppress splenic cytokine release, thereby reducing systemic inflammation. Methods (b): CNI-1493 (8 mg/kg/2swk, IP) or vehicle (saline) was administered twice a week for 4 weeks, starting at 30 weeks of age in female SLE (NZBWF1) and control mice (NZW). At 34 weeks, animals were euthanized and tissues were collected in order to examine splenic and renal inflammation. The spleen and kidney were homogenized using RIPA buffer and the cytokine TNF-a was evaluated using Western blot in these tissues. Results (c): TNF-a expression (normalized to b-actin) was increased in the spleen (36.9±8.7 vs. 22.5±2.8; p=0.02) and renal cortex (8.4±0.4 vs. 3.7±0.5; p=0.03) of SLE mice compared to controls. CNI-1493 had no significant effect on spleen TNF (35.9±4.3), however exacerbated renal cortical TNF (12.5±2.2) in SLE mice. Conclusions (d): Although the results did not support our hypothesis, the data are preliminary and we suspect additional studies are needed to determine the ability of pharmacological stimulation of the vagus nerve to reduce inflammation in SLE mice. Such therapy could potentially benefit hypertensive SLE patients, since chronic inflammation has recently been linked to the development of the disease.
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    Changes In TRPV4 Channel Function In Vasopressin Neurons Of Rats With Hepatic Cirrhosis
    (2016-03-23) Knapp, Blayne; Saxena, Ashwini; Bachelor, Martha; Cunningham, Joseph; Cunningham, Thomas II
    Purpose: Dilutional hyponatremia associated with cardiac and hepatic failure negatively impacts morbidity and mortality of both diseases. Hyponatremia is a consequence of the dysregulation of vasopressin release but is not completely understood. In an animal model of liver failure, the membrane expression of transient receptor potential vanilloid 4 (TRPV4) channels increased in vasopressin-releasing neurons of the hypothalamus. Our hypothesis was that activation of TRPV4 channels with the specific agonist GSK 1019790A produces greater calcium influx in supraoptic nucleus (SON) vasopressin neurons collected from hyponatremic rats with liver failure induced by bile duct ligation surgery or to sham operated controls. Methods: Adult male Sprague-Dawley rats received bile duct ligation surgery or sham ligation surgery. After two weeks, all rats were anesthetized and injected in the SON with an adeno-associated viral 2 (AAV2) vector containing a construct for green fluorescent protein (GFP) driven by a vasopressin-specific promoter. Two weeks later, the rats were anesthetized and the SONs isolated and cultured for Fura-2 ratiometric calcium imaging. Initial experiments had the cultured cells sit overnight prior to imaging; subsequent experiments imaged cells on the same day as their isolation and culturing. SON cells were tested for changes in intracellular calcium produced by the specific TRPV4 agonist GSK 1019790A (20-200 nM). Cells were also tested for its response to a calcium ionophore. In some experiments, data were collected GFP positive (vasopressin) cells. Normalized ratio responses were tested by ANOVA. Results: In these studies using cells cultured for 24-48 hours, 1.5 nM GSK significantly increased calcium influx in GFP-positive cells from sham rats but not from BDL rats (Sham 1.53 ± 0.1, BDL 1.14 ± 0.1, P Conclusion: Our results demonstrate that hyponatremia produces gain in function changes consistent with increased TRPV4 expression. However, these results were strongly influenced by how long the cells were kept in culture.
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    Uterine Perivascular Adipose Tissue Potentiates Contractile Responses in Uterine Arteries from Pregnant rats
    (2016-03-23) Goulopoulou, Styliani PhD; Osikoya, Oluwatobiloba
    Background: Perivascular adipose tissue (PVAT) is the fourth and outer layer of the vascular wall. PVAT has vasoactive effects, mostly via paracrine actions. The effects of PVAT vary with anatomic location; PVAT has anti-contractile effects in peripheral vascular beds in animals and in humans but it potentiates contractions in coronary vascular smooth muscle. Pregnancy is characterized by adipose tissue expansion as well as structural and functional changes in the uterine vasculature. However, the effects of PVAT on uterine artery reactivity during pregnancy are not understood. Hypothesis: We hypothesized that uterine PVAT has a functional role in uterine artery contractile and dilatory responses and this role is modified by pregnancy. Methods: Pregnant Sprague-Dawley rats were sacrificed on gestational day 16 (term=21-22 days). Uterine arteries and their surrounding PVAT were harvested and cleaned for study. Concentration response curves (CRCs) to potassium chloride (KCl, 4.7 – 80 mM) and phenylephrine (PE, 10-9 - 3x10-5 M) were performed using wire myography. CRCs were performed in the presence and absence of the surrounding PVAT (0.1 g) or PVAT-conditioned media. Arteries were incubated with PVAT or PVAT-conditioned media for 30 minutes. To make the media, we incubated 0.4 g of PVAT in 15 ml physiological salt solution for 90 min (37oC - 5% CO2, 95% O2). Results: Uterine arteries incubated with PVAT (+PVAT) had greater contractile responses to KCl compared to control vessels [KCl (30 mM), control: 4.0 ± 0.81 mN vs. +PVAT: 14.7 ± 1.68 mN; KCl (40 mM), control: 14.4 ± 0.68 mN vs. +PVAT: 19.6 ± 0.88 mN, p50, control: 6.0 ± 0.08 vs. +PVAT: 6.3 ± 0.10, p50), control: 6.1 ± 0.08 vs. +PVAT-media: 6.4 ± 0.10, p=0.07]. Conclusions: Our data show that uterine PVAT has a pro-contractile effect on uterine arteries from pregnant rats. We propose that uterine PVAT provides signaling from the outer layer to the inner layers of the vascular wall that determines uteroplacental vascular adaptations to pregnancy.
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    Changes in Gut Hormones in Relation to Cardiovascular Risk Factors One Year After Laparoscopic Gastric Banding Surgery
    (2016-03-23) Jagota, Devina; Franks, Susan; Tijmes, Steven
    Title: Changes in Gut Hormones in Relation to Cardiovascular Risk Factors One Year After Laparoscopic Gastric Banding Surgery Purpose: Obesity rates in the United States continue to rise at an alarming rate, imposing increasingly large health burdens such as cardiovascular disease and diabetes among others. Existing research on gastric bypass surgery has shown that improved gut hormone levels post-surgery are partially responsible for drastic improvements in cardiovascular health. However, similar research studying this effect in laparoscopic gastric banding surgery (LBS) is lacking. The purpose of the current study is to determine whether LBS delivers normalization of gut hormones, specifically ghrelin and GLP-1 and whether that normalization could explain improvements in cardiovascular (CV) indicators of health. Methods: A secondary data analysis was conducted on data from a 1-year prospective study in a community bariatric surgical setting. Bariatric patients who completed baseline (T1) and 12-month post-LBS were included in this analysis (n=29). There were 22 women and 7 men. The average age was 45 years old (sd =11.6) and mean Body Mass Index was 42 kg/m2 (sd =5). Subjects were assessed at before surgery (T1) and 12 months post-surgery (T3) for fasting and postprandial levels of active ghrelin and glucagon like peptide (GLP-1) as well as for indicators of cardiovascular and metabolic health including high density lipoprotein (HDL), lipoprotein (a) (Lipo-a), apolipoprotein A1 (Apo-A1), apolipoprotein B (ApoB), and plasma renin activity (PRA). CV indicators were analyzed at a commercial laboratory. Ghrelin, GLP-1, and PRA were analyzed in-house. Central adiposity at L4L5 was assessed using CT scan. Change scores between T1 and T3 were calculated. Partial correlation was used to examine relationships between change scores in CV indicators with ghrelin and GLP-1, controlling for change in L4L5VAT. Results: The partial correlations of change scores showed that increased levels of postprandial GLP-1 correlated with a reduction in Lipo-a plasma levels (r=-.752, p=.012). The results also showed that increased levels in fasting GLP-1 correlated with a decrease in levels of ApoB in plasma (r=-.686, p=.028). Conclusion: Improvements in secretion patterns 12 months after LBS appears to have a significant relationship to changes in CV risk factors such as Lipoprotein (a) and ApoB. Increased GLP-1 levels appear particularly related to plasma levels of lipoprotein (a) as well as ApoB which could be an indicator of GLP-1 playing an important role in lipid metabolism.
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    Cytoprotective and Anti-Glycation Defenses in Porcine Brain after Cardiac Arrest and Cardiocerebral Resuscitation
    (2016-03-23) Ryou, Myoung-Gwi; Hollrah, Roger; Williams, Jr., Arthur; Scott, Gary; Olivencia-Yurvati, Albert; Mallet, Robert T.; Nguyen, Anh; Nguyen, Anh
    Cardiac arrest (CA) is often lethal, and survivors often face sequelae that greatly impact quality of life due to brain injury inflicted by ischemia-reperfusion. Effective cardiocerebral resuscitation (CCR) is essential for survival and recovery from CA. The complexity of the injury cascades ignited by CA and the presence of the blood-brain barrier challenge the development of pharmacological interventions to protect the brain. Our goal is to identify a blood-brain barrier-permeable intervention that mitigates CA-induced brain damage and, thus, fosters neurological recovery. Pyruvate, a cellular metabolite, energy substrate and antioxidant, has been found to be neuroprotective in a rat stroke model via induction of the cytoprotective cytokine erythropoietin (EPO). Hypothesis: Pyruvate treatment during CCR and post resuscitation decreases brain injury by upregulating cellular defense mechanisms including hypoxia inducible factor-1α (HIF-1α), EPO, the product of HIF-1 α’s gene program, and glyoxalase-1, the principal component of the brain’s defenses against the powerful glycating agent and glycolytic byproduct, methylglyoxal. Methods: Yorkshire swine (25-35 kg, both genders) were subjected to sham protocol (n = 6) or pacing-induced CA (n = 12). CCR was administered by precordial chest compressions (100/min) at 10-14 min CA, and transthoracic countershocks were applied to restore sinus rhythm. NaCl (n = 6) or Na-pyruvate (n = 6) was infused iv (0.1 mmol/kg/min) during CCR and the first 60 min recovery. At 4 h recovery, brain was cross-perfused with 0.9% NaCl, and then brain biopsies were freeze-clamped for biochemical analysis or fixed in 10% formalin for immunohistochemistry. Results: In hippocampus, activity of the oxyradical-sensitive TCA cycle enzyme aconitase fell by 50% (P
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    A Vital role for Median Preoptic AT1a Receptors in the Sustained Hypertension of Chronic Intermittent Hypoxia
    (2016-03-23) Cunningham, J. Thomas; Shell, Brent
    Purpose The hypoxemia from Sleep Apnea (SA) results in hypertension during both the hypoxic sleeping period and the normoxic waking period. This pathophysiological sustained hypertension persists during waking hours and is a source of numerous cardiovascular sequlae. In order to better understand the neurological changes that underlie this disease state, our lab utilizes Chronic Intermittent Hypoxia (CIH) to model the hypoxemia and generate the hypertension of SA sufferers. Previously, our lab has shown that the Median Preoptic Nucleus increases in both neuronal activity and Angiotensin Type 1a Receptor (AT1aR) RNA expression in response to CIH. The MnPO is situated in a critical location that allows it to receive inputs from nuclei outside of the blood brain barrier and provide inputs to regions that control sympathetic outflow, and therefore blood pressure. The ability to modulate sympathetics based upon peripheral inputs coupled with increased activity and AT1aR expression leads us to hypothesize that increased Angiotensin signaling to the MnPO is essential for the sustained component of hypertension from CIH. Methods Male Sprague-Dawley rats were microinjected in the MnPO with either a virus to knockdown AT1aR expression (shAT1a) or a scramble virus (SCR) and instrumented with radio telemetry a week later. Radio telemetry provides continuous recording of cardiovascular variables. After a week of surgery recovery the animals were monitored for a 5 day baseline period before experiencing 7 days of CIH. The morning of the final day, the animals were either perfused with formaldehyde for immunohistochemistry (IHC) or their brains were snap frozen for quantitative PCR. Results Rodents injected in the MnPO with the shAT1a did not exhibit the sustained component of hypertension compared to SCR animals (P Conclusions Overall, this data indicates that the increase in AT1aR expression in the MnPO is essential for the development of the sustained component of CIH hypertension. The shAT1a virus successfully prevents the increase in AT1aR expression and this leads to normal waking blood pressure in the CIH exposed rodents. IHC results indicate that there is less activity in the MnPO and RVLM of the rodents most likely related to reduced sympathetic outflow. This data lends support to optimizing our current treatment regiment through blood brain barrier AT1aR blockers and ACE inhibitors. Future treatment methods could focus solely on preventing Angiotensin as a peptide neurotransmitter in the MnPO to ameliorate neurogenic hypertension.
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    Homer Mediates Vascular Store-Operated CA2+ Entry and is Required for Neointima Formation after Vascular Injury
    (2016-03-23) Jia, Shuping; Wu, Qiong; Williams, Arthur Jr.; Little, Joel; Cunningham, Joseph; Mifflin, Steve; Ma, Rong; Yuan, Joseph; Rodriguez, Miguel
    Occlusive arterial disease (OAD) refers to the pathological obstruction of arteries that become progressively narrowed over time and are eventually blocked due to various risk factors, such as hypertension, diabetes, and atherosclerosis. This chronic arterial damage results from vascular wall remodeling, leading to neointima formation. Store-operated Ca2+ channels (SOCs) and entry (SOCE) play a central role in the vascular smooth muscle cell (VSMC) phenotypic change from contractile to migratory and proliferative states. In the present work, we ask if Homer is a critical molecular component of VSMC SOCE and does Homer mediate VSMC migration/proliferation and neointima formation. Homer binds to transient receptor potential canonical (TRPC) channels and is required for gating of TRPCs, while stromal interacting molecule1 (STIM1) binds to and regulates TRPC and Orai channels as SOCs. We cultured rat aortic VSMCs to increase their SOCE and migration/proliferation, as seen in OAD. Studies were done using small-interfering RNA (siRNA) targeting Homer1, STIM1, and TRPCs. Scratch wound migration assays were performed, and VSMC proliferation was assessed by cell count. In our in vivo OAD model (rat carotid artery balloon injury), the arteries were treated with adeno-associated virus (AAV) encoding short-hairpin RNA (shRNA) targeting Homer1. We found that Homer1 protein expression levels increase in balloon-injured vs. intact VSMCs, similar to known increases in protein expression levels of STIM1, Orai1, and TRPCs. Furthermore, we show that Homer1 binds to Orai1 and that interactions between Homer1 & Orai1/TRPCs and between STIM1 & Orai1/TRPCs markedly increase in injured vs. intact VSMCs. Cultured VSMCs treated with siHomer1 exhibit significant reduction in SOCE (56 ± 4.0%) vs. control (scrambled siRNA), similar to the SOCE reduction seen in siSTIM1-/siTRPC-treated cells. SiHomer1-treated cells also migrate significantly less over the wound surface area (73.3 ± 5.9%), and proliferate significantly less (73.3 ± 4.2%) vs. control, similar to observations seen in siSTIM1-/siTRPC-treated cells. Finally, immunofluorescence staining shows that the increased Homer1, STIM1, and Orai1 protein expression levels are localized in the neointima of the injured carotid artery. Knockdown of Homer1 using AAV-shHomer1 reduces this neointima. These studies provide evidence that Homer is a critical component of VSMC SOCE and neointima formation.
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    Differences in the Kinetics and Spatial Distribution of Actin in the Left and Right Ventricles of Human Hearts
    (2016-03-23) Duggal, Divya; Rich, Ryan; Fudala, Rafal; Gryczynski, Ignacy ; Raut, Sangram; Borejdo, Julian; Guglin, Maya; Campbell, Kenneth; Nagwekar, Janhavi
    The left and right ventricles (LV, RV) are morphologically and physiologically different because they are play vastly different roles in the human circulatory system: the RV pumps blood into the pulmonary system and LV into the systemic systems, both of which offer different resistances to contracting ventricles. It is therefore not surprising that large differences are seen when ventricles are examined on a macroscopic level. This does not mean, however, that the ventricles are different on a molecular level, i.e. that the contractile proteins that are responsible for the ventricular contraction are different in the LV and RV. If this is true it should be possible to develop drugs affecting only one, not both, ventricles. Such drugs are currently not available, and would be of clinical importance because there exist a[J1] number of heart diseases caused by dysfunction of one ventricle only. For example, pulmonary hypertension is mainly due to RV dysfunction. Systolic or diastolic heart failure is mainly due to LV dysfunction. Because of these macroscopic differences, experiments can’t be carried out on the whole organs. Moreover, the whole ventricles (or papillary muscles) contain a large number of contractile molecules (1011-1013). Measurements which originate from such a large assembly yield the average values and all the kinetic information about molecular action is absent from macroscopic data. Similarly, all the details of the steady-state measurements are lost. Therefore, the experiments need to be carried out on a few molecules of contractile proteins: we observed the A-band of a sarcomere - a volume where a force producing interaction between actin and myosin molecules takes place. We measured kinetics and distribution of ~16 actin molecules of isometrically contracting A-bands. Experiments were done ex-vivo – in the A-bands of myofibrils isolated from human non-failing and failing ventricles. We show that the kinetics and the steady-state distribution of actin were different in contracting left and right ventricle of the non-failing human heart. In contrast, these parameters obtained from failing hearts were the same. These results suggest that there exists genuine differences in the way actin interacts with myosin cross-bridges in both ventricles of non-failing hearts, and suggests how the absence of such differences in failing ventricles can be offset.