Browsing by Author "Sturek, Michael Stephen"
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Item Contribution of Perivascular Adipose Tissue to Coronary Vascular Dysfunction(2011-03-10) Payne, Gregory Allen; Tune, Johnathan D.; Bohlen, H. Glenn; Considine, Robert V.; Sturek, Michael StephenThe epidemic of obesity and associated cardiovascular complications continues to grow at an alarming rate. Currently, obesity is thought to initiate a state of chronic inflammation, which if unresolved potentially causes cardiovascular dysfunction and disease. Although poorly understood, release of inflammatory mediators and other cytokines from adipose tissue (adipocytokines) has been proposed to be the molecular link between obesity and coronary artery disease. Furthermore, the anatomic location of adipose has been increasingly recognized as a potential contributor to vascular disease. Importantly, the development of coronary atherosclerosis, a key component of heart disease, is typically found in segments of coronary arteries surrounded by perivascular adipose tissue. Accordingly, the goal of this project was to determine how perivascular adipose tissue affects coronary artery function and elucidate the critical mechanisms involved. Initial studies assessing arterial function were conducted with and without perivascular adipose tissue. Preliminary results demonstrated that factors released by perivascular adipose tissue effectively impaired coronary endothelial function both in vitro and in vivo. This observation was determined to be caused by direct inhibition of nitric oxide synthase (NOS), a critical enzyme for the production nitric oxide. Attenuation of endothelium-dependent vasodilation was independent of changes in superoxide production, smooth muscle response, or peroxide-mediated vasodilation. Additional studies revealed that perivascular adipose-induced impairment of NOS was due to increased inhibitory regulation by the β isoform of protein kinase C (PKC-β). Specifically, perivascular adipose-derived factors caused site specific phosphorylation of nitric oxide synthase at Thr-495. Additional experiments investigated how perivascular adipose-derived factors contributed to coronary artery disease in an animal model of obesity. Results from these studies indicated that perivascular adipose-derived leptin markedly exacerbated underlying endothelial dysfunction, and significantly contributed to coronary endothelial dysfunction through a PKC-β dependent mechanism. Findings from this project confirm epicardial perivascular adipose tissue as a local source of harmful adipocytokines. In addition, perivascular adipose-derived leptin was demonstrated to be a critical mediator of coronary vascular dysfunction in obesity. Together, the results strongly suggest that perivascular adipose tissue is a key contributor to coronary artery disease in obesity.Item Coronary artery disease progression and calcification in metabolic syndrome(2014) McKenney, Mikaela Lee; Sturek, Michael Stephen; Evans-Molina, Carmella; Moe, Sharon M.; Tune, Johnathan D.For years, the leading killer of Americans has been coronary artery disease (CAD), which has a strong correlation to the U.S. obesity epidemic. Obesity, along with the presence of other risk factors including hyperglycemia, hypercholesterolemia, dyslipidemia, and high blood pressure, comprise of the diagnosis of metabolic syndrome (MetS). The presentation of multiple MetS risk factors increases a patients risk for adverse cardiovascular events. CAD is a complex progressive disease. We utilized the superb model of CAD and MetS, the Ossabaw miniature swine, to investigate underlying mechanisms of CAD progression. We studied the influence of coronary epicardial adipose tissue (cEAT) and coronary smooth muscle cell (CSM) intracellular Ca2+ regulation on CAD progression. By surgical excision of cEAT from MetS Ossabaw, we observed an attenuation of CAD progression. This finding provides evidence for a link between local cEAT and CAD progression. Intracellular Ca2+ is a tightly regulated messenger in CSM that initiates contraction, translation, proliferation and migration. When regulation is lost, CSM dedifferentiate from their mature, contractile phenotype found in the healthy vascular wall to a synthetic, proliferative phenotype. Synthetic CSM are found in intimal plaque of CAD patients. We investigated the changes in intracellular Ca2+ signaling in enzymatically isolated CSM from Ossabaw swine with varying stages of CAD using the fluorescent Ca2+ indicator, fura-2. This time course study revealed heightened Ca2+ signaling in early CAD followed by a significant drop off in late stage calcified plaque. Coronary artery calcification (CAC) is a result of dedifferentiation into an osteogenic CSM that secretes hydroxyapatite in the extracellular matrix. CAC is clinically detected by computed tomography (CT). Microcalcifications have been linked to plaque instability/rupture and cannot be detected by CT. We used 18F-NaF positron emission tomography (PET) to detect CAC in Ossabaw swine with early stage CAD shown by mild neointimal thickening. This study validated 18F-NaF PET as a diagnostic tool for early, molecular CAC at a stage prior to lesions detectable by CT. This is the first report showing non-invasive PET resolution of CAC and CSMC Ca2+ dysfunction at an early stage previously only characterized by invasive cellular Ca2+ imaging.Item Diet-induced dyslipidemia drives store-operated Ca2+ entry, Ca2+ dysregulation, non-alcoholic steatohepatitis, and coronary atherogenesis in metabolic syndrome(2010-07-21T20:06:24Z) Neeb, Zachary P.; Sturek, Michael Stephen; Breall, Jeffrey A.; Considine, Robert V.; Obukhov, Alexander; Tune, Johnathan D.Risk of coronary artery disease (CAD), the leading cause of death, greatly increases in metabolic syndrome. Metabolic syndrome (MetS; obesity, insulin resistance, glucose intolerance, dyslipidemia, and hypertension) is increasing in prevalence with sedentary lifestyles and poor nutrition. Non-alcoholic steatohepatitis (NASH; i.e. MetS liver) is progressive and decreases life expectancy, with CAD as the leading cause of death. Pathogenic Ca2+ regulation transforms coronary artery smooth muscle from a healthy, quiescent state to a diseased, proliferative phenotype thus majorly contributing to the development of CAD. In particular, store-operated Ca2+ entry (SOCE) in vascular smooth muscle is associated with atherosclerosis. Genetic predisposition may render individuals more susceptible to Ca2+ dysregulation, CAD, NASH, and MetS. However, the metabolic and cellular mechanisms underlying these disease states are poorly understood. Accordingly, the goal of this dissertation was to investigate the role of dyslipidemia within MetS in the development of Ca2+ dysregulation, CAD, and NASH. The overarching hypothesis was that dyslipidemia within MetS would be necessary for induction of NASH and increased SOCE that would primarily mediate development of CAD. To test this hypothesis we utilized the Ossabaw miniature swine model of MetS. Swine were fed one of five diets for different lengths of time to induce varying severity of MetS. Lean swine were fed normal maintenance chow diet. F/MetS swine were fed high Fructose (20% kcal) diet that induced normolipidemic MetS. TMetS were fed excess high Trans-fat/cholesterol atherogenic diet that induced mildly dyslipidemic MetS and CAD. XMetS were TMetS swine with eXercise. DMetS (TMetS + high fructose) were moderately dyslipidemic and developed MetS and extensive CAD. sDMetS (Short-term DMetS) developed MetS with mild dyslipidemia, but no CAD. MMetS (Mixed-source-fat/cholesterol/fructose) were severely dyslipidemic, exhibited NASH, and developed severe CAD. Dyslipidemia in MetS predicted NASH severity (all groups < DMetS << MMetS), CAD severity (i.e. Lean, F/MetS, sDMetS < XMetS < TMetS < DMetS < MMetS), and was necessary for STIM1/TRPC1-mediated SOCE, which preceded CAD. Exercise ameliorated SOCE and CAD compared to TMetS. In conclusion, dyslipidemia elicits TRPC1/STIM1 SOCE that mediates CAD, is necessary for and predictive of NASH and CAD, and whose affects are attenuated by exercise.Item Effect of coronary perivascular adipose tissue on vascular smooth muscle function in metabolic syndrome(2013-12-19) Owen, Meredith Kohr; Tune, Johnathan D.; Considine, Robert V.; March, Keith Leonard, 1963-; Sturek, Michael Stephen; Witzmann, F. A. (Frank A.)Obesity increases cardiovascular disease risk and is associated with factors of the “metabolic syndrome” (MetS), a disorder including hypertension, hypercholesterolemia and/or impaired glucose tolerance. Expanding adipose and subsequent inflammation is implicated in vascular dysfunction in MetS. Perivascular adipose tissue (PVAT) surrounds virtually every artery and is capable of releasing factors that influence vascular reactivity, but the effects of PVAT in the coronary circulation are unknown. Accordingly, the goal of this investigation was to delineate mechanisms by which lean vs. MetS coronary PVAT influences vasomotor tone and the coronary PVAT proteome. We tested the hypothesis that MetS alters the functional expression and vascular contractile effects of coronary PVAT in an Ossabaw swine model of the MetS. Utilizing isometric tension measurements of coronary arteries in the absence and presence of PVAT, we revealed the vascular effects of PVAT vary according to anatomical location as coronary and mesenteric, but not subcutaneous adipose tissue augmented coronary artery contractions to KCl. Factors released from coronary PVAT increase baseline tension and potentiate constriction of isolated coronary arteries relative to the amount of adipose tissue present. The effects of coronary PVAT are elevated in the setting of MetS and occur independent of endothelial function. MetS is also associated with substantial alterations in the coronary PVAT proteome and underlying increases in vascular smooth muscle Ca2+ handling via CaV1.2 channels, H2O2-sensitive K+ channels and/or upstream mediators of these ion channels. Rho-kinase signaling participates in the increase in coronary artery contractions to PVAT in lean, but not MetS swine. These data provide novel evidence that the vascular effects of PVAT vary according to anatomic location and are influenced by the MetS phenotype.Item The Effect of Omega-3 Fatty Acids on Airway Inflammation, Hyperpnea-Induced Bronchoconstriction, and Airway Smooth Muscle Contractility in Asthma(2012-03-16) Head, Sally K.; Mickleborough, Timothy D.; Gunst, Susan J.; Harrington, Maureen A.; Sturek, Michael Stephen; Tepper, Robert S.; Tune, Johnathan D.Asthma, a chronic inflammatory disease of the airways, affects nearly 25 million Americans. The vast majority of these patients suffer from exercise-induced bronchoconstriction (EIB), a complication of asthma. Although traditionally treated pharmacologically, nutritional strategies provide a promising alternative for managing EIB as the prevalence of asthma may be due in part to changes in diet. Our objective was to determine the effects of novel nutritional strategies on hyperpnea-induced bronchoconstriction (HIB) in asthmatic individuals. HIB uses rapid breathing to identify EIB in a research or clinical setting. Fish oil, a combination of the omega-3 fatty acids eicosapentaenoic acid (EPA) and docsahexaenoic acid (DHA), has been shown to be effective in suppressing EIB. However, its use in combination with other nutritional supplements, the optimal fish oil formula, and its effect on smooth muscle contractility have not been fully explored. An in vivo study (study 1) was conducted in individuals with both asthma and HIB to determine whether a combination of fish oil and vitamin C was more effective than either one alone in alleviating HIB. Pulmonary function was significantly improved with both fish oil and the combination treatment but not with vitamin C alone. In study 2, individuals with both asthma and HIB were supplemented with DHA alone since the optimal formula for fish oil has yet to be ascertained; previous in vitro studies have suggested DHA may be the more potent omega-3 fatty acid in fish oil. However, no significant changes in pulmonary function or airway inflammation were seen with DHA supplementation. For study 3, canine airway smooth muscle tissue was treated with fish oil to determine the in vitro effect of fish oil on smooth muscle contractility. Acute treatment with fish oil relaxed smooth muscle strips that had been contracted with 5-hydroxytryptamine. These minor relaxations in smooth muscle tension with fish oil may represent significant changes at the level of the smaller airways. These studies have confirmed that fish oil represents a viable treatment modality for asthmatic individuals with EIB and suggest that fish oil may influence airway smooth muscle contractility.Item Impaired cardiovascular responses to glucagon-like peptide 1 in metabolic syndrome and type 2 diabetes mellitus(2013-01-30) Moberly, Steven Paul; Tune, Johnathan D.; Mather, Kieren J.; Elmendorf, Jeffrey S.; Considine, Robert V.; Sturek, Michael StephenRecent advancements in the management of systemic glucose regulation in obesity/T2DM include drug therapies designed to utilize components of the incretin system specifically related to glucagon-like peptide 1 (GLP-1). More recently, GLP-1 has been investigated for potential cardioprotective effects. Several investigations have revealed that acute/sub-acute intravenous administration of GLP-1 significantly reduces myocardial infarct size following ischemia/reperfusion injury and improves cardiac contractile function in the settings of coronary artery disease, myocardial ischemia/reperfusion injury, and heart failure. Despite an abundance of data indicating that intravenous infusion of GLP-1 is cardioprotective, information has been lacking on the cardiac effects of iv GLP-1 in the MetS or T2DM population. Some important questions this study aimed to address are 1) what are the direct, dose-dependent cardiac effects of GLP-1 in-vivo 2) are the cardiac effects influenced by cardiac demand (MVO2) and/or ischemia, 3) does GLP-1 effect myocardial blood flow, glucose uptake or total oxidative metabolism in human subjects, and 4) are the cardiac effects of GLP-1 treatment impaired in the settings of obesity/MetS and T2DM. Initial studies conducted in canines demonstrated that GLP-1 had no direct effect on coronary blood flow in-vivo or vasomotor tone in-vitro, but preferentially increased myocardial glucose uptake in ischemic myocardium independent of effects on cardiac contractile function or coronary blood flow. Parallel translational studies conducted in the humans and Ossabaw swine demonstrate that iv GLP-1 significantly increases myocardial glucose uptake at rest and in response to increases in cardiac demand (MVO2) in lean subjects, but not in the settings of obesity/MetS and T2DM. Further investigation in isolated cardiac tissue from lean and obese/MetS swine indicate that this impairment in GLP-1 responsiveness is related to attenuated activation of p38-MAPK, independent of alterations in GLP-1 receptor expression or PKA-dependent signaling. Our results indicate that the affects of GLP-1 to reduce cardiac damage and increase left ventricular performance may be impaired by obesity/MetS and T2DM.Item Novel Roles of p21 in Apoptosis During Beta-Cell Stress in Diabetes(2014) Hernández-Carretero, Angelina M.; Fueger, Patrick T.; Sturek, Michael Stephen; Wek, Ronald C.; Evans-Molina, Carmella; Elmendorf, Jeffrey S.Type 2 diabetes manifests from peripheral insulin resistance and a loss of functional beta cell mass due to decreased beta cell function, survival, and/or proliferation. Beta cell stressors impair each of these factors by activating stress response mechanisms, including endoplasmic reticulum (ER) stress. The glucolipotoxic environment of the diabetic milieu also activates a stress response in beta cells, resulting in death and decreased survival. Whereas the cell cycle machinery (comprised of cyclins, kinases, and inhibitors) regulates proliferation, its involvement during beta cell stress in the development of diabetes is not well understood. Interestingly, in a screen of multiple cell cycle inhibitors, p21 was dramatically upregulated in INS-1-derived 832/13 cells and rodent islets by two independent pharmacologic inducers of beta cell stress - dexamethasone and thapsigargin. In addition, glucolipotoxic stress mimicking the diabetic milieu also induced p21. To further investigate p21’s role in the beta cell, p21 was adenovirally overexpressed in 832/13 cells and rat islets. As expected given p21’s role as a cell cycle inhibitor, p21 overexpression decreased [3H]-thymidine incorporation and blocked the G1/S and G2/M transitions as quantified by flow cytometry. Interestingly, p21 overexpression activated apoptosis, demonstrated by increased annexin- and propidium iodide-double-positive cells and cleaved caspase-3 protein. p21-mediated caspase-3 cleavage was inhibited by either overexpression of the anti-apoptotic mitochondrial protein Bcl-2 or siRNA-mediated suppression of the pro-apoptotic proteins Bax and Bak. Therefore, the intrinsic apoptotic pathway is central for p21-mediated cell death. Like glucolipotoxicity, p21 overexpression inhibited the insulin cell survival signaling pathway while also impairing glucose-stimulated insulin secretion, an index of beta cell function. Under both conditions, phosphorylation of insulin receptor substrate-1, Akt, and Forkhead box protein-O1 was reduced. p21 overexpression increased Bim and c-Jun N-terminal Kinase, however, siRNA-mediated reduction or inhibition of either protein, respectively, did not alter p21-mediated cell death. Importantly, islets of p21-knockout mice treated with the ER stress inducer thapsigargin displayed a blunted apoptotic response. In summary, our findings indicate that p21 decreases proliferation, activates apoptosis, and impairs beta cell function, thus being a potential target to inhibit for the protection of functional beta cell mass.Item Role of Adenosine A1 Receptors in Native Coronary Atherosclerosis, In-stent Stenosis, and Coronary Blood Flow Regulation in Metabolic Syndrome and Exercise(2010-04-08T13:47:08Z) Long, Xin; Sturek, Michael Stephen; Considine, Robert V.; Gunst, Susan J.; Herring, B. Paul; Tune, Johnathan D.Adenosine is widely thought to elicit coronary vasodilation and attenuate smooth muscle cell (SMC) proliferation, thereby providing cardioprotection. We cloned the porcine adenosine A1 receptor (A1R) subtype and found that it paradoxically stimulated proliferation of cultured coronary SMC by the extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) signaling pathways, thus suggesting A1R dysregulation could play a role in coronary artery disease (CAD), restenosis, and regulation of coronary blood flow (CBF). We utilized the Ossabaw swine model of metabolic syndrome (MetS) to test the hypothesis that A1R activation contributes to development of CAD, in-stent stenosis, and CBF regulation. Swine were fed standard chow (Lean) or excess calorie atherogenic diet for over 20 weeks, which elicited MetS characteristics and coronary atherosclerosis compared to Lean. We observed increased A1R in native CAD in MetS, which was reversed by exercise training, and upregulation of A1R expression and A1R-ERK1/2 activation in an in vitro organ culture model of CAD. Intracoronary stent deployment followed by different durations of recovery showed A1R upregulation occurred before maximal in-stent stenosis in vi vivo. More importantly, selective A1R antagonism with 8-cyclopentyl-1, 3-dipropylxanthine (DPCPX)-eluting stents decreased coronary ERK1/2 activation and reduced in-stent stenosis comparable to Taxus® (paclitaxel-eluting stents). A1R antagonism potentiated vasodilatory effects of some vasodilators other than adenosine in porcine coronary microcirculation under basal conditions. Short-term exercise training around stenting prevented stent-induced microvascular dysfunction and attenuated native atheroma in the genetically lean Yucatan swine. Conclusions: A1R upregulation and activation contributes to coronary in-stent stenosis in vivo in MetS, plays a role in the development of coronary atherosclerosis in vitro, and might involve in CBF dysregulation in dyslipidemia and stenting. Exercise training decreased A1R expression in atherosclerosis, reduced native atheroma, and prevented stent-induced microvascular dysfunction. Selective pharmacological antagonism of A1R holds promise for treatment of CAD.Item Role of Voltage-Dependent K+ and Ca2+ Channels in Coronary Electromechanical Coupling: Effects of Metabolic Syndrome(2012-10-19) Berwick, Zachary C.; Tune, Johnathan D.; Basile, David P.; Mather, Kieren J.; Obukhov, Alexander G.; Sturek, Michael StephenRegulation of coronary blood flow is a highly dynamic process that maintains the delicate balance between oxygen delivery and metabolism in order to preserve cardiac function. Evidence to date support the finding that Kv and Cav1.2 channels are critical end-effectors in modulating vasomotor tone and blood flow. Yet the role for these channels in the coronary circulation in addition to their interdependent relationship remains largely unknown. Importantly, there is a growing body of evidence that suggests obesity and its pathologic components, i.e. metabolic syndrome (MetS), may alter coronary ion channel function. Accordingly, the overall goal of this investigation was to examine the contribution coronary Kv and Cav1.2 channels to the control of coronary blood flow in response to various physiologic conditions. Findings from this study also evaluated the potential for interaction between these channels, i.e. electromechanical coupling, and the impact obesity/MetS has on this mechanism. Using a highly integrative experimental approach, results from this investigation indicate Kv and Cav1.2 channels significantly contribute to the control of coronary blood flow in response to alterations in coronary perfusion pressure, cardiac ischemia, and during increases in myocardial metabolism. In addition, we have identified that impaired functional expression and electromechanical coupling of Kv and Cav1.2 channels represents a critical mechanism underlying coronary dysfunction in the metabolic syndrome. Thus, findings from this investigation provide novel mechanistic insight into the patho-physiologic regulation of Kv and Cav1.2 channels and significantly improve our understanding of obesity-related cardiovascular disease.