Cellular & Integrative Physiology Department Theses and Dissertations

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Noninvasive monitoringn of CCl4 induced acute and chronic liver damage in rat by single quantum and triple quantum filtered 23Na magnetic resonance imaging
(2008) Gao, Yong; Bansal, Navin; Babsky, Andriy M.; Kempson, Stephen A.; Basile, David P.
In present study, single quantum (SQ) and triple quantum filtered (TQF) 23Na magnetic resonance imaging (MRI) was used to monitor the severity and progression of CCl4 induced acute and chronic liver damage in rat model. SQ 23Na MRI was proposed to measure the 23Na signal intensity (SI) of total tissue sodium ions, and TQF 23Na MRI was proposed to measure the SI of intracellular sodium ions. In addition, shift reagent aided 23Na and 31P magnetic resonance spectroscopy (MRS) was used to measure in vivo intracellular sodium concentration ([Na+i]), total tissue sodium concentration ([Na+t]) and relative extracellular space (rECS) of liver in the same model. In acute high dose CCl4 intoxication, 24 hours after single dose of CCl4 in 5ml per kg body weight of mixture of CCl4 and oil in 1:1 ratio, SQ 23Na SI increased by 83% and TQF 23Na SI increased by 174% compared to the baseline level. According to SR-aided 23Na and 31P MRS, [Na+i] increased by 188% and [Na+t] increased by 43%. In addition, there was significant decrease in cellular energetic level, represented by ATP/Pi ratio. Histology examination showed pronounced inflammatory response in centrilobular regions, with neutrophiles infiltration, fatty accumulation and swollen hepatocytes. In chronic 8-week experiment, chronic damage was induced by biweekly administration of CCl4 in a dosage of 0.5 ml per kg body weight. From week 1 to week 6, SQ 23Na SI remained relatively constant, and then increased by 15% from week 6 to week 8. TQF 23Na SI progressively increased from week 1 to week 8, totally by 56%. Both SQ and TQF 23Na SI showed significant difference between treated group and control at every week. SR-aided 23Na and 31P MRS experiment showed that, at the end of 8-week CCl4 intoxication, both [Na+t] and rECS were higher than control, by 49% and 47% respectively; however, there was no significant difference for [Na+i] between two groups. Histology examination showed excessive deposition of extracellular matrix. In conclusion, SQ and TQF 23Na MRI appears valuable in the functional assessment of liver in noninvasive approach, and could be a promising diagnostic modality for liver diseases in clinical area.
Regulation of SRF Activity by the ATP-dependent Chromatin Remodeling Enzyme, CHD8
(2009-03-18T18:39:57Z) Rodenberg, Jennifer Marie; Herring, B. Paul; Gallagher, Patricia J.; Pavalko, Fredrick M.; Skalnik, David
Under normal conditions, smooth muscle cells do not replicate, or proliferate, and provide a means of contraction for many internal organs, including blood vessels and the gut. However, under abnormal or disease conditions, such as congenital heart disease and cancer, smooth muscle cells acquire the ability to replicate, to make extracellular matrix proteins and to migrate. Thus, determining how smooth muscle cells regulate these processes is crucial to understanding how the cells can switch between normal and diseased states. Serum response factor (SRF) is a widely expressed protein that plays a key role in the regulation of smooth muscle differentiation, proliferation and migration. It is generally accepted that one way that SRF can distinguish between these functions is through pathway-specific co-factor interactions. A novel SRF co-factor, chromodomain helicase DNA binding protein 8 (CHD8), was originally isolated from a yeast two-hybrid assay. CHD8 is widely expressed in adult tissues including smooth muscle. Data from in vitro binding assays indicate that the N-terminus of CHD8 can interact directly with the MADS domain of SRF. Co-immunoprecipitation assays verified the ability of these two proteins to interact within cells. Adenoviral-mediated shRNA knockdown of CHD8 in smooth muscle cells resulted in statistically significant 10-20% attenuation of expression of SRF-dependent, smooth muscle-specific genes. Similar experiments revealed that knockdown of CHD8 did not affect the SRF-dependent induction of immediate early genes required to promote proliferation. In contrast, knockdown of CHD8 in A10 vascular smooth muscle cells resulted in a marked induction in of apoptosis, characterized by increases in apoptotic markers such as phospho-H2A.X, cleaved PARP and activated caspase-3. These data suggest that CHD8 may play a specific role in modulating SRF’s activity toward anti-apoptotic genes, thereby regulating smooth muscle cell survival.
Major Collateral Vessels Develop from Pre-existing Small Arteries through RAC2/NOX2 Independent Mechanisms
(2009-03-18T18:45:14Z) DiStasi, Matthew Robert; Unthank, Joseph
There is no consensus on which vascular segment or what size of vessels is most important in the process of collateral growth, the degree to which these vessels can enlarge, or the mechanisms that mediate collateral vessel expansion and its impairment. Chapter I identifies the major collateral vessels that develop in response to femoral arterial occlusion in the pig, rat, and mouse hindlimbs for comparison to humans. Pre-existent small named arteries enlarged ~2-3-fold to become the major collateral vessels in each species, these major collaterals displayed characteristics similar to large arteries experiencing flow-mediated outward remodeling, and important differences in vascular wall thickness were observed between rodents and pigs. Chapter II utilized Rac2-/- and Nox2-/- mice to investigate the hypothesis that Nox2-NAD(P)H oxidase is required for major collateral growth subsequent to femoral arterial occlusion. Previous studies suggest bone marrow cell (BMC)-derived reactive oxygen species (ROS) produced by the Nox2 subunit of NAD(P)H oxidase plays an important role in neovascularization and recovery of hindlimb perfusion subsequent to femoral arterial occlusion; but did not investigate collateral growth. The hematopoietic cell restricted protein Rac2 has been shown to bind to and activate Nox2-NAD(P)H oxidase and Rac2-/- and Nox2-/- leukocytes display impaired ROS related functions. The data demonstrated that Rac2 and Nox2 are not essential for major collateral growth, but both are important for the recovery of hindlimb perfusion and preservation of distal tissue morphology. Chapter III investigated BMC and antioxidant therapy in the age-related impairment of collateral growth. Aging, like all cardiovascular disease risk factors is associated with elevated ROS and impaired collateral growth. Studies also suggest BMCs promote collateral growth by secreting paracrine factors but elevated ROS may affect the efficacy of BMCs. The data revealed that neither BMC injection nor antioxidant therapy via apocynin enhanced the process of major collateral artery growth in aged mice.
Contribution of K+ Channels to Coronary Dysfunction in Metabolic Syndrome
(2009-06-24T12:58:39Z) Watanabe, Reina; Tune, Johnathan D.
Coronary microvascular function is markedly impaired by the onset of the metabolic syndrome and may be an important contributor to the increased cardiovascular events associated with this mutlifactorial disorder. Despite increasing appreciation for the role of coronary K+ channels in regulation of coronary microvascular function, the contribution of K+ channels to the deleterious influence of metabolic syndrome has not been determined. Accordingly, the overall goal of this investigation was to delineate the mechanistic contribution of K+ channels to coronary microvascular dysfunction in metabolic syndrome. Experiments were performed on Ossabaw miniature swine fed a normal maintenance diet or an excess calorie atherogenic diet that induces the classical clinical features of metabolic syndrome including obesity, insulin resistance, impaired glucose tolerance, dyslipidemia, hyperleptinemia, and atherosclerosis. Experiments involved in vivo studies of coronary blood flow in open-chest anesthetized swine as well as conscious, chronically instrumented swine and in vitro studies in isolated coronary arteries, arterioles, and vascular smooth muscle cells. We found that coronary microvascular dysfunction in the metabolic syndrome significantly impairs coronary vasodilation in response to metabolic as well as ischemic stimuli. This impairment was directly related to decreased membrane trafficking and functional expression of BKCa channels in vascular smooth muscle cells that was accompanied by augmented L-type Ca2+ channel activity and increased intracellular Ca2+ concentration. In addition, we discovered that impairment of coronary vasodilation in the metabolic syndrome is mediated by reductions in the functional contribution of voltage-dependent K+ channels to the dilator response. Taken together, findings from this investigation demonstrate that the metabolic syndrome markedly attenuates coronary microvascular function via the diminished contribution of K+ channels to the overall control of coronary blood flow. Our data implicate impaired functional expression of coronary K+ channels as a critical mechanism underlying the increased incidence of cardiac arrhythmias, infarction and sudden cardiac death in obese patients with the metabolic syndrome.
ADF/Cofilin Activation Regulates Actin Polymerization and Tension Development in Canine Tracheal Smooth Muscle
(2009-09-03T15:28:09Z) Zhao, Rong; Gunst, Susan J.; Atkinson, Simon J.; Elmendorf, Jeffrey S.; Sturek, Michael S.
The contractile activation of airway smooth muscle tissues stimulates actin polymerization and the inhibition of actin polymerization inhibits tension development. Actin depolymerizing factor (ADF) and cofilin are members of a family of actin–binding proteins that mediate the severing of F–actin when activated by dephosphorylation at serine 3. The role of ADF/cofilin activation in the regulation of actin dynamics and tension development during the contractile activation of airway smooth was evaluated in intact canine tracheal smooth muscle tissues. Two–dimensional gel electrophoresis revealed that ADF and cofilin exist in similar proportions in the muscle tissues and that approximately 40% of the total ADF/cofilin in unstimulated tissues is phosphorylated (inactivated). Phospho–ADF/cofilin decreased concurrently with tension development in response to stimulation with acetylcholine (ACh) or potassium depolarization indicating the activation of ADF/cofilin. Expression of an inactive phospho–cofilin mimetic (cofilin S3E), but not WT cofilin in the smooth muscle tissues inhibited endogenous ADF/cofilin dephosphorylation and ACh–induced actin polymerization. Expression of cofilin S3E in the tissues depressed tension development in response to ACh, but it did not affect myosin light chain phosphorylation. The ACh–induced dephosphorylation of ADF/cofilin required the Ca2+–dependent activation of calcineurin (PP2B). Expression of Slingshot (SSH) inactive phosphatase (C393S) decreased force development and cofilin dephosphorylation. Activation of ADF/cofilin was also required for the relaxation of tracheal muscle tissues induced by forskolin and isoproterenol. Cofilin activation in response to forskolin was not Ca2+–dependent and was not inhibited by calcineurin inhibitors, suggesting it was regulated by a different mechanism. Cofilin activation is required for actin dynamics and tension development in response to the contractile stimulation of tracheal smooth muscle and is regulated by both contractile and relaxing stimuli. These concepts are critical to understanding the mechanisms of smooth muscle contraction and relaxation, which may provide novel targets for therapeutic intervention in the treatment of abnormal airway responsiveness.
The role of SWI/SNF in regulating smooth muscle differentiation
(2009-12-08T19:36:06Z) Zhang, Min; Herring, B. Paul; Firulli, Anthony B.; Pavalko, Frederick M.; Rhodes, Simon J.
There are many clinical diseases involving abnormal differentiation of smooth muscle, such as atherosclerosis, hypertension and asthma. In these diseases, one important pathological process is the disruption of the balance between differentiation and proliferation of smooth muscle cells. Serum Response Factor (SRF) has been shown to be a key regulator of smooth muscle differentiation, proliferation and migration through its interaction with various accessory proteins. Myocardin Related Transcrition Factors (MRTFs) are important co-activators of SRF that induce smooth muscle differentiation. Elucidating the mechanism of how MRTFs and SRF discriminate between genes required to regulate smooth muscle differentiation and those regulating proliferation will be a significant step toward finding a cure for these diseases. We hypothesized that SWI/SNF ATPdependent chromatin remodeling complexes, containing Brg1 and Brm, may play a role in this process. Results from western blotting and quantitative reverse transcription - polymerase chain reaction (qRT-PCR) analysis demonstrated that expression of dominant negative Brg1 or knockdown of Brg1 with silence ribonucleic acid (siRNA) attenuated expression of SRF/MRTF dependent smooth muscle-specific genes in primary cultures of smooth muscle cells. Immunoprecipitation assays revealed that Brg1, SRF and MRTFs form a complex in vivo and that Brg1 directly binds MRTFs, but not SRF, in vitro. Results from chromatin immunoprecipitation assays demonstrated that dominant negative Brg1 significantly attenuated SRF binding and the ability of MRTFs to increase SRF binding to the promoters of smooth muscle-specific genes, but not proliferation-related early response genes. The above data suggest that Brg1/Brm containing SWI/SNF complexes play a critical role in differentially regulating expression of SRF/MRTF-dependent genes through controlling the accessibility of SRF/MRTF to their target gene promoters. To examine the role of SWI/SNF in smooth muscle cells in vivo, we have generated mice harboring a smooth muscle-specific knockout of Brg1. Preliminary analysis of these mice revealed defects in gastrointestinal (GI) development, including a significantly shorter gut in Brg1 knockout mice. These data suggest that Brg1-containing SWI/SNF complexes play an important role in the development of the GI tract.
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.
Protein phosphatase 2A (PP2A) holoenzymes regulate death associated protein kinase (DAPK) in ceramide-induced anoikis
(2010-05-03T19:42:36Z) Widau, Ryan Cole; Gallagher, Patricia J.; Herring, B. Paul; Rhodes, Simon J.; Skalnik, David Gordon
Modulation of sphingolipid-induced apoptosis is a potential mechanism to enhance the effectiveness of chemotherapeutic drugs. Ceramide is a pleiotropic, sphingolipid produced by cells in response to inflammatory cytokines, chemotherapeutic drugs and ionizing radiation. Ceramide is a potent activator of protein phosphatases, including protein phosphatase 2A (PP2A) leading to dephosphorylation of substrates important in regulating mitochondrial dysfunction and apoptosis. Previous studies demonstrated that death associated protein kinase (DAPK) plays a role in ceramide-induced apoptosis via an unknown mechanism. The tumor suppressor DAPK is a calcium/calmodulin regulated serine/threonine kinase with an important role in regulating cytoskeletal dynamics. Auto-phosphorylation within the calmodulin-binding domain at serine308 inhibits DAPK catalytic activity. Dephosphorylation of serine308 by a hitherto unknown phosphatase enhances kinase activity and proteasomal mediated degradation of DAPK. In these studies, using a tandem affinity purification procedure coupled to LC-MS/MS, we have identified two holoenzyme forms of PP2A as DAPK interacting proteins. These phosphatase holoenzymes dephosphorylate DAPK at Serine308 in vitro and in vivo resulting in enhanced kinase activity of DAPK. The enzymatic activity of PP2A also negatively regulates DAPK protein levels by enhancing proteasomal-mediated degradation of the kinase, as a means to attenuate prolonged kinase activation. These studies also demonstrate that ceramide causes a caspase-independent cell detachment in HeLa cells, a human cervical carcinoma cell line. Subsequent to detachment, these cells underwent caspase-dependent apoptosis due to lack of adhesion, termed anoikis. Overexpression of wild type DAPK induced cell rounding and detachment similar to cells treated with ceramide; however, this effect was not observed following expression of a phosphorylation mutant, S308E DAPK. Finally, the endogenous interaction of DAPK and PP2A was determined to be required for ceramide-induced cell detachment and anoikis. Together these studies have provided exciting and essential new data regarding the mechanisms of cell adhesion and anoikis. These results define a novel cellular pathway initiated by ceramide-mediated activation of PP2A and DAPK to regulate inside-out signaling and promote anoikis.
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.
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 Stephen
The 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.

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