• Mechanical stress alters gelatinase expression of cultured murine calvarial osteoblast-like cells

      Nichols, Richard A. Jr.; Department of Oral Biology (Augusta University, 1999-06)
    • MECHANISM OF 12/15 LIPOXYGENASE-INDUCED RETINAL MICROVASCULAR DYSFUNCTION IN DIABETIC RETINOPATHY

      Elmasry, Khaled; Department of Biochemistry and Molecular Biology / Cancer Center (5/22/2018)
      Our earlier studies have established the role of 12/15-lipoxygenase (LO) in mediating the inflammatory reaction in diabetic retinopathy. However, the exact mechanism is still unclear. The goal of the current study was to identify the potential role of endoplasmic reticulum (ER) stress as a major cellular stress response in the 12/15-LO-induced retinal changes in diabetic retinopathy. We used in vivo and in vitro approaches. For in vivo studies, experimental diabetes was induced in wild-type (WT) mice and 12/15-Lo (also known as Alox15) knockout mice (12/15-Lo−/−); ER stress was then evaluated after 12-14 weeks of diabetes. We also tested the effect of intravitreal injection of 12-hydroxyeicosatetraenoic acid (HETE) on retinal ER stress in WT mice and in mice lacking the catalytic subunit of NADPH oxidase, encoded by Nox2 (also known as Cybb) (Nox2−/− mice). In vitro studies were performed using human retinal endothelial cells (HRECs) treated with 15-HETE (0.1 µmol/l) or vehicle, with or without ER stress or NADPH oxidase inhibitors. This was followed by evaluation of ER stress response, NADPH oxidase expression/activity and the levels of phosphorylated vascular endothelial growth factor receptor-2 (p-VEGFR2) by western blotting and immunoprecipitation assays. Moreover, real-time imaging of intracellular calcium (Ca2+) release in HRECs treated with or without 15-HETE was performed using confocal microscopy. Deletion of 12/15-Lo significantly attenuated diabetes-induced ER stress in mouse retina. In vitro, 15-HETE upregulated ER stress markers such as phosphorylated RNA-dependent protein kinase-like ER-regulated kinase (p-PERK), activating transcription factor 6 (ATF6) and protein disulfide isomerase (PDI) in HRECs. Inhibition of ER stress reduced 15-HETE-induced-leukocyte adhesion, VEGFR2 phosphorylation and NADPH oxidase expression/activity. However, inhibition of NADPH oxidase or deletion of Nox2 had no effect on ER stress induced by the 12/15-LO-derived metabolites both in vitro and in vivo. We also found that 15-HETE increases the intracellular calcium in HRECs. ER stress contributes to 12/15-LO-induced retinal inflammation in diabetic retinopathy via activation of NADPH oxidase and VEGFR2. Perturbation of calcium homeostasis in the retina might also play a role in linking 12/15-LO to retinal ER stress and subsequent microvascular dysfunction in diabetic retinopathy.
    • Mechanism of Action of Ristocetin

      Grastie, Miriam Kay; Department of Cell and Molecular Biology (1970-06)
    • The Mechanism of Monomethylfumarate (MMF) as an Anti-psoriatic Agent

      Helwa, Inas; Department of Physiology (2014-09)
      Psoriasis is a chronic hyperproliferative inflammatory skin disorder whose primary etiology is not well understood. Keratinocytes play a pivotal role in the pathogenesis of psoriasis. The fumaric acid ester monomethylfuamarate (MMF) is the bioactive ingredient of the anti-psoriatic drug Fumaderm©, licensed in Germany since 1994. However, the exact mechanism of action of MMF is not yet well understood. Our data showed that MMF dose-dependently inhibited proliferation in primary murine and human keratinocytes and significantly increased the protein expression of the early marker of differentiation K10 and the activity of the late marker of differentiation transglutaminase enzyme. In addition, MMF inhibited mRNA expression of IL-6, TNFα and IL-1α and inhibited the protein expression of TNFα. Recently, the role of oxidative stress in psoriasis etiology has evolved and MMF has been shown to stimulate Nrf2 and mediate its nuclear translocation in other cell types. Therefore, we examined the effect of MMF on Nrf2 expression, localization and downstream effectors in keratinocytes. Nrf2 protein expression and nuclear translocation significantly increased following MMF treatment. Moreover, MMF significantly increased the mRNA expression of the Nrf2- downstream anti-oxidative enzymes, heme oxygense-1 and peroxiredoxin-6. MMF also decreased ROS generation in keratinocytes. Aquporin3 (AQP3) is a glycerol channel expressed in keratinocytes. Earlier studies from our group as well as others have shown that AQP3 plays a role in inducing early keratinocyte differentiation and that the activity of AQP3 correlates with its membranous localization. Therefore, we examined the effect of MMF on AQP3 expression and localization. MMF increased the mRNA and protein 3 expression of AQP3. In addition, MMF stimulated membranous translocation of AQP3 and increased glycerol uptake by keratinocytes. Eventually, we wanted to examine whether Nrf2 plays a role in the expression of AQP3. Our data showed that the Nrf2 stimulator sulforaphane (SFN) increased the expression of AQP3. Thus, our data suggest that MMF exerts its action through Nrf2 stimulation. Nrf2 stimulation helps to regain keratinocyte oxidative balance and may also play a role in inducing AQP3 expression and activity. This provides the molecular basis for the MMF-mediated improvement of keratinocyte differentiation and inhibition of keratinocyte proliferation.
    • The mechanism of poliovirus-induced intracellular alkalinization

      Holsey, Charles Maceo, Jr.; Demartment of Immunology and Microbiology (Augusta University, 1992-12)
    • Mechanisms Driving Innate Regulation Of Immunological Tolerance To Apoptotic Cells Preventing Autoimmunity

      Shinde, Rahul; Department of Neuroscience and Regenerative Medicine (2015-08)
      Innate immune responses to apoptosis are crucial for self-tolerance. Although upstream signals promoting recognition and processing of apoptotic cells have been extensively studied, downstream molecular mechanisms driving innate regulation of apoptotic cell responses are less understood. Here we report an unsuspected discovery that the ligand dependent transcription factor aryl hydrocarbon receptor (AhR) initiates tolerogenic signaling to apoptotic cells and prevents systemic autoimmunity. AhR is known to control xenobiotic stress responses and recently has been linked to modulation of T cell and DC function. In this study, we found that apoptotic cells induced AhR signals in tissueresident MΦs and activation was dependent on DNA from apoptotic cells. AhR was required for apoptotic cell driven immune suppression as deletion of AhR abrogated IL-10, promoting the inflammatory cytokines IL-6 and IL-12, while supplementing IL-10 restored the regulatory phenotype of MΦs. Moreover, inhibition of the AhR pathway fundamentally altered immune responses to apoptotic cells resulting in proinflammatory cytokine production, increased effector T cell responses and abrogation of long-term allograft tolerance to apoptotic cell associated antigens. Further, mice lacking AhR developed spontaneous autoimmunity characterized by excessive macrophage and lymphocyte activation associated with renal pathology. Deficiency of AhR led to breakdown in tolerance with rapid increases in anti-dsDNA and anti-histone antibody responses after chronic challenge with apoptotic cells. Similarly, when SLE-prone mice were treated with AhR antagonist they exhibited significantly elevated humoral auto-reactivity, augmented inflammatory cytokine production in MΦs, intensified autoreactive B and T cells, renal pathology, and mortality; while AhR agonist treatment resulted in significant reduction of autoimmune disease parameters compared to control mice. Collectively, the data demonstrate apoptotic cell activation of AhR is a key mechanism suppressing anti-apoptotic cell inflammatory responses preventing autoimmunity.
    • Mechanisms for Control of Renal Vascular Resistance in Type I Diabetes Mellitus

      Bell, Tracey D.; Department of Physiology; American Heart Association; National Institutes of Health (Medical College of Georgia, 2007-04)
      Glomerular hyperfiltration and an increase in renal blood flow are hallmark characteristics of Type I_ Diabetes Mellitus in the early stages, and are major risk factors for the development of diabetic nephropathy. Previous studies from our laboratory have implicated an important role for the Nitric Oxide system. in mediating this response, because giving nitric oxide synthase inhibitors· 'prevented the increase in renal plasma flow and glomerular filtration rate during diabetes. However, a limitation of. these studies is that single point measurements were taken and may not reflect the time-dependent role of nitric oxide. Therefore, we have developed a more precise method to measure the role of nitric oxide in the chronic control of renal blood flow during diabetes. We measured renal blood flow continuously, 18 hr/day using a Transonic flow probe in control (C) and diabetic (D) rats. Renal blood flow averaged 8.0±0.1 and 7.8±0 ml/min in the .C and D groups, respectively, during the control period and induction of diabetes caused a marked and progressive increase in renal blood flow in the D rats, averaging 10±6% above control on day 1, and 22±3% and 34±1% above control by the end of diabetes weeks 1 and 2. During the control period, glomerular filtration rate averaged 2.1 ±0.1 and 1.7±0.1 ml/min in the C and D groups, respectively. Glomerular filtration rate did not change during the experiment in the C rats, but increased significantly in the D group, averaging 54±21 and 52±19% above control during diabetic weeks 1 and 2 and renal vascular resistance decreased significantly during the diabetic period. There were no significant changes in filtration fraction in either group. Importantly, chronic blockade of nitric oxide completely prevented the increase in renal blood flow and prevented the diabetes-induced hyperfiltration normally associated with diabetes. These data together suggest that nitric oxide is essential for the renal vasodilation caused by onset of type I diabetes and suggest that the renal vasodilation in diabetes occurs primarily at the afferent arteriole. Autoregulation of the afferent arteriole plays an important role in determining glomerular capillary hydrostatic pressure and glomerular filtration. In diabetes, renal autoregulation may be impaired, but the relative roles of myogenic and tubuloglomerular feedback mechanisms in controlling renal blood flow, and the time course of their involvement, is not known. In addition, there is very little known about autoregulatory mechanisms at the very onset of diabetes, before there has been time for renal structural changes to become manifest. Therefore, we designed experiments to establish the role of the myogenic response and tubuloglomerular feedback mechanism in renal blood flow. control at the onset of diabetes. Coupling continuous measurement of renal blood flow using Transonic flow probes and continuous measurement of arterial pressure, we were able to use transfer function analysis to determine the relationship between arterial pressure and renal blood flow .. This type of analysis examines the dynamic ability of the renal vasculature to attenuate, or autoregulate, the influence of the oscillatory power of blood pressure over the range of frequencies at which the myogenic response and tubuloglomerular feedback mechanism operate. In these studies we demonstrated that transfer function gain was negative, indicating effective· autoregulation, in the frequency range of the myogenic (0.1- 0.3 Hz) and tubuloglomerular feedback (0.03-0.06 Hz) mechanisms during control days. However, at the onset ·of diabetes gain increased to positive values and continued through the 2-week diabetic period. Chronic blockade of nitric oxide in diabetic rats normalized the increase in transfer function gain and possibly enhanced the autoregulatory response. Our model provides a novel method to measure the chronic effects of the nitric oxide on renal blood flow control during diabetes. By using this model we have demonstrated that nitric oxide is required for the immediate increase in renal blood flow in diabetes. Furthermore, these data suggest renal autoregulation is impaired at the onset of diabetes and may play a role in the increase in renal blood flow and glomerular filtration rate early in diabetes. In addition, these data together suggest that nitric oxide contributes to the impaired autoregulatory capacity of the renal vasculature.
    • Mechanisms for the dependency of angiotensin II hypertension on interleukin-6

      Sturgis, LaShon C.; Medical College of Georgia (Augusta University, 2007)
      Angiotensin II (Ang II) is involved critically in the development and maintenance of hypertension in both human and animal models. Ang II also is known to stimulate interleukin 6 (IL-6) release, and a recent study by our laboratory demonstrated that Ang II hypertension was attenuated in IL-6 knockout (KO) mice. These data suggest that IL-6 mediates part of the hypertensive actions of Ang II. In addition, Ang II also stimulates production and release of aldosterone, which also has hypertensive actions. Ang II also stimulates the proinflammatory cytokine tumor necrosis factor-alpha (TNF-a.), which can stimulate IL-6 secretion. Therefore, the aim of this project was to determine whether the dependence of Ang II hypertension on 11-6 is du.e to a direct link between Ang II and IL-6 or whether aldosterone and/or TNF-a. are important intermediate factors. In separate studies, we determined whether mineralocorticoid hypertension is IL-6 dependent, the role of IL-6 bioactivity verses IL-6 plasma concentration, and the role of TNF-a. in Ang II hypertension. Mineralocorticoid hypertension was induced by implanting a deoxycorticosterone acetate (DOCA) pellet (1 g/Kg) subcutaneously and giving all animals a solution of 1% sodium chlorid~ (NaCI) and 0.2 % potassium chloride (KCI) to dink for a 14 day experimental period. DOCA-salt treatment increased the mean arterial pressure (MAP) similarly by -30 mm . Hg in both the wr and IL-6 KO mice. However, DOCA-salt treatment did not increase plasma IL-6 concentration in wildtype {WT) mice nor did it increase . IL-6 bioavailability using a bioassay on day 14 of. treatment. There was,. however, a transient increase in plasma IL-6 concentration and bioactivity on day 7 of DOCA-salt treatment in the WT mice. Treating WT mice with Ang II and the mineralocorticoid receptor antagonist, spironolactone, significantly attenuated the Ang II mediated increase in plasma IL-6 concentration on day 7 and day 14 of treatment. Although we cannot explain why the IL-6 response to DOCA was not sustained through 14 days, together these data suggest that that aldosterone plays a role in the increase in plasma IL-6 concentration during Ang II hypertension. However the effect of IL-6 in mediating part of the Ang II hypertension appears d_ue to an interaction with Ang 11-mediated effects and not due to effects mediated by the mineralocorticoid receptor. Similarly, Ang II increased MAP by -30 mm Hg in both the·wr a·nd TN F-a. KO mice by day 14 of treatment. Moreover, which suggests that TN F-a. is not required for Ang II hypertension, Ang II treatment did not increase plasma levels of TN F-a. and TN F-a. infusion did not cause a sustained .increase in IL-6. These data suggest that Ang II may work through an aldosterone, but not TNF-a.- mediated, mechanism to increase plasma IL-6 concentration in Ang II hypertension, but that the role of IL-6 in mediating Ang II hypertension is due to interactions with Ang.ll receptor type 1 (AT1) dependent mechanisms on target tissues.
    • Mechanisms mediating heterogeneous endothelium-dependent relaxation in small arteries from the coronary, mesenteric and skeletal muscle vascular beds in Golden Syrian and cardiomyopathic hamsters

      Clark, Shawn G.; School of Graduate Studies (1998-04)
      V asodilatory substances released from the endothelium are important regulators of vascular smooth muscle tone. Endothelium-dependent relaxation of large arteries is mediated primarily by nitric oxide (NO). Mechanisms mediating relaxation of small arteries (150-250 μm intraluminal diameter; ID) are not well understood. Additionally, endothelium-derived relaxing factors (EDRFs) may differentially modulate vascular tone and · relaxation in arteries from different vascular beds. The mechanisms mediating basal tone and endothelium-dependent vascular relaxation were determined in skeletal (Ske ), coronary (Cor) and mesenteric (Mes) small arteries isolated from male Golden Syrian hamsters. Baseline ID of small arteries was measured before and after pretreatment with inhibitors. Blockade of delayed-rectifying K+ channels CKmJ decreased baseline ID in Ske -small arteries, only. Blockade of large ca++ -dependent K+ channels (BKcJ decreased baseline ID in Cor small arteries, only. Blockade of inwardly-rectifying K+ channels (Km) decreased baseline ID in Cor and Mes small arteries. Therefore, basal 'tone is mediated by KDR channels in isolated Ske, K1R channels in isolated Mes, and K1R and BKca channels in isolated Cor small arteries. Acetylcholine (ACh, 10-9 to 3 x 10-5 M) produced concentration-dependent relaxation in Ske, Cor and Mes small arteries. Cor small arteries demonstrated a lower sensitivity to ACh. Relaxation to ACh was completely abolished after removing the endothelium and was unaffected by inhibition of cyclooxygenase (COX) in Ske, Mes, and Cor small arteries. Inhibition of nitric oxi~e synthase (NOS) significantly reduced maximal relaxation and reduced the sensitivity to ACh in Cor, but had no effect in Ske or Mes, small arteries. High extracellular K+ largely reduced relaxation to A Ch in all ~~ssels. Blockade. of KnR or K1R channels decreased the sensitiyity to ACh in S~e, but had no effect in Cor or Mes small arteries. Blockad~ of ca++ -dependent K+ channels (KcJ ·did iiot. alter relaxation in Mes, significantly reduced relaxation in Ske, and abolished relaxation to ACh in Cor small arteries. These results indicate that relaxation to A Ch is mediated partially by NO and an endothelium-deriveq hyperpolarizingfactor (EDHF), other than NO in Cor small arteries by opening Kea channels. Relaxation to A Ch is mediated by EDHF in Ske small arteries by opening Kea, ~DR and ~R channels. Relaxation to A Ch is mediated by EDHF in Mes small arteries, but the specific K+ channels contributing to relaxation remain unkn.own. Although the chemical identity of EDHF is unknown, it may be a metabolite of arachidonic acid (AA) produced by cytochrome P450 monooxygenase ( cP450). Inhibition of cP450 significantly inhibited relaxation to ACh in all vessels. These results indicate that relaxation to A Ch· that is resistant to inhibition of NOS or COX is dependent on cP450 metabolites in all vessels. In many diseases, mechanisms mediating normal vascular function are altered. Blockade of BKca channels in Cor and Mes small arteries from control hamsters and Cor / small arteries from cardiomyopathic hamsters caused contraction from baseline. Therefore, basal tone is regulated by BKea channels in Cor small arteries from control and cardiomyopathic hamsters and regulation of basal tone by BKea channels is impaired in Mes small arteries from cardiomyopathic hamsters. Concentration-response curves to A Ch were similar between vessels from both groups. COX inhibition decreased relaxation to A Ch in cardiomyopathic, but not control, Cor small arteries. NOS inhibition reduced the sensitivity to ACh similarly in Mes small arteries from both groups;. However, NOS inhibition decreased relaxation to ACh to a lesser extent in cardiomyopathic compared to control hamster Cor small artetjes. NOS inhibition decreased maximum relaxation and reduced the sensitivity to ACh in Cor small arteries from control hamsters, but had no effect on maximum relaxation in vessels from cardiomyopathic hamsters. This ·_indicates that the contribution of NO-mediated relaxation is reduced in- vessels from cardiomyopathic hamsters. Blockade of Kea comple_tely abolished relaxation to A Ch in Cor, but not Mes . . small arteries from both groups. Ther~fore, A Ch~induced relaxation of Cor small arteries from control hamsters is mediated by NO and EDHF via opening Kea channels. AChinduced relaxation of Cor small arteries from cardiomyopathic hamsters is primarily mediated by NO and EDHF, slightly by PG/2 by opening Kea channels. ACh-induced relaxation of Mes small arteries from control and cardio1,:,yopathic hamsters is mediated by NO and EDHF, but does not require opening of Kea channels. Measurement of mean arterial pressure (MAP), mesenteric vascular resistance (MVR) and hindquarter vascular resistance (HVR) was examined in anesthetized control . and cardiomyopathic hamsters after inhibition of NOS · or BKca channels. NOS inhibition increased MAP and MVR to a lesser extent in cardiomyopathic compared to control hamsters and increased HVR in co~trol, but not cardiomyopathic hamsters. Blockade of BKca channels increased MAP and MVR similarly in both groups, but increased HVR significantly less in cardiomyopathic hamsters. These results indicate that regulation of MVR by NOS, and of HVR by both NOS and BKca channels, is reduced.
    • Mechanisms of ANG (1-7) Mediated Control of Blood Pressure in Males and Females

      Zimmerman, Margaret A.; Department of Physiology (2014-07)
      Angiotensin (Ang) (1-7) is a vasodilatory peptide of the renin angiotensin system (RAS). Ang (1-7) levels are greater in females, and Ang (1-7) blunts Ang II-mediated increases in blood pressure (BP) in females compared to males. The molecular mechanism(s) by which Ang (1-7) mediates BP regulation remains largely unknown, although Ang (1-7) has been suggested to increase nitric oxide (NO) levels, suppress proinflammatory markers, and contribute to the BP-lowering effects of RAS-inhibitors. The central hypothesis of my thesis is that Ang (1-7) contributes more to the molecular mechanisms that mediate BP control in females than males. To test this hypothesis, four aims were addressed. Aim 1 tested the hypothesis that the BP in male spontaneously hypertensive rats (SHR) is less sensitive to increases in Ang (1-7) than females. Ang (1-7) levels were pharmacologically increased in male and female SHR, and BP was assessed. However, Ang (1-7) infusion did not alter baseline BP in either sex. Aim 2 tested the hypothesis that Ang (1-7) contributes less to the BP-lowering effects of angiotensin receptor blockers (ARBs) in male than females SHR. To test this hypothesis, BP was measured in male and female SHR in response to an ARB. Males had the greater decrease in basal BP to an ARB than females, although female SHR were more sensitive to ARB-mediated inhibition of Ang II-induced increases in BP. Additional studies indicated that Ang (1-7) contributed to the BP-lowering effect of ARBs to a greater degree in females than in males. vi Aim 3 tested the hypothesis that Ang (1-7) contributes less to NO bioavailability in male than female SHR under basal conditions and following Ang II-hypertension. Ang (1-7) levels were pharmacologically increased or blocked in male and female SHR and the NO pathway was assessed. Renal cortical NO bioavailability was not affected by treatments in either sex. Finally, Aim 4 tested the hypothesis that Ang II infusion will increase renal T cells in both sexes; however, greater Ang (1-7) in females will result in more T regulatory cells (Tregs) relative to male Sprague Dawley (SD) rats. Renal T cells were increased in both males and females following chronic Ang II infusion, however, females exhibited an increase in immune-suppressive Tregs not seen in males. In contrast, males exhibited a greater increase in pro-inflammatory Th17 cells. Inhibition of Ang (1-7) did not alter the sex difference in Tregs, indicating that Ang (1-7) is not responsible for the greater increase in Tregs in females following Ang II-hypertension. In summary, this work examines the role of Ang (1-7) to mediate sex differences in BP regulation, where females are more dependent on Ang (1-7) than males to correct perturbations in the RAS.
    • Mechanisms of Diabetes-Mediated Cerebrovascular Injury in Ischemic Stroke

      Cobbs, Aisha Imani; Department of Physiology (2012-08)
      Diabetes increases the risk of cerebrovascular disease and is a reliable predictor of increased morbidity and mortality following acute ischemic stroke. The objective of the current study was to investigate the underlying mechanisms by which diabetes-mediated vascular dysregulation contributes to greater injury and poor stroke outcomes. We hypothesized that peroxynitrite mediates vascular dysfunction in diabetes by destabilizing the vascular smooth muscle actin cytoskeleton. In addition, we proposed that excess peroxynitrite formation and inflammation during ischemia/reperfusion injury in pre-existing diabetes amplifies the proteolytic activity of matrix metalloproteinases (MMPs), thereby contributing to greater vascular injury (i.e., edema and hemorrhagic transformation) and neurological deficit. Using a modified oxygen-glucose deprivation protocol, we examined the effects of hypoxia on cerebral macrovascular reactivity. We found that peroxynitrite mediates hypoxia-induced loss of myogenic tone and medial thickening in cerebral resistance vessels isolated from type 2 diabetic rats. Furthermore, we demonstrated that reductions in polymerized actin cytoskeletal filaments following hypoxia exposure in these vessels cannot be attributed to peroxynitrite nitration, suggesting that an alternate target or different type of peroxynitrite-mediated protein modification may be involved. Targeting mediators of stroke-induced vascular injury at reperfusion was more beneficial in diabetic animals compared to controls. Acute administration of FeTPPs, curcumin, and minocycline at reperfusion in experimental stroke successfully reduced hemorrhagic transformation in all diabetic animals. This reduction in bleeding was associated with decreased MMP-9 activity in cerebral macrovessels. Administration of curcumin and minocycline attenuated edema formation in these animals. Functional outcomes were also improved in varying degrees by these therapies. Based on the findings of these studies, we concluded that oxidative stress, inflammation, and MMP activity in the cerebrovasculature of diabetic animals play a significant role in stroke pathologies that contribute to worse outcomes. Therefore, the following dissertation research has the potential to reduce the gap in knowledge of how pre-existing diabetes contributes to stroke pathophysiology and will potentially aid in the development of novel therapeutic strategies tailored to the diabetic population.
    • Mechanisms of Diet-Induced Hypertension and Vascular Disease Risk in Dahl Rats

      Spradley, Frank T.; Department of Medicine (2011)
      Dahl salt-sensitive (SS) rats are genetically predisposed to cardiovascular-renal disease. These studies examined cardiovascular-renal outcomes in response to a high-fat diet/normal-salt diet in SS rats. In a separate study, we examined cardiovascular-renal disease risk in SS rats on different standard chow diet/normal-salt diets. We tested the hypotheses that: (1) a high-fat diet induces hypertension and renal injury in SS rats; (2) a high-fat diet enhances aortic vasoconstriction in SS rats; (3) a high-fat diet induces aortic perivascular adipose tissue (PVAT) dysfunction in SS rats; and (4) two standard chow diets, namely AIN-76A and Teklad diet, induce differential vasoconstriction or vasorelaxation phenotypes in aorta and small mesenteric arteries from SS rats. In the high-fat diet studies, rats were provided high-fat diet starting at 12 weeks old. At 16 weeks old, SS rats on the high-fat diet had hypertension and greater renal glomerular and tubular injury than SS-13BN rats. SS rats supplemented with the immunosuppressive drug mycophenolate mofetil (MMF; 30 mg/kg/day, oral) for the duration of the high-fat diet did not develop hypertension. High-fat diet was associated with reduced vasoconstrictive response to angiotensin II and increased acetylcholine-mediated vasorelaxation in SS rats via increased nitric oxide synthase (NOS) function in comparison to SS rats maintained on a normal-fat/normal-salt diet. In regards to PVAT function, high-fat diet increased thoracic aorta PVAT deposition and induced PVAT-mediated blunting of aortic vasoconstriction. In the standard chow diet studies, 16-week old SS rats placed on the AIN or Teklad diet at weaning had similar NOS functional regulation of vasoconstriction and vasorelaxation in large and small arteries. However, by using a diet-switch protocol, we demonstrated that SS rats placed on AIN diet at weaning and changed to Teklad diet at 12 weeks old had reduced NOS-mediated vasorelaxation and reduced NOS buffering of vasoconstriction in small arteries from SS rats, which was not observed in the corresponding diet-switch group. In conclusion, these studies highlight differential renal and vascular responses to a short-term high-fat diet, and even changes in standard chow diet, when genetically predisposed to hypertension.
    • Mechanisms of diet-induced hypertension and vascular disease risk in dahl rats

      Spradley, Frank Travis; Medical College of Georgia (Augusta University, 2011)
      Dahl salt-sensitive (SS) rats are genetically predisposed to cardiovascular-renal disease. These studies examined cardiovascular-renal outcomes in response to a high-fat diet/normal-salt diet in SS rats. In a separate study, we examined cardiovascular-renal disease risk in SS rats on different standard chow diet/normal-salt diets. We tested the hypotheses that: (1) a high-fat diet induces hypertension and renal injury in SS rats; (2) a high-fat diet enhances aortic vasoconstriction in SS rats; (3) a high-fat diet induces aortic perivascular adipose tissue (PVAT) dysfunction in SS rats; and (4) two standard chow diets, namely AIN-76A and Teklad diet, induce differential vasoconstriction or vasorelaxation phenotypes in aorta and small mesenteric arteries from SS rats. In the high-fat diet studies, rats were provided high-fat diet starting at 12 weeks old. At 16 weeks old, SS rats on the high-fat diet had hypertension and greater renal glomerular and tubular injury than SS-138 N rats. SS rats supplemented with the immunosuppressive drug mycophenolate mofetil (MMF; 30 mg/kg/day, oral) for the duration of the high-fat diet did not develop hypertension. High-fat diet was associated with reduced vasoconstrictive response to angiotensin II and increased acetylcholine-mediated vasorelaxation in SS rats via increased nitric oxide synthase (NOS) function in comparison to SS rats maintained on a normal-fat/normal-salt diet. In regards to PV AT function, high-fat diet increased thoracic aorta PV AT deposition and induced PV AT -mediated blunting of aortic vasoconstriction. In the standard chow diet studies, 16-week old SS rats placed on the AIN or Teklad diet at weaning had similar NOS functional regulation of vasoconstriction and vasorelaxation in large and small arteries. However, by using a diet-switch protocol, we demonstrated that SS rats placed on AIN diet at weaning and changed to Teklad diet at 12 weeks old had reduced NOS-mediated vasorelaxation and reduced NOS buffering of vasoconstriction in small arteries from SS rats, which was not observed in the corresponding diet-switch group. In conclusion, these studies highlight differential renal and vascular responses to a short-term high-fat diet, and even changes in standard chow diet, when genetically predisposed to hypertension.
    • Mechanisms of Estrogen Neuroprotection in Stroke

      Raz, Limor; Department of Neurology (2011-04)
      17-β estradiol (17-β-E2) has been implicated to be neuroprotective, yet the mechanisms underlying 17-β-E2-mediated protection against stroke remain unclear. The purpose of the current study was to elucidate the role of 17-β-E2 in NADPH oxidase (NOX2) activation during ischemic reperfusion induction of superoxide (O2 -) in the hippocampus CA1 region following global cerebral ischemia (GCI) and to investigate the post-translational deacetylation of downstream pro-apoptotic factors by 17-β-E2. Using a 4-vessel occlusion model to induce GCI, we showed that neuronal NOX2 localizes to the membrane and that NADPH oxidase activity and O2 - production were rapidly and markedly attenuated by 17-β-E2 following reperfusion, in an estrogen receptor-dependent manner. Inhibition of NADPH oxidase activation via icv administration of a NOX2 competitive inhibitor, gp91ds-tat, strongly attenuated O2 - production and was neuroprotective. The increase of neuronal NOX2 and O2 - following cerebral ischemia was shown to require Rac1 activation, as administration of a Rac1 inhibitor (NSC23766) significantly attenuated these factors following stroke. Interestingly, we found that 17-β-E2 antioxidant ability to diminish neuronal NOX2-induced O2 - generation involves the attenuation of Rac1 activation. We also provide evidence for 17-β-E2 post-translational deacetylation of downstream pro-apoptotic p53 and a reduction of p53 transcriptional target, Puma. Our results revealed that p53 acetylation (activation) is markedly increased in ischemic animals 24h after reperfusion and that 17-β-E2 strongly attenuated that elevation, as well as total p53 protein levels. In support of this suggestion, we also found 17-β-E2 to strongly attenuate ischemia-mediated Puma upregulation, thus interfering with its transcription-dependent function. We further propose that 17-β-E2-induced attenuation of p53 levels may involve an upregulation in p53-Mdm2 interactions and p53 mediated degradation via the ubiquitination pathway. Lastly, we provide evidence showing that treatment with Gp91ds-tat, but not the scrambled tat peptide control, attenuated acetylation of downstream p53 and reduced levels of Puma, thus supporting O2 —p53 crosstalk signaling after stroke. Altogether, our studies reveal a novel, membrane-mediated antioxidant mechanism of 17-β-E2-induced neuroprotection via reduction of neuronal NOX2 activation and O2 - production, while providing evidence for 17-β-E2–mediated deacetylation and inactivation of p53, thereby protecting the hippocampus CA1 against cerebral ischemia.
    • Mechanisms of ET-1-mediated 02~ production in the rat aorta

      Loomis, E. D.; Department of Medicine (2004-06)
      The objectives of this project were to test the hj^othesis that in the rat aorta endothelin-I (ET-I) binds to the ETa receptor stimulating superoxide (O2’ ) production. Furthermore, we wanted to identify the mechanism through which ET-1-mediates O2' production. Chemiluminescent detection of O2” production using probes such as lucigenin has been widely used with enzyme systems, leukocytes, and vascular tissues. Our first goal was to develop a microplate high-throughput protocol for lucigeninamplified chemiluminesence detection of 0 2 'L We have developed a novel adaptation to lucigenin-based assays that allows up to 36 samples to be counted at virtually the same time. Recent studies have shown that NOS 3 can become uncoupled and produce O2* when deprived of its cofactor BH4 . In addition several authors have shown that ONOOoxidizes BH4 in vitro. Using the high-throughput lucigenin assay and dihydroethidine (DHE) staining we have shown that (1) ET-1 is able to stimulate 0 2 "^ production in both endothelium-intact and -denuded vessels through the ETA-receptor, (2) ET-1 stimulates O2’ production through both NAD(P)H oxidase and an endothelial source of NOS, and (3) addition of exogenous tetrahydrobiopterin (BH4 ) and inhibition of peroxynitrite (ONOO-) inhibit ET-I-mediated 0 2 *^ production. Therefore our data have led us to hypothesize that ET-I stimulates O2* production by activating NAD(P)H oxidase through the ETa receptor. O2' production by NAD(P)H oxidase leads to the formation of ONOO- and the degradation of BH4. The loss of BH4 leads to uncoupled NOS which then contributes to ET-1-mediated production. In addition, we have found that (1) ET-1 increases the production of interleukin- 6 (IL-6 ) and (2) ET-1-mediated O2' production adversely affects vascular contractility. Although these consequences do not appear to be due to NOS uncoupling, they help support the role of ET-1 in vascular dysfunction.
    • Mechanisms of Homocysteine-Induced Retinal Ganglion Cell Death

      Ganapathy, Preethi S.; Department of Cellular Biology and Anatomy (2010-12)
      The purpose of these studies was to determine the effect of excess homocysteine on retinal ganglion cell viability. An overview of homocysteine metabolism and the literature concerning homocysteine-induced neurotoxicity is given below, followed by detailed descriptions of the eye, the retina, and retinal ganglion cells.
    • Mechanisms of homocysteine-induced retinal ganglion cell death

      Ganapathy, Preethi S.; Medical College of Georgia (Augusta University, 2010-12)
    • Mechanisms of neuroprotection by estrogen and selective estrogen receptor modulators

      Dhandapani, Krishnan M; School of Graduate Studies (2003-03)