• Macrophage Recruitment Signals Following Unilateral Chorda Tympani Nerve Degeneration

      Cavallin, Melissa Ann; Department of Neuroscience and Regenerative Medicine (2007-02)
      The chorda tympani nerve (CT) innervates taste buds within fungiform papillae. Unilateral transection of the CT causes degeneration of the ipsilateral taste buds and a bilateral increase in activated lingual macrophages. However, dietary Na+ restriction prevents the macrophage response and results in a subnormal neural response to Na+ stimuli by the contralateral, intact CT. Stimulating immune system function with lipopolysaccharide (LPS) restores the bilateral macrophage response to CT section in Na+-restricted rats. This macrophage response is associated with the recovery of normal taste function, suggesting that macrophages affect taste function. Intracellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, and monocyte chemoattractant protein (MCP)-1 are upregulated prior to and during the peak macrophage response suggesting that these molecules are recruitment signals for macrophage entry following CT injury. Macrophage inflammatory protein (MIP)-1α is not significantly upregulated following CT section. Importantly, the increase in VCAM-1 expression is prevented by dietary Na+ restriction, which may partially explain the decreased macrophage response in these animals. However, binding of an antibody against platelet endothelial cell adhesion molecule (PECAM)-1, which is downstream of ICAM-1 and VCAM-1, paradoxically increases macrophage recruitment and does not alter taste function. Other adhesion molecules may be able to compensate for the loss of PECAM-1. The response of the immune system to CT section is diverse and requires the cooperation of many molecules in order to recruit macrophages to maintain normal taste function. ICAM-1, VCAM-1, and MCP-1 are upstream recruitment signals for macrophages that may ultimately affect the function of taste receptor cells.
    • Maintenance of AR Inactivation by S-nitrosylation

      Qin, Yu; Department of Biochemistry and Molecular Biology (2011-04)
      Prostate cancer is the second leading cause of cancer deaths in US men. Unregulated activation of the androgen receptor (AR) is associated with prostate cancer initiation and progression. Post-translational modifications of AR regulate its function, and we propose that nitric oxide (NO) synthase III (eNOS) and its product NO regulate prostate cancer cell growth via S-nitrosylation, a covalent addition of an NO group to a cysteine thiol, of AR. We found that S-nitrosylation levels were reduced in prostate cancer and prostatic intraepithelial neoplasia compared to normal adjacent tissues, and xD;1089-8603 (Linking)15566968
    • Marker Co-Expression Analysis of Initial Cellular Events in the Critical-Size Rat Calvarial Defect Model and the Effect of Bone Morphogenetic Protein-2 (rhBMP-2)

      Capetillo, Joseph F.; Department of Oral Biology (4/15/2016)
      Craniofacial defects can result from congenital malformations, trauma, tumor resection,periodontal disease, post-extraction ridge remodeling, and peri-implantitis. Regenerationof bone is critical to achieving functional and esthetic outcomes in the rehabilitation ofsuch defects. Traditional strategies for osseous regeneration include a multiple ofsurgical techniques utilizing autologous bone, cadaver-sourced allogeneic or xenogeneicbone, synthetic bone biomaterials, barrier membranes, or combinations thereof(Wikesjö, Qahash 2009). The need to enhance the predictability of regeneration inespecially large defects that cannot heal adequately without intervention (critical-sizedefects) has led to recent development of protein- and cell-based technologies.[Introduction, first paragraph]
    • Marketing Emergency Services: The Degree of Involvement Among Nurse Executives

      Byrd, Lura A.; Department of Physiological and Technological Nursing (1986-05)
      The purpese of this study is to determine the degree of invo1vement among nurse executi~es i~ marketi~g emergency s~rvices. Know1edge ther~by obtained will provide identification of cu~rent marketing.trends in emergency services as wel.l as areas ofmarketing content in education programs preparing nurse executfves. A questionnaire~ 11Emergency Services Marketing Activity Survey 11 (ESMAS)~ specific for marketing e~ergency Services was ·adapted from Kotler•s (1975) 11 Systematic Marketing Audit. 11 Items were dev-eloped far·· each major category (marketing environment; marketing system; and ma·rketing activity}'. purported· by Kotler (1975) to be·essentia1 in eva1uating marketing activities. The ESMAS was reviewed by a panel of fie1d experts including facu1ty invo1ved in teaching marketing and finance in hea)th care services. Based on recommendations from the pane1, severa1 items were revised and made less ambiguous. The revi sed vers i on of the ESMAS Questi onna i re was ma i 1 e.9 to Di rectors of Nurs i.ng (DON) and Emergency Department Head Nurses ( EDHN) in 114 Georgia hospitals 1isted by the American Hospital Association Gui de (1985} as· provi ders of emergency servi ces. Study subj ects were asked to respond on a sca 1 e of one to 'seven to .the de·gree to whi eh each marketing emergency services item is a part of their role as nurse executive. Responses were received from 42 DONs and 37 EDHNs. Descriptive information was compi1ed and t-test analysis was done to describe the V involvement of DONs and EDHNs in marketing emergency services and to describe the relationship between the involvement of DONs and EDHNs in their marketing involvement. Involvement was divided into three categories: a rating 1.0-2.9 was considered low involvement, 3.0-4.9 wa? considered moderate involvement,_ and 5.0:7.0 was considered h1gh involvement~ It was·found that·nurses were involved in marketing overall at the moderate level·. There we~e significant differences at the 0.029 level in the involvement of DONs and EDHNs in marketing environment category. There were no significant differences between the involvement of the two groups in marketing system nor in marketing activities categories.
    • Marketing hospital based obstetrical services : the degree of involvement among nurse executives

      Watford, Deborah L.; Master of Science (1987-04)
      The purpose of this study was to determine the degree of involvement among obstetrical nurse executives in marketing hospital-based obstetrical (OB) services. A descriptive nonexperimental design was.used. The "Obstetrical Services Marketing Activity Survey" (OSMAS) and a "General Data Questionnaire" (GDQ) were administered to 90 Directors of Nursing (DONs) for OB Services and 90 Labor and Delivery Head Nurses (LDHNs) in 90 hospitals located in three southern states. The OSMAS was adapted for obstetrical services from an original, unpublished tool entitled "Emergency Services Marketing Activity Survey" (Byrd, 1986). Responses were received from 26 DONs and 23 LDHNs, representing a 27% return rate. On a scale of one to seven, marketing involvement was arbitrarily divided into three categories: 1.0 to .2.9 wa$ designated as a low level of involvement; 3.0 to 4.9 was designated as a moderate level of involve.ment; and 5. 0 to 7. 0 was designated as a high level of inv.olvement. The results of this study indicated that OB nurse executives overall were involved in marketing at the moderate level (4.86 mean). !-Test analysis revealed no significant differences at the .05.level in the degree of involvement bet~een LDHNs and DONs in marketing OB servic_es. Through descriptive analysis, it wa~ found that the health care industry, P.ar~icularly OB.services, is competitive in nature. It was concluded that OB nurse executives are becoming more involved in marketing their hospital-based OB departments~ yet they are not educationally prepared to engage in marketing activities.
    • Maternal-Fetal Bonding and A Previous Spontaneous Abortion

      Elkins, Sharon; Department of Nursing (1985-10)
      Maternal-Fetal Bonding and a Previous Spontaneous Abortion. Sharon Sue Elkins. The purpose of this study was to investigate the relationship between the occurrence of a spontaneous abortion in a prior pregnancy and maternal-fetal bonding· in a. current pregnancy. Cranley•s Maternal;;;Fetal Attachment Sca'le and a demographic questionnaire were completed by 236 pregnant women attending either Lamaze classes or a prenatal clinic .. There was no significant difference found in maternal-fetal bonding, measured by the total score on Cranley•s Maternal-Fetal Attachment Sea 1 e, between wome·n who had experienced a spontaneous abortion .in the previous_ pregnancy and women who had not experienced· a spontaneous abortion in the previous pregnancy. However, on ·subscale (2) 11 interaction with the fetus 11 the scores of the spontaneous abortion group. were significantly lower than the scores of the lnonspontaneous abortion group. A1 s o, the spontaneous abortion group had a significantly greater variance than the nonspontaneous abortion group. No relationship was found between the number of weeks pregnant at the time of the spontaneous abortion and maternal~fetal bonding. -. Social class was found to have the greatest effect on maternal-fetal bonding of all the observed variables. Women in higher social classes had significantly higher total scores on the Maternal-Fetal Attachment Scale. Also-, Caucasian women and younger women had significantly \higher total scores on the Maternal-Fetal Attachment Scale. Women who were the greater number of weeks pregnant also had significantly higher scores ·on two of the subscales, 11 i·nteraction with the fetus" and 11 giving of self ...
    • Mathematical and Stochastic Modeling of HIV Immunology and Epidemiology

      Lee, Tae Jin; Department of Biostatistics and Epidemiology (8/3/2017)
      In HIV virus dynamics, controlling of viral load and maintaining of CD4 value at a higher level are always primary goals for the providers. In recent years, a new molecule was discovered, namely, eCD4-Ig, which mimics CD4 if introduced into the human body and has potential to change existing HIV virus dynamics. Thus, to understand dynamics of viral load, eCD4-Ig, CD4 cells, we have developed mathematical models by incorporating interactions between this new molecule and other known immunological, virological information. We further investigated model based speculations for management, and obtained the level of eCD4-Ig required for elimination of virus. Next, we built epidemiological model for HIV spread and control among discordant couple through dynamics of PrEP (Pre-exposure prophylaxis). For this, an actuarial assumptions based stochastic model is used to obtain the mean remaining time of couple to stay as discordant. We generalized single hook-up/marriage stochastic model to multiple hook-up/marriage model.
    • The Meaning of Life in Organ Transplant Recipients

      Jonason, Anna M.; Department of Physiological and Technological Nursing (1993-05)
      The purpose of this study was to explicate the meaning of life as experienced in a population of renal, cardiac, and liver transplant recipients. The method used was two-fold: A phenomenological design to explore qualities of the lived experience in subjective terms. A questionnaire provided measurable information for corroboration and validation. The theoretical perspective of will to meaning (Frankl, 1969) served as a basis for the study. This view suggests that the search for personal meaning is a primary motivating force for continued survival in human beings. A convenience sample of eleven vital organ transplant recipients participated in the study. Initially, the Life Attitude Profile-Revised (LAP-R) (Reker, 1992) was completed by each participant. This is a multidimensional, Likert-type instrument measuring attitudes toward life. Questionnaire completion was followed by semi-structured interviews. The two sets of data were examined separately. Interviews were analyzed according to phenomenological guidelines set forth by van Kaam (1966), leading to structural definition of the meaning of life for organ transplant recipients. LAP-R data were then analyzed. Analysis culminated in a syncretic integration of findings from both data sources. This provided a rich, contextual description of the indomitability of the human spirit. The meaning of life for organ transplant recipients was a complexity of interconnected aspects, reflecting a paradox of emotions and great intensity. It was at once evolutionary and revolutionary, comedy and tragedy, struggle between dependence and independence, and dream tempered by reality. Important themes described included drawing on internal sources of strength; having the support of family and friends; a desire to help others; acknowledgement of the contributions of a "greater force" to continued survival; some semblance of inner peace; a need to achieve one's purpose in life; and a sense of renewed responsibility for oneself and one's health. Findings from this study afford new insights for clinical nursing. These insights are grounded in improved mutual understanding between persons, which is a critical element for efficient health care planning and effective intervention.
    • A Measure of Satisfaction in Childbirth: The Degree of Women's Fulfillment of Childbearing Expectations

      Cooke, Paulette A.; Department of Nursing (1984-03)
      The ·purpose of .this . study wa~ . to . operationalize the· coJ;icept of . . satisfactiqn (based on· Porter's model). as it pertained to the l~bor and . delivery experi~nce. - 'The goal was to develop and test a tool for measuring a _wo1Jlan' s ·satisfaction with chil_dbirth. in terms. of the difference·· between ~hat was expected .and what· actually occurred.. Satisfaction was . defined as the deg-ree to which expectations were met. A convenience_sample of 50 women from a u.·s. Army hospital . . ' . . participated in this· study_. Of .the saJJiple,_ 44%. (n = 22) were primigrav~das, . . _and 56%_ (n. ~ _28) were multigravidas. A descriptive design· -w~s used for_ reliability a')ld valid~ty estimates~·. The results- of conte~t validity indicated that 4ll of . 'the· itens were consid~red relevant for a measure .· of satisfac-tion with the chilQ.birth experience. There .was 84% agreement ·by experts ob··· item ·(n =-.. :46) .placem~nt into appropriate .subscales . (self, physical care, ·sup_port). The reliability_ coefficient. (Cro11bach' s Alpha) for the Cooke Satisfaction· Scale was • 89. For each subscale, the : reliabilit:J;es (alpha)· were: self,_ .48;, physical care,· .74.;.-and support, .8·6. ·The correlation .for the Cooke Satisfaction Scale (Part A) and the· Marui: and· Mercer Attitude· Scale ·for. convergent construct validity was · .53. The· correlation for, the Cooke Satisfaction Scale (Part A - Par-t ·B) with the Marut and Mercer Scale· for divergent construct. validity· was .1s-.~ On the basi~ of reliability_ and validity coefficients. obtained, it. was concluded that the Cooke Satisfaction Sc·ale is a usefui tool for the · measurement .of ·satisfaction in childl;»earirig .. women.
    • 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.
    • 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.
    • 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 1 Diabetes Mellitus

      Bell, Tracy D.; Department of Physiology (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 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 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 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 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.