Dou, Huijuan; Department of Physiology (1/25/2018)
      A disintegrin and metalloproteinase ADAM17 (tumor necrosis factor–converting enzyme) regulates soluble TNF levels. We tested the hypothesis that aging-induced activation in adipose tissue (AT)-expressed ADAM17 contributes to the development of remote coronary microvascular dysfunction in obesity. We found that the increased activity of endothelial ADAM17 is mediated by a diminished inhibitory interaction with caveolin-1, due to age-related decline in caveolin-1 expression in obese patients and mice or to genetic deletion of caveolin-1. Coronary arterioles (CA) and AT were examined in patients who underwent heart surgery. Excess, ADAM17-shed TNF from AT arteries in older obese patients was sufficient to impair CA dilation in a bioassay in which the AT artery was serially connected to a CA. CA and AT were also studied in 6-month and 24-month lean and obese mice. We found that obesity elicited impaired endothelium-dependent CA dilations only in older patients and in aged obese mice. Transplantation of AT from aged obese, but not from young or aged, mice increased serum cytokine levels, including TNF, and impaired CA dilation in the young recipient mice. In patients and mice, obesity was accompanied by age-related activation of ADAM17, which was attributed to vascular endothelium–expressed ADAM17. Additionally, ADAM17 mediates shedding of JAM-A (junctional adhesion molecule-A). We hypothesized that ADAM17 activation, via increased JAM-A shedding impairs flow mechanosensing and induces abnormal artery remodeling in aging. We found a reduced lumen diameter and increased wall thickness in AT of aged patients. ECs using plasmid JAM-A were aligned to flow direction earlier than GFP treated control cells. Site-directed mutagenesis was employed to generate JAM-A cleavage resistant mutants, we detected soluble JAM-A in the supernatants from cells transfected with plasmid JAM-A, but not from cells expressing mutant JAM-A plasmids. Importantly, soluble JAM-A is significantly increased in the supernatant from cells with combined action of plasmid JAM-A and recombinant ADAM17, when compared to cells with plasmid JAM-A alone. Collectively, our data revealed that age-related reduction in Cav-1 expression and subsequently increased the activity of endothelial ADAM17 led to excess TNF production, which acts remotely to promote coronary arteriole dysfunction. Whereas activation of ADAM17 in vascular endothelium mediates increased JAM-A shedding and causes ECs misalignment. Our data suggest that the combined action of TNF and JAM-A lead to development of CMD and its related vascular remodeling in older obese patients.
    • Adaptive Cerebral Neovascularization in a Model of Type 2 Diabetes

      Schreihofer, Derek A.; Fagan, Susan C.; Ergul, Adviye; Li, Weiguo; Prakash, Roshini; Kelley-Cobbs, Aisha I.; Ogbi, Safia; Kozak, Anna; El-Remessy, Azza B.; Department of Physiology; et al. (2010-01200)
      OBJECTIVE: The effect of diabetes on neovascularization varies between different organ systems. While excessive angiogenesis complicates diabetic retinopathy, impaired neovascularization contributes to coronary and peripheral complications of diabetes. However, how diabetes influences cerebral neovascularization is not clear. Our aim was to determine diabetes-mediated changes in the cerebrovasculature and its impact on the short-term outcome of cerebral ischemia.
    • Adrenal Zona Glomerulosa Targeting in Transgenic Mice

      Parmar, Jeniel; Department of Physiology (2009-12)
      The final step in the production of aldosterone is performed by the enzyme aldosterone synthase (CYP11B2). CYP11B2 is primarily expressed in the zona glomerulosa (ZG) of the adrenal cortex. Adrenocortical expression of CYP11B2 is primarily regulated by circulating levels of angiotensin II (Ang II) and K+, but the molecular mechanisms that control its ZG-specific expression are not clearly defined. Considerable in vitro analyses have been performed towards defining the mechanisms that control CYP11B2 expression. Previous studies from our laboratory and others have identified several c/s-regulatory elements on the 5' flanking promoter region (at -71/64, -129/114, -351/343 and -773/766) that regulate basal expression as well as maximal stimulation of CYP11B2 gene transcription. Moreover, key transcription factors that bind these c/s-regulatory regions including NGFIB, NURR1, SF-1 and COUP-TF have also been identified. Hence, through several in vitro analyses, a considerable evidence exists supporting the contention that these regulatory elements found within the 5' flanking promoter region may control ZG-specific expression of CYP11B2 gene. However, thus far, all evidence is based on in vitro analyses of transcriptional regulation, which does not always depict in vivo occurrences. To initiate our in vivo assessment of CYP11B2 promoter, we began by comparing the DNA sequences between human, mouse, and rat CYP11B2 genes, which interestingly revealed high sequence similarity in the 5' flanking promoter region of the CYP11B2 gene. This result suggested that the cisregulatory regions identified by in vitro analyses likely plays an important role in CYP11B2 ZG-specific gene expression. Therefore, we generated transgenic mouse lines by pronuclear injection of a Transgenic (Tg) DNA construct containing 985 base pairs (bp) of the mouse Cyp11b2 promoter driving expression of a LacZ reporter gene. Importantly, 4 founder Tg mouse lines revealed LacZ expression exclusively in the adrenal ZG. Mice fed a normal sodium diet (0.3 %) and a low sodium diet (0.03 %) showed LacZ mRNA expression exclusively in adrenal tissue. Furthermore, (3-galactosidase protein (the product of LacZ) was localized solely in the ZG of the Tg mice. Hence, the role of the proximal promoter region of the Cyp11 b2 gene was confirmed, in vivo, as this region allowed induction of LacZ exclusively in the adrenal ZG of Tg mice. Moreover, with the expression of LacZ properly restricted to adrenal ZG, we concluded that regions required for Cyp11b2 gene repression in the adjacent inner two zones of the adrenal cortex were also confined within the 985 bp promoter. This regulatory fragment will be an invaluable tool for adrenal ZG targeting of genes believed to play a role in adrenocortical diseases and aldosterone dysregulation. While developing Tg mice, we also focused on characterization and development of novel adrenocortical cell lines. As aforementioned, in vitro culture models have allowed a multitude of studies that have broadened our understanding of normal adrenocortical endocrine function. Primary cultures of adrenocortical cells have been an excellent source for in vitro studies. However, the eventual onset of senescence in primary cultures of cells creates a recurring need for the costly and difficult isolations of fresh adrenocortical cells. Hence, the use of primary cultures has been increasingly supplemented by immortalized cell lines. We utilized an adrenocortical carcinoma to develop a human adrenocortical cell line. We entitled it the human adrenocortical carcinoma cell line clone 15 (HAC15). HAC15 represents only the second human adrenocortical cell line available that exhibits physiological hormonal responses, steroidogenesis, and expression of steroid-metabolizing enzymes. The ability of HAC15 to respond to Ang II, K+, and ACTH makes it the first adrenal cell line capable of responding to the three main physiologic regulators of the adrenal cortex.
    • Angiotensin II Regulation of Aldosterone Synthase

      Nogueira, Edson da F.; Department of Physiology (2009-07)
      Angiotensin II (Ang II) is the major physiological regulator of aldosterone production acting acutely to stimulate aldosterone biosynthesis and chronically to increase the capacity of the adrenals to produce aldosterone. Aldosterone is principally synthesized in the zona glomerulosa of the adrenal by a series of enzymatic reactions leading to the conversion of cholesterol to aldosterone. The major goal of our study was to define the Ang II-induced mechanisms regulating the expression of aldosterone synthase (CYP11B2) in adrenocortical cells. We approached the analysis of the protein synthesis-dependent regulation of this enzyme by defining, through microarray and real time PCR analysis, the transcription factors that are rapidly induced by Ang II incubation of adrenocortical cell models from three species (human, bovine, and rat). The gene list generated by this comparison included: ATF3, BTG2, NR4A1, NR4A2, NR4A3, EGR1, FOS, FOSB, and JUNB. Importantly, pretreatment of H295R cells with cycloheximide had no effect on Ang II induction of these genes, suggesting that they are direct targets of Ang II signaling. Co-transfection studies, used to investigate the role of these transcription factors in the regulation of CYP11B2, determined that out of the nine transcription factors listed above, only the NGFI-B family members (NGFI-B, NURR1, and NOR1) increased expression of CYP11B2. The importance of NGFI-B in the regulation of CYP11B2 was confirmed by the decrease in CYP11B2 expression in the presence of a dominant-negative (DN)- NGFI-B. A pharmacological approach used to characterize the Ang II pathways regulating transcription of NGFI-B family genes suggested that Ang II binding to the AT1R increases activity of protein kinase C (PKC), Ca -dependent calmodulin kinases (CaMK), and SRC kinase (SRC), which act to regulate the expression of the family of NGFI-B genes as well as CYP11B2. In the current study we also analyzed protein synthesis-independent mechanisms regulating CYP11B2 expression. We studied the role of the ATF/CREB family of transcription factors (ATF1, ATF2, CREB, and CREM), which may bind the cAMP response element (CRE) in the promoter region of the CYP11B2 gene. Importantly, analysis of these transcription factors in the human H295R adrenocortical cell line revealed very low expression of CREB in comparison to the other CRE-binding proteins herein studied. We investigated Ang II-induced phosphorylation of these transcription factors, their binding to the promoter region of CYP11B2, and their effect on CYP11B2 expression. Ang II time-dependently induced phosphorylation of ATF1, ATF2, and CREM in H295R cells. The association of these transcription factors with the CYP11B2 promoter region was induced by Ang II and K+. Transfection of siRNA for ATF1, ATF2, and CREM significantly reduced CYP11B2 expression in Ang II-stimulated conditions. Expression of NURR-1 alone or with constitutively active ATF1, ATF2, CREB, and CREM increased the promoter activity of CYP11B2 in H295R cells. In summary, Ang II rapidly induces expression of newly synthesized transcription factors as well as the phosphorylation of transcription factors already present in the adrenocortical cell. These events are followed by increased CYP11B2 expression and, therefore, represent important mechanisms to increase the adrenal capacity to produce aldosterone.
    • Assessment of Renal Ischemia Reperfusion Induced Injury in Male and Female Rats

      Crislip, Gene Ryan; Department of Physiology (2017)
      Acute kidney injury (AKI) is a clinical problem often induced by ischemia reperfusion (IR). Males are reported to have worse outcomes following IR compared to females based on measurements of blood urea nitrogen and creatinine. However, these markers are produced at different levels depending on body mass. The goal of Aims 1 and 2 was to do a complete assessment of the impact of sex on IR to establish a model that displays a sex difference. We measured multiple markers, including inulin clearance which is the gold standard of determining renal function. We determined there is no sex difference in response to IR after 24 hours. However, males had impaired renal function, higher vascular congestion and tubular injury than females 7 days following IR. A consequence of vascular congestion and tubular injury is fluid leakage into interstitial space, which increases renal volume. The goal of Aim 3 was to determine if ultrasound could be used as a tool to detect progressive changes in regional kidney volume following IR. To do this, we compared renal volume measurements with stereological assessment and examined the use of renal volume as an injury marker following IR. We verified the use of ultrasound to monitor renal volume after IR and the changes in volume correlated with the extent of medullary injury. Limiting vascular congestion improves recovery following IR. Pericytes are contractile cells that line the vessels in the renal medulla that are prone to congestion following IR. The goal of Aim 4 was to determine the role of renal pericytes following IR. To do this, we decreased pericytes in rats before IR to determine if this effected injury. We found that lower pericyte density was associated with greater vascular congestion following IR, additionally, males lose more pericytes than females. From these studies, we concluded that there was no sex difference in IR induced injury after 24 hours, however, following 7 days males had poorer recovery than females. We hypothesize that this poorer recovery is attributed to less pericytes in males following IR resulting in the inability to reduce vascular congestion compared to females.
    • Blood pressure impacts the renal T cell profile of male and female spontaneously hypertensive rats

      Tipton, Ashlee J.; Department of Physiology (2014-03)
      Of the 68 million Americans with hypertension, fewer than 46% have their blood pressure (BP) adequately controlled and women are more likely than men to have uncontrolled hypertension. This underscores the critical need for new treatment options; however, this is a challenge due to our lack of knowledge regarding the mechanism(s) driving essential hypertension. T cells have been implicated in hypertension in males. Prior to our work, the role of T cells in hypertensive females had been unexplored. We demonstrate that female spontaneously hypertensive rats (SHR) have a decrease in BP in response to an immunosuppressant, supporting an immune component to their hypertension. We further defined a sex difference in the renal T cell and cytokine profile in SHR. Female SHR have a more anti-inflammatory immune profile in their kidneys than males. To gain insight into the mechanisms mediating sex differences in the immune profile, male and female SHR were gonadectomized. Gonadectomy increased pro-inflammatory markers in both sexes and attenuated anti-inflammatory markers particularly in females. Therefore, while both male and female sex hormones promote an anti-inflammatory immune profile, female ii sex hormones contribute greater to their more anti-inflammatory profile, but do not explain the sex difference. To determine the impact of hypertension on the renal immune profile, experiments measured renal T cells and cytokines in hypertensive male and female SHR, normotensive Wistar Kyoto rats (WKY), and SHR treated with antihypertensive therapy. All T cells and cytokines measured were higher in SHR compared to the same sex WKY. Moreover, antihypertensive therapy decreased renal Tregs only in female SHR. These data suggest that increased BP in both sexes is associated with an increase in renal inflammation; however female SHR have a compensatory increase in renal Tregs in response to increases in BP. TGF-β is a key cytokine regulating Treg and Th17 differentiation and we found that female SHR express more TGF-β than males. Experiments assessed if female SHR possessed a sex hormone or BP-mediated increase in renal TGF- β corresponding with increases in Tregs. We determined that loss of female sex hormones and increased BP in female SHR increase renal TGF-β expression. We conclude that BP status drive sex differences in the renal T cell and cytokine profile of SHR.
    • Cortical gene transcription response patterns to water maze training in aged mice

      Park, Sung-Soo; Stranahan, Alexis M.; Chadwick, Wayne; Zhou, Yu; Wang, Liyun; Martin, Bronwen; Becker, Kevin G.; Maudsley, Stuart; Department of Physiology (2011-06-29)
      Background: The hippocampus mediates the acquisition of spatial memory, but the memory trace is eventually transferred to the cortex. We have investigated transcriptional activation of pathways related to cognitive function in the cortex of the aged mouse by analyzing gene expression following water maze training.
    • Developmental Changes in Hemodynamic Responses and Cardiovagal Modulation during Isometric Handgrip Exercise

      Goulopoulou, Styliani; Fernhall, Bo; Kanaley, Jill A.; Department of Physiology (2010-08-29)
      The purpose of this study was to examine differences in pressor response and cardiovagal modulation during isometric handgrip exercise (IHG) between children and adults. Beat-to-beat heart rate (HR) and blood pressure were measured in 23 prepubertal children and 23 adults at baseline and during IHG. Cardiovagal modulation was quantified by analysis of HR variability. Mean arterial pressure responses to IHG were greater in adults compared to children (P < .05) whereas there were no group differences in HR responses (P > .05). Children had a greater reduction in cardiovagal modulation in response to IHG compared to adults (P < .05). Changes in mean arterial pressure during IHG were correlated with baseline cardiovagal modulation and force produced during isometric contraction (P < .05). In conclusion, differences in pressor reflex response between children and adults cannot be solely explained by differences in autonomic modulation and appear to be associated with factors contributing to the force produced during isometric contraction.
    • Dietary potassium supplementation improves vascular structure and ameliorates the damage caused by cerebral ischemia in normotensive rats.

      Rigsby, Christiné Spring; Pollock, David M; Dorrance, Anne M; Department of Physiology; Vascular Biology Center (2008-03-11)
      ABSTRACT: BACKGROUND: Dietary potassium supplementation in hypertensive rats is cardioprotective. This protection includes a blood pressure independent reduction in the amount of damage caused by cerebral ischemia. Therefore, we hypothesized that dietary potassium supplementation would improve the outcome of ischemic stroke by improving cerebral vessel structure in normotensive rats. METHODS: Wistar Kyoto (WKY) rats were fed a high (HK) or low potassium (LK) diet for six weeks from six weeks of age. At the end of treatment, cerebral ischemia was induced by middle cerebral artery (MCA) occlusion and the resultant infarct was quantified and expressed as a percentage of the hemisphere infarcted (%HI). MCA structure was assessed in an additional group of rats using a pressurized arteriograph. RESULTS: The cerebral infarct was significantly smaller in rats fed the HK diet, compared to rats fed the LK diet (21 +/- 5.4 vs 33.5 +/- 4.8 %HI HK vs LK p < 0.05). Vessel structure was improved in WKY rats fed the HK diet as indicated by an increase in the MCA lumen (298 +/- 6.3 vs 276 +/- 3.9 mum HK vs LK p < 0.05) and outer diameters (322 +/- 7.6 vs 305 +/- 4.8 mum HK vs LK p < 0.05). Wall thickness and area were unchanged, suggesting an outward euthrophic remodelling process. The HK diet had no effect on body weight or telemetry blood pressure. CONCLUSION: These studies are the first to show a beneficial effect of dietary potassium in rats with normal blood pressure.
    • The Effect of Mineralocorticoid Receptor Antagonism on Ischemic Infarct Size

      Rigsby, Christiné Spring; Department of Physiology (2006-12)
      Stroke is the third leading cause of death and the leading cause of long-term disability in the United States, where approximately 88% of stroke occurrences are ischemic in origin. Hypertension is a primary risk factor for stroke. Elevated aldosterone levels have also been identified as a risk factor for stroke, as patients with primary aldosteronism incur increased incidences of cardiovascularrelated pathologies than do patients with essential hypertension. Previous studies from our laboratory have shown that mineralocorticoid (aldosterone) receptor (MR) activation can induce deleterious vascular remodeling and, conversely, blockade of the MR with spironolactone reduces cerebral infarct size in male spontaneously hypertensive stroke-prone rats (SHRSP). It is known from studies in SHRSP that cerebral vessel structure is directly related to infarct size. We hypothesized that chronic spironolactone treatment would alter cerebral vessel structure. Six-week-old male SHRSP were treated with spironolactone for six weeks and passive vessel structure was analyzed using a pressurized arteriograph. Spironolactone treatment prevented cerebral vessel remodeling. From a clinical standpoint, many patients present with pre-existing vascular damage; therefore, we hypothesized that chronic MR antagonism would reverse existing vascular damage. Twelve-week-old male SHRSP were treated as described above. Interestingly, spironolactone treatment partially reversed existing cerebral vessel remodeling. Recent analysis of data from the Framingham Heart Study show that females may be more sensitive to the effects of aldosterone, but few studies looking at MR blockade have been performed in females. Similar ischemic stroke and vascular analysis studies were performed in 12-week-old female SHRSP. Contrary to the male studies, MR antagonism, using spironolactone or eplerenone, did not reduce damage from ischemic stroke or improve vessel structure. MR protein expression was evaluated in cerebral arteries collected from 12-week-old male and female SHRSP using Western blot analysis. Surprisingly, female SHRSP had increased MR expression, compared to male SHRSP. These novel studies uncover an apparent sexual dimorphism in the actions of MR antagonists and expression of the MR in SHRSP. The action of the MR antagonists may be influenced by differential MR expression and this could help to explain the sex difference observed.
    • The Effects of Endocrine Dysfunction on the Cerebrovasculature and Stroke

      Osmond, Jessica Martin; Department of Physiology (2010-02)
      Stroke is the third leading cause of death in the United States and the leading cause of disability among adults.1 According to the American Heart Association Heart Disease and Stroke 2008 Update,1 there is, on average, 1 stroke every 40 seconds in the United States, and every 3-4 minutes someone dies of a stroke. Furthermore, stroke is a major financial burden on the healthcare system, and its prevalence will only continue to increase as the prevalence of its risk factors continues to increase. These risk factors include but are not limited to hypertension, atherosclerosis, diabetes, smoking, and obesity. Currently, therapy for ischemic stroke is very limited. The only approach for acute treatment of ischemic stroke approved by the Food and Drug Administration is clot lysis with tissue plasminogen activator (tPA), based on findings from the National Institute of Neurological Disorders and Stroke recombinant tPA Stroke Study Group demonstrating improvement in clinical outcome with tPA therapy compared to placebo-control patients.2 Patients receiving tPA therapy showed improvements in neurological outcome 3 months after stroke compared to patients receiving placebo. However, the window of treatment for tPA is very small and only a small percentage (<10%) of eligible 2 patients receive therapy. Although efforts to develop safe and effective treatment options for stroke patients continue, a gap remains between promising findings from the bench and positive results in clinical trials. In light of this, it is especially important that research is also focused on gaining better understanding of mechanisms that lead to increased stroke occurrence and increased stroke damage in order to make progress in diminishing the occurrence of stroke.
    • The effects of hypertension on neurovascular unit function and structure

      Iddings, Jennifer Ann; Department of Physiology (2015)
      Functional hyperemia is the regional increase in cerebral blood flow upon increases in neuronal activity which ensures that the metabolic demands of the neurons are met. Hypertension is known to impair the hyperemic response; however, the neurovascular coupling mechanisms by which this cerebrovascular dysfunction occurs have yet to be fully elucidated. The goal of this dissertation project was to test the central hypothesis that hypertension-induced impairments in functional hyperemia are mediated by a specific disruption of communication within the neurovascular unit at the parenchymal arteriole level of the cerebrovascular tree. To test our hypothesis, we measured parenchymal arteriole reactivity, vascular smooth muscle cell Ca2+ dynamics, parenchymal arteriole remodeling and cerebral vascular density in cortical brain slices from normotensive (WKY) and hypertensive (SHR) rats. We found that vasoconstriction in response to the thromboxane A2 receptor agonist U46619 and basal vascular smooth muscle cell Ca2+ oscillation frequency were increased in parenchymal arterioles from SHR. In perfused and pressurized parenchymal arterioles, myogenic tone was increased in SHR. While K+-induced parenchymal arteriole dilations were similar in WKY and SHR, metabotropic glutamate receptor activation-induced parenchymal arteriole dilations were enhanced in SHR. Further, neuronal stimulation-evoked parenchymal arteriole dilations were similar in SHR and WKY. Parenchymal arteriole wall to lumen ratio and wall thickness were increased in SHR. Vascular density was also increased in deeper cortical layers in SHR. Our data indicate that although SHR parenchymal arterioles display vascular remodeling, neurovascular coupling is not impaired in SHR, at least at the parenchymal arteriole level.
    • Endothelin Receptor A Function in Progressive Kidney Disease

      Heimlich, Jonathan B.; Department of Physiology (2014-05)
      Endothelin-1 (ET-1) is a vasoactive peptide that regulates electrolyte and arterial blood pressure homeostasis. For this reason, ET-1 is increased under conditions of high dietary salt intake but also contributes to the progression of a variety of forms of kidney disease associated with hypoxia including sickle cell nephropathy. We previously found that ET-1 is an integral factor in glomerular injury seen during diabetic nephropathy through the activation of the ETA receptor. Recent studies also suggest ET-1 via the ETA receptor mediate the upregulation of reactive oxygen species (ROS) contributing to tissue damage. The overall aim of this work is to understand the role of ETA receptor activation in the development of sickle cell nephropathy and to determine the therapeutic potential of an ETA receptor antagonist in the treatment of renal manifestations associated with sickle cell disease (SCD). Since ET-1 is elevated and hypoxia is a prevalent occurrence in SCD, we utilized several strategies to elucidate the actions of ET-1 via the ETA receptor including ET-1 induction with a high salt diet and a hypoxic challenge to re-create an environment similar to SCD. The first aim was designed to determine whether ET-1 derived from endothelial cells contributes to oxidative stress in the glomerulus of mice subjected to a high salt diet and/or hypoxia. Hypoxia increased glomerular ET-1 mRNA expression in control, but not in vascular endothelial cell ET-1 knockout (VEET KO) mice. Increased superoxide formation was detected in the cortices of mice exposed to acute hypoxia. Under normoxic conditions, mice on a high salt diet had approximately 150% higher glomerular ET-1 mRNA expression compared to a normal salt diet. High salt diet administration also significantly increased glomerular ROS production in flox control, but not in glomeruli isolated from VEET KO mice. In C57BL6/J mice, the ETA receptor selective antagonist, ABT-627, significantly attenuated the increase in glomerular ROS production produced by high salt diet. In addition, chronic infusion of a sub-pressor dose of ET-1 via miniosmotic pumps in C57BL6/J mice significantly increased levels of glomerular ROS that were prevented by ETA antagonist treatment. Finally, three-hour exposure to hypoxia (8% O2) in control C57BL6/J mice significantly increased urinary protein excretion during the 24 hrs following hypoxia, but only in animals on a high salt diet. In conclusion, these data suggest that both hypoxia and a high salt diet increases glomerular ROS production via endothelial derived ET-1-ETA receptor activation and provide a potential mechanism for ET-1 induced nephropathy. The second aim was to test whether ET-1 acting via the ETA receptor contributes to renal injury in a mouse model of SCD. Humanized knockout, knockin sickle mice were used to describe the endothelin phenotype in the kidney. Sickle mice have increased ET-1 mRNA expression in both the cortex and glomeruli compared to heterozygous controls. Sickle animals have increased renal cortex ETA receptor mRNA expression but similar levels of ETB receptor mRNA expression. Radiolabeled ligand binding assays revealed sickle mice had increased ET-1 binding and subsequently increased ETA receptor binding in the renal vessels when compared to control mice. In response to PMA stimulation, sickle mice had increased glomerular ROS compared to controls, which could be prevented by treatment with ABT-627. Protein and nephrin excretion are two urinary markers of renal injury and were both found to be elevated in our mouse model of SCD. Treatment with ABT-627 resulted in significant decreases in both nephrin and protein excretion in SCD mice. Finally, 1 week of ETA antagonism also caused a significant decrease in mRNA expression of NADPH oxidase subunits as well as VEGF mRNA expression, both of which are thought to contribute to pathologic processes in chronic kidney disease. These data indicate a novel role for ET-1 in the progression of SCN, specifically via the ETA receptor in the glomerulus and suggest ETA antagonism to be a viable treatment strategy for SCN. Taken together, these studies reveal a prominent role for the pathologic activation of the ETA receptor in the progression of kidney diseases where ET-1 is upregulated and hypoxia is prevalent. ET-1 is known to contribute to other chronic kidney diseases such as diabetic nephropathy and significant benefit is found through ETA antagonism. These data offer a rationale for a novel treatment modality in renal disease associated with SCD.
    • Evidence for a Developmental Role for TLR4 in Learning and Memory

      Okun, Eitan; Barak, Boaz; Saada-Madar, Ravit; Rothman, Sarah M.; Griffioen, Kathleen J.; Roberts, Nicholas; Castro, Kamilah; Mughal, Mohamed R.; Pita, Mario A.; Stranahan, Alexis M.; et al. (2012-10-11)
      Toll-like receptors (TLRs) play essential roles in innate immunity and increasing evidence indicates that these receptors are expressed in neurons, astrocytes and microglia in the brain where they mediate responses to infection, stress and injury. Very little is known about the roles of TLRs in cognition. To test the hypothesis that TLR4 has a role in hippocampus-dependent spatial learning and memory, we used mice deficient for TLR4 and mice receiving chronic TLR4 antagonist infusion to the lateral ventricles in the brain. We found that developmental TLR4 deficiency enhances spatial reference memory acquisition and memory retention, impairs contextual fear-learning and enhances motor functions, traits that were correlated with CREB up-regulation in the hippocampus. TLR4 antagonist infusion into the cerebral ventricles of adult mice did not affect cognitive behavior, but instead affected anxiety responses. Our findings indicate a developmental role for TLR4 in shaping spatial reference memory, and fear learning and memory. Moreover, we show that central TLR4 inhibition using a TLR4 antagonist has no discernible physiological role in regulating spatial and contextual hippocampus-dependent cognitive behavior.
    • Genetic and gene expression analyses of the polycystic ovary syndrome candidate gene fibrillin-3 and other fibrillin family members in human ovaries.

      Prodoehl, Mark J; Hatzirodos, Nicholas; Irving-Rodgers, Helen F; Zhao, Zhen Z; Painter, Jodie N; Hickey, Theresa E; Gibson, Mark A; Rainey, William E; Carr, Bruce R; Mason, Helen D; et al. (2009-11-13)
      Several studies have demonstrated an association between polycystic ovary syndrome (PCOS) and the dinucleotide repeat microsatellite marker D19S884, which is located in intron 55 of the fibrillin-3 (FBN3) gene. Fibrillins, including FBN1 and 2, interact with latent transforming growth factor (TGF)-beta-binding proteins (LTBP) and thereby control the bioactivity of TGFbetas. TGFbetas stimulate fibroblast replication and collagen production. The PCOS ovarian phenotype includes increased stromal collagen and expansion of the ovarian cortex, features feasibly influenced by abnormal fibrillin expression. To examine a possible role of fibrillins in PCOS, particularly FBN3, we undertook tagging and functional single nucleotide polymorphism (SNP) analysis (32 SNPs including 10 that generate non-synonymous amino acid changes) using DNA from 173 PCOS patients and 194 controls. No SNP showed a significant association with PCOS and alleles of most SNPs showed almost identical population frequencies between PCOS and control subjects. No significant differences were observed for microsatellite D19S884. In human PCO stroma/cortex (n = 4) and non-PCO ovarian stroma (n = 9), follicles (n = 3) and corpora lutea (n = 3) and in human ovarian cancer cell lines (KGN, SKOV-3, OVCAR-3, OVCAR-5), FBN1 mRNA levels were approximately 100 times greater than FBN2 and 200-1000-fold greater than FBN3. Expression of LTBP-1 mRNA was 3-fold greater than LTBP-2. We conclude that FBN3 appears to have little involvement in PCOS but cannot rule out that other markers in the region of chromosome 19p13.2 are associated with PCOS or that FBN3 expression occurs in other organs and that this may be influencing the PCOS phenotype.
    • Genetic mutations cause primary aldosteronism

      Hattangady, Namita G; Department of Physiology (2014-10)
      The human adrenal glands are complex endocrine organs that are physiologically located above the kidney. The cortex of the adrenal gland may be considered as a combination of three different steroidogenic tissue-types which form concentric zones within each adrenal. The three cortical zones include zona glomerulosa (ZG), zona fasciculata (ZF) and zona reticularis (ZR). Each zone, under independent regulation, produces unique steroid(s) which exhibit specific functions. The outermost ZG layer secretes the steroid, aldosterone due to ZG specific expression of aldosterone synthase (CYP11B2). Aldosterone regulates sodium reabsorption, and therefore, blood pressure. Aldosterone production is tightly regulated by the renin-angiotensin-aldosterone system. Thus, aldosterone levels are in direct proportion with renin levels. Other known physiological regulators of aldosterone production include serum K+ and adrenocorticotrophic hormone. A type of endocrine hypertension termed ‘Primary Aldosteronism’ (PA), is characterized by aldosterone secretion under suppressed renin levels. PA accounts for almost 10 % of hypertension. More recently, genetic mutations in an inward rectifying K+ channel (KCNJ5) that occur as both, somatic and germline cases, have been implicated in the pathology of PA. The goal of this dissertation is to define the role of KCNJ5 mutations in PA. In this dissertation, I will summarize my studies that describe the acute and chronic events involved in mutated KCNJ5 mediated aldosterone excess. In addition, I will define a novel mutation in KCNJ5 of germline nature identified at Georgia Regents University. Finally, I will also describe some interesting lessons we learnt from the expression of mutated KCNJ5 in primary cultures of human adrenals. The prevalence of a hereditary form of PA termed as Familial Hyperaldosteronism type III (FH III) is very rare. Thus far, only a few mutations in the KCNJ5 gene, including T158A, G151R, G151E and I157S, are confirmed as causing FH III, following Mendelian genetics. Perhaps the most interesting feature of this disease is the varied phenotype between the different mutations. T158A-affected patients present with massive hyperplasia and require bilateral adrenalectomy. In contrast, patients affected by the G151E mutation have more severe hypertension, although their adrenals are near normal in appearance. In this study we identify a new germline mutation (Y152C). The index case was a 61 year old woman who underwent unilateral adrenalectomy. The patient with the Y152C mutation exhibited a milder hypertension phenotype (like the G151E-affected patient) with extensive hyperplasia (as seen in the T158A-affected patient). In vitro analyses of the Y152C mutation indicated a pathology similar to other known mutations in KCNJ5, including change in conductance to Na+ ions and elevated calcium levels, and increase in CYP11B2 mRNA and aldosterone production. The inherent challenge presented by current studies utilizing constitutive expression of KCNJ5 mutations is the limitation in studying acute temporal events such as post translational modifications of steroidogenic enzymes and transcription factors. To address this issue, we generated a doxycycline inducible cell model system for the T158A harboring KCNJ5 transgene. Herein, we demonstrate a useful system that was amenable to the study of acute and chronic events involved in mutant-KCNJ5 mediated aldosterone excess. Our findings suggest that mutant KNCJ5 increases CYP11B2 expression through the activation of transcriptional activators of CYP11B2. Additionally, this is the first study to demonstrate that mutant KCNJ5 also activates steroidogenic acute regulatory protein (StAR) at the levels of translation and post translational phosphorylation. We also demonstrate calcium channel blocker, verapamil as an efficient blocker of mKCNJ5 mediated aldosterone production. Finally, one of the sharp advantages of our study was the use of primary cultures of human adrenal cells to confirm the effects of mutated KCNJ5. Interestingly, transduction of cells with constitutive viruses for mutant KCNJ5, confirmed an increase in KCNJ5 mRNA, although no change in CYP11B2 expression levels was observed. Pilot data including treatment of primary cells with calcium ionophores indicated that ZF/ZR cells may have a phenotype that is ‘muted’ for calcium mediated pathways. We could also speculate that this may disprove some current hypotheses that APA harboring KCNJ5 mutations may originate from the ZF. Overall, this study has improved our knowledge regarding the pathogenesis of PA caused by KCNJ5 mutations and has identified verapamil as a potentially effective therapeutic strategy in the inhibition of aldosterone excess in this type of PA.
    • A hypothesis concerning a potential involvement of ceramide in apoptosis and acantholysis induced by pemphigus autoantibodies.

      Bollag, Wendy B; Department of Physiology; Department of Medicine; Department of Orthopaedic Surgery; Department of Cellular Biology and Anatomy (2010-06-29)
      Autoimmune diseases affect more than 50 million Americans, resulting in significant healthcare costs. Most autoimmune diseases occur sporadically; however, endemic pemphigus foliaceus (EPF) is an autoimmune skin disease localized to specific geographic loci. EPF, and the related diseases pemphigus vulgaris (PV) and pemphigus foliaceus (PF), are characterized by skin lesions and autoantibodies to molecules found on epidermal keratinocytes. A variant of EPF in patients from El Bagre, Colombia, South America, has recently been reported to be distinct from previously described loci in Brazil and Tunisia epidemiologically and immunologically. As in PF and EPF, El Bagre EPF patients exhibit autoantibodies towards desmoglein-1, a cell adhesion molecule critical for maintaining epidermal integrity. An association of El Bagre EPF with sun exposure has been detected, and ultraviolet irradiation also exacerbates symptoms in PV, PF and EPF. Our hypothesis is that: (1) the autoantibodies generate pathology through an alteration in ceramide metabolism in targeted keratinocytes, resulting in apoptosis and/or cell death and acantholysis, but only when the cell's ability to metabolize ceramide is exceeded, and (2) apoptosis in response to this altered ceramide metabolism is initiated and/or exacerbated by other agents that increase ceramide levels, such as cytokines, ultraviolet irradiation, and senescence.
    • The Implementation of Controlled Physical Training and the Physiological Effects of Exercise in Women with Sickle Cell Disease

      Ramsey, Leigh-Taylor; Department of Physiology (1999-12)
      Sickle cell disease (SCD) comprises a group of genetic disorders of hemoglobin that affect more than 70,000 Americans and is associated with increased morbidity and health care cost (1). W ith improved medical management, individuals with SCD are now surviving well into adulthood. However, adults with SCD are often sedentary and because of medical conservatism are frequently discouraged from exercise fo r fear of precipitating acute vasoocclusive or painful episodes. A review of the literature revealed little baseline data on perceived health status and well-being, physical fitness (cardiovascular and muscular), and body composition in adults with SCD. It is currently unknown whether exercise or physical training (PT) will have beneficial, adverse, or no effect in SCD patients. As a result, exercise recommendations for adults with SCD are vague, and implementation and effectiveness of PT have not been reported. Moreover, there are no reports on the effects of controlled PT on perceived health status, physical fitness, and body composition parameters in patients with SCD. The effect of exercise on inflammatory mediators (i.e., tum or necrosis factor-alpha [TNF-a], interleukin - 6 [IL-6], and C-reactive protein [CRP]) and vasoactive mediators (i.e., nitric oxide metabolites [NOx] and endothelin-1 [ET-1]) has not been reported in patients with sickle cell anemia (SCA). Since changes in these parameters may influence the occurrence of vasoocclusive episodes by increasing adhesion of erythrocytes to the endothelium and by other mechanisms, such as vasoconstriction, it is important to clarify changes that may occur with exercise. Physical training holds the promise of allowing patients with SCD to preserve functional capacity and improve their quality of life, as has been reported for other chronic diseases. Yet, there are no repotted studies assessing the implementation and effectiveness of PT in adults with SCD. The study reported in this thesis provides new information concerning the impact of controlled PT on fitness and well-being in women with SCD. It also provides new information on the effects of three consecutive days of exercise, which might be used in a standard PT program. This project is a first step in the assessment of the role of regular exercise for SCD patients.
    • Increased S-nitrosylation Impairs Contraction and Relaxation in Mouse Aorta

      Choi, Hyehun; Department of Physiology (2011-06)
      S-Nitrosylation is a ubiquitous protein modification in redox-based signaling. This modification uses nitric oxide (NO) to forms S-nitrosothiol (SNO) on cysteine residues. Thioredoxin (Trx) and Trx reductase (TrxR) play a role in limiting Snitrosylation. We hypothesized overall that S-nitrosylation of intracellular signaling molecules impairs contraction and relaxation of vascular smooth muscle cells. Aortic rings from C57BL/6 mice were used to measure vascular contraction and relaxation. The rings were treated with TrxR inhibitors, auranofin or 1-chloro-2,4-dinitrobenzene (DNCB), and/or NO donors, propylamine propylamine NONOate (PANOate) or S-nitrosocysteine (CysNO), to increase Snitrosylation. Contractile responses of aortic rings to phorbol-12,13-dibutyrate (PDBu), a PKC activator, were attenuated by auranofin, DNCB, PANOate, and CysNO. PKCa S-nitrosylation was increased by a TrxR inhibitor and CysNO; concomitantly, PKCa activity and downstream signaling were inhibited as compared to control protein. Vascular relaxation in aortic rings from normotensive (Sham) and angiotensin II (Angll)-induced hypertensive mice was measured after contraction with phenylephrine in the presence or absence of DNCB. DNCB reduced relaxation to acetylcholine (ACh) compared to vehicle, but the antioxidants, apocynin and tempol, normalized DNCB-induced impaired relaxation to ACh in sham aorta. Soluble guanylyl cyclase (sGC) S-nitrosylation was increased by DNCB, and sGC activity (cyclic GMP assay) was reduced in sham aorta. In aortic rings from Angll-treated mice, DNCB did not change relaxation to ACh compared to vehicle. DNCB decreased relaxation to sodium nitroprusside (SNP) in aortic rings from both sham and Angll mice. Total protein S-nitrosylation was enhanced in Angll aorta compared to sham, and TrxR activity was inhibited in Angll aorta compared to sham. These data suggest that PKC is inactivated by S-nitrosylation and this modification inhibits contractile responses to PDBu. TrxR inhibition reduces vascular relaxation via increasing oxidative stress and sGC S-nitrosylation. In Angll-induced hypertensive mice, augmented S-nitrosylation is associated with impaired vasodilation. Thus, TrxR and Snitrosylation may provide a critical mechanism in hypertension associated with abnormal vascular reactivity.