Rationale/ Hypothesis: Reactive oxygen species, including lipid hydroperoxides, play a critical role as second messengers in many physiological processes in the body. Heightened reactive oxygen species production at the time of birth imposes an oxidative stress upon the lung, which may trigger postnatal alveologenesis and physiological lung cell apoptosis in the neonatal rat. Methods: Neonatal rats were subcutaneously injected daily with vehicle (corn oil) or diphenyl-phenyl diamine for the first 6 days of life to study alveologenesis and physiological lung cell apoptosis. Add-back experiments were conducted with a prototypic lipid hydroperoxide, t-butyl hydroperoxide. Main Results: Treatment with diphenyl-phenyl diamine resulted in parenchymal thickening, reduced numbers of secondary crests and enlarged air spaces, all consistent with the inhibition of alveologenesis and reduced physiological lung cell apoptosis. Conclusion: Following an oxidative stress at birth, lipid hydroperoxide formation triggers postnatal alveologenesis and physiological lung cell apoptosis in the neonatal rat.

Rationale/ Hypothesis: Reactive oxygen species, including lipid hydroperoxides, play a critical role as second messengers in many physiological processes in the body. Heightened reactive oxygen species production at the time of birth imposes an oxidative stress upon the lung, which may trigger postnatal alveologenesis and physiological lung cell apoptosis in the neonatal rat. Methods: Neonatal rats were subcutaneously injected daily with vehicle (corn oil) or diphenyl-phenyl diamine for the first 6 days of life to study alveologenesis and physiological lung cell apoptosis. Add-back experiments were conducted with a prototypic lipid hydroperoxide, t-butyl hydroperoxide. Main Results: Treatment with diphenyl-phenyl diamine resulted in parenchymal thickening, reduced numbers of secondary crests and enlarged air spaces, all consistent with the inhibition of alveologenesis and reduced physiological lung cell apoptosis. Conclusion: Following an oxidative stress at birth, lipid hydroperoxide formation triggers postnatal alveologenesis and physiological lung cell apoptosis in the neonatal rat.

The development of plants challenged by environmental stress alters plant architecture through several pathways, including those involving plant hormone responses and reactive oxygen species (ROS) production. Auxin, a phytohormone associated with every aspect of development, and abscisic acid (ABA), a phytohormone involved in abiotic stress responses, both interact with ROS. These ROS are used as secondary messengers to activate transcription of abiotic stress genes, and also in developmental responses such as cell elongation. To understand the mechanisms involved in the abiotic stress response and how the response intersects with auxin, ABA, and ROS, I examined COPPER INDUCED IN LEAVES 1 (<i>CIL1</i>) from <i>Brassica carinata</i> and its Arabidopsis orthologue, AUXIN INDUCED IN ROOTS 12 (AIR12). Expression of both genes increases in response to auxin and recent work has placed both <i>CIL1</i> and AIR12 within a family of plant-specific cytochrome b561 proteins thought to be involved with transmission of ROS signals. This suggests a link between auxin and ROS production resulting from abiotic stress. Antisense <i>CIL1 B. carinata</i> plants produced fewer lateral roots and were resistant to salinity stress during vegetative growth. Mutant air12 plants showed a 50% reduction in lateral root number, lateral root length, and H2O2 root distribution. Growth in the presence of H2O2 was able to restore lateral root length to control levels. In silica analysis of the <i>CIL1</i> and AIR12 amino acid sequences detected an attachment site for glucosylphosphatidylinositol, predicting that the protein is targeted to the extracellular leaflet of the plasma membrane where it could be cleaved and released into the apoplast. Subcellular localization using p35S::GFP-CIL1 and p35S::GFP-AIR12 translational fusions confirmed that CIL1 and AIR12 localize to the plasma membrane and are released into the apoplast. Organ localization of AIR12 using the pAIR12::GFP-AIR12 construct in stably transformed Arabidopsis showed fusion protein accumulation in the apex of the primary root and in the vascular tissue. Fusion protein also localized to cells flanking emerging lateral roots. Investigation of pAIR12::GUS Arabidopsis showed GUS accumulation in the apex of elongating lateral roots. I demonstrate that AIR12 is an extracellular protein and that air12 seedlings are susceptible to salt stress, but not osmostic stress and display increased and decreased sensitivity to ABA during germination and primary root elongation, respectively, suggesting that AIR12 acts downstream of abiotic stress recognition.

The development of plants challenged by environmental stress alters plant architecture through several pathways, including those involving plant hormone responses and reactive oxygen species (ROS) production. Auxin, a phytohormone associated with every aspect of development, and abscisic acid (ABA), a phytohormone involved in abiotic stress responses, both interact with ROS. These ROS are used as secondary messengers to activate transcription of abiotic stress genes, and also in developmental responses such as cell elongation. To understand the mechanisms involved in the abiotic stress response and how the response intersects with auxin, ABA, and ROS, I examined COPPER INDUCED IN LEAVES 1 (<i>CIL1</i>) from <i>Brassica carinata</i> and its Arabidopsis orthologue, AUXIN INDUCED IN ROOTS 12 (AIR12). Expression of both genes increases in response to auxin and recent work has placed both <i>CIL1</i> and AIR12 within a family of plant-specific cytochrome b561 proteins thought to be involved with transmission of ROS signals. This suggests a link between auxin and ROS production resulting from abiotic stress. Antisense <i>CIL1 B. carinata</i> plants produced fewer lateral roots and were resistant to salinity stress during vegetative growth. Mutant air12 plants showed a 50% reduction in lateral root number, lateral root length, and H2O2 root distribution. Growth in the presence of H2O2 was able to restore lateral root length to control levels. In silica analysis of the <i>CIL1</i> and AIR12 amino acid sequences detected an attachment site for glucosylphosphatidylinositol, predicting that the protein is targeted to the extracellular leaflet of the plasma membrane where it could be cleaved and released into the apoplast. Subcellular localization using p35S::GFP-CIL1 and p35S::GFP-AIR12 translational fusions confirmed that CIL1 and AIR12 localize to the plasma membrane and are released into the apoplast. Organ localization of AIR12 using the pAIR12::GFP-AIR12 construct in stably transformed Arabidopsis showed fusion protein accumulation in the apex of the primary root and in the vascular tissue. Fusion protein also localized to cells flanking emerging lateral roots. Investigation of pAIR12::GUS Arabidopsis showed GUS accumulation in the apex of elongating lateral roots. I demonstrate that AIR12 is an extracellular protein and that air12 seedlings are susceptible to salt stress, but not osmostic stress and display increased and decreased sensitivity to ABA during germination and primary root elongation, respectively, suggesting that AIR12 acts downstream of abiotic stress recognition.

Ischemic stroke is characterized by a disruption of blood supply to a part of the brain tissue, which leads to a focal ischemic infarct. The expression and activity of MMP-9 is increased in ischemic stroke and is considered to be one of the main factors responsible for damages to the cerebral vasculature, resulting in compromised blood-brain barrier (BBB) integrity. However, the regulatory mechanisms of MMP-9 expression and activity are not well established in ischemic stroke. Since hypoxia/ischemia and reperfusion generates reactive oxygen species (ROS), I hypothesize that ROS is one of factors involved in up-regulation of MMP-9 expression in brain cells and ROS-mediated effect may occur via MAPK signaling pathway. My study has provided the evidence that ROS is responsible for an increase in MMP-9 expression in astrocytes mediated via MAPK-AP1 signaling pathway. Preliminary studies with an in vitro model of the BBB suggest that inhibition of MMP-9 is a critical component of reducing ROS-induced BBB permeability.

It is well established that hypertension decreases endothelium-dependent vasomotor function, partially by excessive generation and reduced scavenging of reactive oxygen species (ROS). Nevertheless, at appropriate levels, some ROS can act as signaling molecules in the vasculature and contribute to endothelium-dependent dilation. Recent evidence in healthy resistance arteries suggests that the ROS species hydrogen peroxide (H2O2) acts as an endogenous endothelium-dependent dilator through a non-nitric oxide, non-prostaglandin (3NP) pathway. The aim of this study was to investigate the role of endogenous H2O2 in 3NP-mediated endothelium-dependent dilation of rat mesenteric arteries, and the changes that occur in these vessels with essential hypertension. 18-20wk old male spontaneously hypertensive rats (SHR; n=24) had an elevated systolic blood pressure of 198±6mmHg compared to 93±4mmHg (p<0.001) in the age matched normotensive Wistar-Kyoto rat (WKY; n=22). Isolated mesenteric arteries were preconstricted with norepinephrine (NEPI), followed by exposure to increasing doses of the endothelium-dependent dilator acetylcholine (ACh), which revealed vasomotor dysfunction in the SHR (maximal dilation: WKY: 94.8±1.3% vs. SHR: 75.2±2.9%, p<0.001). Incubation of the vessels with the non-specific cyclooxygenase (COX) inhibitor indomethacin (INDO) restored the ACh response in the SHR to the level of the WKY control (area under the curve: WKY: 354.6±8.6 vs. SHR INDO: 350.2±12.2, p>0.05) indicating that the release of constrictory prostaglandins from COX contribute to endothelial vasomotor dysfunction. Co-incubation of vessels with INDO and the nitric oxide synthase inhibitor Nω-nitro-L-arginine (LN) inhibited dilation in SHR (46.2±4.8%, p<0.001) but not in WKY (98.3±1.5%, p>0.05), indicating an elevated 3NP component in WKY over SHR. Further co-incubation with the H2O2 scavenger catalase (CAT), LN, and INDO inhibited the 3NP component to a greater extent in SHR (29.7±3.1%, p=0.062) than in WKY (91.6±2.5%, p<0.05). The responses of SHR and WKY mesenteric arteries to the endothelium-independent dilator sodium nitroprusside, the receptor-mediated constrictor NEPI, and the electrochemical constrictor KCl were no different between LN INDO and CAT LN INDO conditions. These data suggest that endogenous H2O2 has a greater role in mediating endothelium-dependent dilation in the mesenteric resistance arteries of SHR. Interestingly, in SHR, co-incubation with LN INDO improved dilation over LN alone (46.2±4.8% vs 23.3±3.2±, p=0.001), and CAT LN INDO decreased dilation from LN INDO to a similar extent, suggesting that COX-inhibition could be a source of H2O2 for endogenous vasodilation. Western blotting revealed a 54% increase in COX-1 protein expression in the SHR mesenteric arteries (WKY: 1.00±0.18 (n=9) vs. SHR: 1.54±0.17 (n=13), p<0.05), but no difference in the expression of the pro-oxidant enzyme p47phox, and the anti-oxidant enzymes CAT, SOD-1, and SOD-2. Administration of exogenous H2O2 to NEPI preconstricted mesenteric arteries revealed a dose-dependent dilation that was no different between SHR and WKY, and incubation of isolated WKY and SHR mesenteric arteries with CAT reduced the accumulation of H2O2 to a similar extent, as assessed by the H2O2-specific fluorescent dye Amplex Red. In conclusion, endogenous H2O2 is a vasodilator in the mesenteric arteries of SHR and WKY rats in the absence of nitric oxide and prostaglandins. In the SHR, COX-1 inhibition may allow endogenous H2O2 to become bioavailable for vasodilation. This study is the first to show a role for endogenous H2O2 in maintaining endothelium-dependent dilation in hypertensive rat resistance arteries, and provides evidence to support a role for COX-1-inhibition in the increased availability of H2O2 for dilation.

It is well established that hypertension decreases endothelium-dependent vasomotor function, partially by excessive generation and reduced scavenging of reactive oxygen species (ROS). Nevertheless, at appropriate levels, some ROS can act as signaling molecules in the vasculature and contribute to endothelium-dependent dilation. Recent evidence in healthy resistance arteries suggests that the ROS species hydrogen peroxide (H2O2) acts as an endogenous endothelium-dependent dilator through a non-nitric oxide, non-prostaglandin (3NP) pathway. The aim of this study was to investigate the role of endogenous H2O2 in 3NP-mediated endothelium-dependent dilation of rat mesenteric arteries, and the changes that occur in these vessels with essential hypertension. 18-20wk old male spontaneously hypertensive rats (SHR; n=24) had an elevated systolic blood pressure of 198±6mmHg compared to 93±4mmHg (p<0.001) in the age matched normotensive Wistar-Kyoto rat (WKY; n=22). Isolated mesenteric arteries were preconstricted with norepinephrine (NEPI), followed by exposure to increasing doses of the endothelium-dependent dilator acetylcholine (ACh), which revealed vasomotor dysfunction in the SHR (maximal dilation: WKY: 94.8±1.3% vs. SHR: 75.2±2.9%, p<0.001). Incubation of the vessels with the non-specific cyclooxygenase (COX) inhibitor indomethacin (INDO) restored the ACh response in the SHR to the level of the WKY control (area under the curve: WKY: 354.6±8.6 vs. SHR INDO: 350.2±12.2, p>0.05) indicating that the release of constrictory prostaglandins from COX contribute to endothelial vasomotor dysfunction. Co-incubation of vessels with INDO and the nitric oxide synthase inhibitor Nω-nitro-L-arginine (LN) inhibited dilation in SHR (46.2±4.8%, p<0.001) but not in WKY (98.3±1.5%, p>0.05), indicating an elevated 3NP component in WKY over SHR. Further co-incubation with the H2O2 scavenger catalase (CAT), LN, and INDO inhibited the 3NP component to a greater extent in SHR (29.7±3.1%, p=0.062) than in WKY (91.6±2.5%, p<0.05). The responses of SHR and WKY mesenteric arteries to the endothelium-independent dilator sodium nitroprusside, the receptor-mediated constrictor NEPI, and the electrochemical constrictor KCl were no different between LN INDO and CAT LN INDO conditions. These data suggest that endogenous H2O2 has a greater role in mediating endothelium-dependent dilation in the mesenteric resistance arteries of SHR. Interestingly, in SHR, co-incubation with LN INDO improved dilation over LN alone (46.2±4.8% vs 23.3±3.2±, p=0.001), and CAT LN INDO decreased dilation from LN INDO to a similar extent, suggesting that COX-inhibition could be a source of H2O2 for endogenous vasodilation. Western blotting revealed a 54% increase in COX-1 protein expression in the SHR mesenteric arteries (WKY: 1.00±0.18 (n=9) vs. SHR: 1.54±0.17 (n=13), p<0.05), but no difference in the expression of the pro-oxidant enzyme p47phox, and the anti-oxidant enzymes CAT, SOD-1, and SOD-2. Administration of exogenous H2O2 to NEPI preconstricted mesenteric arteries revealed a dose-dependent dilation that was no different between SHR and WKY, and incubation of isolated WKY and SHR mesenteric arteries with CAT reduced the accumulation of H2O2 to a similar extent, as assessed by the H2O2-specific fluorescent dye Amplex Red. In conclusion, endogenous H2O2 is a vasodilator in the mesenteric arteries of SHR and WKY rats in the absence of nitric oxide and prostaglandins. In the SHR, COX-1 inhibition may allow endogenous H2O2 to become bioavailable for vasodilation. This study is the first to show a role for endogenous H2O2 in maintaining endothelium-dependent dilation in hypertensive rat resistance arteries, and provides evidence to support a role for COX-1-inhibition in the increased availability of H2O2 for dilation.

Glutathione (GSH), a 3-amino acid compound is ubiquitously expressed in eukaryotic cells and is the most abundant low molecular weight thiol. The importance of GSH is highlighted by its multitude of effects. Within the vascular wall GSH plays a crucial role as an intracellular antioxidant and it possess the ability to act as a signalling intermediate and store for nitric oxide (NO). The importance of NO and its role in vascular wall homeostasis is well recognized. Within the coronary circulation, NO is the primary dilator of many of the large arteries and the smaller arterioles. In addition to controlling coronary vascular tone, the importance of NO is highlighted by its antithrombotic, antihypertrophic, and antriproliferative effects. During instances of cardiovascular disease and normal aging, increases in the production of reactive oxygen species occur. A portion of the deleterious vascular effects of reactive oxygen species are believed to be due to reduction in NO bioavailability as a result of increased ROS-mediated destruction of NO. Altered GSH production in humans has been demonstrated to reduce endothelial function. Conversely, supplementation with GSH augments endothelium-dependent dilation. The mechanisms by which these alterations in GSH influence vasomotor function have not been resolved. The purpose of the studies within this thesis was to examine the impact of chronic and acute GSH modulations on coronary vascular resistance (CVR) and endothelium dependent dilation. In all experiments vascular reactivity was assessed in the isolated perfused rat heart. The advantage of this technique is that it allows the global coronary vasomotor functioning to be examined. Hearts were allowed to stabilize for 30 minutes to allow for the development of spontaneous coronary vascular resistance, followed by a bradykinin (BK) dose-response curve to assess endothelium-dependent dilation. The coronary circulation was then maximally dilated using an endothelium-independent agonist. In all cases BK-mediated dilation is expressed as a percentage of the endothelium-independent dilation. Chapter 2 of this document examines the chronic nature of GSH depletion and examines whether GSH depletion augments the influence of natural aging. Animals (mean age 33 and 65 weeks) were randomized to receive L-Buthionine-(S,R)-sulphoximine (BSO) in the tap water in order to inhibit GSH synthesis, or regular tap water (normal controls). Following 10 days of BSO treatment, ventricular GSH content was reduced in the BSO group compared to the control (0.182±0.021 vs 2.022±0.084 nmol/mg wet weight, p<0.05) and there was increased ventricular H2O2 content (1.345±0.176 vs 0.877±0.123 pmol/µg PRO, p<0.05). Baseline CVR was significantly reduced in the older animals compared to the adult animals (3.92±0.34 vs 4.76±0.20 and 3.67±0.24 vs 5.12±0.37 mmHg/ml×min-1 in the control and BSO treated groups, p<0.05). Conversely, in the presence of LNAME there was a significant increase in CVR in the adult BSO group (14.15±0.99, p<0.05) compared to all other groups. In the absence of LNAME, maximal dilation (percent endothelium-independent response) was reduced in the older animals compared to the adult animals (77±10.3% vs 95.0±1.0% for older and adult control and 92.7±4.5% vs 98.6±0.6% for the older and adult BSO, main effect of age). In the presence of LNAME the adult BSO group had a significantly reduced sensitivity (EC50) compared to all other groups (-7.39±0.09 Log M, p<0.05). Additionally, adult BSO treated animals had an increase in eNOS protein content. These results demonstrate that chronic thiol depletion resulted in an increased reliance on NO in the adult BSO group only. In chapter 3 the beneficial effects of GSH supplementation on BK mediated dilation were examined. Acute GSH was administered in the perfusate at either 0 (control) or with 10 µM for 2 reasons, 1) this concentration does not reduce basal coronary vascular resistance, allowing for a similar baseline CVR across conditions and 2) the 10 µM concentration is a physiologically relevant concentration of plasma/extracellular fluid GSH. The sensitivity to the endothelial agonist bradykinin was enhanced in the presence of GSH (-8.70±0.16 vs -7.94±0.06 LogM, p<0.01). The GSH effect was not dependent on NO production or utilization by soluble guanylate cyclase (sGC) as the enhanced dilation in the GSH group was maintained despite NOS (LNAME) and/or sGC inhibition. When the hearts were supplemented with a ROS scavenger TEMPOL, enhanced dilation was seen in the control group, but was not further enhanced in the GSH group. The requirement for ROS was best demonstrated when both the CON and GSH groups were supplemented with both TEMPOL and LNAME. This condition resulted in similar sensitivity (-7.76±0.19 vs -7.75±0.17 LogM, p>0.05) and area under the curve (182.33±12.70 vs 170±13.86, p>0.05) between GSH and CON. Thus, it was concluded that the effects of GSH administration requires the presence of ROS and exerts its effect in the microvasculature. The study presented in chapter 4 examined the effects of acute thiol modulation (depletion) on CVR and endothelium-dependent dilation. Previous reports have suggested that a reduction in intracellular GSH causes impaired NO production, and functional data support this contention. However, a majority of the data regarding the effects of thiol manipulation are from endothelial-removed vessels. The following agents were used to reduce GSH: the glutathione reductase inhibitor, BCNU; the thiol oxidizing agent, diamide; the thiol conjugating agent, ethacrynic acid (EA); and a thioredoxin inhibitor (CDNB). Preliminary data revealed that only CDNB (11.46±0.71 mmHg/ml×min-1) and EA (8.61±0.36 mmHg/ml×min-1) caused an elevation in CVR compared to the control (6.73±0.24 mmHg/ml×min-1). Conversely, Diamide and BCNU did not significantly affect baseline CVR, or the BK mediated responses. In the presence of EA, there was an overall blunting of the BK-response curve as observed by reduced EC50 (-7.85±0.07 Log M) and maximal dilation (90.8±1.8 %, percent endothelium-independent dilation) compared to the control group (-8.42±0.08 Log M and 97.7±1.6%). In the presence of CDNB the maximal dilation was 74.4±1.9% and the EC50 was -8.83±0.28 Log M. In addition to altering BK mediated responses, acute thiol depletion with all agents resulted in an increased minimal CVR with significant increases observed in the presence of CDNB and EA. There was a significant correlation with GSH:GSSG ratio and baseline (-0.547, p<0.05) and minimal CVR (r=-0.581, p<0.05). This study demonstrates that modulation of the GSH:GSSG ratio using a variety of agents with diverse mechanisms elicits differential responses within the vasculature. Specifically conjugation of GSH and inhibition of thioredoxin significantly alters BK mediated response, where as BCNU and dimaide did not. These results suggest that a modulation in the GSH:GSSG ratio impairs endothelium-dependent dilation and alters total dilatory capacity (baseline-minimal CVR) and thus may have implications for adequate tissue perfusion. Across all studies there was significant correlation between GSH and GSSG with both baseline and minimal CVR. Therefore it is likely that changes in overall glutathione content plays a role in determining baseline and minimal coronary vascular resistance. These results demonstrate the complexity that manipulations of GSH have on both CVR and endothelium-dependent dilation, and provide mechanistic insight into how changes in GSH alter coronary vascular resistance and endothelium-dependent dilation.

In this study, a reproducible spectrophotometric method was established and applied to measure reductive capacity of various nanomaterials. Reductive capacity had been implicated in the toxicity of nanomaterials, but a standardized measurement method had been lacking until this work. The reductive capacity of nanoparticles was defined as the mass of iron reduced from Fe3 to Fe2 by unit mass of nanoparticles, in an aqueous solution that initially contained ferric ions. To measure the reductive capacity, the nanomaterials were incubated in a ferric aqueous solution for 16 hours at 37 degrees C, and the reductive capacity of the nanoparticles was determined by measuring the amount of Fe3 reduced to Fe2 using a spectrophotometric method. The reagents 1,10-phenanthroline and hydroquinone were used as a Fe2 indicator and a reducing agent respectively for the assay. To standardize this method, various experiments were carried out. For the initial ferric solution, various Fe salts were tested, and Iron(III) sulfate was chosen as Fe salt for the standard method. The measured reductive capacity of nanoparticles was found to vary with the measurement conditions; the measured reductive capacity increased with increasing the Fe/nanoparticle ratio; the measured reductive capacity increased with incubation time and leveled off after 8 hours of incubation. For hydrophobic materials, the surfactant Tween-20 was added so that the particles could be wetted and suspended in the ferric aqueous solution. After incubation, the particles were removed from the solution by either filtration or centrifugation before applying the spectrophotometric method. In addition, optimal pH and minimum time to reach ultimate color intensity were also found. Carbon-based nanomaterials, standard reference material and metal oxides were measured for their reductive capacities with this method and characterized by transmission electron microscopy (TEM), energy dispersive x-ray spectroscopy (EDS), x-ray diffraction (XRD), BET measurement and Raman spectroscopy. For some nanoparticles, the reductive capacity was measured for both the pristine form and the form treated by oxidization or grinding. All carbon-based nanomaterials, except for pristine C60, have a significant reductive capacity while reductive capacity of metal oxides is very low. And it was found that reductive capacity can be increased by surface functional groups or structural defects and reduced by oxidization or heating (graphitization). The reductive capacity of a material can play an important role in its toxicology by synergistic toxic effects in the presence of transition metal ions through the Fenton reaction. Moreover, even without transition metal ions, the ability of a material to donate electrons can be involved in toxicity mechanisms via generation of reactive oxygen species.

Thesis (Ph. D.)--West Virginia University, 2009. / Title from document title page. Document formatted into pages; contains xi, 150 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references.