A Novel Role for Reactive Oxygen Species in the Regulation of RhoA: Implications for Endothelial Permeability and Leukocyte Transmigration

The endothelial lining of the vasculature plays a critical role in regulating the passage of fluid, macromolecules, and cells between the blood and surrounding tissues. Vascular permeability is tightly regulated and is modulated during both physiological and pathological situations. Our laboratory is interested in the mechanisms which regulate vascular permeability and the transmigration of leukocytes during inflammation. The migration of leukocytes across the endothelial barrier is called "leukocyte transendothelial migration" (TEM). Deciphering the mechanisms which regulate TEM is important to understanding and managing inflammatory diseases. My interest in TEM is focused on pathways which involve small GTPases. My specific interest is in the role of Rho GTPases and their regulation by reactive oxygen species (ROS) during leukocyte TEM. Although ROS have been largely seen as mediators of oxidative damage, more recently, ROS have been recognized as necessary components of cell signaling pathways. Importantly, ROS have been shown to play an important role in regulating vascular permeability and TEM. In this dissertation, I show that physiological levels of ROS can directly activate RhoA in cells. In vitro studies had previously identified two critical cysteine residues in the nucleotide binding pocket of RhoA that are oxidatively modified by ROS. My work showed that this oxidative regulation of RhoA can occur in a cellular context. Before these studies, the regulation of small GTPases had almost exclusively focused on regulatory proteins. Importantly, my work identifies direct oxidative modification as a novel way to regulate RhoA activity. I extended this work by investigating the regulation of RhoA by ROS in the context of leukocyte TEM. Based on these findings and my previous work, I hypothesized that leukocyte adhesion to endothelial cells initiates ROS generation and the direct activation of RhoA to promote leukocyte TEM. In preliminary studies, I found that crosslinking of a cell adhesion molecule (ICAM- 1) stimulates RhoA and Rac1 activation. In addition the activation of RhoA appears to be dependent on ROS, as seen by studies of redox-insensitive mutants. The work presented in this dissertation lays the framework for future studies on the role of ROS during leukocyte adhesion and TEM

Inhibition of nitric oxide by reactive oxygen species

1. The aim of this study was to determine the role of the antioxidant enzymes, superoxide dismutase and catalase, in the regulation of vascular tone in isolated rings of rat aorta. 2. In the first part of the study a comparison was made of the ability of superoxide anion to destroy the relaxant activity of basal and acetylcholine (ACh)-stimulated NO. 3. Superoxide dismutase (SOD, 1-300 u ml-1) had no effect on endothelium- denuded rings but it produced a concentration-dependent relaxation of phenylephrine (PE)-induced tone in endothelium-containing rings which was blocked by the NO synthase inhibitor, NG-nitro-L-arginine (L-NOARG, 30 muM). In contrast, SOD (50 u mh-1) had no effect on ACh-induced relaxation. These results demonstrated a selective potentiation of basal but not ACh-stimulated activity of NO by SOD. 4. Superoxide anion generation using hypoxanthine (HX, 0,1 mM)/xanthine oxidase (XO, 16 mu ml-1) augmented PE-induced tone in endothelium- containing but not endothelium-denuded rings. This was likely to have resulted from removal of the tonic vasodilator actions of basally-produced NO by superoxide anion, since it was blocked in tissues treated with SOD (250 u ml-1), NG-mono-methyl-L-arginine (L-NMMA, 30 muM) or L-NOARG (30 muM). Pyrogallol (0.1 mM) had a similar action to HX/XO, but produced an additional augmentation of tone by an endothelium-independent mechanism which was unaffected by SOD (250 u ml-1). In contrast to their ability to almost completely destroy basal activity of NO, HX (0.1 mM)/XO (16 mu ml-1) and pyrogallol (0.1 mM) had no effect on ACh-induced relaxation. Increasing the concentration of HX to 1 mM (keeping XO at 16 mu ml-1) or pyrogallol to 0.3 mM, however, profoundly blocked ACh-induced relaxation and this was prevented by treatment with SOD (250 u ml-1). 5. Treatment with diethyldithiocarbamate (DETCA, 0.1 mM, 1h, followed by washout) to irreversibly inhibit endogenous Cu-Zn SOD, augmented PE- induced tone in endothelium-containing, but not endothelium-denuded rings, and abolished the ability of HX (0.1 mM)/XO (16 mu ml-1) and L-NMMA (30 muM) to augment tone. It was likely that DETCA had led to destruction of basal NO by increasing superoxide anion levels since its actions were reversed by SOD (10-300 u ml-1). 6. In contrast to its ability to completely destroy basal activity of NO, DETCA (0.1 mM) produced only a slight blockade of ACh-induced relaxation. Furthermore, DETCA potentiated the ability of HX (0.1 mM)/XO (16 mu ml-1) or pyrogallol (0.1 mM) to block ACh-induced relaxation and this was prevented by pretreatment with SOD (250 u ml-1). 7. The data suggest that basal activity of NO is more sensitive to inactivation by superoxide anion than ACh-stimulated activity and that endogenous Cu-Zn SOD is vital for the protection of endothelial NO. 8. In the second part of the study the role of catalase in the relaxation induced by sodium azide, hydroxylamine, glyceryl trinitrate and hydrogen peroxide was investigated in isolated rings of rat aorta. (Abstract shortened by ProQuest.)

TRPM2 ion channel trafficking and its role in mitochondrial fragmentation and cell death

Mitochondria play a central role in oxidative stress-induced cell death. By increasing the production of reactive oxygen species, such as H2O2, oxidative stress causes mitochondrial fragmentation and apoptosis. It was hypothesised that Transient Receptor Potential Melastatin 2 (TRPM2) channels play a role in mitochondrial fragmentation and cell death. The rationale behind this hypothesis was the published evidence that oxidative stress stimulates TRPM2 channels, resulting in an increase in the cytosolic levels of Ca2+ and Zn2+, and that both these ions are detrimental to mitochondrial health and cell survival. To test the hypothesis, human umbilical vein endothelial cells (HUVECs) and endothelial cells isolated from wild-type and TRPM2 knock-out mice were used. TRPM2 actions were suppressed using pharmacological agents and small interfering RNA (siRNA). Fluorescent reporters were used to examine changes in intracellular ion distribution and organelle morphology. Molecular biology, biochemical and imaging techniques were used to examine the dynamics of ions and organelles. Exposure of HUVECs to H2O2 or high glucose stress led to TRPM2 activation, resulting in extracellular Ca2+ entry, lysosomal membrane permeability (LMP) and the release of lysosomal free Zn2+. Unexpectedly, this was accompanied by the accumulation of Zn2+ in the mitochondria. The rise in mitochondrial Zn2+ led to extensive mitochondrial fragmentation, mitochondrial outer membrane permeabilisation (MMP) and cell death. Silencing of TRPM2 channels with siRNA prevented intracellular Zn2+ redistribution, mitochondrial fragmentation and cell death. Endothelial cells derived from TRPM2 knock-out mice were resistant to oxidative stress-induced mitochondrial fragmentation. Biochemical and immunostaining experiments revealed an unexpected presence of TRPM2 channels in mitochondria, where they mediated mitochondrial Zn2+ uptake. Accumulation of Zn2+ in the mitochondria led to mitochondrial fragmentation by promoting the recruitment of cytoplasmic Drp1, an enzyme responsible for mitochondrial fission. Taken together, the results of this thesis revealed a novel mechanism for how oxidative stress can cause excessive mitochondrial fragmentation and cell death: the mechanism involves activation of TRPM2 channels leading to increased Ca2+ entry, LMP and release of lysosomal Zn2+; Zn2+ thus released is taken up by the mitochondria, leading to Drp1 recruitment, mitochondrial fragmentation and finally cell death. Since mitochondrial fragmentation is associated with several age-related chronic illnesses, including neuronal (Alzheimer’s, Parkinson’s), cardiovascular (atherosclerosis, myocardial infarction) and metabolic/inflammatory (diabetes) disorders, these results suggest that the TRPM2 channel is a novel target that could be explored for therapeutic intervention of age-related illnesses

Climate Change Issues in Nigeria: A Call For A Sustainable Policy in Agricultural Sector

Climate change has been reported to have been caused by human activities and its impacts on global economy and the citizen is devastating in nature. This paper aims to identify various ways through which farmers in the rural community contribute to climate change. This study intends to contribute to environmental sustainability and farmers’ adoption of cleaner agricultural production practices. This research adopted descriptive survey design type. The methodology includes the use of four stage sampling procedure to select 120 farmers from the study area. Interviewed schedule was used to collect data from the respondents, while the data collected were analyzed using descriptive statistics. The findings revealed that majority (92.4%) engaged in bush burning, 96.7% were involved in deforestation,59 % engaged in soil nutrient depletion activitieswhile 92.4% engaged in drainage construction. All these activities contribute to global warming. Therefore, it is recommended that awareness campaign about the cause of climate change should be organized for the farmers in the zone,while policy should be formulated to encourage farmers to use climate smart agriculture in their production process in order to establish environmental sustainability. Key words: Climate change. Bush burning, Deforestation, Sustainability, Farmers activities Global warmin