The two of major pathways that cause ischemic damage are oxidative stress and inflammation. To decreasing oxidative stress and inflammation, new anti-oxidant and anti-inflammatory agents are tested in ischemic models. In order to study ALRX828C anti-inflammatory properties, an in vivo six-day old air pouch model of inflammation was used to evaluate the anti-inflammatory potential of ALRX828C. Also, the dose response of ALRX828C for TNFα (IC50 = 30 μM) and IL-17 (IC50 = 1.3 μM) were determined by using human peripheral blood mononuclear cell cultures stimulated with ionomycin and PMA. To examine ALRX828C anti-inflammatory effect in neuroinflammation, a neurodegenerative model was used to evaluate its potential. I also showed that reducing oxidative stress with a potent antioxidant, Idebenone in nano-emulsion form, can effectively reduce tissue damage during ischemia in organotypic slice culture subjected to oxygen-glucose depravation (OGD). In conclusion, reducing oxidative stress and inflammation after stroke can reduce ischemic damage substantially.

Regulators of G-protein signaling (RGS) modulate G-protein coupled receptor (GPCR) activity in vascular smooth muscle cells (VSMCs). One such protein, RGS5, has been shown to have selective expression in VSMCs and pericytes, and can inhibit signaling from Gαq and Gαi subunits. Using an RGS5 knockout model, we assessed the functional effect of RGS5 in the constriction and dilation of resistance arterioles. Furthermore, we examined the intracellular lipid interaction of RGS proteins to identify the determinants regulating the biologic function of RGS5. Surprisingly, loss of RGS5 function in mesenteric arterioles had no effect on constriction and dilation of resistance arterioles. Cultured VSMCs showed increased basal ERK1/2 phosphorylation and increased VASP signaling in response to SNP treatment in RGS5KO VSMCs as compared to wild type controls, with no effect on cell proliferation. These data suggest RGS5 may integrate multiple intracellular pathways with competing effects on VSMC contraction.

Nutrient sensing pathways in both the brain and gut decrease hepatic glucose production. Hypothalamic activation of lactate metabolism decreases glucose production, but it is unknown whether the hypothalamus detects circulating lactate to maintain glucose homeostasis. In the gut, lipids decrease glucose production via a neuronal network but the downstream signaling mechanisms are unknown. We tested whether circulating lactate activates central lactate metabolism to decrease glucose production and postulated that duodenal protein kinase C (PKC) acts downstream of lipids to decrease glucose production through a neuronal network. We report that central lactate metabolism is required for the maintenance of glucose homeostasis in the presence of circulating lactate and that activation of duodenal PKC is required for lipids to decrease glucose production. This shows the importance of the brain and gut in the regulation of glucose production, and could pave the way for restoration of glucose homeostasis in disease.

Cholecystokinin (CCK) is a gut peptide involved in the regulation of energy homeostasis by duodenal lipids via a neuronal network. However, it is unknown whether CCK also regulates glucose homeostasis through a neuronal network. Using an in vivo rat model, we demonstrated that duodenal CCK-8 (biologically active form of CCK) can lower glucose production through the activation of a gut-brain-liver axis via CCK-A receptors, and this glucose-regulatory effect is physiologically relevant. Since duodenal lipids can also lower glucose production through a gut-brain-liver axis, we verified that this duodenal-lipid effect is mediated by CCK-A receptor activation. Lastly, in rats fed on a high-fat diet for three days, duodenal CCK failed to suppress glucose production, suggesting a state of CCK-resistance. In summary, these findings revealed that intestinal CCK can regulate glucose homeostasis through a neuronal network and suggest that intestinal CCK resistance may contribute to hyperglycemia in response to high-fat feeding.

The CAPS1 protein was initially discovered as a cytosolic soluble 145kDa protein which was necessary to restore calcium dependent norephinephrine secretion in cracked PC12 cells. Recent findings suggest that CAPS may also play a role in synaptic and dense core vesicle exocytosis as well as in the loading of monoamine neurotransmitters. Recently, studies have implicated CAPS1 in the binding to syntaxin-1. However, no studies have identified the key residues of CAPS1 that facilitate this interaction with syntaxin-1. I show that the binding mode of CAPS1 is independent from that of Munc13 such that CAPS1 requires the full length of syntaxin-1 to bind. Moreover, CAPS1 favors the open conformation of syntaxin-1. Interestingly, the Munc homology (MH) domain of CAPS1 is not critical for this interaction while the C-terminal dense core vesicle binding (DCVB) domain plays an important role. Moreover, truncations to this DCVB domain result in decreased binding to syntaxin-1.

Acute kidney injury (AKI) is associated with renal medullary hypoxia. The medullary thick ascending limb (mTAL) in the renal outer medulla is most susceptible to hypoxic injury, due to marginal O2 supply and high O2 consumption. The objectives of this study were to document the earliest effect of hypoxia (8% O2 for 2.5 hrs) on the mTAL function, and to identify strategies to protect the mTAL from hypoxia. The earliest effect of hypoxia is large water diuresis, due to a fall in the medullary osmolality and increase in vasopressinase. Desmopressin acetate (dDAVP), a synthetic vasopressin analogue resistant to vasopressinase that may also increase O2 delivery, prevented water diuresis. A low dose (0.8mg/kg) of furosemide may significantly reduce the mTAL work without a large excretion of essential electrolytes. Large water diuresis may be diagnostically valuable in detecting renal tissue hypoxia, and dDAVP and furosemide may prevent AKI in the clinical setting.

Acute kidney injury (AKI) is associated with renal medullary hypoxia. The medullary thick ascending limb (mTAL) in the renal outer medulla is most susceptible to hypoxic injury, due to marginal O2 supply and high O2 consumption. The objectives of this study were to document the earliest effect of hypoxia (8% O2 for 2.5 hrs) on the mTAL function, and to identify strategies to protect the mTAL from hypoxia. The earliest effect of hypoxia is large water diuresis, due to a fall in the medullary osmolality and increase in vasopressinase. Desmopressin acetate (dDAVP), a synthetic vasopressin analogue resistant to vasopressinase that may also increase O2 delivery, prevented water diuresis. A low dose (0.8mg/kg) of furosemide may significantly reduce the mTAL work without a large excretion of essential electrolytes. Large water diuresis may be diagnostically valuable in detecting renal tissue hypoxia, and dDAVP and furosemide may prevent AKI in the clinical setting.

Infection with the gut-pathogen Helicobacter pylori is the single, most important risk factor in the development of gastric cancer. Although there is a rising incidence in mortality resulting from this malignancy, the exact mechanism underlying the initiation and progression of bacterial-induced gastric tumorigenesis is still not completely understood. Several studies implicate the activation of the Signal Transducer and Activator of Transcription 3 (STAT3) signaling pathway as a cellular trigger for promoting carcinogenes. In this thesis, I studied the role of the STAT3 signaling pathway in H. pylori mediated tumorigenesis, and attempted to delineate mechanisms involved. I have found that H. pylori activates the STAT3 signaling pathway both in vitro and in vivo, to promote carcinogenesis. Pivotal for H. pylori mediated STAT3 activation are the bacterial effector protein CagA and host receptor components, the gp130 and the IL-6αR subunits. Further investigation into the mechanism of STAT3 induction identified a key role for cholesterol-enriched membrane lipid rafts. Bacterial invasion and CagA injection into host cells was also dependent on lipid raft integrity. Co-fractionation via the use of sucrose gradients, which permits the isolation of lipid rafts, identified H. pylori CagA to be associated with these membrane microdomains. CagA, once injected into the cell, appears to interact with the inner leaflet of the host plasma membrane via a charge association that either directly or indirectly anchors it to the negatively charged anionic lipids in the cytoplasmic membrane. In addition, janus kinases were recruited to rafts upon H. pylori infection. In this thesis, I present a dynamic model of STAT3 activation, which requires the interaction of lipid raft associated proteins, H. pylori CagA and recruited JAKs with non-lipid raft receptor components to support STAT3 signaling. This study is significant since it provides insight into the possible mechanisms by which H. pylori induces gastric cancer and furthermore, it facilitates the development of novel therapeutic targets directed against bacterial induced carcinogenesis.

Heart rate is modulated by the opposing activities of sympathetic and parasympathetic inputs to pacemaker cardiomyocytes in the sinoatrial (SA) node. Parasympathetic activity on nodal myocytes is mediated by acetylcholine-dependent stimulation of M2 muscarinic receptors and activation of Gαi/o signalling. Although, regulators of G-protein signalling (RGS) proteins are potent inhibitors of Gαi/o signalling in many tissues, the RGS protein(s) that regulate parasympathetic tone in the SA node are unknown. Our results demonstrate that RGS4 mRNA levels are higher in the SA node compared to right atrium. Conscious freely moving RGS4-null mice showed a greater extent of bradycardia in response to parasympathetic agonists compared to wild-type animals. Moreover, anaesthetized rgs4-null mice had lower baseline heart rates and greater heart rate increases following atropine administration. Retrograde-perfused hearts from rgs4-null mice also showed enhanced negative chronotropic responses to carbachol, while isolated SA node myocytes showed greater sensitivity to carbachol-mediated reduction in the action potential firing rate. Finally, rgs4-null SA node cells showed decreased levels of G-protein-coupled inward rectifying potassium (GIRK) channel desensitization, and altered modulation of acetylcholine-sensitive potassium current (IKACh) kinetics following carbachol stimulation. Taken together, our studies establish that RGS4 plays an important role in regulating sinus rhythm by inhibiting parasympathetic signalling and IKACh activity. Following these results, we predicted that loss of RGS4 expression and function will result in increased levels of parasympathetic effector activity leading to increased susceptibility to atrial fibrillation. Susceptibility to atrial fibrillation (AF) depends strongly on parasympathetic activity. Since RGS4 inhibits parasympathetic / M2-dependent Gαi/o signalling in the SA node, we explored whether changes in RGS4 levels altered the susceptibility of atrial fibrillation. We found that, RGS4 levels were decreased in atria of tachypaced dogs prior to their development of chronic AF. Moreover, in vivo ECG recordings of anaesthetized mice showed greater susceptibility to AF while optical mapping of isolated atrial preparations using a voltage-sensitive dye revealed greatly increased susceptibility to rotor formation when RGS4 was ablated. Consistent with altered parasympathetic signalling in the myocardium of rgs4-null mice, IKACh evoked by carbachol application were greater in isolated atrial myocytes from rgs4-null mice. These IKACh changes were, as expected, associated with marked action potential duration shortening in response to parasympathetic activation, but not to slower conduction velocities. Together, our findings establish that RGS4 protects atrial tissues from excess parasympathetic signalling that predispose to atrial fibrillation.

INTRODUCTION: Platelets are involved in the inflammatory and thrombotic complications associated with atherosclerosis. Human neutrophil peptides (HNP), released from activated neutrophils, demonstrate inflammatory effects related to lesion development. HNP bind the low-density lipoprotein receptor (LR) family member LRP1 and LRP8 is the only member on platelets. HYPOTHESIS: HNP enhance platelet activation and aggregation through interactions with LRP8. METHODS: Platelet activation and aggregation in response to HNP were determined using flow cytometry and aggregometry. Activation was also examined in the presence of recombinant LRP8 and in LRP8 knockout platelets. RESULTS: HNP activate platelets as determined by P-selectin expression and the formation of microparticles. HNP sensitize platelets enhancing their aggregatory response to ADP. Lastly, LRP8 plays a role in HNP-induced platelet activation. CONCLUSIONS: With an improved understanding of the mechanism by which HNP induce platelet activation, we may be able to devise therapeutic strategies to treat patients with cardiovascular diseases.