Browsing by Subject "Oxidative Stress"

Clinical studies have identified strong correlations between aging and the development of atherosclerosis. In particular, endothelial cell senescence is implicated in age-related changes in vasoreactivity. Oxidative stress is considered the primary source of endothelial cell (EC) senescence in vivo. EC senescence leads to abnormal proliferation of vascular smooth muscle cells, reduced vasoreactivity, enhanced vascular permeability, and greater adhesion of circulating monocytes and lipids. Endothelial senescence often occurs coincident with an inflammatory response within the endothelium. Recapitulating this mechanism of inducing EC senescence in vitro will facilitate a more precise understanding of how aging contributes to endothelial dysfunction and development of vascular diseases, particularly atherosclerosis. Additionally, evidence of vascular remodeling, particularly deposition of fibronectin and stiffening of the vessel wall matrix, is found in both older patients and atheroprone regions. The independent effects of these factors on the function of endothelial cells is poorly understood due to the inability to study them in isolation in vivo. The Truskey lab developed tissue-engineered blood vessels (TEBVs) which recapitulate the structure and function of an arteriole in vitro. These vessels can be fabricated rapidly, perfused immediately after fabrication, and reach functional maturity after a week. Measurements of endothelium-mediated vascular function confirm the presence of a healthy endothelium in the vessels for several weeks after initial fabrication. This in vitro system allows more precise control over the cellular and structural components of blood vessels than is possible with in vivo experiments. Ultimately, the development of a more robust in vitro model for atherosclerosis will contribute to an increased understanding of vascular disease progression and provide a platform for the evaluation of new drugs during preclinical trials. Specific Aim 1: Evaluate the functional effects of stress-induced senescence on TEBVs. Stress-induced senescence reduced endothelium-dependent vessel function and resulted in endothelial cell inflammation with minimal effects on the surrounding hNDFs. Stress-induced senescence was induced in vitro by treatment with hydrogen peroxide. 2-D cells and TEBVs were treated for 5 or 7 days with hydrogen peroxide. Cells in 2-D were stained for p21 to evaluate senescence, as well as key immune cell adhesion markers VCAM-1, ICAM-1, and E-Selectin. To characterize the effects on TEBVs, vasoreactivity in response to an endothelium-independent vasodilator (sodium nitroprusside) and vasoconstrictor (phenylephrine) were quantified, as well as endothelium-dependent vasoreactivity (acetylcholine). Immunostaining of p21 and VCAM-1 expression was also used to confirm that senescence and inflammation were induced in the TEBVs alongside the reduction in endothelium-dependent vasodilation. Specific Aim 2: Evaluate the capacity of stress-induced senescence to increase the monocyte adhesion and foam cell formation in the TEBVs Stress-induced senescence in TEBVs increased adhesion of circulating monocytes and foam cell formation in accumulated monocytes and medial hNDFs. Senescence was induced as in Aim 1, by treating vessels with hydrogen peroxide. The resulting increase in senescence, VCAM-1, and E-selectin increased adhesion of circulating monocytes to the vessel wall. To develop an atherogenesis model, low density lipoprotein was enzymatically modified into a more inflammatory state which is often identified within atherosclerotic lesions. Introducing enzyme-modified low-density lipoprotein (eLDL) alongside hydrogen peroxide treatment further increased endothelial cell activation. There was a significant increase in the percentage of ICAM-1 positive cells when eLDL was applied to endothelial cells alongside H2O2. hNDFs absorbed and retained eLDL, even without H2O2 in the growth media. When TEBVs were exposed to a combination of eLDL, H2O2, and cell-tracker red monocytes, endothelium-dependent vasoreactivity was significantly compromised. Lipid retention within the vessel wall was significant, as was adhesion of monocytes. Specific Aim 3: Evaluate the drug-responsiveness of the TEBV senescence model and the ability of geroprotective agents to reduce senescence-induced vascular dysfunction, monocyte adhesion, and foam cell formation. Development of a physiologically relevant model for vascular senescence can provide a valuable tool for evaluating the efficacy of drugs targeting atherosclerosis, particularly a new class of drugs in development called senolytics. Senolytics, and their sister drugs senomorphics, specifically target senescent cells and transiently disable the anti-apoptotic pathways that prolong their lives, reducing the burden of senescent cells within the tissue. Senomorphics target factors within the senescence-associated secretory pathway (SASP) to reduce cytokine production and inflammation. Dasatinib and quercetin, two senolytics, and tacrolimus, a senomorphic, were tested on CBECFCs growing in 2-D to see if they were effective at reducing the percentage of p21 positive (senescent) cells. Tacrolimus was found to be the best candidate and used in TEBV trials. TEBVs treated with tacrolimus for 48 hours after induction of senescence recovered significantly more endothelium-dependent vasoreactivity compared to vessels left to recover from H2O2 in normal growth media. Additionally, addition of tacrolimus for the duration of hydrogen peroxide treatment had an atheroprotective effect. Adhesion of monocytes and foam cell formation were significantly reduced compared to vessels without tacrolimus. In summary, the work presented here demonstrates that a TEBV model of vascular senescence can be generated in under two weeks using near-physiological levels of hydrogen peroxide. This model can be capitalized upon to model atherogenesis by adding only eLDL and monocytes. We were also able to effectively use the senomorphic tacrolimus to mitigate the effects of senescence on monocyte adhesion and lipid uptake. This system could be used to investigate other senolytics or test the efficacy and toxicity of novel drugs still in development.

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Fluorescent sensors provide a powerful analytical tool for the intracellular detection of metal cations. In some cases, these fluorescent metal-chelating sensors have helped elucidate the function of metal cations within complicated cellular systems. However, most measure or sense changes in the bulk concentration of a metal species and do not respond to those involved in a specific cellular event. For instance, misregulated copper and iron are implicated in neurodegenerative disease and cancer because of their ability to catalytically propagate the formation of the hydroxyl radical through reaction with hydrogen peroxide. A fluorescent sensor that is unresponsive to metal binding until activation by intracellular hydrogen peroxide could potentially pinpoint the location of this oxidative reaction and provide an understanding of the relationship between copper/iron and hydrogen peroxide.

Described here is the development of two fluorescent prochelators that show a selective fluorescence response to iron or copper only in the presence of hydrogen peroxide. A boronic ester masked spirolactam-based prochelator displays a copper-selective turn-on response after oxidation with hydrogen peroxide in organic solvents as determined by absorbance and fluorescence spectroscopy. However, a competing mechanism occurs in aqueous solution due to hydrolytic instability of the masked prochelator and results in a separate copper-dependent turn-on response as verified by liquid chromatography-mass spectroscopy. A second fluorescent prochelator design relies on metal-dependent fluorescence quenching after oxidation of a self-immolative boronic ester in both organic and aqueous solvents. Cellular microscopy studies show that the sensor's fluorescence intensity is unchanged until incubation with exogenous hydrogen peroxide, which resulted in a decreased fluorescent signal that is restored upon competitive chelation. Both of these prochelators provide a template for future applications and designs with improved properties.

Two additional chapters describe the development of a UV-activated iron prochelator and a new fluorescently tagged metal chelator. The UV-activated prochelator is protected with two nitrophenyl groups that are photolyzed with 350 nm light within 10 minutes to reveal a high affinity iron triazole-base chelator. A chelator of this nature may provide protection from UV-induced iron liberation and oxidative stress. A second triazole-based chelator with an embedded coumarin fluorophore was prepared as a potential metal sensor. However, this design showed off-target fluorescence responses, thus it cannot be utilized in its current form for metal detection.

The ability to manipulate the coordination chemistry of metal ions has significant ramifications for the study and treatment of metal-related health concerns, including iron overload, UV skin damage, and microbial infection among many other conditions. To address this concern, chelating agents that change their metal binding characteristics in response to external stimuli have been synthesized and characterized by several spectroscopic and chromatographic analytical methods. The primary stimuli of interest for this work are light and hydrogen peroxide.

Herein we report the previously unrecognized photochemistry of aroylhydrazone metal chelator ((E)-N′-[1-(2-hydroxyphenyl)ethyliden]isonicotinoylhydrazide) (HAPI) and its relation to HAPI metal binding properties. Based on promising initial results, a series of HAPI analogues was prepared to probe the structure-function relationships of aroylhydrazone photochemistry. These efforts elucidate the tunable nature of several aroylhydrazone photoswitching properties.

Ongoing efforts in this laboratory seek to develop compounds called prochelators that exhibit a switch from low to high metal binding affinity upon activation by a stimulus of interest. In this context, we present new strategies to install multiple desired functions into a single structure. The prochelator 2-((E)-1-(2-isonicotinoylhydrazono)ethyl)phenyl (E)-3-(2,4-dihydroxyphenyl)acrylate (PC-HAPI) is masked with a photolabile trans-cinnamic acid protecting group that releases umbelliferone, a UV-absorbing, antioxidant coumarin along with a chelating agent upon UV irradiation. In addition to the antioxidant effects of the coumarin, the released chelator (HAPI) inhibits metal-catalyzed production of damaging reactive oxygen species. Finally a peroxide-sensitive prochelator quinolin-8-yl (Z)-3-(4-hydroxy-2-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)oxy)phenyl)acrylate (BCQ) has been prepared using a novel synthetic route for functionalized cis-cinnamate esters. BCQ uses a novel masking strategy to trigger a 90-fold increase in fluorescence emission, along with the release of a desired chelator, in the presence of hydrogen peroxide.

Outer membrane vesicles (OMVs) are spherical structures that bud from the outer membrane (OM) of bacteria containing OM and periplasmic material. They are known to be produced by all bacteria studied to date and play important roles in inter-bacterial communication, bacterial-host interactions, toxin delivery, survival, nutrient acquisition, and biofilm development. The process of OMV production is known to be genetically regulated and selective cargo packaging into bacterial vesicles has been reported and implicated in many biological processes. While much is known about how cargo gets incorporated into vesicles in eukaryotic systems, the mechanism behind cargo selectivity in bacteria has remained largely unexplored. In this study we aimed to characterize preferential sorting trends in OMV packaging in Escherichia coli under oxidative stress, and investigate the mechanism behind selective sorting into OMVs. Proteomic analysis of outer membrane (OM) and OM vesicle fractions from enterotoxigenic E. coli (ETEC) revealed significant differences in protein abundance in the OMV and OM fractions for cultures shifted to oxidative stress conditions. Analysis of sequences of proteins preferentially packaged into OMVs showed that proteins with oxidizable residues were more packaged into OMVs in comparison with those retained in the membrane. In addition, the results indicated two distinct classes of OM-associated proteins were differentially packaged into OMVs as a function of peroxide treatment and we observed a slight increase in periplasmic content. Implementing a Bayesian hierarchical model, OM lipoproteins were determined to be preferentially exported during stress whereas integral OM proteins were preferentially retained in the cell. We first inquired whether this sorting was due to the need of the cell to discard or retain OM proteins and tested oxidative stress sensitivity of mutants of lipoproteins and integral proteins. We hypothesized that mutants of lipoproteins would not be more sensitive than integral proteins however both groups showed increased sensitivity. Therefore, this did not explain this preferential sorting. We next wondered if selectivity was dependent on gene expression. By mining gene expression databases and performing qRT-PCR we found the sorting to be independent of transcriptional regulation of the proteins upon oxidative stress. We were also able to validate these preferential sorting trends of lipoproteins vs integral proteins using randomly selected protein candidates from the different cargo classes. We also observed that a shift to oxidative stress conditions improved the fitness of bacteria to a secondary oxidative challenge, suggesting the differential sorting resulted in an OMV-mediated remodeling of the OM during stress. Together, our data showed that oxidative stress induced a differential sorting of proteins into OMVs and OM of E. coli and that OMV production might serve as a disposal mechanism for the cell to rid itself of oxidized proteins. Since our data revealed that the preferentially retained proteins were those known to have ties to other cell envelope components, a hypothetical functional and mechanistic basis for cargo selectivity was tested using OmpA as a model. A full-length and a truncated version of OmpA were used to test whether physical tethering to the cell is a determinant for protein retention in the OM. Quantifying OMV protein packaging of both OmpA constructs revealed a basic mechanism for cargo selectivity into OMVs. We show that the untethered version of OmpA was more likely to be exported than the tethered version and that this preferential selection was exacerbated under oxidative stress. The findings of this study provide insight into the dynamics of bacterial cargo selection and membrane remodeling during stress as well as propose and test a mechanism for cargo incorporation in E. coli.

Reactive oxygen species (ROS) are produced endogenously in all aerobes and are induced by environmental stressors. ROS oxidize and disable essential cellular components such as DNA, proteins, and lipid membranes. Exposure to metals, polycyclic aromatic hydrocarbons (PAHs), and some pesticides can induce oxidative stress in marine invertebrates. All aerobic organisms have a network of antioxidants and enzymes to quench ROS and prevent oxidative damage. This dissertation examines antioxidant and oxidative stress biomarkers in endemic molluscs and crabs from two natural extreme environments: deep-sea hydrothermal vents in the Lau and North Fiji Basin, and cold seeps in the Gulf of Mexico. In addition, the acute toxicity and sub-lethal effects of four insecticides and an herbicide are examined in the estuarine blue crab, Callinectes sapidus. Blue crabs are North Carolina's most important fishery species and are frequently found in agricultural drainage ditches, an example of an anthropogenic extreme environment.

Total glutathione, catalase, superoxide dismutase, and lipid peroxidation levels were of the same respective order of magnitude in the two vent gastropods, Alviniconcha sp. and Ifremeria nautilei, and vent mussel, Bathymodiolus brevior. These biomarkers activities were similar to those from previous reports on Mid-Atlantic Ridge mussels, except for ~100-fold higher lipid peroxidation levels among Lau molluscs. Principal component analysis (PCA) of mollusc tissue-specific biomarker levels grouped individuals by species rather than by site.

Biomarker levels in the seep mussels Bathymodiolus childressi, B. brooksi, and B. heckerae were similar across species except for elevated foot and gill cytosolic SOD in mussels from MC-640 compared to those from AC-645. PCA of seep mussel biomarker levels differentiated by species with B. childressi isolated from B. brooksi and B. heckerae. The addition of B. brevior biomarker data to the PCA showed them grouping around B. brooksi and B. heckerae. Bathymodiolus childressi is ancestral to the other species and contains only methanotrophic endosymbionts. Whether symbionts play a role in alleviating possible toxic conditions remains unknown.

Pesticides were acutely toxic to blue crabs in the order of Lambda-cyhalothrin > imidacloprid ≈ aldicarb > acephate ≈ Roundup® (glyphosate). Megalopae were almost always more sensitive to pesticides than early stage juveniles. Commercial formations of pesticides generally showed similar toxicity to active ingredients alone. Exposure to LC₂₀ levels of acephate, aldicarb, imidacloprid and Roundup significantly increased the frequency of juvenile mortality after molting. There was no significant change in total glutathione or lipid peroxidation of exposed megalopae. Lambda-cyhalothrin-, imidacloprid-, and aldicarb-based products have the potential to cause acute toxicity and molting-related mortality in shallow creeks and ditches.

Hydrogen peroxide is used by animals and plants to deter the growth of microbial invaders by inflicting DNA lesions, protein oxidation and lipid membrane modifications. Pathogens protect themselves with enzymes and scavenging proteins. This study investigated population genetic, biochemical and genetic aspects of peroxide survival in Saccharomyces cerevisiae to address its importance for yeast biology and fungal pathogenicity.

Population genetic analyses of DNA sequences from five loci from 103 strains encompassing the known ecological spectrum of S. cerevisiae show that it is a recombining species with three divergent subgroups, which are associated with soil, fruit, and vineyards. Clinical isolates cluster with fruit isolates but are significantly more resistant to peroxide. Clinical isolates are genetically diverse, indicating multiple origins of the pathogenic lifestyle and eliminating the possibility that peroxide resistance is due to shared ancestry rather than it's importance for than its importance in colonizing the host.

Biochemical aspects of peroxide survival were studied in a resistant (high-survival) clinical isolate, a sensitive (low-survival) laboratory strain and their hybrid. Catalase activity and expression levels are indistinguishable among strains. Co-culture assays and growth curve records indicate that a secreted factor improves survival of the laboratory strain and that the phenotypic difference is most pronounced during exponential growth, excluding mechanisms of the General Stress Response effective during stationary phase. Semi-quantitative expression profiles of stress response candidate genes do not differ, suggesting a novel resistance mechanism.

To elucidate the genetic basis of peroxide survival, the hybrid was sporulated and 200 F1 segregants phenotyped and genotyped for oxidative stress candidate genes. Peroxide survival is a dominant quantitative trait and not linked to catalase, peroxidase or superoxide dismutase genes. 1,246 backcross segregants were phenotyped and 93 segregants selectively genotyped using microarrays. A 14-gene locus on chromosome XVI displayed marker-trait association. One gene, RDS2, encodes a zinc cluster protein acting as a regulator of drug sensitivity and contains a non-synonymous polymorphism whose exchange between the parental strains results a 15% decrease in survival in the clinical strain.

This work establishes a novel function for RDS2 in oxidative stress response and demonstrates the effect a quantitative trait nucleotide has on a clinically relevant phenotype.