Mechanisms for Arsenic-Stimulated Sinusoidal Endothelial Cell Capillarization

The vascular effects of arsenic in drinking water are a global public health concern that contribute to disease in millions of people worldwide. However, the cellular and molecular mechanisms for these pathogenic effects of arsenic are not well defined. This thesis examined the hypothesis that arsenic stimulates pathogenic signals through surface receptors on liver sinusoidal endothelial cells (LSECs) to stimulate NADPH oxidase (NOX) activity that is required for arsenic-stimulated LSEC capillarization. In mice and isolated LSECs, we demonstrated that exposure to arsenic promoted capillarization and increased expression of platelet endothelial cell adhesion molecule (PECAM-1) through a time and dose dependent mechanism. Superoxide generating NOX enzyme complexes participate in vascular remodeling and angiogenesis and are central to arsenic stimulated cell signaling. LSEC arsenic exposure increased NOX dependent superoxide generation that was inhibited using gp91ds-tat protein, NSC23766, a Rac1-GTPase inhibitor, or quenched by the intracellular superoxide scavenger, Tempol. These inhibitors also blocked arsenic-stimulated LSEC PECAM-1 expression and defenestration. In vivo arsenic exposures failed to promote LSEC capillarization in p47phox knockout mice. These data demonstrated that arsenic stimulates capillarization through a NOX dependent mechanism. Given that arsenic rapidly activates NOX in vascular cells, we hypothesized that signaling for these responses was receptor mediated. Since arsenic-stimulated LSEC defenestration and capillarization is Rac1 and NOX dependent, we examined whether a g-protein coupled receptor (GPCR) upstream of Rac1 initiated these effects. Pre-treatment LSECs with Pertussis toxin (PTX), an inhibitor of Gi/o, prevented arsenic-stimulated defenestration. Since capillarization is a gain in barrier function, LSEC expression of the sphingosine-1-phosphate type 1 (S1P1) receptor, a major Gi/o linked regulator of endothelial barrier function, and its role in arsenic-stimulated defenestration were investigated. S1P1 was highly expressed in LSECs relative to large vessels. In ex vivo studies, inhibiting LSEC S1P1 with a selective antagonist, VPC23109, blocked arsenic-stimulated superoxide generation, defenestration, and PECAM-1 expression. These data demonstrated that arsenic targets a specific LSEC GPCR to promote vascular remodeling, and the first demonstrating that S1P1 regulates oxidant-dependent LSEC capillarization. Taken together, these data demonstrate that S1P1 activated NOX stimulates LSEC capillarization, which aids in our understanding of mechanisms underlying arsenic-induced liver disease

Thre role of Bach1 in ultraviolet-A mediated human heme oxygenase-1 gene regulation

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Structural, chemical and biological aspects of antioxidants for strategies against metal and metalloid exposure

Oxidative stress contributes to the pathophysiology of exposure to heavy metals/metalloid. Beneficial renal effects of some medications, such as chelation therapy depend at least partially on the ability to alleviate oxidative stress. The administration of various natural or synthetic antioxidants has been shown to be of benefit in the prevention and attenuation of metal induced biochemical alterations. These include vitamins, N-acetylcysteine, α-lipoic acid, melatonin, dietary flavonoids and many others. Human studies are limited in this regard. Under certain conditions, surprisingly, the antioxidant supplements may exhibit pro-oxidant properties and even worsen metal induced toxic damage. To date, the evidence is insufficient to recommend antioxidant supplements in subject with exposure to metals. Prospective, controlled clinical trials on safety and effectiveness of different therapeutic antioxidant strategies either individually or in combination with chelating agent are indispensable. The present review focuses on structural, chemical and biological aspects of antioxidants particularly related to their chelating properties

Glutathione, the redox sensitive transcription factors AP-1 and NF-kB, and early one adenoviral protein in human lung in smoking related lung disease

The major factor in the development of chronic obstructive airways disease (COPD) is cigarette smoking, although not all smokers develop chronic obstructive pulmonary disease. Inflammation and destruction has been shown in the lungs of smokers with chronic obstructive pulmonary disease. Oxidative stress, both from cigarette smoke and oxidants generated endogenously by cellular processes, contribute to the inflammation that occurs in the lungs in chronic obstructive pulmonary disease. It remains unclear why certain individuals appear susceptible to the effects of cigarette smoke and go on to develop inflammation and airflow limitation. The glutathione redox system is an important antioxidant protective system within the lungs, and this system may play a critical role in the development of inflammation. An alteration in the transcription of pro-inflammatory cytokines and mediators is also likely to contribute to the inflammation within the lung. Nuclear factor kappa-B (NF-kB) and activator protein-1 (AP-1) are both redox sensitive transcription factors, and are involved in the regulation of the gene transcription of many pro-inflammatory mediators. Activator protein-1 and nuclear factor kappa-B have a close relationship with y-glutamylcysteine synthetase (y-GCS) (glutamate cysteine ligase, GCL), the rate limiting enzyme in the synthesis of glutathione, with the y-glutamylcysteine synthetase gene containing various elements including an Activator protein-1 binding site. Susceptibility to the effects of cigarette smoke is likely to explain why certain individuals develop chronic obstructive pulmonary disease, and this susceptibility may arise from an earlier viral infection such as adenoviral infection that lies dormant, but which in the face of an oxidant stimulus such as cigarette smoke augments the inflammatory process.The in vivo studies herein have examined glutathione and y-glutamylcysteine synthetase, gene transcription, oxidant/antioxidant imbalance, the redox sensitive transcription factors nuclear factor kappa-B and activator protein-1, and have assessed for the presence of the early one adenoviral protein in human lung in smokers and patients with COPD.The results show similar levels of total glutathione in the lungs of patients with and without airflow obstruction, and decreased y-glutamylcysteine synthetase activity in patients with severe airflow obstruction who have undergone lung volume reduction surgery for emphysema compared to those with no airflow obstruction. Local lung oxidative stress as measured by malondialdehyde, and trolox equivalant antioxidant capacity a marker of systemic oxidative stress did not correlate with lung function. DNA binding of Nuclear factor kappa-B correlated with lung function as measured by percent predicted forced expiratory volume in one second (FEVi), however no such relationship was found with Activator protein-1 DNA binding. Examination for early one adenoviral gene and protein in human lung tissue failed to reveal conclusive results.In conclusion levels of glutathione in human lung tissue, oxidative stress including both lung and systemic oxidative stress, and the DNA binding of activator protein-1 in lung are not related to the degree of airflow obstruction present

Biochemical properties and bioactivities of carbon monoxide-releasing molecules (CO-RMs).

Carbon monoxide (CO), synonymous of the "silent killer", is rapidly emerging as an important and versatile mediator of physiological processes. The study of CO has been hampered by the lack of a means to simulate its release biologically. Current means to replicate the effects of CO include, most notably, the use of CO gas and upregulation of haem oxygenase-1 (HO-1) to generate endogenous CO. Both are limited in their approach and offer only a partial solution. The recent discovery that certain transition metal carbonyls function as CO-releasing molecules (CO-RMs) in biological systems highlighted the potential of exploiting this and similar classes of compounds as a stratagem to deliver CO for research and therapeutic purposes. Initially a large portfolio of CO-RMs was investigated to determine their CO releasing capability. This thesis examines a number of aspects related to the characterisation of a core group of CO-RMs including: a) CORM-3, the prototypic water soluble transition metal carbonyl b) CORM-A1, a water soluble CO-RM without a metal centre c) CORM-319, an iron based water soluble CO-RM and d) CORM-311, an ethanol soluble iron centred CO-RM. Specifically, the study will examine CO-RMs for their ability to: i) release CO ii) suppress LPS-induced nitrite production iii) promote toxicity iv) induce haem oxygenase (HO) activity and HO-1 expression and v) modulate inducible nitric oxide synthase (iNOS) expression. These different aspects of CO-RM characterisation were addressed using biochemical, molecular biology and cell culture techniques. Further work was also carried out determining certain chemical aspects of each CO-RM including the decomposition rate and pH/temperature stability. The study into the CO release of the new CO-RMs emphasizes the versatile potential of the metal carbonyl complexes and related compounds. This research on CO-RMs will help lay the foundations for a novel therapeutic agent based on the delivery of safe and controlled quantities of CO