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

Rural Oklahoma and the nexus of disaster vulnerability, risk, and resilience

The concepts of vulnerability, risk, and resilience have experienced a meteoric rise in academic attention among disaster scholars over the past three decades. Vulnerability operates as the set of social conditions which predispose certain populations or social spaces to inequitable environmental burdens precipitated by disaster. The prolific study of risk has led to a robust set of literatures which tell us how people in a variety of contexts perceive and understand risks associated with natural hazards. Research focusing on disaster resilience elucidate the qualities and behaviors which allow social individuals, groups, and systems to cope and adapt to disaster. While these studies have furthered our collective understanding of what factors contribute to adverse outcomes, or allow certain groups to successfully navigate the dangers of environmental hazards, few have focused on how these concepts operate prior to disasters. Moreover, most disaster research has privileged urban areas. Given our current understanding of climate change, rural areas in the Midwest are positioned to experience some of the most severe socio-economic outcomes. Adapting the Community Capitals Framework to a disaster context, this dissertation uses qualitative methodology to analyze 56 semi-structured, in-depth interviews drawn from two distinct rural areas of Oklahoma to explore the nexus of vulnerability, risk, and resilience. Broadly, a deteriorating, reciprocal, and mutually reinforcing relationship between rural spaces and urban areas have led to a cognitive and geographic rural-urban divide. A combination of politically conservative economic policy, a systematic divestment from social welfare programs, and a combination of rural stoicism and cultural stigma creates, maintains, and exacerbates a condition called community disenfranchisement. Rural communities experience feelings of abandonment and neglect and direct that animosity toward urban areas and government agencies. Perceptions of isolation lead to rural insulation as distal communities seal themselves off from larger institutions. Despite this deleterious relationship, rural communities turn to one another, forging constellations of bonding social capital to stitch together what few resources they do have to cultivate resilience. The current study also offers important implications and best practices for cultivating resilience in rural spaces while demonstrating a need for improving social relations across the rural/urban divide

Influence of Power in Climate Change Media: Framing Strategies and Field Dynamics of Institutional Actors, 1990-2015

This project provides an alternative theoretical perspective from which to assess the influence of power in media narrative construction of politically contentious issues, in this case, climate change. There has been a prolific amount of research addressing the problem of climate change. I argue that most of this research has neglected power as a key dimension in the persistence of climate change inaction. This neglect is a product of myriad factors: conceptual inconsistencies in the definition of “framing”; limitations to conventional analytic frameworks in situating media in larger social and political contexts; properly orienting key political actors and sponsors in the construction of framing strategies; and a lack of assessing the relationship of powerful actors and institutions through time. This project first orients key political actors in relation to one another and their role in the production of climate change discourse. Using Field Theory, I purport to properly situate institutional fields by considering coordinative and oppositional frames in the print news media. This theoretical framework allows me to construct a larger, dynamic constellation of social and political power, departing from traditional “agentic vs. hegemonic” dichotomous standpoints. To substantiate this I investigate a longitudinal analysis of media narratives from 1990-2015. I contend that these data demonstrate that climate change inaction is persistent because of surplus power and resources wielded by elite institutions, allowing them to permeate and elicit support from adjacent institutional fields. This effectively forestalls action in spite of both scientific consensus and public opinion advocating for regulatory intervention.Sociolog

The impact of xanthine oxidase (XO) on hemolytic diseases

Hemolytic diseases are associated with elevated levels of circulating free heme that can mediate endothelial dysfunction directly via redox reactions with biomolecules or indirectly by upregulating enzymatic sources of reactive species. A key enzymatic source of these reactive species is the purine catabolizing enzyme, xanthine oxidase(XO) as the oxidation of hypoxanthine to xanthine and subsequent oxidation of xanthine to uric acid generates superoxide (O2•-) and hydrogen peroxide (H2O2). While XO has been studied for over 120 years, much remains unknown regarding specific mechanistic roles for this enzyme in pathologic processes. This gap in knowledge stems from several interrelated issues including: 1) lethality of global XO deletion and the absence of tissue-specific XO knockout models have coalesced to relegate proof-of-principle experimentation to pharmacology; 2) XO is mobile and thus when upregulated locally can be secreted into the circulation and impact distal vascular beds by high-affinity association to the glycocalyx on the endothelium; and 3) endothelial-bound XO is significantly resistant (\u3e 50%) to inhibition by allopurinol, the principle compound used for XO inhibition in the clinic as well as the laboratory. While it is known that circulating XO is elevated in hemolytic diseases including sickle cell, malaria and sepsis, little is understood regarding its role in these pathologies. As such, the aim of this review is to define our current understanding regarding the effect of hemolysis (free heme) on circulating XO levels as well as the subsequent impact of XO-derived oxidants in hemolytic disease processes

CRISPR/Cas9-Mediated Gene Knock-Down in Post-Mitotic Neurons

The prokaryotic adaptive immune system CRISPR/Cas9 has recently been adapted for genome editing in eukaryotic cells. This technique allows for sequence-specific induction of double-strand breaks in genomic DNA of individual cells, effectively resulting in knock-out of targeted genes. It thus promises to be an ideal candidate for application in neuroscience where constitutive genetic modifications are frequently either lethal or ineffective due to adaptive changes of the brain. Here we use CRISPR/Cas9 to knock-out Grin1, the gene encoding the obligatory NMDA receptor subunit protein GluN1, in a sparse population of mouse pyramidal neurons. Within this genetically mosaic tissue, manipulated cells lack synaptic current mediated by NMDA-type glutamate receptors consistent with complete knock-out of the targeted gene. Our results show the first proof-of-principle demonstration of CRISPR/Cas9-mediated knock-down in neurons in vivo, where it can be a useful tool to study the function of specific proteins in neuronal circuits

Compartmentalized Connexin 43 S-Nitrosylation/Denitrosylation Regulates Heterocellular Communication in the Vessel Wall

Objective-To determine whether S-nitrosylation of connexins (Cxs) modulates gap junction communication between endothelium and smooth muscle. Methods and Results-Heterocellular communication is essential for endothelium control of smooth muscle constriction; however, the exact mechanism governing this action remains unknown. Cxs and NO have been implicated in regulating heterocellular communication in the vessel wall. The myoendothelial junction serves as a conduit to facilitate gap junction communication between endothelial cells and vascular smooth muscle cells within the resistance vasculature. By using isolated vessels and a vascular cell coculture, we found that Cx43 is constitutively S-nitrosylated on cysteine 271 because of active endothelial NO synthase compartmentalized at the myoendothelial junction. Conversely, we found that stimulation of smooth muscle cells with the constrictor phenylephrine caused Cx43 to become denitrosylated because of compartmentalized S-nitrosoglutathione reductase, which attenuated channel permeability. We measured S-nitrosoglutathione breakdown and NOx concentrations at the myoendothelial junction and found S-nitrosoglutathione reductase activity to precede NO release. Conclusion-This study provides evidence for compartmentalized S-nitrosylation/denitrosylation in the regulation of smooth muscle cell to endothelial cell communication. (Arterioscler Thromb Vasc Biol. 2011;31:399-407.

Plasminogen Activator Inhibitor-1 Regulates Myoendothelial Junction Formation

Rationale: Plasminogen activator inhibitor-1 (PAI-1) is a biomarker for several vascular disease states; however, its target of action within the vessel wall is undefined. Objective: Determine the ability of PAI-1 to regulate myoendothelial junction (MEJ) formation. Methods and Results: MEJs are found throughout the vasculature linking endothelial cells (ECs) and vascular smooth muscle cells. Using a vascular cell coculture we isolated MEJ fractions and performed two-dimensional differential gel electrophoresis. Mass spectrometry identified PAI-1 as being enriched within MEJ fractions, which we confirmed in vivo. In the vascular cell coculture, recombinant PAI-1 added to the EC monolayer significantly increased MEJs. Conversely, addition of a PAI-1 monoclonal antibody to the EC monolayer reduced the number of MEJs. This was also observed in vivo where mice fed a high fat diet had increased PAI-1 and MEJs and the number of MEJs in coronary arterioles of PAI-1(-/-) mice was significantly reduced when compared to C57Bl/6 mice. The presence of MEJs in PAI-1(-/-) coronary arterioles was restored when their hearts were transplanted into and exposed to the circulation of C57Bl/6 mice. Application of biotin-conjugated PAI-1 to the EC monolayer in vitro confirmed the ability of luminal PAI-1 to translocate to the MEJ. Functionally, phenylephrine-induced heterocellular calcium communication in the vascular cell coculture was temporally enhanced when recombinant PAI-1 was present, and prolonged when PAI-1 was absent. Conclusion: Our data implicate circulating PAI-1 as a key regulator of MEJ formation and a potential target for pharmacological intervention in diseases with vascular abnormalities (eg, diabetes mellitus). (Circ Res. 2010; 106: 1092-1102.

Lumbopelvic Stability During a Single Leg Step Down Predicts Elbow Varus Torque During Baseball Pitching

During a baseball pitch, energy is transferred from the lower extremities through the lumbopelvic junction to produce ball velocity. Reduced lumbopelvic stability has been associated with elbow injury in pitchers, and commonly ulnar collateral ligament (UCL) tears. The primary biomechanical mechanism of UCL tears is high elbow varus torque. Understanding how decreased lumbopelvic stability influences the development of elbow varus torque could identify risk factors of UCL elbow injury. PURPOSE: Characterize the predictive ability of lumbopelvic stability on elbow varus torque during a baseball pitch. METHODS: NCAA Division 1 baseball players (N=44; 19.6+1.3yrs) participated. Pitchers threw ten fastballs from a mound to a catcher over regulation distance. Elbow varus torque was recorded using an inertial measurement unit and ball velocity was recorded with a radar gun. Pitchers also completed a single leg step down (SLSD) task. Triplanar kinematics were recorded for both legs, pelvis and trunk using inertial measurement units. Statistical analysis consisted of a cluster analysis, principal component analysis (PCA), and a multivariate logistic regression model to determine the relationship between lumbopelvic stability and elbow varus torque. RESULTS: Cluster analysis revealed 2 subgroups of pitchers: Low Torque-High Velocity and High Torque-Low Velocity. PCA analysis indicated 4 patterns of SLSD motion variability (principal components): 1-sagittal plane, 2-transverse plane, 3-frontal plane trail limb, and 4-frontal plane lead limb. Increased transverse plane motion of the trunk and pelvis predicted higher odds of belonging to the High Torque-Low Velocity cluster; trunk [Odds Ratio=2.9 (95%CI:1.1,8.0), p=0.036] and pelvis [Odds Ratio=2.6 (95%CI:1.1,6.0), p=0.031]. CONCLUSIONS: Lumbopelvic motion assessed during the SLSD in pitchers can identify deficits that predict high elbow varus torque and low ball velocity during the baseball pitch. Specifically, higher pelvis and trunk transverse plane motion was associated with pitchers in the High Torque-Low Velocity cluster. The SLSD provides an easily accessible method for coaches and clinicians to identify a potential risk factor related to increased elbow varus torque and UCL injury in pitchers

Evidence for a Complex Relationship Among Weight Retention, Cortisol and Breastfeeding in Postpartum Women

To assess the relationship between cortisol slope, a biologic marker of stress, and postpartum weight retention