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

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

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.

Ageing, physical function, and the diurnal rhythms of cortisol and dehydroepiandrosterone

The present study examined the relationship between ageing, physical function and the diurnal rhythms of cortisol and dehydroepiandrosterone (DHEA). Participants were 36 community dwelling older adults aged between 65-86 years old. Salivary cortisol and DHEA were measured over the course of one day: immediately upon awakening, 30 min later, and then 3 h, 6 h, 9 h and 12 h post-awakening. Participants completed the Nottingham extended activities of daily living index, the Berg Balance Scale and their handgrip strength was assessed. Older participants had a significantly higher cortisol area under the curve (AUC), lower overall DHEA levels, lower DHEA AUC, a decreased diurnal slope of decline and increased cortisol:DHEA ratio. Lower diurnal cortisol levels were associated with poorer performance on the Berg Balance Scale and lower handgrip strength, and those with a flattened DHEA diurnal profile reported less independence in carrying out daily tasks. These associations withstood adjustment for age. In conclusion, this study suggests an association between cortisol, DHEA, ageing and physical function

Arsenic stimulates sinusoidal endothelial cell capillarization and vessel remodeling in mouse liver

Trivalent arsenic [As(III)] is a well-known environmental toxicant that causes a wide range of organ-specific diseases and cancers. In the human liver, As(III) promotes vascular remodeling, portal fibrosis, and hypertension, but the pathogenesis of these As(III)-induced vascular changes is unknown. To investigate the hypothesis that As(III) targets the hepatic endothelium to initiate pathogenic change, mice were exposed to 0 or 250 parts per billion (ppb) of As(III) in their drinking water for 5 weeks. Arsenic(III) exposure did not affect the overall health of the animals, the general structure of the liver, or hepatocyte morphology. There was no change in the total tissue arsenic levels, indicating that arsenic does not accumulate in the liver at this level of exposure. However, there was significant vascular remodeling with increased sinusoidal endothelial cell (SEC) capillarization, vascularization of the peribiliary vascular plexus (PBVP), and constriction of hepatic arterioles in As(III)-exposed mice. In addition to ultrastructural demonstration of SEC defenestration and capillarization, quantitative immunofluorescence analysis revealed increased sinusoidal PECAM-1 and laminin-1 protein expression, suggesting gain of adherens junctions and a basement membrane. Conversion of SECs to a capillarized, dedifferentiated endothelium was confirmed at the cellular level with demonstration of increased caveolin-1 expression and SEC caveolae, as well as increased membrane-bound Rac1-GTPase

Cooperation between CYB5R3 and NOX4 via coenzyme Q mitigates endothelial inflammation

NADPH oxidase 4 (NOX4) regulates endothelial inflammation by producing hydrogen peroxide (H2O2) and to a lesser extent O2•-. The ratio of NOX4-derived H2O2 and O2•- can be altered by coenzyme Q (CoQ) mimics. Therefore, we hypothesize that cytochrome b5 reductase 3 (CYB5R3), a CoQ reductase abundant in vascular endothelial cells, regulates inflammatory activation. To examine endothelial CYB5R3 in vivo, we created tamoxifen-inducible endothelium-specific Cyb5r3 knockout mice (R3 KO). Radiotelemetry measurements of systolic blood pressure showed systemic hypotension in lipopolysaccharides (LPS) challenged mice, which was exacerbated in R3 KO mice. Meanwhile, LPS treatment caused greater endothelial dysfunction in R3 KO mice, evaluated by acetylcholine-induced vasodilation in the isolated aorta, accompanied by elevated mRNA expression of vascular adhesion molecule 1 (Vcam-1). Similarly, in cultured human aortic endothelial cells (HAEC), LPS and tumor necrosis factor α (TNF-α) induced VCAM-1 protein expression was enhanced by Cyb5r3 siRNA, which was ablated by silencing the Nox4 gene simultaneously. Moreover, super-resolution confocal microscopy indicated mitochondrial co-localization of CYB5R3 and NOX4 in HAECs. APEX2-based electron microscopy and proximity biotinylation also demonstrated CYB5R3's localization on the mitochondrial outer membrane and its interaction with NOX4, which was further confirmed by the proximity ligation assay. Notably, Cyb5r3 knockdown HAECs showed less total H2O2 but more mitochondrial O2•-. Using inactive or non-membrane bound active CYB5R3, we found that CYB5R3 activity and membrane translocation are needed for optimal generation of H2O2 by NOX4. Lastly, cells lacking the CoQ synthesizing enzyme COQ6 showed decreased NOX4-derived H2O2, indicating a requirement for endogenous CoQ in NOX4 activity. In conclusion, CYB5R3 mitigates endothelial inflammatory activation by assisting in NOX4-dependent H2O2 generation via CoQ.This work was supported by National Institutes of Health (NIH) R01 awards [R01 HL 133864 (A.C.S), R01 HL 128304 (A.C.S), R01 HL 149825 (A.C.S), R01 HL 153532 (A.C.S), R01 GM 125944 (F.J.S.), R01 DK 112854 (F.J.S.), 1S10OD021540-01 (Center for Biologic Imaging, University of Pittsburgh), 1S10RR019003-01 (Simon Watkins (S.W.)), 1S10RR025488-01 (S.W.), 1S10RR016236-01 (S.W)]. American Heart Association (AHA) [Established Investigator Award 19EIA34770095 (A.C.S.)], Post-doctoral Fellowship 19POST34410028 (S.Y.)]. American Society of Hematology (ASH) Minority Hematology Graduate Award (A.M.D-O.). Junta de Andalucía grant BIO-177 (P.N.), the FEDER Funding Program from the European Union and Spanish Ministry of Science, Innovation and Universities grant RED2018-102576-T (P.N.)

Loss of cardiomyocyte CYB5R3 impairs redox equilibrium and causes sudden cardiac death

Sudden cardiac death (SCD) in patients with heart failure (HF) is allied with an imbalance in reduction and oxidation (redox) signaling in cardiomyocytes; however, the basic pathways and mechanisms governing redox homeostasis in cardiomyocytes are not fully understood. Here, we show that cytochrome b5 reductase 3 (CYB5R3), an enzyme known to regulate redox signaling in erythrocytes and vascular cells, is essential for cardiomyocyte function. Using a conditional cardiomyocyte-specific CYB5R3-knockout mouse, we discovered that deletion of CYB5R3 in male, but not female, adult cardiomyocytes causes cardiac hypertrophy, bradycardia, and SCD. The increase in SCD in CYB5R3-KO mice is associated with calcium mishandling, ventricular fibrillation, and cardiomyocyte hypertrophy. Molecular studies reveal that CYB5R3-KO hearts display decreased adenosine triphosphate (ATP), increased oxidative stress, suppressed coenzyme Q levels, and hemoprotein dysregulation. Finally, from a translational perspective, we reveal that the high-frequency missense genetic variant rs1800457, which translates into a CYB5R3 T117S partial loss-of-function protein, associates with decreased event-free survival (~20%) in Black persons with HF with reduced ejection fraction (HFrEF). Together, these studies reveal a crucial role for CYB5R3 in cardiomyocyte redox biology and identify a genetic biomarker for persons of African ancestry that may potentially increase the risk of death from HFrEF.These studies were supported by NIH grants R35 HL 161177 (to ACS), R01 HL 133864 (to ACS), R01 HL 128304 (to ACS), R41 HL15098 (to GS), R01 GM 122091 (to PHT), GM125944 (to FJS), R01 DK112854 (to FJS), R21 NS112787 (to MF), NS121706 (to YLW), EB023507 (to YLW), F31 HL149241 (to HMS), and F31 HL151173 (to JCG). Support was also provided by American Heart Association grants 19EIA34770095 (to ACS), AHA 18CDA34140024 (to YLW), and 19PRE34380152 (to NTC); the Spanish Ministry of Health (grant FIS PI17-01286); Junta de Andalucía BIO-177 and the FEDER Funding Program from the European Union and CIBERER (U729)-ISCIII (to PN); Department of Defense W81XWH1810070 (to YLW); and Vitalant. This research was supported in part by the University of Pittsburgh Center for Research Computing through the resources provided. Specifically, this work used the HTC cluster, which is supported by NIH award number S10OD028483.Peer reviewe

An innovative integral field unit upgrade with 3D-printed micro-lenses for the RHEA at Subaru

In the new era of Extremely Large Telescopes (ELTs) currently under construction, challenging requirements drive spectrograph designs towards techniques that efficiently use a facility's light collection power. Operating in the single-mode (SM) regime, close to the diffraction limit, reduces the footprint of the instrument compared to a conventional high-resolving power spectrograph. The custom built injection fiber system with 3D-printed micro-lenses on top of it for the replicable high-resolution exoplanet and asteroseismology spectrograph at Subaru in combination with extreme adaptive optics of SCExAO, proved its high efficiency in a lab environment, manifesting up to ~77% of the theoretical predicted performance