Adam C Straub

Publications (16)136.09 Total impact

• Source

  Available from: Scott Johnstone

  Article: Endothelial cell expression of haemoglobin α regulates nitric oxide signalling.

  Adam C Straub, Alexander W Lohman, Marie Billaud, Scott R Johnstone, Scott T Dwyer, Monica Y Lee, Pamela Schoppee Bortz, Angela K Best, Linda Columbus, Benjamin Gaston, Brant E Isakson
  [show abstract] [hide abstract]
  ABSTRACT: Models of unregulated nitric oxide (NO) diffusion do not consistently account for the biochemistry of NO synthase (NOS)-dependent signalling in many cell systems. For example, endothelial NOS controls blood pressure, blood flow and oxygen delivery through its effect on vascular smooth muscle tone, but the regulation of these processes is not adequately explained by simple NO diffusion from endothelium to smooth muscle. Here we report a new model for the regulation of NO signalling by demonstrating that haemoglobin (Hb) α (encoded by the HBA1 and HBA2 genes in humans) is expressed in human and mouse arterial endothelial cells and enriched at the myoendothelial junction, where it regulates the effects of NO on vascular reactivity. Notably, this function is unique to Hb α and is abrogated by its genetic depletion. Mechanistically, endothelial Hb α haem iron in the Fe(3+) state permits NO signalling, and this signalling is shut off when Hb α is reduced to the Fe(2+) state by endothelial cytochrome b5 reductase 3 (CYB5R3, also known as diaphorase 1). Genetic and pharmacological inhibition of CYB5R3 increases NO bioactivity in small arteries. These data reveal a new mechanism by which the regulation of the intracellular Hb α oxidation state controls NOS signalling in non-erythroid cells. This model may be relevant to haem-containing globins in a broad range of NOS-containing somatic cells.
  Nature 10/2012; · 36.28 Impact Factor
• Article: S-nitrosylation inhibits pannexin 1 channel function.

  Alexander W Lohman, Janelle L Weaver, Marie Billaud, Joanna K Sandilos, Rachael Griffiths, Adam C Straub, Silvia Penuela, Norbert Leitinger, Dale W Laird, Douglas A Bayliss, Brant E Isakson
  [show abstract] [hide abstract]
  ABSTRACT: S-nitrosylation is a post-translational modification on cysteine(s) that can regulate protein function and pannexin 1 (Panx1) channels are present in the vasculature, a tissue rich in nitric oxide (NO) species. Therefore, we investigated whether Panx1 can be S-nitrosylated and if this modification can affect channel activity. Using the biotin switch assay, we found that application of the NO donors S-nitrosoglutathione (GSNO) or DEA NONOate to human embryonic kidney (HEK) 293T cells expressing wild type (WT) Panx1 and mouse aortic endothelial cells (mAECs) induced Panx1 S-nitrosylation. Functionally, GSNO and DEA NONOate attenuated Panx1 currents; consistent with a role for S-nitrosylation, current inhibition was reversed by the reducing agent dithiothreitol and unaffected by 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a blocker of guanylate cyclase activity. In addition, ATP release was significantly inhibited by treatment with both NO donors. To identify which cysteine residue(s) were S-nitrosylated, we made single cysteine-to-alanine substitutions in Panx1 (Panx1C40A, Panx1C346A, Panx1C426A). Mutation of these single cysteines did not prevent Panx1 S-nitrosylation; however, mutation of either C40 or C346 prevented Panx1 current inhibition and ATP release by GSNO. This observation suggested that multiple cysteines may be S-nitrosylated to regulate Panx1 channel function. Indeed, we found that mutation of both C40 and C346 (Panx1C40/346A) prevented Panx1 S-nitrosylation by GSNO, as well as the GSNO-mediated inhibition of Panx1 current and ATP release. Taken together, these results indicate that S-nitrosylation of Panx1 at C40 and C346 inhibits Panx1 channel currents and ATP release.
  Journal of Biological Chemistry 10/2012; · 4.77 Impact Factor
• Article: Expression of pannexin isoforms in the systemic murine arterial network.

  Alexander W Lohman, Marie Billaud, Adam C Straub, Scott R Johnstone, Angela K Best, Monica Lee, Kevin Barr, Silvia Penuela, Dale W Laird, Brant E Isakson
  [show abstract] [hide abstract]
  ABSTRACT: Pannexins (Panx) form ATP release channels and it has been proposed that they play an important role in the regulation of vascular tone. However, distribution of Panx across the arterial vasculature is not documented. We tested antibodies against Panx1, Panx2 and Panx3 on human embryonic kidney cells (which do not endogenously express Panx proteins) transfected with plasmids encoding each Panx isoform and Panx1(-/-) mice. Each of the Panx antibodies was found to be specific and was tested on isolated arteries using immunocytochemistry. We demonstrated that Panx1 is the primary isoform detected in the arterial network. In large arteries, Panx1 is primarily in endothelial cells, whereas in small arteries and arterioles it localizes primarily to the smooth muscle cells. Panx1 was the predominant isoform expressed in coronary arteries, except in arteries less than 100 µm where Panx3 became detectable. Only Panx3 was expressed in the juxtaglomerular apparatus and cortical arterioles. The pulmonary artery and alveoli had expression of all 3 Panx isoforms. No Panx isoforms were detected at the myoendothelial junctions. We conclude that the specific localized expression of Panx channels throughout the vasculature points towards an important role for these channels in regulating the release of ATP throughout the arterial network.
  Journal of Vascular Research 06/2012; 49(5):405-16. · 2.65 Impact Factor
• Source

  Available from: Scott Johnstone

  Article: MAPK phosphorylation of connexin 43 promotes binding of cyclin E and smooth muscle cell proliferation.

  Scott R Johnstone, Brett M Kroncke, Adam C Straub, Angela K Best, Clarence A Dunn, Leslie A Mitchell, Yelena Peskova, Robert K Nakamoto, Michael Koval, Cecilia W Lo, Paul D Lampe, Linda Columbus, Brant E Isakson
  [show abstract] [hide abstract]
  ABSTRACT: Dedifferentiation of vascular smooth muscle cells (VSMC) leading to a proliferative cell phenotype significantly contributes to the development of atherosclerosis. Mitogen-activated protein kinase (MAPK) phosphorylation of proteins including connexin 43 (Cx43) has been associated with VSMC proliferation in atherosclerosis. To investigate whether MAPK phosphorylation of Cx43 is directly involved in VSMC proliferation. We show in vivo that MAPK-phosphorylated Cx43 forms complexes with the cell cycle control proteins cyclin E and cyclin-dependent kinase 2 (CDK2) in carotids of apolipoprotein-E receptor null (ApoE(-/-)) mice and in C57Bl/6 mice treated with platelet-derived growth factor-BB (PDGF). We tested the involvement of Cx43 MAPK phosphorylation in vitro using constructs for full-length Cx43 (Cx43) or the Cx43 C-terminus (Cx43(CT)) and produced null phosphorylation Ser>Ala (Cx43(MK4A)/Cx43(CTMK4A)) and phospho-mimetic Ser>Asp (Cx43(MK4D)/Cx43(CTMK4D)) mutations. Coimmunoprecipitation studies in primary VSMC isolated from Cx43 wild-type (Cx43(+/+)) and Cx43 null (Cx43(-/-)) mice and analytic size exclusion studies of purified proteins identify that interactions between cyclin E and Cx43 requires Cx43 MAPK phosphorylation. We further demonstrate that Cx43 MAPK phosphorylation is required for PDGF-mediated VSMC proliferation. Finally, using a novel knock-in mouse containing Cx43-MK4A mutation, we show in vivo that interactions between Cx43 and cyclin E are lost and VSMC proliferation does not occur after treatment of carotids with PDGF and that neointima formation is significantly reduced in carotids after injury. We identify MAPK-phosphorylated Cx43 as a novel interacting partner of cyclin E in VSMC and show that this interaction is critical for VSMC proliferation. This novel interaction may be important in the development of atherosclerotic lesions.
  Circulation Research 05/2012; 111(2):201-11. · 9.49 Impact Factor
• Article: A novel mRNA binding protein complex promotes localized plasminogen activator inhibitor-1 accumulation at the myoendothelial junction.

  Katherine R Heberlein, Jenny Han, Adam C Straub, Angela K Best, Christoph Kaun, Johann Wojta, Brant E Isakson
  [show abstract] [hide abstract]
  ABSTRACT: Plasminogen activator inhibitor-1 (PAI-1) has previously been shown to be key to the formation of myoendothelial junctions (MEJs) in normal and pathological states (eg, obesity). We therefore sought to identify the mechanism whereby PAI-1 could be selectively accumulated at the MEJ. We identified PAI-1 protein enrichment at the MEJ in obese mice and in response to tumor necrosis factor (TNF-α) with a vascular cell coculture. However, PAI-1 mRNA was also found at the MEJ and transfection with a PAI-1-GFP with TNF-α did not demonstrate trafficking of the protein to the MEJ. We therefore hypothesized the PAI-1 mRNA was being locally translated and identified serpine binding protein-1, which stabilizes PAI-1 mRNA, as being enriched in obese mice and after treatment with TNF-α, whereas Staufen, which degrades PAI-1 mRNA, was absent in obese mice and after TNF-α application. We identified nicotinamide phosphoribosyl transferase as a serpine binding protein-1 binding partner with a functional τ-like microtubule binding domain. Application of peptides against the microtubule binding domain significantly decreased the number of MEJs and the amount of PAI-1 at the MEJ. We conclude that PAI-1 can be locally translated at the MEJ as a result of a unique mRNA binding protein complex.
  Arteriosclerosis Thrombosis and Vascular Biology 03/2012; 32(5):1271-9. · 6.37 Impact Factor
• Article: Characterization of the thoracodorsal artery: morphology and reactivity.

  Marie Billaud, Alexander W Lohman, Adam C Straub, Thibaud Parpaite, Scott R Johnstone, Brant E Isakson
  [show abstract] [hide abstract]
  ABSTRACT: In this paper, we describe the histological and contractile properties of the thoracodorsal artery (TDA), which indirectly feeds the spinotrapezius muscle. We used immunolabelling techniques to histologically characterize the TDA while the contractile properties were assessed using pressure arteriography. Our results demonstrate that the TDA is composed of approximately one to two layers of smooth muscle cells, is highly innervated with adrenergic nerves, and develops spontaneous tone at intraluminal pressures above 80 mmHg. The reactivity of the TDA in response to various contractile agonists such as phenylephrine, noradrenaline, angiotensin II, serotonin, endothelin 1, and ATP, as well as vasodilators, shows that the TDA exhibits a remarkably comparable reactivity to what has been observed in mesenteric arteries. We further studied the different components of the TDA response to acetylcholine, and found that the TDA was sensitive to TRAM 34, a blocker of the intermediate conductance potassium channel, which is highly suggestive of an endothelium-dependent hyperpolarization. We conclude that the TDA exhibits comparable characteristics to other current vascular models, with the additional advantage of being easily manipulated for molecular and ex vivo vasoreactivity studies.
  Microcirculation (New York, N.Y.: 1994) 02/2012; 19(4):360-72. · 2.37 Impact Factor
• Article: Pannexin1 regulates α1-adrenergic receptor- mediated vasoconstriction.

  Marie Billaud, Alexander W Lohman, Adam C Straub, Robin Looft-Wilson, Scott R Johnstone, Christina A Araj, Angela K Best, Faraaz B Chekeni, Kodi S Ravichandran, Silvia Penuela, Dale W Laird, Brant E Isakson
  [show abstract] [hide abstract]
  ABSTRACT: The coordination of vascular smooth muscle cell constriction plays an important role in vascular function, such as regulation of blood pressure; however, the mechanism responsible for vascular smooth muscle cell communication is not clear in the resistance vasculature. Pannexins (Panx) are purine-releasing channels permeable to the vasoconstrictor ATP and thus may play a role in the coordination of vascular smooth muscle cell constriction. We investigated the role of pannexins in phenylephrine- and KCl-mediated constriction of resistance arteries. Western blot, immunohistochemistry, and immunogold labeling coupled to scanning and transmission electron microscopy revealed the presence of Panx1 but not Panx2 or Panx3 in thoracodorsal resistance arteries. Functionally, the contractile response of pressurized thoracodorsal resistance arteries to phenylephrine was decreased significantly by multiple Panx inhibitors (mefloquine, probenecid, and (10)Panx1), ectonucleotidase (apyrase), and purinergic receptor inhibitors (suramin and reactive blue-2). Electroporation of thoracodorsal resistance arteries with either Panx1-green fluorescent protein or Panx1 small interfering RNA showed enhanced and decreased constriction, respectively, in response to phenylephrine. Lastly, the Panx inhibitors did not alter constriction in response to KCl. This result is consistent with coimmunoprecipitation experiments from thoracodorsal resistance arteries, which suggested an association between Panx1 and α1D-adrenergic receptor. Our data demonstrate for the first time a key role for Panx1 in resistance arteries by contributing to the coordination of vascular smooth muscle cell constriction and possibly to the regulation of blood pressure.
  Circulation Research 06/2011; 109(1):80-5. · 9.49 Impact Factor
• Article: Spontaneous lung dysfunction and fibrosis in mice lacking connexin 40 and endothelial cell connexin 43.

  Michael Koval, Marie Billaud, Adam C Straub, Scott R Johnstone, Alexander Zarbock, Brian R Duling, Brant E Isakson
  [show abstract] [hide abstract]
  ABSTRACT: Gap junction proteins (connexins) facilitate intercellular communication and serve several roles in regulation of tissue function and remodeling. To examine the physiologic effects of depleting two prominent endothelial connexins, Cx40 and Cx43, transgenic mice were generated by breeding Cx40-deficient mice (Cx40(-/-)) with a vascular endothelial cell (VEC)-specific Cx43-deficient mouse strain (VEC Cx43(-/-)) to produce double-connexin knockout mice (VEC Cx43(-/-)/Cx40(-/-)). The life span in VEC Cx43(-/-)/Cx40(-/-) mice was dramatically shortened, which correlated with severe spontaneous lung abnormalities as the mice aged including increased fibrosis, aberrant alveolar remodeling, and increased lung fibroblast content. Moreover, VEC Cx43(-/-)/Cx40(-/-) mice exhibited cardiac hypertrophy and hypertension. Because VEC Cx43(-/-)/Cx40(-/-) mice demonstrated phenotypic hallmarks that were remarkably similar to those in mice deficient in caveolin-1, pulmonary caveolin expression was examined. Lungs from VEC Cx43(-/-)/Cx40(-/-) mice demonstrated significantly decreased expression of caveolin-1 and caveolin-2. This suggests that expression of caveolin-1 may be linked to expression of Cx40 and endothelial Cx43. Moreover, the phenotype of caveolin-1(-/-) mice and VEC Cx43(-/-)/Cx40(-/-) mice may arise via a common mechanism.
  American Journal Of Pathology 06/2011; 178(6):2536-46. · 4.89 Impact Factor
• Article: Compartmentalized connexin 43 s-nitrosylation/denitrosylation regulates heterocellular communication in the vessel wall.

  Adam C Straub, Marie Billaud, Scott R Johnstone, Angela K Best, Sean Yemen, Scott T Dwyer, Robin Looft-Wilson, Jeffery J Lysiak, Ben Gaston, Lisa Palmer, Brant E Isakson
  [show abstract] [hide abstract]
  ABSTRACT: To determine whether S-nitrosylation of connexins (Cxs) modulates gap junction communication between endothelium and smooth muscle. 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 NO(x) concentrations at the myoendothelial junction and found S-nitrosoglutathione reductase activity to precede NO release. This study provides evidence for compartmentalized S-nitrosylation/denitrosylation in the regulation of smooth muscle cell to endothelial cell communication.
  Arteriosclerosis Thrombosis and Vascular Biology 11/2010; 31(2):399-407. · 6.37 Impact Factor
• Article: Plasminogen activator inhibitor-1 regulates myoendothelial junction formation.

  Katherine R Heberlein, Adam C Straub, Angela K Best, Mark A Greyson, Robin C Looft-Wilson, Poonam R Sharma, Akshaya Meher, Norbert Leitinger, Brant E Isakson
  [show abstract] [hide abstract]
  ABSTRACT: 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. Determine the ability of PAI-1 to regulate myoendothelial junction (MEJ) formation. 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. 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).
  Circulation Research 04/2010; 106(6):1092-102. · 9.49 Impact Factor
• Article: Site-specific connexin phosphorylation is associated with reduced heterocellular communication between smooth muscle and endothelium.

  Adam C Straub, Scott R Johnstone, Katherine R Heberlein, Michael J Rizzo, Angela K Best, Scott Boitano, Brant E Isakson
  [show abstract] [hide abstract]
  ABSTRACT: Myoendothelial junctions (MEJs) represent a specialized signaling domain between vascular smooth muscle cells (VSMC) and endothelial cells (EC). The functional consequences of phosphorylation state of the connexins (Cx) at the MEJ have not been explored. Application of adenosine 3',5'-cyclic monophosphate sodium (pCPT) to mouse cremasteric arterioles reduces the detection of connexin 43 (Cx43) phosphorylated at its carboxyl terminal serine 368 site (S368) at the MEJ in vivo. After single-cell microinjection of a VSMC in mouse cremaster arterioles, only in the presence of pCPT was dye transfer to EC observed. We used a vascular cell co-culture (VCCC) and applied the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (PMA) or fibroblast growth factor-2 (FGF-2) to induce phosphorylation of Cx43 S368. This phosphorylation event was associated with a significant reduction in dye transfer and calcium communication. Using a novel method to monitor increases in intracellular calcium across the in vitro MEJ, we noted that PMA and FGF-2 both inhibited movement of inositol 1,4,5-triphosphate (IP(3)), but to a lesser extent Ca(2+). These data indicate that site-specific connexin phosphorylation at the MEJ can potentially regulate the movement of solutes between EC and VSMC in the vessel wall.
  Journal of Vascular Research 12/2009; 47(4):277-86. · 2.65 Impact Factor
• Article: Oxidized phospholipid species promote in vivo differential cx43 phosphorylation and vascular smooth muscle cell proliferation.

  Scott R Johnstone, Jeremy Ross, Michael J Rizzo, Adam C Straub, Paul D Lampe, Norbert Leitinger, Brant E Isakson
  [show abstract] [hide abstract]
  ABSTRACT: Regulation of both the expression and function of connexins in the vascular wall is important during atherosclerosis. Progression of the disease state is marked by vascular smooth muscle cell (VSMC) proliferation, which coincides with the reduced expression levels of connexin 43 (Cx43). However, nothing is currently known about the factors that regulate post-translational modifications of Cx43 in atherogenesis, which could be of particular importance, due to the association between site-specific Cx43 phosphorylation and cellular proliferation. We compared the effects of direct carotid applications of two oxidized phospholipid derivatives, 1-palmitoyl-2-oxovaleroyl-sn-glycero-3-phosphorylcholine (POVPC) and 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphorylcholine (PGPC), on Cx43 expression and phosphorylation, and on cell proliferation. Since both POVPC and PGPC have been shown to act through different intracellular pathways, we hypothesized that each oxidized phospholipid species could induce differential Cx43 phosphorylation events in the cytoplasmically located carboxyl-terminal region of the protein, which could potentially enhance cell proliferation. Application of POVPC caused a reduction in VSMC Cx43 levels, enhanced its phosphorylation at serine (pS) 279/282, and increased VSMC proliferation both in vivo and in vitro. Treatment with PGPC enhanced VSMC pS368 levels with no associated change in proliferation. These oxidized phospholipid-induced Cx43 post-translational changes in VSMCs were consistent with those identified in ApoE(-/-) mice. Taken together, these results demonstrate that post-translational phosphorylation of Cx43 could be a key factor in the pathogenesis of atherosclerosis.
  American Journal Of Pathology 09/2009; 175(2):916-24. · 4.89 Impact Factor
• Article: Arsenic requires sphingosine-1-phosphate type 1 receptors to induce angiogenic genes and endothelial cell remodeling.

  Adam C Straub, Linda R Klei, Donna B Stolz, Aaron Barchowsky
  [show abstract] [hide abstract]
  ABSTRACT: Arsenic in drinking water is a major public health concern as it increases risk and incidence of cardiovascular disease and cancer. Arsenic exposure affects multiple vascular beds, promoting liver sinusoidal capillarization and portal hypertension, ischemic heart disease, peripheral vascular disease, and tumor angiogenesis. While Rac1-GTPase and NADPH oxidase activities are essential for arsenic-stimulated endothelial cell signaling for angiogenesis or liver sinusoid capillarization, the mechanism for initiating these effects is unknown. We found that arsenic-stimulated cell signaling and angiogenic gene expression in human microvascular endothelial cells were Pertussis toxin sensitive, indicating a G-protein coupled signaling pathway. Incubating human microvascular endothelial cells with the sphingosine-1-phosphate type 1 receptor (S1P(1)) inhibitor VPC23019 or performing small interfering RNA knockdown of S1P(1) blocked arsenic-stimulated HMVEC angiogenic gene expression and tube formation, but did not affect induction of either HMOX1 or IL8. Liver sinusoidal endothelial cells (LSECs) defenestrate and capillarize in response to aging and environmental oxidant stresses. We found that S1P(1) was enriched on LSECs in vivo and in primary cell culture and that VPC23019 inhibited both sphingosine-1-phosphate-stimulated and arsenic-stimulated LSEC oxidant generation and defenestration. These studies identified novel roles for S1P(1) in mediating arsenic stimulation of both angiogenesis and pathogenic LSEC capillarization, as well as demonstrating a role for S1P(1) in mediating environmental responses in the liver vasculature, providing possible mechanistic insight into arsenic-induced vascular pathogenesis and disease.
  American Journal Of Pathology 05/2009; 174(5):1949-58. · 4.89 Impact Factor
• Article: Arsenic-stimulated liver sinusoidal capillarization in mice requires NADPH oxidase-generated superoxide.

  Adam C Straub, Katherine A Clark, Mark A Ross, Ashwin G Chandra, Song Li, Xiang Gao, Patrick J Pagano, Donna B Stolz, Aaron Barchowsky
  [show abstract] [hide abstract]
  ABSTRACT: Environmental arsenic exposure, through drinking contaminated water, is a significant risk factor for developing vascular diseases and is associated with liver portal hypertension, vascular shunting, and portal fibrosis through unknown mechanisms. We found that the addition of low doses of arsenite to the drinking water of mice resulted in marked pathologic remodeling in liver sinusoidal endothelial cells (SECs), including SEC defenestration, capillarization, increased junctional PECAM-1 expression, protein nitration, and decreased liver clearance of modified albumin. Furthermore, the pathologic changes observed after in vivo exposure were recapitulated in isolated mouse SECs exposed to arsenic in culture. To investigate the role of NADPH oxidase-generated ROS in this remodeling, we examined the effect of arsenite in the drinking water of mice deficient for the p47 subunit of the NADPH oxidase and found that knockout mice were protected from arsenite-induced capillarization and protein nitration. Furthermore, ex vivo arsenic exposure increased SEC superoxide generation, and this effect was inhibited by addition of a Nox2 inhibitor and quenched by the cell-permeant superoxide scavenger. In addition, inhibiting either oxidant generation or Rac1-GTPase blocked ex vivo arsenic-stimulated SEC differentiation and dysfunction. Our data indicate that a Nox2-based oxidase is required for SEC capillarization and that it may play a central role in vessel remodeling following environmentally relevant arsenic exposures.
  Journal of Clinical Investigation 01/2009; 118(12):3980-9. · 15.39 Impact Factor
• Article: Low level arsenic promotes progressive inflammatory angiogenesis and liver blood vessel remodeling in mice.

  Adam C Straub, Donna B Stolz, Harina Vin, Mark A Ross, Nicole V Soucy, Linda R Klei, Aaron Barchowsky
  [show abstract] [hide abstract]
  ABSTRACT: The vascular effects of arsenic in drinking water are global health concerns contributing to human disease worldwide. Arsenic targets the endothelial cells lining blood vessels, and endothelial cell activation or dysfunction may underlie the pathogenesis of both arsenic-induced vascular diseases and arsenic-enhanced tumorigenesis. The purpose of the current studies was to demonstrate that exposing mice to drinking water containing environmentally relevant levels of arsenic promoted endothelial cell dysfunction and pathologic vascular remodeling. Increased angiogenesis, neovascularization, and inflammatory cell infiltration were observed in Matrigel plugs implanted in C57BL/6 mice following 5-week exposures to 5-500 ppb arsenic [Soucy, N.V., Mayka, D., Klei, L.R., Nemec, A.A., Bauer, J.A., Barchowsky, A., 2005. Neovascularization and angiogenic gene expression following chronic arsenic exposure in mice. Cardiovasc.Toxicol 5, 29-42]. Therefore, functional in vivo effects of arsenic on endothelial cell function and vessel remodeling in an endogenous vascular bed were investigated in the liver. Liver sinusoidal endothelial cells (LSEC) became progressively defenestrated and underwent capillarization to decrease vessel porosity following exposure to 250 ppb arsenic for 2 weeks. Sinusoidal expression of PECAM-1 and laminin-1 proteins, a hallmark of capillarization, was also increased by 2 weeks of exposure. LSEC caveolin-1 protein and caveolae expression were induced after 2 weeks of exposure indicating a compensatory change. Likewise, CD45/CD68-positive inflammatory cells did not accumulate in the livers until after LSEC porosity was decreased, indicating that inflammation is a consequence and not a cause of the arsenic-induced LSEC phenotype. The data demonstrate that the liver vasculature is an early target of pathogenic arsenic effects and that the mouse liver vasculature is a sensitive model for investigating vascular health effects of arsenic.
  Toxicology and Applied Pharmacology 09/2007; 222(3):327-36. · 4.45 Impact Factor
• Article: Arsenic stimulates sinusoidal endothelial cell capillarization and vessel remodeling in mouse liver.

  Adam C Straub, Donna B Stolz, Mark A Ross, Araceli Hernández-Zavala, Nicole V Soucy, Linda R Klei, Aaron Barchowsky
  [show abstract] [hide abstract]
  ABSTRACT: 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. CONCLUSION: These data demonstrate that exposure to As(III) causes functional changes in SEC signaling for sinusoidal capillarization that may be initial events in pathogenic changes in the liver.
  Hepatology 02/2007; 45(1):205-12. · 11.66 Impact Factor