HIV-1/Cocaine Induced Oxidative Stress Disrupts Tight Junction Protein-1 in Human Pulmonary Microvascular Endothelial Cells: Role of Ras/ERK1/2 Pathway
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{"title"=>"HIV-1/Cocaine induced oxidative stress disrupts tight junction protein-1 in human pulmonary microvascular endothelial cells: Role of RAS/ERK1/2 pathway", "type"=>"journal", "authors"=>[{"first_name"=>"Pranjali", "last_name"=>"Dalvi", "scopus_author_id"=>"54683531300"}, {"first_name"=>"Kun", "last_name"=>"Wang", "scopus_author_id"=>"57198952975"}, {"first_name"=>"Joel", "last_name"=>"Mermis", "scopus_author_id"=>"16402388900"}, {"first_name"=>"Ruoxi", "last_name"=>"Zeng", "scopus_author_id"=>"56086215100"}, {"first_name"=>"Miles", "last_name"=>"Sanderson", "scopus_author_id"=>"56084460500"}, {"first_name"=>"Sara", "last_name"=>"Johnson", "scopus_author_id"=>"57198004034"}, {"first_name"=>"Yuqiao", "last_name"=>"Dai", "scopus_author_id"=>"56084428000"}, {"first_name"=>"Garima", "last_name"=>"Sharma", "scopus_author_id"=>"56084914700"}, {"first_name"=>"Amy O.Brien", "last_name"=>"Ladner", "scopus_author_id"=>"24435972200"}, {"first_name"=>"Navneet K.", "last_name"=>"Dhillon", "scopus_author_id"=>"7003289739"}], "year"=>2014, "source"=>"PLoS ONE", "identifiers"=>{"sgr"=>"84896949572", "pui"=>"372701528", "doi"=>"10.1371/journal.pone.0085246", "pmid"=>"24409324", "scopus"=>"2-s2.0-84896949572", "issn"=>"19326203", "isbn"=>"1932-6203"}, "id"=>"f4e24215-7c89-39d0-804e-19af9dff218a", "abstract"=>"Intravenous drug use (IVDU) is the major risk factor in the development of HIV-related pulmonary arterial hypertension (HRPAH); however, the pathogenesis of HRPAH in association with IVDU has yet to be characterized. Endothelial injury is considered to be an initiating factor for pulmonary vascular remodeling in animal models of PAH. Our previous study shows that simultaneous exposure to HIV-Trans-activator of transcription (Tat) and cocaine exacerbates both disruption of tight junction proteins and permeability of human pulmonary artery endothelial cells compared with either treatment alone. We here now demonstrate that this HIV-Tat and cocaine mediated endothelial dysfunction accompanies with increase in hydrogen peroxide and superoxide radicals generation and involves redox sensitive signaling pathway. Pretreatment with antioxidant cocktail attenuated the cocaine and Tat mediated disassembly of Zonula Occludens (ZO)-1 and enhancement of endothelial monolayer permeability. Furthermore, inhibition of NADPH oxidase by apocynin or siRNA-mediated knockdown of gp-91(phox) abolished the Tat/cocaine-induced reactive oxygen species (ROS) production, suggesting the NADPH oxidase mediated generation of oxidative radicals. In addition, ROS dependent activation of Ras and ERK1/2 Kinase was observed to be mediating the TJP-1 disassembly, and endothelial dysfunction in response to cocaine and Tat exposure. In conclusion, our findings demonstrate that Tat/cocaine -mediated production of ROS activate Ras/Raf/ERK1/2 pathway that contributes to disruption of tight junction protein leading to pulmonary endothelial dysfunction associated with pulmonary vascular remodeling.", "link"=>"http://www.mendeley.com/research/hiv1cocaine-induced-oxidative-stress-disrupts-tight-junction-protein1-human-pulmonary-microvascular", "reader_count"=>9, "reader_count_by_academic_status"=>{"Unspecified"=>1, "Professor > Associate Professor"=>1, "Student > Doctoral Student"=>2, "Student > Ph. D. Student"=>1, "Student > Master"=>2, "Professor"=>2}, "reader_count_by_user_role"=>{"Unspecified"=>1, "Professor > Associate Professor"=>1, "Student > Doctoral Student"=>2, "Student > Ph. D. Student"=>1, "Student > Master"=>2, "Professor"=>2}, "reader_count_by_subject_area"=>{"Unspecified"=>1, "Medicine and Dentistry"=>4, "Agricultural and Biological Sciences"=>2, "Business, Management and Accounting"=>1, "Chemistry"=>1}, "reader_count_by_subdiscipline"=>{"Medicine and Dentistry"=>{"Medicine and Dentistry"=>4}, "Chemistry"=>{"Chemistry"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>2}, "Business, Management and Accounting"=>{"Business, Management and Accounting"=>1}, "Unspecified"=>{"Unspecified"=>1}}, "group_count"=>1}

Scopus | Further Information

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/1340430"], "description"=>"<p>Effect of antioxidants (A) and catalase or SOD (B) on cocaine (1 µM) and Tat (25 ng/ml) mediated barrier dysfunction of HPMECs. Confluent monolayers were grown on collagen-coated Transwell inserts and treated with antioxidant cocktail followed by Tat and cocaine exposure for 6 or 24 hours. Monolayers were then treated with FITC-dextran and after 15 min the fluorescence in the lower compartment was measured and expressed as percentage of basal fluorescence. (C) Effect of antioxidants on Tat and/or cocaine mediated down-regulation of tight junction protein expression in pulmonary arterial endothelial cells. Cells grown on coverslip were immunostained for TJP-1. (D) Quantification of ZO-1 immunofluorescence using ImageJ software. (E) Western blot analysis of ZO-1 in various cellular compartments. Blot is representative of at least three independent experiments with histogram showing the average densitometry analysis normalized to β-integrin for membrane fraction, β-actin for cytosolic fraction and PCNA for nuclear compartment. The values shown are means (±S.E.M.). *P≤0.001 compared to control; <sup>@</sup>P≤0.001, compared to cocaine and Tat treatment.</p>", "links"=>[], "tags"=>["Molecular cell biology", "Cellular types", "Endothelial cells", "Signal transduction", "Signaling cascades", "ERK signaling cascade", "Signaling in cellular processes", "Ras signaling", "Cellular stress responses", "cardiovascular", "Pulmonary vascular diseases", "Vascular biology", "Infectious diseases", "Viral diseases", "hiv", "cocktail", "attenuates", "tat", "cocaine-mediated", "augmentation", "endothelial"], "article_id"=>895573, "categories"=>["Biological Sciences", "Medicine"], "users"=>["Pranjali Dalvi", "Kun Wang", "Joel Mermis", "Ruoxi Zeng", "Miles Sanderson", "Sara Johnson", "Yuqiao Dai", "Garima Sharma", "Amy O’Brien Ladner", "Navneet K. Dhillon"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0085246.g004", "stats"=>{"downloads"=>0, "page_views"=>9, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Antioxidant_cocktail_attenuates_Tat_and_cocaine_mediated_augmentation_of_endothelial_dysfunction_/895573", "title"=>"Antioxidant cocktail attenuates Tat and cocaine-mediated augmentation of endothelial dysfunction.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-01-07 03:32:10"}
  • {"files"=>["https://ndownloader.figshare.com/files/1340429"], "description"=>"<p>(A) HPMECs were treated with various concentrations of apocynin for 30 min followed by exposure to Tat/cocaine for 1 hour. Total ROS formation was measured by DCFDA assay. (B) HPMECs were treated with cocaine and/or Tat for 24 h followed by quantitative mRNA analysis of gp91phox (NOX2) by Real-Time RT-PCR using the SYBR Green detection method. C) Western blot analysis of gp91phox in HPMECs treated with cocaine and/or Tat for 48 h.The upper panel is the representative blot of gp91phox expression and lower panel is the histogram showing the densitometry analyses of 3 independent experiments (mean±SEM). (D) NOX2 mRNA expression and E) ROS generation in HPMECs transfected with scrambled or gp-91<sup>phox</sup> siRNA (5 nM). Cells were loaded with DCFDA for 30 min before 1 hour Tat/cocaine treatment for quantification of total ROS generation. The values shown are means (±SD) of two-three independent experiments. *P≤0.05, **P≤0.01, ***P≤0.001, compared to control; <sup>$</sup>P<0.05 compared with Tat treatment alone, <sup>@</sup>P≤0.05, <sup>@@</sup>P≤0.001, compared to cocaine and Tat treatment.</p>", "links"=>[], "tags"=>["Molecular cell biology", "Cellular types", "Endothelial cells", "Signal transduction", "Signaling cascades", "ERK signaling cascade", "Signaling in cellular processes", "Ras signaling", "Cellular stress responses", "cardiovascular", "Pulmonary vascular diseases", "Vascular biology", "Infectious diseases", "Viral diseases", "hiv", "knockdown", "nadph", "oxidase", "attenuates", "tat", "cocaine-mediated", "ros"], "article_id"=>895572, "categories"=>["Biological Sciences", "Medicine"], "users"=>["Pranjali Dalvi", "Kun Wang", "Joel Mermis", "Ruoxi Zeng", "Miles Sanderson", "Sara Johnson", "Yuqiao Dai", "Garima Sharma", "Amy O’Brien Ladner", "Navneet K. Dhillon"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0085246.g003", "stats"=>{"downloads"=>3, "page_views"=>30, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Inhibition_or_knockdown_of_NADPH_oxidase_attenuates_Tat_and_cocaine_mediated_ROS_formation_/895572", "title"=>"Inhibition or knockdown of NADPH oxidase attenuates Tat and cocaine-mediated ROS formation.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-01-07 03:32:10"}
  • {"files"=>["https://ndownloader.figshare.com/files/1340432"], "description"=>"<p>Phosphorylated ERK was detected by western blot analysis of HPMECs treated with (A) Tat and cocaine for different time intervals as indicated and (B) with cocaine and/or Tat for 1.5 hours. (C) ZO-1 expression analysis in HPMECs pre-treated with U0126 for 30 min followed by Tat and cocaine treatment for 24 hours. Membrane fraction was isolated using compartment protein fractionation kit and ZO-1 was detected by western blot analysis. Lower panels show the average densitometry analysis normalized to β-actin for total cellular extract (A and B) and β-integrin in case of membrane fraction (C) of at least three independent experiments. Mean (±S.E.M.). *P≤0.05, **P≤0.01, ***P≤0.001 compared to control; <sup>#</sup>P≤0.001 compared to cocaine treatment. <sup>$</sup>p<0.001 compared to Tat treatment, <sup>@</sup>P≤0.01, compared to combined cocaine and Tat treatment.</p>", "links"=>[], "tags"=>["Molecular cell biology", "Cellular types", "Endothelial cells", "Signal transduction", "Signaling cascades", "ERK signaling cascade", "Signaling in cellular processes", "Ras signaling", "Cellular stress responses", "cardiovascular", "Pulmonary vascular diseases", "Vascular biology", "Infectious diseases", "Viral diseases", "hiv", "tat", "cocaine-mediated", "erk", "activation", "reverses", "zo-1", "disruption"], "article_id"=>895575, "categories"=>["Biological Sciences", "Medicine"], "users"=>["Pranjali Dalvi", "Kun Wang", "Joel Mermis", "Ruoxi Zeng", "Miles Sanderson", "Sara Johnson", "Yuqiao Dai", "Garima Sharma", "Amy O’Brien Ladner", "Navneet K. Dhillon"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0085246.g006", "stats"=>{"downloads"=>3, "page_views"=>11, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Blocking_Tat_and_cocaine_mediated_ERK_activation_reverses_ZO_1_disruption_in_HPMECs_/895575", "title"=>"Blocking Tat and cocaine-mediated ERK activation reverses ZO-1 disruption in HPMECs.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-01-07 03:32:10"}
  • {"files"=>["https://ndownloader.figshare.com/files/1340431"], "description"=>"<p>(A) Ras activation was assessed by pull-down assay in cells treated with Tat and cocaine for 30 or 60 min. (B) HPMECs were pre-treated with antioxidant cocktail, SU5416 or BD1047 for 5 min followed by Tat and cocaine treatment for 30 min. Representative western blot images are shown with histogram showing the average densitometry analysis of at least three independent experiments. Mean (±S.E.M.), *P≤0.001, compared to control; <sup>@</sup>P≤0.001, compared to cocaine and Tat treatment.</p>", "links"=>[], "tags"=>["Molecular cell biology", "Cellular types", "Endothelial cells", "Signal transduction", "Signaling cascades", "ERK signaling cascade", "Signaling in cellular processes", "Ras signaling", "Cellular stress responses", "cardiovascular", "Pulmonary vascular diseases", "Vascular biology", "Infectious diseases", "Viral diseases", "hiv", "pathway", "tat", "exposed"], "article_id"=>895574, "categories"=>["Biological Sciences", "Medicine"], "users"=>["Pranjali Dalvi", "Kun Wang", "Joel Mermis", "Ruoxi Zeng", "Miles Sanderson", "Sara Johnson", "Yuqiao Dai", "Garima Sharma", "Amy O’Brien Ladner", "Navneet K. Dhillon"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0085246.g005", "stats"=>{"downloads"=>2, "page_views"=>18, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Activation_of_Ras_Raf_Erk_pathway_in_Tat_and_cocaine_exposed_HPMECs_/895574", "title"=>"Activation of Ras/Raf/Erk pathway in Tat and cocaine exposed HPMECs.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-01-07 03:32:10"}
  • {"files"=>["https://ndownloader.figshare.com/files/1340428"], "description"=>"<p>(A) Generation of ROS in HPMECs treated with Tat and/or cocaine was quantified by DCF assay at indicated time-points. (B) ROS production in cells treated with Tat and cocaine in the presence or absence of SU5416 (antagonist of VEGFR-2) or BD1047 (antagonist of sigma receptor) for 1 hour as analyzed by DCF assay. (C) Generation of H<sub>2</sub>O<sub>2</sub> was quantified using Amplex red assay kit. HPMECs were treated with Tat and cocaine in the presence or absence of catalase (10U/ml) or SOD (100U/ml) for 1 hour. (D) Reduction of H<sub>2</sub>O<sub>2</sub> formation on pre-treatment of Tat and cocaine exposed HPMECs with SU5416 or BD1047. (E) Changes in Tat and cocaine-mediated superoxide generation on SU5416 or BD1047 pre-treatment. Formation of superoxide (O<sub>2</sub><sup>−</sup>) was quantified by SOD-inducible cytochrome c reductase assay. The values shown are means (±SD) of at least three independent experiments. *P≤0.05, **P≤0.01, ***P≤0.001 compared to control; <sup>#</sup>P≤0.05, <sup>##</sup>P≤0.01,<sup> ###</sup>P≤0.001 compared to cocaine treatment; <sup>$</sup>P≤0.001 compared to Tat treatment;<sup> @</sup> P≤0.05, <sup>@@</sup> P≤0.001, compared to Tat and cocaine combinational treatment; <sup>&</sup>P≤0.01, <sup>&&</sup>P≤0.001 compared to catalase-treated control; <sup>%</sup>‘P≤0.001, compared to SOD-treated control.</p>", "links"=>[], "tags"=>["Molecular cell biology", "Cellular types", "Endothelial cells", "Signal transduction", "Signaling cascades", "ERK signaling cascade", "Signaling in cellular processes", "Ras signaling", "Cellular stress responses", "cardiovascular", "Pulmonary vascular diseases", "Vascular biology", "Infectious diseases", "Viral diseases", "hiv", "oxidative", "pulmonary", "endothelial", "cells", "tat"], "article_id"=>895571, "categories"=>["Biological Sciences", "Medicine"], "users"=>["Pranjali Dalvi", "Kun Wang", "Joel Mermis", "Ruoxi Zeng", "Miles Sanderson", "Sara Johnson", "Yuqiao Dai", "Garima Sharma", "Amy O’Brien Ladner", "Navneet K. Dhillon"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0085246.g002", "stats"=>{"downloads"=>0, "page_views"=>7, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Enhanced_oxidative_stress_on_treatment_of_pulmonary_endothelial_cells_with_Tat_and_cocaine_/895571", "title"=>"Enhanced oxidative stress on treatment of pulmonary endothelial cells with Tat and cocaine.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-01-07 03:32:10"}
  • {"files"=>["https://ndownloader.figshare.com/files/1340427"], "description"=>"<p>(A) Expression of sigma and dopamine receptors in HPMECs as analyzed by Western blot of total cellular extract. (B) HPMECs were treated with Tat (25 ng/ml) and cocaine (1 µM) for 6 or 24 hours in the presence or absence of SU5416 (antagonist of VEGFR-2) or BD1047 (antagonist of sigma receptor). FITC-Dextran permeability was assessed by using <i>in-vitro</i> vascular permeability assay kit. The values shown are means (±SD) of at least three independent experiments. (C) Membrane fraction was isolated and analyzed for ZO-1 by western blot analysis. Blot is representative of at least three independent experiments with histogram showing (lower panel) the average densitometry analysis normalized to β-integrin (mean ± S.E.M). (D) Representative images showing immunocyto-fluorescence staining of ZO-1. (E) Quantification of ZO-1 immunofluorescence using ImageJ software. The values are represented as fold change compared to untreated control. *P≤0.01, **P≤0.001 compared to untreated control; <sup>@</sup>P≤0.05, <sup>@@</sup>P≤0.01, <sup>@@@</sup>P≤0.001 compared to Tat and cocaine treatment.</p>", "links"=>[], "tags"=>["Molecular cell biology", "Cellular types", "Endothelial cells", "Signal transduction", "Signaling cascades", "ERK signaling cascade", "Signaling in cellular processes", "Ras signaling", "Cellular stress responses", "cardiovascular", "Pulmonary vascular diseases", "Vascular biology", "Infectious diseases", "Viral diseases", "hiv", "tat", "mediated", "endothelial", "dysfunction", "vegfr-2", "sigma", "receptor"], "article_id"=>895570, "categories"=>["Biological Sciences", "Medicine"], "users"=>["Pranjali Dalvi", "Kun Wang", "Joel Mermis", "Ruoxi Zeng", "Miles Sanderson", "Sara Johnson", "Yuqiao Dai", "Garima Sharma", "Amy O’Brien Ladner", "Navneet K. Dhillon"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0085246.g001", "stats"=>{"downloads"=>3, "page_views"=>10, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Attenuation_of_Tat_and_cocaine_mediated_endothelial_dysfunction_in_the_presence_of_VEGFR_2_or_sigma_receptor_antagonists_/895570", "title"=>"Attenuation of Tat and cocaine mediated endothelial dysfunction in the presence of VEGFR-2 or sigma receptor antagonists.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-01-07 03:32:10"}

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