Galina N Antonova

Georgia Health Sciences University, Augusta, GA, United States

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Publications (8)38.57 Total impact

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    ABSTRACT: Multifunctional cytokine transforming growth factor-beta (TGF-β1) plays a critical role in the pathogenesis of acute lung inflammation by controlling endothelial monolayer permeability. TGF-β1 regulates endothelial cell (EC) functions via two distinct receptors, activin receptor-like kinase 1 (ALK1) and activin receptor-like kinase 5 (ALK5). The precise roles of ALK1 and ALK5 in the regulation of TGF-β1-induced lung endothelium dysfunction remain mostly unknown. We now report that adenoviral infection with constitutively active ALK5 (caALK5), but not caALK1, induces EC retraction and that this receptor predominantly controls EC permeability. We demonstrate that ubiquitinated ALK5 and phosphorylated heat shock protein 27 (phospho-Hsp27) specifically accumulate in the cytoskeleton fraction, which parallels with microtubule collapse, cortical actin disassembly and increased EC permeability. We have found that ALK1 and ALK5 interact with heat shock protein 90 (Hsp90). Moreover, the Hsp90 inhibitor radicicol (RA) prevents accumulation of ubiquitinated caALK5 and phospho-Hsp27 in the cytoskeletal fraction and restore the decreased EC permeability induced by caALK5. We hypothesize that specific translocation of ubiquitinated ALK5 receptor into the cytoskeleton compartment due to its lack of degradation is the mechanism that causes the divergence of caALK1 and caALK5 signaling.
    Journal of Cellular Physiology 04/2011; 227(2):759-71. · 4.22 Impact Factor
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    ABSTRACT: Controlling macrophage responses to pathogenic stimuli is critical for prevention of and recovery from the inflammatory state associated with the pathogenesis of many diseases. The adhesion receptor αVβ3 integrin is thought to be an important receptor that regulates macrophage differentiation and macrophage responses to external signaling, but it has not been previously identified as a contributor to macrophage-related inflammation. Using an in vitro model of human blood monocytes (Mo) and monocyte-derived macrophages (MDMs) we demonstrate that αVβ3 ligation results in sustained increases of the transcription factor NF-κB DNA-binding activity, as compared with control isotype-matched IgG(1). Activation of NF-κB parallels the increase of NF-κB-dependent pro-inflammatory cytokine mRNA expression in MDMs isolated from individual donors, for example, TNF-α (8- to 28-fold), IL-1β (15- to 30-fold), IL-6 (2- to 4-fold), and IL-8 (5- to 15-fold) whereas there is more than a 10-fold decrease in IL-10 mRNA level occurs. Upon ligation of the αVβ3 receptor, treatment with TNF-α (10 ng/ml) or LPS (200 ng/ml, 1,000 EU) results in the enhanced and synergistic activation of NF-κB and LPS-induced TNF-α secretion. As additional controls, an inhibitor of αVβ3 integrin, cyclic RGD (10 µg/ml; IC(50) = 7.6 µM), attenuates the effects of αVβ3 ligation, and the natural ligand of αVβ3 integrin, vitronectin, reproduces the effects of αVβ3 activation by an immobilizing anti-αVβ3 integrin mAb. We hypothesize that αVβ3 activation can maintain chronic inflammatory processes in pathological conditions and that the loss of αVβ3 ligation will allow macrophages to escape from the inflammatory state.
    Journal of Cellular Physiology 02/2011; 226(2):469-76. · 4.22 Impact Factor
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    ABSTRACT: Heat shock protein 90 (hsp90) inhibitors inactivate and/or degrade various client proteins, including many involved in inflammation. Increased vascular permeability is a hallmark of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Thus, we tested the hypothesis that hsp90 inhibitors may prevent and/or restore endothelial cell (EC) permeability after injury. Exposure of confluent bovine pulmonary arterial endothelial cell (BPAEC) monolayer to TGF-beta1, thrombin, bacterial lipopolysaccharide (LPS), or vascular endothelial growth factor (VEGF) increased BPAEC permeability, as revealed by decreased transendothelial electrical resistance (TER). Treatment of injured endothelium with hsp90 inhibitors completely restored TER of BPAEC. Similarly, preincubation of BPAEC with hsp90 inhibitors prevented the decline in TER induced by the exposure to thrombin, LPS, VEGF, or TGF-beta1. In addition, hsp90 inhibitors restored the EC barrier function after PMA or nocodazole-induced hyperpermeability. These effects of the hsp90 inhibitors were associated with the restoration of TGF-beta1- or nocodazole-induced decrease in VE-cadherin and beta-catenin expression at EC junctions. The protective effect of hsp90 inhibitors on TGF-beta1-induced hyperpermeability was critically dependent upon preservation of F-actin cytoskeleton and was associated with the inhibition of agonist-induced myosin light chain (MLC) and myosin phosphatase target subunit 1 (MYPT1) phosphorylation, F-actin stress fibers formation, microtubule disassembly, increase in hsp27 phosphorylation, and association of hsp90 with hsp27, but independent of p38MAPK activity. We conclude that hsp90 inhibitors exert barrier protective effects on BPAEC, at least in part, via inhibition of hsp27-mediated, agonist-induced cytoskeletal rearrangement, and therefore may have useful therapeutic value in ALI, ARDS, and other pulmonary inflammatory disease.
    American Journal of Respiratory Cell and Molecular Biology 06/2008; 39(5):551-9. · 4.15 Impact Factor
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    ABSTRACT: Endothelial hyperperme ability leading to vascular leak is an important consequence of sepsis and sepsis-induced lung injury. We previously reported that heat shock protein (hsp) 90 inhibitor pretreatment improved pulmonary barrier dysfunction in a murine model of sepsis-induced lung injury. We now examine the effects of hsp90 inhibitors on LPS-mediated endothelial hyperpermeability, as reflected in changes in transendothelial electrical resistance (TER) of bovine pulmonary arterial endothelial cells (BPAEC). Vehicle-pretreated cells exposed to endotoxin exhibited a concentration-dependent decrease in TER, activation of pp60(Src), phosphorylation of the focal adhesion protein paxillin, and reduced expression of the adherens junction proteins, vascular endothelial (VE)-cadherin and beta-catenin. Pretreatment with the hsp90 inhibitor, radicicol, prevented the decrease in TER, maintained VE-cadherin and beta-catenin expression, and inhibited activation of pp60(Src) and phosphorylation of paxillin. Similarly, when BPAEC hyperpermeability was induced by endotoxin-activated neutrophils, pretreatment of neutrophils and/or endothelial cells with radicicol protected against the activated neutrophil-induced decrease in TER. Increased paxillin phosphorylation and decreased expression of beta-catenin and VE-cadherin were also observed in mouse lungs 12 h after intraperitoneal endotoxin and attenuated in mice pretreated with radicicol. These results suggest that hsp90 plays an important role in sepsis-associated endothelial barrier dysfunction.
    AJP Lung Cellular and Molecular Physiology 05/2008; 294(4):L755-63. · 3.52 Impact Factor
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    ABSTRACT: The nitric oxide receptor soluble guanylyl cyclase (sGC) exists in multimeric protein complexes, including heat shock protein (HSP) 90 and endothelial nitric oxide synthase. Inhibition of HSP90 by geldanamycin causes proteasomal degradation of sGC protein. In this study, we have investigated whether COOH terminus of heat shock protein 70-interacting protein (CHIP), a co-chaperone molecule that is involved in protein folding but is also a chaperone-dependent ubiquitin E3 ligase, could play a role in the process of degradation of sGC. Transient overexpression of CHIP in COS-7 cells degraded heterologous sGC in a concentration-related manner; this downregulation of sGC was abrogated by the proteasome inhibitor MG-132. Transfection of tetratricopeptide repeats and U-box domain CHIP mutants attenuated sGC degradation, suggesting that both domains are indispensable for CHIP function. Results from immunoprecipitation and indirect immunofluorescent microscopy experiments demonstrated that CHIP is associated with sGC, HSP90, and HSP70 in COS-7 cells. Furthermore, CHIP increased the association of HSP70 with sGC. In in vitro ubiquitination assays using purified proteins and ubiquitin enzymes, E3 ligase CHIP directly ubiquitinated sGC; this ubiquitination was potentiated by geldanamycin in COS-7 cells, followed by proteasomal degradation. In rat aortic smooth muscle cells, endogenous sGC was also degraded by adenovirus-infected wild-type CHIP but not by the chaperone interaction-deficient K30A CHIP, whereas CHIP, but not K30A, attenuated sGC expression in, and nitric oxide donor-induced relaxation of, rat aortic rings, suggesting that CHIP plays a regulatory role under physiological conditions. This study reveals a new mechanism for the regulation of sGC, an important mediator of cellular and vascular function.
    AJP Heart and Circulatory Physiology 12/2007; 293(5):H3080-7. · 4.01 Impact Factor
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    ABSTRACT: Severe sepsis is the leading cause of death for patients in intensive care units. Patients with severe sepsis develop multiple organ failure, including acute lung injury (ALI), resulting from a deregulated inflammatory response. Inhibitors of the ubiquitous chaperone, heat shock protein 90 (Hsp90), block the activity of certain proinflammatory mediators in vitro. We hypothesized that Hsp90 inhibitors may ameliorate the inflammation and ALI associated with severe sepsis. To test the hypothesis that Hsp90 inhibitors prolong survival, attenuate inflammation, and reduce lung injury in a murine model of sepsis. Male C57BL/6 mice received either one of two Hsp90 inhibitors, radicicol or 17-allylaminodemethoxygeldanamycin (17-AAG), 24, 12, 6, and 0 hours before receiving a lethal dose of endotoxin (6.75 x 10(4) endotoxin units/g body weight). Outcomes included survival and parameters of systemic inflammation (plasma neutrophil, cytokine, chemokine, and nitrite/nitrate levels), pulmonary inflammation (lung nuclear factor-kappaB and myeloperoxidase activities, inducible nitric oxide synthase expression, inducible nitric oxide synthase-Hsp90 complex formation, and leukocyte infiltration), and lung injury (pulmonary capillary leak and lung function). Mice pretreated with vehicle and receiving endotoxin exhibited 100% 24-hour lethality, a dramatic increase in all parameters of systemic and pulmonary inflammation, increased capillary leak, and reduced lung function. Compared with them, mice receiving either radicicol or 17-AAG before endotoxin exhibited prolonged survival, reduced or abolished increases in systemic and pulmonary inflammatory parameters, attenuated capillary leak, and restored, normal lung function. Hsp90 inhibitors may offer a new pharmacological tool in the management of severe sepsis and severe sepsis-induced ALI.
    American Journal of Respiratory and Critical Care Medicine 11/2007; 176(7):667-75. · 11.04 Impact Factor
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    ABSTRACT: Large (pathological) amounts of nitric oxide (NO) induce cell injury, whereas low (physiological) NO concentrations often ameliorate cell injury. We tested the hypotheses that pretreatment of endothelial cells with low concentrations of NO (preconditioning) would prevent injury induced by high NO concentrations. Apoptosis, induced in bovine aortic endothelial cells (BAECs) by exposing them to either 4 mM sodium nitroprusside (SNP) or 0.5 mM N-(2-aminoethyl)-N-(2-hydroxy-2-nitrosohydrazino)-1,2-ethylenediamine (spermine NONOate) for 8 h, was abolished by 24-h pretreatment with either 100 microM SNP, 10 microM spermine NONOate, or 100 microM 8-bromo-cGMP (8-Br-cGMP). Repair of BAECs following wounding, measured as the recovery rate of transendothelial electrical resistance, was delayed by 8-h exposure to 4 mM SNP, and this delay was significantly attenuated by 24-h pretreatment with 100 microM SNP. NO preconditioning produced increased association and expression of soluble guanyl cyclase (sGC) and heat shock protein 90 (HSP90). The protective effect of NO preconditioning, but not the injurious effect of 4 mM SNP, was abolished by either a sGC activity inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ) or a HSP90 binding inhibitor (radicicol) and was mimicked by 8-Br-cGMP. We conclude that preconditioning with a low dose of NO donor accelerates repair and maintains endothelial integrity via a mechanism that includes the HSP90/sGC pathway. HSP90/sGC may thus play a role in the protective effects of NO-generating drugs from injurious stimuli.
    AJP Heart and Circulatory Physiology 03/2007; 292(2):H893-903. · 4.01 Impact Factor
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    ABSTRACT: Although the existence of hsp90-NOS and hsp90-sGC complexes is now firmly established, their role in many pathophysiological processes remain unclear. These complexes may represent physiological mechanisms aimed at maximizing intracellular cGMP production in response to endogenous or drug-derived NO in endothelial cells and thus affecting permeability, proliferation, migration and apoptosis. Along with minimizing NO scavenging by superoxide and reducing the formation of peroxynitrite, these complexes may also prolong sGC stability by retarding its degradation. Our work and that of others have demonstrated that, depending on the environment, sGC interaction with hsp90 can optimize sGC enzyme activity or modulate sGC survival. This review addresses the functional significance of hsp90 complexes with NOS (eNOS, iNOS) and sGC in endothelial cells relevant for maintaining endothelial barrier integrity and angiogenesis. Structural and functional characteristics of sGC, its expression, transcriptional and post-translational regulation, as they relate to sGC-hsp90 interactions, will also be examined.
    Clinical hemorheology and microcirculation 02/2007; 37(1-2):19-35. · 3.40 Impact Factor