-
[show abstract]
[hide abstract]
ABSTRACT: Oxidant stress caused by pathological elevation of reactive oxygen species (ROS) production in the endothelial cells lining the vascular lumen is an important component of many vascular and pulmonary disease conditions. NADPH oxidase (NOX) activated by pathological mediators including angiotensin and cytokines is a major source of endothelial ROS. In order to intercept this pathological pathway, we have encapsulated an indirect NOX inhibitor, MJ33, into immunoliposomes (Ab-MJ33/IL) targeted to endothelial marker platelet endothelial cell adhesion molecule (PECAM-1). Ab-MJ33/IL, but not control IgG-MJ33/IL are specifically bound to endothelium and attenuated angiotensin-induced ROS production in vitro and in vivo. Additionally, Ab-MJ33/IL inhibited endothelial expression of the inflammatory marker vascular cell adhesion molecule (VCAM) in cells and animals challenged with the cytokine TNF. Furthermore, Ab-MJ33/IL alleviated pathological disruption of endothelial permeability barrier function in cells exposed to vascular endothelial growth factor (VEGF) and in the lungs of mice challenged with lipopolysaccharide (LPS). Of note, the latter beneficial effect has been achieved both by prophylactic and therapeutic injection of Ab-MJ33/IL in animals. Therefore, specific suppression of ROS production by NOX in endothelium, attainable by Ab-MJ33/IL targeting, may help deciphering mechanisms of vascular oxidative stress and inflammation, and potentially improve treatment of these conditions.
Journal of Controlled Release 09/2012; 163(2):161-9. · 5.73 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Reactive oxygen species (ROS) are implicated as injurious and as signaling agents in human maladies including inflammation, hyperoxia, ischemia-reperfusion and acute lung injury. ROS produced by the endothelium play an important role in vascular pathology. They quench, for example, nitric oxide, and mediate pro-inflammatory signaling. Antioxidant interventions targeted for the vascular endothelium may help to control these mechanisms. Animal studies have demonstrated superiority of targeting ROS-quenching enzymes catalase and superoxide dismutase to endothelial cells over nontargeted formulations. A diverse arsenal of targeted antioxidant formulations devised in the last decade shows promising results for specific quenching of endothelial ROS. In addition to alleviation of toxic effects of excessive ROS, these targeted interventions suppress pro-inflammatory mechanisms, including endothelial cytokine activation and barrier disruption. These interventions may prove useful in experimental biomedicine and, perhaps, in translational medicine.
Therapeutic delivery 02/2012; 3(2):263-76.
-
Gerhard Preissler,
Florian Loehe,
Ines V Huff,
Ulrich Ebersberger, Vladimir V Shuvaev,
Iris Bittmann,
Iris Hermanns,
James C Kirkpatrick,
Karl Fischer,
Martin E Eichhorn,
Hauke Winter,
Karl W Jauch,
Steven M Albelda,
Vladimir R Muzykantov,
Rainer Wiewrodt
[show abstract]
[hide abstract]
ABSTRACT: Donor organ shortage represents a major problem in lung transplantation. Donation after cardiac death could help to expand the pool of organs, but the additional period of warm ischemia after cardiac arrest aggravates primary graft dysfunction. The pulmonary endothelium of the graft constitutes an important source and target of reactive oxygen species generated during ischemia and reperfusion. Targeted protection of graft pulmonary endothelial cells by the antioxidant enzyme catalase, conjugated with a platelet/endothelial cell adhesion molecule-1 (PECAM-1) antibody to nanosized particles (anti-PECAM/catalase conjugates), might improve outcome in lung transplantation using donors after cardiac death and prolonged hypothermic preservation.
Left lung transplantation was performed in 18 pigs. Before cardiac arrest, donors received anti-PECAM/catalase, unconjugated component mixture or vehicle solution. After 90-min warm and 18-hr hypothermic ischemia, lungs were transplanted, and function was assessed during 6 hr after reperfusion. Samples of bronchoalveolar lavage fluid and lung tissue were taken thereafter. Six sham-operated animals served as controls.
During 6-hr reperfusion, anti-PECAM/catalase significantly ameliorated graft function, evidenced by major improvements of gas exchange and reduced intrapulmonary shunt fraction. Furthermore, lipid peroxidation, alveolar leakage, and edema formation were reduced in protected grafts. Similarly moderate lung pathology was seen after transplantation.
Augmentation of the antioxidant capacity of graft pulmonary endothelial cells with anti-PECAM/catalase nanoparticles represents a straightforward approach to enable a safe transplantation of prolonged preserved donation after cardiac death lungs. Anti-PECAM/catalase protection alleviated oxidative stress and allowed immediate reconstitution of normal gas exchange and pulmonary microcirculation, a prerequisite for improved graft and patient outcome.
Transplantation 08/2011; 92(4):380-7. · 4.00 Impact Factor
-
Vladimir V Shuvaev,
Marc A Ilies,
Eric Simone,
Sergei Zaitsev,
Younghoon Kim,
Shenshen Cai,
Abdullah Mahmud,
Thomas Dziubla,
Silvia Muro,
Dennis E Discher,
Vladimir R Muzykantov
[show abstract]
[hide abstract]
ABSTRACT: The endothelial lining of the lumen of blood vessels is a key therapeutic target for many human diseases. Polymeric filomicelles that self-assemble from polyethylene oxide (PEO)-based diblock copolymers are long and flexible rather than small or rigid, can be loaded with drugs, and--most importantly--they circulate for a prolonged period of time in the bloodstream due in part to flow alignment. Filomicelles seem promising for targeted drug delivery to endothelial cells because they can in principle adhere strongly, length-wise to specific cell surface determinants. In order to achieve such a goal of vascular drug delivery, two fundamental questions needed to be addressed: (i) whether these supramolecular filomicelles retain structural integrity and dynamic flexibility after attachment of targeting molecules such as antibodies, and (ii) whether the avidity of antibody-carrying filomicelles is sufficient to anchor the carrier to the endothelial surface despite the effects of flow that oppose adhesive interactions. Here we make targeted filomicelles that bear antibodies which recognize distinct endothelial surface molecules. We characterize these antibody targeted filomicelles and prove that (i) they retain structural integrity and dynamic flexibility and (ii) they adhere to endothelium with high specificity both in vitro and in vivo. These results provide the basis for a new drug delivery approach employing antibody-targeted filomicelles that circulate for a prolonged time yet bind to endothelial cells in vascular beds expressing select markers.
ACS Nano 08/2011; 5(9):6991-9. · 10.77 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Reactive oxygen species (ROS) superoxide anion (O(2)()) and hydrogen peroxide (H(2)O(2)) produced by activated leukocytes and endothelial cells in sites of inflammation or ischemia cause endothelial barrier dysfunction that may lead to tissue edema. Antioxidant enzymes (AOEs) catalase and superoxide dismutase (SOD) conjugated with antibodies to platelet-endothelial cell adhesion molecule-1 (PECAM-1) specifically bind to endothelium, quench the corresponding ROS, and alleviate vascular oxidative stress and inflammation. In the present work, we studied the effects of anti-PECAM/catalase and anti-PECAM/SOD conjugates on the abnormal permeability manifested by transendothelial electrical resistance decline, increased fluorescein isothiocyanate-dextran influx, and redistribution of vascular endothelial-cadherin in human umbilical vein endothelial cell (HUVEC) monolayers. Anti-PECAM/catalase protected HUVEC monolayers against H(2)O(2)-induced endothelial barrier dysfunction. Polyethylene glycol-conjugated catalase exerted orders of magnitude lower endothelial uptake and no protective effect, similarly to IgG/catalase. Anti-PECAM/catalase, but not anti-PECAM/SOD, alleviated endothelial hyperpermeability caused by exposure to hypoxanthine/xanthine oxidase, implicating primarily H(2)O(2) in the disruption of the endothelial barrier in this model. Thrombin-induced endothelial permeability was not affected by treatment with anti-PECAM/AOEs or the NADPH oxidase inhibitor apocynin or overexpression of AOEs, indicating that the endogenous ROS play no key role in thrombin-mediated endothelial barrier dysfunction. In contrast, anti-PECAM/SOD, but not anti-PECAM/catalase, inhibited a vascular endothelial growth factor (VEGF)-induced increase in endothelial permeability, identifying a key role of endogenous O(2)() in the VEGF-mediated regulation of endothelial barrier function. Therefore, AOEs targeted to endothelial cells provide versatile molecular tools for testing the roles of specific ROS in vascular pathology and may be translated into remedies for these ROS-induced abnormalities.
Journal of Pharmacology and Experimental Therapeutics 07/2011; 338(1):82-91. · 3.83 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 'Endothelial cells lining vascular luminal surface represent an important site of signaling and injurious effects of reactive oxygen species (ROS) produced by other cells and endothelium itself in ischemia, inflammation and other pathological conditions. Targeted delivery of ROS modulating enzymes conjugated with antibodies to endothelial surface molecules (vascular immunotargeting) provides site-specific interventions in the endothelial ROS, unattainable by other formulations including PEG-modified enzymes. Targeting of ROS generating enzymes (e.g., glucose oxidase) provides ROS- and site-specific models of endothelial oxidative stress, whereas targeting of antioxidant enzymes SOD and catalase offers site-specific quenching of superoxide anion and H(2)O(2). These targeted antioxidant interventions help to clarify specific role of endothelial ROS in vascular and pulmonary pathologies and provide basis for design of targeted therapeutics for treatment of these pathologies. In particular, antibody/catalase conjugates alleviate acute lung ischemia/reperfusion injury, whereas antibody/SOD conjugates inhibit ROS-mediated vasoconstriction and inflammatory endothelial signaling. Encapsulation in protease-resistant, ROS-permeable carriers targeted to endothelium prolongs protective effects of antioxidant enzymes, further diversifying the means for targeted modulation of endothelial ROS.
Journal of Controlled Release 03/2011; 153(1):56-63. · 5.73 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Endothelial targeting of antioxidant enzymes attenuates acute vascular oxidative stress in animal studies. Superoxide dismutase (SOD) and catalase conjugated with antibodies to Platelet-Endothelial Cell Adhesion Molecule-1 (anti-PECAM/SOD and anti-PECAM/catalase) bind to endothelium, accumulate in the pulmonary vasculature, and detoxify reactive oxygen species. In order to define the role of conjugate size in the efficacy and specificity of endothelial targeting, we synthesized anti-PECAM/enzyme conjugates of controlled size (40nm-10,000nm). Binding of anti-PECAM/enzymes to endothelial cells increased with conjugate size from 300nm to 2μm (from 2.5 to 8.5% of bound fraction), and was specific, as conjugates did not bind to PECAM-negative cells. Pulmonary uptake of anti-PECAM/enzyme conjugates injected intravenously in mice also increased from 4.5 to 16% of injected dose for particles from 200 to 800nm. However, control conjugates larger than 300nm showed elevated non-specific pulmonary uptake, indicating that the targeting specificity of anti-PECAM/enzyme conjugates in vivo has a bell-shaped curve with a maximum close to 300-nm diameter. These results show that: i) the size of an antibody/enzyme conjugate modulates efficacy and specificity of targeting, and ii) a size optimum should be defined in vivo to account for parameters that are difficult to model in cell culture.
Journal of Controlled Release 10/2010; 149(3):236-41. · 5.73 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Elevated generation of reactive oxygen species (ROS) by endothelial enzymes, including NADPH-oxidase, is implicated in vascular oxidative stress and endothelial proinflammatory activation involving exposure of vascular cell adhesion molecule-1 (VCAM-1). Catalase and superoxide dismutase (SOD) conjugated with antibodies to platelet/endothelial cell adhesion molecule 1 (PECAM-1) bind specifically to endothelium and inhibit effects of corresponding ROS, H(2)O(2), and superoxide anion. In this study, anti-PECAM/SOD, but not anti-PECAM/catalase or nontargeted enzymes, including polyethylene glycol (PEG)-SOD, inhibited 2- to 3-fold VCAM expression caused by tumor necrosis factor (TNF), interleukin-1β, and lipopolysaccharide. Anti- PECAM/SOD, but not nontargeted counterparts, accumulated in vascular endothelium after intravenous injection, localized in endothelial endosomes, and inhibited by 70% lipopolysaccharide-caused VCAM-1 expression in mice. Anti-PECAM/SOD colocalized with EEA-1-positive endothelial vesicles and quenched ROS produced in response to TNF. Inhibitors of NADPH oxidase and anion channel ClC3 blocked TNF-induced VCAM expression, affirming that superoxide produced and transported by these proteins, respectively, mediates inflammatory signaling. Anti-PECAM/SOD abolished VCAM expression caused by poly(I:C)-induced activation of toll-like receptor 3 localized in intracellular vesicles. These results directly implicate endosomal influx of superoxide in endothelial inflammatory response and suggest that site-specific interception of this signal attained by targeted delivery of anti-PECAM/SOD into endothelial endosomes may have anti-inflammatory effects.
The FASEB Journal 09/2010; 25(1):348-57. · 5.71 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The endothelium is one of the key targets for pharmacological interventions in oxidative stress and thrombosis, two conditions that are notoriously difficult to treat due to limited efficacy and precision of action of current drugs. Design of molecular and nano-devices that deliver potent antioxidant and antithrombotic therapeutic enzymes to the endothelium holds promise to improve the potency, localization, timing, specificity, safety, and mechanistic precision of these interventions. In particular, cell adhesion molecules expressed on the surface of resting and pathologically altered endothelial cells can be used for drug delivery to the endothelial surface (preferable for thrombolytics) and into intracellular compartments (preferable for antioxidants). Drug delivery platforms including protein conjugates, recombinant fusion constructs, and stealth polymer carriers designed to target these drugs to endothelium are reviewed in this article.
Seminars in Thrombosis and Hemostasis 04/2010; 36(3):332-42. · 4.52 Impact Factor
-
Vladimir V Shuvaev,
Melpo Christofidou-Solomidou,
Faiz Bhora,
Karine Laude,
Hua Cai,
Sergei Dikalov,
Evguenia Arguiri,
Charalambos C Solomides,
Steven M Albelda,
David G Harrison,
Vladimir R Muzykantov
[show abstract]
[hide abstract]
ABSTRACT: Oxidative stress underlies diverse vascular diseases, but its management remains elusive, in part because of our inability to selectively detoxify reactive oxygen species (ROS) in pathological sites and our limited understanding which species need to be eliminated. The antioxidant enzymes (AOEs) superoxide dismutase (SOD) and catalase (which decompose and H(2)O(2), respectively), conjugated with an antibody to platelet-endothelial cell adhesion molecule-1 (PECAM-1), bind to endothelial cells and alleviate oxidative stress in cell culture models. Here, we studied the effects of these antioxidant conjugates in mouse models of vascular oxidative stress. Anti-PECAM/catalase and anti-PECAM/SOD conjugates, in contrast to control IgG/AOE conjugates, accumulated in the lungs and vascularized organs after intravenous injection in wild-type, but not PECAM KO mice. Anti-PECAM/catalase, but not anti-PECAM/SOD, protected mice from lung injury induced by H(2)O(2) produced by glucose oxidase deposited in the pulmonary vasculature. Anti-PECAM/catalase also reduced alveolar edema and attenuated decline in arterial oxygen in mice that underwent unilateral lung ischemia/reperfusion, whereas anti-PECAM/SOD was not effective, implying the key role of H(2)O(2) in tissue damage in this pathology. In contrast, anti-PECAM/SOD, but not anti-PECAM/catalase prevented oxidation of tetrahydrobiopterin and normalized vasoreactivity in the vessels of mice rendered hypertensive by pretreatment with angiotensin-II. This outcome agrees with reports implicating superoxide and peroxynitrite in altered endothelium-dependent vasodilatation in hypertension. Therefore, the use of endothelial cell-targeted antioxidants identifies the key specific species of ROS involved in various forms of vascular disease and holds promise for the mechanistically tailored treatment of these pathologies.
Journal of Pharmacology and Experimental Therapeutics 09/2009; 331(2):404-11. · 3.83 Impact Factor
-
Thomas D Dziubla, Vladimir V Shuvaev,
Nan Kang Hong,
Brian J Hawkins,
Muniswamy Madesh,
Hajime Takano,
Eric Simone,
Marian T Nakada,
Aron Fisher,
Steven M Albelda,
Vladimir R Muzykantov
[show abstract]
[hide abstract]
ABSTRACT: The medical utility of proteins, e.g. therapeutic enzymes, is greatly restricted by their labile nature and inadequate delivery. Most therapeutic enzymes do not accumulate in their targets and are inactivated by proteases. Targeting of enzymes encapsulated into substrate-permeable polymer nano-carriers (PNC) impermeable for proteases might overcome these limitations. To test this hypothesis, we designed endothelial targeted PNC loaded with catalase, an H(2)O(2)-detoxifying enzyme, and tested if this approach protects against vascular oxidative stress, a pathological process implicated in ischemia-reperfusion and other disease conditions. Encapsulation of catalase (MW 247 kD), peroxidase (MW 42 kD) and xanthine oxidase (XO, MW 300 kD) into approximately 300 nm diameter PNC composed of co-polymers of polyethylene glycol and poly-lactic/poly-glycolic acid (PEG-PLGA) was in the range approximately 10% for all enzymes. PNC/catalase and PNC/peroxidase were protected from external proteolysis and exerted enzymatic activity on their PNC diffusible substrates, H(2)O(2) and ortho-phenylendiamine, whereas activity of encapsulated XO was negligible due to polymer impermeability to the substrate. PNC targeted to platelet-endothelial cell (EC) adhesion molecule-1 delivered active encapsulated catalase to ECs and protected the endothelium against oxidative stress in cell culture and animal studies. Vascular targeting of PNC-loaded detoxifying enzymes may find wide medical applications including management of oxidative stress and other toxicities.
Biomaterials 02/2008; 29(2):215-27. · 7.40 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Targeting of the antioxidant enzyme catalase to endothelial cells protects against vascular oxidative stress induced by hydrogen peroxide (H(2)O(2))(Am J Physiol 285:L283-L292, 2003; Nat Biotechnol 21:392-398, 2003; Am J Physiol 293:L162-L169, 2007). However, another reactive oxygen species, superoxide anion, is also involved in many forms of vascular oxidative stress, including ischemia/reperfusion, hypertension, and inflammation. To protect endothelium against superoxide attack, we designed and tested antibody-directed targeting of superoxide dismutase (SOD) to the endothelial surface determinant, platelet-endothelial cell adhesion molecule (PECAM)-1. We synthesized anti-PECAM/SOD conjugates that retained 70% of enzymatic activity (superoxide anion dismutation) and specifically bound to endothelial cells, but not PECAM-negative cells. The effect of anti-PECAM/SOD delivery to cells was tested in two distinct models of oxidative stress induced by either extracellular or intracellular generation of superoxide anion. In the first model, anti-PECAM/SOD, but not unconjugated SOD, protected endothelial cells against injury caused by superoxide produced in the medium by hypoxanthine-xanthine oxidase. At the optimal dose, anti-PECAM/SOD provided up to 40 to 50% protection against cell death in this model. In the second model, anti-PECAM/SOD at the optimal dose provided complete protection against necrosis caused by paraquat-induced intracellular superoxide generation. Endothelial targeting of SOD represents a new molecular antioxidant approach that could be used for the management of vascular oxidative stress.
Journal of Pharmacology and Experimental Therapeutics 12/2007; 323(2):450-7. · 3.83 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Vascular drug targeting may improve therapies, yet a thorough understanding of the factors that regulate effects of drugs directed to the endothelium is needed to translate this approach into the clinical domain. To define factors modulating the efficacy and effects of endothelial targeting, we used a model enzyme (glucose oxidase, GOX) coupled with monoclonal antibodies (anti-TM(34) or anti-TM(201)) to distinct epitopes of thrombomodulin, a surface determinant enriched in the pulmonary endothelium. GOX delivery results in conversion of glucose and oxygen into H(2)O(2) leading to lung damage, a clear physiologic endpoint. Results of in vivo studies in mice showed that the efficiency of cargo delivery and its effect are influenced by a number of factors including: 1) The level of pulmonary uptake of the targeting antibody (anti-TM(201) was more efficient than anti-TM(34)); 2) The amount of an active drug delivered to the target; 3) The amount of target antigen on the endothelium (animals with suppressed TM levels showed less targeting); and, 4) The substrate availability for the enzyme cargo in the target tissue (hyperoxia augmented GOX-induced injury). Therefore, both activities of the conjugates and biological factors control targeting and effects of enzymatic cargo. Understanding the nature of such "modulating biological factors" will hopefully allow optimization and ultimately applications of drug targeting for "individualized" pharmacotherapy.
Journal of Controlled Release 05/2007; 118(2):235-44. · 5.73 Impact Factor
-
Mitchell Machtay,
Arnaud Scherpereel,
José Santiago,
James Lee,
Jim McDonough,
Paul Kinniry,
Evguenia Arguiri, Vladimir V Shuvaev,
Jing Sun,
Keith Cengel,
Charalambos C Solomides,
Melpo Christofidou-Solomidou
[show abstract]
[hide abstract]
ABSTRACT: Since oxidative injury is implicated in radiation-induced tissue damage to the lung, we studied systemically administered polyethylene glycol (PEGylated) antioxidant enzymes (AOEs) as pulmonary radioprotectors in mice.
C57/bl6 Mice received 13.5 Gy single-dose irradiation to the thorax. One cohort also received 100 microg of a 1:1 mixture of PEG-AOEs {PEG-catalase and PEG-superoxide dismutase (SOD)} intravenously, pre-irradiation and subgroups were evaluated at variable time-points for inflammation and fibrosis. Potential for AOE tumor protection was studied by thoracic irradiation of mice with Lewis lung carcinoma.
At 48 h post-irradiation, control irradiated mice had marked elevations of tissue p21, Bax and TGF-beta1 in lungs, not seen in irradiated, PEG-AOE-treated mice. TUNEL staining of lung sections was performed at just one time-point (24 h post-irradiation) and revealed a decrease in apoptotic cells with AOE treatment. At four months post-irradiation, these mice had significantly increased pulmonary fibrosis as measured by hydroxyproline content. Mice treated with PEG-AOE prior to irradiation had 4-month hydroxyproline levels that were similar to that of unirradiated controls (p = 0.28). This corresponded to less pulmonary fibrosis as visualized histologically when compared with mice irradiated without AOEs. PEG-AOEs did not prevent post-irradiation pulmonary inflammation or lung cancer response to irradiation.
A mixture of PEG-SOD and PEG-CAT successfully diminished radiation pulmonary fibrosis in mice. There was also a corresponding effect on several early biomarkers of lung injury and decreased apoptosis. There were no significant effects on acute pneumonitis or tumor protection.
Radiotherapy and Oncology 12/2006; 81(2):196-205. · 5.58 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Targeted drug delivery to endothelial cells lining the vascular lumen will provide effective, precise and safe therapeutic interventions for treatment of diverse disease conditions. Rational design of such drug delivery systems (DDS) includes the following intertwined tasks: 1) selection of proper target determinants on endothelial surfaces, such as cell adhesion molecules, ectopeptidases, or caveolar antigens; 2) production of affinity ligands useful for targeting, such as affinity peptides, antibodies, or their fragments; 3) selection and adopting of suitable delivery vehicles (such as liposomes or polymer nanocarriers); and 4) formulation of DDS with optimal targeting and therapeutic features. Important therapeutic features of DDS include: 1) sufficient targeting effectiveness, circulation time, and safety (i.e., lack of systemic and local adverse effects); 2) precise subcellular localization of drugs targeted to endothelial cells; and 3) adequate amplitude, kinetics, and duration of effects. This review utilizes examples of DDS-mediated interventions in vascular inflammation, oxidative stress, and thrombosis and analyzes them in an attempt to create design parameters that best regulate the pharmacological and therapeutic features of DDS that target endothelial cells.
Molecular Interventions 05/2006; 6(2):98-112. · 4.59 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Endothelial dysfunction is considered to be the earliest event in atherogenesis. Oxidative stress, inflammation, and apoptosis play critical roles in its progression and onset. Lipid peroxidation, which occurs during oxidative stress, results in the formation of lipid hydroperoxide-derived bifunctional electrophiles such as 4-hydroxy-2(E)-nonenal that induce apoptosis. In this study, recently identified lipid hydroperoxide-derived bifunctional electrophiles 4-oxo-2(E)-nonenal (ONE; 5-30 microm) and 4,5-epoxy-2(E)-decenal (EDE; 10-20 microM) were shown to cause a dose- and time-dependent apoptosis in EA.hy 926 endothelial cells. This was manifest by morphological changes, caspase-3 activation, and poly(ADP-ribose) polymerase cleavage. Bifunctional electrophiles caused cytochrome c release from mitochondria into the cytosol, implicating a mitochondrial pathway of apoptosis in the endothelial cells. The novel carboxylate-containing lipid hydroperoxide-derived bifunctional electrophile 9,12-dioxo-10(E)-dodecenoic acid was inactive because it could not translocate across the plasma membrane. However, its less polar methyl ester derivative (2-10 microM) was the most potent inducer of apoptosis of any bifunctional electrophile that has been tested. An acute decrease in intracellular glutathione (GSH) preceded the onset of apoptosis in bifunctional electrophile-treated cells. The ability of ONE and EDE to deplete GSH was directly correlated with their predicted reactivity toward nucleophilic amino acids. Liquid chromatography/mass spectrometry methodology was developed in order to examine the intracellular and extracellular concentrations of bifunctional electrophile-derived GSH adducts. Relative intracellular/extracellular ratios of the GSH adducts were identical with the rank order of potency for inducing caspase 3 activation. This suggests that there may be a role for the bifunctional electrophile-derived GSH adducts in the apoptotic response. N-Acetylcysteine rescued bifunctional electrophile-treated cells from apoptosis, whereas the GSH biosynthesis inhibitor d,l-buthionine-(R,S)-sulfoximine sensitized the cells to apoptosis. These data suggest that lipid hydroperoxide-derived bifunctional electrophiles may play an important role in cardiovascular pathology through their ability to induce endothelial cell apoptosis.
Free Radical Biology and Medicine 12/2005; 39(9):1162-76. · 5.42 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The streptavidin-biotin system may be used to synthesize immunoconjugates for targeted delivery of drugs, including therapeutic enzymes. The size of antibody-enzyme conjugates, which is controlled by the extent of biotinylation and molar ratio between the conjugate components, represents an important parameter that in some cases dictates subcellular addressing of drugs. This chapter describes the methodology of formation and characterization of polymeric immunoconjugates in the nanoscale range. A theoretical model of streptavidin conjugation based on general principles of polymer chemistry is considered. Factors that influence size and functional characterization of resulting polymer conjugates, as well as advantages and limitations of this approach, are described in detail. The protocols describe the formation of immunoconjugates possessing an antioxidant enzyme, catalase, directed to endothelial cells by anti-platelet endothelial cell adhesion molecule antibodies. However because of the modular nature of the streptavidin-biotin crosslinker system, the techniques herein can be easily adapted for the preparation of nanoscale immunoconjugates delivering other protein drugs to diverse cellular antigens.
Methods in molecular biology (Clifton, N.J.) 02/2004; 283:3-19.
-
[show abstract]
[hide abstract]
ABSTRACT: The streptavidin-biotin system may be used to synthesize immunoconjugates for targeted delivery of drugs, including therapeutic
enzymes. The size of antibody-enzyme conjugates, which is controlled by the extent of biotinylation and molar ratio between
the conjugate components, represents an important parameter that in some cases dictates subcellular addressing of drugs. This
chapter describes the methodology of formation and characterization of polymeric immunoconjugates in the nanoscale range.
A theoretical model of streptavidin conjugation based on general principles of polymer chemistry is considered. Factors that
influence size and functional characterization of resulting polymer conjugates, as well as advantages and limitations of this
approach, are described in detail. The protocols describe the formation of immunoconjugates possessing an antioxidant enzyme,
catalase, directed to endothelial cells by anti-platelet endothelial cell adhesion molecule antibodies. However because of
the modular nature of the streptavidin-biotin crosslinker system, the techniques herein can be easily adapted for the preparation
of nanoscale immunoconjugates delivering other protein drugs to diverse cellular antigens.
Key WordsImmunoconjugates–vascular immunotargeting–polymerization–nanoscale carrier–catalase–streptavidin–biotin–dynamic light scattering–drug delivery
12/2003: pages 3-19;
