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Microsoft word - akhtar et al_revised manuscript

JBC Papers in Press. Published on September 16, 2010 as Manuscript M110.126789
OXIDATIVE AND NITROSATIVE MODIFICATIONS OF TROPOELASTIN PREVENT
ELASTIC FIBER ASSEMBLY IN VITRO*
Kamal Akhtar1, Thomas J. Broekelmann2, Ming Miao3, Fred W. Keeley3, Barry C. Starcher4,
Richard A. Pierce1, Robert P. Mecham1,2, and Tracy L. Adair-Kirk1
Departments of Medicine1, and Cell Biology and Physiology2, Washington University School of
Medicine, St. Louis, Missouri, 63110, Molecular Structure and Function Program3, Hospital for Sick
Children, Toronto, Canada M5G 1X8, Department of Biochemistry4, University of Texas Health Center
at Tyler, Tyler, Texas 75708 Running head: Oxidants prevent elastic fiber assembly in vitro Address corresponds to: Tracy Adair-Kirk, 660 South Euclid Avenue, Box 8052, St. Louis, MO 63110 Tel: 314 454 7458; Fax: 314 454 5919; E-mail: tkirk@dom.wustl.edu Elastic fibers are extracellular structures
function of these tissues. Elastic fibers consist of that provide stretch and recoil properties of
two major components, elastin and microfibrils. tissues, such as lungs, heart, and skin. Elastin
Elastin is an amorphous component that is the predominant component of elastic
comprises most (>90%) of the mass of the fibers. Tropoelastin (TE), the precursor of
mature elastic fiber. Microfibrils are fibrillar elastin, is synthesized mainly during late fetal
components that are rich in acidic glycoproteins, and early postnatal stages. The turnover of
such as fibrilins (Fbns), fibulins (Fblns), and elastin in normal adult tissues is minimal.
microfibril-associated glycoproteins (MAGPs). However, in several pathological conditions
Other components, such as glycosaminoglycan often associated with inflammation and
(GAGs), lysyl oxidase (LOX), and other elastin oxidative stress, elastogenesis is re-initiated,
binding proteins are also present in elastic fibers but newly synthesized elastic fibers appear
abnormal. We sought to determine the effects
Elastogenic cells, such as fibroblasts and of reactive oxygen and nitrogen species
smooth muscle cells, secrete elastin as a soluble, (ROS/RNS) on the assembly of TE into elastic
precursor protein called tropoelastin (TE). fibers. Immunoblot analyses showed that TE
Assembly of monomeric TE into elastic fibers is is oxidatively and nitrosatively modified by
a multi-step process. TE monomers self- peroxynitrite (ONOO-) and hypochlorous
associate/coacervate into aggregates which are acid (HOCl) and by activated monocytes and
deposited onto pre-existing microfibrils. TE macrophages via release of ONOO- and
interacts with a number of different matrix HOCl. In an in vitro elastic fiber assembly
proteins during the assembly process. For model, oxidatively-modified TE was unable to
example, Fbln-4 and Fbln-5 facilitate TE form elastic fibers. Oxidation of TE enhanced
alignment for crosslinking and deposition of coacervation, an early step in elastic fiber
crosslinked TE aggregates onto microfibrils. assembly, but reduced crosslinking and
Microfibrillar components (MAGP-1, Fbn-1 and interactions with other proteins required for
Fbn-2) align TE aggregates to undergo further elastic fiber assembly, including fibulin-4,
crosslinking to form mature elastic fibers (5-9). fibulin-5, and fibrillin-2. These findings
Elastin is mainly synthesized during late establish that ROS/RNS can modify TE and
fetal and early postnatal stages and its turnover that these modifications affect the assembly of
in normal adult tissues is negligible. However, in elastic fibers. Thus, we speculate that
cardiovascular and pulmonary diseases, such as oxidative stress may contribute to the
atherosclerosis and emphysema/chronic abnormal structure and function of elastic
obstructive pulmonary disease (COPD), fibers in pathological conditions.
excessive degradation and/or inefficient repair of Elastic fibers are complex, insoluble elastic fibers results in compromised tissue extracellular matrix structures that are abundant function. New elastin synthesis occurs in these in tissues such as the heart, arteries, skin, and pathological conditions suggesting elastic fiber lungs. They provide architectural support, as repair mechanisms are activated. However, the well as the stretch and recoil required for normal integrity and organization of the elastic fibers is Copyright 2010 by The American Society for Biochemistry and Molecular Biology, Inc.
disrupted and the network discontinuous (10- Millipore Corporation (Bedford, MA). 14). These data suggest that there is aberrant Hypochlorous acid (HOCl), H2O2, uric acid assembly of newly synthesized TE into elastic (UA), lipoic acid (LA), E. coli lipopolysaccharide (LPS), N-formyl-Met-Leu- Oxidative stress has been implicated in the Phe (fMLP), and protease inhibitor cocktail were pathogenesis of several cardiovascular and purchased from Sigma-Aldrich (St. Louis, MO). pulmonary diseases. Oxidants can be generated The anti-elastin BA4 monoclonal antibody was by external factors such as cigarette smoke, or purchased from Abcam Inc (Cambridge, MA). internal factors such as inflammatory cells, and The anti-V5 antibody was purchased from mitochondrial respiration. These systems Invitrogen (Carlsbad, CA). The TRITC- produce various reactive oxygen and nitrogen conjugated anti-mouse, FITC-conjugated anti- intermediates, such as superoxide anion (O2¯), rabbit, and HRP-conjugated anti-mouse and hydrogen peroxide (H2O2), hydroxyl radical anti-rabbit secondary antibodies were purchased (•OH), nitric oxide (NO), nitrite (NO -2) and
from Jackson ImmunoResearch Laboratories peroxynitrite (ONOO-) (15-17). When free
(West Grove, PA). radicals exceed endogenous antioxidant Cell Culture—CHOKI cells stably capacity, they modify proteins, lipids and expressing a 6xHis-tagged fragment of human nucleic acids. For example, carbonyl formation Fbn-2 encompassing amino acids 1-1114 (7) on side chains of specific amino acids (Lys, Arg, were maintained in Ham's F-12 supplemented Pro, and Thr) occurs during oxidative stress, with 10% FBS, penicillin (100 U/ml), whereas 3-nitrotyrosine (N-Tyr) formation is a streptomycin (100 μg/ml), 4 mM L-glutamine, common modification that occurs as a result of hygromycin B (100 µg/ml), and zeocin nitration. These modifications have also been (100µg/ml). CHOKI cells stably expressing reported in many diseases, including COPD, V5/6xHis double-tagged full-length rat Fbln-4 or atherosclerosis and other cardiovascular diseases V5/6xHis double-tagged full-length rat Fbln-5 (kind gifts from Dr. H. Yanagisawa (9) were There is evidence that matrix proteins can be maintained in the same media. Human retinal modified by reactive oxygen species (ROS) pigmented epithelial ARPE-19 cells (American resulting in an alteration of protein structure and Type Tissue Culture, Rockville, MD) were function. For example, oxidative modifications maintained in DMEM supplemented with 10% to collagen cause a change in the elasticity of the FBS, penicillin (100 U/ml), streptomycin (100 skin, as well as stiffer and more brittle cartilage μg/ml), and 4 mM L-glutamine. (31,32). However, few studies have examined Purification of Proteins—Full-length bovine the effects of oxidation of elastin (33,34). We TE (35) and full-length mouse MAGP1 (36) hypothesized that oxidants generated during were expressed as 6xHis fusion proteins in M15 oxidative stress in pathological conditions can E. coli and purified as previously described modify newly synthesized TE and impair the using Ni-NTA agarose beads (Qiagen Inc, assembly of TE into elastic fibers. To test this Valencia, CA). The purified proteins were hypothesis, we investigated the effects of dialyzed against 50 mM glacial acetic acid, oxidizing agents on TE and the effects of lyophilized and further purified by reverse phase oxidized TE on different steps of elastic fiber high performance liquid chromatography. assembly. We found that oxidants modified TE Fractions containing TE or MAGP1 were pooled and that oxidized TE was not assembled into and lyophilized. The lyophilized proteins were resuspended in sterile water and subjected to amino acid analysis and immunoblot analysis EXPERIMENTAL PROCEDURES
using the appropriate anti-elastin or anti- Reagents and Antibodies—ONOO-, the anti-
The human Fbn-2 fragment, full-length rat N-Tyr polyclonal antibody, protein A/G plus- Fbln-4, and full-length rat Fbln-5 were purified agarose beads and the OxyBlot Protein from conditioned media of transfected stable Oxidation Detection Kit were purchased from CHOKI cell lines as previously described (7,9). Cells were maintained confluent for one week in System (Amersham Pharmacia, Piscataway, NJ), serum-free SFM4-CHO media (Hyclone, and subsequent autoradiography. Waltham, MA), and secreted Fbn-2 fragment, Isolation of Mouse Peritoneal Macrophages Fbln-4 and Fbln-5 were purified from the and Human Monocytes—Eight-week-old conditioned media using Ni-NTA agarose beads C57BL/6 mice obtained from Taconic Farms following the manufacturer's protocol. The (Germantown, NY) were housed in a pathogen- purified Fbn-2 fragment, Fbln-4, and Fbln-5 free animal facility under the veterinary care of were then dialyzed against buffer containing 50 the Department of Comparative Medicine at mM Tris (pH7.5), 150 mM NaCl, and 1 mM Washington University School of Medicine. EDTA and subjected to amino acid analysis and Resident peritoneal mouse macrophages were immunoblot analysis using an anti-Gly antibody isolated as previously described (38). Saline was for Fbn-2 (7) or the anti-V5 antibody for Fbln-4 injected into the peritoneal space and the lavage fluids of 3-4 mice were pooled. The ONOO-, HOCl, and H
predominance of macrophages in the fluid 2O2 Exposure of TE— Aliquots of ONOO- in 0.3 N NaOH were stored
(>95%) was confirmed by Wright-stained at -80°C, and immediately prior to each assay, cytospins. Experiments were performed on at the concentration of ONOO- was determined
least four independent cell isolations. All spectrophotometrically at 302 nm (εM= 1,670 M- procedures were approved by the Washington 1 cm-1) and diluted in 0.01 N NaOH. Aliquots of University School of Medicine Animal Studies HOCl were stored at -20°C, and immediately Committee and were performed in accordance prior to each assay, the concentration of HOCl with the Animal Welfare Act and the Guide for was determined spectrophotometrically at the Care and Use of Laboratory Animals. 292nm (εM= 1,670 M-1 cm-1) (37). An 8.8 M Human monocytes were isolated as stock of H2O2 was stored at room temperature. previously described (38). Briefly, 60 mL of HOCl and H2O2 were diluted in sterile water. TE whole blood was obtained by venipuncture from was diluted in Tris-buffered saline (50 mM Tris, healthy adult human volunteers and layered on a pH 7.5 and 150 mM NaCl), and ONOO-, HOCl,
Histopaque (Sigma-Aldrich, St. Louis, MO) or H2O2 was added while vortexing. The gradient. Cells that sedimented at the interface solutions were incubated at room temperature were collected, washed several times with PBS, for 5 min. The pH was monitored to make sure and the percentage of monocytes assessed by that each reaction was performed at neutral pH. Wright-stained cytospins (38). Experiments Detection of Oxidative and Nitrosative were performed on at least four independent cell Protein Modifications—Oxidation of TE was isolations. This study was reviewed and detected by using the OxyBlot Protein Oxidation approved by the Washington University School Detection Kit (Millipore) according to the of Medicine Human Studies Committee. manufacturer's recommendations. Treated and Volunteers gave informed, written consent. untreated TE was derivatized with 2,4- Activation of Monocytes and dinitrophenylhydrazine (DNP), separated on an Macrophages—Macrophages and monocytes 8% SDS polyacrylamide gel under reducing (1x105) were diluted in DMEM containing
conditions, transferred to a Immobilon-P PVDF protease inhibitor cocktail and incubated with 1 membrane (Millipore), and detected by µg TE in the presence or absence of 10 µM immunoblotting using an anti-DNP antibody and fMLP plus 100 µg/ml LPS for 15 min at room an HRP–conjugated anti-rabbit  secondary temperature. In some experiments, 1 mM uric antibody. Nitration of TE was detected by acid, the ONOO--specific scavenger, 1 mM
separation of treated and untreated TE on an 8% lipoic acid, the HOCl-specific scavenger, or both SDS polyacrylamide gel under reducing were incubated with the cells prior to the conditions, transfer onto PVDF membranes, and addition of activators. After 15 min incubation, incubation of the membranes with an anti-N-Tyr the solutions were centrifuged to pellet the cells antibody, followed by HRP–conjugated anti- and the supernatants containing TE was rabbit secondary antibody. Blots were developed analyzed for oxidative and nitrosative using the ECL Plus Western Blotting Detection modifications as described above. TE was incubated with cells in the absence of activators immunofluorescence microscopy using the anti- as a negative control. elastin BA4 monoclonal antibody, followed by TRITC-conjugated anti-mouse secondary were scanned and analysis of the optical antibody. Nitration of TE was confirmed using densities was performed using the public domain the rabbit anti-N-Tyr antibody, followed by a NIH Image program (developed at National FITC-conjugated anti-rabbit secondary antibody. Institutes of Health and available at Coacervation Assay—Coacervation was http://rsb.info.nih.gov/nih-image/). The data carried out as previously described (41). TE was represents the mean ± SE from at least five exposed to ONOO- as described above and
independent experiments. All quantification was coacervation was monitored by increasing the done on exposures in which individual bands solution temperature at a rate of 1°C/min with were not yet saturating. constant stirring at 1000 rpm and measuring the absorbance at 440 nm using a Shimadzu UV- Immunoprecipitation of TE was performed as 2401PC UV-visible recording mentioned elsewhere (39). Briefly, unmodified spectrophotometer equipped with temperature and ONOO--modified TE were incubated with
controller. TE exposed to equal amount of the anti-N-Tyr antibody in buffer containing 50 NaOH was used as a carrier alone control. mM Tris-HCl (pH 7.5), 150 mM NaCl, and 1% Desmosine Analysis—Crosslinking of TE NP-40 at 4°C overnight with gentle agitation. was measured by the formation of desmosine. Protein A/G plus-agarose beads were added to ARPE-19 cells were plated on 100 mm dishes each tube and incubated for 1 hr at 4°C with and maintained confluent for 8-10 days, after gentle agitation. The immune complex was which oxidized and unoxidized TE were added centrifuged and the supernatant containing at a final concentration of 0.1 mg/ml in normal unbound, unmodified TE was collected. The growth media described above. Cells were beads with bound, nitrated TE were washed incubated with the TE for 16 hr at 37°C, washed three times with 50 mM Tris-HCl (pH 7.5) with with PBS, and scraped into 1 ml water. The 400 mM NaCl and once with 10 mM Tris-HCl samples were centrifuged and the pellet was (pH 6.8). SDS-PAGE sample buffer containing hydrolyzed in 6 N HCl overnight at a constant β-mercaptoethanol was added to both temperature of 110°C. The desmosine content in supernatant and pellet and boiled for 10 min. hydrolysate was determined by Samples were adjusted for equal volume, radioimmunoassay (42,43) and normalized to separated on a 6% SDS polyacrylamide gel, and transferred onto Immobilon-PVDF transfer Solid-Phase Binding Assay—Solid-phase membranes (Millipore). Oxidized, binding of TE by Fbln-4, Fbln-5, Fbn-2, and immunoprecipitated and unoxidized, soluble TE MAGP1 was analyzed as previously described were detected using an anti-elastin antibody an (7,9). Flat-bottomed microtiter plates (Costar, HRP–conjugated anti-rabbit  secondary, and the NY) were coated with 1 µg/well of unmodified, ECL Plus Western Blotting Detection System, ONOO--modified, or HOCl-modified TE in 10
and subsequent autoradiography. mM carbonate buffer (pH 9.2) at 4°C overnight. In Vitro Elastic Fiber Assembly Assay— The plates were rinsed with PBS, blocked with Incorporation of exogenously added TE into the nonfat dry milk in PBS, and incubated with microfibrils of ARPE-19 cells was performed as soluble ligands (Fbn-2, Fbln-4, Fbln-5, or previously described (40). ARPE-19 cells were MAGP1) in blocking buffer at 37°C for 3 hr. In plated on four-well chamber culture slides and the case of Fbln-4 and Fbln-5, binding was maintained at confluency for 8-10 days, after carried out in the presence of 2 mM CaCl2. The which oxidized and unoxidized TE were added plates were then washed with PBS and incubated at a final concentration of 0.1 mg/ml in normal with primary antibody (anti-V5 antibody for growth media described above. Cells were Fbln-4 and Fbln-5, anti-Gly antibody for Fbn-2 incubated for 1 hr or 16 hr at 37°C and then (7); and anti-MAGP1 antibody for MAGP1 (36) fixed with ice-cold methanol. TE deposition for 2hr at room temperature, followed by HRP- onto pre-existing microfibrils was detected by conjugated secondary antibody. Binding of ligands was quantified using the ABTS activated inflammatory cells could modify TE. Peroxidase Substrate System (KPL, Purified TE was incubated with resident mouse Gaithersburg, MD) and measuring the peritoneal macrophages or human monocytes in absorbance at 410 nm. Equal coating of oxidized the absence or presence of LPS and fMLP, and unoxidized TE of the microtiter plate was which will activate the cells. Since activated confirmed using the BA4 anti-elastin antibody monocytes and macrophages produce proteases followed by HRP-conjugated anti-mouse that could degrade TE as well as ROS/RNS in secondary antibody (data not shown). response to LPS and fMLP, the incubation was Statistical Analysis—All statistical analysis performed in the presence of protease inhibitor was performed with the SPSS 13 program. cocktail. Incubation of TE with monocytes (Fig.
Paired Student's t test was used to analyze the 2A) or macrophages (Fig. 2B) alone failed to
relationship between unmodified and modified induce carbonyl formation of TE. In contrast, in conditions. Data are representative of at least the presence of LPS and fMLP, activated three independent experiments expressed as monocytes and macrophages induced the mean ± standard error. A P value of less than oxidation of TE, as determined by OxyBlot 0.05 is considered significant. To determine which ROS/RNS produced by the activated cells induced the carbonyl formation of TE, cells were activated in TE is a Substrate for Oxidation and presence of uric acid (UA), a ONOO--specific
Nitration by Reactive Oxygen and Nitrogen scavenger, lipoic acid (LA), a HOCl-specific (ROS/RNS) Species—Elastin synthesis in normal scavenger, or both. A significant reduction in the adult tissues is negligible, however, in aging and oxidation of TE was observed when either UA several cardiovascular and pulmonary diseases, or LA was present in both macrophage and elastin synthesis is reinitiated in attempts to monocyte cells ( 50% decrease, p<0.05). repair damaged elastic fibers (10-14). Since Oxidation of TE was further reduced when cells elastogenesis occurs in an oxidative were activated in the presence of both UA and environment, we examined whether TE becomes LA ( 80% decrease, p<0.05). This reduction modified by exposure to ROS/RNS. Purified was not due to precipitation and/or degradation recombinant TE was exposed to ONOO-, HOCl,
of TE in the presence of UA or LA, as equal or H2O2 and modifications to TE were examined amounts of TE were detected in each lane by immunoblot analysis. Concentrations of following reprobing of the blot with the anti- oxidants used were in a range similar to that elastin antibody (Fig. 2). These data suggest that
produced by inflammatory cells (44-46). ONOO-
activated macrophages and monocytes release exposure of TE induced N-Tyr (Fig. 1A) and
both ONOO- and HOCl, which in turn can
carbonyl formation (Fig. 1B) of TE in a dose-
oxidatively modify TE. dependent manner. Neither oxidation nor Oxidized TE is not Assembled into Elastic nitration of TE was detected in unmodified Fibers—To determine whether oxidative samples. Exposure of TE to HOCl also induced modifications of TE alter elastic fiber assembly, carbonyl formation (Fig. 1C), but H2O2
we used an in vitro model system for elastic exposure did not (Fig. 1D). Exposure to ONOO-,
fiber assembly (40). ARPE cells secrete and HOCl or H2O2 did not induce degradation or organize microfibrillar components, including precipitation of TE, as shown by anti-elastin blot multiple isoforms of fibulins (Fbln), fibrillins (Fig. 1). These data indicate that ONOO- and
(Fbn), and matrix-associated glycoproteins HOCl induces oxidative and nitrosative (MAGPs), into the extracellular matrix, but do modifications to TE. not produce TE. However, when TE is provided Activated Macrophages and Monocytes in the culture medium, the TE is assembled with Oxidatively Modify TE via Release of ONOO-
microfibrils to form elastic fibers, which can be and HOCl—Since injury-induced elastogenesis detected by immunofluorescence using an anti- often occurs in inflammatory conditions, we elastin antibody. Consistent with previous examined whether ROS/RNS generated by findings (40), deposition of unmodified TE into the microfibril-rich matrix of ARPE cells occurs crosslinking. ARPE cells incubated with ONOO-
first as small globules at 1 hr (Fig. 3A) and then
-modified TE showed significantly decreased redistributes into elastin-containing desmosine/total protein ratio ( 45% reduction,
microfilaments over time (Fig. 3B). Similar to
p<0.05) compared with treatment with unmodified TE, ONOO--modified TE was
unmodified TE (Fig. 4B). Supporting the
deposited on the microfibril-rich matrix as immunofluorescence data (Fig. 3), these results
globules at 1hr after addition to ARPE cells suggest that oxidized TE has decreased ability to (Fig. 3D). In contrast to unmodified TE,
assemble into microfilaments. however, ONOO--modified TE organized into
Oxidation of TE Inhibits its Association with larger globules (Fig. 3E) after 16 hr incubation.
Fbln-4 and Fbln-5—Fbln-4 and Fbln-5 interact Incubation of unmodified or ONOO--modified
with TE and facilitate crosslinking of TE TE in the absence of cells resulted in a fairly monomers to form aggregates and deposition of uniform distribution of TE with few small aggregates onto microfibrils. To determine particulates (data not shown). These data suggest whether oxidative modifications of TE alter its that the formation of larger globules of ONOO--
interactions with Fbln-4 and Fbln-5, we modified TE was cell-mediated and not merely examined the binding of Fbln-4 and Fbln-5 with due to enhanced precipitation of oxidized TE. unmodified and ONOO--modified TE using a
Staining with anti-N-Tyr antibody was solid phase binding assay. Both ONOO--
performed to confirm the modification induced modified and unmodified TE bound to the by ONOO- (Fig. 3F), which is absent in
microtiter plates as determined using an anti- unmodified sample (Fig. 3C). Similar results
elastin antibody (data not shown). Fbln-4 bound were obtained when TE was modified with to unmodified TE in dose dependent manner. HOCl (Supplemental Fig. 1). These data
However, Fbln-4 showed a significant reduction suggest that oxidative modifications of TE ( 35% decrease, p<0.005) in binding to ONOO--
prevent its assembly into elastic fibers. modified TE as compared to that of unmodified Oxidation of TE Decreased the optimal TE at 5 μg/well (Fig. 5A). Fbln-5 showed a
Coacervation Temperature—During elastic fiber significant reduction ( 50% decrease, p<0.005) assembly, TE monomers undergo self- in binding to ONOO--modified TE as compared
association through interactions between to that of unmodified TE at 5 μg/well (Fig. 5B).
hydrophobic domains of TE to form aggregates Similar results were obtained when TE was through a process called coacervation. These modified with HOCl (Fbln-4, 35% reduction; aggregates are then deposited on pre-formed Fbln-5, 50% reduction) (data not shown). There microfibril to form elastic fibers. Since oxidized was minimal binding of Fbln-4 and Fbln-5 to TE formed large aggregates and did not form wells coated with non-fat milk alone. These data elastic fibers (Fig. 3), we examined whether
suggest that oxidation of TE prevents elastic oxidative modifications of TE alter the process fiber assembly, in part by altering interactions of coacervation. Similar to previous studies (47) with Fbln-4 and Fbln-5. unoxidized recombinant bovine TE coacervated Oxidation of TE Inhibits Binding with Fbn-2 at 42°C (Fig. 4A). However, oxidation of TE
but not with MAGP1—Microfibrils are induced by ONOO- exposure reduces the
composed of Fbn, MAGPs and several other coacervation temperature by almost 10°C (Fig.
components. Others and we have shown that TE 4A). These data suggest that oxidation of TE
directly interacts with N-terminal of Fbn-2 and increased the propensity for self-aggregation. full-length MAGP1 (7,48) and that these Oxidative Modifications of TE Inhibit interactions play a role in elastic fiber assembly. Crosslinking—Elastic fiber assembly involves To further decipher the mechanism of inhibition crosslinking of soluble TE monomers into of elastic fiber assembly by oxidation of TE, we insoluble functional polymers. To determine examined whether oxidative modifications of TE whether oxidative modifications affect alter its interaction with Fbn-2 and MAGP1 crosslinking of Lys residues of TE, we used the using a solid phase binding assay. Fbn-2 bound in vitro model system for elastic fiber assembly to unmodified TE in dose dependent manner. and measured desmosine formation, a marker of However, Fbn-2 showed a significant reduction ( 55% decrease, p<0.005) in binding to ONOO--
of TE monomers to form appropriately-sized, modified TE as compared to that of unmodified larger aggregates and mediate the deposition of TE at 12 μg/well (Fig. 6A). Similar results were
the TE aggregates onto pre-existing microfibrils obtained when TE was modified with HOCl, but (step 2). Through interactions with microfibril to a lesser degree ( 25% reduction) (data not components, the TE aggregates become aligned shown). In contrast, MAGP1 bound equally to with precise positioning so that crosslinking unmodified and ONOO--modified (Fig. 6B) or
domains are juxtaposed. Finally, LOX facilitates HOCl-modified TE (data not shown). There was crosslinking and the generation of mature elastic minimal binding of Fbn-2 or MAGP1 to wells fibers (step 3) (8,49,50). At each of these steps, coated with non-fat milk alone. These data critical amino acids in TE have been identified, suggest that oxidation of TE alters its interaction many of which could be susceptible to oxidative with critical components of microfibrils, and modifications and thus could affect elastic fiber thus prevents elastic fiber assembly. assembly at various steps. Oxidative Modifications to TE Promote DISCUSSION
Coacervation—An initial step in elastic fiber assembly is coacervation, which takes place Many studies have focused on elastic fiber through the interaction between the hydrophobic destruction as the critical process leading to domains of TE, such as repetitive sequences of inflammatory and degenerative changes in GVGVP, GGVP, GVGVAP, and PGAIPG (Fig.
several cardiovascular and pulmonary diseases. 7, step 1). In addition the process of
Our studies suggest that oxidative modification coacervation is highly influenced by the overall of TE also plays a role in the development of charge state of the TE molecule (2,51-54). For these diseases by preventing elastic fiber example, the interaction of negatively charged assembly and repair. We found that TE is molecules such as GAGs with positively modified by ONOO- and HOCl released by
charged Lys residues of TE molecules has been activated macrophages and monocytes, and that shown to promote coacervation at lower modified TE was unable to incorporate into pre- temperatures by interfering with the charge existing microfibrils and thus cannot assemble interactions (2,25, 50,55,56). We speculate that into elastic fibers. In these studies, we detected oxidation neutralizing the Lys residues of TE, N-Tyr and carbonyl formation on Lys, Arg, Pro, allowing the hydrophobic domains to more or Thr (Fig. 1 & 2). However we cannot rule out
easily interact and thus promote coacervation at the possibility that modifications to other amino lower temperature. acid residues could occur during oxidative It has been postulated that Fbln-4 and/or stress. TE contains several amino acids that are Fbln-5 interactions with TE regulate the size of potentially susceptible to oxidative modification, the TE aggregates and play a role in the including Tyr, Lys, Arg, Pro, Thr, Cys, Met, deposition of aggregates onto microfibrils by Val, Leu, and Phe residues (1). Thus, forming a ternary complex with TE and Fbns in modifications to any of these residues could alter the microfibrils (8,57,58). Oxidation of TE the function of TE and contribute to the aberrant reduced the interactions with Fbln-4 and Fbln-5 elastic fiber assembly seen in several pathologic (Fig. 5) and resulted in the formation of larger
conditions. Additional studies using mass aggregates (Fig. 3E). Thus, by blocking the
spectrometry are needed to determine the amino interactions between TE and Fblns-4/-5, acid residues that are modified by ROS/RNS, oxidative modifications to TE could block and determine which of these modifications elastic fiber assembly by promoting uncontrolled impact elastic fiber assembly. coacervation of TE monomers and/or by Elastic fiber assembly is a multistep process disruption of the ternary complex required for (Fig. 7) in which TE monomers are secreted
deposition of TE onto the microfibrils. from elastogenic cells and self- Oxidative Modifications to TE Could Inhibit assemble/coacervate to form microaggregates Deposition onto Elastic Fibers—Another that interact with Fbln-4 and Fbln-5 (step 1). potential mechanism by which oxidative Fbln-4 and Fbln-5 help facilitate the crosslinking modifications to TE could alter elastic fiber assembly is that modifications to the C-terminal is present in several cardiovascular and domain of TE could affect deposition of TE pulmonary diseases, suggesting a lack of aggregates onto microfibrils (Fig. 7, step 2). The
efficient assembly/repair of elastic fibers where C-terminal domain of TE is highly conserved oxidative stress is elevated (10-12). Our results across species. Removal of C-terminal domain show that oxidative modification of TE alters reduces the ability of TE to assemble into elastic elastic fiber assembly in an in vitro elastic fiber fiber (4). This region contains two Cys residues assembly model. When unmodified TE is added that form a disulphide bond. Disruption of the in this system, it becomes incorporated into the disulphide bond reduces the ability of TE to pre-existing matrix deposited by the ARPE cells. assemble into elastic fiber (4,59). In addition, In contrast, when ONOO--modified TE is added,
the C-terminal region contains a positively- the oxidized TE remains as large aggregates and charged RKRK sequence, also shown to play a does not assemble into microfilaments. This role in elastic fiber assembly. Thus, oxidation of scenario may occur in vivo in settings of high the Cys, Lys and/or Arg residues in the C- oxidative stress and increased synthesis of TE, terminal domain could potentially be another as seen in several cardiovascular and pulmonary mechanism by which oxidation of TE prevents diseases. Just as likely to occur in vivo are elastic fiber assembly. oxidative modifications to the pre-existing Oxidative Modifications to TE Inhibits elastic fibers, which too could impact elastic Crosslinking—During crosslinking of TE, four fiber repair. Using our in vitro model system, we Lys residues in KA (Lys-Ala) crosslinking found that when the pre-existing ARPE matrix domains are aligned to be in close proximity for was exposed to ONOO- prior to the addition of
processing by LOX to form tetrafunctional unoxidized, "newly synthesized" TE, the crosslinks of desmosine or isodesmosine (60). unmodified TE was unable to incorporate into Hydrophobic amino acid residues (Tyr, Phe, Ile, the oxidized matrix (data not shown). This or Leu) next to the Lys residues play a critical observation suggests that in addition to role in maintaining the optimal modifying TE, oxidants generated during microenvironment for proper crosslinking (8, pathological condition can also modify other 47-52) (Fig. 7, step 3). We found that oxidative
proteins involved in elastic fiber assembly, such modification of TE inhibits desmosine formation as LOX, Fblns, Fbns, and might contribute in (Fig. 4B). There are several possible
the production of abnormal elastic fibers. explanations of how oxidation of TE resulted in In summary, this study was designed to decreased crosslinking: first, oxidative investigate the effects of oxidative stress on the modifications to the Lys residues of TE could assembly of TE into elastic fibers. We found that render the Lys residues incapable of TE is susceptible to oxidative modifications by crosslinking; second, juxtaposed Tyr or Phe ONOO- and HOCl released from activated
residues could become modified, resulting in monocytes and macrophages, and that oxidized failure of alignment of the crosslinking domains TE cannot assemble into elastic fibers, at least in for crosslinking; and third, oxidation of TE part by inhibiting critical interactions with could inhibit crosslinking by interfering with the microfibrillar components, including Fbln-4/-5 formation of the ternary complex with Fblns-4 and Fbn-2, required for elastic fiber formation. (Fig. 5) and Fbns (Fig. 6A), and thus prevent of
We believe that these studies provide new elastic fiber assembly. insights into mechanisms by which abnormal Potential Consequences of Oxidative Stress elastic fibers are generated, which in turn play a on Elastic Fiber Assembly—Evidence of role in the development of various abnormal assembly of newly synthesized elastin cardiovascular and pulmonary diseases. REFRENCES
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*We would like to give our sincere thanks to Dr. Robert M. Senior (Washington University, St. Louis) for critical reading of the manuscript. Dr. Hiromi Yanagisawa (University of Texas Southwestern Medical Center, Dallas) is acknowledged for kindly providing us CHO cells expressing Fbln-4 and Fbln-5. This work was supported by the Alpha-1 Foundation, National Institutes of Health (P50HL084922), and the Alan A. and Edith L. Wolff Charitable Trust/Barnes-Jewish Hospital Foundation.

The abbreviations used are: TE, tropoelastin; ROS, reactive oxygen species; RNS, reactive nitrogen
species; ONOO-, peroxynitrite; HOCl, hypochlorous acid; N-Tyr, 3-nitrotyrosine; COPD, chronic
obstructive pulmonary disease; Fbln, fibulin; Fbn, fibrillin; MAGPs, microfibril associated glycoproteins; LOX, lysyl oxidase; LPS, lipopolysaccharides; fMLP, N-formyl-Met-Leu-Phe; UA, uric acid; and LA, lipoic acid. FIGURE LEGENDS
Fig. 1. Tropoelastin (TE) is modified by reactive oxygen and nitrogen species. Recombinant bovine TE
(1µg) was exposed to indicated concentrations of ONOO- (A and B), HOCl (C), or H2O2 (D) and
subjected to immunoblot analyses for nitrosative modifications using an anti-nitrotyrosine (N-Tyr)
antibody (A) and oxidative modifications using OxyBlot protein oxidation detection kit (B-D). The same
membranes were also probed with an anti-elastin antibody to control for loading. Data are representative
of at least three independent experiments.
Fig. 2. Activated monocytes and macrophages oxidatively modify TE via release of ONOO- and HOCl.
Mouse peritoneal macrophages (A) and human monocytes (B) were incubated with TE in the absence or
presence of combination of LPS (100 µg/ml) and fMLP (100 mM). Activation was also performed in the
presence of the ONOO--specific scavenger uric acid (UA), the HOCl-specific scavenger lipoic acid (LA),
or both scavengers. Carbonyl formation on TE was detected by OxyBlot analysis. The same membranes
were also probed with an anti-elastin antibody to control for loading. The immunoblots were scanned and
quantified using the NIH image program. The data represents the mean ± SE from at least five
independent experiments. *P<0.05 compared to cells activated with LPS and fMLP.
Fig. 3. Oxidative modification of TE prevents elastic fiber assembly. ARPE cells were incubated with 100
µg/ml unmodified (A-C) and ONOO--modified (D-F) TE for 1 hr (A, D) or 16 hr (B-C, E-F).
Incorporation of TE in the pre-existing microfibrillar matrix was detected by immunofluorescence using
an anti-elastin antibody. N-Tyr modification of TE induced by ONOO- exposure was confirmed by
immunostaining using the anti-N-Tyr antibody (C, F). Images (40x) are representative of at least five
independent experiments.
Fig. 4. Oxidative modification of TE promotes coacervation, but decreases crosslinking. (A) Oxidized and
unoxidized TE was dissolved in 50 mM Tris, 150 mM NaCl and kinetics of coacervation was monitored
at 400 nm. (B) ARPE cells were maintained at confluency for 8-10 days and then incubated with 100
µg/ml unmodified or ONOO--modified TE for 16 hr. Crosslinking of TE was determined by desmosine
analysis. Desmosine was quantified by radioimmunoassay (42,43) and total protein was determined by
amino acid analysis. Results are displayed as the mean ratio of pmol desmosines/mg protein of at least
five independent experiments ± SE. *P<0.05 compared to unmodified TE.
Fig. 5. Oxidative modification of TE reduced binding to fibulin-4 (Fbln-4) and fibulin-5 (Fbln-5). A 96-
well non-tissue culture plates coated with 1 µg/well of unmodified (UnOx-TE; closed circle) or ONOO--
modified (Ox-TE; open circle) were incubated with indicated concentrations of V5-tagged Fbln-4 (A) or
Fbln-5 (B) at 37°C for 3 hr. Following washing, bound Fbln-4 or Fbln-5 was detected using an anti-V5
antibody, HRP-conjugated secondary antibody, and the ABTS Peroxidase Substrate System, and
measuring the absorbance at 410 nm. Data represents the mean of at least three independent experiments
done in duplicate ± SE. *P<0.05, **P<0.005 compared to unmodified TE.
Fig. 6. Oxidative modification of TE reduced binding to fibrillin-2 (Fbn-2) but not to MAGP1. A 96-well
non-tissue culture plates coated with 1 µg/well of unmodified (UnOx-TE; closed circle) or ONOO--
modified (Ox-TE; open circle) were incubated with indicated concentrations of Fbn-2 fragment (7) (A) or MAGP1 (B) at 37°C for 3 hr. Following washing, bound Fbn-2 or MAGP1 was detected using an anti-Gly antibody for Fbn-2 (7) or anti-MAGP1 antibody, HRP-conjugated secondary antibody, and the ABTS Peroxidase Substrate System, and measuring the absorbance at 410 nm. Data represents the mean of at least three independent experiments done in duplicate ± SE. *P<0.05, **P<0.005 compared to unmodified TE. Fig 7. Model of elastic fiber assembly. (1) TE is transported to assembly sites on the plasma membrane where it self assembled via coacervation into aggregates that are cross-linked by a LOX. Hydrophobic domains of TE containing Pro residue assist in the process of coacervation. Interaction with Fbln-4 and/or -5 may facilitate cross-linking or possibly help limit the size of the TE aggregates. (2) The aggregates are then transferred to extracellular microfibrils, which interact with the cell through integrins. Fbln-4 and/or -5 assist the transfer of elastin aggregates on to the microfibril while C-terminal domain of TE that contains Cys and terminates into Lys residues direct its association with microfibrils. (3) Elastin aggregates on the microfibril align and are further cross-linked by LOX to form the mature elastic fiber. Tyr, Phe and Leu residues contribute in the alignment of TE aggregates on microfibril.

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